Foodborne Disease Handbook Second Edition, Revised and Expanded Volume 4: Seafood and Environmental Toxins
edited by
Y. H. Hui Science Technology System West Sacramento, California
David Kitts University of British Columbia Vancouver, British Columbia, Canada
Peggy S. Stanfield Dietetics Resources Twin Falls, Idaho
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D E K K E R
MARCEL DEKKER, INC.
NEWYORK BASEL
ISBN: 0-8247-0344-8
This book is printed on acid-free paper. Headquarters Marcel Dekker, Inc. 270 Madison Avenue, New York. NY 10016 tell 212-696-9000; fax: 212-685-4540 Eastern Hemisphere Distribution Marcel Dekkcr AG Hutgasse 4, Postfach 812, CH-4001 Basel, Switzcrland tel: 41-61-261-8482: fax: 41-61-261-8896 World Wide Web http:llwww.dekker.com
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Introduction to the Handbook
The Foodborne Disease Handbook, Second Edition, Revised and Expanded, could not be appearing at a more auspicious time. Never before has the campaign for food safety been pursued so intensely on so many fronts in virtually every country around the world. This new edition reflects at least one of the many aspects of that intense and multifaceted campaign: namely, that research on food safety has been very productive in the years since the first edition appeared. The Handbook is now presented in four volumes instead of the three of the 1994 edition. The four volumes are composed of 86 chapters, a 22% increase over the 67 chapters of thefirstedition. Much of the information in thefirst edition has been carried forward to this new edition because that information is still as reliable and pertinent as it was in 1994. This integration of the older data with the latest research findings gives the readera secure scientific foundation on which to base important decisions affecting the public’s health. We are not so naive as to think that only scientific facts influence decisions affecting food safety. Political and economic factors and compelling national interests may carry greater weight in the minds of decision-makers than the scientific findings offered in this new edition. However,if persons in the higher levelsof national governments and international agencies, such as the Codex Alimentarius Commission, the World Trade Organization, the World Health Organization, and the Food and Agriculture Organization, who must bear the burden of decision-making need and are willingto entertain scientific findings, then the information in these four volumes will serve them well indeed. During the last decade of the previous century, we witnessed an unprecedentedly intense and varied program of research on food safety, as we have already noted. There are compelling forces driving these research efforts. The traditional food-associated pathogens, parasites, and toxins of forty years ago still continue to cause problems today, but newer or less well-known species and strains present extraordinary challenges to human health. These newer threats may be serious even for the immunocompetent, but for the immunocompromised they can be devastating. The relative numbers of the immunocompromised in the world population are increasing daily. We include here not just those also the elderly; thevery young; affected by the human inmunodeficiency virus (HIV), but the recipients of radiation treatments, chemotherapy, and immunosuppressive drugs; paiii
iV
Introduction to the Handbook
tients undergoing major invasive diagnostic or surgical procedures; and sufferers of debilitating diseases such as diabetes.To this daunting list of challenges must be added numerous instances of microbial resistance to antibiotics. Moreover, it is not yet clear how the great HACCP experiment will play out on the worldwide stage of food safety. Altruism and profit motivation have always made strange bedfellows in the food industry. It remains to be seen whether HACCP will succeed in wedding these two disparate motives into a unifying force for the benefitofall concerned-producers, manufacturers, retailers, and consumers. That HACCP shows great promise is thoroughly discussed in Volume 2, with an emphasis on sanitation in a public eating place. All the foregoing factors lend a sense of urgency to the task of rapidly identifying toxins, species, and strainsof pathogens and parasites as etiologic agents, and of determining their roles in the epidemiology and epizootiology of disease outbreaks, which are described in detail throughout the Foodborne Diseuse Handbook. It is very fortunate for the consumerthat there existsin the food industry a dedicated cadre of scientific specialists who scrutinize all aspects of food production and bring their expertise to bear on the potential hazards they know best. A good sampling of the kinds of work they do is contained in these four new volumes of the Hmdbook. And the benefits of their research are obvious to the scientific specialist who wants to learn even more about food hazards, to the scientific generalist who is curious about everything and who will be delighted to find a good sourceof accurate, up-to-date information, and to consumers who care about what they eat. We are confident that these four volumes will provide competent, trustworthy, and timely information to inquiring readers, no matter what roles they may play in the global campaign to achieve food safety.
Y. H. Hui J. Richard Gorhrrm David Kitts K. D. Murre11 Wui-Kit Nip Merle D. Pierson Syed A. Scrttar R. A. Smith David G. Spoerke, Jr. Peggy S. Stnnjield
Preface
Most people prefer to think of marine environments farthest removed from human settlements as being pristine and pure; however, much of the ocean is under attack on several fronts. One of the foci of this fourth volume of the Foodborne Disease Hundbook is the topic of pollutants, which are being deposited in the oceansin unprecedented quantitiesas raw sewage (often with viable pathogens and parasites), industrial effluents containing toxic or radioactive chemicals, trash and garbage, pesticides in runoff from crop lands, and top soil, one of our most valuable natural resources. Some of these potentially harmful organisms and chemicals enter the food webs of freshwater and marine organisms. Someof these organisms are harvested for human food and have the capacity and tendency to concentrate and sequester in their bodies many of the chemicals and pathogens present in the aquatic environments. The methods of how harmful organisms or hazardous chemicals are detected, analyzed, and identified, and how they can affect human health, are thoroughly reviewed in this volume. In contrast, some marine organisms do not collect toxins from the environment but rather produce their own toxins as a part of normal metabolic processes. When such fish are usedas human food, the result can belife-threatening.Thisvolumediscussesthe species and toxins of importance, analytical methods, and epidemiological aspects of intoxication. The seafood processor was one of the first food industries required to implement the HACCP principles. The development of the seafood HACCP program and its benefits to the consumer are discussed in this volume. A cloud of controversy hovers over the concept of food irradiation. In this volume we present informative facts needed for the reader to come to an enlightened conclusion about the safety of food irradiation. We caution that no matter how successful irradiation might be, mishandling after irradiation treatment makes the product unsafe. We also discuss the continuing need to adhere to HACCP principles and essential sanitary standards that make it possible for HACCP to work. Many, perhaps even most, of the food toxicity issues addressed in this volume are subtle and unknown to most of us. For example, the use of food additives, radioactive isotopes, pesticide chemicals applied to our crops, toxicants occurring naturally in some of our foods, and the therapeutic and growth-promoting drugs fed to domestic animals V
Vi
Preface
are important topics of food toxicology. In addition, plasticizers i n many kinds of vessels used for food storage and handling mayin some instances be a source of toxic chemicals. Most consumers are quite unaware of rat hairs, beetle setae, and fly eggs in their food, and less aware of what effect, if any, such adulterants might have on their health. The editors and contributors to the fourth volume of the Foodborne Disease H L U I ~ book have provided an abundance of facts and supporting explanatory information enabling readers to make confident decisions about their health. Moreover, all sectors of the food industry have the tools needed to apply the sanitary practices and HACCP-driven safeguards that will result in the prevention of foodborne diseases and helpto make available wholesome foods for all consumers around the world. Volume 4 is a composite of current information and policies that enable proper risk-assessment decisions to be made regarding potential food toxicants derived naturally in the environment or through agricultural production and food processing practices.
Y. H . Hui David Kitts Peggy S. Stcrt1jieId
Contents
Itltroduction to the Handbook Pwfrtce Contributors Contents of Other Vnlurnes
...
ill
I’
Xi
...
.v11 l
I. Poison Centers 1.
SeafoodandEnvironmentalToxicantExposures:TheRoleofPoisonCenters David G. Spoerke, Jr.
11.
Seafood Toxins
2.
Fish Toxins Bruce W. Hctlstertd
1
23
3. Other Poisonous Marine Animals Bruce W. Hdstead
51
4. Shellfish Chemical Poisoning L w d m E. Llewellyn
77
5. Pathogens Transmitted by Seafood
109
Russell P. Herwig
6. Laboratory Methodology for Shellfish Toxins Dcwid Kitts
183
Vii
Viii
7.
Contents
Ciguatera Fish Poisoning Yoshitsugi Hokama and Joanne S. M. YoshikLrwa-Ebesu
209
8. Tetrodotoxin Joanne S. M. Yoshikawa-Ebesu, Yoshitsugi Hokama, and Tamao Noguchi
253
9.
287
Epidemiology of Seafood Poisoning b r a E. Fleming, Dolores Katz, Judy A. Bean, and Roberta Hammond
10. The Medical Management of Seafood Poisoning Donna Glcrd Blythe. Eileen Hack, Giavanni Washington, and b r a E. Fleming
31 1
11. The U S . National Shellfish Sanitation Program
32 l
Rebecca A. Reid and Timothy D. Durance 12. HACCP, Seafood, and the U.S. Food and Drug Administration Kim R. Young, Miguel Rodrigues Kamat, and George Perry Hoskin
339
111. Environmental Toxins
13. Toxicology and Risk Assessment Donald J. Ecobichon
347
14. Nutritional Toxicology David Kitts
379
15. Food Additives Laszlo P. Somogyi
447
16. Analysis of Aquatic Contaminants Joe W. Kiceniuk
517
17. Agricultural Chemicals Debra L. Browning and Carl K. Winter
537
18. Radioactivity in Food and Water Hank Kocol
557
19. Food Irradiation Hank Kocol
57 1
20.
Drug Residues in Foods of Animal Origin Austin R. L m g and Jose E. Roybol
579
21.
Migratory Chemicals from Food Containers and Preparation Utensils Yvonne V. Yutrn
599
Contents
ix
22. FoodandHardForeignObjects:AReview J. Richard Gorham
617
23.Food,Filth,andDisease:AReview J. Richard Gor-ham
627
24.
Ides
Food FilthandAnalyticalMethodology:ASynopsis J. Richnrd Gorhnm
639
645
This Page Intentionally Left Blank
Contributors
Judy A. Bean Children's Hospital of Cincinnati, Cincinnati, Ohio,and National Institute of Environmental Health Sciences (NIEHS) Marine and Freshwater Biomedical Sciences Center, University of Miami Rosensteil School of Marine and Atmospheric Sciences, Miami, Florida
Donna Glad Blythe Department of Epidemiology and Public Health, University of Miami School of Medicine, Miami, Florida Debra L. Browning Food Safe Program, Department of Food Science and Technology, University of California-Davis, Davis, California Timothy D. Durance Food, Nutrition, and Health, University of British Columbia, Vancouver, British Columbia, Canada Donald J. Ecobichon Department of Pharmacology and Toxicology, Queen's
Univer-
sity, Kingston, Ontario, Canada
Lora E. Fleming Department of Epidemiology and Public Health, University of Miami School of Medicine, and National Institute of Environmental Health Sciences (NIEHS) MarineandFreshwaterBiomedicalSciencesCenter,University of MiamiRosensteil School of Marine and Atmospheric Sciences, Miami, Florida
J. Richard Gorham
Department of Preventive Medicine, Uniformed Services University of the Health Sciences, Bethesda, Maryland
Eileen Hack Department of Epidemiology andPublicHealth,University School of Medicine, Miami, Florida Bruce W. Halstead
of Miami
InternationalBiotoxicologicalCenter,WorldLifeResearchInsti-
tute, Colton, California
Roberta Hammond State of Florida Department of Health, Tallahassee, Florida Russell P. Herwig School of Fisheries, University of Washington, Seattle, Washington xi
xii
Contributors
Yoshitsugi Hokama Pathology Department, JohnA. Burns School of Medicine, University of Hawaii, Honolulu, Hawaii George Perry Hoskin Office of Seafood, Division of Science and Applied Technology, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C.
Miguel Rodrigues Kamat Office of Seafood, Division of Programs and Enforcement Policy, Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration, Washington, D.C.
Dolores Katz Department of EpidemiologyandPublicHealth,UniversityofMiami School of Medicine,Miami,andState of FloridaDepartment ofHealth,Tallahassee, Florida
Joe W. Kiceniuk Department of Chemistry, Dalhousie University, Halifax, Nova Scotia, Canada
David Kitts
Food, Nutrition, and Health, University British Columbia, Canada
of British Columbia, Vancouver,
Hank Kocol* HealthPhysicist,Roseville,California Lyndon E. Llewellyn Australian Institute of Marine Science, Townsville, Queensland, Australia
Austin R. Long
Pacific Regional Laboratory Northwest, U.S. Food and Drug Administration, Bothell, Washington
TamaoNoguchi RebeccaA.Reid
Laboratory of Food Hygiene, Nagasaki University, Nagasaki, Japan Department of Fisheries and Oceans, Vancouver, British Columbia,
Canada
Jose E. Roybal AnimalDrugsResearchCenter,
U.S. Food and DrugAdministration,
Denver, Colorado
Laszlo P. Somogyi ConsultingFoodScientist,Kensington,California DavidG. Spoerke, Jr. BristleconeEnterprises,Denver,Colorado Giavanni Washington Department of Epidemiology and Public Health, University Miami School of Medicine, Miami, Florida
of
Carl K. Winter Department of Food Science and Technology, Universityof CaliforniaDavis, Davis, California
Joanne S. M. Yoshikawa-Ebesu Oceanit Test Systems, Inc., Honolulu, Hawaii Kim R. Young Office of Seafood, Divisionof Programs and Enforcement Policy, Center for Food Safety and Applied Nutrition,U.S. Food and Dmg Administration, Washington, D.C. Yvonne V. Yuan School of Nutrition,RyersonPolytechnicUniversity,Toronto,Ontario, Canada
* F o n w r c!ffi/itrtiorz: California Department of Health Services, Roseville, California.
Contents of Other Volumes
VOLUME 1: BACTERIAL PATHOGENS
I. Poison Centers 1.
The Role of U.S. Poison Centers in Bacterial Exposures David G. Spoerke, Jr.
11. Bacterial Pathogens 2 . Bacterial Biota (Flora) in Foods Jut1re.s M. Joy 3. Aerotmtzns hydrophiltr Carlos Aheytcr, Jr., Srmuel A. Palumbo, cltrd Gertrrd N. Steltnrr, Jr.
4. Update: Food Poisoning and Other Diseases Induced Kcrthleetl T. Rtrjkowski cwd Jcrtnes L. Smith
by Bacillus cereus
5. B r u c e l l ~ ~ Shirley M. Hullitrg nrtd Edward J. Youtrg 6.
Campylobncter jejutri Dorz A. Frtrtzco ntzd Charles E. Williatm
7.
Clostritliunr botulirwn Johtr W. Austitl erne1 Karen L. Dodds
8.
Clostridium pelfiingetzs Dorotl1.v M. Wrigley xiii
xiv
Contents of Other Voolumes
9. Eschericlritr coli Mcrrguerite A. Neil, Phillip I. Tarr, David N. Toylor, arld Mtrrcio Wolf 10. Listerirr rrrormytogerres
Cutherirre W. Dortnelly 11.
Bacteriology of Salrnorwllcr Robill C. Anderson crnd Richlrrd L. Ziprirl
12. Salmonellosis i n Animals Dmid J. Nisbet nrrd Richrrrd L. Zipritr
13. Human Salmonellosis: General Medical Aspects Richcrrd L. Zipritr trtrd Michrrel H. Hrme 14. Shigelltr
Allthotry T. Muurelli rrr~dKeith A. Lntrrpel 15.
Strrl~hyloc,occust1ureu.s Scott E. Mcrrtirr, Eric R. Myers, artd Jokrr J. I m d o l o
16. Vibrio cholertre Charles A. Kaysner rrnd June H. Wetherirlgtorl 17.
Vibrio prrrtrhtremolytic~~.s TuuTjyi Clmi t r r d Johrr L. Pace
18.
Vilwio vulnificus Arlc1er.v Dtrlsgntrr-d,Lise H@i,Debi Lirtkous, turd Jcmes D. Oliver
19.
Yersirritr Scott A. Mirlrrick, M i c h e l J. Smith, Steverr D. Wengant, mrd Peter Ferrg
111. Disease Surveillance, Investigation, and Indicator Organisms
20. Surveillance of Foodborne Disease EW[JII C. D. Todd 21.
22.
Investigating Foodborne Disease D d r L. Morse, Grrtllrie S. Birkhecrd, crrrd Jock J. Guwvich Indicator Organisms in Foods Jtrrtlrs
M. Jcry
xv
Contents of Other Volumes
VOLUME 2: VIRUSES, PARASITES, PATHOGENS, AND HACCP
I. Poison Centers 1.
The Role of Poison Centers in the United States Dmid G. Spoerke, Jr.
11. Viruses 2. Hepatitis A and E Viruses There,sa L. Cromearls, Michael 0. Favoro1~,Omnntr V. Naitrcrtr, Mtrrgo1i.s
trtld
Htrrold S.
3. Norwalk Virus and the Small Round Viruses Causing Foodborne Gastroenteritis Hrrzel Appleton
4. Rotavirus Syed A. Strttw, V. Sustrrl Spritrgthorpe, raid Jmorl A. Tetro 5.
Other Foodborne Viruses Syecl A. Sattrrr trtrci Jcrsotr A. Tetra
6. Detection of Human Enteric Viruses in Foods Lec~-A~Irr JtrYkus 7. Medical Management of Foodborne Viral Gastroenteritis and Hepatitis Su:trnne M. Matsui trnd Rtrmsey C. Cheurrg 8. Epidemiology of Foodborne Viral Infections Thomas M. Liithi
9. Environmental Considerations in Preventing the Foodborne Spread of Hepatitis A Syed A. Srrttar a t d Scrbtrh Bidawid 111. Parasites 10. Taeniasis and Cysticercosis Zbigrriew S. Pawlowski m d K. D. MLrrrell
11.
12.
Meatborne Helminth Infections: Trichinellosis William C. Crrnlpbell Fish- and Invertebrate-Borne Helminths
John H . Cross
xvi
Contents of Other Volumes
13. Waterborne and Foodborne Protozoa Rotlrrld Frryer. 14. Medical Management Prrul Pro& 15. Inmunodiagnosis of Infections with Cestodes Bruno Gottsteitl
16. Immunodiagnosis: Nematodes H . Ray Gamble
17. Diagnosis of Toxoplasmosis A I m M.Johnsow rrtld J. P. DuDey 18. Seafood Parasites: Prevention, Inspection, and HACCP Atw M.Adams n t d Debra D. DeVlieger
IV.
HACCP and the Foodservice Industries
19. Foodservice Operations: HACCP Principles 0. Peter Stlyder, Jr.
20. Foodservice Operations: HACCP Control Program 0. Peter Snyder, Jr. It1de.v
VOLUME 3: PLANT TOXICANTS
I. Poison Centers 1.
U.S. Poison Centers for Exposures to Plant and Mushroom Toxins Dovid G. Spoerke, Jr.
11. Selected Plant Toxicants
2. Toxicology of Naturally Occurring Chemicals in Food Ross C. Beier crnd Herbert N. Nigg 3. Poisonous Higher Plants Doreen Grcrce L m g crttd R. A. Smith 4. Alkaloids R . A. Smith
Contents of Other Volumes
5.
Antinutritional Factors Related to Proteins and Amino Acids It-vitl E. Liener
6. Glycosides W d t r r Mujcrk und Miclruel H . Berm
7. Analytical Methodology for Plant Toxicants Alister David Muir 8. Medical Management and Plant Poisoning Robert H. Poppetlgcr 9. Plant Toxicants and Livestock: Prevention and Management Michcrel H. R d p k s 111. Fungal Toxicants 10. Aspergillus
.&$a Kozakiewicz 11.
Clnviceps and Related Fungi Gretchen A. Kuldau and Charles W. Bacotl
12. Fusarium Walter F. 0. Murcrsrrs 13.
Perricilliutu John 1. Pitt
14.
Foodborne Disease and Mycotoxin Epidemiology Sara H d e Hen? and F. Xavier Bosclr
15.
Mycotoxicoses: The Effects of Interactions with Mycotoxins Heather A. Kosllitlsky. Adrietrtre Woytowich, crtrci George G. Kl1rrchatourirrn.s
16.
Analytical Methodology for Mycotoxins James K. Porter
17. Mycotoxin Analysis: Immunological Techniques Futl S. QILI 18.
Mushroom Biology: General Identification Features Drrvid G. Spoerke, Jr.
xvii
xviii
Contents of Other Volumes
19. Identification of MushroomPoisoning(Mycetismus),Epidemiology, and Medical Management David G. Spoerke, Jr. 20. FungiinFolkMedicineandSociety David G. Spoerke, Jr.
Seafood and Environmental Toxicant Exposures: The Role of Poison Centers
Epidemiology 1 AAPCC A. 2 B. Who staffs poison a center'? 3 C. Whattypes of callsarereceived? 4 D. How are calls handled? 5 E. What references are used'! 6 F. How are poisoncentersmonitoredfor quality? 7 G. Professional and public education programs 7 Related H. toxicology organizations 7 I. International affiliations IO J. Toxicology and poison center Web sites IO 11. PoisonInformationCenters intheUnitedStates 10 References 2 l I.
1.
EPIDEMIOLOGY
Epidemiological studies aid treatment facilities in determining risk factors and who becomes exposed, and in establishing the probable outcomes of various treatments. A few organizations have attemptedto gather such information and organizeit into yearly reports. The American Associationof Poison Control Centers (AAPCC) and some federal agencies work toward obtaining epidemiological information, while the AAPCC has an active role in assisting with the treatment of exposures Epidemiological studies assist government and industry in determining package safety, effective treatment measures, conditions of exposure, and frequency of exposure. Studies on seafood and environmental exposures provide information on the type of people most commonly involved in exposures. Are they children, adults at home, outdoorsmen, industrial workers, or blue-collar workers. Studies can also tell us which bacterial species are most commonly involved. What symptoms are seenfirst, what is the onset
Spoerke
2
of symptoms, and are theirany sequalae may also be determined and compared to current norms. A.
AAPCC
1. What Are Poison Centers and the AAPCC? The group in the United States concerned, on a daily basis, with potential poisonings due to household agents, industrial agents, biologics, and food poisoning (including seafood poisoning) is the AAPCC. This is an affliliation of local and regional centers that provides information to health care professionals and the lay public concerning all aspects of poisoning. These centers also refer patients to treatment centers. This group of affiliated centers is often supported by government and private funds, as well as industrial sources. in the late 1950s; the first is thought to have been in Poison centers were started the Chicago area. The idea caught on quickly andat the peak of the movement there were hundreds of centers throughout the United States. Unfortunately there were few or no standards for what could be called a poison center, including the type of staff, hours of operation, or information resources. One center may have hada dedicated staff of doctors, pharmacists, and nurses trained specifically in handling poison cases, while another had nothing but a book on toxicology in the emergency room or hospital library. In 1993 the Health and Safety Code (Sec. 777.002) specified that poison centers provide 24-hour service for public and health care professionals and meet the requirements established by the AAPCC. This action helped the AAPCC standardize the staffs and activities of the various poison centers. The federal government does not fund poison centers, even though for every dollar spent on poison centers there is a savings of $2-$9 in unnecessary expenses (1,2). The federal agency responsible for the Poison Prevention Packaging Act is the U.S. Consumer Product Safety Commission (CPSC). The National Clearinghouse for Poison Control Centers initially collected data on poisonings and information on commercial product ingredients and toxic biological agents. For several years the National Clearinghouse provided product and treatment information to the poison centers. At first most poison centers were fundedby the hospital in which they were located. As the centers grew in size and the numberof calls increased, both city and state governments took on the responsibility of contributing funds. In recent years local governments have found it very difficult to fund such operations and centers havehad to look to private industry for additional funding. Government funding may take several forms, either as a line-item on a state’s budget, as a direct grant, or moneys distributed on a per call basis. Some stateswith fewer residents may contractwith a neighboring state to provide services to its residents. Some states are so populous that more than one center is funded by the state.Industrialfunding also varies,sometimes as a grant,sometimes as paymentfor handling the company’s poison and drug information-related calls,and sometimes as payment for collection of data regarding exposure to the company’s product. Every year the AAPCC reports a summary of all kinds of exposures.
2. Regional Centers The number of listed centers has dropped significantly since its peak of more than 600. Many centers have been combined into regional organizations. These regional poison centers provide poison information, telephone management, and consultation, collect pertinent data, and deliver professional and public education. Cooperation between regional poison
The
of Poison Centers
3
centers and poison treatment facilities is crucial. Regional poison information centers, in cooperation with local hospitals, should determine the treatment capabilitiesof the hospia working relationship with their analytical toxicoltals in the region and identify and have ogy, emergency and critical care, medical transportation, and extracorporeal services. This evaluation should be done for both adults and children. A“region” isusually determined by stateauthorities in conjunctionwith local health agencies and health care providers. Documentation of these state designations must be in writing unless a state chooses (in writing) not to designate any poison center or accepts a designation by other political or health jurisdictions. Regional poison information centers should serve a population base of more than one million people and must receive at least 10,000 human exposure calls per year. The number of certified regional centers in the United States is now less than 50. Certification as a regional center requires the following: Maintain a 24 hourslday, 365 dayslyear service. Provide service to both health care professionals and the public. Have available in the center at all times at least one specialistin poison information. Have on call by telephone at all times a medical director or qualified designee. Readily accessible service by telephone from all areas within the region. Comprehensive poison information resources and comprehensive toxicology information covering both general and specific aspectsof acute and chronic poisoning should be available. A list of on-call poison center specialty consultants. Writtenoperationalguidelines that provideaconsistentapproachtoevaluation, follow-up, and management of toxic exposures should be obtained and maintained. These guidelines must be approved in writing by the medical director of the program. A staff of certified professionals answering the phones (at least one of the persons on the phone has to bea pharmacist or nurse with 2000 hours and 2000 cases of supervised experience). A 24 hourdday physician (board certified) consultation service. An ongoing quality assurance program. Othercriteria,determined bythe AAPCCandestablished with membershipapproval. The regional poison information center must be an institutional lnember ingood standing of the AAPCC. Many hospital emergency rooms still maintain a toxicology reference such as the POISINDEXB system to handle routine exposure cases, but they rely on regional poison centers to handle most of the calls in their area.
B. Who Staffs a Poison Center? The staff of poison centers varies considerably from center to center. The three professional groups most often involved are physicians, nurses, and pharmacists. Who answers the phones is somewhat dependent on thelocallaborpool, moneys available, andthe types of calls being received. Other persons who answer the phone include students in medically related fields, toxicologists, and biologists. Persons responsible for answering the phones are either certifiedby the AAPCC or arein the process of obtaining the certifi-
4
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cation. Passage of an extensive examination on toxicology is required for initial certification, with periodic recertification required. Regardless of who takes the initial call, there is a medical director and other physician backup available. These physicians have specialized training or experience in toxicology and are able to provide in-depth consultations for health care professionals calling the center.
1. Medical Director A poison center medical director should be board certified in medical toxicology or in internal medicine, pediatrics, family medicine, or emergency medicine. The medical director should be ableto demonstrate ongoing interest and expertise in toxicology as evidenced by publications, research, and meeting attendance. The medical director must have a medical staff appointment at a comprehensive poison treatment facility and must be involved in the management of poisoned patients.
2. ManagingDirector The managing director must be a registered nurse, pharmacist, or physician, or hold a degree in a health science discipline. The individual should be certified by the American Board of Medical Toxicology (for physicians) orby the American Board of Applied Toxicology (for nonphysicians). The director must be able to demonstrate ongoing interest and expertise in toxicology.
3. Specialists in PoisonInformation These individuals must be registered nurses, pharmacists, or physicians, or be certified by the AAPCC as a specialist in poison information. Specialists in poison information must complete a training program approved by the nledical director and must be certified by the AAPCC as a specialist in poison information within two examination administrations of their initial eligibility. Specialists not currently certified by the associqt' L Ion must spend an annual average of no less than 16 h o u d w e e k in poison center-related activities. Specialists currently certified by the AAPCC must spend an annual average of no less than 8 hourdweek. Other poison information providers must have sufficient background to understand and interpret standard poison information resources and to transmit that information understandably to both health professionals and the public.
4. Consultants I n addition to physicians specializing in toxicology, most centers also have listsof experts by in many other fields as well. Poison center specialty consultants should be qualified training or experience to provide sophisticated toxicology or patient care information in their area(s) of expertise. In regard to seafoodor environmental toxins, this would include specialists in pesticides, heavy metals, botanical exposures, marine toxins, and hydrocarbons, just to name a few. These experts should be willing to donate their expertise in identifying and handling cases within their specialty. Most poison centers do not have the money to pay a wide variety of consultants.
C. What Types of Calls Are Received? All types of calls are received by poison centers, most of which are handled immediately, while others are referred to more appropriate agencies. Which calls are referred depends
The Role of Poison Centers
5
on the center, its expertise, and the appropriateness of a referral. Below are lists of calls which generally fall into each group Remember, there is considerable variation between poison centers; if there is doubt, call the poison center and they will tell you if your case is more appropriately referred. Poison centers do best on calls regarding acute exposures. Complicated calls regarding exposure to several agents over a long period of time which produce nonspecific symptoms are often referred to other medical specialists, to the toxicologist associated with the center, or to an appropriate government agency. The poison center will often follow-up on these cases to track outcome and the type of service given. Types of calls usually accepted Drug identification Actual acute exposure to a drug or chemical Actual acute exposure to a biological agent (plants, mushrooms, various animals) Information regarding the toxic potential of an agent Possible food poisonings (including seafood poisoning) Exposure to environmental toxins Types of calls often referred Questions regarding treatment of a medical condition (not poisoning) Questions on common bacterial, viral, or parasitic infections General psychiatric questions Proper disposal of household agents such as batteries, bleach, insecticides Use of insecticides (e.g., which insecticide to use, how to use it) unless related to a health issue (e.g., a person allergic to pyrethrins wanting to know which product does not contain pyrethrins)
1. Data Collection Records of all calls/cases handledby the center should bekept in a form that is acceptable as a medical record. The regional poison information center should submit all its human exposure data to the association’s National Data Collection System. The regional poison information center shall tabulate its experience for regional program evaluation on at least an annual basis. N. AAPCC Toxic Exposure Surveillmce Systern (TESS) In 1983theAAPCC formed TESS from the former National Data Collection System. Currently TESS contains nearly 16.2 million human poison exposure cases. Sixty-five poison centers, representing 181 3 million people, participate in the data collection. The information has various uses for both governmental agencies and industry, providing data for product reformulations, repackaging, recall, bans, injury potential, and epidemiology. The summation of each year’s surveillance is published i n the late summer or fall in the Arnericm Jolrrrlal of E~nerge~lcy Mecliciwe.
D.
How Are Calls Handled?
Most poison centers receive requests for information via the telephone. Calls come from both health care professionals and consumers. Only a few requests are received by mail or in person, these are often medicolegal or complex cases. Most centers can be reached as well as by a local number. Busy by a toll-free phone number in the areas they serve, centers have a single number that rings on several lines. Calls are often direct referrals from the 91 1 system.
Spoerke
6
Poison information specialists listen to the caller, recording the history of the case on a standardized form developed by the AAPCC. Basic information such as the agent involved, the amount of the agent, time of ingestion, symptoms, previous treatment, and currentconditionarerecorded, as well as patient information such as sex, age, phone number, who is with the patient, relevant medical history, and sometimes patient address. All information is considered a medical record, and is therefore confidential. The case is evaluated (using various references) as Infonnation only, no patient involved Harmless and not requiring follow-up a follow-up call is given Slightly toxic, no treatment necessary but Potentially toxic, treatment given at home and follow-up given to case resolution Potentially toxic, treatment may or may not be given at home, but it is necessary for the patient to be referred to a medical facility Emergency, an ambulance and/or paramedics are dispatched to the scene. Cases are usually followed until symptoms have resolved. In cases where the patient is referred to a health care facility, the receiving agency is notified. The history is relayed, toxic potential discussed, and suggestion for treatment given.
E. What ReferencesAreUsed? References used also vary from center to center, but virtually all centers use a toxicology system called POISINDEXB which contains lists of products, their ingredients, and suggestions for treatment. The systeln is compiled using medical literature and toxicology specialists throughout the world. Biological products such as plants, insects, mushrooms, and animal bites etc are handled similarly. There is an entry for each individual plant containing a description, the toxic agent present, potential toxic amounts, and so forth. The physician or poison information specialist is then referred to a treatment protocol that may be for a general class of agents; for example, exposure to malathion is referred to a protocol on organophosphate pesticides. An unknown skin irritation or potential infection would deserve a consult with an infectious disease specialist. Questions involving specific agents, such as lead or mercury, are directed to individual treatment protocols. POISINDEXB is available on microfiche, CD-ROM, over a network, or on a mainframe. It is updated every 3-months. Various texts are also used, but much of this infomation is already i n POISINDEXB. It is often difficult to identify some potentially toxic marine animals using a description given over the phone, so often the assistance of a marine biologist is used. If a type of marine food poisoning is involved, the help of an infectious disease consultant and an epidemiologist may be requested. Some poison centers have more experiencewith certain types of poisonings than do others, so often one center will consult another. These are often more complex cases, or cases involving centers in the same region. For example, a poison center in Utah may consult with one in California or Hawaii concerning a lionfish envenomization. A recent trend has been for manufacturers to contract with one poison center to provide poison information services for the whole country. Product information is given
The Role of Poison Centers
7
to only that center and exposures throughout the country can only be handled effectively in that one center.
F. How Are Poison Centers Monitored for Quality? Most poison centers havea system of peer review in place. One person takes a call, another reviews it. Periodic spot reviews are done by supervisory and physician staff. General competence is ensured by certification and recertification via examination of physicians and poison infonnation specialists.
G. Professional and Public Education Programs The regional poison information center provides information on the management of poiat soning to health professionals throughout the region. Public eduction programs aimed educating both children and adults about poison safety and potential dangers should be provided. In thepast, some centers provided stickers or logos suchasOfficer Ugh, Safety Sadie, and Mr. Yuck that could be placed on or near potentially toxic substances. While the intent was to identify potentially toxic substances that children should stay away from, the practice has been much curtailed because in some cases the stickers actually attracted children. Poison prevention week is held each yearin the spring. National attention is focused on the problem of potentially toxic exposures. During this week many centers run special programs for the public. This may include lectures on prevention, potentially toxic agents in the home, potentially toxic biological agents, or general first aid methods. Although an important time for poison centers, public and professional education is a year-round commitment. Physicians are frequently involved in medical toxicology rounds, journal clubs, and lectures by specialty consultants. Health fairs, school programs, and various men’s and women’s clubs are used to educate the public. The extent of these activities is often determined by the amount of funding from government, private organizations, and public donations.
H. Related Toxicology Organizations ACGIH American Conference of Governmentaland IndustrialHygienists Address: Kemper Woods Center; Cincinnati, OH 45240 Phone: 5 13-742-2020 FAX: 5 13-742-3355 ABAT American Board of AppliedToxicology Address: Truman Medical Center, West, 2301 Holmes St., Kansas City, MO 64 108
Phone: 8 16-556-3 1 I 2 FAX: 8 16-88 1-6282 AACT AmericanAssociation of ClinicalToxicologists Address: c/o Medical Toxicology Consultants, Four Columbia Dr., Suite810, Tampa, FL 33606 AAPCC AmericanAssociation of PoisonControlCenters
8
Spoerke
Address: 3201 New Mexico Ave. NW, Washington, DC 20016 Phone: 202-362-72 17 FAX. 202-362-8377 ABEM AmericanBoard of EmergencyMedicine Address: 300 Coolidge Rd., East Lansing, MI 48823 Phone: 5 17-332-4800 FAX. 5 11-332-2234 ACEP American College of Emergency Physicians (Toxicology Section) Address: P.O. Box 6 I99 1 I , Dallas, TX 7526 1-991 1 Phone 800-798- 1822 FAX: 214-580-2816 ACMT American College of Medical Toxicology (formerly ABMT) Address: 777 E. Park Dr., P.O. Box 8820, Harrisburg, PA 17105-8820 Phone: 717-558-7846 FAX: 7 17-558-7841 e-mail:
[email protected] (Linda L. Koval) ACOEM AmericanCollege of OccupationalandEnvironmentalMedicine Address: 55 West Seegers Rd., Arlington Heights, IL 60005 Phone: 708-228-6850 FAX: 708-228- 1856 ACS Association of ClinicalScientists Address: Dept. of Laboratory Medicine, University of Connecticut Medical School, 263 Fannington Ave., Farmington, CT 06030-2225 Phone: 203-679-2328 FAX: 203-679-2328 ACT AmericanCollege of Toxicology Address: 9650 Rockville Pike, Bethesda, MD 20814 Phone: 301 -57 1 - 1 840 FAX: 301-571-1852 AOEC Association of OccupationalandEnvironmentalClinics Address: IO10 Vermont Ave. NW, #513, Washington, DC 20005 Phone: 202-347-4976 FAX: 202-347-4950 e-mail:
[email protected] ASCEPT AustralianSociety of ClinicalandExperimentalPharmacologistsand Toxicologists Address: 145 Macquarie St., Sydney N.S.W 2000 Australia Phone: 61-2-256-5456 FAX: 6 1-2-252-33I O BTS BritishToxicologySociety Address: MJ Tucker,ZenecaPharmaceuticals,22B 1 1 Mareside,Alderley Park, Macclesfield, Cheshire, SKI0 4TG UK Phone: 0428 65 5041 CAPCC CanadianAssociation of PoisonControlCenters Address: Hopital Sainte-Justine, 3 175 Cote Sainte-Catherine, Montreal, Quebec H3T 1 C5, Canada Phone: 5 14-345-4675 FAX: 5 14-345-4822
The Role of Poison Centers
9
CSVVA (CEVAP) Center for the Study of Venoms and Venomous Animals Address: UNESP, Alarneda Santos, N 647, CEP 01419-901, Sa0 Paulo. sp, Brazil Phone 55-0 1 1-252-0233 FAX: 55-01 1-252-0200 EAPCCT EuropeanAssociation of PoisonControlCenters Address: J. Vale, National Poisons Infornlation Centre, P 0 Box 81898 Dep, N-0034 Oslo, Norway Phone: 47-260-8460 HPS HungarianPharrnacologicalSociety Address: Central Research Institute for Chemistry, Hungarian Academy of Sciences, H- 1525 Budapest, P.O. Box 17, Pusztaszeri ut 59-67 Phone: 36- 1- 135-21 12 ISOMT International Society of Occupational Medicine and Toxicology Address:USCSchool of Medicine,222OceanviewAve.,Suite 100, Los Angeles, CA 90057 Phone: 213-365-4000 JSTS JapaneseSociety of ToxicologicalSciences Address: Gakkai Center Building, 4-16, Yayoi 2-chome, Bunkyo-ku, Tokyo 113, Japan Phone: 3-38 12-3093 FAX: 3-3812-3552 SOT Society of Toxicology Address: 1101 14th St., Suite 1100, Washington, DC 20005-5601 Phone: 202-371 - 1393 FAX: 202-37 1- 1090 e-mail:
[email protected] SOTC Society of Toxicology of Canada Address: P.O. Box 517, Beaconsfield, Quebec H9W 5V1, Canada Phone: 5 14-428-2676 FAX: 5 14-482-8648 STP Society of ToxicologicPathologists Address: 875 Kings Hw., Suite 200, Woodbury, NJ 08096-3172 Phone: 609-845-7220 FAX: 609-853-041 1 SSPT Swiss Society of PharmacologyandToxicology Address: Peter Donatsch, Sandoz Pharma AG, Toxicologue 88 1/130, CH4132 Muttenz, Switzerland Phone: 41-61-469-5371 FAX: 41-6 1-469-6565 WFCT WorldFederation of Associations of ClinicalToxicologyCenters and Poison Control Centers Address:CentreAnti-Poisons,HopitalEdonardHerriot, 5 p1 d’Arsonva], 69003 Lyon, France Phone: 33 72 54 80 22 FAX: 33 72 34 55 67
Spoerke
10
1.
InternationalAffiliations
The AAPCC and its members attend various world conferences to learn of toxicology problems and new methods used by other agencies. An especially close relationship has formed between the American and Canadian poison center associations. Once a year the AAPCC and CAPCC hold a joint scientific meeting and invite speakers and other toxicology specialists from throughout the world to attend.
J. Toxicology and Poison Center Web Sites Association of Occupational ctnd Environmental Clinics: This group is dedicated to higher standards of patient-centered, multidisciplinary care emphasizing prevention and total health through information sharing, quality service, and collaborative research. Address:
[email protected] Finger Lrrkes Regional Poison Center. Address:
[email protected] Mediccll/Clinical/Occupntional Toxicology Professionctl Groups A list of primarily U.S. professional groups interested in toxicology. There is a description of each group, addresses, phone numbers, and contact names. Keyword: poison centers, toxicology Address: http://www.pitt.edu/-martint/pages/motoxorg.htm Poison Net A mailing list dedicated to sharing information, problem solving, and networking in the areas of poisoning, poison control centers, hazardous materinot als, and related topics. The list is intended for health care professionals, the lay public. The moderators do not encourage responses to individual poisoning cases from the public. Keywords: poisoning, poison control centers
II. POISON INFORMATION CENTERS IN THE UNITED STATES The Poison Control Center telephone numbers and addresses listed below are thought to be accurate as of the date of publication. Poison Control Center telephone numbers and addresses may change. The address and phone number of the Poison Control Center nearest you should be frequently checked.If the number listed doesnot reach thepoison center, contact the nearest emergency service, such as9 l 1 or your local hospital emergency room. The author disclaims any liability resulting from or relating to any inaccuracies or changes in the phone numbers provided below. This information should NOT be used as a substitute for seeking professional medical diagnosis, treatment, and care. (* Indicates a regional center designated by the American Association of Poison Control Centers.)
11
The Role of Poison Centers ALABAMA
ARKANSAS
Birrningharrr
Little Rock
Regional Poison Control Center* Children's Hospital of Alabama 1600 Seventh Ave., South Birmingham, AL 35233-171 1 800-292-6678 (AL only) 205-933-4050
Arkansas Poison and Drug Information Center University of Arkansas College of Pharmacy 4301 West Markham, Slot 522 Little Rock, AR 77205 800-482-8948 (AR only) 501-661-6161
Tusccrloosu
Alabama Poison Control System, Inc. 408A Paul Bryant Dr. East Tuscaloosa, AL 3540 1 800-462-0800 (AL only) 205-345-0600 ALASKA Arlchorqe
Anchorage Poison Center Providence Hospital P.O. Box 196604 3200 Providence Dr. Anchorage, AK 995 19-6604 800-478-3193 (AK ody) Ftrirbartks
Fairbanks Poison Center Fairbanks Memorial Hospital 1650 Cowles St. Fairbanks, AK 99701 907-456-7 I X2 ARIZONA Pkoetl~s
Samaritan Regional Poison Center'@ Good Samaritan Medical Center 1130 East McDowell Rd., Suite A-5 Phoenix, AZ 85006 602-253-3334 7irc.son
Arizona Poison and Drug Information Center* Arizona Health Sciences Center, Room 1 l56 1501 N. CampbellAve. Tucson, AZ 85724 800-362-0101 (AZ only) 602-626-6016
CALIFORNIA Fresrlo
Frcsno Regional Poison Control Center" Fresno Community Hospital and Medical Center 2823 Fresno St. Fresno, CA 93721 800-346-5922 (CA 011ly) 209-445- 1222 Los Angeles
Los Angeles County University of Southern California Regional Poison Center* 1200 North State. Room I 107 Los Angeles, CA 90033 800-825-2722 2 13-222-3212 Orarlge
University of California lrvine Medical Center Regional Poison Center* 101 The City Dr. South Route 78 Orange, CA 92668-3298 800-544-4404 (CA only) 7 14-634-5988 Richmorld
Chevron Emergency Information Center 15299 San Pablo Ave. P 0. Box 4054 Richmond, CA 94804-0054 800-457-2202 510-233-3737 or 3738
Spoerke
72 S~lc.rtrrnento
Regional Poison Control Center* University of California at Davis Medical Center 2315 Stockton Blvd.. Rm. HSF-124 Sacramento. CA 95817 800-342-3293 (northern CA only) 9 16-734-3692 Scrn Diexo
San Diego Rcgional Poison Center* University of California at San Diego Medical Center 225 Wcst Dickinson St. San Diego, CA 92013-8925 800-876-4766 (CA only) 6 19-543-6000 Son Frt~nci.sc.o
San Francisco Bay Area Poison Center* San Francisco General Hospital 1001 PotreroAve., Rm. IE86 San Francisco, CA 94122 800-523-2222 4 15-476-6600 Son Jose
San Jose Regional Poison Center Santa Clara Valley Medical Center 751SouthBascomAve. San Jose, CA 95128 800-662-9886, 9887 (CA only) 408-299-51 12,51 13, 51 14 COLORADO Den\vr
Rocky Mountain Poison Center* 1010 Yosernite Circle Denver. CO 80230 800-332-3073 (CO only) 303-629- 1 123 CONNECTICUT
DELAWARE Wilnrington
Poison Information Center Medical Center of Delaware Wilmington Hospital 501 West14thSt. Wilmington, DE19899 302-655-3389
DISTRICT OF COLUMBIA Wt~S1Iill~tOll
National Capital Poison Center* Georgetown University Hospital 3800 Reservoir Rd. NW Washington, DC 20007 202-625-3333
FLORIDA Jtrcksonville
Florida Poison Information Center University Medical Center 655 West Eighth St. Jacksonville, FL 32209 904-549-4465 or 764-7667 Tnllohtrssee
Tallahassee Memorial Regional Medical Center 1300 Miccosukk Rd. Tallahassee, FL 32308 904-68 1-541 1 Tu~npo
Tampa Poison Information Center* Tampa General Hospital Davis Islands P.O. Box1289 Tampa, FL 33601 800-282-3171 (FL only) 813-253-4444
~tJ'Llllill,~fOII
Connecticut Poison Control Center University of Connecticut Health Center 263 Farmington Ave. Farmington, CT 06030 800-343-2722 (CT only) 203-679-3456
GEORGIA Atlontcr
Georgia Regional Poison Control Center* Cerady Memorial Hospital
13
The Role of Poison Centers
80 Butler St. SE Box 26066 Atlanta, C A 30335-3801 800-282-5846 (CA only) 404-61 6-9000 Macon
Regional Poison Control Center Medical Center of Central Georgia 777 Hemlock St. Macon, CA 3 I208 912-744-1 146, 1100, 1427 Savannah
Savannah Regional Poison Control Center Memorial Medical Center Inc. 4700 Waters Ave. Savannah, GA 31403 912-355-5228 or 356-5228
Chicago, IL 606 12 800-942-5969 (Northeast IL only) 3 12-942-5969 Normal
Bronlenn Hospital Poison Center Virginia at Franklin Normal, IL 61761 309-454-6666 Springfield
Central and Southern Illinois Poison Resource Center St John’s Hospital 800 East Carpenter St. Springfield, IL 62769 800-252-2022 (IL only) 217-753-3330 Urlmna
HAWAII
Honolulu
Kapiolani Women’s and Children’s Medical Center 1319 Punahou St. Honolulu, HI 96826 800-362-3585, 3586 (HI only) 808-941 -4411
IDAHO Boi.W?
Idaho Poison Center St Alphonsus Regional Medical Center 1055 North Curtis Rd. Boise, ID 83706 800-632-8000 (ID only) 208-378-2707
National Animal Poison Control Center University of Illinois Department of Veterinary Biosciences 2001 South Lincoln Ave., 1220 VMBSB Urbana, IL 61801 800-548-2423 (Subscribers only) 217-333-2053
INDIANA
Indinnapolis
Indiana Poison Center* Methodist Hospital 1701 North Senate Blvd. Indianapolis, IN 46202-1 367 800-382-9097 3 17-929-2323
IOWA ILLINOIS Chiccrgo
Chicago and NE Illinois Regional Poison Control Center Rush Presbyterian-St. Luke’s Medical Center 1653 West Congress Pky.
Des Moines
Variety Club Drug and Poison Information Center Iowa Methodist Medical Center 1200 Pleasant St. Des Moines, IA 50309 800-362-2327 5 15-24 1-6254
Spoerke
14 IOM'U
Cih
University of Iowa Hospitals and Clinics 200 Hawkins Dr. Iowa City, IA 52246 800-272-6477 or 800-362-2327 (IA only) 3 19-356-2922 Cih St. Luke's Poison Center St. Luke's Regional Medical Center 2720 Stone Park Blvd. Sioux City, IA 51 104 800-352-2222 (IA, NE, SD) 712-277-2222
Si0lt.X
KANSAS
Lolii.s\~ille
Kentucky Poison Control Center of Kosair Children's Hospital 315 East Broadway P.0 Box 35070 Louisville, KY 40232 800-722-5725 (KY only) 502-589-8222
LOUISIANA Hou~ncc
Terrebonne General Medical Center Drug and Poison Information Center 936 East Main St. Houma, LA 70360 504-873-4069
Karrscrs C i h
Monroe
Mid America Poison Center Kansas University Medical Center 39th and Rainbow Blvd., Rm. B-400 Kansas City, KS 66160-7231 800-332-6633 (KS only) 9 13-588-6633
Louisiana Drug and Poison Information Center Northeast Louisiana University School of Pharmacy, Sugar Hall Monroe, LA 7 1209-6430 800-256-9822 (LA only) 3 18-362-5393
7opeka
Stormont Vail Regional Medical Center Emergency Department 1500 West 10th Topeka, KS 66604 9 13-354-6100 Wicllitcl
Wesley Medical Center 550 North Hillside Ave Wichita, KS 67214 3 16-688-2222
MAINE
Portlnnd
Maine Poison Control Center Maine Medical Center 22 Bramhall St. Portland, ME 04102 800-442-6305 (ME only) 207-87 1-2950
MARYLAND KENTUCKY Baltinrore
Ft. Tllovra.7
Northern Kentucky Poison Information Center St Luke Hospital 85 North Grand Ave. Ft. Thomas, KY 41075 5 13-872-51 1 1
Maryland Poison Center* University of Maryland School of Pharmacy ' 20 North Pine St. Baltimore, MD 21201 800-492-2414 (MD only) 410-528-7701
75
The Role of Poison Centers MASSACHUSElTS Boston
St. Pa111
Center
Massachusetts Poison Control System* The Children’s Hospital 300 1222 800-222Longwood Ave. Boston, MA 021 15 800-682-9211 (MA only) 617-232-2120 or 735-6607
Minnesota1 Regional Poison Center* Medical St Paul-Ramsey 640 Jackson St. St Paul, MN 55101 (MN only) 612-221-2113
MISSISSIPPI MICHIGAN Adriurr
Bixby Hospital Poison Center Emma L. Bixby Hospital 818 Riverside Ave. Adrian, MI 4922 1 517-263-2412 Detroit
Poison Control Center Children’s Hospital of Michigan 3901 Beaubien Blvd. Detroit, MI 48201 800-462-6642 (outside metropolitan Detroit) 3 13-745-571 1
Juck~ort
University of Mississippi Medical Center 2500 North State St. Jackson, MS 39216 60 1-354-7660 Huttieshrg
Forrest General Hospital 400 S 28th Ave. Hattiesburg, MS 39402 601-288-4235
MISSOURI Kcrrtsus City
Blodgett Regional Poison Center 1840 Wealthy St. S.E. Grand Rapids, MI 49506 800-632-2727 (MI only)
Poison Control Center Children’s Mercy Hospital 2401 Gillharn Rd. Kansas City, MO 64108-9898 8 16-234-3000 or 234-3430
Kolonltrzoo
St. Louis
Grmd Rupids
Bronson Poison Information Center 252 East Lovell St. Kalamazoo, MI 49007 800-442-41 12616 (MI only) 6 16-341 -6409
Regional Poison Center* Cardinal Glennon Children’s Hospital 1465 South Grand Blvd. St. Louis, MO 63104 800-392-9111 (MO only) 800-366-8888 (MO, West IL) 3 14-772-5200
MINNESOTA Minneupolis
Hennepin Regional Poison Center* 701ParkAve. S. Minneapolis, MN 55415 612-347-3144 612-347-3141 (Petline)
MONTANA Derwer
Rocky Mountain Poison and Drug Center Denver, CO 80204 800-525-5042 (MT only)
16
Spoerke
NEBRASKA
NEW MEXICO
Olll(1hLI
Albuquerque
The Poison Center* Children's Memorial Hospital 8301Dodge St. Omaha, NE 681 14 800-955-9119 (WY, NE) 402-390-5400, 5555
New Mexico Poison and Drug Information Center* University of New Mexico Albuquerque, NM 87 13I 800-432-6866 (NM only) 505-843-2551
NEVADA NEW YORK Lcrs Vrp1.s
Hurnana Hospital-Sunrise" 3 l86 Maryland Pky. Las Vcgas, NV 89109 800-446-6I79 (NV only) RPilO
Washoe Medical Center 77 Pringle Way Rcno, NV 89520 702-328-4 144 NEW HAMPSHIRE Lellailol~
New Hampshire Poison Center Dartmouth-Hitchcock Medical Center 1 Medical Center Dr. Lebanon, NH 03756 800-562-8236 (NH only) 603-650-5000 NEW JERSEY
Newcrrk New Jersey Poison Information and Education Systems* 201 LyonsAve. Newark, NJ 071 12 800-962-1253 (NJ only) 20 1 -923-0764 Phi1lip.shur.g
Warren Hospital Poison Control Center 185 Rosberg St. Phillipsburg. NJ 08865 800-962- 1253 008-859-6768
Buffalo
Western New York Poison Control Center Children's Hospital of Buffalo 219 Bryant St. Buffalo, N Y 14222 800-888-7655 ( N Y only) 7 16-878-7654 Mineolu
Long Island Regional Poison Control Center" Winthrop University Hospital 259 First St. Mineola, NY 11501 5 16-542-2323, 2324, 2325 New York C i v
Ncw York City Poison Control Center* 455 First Ave., Rm. 123 New York, NY 10016 2 12-340-4494 2 12-764-7667 Npck Hudson Valley Regional Poison Center Nyack Hospital 160 North Midland Ave. Nyack, NY 10920 800-336-6997 (NY only) 914-353-1000 Rochester
Finger Lakes Regional Poison Control Center University of Rochester Medical Center 601 Eltnwood Ave. Rochester, NY 14642 800-333-0542 (NY only) 7 16-275-515 1
17
The Role of Poison Centers Syrmuse
DAKOTA
Central New York Poison Control Center SUNY Health Science Center 750 E Adams St. Hospital Luke’s St NY 13210 Syracuse, 800-252-5655 Fargo, 3 15-476-4766
NORTH CAROLINA Ashevillc
Western North Carolina Poison Control Center Memorial Mission Hospital 509 Biltmore Ave. Asheville, NC 28801 800-542-4225 (NC only) 704-255-4490 or 258-9907 Chcrrlotte
Carolinas Poison Center Carolinas Medical Center 100 Blythe Blvd. Charlotte, NC 28232-2861 800-848-6946 704-355-4000 Durlzom
Duke Regional Poison Control Center P.O. Box3007 Durham, NC 27710 800-672-1697 (NC only) 919-684-811 1 Greensboro Triad Poison Center Moses H Cone Memorial Hospital 1200 North Elm St. Greensboro, NC 2740 l - 1020 800-953-4001 (NC only) 919-574-8105
NORTH
F0 rg o Poison Dakota North
Center
720 North 4th St. ND 58122 800-732-2200 (ND only) 701-234-5575
OHIO Akron
Akron Regional Poison Center 281 Locust St. Akron, OH 44308 800-362-9922 (OH only) 216-379-8562 C~lrlron
Stark County Poison Control Ccnter Timken Mercy Medical Center 1320 Timken Mercy Dr. NW Canton, OH 44667 800-722-8662 (OH only) 2 16-489- 1304 Cincinrlari
South West Ohio Regional Poison Control System and Cincinnati Drug and Poison Information Center* University of Cincinnati College of Medicine 231 Bethesda Ave., ML #l44 Cincinnati, OH 45267-0144 800-872-51 1 1 (Southwest OH only) 5 13-558-51 1 1 Cleveland
Greater Cleveland Poison Control Center 2074 Abington Rd. Cleveland, OH 44106 2 16-23 1-4455
Hickory
Colundm.7
Catawba Memorial Hospital Poison Control Center 810 Fairgrove Church Rd. SE Hickory, NC 28602 704-322-6649
Central Ohio Poison Center* 700 Children’s Dr. Columbus, OH 43205 800-682-7625 (OH only) 614-228-1323
Spoerke
18 Dayton
West Ohio Regional Poison and Drug Information Center Children’s Medical Center One Children’s Plaza Dayton, OH 45404- 18 15 800-762-0727 (OH only) 5 13-222-2227 Loruin
County Poison Control Center Lorain Community Hospital 3700 Kolbe Rd. Lorain, OH 44053 800-821-8972 (OH only) 216-282-2220
940 NE 13th St. Oklahoma City, OK 73 104 800-522-4611 (OK only) 405-27 1-5454
OREGON Portland
Oregon Poison Center Oregon Health Sciences University 3 181 SW Sam Jackson Park Rd. Portland, OR 97201 800-452-7165 (OR only) 503-494-8968
Sandusky
Firelands Community Hospital Poison Information Center 1101 Decatur St. Sandusky, OH 44870 419-626-7423 Toledo
Poison Information Center of Northwest Ohio Medical College of Ohio Hospital 3000 Arlington Ave. Toledo, OH 49614 800-589-3897 (OH only) 419-381-3897 Yuungsfown
Mahoning Valley Poison Center St Elizabeth Hospital Medical Center 1044 Belmont Ave. Youngstown, OH 44501 800-426-2348 (OH only) 21 6-746-2222 Zanesville
Bethesda Poison Control Center Bethesda Hospital 2951 Maple Ave. Zanesville, OH 43701 800-686-4221 (OH only) 614-454-4221 OKLAHOMA
PENNSYLVANIA Hershey
Central Pennsylvania Poison Center* Milton Hershey Medical Center Pennsylvania State University P.O. Box 850 Hershey, PA 17033 800-521-6110 717-531-611 1 Lancaster
Poison Control Center St. Joseph Hospital and Health Care Center 250 College Ave. Lancaster, PA 17604 7 17-299-4546 Philadelphia
Philadelphia Poison Control Center” One Children’s Center 34th and Civic Center Blvd. Philadelphia, PA 19104 215-386-2100 Pittsburgh
Pittsburgh Poison Center* One Children’s Place 3705 Fifth Ave. at DeSoto St. Pittsburgh, PA 15213 41 2-68 1-6669
Oklahonlu C i h
Williamsport
Oklahoma Poison Control Center Children’s Memorial Hospital
The Williamsport Hospital Poison Control Center
79
The Role of Poison Centers
777 Rural Ave. Williamsport, PA 1770I 717-321-2000
RHODE ISLAND
800 East 21st St. P.O. Box 5045 Sioux Falls, SD 57 117-5045 800-952-0123 (SD only) 800-843-0505 (IA, MN, NE) 605-336-3894
Providence
Rhode Island Poison Center* 593 Eddy St. Providence, RI 02903 40 144-5727
SOUTH CAROLINA Charlotte
Carolinas Poison Center Carolinas Medical Center 1000 Blythe Blvd. Charlotte, NC 28232-2861 800-848-6946 Cohonhia
Palmetto Poison Center University of South Carolina College of Pharmacy Columbia, SC 29208 800-922-11 17 (SC only) 803-765-7359
SOUTH DAKOTA Aberdeen
Poison Control Center St Luke’s Midland Regional Medical Center 305 S. State St. Aberdeen, SD 57401 800-592-1889 (SD, MN, ND, WY) 605-622-5678 Rapid Cif?, Rapid City Regional Poison Control Center 835 Fairmont Blvd. P.O. Box 6000 Rapid City, SD 57709 605-341-3333 Sioux Fcrlls McKennan Poison Center McKennan Hospital
TENNESSEE Knoxville
Knoxville Poison Control Center University of Tennessee Memorial Research Center and Hospital 1924 Alcoa Hwy. Knoxville, TN 37920 615-544-9400 Memphis
Southern Poison Center, Inc. Lebanheur Children’s Medical Center 848 Adams Ave. Memphis, TN 38103-2821 901 -528-6048 Nashville
Middle Tennessee Regional Poison Center, Inc. 501 Oxford House 1161 21st Ave. S, B-IOIVUII Nashville, TN 37232-4632 800-288-9999 (TN only) 615-322-6435
TEXAS Conroe
Montgomery County Poison Information Center Medical Center Hospital 504 Medical Center Blvd. Conroe, TX 77304 409-539-7700 Dallas
North Central Texas Poison Center* Parkland Memorial Hospital 5201 Harry Hines Blvd. P.O. Box 35926 Dallas, TX 75235 800-441-0040 (TX only) 214-590-5000
Spoerke
20
El Paso El Paso Poison Control Center
Thomas General Hospital 4815 Alameda Ave. El Paso, TX 79905 915-533-1244 Gnlveston
Texas State Poison Control Center University of Texas Medical Branch 8th and Mechanic St. Galveston, TX 77550-2780 800-392-8548 (TX only) 7 13-654-1701 (Houston) 409-765-1420 (Galveston) Lubbock
Methodist Hospital Poison Control 3615 19th St. Lubbock, TX 79413 806-793-4366
Box 67 Charlottesville, VA 22901 800-45 1- 1428 (VA only) 804-924-5543 Richmond
Virginia Poison Center Virginia Commonwealth University MCV Station Box 522 Richmond, VA 23298-0522 800-552-6337 (VA only) 804-786-9123
WASHINGTON Washington
P.O. Box 5371 Seattle, WA 98105-0371 800-732-6985 (Within WA) 206-526-2121
UTAH Salt Lake C i h
Utah Poison Control Center* Intermountain Regional Poison Control Center 410 Chipeta Way, Suite 230 Salt Lake City, UT 84108 800-456-7707 (UT only) 801-581-2151
VERMONT Burlington
Vermont Poison Center Medical Center Hospital of Vermont 11 1 Colchester Ave. Burlington, VT 05401 802-658-3456
WEST VIRGINIA Chcrrleston
West Virginia Poison Center* West Virginia University 31 I O MacCorkle Ave. SE Charleston, WV 25304 800-642-3625 (WV only) 304-348-4211 Porkershurg
St. Joseph’s Hospital Center 19th St. and Murdoch Ave. Parkersburg, WV 26 101 304-424-4222
WISCONSIN VIRGINIA Cl~trlorte.sville
Blue Ridge Poison Center* University of Virginia Health Sciences Center
Mndisorr
Regional Poison Control Center University of Wisconsin Hospital 600 Highland Ave. Madison, WI 53792 608-262-3702
21
The Role of Poison Centers Milwaukee
Poison Center of Eastern Wisconsin Children’s Hospital of Wisconsin Poison Wisconsin The West Ave. 9000 Hospital Memorial Children’s Box 1997 P.O. Milwaukee, Dodge8301 W1 53201 414-266-2222
WYOMING Omoha
Center” St.
681 Omaha, NE 14 800-955-91 19 (WY, NE) 402-390-5400. 5555
REFERENCES DL Harrison, JR Draugalis, MK Slack, PC Langly. Cost effectiveness of regional poison control centers.ArchlnternMed156:2601-2608,1996 2. CPSC. CPSC Chairman Ann Brown suggests information technology study to support work of poison ccnters. News release 94-047, March 15, 1994. 1.
This Page Intentionally Left Blank
2 Fish Toxins
I. 11.
111.
1V.
V. VI. VI1. VIIl.
1.
Introduction 23 Toxigcnesis 24 DystrophicationandToxigenesis 24 Icthyosarcotoxic Fish 25 A. Lampreys and hagfish: cyclostome fish 25 B. Sharks,skates,rays,andchimaeras:elasmobranchfish C. Ciguatoxic fish 28 D. Clupeotoxic fish 33 E. Gempylotoxic fish 35 Scombrotoxic F. fish 35 G. Tetrodotoxic 37 fish Icthyootoxic Fish 40 Icthyohcmotoxic Fish 42 Icthyohepatotoxic Fish 43 Icthyoallyeinotoxic(Hallucinogenic)Fish 44 Acknowledgment 45 References 45
26
INTRODUCTION
Fish toxins are of two types: the small, molecular oral biotoxins that are poisonous to eat, and the large molecular venoms that are injected into the body by means of a specialized device known as the venom apparatus. Thus all venoms are poisons, but not all poisons are venoms. In this chapter, only the oral biotoxins are discussed in regard to the names of thetransvectors,theirgeographicaldistribution,clinicalcharacteristics,andabrief statement of the toxin involved. Reports of food poisoningof marine origin are increasing in frequency and outbreaks appear to be spreading geographically. Part of this increase may be credited to heightened awareness, more travel to areas of the world where marine toxicity is endemic, and a greater opportunity for exposure to oral fish toxins, There also has been an increase in
23
Halstead
24
the importation of toxic marine food products into North America, Europe, Russia, Taiwan, Japan, and elsewhere. However, travel and awareness are only two facets of the overall epidemiology of the marine food biotoxication problem. There is evidence that suggests that pollution may be an added factor that needs to be taken into consideration.
II. TOXIGENESIS Toxicity in marine organisms is the result of a progression of biochemical events taking place in the bodyof the target organism. Various combinations of atoms of carbon, hydrogen, oxygen, nitrogen, chlorine, sulfur, and phosphorus are synthesized by the organism into complex biotoxin molecules thatmay have extreme complexity and toxicity. The process by which this is accomplished is referred to as biogenesis, biosynthesis, or, more specifically, as toxigenesis. In actuality, very little is known about the precise chemical processes involved. There is a growing amountof chemical data that suggests that in some instances marine bacteria play a role in toxin biosynthesis. Oral marine biotoxins may develop in the bodies of marine organisms as a result of naturallyoccurringprecursorchemicalagents, or theymay develop as a resultof human-induced chemical pollutants. In either case, the resulting biotoxins are capable of producing serious public health problems. These problems may occur in endemic areas wherever marine biotoxins exist or in far-removed areas to which toxic marine products have been transported. The increase of marine biotoxications worldwide are of grave economic and public health significance.
111.
DYSTROPHICATION AND TOXIGENESIS
There is an aspect of ecotoxicology that appears to have a bearing on the topic of foodborne marine intoxicants, that is, the matter of ocean eutrophication and its resultant dystrophication due to pollution. The process of ocean eutrophication is a phenomenon that has been well documented (1 - 3 ) . Eutrophication is a process of nutrient enrichment involving ocean ecosystems. Dystrophication generally is looked upon as a posteutrophication process in which there is oxygen depletion resulting from the actionof aerobic bacteria upon organic matter accompanied by other poorly defined chemical alterations in the marine environment. These two processes generally are looked upon as an aging activity in a body of water. A vast array of chemical agents, military and industrial pollutants, pesticides, and heavy metals are entering the marine environment and contributing to the eutrophication process. This ocean enrichment process is takingon global proportions and is of growing concern to marine toxicologists. More detailed information on this subject has been published in Ref. 4. Current evidence suggests thatthe combined onslaught of all of these chemical substances entering the ocean environment undoubtedly contributes to the degradative enrichment process. This involves a series of chemical and physical vector forces that presently appearto defy analysis. The ever-increasing chemical contamination of the ocean environment strongly suggests that the growing number of outbreaks of oral intoxications may be related events.
Fish Toxins
25
The bacterial degradation of organic matter by proteolytic bacteria produces a decrease in dissolved oxygen and may increase the growth of sulfate bacteria and the production of hydrogen sulfate and sulfur. Pollutants of various types may upset the phytoplanktoniccycleandaltertheworkofchemicalmediators,resulting ineutrophicationand dystrophication. All of these factors may cause an increasein toxic phytoflagellate blooms and various forms of bacterial toxigenesis. Yasumoto et al. (5,6)and Noguchi etal. (7) reported that the bacterial Psc~1rrlorr1orrcrs and Vibrio species were found in association with toxic pufferfish and toxic phytoflagellates. The investigators concluded that the bacteria were responsible for the production of tetrodotoxin and saxitoxin. Kotaki et al. (8) isolated the bacteriunl Mornellrr species and concluded that it was responsible for the biosynthesis of saxitoxin in cultures of the toxic dinoflagellate P r [ ) t ~ ) ~ o t ~ (Gorryauleu) ~ ~ l l l ~ r . ~ tcrn~crrensisis. The studies cited above provide substantial evidence that microbial organistus are responsible for the production of such toxins as tetrodotoxin, saxitoxin, and some of their congeners. It is possible that the ciguatoxin complex, palytoxin, and probably other marine toxins yet to be identified may also be the products of bacterial activity acting in association with a variety of marine plants and animals (5,6,9-21); Aubert feels that it is difficult to explainthetoxigenesis of phytoplankton by any other means than by bacteria (M. Aubert, personal communication, 1991). Bacterial toxigenesis has now become an area of major epidemiological concernin dealing with outbreaksof organic marine biotoxications. There are various waysof classifying food-borne outbreaks resulting from the ingestion of toxic fishes. Here the outbreaks are arranged phylogenetically accordingto the fish transvectors involved. All of the fish presented in the sections below are members of the phylum Chordata.
IV.
ICHTHYOSARCOTOXIC FISH
Ichthyosarcotoxic fish are those that contain a poison within the flesh of the fish (i.e., the musculature, viscera, skin, or slime) which, when ingested, causes a biotoxication. This category should not be confused with that which causes ichthyootoxism, in which the poison is restricted to the gonads or roe of the fish.
A.
LampreysandHagfish:CyclostomeFish
The cyclostome fish are members of the class Agnatha. The cyclostomes, which include thelampreysand hagfish,are a group of fishlikevertebrateshavinganeel-likeform, cartilaginous or fibrous skeleton, no definite jaws or bony teeth, and a primitive type of cranium. There are no pelvic girdles, paired limbs or true ribs. There are 6-14 pairs of gill pouches opening either directly into the pharynx or into a separate respiratory tube. Only a single nostril is present. Because of their structural simplicity, cyclostomes generally are considered the most primitive of true vertebrates. The hagfish are strictly inhabitants of temperate and subtropical inshore marine waters of the Atlantic and Pacific Oceans. Hagfish are members of the family Myxinidae. The skin of the hagfish is richly supplied with large mucous cells. A large hagfish is said to be capable of filling a twogallon bucket with slime. The slime is reputed to be toxic. Hagfish are rarely eaten as food.
26
Halstead
Representative Species Family: Myxinidae (hagfish) Species: Myxiwe glutinosa Linnaeus. Atlantic hagfish. Length 31 in. (79 cm). Distribution: North Atlantic. Family: Petromyzonidae (lampreys) Species: Petmtnyzon rnarinus Linnaeus. Sea lamprey. Length 33 in. (84 cm). Distribution: Coasts and rivers of both sides of the Atlantic, rivers of the Mediterranean.
Cyclostomc Poisoning Clinicd Chrrrrrcteristics: Poisoning from cyclostomes is rare because they seldom are eaten. The slime is said to be toxic to eat. Symptoms consist of nausea, vomiting, dysenteric diarrhea, tenesmus, abdominal pain, and weakness (22-26). There are no recent accounts of cyclostome poisoning. Treetrttnent:Treatment is symptomatic. See Refs. 27-29. Prevetltion: Most cyclostome poisonings are said to be caused because of failure to deslime the fish. For prevention, some authors claim that if the fresh fish is covered with salt and left in a concentrated brine solution for several hours prior to cooking, the fish is safe to eat (30,31).
B. Sharks, Skates, Rays, and Chimaeras: Elasmobranch Fish The elasmobranch fishes include the sharks, skates, rays, and chimaeras, all of which are members of the class Chondrichthyes. The poisoning is referred to as elasmobranch poisoning when it involves sharks, skates, or rays. When involving chimaeras, also known as the elephantfish or ratfish, the intoxication is known as chimaera poisoning. The sharks, skates, and rays are fishlike vertebrateswith well-developed lower jaws and bony teeth; two pairs of appendages supported by pectoral and pelvic girdles; a cartilaginous skeleton that, while more or less calcified, lacks any true bone; scales, essentially toothlikeinstructure,known as placoidscales;twonostrils,eachsingleandpartially subdivided; and blind olfactory sacs, not opening into the mouth. The posterior end of the vertebral column is either straight or heterocercal. A sympathetic nervous system, pancreas, spleen, and contractile arterial cone are present. There is a series of two, three, or more heart valves and a swim bladder is present. There are five to seven pairs of gills and five to seven gill clefts, each of the latter opening separately to an exterior dorsal fin or fins, and spines, if present, are rigid and not erectile.The skin is with or without dermal denticles, teeth are numerous, and the upper jaw or palatoquadrate cartilage is not fused to the cranium, although it may be attached locally to it. The nostril cartilage is fused to the cranium. At least some of the vertebrae of the trunk region have transverse ribs and the two halves of the pelvic girdle are fused into a single bar. The anus and urogenital canals discharge into a common cloaca and the males are without prepelvic or frontal tenacula.
1, Poisonous Sharks Representrrtive Species Family: Isuridae (mackerel sharks) Species: Crr~-chcrrodoncxrrchrirrs (Linnaeus). Great white shark. Length 20 f t (6 m).
Fish Toxins
27
Distribution: Cosmopolitan: tropical, subtropical, and warm temperate seas worldwide. Family: Carcharhinidae (requiem sharks) Species: Ccrrchcrrhinus me1mopteru.s (Quoy and Gaimard).Black-tip reef shark. Length 6.5 ft (2 m). Distribution: Tropical-Indo Pacific region. Species: Carchnrhitrus amboinet?.sisMuller and Henle. Pig-nosed Shark. Length 82 in. (2.8 m). Distribution: Eastern North Atlantic, Indo-Pacific oceans, South Africa, Madagascar, Gulf of Aden: Indian Ocean, and Australia. 12 f t Species: Cerrcherr11inu.s Ieucas (Valenciennes).Bullheadshark.Length (3.6 m). Distribution: Warm waters of the Atlantic, Pacific, and Indian Oceans. Family: Hexanchidae (cow sharks) Species: Heptranc1riLr.s perlo (Bonnatere). Seven-gilled shark. Length 6.3 ft (2 m). Distribution: Atlantic, Mediterranean, South Africa, and Japan. Family: Dalatidae (sleeper sharks). Species: Sormiosus tnicrocephcrlus (Bloch and Schneider). Greenland shark. Length 6.3 ft (2 m). Distribution: Arctic Atlantic, North Sea. east to the White Sea, and west to the Gulf of St. Lawrence. Family: Sphyrnidae (hammerhead sharks). Species: Sphyrmr ;ygaetrcr (Linnaeus). Scalloped hammerhead shark. Length 14 ft (4 m). Distribution: Tropical to warm temperate belt of the Atlantic and Pacific Oceans.
The elasmobranch form of icthyosarcotoxism is most commonly caused by eating sharkliversandtheflesh of some of the tropical sharks. Skates and rays are seldom involved in food poisoning. Clinicd Characteristics: The ingestion of fresh Greenland shark flesh is toxic to both dogs and man. The symptoms consist of nausea, vomiting, diarrhea, difficulty in walking, convulsions, respiratory distress, and muscular twitching. The local natives gradually build up a tolerance to the poison. The toxicity of Greenland shark poisoning is believed to be due to large amounts of trimethylamine oxide (32). Serious intoxications have resulted from eating the tropical sharks Ctrrchcrroclon curcherrim, C(rrcharhinus tnekrnopterus, Ccwcharhinus amboinensis, Ccrrckrrrhitrus leuc m , He~)trernchinsperlo,and Sphyrnrr zygtrerrcr. The symptoms resulting from the ingestion of shark liver may be severe, developing within 30 minutes after eating. The symptoms consist of nausea, vomiting, diarrhea, abdominal pain, headache, joint aches, tingling about the mouth, and a burning sensation of the tongue, throat, and esophagus. As time progresses, the symptoms involving the nervous system may worsen, resulting in muscular incoordination and difficulty in breathing due to muscular paralysis, followed by coma, and finally death. Ingestion of the flesh of certain species of tropical and arctic sharks may be dangerous to eat, butthe symptoms usually aremild,consistingmainlyof a gastrointestinal upset.
Halstead
28
Trecrtnw1t: Treatment is symptomatic. See ciguatera fish poisoning. See Refs. (2729).
Prevetrtion: Avoid eating the liver of any shark unless it is known for certain to be edible. The livers of tropical and arctic sharks are known to be especially dangerous to eat. The flesh of tropical and arctic sharks is potentially poisonous and should be eaten with caution.
2. Poisonous Chimaeras The chimaeroids-elephantfish or ratfish-differ from the sharks, skates, and raysin that they have only four pairs of gills and four pairs of gill clefts, with only one opening to the exterior on each side of the head. The dorsal fin and spine are erectile. In the adult, the skin is naked, without dermal denticles. Teeth are represented by six pairs of grinding plates; the upper jaw or platorate cartilage is fused with the cranium and the rostral cartiof the lage is articulated to the cranium, not fused. Ribs are lacking and the two halves pelvic girdle separate. There is no cloaca, and the urogenital aperture is distinct from the anus and posterior to it. The males have an erectile prepelvic tenaculum, usually with a frontal tenaculum on the head (33).
Represewtative Species Family: Chimaeridae (Chimaeras) Species: Chirnrrercr ttwt1stro.w Linnaeus. Length 39 in. European chimaera. Distribution: North Atlantic and Mediterranean.
( 1 m).
Clrimcrerrr Poisotlirzg The musculature and visceraof some of the elephantfish and ratfish have been found to be toxic, but the nature of the chimaera poison is unknown. Cliwiccrl Chcrructeristics: The symptomatology of chimaera poisoning in humans is unknown. Treatmetzt: Treatment is symptomatic. Prevention: Chimaeras should not be used for human consumption.
C. Ciguatoxic Fish Ciguatoxic fish cause oneof the most widespread and seriousforms of ichthyosarcotoxism known. More than 400 species of fish are alleged to have transvectored the ciguatoxin complex poisons that serve as the causative toxins in ciguatera fish poisoning. The fish involved are for the most part tropical or warm, temperate zone reef or inshore species found between 35"N latitude and 34"s latitude in the Caribbean and tropical Pacific and Indian Oceans. Occasionally, offshore fish may be involved, but by far the bulk of the outbreaks have occurred in insular areas. Historically, ciguatera-like symptoms have resulted from eating marine turban shells (Turbo picrr) in the Caribbean (25,34,35), and similar outbreaks have been caused by eating coconut crabs in Tahiti and in the Ryukyu Islands (36,37). No freshwater fish have been incriminated. Many of the ciguatoxic fish are valuable food fish that on occasion become toxic within a few hours of ingesting toxic dinoflagellates or algae in association with toxic dinoflagellates. Carnivorous fish may become toxic as a result of ingesting toxic herbivores. Thus ciguatera is a toxic food chain problem. Once a fish becomes poisonous, the
Fish Toxins
29
toxicity within the body of the fish may continue for a period of many years. One of the species of dinoflagellates .that has been incriminated in ciguatera fish poisoning is Gambierdiscus tosicus Adachi and Fukuyo. Several other dinoflagellate species are highly suspect as causative agents of ciguatera fish poisoning, including Ampl~idiniutncnr'tertre Hulbert, Ostreopsis ovtrt~Fukuyo, Prorocentrum C ' O I ~ C L I V U IFukuyo, I? P. limtr (Ehrenberg) Dodge, and P. me.~iccrnu~r~ Tafall (38). There is growing evidence that suggests that the in a symbiotic primary causative agent in this toxicity cycle may be bacteria that live relationship with dinoflagellates or possibly macroalgae (see Sec. 111). Ciguatoxic fish are a group of phylogentically diversified species, most of which are members of theclassOsteichthyes,thebonyfish.Thebiology of thesefishis as diversified in habitat, habits, feeding, and reproduction as it is in morphology. Consequently, it is impossible to present a stereotyped characterization of a ciguatoxic fish. Moreover, you cannot detect a ciguatoxic fish by its appearance. In one part of an island, any given fish species may be edible, whereas on the opposite side of the island or on an adjacent reef, the same species may be deadly poisonous. This is the major problem in ciguatera fish poisoning. The members of the class Osteichthyes are characterized as having a skeleton, in part or all with true bone; the skull has sutures; and the teeth are fused to the bones. The soft fin rays usually are segmented. Nasal openings on each side usually are double and more or less dorsal in position. The biting edge of the upper jaw usually is formed by a functional lung dermal bones, the premaxillae and the maxillae. A swim bladder or usually is present. There is an intestinal spiral valve in only a few lower groups. Internal fertilization is relatively rare, and there is a pelvic copulation device in only one group (phallosthoids). The embryos are not encapsulated in a case (39).
Representdve Species An attempt to list all of the ciguatoxic fish species that have been incriminated to date would not be feasible; consequently, only a small representative group of species is listed below. The fish are arranged in alphabetical order according to their family names and within the family by their generic names. Family: Acanthuridae (surgeonfish) Species: A c c r t h m s lineatus (Linnaeus). Striped surgeonfish. Length 7 in. (18 cm). Distribution: Indo-Pacific. Family: Balistidae (leatherjackets, filefish, triggerfish) Species: AIutera scriljttr (Osbeck). Scribbled filefish. Length 19 in. (50 cm). Distribution: All warm seas. Species: Btrlistoitfes conspicillurn Bloch and Schneider. Clown triggerfish (Fig. 1 1). Length 13.7 in. (35 cm). Distribution: Tropical Pacific from Polynesia to Madagascar, China, Japan. Family: Carangidae (jacks, pompanos) Species: Ctrrtrn.~ hi[)i)os(Linnaeus). Jack, crevalle. Length 29.5 in. (75 cm). Distribution: Tropical Atlantic. Cuvier. Blue jack. Length 25.5 in. (65 cm). Species: ccrrcrnx melatn~~pgus Distribution: Tropical Pacific. Family: Lutjanidae (snappers) 35.5 in. Species: Lutjarzr4.s bohtrr (Forskil).Redtwo-spottedsnapper.Length (90 cm).
30
Halstead
Distribution: Tropical Indo-Pacific. Species: Lutjrrnus gibbus (Forskil). Humpback snapper. Length 15.5 in. (40 cm). Distribution: Tropical Indo-Pacific. 19.5 in. Species: Lutjatzus vaigiensis (Quoy and Gaimard). Redsnapper.Length (50 cm). Distribution: Indo-Pacific. Family: Mugilidae (mullets). Species: CIlelow vcligiensis (Quoy and Gaimard). Mullet. Length 12 in. (30.5 cm). Distribution: Indo-Pacific. Species: Mugil cepkrr1u.s Linnaeus. Mullet. Length 12 in. (30.5 cm). Distribution: Cosmopolitan in warm temperate seas. Family: Mullidae (goatfish, surmullets) Species: Mulloidicl~thysc/ur(jIatrrma (Forskil). Goatfish. Length 13 in. (35 cm). Distribution: Indo-Pacific. Species: Parupet~euschtyser:\ldros (Lacepide). Goatfish. Length 13 in. (33 cm). Distribution: Indo-Pacific, East Africa. Family: Muraenidae (moray eels) Species: Gytnnothortr.u javcrrlicus (Bleeker). Giant brown moray eel. Length 5 ft (1.5 m). Distribution: Indo-Pacific. Species: Gymtlothorc/.r rnelecrgris (Shaw and Nodder). White-mouthed moray eel. Length 39 in. ( I m). Distribution: Indo-Pacific, Japan. Family: Scombridae (tunas, mackerels, albacore) solrrttdri (Cuvier). Wahoo. Length 78 in. (2 m). Species: Acc~t~thocybiun~ Distribution: Circumtropical. Species: Scombemmorus crrvcrllrr (Cuvier). King mackerel. Length 59 in. (1.5 m). Distribution: Tropical Atlantic. Family: Serranidae (sea basses, grouper) Species: Cephnlopholis nrgus (Bloch and Schneider). Peacock grouper. Length 20 in. (51 cm). Distribution: Indo-Pacific. (Forskil). Brown nnarbled grouper. Length 23.5 Species: E~~ine~~halus.fu.scoglrttr~tu,s in. (60 cm). Distribution: Indo-Pacific. Species: Mycterol~erccr vetzertow (Linnaeus).Poisonousgrouper.Length35 in. (90 cm). Distribution: Western tropical Atlantic. Species: Plectroponrrrs olipccrrlthus Bleeker. Blue-lined coral grouper. Length 2 1 in. (55 cm). Distribution: Indo-Pacific. Species: Vrrriola louti (Forskil). Lyretail grouper. Length 23 in. (60 cm). Distribution: Indo-Pacific. Family: Siganidae (rabbitfish). Species: Sig~rnlrslinearus (Valenciennes). Rabbitfish. Length 1I in. (30 ~111). Distribution: Indo-Pacific. Species: Sigrrrrrrs prrellus (Schlegel). Rabbitfish. Length 10 in. (27 cm). Distribution:Indo-Pacific.
Fish Toxins
31
Family: Sphyraenidae (Barracuda) Species: Sphyraetu~hnrrczcudcl (Walbaum). Great barracuda. Length 5.2 ft (1.6 m). Distribution: All warm seas, except eastern Pacific.
Cigutrtern Fish Poisotlitlg Ciguatera fish poisoning results from the ingestionof any of a large variety of shore and reef fish which are usually subtropical or tropical in their distribution. The degree of freshness of the fish has no bearing on its toxicity. The victim becomes poisoned as a result of ingesting a toxin within the flesh of the fish. Ciguatera is not a form of ordinary bacterial food poisoning. Ciguatera fish poisoning involves a complex of poisons: ciguatoxin, molecular formula CI,,,HXhOll,, molecular weight 1 1 12, median lethal dose (LDS,,) 0.45g/kg mouse intraperitoneal (IP) (40-45); maitotoxin, molecular formula ClhlhH2?cS?07J, approximate molecular weight 3396.1, LDcotoxicity 0.13 g/kg mouse IP (46); scaritoxin, molecular formula and toxicity unknown (47). Ciguatoxin and maitotoxin are two of the most toxic marine poisons known. Ciguatoxin acts by increasing the membrane permeability to sodium ions of excitableneurons by openingthevoltage-dependentsodiumchannels(43,48,49).Repeated doses of ciguatoxin to mice on an experimental basis have been found to produce severe ultrastructural morphological changes in the cardiac muscle cells and endothelial lining cells of blood capillaries in the heart. Damage to the capillaries was followed by effusion of serum and erythrocytes into the interstitial spaces of the myocardium. Swelling of the endothelial lining cells of capillaries caused narrowingof the lumen and accumulation of of cardiac blood platelets in capillaries, which resulted in multiple single-cell necrosis muscle cells (50). These experimental findings in mice may possibly explain some of the clinical cardiac findings in individuals that have suffered multiple exposures to ciguatera fish poisoning. The most toxic part of the fish is usually the liver, followed by the intestines, then the testes, ovaries, and the muscle. As noted in Sec. IV.C, the toxin originates in the food web of the fish. There is no evidence of a seasonalincidenceinciguatoxicity,butthespawning season in some of the larger predacious fish may be a more dangerous period than the other seasons of the year. There is no way to detect a ciguatoxic fish by its appearance. Clitliccrl Chcrrcrcteristics: Ciguatera fish poisoning produces a constellation of 175 gastrointestinal, cardiovascular, and neurological symptoms, some of which are pathognomonic for the disease. The onset of the poisoning may occur within minutes and up to 48 hours after the fish is ingested. The initial symptoms generally consist of paresthesias and tingling or numbness of the lips, tongue, and extremities. The neurosensory symptoms may be accompanied by nausea, abdominal pain, vomiting, diarrhea, salivation, general malaise, and muscle and joint pain. The gastrointestinal symptoms are present in about 75% of cases, but usually resolve within 24 hours. There is a neurosensory symptom that is of diagnostic importance in ciguatera poisoning: the reversal of temperature sensation in which cold objects (water, ice, etc.) feel hot, produce a stinging sensation, or are painful upon contact. Warm objects may feel cold. This temperature-reversal sensation appears in more than 89% of cases. Cardiovascular symptoms usuallyconsist of bradycardia and hypotension, which later may change to
32
Halstead
tachycardiaandhypertension.Cardiovascularsymptomsgenerallyresolvewithin 48 hours, but may persist for several weeks. Neurological symptoms of perioral and extremity paresthesias, ataxia, pruritus, mental depression, hysteria, maculopapular skin eruptions, blisters, desquamation, loss of hair and nails, cranial nerve palsy, vertigo, tremors, chills, headache, sweating, dysurea, hiccups, visual blurring, superficial hyperesthesia, motor weakness, respiratory distress, myalgia, arthralgia, temperature reversal, hyporeflexia, metallic taste, loose or painful sensation of the teeth, and extraocular muscle pain Inay be present. Their occurrence and duration vary with the individual. Seldom is fever present. Extreme muscle weakness is a common complaint. Paralysis of the extremities may occur. Physical findings are variable and nonspecific in ciguatera poisoning. The severity of the case varies withthe individual’s sensitivity to the toxin, the toxicity of the fish, and the amount of toxic fish eaten. Recovery varies greatly from case to case, and can be from 48 hours to several days, weeks, months, and, in some cases, years. Fatalities are uncommon, but do occur. Fatalities may be due to cardiovascular collapse or respiratory paralysis. Exposure to the ciguatoxin complex does notconfer immunity. There are no accurate statistics worldwide as to the incidence or mortality rate of ciguatera fish poisoning. There is a gross underreporting of outbreaks even in regions in which ciguatera is endemic. The subject of ciguatera fish poisoning has been under intensive investigation by numerous workers over an extended period of time (25,483 190). Treatment: The treatment of ciguatera fish poisoning is largely symptomatic, but thereare a fewspecificmodalitiesthatmaybeespeciallyhelpful.Theidentity of the fish is of minor value because about 300 different species of tropical reef fish have been incriminated thus far and the amount of toxin in the fish varies greatly from one specimen to the next. The diagnosis of ciguatera fish poisoning is based on the symptomatology presented. Gastric lavage or vomiting induced by sticking ones finger down the throat, or using apomorphine or ipecac, should be done as soon as possible. This should be followed by the administration of a slurry of charcoal to absorb the poison in the intestinal tract. Nausea and vomiting can be controlledby using an antiemetic drug such as prochlorperazine. Hypotension can usually be helped with the useof a pressor drug such as dopamine or dobutamine. Calcium gluconate may also be helpful in treating the hypotension and myocardial insufficiency. Bradycardia may be controlled with the use of atropine. Cool showers and the use of hydroxyzine may be helpful in relieving the pruritus. Intravenous sodium ascorbate (25 g diluted in 250 1111 of normal saline per day for10 days) and vitamin B complex have been ernployed in relieving someof the toxic effectsof ciguatoxin. Mannitol has been found to provide symptornatic relief in many cases (121,130). The fruit of the nono tree (Morinda citrijiolia Linnaeus) has been used for centuries by South Pacific islanders to treat the symptoms of ciguatera fish poisoning ( 2 ) . The juice of this fruit is now sold in the United States and elsewhere throughout the world under the trade name “Noni.” Theusual dosage is 3-4 ounces of the juice per day. The product is nontoxic and should be tried. A variety of other therapies have been employed, but none of them have been shown to be completely effective. See Refs. 27-29. Preverlrion: There is no reliable methodof detecting a ciguatoxic fish by its appearance. However, there are a few basic guidelines that are helpful. Such large, predacious reef fish as snapper, barracuda, grouper, and jacks should be eaten with caution. The larger the fish is the greater is the potentialof ciguatoxication if the fish iscaptured in an endemic
Fish Toxins
33
region. Large fish generally accumulate the toxic products fromthe various trophic levels in the food chain below them. Ciguatera is essentially the end resultof a toxic biochemical magnification problem in which humans are the final recipient of the toxic agents from all of the marine organisms in the trophic levels below them. When catching fish in a suspected ciguatoxic region, it is always advisable to seek the advice of the locals as to the edibility of the fish. The viscera (i.e., liver, gonads, and intestines) of many tropical reef fish are toxic and should not be eaten. Such ordinary cooking procedures as frying, baking, boiling, or drying do not render a fish safe to eat. Ifin doubt concerning the toxicity of the fish, eat only a small amount and wait for a period of several hours before eating additional quantities of the fish. Tropical moray eels frequently are toxic, maybe deadly, and should not be eaten. Offshore fish generally are safer to eat than inshore reef species. Bioassay methods have been used to detect ciguatoxic fish utilizing a variety of test animals,includingbrineshrimp,ants, flies, cats,dogs,mongooses,rats,guineapigs, plants, chickens, frogs, and so on. See Ref. 48 for a review of these bioassay methods. Cats have been found to be extremely sensitive to ciguatoxin. All of these bioassay methods tend to be cumbersome, time consuming, and, in most instances, expensive. Several immunoassay methods have been developed for the detection of ciguatoxic fish. One of thefirst of these techniques utilized a radioimmunoassay method for the detection of minute quantities of antigens and antibodies (91-94). This test was refined further into an enzyme immunoassay method (95) that was found to be easier to run, less expensive, more feasible for screening all sizes and varieties of fish, and could be used to test liver and musculature. A more inexpensive, but still reliable assay is the stick test developed by Hokama (79,96,97) and Hokama et al. (98). The stick test measures ciguatoxin and polyether compounds, including okadaic acid. The stick test has been used commercially to a limited extent in Hawaii. The stick test appears to be the most reliable and practical assay method currently available for the screening of ciguatoxic fish. Unfortunately the stick test is generally not available in most endemic ciguatoxic regions of the world.
D. Clupeotoxic Fish Clupeotoxic fishare members of theorderClupeiformeswhichincludes theherrings, anchovies, and related species. Clupeiform fishes become poisonous after eating toxic dinoflagellates such as Osrreopsis siarnensis Schmidt (99). Undoubtedly other species of dinoflagelllates are also involved but have yet to be incriminated. This fom1 of poisoning is rare and resembles ciguatera, but it is very rapid in its action and has a high mortality rate. The membersof the order Clupeiformes include the families Clupeidae, the herrings; Engraulidae, the anchovies; Elopidae, the tarpons; Albulidae, the bonefish; Pterothrissidae, the deep-sea bonefish; and Alepocephalidae, the deep-sea slickheads. However, the families most commonly incriminated in human clupeotoxications are members of the Clupeidae and Engraulidae. The clupeiform fish are characterized as follows. They are softrayed fish with the anterior vertebrae simple, unmodified, and without auditory ossicles; symplectic bone is present and there are no interclavicles.The recessus lateralis is present, and the infraorbital canal merges with the preopercular canal within a chamber of the neurocranium. Parasphenoid teeth are absent. There is no large foramen on the anterior ceratohyal, the parietals are separated by the supraoccipital, and the opercular bones are
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distinct. The pharyngeal bones are simple above and below,with the lower not falciform. The bones of the jaws are developed; the maxillary is broad, always distinct from the premaxillary, and forms part of the margin of the upper jaw. There are no barbs. The shoulder girdle is well developed and connected withthe cranium by a bony posttemporal. a slit behind the fourth. The air bladder,if present, hasa pneumatic There are four gills with duct. The dorsal and anal fins are without true spines; the ventral fins are abdominal and the adipose fin can be present or absent. This is a large group of fish comprising most of themarinesoft-rayedfish.Mostareplanktonfeederswithnumerous,longgillrakers (39.100).
Representcltive Species Family: Clupeidae (sardines, herrings) Species: Clupmodon thrissa (Linnaeus). Thread herring. Length 9 in. (25 cm). Distribution: Indo-Pacific, China, Japan, Korea. Species: Cluperr sprnttus Linnaeus. Sprat. Length 6 in. ( 1 S cm). Distribution: Northeastern Atlantic, Mediterranean. Species: Hcrrewgulrr ovnlis (Bennett). Sardine. Length 6 in. ( I S cm). Distribution: Indo-Pacific, Red Sea. Species: Opisthot?etncr oglinum (LeSueur). Atlantic thread herring. Length 9 in. (25 cm). Distribution: West Indies. north to Cape Cod. Family: Engraulidae Species: Etrgrcrulis et~crnsicholus(Linnaeus). Anchovy. Length 7 in. (20 cm). Distribution: Eastern Atlantic and Mediterranean. Species: Thrissincr baelmrcr (Forskil). Anchovy. Length 4 in. ( 12 cm). Distribution: Indo-Pacific, Red Sea, enters river mouths.
Clupeotoxications result from eating clupeiform fish such as sardines, herring, and anchovies. Most poisonings have occurred in tropical island areas and were caused by eating fish captured close to shore. It is believed that clupeotoxism is seasonal and most likely to occur during the summer months. Clupeotoxin has been isolated (99), but its molecular structure has not been elucidated. Clirzicul Clrcrrrrc.ter-istics: The symptoms and signs of clupeotoxism are distinct and usually violent. The first indication of an intoxication is a sharp metallic taste that may be present immediately following ingestion of the fish, followed by nausea, dryness of the mouth, vomiting, malaise, abdominal pain, and diarrhea. The gastrointestinal upset may be accompanied by a feeble pulse, tachycardia, chills, cold and clammy skin, vertigo, a drop in blood pressure, cyanosis, and other evidences of vascular collapse. Within a very short period of time, or concurrently, a variety of neurological disturbances ensue, such as nervousness, dilated pupils, violent headaches, numbness, tingling, hypersalivation,musclecramps,respiratorydistress,progressivemuscularparalysis,convulsions, coma, and death. Death may occur in less than IS minutes. Ferguson (101) claimed that the poison was so rapid in its action that natives died while in the very act of eating the yellow-billed sprat (Clulwn tlrrissrr). Pruritus and various typesof skin eruptions, including desqualnation and ulcerations, have been reported in v i c t i m that have survived. There are no accurate statistics available regarding the mortality rateof clupeotoxism, but judg-
Fish Toxins
35
ing from the documented case reports, the fatality rateis very high and the victims generally die within minutes to hours. It is believed that clupeotoxisln in some instances may be related to ciguatera poisoning, but this has not been documented. Some cases appearto exhibit ciguatera-like symptoms. Since clupeoid fish are primarily plankton feeders, it is likely that some of these fish are ingesting highly toxic dinoflagellates. Tretrtnzent: Follow thetreatmentrecommendedforciguaterafishpoisoning.See Refs. 27-29. Prcventiorr: There are no reliable methods of detecting a clupeotoxic fish and preventing intoxication. Outbreaks of this intoxication are rare and there are insufficient data concerning the nature of the poison. No screening methods have been developed for the testing of clupeotoxic fish. Most of the clupeotoxic fish are generally valuable food fish.
E. Gempylotoxic Fish The gempylids, escolars, or pelagic mackerels are a small group of predacious oceanic fish. They have a band-shaped body, large. sharp teeth, and are distinguished from the true mackerels by the complete absence of a later keel or ridge on the caudal peduncle. Two dorsal finsarepresent,thefirst of which is spinous and longer thanthesecond. Gempylids produce an oil that has a pronounced purgative effect.
Representcrtive Species Family: Gempylidae (castor oil fish) Species: Rrrvcttus pr-etiosus Cocco. Castor oil fish. Length 4.2 ft (1.3 m). Distribution: Tropical Atlantic and Indo-Pacific.
Gernpylicl Poisonirrg The gempylid poisoning formof ichthyosarcotoxism is caused by ingesting the flesh or sucking the rich, oily bones of the fish. People suffering from constipation in the South Pacific islands use gempylid fish for its relief. Clinical Clrnrcrcteristics: Ingestion of theoil contained i n theflesh and bones of gempylid fish causes diarrhea, which, although pronounced, is generally without pain or cramping (26,102,103). No other untoward effects have been reported. Gempylotoxisln has also been referred to as gempylid diarrhea. The oil is similar to castor oil, comprised mainly of oleic acid, but it has different pharmacodynamic properties ( 104,105). Treutmerlt: No treatment is required. Prew1tiotl: Avoid eating gempylid fish.
F.Scombrotoxic
Fish
Scombrotoxism,orscombroidpoisoning, is caused by perciform fish of thesuborder Scombroidei, all of which are members of the single family Scombridae, the tunas and related species. One of the members of the order Beloniformes, family Scomberesocidae, the Japanese saury (Cololabis s a i r . c ~ )has also been incriminated in sombroid poisoning. Recently, such otherfish species as mahi-mahi, jack, bluefish, herring, sardines, and anchovies have been reportedto cause scombroid poisoning (87). Scombroid poisoning accounts for about 5% of food-borne poisonings i n the United States. Scombroid fish are characterized by their adaptation for swift locomotion, having
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a sharp profile anteriorly and a slender tail with a widely forked caudal. There is a series of detached finlets on the back behind the second dorsalfin and on the undersurface behind the anal fin, a feature that distinguishes them from most other fish. Scombroids have two dorsal fins, of which the first is composed of spines and the second of soft rays. The fins fit into grooves or depressions on the body, the bones of the head lie flat, and the gill covers fit tightlyagainstthesides. Thescalesare usuallysmall,thin, andmetallicin appearance, offering a minimum of friction in the water. Scombroids are the epitome of grace, form, and speed. Tunas and related species are largely oceanic, migrating great distances through the open seas. Mackerels are largely inhabitantsof littoral waters. Scombroids are distributed widely throughout all temperate and tropical seas, and some occasionally are found in arctic and antarctic waters. They usually swim in large schools. Scombroids generally swim near the surface of the water during spawning season. During the warmer months they approach the shore, but retire to deeper water during the cold months. They feed on to the surface at night. the plankton swimmingin the deeper water during the day, and rise Scombroids are predatory and voracious feeders. In addition to plankton, they also feed on a wide variety of moderate-size fish. Some of the tunas can be found at depths of 200 m or more, whereas other scombroids seldom descend below 40 m . The sauries of the family Scomberesocidae resemble the needlefish, but have short jaws and are identified by a series of five to seven finlets following both the dorsal and anal fins. There are four species and none attain a length much beyond 35 cm. Sauries inhabit temperate and tropical waters. They arevery abundant in some regions and constitute an important foodfisheryin Japan. Sauries feed on planktonandsmall fish. The sauries also have been incriminated in scombroid fish poisoning, but outbreaks have been limited to Japan. Repr~ser~tcrtive Species Family: Scomberesocidae (sauries) Species: Cololabis saircr (Brevoort). Saury. Length 12 in. (30 cm). Distribution: Japan. Family: Scombridae (tunas. nmckerels, albacore) Species: Euthgnm~.s peltrrtis (Linnaeus). Skipjack. Length 19 in. (50 cm). Distribution: Cosmopolitan in warm seas. Species: Scolllber. jcrpotzic~~ Houttuyn. Pacific mackerel. Length 15 in. (40 cm). Distribution: Cosmopolitan in warm seas. Species: Thunnlr.~tl1gtIr~u.s(Linnaeus). Bluefin tuna. Length I O ft (3 m). Distribution: Cosmopolitan in subtropical and temperate seas.
Sconlhroicl (Histtmirle) PoisorlirlR Scombroid poisoning is caused by the improper preservation of scombroid fish or other fish species that results in certain bacteria, mainly species of the family Enterobacteriaceae ( C l o . ~ f r i d i wLrrctobmillus, ~, Prote~rs,Vibrio), acting on histidine in the muscle of the fish convertingit to histamine.The toxicity of histamine is enhanced by the presence of certain potentiators (e.g., cadaverine and putrescine) that act by inhibiting intestinal histamine-metabolizing enzymes. The enzyme inhibition increases the intestinal uptake of unmetabolized histamine (106-108). Histamine ingested by itself generally is much less toxic. Scombroid poisoning is the most common form of ichthyosarcotoxism and occurs
Fish Toxins
37
throughout the world wherever scombroid fish are eaten. Moreover, it is the only form of ichthyosarcotoxism in which bacteria play an active role i n toxin production within the body of the fish. Clirlical Chrrracteristics: The symptoms of acutescombroidpoisoningresemble those of histamine intoxication. The symptoms are characteristic and appear with almost monotonous consistency. Toxic scombroid fish frequently can be detected immediately upon tasting the fish. Victims state that the fish has a sharp or peppery taste. Symptoms usually occur within a few minutes after ingestion of thetoxin and consist of intense headaches, dizziness, throbbing of the carotid and temporal vessels, epigastric pain, burning of the throat, cardiac palpitation, rapid and weak pulse, dryness of the mouth, thirst, inability to swallow, nausea, vomiting, diarrhea, and abdominal pain. Within a short time a generalized erythema and an urticarial eruption may develop, covering the entire body and accompanied by a severe pruritus. The face of the individual becomes swollen and flushed, the eyes become injected, and coryza develops. In severe cases there may be bronchospasm, suffocation, and severe respiratory distress. Various other minor discomforts such as fever, chills, malaise, tremors, metallic taste, and cyanosis of the gums and tongue may occur. There is a danger of shock, and deaths have been reported. However, the acute symptoms generally are transient, lasting only 8-12 hours (48,108-1 1l ) . Treatment: The treatment of scombroid poisoning is largely directed toward relieving the symptoms of the histamine reaction. This form of poisoning is generally selflimiting and fatalities are rare. Minor intoxications can usually be treated with diphenhydramine (Benedryl). Scombroid poisoning may cause respiratory distress, in which case the victim should be taken immediately to an emergency treatment center. Epinephrine is the treatment of choice for respiratory problems in this instance, but it should be used withcaution in olderindividuals with a historycardiacproblems.SeeRefs.(27-29, 92,l 12-1 15). Prevenrion: Scombroid fish and other related species believed to cause this form of ichthyosarcotoxism should be refrigerated promptly or eaten soon after capture. It has been shown that the histamine content in some of the scombroid fish increases from 0.09 mg/100 g of tissue to about 95 mg/ 100 g of tissue when kept at room temperature (20°C25°C) for about10 hours ( 1 16).Toxic scombroid fish cannot always be detectedby appearance or odor. The histamine content in the flesh may be very high with little or no evidence of putrefaction. Scombroid or any other fish having a sharp or peppery taste should be discarded. Scombroid fish with histamine levels greater than 20 mg/ 100 g should be discarded.
G. Tetrodotoxic Fish Tetrodotoxications constitute one of the most violent formsof marine biotoxications. This type of ichthyosarcotoxism commonly is designated as tetrodon or puffer poisoning. The causative transvectors of the poison tetrodotoxin are members of the order Tetraodontiformes, formerly the Plectognathi, which includes the families Tetraodontidae, the puffers; Diodontidae, the porcupinefish; Canthigasteridae, the sharpnosed puffers; and Molidae, the molasor ocean sunfish. The order also includes such other fish families asthe spikefish, filefish, trunkfish, three-toothed fish, and triggerfish, but these are not included in any fish mayalsotransvectany or all of the degree of detail in this chapter. These same ciguatera complex of poisons (e.g., ciguatoxin, maitotoxin, or possibly palytoxin). For
38
Halstead
the 111ost part, this section focuses on the fish families Tetraodontidae, Diodontidae, and Canthigasteridae. Tetrodotoxications are the resultof ingesting a poison known as tetrodotoxin, molecular formula C,,H,,N,O,, molecular weight 319, and LDS,, toxicity in mice is I O pg/kg ( 1 17,118). Tetrodotoxin actsby preventing nerve conductionby an extremely specific and reversible blockage of the inward movement of sodium ions through the cell membrane of an activated neuron (48,119-121). The tetraodontiform fish are characterized by the absence of parietal, nasal, or infraorbital bones, and usually have no lower ribs; the posttenlporal region is present and is siniple and fused with the pteroticof the skull; the hyomandibular and palatine are firmly attached to the skull. Gill openings are restricted. The maxilla is usually firmly united or fused with the premaxilla. Scales are usually modified as shields or plates. The lateral line may be present or absent, and sometimes is multiple. The swim bladder is present except in molids, and there are 16-30 vertebrae. Tetraodontifornles can produce sounds by grinding the jaw teeth or the pharyngeal teeth or by vibrating the swim bladder. The stomach of some of these fish is highly modified to permit inflation to an enormous size. Fish with this ability are commonly called puffers. Inflation is caused by gulping large quantitiesof water into aventral diverticulum of the stomach when the fish is frightened or annoyed. Deflation occurs by expelling the water. If the fish is removed from the water, inflation can occur with air (39,122).Puffers feed mainly on corals and mollusks, but tend to be omnivorous. A wide range of phylogenetically unrelated aquatic organisms are now known to transvect tetrodotoxin, including starfish, gastropod mollusks, the Australian blue-ringed octopus, tropical reef crabs, gobyfish, and a variety of freshwater amphibians. This subject has been reviewed in greater depth elsewhere (48).
Representcrtive Species There are a large numberof fish species incriminated in transvectoring tetrodotoxin, but only a few representative fish are listed below. Family: Canthigasteridae (sharp-nosed puffers) Species: Canthigaster rivuhtus (Temminck and Schlegel). Rivulated Goby. Length 3 in. ( I O cm). Distribution: Indo-Pacific, Japan. Family: Diodontidae (porcupinefish) Species: Chilonzycteru.7 c!jjitziirlis Giinther. Porcupinefish. Length 6 in. ( 1 7 cm). Distribution: Southern California, Galapagos Islands, Hawaii, Japan. Species: Diodorl hystri.r Linnaeus. Porcupinefish. Length 35 in. (90 ctn). Distribution: All warm seas. Family: Tetraodontidae (puffers, blowfish, fugu) Species: Arothron lzisjdus (Linnaeus). White-spotted puffer. Length21 in. (53 cm). Distribution: Indo-Pacific, Panama, Japan, Australia, South Africa, Red Sea. Species: Arothron meleagris (LacCpkde). Guineafowl puffer. Length 13 in. (33 cm). Distribution: West coast of Central America, Indo-Pacific. Species: Fugu Imrddis (Temminck and Schlegel). Fugu. Length 14 in. (35 cm). Distribution: China, Japan. Species: kcgocephcc/u.s luncrris (Bloch and Schneider).Puffer.Length 12 in. (35 Clll).
Fish Toxins
39
Distribution: Indo-Pacific, Red Sea, China, Japan, eastern coast of Africa. Species: Sldlaeroides umul~/tu.s(Jenyns). Puffer. Length 1 I in. (28 cIl1). Distribution: California to Peru, Galapagos Islands. Species: S~h7er.oide.vrnncu1rrtu.s (Bloch and Schneider). Botete. Length 10 in. (25 cm). Distribution: Atlantic coast of the United States south to Guiana. Species: Tettwodorl lineatus Linnaeus. Puffer. Length 18 in. (46 cm). Distribution: Rivers of northern and western Africa.
Tetrorlorr (Puffer.) Poisoilirlg Method ofIntmication: Puffer poisoning or tetrodon poisoning is caused by ingesting the flesh, viscera, or skin of toxic tetraodontiform fishes (i.e., sharp-nosed puffers, puffers, porcupinefish, and other related fish). Certain goby fish, gastropod mollusks, and tropical reef crabs are also capable of transvectoring tetrodotoxin and causing a biotoxication. Puffer fish are more dangerous to eat imnlediately prior to and during the reproductive season, during which time the poison content in the body of the fish increases with the gonadal activity. The skin, liver, ovaries, and intestines arethe most toxic portions of the fish. The tnusculature of the fish usually is safer to eat than other parts of the fish, but at times it also may be toxic. The toxicityof the fish cannot be determinedby its appearance, freshness, or size since even small puffers may contain sufficient poison to be lethal. Puffer and fugu poisoning continuesto be the major causeof fatal food intoxications in Japan, where puffer meatis looked upon as a gourmet delicacy. The sale of toxic puffers is carefully regulated by public health authorities in Japan, butthis has not prevented periodic outbreaks of fatal food poisoning. TheU.S. Food and Drug Administration (FDA) recently has permitted the importation of Japanese puffers for sale in fugu restaurants in the United States (25,87), but this does not guarantee the safety of puffer products. All puffers are potentially toxic unlessthey have been cultivated artificially (123-125). There is evidence that several different strains of marine bacteria may play an important role in the biosynthesis of tetrodotoxin in the body of the fish (5,7). Clinicd CharLlcteri.7tic.s:The onset and symptomatology in puffer poisoning varies greatly depending upon the person and the amount of poison ingested. Initial symptoms usually consist of paresthesias of the lips and tongue, malaise, pallor, dizziness, and ataxia that develop within 10-45 minutes after ingestion of the fish, but may occur as much as 3 hours or niore after ingestion. The paresthesias are described as a tingling or a prickly to the fingers and toes, and gradually develops sensation that may subsequently spread into numbness.In severe cases,the numbness may involve the entire body, which has been described by victims as a floating sensation. Hypersalivation. profuse sweating, extreme weakness, precordial pain, headache, subnormal temperatures, hypotension, and a rapid, weak pulse usually appear early in the succession of symptoms. Nausea, vomiting, diarrhea, and epigastric pain frequently are present. The pupils are constricted during the early part of the intoxication, but later become dilated.As the disorder progresses,the pupillary and corneal reflexes are lost. Shortly after the development of the paresthesias, respiratory symptoms become a prominent part of the clinical picture. Respiratory distress, as noted by an increased rate of respiration, movements of the nostrils, and shallow respiration, generally is observed. Respiratory distress later becomes very pronounced, and the lips, extremities, and body become intensely cyanotic. Petechial hemorrhages involving extensive areas of the body, blistering, and subsequent desquamation may occur. Muscular twitching, tremor, andloss
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of motor coordination become progressively worse and finally terminate in extensive muscular paralysis. The first areas to be involved areusually the throat and larynx, resultingin aphonia, dysphagia, and, later, complete aphagia. The muscles of the extremities become of the patient paralyzed and the patient is unable to move.As the end approaches, the eyes become fixed and glassy, and convulsions may occur. The victim may become comatose, but in most instances the patient remains conscious and the mental faculties remain acute until shortly before death. The fatality rate of puffer poisoning is about 59% in untreated cases. There are no accurate mortality statistics worldwide. Treutmerlt: Early treatment seeks to remove the gastric stable toxin, which is partially inactivated by alkaline solutions. If the victim is seen within 3 hours of ingestion, 1 L 2%sodiumbicarbonate.Thisis gastriclavageshouldbeperformedwithatleast followed with activated charcoal in sorbitol solution. If the victim has difficulty swallowing or breathing, or is not alert, intragastric manipulation should be preceded by endotracheal intubation. Supplemental oxygen and ventilatory assistance should be promptly instituted as respiratory paralysis progresses. The physician should rememberthat the paralyzed victim may be fully conscious and should offer the victim frequent verbal reassurances. Hypotension induced by tetrodotoxin may require the intravenous (IV) administration of crystalloid fluid augmentation. Bradyarrhythmias generally respond to atropine (0.5 mg IV up to 2.0 mg). Severe heart block may require the placement of a temporary transvenous pacemaker. While a minor intoxication may be limited to paresthesias, all victims should be observed for at least 8 hours to detect deterioration, particularly respiratory failure. Under no circumstances should anyone with dysphagia be given liquids by mouth. The fruit of the nono tree (Morindcr citrijola Linnaeus) has been used for centuries by South Pacific islanders to treat the symptoms of ciguatera fish poisoning (2) and may be helpful in the treatment of tetrodon poisoning. The juice of the fruit is now sold in the United States and elsewhere throughout the world under the trade name “Noni.” The usual dosage is 3-4 ounces of the juice per day, or four capsulesof the concentrate. The product is nontoxic and should be tried. See Refs. 27-29. Preverltion: If one follows the old Mosaic sanitary laws in Deuteronomy 14:9- 10eliminate all scaleless fish from the diet-then puffer poisoning will never be a problem. If one is living in Japan and has a desire to eat fugu, he should purchase the fish from a first-class, authorized restaurant with a licensed puffer cook. However, even following this procedure will not absolutely guarantee food safety. Eating puffers, at best, is a game of Russian roulette. In any event, the skin and viscera of the fish should never be eaten. No cooking or drying procedure destroys the poison.
V.
ICHTHYOOTOXIC FISH
Ichthyootoxism is one of the lesser-known forms of fish poisoning. Ichthyootoxic fish constitute a group of fish that produce a poison generally restricted to the gonads. This group of toxic fish does not include the ichthyosarcotoxic puffers because the poison in puffers is distributed widely throughout the body. The musculature and other partsof the body in ichthyootoxic fish generally are safe to eat. Some of these fish are found only in freshwater. There does not appear to be any particular phylogenetic affinity other than the fact that the fish involved are all members of the class Osteichthyes, the true bony
Fish Toxins
41
fish (see Sec. 1v.C for a description of the class Osteichthyes). Most of the intoxications resulting from the ingestion of ichthyootoxic fish occur during the reproductive season, during which the gonadal activity of the fish is at its peak.
Repre.sewtative Species Family: Acipenseridae (sturgeons) Species: Huso huso (Linnaeus). Sturgeon. Length 6 ft (1.8 m). Distribution: Black Sea, Sea of Azov, Caspian Sea, Mediterranean Sea, and rivers that drain into these seas. Family: Lepisosteidae (gars) Species: Lepisosteus tristoechus (Bloch and Schneider). Alligator gar. Length 19 ft (6 m). Distribution: Rivers of Cuba, bays and coastal waters of the Gulf of Mexico. Family: Esocidae (pikes) Species: Esox lucius Linnaeus. Northern pike. Length 48 in. ( I .2 m). Distribution: Freshwaters of Europe, northern Asia, and North America. Family: Cyprinidae (minnows) Species: Brrrbus barbus (Linnaeus). Barbel. Length 35 in. (89 cm). Distribution: Freshwaters of northern and central Europe. Species: Schizothorctx irltermedius McClelland. Snow trout, marinka. Length 18 in. (46 cm). Distribution: Freshwaters of central Asia. Species: Tirlccrtinccz (Linnaeus). Tench. Length 24 in. (64 cm). Distribution: Freshwaters of Europe. Family: Stichaeidae (pricklebacks) Species: Stichaeus grigorjewi Herzenstein. Japanese prickleback. Length 20 in. (51 cm). Distribution: Freshwaters of Japan and Korea. Family: Cottidae (sculpins, cabezon) Species: S~.orl.’aenichtll?,smrrrmorrrtus (Ayres). Cabezon. Length 30 in. (76 cm). Distribution: Pacific coast of North America.
Ichthyootoxism (Fish Roe Poisoning) Mechanism of Intoxication: Ichthlyootoxism, or fish roe poisoning, results from ingestion of various salt- and freshwater fish of Europe, Asia, and North America, and to a lesser extent, the tropics. The of roemany freshwater and estuarine fish of eastern Europe and Asia are dangerous to eat during their reproductive period,usually March-June. Most ichthyootoxicfisharemembers of thefreshwaterCyprinidaeminnowgenera Burbus, Schizothorclx, and Tinca, found in Europe and Asia, and the genus Srichaeus of the family Stichaeidae, found in Japan and Korea. These fish have caused innumerable intoxications in Europe and Asia. A Pacific North American species of the Cottid genus, Scorpaenichthys, also has produced intoxications. Although cooking is said to destroy most ichthyootoxins, it cannot be relied upon as a completely safe procedure since the poison in some fish appears to be resistant to of ichthyootoxic fish generally are safe to cat during heat. The musculature and other parts the reproductive season. The chemical nature of ichthyootoxins is unknown. For a more comprehensive review of this topic, see Refs. 25 and 48. Cliniccrl Chctracteristics: Symptoms develop soon after ingestion oftheroeand
Halstead
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consist of abdominal pain, nausea, vomiting, diarrhea, headache, fever, bitter taste in the mouth, dryness of the mouth, intense thirst, a sensation of constriction of the chest, cold sweats, irregular pulse, low blood pressure, cyanosis, pupillary dilation, syncope, chills, dysphagia, and tinnitus. In severe cases there may be muscle cramps, paralysis, coma, and death.Barbus roe usually does not cause death, but fatalities have resulted from eating Schizothom.r roe ( 5 3 ) . Treatment: Treatment is symptomatic. There are no known antidotes. Prevention: Avoid eating the roe of any fish during the reproductive season unless you have positive knowledge that the roe is safe to eat. This preventive advice is particularly pertinent to the freshwater and brackish water fish of Europe andAsia and all tropical marine species. Cooking fish roe cannot be relied upon to inactivate ichthyootoxins.
VI.
ICHTHYOHEMOTOXIC FISH
Ichthyohemotoxic fish consist of a variety of different species of eels. All of the fish of this group are membersof the order Anguilliformes (Apodes). The members of this group are characterized by an eel-like body, abdominal pelvic fins (when present), and an air bladder connected with the intestine by a duct. Gill openings are narrow or slitlike. The scales, if present, are cycloid. The dorsal and analfins are very long and usually confluent (1 26). The research on ichthyohemotoxicfish, or fish having toxic blood, reveals that there isverylittleclinical infomlation involving humans. Hemotoxins are largely parenteral poisonsandseldomtoxicwhentaken by mouth. Verylittle is known concerning the chemical nature of these posions.
Representcltive Species Family: Anguillidae (freshwater eels) Species: Anguilla anguilla (Linnaeus). Common European eel. Length 39 in. ( I m). Distribution: Europe, fresh- and saltwater rivers. Family: Congridae (conger eels) Species: Conger conger (Linnaeus). Conger eel. Length 79 in. (2 m). Distribution: Atlantic Ocean, Mediterranean Sea. Family: Muraenidae (moray eels) Species: Muraetza helena (Linnaeus). Moray eel. Length 59 in. (1.5 m). Distribution: Eastern Atlantic and Mediterranean Sea.
1chtk~ohemoto.rism~to.ris~n Mechanism of Intoxication: Ichthyohemotoxins are largely parenteral poisons, although there are a few instances on record in which individuals have become intoxicated due to ingestion of large quantities of the poison by mouth. Most of the ichthyohemotoxic or fish generally are recognized as good food fish. However, ingestion of fresh blood serum from these fish may cause food poisoning. This is a rare form of marine food poisoning. Clinical Characteristics: Very littleis known concerning the symptomatology of ichthyohemotoxism in humans. Fish serum intoxications may be of two types: syste1nic9 a form that results from drinking fresh, uncooked fish blood, and topical. The symptolns of the systemic form consist of diarrhea, bloody stools, nausea, vomiting, hypersalivation,
Fish Toxins
43
skin eruptions, cyanosis, apathy, irregular pulse, weakness, paresthesias, paralysis, respiratorydistress,andpossiblydeath.Forthetopicalform,thereisasevereinflammatory response when raw eel serum accidentally comes in contact with the eye or the tongue. Oral symptoms consist of burning, redness of the mucosa, and hypersalivation. Ocular contact invokes a severe burning sensation and redness of the conjunctivae, lacrimination, and swelling of the eyelids. Eye irritation may persist for several days. Usually recovery is spontaneous. For a more comprehensive discussion of this subject, see Ref. 48. Treatment: Treatment is symptomatic. There are no known specific antidotes. Prevewtiow: Care should be takenin the handlingof eel blood. Raw eel blood should not be ingested. Cooking is said to destroy the toxic properties of eel blood.
VII. ICHTHYOHEPATOTOXIC FISH The livers of certain edible species of fish are sometimes found to be toxic to eat. Most of the outbreaks of ichthyohepatotoxism have occurred in Japan. Nothing is known concerning the chelnical nature of the poisons involved. It is believed that in some instances the intoxications are due to hypervitaminosis A. The fish involved are members of the class Osteichthyes.
Representdve Species Family: Scombridae (tunas, mackerels, albacore) Species: Scornberomorus niphonius (Cuvier and Valenciennes). Japanese mackerel. Length 39 in. ( 1 m). Distribution: Japan, Korea, China. Family: Serranidae (Sea bass, grouper) Species: Stereolepis ischinagi (Hilgendorf). Sea bass. Length 78 in. ( 2 m). Distribution: Japan, Korea.
I c h t h ~ o h e ~ ~ ~ t ~(Fish t ~ ~ Liver i s t r /Poisoning) Symptoms of ichthyohepatotoxism appear within 30 minutes- 12 hours after ingesting the fish liver. The initial symptoms consist of nausea, vomiting, fever, and headache. The headache may be very severe and is said to be intensely aggravated by the slightest movement of the body, head, or eyes. A mild diarrhea may be present, but abdominal pain generally is absent. The face of the victim usually becomes flushed and edematous, and a macular rash having large patchy erythematous areas develops. Within 3-6 days, desquamation appears. Large areas of skin may peel off around the nose, mouth, head, neck, and upper extremities, and gradually extends over the entire body. Epilation may 30 days. Vesicular formation of the oral result. Desquamation may continue for about mucosa and bleeding from the lips may occur. Orbital pain, joint aches, and cardiac palpitation with a rapid pulse may be present. Victims have complained of a slippery sensation on the tip of the tongue. Most of the more acute symptoms disappear in about 3-4 days. Residual symptoms consist of chapping of the lips, stomatitis, and mild hepatic dysfunction. Recovery usually is uneventful. No fatalities have been reported. The liver may be enlarged, but no jaundice has been observed. Treatment: Treatment is symptomatic. There are no specific antidotes. Prevention: Care should be taken in eating fish livers. In general, the liver is one of the most dangerous parts of a fish to eat. If a fish is poisonous, a greater concentration
Halstead
44
of the poison is likelyto be foundin the liver than almost any other part of the fish. Cooking does not destroy the poison. Most outbreaks of ichthyohepatotoxism have resulted from eating fish livers that have been sauted or in soup. The toxicity of of fish liver cannot be determined by its appearance. It is recommended that fish livers be eliminated from the diet unless there is reliable information that it is safe to eat.
VIII. ICHTHYOALLYEINOTOXIC (HALLUCINOGENIC) FISH Ichthyoallyeinotoxism, or hallucinogenic fish poisoning, is caused by ingesting certain types of reef fish known to occur in the tropical Pacific and Indian Oceans. This biotoxication may result from eating either the head or flesh of the fish. The source and chemical nature of the poison is unknown. Most of the fish species incriminated in ichthyoallyeinotoxism also are involved in ciguatera fish poisoning. Whether there is a relationship between these two types of intoxications is not known. All of the hallucinogenic fish are members of the class Osteichthyes.
Representative Species Family: Kyphosidae (sea chubs) Species: Kyphosus citwrmcetIs (Forskil). Sea chub. Length 20 in. (50 cm). Distribution: Indo-Pacific. Family: Mugilidae (mullets) Species: Mugil cephalus (Linnaeus). Common mullet. Length 12 in. (30 cm). Distribution: Cosmopolitan. Family: Mullidae (Goatfish, surmullets) Species: Upetleus arge Jordan and Evermann. Goatfish. Length 12 in. (30 cm). Distribution: Indo-Pacific.
Ichtkgocrllyeinott,.risrn (Hdlucinogenic Fish Poisoning) Mech~rnistnqf Intoxication: Ichthyoallyeinotoxism, or hallucinogenic fish poisoning, is caused by eating the flesh or head of certain species of toxic reef fish, producing hallucinations. The poison reputedly is concentrated in the head of the fish, which is said to be the most dangerous part of the fish to eat. The nature of the poison is unknown. This type of biotoxication is sporadic, uncommon, and completely unpredictable. You cannot detect a hallucinogenic fish by its appearance. The poison is not destroyed by cooking. Clirlicrrl Chnrcrcteristics: The poison affects primarily the central nervous system. 2 hours after ingestion of the fish, continue The symptoms may develop within minutes to for about 24 hours, and then gradually subside. Symptoms consist of dizziness, loss of equilibrium, lack of motor coordination, hallucinations, and mental depression.A common complaint of the victim is that it feels as though “someone is sitting on my chest,” or there is a sensation of a tight constriction around the chest. The conviction that they are going to die or other frightening nightmares are characteristic aspects of the clinical picture. Other complaints consist of itching, burning of the throat, muscular weakness, and, rarely, abdominal distress. No fatalities have been reported. This form of poisoning is generally mild. Treatment: Treatment is symptomatic. No specific antidote is available. Prevention: Caution should be exercised in eating those species of reef fish that
Fish Toxins
45
have been incriminated in ichthyoallyeinotoxism. When possible, natives should be consulted before eating the fish in tropical areas. Hallucinogenic fish cannot be detected by their appearance.
ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the technical assistance Medrano in the preparation of this chapter.
of Leonette C.
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ND Lewis.Diseaseanddevelopment:ciguaterafishpoisoning.Soc SCi Med23:983. 1986. 86. p Gopalakrishnakone, CK Tan. Progress in Venom and Toxin Research. Singapore: National University of Singapore, 1987. 1991. 87. FE Ahmed, ed. Seafood Safety. Washington, DC: Natronal Academy Press, 88. GM Calvert. The recognition and management of ciguatera fish poisoning. In: DM Miller. ed. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, 1991, pp 1- 1 1. 1991. 89. DM Miller, cd. Ciguatera Seafood Toxins. Boca Raton, FL: CRC Press, FE Russell, NB Egen. Ciguatcric fishes, ciguatoxin (CTX) and ciguatera poisoning. Toxicol 90. 1991. ToxinRev10:37-62, 91. J Laigret, G Bereziat, G Cuzon. J Polonovski. Etude comparative des acides gras extraits de s provenant de zones toxicogeneset non toxicogenes du lagon poissons C t e t l o d l ~ r m striurus de Tahiti. Bull Soc Pathol Exot 66:235-239, 1973. of ciguatoxin. 92. Y Hokama, AH Banner, D Boyland. A radioimmunoassay for the detection Toxicon15:317-325,1977. 93. PF Parc, R Ducousso, S Chanteau, E Chungue, R Bagnis. Problemes poscs par la detection ilnmunologique dc la ciguatoxine dam les tissus pisciaires [in Frcnch]. Med Oceanogr 14: 1-4,1980. 94. S Chanteau, I Lechat, F Parc, R Bagnis. Essai de detectionladeciguatoxine par une methodc immunoenzymatique [in French]. Bull Soc Pathol Exot 74:227-232, 1981. DC Baden,TYasumoto,MNukina, PJ 95. YHokama,LHKimura, MAAbad,LYokochi, Scheuer, Y Shimizu.An enzyme ilnlnunoassay for the detection of ciguatoxin. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 307-320. of ciguatoxin and related 96. Y Hokama. Simplified solid-phase immunobead assay for detection polyethers. J Clin Lab Anal 4:213-217, 1990. 97. Y Hokama. Immunological analysisof low molecular weight marine toxins. J Toxicol Toxin Rev 10:l-35, 1991. 98. Y Hokama, SAA Honda,MN Kohayashi, LK Nakagawa, AY Asahina, JT Miyahara. Monoclonal antibody (MAb) in detection of ciguatoxin (CTX) and related polyethers by the stickenzyme immunoassay (S-EIA) in fish tissues associated with ciguatera poisoning. In: S Natori.KHashimoto, Y Ueno. eds. MycotoxinsandPhycotoxins '88. Amsterdam:Elsevier Science, 1989, pp 303-310. 99. T Yasumoto, M Satake, Y Onuma, J Roux. Toxins involved in ciguatera, clupcotoxism and shark poisoning. Fifth Indo-Pacific Fish Conference, Noumea. New Caledonia, November 1997. 100. DS Jordan, BW Evermann. The Fishes of North and Middle America: A Descriptive Catalogue. Part 1. Washington,DC:SmithsonianInstitution.1896. 101. W Ferguson. On the poisonous fishes of the Caribbean Islands. Trans R SOC Edinb 9:6579, 1823. 102. RT Lowe. A History of the Fishes of Madeira. London: Bernard Quaritch, 1843. 103. A Kramer. Der purgierfisch der gilbcrtinsein [in German]. Globus 79( 12): 181-1 83, 1901, 104. Dl Macht, J Barba-Gose. Pharmacology of Rlr,Ietrlr.spreriosus, or "caster-oil fish." Proc Soc Exp Biol Med 28:772-774, 193 1. 105. Dl Macht, J Barba-Gose. Two new methods for pharmacological comparison of insoluble purgatives. J Am Pharm Assoc 20:556-564, l93 1, 106. SL Taylor, J Hui, DE Lyons. Toxicologyof scombroid poisoning. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 417-430. 107. HA Frank, DH Yoshinaga. Histamine formation in tuna. In: EP Ragelis, ed. Seafood Toxins. Washington, DC: American Chemical Society, 1984, pp 443-451. 1 08. SL Taylor. Histamine food poisoning: toxicology and clinical aspects. CRC Crit Rev Toxicol 17:92,1986. 109. T Kawbata, K Ishizaka, T Miura. Studies on the food poisoning associated with putrefaction 85.
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of marine products. I. Outbreaks of allergylike food poisoning caused by "samma sakuraboshi"(driedseasoned saury) andcannedseasonedmackerel.BullJpnSoc Sci Fish21: 335-340,1955. 1 IO. GM Dack. Food Poisoning, 2nd ed. Chicago: University of Chicago Press, 1956. 1 1 1. HBouder.ACavallo,JBouder.Poissonsveneneuxetichtyosarcotoxisme.BullInst Oceanogr 1-66, 1962. 112. JAWilliamson, PJ Fenner, JW Burnett, JF Filkin.cds.VenomousandPoisonousMarine Animals: Medical and Biological Handbook. Sydney: Universityof New South Wales Press. 1996. 113. JMeier, J, White.eds.ClinicalToxicology ofAnimalVenomsandPoisons.BocaRaton, FL:CRCPress,1995. 114. BW Halstead. Dangerous Marine Animals, 3rd ed. Ccntreville, MD: Cornell Maritime Press. 1995. 115. PSAuerbach, ed. WildernessMedicine.Management of WildernessandEnvironmental Emergencies, 3rd ed. NewYork:Mosby,1995. 116. E Geiger. Histamine content of unprocessed and canned fish. A tentative method of qualitative determination of spoilage. Food Res 9:293-297. 1944. 1 17. K Tsuda, R Tachikawa, K Sakai, C Tammura,S Ikuma, 0 Amakasu, M Kawamura, S Ikutna. O n the structure of tetrodotoxin. Chem Pharmcol Bull 12:642-645, 1964. 1 18. RBWoodward.Thestructureoftetrodotoxin.PureApplChem 9: 49-74,1964. 119. T Namhashi. Mode of aclion of dinoflagellate toxins on nerve membranes. In: VR LoCicero. ed. Proceedings of the First International Conference on Toxic Dinoflagellates. Wakefield, MA: Massachusettes Science and Technology Foundation, 1974, pp 395-402. I n : EP Ragclis, ed. Seafood 120. HS Mosher, FA Fuhrman. Occurrence and origin of tetrodotoxin. Toxins. Washington, DC: American Chemical Society, 1984, pp 333-341. 121. G Strichartz,NCastle.Pharmacologyofmarinetoxins.In: S Halland G Strichartz, eds. Marine Toxins. Washington. DC: American Chemical Society, 1990, pp 2-20. 122. JC Tyler. Osteology, Phylogeny, and Higher Classification of the Fishes o f the Order Plectognathi (Tetraodontiformes). Seattle: U.S. Department of Commerce. 1980. 123. TMatsui, S Hamada, S Konosu. [inJapanese].Bull Jpn Soc SciFish 47535, 1981. 124.TMatsui, S Hamada, K Yamamori.[inJapanese].BullJpnSocSciFish 48:l 179. 1982. 125. T Matsui, S Hamada,C Shimizu. [in Japanese]. Bull Jpn Soc SciFish 48:253, 1982. 126. LS Berg. Classification of Fishes Both Recent and Fossil. Ann Arbor, MI: J. W. Edwards, 1947.
3 Other Poisonous Marine Animals
1. 11.
Ill.
Introduction S I lnvertebratcs S 1 A. Poisonous marine Protista (Protozoa) S1 B. Poisonous cnidarians (Coelenterata) 53 C. Poisonous echinodcrms (sea cucumbers, sea urchins) D. Poisonous mollusks 56 63 E. Poisonousarthropods: crabs andlobsters Vertebrates 66 Poisonous marine turtles B. Poisonous marine tnammals Acknowledgtnent 72 References 72
A.
1.
54
67 68
INTRODUCTION
Although toxic marine fishes, paralytic shellfish poisoning, diarrhetic shellfish poisoning, brevitoxic (neurotoxic) shellfish poisoning, and amnesic shellfish poisoning probably account for the bulk of the foodborne intoxications caused by marine organisms, there are a variety of other toxic marine organisms capableof causing severe oral intoxications and even fatalities. This chapter deals with someof these lesser-known marine biotoxications. The topics discussed have been arranged phylogenetically.
II. INVERTEBRATES
A.
Poisonous Marine Protista (Protozoa)
The phylum Protista, or Protozoa, consists of single-celled microscopic organisms, most of which are free livingand inhabit an aquatic environment. Afew live i n the body fluids of other animals. Most protistans liveas independent cells, but some are grouped as colonies. Marine protistans poisonous to man are largely members of the class Mastigophora of the order Dinoflagellata. Since dinoflagellates exhibit both animal and plant characteris51
52
Halstead
tics (i.e., motility and chlorophyll), theyare claimed by bothzoologistsandbotanists. Hence they sometimes are referred to as plant-animals. They also are designated as phytoplankton. They abound in neritic waters and i n the high seas, ranging from the tropics to polar oceans. Dinoflagellates form an important part of the ocean plankton as synthetic producers of carbohydrates, proteins, and fats. During their periodic maxima, they may cause yellow, brown, green, black, red, or milky local discolorations of the sea. The “blooming” of thesetoxic plankton in excessive numbers frequently causes the mass mortality of the fish and other animals livingin the region. Phytoplankton blooms often areassociated with weatherdisturbancesorweatheralterationsthatbringabout changes in water masses or upwellings. Conditionsmost favorable for the growth of dinoflagellates are found more oftenin coastal waters than far offshore. Dinoflagellate blooms can causeseriouseconomiclosses i n a regionbecause of theirtoxicityandthemass mortalities of fish they cause. Toxic dinoflagellates play a major role as transvectors of poisons that are ingested by a variety of mollusks, causing paralytic, brevitoxic (neurotoxic), and diarrhetic shellfish to.ricus (Adachi and Fukuyo) serves as a poisoning.Thedinoflagellate Gtrmhie~~cli.scu.s transvector of theciguatoxincomplexinciguaterafishpoisoning. In addition,several other species of dinoflagellates are suspected as causative agents in ciguatera fish poisoning; they are A~nphidiniumcarterere (Hulbert), Ostreopsis ovuttr (Fukuyo), Prorocerltrurn conccrvum (Fukuyo), P. l i m r (Ehrenberg), and P. tuexicmrm (Tafall) (1).
Family: Pfiesteriaceae (dinoflagellate). Species: Pfiestericr piscicidn (Steidinger, Burkholder, et al.) Pfiesteria dinoflagellate. Distribution: Maryland, south along the southeastern coast of the United States.
Over the past 20 years there has been an increase in toxic dinoflagellate blooms worldwide which has resulted in mass mortalities of shellfish and finned fish capable of adversely affecting human health. One of the worst of these toxic blooms was caused by a new species of dinoflagellate known as Pfiesteritr pisciciclum, first reported by Burkholder et al. (2) in 1992. It was later determined by Steidinger et al. (3) that this dinoflagellate was a new species, a new genus, and a new family of the order Dinamoebales. This dinoflagellate is unique because of its polymorphic multiphasic life cycle. The dinoflagellate ranges in size from 5 to 250 pm. Moreover, the dinoflagellate produces potent toxin(s), the molecular structure of which has not been elucidated. It is estimated that this dinoflagellate has produced a mass mortality of more than one billion invertebrates and fish along the Atlantic coast involving the Pamlico and Neuse estuaries in North Carolina and the lower shores of Maryland. The cause of the toxic dinoflagellate bloom was due directly to excessive pollution by hog, chicken, and other untreated wastes that were being dumped into the rivers. The dinoflagellate requires fresh finfish or their excreta for excystment and the release of its potent neurotoxin(s). Mechnrrism of Intoxiccrtiorz: Human exposure to Pjiestericc may be in the form of an aerosol or water containing the toxic dinoflagellate or contact with the bodies of fish or shellfish that have been killed by Pfiesteria. Fish that have been killed by Pfiesteria have skin lesions over their bodies and should not be handled.
Other Poisonous Marine Animals
53
Cli~itwIC I ~ ~ ~ r ~ ~ ~ t eContact r i . s t i (with ~ ~ :Pjesteriu toxin may result in paresthesias of the extremities and circumoral area, joint and muscle aches, headaches, itching, nausea, vomiting,abdominalpain,memoryloss,disorientation,sweating,respiratorydistress, emotional changes, and skin lesions (4-6). Twtrtmetrt: The treatment is symptomatic. There is no known antidote. Pretvntiotl: Avoid handling any dead or dying fish having skin lesions in water contaminated by Pfiestcviel. Do not swim in estuaries or coastal waters contaminated by Pje.ster.it/.Persons working with cultures of Pjiesterin should be properly covered with protective clothing and masks to avoid contact with either water or air contaminated with Pje.steri(r toxin. Contaminated fish should not be eaten.
B. Poisonous Cnidarians (Coelenterata) Cnidarians or coelenterates are best known for their stinging abilities. However, i n this chapter, only their toxicity is discussed as it relates to biotoxications by ingestion. Cnidarians. or coelenterates, are simple metazoans having primary radial, biradial, or radiobilateral symmetry. They are composed essentially of two epithelial layers and an internal cavity, the gastrovascular cavity or coelenteron, which opens only throughthe mouth. Another dominant characteristicof the group is the presence of tentacles equipped with stinging nematocysts. The group is characterized further by showing a remarkable degree of polymorphism, having an alternation of generations with a sexual and asexual phase, as well as a specialization of individual polyps as in the siphonophores. A single species may present a variety of forms of either the sessile polyp or the free-swimming medusoid type. For at least a part of their life span, most coelenterates are attached or sedentary. The phylum Cnidaria (Coelenterata) includes three classes: Hydrozoa, the hydroids; Scyphozoa, the jellyfish; and Anthozoa, the sea anemones, corals, and alcyonarians. Apparently hydroids are not used as food. Jellyfish commonly are eaten in Japan and elsewhere, and there have been no reported cases of poisonings. The nematocysts of hydroids and jellyfish contain proteinaceous toxins that are inactivated by heating and gastric juices. However, oral biotoxications have resulted from the ingestion of sea anemones in the Philippines, New Guinea, and Samoa. Palytoxin, which is produced by anthozoan Ptrlyfhoa species. is not discussed in this section because palythoans generally are not considered to be a foodstuff. However, palytoxin has been found to be transvectored by fish and other organisms (7,8).
Represmttrtive Species Family: Actiniidae (sea anemones) Species: Phy.sobr~crchiacInu~~Itr.si(Kent). Lulnane (Samoa). Diameter 2 in. ( 5 cm). Distribution: Indo-Pacific. Family: Actinodiscidae (sea anemones) Species: Rhotfrrctis howc~si(Kent). Matalelei (Samoa). Diameter 4 in. (30 cm). Distribution:Indo-Pacific. Family: Stoichactiidae (sea anemones) Species: Rcrdicrr~tl~u.s p t r u r n o t e r r s i s (Dana). Matamala samasama (Samoa). Diameter 3 in. (8 cm). Distribution:Indo-Pacific.
Halstead
54
Sea Anemone Poisoning Intoxications resulting from the ingestion of poisonous sea anemones in Samoa and other parts of the tropical IndoPacific region have been reported by Farber and Lerke (9) and Martin (lo), and are discussed by Halstead ( 1 1.12) and Hashimoto (13). The nature of the poison is unknown. Mechanistn of Intoxication: Sea anemones commonly are eaten in Samoa and elsewhere in the tropical Indo-Pacific region,but they generally are cooked. Rhodncris howesi (Matalelei) andPhysobmchia douglasi(Lumane) generally are consideredto be poisonous when raw, but safe to eat when cooked. Rarlionthus pnurnotensis (matamala samasama) and some of the other members of this genus are considered to be poisonous to eat raw or cooked. Small children are frequent victims of sea anemone poisoning intheIndoPacific region. Clinical Chorcrcmiytics: The initial symptoms of sea anemone poisoning consist of acute gastritis with nausea, vomiting, abdominal pain, cyanosis, and prostration. Shortly after ingestion of the sea anemones, the victim may become comatose, which may last a period of 36 hours or more. During this comatose period, the superficial reflexes may be absent. The blood pressure and pulse remain normal. Pulmonary edema has been reported. The patient may go into profound shock, and death usually ensues. Trecrtment: The treatment is symptomatic. There are no specific antidotes. Prevention: The safest procedure for prevention is to avoid eating sea anemones.
C.
Poisonous Echinoderms (Sea Cucumbers, Sea Urchins)
Echinoderms include the starfish, sea urchins, and sea cucumbers. They are all members of the phylum Echinodermata. The members of this group are characterizedby their radial symmetry, a body with usually five radii around an oral-aboral axis, calcareous platesthat form a more or less rigid skeleton, or plates and spicules embedded in the body wall. Spines and pedicellariae are presentin the asteroids and echinoids,but are absent in some of the others. The coelom is complex and includes a water vascular system with tube feet. The digestive tract may or may not include an anus. The sexes usually are separate. Echinoderms, with the exception of a few planktonic holothurians, are all benthic and all are marine. The phylum Echinodermata is divided into four classes: Asteroidea, the starfish; Ophiuroidea, the brittle stars; Holothuroidea, the sea cucumbers; and Echinoidea, the sea urchins, heart urchins, and sand dollars. Some species of starfish are reported to be toxic, but little is known concerning the nature of the poisons or their effects on humans. Poisonings have resulted from eating sea cucumbers and the ovaries of sea urchins.
1. Poisonous Sea Cucumbers Sea cucumbers are free-living echinoderms having an elongate, wormlike or sausagelike body, without free arms but with a series of tentacles circling the mouth, located at the anterior end of the body. The intestinal tract is long and looped, terminating in an anus at the posterior end. The skeleton consists of variable-size, irregularly arranged plates embedded in the skin. Tube feet are present but are not situated in a furrow. I n some species of sea cucumber, a number of white,pink,orredtubulesareattached to the common stem of the respiratory trees. These are the so-called organs of Cuvier. or Cuvierian tubules. If the sea cucumber is irritated, the Cuvierian tubules are discharged through the anus. Upon contact with the water, they swell and elongate into sticky slender threads
Other Poisonous Marine Animals
55
that serve to entangle the predator. Only part of the tubules are emitted at any one time, and the expelled tubules are soon replaced by new ones. In some species, the organs of Cuvier are quite toxic, containing large concentrations of holothurin. Holothurians are sluggish creatures, generally moving over the bottom of the sea by means of rhythmic contractions of the body. The tube feet are used principally as organs of attachment. Sea cucumbers are not important i t e m of food for fish, but they are used as food by many Pacific islanders and Asians. Sea cucumbers are sold commercially under the names of “trepang” and “beche-de-mer.” They are prepared by boiling them, causing them to eviscerate, shorten, and thicken. After thorough drying, the trepang is ready for marketing. Trepang is used for flavoring soups and stews.
Represewt~rtiveSpccirs Family: Holothuridae (sea cucumbers) Species: Holotkuriu cwgus (Jaeger). Sea cucumber. Length 12 in. (30 cm). Distribution:Indo-Pacific. Species: Holothuria tl~b1do.w(Gmelin). Sea cucumber. Length 10 in. (25 cm). Distribution: Mediterranean and adjacent Atlantic Ocean.
Scrr C~vnr1x.rPoisorling Very little information is available on the clinical effects resulting from the ingestion of poisonous sea cucumbers. Mechnisrn of I~rto.vicrrtion:Intoxications have been reported from eating poisonous sea cucumbers (14,15). The causative toxins in seacucumbersarecalledholothurins, of sugars, steroid which involve a complex of saponins. Saponins are complex compounds moieties, or triterpenoid moieties and are characterized by forming a durable foam when their water solutions are shaken. See Refs. 12, 13. 16, and 17 for a more detailed discussion of the chemistry of holothurins. Clirlictrl Clltrmcteristics: Reported symptoms of dermal contact with sea cucumber poison are burning pain, redness, anda violent inflammatory reaction. Liquid ejected from the visceral cavityof some species, when contacting the eye, may cause blindness. Nothing appears to be known concerning the symptoms resulting from the ingestion of poisonous sea cucumbers, but fatalities have been reported ( 18-20). Treatrncnt: Treatment is symptomatic. Pharmacological studies suggestthat anticholinesterase agents may be effective in the event of ingestion of holothurin (21). Prevention: Check with the locals prior to eating sea cucumbers. This is particularly important in tropical regions.
2. Poisonous Sea Urchins Sea urchins are free-living echinoderms having globular, egg-shaped, ora flattened body. The viscera are enclosed within a hard shell, or test, formed by regularly arranged plates carrying spines articulating with tubercles onthetest.Betweenthespinesaresituated three-jawed pedicellariae, which are of interest to the venonlologist and have been described at great length elsewhere ( 1 l , 12). Tube feet are arranged in 10 meridian series rather than in furrows. A double porein the testcorresponds to each tube foot. The intestine is long and coiled, and an anus is present. The gonads are attached by mesenteries to the inner aboral surface of the test. The mouth, situatedon the lower surface, turns downward and is surrounded by five strong teeth incorporated i n a complex structure called “Aristotle’s lantern.” Sea urchins move by means of spines on the oral side of the test.
56
Halstead
Represerztc/tive Species Family: Echinidae (sea urchins) Species: P~rracet1trotu.slividus (Lamarck). European sea urchin. Diameter of test 3 in. (7 cm). Distribution: Atlantic coast of Europe, Azores, West Africa. Family: Toxopneustidae (sea urchins) Species: Tri/meusres g r d l l a (Linnaeus). Sea urchin. Diameter of test 4 in. (10 cm). Distribution: Indo-Pacific, Japan, East Africa, Australia. Secr Urchin Poisonitlg
Several species of European and Indo-Pacific echinoids serve as commercial food sources. Only the gonads are eaten, either raw or cooked. During the reproductive season, generally the spring and summer, the ovariesof certain species of sea urchins are reported to develop toxic products that are injurious to man (8,1 1 -13,22-25). Mecllcrnisrn o f Itltoxiccrtiorl: Sea urchin poisoning is the result of ingesting toxic sea urchin gonads. The chemical nature of the poison is unknown. Cliniccrl Characteristics: The symptoms of sea urchin poisoning consist of general epigastric distress, nausea, diarrhea, vomiting, severe migrainelike headaches, and swellto be an allergic type of reaction in some ing of the lips and mouth. There is believed cases. Ttwtmerzt: Treatment is symptomatic. There is no known antidote. Prevewtior1: Care should be taken when eating the ova of known toxic species of sea urchins during the reproductive season. It is especially important to contact the local inhabitants as to the edibility of sea urchin eggs, especially in tropical regions.
D. Poisonous Mollusks Mollusks have been incriminated in a number of types of oral food intoxications aside from paralytic shellfish poisoning, diarrhetic shellfish poisoning, and amnesic shellfish poisoning. Mollusks are members of the phylum Mollusca. Mollusks are unsegmented invertebrates with a soft body and usually secreting a calcareous shell. A muscular foot is present that may be modified to serve various functions. Covering at least a portion of the body is soft skin, the mantle, the outer surfaceof which secretes the shell. Respiration is by means of gills or a modified primitive pulmonary sac. Jaws are present in some species. In four of the five classes, food is obtained by the use of a rasplike device called a radula. In the cone shells and a few others, the radula ribbon is lost and the teeth are modified into hollow, harpoonlike structures that may contain venom. The phylum Molluscais generally divided into five classes: Amphineura, the chitins; Scaphopoda, the tooth shells; Gastropoda, the snails and slugs; Pelecypoda, the bivalves (scallops, oysters, clams); and Cephalopoda, the octopuses, squids, and cuttlefish. Most of the toxic species of mollusks are gastropods, pelecypods, and, rarely, cephalopods.
1. Poisonous Gastropods: Abalone Abalone are gastropod mollusks that have a rough, horny coating that quite frequently is hidden by a thick cover of algae and other growth. The spire of the shell is flattened and the epipoda is bordered with a fringe and tentacles that project around the margin of the shell. Along the margin of the shell in older specimens, there is a single row of holes through which feelers may project and from which water passing over the gills is discharged. The living mollusk projects its head out from under the edge of the shell in the
Other Poisonous Marine Animals
57
area where therow of holes terminate. The tip of the broad muscular foot is pointed backward from under the spiral. The lining of the shell is pearly iridescent. Abalone shell has been used extensively as a source of mother-of-pearl i n the manufacture of buttons. ornaments, and trinkets. The muscular foot is used i n the preparation of soups, chowders, and as steaks. The toxic substance foundin the viscera of abalone is believed to originate through their food web, namely, certain species of seaweeds belonging to the genus L)r~.srrraresticr (26).
Represmtrrtitv Species Family: Haliotidae (abalone) Species: Hrrliotis di.sc~u.s(Reeve). Abalone. Length 6 in. (15 cm). Distribution: Japan.
Abnlorle Viscer-lr Poisorrirrg Abalone poisoning has been reported fro111 eating Hrrliotis di.scw.s and H. siebolcli in Japan (27). Mechcrrrisrrr of 1rlto.riccrtiotr: Poisonings from abalone are theresult of eating the viscera of the mollusk. The custom of eating the entire mollusk is practiced in Oriental countries. Elsewhere, only the muscular foot (abalone steak) is eaten. The tnuscular foot generally is safe. Although only two species of Japanese abalone, H r r l i o t i s discus (Reeve) and H. sieboldi (Reeve), have been incriminated, the viscera of other species of abalone are suspect. Clirriccrl Clrrrmc.tr.ri.stics: The symptoms of abaloneviscerapoisoninghavebeen described as a sudden onset of a burning or stinging sensation over the entire body, followed by an urticarial rash, itching, erythema, pain in the face and extremities, edema. and subsequent development of skin ulceration. It has been observed that the skin lesions are limited to those parts of the body exposed to sunlight, and there is a distinct boundary between covered and exposed parts of the body (26). Tretrtmerrt: Treatment is symptomatic. Pre\wtion: Do not eat the viscera of the abalone. Only the muscular foot or steak should be eaten.
2. Poisonous Gastropods: Turban Shells The turban shells are members of the gastropod mollusk family Turbinidae. They
are related to the top,or trochus, shells of the Trochidae. As their name implies, they generally are conical or top-shaped, spiral, and have a pearly luster on the inner surface of the shell. The turban shells are largely algae feeders. Some of the turban shells have bee11 found to be toxic. Rel~r-e.~~~rltrrtitl~~ Species
Family: Turbinidae (turban shells) Species: Turho crrgyro.sfor,lu.s (Linnaeus). Turban shell. Dialneter 3 in. (8 ~ 1 1 1 ) . Distribution: Indo-Pacific. Species: Tur-hoIr1rrrwwrrrfus (Linnaeus). Turban shell. Dialneter 8 in. (20 cm). Distribution: Indo-Pacific.
T ~ r l ~Shell r t ~ Poisorrirlg Human outbreaks have beenreportedfromeatingturbanshellstaken at M;lrcus Island, the Marianas Islands, and the Western Pacific (12,13.28). Several toxic sLIbstallces
Halstead
58
have been isolated from 7'. trrggrostottlus (Linnaeus), butthe chemical nature of these compounds hasnot been fully determined.A poison has been isolated from T. rwI-tt1oI-utu,s (Linnaeus) thatisbelieved to beidentical to ciguatoxin (29). Saxitoxinhas also been found in the gut of this turban shell. Mechmism of Itltosicertion: The toxic substances apparently are foundin the midgut gland and the gut of theturbanshell and apparently are obtained by feeding on toxic algae. Ingestion of the whole mollusk may cause poisonings in humans. Ciguatoxin and saxitoxin are also believed to occur in these mollusks. See Chapter 2 for further information on ciguatoxin and saxitoxin. Clillicd ChNrcrL.teI-iSt1'c.s: The synlptoms of turban shell poisoning consist of gastrointestinal upset, nausea, vomiting, diarrhea, fatigue, temperature-reversal sensation, and pruritus. In general, the symptoms resemble those of ciguatera fish poisoning (28). TI-ecrttnerlt:Treatment is symptomatic. See Chapter 2, Sec. 1V.C on ciguatera fish. PI-evention: The turban shells are believedto be safe to eatif the viscera are removed. However, since ciguatoxin and saxitoxin Inay be present, to prevent poisoning one should be extremely cautious when eating these shellfish. It is advisable to check with the local inhabitants concerning their edibility.
3. Poisonous Gastropods: Whelks The whelks area large and aggressive familyof carnivorous gastropod mollusksthat range from tropical to polar seas. They have a vertical distribution that ranges from the littoral zone to great depths. Their shells come in a variety of shapes, sizes, and colors. Whelks commonly are observed clambering about on rocks or plowing their way through mud, sand, or gravel, with the muscular foot largely buried in the bottom. When at rest, the mollusk retracts the foot and thereby closes the aperture with a horny operculum. Most whelks are more scavengers than active predators, feeding on dead fish and other scraps, but they tend to shun anything in an advanced state of decay. Some of the whelks feed on live mollusks, and at times may cause considerable damage to oyster beds. Whelk poison is said to be concentrated i n their salivary glands.
Represeutdve Species Family: Buccinidae (whelks, ivory shells) Species: Bcrbylnrlirr jrrpotIiccr (Reeve). Japanese ivory shell. Length 3 in. (7 cm). Distribution: Japan. Species: Neptrrneer to~tiglrrr(Linnaeus). European whelk. Length 4 in. (10 cm). Distribution: Northern Europe. Species: Nepturlerr inteI-scwlpttr (Sowerby). Whelk. Length 6 in. ( I S cm). Distribution: Japan.
Whelk Poisotlitlg Several outbreaksof poisonings fromthe ingestion of the Japanese ivory shell (Btrbylotlicr jcrponi~tr)have been reported in Niigata, Japan (12,13,30,3 1). Human intoxications have been reported by Kanna and Hirai (32) for Neptunea itrtersarlptcr in Japan. Fange has reported finding the toxic substance tetrarnine i n the salivary glands of N. crllticqLdcr from Sweden (33-3). No clinical data were presented in the Swedish reports. M e c j ~ c r j l ic!fActiotl: .~~ Whelk poison is believed to be restricted to the salivary glands of the mollusk. Intoxication results when these glands are ingested in whole shellfish in the raw, cooked, or canned state.In B. jqmliccr, the poison was found to be concentrated in the lnidgllt gland. The poison found in B. jtrporlictr is called surugatoxin and has been
Other Poisonous Marine Animals
59
found to be a potent mydriatic, ganglion blocker, and hypotensive agent (36). Its molecular formula is C22Hl,,BrN,0,,, nlolecular weight 660 (13). Paralytic shellfish poison also has been detected in the digestive glands of N . decemcostlrta (Say) taken in eastern Canada (37). Clirlicrrl Cl~crmcterisfics: The toxic substance present in poisonous whelks istetramine. Tetramine is an autonomic ganglionic blocking agent. Intoxication from tetramine may result in nausea, vomiting, anorexia, weakness, fatigue, faintness, dizziness, thirst, dysuria, mydriasis, aphasia, numbness, photophobia, impaired visual accommodation, and dryness of the mouth. Trecrtrnent: Treatment is largely symptomatic. Preverltiorl: Poisonous whelks are said to be safe to eat if the salivary glands are removed.
4. Poisonous Bivalve Mollusks The pelecypods, or bivalves, have their bodies laterally compressed and are surrounded entirely by the lateral mantle folds, which are greatly enlarged and covered bya longitudinally divided shell. They lack a proboscis and a radula. The byssus gland, which is found at the base of the foot, secretes sticky threads that harden and serve as mooring lines to the substratum. The byssus threads of mussels are a good example of this attachment mechanism. The foot in bivalves is used as a burrowing organ. The mantle cavity lies on both sides of the foot and contains a pair of modified and enlarged gills, or ctenidia, also known as branchia. The stomach is associated with a crystalline style. The nervous system is simple and concentrated in the posterior portion of the body. Most bivalves are dioecious, and fertilization takes place in the water or in the mantle cavity. Pelecypods are largely filter feeders. In addition to paralytic, amnesic, brevitoxic (neurotoxic), and diarrhetic shellfish poisoning, there are other forms of bivalve shellfish intoxications, including callistin, venerupin, giant tridacna clam, and scallop poisoning.
5. Poisonous Bivalve Mollusks: Callistin Shellfish The genus Ctrllista belong to the family Veneridae, a group of bivalve mollusks of the class Pelecypoda. The members of this genus have an ovate shell that is acunlinate on the back and rounded at the front. The surface of the shell in Ccdlista brevi.si~~hotrrrtrr is marked by rough growth lines and is covered with a smooth and polished periostracum, under which light purplish-brown rays are seen on the yellowish background in young specimens. Members of the genus Cullisfa have been incriminated in human intoxications.
Repre.sewtative Species Family: Veneridae (Venus clams) Species: Ccrllisftr hrevisiphotlcrtrr (Carpenter). Japanese callista. Length in. 6 (15 cm). Distribution: Japan.
The history of callistin shellfish poisoning appears to be of recent origin. The first report of an outbreak of poisoning was by Asano et al. (38) and was caused by eating C. brevisil,hnntrttr taken in the vicinityof Mori, Hokkaido, Japanin 1950. There wasa second report of an occurrence in the same region in 1953. The Mori Health Ccnter subsequently banned the sale of this shellfish.
Halstead
60
Mechcrnistn ofPoi.soning: Callistin shellfish poisoning results from eating the ovaries of the Ctrllistn bivalve, which contains a high concentration of choline. The shellfish are said to be toxic only during the spawning season, May-September. Clirricrrl Charcrcteristics: The onset of symptoms generally occurs within 1 hour after ingestion of the toxic shellfish. The chief symptoms are itching, flushing of the face, urticaria, a sensation of constriction in the chest, epigastric and abdominal pain, nausea, vomiting, dyspnea, cough, asthmatic manifestations, hoarseness, paralysis or numbness of the throat, mouth, and tongue, thirst, hypersalivation, drop in blood pressure, increase in pulse rate, leucocytosis, sweating, chills, and fever.In general, this biotoxication resembles a severe allergic reaction. No fatalities have been reported. Treatnrent: Treatment is symptomatic.. Antihistamines have been recommended. Pretrntiow:The callistin shellfish are saidto be safe to eat if the ovaries are removed. The ovaries can be identified by smearing a small amount of the whitish- or yellowishcolored gonads ona glass slide and observing the material undera microscope. The female ovary can be identified by the white, granular-appearing ova. The male gonadal substance appears as a milky paste. Cooking does not destroy the toxic principalof callistin shellfish poison. The freshness of the shellfish is not a factor in the occurrence of this disease.
6. Poisonous Bivalve Mollusks: Venerupin Shellfish Venerupin shellfish poisoning was named after the bivalves Dosinicl jcrporliccr and Tq’es serniclecusscrtr~,which are members of the pelecypod family Veneridae. In addition, the Japanese oyster (Crtmostrecr g i p s ) , a bivalve of thefamilyOstreidae,hasalsobeen incriminated as a causative shellfish species. The toxicity of the bivalves is believed to be due to the transvectoring of one or several toxic speciesof dinoflagellates of the genus Prorocerrtrum. One of the agents is thought to be P. rrltrric.le-lebouric~e(12).
RL~l~resenttrtitle Species Family: Ostreidae (oysters) Species: Crcrssostrecr gigas (Thunberg). Oyster. Length 25 in. (25 cm). Distribution: Japan, British Columbia, southern California. Family: Veneridae (Venus clams) Species: Dosinitr jcporriccr (Reeve). Japanese dosinia. Length 3 in. (7 cm). Distribution: Japan. Species: Ttrpes semidecussata (Reeve). Japanese littleneck. Length 2 in. ( 5 cm). Distribution: Japan, British Columbia south to California.
Vcrrerrrpir~Slrellfislr Poisorrirrg Outbreaks of venerupin shellfish poisoning were first reported i n 1957 near Niigata, Japan, and later in the Kanagawa and Shizuoka Prefectures, Japan, during the months of January-April ( 1 3,39). There is no record of this type of shellfish poisioning occurring elsewhere, despite the fact that the species of shellfish involved are found in other parts of Japan and have been introduced into the United States. There donot appear to be any recent outbreaks reported. M t 4 ~ a r r i s mc!fItrtc).~ictrtion:Venerupin poison appears to be transvectored by a toxic species of dinoflagellate of the genus Prorocentrur?~,which is ingested by the shellfish and concentrated in the digestive gland or “liver” of the mollusk. The chemical nature of the poison is unknown. Clinicer/ Cl?nmctPri,stic.s:The symptoms of venerupin, or asari, shellfish poisoning
Other Poisonous Marine Animals
61
usually develop within48 hours of ingesting the shellfish.The initial symptoms are nausea, gastricpain,vomiting,constipation,headache,andmalaise.Bodytemperatureremains normal. Within 36 hours, additional symptoms such as nervousness, hematemesis, and bleeding from the mucous membranes of the nose, mouth, and gums develop. Halitosis is a dominant part of the clinical picture. Jaundice, petechial hemorrhages, and ecchymoses of the skin generally are present, particularly about the chest, neck, and upper portion of the arms and legs. Leucocytosis, anemia, retardation of blood clotting time, and evidence of liver dysfunction have been noted. The liver generally is enlarged, but painless. In fatal cases, the victim becomes extremely excitable, delirious, then comatose. There is no evidence of paralysis or other neurotoxic effects as in paralytic shellfish poisoning. The low mortality rate has been credited to early diagnosis and prompt medical care.In severe cases, the victim usually dies within 1 week. Recovery is extremely slow and the victim remains in a weakened condition for an extended period of time. The fatality rate is about
33%. Trecltment: Treatment is symptomatic, buttheuse of IV glucose, vitamins B, C, and D, and insulin have been recommended. Prevetltim: ShellfishtakenintheShizuokaandKanagawaPrefectures,Japan, should not be eaten during the months of January-April. Ordinary cooking procedures do not destroy the poison. Toxic shellfish cannot be detected by their appearance. The only certain method to determine the safety of the shellfish is to prepare tissue extracts and test them on laboratory animals.
7. Poisonous Bivalve Mollusks: Tridacna Clams Some species of giant c l a m of the family Tridacnidae have shells nlore than 39 in. (1 m.) in length. Tridacna clams are recognized by their massive shells and large irregular teeth set on a broad hinge plate; they can be found burrowed or wedged into coralon the bottom of tropical seas. The shape and color of the shell often blends in with the coral rock, but the giant clam is detected quickly by the brilliant coloration of its siphon edges and mantle. Two species of the giant tridacna clams have been incriminated in French Polynesia in human intoxications, which clinically resemble ciguatera fish poisoning. Representntive Species Family: Tridacnidae (tridacna clams) Species: Triductra gigas (Linnaeus). Tridacna clam. Length 54 in. (137 cm). Distribution: Indo-Pacific. Species: Tridclctlo tmrima (Roding). Tridacna clam. Length 14 in. (35 cm). Distribution: Indo-Pacific.
TridLtcm Shelljsh Poisowing Tridacna shellfish poisoning clinically resembles ciguatera fish poisoning (see Chapter 2 . Sec. 1V.C). The symptoms consist of gastrointestinal, vasomotor, and various disturbances, including a loss of motor coordination. Bagnis (40) provides the most complete account of tridacna shellfish poisoning, which involved33 people and a numberof domestic animals that had eaten TridLroln nrnxirncr at Bora-Bora, Society Island, French Polynesia. The poison involved was believed to be of the ciguatoxin complex. Trecrtment: Treatment is symptomatic (see Chapter 2, Sec. 1V.C). Prrvention: It is advisable to check withthelocalinhabitantsprior toingesting tridacna clams.
62
Halstead
8. Poisonous Bivalve Mollusks: Scallops Representative Species Family: Pectinidae (scallops) Species: Patinopectin yessoensis (Jay). Scallop. Length 6.3 in. (160 mm). Distribution: Northern Japan.
Sccrllop Poisoning Yasumoto et al. (41 ), Murata et al. (42), and Okada and Niwa (43) reported biotoxications in 1985 resulting from the eating of toxic scallops (Prrtinopectin yessoensi.s) harvested from northeast Japan. They isolated two toxins called pectenotoxin (PTXI and PTXI-5). PTXI was found to be a major component of dinophysotoxin (DTX) isolated from the mussel Mytilis eclulis taken from the northeast coast of Japan (41). Dinophysotoxin derives its name because of its association with the dinoflagellate Dinophysis forti;. Dinophysotoxin has also been found in toxic mussels found in Bantry Bay, Ireland (43). Another toxin, yessotoxin (YTX), has also been isolated from P. yessoensis by Murata et al. (42). Mechcrnism sf’ Action: Scallop poisoning is caused by eating poisonous scallops containing pectinotoxin or yessotoxin. Clinicnl Cl~trr.ncteri.stics:The symptoms generally consist of nausea, vomiting, and diarrhea. Neurological disturbances are generally not present. Trecrtnrent: The treatment is symptomatic. There is no known antidote. Prevention: Check with the local public health authorities if there is any question as to their edibility.
9. Poisonous Cephalopods Cephalopods are nlollusks of the class Cephalopoda that have bilateral symmetry and a well-developed head containinga circumoral crown of mobile appendages bearing suckers and/or hooks (exceptin Nrrutilus). The mouth has chitinous, beaklike jaws and a chitinous tonguelike radular band of teeth. The shell is variously modified, reduced, or absent and is enveloped by the mantle; an external shell occurs only in Nmrtilus, which is restricted to the Indo-Pacific area. They are soft-bodied animals, with their primary skeletal features consisting of a cranium-a mantle/fin support comprised of a cuttlebone or gladius. The central nervous system is highly developed and is associated with well-organized eyes. A funnel or siphon tube expels water from the mantle or body cavity. The coloration of cephalopods is variable, depending upon the species and habitat. Most cephalopods have numerous chromatophores and iridocytes i n the skin to accommodate rapid changes in colors and patterns. The size of cephalopods varies greatly, from less than 1 in. (2.5 cm) to 70 ft (20 m) in length and weighing over 1 ton. Locomotion is achieved by drawing water into the mantle cavityand expelling it i n a jetlike manner through the funnel. Octopi are sometimes observed crawling alongthe bottom of the sea on their a r m . Cephalopods are largely predatory and feed carnivorously on crustaceans, bivalves, and fish. Human intoxications resulting from the ingestion of octopus and squid have been reported. Repwsentrrtil>e Species Family: Ommastrephidae (squid) Species: Ommnstrephes slotrni p~rc$c~u,s(Steenstrup). Pacific squid. Length 12 in. (30 cm) Distribution: Pacific Ocean. Family: Octopodidae (octopi)
Other Poisonous Marine Animals
63
Species: Octopus vulgcrris (Lamarck). Octopus. Length 32 in. (80 cm). Distribution: Warm temperature and tropical waters, all oceans.
Cepkolnpod Poisorritrg Intoxications resulting from the ingestion of cephalopods generally are rare. However, a series of 779 outbreaks involving 2974 persons occurred in Japan from 1952 to 1955 caused by the ingestion of squid and octopus. Fatalities were reported (44). Apparently there have been no recent outbreaks reported. Meckrrrrisnr of 1rrto.ricrrtiorr:The nature of the poison involved in cephalopod poisonings is unknown. It is unknown whether bacteria may have playeda role in these cephalopod outbreaks. ClitriccrlCIr(1rcrcteristic.s: Symptoms did not develop in the Japanese outbreaks of cephalopod poisoning until about 10-20 hours after ingestion of the squid or octopus. The symptoms consisted of nausea, vomiting, abdominal pain, diarrhea, low-grade fever, headache, chills, weakness, dehydration, paralysis, and convulsions. Neurological sympa toms were not a dominant part of the clinical picture. Recovery was generally within period of 48 hours. In most instances, cephalopod poisoning resembles severe gastroenteritis, but death may occur. Trecrtrtrerrt: Treatment is symptomatic. There are no known specific antidotes. Prevetrtiorl: TheJapaneseoutbreaks of cephalopodposioningoccurredwithout warning and there was no evidence in the appearance or taste of the cephalopods. Both of the species of squid and octopus incriminatedin the Japanese outbreaks are eaten commonly throughout the world without any ill effects.
E. Poisonous Arthropods: Crabs and Lobsters The phylum Arthropodais the largest single group within the animal kingdom, containing more than 800,000 species. Arthropods are characterizedas having a body usually divided into a head, thorax, and abdomenof like os unlike somites, which are variously fusedand with each segment bearing a pair of jointed appendages. The exoskeleton is chitinous and is molted at intervals. The digestive tract is complete and divided into fore, mid-, and dorsally. The hindgut.The body spaces serve as ahemocoel,and theheartislocated phylum Arthropoda is divided intoa large numberof classes, but only twoof them, Merostomata, which includes the horseshoe crabs, and Crustacea, which includes the lobsters, crayfish, and crabs, are pertinent to toxicologists.
1. Poisonous Horseshoe Crabs The class Merostomata includes the horseshoe crabs or king crabs, which are characterized by an arched cephalothorax, a horseshoe-shaped carapace, a wide unsegmented abdomen possessing three-jointed chelicerae, pedipalpi, and six-jointed legs. They inhabit coastal waters with sandy or muddy bottoms at depths of less than 6 fathoms. Horseshoe crabs are scavengers and feedon polychaete worms, mollusks,a variety of small marine animals, and T d r y i e u s can be and algae. The Asiatic members of the genera Ctr~citroscorpir~s very toxic to eat.
Rcpreserrttltive Species Family: Xiphosuridae (horseshoe crabs, king crabs) Species: Crrrcirroscor1~ilr.srotrrrrrlictrudrr(Latreille). Asiatic horseshoe crab. Length 13 in. ( 3 3 cm).
Halstead
64
Distribution and habitat: Philippines, Indonesia, Malaysia. Species: Tdlypleu.7 gigas (Muller). Asiatic horseshoe crab. Length 19 in. (50 cm). Distribution: Torres Straits, Vietnam, east coast of Bay of Bengal.
Horseshoe Crcrh Poisoning Human intoxications from eating Asiatic horseshoe crabs have been reported by Smith ( 4 3 , Soegiri (46), Waterman (8,47,48), and Banner and Stephens (49). Mostof the reported outbreaks have occurredi n Thailand, but they probably occur elsewherein Southeast Asia wherever these horseshoe crabs are endemic. Horseshoe crab poisoning is referred to as mimi poisoning in Thailand. Mrchmism of Intmicrrtion: Asiatic horseshoe crab poisoning is caused by eating the unlaid green eggs, flesh, or viscera during the reproductive season. Despite their periodic toxicity, the large masses of unlaid green eggs are highly prized by Asiatic peoples (47,48). The poison in Asiatic horseshoe crabs is believed to be chemically identical to saxitoxin (50). Cliniccl/ ChLlrac.teristic,s:The onset of symptoms in Asiatic horseshoe crab poisoning usually occurs within 30 minutes of ingestion of the poison. The initial symptoms consist of nausea, vomiting, abdominal cramps, headache, dizziness, slow pulse rate, decreased body temperature, aphonia, cardiac palpitation, numbness of the lips, parethesias of the lower extremities, and generalized weakness. More-severe symptoms may occur in rapid succession: aphonia, sensation of heat in the mouth, throat, and stomach, inability to lift the arms and legs, generalized muscle paralysis, trismus, hypersalivation, drowsiness, and loss of consciousness. The mortality rate is unknown but is said to be very high. Death, when it occurs, takes place within a period of 16 hours. Trecrtmetzt: Treatment is symptomatic. Prevention: Although Asiatic horseshoe crabs commonly are eaten in many parts of Southeast Asia, they should be avoided during the reproductive season.
2. Poisonous Tropical Reef Crabs The class Crustacea includes the lobsters, crayfish, and crabs. Most of the known toxic reef crabs are members of the crustacean family Xanthidae. The xanthid crabs have a carapace that is transversely oval, hexagonal, or subquadrate, rarely subcircular, and almost always broader than long. The front of the carapace tends to be broad and is never produced in the form of a rostrum. The legs of the crab are of the ambulatory type rather than the swimming type. The family Xanthidae is found throughout the tropical littoral, inhabiting coral reefs. Most of the family feeds on a variety of foods, including algae. Some of the xanthid crabs have been foundto be extremely toxic and have caused human fatalities. Representative Species Family: Xanthidae (mud or reef crabs) Species: Atergutis yoridus (Linnaeus). Reef crab. Width of carapace 2 in. (5 cm). Distribution: Indo-Pacific. Species: Corpilius rntrculntlts (Linnaeus). Spottedreef crab. Width of carapace 4 in. (10 cm). Distribution:Indo-Pacific. Species: Demanin toxiccl (Garth). Poisonous reef crab. Width of carapace 2 in. ( 5 cm).
Other Poisonous Marine Animals
Distribution: Indo-Pacific. ?tl Reef crab. Width of carapace 2 in. Species: PlotypodiL/ ~ 1 y ( 1 1 1 ~ ~ 0 1 7 (Ruppell). cm). Distribution: Indo-Pacific. Species: Zozytmrs aeneus (Linnaeus). Width of carapace 3 in. (7 cm). Distribution: Indo-Pacific.
65
(5
Tropicul Reef Cmb Poisoning Reports of human biotoxications from tropical reef crabs have appeared at infrequent intervals. Most of these intoxications have occurred in the tropical Indo-Pacific region. References 2, 13, and 5 1-65 contain reports on poisonings by tropical reef crabs. Mechrrrlisrn of Itmxiccrtion: The poison contained in Zozymus aeneus, according to Refs. 8 and 66-68, appeared to be chemically identical to saxitoxin. Yasumoto et al. (697 I ) found saxitoxin, neosaxitoxin,and gonyautoxins to be present in various other species of tropical reef crabs including Nemanthias impressus (Lamarck), Actaeodes tornerltosus (H.MilneEdwards), Eriphicr scahricwltr (Dana), Pi1ur~111u.s vespertillo (Fabricius), Schi:.ophrys asperu (H. Milne Edwards), andThnlarnita species, and Halstead and Schantz (72) found themin Perctwn plani,s.sirnum(Herbst). Yasurnuraet al. (73) found tetrodotoxin in the reef crab Zozymus c m c u s . Clirlicnl Chcrrcrcteristics: The symptoms of tropical reef crab poisoning consist of paresthesias, muscular paralysis, aphasia, nausea, vomiting, and collapse. Death may occur within 2 hours to several days (66). The symptoms may resemble those of paralytic shellfish poisoning or tetrodotoxism (see Chapter 2, Sec. 1V.G.). Treatment: Treatment is symptomatic. Prevention: The eating of tropical reef crab is at best an extremely hazardous game of Russian roulette. Tropical reef crabs should not be eaten.
3. PoisonousCoconut Crabs Coconut or robber crabs (Birgus lntro) are land hennit crabsthat are equipped with powerful pincers capable of cutting coconuts from palm trees and opening them. Coconut crabs are capable of climbing palm trees and can do so with considerable agility. Under most circumstances, coconut crabs are eaten and looked upon as a great delicacy, but under certain conditions they may be toxic and may cause fatalities.
Representcltive Species Family: Coenobitidae (coconut crabs) Species: Birgus lntro (Linnaeus). Coconut crab. Length of carapace 5 in. (13 cm). Distribution: Indo-Pacific.
Coconut Crnb Poisonirlg Human biotoxications from coconut crabs have occurred from time to time in the Tuamotu and the Ryukyu Islands in the Indo-Pacific region (8,66,74). Mechanism ofI7?ro.rimrion: Coconut crabs commonly are eaten throughoutthe IndoPacific region, but under certain circumstances they may become extremely poisonous. It is believed that these crabs become toxic by feeding on the roots of certain toxic terrestrial plants. Native islanders in the Tuamotus have suggestedthat one of these toxic plants may be the “Piratea” [Ceoclosrmbrcrcul$ercr (Forster)], a member of the family Hernandiaceae, found in swampyareas on variousPacificatolls (74).Hashimoto(13)reported
Halstead
66
several other poisonous plants that the coconut crab may be feeding on in the Ryukyus, such as Diospyros tt~rrritimrr(Blume Bijdr) of the family Ebenaceae, Herncmdiu .so~~or(r (Linnaeus) of the family Hernandiaceae, and Aloccrsicr mtrcrorrhizc~(Linnaeus) Schott of the family Araceae. Clirlicrrl CI1omcteristics: The symptoms of coconut crab poisoning consistof a violent gastrointesintal upset, headache, chills, joint aches, extreme exhaustion, and muscular weakness. Deaths have been reported. Treatment: Treatment is symptomatic. Prevention: If there is the slightest question concerning the edibility of the coconut crab, avoid eating it. Unfortunately these crabs generally are considered edible anda great delicacy, and reliable local information is difficult to obtain to prevent poisoning. 4. Poisonous Lobsters The term lobster as used in the vernacular sense may include any member of the families Homaridae, the true lobsters; Palinuridae, the spiny lobsters; Scyllaridae, the slipper or shovel lobsters; and the deep-sea lobsters,all of which are members of the arthropod class Crustacea. The true and spiny lobsters are of commercial importance. Lobsters have a rigid, segmented exoskeleton, andfive pairs of legs, one or more pairsof which are modified into pincers or chelae, with the chela on one side usually larger than on the other. The eyes are on movable stalks, and there are two pairs of long antennae. Several pairs of swimmerets are on the elongated abdomen. A flipperlike tail is used for swimming; flexure of the tail and abdomen are used to propel the animal backward. All lobsters are marine and bottom dwelling. Lobsters are nocturnal in habit and scavenge for dead animals, fish, invertebrates, and seaweed. A number of poorly documented accounts have indicated that someof the spiny lobsters in French Polynesia have caused human intoxications. The exact species involved have not been documented (8.74).
Representrrtive Species Family: Palinuridae (spiny lobsters) Species: Paliwurus species. Spiny lobster. Length variable, about Distribution: Indo-Pacific.
12 in. (30 cm).
Lobster Poisorlirlg Lobster poisoning generally is considered to be a rare occurrence and apparently nothing is kllown concerning the nature of the poisons involved or the clinical characteristics of the intoxications they produce. It is assumed that the poisons involved originate through the food chain of the lobster, similar to the toxic tropical reef crabs which inhabit the same reef environment and have similar eating habits. Clirlictrl Cl7arcrcteristics: The clinical characteristics are unknown. Trmment: Treatment is symptomatic. prel~ptltiotl:Data on prevention of lobster poisoning are extremely meager. The best policy is to check with the local inhabitants.
111.
VERTEBRATES
Vertebrate fish poisonings are covered in Chapter 2, so this discussion is limited to poisonous marine vertebrates other than fish, that is, marine reptiles and mammals.
Other Poisonous Marine Animals
A.
67
PoisonousMarineTurtles
Marine turtles are reptiles of the order Chelonia (Testudinata) and are characterized by a broad body encased in a bony shell comprised of a rounded dorsal carapace and a flat ventral plastron that are joined at the sides and coveredby polygonal laminae (i.e., Scut% scales, or leathery skin). The jaws are edentulous and equipped with horny sheaths. The quadrate bone is united to the skull. The ribs are fused to the shell, and the sternum is absent. All turtles, tortoises, and terrapins are oviparous in their reproduction. There are five species of marine turtles that have been reported as poisonous to man.
Represetrtcrtive Species Family: Chelonidae (marine turtles). Species: Ccrretrcrcvrrettrr gigcrs (Deraniyagala). Loggerhead turtle. Length of carapace 47 in. (1.2 m). Distribution: Tropical Pacific and Indian Oceans. Species: Cl?e/orlinmyclcrs (Linnaeus). Green sea turtle. Length of carapace 47 in. (1.2 m). Distribution: All tropical and subtropical oceans. Species: Et-etmockelys irnhricntrr (Linnaeus). Hawksbill turtle. Length of carapace 35 in. (89 cm). Distribution: All tropical and subtropical oceans. Family: Derlnochelidae (leatherback turtles) Species: Dertnochelys coricrcea (Linnaeus). Leatherback turtle. Length of carapace up to 9 ft (2.7 m). Weight of about 1500 Ib (700 kg). This is the largest of all the turtles. Distribution: Largely circumtropical. Family: Trionychidae (soft-shelled turtles) Species: Peloche/ys Dibrotri (Owen). Soft-shelled turtle. Lengthof carapace 6 ft (1.8 m). Distribution: Rivers and coastal areas of Southeast Asia.
Moritre Turtle Poisonins (Cllelotlito~~icrrtio~~) Marine turtles have been reported as poisonous to eat at sporadic intervals over the years. Unfortunately very little specific information is available concerning manyof these outbreaks. References 8,12,52,66,75-82 report on turtle poisoning (chelonitoxications). Mechcrrrisnl o f htoxiccrtiotr : The origin of turtle poison (chelonitoxin) is unknown, of the opinion that the poison but most investigators who have studied the problem are originates in the food chain of the turtle. Most marine turtles appear to be omnivorous, and feed on marine algae, among other things. It is believed that they become poisonous in humans by feeding as a result of feeding on toxic algae. Turtle poisoning may be caused on the flesh, fat, viscera, or blood of various species of tropical sea turtles. The chemical nature of chelonitoxin is unknown. Clitlicd Clrnrcrcteristics: The symptoms of chelonitoxication appear to vary with the amount of turtle ingested and the individual. Symptom develop within a few hours to several days after ingestion. The initial symptoms consist of nausea, vomiting, diarrhea, of the exepigastric pain, tightness of the chest, pallor, tachycardia, sweating, coldness tremities, and vertigo. Frequently there is reported an acute stomatitis, consisting of a dry, burning sensation of the lips, tongue, and lining of the mouth, and thirst. Swallowing
68
Halstead
becomes very difficult, and hypersalivation may be pronounced. The tongue develops a white coating, and the breath becomes very foul. The tongue later may develop pinheadsize, reddened pustules. The oral symptoms may be slow to develop, buttheyusually become very severe after several days. The lingual pustules may persist for several months or may break down into ulcers. Some victims develop a severe hepatomegaly with right upper quadrant tenderness. The conjunctivae become icteric. Headaches and a feeling of “heaviness of the head” are reported. Deep reflexes may be diminished. Somnolence is one of the pronounced symptoms present in severe intoxications, and is usually indicative of an unfavorable prognosis. At first the victim is difficult to awaken, then becomes comatose, which is followed rapidly by death. The symptoms presented are typicalof hepatorenal disease. The clinical characteristics of marine turtle poisoning are discussed in Refs. 12,77,78,8I , and 83-85. Trentment: Treatment is symptomatic. There is no known antidote. Prevention: There are no reliable external characteristics indicativeof a toxic marine turtle to aid in prevention of poisoning. Natives frequently will test suspect turtle meat by feeding samples of it to dogs or cats. It is advisable to seek the advice of the local inhabitants before eating marine turtles.
B. PoisonousMarine Mammals Mamnals are characterized by bodies that are covered with hair and skin containing various types of glands. The skull possesses two occipital condyles. The jaws usually have differentiatedteeththatarecontained in sockets. The limbs are adapted variously for walking, climbing, burrowing, swimming, or flying. The feet have claws, nails, or hoofs. The heart is four-chambered, with only a left aortic arch. The lungs are large and elastic. There is a diaphragm between the thoracic and abdominal cavities. The male has a penis and fertilization is internal. The eggs are small or minute and usually are retained in a uterus for development. The females have mammary glands that secrete milk to nourish the young. Body temperature is regulated. Several orders of marine lnamnals have been found to be toxic to eat: the order Cetacea, which includes whales, dolphins, and porpoises; Pinnipedia, which includes walruses and seals; and Carnivora, which includes the polar bear.
1. Poisonous Cetaceans There are only four species of cetaceans (whales, dolphins, and porpoises) that have been reported as toxic to man: the sei whale (Brrlcrenoptercr borealis), white whale (Delphinq>terus leucrrs), sperm whale (Physeter crrtodorl), and Southeast Asiatic porpoise (Neophoerrem phocnenoide). Members of the family Bulnenopteridrre are the finback whales, which includes the sei whale. Some of the members of this family are among the largest of living animals. The largest member of the group, the blue or sulphur-bottom whale [Sibbddus 1nmculu.s (Linnaeus)], attains a length of 98 ft (30 m ) and a weight of 110 tons ( 1 12.500 kg). This is one of the two families of baleen whales i n which the embryonic teeth are replaced by baleen plates in the adult animals. Finback whales frequently are called rorquals, which refers to a whale having folds or pleats. The rorquals are equipped with longitudinal furrows, usually 10 to 100 in number, that are present on the throat and chest. These furrows increase the capacity of the mouth when it is opened. The members of this family are the
Other Poisonous Marine Animals
69
fastest swimmers of the baleen whales. Their food consists largely of euphausiid shrimp, copepods,amphipods,andotherzooplankton.Whalesareconsideredamongthemost healthy of all living mammals since evidence of pathology is seldom observed. The liver of the sei whale is considered to be toxic to eat. The family Monodontidae, or white whales, consists of only two species, the white whale (Dell,hirlcrl,ter-lrs leuccrs) and the beaked narwhal (Momdon monocerosj. They inhabit arctic seas and sometimes ascend rivers. Eating white whale has produced fatalities (86,87). There is no information available concerning the toxicity of the narwhal; apparently they are used for human food at times, but their meat is usually fed to sled dogs. White whales have a body shape similar to members of the Delphinidae. The snout is blunt and there is no beak. There are no external grooves on the throat. White whales usuallylive in schools, sometimes consisting of more than 100 individuals. They feed mainly on benthic organisms, cephalopods, crustaceans, and fish. White whales are of economic importance and are hunted mainly for their skins, which are sold as "porpoise leather." The family Physeteridae, or the sperm whales, inhabitsall oceans. The sperm whale (Physeter-cntodon) attains a large size of up to 65 ft (20 m ) and 55 tons (55,880 kg) in weight. The sperm whale is said to be the only cetacean with a gullet large enough to swallow a human. The characteristic featuresof the sperm whale areits tremendous barrelshaped head and the underslung jaw. The sperm whale feeds on squid, cuttlefish, fish, and elasmobranches. Theoil and fesh of the sperm whale have been reported to be toxic, but the documentation of this is poor (88). The family Delphinidae includes the dolphins and porpoises. They inhabit all oceans and the estuaries of many large rivers; some species may ascend rivers for great distances. Some species seem to prefer warm coastal waters; none of them are found in the polar regions. The term dolphin generally refersto small cetaceans having a beaklike snout and a slender streamlined body, whereas the term porpoise refers to small cetaceans having a blunt snout and a rather stout, stocky body. Their food consists of fish, crustaceans, and cuttlefish. The Asiatic porpoise( N e o l h ) c m n a phocaenoicle.~)ascends estuaries and rivers has been reported as toxic (89-91). for more than 1000 miles (1600 km). Neophocaer~~~
Family: Balaenopteridae (sei whales) Species: Bcrlcrenoptertr borecrlis (Lesson). Sei whale. Length 60 ft (1 8 m). Distribution: Atlantic Ocean, south to the Gulf of Mexico; Pacific Ocean, Bering Sea south to Baja California. Family: Monodontidae (white whales) Species: Delphitlnpter-us 1errccr.s (Pallas). White whale. Length l8 ft (5.5 m). Distribution: Arctic and subarctic seas. Family: Physeteridae (sperm whales) Species: Physeter- ctrtodon (Linnaeus). Sperm whale. Length 60 ft ( 1 8 m). Distribution: Polar, temperate, and tropical seas. Family: Delphinidae (dolphins, killer whales, and pilot whales) Species: Asiatic porpoise [Neophoc-amrrphocvenoides (Cuvierj]. Length 5 f t (1.5 m) for Asiatic porpoise. Distribution: Coastal areas, estuaries, rivers, and lakes of China.
Halstead
Some cetaceans havebeen reported as poisonous to eat including Baltrenoptercr borealis (92), Delphinrryterus 1eucrr.s (86,87), Physeter. catorlot1 (88), and Neophoccretm pkocmrtoides (89-91,93). The subject of poisonous cetaceans hasbeen reviewed by Halstead ( 12.8 1). Mechanism of Intoxication: Sei whale. The liver ofthesei whale (Bcrlcrettopter(t borealis) is reported to be toxic to cat. 1euccr.y) is White whale. The viscera and meat of the white whale (Del~hinrrl~terus in fatalities. reported to be toxic and may result Sperm whale. The oil and meat of the sperm whale (Physeter ccrrodon) may be poisonous in some localities. Asiatic porpoise. The liver, other viscera, and muscle of the Asiatic porpoise (Neophourettcr phocrrettoides) are reported to be poisonous and may cause death. The nature of the poisons has not been determined, but it is suspected that in some instances vitamin A may be the causative agent. Clitticcl1 CIlctracteristics: Seiwhalepoisoning.Theonset of symptomsbegins within 24 hours after ingesting the liver and consists of severe occipital headaches, neck pain, flushing of the face, nausea, vomiting, abdominal pain, diarrhea, fever, chills, photophobia, tearing, and erratic blood pressure. After several days, the victim's lips become dryanddesquamationdevelopsaroundthemouth,graduallyspreadingto the cheeks, forehead,andneck.Thedesquamationusuallydoes not involvetheentirebody.Although jaundice is not reported, there is evidence of liver impairment. Acute symptoms generally subside within 2 days, but the desquamation may continue for a longer period of time. White whale poisoning. Ingestion of the flesh of the white whale may cause death, but little appears to be known concerning the clinical characteristics. Sperm whale poisoning. No information is available on the clinical characteristics of sperm whale poisoning. Asiatic porpoise poisoning. The symptoms of Asiatic porpoise poisoning consist of abdominal pain, nausea, vomiting, bloating, swelling and numbness of the tongue, loss of vision, cyanosis, numbness of various areas of the body, hypersalivation, greenish tinge to the saliva, and muscle paralysis. Death may be rapid, and the fatality rate is said to be very high. Treatment: The treatment of cetacean poisoning is symptomatic. The nature of the poison is unknown. Prelwttion: T o prevent cetacean poisoning, the liver of the sei whale should never be eaten. The flesh of the white whale should never be eaten. The oil and flesh of the sperm whale reportedly are toxic in some areas and should be eaten only with extreme caution. The Asiatic porpoise viscera and flesh should not be eaten.
2. PoisonousWalrusesand Seals Walruses and seals are members of the mammalian order Pinnipedia. The pinnipeds are a group of marine mammals havinga spindle-shaped body and limbs modified into flippers for aquatic locomotion. The toes are included into webs, and the tail is very short. The males usually are larger than the females. The livers of walruses and certain species of seals may at times be poisonous.
Other Poisonous Marine Animals
71
Family: Odobenidae (walruses) Species: 0doI7en~1.srostncrrus (Linnaeus). Walrus. Length 1 1 f t (3.5 m). Distribution: Arctic Ocean, northeast coast of Siberia, northwest coast of Alaska, north to northwest of Greenland, Ellesmere Island. Family: Phocidae (seals) Species: EriSncrthus bnrbotus (Erxleben). Bearded seal. Length 9 ft (2.7 111). Distribution: Inhabits the edge of the ice along the coasts and islandsof arctic North Atnerica and northern Eurasia. Species: N(@10ccr ciwerctr (Peron). Australian sea lion. Length 8 ft (2.4 m). Distribution: South and southwest coasts of Australia. Species: Prrstr hi.s~,irr'tr(Schreber). Ringed seal. Length 4.5 ft (1.4 111). Distribution: Circumboreal, near the edge of the ice, to the North Pole.
Wdrm and S e d Poisot1ing Walruses (Od0berlrrs ro.smrru.s)have been reportedto have toxic livers(94). Several reports have also appeared on the toxicity of the liver of the bearded seal (Erignathus barbatus) (95-100). The Australian sea lion (Neophoca cinerea) hasbeen reported to have poisonous flesh and allegedly has caused produced deaths in humans and dogs (101,102). Mechmism of Itltoxicntiott: Walrus and seal poisoningis usually causedby ingesting the livers of the rogue bull walrus (Odobetlus) or the bearded seal (Erignr~thus),or the flesh of the Australian sea lion (Neophoca).Intoxications are believed to be caused by an excessive intake in vitamin A, which is present in the liver. However, in the case of the Australian sea lion, the nature of the poison is unknown. Clitliccrl Clr~rrcrcteristics:The symptoms of walrus and seal poisoning are said to be sirnilar to that of polar bear poisoning (see Sec. III.B.3). Tretrtnrent: The treatment of walrus and seal poisoning is similar to that for polar bear poisoning (see Sec. III.B.3). Preverltion: There is no reliable method of detecting a toxic walrus or seal by visual examination. Avoid eating walrus and seal livers and the flesh of Australian sea lions.
3. PoisonousPolar Bears Polar bears [Tllcllrlrctos tntrrifitnus (Phipps)] are marine carnivoresof the class Mammalia thatarecharacterizedbyhavingfourorfivetoes;claws;mobilelimbs;completeand separate radii and ulnas, tibias and fibulas; small incisors; and canines that are slender fangs. The only marine carnivore toxic to man is the polar bear. Most rnanmologists are of the opinion that there is only a single species. Relwesertttrtive Species Family: Ursidae (bears) Species: Tlln1arcto.s rnnritirnus (Phipps). Polar bear. Length 8 ft (2.5 m). Distribution: Arctic, circumpolar.
Polar Berrr Poisoning The biogenesis of the poison present in the polar bear has not been established, but it is believed that the toxin is due to the presence of excessive quantities of vitamin A.
72
Halstead
Meckcrnisnr cfb~toxic~rrion: Polar bear poisoning is caused by eating the liver or kidneys, which apparently concentrate large quantities of vitamin A. Clirzicd ChcrrLlc,reristics:The symptoms of polar bear poisoning usually begin about 2-5 hours after ingesting either the kidneys or the liver. The predominant symptoms are intense throbbing or dull frontal headaches, nausea, vomiting, abdominal pain, dizziness, drowsiness, irritability, weakness, muscle cramps, visual disturbances, and collapse. The headaches may become intense during the first 8 hours and may cause insomnia since they are aggravated by lying down. Gradually the headache lessens in severity and may disappear by the following day. Numerous cases have been cited i n which desquamation occurred on various parts of the body, particularly the face, arms, legs, and feet. Tonic a case in which photosensitiand clonic convulsions may be present. Sutton (103) reported zation was a prominent symptom. If fatalities do occur from these intoxications, they are rare. The amount of liver ingested appears to have a direct bearing on the severity of the symptoms. Eskimos believethat ingestion of polar bear liver may result in depigmentation of theskin.However,investigationfails to substantiatethisbelief (104). The clinical characteristics of polar bear poisoning are discussed in Refs. 12,96,99,103, and 105-1 IO. Trear~nent:Treatment of polar bear poisoningis symptomatic. Emetics and laxatives promptly administered sometimes are useful in relieving the severity of the symptoms. The clinical manifestations gradually disappear after ingestion of the toxic meat has been discontinued. Prevelltion: There is no reliable method of detecting toxic polar bear liveror kidneys by visual examination. The age of the bear seems to have no bearing on the edibility of the meat. The liver from cubs has been known to cause intoxication ( 1 1 I ) . In general, it is best to discard polar bear liver or kidneys. If these parts are eaten,they should be eaten in amounts of less than a 0.5 Ib (230 g).
ACKNOWLEDGMENT It is with deep appreciation that I acknowledge the technical assistance Medrano i n the preparation of this chapter.
of Leonette C.
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VK Pillai. MB Nair, K Ravindranathan, CS Pitchumoni. Food poisoning due to turtle flesh. J Assoc Phys India 10(4):181-187, 1962. V Stefansson. My Lifc with thc Eskimo. New York: Macmillan, 1924, pp 32-35. V Stefansson. Arctic Manual. New York: Macmillan, 1944, pp 98-283. Y Sahashi. Nurtritive value of sperm whale oil and finback whale oil. Sci Papers Inst Phy Chem Res 20(416):245-253, 1933. DJ Macgowan. Porpoise poison. Med Rcp Imp Marit Customs China (27):12. 1884. DJ Macgowan. Poisonous fish in China. Bull US Fish Comm 6: 130- 13 I , 1887. BE Read. Chinese Materia Medica: Fish Drugs. Peking: Peking Natural History Bulletin, 1939 M Mizuta, T Ito, T Murakami, M Mizobe. Mass poisoning from the liver of Sawara and Iwashikujira [English translation from Japanese]. Jpn Med J 1710:27-34, 1957. DJMacgowan.Poisonousfishandfish-poisoninginChina.ChineseRecMissionJ 17(2): 45-49,1886. FH Fay. Carnivorous walrus and some arctic zoonoses. Arctic 13: 111-122, 1960. J Lindhard. Sundhedsforholdene paa "Danmark-Expeditionen" [in Danish]. Hospitalstidcnde 325355347, 1910. A Krogh, M. Krogh. A study of the diet and metabolism of Eskimos[in Norwegian]. Meddel om Gronland 51:11-52, 1913. EO Jordan. Food Poisoning and Food-Borne Infection. Chicago: University of Chicago Press, 1931,pp55-64,1931. K Rodahl, T Moore. The vitamin A content and toxicity of bear and seal liver. Biochem J 37:166-168,1943. K Rodahl. Toxicity of polar bear liver. Nature 164(4169):530-531, 1949. R Rausch. The toxicity of polar bear liver. Unpublished, 1956. WH Leigh. Reconnoitering voyages and travels with adventuresin the new colonies of South Australia. In: Reconnoitering Voyages and Travels with Adventures in the New Colonies of South Australia, Cornhill, London: Smith, Elder, and Co. 1839, p 164. JB Cleland. Injuries from animals. Med J Aust 2(22):491-492, 1942. RL Sutton. Is polar bear liver poisonous? JAMA 118: 1026, 1942. V Stefansson. The Friendly Arctic: The Story of Five Years in Polar Regions. New York: Macmillan,1921. H Khl. Kann die Liber der Fleischfresser gifting sein'? In: R. Ostertag-Stuttgart, ed. Zeitschrift fur Fleisch-und Milchhygiene. Berlin: Verlagsbuchhandlung von Richard Schoetz, 1929; pp 45-49. 0 Boje.Toxin i n thefleshoftheGreenlandshark[inNorwegian]Mcddelom Granland 125(5):1-16,1939. JK Doutt. Toxicity of polar bear liver. J Mammal 21:356-357. 1940. RL Sutton. Is polar bear liver poisonous? JAMA 118: 1026. 1942. W Beckcr, C Klotzsche. Die hypervitaminose A [in German]. Arztl Woch 24545-550, 1955. H Jcghers, H Marraro. Hypervitaminosis A: its broadening spectrum. Am J Clin Nutr 6:335339,1958. EK Kane. Arctic Explorations: The Second Grinnell Expedition in Searchof Sir John Franklin, 1853, '54, '55. Philadelphia: Childs and Peterson, 1856.
4 Shellfish Chemical Poisoning
I. Introduction 78 11. Paralytic Shellfish Poisoning 79 A. Causativc toxin and its source 80 B. Molecular mechanism of action 81 C. Toxin uptake 82 D. Toxin metabolism, transport, and elimination 82 E. Treatment 83 111. Diarrhetic Shellfish Poisoning 84 A. DSP-causing agents and their sources 84 B. Known molecular pharmacology 85 C. Cellularimpactsofokadaicacidactions 85 D. Organandtissueeffects of DSTs 86 E. Toxinuptakemetabolism,transport,andelimination F. Treatment 86 IV. Neurotoxic Shellfish Poisoning 87 A.Thecausativetoxinsandtheirorigins 87 B. Molecular pharmacology of the toxin 87 C. Toxin uptake, metabolism, and distribution 89 D. Toxin elimination 89 E. Whole animal effects 90 F. Treatment for NSP 90 V. Amnesic Shellfish Poisoning 90 A. Causative toxin and its origin 91 B. Molecular target of domoic acid 92 C. Domoicaciduptakebythegut 92 D. Toxin distribution and elimination 92 E. Pathology 93 F. Treatments for ASP 93 VI.MinorShellfishToxins 94 A. Cyclized, peptidic hepatotoxins 94 B. Tetrodotoxin 95 of Toxins 95 VII.CO-Occurrence
86
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VIII.
ShellfishResponse t o Toxins 96
IX. Effect on Toxins by ShellfishPreparations07 X. Concluding Remarks 97 Acknowledgment 98
References 98
1.
INTRODUCTION
Molluscan shellfish have long been a gastronomic treat for humans, a delight that has tragically for some, ended in poisoning and even death. The cause of these poisoning’s can be twofold. First, poor food hygiene may lead to bacterial spoilage of the shellfish, resulting in human illness such as botulism( 1 ), a type of poisoning not unique to shellfish. The second cause, which rarely occurs i n other food sources and is the subject of this chapter, results from the shellfish sequestering toxic compounds. Many shellfish gain sustenance by sieving the water column and feeding upon the minute organisms therein. At times the organisms comprisingthe shellfish’s diet are toxic, so as well as nutrients, shellfish consume compounds that are seemingly harmless to them (2.3) but are toxic to those organisms further up the food chain. These toxic microorganisms are present in the environment as a matter of course, but are usually in numbers too low to present a problem. Their populations do on occasion bloom, intoxicating exposed shellfish to levels harmful tothe consumer. These toxins can remain withinthetissues of theshellfish for days, weeks, or months after exposure of the shellfish to the toxic microalgae. Therearefourmajor shellfish poisoningsyndromescaused by bioaccumulated toxins: paralytic shellfish poisoning (PSP), diarrhetic shellfish poisoning (DSP). amnesic shellfishpoisoning(ASP),andneurotoxic shellfish poisoning(NSP).Themicroalgal blooms thatgiverise to toxicshellfishareincreasing i n frequencyandimpactona globalscale (4). This has beenattributedtofactorssuchasglobalwarming (5,6) and increasedhumanimpactoncoastalwaters,includingagriculturalfertilizerrunoffand wastewater discharge (7,8). Globalization of the toxic shellfish problem may also be due, i n part, to the deposition of toxic microalgae from the ballast water of international shipping into areas where it lacks competitors and thus is able to flourish (9). Evidence of this global increase is the occurrence of toxic algal blooms and associated human intoxications in regions with no recorded history of such events. For instance, New Zealand experienced an NSP episode in 1993 (10) that was the first ever occurrence of this malady outside of the Americas. The public health impact of shellfish poisoning in naive regions is magnified because the local health authorities are inexperienced at recognizing the event unfolding before them, thus delaying implementation of suitable countermeasures. The shellfish that primarily cause human intoxication are the bivalve molluscs. This class of molluscs includes clams and oysters and is characterized by a two-piece hinged shell that protects the soft body. Some of the species reported i n shellfish poisonings are listedinTable 1. Thetoxinsaregenerallyaccumulated i n thedigestivegland,which counts among its functions excretion of digestive enzymes and some nutrient absorption, and in the siphon, the tubular organ that draws water into the animal’s body cavity and gut. Many adult bivalves attach to a solid surface or live in the sediment. This makes them ideal for farming as the main requirements for their growth are a suitable substrate
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Shellfish Chemical Poisoning
Table 1 ShellfishSpeciesConfirmed to HaveCausedHuman Intoxications and the Syndromes Observed
Species
Shellfish poisoning syndrome
Example references
NSP PS P PSP ASP DSP DSP DSP DSP DSP PSP PSP PSP PSP PS P PSP ASP
10 11 12 13 14.15 16 17 15 15 18 19 11 20 21
22 23
and high-quality water from whichto feed. However, farmingcan also exacerbate shellfish toxicity as it concentrates the shellfish population, magnifying the toxic effectof an algal bloom. Rapid international freight and improved shipping technology mean that shellfish can be exported to many countries from a single region. Thus it is possible, that a shellfish poisoning event may occur far from where the product originated. As an example, the Galician Rios in Spain supplies approximately 40% of all European shellfish (24). Shellfish 200 people being hospitalized all over intoxication i n this one region led to more than Europe (25). For these reasons, monitoring shellfish toxicity has become a responsibility incumbent upon industries and government in virtually every region of the world. These programs ensure that shellfish bound for export and domestic consumption, and, at times, those imported. are either toxin free or contain toxin levels far below that which will cause human illness (26,27). These efforts have dramatically decreasedthe possibility of people being poisoned, but the safeguards arenot absolute. Public health authorities, other health professionals. government authorities, and shellfish farmers should therefore be aware of the various shellfish toxins, their effects, and etiology.
II. PARALYTIC SHELLFISH POISONING Paralytic shellfish poisoning (PSP) is the longest studied shellfish poisoning syndrome, with the toxic principle being recognized early this century (28,29). Human intoxications PSP is an are now global (Figure l ) , and i n some countries i n the Asia-Pacific region, almost annual concern (57). As the name suggests, paralysis is a prominent symptom of PSP. In theearlystages of theintoxication,victimstypicallyexperiencetinglingand numbness of the mouth, tongue, face, and extremities. Accompanying these effects may
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;9
\
V
Fig. 1 Regions of the world from where shellfish to havc caused human intoxication havc originated. (Amnesic shellfish poisoning A;diarrhetic shellfish poisoning m; neurotoxic shellfish poisoning V; paralytic shellfish poisoning ). (16,20.22,23,30-56).
be nausea and vomiting. Hypertension is usually evident in PSP victims (S8,59).In severe cases, the patient will exhibit advanced neurological dysfunction suchas ataxia, weakness, dizziness, a sense of dissociation, followedby complete paralysis. Thereis a compounding effect of central nervous system depression, rendering the diaphragm nonfunctional (60), and death mayresult from cardiorespiratory failure. Mortality rates from PSP have reached 40% (22).
A.
Causative Toxin and Its Source
PSP is caused by saxitoxin (STX; C,,,H17N704; molecular weight 299) and its chemical relatives (Figure 2); this family of toxins will be referred to here as the paralytic shellfish to guinea pigs at only S pg/kg when toxins (PSTs). STX is highly toxic, being lethal injected intramuscularly (61) and mice when injected intraperitoneally (29). PSTs are tricyclic molecules with the 1,2,3- and 7,8,9-guanidino groups of STX possessing ~ K , Jof 1 1.3 and 8.2, respectively (62-64). Thus, at physiological pH, the 1,2,3- guanidino carries a positive charge, whereas the 7,8,9-guanidino group is partially deprotonated (65). This polar nature of STX makes it readily soluble in water and lower alcohols but insoluble in organic solvents. It is extremely stable in solution at neutral and acidic pHs, even at high temperatures. However, alkaline exposure will oxidize the toxin to an inactive derivative (66). Both marine and freshwater microalgae produce PSTs. In freshwater, blue-green to freshalgae (Anlrbtrerltr circirdis) producePSTsandcantransfertheirtoxins water shellfish (Altrthyrilr conclollr) (69), although no reports exist of PST intoxication via thisroute.MarineshellfishsequesterPSTsfrommotilemarinemicroorganismscalled dinoflagellates, particularly Ale.randrium catenelllr, A. n~ir~lrtut?~, A. oster!feldii, A . tcrmcrreme, Gymnoclitliw?? ccrtencrturn, and Pyrodit~iuml~trharner~sew r . cor?IpressmI. At this
81
Shellfish Chemical Poisoning
STX neoSTX
B1 Gonyautoxin 2 Gonyautoxin 3 decarbamoylSTX
RI
R4 RZ
R3
H OH H H H
H H H H
H H H
H H
OSOS
OSOY
H H
H H H
H
soy
’ In this derivative, a proton replaces of all the structure beyond the wavy line, including R+ Fig. 2 Thc structure of saxitoxin and o f
naturally
occurring chemical variants (62,63,67,68).
time there is a debate concerning the possible production influence on PST production (70,7 I).
of PSTs by bacteria or their
B. Molecular Mechanism of Action PSTs prevent sodium ions from passing through the voltage-sensitive sodium channel (VSSC), binding to the channel with nanomolar affinity (72,73). This large protein, of approximately 260 kDa, spans cell membranes mainly in nerve and muscle. Depolarization of the cell membrane initiates a conformational change in the VSSC, opening a pore that selectively transports sodium ions into the cell. The VSSC is a multisubunit protein, with the largest subunit andthat which contains the pore, the a-subunit, containing four internal of these sequence repeats, it is hypothesized amino acid sequence repeats. Within each that six transmembrane a-helices assemble around the central ion transporting pore (74). Depending on the tissue and VSSC isoform, other smaller proteins, the P-subunits, attach to the a-subunit and affect channel properties such as the speed of ion conduction and channel activation and inactivation rates (75,76). To date, VSSCs have only been detected in animals, occurring in all vertebrates and in most invertebrate phyla, including molluscs (77), jellyfish (781, and flatworms (79). PSTs bind at the opening of the pore that allows the ions to pass through the VSSC (80,81). Whether ion passage is hindered by physical occlusion of the pore, or by an allosteric modulation of the pore’s structure making it unable to conduct ions, is still not fully resolved. Modified STX can havea dramatically altered ability to bind to the VSSC. For instance, the sulfated PST, B 1 (Figure 2), has an affinity 400 times less than STX for the VSSC rat skeletal muscle (73) and neoSTX (Figure 2) is four fold better than STX at binding to the same channel. Crucial also to the ability of STX to bind to the VSSC
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is the charge state of the 7,8,9-guanidino group, with its deprotonation preventing it from ligating to the channel (65). Mammals also express several VSSC isoforms to which STX can bind with only micromolar affinity. They exist in lnatnmalian sensory neurons (82), cardiac muscle(83). skeletal nluscle in the early stages of development (84), and experimentally denervated skeletal muscle (85). Differences in VSSC amino acid sequences underlie this diversity in toxin sensitivity, and mutation of a single amino acid can convert a toxin-insensitive VSSC to a channel that is easily blocked by STX (86).
C. ToxinUptake STX uptake from the gut appears to be quite efficient since no toxin is eliminated i n the feces of test animals (58,87,88). Absorption of drugs and toxins from the mammalian gut mainly occurs by passive processes and not via the specific active transporters for nutrients (89). Passive uptake across the gut epithelium favors nonionized and lipophilic molecules that readily penetrate cell membranes.It is curious thenthat the cationic STX is efficiently absorbed. Of importance for ionizable compounds the likePSTs, the pH of the gut environment affects toxin uptake by altering the charge state of the toxin. For example, since most absorption occurs in the alkaline intestine, in this part of the gut STX will lose a proton from the 7,8,9-guanidiniunl, making it less polar and therefore approaching a state more amenable to diffusion across the lipid bilayer. Also, the overall charge state in some PSTs can be modified by the presence of anionic sulfate groups, which can negate the positive charges provided by the guanidino groups. Passive absorption of charged, hydrophilic compounds occursin the gut by diffusion through the tight intercellular junctions of the epithelium (89). This process, called paracellular diffusion, is restricted to molecules of approximately 5 2 0 0 Da and is unaffected by pH. This exclusion limitisnotabsolute andmaybeaffected by agentsthatsequester calcium and magnesium. The complex biological matrix in which the toxin is ingested may contain natural ion chelating agents, making it easier for PSTs to enter the bloodstream via this route. Although paracellular diffusion is much less efficient than uptake through the epithelial cells, it may be a significant route of toxin absorption, as STX is not much greater than the size exclusion limit, even if not affected by ion chelators. Also, those PSTs significantly smaller than the parent STX molecule, for example, decarbamoylSTX (Figure 2; molecular weight 256), would be better able to pass through the tight junctions.
D. Toxin Metabolism,Transport,andElimination The first point where PSTs may be modified after ingestion is in the gut itself. Gastric acid can convert small amounts of the less toxic sulfated PSTs to more potent compounds (90). This may prove significant if a very large amount of these sulfonated toxins is ingested and critical amounts of the highly toxic STX or neoSTX are produced. Once in the blood, STX is returned to physiological pH, and thereforeto a predorninantly doubly charged and highly toxic state. With STX again being quite polar, there is little possibility of it crossing the blood-brain barrier, which is best traversedby lipophilic agents. This is borne out by a recent study where no toxin reached the brain of cats that received 2.7 pg/kg STX (87). Curiously though, at higher doses ( I O pg/kg) in this same study, STX did enter the brain. This dose dependence of STX’s ability to invade the
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brain may explain earlier reports of STX exerting central effects (60.91,92). One other explanation for this unexpected observation may be a process where charged drugs, to which STX can be equated, binds to plasma proteins (89), some of which may be actively taken across the blood-brainbarrier, carrying STX along with them. PST’s effects on central systems can also be achieved upon regions of the brain not encapsulated by the impermeant blood-brain barrier, such as the circumventricular region. Toxin is eliminated predominantly via the urine, with no elimination of PSTs via the feces (58,87,88). Excretion of PSTs occurs rapidly,no matter how the toxin is administered. For instance, the bulk of IV administered saxitoxinol, a chemically reduced STX, and STX was urinated by rats within hours of injection (66,88). In humans, the half-life after oral ingestion of toxic shellfish is approximately I O hours (58). Metabolic conversion of PSTs while passing through mammals has been little studied, especially in humans. Examination of the serum and urine of human PSP victims revealed a significant increase in Cl compared to its relative gonyautoxin-2, which differ only in an additional sulfate on the C type PST (58). This sulfation of gonyautoxin-2 in humans contrasts with rats, where there was no metabolism of STX or saxitoxinol during their-passage into the rat’s urine (66,88).
E. Treatment Victims should undergo immediate gastric evacuation to remove toxic contents unyet digested and absorbed. This can be achieved not only orally, but also by enemas to clear the intestines. This will also decrease the impact of any conversion of lesser toxic PSTs in the gut. Artificial ventilation for victims of severe PSP is the only recommended treatment to date. Symptomatic relief should accompany all other efforts. The dominant strategy for developing a medicinal treatment has been to use antibodies developed to STX, a carrier protein to make an antigenic epitope, that created against toxin conjugated to may sequester PSTs and out-compete the sodium channel to bind the STX (93-96). If the antibody has a significantly lower affinity than the sodium channel for STX, which was indeed the case with one antibody that possessed a micromolar affinity for the toxin (95). then it must compete for thetoxin by weight of numbers. This would requires administration of large amounts of the antibody. Another limitation of this approach is that the antibodies may be quite specific to single PSTs such as STX; other PSTs to which the victim was exposed maybe able to bypass the antibody and still act. A mixtureof antibodiesmay be necessary if this approach is to succeed. Despite these potential problems, there has been some successi n animal modelswhen treated with STX antibodies, although they have not been tested on PST mixtures (93-96). Since it seems that STX can cross the blood-brain barrier at high doses, antibodies will be ineffective in inhibiting this pool of toxin since they cannot cross the blood-brain barrier. Surprisingly, in guinea pigs, the potassium channel blocker 4-aminopyridine (4-AP) effectively counteracted the effectsof lethal IV doses of STX (61). This drug reverted the loss of blood pressure and enhanced neuromuscular transmission to allow the diaphragm to again function. Large dosesof 4-AP were necessary, however, and may cause serious side effects such as seizures and convulsions. If administered in a hospital, steps can be taken to ameliorate these side effects. A clue to an alternative drug strategy is present in a recent study on cats (87). Cats given lethal doses of STX were kept alive forthe duration of the experiments by continuous administration of the adrenergic agonist, dobutamine, in conjunction with mechanical
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ventilation. In these animals, because breathing was assisted, cardiac rather than respiratory arrest was the cause of death. Dobutanline increases cardiac contractile strength and volume, and providing it along with artificial ventilation, as was done with the cats, may be a suitable emergency medical strategy for PSP victims (87).
111.
DlARRHETlC SHELLFISH POISONING
The firstreport of what became known as diarrhetic shellfish poisoning (DSP) was in 1978 in the Tohoku district in Japan (IS). Since then reports of DSP have emerged from every continent except Africa and Australia (Figure1 ). DSP has never resultedin a human fatality, despite the causative toxins being lethal to mice when injected intraperitoneally (LDi0 of 192 pg/kg) (97). Diarrhea is the major symptom exhibited by victims as well as other gastrointestinal upsets such as vomiting, nausea, and abdominal cramps. These (IS). symptoms may become so severe as to incapacitate the patient
A.
DSP-Causing Agents and Their Sources
The major causative toxin for DSP is okadaic acid (OA) and its relatives, the dinophysistoxins (Figure 3). To be consistent throughout this chapter, the suite of DSP-causing toxinswillbereferredto asdiarrheticshellfishtoxins(DSTs).Okadaicacidderivesits name from the organism from which it was first isolated, the sponge Herlichondricr okcrh i , andwashypothesizedtobeproduced by microorganisms(97).Okadaicacid (C4,HtjXOl3; molecular weight 804) isa complex lipophilic polyether, readily soluble in many organic solvents (99) and sensitive to degradation by acid or base exposure (48). DSP isa somewhatconfusingsyndrome inthatseveralstructurallydistinctpolyether toxin families, the yessotoxins and pectenotoxins, often occur i n shellfish alongside OA and the DTXs and are also referred to as DSTs. Only dinophysistoxin and OA are truly diarrheagenic (IOO), and will be focused upon here, but the other toxins confuse toxicity monitoring results because of their effects on mice used i n the regulatory bioassay. Pectenotoxin is clearly an hepatotoxin (IOO), whereas how yessotoxin mediates its toxicity is still unclear (101); experimental animals injected with it die from cardiac failure (102).
H acid Okadaic Dinophysistoxin-l Dinophysistoxin-2
H H H
CH3 CH3
CH3 CH3 H
Fig. 3 Structure of' thepolyether diarrhetic shellfish toxins, okadaic acid and dino1Jhysistoxins (48,97.98).
Shellfish Chemical Poisoning
B.
85
Known Molecular Pharmacology
Okadaic acid is a very potent inhibitor of several classes of serinekhreonine phosphatase, acting at nanomolar and even picornolar concentrations (107-109). These enzymes are a variable subunit that then multisubunit proteins targeted to their substrate protein by forms a heterodimer with a structurally invariant subunit which catalyses the dephosphorylation of serine and threonine residues ( 1 IO). Serine-threonine phosphatases are subclassified depending upon their substrate specificity, their requirement for cofactors such as to several inhibitory peptides and divalent cations and calmodulin, and their sensitivity okadaic acid itself. OA-sensitive subclasses are PP-l and PP-2A, whereas PP-2C is unaffected by OA,andmicromolaramounts ofthetoxin arerequiredtoinhibitPP-2B ( l 0 9 , l I l , I 12). It is the catalytic subunits of PP-l and PP-2A that are affected by OA, mainly by noncompetitive inhibition (1 13,l 14). These two isoforms are highly homologous, explaining the similarity in their OA sensitivity. The homology is not complete and the differences i n amino acid sequence cause subtle differences in their affinity for OA. A sequence of four amino acids containing two acidic residues from rabbit PP-l can be a replaced by the corresponding amino acids from rabbit PP-2A, which contain instead cysteine and a basic amino acid, arginine. This increased the toxin sensitivity of the chimeric mutant so that it approached that of PP-2A from which the arginine-cysteine sequence was derived ( 1 15). The carboxylic acid of OA is essential to its activity, as demonstrated by methyl esterification, a modification which abolishes OA's abilityto inhibit its target phosphatases ( I 16,l 17). Also, oxidation of the 27-OH greatly reduced its abilityto inhibit PP-2A. From molecular modeling, this portion of molecule is not involved in bonding to the enzyme, but is more likely to be involved in binding the phosphate substrates (1 18). Apart from its acute toxicity, chronic exposure to OA may have a genotoxic effect. At low concentrations. OA forms DNA adducts in mammalian cell lines, between 2 and 100 adducts per 10' bases, a phenomenon not observed at higher concentrations of 2 10 nM ( 1 19). Regular dietary exposure to OA levels that may not elicit DSP may therefore have a teratogenic effect.
C. Cellular Impacts of Okadaic Acid Actions In those cells that take up DSTs, the toxins cause a buildup of phosphorylated proteins. Phosphatases are counterbalanced within the cell by protein kinases,which introduce phosphate groups onto proteins. Since the phosphorylation state of an enzyme, or receptor, often governs whether it is active or not, this interplay between kinases and phosphatases regulates cell function and manyof the cascades which underlie cellular processes. Further, of a kinases and phosphatases can act upon each other. For example, dephosphorylation kinase renders it inactive and phosphorylation of a phosphatase may switch the enzyme into action ( I 1 I ). In the presence of OA, protein kinases continueto function, but in those pathways containing PP- I and PP-2A, which are inhibited, phosphorylated proteins will become overabundant. Thus enzymic cascades and membrane receptors may continue to
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function long beyond their optimal time period, or may not switch on again until the OA is eliminated. Because OA has an effect on such a crucial enzyme, it may result in many effects within the same cell. This, of course, depends upon the efficiency of toxin uptake by the cell and the metabolic status of the cell when the toxin is absorbed, which would govern which subunit the PP-1 and PP-2A catalytic subunits would be dimerized to at the time. Multiple effects can be shown with vero cells, a monkey kidney cell line, where OA inhibited protein, DNA, and RNA synthesis, all at nanomolar concentrations ( 1 20). Certain cell types are also more sensitive to OA than others. For example, in culture, neuronal cells are affected at subnanomolar concentrations, whereas astrocytes and fibroblasts (121) require much more toxin for an effect to be realized. Another case in point would be rat hepatocytes, which require micromolar concentrations of OA for cytotoxicity to be observed ( 122). The role of celltype and cell susceptibility is also demonstrated by the differences between cellular and in vitro effects. Protein synthesis in a cell line was inhibited by SO nM OA, whereas in in vitro studies, generic protein synthesis using a rabbit reticulocyte lysate system was almost 10,000-fold more sensitive to OA, being inhibited by SO% at a concentration of 6.5 pM (120).
D. Organ and Tissue Effects of DSTs DSTsproducetwomajorphysiologicalresponses totheirmolecularactions: an acute effect-diarrhea-and a potentially chronic effect-tumor promotion. Intraperitoneal injection of DTX- 1 desquamates intestinal epithelia (100,123). This, in conjunction with induction of excessive fluid secretion in the intestine ( 124), underlies many of the gastrointestinal effects seen in DSP victims. DSTs alone do not induce tumor formation, but when applied to the skin of mice after a tumor-initiating substance, dimethylbenzanthracene, virtually all test animals developed skin tumors of varying malignancy ( 125,126).
E. Toxin Uptake Metabolism, Transport, and Elimination The sensitivity of these toxins to acid and base exposure would be expected to impact upon the stability of the DSTs while in the human gut before absorption, but the presence of DST degradation products in fecal material has never been reported. If not degraded to a form chemically different from the parent DST, the lipophilicity of the toxins would allow them to passively diffuse through the gut epithelium and toxin uptake would be expected to be quite efficient. Oral administration of tritiated OA to mice resulted in its In pregnant rats, 7% of distribution to liver (2%), kidney (2%), and blood (6%) (127). administered OA crossed the placenta and contaminated the fetal pups (127). Pregnant victims of DSP must receive special care because if DST canalso cross the human placenta it would be concentrated in a single fetus. OA was eliminated in urine, with almost 8% of the administered tritiated OA being detected in the urine within 24 hours (127). Curiously, OA has a secondary effect within the gut of increasing epithelial permeability via paracellular diffusion (128), which may increase uptakeof DSTs themselves or of hydrophilic compounds accompanying these toxins.
F. Treatment DSP is rarely life threatening and usual treatment is to make the patient as comfortable as possible for the durationof the intoxication. Symptomatic treatment for severe diarrhea,
Shellfish
87
such as fluid replacement, should be employed. Because epithelial destruction by the DSTs contributes to 111uch of the diarrhetic effect, administration of classic antidiarrheals is unlikely to provide any relief.
IV. NEUROTOXIC SHELLFISH POISONING The Gulf of Mexico has a long history of toxic microalgae blooms that cause massive fish kills and respiratory irritation in humans. It was eventually recognized that the toxic to induce what agent from these blooms could also be transmitted to humans via shellfish NSP wasrestrictedfor becameknownasneurotoxicshellfishpoisoning(NSP)(129). manyyearstotheAmericasuntiltheearly1990s,whencaseswerereportedinNew Zealand and Australia (Figure l ) . Victims of this syndrome exhibit many of the same symptoms as people who suffer fromthe fish-derived seafood poisoning syndrome. ciguatera. Typical symptoms are tingling in the face, throat, and digits. dizziness, fever, chills, muscle pains, abdominal cramping, nausea, diarrhea, vomiting, headache, reduced heart rate, and pupil dilation (50). There have been no recorded deaths from NSP, although the causative toxin is fatal to test mammals ( 1 30- 132) when administered by various routes, including orally.
A. The Causative Toxins and Their Origins Like the PSTs and DSTs, the compounds that cause NSP originate from dinoflagellates. Inthis case, the culprit alga is Ptychodiscus hruvis (formerly known as Gyrrrnodiniunr b r e w ) ,which gives it name to the toxins, the brevetoxins. Many naming conventions have appeared over the years with regard to brevetoxins. but here we will refer to the toxins as PbTxs, an abbreviation derived from their taxonomic origin. PbTxs are lipophilic, 10to I I-ring polyether compounds (Figure 4) which can be divided into two classes. Type 1 brevetoxins contain l I hexameric rings except for two 7-membered and one 8-membered ring. Type 2 brevetoxins possess only IO rings with one more 8-membered ring than type l PbTxs and an unusual 9-membered ring. They also differ from type 1 PbTxs inthat they have a terminal pentameric ring ( 133). Decomposition of both types of brevetoxins is accelerated in aqueous solutions with pHs greater than 10 and less than 2 ( 135). When dry, however, PbTxs are extraordinarily stable with PbTx-2 and -3 being stable in this state to 300°C ( 1 3 5 ) . I n rats, PbTx-2 and -3 possess LDS,,s of 60 and 200 pg/kg via the intravenous route ( 130,131 ). Similar LD,,, values are obtained when administered intraperitoneally (131). but the toxin’s effectiveness is markedly reduced when ingested, with the respective oral LD,,,s being S20 and 6600 yg/kg ( 13 1 ).
B. Molecular Pharmacology of the Toxin PbTxs bind to the voltage-sensitive sodiunl channel described i n detail in Sec. 11. It binds, however, to a different region of the VSSC than that via which the PSTs act and elicits a very different effect. The VSSC binding site for PbTxs involves both the first and fourth of the four homologous domains within the a-subunit ( 136,137), and quite likely part of one of the extracellular loops of the channel ( 138). Theaffinity of the toxins for theVSSC is nanomolar ( 139,140).and unlike with the PSTs, toxin-insensitive channel isoforms have
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Me
H0 R
0
Fig. 4 Brevetoxins type 1 (A) and 2 (B) and some of the chemical variations that nature (133,134).
may occur in
yet to be reported and mutations of the channel which affect brevetoxin binding have not been reported. PbTxs shift the activation voltage of the VSSC to more negative potentials (141144). This puts the channel nearer the triggering threshold where the change in potential difference across the cell membrane initiates the seriesof conformational changesto open and allow conduction of sodium ions. Inactivation of ion conduction is also believed by some to be slowed by brevetoxins (132,141). The end result of either of these two effects, or their combined effect, is to lengthen the mean open time for the VSSC, allowing a higher overall number of sodium ions to enter the cell. This hyperexcitability of cells reliant upon action potentials can lead directly to cellular malfunction. The additional sodium loadmay also overwhelm theprocesses,such as the N a + / K ' transporter,that maintain the concentration gradient of sodium across the cell membrane, providing the
Shellfish
89
electromotive force for action potentials. PbTx affected VSSCs also display subconductance states; that is, the rate of ion flow through the channel is reduced (141,145). The VSSC might not be the only receptor affected by the brevetoxins, because other smallpopulations of brevetoxinbindingsiteshavebeenobserved(138,139,146,147). These other binding sites were not identified, but the candidates include the nicotinic receptor-ionophore complexof the neuromuscular junction( 148)and the aryl hydrocarbon receptor, which is a ligand-activated transcription factor (149).
C. Toxin Uptake,Metabolism,andDistribution The lipophilic PbTxs easily pass through the gut epithelia into the bloodstream. Oral administration of PbTx-3 to rats resulted in almost all of the toxin being absorbed ( 1 SO). and within hours SO% of the toxin had accumulated equally in the liver and stomach. The ingested toxin was also readily distributed to the intestine (14%), heart (8%').and kidneys (695). Of the other tissues tested-spleen, lung, fat, muscle, plasma, testes, brain, and skin-no more than 4% of toxin appeared. Although IV administration of PbTx-3 results in a different pattern of tissue distribution in rats to that after ingestion, what is revealed is that uptake of brevetoxin is incredibly fast, with it disappearing from the blood within 1 minute of injection (IS1). Within 1 hour of the IV PbTx-3 injection, almost 70% of the 20% in the liver, and the remainder radiolabeled toxin appeared in the skeletal muscle, in the intestinal tract ( 1S 1 ). Orally ingested compounds first pass through the liver for detoxification, but in some cases, the compounds may attack the liver. In the case of the brevetoxins,thisisindeedthecase,wherePbTx-3affectedmouseliverefficiency by ( 152). enhancing sodium entry into liver cells, thereby inhibiting oxygen consumption Therefore the large anlount of PbTx that resides in the liver after its uptake could result in significant and possibly long-term liver dysfunction in NSP victims. The lipophilic nature of the brevetoxins would allow them to not only easily cross the gut epithelium, but also the brain barrier. This supposition is supported by a study with the chemically and pharmacologically related toxin ciguatoxin, which binds to the same sitc on the VSSC as the brevetoxins and elicits the same effects (153), which can induce many effects in the brains of mice (154). Curiously though, with PbTx-3, very little toxin reached the brain of rats, getting to no more than approximately 1% of h e toxin dose at any one time ( 150, IS l ) . PbTxs are vulnerable to metabolic conversion in mammals. This was demonstrated after rats were orally dosed with radioactive PbTx-3 and fecal extracts contained several radioactive compounds apart from the parent toxin, indicating some modification of the toxin had occurred during its movement through the body ( IS I ) .
D. Toxin Elimination PbTxs exit the lnamnalian body by both urination and defecation. In experimental m a n mals, the route of'toxin administration can affect which of the two routes will predominate. After IV administration of radiolabeled brevetoxin, 75% of the toxin was eliminated via the feces, whereas only 14% exited via the urine and the remainder was still in the rat's body after 6 days ( 15 I ) . After oral dosing with PbTx-3, however, some of the toxin is not absorbed, passing directly through the gut to be eliminated with the feces. Equivalent amounts of the toxin were eliminated in both the urine and feces, but because feces in-
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cludes unabsorbed toxin, it was suggested thaturinaryeliminationmight be dominant over the fecal route (150). The symptoms produced by the toxin may therefore accelerate in test mammals (135) and its elimination by increasing defecation and urination rates humans.
E. WholeAnimal Effects Intravenous injection of PbTx into anesthetized dogs caused apnea, bradycardia. uncontrolled spontaneous skeletal muscle twitching, and tonic contractions. Heart rate and blood pressure immediately dropped upon injection, but recovered quickly. Doses greater than 80 pg/kg causedrespiratory arrest in dogs ( 1 SS). This short-lasting bradycardia, hypotension, and respiratory inhibition is known as the Bezold-Jarish refex and also occurs in anesthetized cats given PbTx (156j.In awake rats, the respiratory rate is depressed, which the animal compensates for by increasing breath volume (157). A substantial decrease in core and peripheral body temperatures can occur in mammals given a brevetoxin (157). Thisobservation, in conjunction withtheneurologicaldysfunctionsuchasataxiaand simultaneousseizures of limbpairs,indicatesbrainandspinalchordinvolvement (156,157).
F. Treatmentfor NSP As with PSTs, antibodies to PbTxs have been investigated as a means of treating NSP victims. Pretreatment of rats with goat polyclonal antibodies directed toward PbTx-3 prevented any NSP symptoms i n rats after injection with PbTx-2 (130,157). Immediate injection of PbTx antibodies after injection with PbTxalsoprotectedthe animals from the toxin’s effects (130), except for some subtle emanations of toxicity such as mild ataxia. This was somewhat surprising since brevetoxin disappears from the blood so quickly and wouldnotbe exposed to the antibody. Brevetoxin is a reversible toxin ( I 5 8 ) , andan antibody may manage to sequester the toxin during the periods that it is dissociated from the receptor and before it reassociates. This is unlikely to be a suitable route for treating NSP victims, since quitea long time is likelyto occur between intoxication and realization by the victim that they may need medical attention.
V.
AMNESIC SHELLFISH POISONING
In 1987. on Prince Edward Island in Canada,an unusual shellfish poisoning event unfolded involving the blue mussel(Mytilus erlulis), a shellfish implicated in many different poisoning syndromes (TableI ) (46). Of the symptoms manifestedin the victims, themost curious was the effect on memory, an effect that lasted days or longer, which led to the syndrome being dubbed amnesic shellfish poisoning (ASP). Almost 200 people were affected and unfortunately, 3 of the victims died (159). Of interest, approximately 50,000 people are believed to have consumed mussels from the same batch that caused the poisoning (160). There have been few ASP events since (Figure l ) . Other symptoms experienced by ASP victims include nausea, vomiting, headache, diarrhea, and abdominal cramps. Neurological symptoms may follow, such as confusion, memory loss, and disorientation (161 ). I n severe cases, seizures, followed by coma and death may occur.
Shellfish
A.
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Poisoning
Causative Toxin and Its Origin
Because of the unusual nature of the s y m p t o m and the number of people affected in the first ASP event, Canadian authorities mobilized a substantial scientific team to ascertain whether the cause of the intoxication was anthropogenic or natural. They discovered that domoic acid (DA) (Figure3 , a known neuroexcitatory toxin, was the cause of the poisonings and had been accumulated in the shellfish from a diatom Pseurlo-t/it:schia (previously Nit:sc.hitr) ~nrrrget~s forma rtrultiseries ( 13,166). DA derives its name from the first organChotlclricr nrmtrttr, locally known ism in which it was found, the macroscopic red alga, as domoi (163). It was originally purified as the antihelminth in a long-used traditional medicine (163). This use, coupledwith the observation that approximately 50,000 people ingested DA-contaminated shellfish in Canada in 1987, giving a morbidity rateof approximately 0.4% and a mortality rate of 0.006%, indicates that DA is not very toxic compared to other shellfish toxins. Other DA-producing diatoms include Pseuno-rritcsc,hia seritrtcr, P. rtrultiseries, P. trustrnlis, P. psc.uek)cleliccrtis.sirtItr, P.clPlic.trti.ssitnci, and P. tur&ldcr (167-17 I). Of concern is an outbreak of DA poisoning in seabirds in California in 1991, where the vector was not shellfish but anchovies ( I7 1 ), a widely eaten organism not normally subjected to toxicity monitoring. Thus a different pathway exists that may lead to an ASP-like outbreak. Domoic acid (C,,H:,NO,,; molecular weight 31 I ) is a tricarboxylic acid that differs very little from the glutamate receptor agonist kainic acid, both being cyclized analogues
2 C O O H
'PCOOH
A
B
C
D
E
F
Fig. 5 Domoicacid ( A ) andthevariousanaloguesknown to date withregions o f isomerism depicted with the circular ;~rrows ( 162- 165). Note that not a l l of these compounds occur i n shellfish but arc specific to thc macroscopic red algae ( 164). Kainic acid, the ncurotoxin that defines the subset o f glutanmtc receptors activated by domoic acid and its analogues, is depicted in F.
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of L-glutamate (Figure 5 ) . DA is only weakly toxic to mammals, having an LDS,)in mice (intraperitoneally) of 3.6 mg/kg (172).It is a polar molecule, insolublein organic solvents, but soluble in water (-8 g/L) and slightly soluble in methanol (173). This polarity arises from three carboxylic acids and an amino group in the pentameric ring, which have pK,s of 2.1, 3.7, 5.0, and 9.8, respectively (163,174). The ionic state of DA affects its overall toxicity, with intraperitoneal DA injection into mice in an acidic vehicle significantly less toxic than at physiological pH( 175).
B. Molecular Target of Domoic Acid Domoic acid was recognized many years ago as a potent amino acid neuroexcitant (176), mainly through its structural similarity to kainic acid, a compound used to delineate a subclass of ionotropic glutamate receptors (IgluRs). IgluRs are ligand gated, neuronal ion channels, which respond to L-glutamate by opening and allowing the passage of cations. IgluRs trigger many intracellular cascades either directly by their introduction of cations or indirectly by the second messengers produced by these cascades, such as cyclic AMP or reactive oxygen species (177,178). IgluRs have been subdivided depending upon their sensitivity to three compounds: NMDA, AMPA, and kainate. It is primarily the kainate IgluRs that are sensitive to DA, although there is some evidence that AMPA IgluRs may also respond to the toxin (179). From the amino acid sequences of IgluRs cloned to date, hydrophobicity plots predict there are four transmembrane regions with both the carboxy and amino tails of the subunits being intracellular. The molecular diversity of the IgluRs arises not just from their differences in amino acid sequences, but also from the fact that they are made up of five, sometimes different, subunits which combine to surround the central ion-conducting pore (180,181). It is possible that domoic acid may affect other kainate-sensitive glutamate systems. For example, kainateis a nontransported competitive inhibitorof an excitatory amino acid transporter, EAAT2 ( 1 82). SinceDA can access the kainate binding site in kainate IgluRs, it is possible that it may also inhibit this enzyme.
C. Domoic Acid Uptake by the Gut The hydrophilicity and ionic properties of DA make it difficult for it to cross cell membranes in the gut epithelium. Evidence for the poor uptake efficiencyof DA by the human gut is its low morbidity rate. In fact, orally administered DA in both rats and mice was virtually completely eliminated in the feces and none of it made it to the urine (172). Since it possesses both basic and acidic pK,s, it can exist in charged states in both the stomach and intestines. For instance, in the stomach, a variety of charge forms from deprotonation of the various carboxylic acids can exist, whereas in the small intestine, all 3carboxylics will be charged while a pool of DA also bearing a positive charge on the imino will develop. The most likely route then for DA uptake is paracellular diffusion, an inefficient and size-selective method.
D. Toxin Distribution and Elimination Despite DA’s poor ability to cross the gut epithelium, it obviously can enter the human bloodstream and elicit drastic effects in ASP victims. It does not have much time to do this though, as it has been shown in monkeys that IV DA disappears from the blood very
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quickly, with 60-90% of the injected dose appearingin the urine within 6 hours of injection (183). Once DA has successfully entered the bloodstream it returns to physiological pH and all of the carboxylic acids will be charged, significantly hinderingit from crossing the blood-brain barrier, althoughnot absolutely (160). However, someof the human brain lacks the blood-brain barrier (184), exposing it to absorbed DA. After attacking these brain regions there may be a flow-on effect, whereby trauma in these exposed regions of the brain triggers excessive glutamate release within the brain and causes general excitotoxicity.Kainate-receptoractivationleads to activation of other IgluRs, with NMDAreceptor activation being the major cause of cellular death in cultured cerebellar granule neurons (185). DA that does enter the blood is primarily eliminated from mammals in their urine ( 1 86). Injection of radiolabeled DA into rats resulted in virtually the entire toxin dose being urinated within several hours of injection. Little metabolism of DA occurs during its time in the body because, in this same study, all of the radiolabel in the urine was associated with the parent domoic acid.
E. Pathology Kainate IgluRs are a prominent receptor family in the human brain, and since the bloodbrain barrier does not enclose all of the human brain,it is vulnerable to DA attack resulting in lesions and other neuropathologies (161,184,187). In mice, high doses were necessary to inflict observable damage upon these unprotected regions of the brain, the so-called circumventricular region. Lesions were generally localized but did extend a short way into neighboring regions of the brain ( 1 84), which may reflect the flow-on effect of DA neurotoxicity discussed above.Some animal models may be more susceptible to this effect. For instance, rats receiving an intraperitoneal injection of DA suffered significant lesions in most areas of the central nervous system (CNS), including cerebrum, cortex, hippocam( 1 88). As alluded to pus, hypothalamus, olfactory system and septum, and even the eye above, small amounts of DA can permeate the blood-brain barrier, albeit very slowly, and when it does, it can inflict additional neurological lesions apart from the circumventricular region (160).
F. Treatmentsfor ASP Symptomatic treatment and life support are the only recommended treatment. There are clues in some studies, however, of medicinal strategies that may be employed. The most direct strategy is the use of a kainate IgluR antagonist, CNQX, which ameliorated DA’s action on retinal tissue (1 89). And because kainate receptor activation by DA can elicit neuronal damage by their activation of the NMDA class of IgluRs, it is not surprising that the competitive NMDA receptor antagonist, D( -)-2-amino-5-phosphonopentanoic acid, and non-NMDA receptor antagonist, NBQX, greatly reduced domoate toxicity by almost 80% (185). Dextromethorphan is also another NMDA IgluR receptor antagonist that showed promise in mice as a DA antidote (190). Alternative pharmaceutical strategies may arise from inhibition of systems peripheral to the IgluRs. Pretreatment of rats with diazepam, a benzodiazepine sedative and anesthetic, can reduce DA-induced convulsions in rats at 5 mg/kg but achieved little else at this dose (191). Its efficacy if administered after DA intoxication was not explored. Kynurenic acid, a tryptophan metabolite found in the mammalian brain, protected mice
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from DA toxicity for several hours, even when administered some time after the onset of motor seizures (192). Gastric lesions, which appear after DA administration in mice, were also significantly reduced by kynurenic acid (193). Administration of tryptophan and the organic acid transport blocker, probenecid, augmented the protective effects of kynurenic acid (190). Activation of the 5-HT1A subclass of serotonin receptors in rat hippocampi by the drug 8-hydroxy-dipropylaninotetralinnegated most of the effects of DA that was injected directly into the hippocampus (194).
VI. MINOR SHELLFISH TOXINS Several peptidic hepatotoxins, previously considered to occur only in freshwater microalgae. have now been found in marine shellfish and phytoplankton. They have not, as yet, been implicated in any shellfish poisoning, but we should be wary of their potential to causehumanintoxication.Also, the long-knownmarine toxintetrodotoxin,which has caused human intoxications from gastropod nlollusc ingestion rather than bivalve molluscs, has been found in a dinoflagellate known to be a source of PSTs. This raises the possibility that i t may toxify bivalve shellfish. In this section, these two toxins will be briefly summrtl-ized.
A.
Cyclized, Peptidic Hepatotoxins
Microcystins are cyclic heptapeptides from species
of thefreshwaterblue-grcenalgae
Microcystis (Figure 6), long known as hepatotoxins (197). The amino acid composition of the microcystin may vary, but the presence of the novel hydrophobic amino acid, 3amino-9-methoxy- IO-phenyl-2,6,8 trimethyl deca, 4,6 dienoic acid (ADDA)is essential to its bioactivity. Like okadaic acid, microcystins are potent inhibitors of the serine/threonine protein phosphatases, binding to the same site on the enzymes as OA (198j, and also act as tunlorpromoters (199). Althoughacutehumanintoxication bythesepeptidesafter eating marine shellfish has not been reported, they have been detected in the blue mussel
Me 0
Fig. 6 The structure of the cyclic heptapcptide toxin. microcystin (195,196). Thc side chain of the essential and unique amino acid. ADDA is to thc left of the molecule.
95
Shellfish Chemical Poisoning
(Mytilus edulis) (200), a shellfish responsible to date for ASP, DSP, and PSP (Table l), and other commercial marine bivalves (201). We must be wary of their presence in the marine environment because chronic exposure to subacute doses of microcystins may impact upon human health, especially their potential role in cancer development.
B. Tetrodotoxin Tetrodotoxin (TTX) (Figure 7), is the famous pufferfish poison, highly prized in the fugu tradition in Japan. Human intoxication and death has occurred after consuming mollusks as in all of infested with this toxin. although the mollusks involved were not bivalves, the shellfish poisoning syndromes described above, but were gastropods. Seventeen Taiwanese in 1994 were poisoned after eating samples of Nrrs.sari~r.scustus and N. conoiddis, with one elderly victim dying, although most were probably from complicationsthat arose after the initial effects of the poisoning had abated (205). TTX competes with a comparable affinity for the same binding site on the VSSC as the PSTs, acting in the same manner as PSTs. Despite these similarities in their molecular pharmto elicit the same effects acology, TTX is structurally dissimilar to the PSTs (Figure 7), but like the PSTs, they possess a guanidino group (PK,~8.8) essential to its toxicity (202-204). The possibility of TTX intoxication from bivalve shellfish is of concern because of a recent report that thedinoflagellate Alexmdriurn n l i m t w r l , a dinoflagellateknown to havecausedPSP intoxicationviabivalveshellfish,mayalsoproduce TTX(206).Thisdiscoverywas made due to the fact that toxic individuals of the commercial edible scallop, Patirzopecten yessoerlsis, contained TTX along with PSTs. Thus TTX may abecause of PSP and analysis of culprit samples is necessary to confirm that PSTs and not TTX were the causative toxin.
VII.
CO-OCCURRENCE OF TOXINS
From Table 1, it can be seen that some shellfish, such as Mytilus edulis, are responsible for more than one type of shellfish poisoning. The conditions that initiate and maintain a bloom for one toxic microalgae may be just as suitable for other toxic species and so a bloom may contain several speciesof toxin-producing organisms. Also, slow detoxification of one toxin from a shellfish may overlap with a bloom of another toxic species, adding to the shellfish’s toxin load. Thus it is possible that people may experience more than one type of intoxication, making the diagnosis of such an event far more difficult
pK, = 8.8 Fig. 7 The neurotoxin,tetrodotoxin (202-204)
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and the treatment even more problematic. In fact, both diarrhetic and paralytic shellfish toxins have co-occurred in mussels from Spanish waters (24). A potential complication of the co-occurrence of toxins is the effect by okadaic acid on paracellular diffusion. As described above, this process is the means by which molecules traverse the gut epithelia through the tight junction barrier, allowing small hydrophilic molecules not amenable to passing through cellular barriers to enter the human system. Okadaic acid has been found to increase this permeability ( 1 28) which may not only affect its own uptake, but also of hydrophilic toxins such as the PSTs or domoic acid with which it may co-occur.
VIII. SHELLFISH RESPONSE TO THE TOXINS Many of the factors that govern the ability of a toxin to be taken up across the human gut also influence the ability of shellfish to accumulate the toxin. Domoic acid, which is poorly taken up across the gut epithelium, is also very poorly accumulated by shellfish (207). Lipophilic compounds like the brevetoxins, however, which are retained in the fat tissue of mammals for long periods (151), may be expectedto be accumulated effectively and retained by shellfish for long periods after a toxic algal bloom. This is indeed the case, with detectible levels of brevetoxin remaining in shellfish some months after the toxic dinoflagellate Prychodiscus brevis had disappeared (208). Poisonous shellfish do detoxify when no longer exposed to the intoxicating organism, with the rateof detoxification being species dependent and influenced by salinity, water temperature, and the size of the shellfish (207,209-21 1). Different life stages of toxifying microalgae can produce differentamounts oftoxin.Gametes,zygotes,and thefirstfew stages of vegetatively growing cellsof the DA-producing diatomP.seud~-tzit,-.schie/ purrpws f. rwltiser'ies do not produce domoic acid (212). The age structure o f a toxic algae bloom influences the amount of toxin a shellfish is exposed to. What effect then. do these toxins have on the shellfish themselves. By definition, shellfish that accumulate these toxins must be resistant to some degree, as the compounds often reside in the animals' tissue for long periods of time. Shellfish do possess the receptors that are inhibited by some of these toxins. The mollusk Aplysicr is known to have kainate-sensitive glutamate receptors (213) and a VSSC (77). Resistance may arise from the possession of insensitive isoforms of the enzymes and receptors targetedby the toxins. As has been discussed, simple mutations of the VSSC can result in much reduced PST sensitivity, and a naturally occurring mutation may underlie the apparent resistance of shellfish nerves to PSTs (2,3). Similarly, a single point mutation in a serine-threonine protein phosphatase can cause a 50-fold decrease in the sensitivity of this enzyme to OA (214). The toxin sensitivity of these receptors i n toxin-accumulating species of shellfish has yet to be elucidated. Alternatively, physical separation of the toxin away from its active site, such as in the digestive gland, may confer resistance. Depuration rates of particular toxins are complicated by their metabolic conversion within the shellfish which may modify their chemistry. Incubation of sulfated PSTs with homogenates of different tissues of the scallop (Plmqm~retrnrrrRe1lcrnicv.s) and littleneck clam (Prororhcrca stcrmitwa) desulfated the toxins, converting them to the most potent PST, saxitoxin (215,216). This transformation would remove a negative charge from the molecule, changing its overall charge state and therefore its pharmacokinetics in the shellfish.
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IX. EFFECT ON TOXINS BY SHELLFISH PREPARATIONS The means of preparing shellfish may impact on the amount and state of the toxins that are ingested by potential victims. Raw shellfish, which are commonly consumed, would obviously bethe most dangerous way to eat any toxic shellfish. Some of the shellfish toxins are heat stable and water soluble, such as the PSTs and domoic acid. One would expect therefore that boiling of shellfish in water, as in the making of soup, would extract the toxin. Although this possibility has not been studied with shellfish, anecdotal evidence comesfromJapanesecaseswhereparticularcrabswhichcanaccumulatePSTswere cooked as part of miso soup and consumersof the broth died from PSP (217). In contrast, research with the hepatopancreas of the lobster (Hotntrrus arner-iccrnus), which can accumulate PSPs, showed that boiling removed very little of the PSTs present in the samples (218). Steaming, however, which caused a loss of tissue water, did significantly reduce the toxicity. It may be that the toxin is biochemically anchored in the tissue and not easily extracted by cooking, withthis anchorbeingpresentonly in somespecies. A similar scenario can be raised with lipophilic shellfish toxins and their possible extraction from shellfish tissues into cooking oils.
X.
CONCLUDING REMARKS
It must be remembered that the toxins described herin are naturally occuring compounds, unlikely to have evolved for the purpose of killing humans. Rather,they have some natural function, or are the physiological resultof the organism’s metabolism,not yet fully understood. Their occurrencc in nature is probably underestimated, only being detected after our attention has been attracted by some event such as a human intoxication. efficacy Individual variation in the healthof shellfish consumers can impact upon the of shellfish toxins in humans. For instance, people who suffer stomach and intestinal ailments that affect gut pH may experience different pharmacokinetics with regard to the toxins, possibly improving the efficiency of toxin uptake. Medication given to counteract these ailments, such as drugs to inhibit acid production, could have the opposite effect. Also, the existenceof an ulcer may provide a direct method for toxin entry into the bloodstream for toxins such as domoic acid which are taken up poorly across the gut wall. The occurrence of shellfish poisoning may also be lower than the reality. Mild poias sonings may notbe recognized by victims as poisonings, but rather self-diagnosed generic food poisoning or as an allergic response. The general community is often more aware of these two ailments than shellfish poisoning, and unless they present themselves to a shellfish toxin-aware health professional. the intoxication may never be identified. The adage thatthe world is getting smaller due to modern technology is as true with the food we eat as with anything else. Shellfish have increasingly become a staple of the global diet. Toxins transmitted from the environmentto the consumer are a danger faced today by many people in many countries, not just those which traditionally have a seafood diet. A shellfish poisoning eventnot only affects the victims and their loved ones, but can have an economic impact due to closure of the shellfish industry and a flow-on effect to the whole seafood industry due to reduced consumer confidence in general seafood safety. Steps have been taken in many countries to ensure that their shellfish are safe. These include monitoring of water supplies for harmful microorganisms and direct
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toxicity testing of selected shellfish. These monitoring studies have upper limits that are usually far below the real acute toxic dose, but little is known about the effects of chronic consumption of some of thesetoxins.Increasedsensitivity of analyticalmethodsand expansion of surveys to other members of the marine and aquatic environment may extend our knowledge of the actual taxonomic distribution of these toxins and fully measure how exposed humans are to these compounds. In due course, it will also reveal the role the toxins play in the physiology and ecology of the organisms.
ACKNOWLEDGMENTS This is contribution number 987 of the Australian Institute of Marine Science.
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165.LZaman, 0 Arakawa,A Shimosu, Y Onoue, S Nishio, Y Shida, TNoguchi. Two new isomers of domoic acid from a red alga, Clzmdrier arrncctcc. Toxicon 35:205-212, 1997. 166. SS Bates,JCBird, ASWDeFreitas,RFoxall,MGilgan, LA Hanic,GRJohnson, AW McCulloch, P Odense, R Pocklington, MA Quilliam, PG Sim, JC Smith. DV Subba Rao, Nir:.sc/ricr purlgerls as the primary source ECD Todd, JA Walter, JLC Wright. Pennate diatom of domoic acid, a toxin in shellfish from eastern Prince Edward Island, Canada. Can J Fish Aqaut Sci 46:1203-1221, 1989. 167. DV Subba-Rao, MA Quilliam, R Pocklington. Dornoic acid-a neurotoxic amino acid produced by the marine diatom Nit3chirr p~cngc’nsin culture. Can J Fish Aquat Sci 45:20762077.1988. 168. DL Garrison, SM Conrad, PP Eilers, EM Waldron. Confirmation of domoic acid production by Pseudonitz.shia ctusfr.cr/is (Bacillariophyceae) cultures. J Phycol 28:604-607, 1992. 169.MCVillac, DL Roelke, FP Chavez, LA Cifuentes. CA Fryxell. P.srrr~lor~ir~sc~hin trrrstrcrlis Frenguelli and related species from the west coast of the USA: occurrence and domoic acid production. J Shellfish Res 12:457-465, 1993. Nitzschicr psercr/oc/e~/ic~rrtissir,,N-asource of 170. JL Martin, K Haya, LE Burridge, DJ Wildish. domoic acid in the Bay of Fundy, eastern Canada. Mar Ecol Prog Scr 67:177-182, 1990. 171. L Fritz, MA Quilliam, JLC Wright. An outbreak of domoic acid poisoning attributed to the uustr.cr1i.s. J Phycol 28:439-442, 1992. pennate diatom P.seLrd~)~lir~.s~/liu 172.FIverson.JTruelove,ENera,LTryphonas,JCampbell,ELok.Domoicacidpoisoning and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract. Food Chem Toxicol 27:377-384. 1989. 173. M Falk, PF Seto, JA Walter. Solubility of domoic acid in water and in non-aqueous solvents. Can J Chem 69: 1740- 1744, 1991. of domoicacid.Can J Chem67: 174.MFalk,JAWalter,PWWiseman.Ultravioletspectrum 1421-1425,1989. 175. MS Nijjar, MS Madhyastha. Effect of pH on domoic acid toxicity in mice. Mol Cell Biochem 167:179-185,1997. 176.TJBiscoe,RHEvans, PM Headley,MMartin.JCWatkins.Domoicandquisqualicacids 255: 166-167, aspotentaminoacidexcitantsoffrogandratspinalneurones.Nature 1975. 177. EK Michaelis. Molecular biology of glutamate receptors inthe central nervous systeln and their role in excitotoxicity, oxidative stress and aging. Prog Neurobiol 54:369-415. 1998. 178. S Ozawa, H Kamiya, K Tsuzunki.Glutamatereceptorsinthemammalianccntralnervous sytem. Prog Neurobiol 54581-618, 1998. 179. JA Larm, PM Beart, NS Cheung. Neurotoxin domoic acid produces cytotoxicity via kainateand AMPA-sensitive receptors in cultured cortical neurones. NeurochemIn1 3 1 :677-682, 1997. 180. WWisden,PH Seeburg. Mammalian ionotropic glutamate receptors. Curr Opin Neurobiol 3:291-298,1993. 18 1. S Nakanishi. Molecular diversity of glutamate receptors and impkations for brain function. Science258:597-603,1992. 182. RJ Vandenberg, JL Arriza, SG Amwa, MP Kavanaugh. Constitutive ion fuxes and subslrate 1. 1995. binding domains of human glutamate transporters. J Biol Chem 270:17668-1777 183. J Truelove, F Ivcrson. Serum domoic acid clearance and clinical observations in the CynOmOlgus monkey and Sprague-Dawley rat following a single i.v. dose. Bull Environ Contaln Taxicol 52:479-486, 1994. 184. JE Bruni,RBose,CPinsky.GBGlavin.Circumventricularorgalloriginofdomoicacidinduced neuropathology and toxicology. Brain Res Bull 26:419-424, 1091. 185. FW Berman, TF Murray. Domoic acid neurotoxicity in cultured cerebellar granule neurons is mediated predominantly by NMDA receptors that are activated as a consequence of exitatory amino acid release. J Neurochem 69:693-703, 1997.
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~ ,Hierlihy. Renal clearance of domoic acid in the rat. Food Chcm Toxicol 10: 186. CA S U Z USL 701-706. 1993. 187. L Tryp[lollas, F [verson. Neuropkithologyof excitatory neurotoxins: the domoic acid model. Toxicol Pathol 18: 165- 169. 1990. 188. L Tryphonas. J Truelove, F Iverson. Acute parenteral neurotoxicity of domoic acid i n c p o 1990. lnolgus monkeys. Toxicol Pathol 18:297-303. to excitatory amino acidsand excite189. CD Zcevalk, WJ Nicklas. Nitric oxide in retina: relation toxicity. Exp Eye Res 58:343-350, 1994. 190. R Base, C Pinsky, GB Glavin.Sensitive murine model and putative antidotes for behavioural toxicosis from contaminated mussel extracts. Can Dis Weekly Rep 16:91-98. 1990. 191. S NakajiIna, JL Potvin. Neural and behavioural effects of domoic acid, an amnesic shellfish toxin, i n the rat. Can J Psycho1 46:569-581, 1992. 192. C Pinsky, GB Glavin, R Bosc. Kynurenic acid protects against neurotoxicity and lethality of toxicextractsfromcontaminatedAtlanticcoastmussels.ProgNeuropsychophannacol BiolPsychiatry 13595-598, 1989. 193. GB Glavin, R Base, C Pinsky. Kynurenic acid protects against gastroduodenal ulcerationi n mice injected with extracts from poisonous Atlantic shellfish. Prog Neut.opsychopharmaco1 BiolPsychiatry 13569-572, 1989. 194. SK Shartna, K Dakshinamurti.Suppressionofdomoicacidinducedseizuresby%(OH)DPAT. J Neural Transm 93:87-98, 1993. 195. DP Boles, AA Tuinman, PL Wessels, CC Viljoen, H Kruger, DH Williams, S Santikarn, RJ Smith, SJ Hammond. The structure of cyanoginosin-LA, a cyclic heptapeptide toxin from the cyanobacterium Micrmysris nerugirtosa. J Chcm Soc Perkin Trans 1:231 1-2318, 1984. S Santikarn, RJ Smith. JCJ Bru-na, DH 196. DP Botes, PL Wesscls, H Kruger, MTC Runnegar. Williams. Structural studies on cyanoginosins-LR,-YR, -YA, and -YM, peptide toxins from cyanobacterium M i c w c w i s ~ e r t c g i ? ~J .Chem ~ ~ . Soc Perkin Trans 1 :2747-2748, 1985. 197. WW Carmichael. Thc toxins of cyanobacteria. Sci Am 270:78-86, 1994. 198. C MacKintosh, KA Bcattie, S Klumpp, P Cohen, CA Codd. Cyanobacterial rnicrocystirl-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both manmnls and higher plants. FEBS Lett 264: 187-192, 1990. 199. R Nishiwaki-Mntsushima, S Nishiwaki, T Ohta, S Yoshizawn, M Suganuma, K Harada, MF Watanabe, H Fujiki. Structure-function relationships of microcystins, liver tumor promoters. in interaction with protein phosphatase. Jpn J Cancer Res 82:993-996, 1991. 200. DE Williams, SC Dawe. ML Kent, RJ Andersen, M Craig, CFB Holmes. Bioaccumulation and clearance of microcystins from salt water mussels, Mytilus ed~tlis,and in vivo evidence for covalently bound microcystins in mussel tissues. Toxicon 35: 1617-1625, 1997. RJ Andersen, CF Holmes. Identification 201. DZX Chen, MP Boland, MA Smillie, H Klix, C Ptak, of protein phosphatase inhibitorsofthe microcystin class in the marine environment. Toxicon 31:1407-1414, 1993. 202. K Tsuda, S Ikutna, M Kawamura, R Tachikawa, K Sakai. Tetrodotoxin. VII. On the structure of tetrodotoxin and its derivatives. Chem Pharrn Bull 12: 1357-1 374, 1964. 203 T Goto, Y Kishi, S Takahashi. Y Hiratn. Tetrodotoxin. Tetrahedron 21:2059-2088, 1965. 204 RB Woodward. The structure of tetrodotoxin. Pure Appl Chem 9:49-74, 1964. 205. C Yang, K Han, T Lin, W Tsai, J Dcng. An outbreak of tetrodotoxin poisoning following gastropod mollusc consumption. Hum Exp Toxicol 14446-450, 1995. 206. M Kodama,S Sato, S Sakamoto, T Ogata. Occurrenceof tetrodotoxin in Alesnndrilrnr fctr)tNrerrse, a causativedinoflagellateofparalyticshellfishpoisoning.Toxicon34:1101-1105, 1996. 207. GD Wohlgeschaffen, KH Mann, DV Subba Rao, R Pocklington. Dynamics of the phycotoxin dolnoic acid: accumulation and excretion i n two comnercially important bivalves, J Appl Phycol 4297-310, 1992. 208. H Ishida, N Muralnatsu, T Kosuge, K Tsuji. Studyof neurotoxic shellfish poisoning involving
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210. 21 1.
212.
213. 214. 215. 216. 217. 218.
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New Zealand shellfish,CrcrssosrrecI gigrts. In: T Yasumoto, Y Oshima,Y Fukuyo, eds. Harmful and Toxic Algal Blooms. Paris: UNESCO, 1996, pp 491-494. J Blanco, A Morono, J Franco, MI Reyero. PSP detoxification kinetics in the mussel Mytilus gefllul”nvirlcicr1is. One- and two-compartment models and the effect of some environmental variables. Mar Ecol Prog Ser 158:165-175, 1997. W Silvert, DV Subba-Rao. Dynamic model of the flux of domoicacid, a neurotoxin, through a Mxtilus edulis population. Can J Fish Aquat Sci 49:400-405. 1992. W Silvert, AD Cembella. Dynamic modelling of phycotoxin kinetics in the blue mussel, Mytilus rdulis, with implications for other marine invertebrates. Can J Fish Aquat Sci 52: 521-531,1995. DV Subba Rao, ASW De Freitas, MA Quilliam, R Pocklington, SS Bates. Rates of production of domoic acid, a neurotoxin amino acid in the pennate marine diatom Nir:.schier purtgerls. In: E Graneli, B Sundstrom.L Edler, DM Anderson, eds. Toxic Marine Phytoplankton. New York: Elsevier, 1990, pp 413-417. LE Trudeau, VF Castellucci. Excitatory amino acid neurotransmission at sensory-motor and interneuronal synapses of AplysicI ccrlifornica. J Neurophysiol 70: 1221- 1230, 1993. L Zhang, Z Zhang, F Long, EYC Lee. Tyrosine-272 is involved in the inhibition of protein 1, 1996. phosphatase-l by multiple toxins. Biochemistry 35: 1606-161 in YShimizu,MYoshioka.Transformationofparalyticshellfishtoxinsasdemonstrated scallop homogenates. Science 21 2547-549, 1981. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck Biochem Biophys Res Commun 114:465-472, 1983. clam (Prorothncrr srcu~rir~ecr). Y Hashimoto. Marine toxins and other bioactive marine metabolites. Tokyo: Japan Scientific Societies Press, 1979, pp 53-55. JF Lawrence, M Maher, W Watson-Wright. Effectof cooking on the concentrationof toxins associated with paralytic shellfish poisonin lobster hepatopancreas. Toxicon 32:57-64, 1994.
5 Pathogens Transmitted by Seafood"
I. Introduction 109 11. BacterialandViralPathogensAssociatedwithRawandUnderprocessedShellfish
121
Bacterial and Viral Pathogens Primarily Associated with Improper Processing or Handling of Seafood 159 of Seafood-BornePathogens 164 IV. Development ofRapidMethodsforDetcction 165 V. ConclusionsandRecommendations VI. Additional Sources of Information: World Wide Web Sites for Pathogens Associated with Seafoods 17 1 References 172 111.
1. A.
INTRODUCTION Overview of Seafood-Borne Disease
Risks and hazards are associated with all forms of human activities. While humans may choose to minimize or eliminate some risks by changing or eliminating a certain type of behavior or activity, all humans must consume food and water to live. In some regions of the world, the consumptionof food and watercan be, unfortunately, routinely associated with exposure to pathogenic organisms. Inhabitants of developed countries generally assume that their supply of food and water is safe, and government regulatory and public health agencies are charged with the task of ensuring the safety of these items. In many regions, however, infectious disease is a wayof life, responsible for the deathsof millions of people each year in developing countries. Part of this infectious disease is associated with the transmission of pathogenic microorganisms in the food and water that is consumed. The World Health Organization( I ) reported that in 1997, of a global total of 52.2 million deaths, 17.3 million or one-third were due to infectious and parasitic diseases.
* This chapter is dedicated to the educational and scientific contributions of John Liston and Jack R. Matches, two former faculty members and microbiologists in the School of Fisheries, University of Washington. 109
110
Hetwig Other and unknown causes 9%
Infectious and parasitic diseases 43%
9%
Perinatal and maternal causes 10%
Fig. 1 Major causes of death in developing countries (1).
The leading causes of death from infectious diseases were acute lower respiratory infections (3.7 million), tuberculosis (2.9 million), diarrhea (2.5 million), H I V / A T D S (2.3 million), and malaria (1.5-2.7 million). Diarrheal and parasitic diseases may be associated with the consumption of contaminated water and food. Major differences in the causes of human death exist between developing and developed countries (Figs. 1 and 2). Infectious and parasitic diseases cause43% of the deaths in developing countries and approximately 1% of the deaths in developed countries. An excellent choice of food for protein and other essential nutrients is seafood. In many regions of the world seafood and other aquatic products are a major portion of the diet, while in other regions of the world seafood is viewed as a delicacy or something
Diseases of the circulatory system 46% Fig. 2 Major causes of deathin developed countries (1).
Pathogens Transmitted by Seafood
111
unusual. Besides supporting the healthy livesof millions of people, seafood is also capable of pathogenic of supporting the growth of or acting as a vehicle for the transmission organisms. The principalbiologicalagentsthat cause seafood-borne disease are bacteria, viruses, and parasites. The major human diseases caused by fish-borne parasites are trematodiasis, cestodiasis, and nematodiasis. Worldwide, but primarily in developing countries, trematodes are the most important food safety hazard linked to fish and fishery products. The trematodes infecting the greatest number of people are species of the genera Clonorchis, Opisthorchis, and Parvrgonirnus (2). The World Health Organization (2) estimates that tens of millions of people are affectedby fish-borne trematodes. Most of these people live in developing countries. Parasites area minor problem for seafood consumersin most developed countries. The focus of this chapter is on the bacteria and viruses associated with aquatic foods that are pathogenic to humans, although the reader should understand that parasitic infections are themajor problem associated with the consumption of seafood in many regions of the world. Bivalve mollusks present a much greater risk of infecting humans with bacterial and viral pathogens than do crustaceansandfinfish.Thegreatestnumberofseafoodassociated disease outbreaks and cases in developed countries are causedby the consumption of raw or insufficiently cooked bivalve mollusks that were harvested from waters contaminated with human sewage or waters wherea fraction of the indigenous microflora are pathogenic to humans. These two types of contamination are associated with enteric bacteria and viruses (Escherichiu coli, Salmonella, hepatitis A, Norwalk-like viruses) and bacteria naturally found in surface water and sediments (Vibrio vuln$cu.s, V. pcrrclhaetnolyticu.7, V. cholerae, Aercmo11c1.s hydrophila, Clostridiutn Dotdinurn). Another group of pathogenic microorganisms (suchas Stclphvlococcus aureus) are primarily introduced into fishery products by humans who handle or process seafoods after harvesting.
B. Consumption of Seafood and the Rise in Aquaculture 1. Increasing Consumption of Seafood In recent years the supply of fish has continued to increase steadily and in 1995 the total world production of finfish, crustaceans, and mollusks from capture fisheries and aquaculture reached 1 12.9 million metric tons. Much of the increase in annual aquatic production is attributable to aquaculture. For cultured finfish and shellfish, the annual contribution to total finfish and shellfish rose from 11.7% in 1989 to 18.5% i n 1995. For food fish, more than one-quarter of the total world supply was derived from aquaculture. Aquaculture is one of the fastest growing food-producing sectors, providing an acceptable supplement and substitute for wild fish and plants. The relative importance and role of aquaculture in different countries varies widely. I n 1995, for example, more than 60% of the total aquatic production in China came from aquaculture. This was nearly twice that seen in France, India, Republic of Korea, and the Philippines. A considerably lower contribution was reported in Thailand (13%), Norway (9%). and the United States (7%) (3).
2. The Safety of Aquaculture Products There are increasing concerns related to the safetyof food products from aquaculture and studies are now focusing on these concerns. Are there greater hazards associated with aquaculture products versus wild-caught products? Are different pathogens associated with
Herwig
112
Table 1 Prevalence of Snlrrrorlrllrr i n AquaculturalSpecies,’
Country Product Tilapia Catfish Catfish Eel
Prawns
Reference % Positive Africa South U.S.
us. Japan Philippines
55 3 21 26
187 188 1x9 190
16
191
aquacultural products? The greatest amountof aquaculture is done i n developing countries i n tropical and subtropical regions of the world, yet most of the scientific literature and research related to seafood safety has been performed i n “Western” countries with seafood captured or raised in temperate waters. Historically, cultured fish have not been considered important vectors of human pathogens. This situation may be changing, partly due to increasing animal densities as a consequence of a rapidly growing industry and partly due to increasing awareness by health care providers of pathogens in aquatic species that may result in human illness (4). Not surprisingly, bacteria that are known to be pathogenic to humaw have been isolated from locations where aquaculture products are produced and from products that have been sent to the marketplace. Table 1 lists the results from several studiesas summarized by D’ Aoust (5) that indicate the prevalence of Srrlnlowello i n different aquacultural species, including tilapia, catfish, eel, and prawns. A more recent review (6) summarized the association of pathogenic bacteria with cultured fish (Table 2). Although evidence has been published showing the presence of a variety of pathogenic organisms in culturedfishandshellfish,whetherthere is a proportionately
Striped bass hybrid
Pathogens Transmitted by Seafood
113
larger number of cases or outbreaks of seafood-borne disease associated with cultured animals has not been established. More research is required in this emerging sector of seafood production.
C. Cases and Outbreaks: Statistics and Government Agencies to Oversee Seafood Safety 1. United States-Developingan Interest in Shellfish Sanitation and the Food Safety Initiative Many i n the public may perceive food safety issues as something completely new, but several federal, state, and local agencies have been concerned with these issues for many years. Regarding seafood safety, there is a lengthy history of government involvement i n the safety of shellfish. Most of the pathogenic bacteria and viruses associated with seafood-borne disease are associated with the consumption of bivalve mollusks. The early history of shellfish-borne disease and the establishment of shellfish sanitation regulations in the United States is summarized i n the manual of operations for the National Shellfish Sanitation Program (7). While the exact cause or microbiology of disease was not known at the time, public health controls on shellfish became a national concern in the United States in the late 19th and early 20th centuries. At that time, public health officials noted a large number of illnesses associated with the consumption of raw oysters, clams, and mussels. Shellfish-associated disease outbreaks were also recorded in Europe. During the winter of 1924-1925, widespread outbreaks of typhoid fever occurred in New York, Chicago, and Washington, D.C. These outbreaks were traced to oysters that had been contaminated with sewage. Local and state public health officials and the shellfish industry became so alarmed that they requested that the surgeon general of the United States Public Health Service develop the controls necessary to ensure a safe supply of shellfish. The surgeon general called a conference in 1925 of representatives from state and municipal health authorities, state conservation commissions, the Bureau of Chemistry [later to become the Food and Drug Administration (FDA)], the Bureau of Commercial Fisheries [now called the National Marine Fisheries Service (NMFS)], and the shellfish industry. TheNationalShellfishSanitationProgram(NSSP)developedfromtheprinciplesand shellfish controls formulated at this conference. To strengthen the mission of the NSSP. in 1982officialsfrom22statesformedtheInterstateShellfishSanitationConference (ISSC). The ISSC allows state regulatory officials to establish uniform guidelines and to exchange information about the sources of safe shellfish. The ISSC adopted the NSSP manual of operations and has established procedures that enable it to adopt changes i n the manual (7). The Centers for Disease Control and Prevention (CDC) is largely responsible for collecting and tabulating data related to prevention and control of disease, injury, and disability in the United States. Alerts and information about current foodborne disease outbreaks and cases are disseminated in a publicationcalled Mnrbidify and Morralip Weekly. The most recent publications that summarize the numbers and causes for foodborne and seafood-related disease outbreaks are the “Summary of Notifiable Diseases, United States 1997” (8) and “Surveillance for Foodborne-Disease Outbreaks-United States, 1988-1992” (9). Data related to shellfish-borne disease was previously collected by the FDA’s Northeast Technical Services Unit (NETSU). The NETSU data were used
114
HeMg
and cited in previous reviews (10,ll). While the NETSU considers reports of outbreaks (two or more persons who become ill after consumption of a common food) and individu cases, the CDC database usually includes only outbreaks. In addition, the CDC data are collected exclusively through the voluntary submission of outbreak forms from state public health departments. The NETSU data are considered more inclusive and precise than the CDC data for shellfish-borne disease (10). In 1994, Rippey(1 1) published a review that summarized the infectious diseases associated with molluscan shellfish consumption. The data presented were related to reports presented through 1990, and as thorough as he could be, Rippey admitted that his data represented only a small portion of the actual number of cases that occur annually because of the weakness of the reporting system and the nature of most foodborne disease. Unfortunately, data collection related to shellfish-born disease is no longer being performed by NETSU, and no other agency in the United States has formally begun to collect this data in a similar manner. The result of these weaknesses is that the data for the number of cases of seafood-borne illness in the United States are most likely poorer and less representative than that collected 10 years ago. With these caveats in mind, data reported by the CDC suggest that among the dif ent kinds of food that are tabulated, there are a significant number of foodborne-disease outbreaks (25%) and a smaller fractionof cases that are associated with seafood (Figs.3 and 4). For the purposes of this review, seafood is considered “shellfish” and “other fish” in the categories that are listed in the CDC data. The data suggest that seafood has fewer cases associated with each outbreak compared to other typesof foods. From 1988 to 1992, 6095 foodborne disease outbreaks representing 77,373 cases were reported to the CDC. Seventy-five deaths were reported to be caused by foodborne disease (9). The outbreaks reported include only a fraction of the cases of foodborne disease that occur each year. For the average of 15,500 cases and 14 deaths reported each year by the CDC’s surveillance system, there are an estimated 6 million cases that actually occur each year (12). The etiology was not determined for 59% of the reported outbreaks. Most of the outbreaks that had an unknown etiology had an incubation period of 15 hours or more, suggesting an infectious agent. Manyof these outbreaks may have been caused by viruses (9). The capability of testing serum for antibodies to foodborne viruses is not widely available.
6,095 outblreah
Fig. 3 Distribution of foodborne disease outbreaks in the United States, 1988-1992 (9).
115
Pathogens Transmitted by Seafood
Shellfish
Other Fish
1Yo
2%
Other Foods 97%
Fig. 4 Distribution of foodborne disease cases in the United
States, 1988-1992 (9).
Shellfish-borne and “otherfish” diseases represented 1% and 2%of the total number of cases, but 2% and 23% of the outbreaks, respectively. In general, seafood-borne disease outbreaks are characterized by a small number of cases. On the other hand, an “outbreak” represented by only a single case is not reported in the CDC data. The responsible pathogen is not identified in more than half of the foodborne disease outbreaks that are reported to the CDC. During this 5-year period there were no cases associated with “other fish” that were attributable to Vibrio cholerae, V. parahuemolyticus, and V. vulniJcus. Examining outbreaks that were associated with shellfish during this same period, these three pathogens together caused one to four outbreaks (9). In 1988, a multistate hepatitis A outbreak with 61 cases was caused by eating raw oysters (13). An outbreak of V. cholerae occurred in Guam, an island in the U.S. Pacific territory, was caused by eating contaminated reef fish. In this single outbreak, 26 people became ill and 1 died. In 1991, there were two outbreaks of V. chokrae caused by tainted food imported to the United States. One outbreak with two cases was attributed to crabs that were imported illegally from Ecuador (14). Most of the disease outbreaks and cases associated with seafood, as reported by the CDC (9), are caused by chemical agents such as scombrotoxin, ciguatoxin, and paralytic shellfish poisoning. These seafood-borne disease agents are discussed elsewhere in this book. During the past 3 years there has been growing public concern about the safety of the food supply in the United States. Much of this concern was ignited by outbreaks caused by the emerging pathogen E. coli 0157:H7. In May 1997, a report called “Food Safety from Farm to Table: National Food-Safety Initiative” was presented to President Clinton. This report presented an intergovernmental agency strategy to prevent foodborne disease and included plans to develop elements of an improved foodborne disease surveillance system. The first component of the new system is the Active Foodborne Disease Surveillance System, known as FoodNet, a collaborative effort between the CDC, FDA, and the U.S. Department of Agriculture (USDA), and selected counties in seven states (California, York, and Oregon)participating in Connecticut, Georgia, Maryland, Minnesota, New CDC’s Emerging Infections Program. FoodNet is designed to conduct population-based
116
Herwig
active surveillance of seven bacterial foodborne pathogens (Saln~onella, ShipAlcr, C m 7 p ~ lobacter, E. coli 0157:H7, Listeria, Yersirlia, and Vibrio),and to determinethe magnitude of diarrheal illnesses and the proportion of these illnesses that are attributable to foods. The population of these seven FoodNet sites i n 1997 was 20.3 million people (7.7% of the U.S. population). The objectives of FoodNet are to (a) describe the epidemiology of new and emerging bacterial, parasitic, and viral foodborne diseasesof national importance, (b)morepreciselydeterminethefrequency andseverity of foodbornediseases in the United States, and (c) determine the proportion of foodborne disease caused by eating specific foods. To addresstheseobjectives,FoodNetconductsactivesurveillanceand related studies: a population survey, a physician survey, and a case-control study of E. coli 0157:H7 infections ( 1 516). A positive outcome from the initiation of FoodNet is perhaps a more realistic estimate about the incidence of foodborne diarrheal disease in the United States. Data from FoodNet suggest that the incidence of diarrheal illness in the United States is about 1.4 episodes/person/year, or some 370 million episodes each year. If only 25% of diarrheal disease is food related, the burden of foodborne diarrheal disease in the United States far exceeds current estimates (16). In theUnited States, four federal agencies play major roles in carrying out food safety regulatory activities: the FDA, the Food Safety and Inspection Service (FSIS) of the USDA, the Environmental Protection Agency (EPA), and the NMFS. Seafood safety is under the jurisdiction of the FDA, EPA, and NMFS. The FDA has jurisdiction over domestic and imported seafoods thatare marketed in interstate commerce. The FDA’s Center for Food Safety and Applied Nutrition (CFSAN) seeks to ensure that seafoods are safe, sanitary, nutritious, wholesome, and honestly labeled. The CFSAN also has control over seafood processing plants. The EPA establishes tolerances for pesticide residues in seafoods, and is responsible for protecting against other environmental chemical and microbial contaminants in water that might threaten the safety of seafoods. The NMFS conducts a voluntary seafood inspection and grading program that is primarily a food quality activity (16). The seafood industry is in the early stages of transitioning to hazard analysis critical control point (HACCP) programs. It is generally accepted in the food science community that the use of HACCP programs i n all aspects of food production, processing, and distribution is proactive and an excellent approach toward food safety. In 1995, the FDA issued itsfinalruleon HACCP programs for seafood. Written HACCP plans for seafood are now required, and must be specific for each processor and type of seafood. In response to the need to train members of the seafood industry i n HACCP procedures, the National Seafood HACCP Alliance for Training and Education was created. Thisand other organizations provide HACCP training courses and model plans that can be used as templates for developing specific plans ( I 6).
2. Asia: The Importance of Seafood in Their Diets The people of Asian countries consume a larger portion of fish and fishery products in theirdietsthancitizensinnlostWesterncountries,thereforeseafood-bornediseaseis generally a much larger percentage of the overall problems associated with foodborne disease. I n 1996, Lee et al. (17) published an epidemiological study of food poisoning in Korea and Japan. From 1981 to 1990, the most commonly incriminated vehicles of foodborne illness in Korea were fish and other seafood (31.8%). meat and animal products (25.0%), grainsandvegetablesincludingmushrooms(17.5%),andcompoundfoods
Pathogens Transmitted by Seafood
117
( 1 8.3%). During the same time,in Japan the most comnlon vehicles were seafood ( 2 I .7%), meat and animal products (3.6%),and graimand vegetables (14.6%). Amongthe seafoods, the major causes of food poisoning in Korea were shellfish (9.3% of total) and puffer fish (5.0%).In Japan, shellfish and puffcr fish accounted for 6.9% and 2.8%, respectively, of the total number of disease outbreaks. In Korea, of the 115 deaths from food poisoning from 1981 to 1990, 42.6% were caused by seafoods, 13.1%J by meat and animal products, and 33.1% by grains, vegetables, and mushrooms. In Japan, of the 106 deaths from food poisoning during the same period, 62.3% were caused by seafoods and 22.5% by grains, vegetables, and mushrooms. No fatalities were attributed to meat and animal products. In Korea, 58.6% of the outbreaks of food poisoning from 1981 to 1990 were due to bacteria, 18.6% were due to toxic compounds, and 23.3% were due to unknown causes. In Japan, 61.3% of food poisoning cases were due to bacteria, 23.5% were due to toxic compounds,and 15.2% weredue to unknowncauses.Thefollowing bacterialspecies were incriminated in Korea: Vibrio spp. (37.6%), Strlrrrotrellcr spp. (23. I %), other species (17. I %), S t t r ~ ~ l r ~ l o ~ ~ (14.9%), o c c u s and E. coli (6.8%). In Japan, the bacterial foodborne problemswere Vibrio spp.(47.3%). Strrl,l,ylococclrs spp.(24.8%), Salmowellcr spp. (14.8%), and other species(9.6%).With data such as these,it is understandable why there needs to be research related to seafood safety in these countries (17).
D. Microbial Classification and Identification of Pathogens 1, Prokaryotes (Bacteria and Archaea) A revolution in describing the taxonomy of microorganisms has occurred over the past 20 years, largely initiated by the contributions by Carl Woese and his coworkers (182 1 ). The taxonomy of microorganisms is now based on the phylogenetics. or evolution, of organisms rather than on interpretation of the results of phenotypic properties. The phylogeny of microorganisms can be inferred by finding the changes that have occurred i n molecules that act as chronometers of evolutionary history over thousands of years. The 16s rRNA (ribosomal RNA) and 30s rRNA are the molecules of choice today. Since the fossil record for microorganismsis extremely poor and virtually nonexistent, the relationship between microorganismsis now determinedby sequencing representative molecules. After the sequences are obtained, the phylogenetic relationship is suggested by performingintensivecalculations withtheaid of personalormainframe computers. A variety of algorithms have been developed to create phylogenetic trees, also known as "trees of life." The work by Woese and others clearly illustrate that there are three major groups or domains of organisms, called the Btrcteritr, Arclraea, and Errkatptr. The natnes A ~ ~ J ~and I CErrkrrryr YI were formerly known as Archaebacteriaand Eukaryotes, in use and found in the literature today. Besides respectively, and these older names are still observing the three major trunks that can be determined by sequencing the representative molecular chronometers, these three domains of life have major differences in their cellular biochemistry and genetic organization. The 16s rRNA sequences and phylogenetics for many of the bacterial food pathogens have been determined and are available fromvariety a of electronic database servers. One of the best resources for phylogenetic analysisand 16s rRNA sequences is the Ribosomal Database Project (RDP) (22) located at Michigan State University. The URL for their web site is www.cllle.lllsu.edt1/RDP/.
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118
Of interest, most of the seafood-borne bacterial pathogens cluster into two phylogenetic groups and arenot widely distributed throughout the "tree of life." No human microbial pathogens are found in the Archaea. Bacterial pathogens associated with seafood are members of the y (gamma) subdivision of the Proteobnctericr and the gram-positive bacteria (Fig. 5). Besides the basic scientific curiosity about the phylogenetic position of seafood-borne pathogenic bacteria, this information can also be used to develop molecular probes and polymerase chain reaction (PCR) primers. Protocols developed for the phylogenetic analysis of microorganisms are used to examine the composition or identity of organi s m present on a food or environmental sample without havingto culture or grow organisms on media. y SubdivisionProteobucteriu Vibrio group Vibrio cholerue subgroup Vibriocholerae Vibrio vulnificus Vibriofisheri assemblage Vibrioparahaemolyticus Aerornonus group Aerornonas hydrophilu subgroup Aeromonas hydrophila
Enterics and relatives Eschericl~iu-Salmor~ell~~ group Escherichiacoli
Salmonellaspecies Yersiniu group Yersiniaenterocolitica Plesiomonas shigelloides Catnpylobacter and relatives Canlpylobacter fetus subgroup Campylobacter jejuni
Gram positive phylum Clostrium and relatives Clostridium botulinum subgroup Clostridiumbotulinum Bacillus-Lactobacillus-Streptococcu~. subdivision
group
StUphY~OCOCCUS
Staphylococcus aureus Listeriu-Bruc/lot}fri~~ Group Listeria monocytogenes
Streptococci Streptococcusiniae
Fig. 5 Phylogeneticposition of pathogenicbacteriaassociatedwith sedood. No111ellclalure for the lnajor groups and subgroups are thc t e r m used by the Ribosomal Database Project ( 2 2 ) .
Pathogens Transmitted by Seafood
119
The discovery of foodborne bacterial pathogens was made possible by the ability to culturetheorganismsonbacteriologicalmedia.Today, therapididentificationand differentiation of many foodborne pathogens is possible because of molecular genetic and immunologic methods. Nevertheless, for the development of theserapid tools, a pure culture of the pathogenic organism was required, meaningthat the culture was most likely initially isolated, characterized, and grown on bacteriological media in the laboratory. In other words, if the pathogenic organism has not been cultured, it has not been identified or implicated as causing seafood-borne illness.
2. Viruses In addition to the bacteria that may cause disease, animal viruses that can cause human illness are associated with seafood. Compared to bacteria, viruses are small, ranging from 25 to 75 nm, and are therefore not observable under the light microscope. While animal viruses come in a variety of shapes, mostof those associated with food are spherical. The of DNA or RNA, but most of the genetic material of animal viruses may be composed food viruses contain RNA, usually single stranded. Virus particles do not have any metabolism of their own. but require living animal host cells for replication. to host by direct or indirect transmission. Direct transmission, Viruses pass from host also known as contact transmission,is probably the most common way that a virus particle passes from one host to another. For example, direct transmission of viruses that cause gastrointestinal disease occurs through an anal-oral route, usually by hands that are contaminated with fecal material containing the infectious virus. Indirect transmission of viruses may occur by (a) vectors, which are intermediate animals within which the viruses are transported; and Inay multiply; (b) fomites, inanimate objects on which the viruses (c) vehicles, foods and water which may transport the viruses. Viruses that have indirect modes of transmission are required to be more stable and durable compared to those that can only be transmitted directly. Viruses are classified using a different scheme compared to other microorganisms and other higher forms of life. A “tree of life” that consolidates and organizes all of the different viruses does not exist.The primary viruses associated with seafoods are members of two viral families, the Picortuviridue and the Crdiciviriche. The Picortmviridcre are snlall single-stranded RNA viruses comprising some of the important pathogens of humans and animals. These viruses are small, having an icosahedral structure, and their nucleic acid consists of positive sense, linear, single-stranded RNA. The Cdiciviridw is also a positive sense, linear, single-stranded family of viruses. Fig. 6 shows the taxonomic groups of viruses associated with foods using the taxonomic nomenclature as described by the International Committee on the Taxonomy of Viruses (23). Six kindsof viruses causing infectionin humans are associatedwith the consumption of seafood: hepatitis A, human caliciviruses (Norwalk virus and Norwalk-like virus), hepatitis E, astrovirus, group A rotavirus,and human adenovirus. Human poliovirus in seafoods is primarily of interest for historic reasons and since this virus is routinely used in laboratory experiments with seafood. The morphologies of human viruses and the detection of these viruses is often performed with theaid of an electron microscope. Samples containing virus particles are stained with a solution containing an electron-dense compound that creates a “negativestain” image that can be observed under an electron microscope. Fig. 7 displays the electron micrographs of representative viruses that may cause seafood-borne disease.
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Positive Stranded Single StrandedRNA Viruses Family Picornaviridae Genus Enterovirus human poliovirus Genus Hepatovirus hepatitis A (HAV) Family Caliciviridae Genus Calicivirus human caliciviruses, alsoknownas small round structured viruses (SRSV) Norwalk virus Norwalk-like viruses, including Hawaii strain, Snow Mountain strain, Taunton strain, Southampton strain Hepatitis E Family Astroviridae Genus Astrovirus Small Round Viruses (SRV) (probable classification) Double Stranded RNA Viruses Family Reoviridae Genus Rotavirus group A rotavirus Double Stranded DNA Viruses Family Adenoviridae Genus Mastadenovirus human adenovirus Fig. 6 Viruses and virus families associated with food. Viruses that are most frequently associated with seafood are in boldface print. Taxonomic nomenclature as described by the Sixth Report by the International Conunittee on Taxonomy of Viruses (23).
While hepatitis A, the Norwalk viruses, and other viral pathogens are thought to cause many outbreaks and cases of gastroenteritis and hepatitis in humans, the good news for food lnicrobiologists and the seafood industry is that there are many human infectious viruses that are not known to be transmitted by seafoods to humans. These include human immunodeficiency virus (HIV), herpesvirus, Hantavirus, rabies virus, rhinoviruses causing the common cold, and the agent of bovine spongiforrn encephalopathy. The last disease agent is not a virus, but a prion.
E. Summary of Clinical Presentations of the Diseases Table 3 summarizes the symptoms,infectivedose,incubationperiod,andduration of seafood-borne diseases. The time intervals listed for the incubation period and the duration of disease are approximations and vary between individuals, episodes, pathogenic strains,
ransmitted Pathogens
h
by Seafood
727
Fig. 7 Electronmicrographs of representative viruses associated wlth seafood (A) calicvirus, (B) astrovirus, (C) human rotavirus, (D) human adenovirus. Micrographs were electronically retrieved from the Universal Virus Database approved by the International Committee on Taxonomy of Viruses. This site is maintained at the Australian National Universlty.
and the numberof bacteria or virus particles consumed. In this table, pathogenic organisms are separated from each other based on the types of symptoms that are commonly observed. For some of the pathogens the infective dose is based on feeding trials with human volunteers and/or the number of organisms that were enumerated on the incriminating or may item of food. Most of the foodborne pathogens cause a diarrheal disease that may not be accompanied by vomiting and nausea, but some seafood-borne pathogens may cause septicemia or neurologic effects that may lead to death. Additional details about the individual seafood-borne pathogens are provided below.
II. BACTERIAL AND VIRAL PATHOGENS ASSOCIATED WITH RAW AND UNDERPROCESSED SHELLFISH A.
Bacteria
In developed countries, most of the problems associated with seafood are caused by the consumption of raw, underprocessed, or mishandled shellfish, particularly bivalve mollusks. These invertebrates concentrate microorganisms that arein surrounding waters by a filter-feeding process. Someof the pathogenic organisms that are concentrated are indigenous to the aquatic environment, while other pathogens are introduced from terrestrial sources or by the fecal pollution of humans or other warm-blooded animals. After fish
Table 3 Symptoms, Infective Dose, Incubation Period. and Duration of Seafood-Borne Diseasesa Organism
Symptoms
Infective dose
Upper ~~a.stroiritestirin1 tract syniptoins (riatisea, \vmiirirrg) occur Jirsr or predomiriute Sraph~lococci~.s nurect.s Nausea. vomiting, diarrhea. Less than I mg of toxin will abdominal pain produce symptoms.
Incubation period or onset time to symptoms 1-6 hour, mean 2-4 hours
Loic,er ,qa.rrstroiritesfinof rmcr symprotns (abdominal cramps, diarrhea) occiir first or predominate 1- 10 cells 8-72 hours Snlmoriella spp. Abdominal pain, diarrhea, vomiting, nausea Some serotypes are highly in- Variable. depending on seroEscherickia coli Abdominal pain. diarrhea. fective for infants and nausea; some strains may type young children. E. coli is a cause watery or bloody dinormal inhabitant in the arrhea. gut of humans and other mammals. 18-36 hours Cunipylohucter jejLoii Small, perhaps less than 200 Abdominal pain. diarrhea. vomiting. nausea cells 24-48 hours Yersiiiia enterocolitico Fever and abdominal pain are Unknown. but thought to be the primary symptoms; fregreater than 10' cells quently have diarrhea and/ or vomiting; may imitate flu and acute appendicitis. Vihrio cholerue (01 8-96 hours 10" cells with production of Abdominal pain. diarrhea, and 0 1 3 9 ) cholera toxin (CT). Much vomiting, nausea. "rice walower number if stomach ter" stool acid is neutralized 18-36 hours Suspected to be 10"-10' cells Vihrio cholerue (non1. Abdominal pain. diarrhea. vomiting. nausea 01 and non-0139) 2. Extraintestinal infection such as septicemia. wound infections. ear infections 3-76 hours Diarrhea, abdominal pain. Vihrio parahuenrolyti10-10- Kanagawa positive cells CllS vomiting, nausea
Duration of disease
6
2-3 days
5 days Variable. depending on serotype
7-10 days 3-21 days
Varies between individuals, mild, watery diarrhea to acute diarrhea Diarrhea may be quite severe lasting 1 week
X Illness usually mild, with duration of 3-5 days
2
aF
L
v.
e,
U2
0
Pathogens Transmitted by Seafood
-d 0 0
d
c
a
I
r-
-
e,
0
D
124
3
S .M
Henvig
3
S .-
Pathogens Transmitted by Seafood
Hewig
126
and shellfish are harvested, pathogenic microorganisms may also be introduced when the food is processed and transported. y-Proteobacteria and gram-positive A variety of bacteria, including members of the organisms, are associated with shellfish-borne disease in humans. Table 4 lists the Gram’s stain reaction, cellular morphology, major phenotypic characteristics, and the natural hab tats where these pathogenic bacteria are normally found. For many of these organisms, more thorough and detailed reviews about the individual genera and species have been published. In the following sections I will summarize the major features of each of the pathogenic organisms and briefly outline protocols that are routinely used to detect and enumerate these pathogens. Additional details can be found in the citations listed.
1. Vibrio Species Of the several species that comprise the genus Vibrio, three species areof primary concern in seafood-borne disease:V. cholerae, V. parahaemolyticus,and V. vulnijicus. To a much lesser extent, some additional speciesof Vibrio have been suggested to cause human disease such as wound infections and gastroenteritis, includingV. alginolyticus, V. carchariae, V . cincinnatiensis, V. damsela, V. jluvialis, V. fumissii, V. hollisne, V. metschnikovii, and V. mimicus (24). Fig. 8 shows the distribution of cases caused by Vibrio species in the United States from 1989 to 1998 (M. Glatzer, personal communication). During this Vibrio species were attributed same period89%of the deaths caused by seafood-associated to V. vuln$cus (Fig. 9) (M. Glatzer, personal communication). (Since the database was established primarily to follow the number of cases associatedV.with vulnijicus, the number of deaths and cases associated with the other Vibrio species should be examined with caution.) All species of the genus Vibrio are gram-negative rods that are often motile with either peritrichous or single polar flagella. They are facultative anaerobes with both fermentative and respiratory metabolisms. In cultural studies, members of the genus Vibrio are frequently isolated from estuarine water, sediments, and biota. Many strains are chitinolytic, capable of hydrolyzing chitin, a biopolymer composed of monomers of N-acetyld-glucosamine (25). Chitin is found in the exoskeleton of arthropods and in certain fungi. In aquatic environments, strains of Vibrio have been foundin close association with crusta-
V. flwralis V V. mrmrcus
-
3%
alginolyticus 1%
5%
V. vulnificus
55%
parahaemolytlcus 16%
Fig. 8 Cases of Vibrio species causing seafood-borne illness in the United States, 1989-1998 m. Glatzer, personal communication).
127
Pathogens Transmitted by Seafood V. parahaernolyticus -
V. cholerae 6%
!io/.
V. vulnificus
89%
Fig. 9 Deaths caused by seafood-associated Vibrio species in the United States, 1989-1998 (M. Glatzer, personal communication).
ceans. These include crabs, shrimp, and lobsters, seafoods that are of great importance for commercial and recreational harvesters. The reported incidence of potential pathogenic species of Vibrio in fish and shellfish is relatively high. Unlike many of the other pathogenic bacteria listed in this chapter, pathogenic and nonpathogenic strains of the genus Vibrio are autochthonous microorganisms in freshwater, estuarine, and the marine environment. U.
Vibrio cholerue
Disease. Vibrio cholerue is the causative agent responsible for the disease called cholera and in the most severe form is sometimes called cholera gravis. Cholera has killed many people and it continues to be a problem in many developing countries. Outbreaks, epidemics, and pandemics of cholera have been recorded throughout history. Since 1817, the world has been affected by seven pandemics of cholera (26). Specific distinctions in the genus are made based on the production of cholera enterotoxin, also known as cholera toxin (CT), serogroup, and the potential for epidemic spread. The basis of serotyping V. cholerue is the lipopolysaccharide somatic (0)antigen. Until recently, the distinction was simple, V. cholerue 0 1 that produced CT were associated with the epidemics and all other strains were nonpathogenic or occasional pathogens. Now, two serogroups-01 and 0139-are associated with epidemic disease, but not all strains of these serogroups produce CT. These strains do not produce cholera, and are not pathogenic (24). V. cholerue strains of the 0 1 serogroup that produceCT have long been associated with pandemic cholera. CT-negative V. cholerae strains have been occasionally isolated in cases of diarrhea or extraintestinal infections. This serogroup can be further divided into two serogroups called Ogawa and Inaba. V. cholerue 0 1 can also be divided into two biotypes, classical and El Tor. The causative agents of the first four pandemics are not known, but those of the fifth and sixth pandemics are due to the classical biotype of V. cholerue 0 1 . For the past 20 years the classical biotype has only been isolated in Bangladesh. The causative agent of the present, seventh pandemic cholera, the El Tor biotype of V. cholerue, began in 1961 in Sulavesi, Indonesia. The seventh pandemic extended across Asia into the Middle East during the 1960s and through Africa, southern Europe, and the Pacific Islands during the 1970s.This pandemic reached South America
128
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in January 199 I . Within a year a majority of countries in South and Central America were affected (26).Until the emergence of the 0139 serogroup, all isolates that were identified as V. cholerere on the basis of biochemical tests, but were negative for 01 serology were referred to as “non-01.” By late 1992,a new cholera epidemic was caused by the emergence of a new serotype, V. cholerrre 0139,also known as V. cholerrre Bengel (24,26). V. cholercw 0139, which emerged in theBay of Bengalarea i n 1992 hassince been detected in 10 countries. This new strain continues to be confined to southeast Asia (Bangladesh, China, India, Indonesia, Malaysia, Myanmar, Nepal, Pakistan, Sri Lanka, Thailand). Upuntil 1994,imported cases were reported in Estonia, Germany. Hong Kong, Japan, Korea, Singapore, Switzerland, Thailand, and the United States (27).V. cholerrre 0139 appears to be a hybrid of the 01 and non-Ol strains. The clinical presentation and modes of transmission of V. cholercre 0139 are the same as V. cholerere 0 I . In important virulence characteristics, V. cholerne 0 1 39 is indistinguishable from V. cholercrc 01 El Tor strains (28). However, this organism does not produce the 0 1 lipopolysaccharide and has a polysaccharide capsule that is similar to non-Ol strains (24). Cholera remains a global threatand one of the key indicators of social development. While the diseaseno longer poses a threat to countries with a minimum standardof healthy living, it remains a challenge in countries where accessto safe drinking water and adequate sanitationcannotbeassured.Almosteverydevelopingcountryisnowfacingeither a cholera outbreak or the threat of an epidemic (27). In India, cholera is estimated to be responsible for the deaths of 20 million people during this century. Since1991,the cholera pandemicin South America hasbeen responsible for the deaths of 10,000people. Death from cholera is caused by extreme dehydration and loss of electrolytes. An individual with cholera may lose up to 20 L of liquidlday and the tnortality rate may be 30-50% if an infected person is untreated (26). Three major types of V. cholerrre are recognized and are differentiated based 011 serology: 01, 0139,and non-OI/non-0139. The 0 1 serotypeisdivided into classical (nonhemolytic) or El Tor (hemolytic) types. V. c h o l e r m 01 causes cholera, which may be a mild case of diarrhea or a life-threatening disorder. The more recently discovered serotype, 0139,is very similar to 01. Non-Ol V. cholerere causes a less severe disease, usually a gastroenteritis or soft-tissue infection and septicemia. Most strains of non-01/ non-0 139 V. cholertre are non pathogenic (24,29). The symptoms associated with V. cholerne 01 range from a mild, watery diarrhea to an acute diarrhea with rice water stools. The mechanisms for pathogenesis have been well described and include two important aspects, colonization of the small intestine and of intracellular cyclic adenoproduction of cholera toxin. The toxin causes increased levels sine monophosphate (CAMP) and the secretion of water and electrolytes (Na‘, K - , Cl-, bicarbonate) into the lumen of the small intestine (26).
Ecology. Cholera is a disease that is primarily waterborne, but food that has contacted contaminated water may also carry organisms of V. c~l~olcrrre 01. The food that has been most frequently implicated in outbreaks is seafood, both molluscan shellfish and crustaceans. Estrada-Garcia and Mintz (30)showed that seafood was the most commonly implicated vehicle in foodborne cholera outbreaks around the world, with more than 12 outbreaks associated with seafood since 1961 (Table 5 ) . The primary reservoir for V. (Aolerrre is infected humans. Short-tertn carriage of this pathogen by humans is important in transmission of the disease. Persons with acute cholera excrete 107-10s V. cholerrre cells per gram of stool. People who are excreting
nd
Pathogens Transmitted by Seafood
129
Table 5 SeafoodsImplicatedinCholeraTransmissiond
Location Scverlrh pcrrlderrlic
1973 1974 I974 1977 1978 1982 1994
Raw shellfish Raw and undercooked shellfish Salted raw fish Raw salted fish and clams Steamed prawns Cooked squid "Samba1 sotong" Rawfish
198 199 200 20 1 35 36 202
1991 1991 1991 1992
Raw seafood ceviche Cooked crab Cooked crab Shrimp and fish
203 14 204 205
Louisiana Louisiana
1978 1986
34 206
Colorado
1988
Cooked crab Cooked crab and shrimp, raw shrimp and crab Raw oysters
Italy Portugal Guam Gilbert Islands Singapore Singaporc Italy L n r i r ~Arrlericrr epirlenlic
Ecuador Ncw Jersey (Ecuador) New York (Ecuador) California (Pcru) U.S. Gldf Cotr.st
207
large volumes of diarrhea may easily release a trillion viable and pathogenic cells in I O L of diarrhea. Asymptomatic carriers may live in the same household as an individual suffering from acute illness. In various studies 4-22% of individuals may be asymptomatic carriers. Although there is evidence that V. cholerne can survive and multiply in the environment (31), the rates of isolation of CT-producing V. cholera 0 1 from the environment correlate primarily with the degree of sewage contamination (29). Several reports have illustrated the close association between Vibrio species and crustacean zooplankton. While most research related to public health has focused on the shellfish that humans purchase and consume, smaller planktonic animals may be causing very significant problems for humans. Huq et al. (32) described a simple filtration method to remove plankton-associated V. cholercre in raw water supplies in developing countries. In laboratory experiments, the bulk of the plankton was removed with a filter constructed from either nylon net or sari material, the latter being inexpensive and readily available in Bangladeshi villages. Seafood may be contaminated if harvested from water polluted by sewage or from environments where V. cholerne 0 1 occurs naturally independentof human fecal contamination. In artificially contaminated oysters and clams, V. ckolerne 0 1 survived for more than 3 weeks when refrigerated (33). Crabs boiled for less than 10 minutes or steamed for less than 30 minutes may still harbor viable V. choler-ne 0 1 organisms (34). In the United States, the first cholera outbreak associated with seafood occurred in Louisiana. Eleven cases of cholera were associated with the consumptionof home-cooked crabs that were either boiled or steamed. Secondary contamination of seafood has been reported. Steamed prawns (35) and cooked squid left for at least 4 hours at room temperature (36) were implicated in two cholera outbreaks in Singapore. In businesses that maintain fish live in aquaria for human consumption, the aquaria
130
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water may contain pathogenic organisms such as V. choler.cre. Kam et al. (37) described the investigation of 12 cholera cases in Hong Kong. Microbiological investigations demonstrated that contaminated seawater in fish tanks that were used for keeping seafood alive was the most likely vehicleof transmission. The source of seawaterin some facilities Inay be contaminated. Restaurant owners in Asia who serve seafood often receive seawater for their holding tanks from “water vehicles.” The operators of these vehicles Inay draw seawater from polluted harbors or typhoon shelters, protected anchoring areas that provide shelter for fishing junks and other small vessels during monsoons (38). Estrada-Garcia and Mintz (30) listed several factors that make seafood a possible also important for the other Vibrio vehicle for cholera transmission. These factors are species that are pathogenic to humans: In many parts of the world seafood is consumed raw or undercooked. Seafood may be contaminated with V. cholcrcre in the aquatic environment. V. cholercre persists for many weeks in seafood, even if refrigerated (39) and multiplies rapidly if introduced onto cooked seafood (40). Therefore secondary contamination may be a problem. V. cholernc adheres to chitin, and chitin-absorbed bacteria seem to be more resistant to variations in pH and heat than free-living bacteria (41). V. cholercle colonize the surfaceof copepods and may concentratein the gastrointestinal tracts of animals that ingest copepods (42). MicrobiologicalProcedures.Traditionalmicrobiologicalproceduresfor
V. chol-
erne and other pathogenic Vibrio strains are well established and have changed little in
recent years. Most species of Vibrio grow well at alkaline pH and a key step in the protocols istheinitial enrichment in alkaline peptone water. Enrichments are streaked onto thiosulfate citrate bile salt (TCBS) agar and typical colonies are tested with a series of biochemical and physiological tests. Fig. 10 summarizes the protocol for V. cholercre (43). The key confirmation for the identificationof V. cholercre 0 1 is agglutination in polyvalent antisera. Antiserum against 0 1 3 9 is now available.
b.
Vibrio vulniJcus
Disease. Vibrio vultlificus isthe mostseriouspathogenic Vibrio species in the United States, responsible for 95% of the seafood-related deaths. A disease caused by V. vultzificus was first described by Roland in 1970 (44) who detailed the case of endotoxic syndrome and leg gangrene acquired in New England coast water. The etiologic agent was misclassified as V. parnhaernolvticus (44). Investigators later examined their collections of lactose-positive vibrio strains and a new species of Vibrio, V.
[email protected], was declared in 1979 because the pathogen could produce cutaneous lesions (vulnificus, meaning wounds in Latin) (45,46). In the state of Florida, this organism is the leading cause of foodborne deaths. While most of the cases that have been documented are from the southeast region of the United States,V. vulngcus was recently found in the waters and shellfishin Europe. In 1996, Dalsgaard et al. (47) reported the first clinical and epidemiological data about a series of V. drzificus infections in northern Europe. Arias et al. (48) presented the first report of the detection of V. vult$ficus naturally present in seawater and edible shellfish along the Spanish Mediterranean coast. This pathogenic bacterium may be Inore widespread in temperate and tropical waters than previously thought. v . \)uln.$cu.y manifests itself in three fornls of human disease, with two forms of seafood-borne disease having a very high rate of mortality. First, v. v u i t l i f i c w lnay cause
131
Pathogens Transmitted by Seafood
Vibrio cholerae Oysters
Oysters
Weigh 25 g sample and add 225 ml of APW Homogenize for 2 min
Weigh 50 g sample and add450 ml Alkaline Peptone Water (APW) Homogenize for 2 min
Split homogenatein half
250 m1 anddilutions
Than Foods Other
Prepare serial dilutionsof homogenate 250 ml anddilutions
t
Incubate homogenate and APW dilutions 6-8 h, 16-24 h at 35-37°C
Incubate homogenateand APW dilutions 6-8 h, 16-24 h at 42°C
Streak all enrichments onto Thiosulfate Citrate Bile Salts (TCBS) agar (and, optionally, Modified Cellobiose PolymyxinB Colistin (mCPC) agar) TCBS is incubated 18-24h at 3537°C; mCPC is incubated 18-24 h at 39-40°C
t
Pick typical colonies onto 1%Tryptone + 1% NaCl (T,N,) or Trypticase Soy agar (TSA) + 1.5% NaCl Incubate 12-24h at 35-37°C
I
t
Perform preliminary biochemicaland physiological tests Triple Sugar Iron (TSI) Kligler Iron Agar (KIA) Arginine Glucose Slant (AGS) 1% Tryptone + 3% NaCl TIN^), 1% Tryptone + 0% NaCl (TINo) Gelatin Agar (GA), Gelatin Salt (GS) Hugh-Leifson glucose broth Oxidase Gram stain
t
Confirm V. cholerae 01, V. cholerae 0139, V. cholerae non-01, V. cholerae non-0139, and V. mimicus using serological and biochemical tests
Fig. 10
Enrichmcnt.isolation,andidentificationprotocol
for Vihrio cholerrre (43).
Henvig
132 Table 6 Characteristics Among Patients with
Vibrio vulnificus Infections in the United States,
1988-1996O
Gastroenteritis septicemia Wound Characteristic Primary Median age, years (range) Males (9%) Fever Diarrhea Abdominal cramps Nausea Vomiting Shock Localized cellulitis Bullous lesions Hosuitalized
infection 54 (24-92) 89 91 58 53 59 54 64
-
49 97
35 (0-84) 57 57 100 84 71 68 0
0 65
59 (4-91) 88 88
-b
30 91
89
' Data presented by Shapm et al. (49). Characteristlc not associated with syndrome.
a primary septicemia often resulting from the consumption of raw oysters. The mortality rate for this form is about 60%. Second,V. vulnijicus may cause wound infections on the skin. This disease may be causedby organisms present in seawater andlor shellfish, and has a mortality rate of 20-25%. The third form of the disease is a gastroenteritis that rarely causes death. Table 6 summarizes the characteristics among patients with V. vulnacus infections in the United States from 1988 to 1996 (49). V. vuZn$cus septicemia is nearly always associated with the consumption of raw oysters. Previous reports have shown the prevalence of raw oyster consumption in the general United States population to be around 17%, with a prevalence reaching as high as 32% in coastal states such as Florida(50). Data clearly indicate the hazards associated with the consumption of raw shellfish, particularly oysters, harvested from the Gulf Coast of the United States. Oysters causingV. vulnijkus disease have been harvestedin Louisiana, Florida, and Texas (Fig. 11). Although many of the shellfish-associated cases of V. Unknown
Fig. 11 States of harvest for shellfish associated withVibrio vulnificuscases (M. Glatzer,Personal communication).
Pathogens Transmlfted by Seafood Unknown
133
9 states
Anzcna
2%
New York 2%
Florida
38%
Alabama
Texas
9%
11%
Fig. 12 States of consumption of shellfish for cases associated with Vibrio vuln$cus personal communication).
(M.Glatzer,
vulnijicus occurred in these harvest states, there were also a significant number of cases as California, and states that in states where there are major population centers, such receive shipmentsof shellfish from the Gulf (Fig.12). These data also illustrate the complexity of the problems that are faced by federal and state public health officials in dealing with the interstate transport of raw shellfish distributedto retail and food service establishments. Nearly three-quarters of the cases of V. vulnifcus disease are associated with the consumption of shellfish in restaurants (Fig. 13). The common symptoms are fever, chills, nausea, and hypotension (low blood pressure). Symptoms associated with gastroenteritis are often present, but are less common. An unusual symptom that also occurs is the development of secondary lesions that are often found on the extremities. These lesions may develop into necrotizing fasciitis or vasculitis that may require the amputation of limbs or surgery. Fortunately infection with V. vulnijicus is not a serious problem for healthy people; nearly all serious and deadly Unknown Miscellaneous
Party
4%
4%
5%
qestaurant 72% Fig. 13 Location of shellfish consumption for cases associated with Vibrio vuln$cus (M. Glatzer, personal communication).
134
Herwig
infections occur in people with an underlying disease that causes an elevated level of iron of patients in the blood or in those who are immunosuppressed. About three-quarters with raw oyster-associated V. vulnificus primary septicemia have preexisting liver disease ( 5 1S 2 ) . In Florida, the reported rateof raw oyster-associated V. vuln$cu.s primary septicemia is 60 times greater among personswith liver disease thanin those without liver disease (53). Liver or blood-related diseases may predispose an individual to serious V. vulrl$cr~ septicemia. In recentyears,additionalwork has indicated that patients who are immunosuppressed are also susceptible to V. vuln$cus and V. ckolerue infections. Raw oyster-associated V. vulnificus septicemia has been reported in at least one patient with HIV infection (54) and a fatal case of V. cholerae non-01 sepsis was described i n a 51-year-old patient who was undergoing chemotherapy for leukemia and reported consuming raw oysters 48 hours before he became ill ( 5 5 ) . A report (56) describing the acquisition of V. vulr~ijcus septicemia from wounds in two patients with solid organ transplants supports the notion that immunosuppressed persons are at increased risk for severe Vibrio infections. Some patients with underlying liver diseases continue to consume raw oysters even after they are informed about the risks. Tayloral. et (57) studied the raw shellfish consumption practices of patients with liver disease being seen in the outpatient gastroenterology clinic at Walter Reed Army Medical Center. One-fifth of patients with known liver disease, even when previously informed about the risks, reported eating raw shellfish. In 199 I , California was the first state to require restaurants and other establishments that serve or sell Gulf Coast oysters to warn prospective customers about the possible risks. Florida and Louisiana also use these warning regulations. In October 1995, the ISSC rejected an FDA proposal to ban the sale of raw oysters obtained from the Gulf of Mexico during warm-weather months. Instead, oyster harvesters are now required to refrigerate oysters within 6 hours after harvesting from this region. The U.S. government has received pressure from national consumer groups to set a standard requiring the Gulf Coast shellfish industry to eliminate pathogenic bacteria, such as V. ~ ~ u l n ~ j i cfrom r ~ s ,oysters. The consumer groups notethat technologies are available that can eliminate this organism. A mild heat pasteurization technology is offered by AmeriPure Oyster Companies of Empire Louisiana. Another technology that appears to be promising, a high-pressure treatment, is also being developed (58).
Ecology. V. vulnificw is a normal inhabitant of the estuarine environment, having of the United States, the Gulf of Mexico, and in estuarine been isolated on both coasts environments around the world. The ecology of V. \~uh$cu.sis very similar to V. pcrrcrhrremolvticus. As filter-feeding shellfish, oysters concentrate the number of microorganisms found in the water column in their gut. For example, one study found a nearly 10,000-fold increase in concentration, from 7 colony forming units (cfu)of V. vuln[jicu.s in seawater to 10" cfu/g of oyster. As found with other marine vibrios, there is no correlation between the presence of V. vulrlificus and fecal coliforms. Therefore, measuring thelevel of E. coli or fecal coliforms is not a good monitoring method for V. vulrlificus. Some of the highest concentrations of V. vulniJcus have been found in the intestines of finfish (59). V. vulnificus also undergoes seasonal population variations correlated with temperature. The data of Motes et al. (60) clearly show the relationship between temperature and the levels of V. vulr1ificu.s in oysters. During warm-weather months,when more than 85% of the shellfish-associated V. vuln$cus occur, most probable number (MPN) counts were usually 103-10'/g of oyster meat. During cold-weather months,when infections have not
Pathogens Transmitted by Seafood
135
been reported, MPN counts were less than IO/g. Gulf Coast data suggest that the number of V. wtln[ficus organisms i n oysters is strongly correlated with water temperature until the temperature reaches 26”C, above which there appears to be no additional increase in the number of bacteria. Seasonal temperature change explains most of the variability in the Vibrio levels in the Gulf. Salinity explains an additional 10% of the variability in these levels and also explains the differences between sites. Shapiro et al. (49) summarized data that was collected by the Gulf Coast Vibrio Surveillance System, which includes public health agencies in the states of Florida, Alabama, Louisiana, and Texas. Using data collected by the CDC, they performed oyster trace-backs and examined the temperature of the place of harvest. For primary septicemia infections caused by V. vulrz(ficus, they found that all of the implicated oysters were harvested in the Gulf of Mexico when water temperatures were greater than 22°C. Among the 72 traced cases between 1988 and 1995, only 3 infections occurred in persons who consumed oysters harvested in waters with temperatures less than 20°C. While water temperature can serve to predict infections by V. vuh~[ficus,Shapiro et al. (49) did not prove that higher temperature is the cause of the increased incidence of V. vuln$cus infections. Unfortunately for the oyster industry in the Gulf, their data support the strong association between “summer harvesting” and illness, but summer harvesting represents 6 months of the year (May-October). Since 1972, oyster harvesting during this period has increased from 15% to 40% of the total annual production. A simple response to this problem, such as a closure of the harvest from May to October, would resultin a severe economic impact to the Gulf Coast oyster industry. Methods to reduceV. vuln$cus in live oysters after harvest include depuration(also called relaying), irradiation, and heat treatment. Gamma irradiation, although effective in reducing V. vuln$clrs in oysters in doses of 1.O- 1.5 kGy, also increases oyster mortality during storage. Also, this process is not approved by the FDA. Conventional depuration of oysters with an indigenous microflora of V. vulw$cus has been unsuccessful (61,62) and if conducted at temperatures higher than 21°C may actually increase the number of V. vuln$cus in oysters. Motes and DePaola (63) evaluated suspension relaying of Gulf Coast oysters to offshore waters that are normally free of V. vuln$cus. They observed a decline of V. v~rlr~$cus in relayed oystersthat was suggested tobe associated with exposure to high-salinity environments essentially devoid of V. v u l ~ z $ c ~ ~ s . Besides using methods to reduce the populations of potentially pathogenic v. vu/r~$cus cells in oysters, procedures can be used to tninilnize the increase in v. vuln(fifi~us numbers after harvest. Reducing the time oysters remain outside of refrigeration can decrease consumer exposure to high numbers of V. vulrz$cus; the oysters must be cooled immediately after harvest to eliminate postharvest growth of V. vuln$cus. Under typical Gulf Coast industry practices, oysters are held on the deck of harvest vessels without refrigeration or icing until the vessel docks. The NSSP does not require shellstock to be refrigerated on harvest vessels. By the summer of 1995 most Gulf Coast states had regulations on the time shellstock can remain outside refrigeration that are more restrictive than those set forth in the NSSP manual (64).
MicrobiologicalProcedures. Theenrichment,enumeration,andisolation procedures for V. vuln$c~u are very similar to those used for V. parcrhaemolyticus. Many studies attempt to perform enumerations for both Vibrio species using the same samples. Mostprotocolsbeginwith an enrichment step in alkalinepeptonewater,followedby plating onto a selective and differential medium called TCBS agar. Similarto V. cholerae,
Herwig
136
V. vu1rlijicu.s can grow under alkaline conditions. Inrecentyears, however, a modified cellobiose polymyxin B colistin (mCPC) agar has replaced the use of TCBS for isolating V. vulnificus. Suspected colonies are examined in a variety of biochemical tests. Fig. 14 outlines the enrichment and identification procedures for V. vulrzijicus and V. purcrhnetno-
lyricus (43). Some V. vultz@xs researchers believe that the environment contains a very diverse population of V. vultlificw strains and that only a certain subset appears to be associated with human disease(65,66).To examinethe diversity that exists within the species, molecular methods have been used including restriction fragment length polymorphism (RFLP) and ribotyping. Tamplin etal. (65) described RFLP and biotype profiles for environmental
Vibrio vuinificus
Vibrio parahaemolyticus
Weigh 50 g of sample and add 200 ml of Phosphate Buffer Saline(PBS)
Weigh 50 g of sample and add 200 ml of 2-3% NaCl solutionor Phosphate Buffer Saline (PES)
Prepare serial hutions in PBS
PreDare serial dilutions in saline or PBS
Inoculate Alkaline PeptoneWater (APW) MPN Incubate 12-16 h at 3537°C
Inoculate APW or Alkaline Peptone Salt broth (APS) MPN Incubate 16-18 hat 3537°C
t
Streak enrichments onto Modified Cellobiose Polymyxin B Colistin (mCPC)agar Incubate 18-24 hat 39-40°C
+
Streak enrichments onto Thiosulfate Citrate Bile Salts (TCBS) agar Incubate 18-24 hat 35-37°C
Pick typical colonies onto l%Tryptone1% NaCl (TINl agar) or Trypticase Soy agar + 1% NaCI, and Gelatin agar (GA) or Gelatin Salt agar (GS) Incubate 12-24 hat 35-37°C
+
Perform prelimmary biochemical testing to differentiate species Gram stain Oxidase Motility Arginine Gelatin Salt (AGS) Triple Sugar Iron (TSI) 0/129 vibriostat sensitivity ONPG test
Perform additional physiological, biochemical, serological, and pathogenicity tests
Fig. 14 f?ruiyticlls
Pathogens Transmitted by Seafood
137
and clinical V. vlrlw$c.u.s strains. They found high levels of variation in RFLP profiles among 53 clinical and 78 environmental isolates as described by pulsed-field gel electrophoresis. In contrast, ribotype profiles showed greater similarity. Jackson et al. (66) presented evidence that V. vuln;fic~rsinfections result in the proliferation of a single pathogenic strain, not a mixture of strains, and that high-risk individuals may be susceptible torelativelylowconcentrations of V. r~uln~ficus, as low as 10' cells/g of oyster if the pathogenic strain is present. Characterization of V . 1~1rl11$crrs strains has led to subdivision of the species into two biotypes which are now suggested to be basedon serological properties and host range, rather than the more variable biochemical properties. Biotype 1 strains are pathogenic for humans, exhibit several immunologically distinct lipopolysaccharide(LPS) types, and are indole positive (67). Biotype 2 strains appear to be virulent for both humans and eels and express a conmon LPS type, and the majority of biotype 2 strains are indole negative (67,68).
c'. Vihrio ~~nr~r11tremolytic'rl.s Disease. The firstreport of a V. pnr~rhaernolyticusfoodborneoutbreakwas in 1950. This organism was first isolated in 1953 as the causative agent for food poisoning i n Osaka, Japan (69). In some regions of the world V. parnkaernolyticw is the leading cause of gastroenteritis associated with food. In 1994, 102 outbreaks of foodborne disease involving 4726 cases were reported to the Taiwan Department of Health. Of these outbreaks, 72.5% were caused by bacterial pathogens with V. partrknemolyticus responsible for 56.7%, Stcll'h?.lococc.lrs mre1r.s 20.3'76, Bcrcilllrs cereu.s 14.9%, and Salmonella spp. other than S. typhi and S. pcrrcrtyphi 8.1% (70). V. ~'c~r~~hrrenrol~ticus has been the leading high proportion (54%) of the cause of foodborne illness i n Taiwan for many years. A large outbreaks, defined as having more than 50 cases, was associated with commercial lunch-boxes suppliedto elementary and juniorhigh schools. In Japan, V. pczr~rkezernolyticus is the leading cause of foodborne disease, with as much as 70% of the bacterial foodborne disease i n the 1960s being caused by this organism. Most of these outbreaks were associated with the consumption of seafood. Whereas most Japanese outbreaks involve fish, in the United States outbreaks primarily involve crab, shrimp, lobster, and oyster. The largest outbreak caused by V. p r o hrlenlo1yticu.s occurred in the summer of 1978 and affected 1133 of 1700 people attending a dinner in Port Allen, Louisiana, in which boiled shrimp was served. The shrimp were boiled in the morning then repacked i n the wooden crates in which they had been stored before they were cooked. The boiled shrimp were stored in an unrefrigerated truck until they were served approximately 8 hours later. In the last 2 years, two major outbreaks caused by V. pcrrtrhrrenro1~ticu.shave occurred in the United States. Thereis some concern among regulatory officials and within the industry about whether the recent outbreaks are an anomaly or the start of a new trend. During July-August 1997, the largest outbreak i n North America in recent years of confirmed V. ~~nrcrhaemo1~~ticu.s infections occurred (7 1). Illness in 209 people was associatedwith eating raw oysters harvested from California, Oregon, and Washington in the United States and British Columbia in Canada. One person died from the outbreak. During this outbreak, most ill people had no underlying illness. There was a suggestion that a slightly higher than normal water temperature contributed to this outbreak. Mean Pacific coast sea surface temperatures recorded by the U.S. Navy ranged from 12°C to 19°C from May through September. These temperatures were I"C-5"Chigherthanthe temperaturesfor the sameperiod in1996.Thelargest
138
Henvig
previous outbreak of V. PLrrah~lerno1yticu.sinfections reported in North America occurred in 1982 and resulted in 10 culture-confirmed cases (71). In 1998, an even larger V. purerIlnernolyticus foodborne outbreak occurred along the Gulf Coast. This outbreak caused illness in more than 400 people (M. B. Glatzer, personal communication). The symptoms associated with V. ~~arrrhtret~~ol~ticr~,s are primarily diarrhea and abdominal cramps with fever, nausea, and vomitingto a lesser extent. The infectious doseis thought to be large, somewhere between lo5and IO’ cells. Pathogenic V. ~~~rrahnemolyticus strains, which cause acute gastroenteritis after consumption of raw or partially cooked seafoods, have been known to produce thermostable direct hemolysin (TDH), also known as the Kanagawa phenomenon, or TDH-related hemolysin (TRH), orboth TDH and TRH (72,73). Most clinical isolates (96.5% of 2720 strains) have been Kanagawa phenomenon positive, whereas 99% of 650 environmental and food cultures have been found to be Kanagawa phenomenon negative (74,75). Suthienkul et al. (76) used PCR to analyze genes encoding TDH (tdl1) and TRH (tr.11) in 489 clinical strains and found that 81 % of the 489 strains were TDH’TRH-, 5.5% were TDH-TRH’, 2% were TDH-TRH’, and 11.5% were TDH-TRH-. Hence the investigators thought that identification of V. parahnemol~ti~us, irrespective of its virulence factors, is necessary for public health (77). V. ~~a~~rhnemolyticus is not a reportable disease in all states. During the 1997 outbreak,publichealthofficials in Washington,California,andBritishColumbiabecame aware of the outbreak through routine reporting. An editorial note in Morbidity mcl Mortality Weekly (71) suggested that all states should consider making V. ~~trr~rh~retrrolyticus and other vibrioses reportable.
Ecology. V. ~~arc~haerrrolyticus is a naturallyoccurringbacteriuminestuaries around the world. It is found in sediments, plankton, and in a variety of fish and shellfish. Pioneering work performed in the 1970s in Rita Colwell’s laboratory clearly documented the ecology of this human pathogen in the Chesapeake Bay (78). V. ~~arahaemolyticus was found to have a strong association with zooplankton during the warm summer months, and appeared to “overwinter” in the sediments during the cooler periods of the year. Most Vibrio species are chitinolytic, and thus are involved with the biodegradation of crustacean exoskeletons and other forms of chitin (79). Several studies have not found a correlation between the number of V. pLrrCrhClern[)lyti(.u,~ and fecal coliforms in environmental samples (61$0). V. yrrrcrhc~emolyticus has a remarkably rapid growth time, as short as 8 or 9 minutes at 37°C in bacteriological media. Even in seafoods, generation times of 12-18 minutes havebeenreported(24).Withsuch a shortgenerationtime,thisspeciescangrowto remarkably high concentrations i n a relatively short time. Recently another type of shellfish has been implicated as a vehicle in seafood-borne V. f,crr~rhnemolyticusdisease. Bean et al. (81 ) demonstrated that the consumption of cooked crayfish was associated with V. pcrrcrhaemolyticus infection. Crayfish are commercially harvested along the Gulf Coast of the United States. Of interest, no crayfish consumption was reported in V. vuln$cus infections. Crayfish have been described as “nouvelle cuisine” and are becoming an increasingly popular seafood item in many areas outside the Gulf Coast region. MicrobiologicalProcedures. Procedures verysimilartothoseusedfor V. vulr1ificu.s are used for V. parahaemolyticus (Fig. 14) (43). Sometimes additional amounts of NaCl are included in the diluent or enrichment broths used for V. ~~~rrrhaemolyticus. Isolates of V. pcrrahcretnolyticus can be serotyped according to their somatic (0)and capsular
Pathogens Transmitted by Seafood
139
(K) antigens, based on a scheme developed by Sakazaki et al. (82) who exanlined 2720 strains. Presently there are 120 antigens and 59 K antigens. Although many environmental and some clinical isolates cannot be typed bythe K antigen, the majority of clinical stains have recognized 0 types. While the antigenic characteristic is helpful in identifying and monitoring different strains, there appearsto be no correlation between serotype and virulence. Not all strains of V. / ~ r ~ ~ r r l ~ r ~ e t ~cause r ~ ) / yhuman t i c ~ ( . ~disease.I n many environmental studies pathogenic strainsof V. pcr~rrhcrenrolytit~us are never orrarely found. To enumerate or identify pathogenic strains, special attention is paid to finding strains that produce a hemolysin, called TDH (thermostable direct hemolysin) or Kanagawa hemolysin. A special blood agar called Wagatsuma agar (83) can be used to detect the production of this special hemolysin. but i n recent years a gene probe has been used to detect tdh genes. Primer sets for specific amplification of the TDH gene fragment have been described, and by using a preenrichment step i n alkaline peptone water for 8 hours, I O cells can be detected (84). Kaysner et al. (85) concluded that the urease-positive biotype is a useful marker for identifying potentially pathogenic strainsof V. ptrrnhtrernolyticlrs isolated from mollusks grown in the Pacific Northwest. This phenotype may only be useful for a preliminaryscreen,since all TDH-positivestrainswereureasepositive,however, notall urease-positivestrainsproducedTDH.Theassociation of positiveureaseactivityand TDH. has not been observed, however,in strains isolated from other regionsof the world (86). Chen and Chang (86a) described a rapid method for detecting V. /,rr~rrhaemo/yticrrs in oysters by immunofluorescencemicroscopy.Antibodieswerepreparedagainsttwo outer membrane proteins of V. pcrmhrretrrolyticus and an indirect staining method was employed. It was suggestedthat this could beused as a rapid screen ofV. ~~tr~crhLlerr~o!\‘ricu.s in oysters, with a presumptive positive result obtainable within 24 hours. Some problems remained, however, with cross-reactions between closely related but generally nonpathogenic organisms, such as V. crlgir~olyticrrs.
2 . Salmonella (1. Diserrse. Species of Soltwtrelltr are gram-negative, facultative anaerobic, nonspore-forming, peritrichously flagellated rods that fernlent glucose and utilize citrate as a sole sourceof carbon. Some species arenot motile. S c h r o t w l l t r spp. are very closely related ~ ~ ~ r rspecies e. of Srrlrrronellcr are to E. coli and are lnenlbers of the E l l t e ~ o b t r c ~ t e r - i ~ ~Several recognized and hundreds of different serotypes have been identified. The nomenclature of the Salmonella group has changed over the years and is based on a taxonomic scheme that uses biochemical and serologic infomiation and on the principles of numerical taxonomy and DNA/DNA homology (87). Manywould argue that all of the different “species” and strains are actually a single species with numerous subspecies. The three species that are generally recognized on a biochemical basis include S. typhi, S. cholertresui.~,and S. erlteritidis (88). Theserotypes are identified according to the Kauffnlann-White antigenic scheme and are segregated based on their somatic (O), capsular (Vi), and flagellar (H) profiles. Serologic tests are complex and labor-intensive techniques involving the agglutination of surface antigens with Salmonella-specific antibodies. Before 1950 typhoid fever, the disease caused byS. phi, was the primary infection caused by Salttrorwlltr in Western countries. This disease was the most frequent form of water-borne disease, and thousands of people died of typhoid fever during the early part
140
Hewig
of this century. Most of the outbreaks occurring earlier this century were related to S. hphi in the consumption of raw mollusks. At present, nontyphoid Srrltnonella spp. are one of the leading causes of bacterial foodborne disease in the United States and in many other non-Asian countries. Scrlrnorrell~ is associated with shellfish that is harvested from contaminated water or with seafoods that may have come in contact with other meats, poultry, or dairy products during preparation. The acute symptoms of the disease, called salmonellosis, are nausea, vomiting, ab1-2 days. dominalcramps,diarrhea,fever,andheadaches.Acutesymptomsmaylast Some people may suffer more chronic symptoms including arthritis-like symptoms that may follow 3-4 weeks after the onset of the acute symptoms. The infectious dose may be low, withas few as 15 cells causing the disease. As with other gastrointestinal diseases, the severity of the infection depends on the health and age of the person and strain differences within the genus.All age groups are susceptible, but symptoms are most severe in the elderly, infants, and the infirm. AIDS patients frequently suffer salmonellosis (estimated at 20-fold more than the general population) and suffer from recurrent episodes. The disease is caused by penetration and passage of Srrlrnonella organisms into the epithelium of the small intestine where inflammation occurs. There is evidence that an enterotoxin may be produced (87). Strlnwnella is among the leading causes of foodborne illness in the United States. It has been estimated that 2-4 million cases of salmonellosis occur in the United States each year. The CDC estimates that 75% of S. enterifidis outbreaks are associated with the consumption of raw or inadequately cooked Grade A eggs (87).
h. Ecology. Salmonellu arewidelydistributed amonganimals,particularlydomesticatedpoultryandswine,and in the environment.Environmentalsourcesinclude water,soil,insects,factorysurfaces,kitchensurfaces,animalfeces,andwildanimals. Scdrnor~ellaare primarily a problem associated with raw meats, poultry, and eggs, but they are also associated with milk and dairy products, fish, shrimp, frog legs, sauces and salad dressing, and manufactured foods that have ingredients that include the above.SLrlmonella species can be readily isolated from fish from aquaculture farms. Table 1 lists the prevalence of S d r n o r w l l c r in aquaculture farms in South Africa, the United States, Japan, and the Philippines ( 5 ) . There is evidence that Scrlrnonelkr is frequently isolated from these operations. Many aquaculture farms may be using antibiotics to safeguard the health of farmed fish and shellfish. c. Microhiologictrl Procedures. Traditionalmethodsfortheenrichment,isolation, and identification of Sdrnonella often include several steps or transfers and may take more than a week to perform. Usually, a preenrichment step in lactose broth precedes enrichment steps in tetrathionate broth and Rappaport-Vassiliadis (RV) broth. These steps are followed by plating on selective media that may include bismuth sulfite agar, xylose lysine deoxycholate agar, and hektoen enteric agar, biochemical screening, and serotyping. Fig. 15 describestheprotocolrecommended by the FDA.Recentstudiesindicate that modified semisolid Rappaport-Vassiliadis (MSRV) broth is very effective at yielding results that are better than or equal to those obtained with RV broth (89).In the last 10 years several research laboratories and commercial firms have described more rapid screening methods (90). Several of these methods have been recognized and approved by international agencies.
147
Pathogens Transmitted by Seafood Weigh 25 g of tissue Add 225 ml Lactose broth and blendfor 2 minutes
+
i
Transfer to wide-mouth, screw cap jar (500 ml and let stand for 60 min at room temperature
*
Mix well, adjust pH, if necessary to 6.8 0.2 Add up to 2.25 ml of surfactant (Tergitol Anionic 7 or Triton X - l 00)
+
Loosen jar lids and incbbate 24 k 2 h at 35°C
Transfer 0.1 ml of mixture
Rappaport-Vassiliadis (RV) broth Incubate 245 2 h at 42 rt 0.2%
Tetithionate (TT) broth Incubate 24 rt 2 h at 43f 0.2%
+
Transfer loopful from TT and RV broths onto: Bismuth Sulfite (6s) agar Xylose Lysine Deoxycholate (XLD) agar Hektoen Enteric (HE) agar Incubate 24 f 2 h at 35°C
/
Pick 2 or more colonies from each selective agar Pick typical colonies onBS agar at 24 rt 2 h, 48 f 2 h Transfer to Triple Sugar Iron (TSI) agar, Lysine Ironagar Incubate 24 k 2 h at 35°C
+
Apply biochemical and serological tests to presumptive Salmonella from TSI agar Urease test Polyvalent flagellar(H) test Lysine decarboxylase broth Phenol red dulcitol broth KCN broth Malonate broth Indole test Polyvalent somatic (0) test Phenol red lactose broth Phenol red sucrose broth Methyl Red-Voges Prokauer tests Simmons citrate
Fig. 15
Enrichmentandisolationprocedurefor
Salnronella
species
(90).
Herwig
142
3.
Escherichia coli
(1. Disecrse. The bacterial species E. coli has been described and known for many years. The organism is a common inhabitant of the facultative anaerobic microflora found in the intestinal tracts of humans and other warm-blooded animals. E. coli is a gramnegative, motile rod, capable of fermenting lactose and other sugars at 35°C. This organism is a member of the family of bacteria known as the Enterobacteriaceae. Isolates can be serologically differentiated on the basis of three major surface antigens, the somatic (O), flagellar (H), and capsule (K) antigens. Most of the strains found i n humans and in contaminated water samples are avirulent. Only certain strains of E. coli are pathogenic to humans and these strains are referred to as theenterovirulent E. coli (EEC) group. Four classes of E. coli are generally recognized: (a) enterotoxigenic E. coli (ETEC), (b) enteropathogenic E. coli (EPEC), (c) enterohemorrhagic E. coli (EHEC). primarily of the serotype 0157:H7, and (d) enteroinvasive E. coli (EIEC) (91). In addition to these four classes, diffuse-adhering E. coli (DAEC) and enteroaggregative E. c d i (EaggEC) strains have been recently recognized and associated with diarrhea in children in certain parts of the world (92). ETEC causes gastroenteritis andis sometimes called travelers’ disease. The infective dose is IO’ or more cells, possibly less for infants. This E. coli class is not a problem in countries with high sanitary standards. Humans are the principal reservoir of ETEC that cause human disease. EPEC causes watery or bloody diarrhea primarily i n infants, with few cases reported for adults. The most common foods associated with this disease are raw beef and chicken, and infant formula prepared with contaminated water. Humans are an important reservoir for EPEC. EHEC, primarily associated with serotype 0157:H7, causes hemorrhagic colitis, a disease that can be particularly severe for infants and young children. The vehicle generally associatedwith this disease is raw or undercooked ground beef. Cattle appear to be the natural reservoir for E. coli 0157:H7. EIEC causes bacillary dysentery, a mild form of dysentery, where there is the appearance of blood and mucous i n stools. The infective dose for EIEC is thought to be low and it is associated with food contaminated with human feces, since humans are a major reservoir.
h. Ecology. E. coli andothercloselyrelatedentericbacteriaarenonnalinhabitants of the guts of humans and other warm-blooded animals. Pathogenic strains of E. c d i may be a problem in shellfish and fish that are harvested from water containing human and other fecal pollution. Even though there are few reported cases of E. coli causing shellfish-borne disease, E.coli is a useful organism for monitoring the environment and shellfish for fecal contamination.
c. Mic.rohiolo~~iic.LI1 Procedures. Proceduresfor the enumeration of E. coli and fecal coliforms have beenusedformanyyears. The bacteriological quality of surface water, drinking water, and shellfish growing water is generally determined and regulated based on the presence and levels of fecal coliform and E. coli. The term “fecal coliform” is described by an operational definition, that is, by the ability to ferment and produce gas in particular media, at an elevated temperature, within a specific time. Classical enumeration procedures for fecal coliforms include the inoculation of a series of broth tubes for initial enrichnlent, followedby the transfer of positive tubes into more selective media. Using a series of dilutions of broth tubes results in the calculation of an MPN based on the number of positive tubes overa series of three dilutions. Conventional MPN procedures for total and fecal coliforms and E. coli require several days for incubation of the inoculated
Pathogens Transmitted by Seafood
143
media and aliquots from positive tubes from one type of medium to another at very specific times. Fig. 16 describes the conventional MPN procedure beginning with a presumptive MPNs (91). To test using lauryl sulfate tryptose broth and brilliant green lactose broth perform the fecal coliform test, small aliquots from positive lauryl sulfate tryptose tubes are transferred toEC broth tubes. Positive tubes from this step are streaked onto a differential medium and later presumptive colonies of E. coli are examined in a series of biochemical tests (Fig. 16). For the enumeration of fecal coliforms in shellfish growing waters A-
Weigh 50 g tissue, add450 ml phosphate-buffered dilution water Blend for 2 minutes
t
Prepare declmal dilutionsof blended material
Inoculate Lauryl Sulfate Tryptose(LST) MPN tubes Incubate for 48 ? 2 h at 35°C Examine after 24 rt 2 h and again at 48 k 2 h
+
Tubes are scored positiveif gas is present Transfer loopful from positive tubesto Brilliant Green Lactose Bile (BGLB) tubes
t
Calculate coliform MPN based on positve BGLB tubes, those with gas
EC Broth Method for Fecal Coliforms and Confirmed Test for E. coli Transfer loopful from positive LST tubes from the PresumptiveTest to EC tubes Incubate 48 rt 2 h at 45.5"C Examine after24 rt 2 h and again at 48 rt 2 h
+ +
Tubes are scored positive if gas is present
Streak loopful from positivetube to Levine's Eosin Methylene Blue (L-EMB) agal Incubate 18-24 hat 35°C
Transfer suspected E. coli colonies to Plate Count agar slants Perform biochemicaltests Gram stam Indole production Voges-Proskauer (VP)-reactive compounds Methyl red-reactwe compounds Citrate utilization Gas lrom lactose
Calculate E. coli MPN based on propbrtionof EC tubes that contain E. coli
144
Herwig
+ + +
Collect water sample
Prepare decimal dilutions
Inoculate A - l MPN tubes Incubate 3 h at 35"C, followed by 21 h at 44.5%
Tubes are scored positiveif gas is present
Fig. 17 MPN protocolusing A - l medium for shellfish growing waters (91).
1 brothcanbeused. Water samples are incubated at 35°C for 3 hours before theyare moved into a 44.5"C water bath incubator (Fig. 17). A newer variation of the conventional protocol incorporates the use of a fluorogenic substrate, 4-methylumbelliferyl-~-D-glucuronide (MUG), thatwillfluoresce under UV light when the enzyme P-glucuronidase cleaveslnethylutnbelliferonefromthesubstrate(Fig. 18). Achromogenicsubstrate 5 bron~o-4-chloro-3-indoyl-~-D-glucuronide (S-GLUC or BCIG) can also be used. These substrates have been incorporated into several selective media for the Enterobacteriaceae for rapid detection of E. coli (93). Chromogenic and fluorogenic substrates are used in several commercially available systems, such as those with the trade name Petrifilm, for the enumeration of coliforms and E. coli (94). At the present time there are no simple procedures available for the direct cultivation of EPEC, ETEC, and EIEC from foods. Weigh 25 - 50 g shellfish meat and add phosphate-buffered dilution water Blend for 2 minutes
PreDare serial dilutionsof blended material
Incubate Lauryl Sulfate Tryptose(LST) tubes as in Conventional MPN Procedure
I
t
Transfer loopful from positive LST tubes to EC-MUG broth tubes Incubate 24 h at 44.5 f 0.2%
t
Determine fluorescencein tubes All tubes that fluoresceare positive Calculate E. coli MPN
Fig. 18 EC-MUG method for dctermining E. coli MPN i n shellfishnle:l(s (91 ).
Pathogens Transmitted by Seafood
145
Since Shiga toxin-producing E. coli (STEC), especially serotype 0157:H7, arewell recognized as in1portallt food pathogens, several selective media have been described for these in detail in regulatory manuals organisnls (89). The conventional procedures are described such as the Bacteriological Analytical Manutrl (9 l), Oflcial Metl1od.s of Anc4wi.s (?fAoAC IilterilatiollLl[(95), and Recornmended Procedures for the Ex-nminarion of Sea W m r and Shellfish (96).
4.
Campylobacter jejuni
(1. Diseclse. Ctlinpglobacter jejuni is a gram-negative,curved,andmotilerod.It is a nlicroaerophilic organism requiring reduced concentrations of atmospheric oxygen for growth. Because of its microaerophilic requirements, optimum conditions for growth are achieved in the laboratory when 3-5% oxygen and 2-10% carbon dioxide are Provided. Before 1972 and after techniques were developed to culture this Organism from i primarily associated with abortion and enteritis feces, it was thought that C. j e j ~ n was in sheep and cattle. In recent years, however, C. jejuni is believed by some to bethe leading cause of bacterial diarrheal disease in the United States, causing more cases than S c h n o n e l k ~and Slligellrr combined (97,98). C. jejuni andothercloselyrelatedspecies,forexample, C. lari, C. coli, and C. }~?'ointestir~e~lis, cause diarrhea that may be watery or sticky and may contain blood and fecal leukocytes. It is believed that 99% of cases are caused byC. jejuni. Other symptoms that are often present include fever, abdominal pain, nausea, headaches, and muscle pain. The illness usually occurs 2-5 days after ingestion of contaminated food or water and than usually lasts 7-10 days. The infective dose is considered to be small, perhaps less 200 organisms (99). The pathogenic mechanisms for C. jejuni are still not completely understood. Death from C. jejuni is extremely rare, but there are reports of abortion induced by the organism. Some investigators suspect that C. jejuni is the leading cause of gastroenteritis in the world (100).
b. E c o l ~ ~ q y .C. jejuni isnotusually found in healthyhumans, but it is found in healthy farm animals, birds, and flies. In foods, C. jejuni is frequently associated with raw chicken; raw milk is also a source. C. jejuni is sometimes found in surface waters, such as streams and ponds. Shellfish have been implicated as a vehicle in human cases of C. jejunienteritidis (101,102).In 1993,69% of investigated mussels and 27%of oysters traded in the Netherlands were found to be contaminated with Ccrtnpylobacterspp. (1 03). Initial and later testing suggestedthat most of the isolates were C. lari. C. lari is a thermophilic species that was first isolated from gulls and subsequently from other avian species, dogs, cats, and chickens. Throughout the world, C. jejuni is the most significant known agent of human gastroenteritis. C. lari is isolated much less frequently from clinical samples, but is also recognized as a causative agent of gastroenteritis in humans (104). Studying the distribution of Cnrnpylobacter along the West Coast of the United States over an 8-year period, Abeyta et al. ( I 02,105) indicated that Camnpylobacter are well distributed in shellfish growing waters. In temperate aquatic environments, Campglobacter may exhibit a seasonality in its population numbers. Wilson and Moore (106) reported that few Cmlpylobacter were recovered between May and August; most were foundin the cooler winter months (November-March). Less than 6% of the shellfish examined between May and August contained C~rmpylobacter.Thermophilic Campylobocter spp. were detected in 42% of the shellfish. The greatest percentageof these (57%) were urease-positive thermophilic campylobacters
Herwig
146
(UPTC), atypical types that do not appear to be closely associated with domestic or farm animals or man. Their significance in foodborne human diseaseis probably minimal. These atypical Ccrmpvlobncters have not been commonly reported elsewhere. This may be the result of misidentification as C. coli.
c. Microbiologicc~lProcedures. Cc~mpylobcrc~ter areusuallypresentin highnumbers in the diarrheal stools of individuals, but isolation requires antibiotic media and a special microaerophilic atmosphere. Crrmpylobcrcter was difficult to culture until protocols for using the appropriate gas atmospheres were developed for enrichment and isolation of this organism. Ccrmpylobc~cterwill grow only under microaerophilic (microaerobic) conditions which can be supplied using special gas mixtures purchased from gas vendors or by using special “gas-paks” that generate the proper atmosphere within an anaerobe jar. More recently Abeyta et al. (105) reported that selected enrichment broths supplemented with the commercially available enzyme Oxyrase were evaluated for recovery of Ce~mpylobercterunder nornlal atmospheric conditions from shellfish. Results indicated that the Oxyrase enzyme was useful. Growth of the organism is not very fast, and isolation of C. jejurzi from food may take several days to a week. Water samples can be tested for the presence of Ce~mpylobc~ctrrby filtering samples through a 0.45 pm positively charged filter and adding the filter to an enrichment broth, a Bolton broth withantibiotics.Afterincubation in the enrichment broth, aliquots are streaked onto selective medium and incubated under microaerophilic conditions. Presumptive Cclmpylobacter colonies are tested with a variety of physiological and biochemical tests. Shellfish and other fishery products are examined in a similar fashion, except that the samples are placed in a stomacher or blendedto prepare a homogenate. Fig. 19 shows the suggested protocols for testing for Ccrmpylobcrcrer in water and shellfish samples as suggested by Hunt et al. (97). 5. Listeria monocytogenes cl. Disease. Listericl monocytogerws is a gram-positiverod,motile by means of peritrichousflagella.Theorganism is not a spore former. Some studies suggest thata significant number of humans Inay be intestinal carriersof this organism. It has been found in a variety of mammals, both domesticated and wild, a number of different bird species, and in some species of fish and shellfish (107,108). Lennon et al.(109) described a perinatal outbreak in Auckland, New Zealand, and hypothesizedbut did not prove an association with raw fish or shellfish. Schlech (1 IO) provided an extensive review on listeriosis and suggested that there has been a significant increase in the incidence of the disease. Listeriosis is an atypical foodborne pathogenof public health concern because of the severity and the nongastroenteric nature of the disease. While a relatively unco1nn1on disease, the high mortality rate suggests the need for continued vigilance and study. Listeriosis is clinically defined when the organism is isolated from blood, cerebrospinal fluid, or from otherwise sterile sites. The symptoms and outcome of listeriosis can be very serious and include septicemia, meningitis, encephalitis, and intrauterine or cervical infections in pregnant women which may result in spontaneous abortion or stillbirth. The onset of these disorders is usually preceded by influenza-like symptoms such as nausea, vomiting, and diarrhea. The onset time of serious forms of listeriosis is unknown but may range from a few days to 3 weeks
(107,108). The infective dose for
L. mo~zoc~ytogenes is unknown but is believed to vary with
147
Pathogens Transmitted by Seafood Water Samples Filter 2 - 4 liters through 0.45 pm positively-charged filters
t
Add filter to 100 m1 of Bolton broth with antibiotics Incubate 3 h at 30"C, followed by 2 h at 37°C under microaerobic conditions
+ +
Incubate, with shaking28 h at 42 C, without shaking 48 h at 42°C
I
t
Streak enrichmentsonto Abeyta-Hunt-Bark agar or Modified Campylobacter Blood-Free Selective agar base (CCDA) using anerobic jars under microaerobic conditions at 37-42°C
Test presumptive Campylobacter colonies with physiological and blochemical tests Hippurate hydrolysis Antibiotic sensitivities Nitrate reduction H2S from cysteine
Triple Sugar Iron (TSI) Temperature range of growth 3.5% NaCl
Glucose utilization MacConkey agar Glycine
Shellfish Samples Measure 100-200 g shell liquor and meat Blend at low speedor stomach for 60 seconds
Remove 25 ghomogeGate for sample analysis Add to 225 Bolton broth(1:l 0 homogenate)
t
+
Remove 25 g of i:l 0 homogenate Add to 225 Bolton broth (1:lOO homogenate)
Incubate 4 h at 37°C - for samples producedor processed c10 days previously OR Incubate 3 h at 30°C, followed by 2 h at 37°C - for frozen samplesor samples produced or processed 2 10 days previously
t
Incubate, with shaking28 h at 42°C: without shaking 48 h at 42°C
Streak enrichments onto Abeyta-Hunt-Bark agar'or Modified Campylobacter Blood-Free Selective agar base (CCDA) using anerobic jars under microaerobic conditions at 37-42°C
Test presumptive Campylobacter colonies with physiological and biochemical tests
Fig. 19 Protocols for examining water and shellfish salnples for Cunrpylohactcr species (97)
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148
the strain and the susceptibility of the human. Fewer than 1000 organisms can cause the disease.Ingested L. tnonocytogenes invades thegastrointestinalepithelium,entering monocytes, macrophages, or polymorphic leukocytes. The presence of L. rrrorrocytoSerlc’s in phagocytic cells permits access to the human brain and probably allows for migration tothe fetus in pregnant women. The pathogenesis of L. rrror~ocyrogerrescenters on its ability to survive and multiply in phagocytic host cells (107,108).
b. Ecology. L. monoc.ytogerles differs in many aspects from most other foodborne pathogens because it is ubiquitous and is resistant to diverse environmental (and food processing) conditions such as low pH and high NaCl concentrations. This pathogen is microaerobic and psychrotrophic. Two properties that contribute to its widespread distriit bution in the environment are its ability to survive for long periods of time, although is not a spore former, and its ability to grow at low temperatures. L. ttrorlocyroserlc’.s is widely distributed in a number of mammalian, avian, fish, and shellfish species (107,108). The organism appears to be widely distributed in freshwater and less so in bottom sediments. Colburnet al. (1 1 1) conducted a study to determine the incidence of Listeritr species in freshwater tributaries draining into Jumboldt-Arcta Bay in California. Lister-icr species were detected in 8 1% of freshwater samples and30% of sediment samples. Whether these organisms are indigenous to freshwater or are the result of runoff from livestock found in the vicinity of the rivers is uncertain. The organism is very hardy and resists the effects of freezing, drying, and heat. L. tnowocytogetzes has been associated with raw milk, cheeses (primarily soft varieties), ice cream, raw vegetables, fermented raw-meat sausages, poultry, raw meats, and raw and smoked fish. Seafoods, particularly ready-to-eat forms, have been frequently reported to be contaminated with L. rtrotloc~togewes( 1 12-1 14). The organism has the abilityto grow in temperatures as low as 3OC, permitting growth i n refrigerated foods. This characteristic may be a particular problem in ready-to-eat foods, such as cold smoked salmon and cooked fish products, since these products are not further heat processed before they are consumed. A relativelyhigh incidence of the organism (6-369’0) has been found in these products ( 1 13).
c. Microbiological Procedures. The traditional methods of analyzing food for L. monocytogenes are complex and time consuming, requiring a total time of 5-10 days. Protocols ( 1 15) are outlined in Fig. 20. Sample processing begins withan enrichment step using trypticase soy broth which is supplemented with yeast extract, phosphate salts, and pyruvic acid. Selective agents (acriflavin, nalidixic acid, and cyclohexamide) are added to the enrichment. Aliquots from the enrichment are streaked onto selective media and then presumptive L. rr7orlocytogerle.s colonies are examined in several biochemical and physiologicaltests.Finally,serologic,mousepathogenicity,andtheChristie,Atkins, Munch-Peterson (CAMP) testare performed. In recentyears,specificnonradiolabeled DNA probes have been developed which allow for simpler and faster confirmation of suspect isolates. Agersborg et al. (1 16) detected L. nrot1ocytoRene.s in seafood products using PCR and primers for fragments of the lysteriolysin 0 (/dy) gene and forthe invasionassociated protein gene (icrp). They were able to detect one to five L. trrorlocytogerlescells in S g of product in 55 hours.
6.
Yersinia enterocolitica
N. Disease. Thegenus Yersinicr hasthreepathogenicspeciesforhumans: Y. ellterocolitic.cr, a common intestinal pathogen in humans; Y. pseuclotuberculnsis, an intestinal pathogen in rodents which occasionally infects humans; and Y. pestis, the infectious agent
149
Pathogens Transmitted by Seafood Weigh 25 g of sample and add 225 ml of Enrichment Broth (EB) [Trypticase Soy Broth with Yeast Extract (TSBYE) supplemented with monopotassium phosphate, disodium phosphate, pyruvic acid]
I
+
Blend or stomach. Transfer to 500 ml flask Incubate 4 h at 30°C
+
Add selective agents acriflavin, nalidixic acid,and cycloheximide, and incubate additional44 h for a total of 2 days at 30°C
At 24 and 48 h, streak EB culture ontoboth Oxford Medium (OXA) and Lithium Chloride Phenylethanol Moxalactum (LPM)agar or LPM + esculin/Fe3+ agars. PALCAM agar may be substituted for LPM agars Incubate OXA andPALCAM media for 24-48 h at 35°C Incubate LPM mediafor 24-48 h at 30°C
Streak typical Listeria colonies from OXA and LPM (or PALCAM) onto TrypticaseSoy Agar with Yeast Extract (TSAYE)
+ +
Perform biochemical and physiological tests Cellular morphology and motility Catalase Gram stain Carbohydrate testing Hemolytic activity
Perform serological, mouse pathogenicity,Christie-Atkins-Munch-Peterson (CAMP) test
of bubonic and black plague. As members of the Enterobacteriaceae, Yersini(l are small, rod-shaped, gramncgative. oxidase-negative, facultative anaerobes that ferment glucose. Y. cwter’ocdificlr is often isolated from clinical specimens such as wounds, feces, sputum, and lymph nodes. It is not considered part of the normal human microflora. Y. entewcolirictr is a heterogeneous species, being separated into several subgroups according to biochemical activity and somatic (0)lipopolysaccharide antigens ( 1 17- 119). Yersiniosis in humans is characterized by synlptoms of gastroenteritis with diarrhea and/or vomiting. Most systematic infections occur in children, especially those less than 5 years old. Fever and abdominal pains are the hallmarks of this disease, with the organism causing physicians to misdiagnose appendicitis. Crohn’s disease, and mesenteric lymphadenitis. Most symptomatic infections result in self-limiting diarrhea. Yersiniosis may also give rise to a variety of autoimmune complications including reactive arthritis, erythema nodosum, iridocylitis, Homerulonephritis, carditis, and thyroiditis. These diseases have been reported to follow acute infection ( 1 19). This outcome is infrequent before the age of I O years and
750
Herwig
occurs most often in Scandinavian countries. Yersiniosis is a rare disease in the United States, but appears to be a more common disease in northern Europe, Scandinavia, and Japan. b. Ecology. Y. etlterocoliticcl hasbeenfound in a variety of environmentsand has been isolated from the intestinal tracts of many different mammalian species, as well as from birds, frogs, fish, flies, fleas, crabs, and oysters ( 1 19). Y. etlterocoliticn can be found in meats, raw milk, fish, and oysters. This organism is also commonly found in a variety of terrestrial and freshwater habitats, including soil, vegetation, lakes, rivers, wells, and streams. Pigs are the only animal species from which Y. enterocoliticcr of biovar 4 serovar 0 3 , the variety most commonly associated with human disease, has been isolated with any degree of frequency. Individual isolates of Y. erlterocditicxr from pigs and humans appear identical with each other in terms of serovar, biovar. RFLP of chromosomal and plasmid DNA, and virulence determinants ( 1 17). Y. enterocoliticrr, including pathogenic strains, can survive for extended periodsin the environment, such as in soil, vegetain rivers than tion, streams, lakes, wells, and spring water (120). Its survival was lower i n soils; Chao et al. ( 120) suggest that the presence of protozoans in the aquatic environment are responsible for their enhanced decline. The relatively low optimum temperature for growth may account for the higher incidence of yersiniosis in temperate regions of the world and the tendency for infections to peak during late autumn and winter where Y. crlterocoliticrr is endemic (121 ). c. Mic.robiologicer1 Procedures. If highconcentrations of Y. erlteroc-oliticcr are 36-48 hours;however, suspectedthen thisspeciescanbepresumptivelyidentifiedin confirmation or enrichment of samples for Y. enterorditiea may add another 2-3 weeks to the procedure. Fig. 2 1 summarizes the protocol ( 1 18) for the enrichment and isolation of Y. enterocoliticcr. Samples for enrichment are placedin peptone sorbitol bile broth and incubated for 10 days at 10°C. This step takes advantageof the ability of Y. erlterocoliticcr to grow at lower temperatures. Following the enrichment step, samples are streaked onto selective agars and presumptive colonies are tested through a series of differential media, physiological, and biochemical tests. Some investigators have recommended the use of two enrichment media, suggesting thatnosingle procedure will recover all pathogenic serotypes. The second enrichment method uses irgasan ticarcillin chlorate broth followed by plating on Snlrnot~ellrr-Shi~~e/ler agar (89).If high countsof this organism are suspected, aliquots of the homogenized sample can be directly plated on selective media and later colonies can be probed with a virulence gene probe. Determination of pathogenic strains requires additional steps and may include an autoagglutination test, the low calcium response Congo red agarose virulence test, and the crystal violet binding test. Other tests include DNA colony hybridization with probesto detect virulence factor genes. and intraperitoneal infection of adult mice pretreated with iron dextran and desferrioxamine B. Screening isolates for invasive potential usingan in vitro HeLa cell assay is alsoan effective procedure for determining pathogenic strains.
7 . Plesiomonas shigelloides cr. Disetrse. P1esionIoncr.s.skigelloitle.s is a motile,oxidase-positive,catalase-positive, facultative anaerobic, gram-negative rod that has been isolated from freshwater, freshwater fish, and shellfish and from many types of domesticated and wild animals. The genus are consists ofa single species. Manyof the phenotypic properties found in P. .shi,~e/loidr.s shared with the genera Vibrio and Aerornonas. For example, P. .shic~e//oicfe.es is Silnilar to
751
Pathogens Transmitted by Seafood Weigh 25 g of sample, add 225 ml Peptone SorbitolBile Broth (PSBB) Homogenize 30 seconds
t
Incubate 250 ml of homogenate for 10 days at 10°C OR
1
If high counts of Yersiniasuspected, spread-plate0.1 r n l of homogenized sample onto Macconkey agar or Celfsulodin-lrgasan-Novobiocin(CIN) agar, and transfer 1.O ml of homogenate to 9.0 ml solution of 0.5% KOH in 0.5% saline and spread-plate onto MacconkeyAgar and CIN Agar Incubate for 24 h at 30°C
Perform colony hybridization withYersinia virulence gene probe Transfer 0.1 PSBB of enrichment to 1.0 ml solution of 0.5% KOH in 0.5% saline and mix Transfer 0.1 ml enrichment to 1.O ml to 0.5% saline Streak from each saline tube onto Macconkey agar and CIN agar Incubate 24 h at 30°C
4-J
Pick presumptive Yersinia colonies and inohlate Lysine Arginine Iron (LAI)agar slants, Christensen's Urea agar,and Bile EsculinAgar Incubate 48 h at room temperature
t
Cultures yielding typical reactions onLA1 agar are streaked onto Anaerobic Egg Yolk (AEY) agar Incubate at room temperature
Perform gram stain, biochemical,and other physiological tests Lysine, arginme. ornithine decarboxylase Phenylalanine deaminase Motility at 2226°C and 3537°C Acids from: mannitol, sorbitol, cellobiose, adonitol. Inositol, sucrose, rhamnose, raflinose, meliblose. salicin, trehalose, xylose Simmons citrate Indole Voges-Proskauer test Llpase beta-D-glucosldase test Pyrazinamidase
152
Herwig
Vibrio spp. in its susceptibility to the vibriostatic agent O/ 129. Like Aerornontrs species, P. shigelloides does not have a requirement for sodium and is unable to grow in 6% NaC1. Humans and other animals may be carriersof the organism, showing no overt sylnptoms of disease. Much of the earlier information about this pathogen came from outside of the United States, but in recent years studies from the United States are supporting the role of this organism as a pathogen that causes diarrhea. Many of these cases are associated with the consumption of raw bivalve mollusks or with foreign travel (122,123). p . shigel1oide.s may cause a mild self-limiting gastroenteritis with fever, chills, abdominal pain, nausea, diarrhea, and vomiting with symptoms beginning 20-24 hours after consumption of the contaminated food or water. The infection may cause diarrhea for 12 days in healthy adults. There may be high fever and chills, and protracted dysenteric symptoms in infants and children under 15 years of age. The infectious dose is presumed to be quite high and is thought to be more than one million organisms. In healthy people, gastrointestinal illness caused by P. skigelloides infection may be so mild that infected individuals do not seek medical treatment. consequently the number of cases associated with this organism is assumed to be vastly underreported (124,125). A cluster of cases occurred in North Carolina in November 1980 following an oyster roast. Thirty-six of 150 people who had eaten roasted oysters experienced nausea, chills, fever, vomiting, diarrhea, and abdominal pain beginning 2 days after the roast. The averag duration of the symptoms was 2 days. P. skigelloides was recovered from oyster samples and patient stools (126). b. Ecology. P. shigelloides is a gram-negativerod thathas beenisolatedfrom freshwater, freshwater fish, and shellfish and from many types of domesticated and wild aninlals ( 123,127-130). Most human infections caused by this organism are suspected to be waterborne. The organism may be found in unsanitary water that is used for drinking water, recreational water, or water used in food processing. Studies indicate a seasonal effect from environmental sources, with an increase i n the reported casesof diarrhea during the warmer months ( 127,128).
c.MicrobiologicalProcedures. P. s1~igelloide.scan beculturedonInany of the media used for isolation and enrichment ofthe enteric bacteria. This speciesis tolerant of bile salts and brilliant green. Because lactose is generally slowly fermented, P. shigelloides appears as lactose negative on solid media. Because of a lack of competitiveness, enrichment techniques may be of limited usefulness (13 l). Procedures for the enrichment and isolation of P. shigelloides are not standardized and have not been published by the FDA ( 132). Koburger and Wei (124) suggesteda protocol where samples are diluted and plated onto Inositol brilliant green bile salt agar and PL agar. One to 10 g of sample are added to 90 m 1 of (etrathionate broth. Following a 24-hour incubation period, suspected colonies are examined in differential media and the enrichment broth is streaked onto the isolation media. Fig. 22 outlines the procedure (124). P. sl~igelloidescan be differentiated from Aeromonas spp. using a series of biochemical tests.
8. Aeromonas hyrophila and Other Aeromonas Species
N. Disease. Aerotnonu h y h p k i l n andother Aerornonas speciescanoften be isolated from food and the environment. CertainAerolnonus species are human pathogens that may cause gastroenteritis. Among the suspected foods are prefrozen or inadequately cooked seafood and oysters (133). Based on hybridization studies, the taxonomy and speci-
Pathogens Transmitted by Seafood
153
Add 10 g of sample to 90 ml Tetrathionite Broth Incubate at 40°C for 24 h
+
Streak onto duplicate platesof two of the following selective media Incubate at 35°C for 24 h Macconkey agar Billiant Green Lactose Bileagar PL agar Salmonella shigella agar Inositol Brilliant GreenBile Salts agar
+ +
Pick three typical coloniesfrom each of the selective media into Triple Sugar Iron agar and Inositol Gelatin deeps Incubate at 35°C for 24 h
Perfom oxidase test and gram stain from TSI slant
ation of the genus A P I Y ) I ) I Ounderwent II~ revision in 1980s. The taxonomy of the genus is still evolving and still confusing. Since DNA-DNA hybridization is not routinely employed in most clinical laboratories. most still rely on a series of phenotypic tests to differentiate Aeronlo/m species. The species name A. hydrophilrr has been broadly used i n the older literature to refer to the whole group of mesophilic aeromonads, but it should he more narrowly definedby the phenotypic characterizationof Popoff or DNA-DNA hybridization. If the speciation of a strain is in question, or if an isolate has not been thoroughly Aer.otnmm sp. or aeromocharacterized, then investigators should refer to the isolate as nads. Based on phenotypic testing and definitions, the generally accepted speciesof Aeromo)uI.s associated with diarrhea in humans are A. hydrophiltr, A. ( ~ r v i wA., \ ~ c ~ l n t r ibiovar i sobricl and biovar lvrorrii ( 134,135). Two types of gastroenteritis have been associated with A. hydrophila, a cholera-like illness with “rice water” diarrhea, and a dysenteric of illness characterized by loose stools containing blood and tnucus. The infective dose the organism is unknown, but SCUBA divers who have ingested small amounts of watcr have become ill and A. hydrophilu has been isolated from their stools ( 1 36).
b. E ~ o l o ~ y y .AcJromo1russpp. are commonly associated withfish and seafood and arewidelydistributed in the aquatic environment (137). Most of the published studies have dealt with A. /ryhophi/u, a bacterial species that is present in all freshwater environments and in estuaries. Some strains are capable of causing disease in fish and amphibians as well as humans. who may acquire infections through open wounds or by ingestion of organisms in food orwater. A. /rydrop/riI~rhas beenfrequentlyisolatedfromfishand shellfish. It has also been found in market samples of red meats and poultry. There are
154
Herwig Prepare dilutionsor use 25 gof original samplefor enrichments For dilutions, weigh 25 g and blend with 225 ml of 0.1% peptone water
+
For enrichments and MPN procedures, inoculate sample into Alkaline PhosphateWater or Tryptic Soy Broth Containing30 mg/L ampicillin Incubate at 28°C for 24 h
Streak enrichmentsor MPN tubes onto Starch Ampicillin (SA) agar or Bile Salts Brilliant Green Starch (BBGS) agar Incubate at 28°C for 18 to 24 h
+ + +
Flood the surface of the agar plates with Lugol's iodine solution Count typical colonies that have surrounding zones of starch hydrolysis
Pick typical colonies and streak onto nutrient agar that does not contain carbohydrate, such as Nutrient agar, TrypticSoy agar Streak DNase testagar
Perform gram stain, catalase test, resistance to 0/129 vibriostatic agent Inoculate Kaper's medium and observe reactions
relatively few published cases in which foodborne gastroenteritis.
Aerotnolm species have been associated with
c. Mictnbiological Procedures. Protocolsfor the enrichmentandisolation of pathogenic species of Aerovronm have not been officially accepted by the FDA i n the United States. Media for isolation of aeromonads usually exploit a resistance to ampicillin. Palumbo et al.(138) described a protocol for the isolation and identificationof Aerorwrltrs species from food (Fig. 23). Food samples can be blended and diluted i n peptone water and spread onto a surfaceof starch ampicillin agar or bile salts brilliant green starch agar. The latter medium is suggested by investigators who formulated it for samples i n which a large number of Proteus spp. might be encountered. Typical colonies on these media are identified after incubating by flooding the surface with an iodine solution. If low numbers of A e r o ~ n ~spp. ~ ~are m expected, food samples can be inoculated into enrichment broths or a seriesof MPN tubes with alkaline peptone water or tryptic soy broth containing ampicillin. After 24 hours, aliquots from the enrichments are streaked on the plating media. Suspected colonies from the plating media canbe tested for additional phenotypic proper-
Pathogens Transmitted by Seafood
155
ties to identify whether A. hyiro~philaor other Aewtw11m species were isolated. Species identification is confirmed by performing a series of biochemical tests (125,138).
B. Viruses There are four major categories of viruses that cause gastroenteritis in humans: rotavirus, enteric adenovirus, calicivirus (that includes Norwalk virus and its relatives). and astrovirus. Hepatitis virus is a foodborne virus whose symptoms and signs are associated with liver disorders. Raw shellfish havebeen implicated in outbreaks of foodborne viral gastroenteritis in the United States, Europe, and Australia (139-143). Outbreaks have occurred following the consulnption of shellfish harvested from waters contaminated with human sewage (143,144). Mostof the viruses associated with seafood-borne disease are calicivirus and hepatitis A. Table 7 lists the structural features and diseases caused by viruses associated with foodborne disease. Unlike most bacteria, viruses are not easily inactivated by normal cooking procedures. Shellfish tissue may "protect" the virus and individuals may become ill after consuming cooked shellfish that are contaminated with enteric viruses. To study the extent of the hazard presented by oysters contaminated with virus, DiGirolamo et al. (145) contaminated samples of whole and shucked Pacific oysters with IO' plaque-fonning units of poliovirus/ml and heat processed the oysters in four ways: stewing, frying, baking, and steaming. Results indicated that a significant portion of virus in oysters withstood these methods of processing. The survival rate varied from 7% to 10%. Cooking experiments performed with contaminated mussels revealed that S minutes after the opening of the mussels' valves, rotaviruses and hepatitis A virus could still be recovered in the steamed shellfish (146). While depuration or relaying shellfish may be a satisfactory method for removing pathogenic bacteria from these animals, such methods must be used with caution for the removal of human virus. Power and Collins (147) studied the elimination of poliovirus, E. coli, and a coliphage from the common mussel (Myrillrs cdulis). In their experiments. the relative rates of elimination during depuration were E. c w l i > coliphage > poliovirus. It was concluded that E. coli cannot beareliableindicator of viruseliminationfrom mussels. A dramatic experiment that showed the potential danger of relying on indicator bacteria and depuration was performed in Australia. Following the widespread outbreaks of oyster-associated gastroenteritis in Australia in 1978. several programs were introduced to minimize the occurrence of further outbreaks. One program included the depuration of oysters and the use of human volunteers, as an interim measure, to test samples before sale to the public. After eating Georges River oysters that were thought to be safe. S2 volunteers became ill and Norwalk virus was foundto be the cause of the infection. Depuration, as carried out in pollution-free water for 7 days, was therefore concluded to be an unsatisfactory method ( 148).
1. Hepatitis A Virus (1. Disctrw. HepatitisAvirus (HAV) is also referred toas Heptovirus A. and i n older literature as infectious hepatitis. Five kinds of hepatitis are recognized: A, B. C, D. and E. Hepatitis A and E are of concern with food, but type E is not found inNorth America. HAV is classified with the enterovirus group of the Picornaviridae family. Like other enteric viruses, HAV is transmitted by the fecal-oral route. The most common form
Herwig
156
Table 7 Structural Features and Symptoms of Human Viruses Associated with Seafoods,’
Virus Virus most ,frequently trssociaterl with seafood:
Hepatitis A
Calicivirus [includes Nonvalk virus and other small round structured viruses (SRSV)]
diameter, 27virus, round nm icosahedral symmetry, not enveloped, single stranded RNA, genome length of 7500 nth 35-39 nm diameter. round virus, icosahedral symmetry, not enveloped, one or two structural proteins, single stranded RNA, genome length of 7700 nt
Virus less ,frequently associated with seufood: 80 diameter, nm double-
Human adenovirus
Human poliovirus
shelled wheel-like capsid, icosahedralsymmetry.11 segments of doublestranded RNA, genome length of 18,500 nt 27-30 nm diameter, round surface and pointed star with 6 points, polyhedral symmetry, several structural proteins, single stranded RNA, genome length of 6800-7900 nt 75 nm diameter, not enveloped, fastidious growth in culture, double-stranded DNA, helper or satellite virions may be present, genome length is 30000360000 nt 28-30 nm diameter, round virus, icosahedral symmetry, not enveloped, single stranded RNA, genome length of 7400 nt
Structure and disease symptoms from sunlrnarles present (208). ” n t = nucleotides.
Epidemics of acute diarrhea and vomiting in older children and adults, often food or water borne
Major cause of severe dehy-
Rotavirus (group A)
Human astrovirus
Fever, malaise, nausea, anorexia, abdominal discomfort, followed by jaundice
111
drating diarrhea in infants and young children
Watery endemic diarrhea of children, in day care ccnters, some disease outbreaks, role in HIV-related diarrhea
Endemic diarrhea of infants and young children
Cause of polio in humans
Murphey et al. (23) and Blacklow and Herrmann
Pathogens Transmitted by Seafood
157
of transmission is person to person by fecal contamination. The incubation period for HAV ranges from 10 to 50 days with a mean of approximately 30 days, depending upon the number of virus particles consumed. The infectious dose is thought to be 10-100 particles. The virus infects the liver producing a debilitating, low-mortality disease that sometimes includes jaundice. The infected person is characterized by a sudden onset of fever, malaise, nausea, anorexia, and abdominal discomfort, followed by several days of jaundice. Death from HAV isveryrare. Most adultshaveanimmunity to HAV that provides lifelong protection against reinfection (23,149,150).
b. Ecology. HAV is an entericvirusthatiscloselyassociatedwithinfectedhumans and fecal contamination. Less than 10% of HAV cases are food associated or water associated. With foods, it is most frequently associated with bivalve mollusks, but it may also be found in foods associated with a large amount of handling, such as salads. In the later association, contamination may occur during preparation by a food handler who is infected with HAV. In the United States, between 5000 and 35,000 cases of food poisoning caused by HAV are estimated to occur each year. One of the more noteworthy outbreaks associated with seafood includes an outbreak in Sweden in 1955. Oysters held in a harbor awaiting sale around Christmas were contaminated with fecal pollution from a toilet that was over theharbor.Oystersfromtheharborwereeatenrawand629peoplebecame ill. This outbreak was the first report of HAV transmission by shellfish. In 1988 an HAV outbreak occurred in Shanghai, China. Clams taken from water that was contaminated with human sewage were eaten raw, resulting in nearly 300,000 cases of HAV infection (126). c. Microbiologiccrl Procedures m c l Detectiorl. Thediagnosis of an HAVinfection can be performed by detecting antibodies to HAV in human serum (126).
2. Human Calicivirus (Norwalk and Norwalk-Like Viruses) (1. Disetrse. Thehumancaliciviruses,whichincludeNorwalk-likevirusesorthe Norwalk family of viruses, are called small, round, structured viruses (SRSV) i n Europe. These viruses are 35-39 nm in diameter and are a serologically related group of viruses. Norwalk viruses are classified in the Caliciviridae family, and are positive sense, linear, single-stranded RNA viruses. Norwalk viruses cause gastroenteritis, typically with diarrhea and vomiting, with an incubation period of 24-48 hours and a duration of infection lasting from 24 to 60 hours. The disease caused by Norwalk viruses is often referred to as viral gastroenteritis. The infectious dose is thought to be low. The attack rate for Norwalk viruses is particularly high; often more than 50% of individuals who consulne contaminated food become ill. Humans appear to be susceptible to repeated infectionwith the same strain since the virus does not elicit a strong immunological response (23,149.150).
b. Ecology. Some investigatorssuspectthatNorwalkvirusesmaybeamongthe leading agents causing foodborne disease. Water is the most common source of outbreaks, but raw or insufficiently cooked bivalve mollusks have also been recognized as vehicles for transmission. Food outbreaks are often associated with the consumption rawofshellfish. In 1978 morethan 2000 people who ate oysters were infected in New South Wales, Australia. Some groups who consumed oysters showed attack rates of 85% (140). In December 1994 and January 1995, Florida experienced the largest outbreak of oyster-associated gastroenteritis ever reported. The largest shellfish and trout harvest for the Apalachicola Bay fishing industry occurs for the New Year’s holiday, when oyster
158
Herwig
roasts are a tradition. Of 223 oyster eaters, 58% became ill, compared with 3% of nonoyster eaters. Most oyster eaters ate only cooked (grilled, stewed, or fried) oysters. Oyster eaters who reported eating thoroughly cooked oysters were as likely to become ill cornpared to those who ate raw oysters. The outbreak demonstratedthat oysters cooked to the point where consumers consider them done or overdone werestill capable of transmitting enough virus to cause disease in a substantial proportion of people. The outbreak may have resulted from overboard dumpingof feces during a cornmunity outbreak of diarrheal illness. McDonnell et al. (15 1) suggested that using fecal coliform monitoring to define water quality is inadequate. Contamination of oysters with SRSV occurred during a time when routine water quality monitoring indicators were in acceptable ranges both in Florida and Texas. c. Mic~rohiologicdProceclrrres N I I ~Detectiorl. Norwalkvirusescanbedetected using serologic methods, but in recent years gene probes and PCR amplification methods have become increasingly more popular. While the DNA molecular procedures are theoretically very sensitive and rapid, nluch of the research effort has been directed toward developingmore efficientextractionproceduresandreducingtheproblemsassociated with inhibitory compounds that are present i n complex food matrices. I n 1996. Atmar et al. (152) described a multicenter, collaborative trial that was performed to evaluate the reliability and reproducibility of a reverse transcription PCR method ( 153) for the detection of Norwalk virus in shellfish tissues. The sensitivity and specificity of the assays were 85% and 9170, respectively, when results were detcrmined by visual inspection of ethidium bromide-stained agarose gels. The test sensitivity and specificity improved to 87% and 100%afterconfirmation by hybridizationwith a ctioxigenin-labeled,virusspecific probe.
3. Group A Rotavirus CL Disctrsc.. Rotaviruses are members of the Reoviridae family. They have a genome consisting of l l double-stranded RNA segments surrounded by a distinctive twolayered protein capsid. Particles are 80 nm in diameter. Six serologic groups have been identified, three of which (A, B, and C ) infect humans. Rotaviruses cause acute gastroenteritis. The most widesprcad group A rotavirus is also known as infantile diarrhea, winter diarrhea. acute nonbacterial infectious gastroenteritis, and acute viral gastroenteritis. Rotavirus infection is the most common cause of dehydrating diarrhea in children in the United States. Rotavirus, the most important cause of pediatric gastroenteritis in the United States, is responsible for an estimated one-third of all hospitalizations for diarrhea in children less than 5 years of age (154). Rotavirus gastroenteritis is a mild to severe disease characterized by vomiting. watery diarrhea, and low-grade fever. The infective dose is presumed to be low, from 10 to 100 virus particles. Since a person with a rotavirus diarrhea excretes large numbers of virus particles ( IOs-lO"' particles/ml of feces), infectious doses can easily be acquired from contaminated hands or utensils ( 154,155).
h. Ecology. Rotavirusesarctransmitted by thefecal-oralroute.Person-to-person contact is thought to be the most common means of transmission. Rotaviruses are very stable in the environment ( 154,155). These viruses do not appear to be the most frequent virus type associated with seafood.
Pathogens Transmitted by Seafood
159
c. Microbiolo,yiccrl Procedures ~rrrdDetection. Specificdiagnosis of the disease is made by identification of the virus in the patient's stool. Enzyme immunoassay is the test most widely used to screen clinical specimens, and several commercial kits are availableforgroupArotavirus.Electronmicroscopyandpolyacrylamidegelsareusedin some laboratories. More recently a reverse transcription PCR (RT-PCR) method has been developed to detect all three groups of human rotavirus (156-158).
4. Other Viruses:HumanAstrovirus,HumanAdenovirus, and Poliovirus Other viruses may also be found associated with shellfish, including human astrovirus, human adenovirus, and poliovirus. These viruses have notbeenreported as being frequently associated with seafood.
111.
BACTERIAL AND VIRAL PATHOGENS PRIMARILY ASSOCIATED WITH IMPROPER PROCESSING OR HANDLING OF SEAFOOD
A.
Bacteria
1.
Clostridium botulinum
(7. Diserrsc~. Foodbornebotulism, as distinguishedfromwoundbotulism andinfant botulism, is an extremely severe form of food poisoning caused by the ingestion of foods containing a potent neurotoxin that is formed during growth of Clostridium botulir u m . The toxin is heat sensitive and can be destroyed if food is heated to 80°C for 10 minutes or longer. The incidence of the disease is low, but the mortality rate is very high if the infected patient is not treated quickly. Approximately 10-30 outbreaks are reported in the United States each year, and these are mostly associated with inadequately processed home-canned foods. Some cases of botulism may go undiagnosed because symptoms are transient or mild or are misdiagnosedas Guillain-Barr6 syndrome. Seafood products have been involved in botulism outbreaks ( 126,159,160). C. b o t r r l i r r u r r r is a gram-positive, obligately anaerobic, spore-forming rod that may produce a neurotoxin. Seven types of C. botulirzrrrn (A, B, C, D, E, F, and G) are recognized, based on the antigenic nature of the toxin produced. Types A, B, E, and F cause human botulism, and typeE is mostly associated with seafoods in the United States. Different types of C. botrrlitrum are found in different areas of the world. Overall, type E is infrequcntly found around the world, but predominates in northern regions and in most temperate aquatic environments. This type is found in high numbers around the Great Lakes and in the coastal areas of Washington and Alaska. Along the Pacific coast of the United States, however, the prevalent type shifts from E to A and B below latitude 36"N. In Europe, type B predominates in the aquatic environments around the United Kingdom, while in other aquatic environments, type E is most frequently found. Surveys of Asia report high levels of type E spores around the Caspian Sea and in the northern areas of Japan. I n tropicalareas of Asia,typesCandDarefoundathigherlevelsinaquatic environments thantype E. Nanogram quantities of botulinum toxin can cause illness. Onset of symptoms in foodborne botulinum usually occur 18-36 hours after ingestion of the toxic food, although this can vary from 4 hours to 8 days. Early signs of intoxication
160
Herwig
include marked lassitude, weakness, and vertigo, usually followed by double vision and progressive difficulty in speaking and swallowing. Difficulty in breathing, muscle weakness, abdominal distention, and constipation may also occur ( 126,159,160). Storage of fresh and processed fish using modified atmospheric packaging (MAP) has gained widespread application in Europe. The technology has been approached more cautiously in North America because of the risk associated with C. borulinum E. The conditions often encountered in MAP fish products are conducive to the growth andtoxin production of C. borulinurrr E evenat temperatures as low as 33°C (161 ). Mild temperature abuse conditions of MAP fish products are of additional concern. Furthermore, the restricted growth of normal spoilage organisms may enhance the growth of C. horulitrum. These possibilities were shown by Eklund ( 162) in sallnon inoculated with C. b ~ t u l i t ~ ~ t t ~ spores and stored under MAP conditions. While spoilage was delayed for 10 days, toxin production occurred earlier. Whether toxin production precedes or follows spoilage is debated by some, but there is little question that with mild temperature abuse, botulinum toxin can be detected prior to organoleptic deterioration. To ensure the safety of MAP fish, strict temperature control at low temperatures (
h. Ecology. Vegetative organisms and spores of C. botulir~utr~ arewidelydistributed in nature. The organism requires an anaerobic environment for growth,but the spores are resistant to a wide variety of environmental perturbations. Organisms and spores have of streams, lakes, and been found in forest soils, cultivated soils, the bottom sediments coastal waters, in the intestinal tracts of fish and mammals, and in the gills and viscera of crabs and other shellfish. Since C. botulirlutn is so widely distributed i n nature, the food industry and food consumers have learned to take measures to prevent the growth of C. botulit7um cells from spores and toxin production. Two major mechanisms are used to prevent foodborne botulism: (a) maintaining the pH at less than 4.6 and (h) utilizing a heat treatment during food processing to kill C. boruliturrrr spores and vegetative cells. Smoked fish, both hot and cold smoked (e.g., Kapchunka) have caused outbreaksof type E botulism (126,159,160). In 1987, eight cases of type E botulism occurred, two in New York City and six in Israel. All eight patients had consumed Kapchunka, an uneviscerated, salted, air-dried, whole whitefish. The product was made in New York and transported by individuals to Israel. All eight patients developed symptoms within 36 hours of consuming the Kapchunka. One patient died, two required breathing assistance, three were treated with antitoxin, and three recovered spontaneously. The Kapchunka contained high levels of botulinum type E toxin, despite the high levels of salt that should have prevented the growth of the organisms. One possible explanation was that the uneviscerated fish contained low levels of salt when air dried at room temperature, became toxic, and then were rebrined. of Kapchunka Regulations were publishedto prohibit the processing, distribution, and sale and Kapchunka-type products in the United States (164).
c. Microbiologiccrl ~ n Detection d Plncec1ure.s. Examiningsuspectfoodsforbotulinum toxin involves using procedures to detect and identify the toxin. The most widely accepted method is to inject extracts of the food into passively immunized mice, called the mouse neutralization test. This animal assay takes 48 hours to complete. The severe and sometimes fatal outcome caused by C. botulinunr toxins requires rapid identification of the neurotoxin so treatment can begin and public health agencies can minimize the spread of the illness. Current animal tests and other bioassays may take days to complete, too long a time when the seriousness of the consequences are considered. Following this
Pathogens Transmitted by Seafood
161
assay, suspect food can be cultured in an enrichment medium for the detection and isolation of C. botulinurn. The microbiological procedure takes at least 7 days to complete. Fig. 24 outlines a protocol (159) for screening smoked fish for viable vegetative cells or spores of C. botuliwum.
2. Staphylococcus aureus (1. Disease. Srcrl,h),lococcus clweus foodpoisoning is caused bythemicrobial production of a toxin. The onsetof symptoms is usually very rapid and acute, but depends on the amount of toxin that is consumed and the susceptibility of the individual to the toxin. The symptoms include nausea, vomiting, retching, and abdominal cramps. Recovery generally takes 2 days, but severe cases may take longer. Death from staphylococcal food poisoning is very rare. The infective doseis less than 1 pg of toxin, a level that is achieved when S. nureu.7 populations exceed 1 Os/g ( 132,165,166). Staphylococcal food poisoning is not particularly associated with seafood or fisheries products, except when seafood isan ingredient in the preparationof salads. It is not associated with raw shellfish. Foods that require considerable handling and are maintained at warm temperatures are frequently involved in staphylococcal food poisoning (165,166).
b. Ecology. Humans and other warm-blooded animals are the primary reservoirs for S. clweus. This organism is normally present in the nasal passages, throats, hair, and skin of more than 50% of individuals. Foods that are maintained below 7°C or above 60°C will not support the growth of S. nureus (132,165,166). C. Microbiologicd Procedures. S. ( I U ~ ~ MareS gram-positivecocci that occur in pairs, short chains, or grape-like clusters. Some strains producea heat-stable protein toxin thatcausesillness in humans.Fig. 25describes a directplatingmethodandan MPN method for the enumeration of S. [rureu.s in food samples (165).
Place smoked fishin water-tight plastic bag andadd Trypticase Peptone GlucoseYeast Extract (TPGY) broth Remove air from bag and seal Incubate 5 days at 28°C
Remove 20 ml of enrichment and centrifuge Adjust pH if necessary I
Add 5% solution of trypsin to culture broth
t
Prepare dilutionsof trypsinized and nontrypsinized broth and Inject mice Observe mice for botulism and record observations Death of mice is presumptive evidence
+
Confirm botulism toxin using mice protected with toxin antisera
Fig. 24 Protocol for testing smoked fish for C/o.stridium hotulinunl and production of toxin (159).
162
Herwig Direct Plate Count Method Weigh 50 g of sample and add 450 ml of Phosphate Buffer Saline (PBS) Blend for 2 minutes
+ +
Using 1 ml of homogenate, inoculate 3 platesof Baird-Parker agar by spreadplating Incubate 45-48 hat 35°C
Count colonies tybicalfor S. aureus
Select more than one colony of each type and test for coagulase
t
Perform additional biochemical tests Catalase Glucose, anaerobic utilization Mannitol, anaerobie utilization Lysostaphm sensitivity Thermostable nuclease production
MPN Method
+ + +
Prepare sampleas above
Using blended sample, inoculate MPN tubes containing Trypticase Soy Broth (TSB) + 10% NaCl and 1% sodium pyruvate Incubate 48 k 2 h at 35°C
Transfer loopful from each turbid tube to Baird-Parker agar Incubate 48 h at 35°C
Select colonies typicalfor S. aureus test for coagulase and perform additional biochemical tests
3. Streptococcus iniae
N. Disease. Thegenus Str'eytoL'oc'c'u.7 is comprised of gram-positive,microaerophilic cocci that are not motile and occur in chains and pairs (25). S. iwiw is a pathogen for aquatic animals, but it was recently reported to cause serious disease in humans. The pathogen is associated with live finfish culture and aquaria. Frequently, in seafood markets
Pathogens Transmitted by Seafood
163
and restaurants in Asia or in Asian markets locatedin Western countries,finfish are maintained live in aquaria. Humans that contact these fish may be exposed to S. itliere. h. Ecology. S. itliere was firstreportedin1976 to causesubcutaneousabscesses in Amazonfreshwaterdolphins at aquariums in San Francisco and New York. Later, meningoencephalitis outbreaks caused by streptococci have been recognized in a variety of cultured finfish, including tilapia, yellowtail, rainbow trout, and coho salmon. S. itliae may colonize the surfaceof fish or cause invasive disease associated with 30-50% mortality rates (167-169). S. itliere is a bacterium recently linked to intensive recirculation tank systems in the United States. A virulent strain has been isolated from humans: this infected several customers who purchased live Tilupia sp. and presumably became infected through an open wound while cleaning the fish. While most of the problem are associated with skin lesions, systemic infections have been reported in Canada (167,168). During the winter of 1995-1996 in the Toronto area, a cluster of four cases of invasive S. itliae infection occurred in peoplewho hadrecentlyhandledfresh,wholefishfrom a farm.Results of surveillance identified a total of nine patients with invasive S. itzie~einfection (cellulitis of the hand in eight and endocarditis in one). All the patients had handled live or freshly killed fish, and eight had percutaneaus injuries. Six of the nine fish were tilapia, which are commonly used in Asian cooking. All of the patients were of Asian descent and all the patients reported preparing whole, raw fish. Eight patients recalled injuring their hands by puncturing the skin with the dorsal fins, a fish bone, or a knife used to clean the fish. Fish were purchased from markets where they were maintained live and were killed and gutted when purchased. The head, tail, and fins were left on the fish carcass. Cellulitis developed within 16-24 hours after the injuries.All of the patients responded to antibiotics within 2-4 days. The investigators concluded that S. irzine can produce invasive infection after skin injuries during the handlingof fresh fish grown by aquaculture. Using molecular typing methods, the authors identified a clone of S. itline that causes invasive disease in both humans and fish ( 167- 169). This organism may be an emerging pathogen since it was only recently identified as a pathogen in fish produced by the aquaculture industry.
c. Microhiologicd Procwlures. Routineexamination of patients,fish,andfishrearing ponds and aquaria for S. irlicre is not performed. Since the genus Streptococcus is of routine clinical importance, protocols for the identification of S. itlirre can be easily performed. Characteristics used to identify streptococci as S. itliere are that they are phemolytic ontrypticasesoyagarwith 5%) sheep's blood; they are not groupable with Lancefield groups A-V antiserum; they are susceptible to vancomycin; they do not produce gas, are nonmotile, are positive for pyrolidonyl arlamidase and leucine aminopeptidase; and they producc negative results on bile esculin, Voges-Proskauer, and hippurate tests. Not surprising for an environmental organism, most strains grow at 10°C but not at 4S"C, and they do not grow in 6.5% NaCl. Surface swabs from fish can be inoculated onto colistin-nalidixic acid blood agar and incubated at35°C in 5% CO: for 18-24 hours ( 158,169). Public health agencies and officials need to be aware of S. itzicre, a pathogen emerging in the past few years. B. Viruses Viral pathogens described in a previous chapter section may also be associated with seafood that has been improperly processed or handled. These pathogens primarily include
Henvig
164
hepatitis A, human calicivirus, and group A rotavirus. Viral pathogens may be introduced by fecal contamination of the seafood from an infected food handler or from processing waterorfoodingredients thatare contaminated withfecalmaterial. In addition,viral pathogens that are present on raw products may not be inactivated if the contaminated seafood receives insufficient cooking.
W. DEVELOPMENT OF RAPID METHODS FOR DETECTION OF SEAFOOD-BORNE PATHOGENS Much has been published and discussed about the development of rapid methods for the detection of pathogenic microorganisms. While a “dip stick,” “early pregnancy” style prototype kit is desired by regulatory agencies and food processors, the science for this technology is not presently at a stage for use on most pathogens, but recent work appears to be very promising. Improvements in sensitivity and speed of analysis have occurred. Most of theimprovementsandresearchefforts inrecentyearshavefocusedonPCR technologies. Hill (170) and Scheu et al. (17 1 ) provided extensive reviews on PCR and its applications for detectionof foodborne pathogens. Muchofthe difficulty in implementing PCR for the analysis of food samples lies in the problems associated with preparation and purification of DNA templates from food matrices. If most of these problems can be resolved, PCR technologies offer an investigator the opportunity to quickly identify specific pathogenic species or strains and the presence of specific genes associated with pathogenesis. DNA canbe extracted from food samples and thereis no need to perform enrichmentsteps.Hill (170) also listsmanyPCRprimers,amplificationconditions,and references for detecting a wide variety of foodborne pathogens. Limitations of the PCR method have been discussed in previous reviews ( 170,171) and articles. Two major limitations are the differentiation of living and nonliving cells, and the inhibitionof the PCR by food. PCR protocols maynot differentiate between viable and nonviable microorganisms. Amplification methods only indicate whether the target sequence of DNA is present, and not whether the organisms are alive or dead. DNA from dead microorganisms may leadto false-positive results. One way to minimize the detection of dead cells is a preenrichment step prior to the PCR analysis. This step increases the sensitivity of the test and restricts detection to culturable cells (172). The presence of intact RNA as a sign of active metabolism mayalso be a valuable indicator of the viability of the microorganisms (171). Messenger RNA (mRNA) can be detected as the template for be difficult to RT-PCR. Cells need to be rapidly extracted, howcver, and intact mRNA may isolate. Ribosomal RNA (rRNA) maybe a better target for analysis because of its higher stability. For most investigators, working with RNA is more difficult than working with DNA. A large problem in using PCR methods with foods is the apparent presence of PCR inhibitorsinfoods (171). False-negativeresultscanoccurforvariousreasons: (a) the presence of substances that chelate divalent magnesium cations, ions that are necessary for DNA polymerase, (h) degradationof nucleic acid targets or primcrs through nucleases, (c) substances that directly inhibit DNA polymerase. To reduce false-negative results it is very important for the investigatorto include the appropriate controls when developing and using PCR protocols. While most publications have described the detection of single pathogenic species
Pathogens Transmitted by Seafood
165
or strains, detection of several pathogens may be possible with a single test. For example, Wang et al. (173) showed the development of a protocol using universal culture medium and the same PCR conditions for detecting more thana dozen foodborne bacterial species in foods. Internal probe hybridization and nested PCR procedures were used as confirmation. Short enrichment culture procedures without DNA isolation may be the best approach because they are easy to perform and have a high sensitivity. Table 8 lists a variety of PCR systems that have been developed for the detection in seafood products. In several investigations, seafood of bacterial and viral pathogens was among the different foods for which the molecular method was developed. A more comprehensive table that was published by Scheu et al. (171) lists seafoods and other types of foods. For many of the listed protocols the detection limit is extremely low, between 1 and 100 cells/g. While PCR methods are very attractive because of their sensitivity and speed, especiallywhen compared to traditional enrichment and cultural methods, these methods may not be practical for routine analyses performed by the quality control laboratories present in seafood companies and other small agencies. Several of the protocols use radioactive probes and require the skills of laboratory personnel specially trained in molecular techniques such as PCR, gel electrophoresis, and restriction digests of DNA.
V.
CONCLUSIONS AND RECOMMENDATIONS
A.
E. colias anIndicator Organism
Routine monitoring for fecal coliforms or E. c ~ o l iare inadequate for protecting the public againstmostseafood-bornebacterialandviralpathogens.Depuration of contaminated shellfish in clean water may rcmove bacterial contaminants, butmaynot be a reliable method for the elimination of viral contamination (106). Several authors have suggested that routine screening of bivalves for the presence of health-significant enteric viruses before public consumption may help to prevent disease outbreaks among shellfish consumers. Most current regulations concerning the contamination of shellfish and their growing waters are based solely on bacteriological standards, primarily E. coli or fecal coliforms. This aspect is of concern because many bacterial organisms failto provide a reliable indication of the virological quality of the bivalve shellfish and their waters (146).
B. Consuming Raw Seafood The consumption of raw animal protein is inherently an activity with potential risk and consumers should be informed about the types of seafood and processing procedures where the risks maybe unacceptable. If people refused to consume raw animal protein and regulatory agenciesprohibitedthesale of suchproducts in foodserviceestablishments,the number of cases and outbreaks associated with infectious bacteria would be greatly reduced. Many people, particularly those with liver or other blood disorders, become seriously ill and diewhen they consume raw oysters. In the United States, three states (Florida, California, and Louisiana) require warning labels or signs where raw oysters are sold. Fig. 26 shows the warning labels that are required in two states. Should this labeling be required i n all restaurants and stores that sell raw oysters? Some would argue that widespread warnings would make little difference. The long and active campaign against cigarette smoking serves as a vivid example. Klontz et al. (174) suggested that the consumer
Table 8
PCR Systems for the Detection of Bacterial and Viral Pathogens in Seafood Productsq
Organism Bacterial pathogetis Clostridium hotuliriuni A3.E
E. coli 0157
E. coli enteroinvasive Listeria nionocyrogeries L. monocytogerzes L. monocytogetzes
Type of sample Milk, meat juice. canned tuna, mushrooms. sausage Raw hamburger. raw milk, raw oysters, bean sprouts Seafood. greens, dairy products 250 different food types Cold smoked salmon Meat products, vegetables. seafood
Amplified gene
Detection system
Detection limit
Reference
Botulinum neurotoxin genes
Gel
4 cfu/g following 48hour enrichment
209
sltl, sltII (vertoxin 1 and 2 genes)
Immunoinagnetic separation. multiplex PCR. gel Restriction enzyme digestion, gel Gel Nested PCR, gel
1 - 10 cells/g
210
10’ cfu/ml
21 1
100 cfu/ml 100 cfu/g
212 213
Gel
1 cfu/g following nonse-
214
lective and selective enrichments 2 x 10’to 2 x IO’cfu/g
215
40 cells
216
I - 10 cells/g after 3hours enrichment
217
Less than 40 cells/g
218
EIEC virulence plasmid Listeriolysin 0-gene prfA (transcription activat-
ing protein) h(vA (IisA. literiolysin 0 gene), iap (invasion associated protein) 16s rDNA
Salnioriella
Beef, fish, pork samples
Salnionella typlii
Salrnoriella spp.
Crabmeat salad, fried chicken. peppersteak, ratatouille salad, smoked mussels Oysters
himA
Salniotiella spp.
Oysters
1iti.s
5 s rDNA-23s rDNA spacer region
gene
Gel and “P-labeled oligonucleotide probe Gel
Gel and Southern blot. dot blot, radioactive ohgonucleotide probe Gel and radioactive oligonucleotide probe
X
8.s
Gel, Southern blot, fluorescein labeled oligonucleotide probe Multiplex PCR h/yA amplicon as internal control, gel nested PCR. gel Gel and radioactive oligonucleotide Nested PCR. gel Restriction endonuclease digestion of amplicons. gel
Vihrio cliolerue
Oysters. crabmeat, shrimp. lettuce
Cholera toxin operon
V. cholerae
Raw oysters
l i l y 4 hemolysin gene ctx cholera enterotoxin
Vihrio puruhaeriiolyticics
Shellfish
Cloned DNA fragment
Fish. water Oysters
2 3 s rDNA Cytol ysin
Oysters and clams
5‘ noncoding end
RT-PCR. gel
Oysters
RT-PCR, gel
Oysters, shellfish
Pan-enterovirus HAV capsid Polymerase gene
Oysters, clams
Polymerase gene
Shellfish
Polymerase gene
Virol pcifhogens Enteroviruses. polioviruses Enteroviruses. poliovirus 1, hepatitis A virus Enteroviruses, polio virus I, HAV, Nonvalk virus Nonvalk virus
Round structured viruses
&Tablemodified after Scheu et al. (171).
RT-PCR, restriction endonuclease digestion, gel RT-PCR, restriction endonuclease digestion, gel RT-PCR, gel, Southern blot, internal oligonucleotide probe
10 cfulg following an 8hour enrichment
219
30 cfulg 3 cfulg after 6-hours enrichment
220
9 cfu/g after 3-hours enrichment 12- 120 cells/PCR 10’ cfulg after 24-hours enrichment
221
2 pfulg 0 pfu/polio virus/PCR 295 pfu HAVIPCR OL-lO” pfulml
48,222 223
224 225.226 227
228 229
168
Henvig
Florida(FloridaAdministrativeCode
10D-3.091 ( 6 ) a.)
“Consumer information: There is risk associated with consuming raw oysters or any raw animal protein. If you have chronic illnessof the liver, stomachor blood, or have immune disorders, you are at greater riskof serious illness from raw oysters, and should eat oysters fully cooked. If unsure of your risk, consult a physician.”
California(CaliforniaCodeofRegulationsTitle
17)
“Eating raw oysters may cause severe illness and even death in persons who have liver disease (for example alcoholic cirrhosis), canceror other chronic illnesses that weaken the immune system. If you eat raw oysters and become ill, you should seek immediate medical attention. If you are unsure if you are at risk, you should consult your physician.” Fig. 26 Warning labels for raw oysters from Florida and California.
of raw oysters is, by nature, a “risk-taker.” Performing a survey in Florida, they found that the prevalence for eating raw oysters was higher for men18-49 years old. The prevalence was even higher for persons who reported being cigarette smokers or acute or chronic alcohol drinkers.
C. Transport of Seafood Fresh, raw, and potentially contaminated seafood is often transported long distances from the location of harvest or processing. Physicians, emergency room, and other medical personnel who work in these distant areas need to be aware of seafood-borne disease. Several isolated cases have been reported where individuals who consumed contaminated seafood in “nonseaside” communities have died from V. vulnijcus. For example, a 61year-old man with adult-onset noninsulin-dependent diabetes died after consuming raw oysters in Louisville, Kentucky that were harvested in Florida ( 175). In Oklahoma, another man with underlying liver disease and a history of eating raw oysters died within15 hours of arriving at the hospital of V. vuln$cus sepsis (176). For V. vuln$cus infections, early treatmentwithantibioticshasbeenshown to improvesurvivalinsepticemicpatients ( 177,178), so prompt recognition of the infection by physicians is critical to reduce mortality.
D.
V, parahaemolyticus and V. vulnificus as Notifiable Diseases
In the United States, federal and state public health agencies need to consider adding V. I’““ahaernol~,ricu.~ and V. vuln$cus to the list of infectious diseases that are notifiable. In 1997,52 infectious diseases were designated notifiable as at the national level in theUnited States (8). Of these, only a few are associated with food, including botulism, cholera, cryptosporidiosis, E. coli 0157:H7, hepatitis A. salmonellosis,shigellosis,andtyphoid fever. Somewhat ironic is the fact that, in the United States during the past I O years, the
Pathogens Transmitted by Seafood
169
number of deaths caused by V. w l t ~ j f i c x exceeded s the number caused by foodborne botulism, yet V. \wln(fifiCv.sis not notifiable to the CDC. Also, reporting of nationally notifiable diseases to the CDC by individual state governments is voluntary, not mandatory. Reporting is only mandated at the state and local levels. Thus the list of diseases that are notifiable varies slightly between states. The FoodNet program in the United States is a good start, since all Vibrio species are reported, but only a limited number of states and counties are participating in this new program.
E. International Trade in Seafood and Public Health Many species of raw and processed fish and shellfish are being imported and exported from many different countries around the world. Regulatory agencies face a very difficult challenge in attempting to keep all of the imported, along with domestically produced seafood products, safe for human consumption. Consumer education programs need to inform the public about the potential risks and the procedures that can be followed in the home and in retail establishments to reduce or minimize possible seafood disease risks. The United States imports more than 50% of the total consumed seafood from 172 countries, originating from about 6000 foreign firms (179). In December 1997, the FDA required ail seafood processors to adopt a quality assurance program based on the HACCP concept. These measures arealso being applied to seafood importers. HACCP focuses on preventing hazards ratherthan relying on spot-checks and the random sampling of products at the endof the production line. Under HACCP each food processor and importer prepares a plan for identifying and monitoring the steps of their operation that may contribute hazards to human health. While the seafood industry, regulatory agencies, seafood technologists, and scientists embrace the principals and objectives of HACCP, the effectiveness of these measures i n reducing the incidence of seafood-borne pathogenswill probably not be known for several years. The dauntingtask faced by the FDA was highlighted in April 1998 when the U.S. General Accounting Office (GAO) released a report entitled “Food Safety: Federal Efforts to Ensure the Safety of Imported Foods are Inconsistent and Unreliable.” The GAO report stated that the FDA inspects less than 2% of all food imports, including seafood imports. With such a low level of inspection, the proactive approach of HACCP may be the only logical way to keep seafood, and other food commodities, as safe as reasonably possible for human consumption.
F. The Rise of Aquacultured Seafood Freshwater and marine aquacultured products are becoming increasingly important in the production of seafood. Studies may need to be performed to see if new bacterial and viral pathogens are associatedwith these products.To date,no one has foundthat these products al. are inherently more hazardous thanwildfish and shellfish. For example, Pullela et ( I 80) examined the nature and number of indicator and pathogenic microbes in fish reared using recirculating and nonrecirculating freshwater systems. L. monocyfogenes, Y. enterocolitictr, E. coli 0 157:H7, and Salmonella spp. were not isolated. C.botulinunr, however, was isolated from all the aquacultured fish sampled except pacu and tilapia grown in a recirculating aquaculture system.
170
Herwig
G. Importance of Environmental Quality For the survival and success of their industry, fish and shellfish harvesters and processors have a stake in water and sediment quality. This chapter focused on microbial pathogens that are associated with seafood. Many of these pathogens are introduced by pollution into fish and shellfish in the aquatic environment. The better the environmental quality where the seafood is harvested, whether in an aquaculture farm, an intertidal beach, or in the open sea, the less likely that microbial or chemical contaminants will cause problems for the seafood consumer.
H. Passive Transport and Introduction of Pathogenic Organisms The transport of pathogenic microorganisms from one part of the world to another in the ballast water and sewage of large ships is alarming and probably requires consideration. Most of the attention by the popular press in recent years has focused on the transport of exotic species of invertebrates and plants, suchas zebra mussels and European green crabs, from one part of the world to another. McCarthy and Khambaty ( 18 1) recovered toxigenic V. clzolerne 01, serotype Inaba, biotype El Tor from ballast, bilge, and sewage water from five cargo ships docked in ports of the U.S. Gulf of Mexico. Four of these ships had taken on ballast water in cholera-infected countries; the fifthtookonballastina noninfected country. Isolates examined by pulsed-field gel electrophoresis were indistinguishable from the Latin American epidemic strain, C6707. On the basis of their findings, the FDA recommended that the U.S. Coast Guard issue an advisory to shipping agents and captains requesting that ballast waters be exchanged on the high seas before entry of ships into U.S. ports. More recently, following the largest V. p c l m h n e m n l ~ t i coutbreak ~~.~ in North America in many years, in which more than 400 cases were reported, the FDA began an investigation of shellfish contamination causedby ballast water discharged from ships. This suspicion was spurred by the fact that the serotype isolated(03:K6) is common in Japan, India, and Taiwan, but had never before been detected in Texas waters (182).
1.
Development of Test Kits
The development of rapid and relatively simple test kits for seafood-borne pathogens, biotoxins, and hazardous chemicalsis an area of research that needsthe support of governments, the research community, andthe seafood industry. T o help importers monitor shipments for HACCP compliance, several companies have developed rapid test kits. For example, Neogen offers akit that tests histamine levels using an enzyme-linked immunosorbent assay (ELISA) and yields visual color results in 1 hour (183). Colwell and her associates at the University of Maryland have developed protocols and kits for the rapid detection of V. cholerne 01 and V. cholerne 0139 (184-1 86), and a kit is now commercially available (Cholera SMART).
J.
Relative Risks Associated with Consumption of Seafood
Although a varietyof foodborne diseases are associated withfish and shellfish, the number of human fatalities that occur each year, particularly in developed countries, from the
171
Pathogens Transmitted by Seafood
Table 9 WorldWideWebSitcswithInfortnationaboutPathogensAssociated with Seafoods
URL
Site Description ~~
U.S. Food and Drug Administration Seafood Information and Resources. Center for Food Safety and Applied Nutrition Thc Bad Bug Book The National Food Safety Database Centers for Disease Control and Prevention Emerging Infectious Diseases. A peer-reviewed journal that is available on the Internet, produced by the National Center for Infectious Diseases, CDC FoodNet Interstate Shellfish Sanitation Commission Seafood Network Infortnation Center (SeafoodNIC), Sea Grant Extension Program, University of California, Davis National Scafood HAACP Alliance for Training and Education
~
~~
~~~~
www.fda.gov vm.cfsan.fda.gov/seafood 1 .html
vm.cfsan.fda.gov/-mow/intro.httnl
www.foodsafety.org www.cdc.gov www.cdc.gov/ncidod/EID
www.cdc.gov/ncidod/dbmd/foodnet
www.issc.org seafood.ucdavis.edu
seafood.ucdavis.edu/haccp/ha.htnl
consumption of fish and shellfish is extremely low compared to the mortalities caused by other infectious and noncommunicable diseases. Other routine activities performed daily by humans around the world are much more hazardous than consuming seafood. Large differences certainly exist between developed and developing countries. For example, in developed countries infectious and parasitic diseases account for 1% of the deaths each year. In developing countries this figure is much larger, 43%. Some of these infectious disease agents may be associated with seafood.
VI.
ADDITIONAL SOURCES OF INFORMATION: WORLD WIDE WEB SITES FOR PATHOGENS ASSOCIATED WITH SEAFOODS
Several agencies in the United States provide large amounts of information for the consumer or educator interested in seafood safety and the characteristics of seafood-borne pathogens. Much of this information is freely available on the Internet or by contacting the agencies by telephone or mail. Table 9 lists the URL for several of the sites related to seafood safety.
ACKNOWLEDGMENTS I would like to acknowledge the assistance of Robyn Estes and Jennifer McLarnan, who searched the University of Washington libraries for the nun1erous articles that were re-
Herwig
172
quired to prepare this review. Unpublished data about the number of cases of shellfishborne disease caused by Vibrio species were kindly provided by Marc Glatzer, shellfish specialist at the U.S. Food and Drug Administration, Tallahassee, Florida.
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methods for the detection of Listeria morm-ytogerres in cold-smoked salmon. Appl Environ Microbiol 622322424, 1996. C Niederhauser, C Hofelein, J Luthy, U Kaufmann, HP Buhler, U Candrian. Comparison of “Gen-Probe”DNAprobeandPCRfordetection of Listericc nronocytogenes innaturally contaminated soft cheese and semi-soft cheese. Res Microbiol 144:47-54, 1993. K Wernars, CJ Heuvelman, T Chakraborty, SH Notermans. Use of the polymerase chain reaction for direct detection of Listeria rrlonocytogews in soft cheese. J Appl Bacteriol 70: 121-126,1991. Q Zhu, CK Lim, YN Chan. Detection of Saln~or~ella tyyhi by polymerase chain reaction. J Appl Bacteriol 80:244-25 1, 1996. AK Bej, MH Mahbubani, MJ Boyce, RM Atlas. Detection of Scrlrmnella spp. in oysters by PCR. Appl Environ Microbiol 60368-373. 1994. DD Jones, R Law, AK Bej. Detection of Seclrrwnelltr spp. in oysters using polymerase chain reaction (PCR) and gene probes. J Food Sci 58:1191-1197, 1993. WH Koch, WL Payne, BA Wentz, TA Cebula. Rapid polymerase chain reaction method for detection of Vibrio cholerne in foods. Appl Environ Microbiol 59:556-560, 1993. YH Shangkuan, YS Show, TM Wang. Multiplex polymerase chain reaction to detect toxigenic Vibrio cholerae andtobiotype Vibrio choleme 01. JApplBacteriol79:264-273, 1995. CY Lee, SF Pan, CH Chen. Sequence of a cloned pR72H fragment and its use for detcction Iof Vibrio parcthaenlolyticus in shellfish with the PCR. Appl Environ Microbiol 61:131 1317,1995. CR Arias, E Garay, R Aznar. NestedPCR method for rapid and sensitive detectionof Vibrio wlnijicus in fish, sediments, and water. Appl Environ Microbiol 61:3476-3478, 1995. WE Hill, SP Keasler, MW Trucksess, P Feng, CA Kaysner, KA Lampel. Polymerase chain reaction identification of Vibrio vulnijicus in artificially contaminated oysters. Appl Environ Microbiol 57:707-711. 1991. of a method for detectionof enteroviruses DN Lees, K Henshilwood, WJ Dore. Development as amodel.ApplEnvironMicrobiol 602999-3005, inshellfishbyPCRwithpoliovirus 1994. LA Jaykus, R De Leon, MS Sobsey. Application of RT-PCR for the detcction of enteric viruses in oysters. Water Sci Techno1 27:49-53, 1993. H Chung, LA Jaykus, MD Sobsey. Detection of human enteric viruses in oysters by if! vivo and in vitro amplification of nucleic acids. Appl Environ Microbiol 62:3772-3778, 1996. RL Atmar, TG Metcalf, FH Neill, MK Estes. Detection of enteric viruses in oysters by using the polymerase chain reaction. Appl Environ Microbiol 59:631-635, 1993. V Gouvea, N Santos, M do Carmo Timenetsky, MK Estcs. Identification of Norwalk virus in artificially seeded shellfish and selected foods. J Virol Meth 48:177-187, 1994. C1 Gallimore, DW Brown.Detectionofsmallround DN Lees, K Henshilwood, J Green, structuredviruses inshellfishbyreversetranscription-PCR.ApplEnvironMicrobiol61: 4418-4424,1995.
6 Laboratory Methodology for Shellfish Toxins
Introduction I83 11. Methods of PSP Testing185 A. Bioassay methods 185 B. Chemical methods 190 C. Immunological assays 192 111. Domoic Acid Testing 194 A. Bioassays 195 B. High-performance liquid chromatography (HPLC) C. Capillary electrophoresis 197 Gas chromatography D. 197 E. Radioassays 197 F. Enzyme-linked immunosorbent assay (ELISA) 197 IV. Conclusions 201 References20 1 I.
1.
196
INTRODUCTION
Paralytic shellfish poisoning (PSP) occurs following the consumption of shellfish that have been exposed to toxic marine dinoflagellates and contain a potent source of neurotoxins, called paralytic shellfish toxins (PSTs) ( l ) . PSTs consist of at least 18 different toxins that can be divided into subclasses containing hydrogen atom (e.g., saxitoxin) or hydroxyl (neosaxitoxin-relatedtoxins)groupsontheR1position.Gonyautoxinstoxins (GTXs), are characterized by 1 l-hydroxy-saxitoxin sulfate derivatives (Fig. 1). Dinoflagellates are important ocean plankton that contribute to the foundation of the marine food chain as a primary source of carbohydrates, proteins, and lipids. The organisms are microscopic, single-cell photosynthetic algae, which during periodic maxima produce local discolorations of coastal waters. Shellfish poisonings are difficult to control because of the unpredictable and sporadic occurrence of the dinoflagellates which produce PSTs. The initial 183
184
Kitts
NH
Toxin
H H H
H OH H H H H H
H H
Saxitoxin Hc-N-OH'-saxitoxin H dc2saxitoxin saxitoxin doc3 Saxitoxin ethanoic acid OH Neosaxitoxin H c-N-OH-neosaxitoxin OH H dc OHneosaxitoxin OCONH2 GonyautoxinH 1 gonyautoxin-l OH dc H Gonyautoxin-2 gonyautoxin-2 H dc H Gonyautoxin-3 gonyautoxin-3 H dc OHGonyautoxin-4 gonyautoxin-4 OH dc H Goilyautoxin-5 OH Gonyautoxin-6 Cl c2
c3 c 4
RI
R2
H
CHzCOOH OCONH2 H
R3
I4
H3
OH
oso;
oso; OCONHZ oso; oso; OH OCONH2 oso; oso; OH
oso3' H H H H H
H H OH OH
oso;
H
oso3-
OH OCONH2 OH
oso; H
oso; H
OCONHSOY
ocoNHso; OCoNHso3' 0coNHso; OCONHSOi 0coNHso;
IC-N-OH:carbamoyl-N-hydroxy 2dc:decarbamoyl 'doc:deoxycarbamoyl Fig. 1 Saxitoxinandrelated PSP isomersassociatedwithparalyticshellfishpoisoning.
relationship between PSTs and the presence of plankton from the genus Gntzy1ulnx was reported by Sommer et al. (2). In fact, toxic dinoflagellate blooms occur sporadically in large numbers from as many as 20 species of dinoflagellates throughout the world, with the greatest frequencies occurring in the north and south temperate zones (3). Outbreaks of PSP occur in both protected and unprotected waters in which conditions arc prone to an upwelling of nutrient-rich water often associated with organic loadingof seawater from run off or sewage disposal (4), or unique changesin salinity, temperature, light, and water turbulence. Seasonal outbreaks of red tide vary in coastal regions, as evidenced by the
Laboratory Methodology for Shellfish Toxins
185
reported outbreaks between mid-May and late October alongthe Pacific Coast,in contrast to the period of Inid-July to late September in Atlantic Canada (5).Dinoflagellates represent a rich source of food for bivalve shellfish and changes in water conditions or the presence of predators,suchasdiatoms, will change thedinoflagellatepopulation.The principle group of shellfish associated with PSP outbreaks has been the bivalve mollusks, such as clams, mussels, scallops, and oysters. These species accumulate PSTs from poison011s dinoflagellates in the hepatopancreas and are then metabolized, excreted, or released within several weeks. One exception is the Alaskan butter clam (Srrsidorrlus Lqi,qtrrltc’/r.s), which mobilizes PSTs from the hepatopancreas to the siphon, where they can remain for of becolnillg several years (6). Shellfish become toxic with PSTs only after a few days exposed to a red tide bloom or extremely high concentrationsof poisonous dinoflagellates i n cohabitat waters. Other intertidal organisms such as gastropods, periwinkles. whelks, and certain crab specieshave also been foundto contain PSP (7-9). The blooms of Gor~yt r c r k r . ~species are often referred to as red tide b l o o m due to the color produced from a xanthophyll derived from light-harvesting antenna ( I O ) and the fact that the bloolns are 90-95% monospecific tothisspecies of dinoflagellatethatreachasninnyas 20.00030,000 organisndml of seawater. The distribution of PSP in shellfishisvariableand I n the case with red depends on the species of bivalve and the specific tissue involved. tide blooms, the correct environmental conditions produce a generation of algae that produce domoic acid poisoning, also referredto as amnesic shellfish poisoning (ASP). Subsequently, shellfish and finfish which feed by filtering seawater concentrate the algae and accumulate domoic acid toxin, principally i n hepatopancreatic tissue. As is the case with other sources of seafood toxins, the principle agent is confined to the viscera. Shellfish poisoning represents both a serious public health concern as well as having potentially devastating economic consequences where shellfish harvesting, tourism, recreation, and restaurants are important localindustries.Thistypeofpoisoningcannotbe completely controlled or prevented and the absence of an antidote has resulted in temporary incapacitating illness and even death. Although the number of mortalities attributed to PSP following consumption of contaminated shellfish is relatively low, the control of shellfish poisoning is largely the result of extensive monitoring procedures principally controlled by government health bodies. Since there is very little information on the predictors of when and where sourcesof the toxins may occur, PSP and ASP monitoring are imperative to ensure a safe and viable industry. This chapter describes the different laboratory methods that have been used to detect shellfish poisons and estimate toxicity.
II. METHODS OF PSP TESTING
A.
Bioassay Methods
1. Mouse Bioassay (Mouse Lethality Technique) The standard tnouse bioassay, initially developed by S o n m e r and Meyer ( l ) , is currently the official method for measuring shellfish toxicity ( 1 1). The procedure measures total toxicity of a shellfish sample by challenging, with intraperitoneal injection, white Swiss mice of standard size ( 1 8-22 g) and sex (mal?) with an acid extract of homogenized shellfish tissue (Table I ) . The mouse bioassay, which was adopted in 1965 as an AOAC method in Canada. remains the accepted procedure for the assessment of PSP toxicity. Time to death is recorded for 3-5 mice to screen samples and in 10-12 animals to assay specific samples expected to contain PSTs. The survival time of mice administered acidic
Kitts
186
Table 1 ProceduralInformation for BioassayandChemicalPSTDctection Prmcipal
Assay
Reference
Houselly Chlcken emhryo
Elcctrophoresis Thin layer chromatography HPLC
HA-ISP-MS
Total toxlclty (mean mousc death tllnc) LD,,, in housclly Total toxlcity ( % mortality) Morphology or vlability o f N2A cell enzymatic metabolism Separatm on cellulose acetate strips TLC wlth lluorescent detcctlon (KOH + H@:) HPLC-cyanocolurnn separation; perlodate Ruorcsccnt derivative Ion spray nuss spectrometry Capillary electrophorcSIS with UV detection
CE-UV
STX
saxitoxin;Neo
=
Limitations
=
neosaxitoxin: GTX
Sensitivlty 30 pg/ 100 g shelllish 0 . 2 pg PSP/mI 20 pg/100 g shelllish 0.11 pg STX 3.0 pmole/ml STX 2 pg STX 2 pg/100 g tissue 0.4 pg/ 100 g tissue No quantltation
0.5 ng STX, GTX-2. GTX-3. 8.0 ng NCO.GTX- 1-4
0.1 pM STX 5.0 pM STX, 5.6 I.IM Nco-STX
=
CV = -+ 20%intcrlah varlation = 60-70% Microtechnique 96-hour Incubation time Effects o f extraction procedure unknown
2.13.1618.2 1 23 24 3 1-36
S5
Volume limitations
45
Nonuniform Iluoresccnce intensities for all PSTs limited t o laboratory environment Spcc~alizcdequipment
50-54
60-6 1
56
gonyautoxlns.
shellfish extracts is indicative of the presence and the toxicity of PSTs in the sample, which in turn is expressed in mouse units. One mouse unit is defined, as the amount of poison that will kill a 20 g mouse in 15 minutes with symptoms of paralysis or respiratory failure. The use of the mouse unit (MU) as a measure of toxicity was derived initially before the availability of a reference saxitoxin standard and was calculated from the following expression: log dose (MU) = 145/r - 0.2 where MU
=
mouse units, and
t = time in minutes.
MU/ Shellfish quarantines are put in place when toxicity from PSTs reaches 400 100 g shellfish. Human illness has been reported at 600 MU/100 g shellfish and death caused by PSP can occur at 3000 MU (12 ) . Schantz et al. ( 13) were successful in preparinga highly purified source of saxitoxin from the digestive glands of toxic mussels (Myrilus ctrlifrrtrimlus) and also from the siphons of toxic Alaska butter clams (Sc1.ridoma.sgigcrnteus). The reference saxitoxin standard was determined to elicit a toxicity of 5500 -+ 500 MU/mg and was subsequently used as the reference standard for the official mouse lethality bioassay. One mouse unit was determined to be equivalent to 0.18 pg saxitoxin dihydrochloride poison required to kill a mouse in approximately 10-20 minutes. For example, a value greater than 70-80
Laboratory Methodology for Shellfish Toxins
187
pg PST/100 g fresh shellfish is considered unfit for human consumption in Canada. Under permit for canning purposes, shellfish may be taken from areas with PST levels of 80160 pg/100 g tissue, since the canning process may reduce the toxin content by 90% when analyzed by the AOAC method (14). The lethal PST dose for humans is I O mg of toxin from consumption of shellfish ( 15). This value is obtained from mouse units calcuto five mice are referred to lated to the nearest 5 seconds. Mean time to death for three in Sommer’s table, and the total toxicity from the lethality time is standardized against a known saxitoxin standard solution. Such factors as strain and sex of the mice, route of administration. and pH or presence of ions such as sodium have been evaluated on the acute median lethal dose (LD,,,) of saxitoxin (16,17). Examplesof calculated toxicity from three variable mouse death times are shown below: Example I : Average weight of mice (g) = 24.97 = I . l 1 MU Death time (min) = 4.27 = 2.32 MU Corrected mouse units
= 1.1 1
Toxicity = 2.575 MU
* 0.18 pg poison/MU
4:
2.32 = 2.575 MU Q
100 g meat
= 92.7 yg PST/ IO0 g meat
Example 2: Average weight of mice (g) = 24.50 = 1.10 MU Death time (min) = 7.906 = 1.25 MU Correctedmouseunits
= 1.10
Toxicity = 1.375 MU
x.
:C
1.25 = 1.375 MU
0. I8 pg poison/MU
:k
100 g meat
= 49.5 yg PST/100 g meat
Example 3: Average weight of mice (g) = 24.30 = 1 . I O MU Death time (min) = 1.82 = 9.50 MU Corrected mouse units
= 1.82 * 9.50 = 10.45 MU
Toxicity = 10.45 MU
:k
= 376.2
0.18 pg poison/MU
Q
100 g meat
yg PST/ 100 g meat
Female mice are more susceptible than male mice at higher doses of toxin, with the LDS,) per mouse being directly related to the average body weight of the test animal. Acute LD,(, values determined for oral (263 pg/kg), intraperitoneal (10 pg/kg), and intravenous (3.4 pg/kg) ingestion demonstrated the relative toxicity of PST for different routes of exposure. Increases in pH above 4.0, or the addition of sodium ions,will reduce intraperitoneal toxicity of saxitoxin, but has less effect on the oral or intravenous toxicity. Conversely, boiling extracts at low pH levels( 1.S-4.0) for up to I O minutes can have no effect on mowe toxicity (14). The volumeof injection medium containing the toxic lnaterial was that the shown to have no effect on the toxicity. It was concluded from these findings median time to death of mice exposed to low concentrations of PST as a criterion of toxicity was not reliable at pH values above 4, or in the presence of sodium ions above a 0.1 M concentration following intraperitoneal exposure. Further studies by Park et al. ( 1 8) have confirmed the importance of the final pH of the sample extract, especially when
188
Kitts
samples are stored for various times. Storage of standard solutions for as long as 4 weeks at 4°C and pH 6.0 produced a SO% decrease in toxicity, even after adjustment to pH 2 prior to testing. The limit of sensitivity for the mouse bioassay is approximately 30 pg/ 100 g shellfish tissue, with an accuracy of approximately 20% of the maximum allowable content (e.g., 80 pg saxitoxin equivalents/ 100 g sample) (19), a level set by both North American and European communities (20). Other researchers have claimed that the lnouse bioassay may underestimate marginally toxicshellfish by as much as 60% (21), while interassay variation in sample analysis can reach 2 2 0 % (22). The errorin toxicity estimation depends on the mice being within the 18-20 g weight range, and the subjectivity of estimating time to death. Moreover, the mouse bioassay is limited to accurately deternlining the potential toxicity of shellfish extracts containing sulfamate toxins, since the conditions of sample preparation are not sufficiently acidic to ensure complete hydrolysis of the toxin complex to a corresponding carbamate state. In addition to the technical limitations. the mouse bioassay requires large mouse colonies to supply mice for testing with a relatively uniform body weight. The lethality procedure is relatively expensive becauseof the need to maintain a specific mouse colony and therefore it is not suitable for use in field conditions. The method, albeit the only true indicator of total toxicity, represents an unacceptable procedure for animal welfare groups. which has resulted in further pressure for its replacement with a more humane method for reliably monitoring PST in large numbers of contaminated shellfish samples.
2. Other Bioassays There have been many attemptsto develop alternative bioassay methods for PST measurement and screening, including houseflies (23), chicken embryos, brine shrimp and selected microbial assays (24), selected microbial bioassays, and tissue biosensors (25-27). The housefly and chicken embryo assays have noteworthy limitations concerning sensitivity, relatively long incubation periods, and specialized techniques which preclude their use as alternativestothemousebioassay.Aradiolabeldisplacementassaywasproposed by Davio and Fontelo (28), which was based on the affinity of saxitoxin and other paralytic toxins to compete for nerve membranes. Samples were mixed with tritiated saxitoxin and incubated witha suspension of rat brain membranes. Following incubation, the mernbranes were washed and radjoactivity was determined. The concentration of PST in the sample was inversely proportional to the radioactivity recorded by liquid scintillation counting. This assay was reported to be very sensitive ( 1 000 times that of the mouse bioassay); however, the method did not measure total toxicity and there was a problenl finding shellfish samples sufficiently free of PSTs, to be run as negative controls. Similar solid-phase, radioreceptor binding assays have been developed for estimating cumulative PST toxicity (29,30). Good agreement i n estimating saxitoxin equivalents over a wide concentration range (0.5 pM-0.1 mM) of saxitoxinusingboththemousebioassay ( r = 0.946) and HPLC ( r = 0.878) has been reported with dinoflagellate toxins (30). Furthermore, this radioreceptor assay also correlated very well (0.967) with HPLC results on PST-contaminated clams, mussels, and oysters over a concentration range of 40-8000 pg STX/100 g shellfish. A tissue biosensor procedure designed to rapidly (e.g., S min) measure small quantities of PSTs (e.g., S fg STX) has also been reported (26). Thebiosensor, composed of a sodium electrode which covers a frog bladder membrane, measures the affinity of various shellfish toxins to block sodium channels. The active transfer of sodium across a frog bladder from the internal to external side of the membrane is detected by a sodium electrode within a flow cell (25,27). There is little information available concerning the
Laboratory Methodology for Shellfish Toxins
189
influence of marine conditions (e.g., salt concentration, temperature) on the perfornxmce of this procedure. An extension of the tissue culture methodology concept involving rapidly growing mouse neuroblastoma cells was initially developed by Kogure et al. ( 3 1 ). Similar strategies have been developed using bacterial cultures (32). The channel blocking potential of saxitoxin and related toxins is used to antagonize the toxic effects of added known agents (e.g., ouabain and veratridine) which stimulate sodium influx and depolarize the action potential on excitable membranes. The effect of the presence of PSTs enables nerve cell or bacterial growth to continuein culture. Evaluation of both the morphology and viability of neuro-2A (N2A) cells was reportedto be simple, inexpensive, and more sensitive than chromatography methods for saxitoxin and gonyautoxin (31). Jellett et al. (33,34) automated this method by using a microplate reader to measure absorbances of crystal violet from vital stained neuroblastonla cells exposed to both pure saxitoxin standard and acidic shellfish extract. The extract was processed through an LC-l8 SPE (Supelco) cartridge. Detectionlimitswere as low as 10ngsaxitoxinequivalentsandcorrelatedverywell with the mouse bioassay over a wide range of toxin concentrations (0-5500 pg/IOO g). Subsequent studies by Manger et al. ( 3 5 ) employed cultured neuroblastoma cells (N2A, mouse cells) using a microtiter plate format; however, this protocol relied on enzymatic colorimetric end-point measurements to assess PST toxicity. The neuroblastoma cells me(3-[4,5-dimethylthiazol-2-yl]2,5-diphenyltetrazoli~~1~~) tabolizethereducingagentMTT when positively correlated with the concentration of saxitoxin standard in a concentration range of 0-0.6 ng. Moreover, these researchersalso demonstrated that this bioassay would work well with tissue extracts collected from crab viscera and produced results i n close agreement with the standard mouse bioassay (36). A detailed comparison of the sodium channel cytotoxicity assay with the traditional mouse bioassay for PST detection in shellfish has been reported (37).
3. Macroscopic and Biotechnology Methods Toxin-producing dinoflagellates can be monitored using a light microscope and, therefore, in contrast to the mouse bioassay. may provide an early warning signal of impending toxic blooms. This allows cultured shellfish to be harvested or salmon ranchesto be moved away prior to an infestation of toxic algae (38). PST production is a stable characteristic in selected strains of dinoflagellates; however, training is required to distinguish toxic from nontoxic strains under light microscopy. A specific oligonucleotide gene probe was developed to recognize regions of DNA that are unique to any given organism or group of related organisms. It was postulated that using DNA probesto recognize toxic dinoflagellates could provide a rapid and cost-effective early warning method for the presence of impending red tide blooms. Specific DNA probes were also considered useful to serve as molecular tags for tracking the movement of toxic blooms, or for studying the dynamics of phytoplankton growth and succession in nlarine waters. Specific molecular weight soluble proteins have been identified in both crabs and clams (8,39,40) and correspond to estimates of toxicity using the mouse bioassay or fluorescence detection. The induction of a specific 79 kDa, protein in crabs exposed to saxitoxin injections was used as the basis for ELISA and Western blotting methods to screen clam and crab tissue for PSTs (41). Finally, Fairey et al. (42) published a gene manipulation procedure whereby a c,fOs luciferase reporter gene construct was cloned in an N2A mouse neuroblastoma cell line to enable a direct cytotoxicity measurement, utilizing a luciferase catalyzed light gen-
790
Kifts
eration as an end-point measurement. This procedure enabled sensitive detection of saxitoxin over a range of 1-10 ng/ml. As is the case with other cell cytotoxicity or receptor binding assays, this method does not provide information on the identity of the chemical toxin and therefore would work well in parallel with HPLC methods.
B. Chemical Methods A chemical approach to monitoring PSTs in shellfish was first developed by Bates and Rapoport (43) and Bates et al. (44) using a fluorometricmethod.Thisprocedure also provided the basis for several subsequent chemical assays, including thin-layer chromatography (TLC) ( 4 3 , low-pressure liquid column, and HPLC methods. The method used an oxidation step with hydrogen peroxide under alkaline conditions to produce a fluorescent derivative of saxitoxin, 8-an~ino-6-hydroxymethyl-2-i1~~i1~opurine-~(2H)-propionic acid. Although based on the separation of toxins on a weak cation exchange resin eluted with sulfuric acid, a major limitation of this method is that only saxitoxin is measured, and while some workers havehad success withthe method, the results can underestimate toxin content by as much as 65% compared to the mouse bioassay (46). The reason for the underestimation of toxicity is due to the fact that other PSTs which are less likely eluted from the weak ion-exchange column dueto the high polar nature have limited florescence. Moreover, only saxitoxin is measured relative to the known standard. For example, the carbamate and sulfocarbamoyl PSTs are two of the most predominant isomer sources in some shellfish; however, while the carbamate toxins the are most toxic, the sulfocarbamoyl toxins are the least toxic (47). Thus a situation for underestimating PSTs occurs if there are other PSTs present in samples other than saxitoxin which exhibit specific toxicities. The finding by Sullivan et al. (48) that metabolic transformation of PST occurs in the littleneck clan1 demonstrates the potential for multiple fornx of PST. Halstead (3) reported that the success of the batch fluorometric method based on the strong adsorbability of saxitoxin on acidic ion-exchange columns is due to the fact that saxitoxin was the primary saxitoxinpresentincontaminatedshellfish. It is important tonote,however,thatthe method of Bates and Rapoport distinguishes between acceptable and unacceptable shellfish with some false-positive but no false-negative results (49). A number of researchers have demonstrated that PST can be derivatized to yield fluorescent compounds, although the degree of fluorescence is extremely variable (5052). The N- 1 hydroxy toxins, which include neosaxitoxin and GTX- 1 and -4, have lower fluorescence(Fig.2a), yet displayhighspecifictoxicities(Fig.2b).Otherprocedures (54), capillary electrophoresis ( 5 5 ) , and HPLC including TLC (45.53), cellulose (50,52,54,56) are based on the measurement of fluorescence. The HPLC method offers in the sample and isInore the potential for identifying individual PST isomers present sellsitive than theInOuse bioassay. HPLC procedures represent sensitive and accurate techniques which are suitable for handling a large number of samples for routine monitoring. Sullivanet al. (57) reported a method that involvedpreparation of an acidextractof shellfish followed by ion interaction chromatography using a polystyrene divinyl benzene resin column. A binary gradient HPLC method witha postcolumn oxidation derivatization and fluorescence detection enabled the detection of nanogram quantities of the N-1 hydroxyl toxins. I n general, the detection chemistry for this method is based on the oxidation of all toxins with periodate under alkaline conditions and detection of fluorescent products with two chromatographic separations. Good correlation between the HPLC method and the InOuse bioassay requires correction for the contribution of each PST toward the total
191
Laboratory Methodology for Shellfish Toxins 2 r
Type of Toxin 1.2
B
Type of Toxin
Fig. 2 Relative fluorescence (A) and toxicity (B) of different PSP. (From Ref. 54.)
toxicity (58). A semiautomated method employing thefluorescenceresponseusingan autoanalyzer system was also successful with good accuracy (CV = 9.5%) for samples 260 pg/lOO g and sensitivity 10 pg/IOO g toxin for screening samples (59). This procedure was proposed to be coordinated with the mouse bioassay for initial screening of samples performed by the semiautomated method and subsequent confirmationof samples total in a range of 60-250 pgI100 g shellfish meat tested using the mouse bioassay for
792
Kitts
toxicity. Further advancements in lowering detection limits for low molecular weight marinetoxins,includingsaxitoxin,havebeenreportedusingionspray(60),electrospray (61), capillary electrophoresis mass spectrometry (62,63). The two quanidinium groups on the saxitoxin molecule provide the charged sites for monitoring detection of saxitoxin at a fragment ion spectrum of 300 m/:. Principal ions for detection arett1/: 300 (saxitoxin), m/:316 (neosaxitoxin), m/: 380 (gonyautoxin-5), and m / z 396 (gonyautoxin-6). Using atmospheric pressure ionization and combined liquid chromatography-mass spectrometry analysis, a detection of 30 pg saxitoxin has been achieved, which represents a saxitoxin concentration detection limit of 0.1 FM/ 1 p1 injection (compared to 0.0 14 pM and 20 pM injection for HPLC) (57). The detectionlimit for saxitoxin using electrospray ionizationis similar to ion spray when flow injection analysis is used (61). Moreover, there is very little difference in the PST spectrum between the ion spray and electrospray methods.
C. Immunological Assays Immunological methods that have shown promise for monitoring PSTs include radioimmunoassay (64) and direct (65) and indirect (66-68) ELISA assays (Table 2). The direct ELISA developed by Usleber et al. (69) has been used in dipstick (70,71) and immunofiltration (69) formats. The small molecular size common to all PSTs requires that appropriate conjugates are used to covalently attach haptenic components to functional groups of toxins for generating appropriate immunogens required for generating specific antibodies. The coupling of amine functional groups of bovine serum albumin to purified PST, derived from clam siphon glands using formaldehyde, was first reported by Johnson et al. (72) and used by Carlson et al. (64) and Chu and Fan (66) in developing a radioimmunoassay format. The high specificity for saxitoxin and low cross-reaction with NI-hydroxylated toxins (2-10% cross-reactivity with neosaxitoxin) using this approach resulted in a potential severe underestimation of toxin content in PST-contaminated shellfish where toxicity was in fact attributed to PSTs otherthan saxitoxin. Similar limitations were present with very specific polyclonal (66-68) and monoclonal (73,74) ELISA assays. Antisaxitoxin sera generated using a glutaraldehyde-reacted saxitoxin-polyalanine carrier produced some cross-reactivity with neosaxitoxin, GTX-2 and -3, and mixtures of N-21 sulfocarbamoyl derivatives in both contaminated shellfish and dinoflagellate organisms that were fourfold more sensitive than the mouse bioassay and twofold more sensitive than HPLC (67). Immunological assays using the ELISA format have reported STX detection limits of about 3-4 ng/g of tissue in mussels and clams (69). Efforts to overcome the limitation of specificity of PST antisera in the faceof diverse numbers of PST isomers and variability i n PST composition of different shellfish due to metabolic interconversions were made by Chu et al. (68), with the generation of antibodies to neosaxitoxin instead of saxitoxin. This polyclonal antiserahad a relatively greater cross-reactivity with saxitoxin (up to 50% depending on the ELISA format). Despite the improvements using greater cross-reacting antibodies derived from neosaxitoxin. the fact still remains that ELISAs are limited by the underestimation of some toxic samples (e.g., dc-GTX-1-4 are present in significant quantities). Other researchers have used a glucose oxidase conjugate to facilitate measuring very small quantities of neosaxitoxin standard by employing periodate coupling and have reported success at generating specific antibodies for both neosaxitoxin and GTX1 and -4, with cross-reactivity of 3.4%, 3.4%, 0.1%, and 0.04% for saxitoxin, GTX-1 and -3, descarbamoylsaxitoxin, and N-sulfocarbamoylsaxitoxin, respectively (75). As a consequence of cross-reactivity with different PST analogues, detection limits ranged from
x
Laboratory Methodology for Shellfish Toxins e,
V
C
E
2
2
E n
Y
W
U
C
U
6
2
e,
W
.d
U
.-
e,
W
3
Q
8
m
a
M
E
3
m
.
x
.-> .-m U
e,
m
.d
m
U
G
5
e,
-a .3
‘C a
C M 0 C
2 E W
Kitfs
194
17 to 22 pg/ml for both neosaxitoxin and GTX, 0.58 to 1.4 ng/ml for saxitoxin, 1.45 ng/ m1 for GTX-2 and -3, and 12 to 61 ng/ml for dcSTX and C l and 2, respectively. Partialsuccess inaccuratelymeasuringPSTsinbothspecificPSTmixturesand PST-contaminated shellfish from different species has been accomplished on solid-phase systems using specific STX or neoSTX antibodies with either STX-HRP or neoSTX-HRP conjugate markers (76,77). In conclusion, a requirement for an ELISA method that will respond to all PSTs including the C group of toxins remains a primary challenge in diagnostic test development.
111.
DOMOIC ACID TESTING
Domoic acid is a neurotoxin that has been detected in various seafoods including mussels, razor clams, and anchovies (78-81). In addition to domoic acid, a number of cis-trrrns isomers, collectively referred to as isodomoic acids, have been identified (Fig. 3) (82), althoughtoxicitiesarerelativelylower (81). Domoicacid is arare,watersoluble but natural, amino acid that is structurally related to kainic acid, a potent excitotoxin that produces its effects via an interaction at specific glutamic acid receptors (83). The toxin, which acts as an agonist to glutamate, expresses neurotoxicity through an interaction at kainate receptors (84). The toxin is produced by at least two speciesof red algae, Chondrin urrnot(1 (85)and Alsidiurn cor-allium (86),and isa secondmetabolite of the diatom Nirzschin pungens (87). N. purlgens is widely distributed in coastal waters of the Atlantic, Pacific and Indian Oceans. However, not all strains have been demonstrated to produce domoic acid (88). Grimmelt et al. (80) reported that more than 90% of total domoic acid was recovered in mussel hepatopancreas tissue, related to the content of cholorphyll a, a useful marker for algae biomass. Domoic acid cannot be destroyed by cooking or freezing, but reports
A
B FH3
Fig. 3 Do~noicacid (A) and the diastereoisomer of dornoic acid (B).
Laboratory Methodology for Shellfish Toxins
195
have disclosed that it is possible to wash out the toxin by flushing contaminated mussels with uncontaminated water for 18 days (80). The governmentof Canada has a safetylevel for domoic acid setat 20 ppm in wet mussel tissue. The consumptionof mussels containing less than this amount is unlikely to pose a health hazard (89). Domoic acid,a potent agonist of glutamatergic kainate receptors located in the dorsal hippocampus of the brain (84) produces symptoms of gastrointestinal and neurologic disorders (e.g., temporary or permanent memory loss). The intoxication syndrome is known as amnesic shellfish poisoning (ASP) and includes symptoms of vomiting, diarrhea, and/ or abdominal cramps within 24 hours, memory loss, confusion and disorientation, seizures, coma, and cranial nerve palsies within 48 hours (90). As with PSTs, there is no antidote for treatment of domoic acid poisoning. Initial physical symptomsof domoic acid poisoning occur within 0.5-24 hours, depending on the severity of the intoxication (91). Physical symptoms include vomiting, diarrhea, and headache, with more severe intoxications causing excessive bronchial secretions, difficulty in breathing, coma, permanent memory loss, and even death. The first record of foodborne illness attributed to domoic acid in North America occurred in a Canadian maritime provincein 1987, when numerous people became ill and three elderly people died from amnesic shellfish poisoning (92). Until 1987 the only shellfish poisoning reported in the maritime area with regularity was PSP. Leftover mussels were assayed using the mouse bioassay for PST; however, test mice displayed involuntary scratching of their shoulders with their hind legs which was not typical of PSP symptoms (90). The source of the outbreak was traced to three river estuaries on Prince Edward Island. Wild and cultivated mussel samples were taken for laboratory analysis for potential toxic agents, including bacteria, viruses, and chemical residues. No bacterial or viral pathogen, heavy metals, polychlorinated biphenyls, or pesticides were detected in the samples to (79). The domoic acid concentrations in the leftover mussel samples ranged from 31 128 mg/ 100 g of mussel tissue. Estimates of total domoic acid ingested by the patients ranged from 60 to 290 mg (79). Illnesses reported in past years in this area were traced to consumption of wild shellfish that had been harvested in toxic areas where warning notices had been posted. The outbreak of domoic acid poisoning occurred on Prince Edward Island in November 1987. Histologic studies of brain samples collected from those killed by the poisoning revealed neuronal necrosis or cell loss and astrocytosis, particularly in the hippocampusandtheamygdaloidnucleus(93).Subsequent tothe dolnoicacid outbreak in eastern Canada were two outbreaks reported on the West Coast of the United States. In September 1991, a high incidence of sick and dying pelicans in Monterey Bay was traced to domoic acid-contaminated anchovies. A similar outbreak occurred in November 1991 on the Long Beach peninsula in Washington State with razor clams. Most of the domoic acid appeared to reside in the gut lumen of mussels, with small alnounts found in the intracellular compartments (94). Meat of small mussels contained relatively more toxin per unit weight than large mussels because of their relatively large digestive glands.
A.
Bioassays
The original method used for domoic acid detection was based on the mouse bioassay for PST (95). The overall behavioral signs are quite distinct from the paralysis associated with PSP, but yield compatible standard curves (95). Mice injected intraperitoneally with is similar an acid extract derived from boiled, whole mussels for domoic acid detection
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Kitts
to the PST assay but produces an atypical response that is characterized by scratching, convulsions, and death. I n previous studies that used the mouse bioassay, mice were observedfor4hoursafterintraperitonealextractinjectionforbehavioralresponses (scratching) oruntil death. Onlyrarely does the animal die morethan 3 hours postinjection. False positives can occur in mice that show decreased locomotor activity and recovery to normal I hour after injection (80). There is some biological variability in the toxicity estimates of domoic acid when using onset of scratching or even time to death in different mice. In contrast to PST, mouse weight is not a critical factor in estimating the toxic effects of domoic acid-contaminated extracts (80). In mussels the method produces a noeffect level of 24 ppm and 94 ppm for death in mice when performed intraperitonally. An LDio of 83.5 pg domoic acid/mouse ( 5 mg/kg) has been reported for intraperitoneal injections (80). When administered orally, domoic acid doses between 35 and 70 mg/kg (or ppm) body weight are required to produce toxic symptoms inboth mice andrats. Besides havinga degree of similarity with the human clinical situation, the mouse bioassay is rapid for sample screening. However, becauseof the low sensitivity of this test (40 pg/ g drained wet tissue), below the current legal limit (20 pg/g tissue) for edible tissue, this procedure is not used.
B. High-Performance Liquid Chromatography (HPLC) Due to the limitations of the mouse bioassay, shellfish extracts are routinely analyzed for domoic acid using HPLC methods. In addition to the standard AOAC method for sample extraction of shellfish material used in the conventional mouse bioassay (95,96), which recovers only 70-80% domoic acid, an additional extraction method was developed which includes a cleanup procedure prior to HPLC analysis using a C l 8 solid-phase extraction. Two isocratic mobile phases including a 10% acetonitrile with 0.1%1trifluoroacetic acid (92) phase and a 12% acetonitrile phase containing 2% orthophosphoric acid (97) were developed, with a conmon UV-detection wavelength step (242 nm). Rapid analysis (S minutes) and excellent sensitivity(0.5 pg/g of toxin i n shellfish tissue) enable these methods to meet the demands of high sample volume turnover in laboratories as well as being below the tolerance limit of 20 pg/g of tissue set by regulatory agencies. Two certified reference materials for domoic acid quantitation consist of an instrument calibration solution (DACS- 1 ) released in May 1989 and a mussel tissue reference material obtained from Mstilus ecldis, released in August 1989 from the Marine Analytical Chemistry Standards Program.Thesestandardizedextractshave been of greatassistanceinprovidingboth quality control for domoic acid analysis in different laboratories and confirmation ofsymptoms of intoxification in mouse bioassays (98). The extraction and cleanup procedure for tissue domoic acid determinations has also been reported (99). Due to the complex differences in sample matrix for crab viscera, anchovies, and even porpoise stomach contents, the problem of extraneous and unidentifiable peaks has been reported (81). A relatively simple NaCl sample cleanup and extraction procedure overcomes this limitation, yielding a detection limit of approximately 0.5 ppnl (81). Other methods have used a fluorenylmethoxycarbonyl derivative of domoic acid for monitoring trace amounts of domoic acid in seawater and phytoplankton and have reached minimum detection limits of 15 pg/ml (100). HPLC methodologies have also been applied to urine and feces from monkeys and blood serum from humansin a study reported by Lawrence (101). Although the procedure was not appropriate for urine and blood samples that required additional cleanup before analysis, it did work well for domoic acid analysis i n feces at concentrations greater than 1 mg/kg ( 1 ppm). Blanchard and Tasker( 102) also usedH P I X for donloic acid determina-
Laboratory Methodology for Shellfish Toxins
197
tion in the serum of rats, pigs, and rainbow trout, with a detection limit of 0.20 ppm in serum. The assay demonstrates a high degree of reproducibility and reasonable sensitivity. of HPLC/mass spectrometry methods are extremely sensitive for the quantitation domoic acid (60,103-105). The ion spray mass spectrum is characterized by a prominent peak resulting from protonated molecules at tn/z 312. Detection limits as low as 100 ng/ g of shellfish have been reported using reverse-phase liquid chromatography coupled with ion spray mass spectrometry.
C. Capillary Electrophoresis Mussel tissue homogenates extractedwith water and processed further usingan octadecylsilica solid-phase extraction cartridge prior to capillary electrophoresis (CE) has proved successful. Using mussel extracts spiked with domoic acid at 3.91-500 ppm, electropherograms showed a strong linear relationship between peak area and domoic acid concentration (106). For domoic acid determination i n contaminated tissue, the treated extract was mixed with a known amount of domoic acid or with water. Domoic acid concentration was calculated from the difference in peak areas of the electropherograms. The detection limit for domoic acid inwettissue was I O ppm. Removal of lipids from the shellfish extract facilitates electroendonosis and shortens analysis time. A modification of the HPLC procedure using capillary electrophoresis has also been reported by Pleasance et al. (62).
D.
Gas Chromatography
Attention has also been given to developing a gas chromatographic/mass spectrometry method employing a two-step reactionof domoic acid to N-trifluoroacetyl-0-?er!-butyldinlethylsilyl prior to analysis (107), or derivatization of domoic acid with dimethylfornlamide dimethyl acetal to produce a volatile derivative of domoic acid, namely, N-formyl0-methyl domoic acid,in a single reaction step (108). Confirmation of domoic acid using the several diagnostic ions i n the mass spectrum was reported to be sufficiently below regulatory levels of 20-30 ppm for dornoic acidin razor clams, blue mussels, and Dungeness crabs (108). As with the HPLC/mass spectrometry methods, which require sophisticatedequipment,gaschromatographymethods atthepresenttimearealsolimited to access to official analytical laboratories.
E. Radioassays The generation of domoic acid antisera was first reported by Newsome et al. (109) using a rabbit polyclonal antidomoic acid serum in a tritiated radioimmunoassay format. Radioimmunoassays are highly sensitive and accurate, but the cost of radiotracers and radioactive waste disposal is high. Furthermore, radioimmunoassays are not applicable to field tests because of the possibility of radioactive contamination. In addition to radioimmunoassay, a competitive radioreceptor assay employing a recombinant kainate glutamate re(5 nM) and varying concentrations of domoic acid has ceptor with tritiated kainic acid been developed ( 1 IO). The detection limit for domoic acid using the cloned receptor was approximately 1 nM, with an ICso of 3.4 nM (1.02 ng/ml).
F. Enzyme-Linked lmmunosorbent Assay (ELISA) Newsome et al. (109) first raised antiserum against domoic acid by conjugating domoic acid to BSA using ethyl dimethylaminopropyl carbodiimide hydrochloride (EDC) reagent. The antibodies raised i n rabbits were also formatted for ELISA. No cross-reactivity was found with glutamic acid, aspartic acid, kainic acid, and saxitoxin. The ELISA method
Kitts
198
proved to be more sensitive than radioimmunoassay for domoic acid quantitation i n rat urine (40 ng/ml-2.6 yg/ml), but it producedinconsistentresultswithvariousratand monkey antiserum dilutions. Subsequent studies compared antisera obtained i n mice with domoic acid immunogen conjugates derived from ovalbumin (OVA), keyhole limpet hemocyanin (KLH), and BSA carrier proteins using either EDC or N-hydroxysuccinimide
1.OE-2 1.OE-3 1.OE-4 1.OE-5 1.OE-6 1.OE-7 1.OE-6 1.OE-9
Dilution of Anti-OVA-DA
0.W
0.04
0.08
0.12
KLH-DA (ug/rnL)
Fig. 4 (A) Different dilutions and affinity absorbance readings of anti-OVA-Dornoic acid. -X = RI-antisera; -C = R2-antisera. (B) Influence of differentcoatinglevelsof KLH-Dornoic acid antisera on affinity of detection. '- = RI-antisera; -f = R2-antisera. ~
Laboratory Methodology for Shellfish Toxins
199
reactions ( 1 1 l ) . Both conjugation efficiencies as well as serum titers were superior with ovalbumin and KLH, with OVA displaying the highest affinity for free domoic acid in a competitive ELISA. The antiserum did not cross-react with kainic acid, glutamic acid, aspartic acid, or saxitoxin, and when formatted for a competitive ELISA produced lower limits of accurate determinations of 0.2 pg/ml domoic acid in urine, 0.25 pg/ml domoic acid i n plasma, and 10 pg/ml domoic acid in milk. It was concluded that the concentration
S
50
0
100
150
200
250
Free Domoic Acld (nglml)
% Inhibition = 12.1286 log (Dornolc Acid] + 3.78298
r = 0.9929
1
10
100
1“
Domoic Acid (nglmL)
Fig. 5
(A) Performance of different antisera in competitive ELISA for domoic acid using KLHDomoicacidcoating of 0.025 pg/ml (a) and 0.050 pglml (b). f = R1-antisera; t f = R2antisera. (B) Standard curvc for competitive ELISA for domoic acid.
Kitfs
200
of domoic acid in body fluids of individuals with ASP could be determined directly using this method. The anti-OVA domoic acid was also used for domoic acid determination in mussel extracts(1 12). Spike-recovery experiments showed that domoic acid concentration in both aqueous and acid mussel extracts could be measured to within 8% of the actual value using competitive ELISA. The ELISA yielded a domoic acid detection limit of 0.5 pg/g of extract and correlated(r = 0.96) with the standardHPLC method. The competitive ELISA also detected domoic acid for several samples that were undetected by HPLC. Due to the limited amount of serum expected to be recovered from mice, domoic acid antibodies can be raised in rabbits and the ELISA for domoic acid can be standardized according to the methods reported previously (111,112). The anti-domoic acid titers are made in buffers resulting in reductions in absorbances as the dilution of titers increases (Fig. 4a). In the example given herein, with rabbit antisera, the optimal domoic acid antisera dilution waslo-'. Determining the optimal coating level of antisera on the microtiter plate is also essentialfor obtaining optimal absorbance readingsof toxin, which can vary depending on individual antisera (Fig. 4b). In this example, the coating level for antisera obtained for rabbit 1 (R-l) was approximately 0.025 lg/ml, compared to 0.05 pg/ml for rabbit 2 (R-2). Using the KLH conjugate, tests with plates coated with KLH alone should be confirmed for a negative control, thus providing absorbance readings of 0.00 and confirming that the domoic acid antisera derived using this conjugate does not react with the KLH protein carrier. The reactivity of the antisera to the antigen is also especially important in a competitive ELISA and will vary depending on the coating concentration of KLH-domoic acid antisera (Fig. 5a). In the present example, inhibition was less at the 0.05 pg/ml coating level than at 0.025 pg/ml. Both coating levels demonstrate that the more free domoic acid available, the greater the reduction in absorbance and thus the more competitive the reaction. Anti-domoic acid obtained fromR-2 was more sensitive,
n
80
M
2 70 v
z2
60
U
8
.W
50
e 40 0
0
U
4
GtJ
30 20
U
8 10 0
l
2
3
4
5 6 7 SampleNumber
8
9
1
0
Fig. 6 Comparison of domoic acid determinations using HPLC,Mouse antibody ELISA andRabbit antibody ELISA. W = HPLC; = Mouse ELISA; 0 = Rabbit ELISA.
Laboratory Methodology for Shellfish Toxins
201
hence a small change in the concentration of free domoic acid results in a large change in the percent inhibition. A typical domoic acid ELISA standard curve using the aforementionedcoatingconcentrationandantiseratiterisshown i n Fig.5b.Oncomparingthe different estimates of dotnoic acid concentration in sea mussel extracts by HPLC and the two ELISA methods, it can be seen that both the mouse and rabbit ELISA procedures are in good agreement with the HPLC method (Fig. 6). In most cases, as reported earlier (1 l?), ELISA results for domoic acid are higherthan HPLC measurements, but the rabbit ELISA correlates well with the HPLC results ( r = 0.964). The rabbit antisera, however, gives relatively higher readings for domoic acid than the mouse antisera, although good agreementexistsbetweenthetwoprocedures ( v = 0.987). Againthehigherreadings obtained using ELISA compared to HPLC may be due to the presence of domoic acid isomers that cross-react with either antisera; possibly Inoreso than with the rabbit antisera.
IV.
CONCLUSIONS
The persistent and yet unpredictable nature of shellfish contamination with PSTs or domoic acid poisons has resulted in an absolute requirement for routine testing of shellfish harvested for human consumption. With the development of reliable, practical, and costeffective monitoring procedures performed by both government laboratories and shellfish processors, the current problems of monitoring vast areas of coastline would be reduced, and a sound quality control standard for shellfish poisons in processed shellfish could be established. With the greater testing made possible by alternative methodsto the traditional mouse assay comes an enhancement of the process of risk assessment which translates into increased public confidence in the safety of the product. These important changes i n quality control assessment and public confidence could also demand greater market value of shellfish resources. It is a fact that the public reacts adversely to marine products in general when there is news of a PSP or ASP outbreak. Adverse publicity, although specific to the bivalve mollusk industry, can also have an impact on the fish industry as well. More efficient and practical monitoring methods for contaminated shellfish could further reduce the risk of food poisoning. Finally improvements in routine monitoring of seafood toxins in shellfish by more efficient and reliable methods could also contribute to a greater utilization of the entire shellfishery without overharvesting of selected areas designated as safe.
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66. 67.
Kitts
mussel samples gathered near Prince Rupert, British Columbia. In: DL Taylor, HH Seliger, cds. Toxic dinoflagellate blooms. New York: Elscvicr, 1979, pp. 399-402. S Hall, PB Reichardt. Cryptic paralytic shellfish toxins. In: EP Ragelis, ed. Seafood toxins. Washington, D.C.: Atncrican Chemical Society, 1984, pp. 1 13-1 24. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck clam (Prorothcrur srominecl). Biochem Biophys Res Cotntnun 114:465-472, 1983. SS Hall, PB Rcichardt, RA Neve. Toxins cxtracted from an Alaskan isolateof Protogoqwclrrr sp. Biochem Biophys Res Commun 97:649-653, 1980. JJ Sullivan, WT Iwaoka. High pressurc liquid chromatographic determinationo r toxins associated with paralytic shellfish poisoning. J Assoc Anal Chem 66:297-303, 1983. JF Lawrence. C Menard. Liquid chromatographic determination of paralytic shellfish poisons in shellfish after prechrotnatographic oxidation. J Assoc Anal Chem 74:1006-1012, 1991. JF Lawrence. C Menard, CF Charbonncau. SS Hall. A study of ten toxins associated with paralytic shellfish poison using chromatographic oxidation and liquid chromatography with fluorescence detection. J Assoc Anal Chcm 74:404-409. 1991. of gonyaulax toxins and other NH Shoptaugh, LJ Buckley, M Ikawa, JJ Sasner. Detection guanidine compounds on thin-layer silica gel chromatograms. Toxicon 16:509-513, 1978. JJ Sullivan, MG Simon, WT Iwaoka. Comparison ofHPLCandmousebioassaymethods for determining PSP toxins in shellfish. J Food Sci 48: 13 12- I3 15, 1983. WE Fallon, Y Shimizu. Electrophoretic analysis of paralytic shcllfish toxins. J Environ Sci HcalthA12:455-464,1977. P Thibault, S Pleasance, MV Laycock. Analysis of paralytic shcllfish poisons by capillary electrophoresis. J Chromatogr 542483-501, 1991. JJ Sullivan, MM Wekell, LL Kentala. Application of HPLC for the determination of PSP toxins i n shellfish. J Food Sci 52:26-29. 1985. JJ Sullivan, WT Iwaoka, J Liston. Enzymatic transformation of PSP toxins in the littleneck clam (Prororhcrcrr stumimrr). Biochctn Biophys Res Cotntnun 1 14:465-472, 1983. J Jonas-Davies, JJ Sullivan,LL Kcntala, J Liston, WT Iwaoka, L Wu. Semiautomated method for the analysis of PSP toxins in shellfish. J Food Sci 49: 1506-1509, 1516, 1984. MA Quilliurn, BA Thomson, GJ Scott, KWM Siu. Ion-spray mass spectrometry of marine neurotoxins. Rapid Cotntnun Mass Spectrom 3:145-150, 1989. HB Hincs. Electrospray ionization of sclcctcd low-molecular-weight natural biotoxins. Biol Mass Spectrotn 22:243-246, 1993. S Pleasance. P Thibault, J Kelly. Comparison of liquid-junction and coaxial interfaces for capillary electrophoresis-mass spectrometry with application to compounds of concern to the aquaculture industry. J Chromatogr 591:325-339, 1992. S Pleasance, SW Ayer, MVLaycock,PThibault.Ionspraymassspectrometryofmarine toxins. 111. Analysis of paralytic shellfish poisoning toxins by flow-injection analysis, liquid chromatography/mass spectrometry and capillary electrophoresis/tnass spectrometry. Rapid Cotnmun Mass Spectrotn 6:14-24, 1992. RE Carlson, ML Lever, BW Lee, PE Guire. Development of immunoassays for paralytic shellfishpoisoning:aradioimmunoassayforsaxitoxin.In:RagelisE, ed. Seafoodtoxins. Washington, D.C.: American Chcmical Society, 1984, pp. 18 1- 191. E Usleber, E Schneider, G Terplan. Direct enzyme immunoassay i n microtitration plate and test strip format for the detection of saxitoxin in shellfish. Lett Appl Microbiol 13:275-277, 1991. in shellfish. FS Chu, TSL Fan. Indirect cnzymc-linked immunosorbcnt assay for saxitoxin J Assoc Anal Chcrn 68:13-16, 1985. A Cembella, Y Parent, D Jones, G Lambourcau. Specificity and cross-reactivity of an adsorption-inhibition enzyme-linked immunoassay for the detection of paralytic shellfish toxins. In: E Graneli, B Sundstrom, L Edler,DM Anderson, eds. Toxic marine phytoplankton. New York: Elsevier Science. 1989, pp. 339-344.
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68. FS Chu,X Huang. S Hall. Production and characterization of antibodies agatnst neosaxitoxin. J AOAC Int 75:341-345, 1992. 69. E Usleber, E Schneider, G Terplan. Direct enzyme imnunoassay in microtitration plate and test strip format for the detection of saxitoxin in shellfish. Lett Appl Microbiol 13:275-277, 1991. of 70. E Schneider. E Usleber, G Terplan. Test strip enzyme immunoassay for the detection saxitoxin. Food Agric lmtnunol 3:103-104, 1991. 71. E Usleber, E Schneidcr, G Tcrplan. MVLaycock. Two formats of enzyme immunoassay for the detectionof saxitoxin and other paralytic shellfish poisoning toxins. Food AdditContam12:405-423,1995. 72. HM Johnson, PA Frcy, RAngelotti, JE Campbell, KH Lcwis. Haptenic properties of paralytic shellfish poison conjugated to proteins by formaldehyde treatment. Proc Soc Exp Biol Med 117:425-430,1964. 73. RC Huot, DL Armstrong. TC Chanh. In vitro and in situ inhibition of the sodium blocker saxitoxin by lnonoclonal antibodies. J Toxicol Environ Health 27:381-394, 1989. 74. R Hack, V Rcnz, E Martlbauer, G Terplan. A monoclonal antibody to saxitoxin. Food Agric Ilntnunol2:47-48,1990. 75. C Burk, E Usleber, R Dietrich, E Martlbauer. Production and characterization of antibodies against neosaxitoxin utilizing a novel immunogen synthesis procedure. Food Agric Immunol 713155322.1995. 76. H Huang,K-HHsu,FS Chu. Direct cotnpetitive enzyme-linked inmunosorbent assay for saxitoxin and neosaxitoxin. J Agric Food Chetn 44: 1029-1035, 1996. in naturally 77. FS Chu, KH Hsu, X Huang,RBarrctt,CAllison.ScreeningofPSPtoxins occurring sarnplcs with three different direct competitive ELISA. J Agric Food Chem 44: 4043-4047,1996. EW Dyer, DJ Embree, M Falk. 78. CJ Bird, RK Boyd, D Brewer, CA Craft, ASW deFreitas. MC Flack, R Foxall, C Gillis, M Greenwell, WR Hardstaff, WD Jamieson. MV Laycock, P Leblanc, NI Lewis, AW McCulloch, GK McCully. M McInemey-Northcott, AG Mclnnes. JL McLachlan. P Odensc, D O’Neil, VP Pathak, MA Quilliam, MA Ragan, PF Seto, PG Sim, D Tappen. P Thibault, JA Walter, JLC Wright, AM Backman, AR Taylor, D Dewar. M Gilgan, DJC Richard. Identification of domoic acid as the toxic agent responsible for the P.E.I. contaminated mussel incident. Technical report no. 56. Atlantic Research Laboratory. 1988, pp. 86. 79. JLC Wright, RK Boyd, ASW deFrcitas, M Falk, RA Foxall, WD Jamieson, MV Laycock, AW McCulloch, AG McInnes, P Odense, V Pathak, MA Quilliam, MA Ragan. PG SinP Thibault, JA Walter, M Gilgan, DJA Richard,DDewar.Identification o f domoicacid, a ncuroexcitatory amino acid, in toxic mussels from eastern P.E.I. Can J Chetn 67: 48 1-490, 1989. S Wager, GR Johnson,JF Amend. Relationship 80. B Grimmelt, MS Nijjar. J Brown, N MacNair. between dolnoic acid levels in the blue mussel (Mytilu.7 c d d i s ) and toxicity in mice. Toxicon 28:501-508.1990. 81. CL Hatfield, JC Wekell, EJ Gnuglitz, HJ Barnctt. Salt clean-up procedure for the determination of dotnoic acid by HPLC. Nat Toxins 2:206-21 I , 1994. 82. JLC Wright, M Falk, AG Mclnnes, JA Wnltcr. Identification of isodomoic acid D and two geometrical isomers of domoic acid i n toxic mussels. Can J Chem 68:22-25, 1990. 83. JT Slevin. JF Collins, JT Coyle. Analoguc interactions with the brain receptor labeled by [‘H] kainic acid. Brain Res 265:169-172, 1983. 84. GDebonnel,LBeauchesnc,CDCMontigny.Domoicacid,thealleged“musseltoxin,” might produce its neurotoxic effect through kainate receptor activation: an electrophysiological study in the rat dorsal hippocampus. Can J Physiol Pharlnacol 6729-33. 1989. 85. K Daigo. Studies on the constituents of Clzor~clr-itrnnrratcc. 11. Isolation of an antihelnlintical constituent. Yakugaku Zasshi 79353-356, 1958.
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86. G Impellizzeria, S Mangiafico, G Oriente. M Piattelli. S Sciuto, E Fattorusso. S Magno, C Santacroce, D Sica. Amino acids and low molecular weight carbohydrates of some marine redalgae.Phytochemistry14:1549-1557,1975. 87. D Subba Rao, MA Quilliam. R Pocklington. Domoic acida neurotoxic amino acid produced bythemarinediatom Nitzchicc pungetts in culture. Can JFishAquatSci45:2076-2079, 1988. 88. MA Quilliam, JLC Wright. The amnesic shellfish poisoning mystery. Anal Chem 61: 1053A1989. 1054A.1056A.1058A-l060A, 89. HBS Coacher. Outbreak of mussel poisoning i n Canada. Identification and determination of a new toxin in Prince Edward Island mussels. Food Lab Ncwslett 1332-33, 1988. An outbreak of toxic encepha90. T Perl, L Bedard. T Kosatsky, J Hockin. EDC Todd. R Retnis. lopathy caused by eating mussels contaminated with dotnoic acid. N EnglJ Med 32217751780,1990. 91. EDC Todd. Domoic acid and amnesic shellfish poisoning-a review. J Food Protect 56:6983, 1993. 92. MA Quilliam, PG Sim, AW McCulloch. High performance liquid chromatographyofdomoic J EnvironAnal acid,amarineneurotoxin.withapplicationtoshellfishandplankton.Int Chem 36: 139- 154, 1989. 93. J Teitelbaum, R Zatorre. S Carpenter, D Gendron. A Evans, A Gjedde, N Cashman. Neurologic sequelae of dotnoic acid intoxication due to the ingestion of contaminated mussels. N Engl J Med 3221781-1787, 1990. 94. 1 Novaczek, MS Madhyastha. RF Ablett. A Donald, G Johnson, MS Nijjar.Sims. DE Depuration of domoic acid from live blue mussels (Mytillts rchrlis). Can J Fish Aquat Sci 49:3 12318. 1992. 95. AOAC.Officialmethodsofanalysis.Arlington.VA:AssociationofOfficialAnalytical Chemists,1984. L Tryphonas, J C:unpbell, E Lok. Domoic acid poisoning 96. F Iverson, J Truelove, E Vera, and mussel-associated intoxication: preliminary investigations into the response of mice and rats to toxic mussel extract. Food Chem Toxicol 273777384, 1989. 97. JF Lawrence, CF Charbonneau, C Menard, MA Quilliam, PC Sim. Liquid chromatographic determination of dotnoic acidi n shellfish products using the AOAC paralytic shellfish poison extraction procedure. J Chromatogr 462349-356, 1989. 98. WR Harstaff,WDJamieson. JE Milky, MA Quilliam. PG Sim. Referencematerialsfor dotnoic acid, a marine neurotoxin. J Anal Chem 338520-525. 1990. 99. MA Quilliam. MXie,WR Hardstaff.Arapidextractionandclean-upprocedureforthe determination of dotnoic acid in tissue samples. Tcchnical report 64. Ottawa: National Research Council of Canada, Institute for Marinc Biosciences, I99 I . 100. R Pocklington. JE Milky, SS Bates, CJ Bird. ASW deFreitas, MA Quilliam. Trace deternmination of donloic acid in seawater and phytoplankton by high performance liquid chromatography of the fluorenyllnethoxycarbonyl (FMOC) derivative. Int J Environ Anal Chem 38:351368.1990. 101. J Lawrence. Determination of domoic acid i n seafoods and in biological tissues and fluids. CanDisWklyRep16(supplIE):27-31, 1990. 102. J Blanchard. R Tasker. High-performance liquid chrotnatographic assay for domoic acid in serum of different species. J Chrotnatogr 526546-549. 1990. 103. MA Quilliam, BA Thomson, GJ Scott. KWM Siu. Ion-spray mass spectrometry of marine neurotoxins. Rapid Colnmun Mass Spectrom 3: 145-150, 1989. 104. P Thibault. J Walter. Identification of domoic acid, a neuroexcitatory amino acid. in toxic mussels from eastern Prince Edward Island. Can J Chem 67:481-490, 1989. D Lewis. Comparison ofUV absorption and electrospray 105. JF Lawrence, BP-Y Lau, C Cleroux. mass spectrometry for the high performance liquid chromatographic determination of dolnoic 659:1 19-126. 1994. acid in shellfish and biological samples. J Chromatogr
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106. AL Nguycn, JHT Luong, C Masson. Capillary electrophoresis for detection and quantitation of domoic acid in mussels. Anal Lett 23: 1621 -1634, 1990. 107. S Pleasance, M Xie, Y LeBlanc, MA Quillium. Analysis of domoic acid and related compoundsby mass spectrometryandgaschrotnatography/massspectrometryasN-trifluoroacetyl-0-silyl derivatives. BiomedEnvironMassSpectrom19:420-427,1990. 108. SW Hadley, SK Braun, MM Wekrll. Confirmation of domoic acidas an N-formyl-0-methyl derivative in shellfish tissues by gas chrotnatography/tnass spectrometry. I n : F Shahidi. Y Jones, DD Kitts, eds. Seafood safety. processing and biotechnology. Lancaster, PA: Technomic Publishing, 1997, pp. 25-32. 109. H Newsome. J Truelove, L Hierlihy, P Collins. Determination of domoic acid in serum and urine by immunochemical analysis. Bull Environ Contam Toxicol 47329-334, 1990. 1 IO. FM Van Dolah, JF Doughtie, DR Hampson, JS Ramsdell. High capacity receptor assay for domoic acid: use of a recombinant glutamate receptor produced in a baculovirus expression system. In: Forbes JR. ed. Proceedings of the 4th Canadian Workshop on Harmful Marine Algae. Can Tech Rep Fish Aquat Sci 2016:63-64, 1994. I 1 1. DS Smith, DD Kitts. A competitive enzyme-linked immunoassay for dotnoic acid determination in human body fluids. Food Chetn Toxicol 32: 1147-1 154, 1995. 112. DS Smith, DD Kitts. Enzyme immunoassay for the determination of domoic acid in mussel extracts. J Agric Food Chem 433677371, 1995.
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7 Ciguatera Fish Poisoning Yoshitsugi Hokama Universih of Hawaii, Honolulu, Huwaii
Joanne S. M. Yoshikawa-Ebesu Oceanir l e s t System,
Honoluluv Hnnwii
I. Introduction 210 11. History 210 111. Biology2 1 1 IV. Chemistry213 V. Pharmacology216 VI. Epidemiology 2 18
VII.
VIII. IX.
X.
A. Geographic distribution 2 18 Incidence B. 21 8 C. Fish associated with ciguatera poisoning 219 1 DetectionMethods22 A. Bioassay 221 B. vitro Inbioassay 223 C. In vitro cell assay 225 D. Immunoassay 226 Membrane E. immunobead assay 227 Chemical F. methods 235 G. Summary 235 Clinical 235 Pathology 237 A. Human 237 B. Animal studies 238 Therapy 242 A. Earlier treatment 242 Recent B.treatment 242 C. Summary 244 References 244
209
210
1.
Hokama and Yoshikawa-Ebesu
INTRODUCTION
This chapter covers one of the common foodborne diseases associated with consumption of fish from coral reefs and the near-shore environment of the oceans in the tropics and in some instances the semitropics to depths down to 60 m. The region is circumglobal between the Tropic of Cancer and the Tropic of Capricorn. Seafood poisoning encompasses a broad spectrum initiated by a variety of marine toxins. Some of the better known seafood poisonings include paralytic shellfish poisoning (PSP); scombroid poisoning; pufferfish poisoning; amnesic shellfish poisoning (ASP); ciguatera poisoning; and diarrhetic shellfish poisoning (DSP). PSP is associated with dinoflagellates producing saxitoxins and gonyautoxins, which accumulate in shellfish during heavy dinoflagellate blooms at certain timesof the year ( 1,2). Scombroid poisoning results from the spoilageof fish tissues due to bacterial contamination and subsequent production of the enzyme decarboxylase, which converts amino acids to amines (e.g., histidine to histamine), thus inducing clinical changes mimicking histamine-like attacks. This poisoning isassociatedwithpoor or inadequate fishpreservationandstorageprocesses (3,4). Pufferfish poisoning is associated with tetrodotoxin (TTX) found in fish of the order Tetraodontoidea and in certain crustacea (5,6), and is probablyof bacterial origin (7,8). Tetrodotoxin is also found in the newt and certain speciesof octopus. ASP is caused on St. Andrew‘s by domoic acid and was first reported in a mussel poisoning outbreak Island, Canada, in late 1987 (9). The neuroexcitotoxin (domoic acid) produced by Pseudonitizches was identified as the causative factor. DSP causes severe diarrhea in patiem consuming shellfish or fish containing okadaic acid or its analogs (IO, 1 I ) . The emphasis in this chapter concerns the seafood poisoning known as ciguatera.It is becoming a major problem as fish consumption in the United States increases and the fishing industries of endemic areas (tropics, semitropics, and temperate zones) increase their export and distribution of fish worldwide.
II. HISTORY Fish poisoning probably dates back to antiquity. It was cited in Homer’s Odyssey (800 B.c.) and was observed during the time of Alexander the Great (356-323 B.c.), whose armies were forbiddento eat fish in order to avoid the accompanying sickness and malaise that could threaten his conquests (12 ) . Ciguatera poisoning was described as early as 1606 in the South Pacific island chain called New Hebrides (13). A similar outbreak there and in nearby New Caledonia was reported by the famous English navigator Captain James Cookin 1774 (14). He described the clinical symptomsof his sick crew, symptoms that coincide with the clinical manifestations described today for ciguatera poisoning (l5,16). In addition, viscera from the same fishes given to Cook’s crew were also given to pigs, causing their deaths (14). The term ciguateru originated in the Caribbean to designate intoxication induced by the ingestion of the marine snail, Turbo livonn pica (called cigucr), described bya Cuban ichthyologist. Today the term is widely accepted to denote a particular type of fish poisoning resulting from the ingestion of certain fish (primarily reef fishes) encountered around islands in the Caribbean, the Pacific, and elsewherein the tropics. Current information points to at least one of the many polyether toxins, such as okadaic acid, palytoxin, or maitotoxin, which are structurally closely associated with ciguatoxin, as also being
Ciguatera Fish Poisoning
21 1
Table 1 ClinicalSymptomsAssociatedwithCiguateraFishPoisoning
Category or system Digestive
Neurological
Cardiovascular
General
Nausea, often followed by vomiting; diarrhea; painful defecation; abdominal pain and cramps. Symptoms generally abate after 24 hours, leaving an asthenic and dehydrated patient. Dysesthesia, principally with sensitivity to cold, temperature reversal; paresthesia, painful tingling of the palms of the hands and soles of the feet on contact with cold water; superficial hyperesthesia, with sensation of burning and electrical discharge: often mydriasis is present; patellar and achilles reflexes sometimes diminished. Neurological symptoms generally persist for 1 week. It is not unusual to see contact dysesthesia lasting a month. Pulse slow (35-50 beatdmin), often irregular pulse, low arterial pressure (heart sounds distant); EKG may show dysrhythmia from sinus bradycardia (slow heart beat) to bursts of supraventricular or ventricular extra systoles (rapid systolic heart beat). Ventricular tachycardia, excessively rapid action o f thc heart, may also occur. There may also be a 1st A-V (atricular-ventricular) block. Cardiovascular disorders usually disappear in 48-72 hours and may be mistaken for a heart attack. Toxins from carnivorous fish tend to cause cardiovascular problems. Asthenia, making it difficult to walk, keeping the patient in bed for several days; arthralgia, especially of knee, ankle, shoulder, and elbow; dorsolumbar stiffness. myalgia, especially leg muscles; headache; marked and constant chilliness, but no problems of thermal regulation; lipothynlia and dizziness; itching 2-3 days after onset and may persist for many days; oliguria, sometimes during the first 48 hours.
associated with ciguatera ( 1 1,I7,18). The characteristic clinical symptoms are described in Table l and are discussed further in the clinical section. Since the 1970s, muchprogresshasbeenmade in ciguateraresearchbyseveral investigators (17-27), encompassing all phases of the problem. These areas, covered in this chapter, include biology; chemistry, including the closely related polyethers; pharmacology; clinical; epidemiology; immunological analysis, or the testing and detection of the major toxin ciguatoxin and its related polyether toxins, and other biological testing methods for assessing ciguatoxin and related polyethers; pathology; and therapy.
111.
BIOLOGY
Fish poisoning outbreaks occurred throughout the tropical regions of the Pacific before, during, and after World War 11. It became a serious problem for the military during the war because outbreaks of ciguatera occurred among the troops stationedin various areas endemic for ciguatera poisoning, including Midway, Wake, Guam, Johnson Island, French Polynesia, New Caledonia, the Marshalls, and the Marianas. Other tropical areas of the world with ciguatera include the Caribbean, Gulf of Mexico, and Indian Ocean. In the in postwar years, investigators from the University of Hawaii, Japan, and Tahiti went search of the etiological factor causing ciguatera poisoning.
212
Hokama and Yoshikawa-Ebesu
Banner et al. (28) were pioneers in the study of the ecology and biology of ciguatera fish poisoning. Randall (29), in his investigations of toxic fish, showed that all fish of the same species in the same area were not necessarily carriers of the toxin, and hence the toxin was not produced by the fish species. Therefore the toxin in the fish must be derived from their food source. This was the beginning of the development of the food chain concept shown i n Fig. 1. The sequence of this concept follows the scheme: dinoflagellates or other sources (bacteria, invertebrates, macroalgae) are consumed by herbivores or omnivores, which in turn are eaten by carnivores that store the toxins. Humans and other mammals develop ciguatera poisoning as a result of eating the contaminated fish. Theetiology of ciguaterapoisoningwasestablishedwhenYasumotoet al. ( I O ) discovered a dinoflagellate resembling a Diplopstrlis sp. as the initiator of the food chain disease. Later the organism was correctly identified as Gumhetdiscus toxicus, a new species of the family Dinophyceae (30). The species was named after its origin of discovery, the French Polynesian Islandsof Gambier in the South Pacific (10.30). Studies have shown the food chain concept to be correct. Campbell et al.(3 1) showed an outbreak of ciguatera poisoning in a species of herbivore (Ctenock~wtus sp., kole). Examinationsof gut smears stained with anti-CTX fluorescence dye showed that 95% of the gut contents contained numerous G. toxicus. All tissues tested positive for ciguatoxin in the stick enzyme immunoassay (S-EIA). Yasumoto et al. ( 1 0) demonstrated G. tosicus in the gut of herbivores. More recently, chemically characterized congeners of ciguatoxin were found i n moray eel tissues (32). The precursor for these two oxidized congeners appears to be C T X K , characterized and obtained from G. toxicus (33,34). G. toxicus, a benthic organism collected from the natural environment yielded both ciguatoxin and maitotoxin, but when culturedi n the laboratory, ciguatoxin production was essentially nil. Maitotoxin, a water-soluble, polyhydroxy-containing compound and one of the most potent toxins (0.125 pg/kg LD,,,,in mouse), was obtained in moderate yields (35). The inability of G. toxicus to produce ciguatoxin in culture may be because of its need for a symbiotic relationship with red-brown alga in nature. G. toxicus was found i n large blooms at certain timesof the year. Among the macroalgae associated withG. toxicus were Tur-hinnria sp., Jcrrzirr sp., Sypridicr sp., andBpopsis sp. (19,36-38). G. rosicus adherence to macroalgae and release for movement in calm water appears to be regulated by a substance(s) produced by the macroalgae (39). Other organisms of significant interest
GAMBIEKDISCUS TOXICUS
Fig. 1 Transmission path of ciguatera toxin from the lnarinc dinoflagellate through herbivorous and carnivorous fishes to man.
Gnl,lbie,.rli.sc.lr.s tosicws
Ciguatera Fish Poisoning
213
Fig. 2 Life cycle of G. toxicus. A) Motile, free-swimmingG. toxicus; B) Precyst: immobile, dark, dense intracellular pigment, initial secretion of mucoid sheath; C ) Cyst: adherence to petri dish bottom, thick mucoid sheath, dark centrally located pigment; D) Exocyst: emergence from thick mucoid wall; E) Division of exocyst; about 20 divisions per exocyst; and F) Conditions unknown, 4-6 months. Prorocentrurn species and coral(Hulichoninvolved in ciguatera poisoning (4,40) include dria sp.) producing okadaic acid and its congeners, and recently Ostreopsis sp. elaborating palytoxin (41,42), which was originally discovered in Pulythou (43). The geographic distribution ofG. toxicus is circumglobal and primarily in the tropics. It has been found in the Pacific and Caribbean by numerous investigators(44).Growth (40,000 of G. toxicus in nature is subject to changes in the environment. Heavy growth cells/L of seawater) in the environment is spotty: in some areas, growth is nonexistent and in other areas growth is lowto moderate (100-1000 cells/L). The proper amount of sunlight, temperature(24OC 2 4"C), salinity (28-40 ppm),and appropriate nutrients are necessary for growth. Salinityof less than 2.8% generally showed no growth; that is, the presence of freshwater is deleterious for G. toxicus growth. No cyclic pattern for growth was observed in a 2-year, monthly collection of G. toxicus from Waianae Boat Harbor, Hawaii (45). In this study, the physical parameters (dissolved oxygen, salinity, pH, temperature, and water) remained constant throughout the study. The only significant finding was the lack of growth of the associated macroalga(Bryopsis sp.), which led to difficulty in finding G. toxicus during this period of no algal growth. The asexual life cycle of G. toxicus was examined in vivo and in vitro (45). The life ofcycle G. toxicus from Waianae Boat Harbor is shown in Fig. 2. It was initially cultured successfully by Yasumoto et al. (37). Since then, numerous laboratories have grownG. toxicus for biological and chemical studies. A comprehensive compilation of G. toxicus and other dinoflagellate literature is presented by Brusle (44).
W.
CHEMISTRY
The isolation, purification, and determination of the structure of the causative factor associated with ciguatera poisoning have constituted a tedious and long-term study initiated by Professor P. J. Scheuer et al. in Hawaii (23-25,46,47). The major obstacle in structural
214
Hokama and Yoshikawa-Ebesu
determination was the source of the natural material in fish, which was scarce even in the most toxic fish, G!'rnnothorclxju\,anicus (one moray eel liver yields about 1 btg purified ciguatoxin). Nevertheless, following the first isolation and characterization in 1967 (23), purified ciguatoxin (0.45 pg/kg LD5[,intraperitoneally in mouse) was obtained and the structure determined (23-26,28). It is a polyether of lipid nature consisting of several hydroxyl groups, solublein organic solvents (chloroform, diethyl ether, methanol, ethanol, 2-propanol,andacetone),withamolecularweight of 11 11.7 5 0.3 and an empirical fornlula of C,,H,,NO,, (47). Thepolyether nature of CTX was noted following the demonstration by Yasumoto et al. (48) that a compound (toxin PII) isolated from Prorocentrutn limL/ had all the biological and TLC (thin layer chromatography) characteristics of ciguatoxin. However, this compound was later identified as okadaic acid, a polyether that was earlier isolated from a sponge ( / f d i c h o n d r iokadui) ~~ by Scheuer's group ( 17). Brevetoxin (49) and many monocarboxylic antibiotics (50) isolated from Streptomyc~sbelong to this group of polyethers. The polyether antibiotics all possess ionophoretic properties affecting thepemleability of variouselectrolytesthroughthecellmembranes.Notsurprisingly, many of the dinoflagellate toxins (brevetoxin, okadaic acid, maitotoxin, and ciguatoxin) appear to have this same effect on various electrolytes (Na', K ' , Gal-), although the precise mechanisms have not all been ascertained. In the late 1980s and early 1990s, the structures of ciguatoxin from moray eel tissues andG. toxicus cultures were determined and reported by Yasumoto and Murata(51). The initial report was presented in 1990 (52). Fig. 3 shows the structure of CTXlB (from morayeel tissues) and CTX4B (from G. to.ricu.s). The yield from 125 kgof Gymr~othorrrxjmonicus viscera was 0.35 mg of pure CTX (molecular weight 11 11.5843 t 0.0053, formula C , , ~ ~ H ~ ~Several ~ O ~ c ~thousand ). liters of G.
37
1 R1 =
( f m Gabbierdiscus toxicus)
R2 = H Fig. 3 Structures of ciguatoxin from G . tosicus (CTX 4B) andlnoray eel liver (CTX1).
Ciguatera Fish Poisoning
215
to.ricus culture yielded 0.74 pg of pure CTX4B (the less polar congener of CTX), with a molecular weightof 1061S 8 4 and the formula C,,H,,O,, (33,5 1,52). Prior to the isolation and identification of CTX4B from cultured G. toxicus, gambiertoxin was only isolated from collecting wild, natural-growing G. toxicus. Yasumoto et al. (33,5 1,52) have reported more than 19 ciguatoxins that are associated with either ciguatera poisoning in fish or G. toxicus from the Pacific. The one major and 10 minor ciguatoxins (polyether isolates) from G. toxicus are referred to as gambiertoxins, while the three major and numerous minor ciguatoxins from three species of fish are called ciguatoxins. Table 2, taken from Legrand et al. (33) and partially updated by Yasumoto's group (personal communication), shows the number of ciguatoxin congeners characterized to date. Other polyether compounds isolated from G. toxicus are of significance. Gambieric acids isolated from the culture medium have shown potent antifungal properties (54) and G. toxicus culture medium weak toxic properties. The drawback is that the yields from Table 2 Ciguatoxins from Various Specics of Fishand G.
Code no.
MW
IA IB(CTX)
1111
1c 2A 1 2A2 2A3 2B 1 2B2 2c
I023
G. tosicm
tosicus
Scnrus Lutjonus 6ol1nr gihhtts (flesh) (viscera) (viscera) 0 e
1057 1095 1057 1061 1095 1061
e
3c 3C
3D
"4A *4B(GT-4B) 4C C-CTX- 1 C-CTX-2
(Caribbean) 0 e e
e
e e
e
e
e e e e
1077 l085
e
0 e
1080**
e
1062"'@ 1023 94 1 I095 1023 l06 1 1023 1061
e
106l
Crrnrrln latus
e
1095
3A 3B 3C 3C
Gymothoros jovor1icu.s
e
e
e e e
e e
e
e
e
e
e
0 1 142 +*l142
e
e ~
+ Carlhhean C-CTX: From Cumrl.r Intrlts, MW I 142 Da, clcctrophyslological studies (neuromuscular and cardiac tlssue) rcsumhle Pacilic CTX: sonic differences detected (53). 0 : M L I J Ot ~o x ~fraction (from Ref. 3 2 ) . * : Stcreo isomcr ** : Two recent CTX congners Isolatcd from moray eel tissucs ( 3 2 ) .
Hokama and Yoshikawa-Ebesu
216
are poor: yields of these toxins are very limited and generally only in the low microgram quantities. Nevertheless, if they could be synthesized in the laboratory, their use against fungal diseases would be of great significance. Lewis et al.( 5 5 ) isolated, purified, and determined ciguatoxin structures from moray eel, confirming Murata et al.'s (52) findings in 1990. Allof these chemical studies demonstrated the diversity of ciguatoxins that contribute to the clinical entity called ciguatera poisoning. Also contributing to these variabilities are the genetic and ethnic makeups of the individuals consuming ciguatoxin-contaminated fishes. Thus the diverse clinical findings in ciguatera poisoning appear to be compatible with the variation of chemical structures described in the study of ciguatoxin chemistry in the marine environment (33,51). Furthermore, this isolation, purification, and structural determination of ciguatoxin was a major breakthrough in ciguatera research, making possible the potential synthesisof ciguatoxin and the preparation of epitopes for better immunological assay developments. Two ciguatoxins were recently isolated in the Caribbean from horse-eye jack (Cmm x latus), a species associated with ciguatera poisoning(53). These two ciguatoxins were considered diastereomers and differed fromCTXlB, the major toxin isolated from moray eel viscera (52). Whether these toxins are related to the numerous minor ciguatoxins of Gyrnnot1mu.r sp. and L U ~ ~ U Isp. I Uremains S to be determined. Caribbean ciguatoxin was found to have similar biological toxicity in the mouse. At this date, it is much too early to concludethat Caribbean ciguatoxin differs significantly from Pacific ciguatoxin. Immunologically, studies with sheep anti-CTX and antibody prepared with ciguatoxin coupled to human serum albumin reactedvery well with tissues of Lutjrrrzus sp., a species involved in a large outbreak in the Caribbean (56). Recently,two newciguatoxinanalogs,2J-dihydroxyCTX3Cand 5 l-hydroxy C T X X , were isolated from moray eel (G. J a \ w ~ i c u sby ) Satake et a1 (32). The precursor CTX3C is a minor analog originating as a component of G. tmicus (34). Synthesis ofCTX1Bis presently being attempted by two laboratories in Japan, with one laboratory starting at the west sphere and the other at the east sphere of the C T X l B molecule (see Fig. 3). Both synthetic spheres (the west consisting of the A, B, and C rings; and the east consistingof J, K, L, and M rings) have been tested with the monoclonal antibody to ciguatoxin (MAb-CTX). Only the east rings J, K, L, and M have shown activity with the stick-enzyme immunoassay (S-EIA) procedure (57).
V.
PHARMACOLOGY
In an earlier pharmacologic study (an assessment of ciguatoxin with rabbit intestine suspended in tyrode solution) the resulting contraction of the muscle was shown to be greater when the blood was initially primed with ciguatoxin, prior to the addition of acetylcholine plus blood with glucose (58). Since no information of the ciguatoxin preparation was given, the data appears questionable in light of today's information on the chemistry of ciguatoxin. Subsequently, Rayner (59) in 1972, with partially purified ciguatoxin preparation, showed that the physiological effect of ciguatoxin was not due to an anticholinesterase action. Instead, ciguatoxin evolves its action through the voltage-dependent sodium channel (60,61). Ciguatoxin is a novel type of Na- channel toxin (60,61). The primary receptor site of ciguatoxin action is the fifth domain of the Na' channel, similar in this
Ciguatera Fish Poisoning
21 7
respect to the primary receptor site of brevetoxin (61). Ciguatoxin and brevetoxin (PbTx) share a common receptor site on the neuronal voltage-dependent Na’ channel (62), although ciguatoxin has a 20-50 times greater affinity for the Na- channel than brevetoxin. Whereas the varied clinical symptoms of ciguatera would suggest involvement of different toxins (15,64), ciguatoxin alone exerts its action on many tissue target sites. I n nervous tissues, ciguatoxin causes a tetrodotoxin-sensitive increase in sodium ion permeability and depolarization of the resting membrane (59). Depending on the magnitude of thedepolarization,theconsequence can be anincreaseinexcitability of theneuronal membrane or a depolarizing type of conduction block at high concentrations. Lower doses of ciguatoxin have marked effects on both the respiratory and cardiovascular systems. Although ciguatoxin has some neuromuscular blocking properties, the respiratory arrest induced by a lethal dose results mainly from depression of the central respiratory center (65).On the cardiovascular system, the effect is often biphasic: hypotension with bradycardia followedby hypertension and tachycardia (66). The hypotension and accompanying bradycardia are readily antagonized by the anticholinergic agents atropine, hexamethonium, and hemicholiniutn (67), whereas the tachycardia and hypertension are mediated by the sympathetic nervous system and suppressed by the adrenergic blockers propranolol and reserpine (67). The effects of ciguatoxin on smooth muscles are quite complex and depend on the predominant autonomic innervation and the postsynaptic receptor function. For example, on isolated guinea pig tissues, ciguatoxin enhances the inhibition of the taenia caecum (68) and excitation of the vas deferens (69). These effects are associated with norepinephrine release and antagonized by guanethidine, propranolol, reserpine, and tetrodotoxin. In contrast, ciguatoxin induces a sustained contraction of the ileum that is potentiated by eserine and completely blocked by atropine (70). Similar effects on excitation and inhibitionare observed i n other tissues. Ciguatoxin contracts the thoracic aortic strip of the rabbit (67) and relaxes the spontaneously contracting rat jejunum (71). Many of these effects of ciguatoxin can best be explained by the depolarization of nerve terminals and the resultant release of lleurotransmitters. Recent examinationof various crude methanol-soluble extracts of a herbivore (Cterloclltretus stripsrrs) and a carnivore (Serioltr clurtrerili) demonstrated that the pharmacologic use of guinea pig tissues (atrium and taenia caecum) in vitro was of great value in the characterization of ciguatoxin-like or maitotoxin-like toxins. C. srrigosus flesh extracts showed maitotoxin characteristics, whereas S. clunlcrili extracts showed ciguatoxin-like activity in the atrium and taenia caecum studies (72). I n electrophysiological studies with rats and human paticnts affected with ciguatera poisoning, Cameron et al (73) obtained findings indirectly suggesting that ciguatoxin acts on tnammalian nerves by prolonging sodium channel actions. The paradoxical sensory discomfort experiences in paresthesia and dysesthesia are most likely a result of an exaggerated and intense nerve depolarization occurring i n peripheral small A delta myelinated and C-polymodel nociceptor fibers (73,74). Other experimental studies of positive-CTX extracts in animals included (a) inhibition of the accunlulation of y-aminobutyric acid and dopamine (60); (b) prolonged sodium channel activation in rats and humans (73,75); (c) increased frequency of depolarization (75,76);(d) effect on myelinatednerve fibers (75);(e) release of norepinephrine (68,75,77); (f) release of intracellular calcium (78); (g) causeof nodal swelling (76); (h) release of acetylcholine (79) from NG 105- 15 neuroblastoma and glioma hybrid cells; (i)
Hokama and Yoshikawa-Ebesu
218
synaptic vesicle depletion (80); (j) fragmented nerve terminal, frog motor nerve terminals (80); and (k) positive inotropic effects in vitro in nmnlnalian atrial tissue assays (81-83). With the exception of the use of pure ciguatoxin preparations, results of crude ciguatoxin preparations used in many of the earlier pharmacologic analyses are questionable because of contaminating compounds.
VI.
EPIDEMIOLOGY
A.
Geographic Distribution
As indicated in the introduction, ciguatera poisoning is found worldwide between latitudes 35"N and 35"s circumglobally. The major endemic areas where most studies have been done are the Caribbean and the Pacific islands, including the lands adjacent to the Indian Ocean and the Gulf of Mexico. Archipelagos of the tropics considered to be safe from ciguatera poisoning are probably nonexistent. The travels of ships today and the periodic destructive hurricanes in the Pacific and Caribbean have contributed to the distribution and increase of the toxic dinoflagellate G. foxicws (84,85). For example, the presence of G. toxicus i n the state of Hawaii may be attributed to troop transports and warships coming from endemic areasof the Pacific to Pearl Harbor, which may have carried cysts or dinoflagellates on the bottoms and sides of ships and inballastwaterreleasedfromships during World War 11. Two major hurricanes in the state of Hawaii, Iwa (1982) and Iniki ( 1991), have further contributed to ciguatera. The increase i n knowledge of ciguatera i n recent years has also heightened awareness of ciguatera.
B. Incidence Ciguatera incidences have been reported throughout the world. The most extensively studied area has been French Polynesia by Bagnis and Legrand (85). In French Polynesia, the culprit involved, G. toxicus, was discovered by Yasurnoto et al. (22). Ciguatera poisoning has been recognized inmost of these epidemiological surveys, in which it is believed that only 20% of the total cases have been reported. In the Pacific, the epidemiology of ciguatera poisoning has received considerable attention becauseof the efforts of the South Pacific Commission (SPC). The countries compiled in Table 3 comprise what is probably the most studied area for the incidence of ciguatera poisoning. The Federated Statesof Micronesia, Palau, Puerto Rico, and Hawaii share relatively low incidencesof toxicity from fish in contrastto French Polynesia. In parts of the Central Pacific, the incidences of ciguatera poisoning are generally higher than i n Hawaii, but much lower than the French archipelagos (90). The incidencesof ciguatera poisoning in the region of the Indian Ocean are relatively low and nonexistent on some islands, such as Madagascar, Comores, Ceylon, and Indonesia.Arecentincident of interest, however, occurred in Madagascar, where 98 of 500 people died after eating the flesh of a shark. Clinical symptoms were not definitive for ciguatera poisoning, and with limited tissues, two new liposoluble compounds, designated carchatoxin A and B, were isolated (91,92). Reunion Island appeared to be unfavorable for toxic algae growth, but its rate of intoxication for ciguatera poisoning is the highest with 13.4 cases/100,000. These incidences may be due to imported fishes (93).
ses/
Ciguatera Fish Poisoning
279
Table 3 Numhcr of Incidences i n French Polynesia, the Micronesian Islands, and Other Arcas
Country 4.8 Federated States 1982-1987 of Micronesia 1982234.9 Marshall Islands 19871982-74.8 Northern Mariana Islands Palau 1960Polynesia French 807 Australia 100 Society S00 Tuamotu I700 Marqucsas 4300 Gambier 7.8 State of Hawaii RicoPuerto
x-l 1
SPC (86) l987
1984
SPC (86) SPC (86) SPC (86) Danielson Danielson and Bagnis 1985 ( 8 8 )
Health 1970-1995 Department of I996
(87)
(unpuhlished data) et Dcmotta al. (89)
I n the Caribbean, ciguatera poisoning varies with the islands involved. The incidence of ciguatera poisoning is low to moderate in Puerto Rico (89), high in St. Barthelemy (94) and high in the Virgin Islands(95). Ciguatera poisoning toxicities were also relatively high in the Dominican Republic and Cuba. Countries such as the United States and Canada have reported sporadic cases of ciguatera poisoning. In the United States, the major states reporting ciguatera poisoning are Hawaii and Florida. Other reports have resulted from importation of fish from endemic areas such as the Caribbean, and in some instances, from the Pacific. Ciguatera outbreaks also occur among tourists returning from endemic areas.
C. Fish Associatedwith Ciguatera Poisoning Ciguateric fish are generally restricted to species feeding on algaeor detritus around tropical reefs (96),especially surgeon fish (Ac,trrlt/rrrritltre)and parrotfish (Smridcre).Carnivores such as snappers(Lrr{jcr/riclcre).jacks (Ctlrcrngitltre),wrasses (Orbrickre),groupers, sea bass, rock cod (Serrcuridtre), moray eels (Murcre/ridcre), triggerfish (Bcrlisricicre),and barracudas (Sl)h!.rcrPnicitre) feed on the herbivores (97). For example, an outbreak on the island of Kauai occurred when a large bloom of G. r o x i u r s among the green algaBrycpvis contaminated the surgeon fish, Ctcwocheretrrs strigo.s~rs,causing an outbreak of ciguatera poisoning following consumption of C. srripsus. G. t0.ricv.s was found in 95% of the gut contents of the surgeon fish when examined with sheep anti-CTX fluorescent-labeled antibody(3 I ). The fishreportedlyinvolved in ciguaterapoisoningarelisted in Table 4.These fishes represent the major causesof ciguatera poisoning in particular regionsof the tropics. In a l l of the regions reported, the families of fish presented are similar. These include moray eels, snappers. groupers, wrasses, barracuda, amberjacks, jacks, surgeonfish, and triggerfish, and prilnarily represent tropical reef fish.
ion
Hokama and Yoshikawa-Ebesu
220 Table 4
FishesIlnplicated in CiguateraPoisoningfromVariousRegions
Geographical American Samoa
Australia
Caribbean (Virgin Islands) Florida
Vcrnoux and Lejeune ( 100) Lawrence et al. (101)
French Polynesia
Hawaii
Ciguatera Fish Poisoning
VII. A.
22 1
DETECTION METHODS
Bioassay
it was imperative to have an assay with specificity, sensitivity, and simplicity of application. Various invertebrate and vertebrate animals have been used for bioassays (28). Later, when extraction procedures were developed and the toxins were shown to be lipids, chemical procedures using fluorescent markers were utilizedfortheisolationandpurification of thetoxins.Purification of ciguatoxinswas achieved i n the late 1960s and late 1980s, enabling the development of immunological procedures (105,106). Banner et al. (28) examined a nurnber of animal species, from the lower forms to mammals. Some of the phyla examined included Protozoa, Ciliates, Arthropoda (CrustaceaandInsecta),Mollusca,Echinodermata,Chordata,Amphibia,Reptilia,Aves,and Mammalia. These animals were fed pieces of raw flesh by voluntary or force feeding, and in some cases were fed residues of alcoholic extracts (28). In feeding experiments with crayfish, significant physiological changes were observed, ranging from paralysis to death. The Africanland snail gave questionable results. The oral feedingof raw and dried toxic O r r j a n u s bohtrr showed variable reactions i n the mouse. The 36 otherspecies that were used demonstratednoresponses. Inlight of the present knowledge of the chemistry and concentration of ciguatoxin in fish tissue, thc results obtained by Banner et al. (28) were to be expected. Oral administration of raw or dried toxic fish flesh to mamn1als requires a larger dose of toxins over long periods to demonstrate any effect. Based on the mouse bioassay for PSP. Banner et al. (28) turned to intraperitoneal injection of toxic fish extracts in mice with some success, but still adhering to the fact that nlan becomes intoxicated via the oral route, they turned to the mongoose (Herpestc~s rnur7go) for the feeding assay. The mongoose can be fed up to 15% of its body weight of raw fish, dried fish, or oil extracts of fish incorporated in raw eggs. Unlike cats. 111011gooses retain all of the food consumcd. Cats tendedto regurgitate fish tissue, so the accuracy of the amounts eaten was questionable. The clinical response to toxic fish following consumption by the mongoose showed similarities to that of humans. The responses were classified into five stages for a single feeding followed for48 hours: no reaction, 0; slight weakness andflexing of the forelimbs, 1 ; slight motor ataxia, pronounced fiexionof the forelimbs and weaknessof the hind limbs, 2; moderate motor ataxia, weakness and partial paralysis of limbs and muscles, 3; acute motor ataxia and extreme weakness, limited movement, or coma, 4; death, 5. All responses 3,4, and 5 were solnetimes accomwere accompanied by loose stools (diarrhea); responses panied by extensive salivation and trembling. Although in the acute cases symptoms were neurological, no histological changes were observedin postmortem examination with light microscopy. Postmortem examination revealed some animals with gastric ulcers, but this phenomenon was also noted earlier in natural, wild, untreated mongooses. Animals usedinciguaterapoisoningbioassaysincludecat,mongoose,chicken, mouse, mosquito, and fish. Cats (Fdi.sctrtrrs) have been shown to be highly sensitive to toxic fishes, and kittens have been used as animal models in bacterial food poisoning. However, for humane reasons, the use of cats has been discouraged in the United States. Nevertheless, in earlier studies, cats were used in other countries (Tahiti, Japan) because of their sensitivity to toxic fish, which permitted the use of small fish samples without extraction procedures. Hypersalivation i n cats was one of the significant physiological 111 the study of ciguatera poisoning,
222
Hokama and Yoshikawa-Ebesu
reactions to ciguateric fish. However, regurgitation of the fish satnples caused difficulties i n obtaining accurate results. In addition, Inaintaining proper and clean housing facilities was a problem (28,107,108). Chicks (Gallus g ~ l l u ssp., G. donresticus) were used as an assay method for ciguatera poisoning assessment by administration of fish extracts via intravenous, intraperitoneal, or intratnuscular methods (58,109). Administration of extracts by intramuscular injection in hens demonstrated pathomorphological changes in the nervous system(58). Oral administration of toxic fish tissues or extracts to chicks induced hypersalivation, loss of weight, decrease in rectal temperature, and acute motor ataxia. Chicks appeared to be two IO five times more sensitive to toxic fish than mice. Chicks have proved to be satisfactory animal models for both feeding and injection assays for fish poisoning. They are readily available, easy to manage, relatively small in size and unlikely to regurgitate. They are highly sensitive to fish toxins and the effects of the toxin are cumulative. However, some negative aspects of using chicks have been pointed out by several investigators, including those using the chick assay (109). The effects of crude ciguatera toxin extracts fed to red snapper (Lufjcr1rr4.sh l r r r r ) were first determined in 1960 ( 1 10).In this study it was demonstrated that the toxin accumulated and was retained in the tissues of the snapper with no adverse effect to the fish. However, fish fed ciguatoxin and maitotoxin showed behavioral changes. Maitotoxin was G. to.vic~us-led fish the probable cause of the behavioral pattern, since experiments with showed patholnorphological changes i n the liver of S~rr.cr~ru.s cwbrillrr ( 1 1 1 ). Feedin, experiments withvariousfishspeciesweredifficulttoassesssincethedinoflagellate G. to.ric~4.scan produce several toxins in addition to CTX4B and CTX4C, maitotoxin and severalminorpolyethertoxins (33). Consequently, toattributetissuechangesonlyto ciguatoxin alone may be misleading. The results could also be due to the species of fish used for the particular assay: the use of freshwater and nontoxic reef fishes (not exposed to G. t o s i c u s in nature) could contribute to nlisleading results. More recent studies using fish and dinoflagellate extracts have demonstrated that fish are sensitive to IP administration but less sensitive than mouse. Mosquitoes (Aedes L w g y 7 t i i ) havebeen usedextensively i n FrenchPolynesia to assess crude ciguatera fish extract for toxicity ( 1 12). Crude fish extracts are injected into the intrathoracic cavity and the mosquito LD5,,of the fish toxicity expressed as grams of fish/mosquito. Significant correlations between toxicity in mosquito, mouse, and cat, and toxicity in man have been shown (108). Not considering the extraction preparation for each fish, the mosquito test is time consuming, but cost saving. It was considered the most reliable bioassay in French Polynesia, but unfortunately, as with all bioassays, unless pure material is tested, the test has no specificity, and the effects of CTXl alone cannot be demonstrated i n a crude mixture of unrelated toxins. Invertebrates have been used in assay systems for qualitative and semiquantitative assessment of fish toxicity. One advantage of their use is that their testing only requires small amounts of materials. The invertebratesutilized by some investigators have included brine shrimp, crayfish, flies, and mosquitoes (28,l 12- 1 16). I n these procedures, the technique is important to the success of using these assay organisms. Mice have been the major animal used to assess ciguatera poisoning. Because oral feeding in both mice and rats showed no promise, investigators (28) use the intraperitoneal route i n the nlouse assay. I n their purification studies, Banner et al. (28) showed that the active toxin was in the diethyl ether fraction and not the polar, water-soluble fraction in
Ciguatera Fish Poisoning
223
the intraperitoneal mouse assay. Most investigators now use the standardized procedure l/t), where t = hours. One for mouse toxicity with the formulation: MU = 2.3 log (1 mouse unit (MU) is the concentration of toxic extract injected intraperitoneally which kills a 20 g mouse in 24 hours. It has been found that with highly purified moray eel CTXIB, 1 MU = 7-8 ng/mouse. With crude fish extracts, the procedure (with modifications within each laboratory) is as follows: Swiss Webster mice weighing 20-25 g are utilized to assess the toxicity of fish extracts. One hundred milligrams of crude fish extract are resuspended i n 1 m l of 1% Tween 60 in saline and injected intraperitoneally into mice. Symptoms displayed by the nlouse are observed at 30 minutes, 1, 2, 4, 6, 8, 24, and 48 hours after injection and rated on a scale of 0-5 according to toxicity (Table 5 ) . The estimation of ciguatoxin by mouse units from known cases of ciguatera fish obtained from the Hawaii Department of Health (HDOH) ranges from 7 to 9 ng (1 17). The mouse test, like the other bioassays presented a lack of specificity, since any toxic factor in fish extracts to which the mouse is sensitive will cause death.
+
B. In Vitro Bioassay The in vitro bioassay to assess marine toxins has been of great interest in the past decade because of its sensitivity and use of minimal amounts of material. However, these test systems require tissue culture techniques and special physiological instrumentation ( 1 IS, 119). Nevertheless, these assays give some measure of specificity, since the actions are at the sites of the sodium channel of neuroblastoma cells or guinea pig atrial tissue (82). For the guinea pig atrial assay (82), 100 mg of each fish extract is resuspended in 1 1111 of Krebs-carbonate solution. One hundred microliters of the suspension is tested on the guinea pig atria. Subsequent inotropic and chronotropic actions are noted, in addition to the extract's pharmacological responseto tetrodotoxin (TTX), verapamil, and the adrenergic blockers (propranolol and phentolamine). The inhibitors TTX and verapamil, and p1 of a IO" M the adrenergic blockers are given after the inotropic responses at 12.5 concentration (68).
Table 5
MouseToxicityAssayScoring
ToxicityDescription 0 1
of visible clinicalsymptoms
in mouse after extract injection
No ill effect 15-60 minutes: muscle contraction in lower back area (flexion), increased respiration.
immobile (inactive), recovery as I , but recover in 2-3 hours, pilo-erection 3 Rccoverin12-24hours:same as 2,musclecontraction,paralysis in extremities (usually hind legs), rapid and irregular breathing, immobile, closed eyes, pilo-erection, slight cyanosis (tail) Symptoms 4 as in 3, but death within 24-18 hours,' 5 Symptoms of 3 and 4, death in lessthan 6 hours
Same 2
.' 1 MU. death withln 24 hours In 20 g mouse; contains 7-9 ng ofciguatoxln in the sample o f 100 mg of crude
cxtract/mousc injectcd (IP) (estimated from HDOH contirmcd clguatcric fish extracts).
Hokama and Yoshikawa-Ebesu
224
The guinea pig atrial in vitro assay, in spite of the specialized techniques involved (including dissection of the atria, left and right, from the heart), is an excellent means of studying marine toxins, especially crude marine toxin extracts. Once inotropic standard patterns are obtained for each pure toxin and the effects of various chemical inhibitors are ascertained, the inotropic patterns of crude fish extracts with inhibitors can be compared with the standardized patterns of the pure samples. Thus CTXlB and other ciguatoxin-like toxins (okadaic acid and brevetoxin) show similar inhibition with tetrodotoxin. That is, ciguatoxin, okadaic acid, and brevetoxin can be readily distinguished from palytoxin and maitotoxin. Sodium channel effector toxins can be distinguished from calcium channel effectors by use of appropriate inhibitors to the inotropic response. Figure4 representsclassicalinotropicpatterns of pureciguatoxin,okadaicacid,palytoxin,andbrevetoxin with or without TTX inhibition.
l
TTX
TTX
Ver
Rinse
Rinse
Fig. 4 Inotropic pattcrns for PTX, CTX. OA and PbTx-3 in the guinea pig atrial assay. a) Pure PTX (240 ng): blocked by verapalnil (ver) but not by tetrodotoxin (TTX). b) Crude CTX (3.2 ng): blocked by TTX. c) Crude OA (1 pg): blocked by TTX. d) Pure PbTx-3 (20 ng): blocked by TTX.
Ciguatera Fish Poisoning
225
C. In Vitro Cell Assay
1. Neuroblastoma Assay Directed cytotoxicity to sodium channels of neuroblastoma cells has been established for purified ciguatoxins, brevetoxins, saxitotoxin, and crude seafood extracts, including finfish as well as from thePacific ( 1 19). Mouseneuroblastoma extracts from the Caribbean (neuro-2a, ATCC) were grown in RPM1 complete medium supplemented with 10% heatinactivated fetal bovine serum, 2 mM glutamine, l mM sodium pyruvate, 50 pg/ml streptomycin, and 50 U/ml penicillin. Actively growing cultures were maintained at 37" C in a humidified 5 % CO: atmosphere. For bioassay, 96-well plates were seeded with 1 X 10' neuroblastoma cells in 200 p1 growth medium and incubated for 24 hours. Culture wells then receive 10 p1 each of the test sample, 10 mM ouabain, and 1 mM veratridine. Toxin samples were tested in replicates of four at various concentrations of the sample. A minimum of 15 wells were processed as ouabadveratridine controls and 5 wells were evaluated as untreated controls (cells only). Purified brevetoxin (PbTx-l) was included as a positive control of sodium channel enhancing activity in conjunction with ciguatoxic finfishextractassays.Nonspecificcellactivitywasassessed by addition of samples and PbTx-l, but no ouabain and veratridine. Cultures were then incubated either 4 or 22 hours for detection of sodium channel enhancing activity or 24-48 hours to assess sodium channel inhibitory activity. MTT (3[4,5-din~ethylthiazol-2-yl]-2.5-diphenyl-tetrazolium) was used to assess cytotoxicity by replacing the overlying medium with 60 p1 of a 1 :6 dilution of MTT stock ( 5 mg/ml PBS, pH 7.4) in complete culture medium. Plates were then incubated for 15 minutes at 37°C or until sufficient formazan deposits were observed in untreated control wells.The overlying 100 p1 of DMSO andtheplatesreadat medium was then replaced in each well with 570 nm in an ELISA plate reader. This procedure measures the viable active cells via mitochondrial dehydrogenase reduction of the colorless MTT to formazan to give a purplish color read at 570 nm. The IDSo for PbTx-l was detected at 250 pg and the ID5,, for purified ciguatoxins were detected at 1 pg for CTXl and 3 pg for CTX3C at 7 hours incubation. CTXl was isolated from moray eel livers and CTX3C fromG. toxicus culture as a minor component. This cell bioassay isof value for marinetoxin studies and may replace mouse assays when the use of live animals for experimental studies becomes prohibited. Measures of specificity and sensitivity for sodium channel activator and inhibitor compounds were excellent. In this respect, it was very similar to the guinea pig experiments, but appeared to have a greater sensitivity. 2. Other Cells In addition to neuroblastoma cells, HeLa cells and fibroblastic malnmalian kidlley cells have been used to evaluate ciguatoxicity (120,121). 3. Radiolabeled Binding Assay Since ciguatoxin and brevetoxin sharea common receptor in the sodium channel (domain S), although with a difference in affinity (CTX1 > PbTx), with the use of labeled brevetoxin UH-PbTx-B), ciguatoxin can be quantitated by competitive binding assay with sodiumchannel-containingproteins.Thishasbeenshown by severalinvestigators (1 18,122) using isolated rat brain synaptosome with 3H-PbTx-B and the CTX3C congener of CTXlB isolated from G. tosicus ( 1 18). This procedure requiresa small amount of fish
226
Hokama and Yoshikawa-Ebesu
extract and the method is rapid, simple, and has a high sensitivity. Although the method of the is useful for research, it may be impractical for large-scale fish screening because method’s use of a beta counter and radiolabeled compounds.
D. Immunoassay 1. Radioimmunoassay The first successful detection of a low nlolecular weight polyether toxin, ciguatoxin, directly from contaminatedfish tissues was reported in 1977 using radioimmunoassay (RIA) with a sheep antibody prepared with purified moray eel ciguatoxin conjugated to human serum albumin (HSA) as carrier (26). Subsequently the RIA was used to screen 5529 Seriolo dutnerili (kahala), a species associated with ciguatera outbreaks in Hawaii from 1979 through part of 1981 (123). The 15% of fishtestingborderline or positivewere discarded and the remaining 85% of fish scoring negative were sold commercially with no false negatives. Although the RIA procedure proved effective, its complexity and cost suggested seeking alternative immunological ciguatera screening methods.
2. Enzyme Immunoassay An enzyme immunoassay (EIA) was subsequently established using sheep anticiguatoxin (anti-CTX) coupled to horseradish peroxidase, based on the same principle used in the of expensive isoRIA (26). This method, although economically feasible because the use to be tedious. Consequently this tope counting instruments was eliminated, was found EIA method was abandoned and was followedby the stick-enzyme immunoassay (S-EIA) employing sheep anti-CTX and monoclonal antibody to ciguatoxin (MAb-CTX) coupled to horseradish peroxidase. This method has been used extensively for surveys of ciguateraendemic areas and clinical confirmation of documented ciguatera poisoning for the Hawaii Department of Health ( 1 24- 129).
3. lmmunobead Assay The application of colored polystyrene particles coated with MAb-CTX as markers for direct detection of ciguatoxin adsorbed onto organic correction fluid-coated bamboo paddles was instituted in 1990 (130). In this solid-phase imlnunobead assay (SPIA), the organic correction fluid coat bound both polar and nonpolar components onto the bamboo paddle sticks. Numerous documented ciguateric fish from the HDOH and reef fishes surveyed in Hawaii were assessed using this procedure (1 3 1,132).
4. Summary The compilation of data of HDOH fish from ciguatera poisoning-implicated fish tested by RIA, S-EIA, and SPIA is shown in Table 6. The data include both the fish implicated in ciguatera poisoning as compiled by the HDOH and the routinely tested Hawaiian reef fishes generally associated with ciguatera poisoning in Hawaii. The major species tested included Ctenochuetus strigosus (kole) andSeriolrr dumerili (kahala). Ciguatera poisoning in patients was clinically documented by physicians or epidemiologists from the Department of Health. Of the 176 ciguatera-implicatedfish tested with these assays, 171 (97.2%) were found to be borderline or positive, while 5 (2.8%) were found to be negative (false negative). These procedures demonstrated a high degree of sensitivity with both sheep anti-CTX and MAb-CTX. On the other hand, the examination of Hawaiian reef fish of unknown toxicity demonstrated a fair to moderate degree of specificity with the MAb-
Ciguatera Fish Poisoning
227
Table 6 Compilation of the Assessments of Test Procedures Prior to the
MLA
Results
delinel Number used Procedure Radioimmunoassay (RIA)
Stick-enzyme 16 immunoassay (S-EIA)
Antibody of fish Reference Negative Positive
Sheep anti-CTX
Sheep anti-CTX
HDOH 46 Routine samples S. dlollerili 5596 Other Species 766 HDOH
824 (14.7%) 17 (2.2%) 16
2 (4.4%)
154
4112 (85.3%) 749 (97.8%) 0
( 100%)
Routinc samples various species 574 HDOH MAb-CTX 83 Routine samples
c. strigosis 712 S. durwrili
168
44
(95.6%)
168 Other species 3539
Solid-phase HDOH MAb-CTX inmunobcad assay (SPIA)
31
Routine samples various species 482
212 (36.9%) 81 (96.8%)
362 (63.1%) 2 (2.4%)
392 (55%) 82 (49%) 1720 (48.6%) 30 (96.8%)
320 (45%) 86 (51%) 1819 (51.4%)
26 1 (54.1qJ)
22 1 (45.9%)
1
(3.2%)
Both State of Hawaii Department o f Health (DOH) implicated toxic tishcs and routine samples of reef fishes arc included. The MIA data are prcscntcd 111 detail i n Tdbles 7 and 8.
CTX in the S-EIA and SPIA procedures showing 45.0-5 1.4% for the unknown fish. This is due in part to nonspecific binding to the coated bamboo stick. This moderatespecificity has limited the commercial use of these tests. However, following extraction, 70% of the borderline and positive extracts of unknown fish tested in the mouse and the guinea pig assays provedto be toxic. The sheep anti-CTX proved to give the best results in specificity (85.3-97.8%) and sensitivity (95.6%) in the RIA procedure in assessment of S. dutnerili and other reef fishes. The lack of a higher specificity suggested a search for a better solidphase medium for binding ciguatoxin and related polyethers.
E. Membrane lmmunobead Assay The membrane inmunobead assay (MIA) technique is based on the same immunological principles used to develop the SPIA ( 130), using a monoclonal antibody to purified moray
Hokama and Yoshikawa-Ebesu
228
eel ciguatoxin (CTX-I) (13 1,132), colored polystyrene beads, and a hydrophobic membrane laminated onto a solid plastic support. The membrane only bindsto lipids and generally not to polar or water-soluble compounds, unlike the liquid paper-coated sticks. The polyether toxins bind to the hydrophobic polyvinylidene fluoride (PVDF) melnbrane and are then detected with the MAb-CTX coated onto colored polystyrene beads. The intensity of the color on the membrane correlates to the concentration of toxin.
1. Preparation of Monoclonal Antibody to Purified
Ciguatoxin (MAb-CTX) Highly purified ciguatoxin was obtained through the courtesy of Professor P. J. Scheuer of the University of Hawaii Department of Chemistry. The toxin was isolated from LW)doiltis (G?.mrlothor.rrx)jrrvmicus (moray eel) livers and consisted of two interchangeable isomers (24).
2. Coupling of CTX1to Human Serum Albumin Purified ciguatoxin ( I pg) was coupled to human serum albumin (HSA)( 1 mg) in 7.5 t n l of 0.05 M phosphate buffered saline (PBS) by the carbodiimide procedure (133). Following exhaustive dialysisof the protein conjugate against salineat 4"C, the conjugate was precipitated with cold acetone in a ratio of 4: 1 (acetone:conjugate). The precipitate was centrifuged, rinsed twice with cold acetone, suspended in saline, and then used for the innnunization of the BALB/c mice. After pervaporation, the acetone phase was analyzed for free ciguatoxin. These steps were critical for the immunization process (133).
3. Immunization of BALB/c Mice Three BALB/c mice, 10 weeks of age, were injected intraperitoneally once a week for 3 consecutive weeks with 0.1 1111 of the CTX/HSA conjugate. A booster was given in the sixth week, 3 days prior to sacrificing the aniruals. Each nlouse received a total of 80100 ng CTX/80-100 pg HSA and 0.2 1111Freund's complete adjuvant (FCA) subcutaneously for each third injection of 0.1 ml of the conjugate.
4. Fusion Step The nonimlnunoglobulin synthesizing mouse myeloma cells selected for fusion were those previously reported by Kearney et al. (134), designated PBX63-Ag8.656B. These cells were grown in Dulbecco's modified eagles medium (DMEM) supplemented with 10% fetal calf serum. Myeloma cells in the logarithmic growth phase were used for fusion. The fusion of the sensitized cells from the BALB/C mice with the myeloma cells was doneby the method of Kennet etal. ( 135-137). These procedures included hybridoma selection, media preparation, and limiting dilution methods. The original active hybridonla containing reactive anti-CTX was designated 5C8. Analysis of 5C8 with commercial immunofluorescence-labeled goat anti-mouseimmunoglobulinisotypes(SigmaChemical Co., St. Louis, MO) demonstratedthe following hybrid cells: IgM,85%; IgG,, 55%; IgC?,,, 14%;IgG2h,3%: and possibly IgG3.This suggested a mixture of clones since the original myeloma produced no immunoglobulins. Nevertheless, 5C8 supernatants and ammonium sulfate fractions(50% precipitates) were used successfully in numerous studies from 1987 to 1994 using a variety of test procedures (124-132). Throughout theyearsfrom1987 to 1996,subculturing of 5C8 causedgradually diminishing activity of the MAb-CTX. Subsequent ELISA isotyping of different lots of SC8 (50% anlmonium sulfate fractions) showed the loss of the IgM, IgG,,,, and IgG2,, isotypes and the appearance of IgG+ The first recloning of 5C8 resulted in six viable,
Ciguatera Fish Poisoning
229
moderately active clones out of 100 plated. The clone designated 2E6 was further cloned by limiting dilutions resulting i n 1 1 (6%) clones with good activity. All of the good and moderately active clones were frozen and stored at -80°C. One of the highly active clones, designated 1C6, has been further cultured and used. Purification by gel affinity protein G column and isotyping in the ELISA procedure demonstrated equal amounts of IgG, and IgG; in the clone IC6. The analyses for sensitivity and specificity were taken from Morgan et al. (138): Sensitivity =
positive (HDOH) fish implicated in ciguatera detected by MIA X 100 total number of positive fish implicated in ciguatera ~
Specificity =
fish presumably without ciguatera toxins, negative in MIA X 100 total number of fish tested, presumed to be negative for ciguatera
5. lmmunobeadPreparation The clone designated 1C6 was selected and its cultured supernatants collected and diluted 1 :20 in saline in a final suspension of 0.1 % colored polystyrene beads. The 1 :20 dilution of antibody was selected as the optimal antibody concentration following titrationof each batch of monoclone IC6 cultured supernatants with the end point determined as approxinlately I : 100 dilution. The 1 :20 dilution circumvents the ‘‘hook effect” ( 139) and appears to be able to detect ciguatoxin at levels as low as 80 pg/g of fish (80 ppt). Sensitivity can be enhanced by increasing the MAb-CTX concentration. Polystyrene beads of blue (0.314 pm in diameter) and red (0.124 pm in diameter) colors were used i n a ratio of 3 : 1. These bead sizes were found to be satisfactory in suspension since larger-size beads, 20.8 pm in diameter, tended to settle readily to the bottom of the vial. The use of a single color of beads was satisfactory,but the combination of two different bead colors gave a deeper hue. Polyvinylidene fluoride membrane was laminated onto a plastic support stick (Fig. 5). Other materials and chemicals used in the MIA include test tubes (8 mm inside diameter 75 mm length), a punch biopsy tool or sharp razor to obtain fish samples, methanol for soaking fish samples, a suspension of 0.1% colored microbeads (0.3 14 pm and 0.124 pm diameter) coated with MAb-CTX. Standard control for positives consisted of pooled crude extracts of HDOH implicated fish from clinically diagnosed ciguatera poisonings. The of Hawaiian reef fishes implicated inciguatera presence of ciguatera in these crude extracts poisoning was previously examined by Hokalna et al. ( 1 30) and Manger et al. ( 1 19) in the neuroblastoma cell bioassay. In addition, the presence of ciguatera was previously
80 mm (plastic suppon)
T 7
~
1
1-1
+OSmrn 20 mm
(membrane) Fig. 5 MembranestickforMembraneImlnuunobeadAssay (MIA) withdimensionsgiven.The membrane portion of the stick is directly laminated onto the longer plasic support.
Hokama and Yoshikawa-Ebesu
230
established at 4 ng/ 12.5 mg of crude extract by itnmunoinhibition of the MAb-CTX with highly purified C T X l B (140).
6. Membrane lmmunobead Procedure The concept of the MIA is similar to that of the solid-phase immunobead assay (SPIA): colored immunobeads will adhere to ciguatoxin (antigen) on the membrane portion of the plastic stick previously exposed to the antigen; positive samples will show visible color changes, while negative ones will not (Fig. 6). A fish tissue sample (approximately S 2 3 mg) cut with a punch biopsy tool or razor blade is placed along with 0.5 ml of methanol and a membrane stick into a test tube. After soaking for 20 minutes, the stick is removed from the test tube and thoroughly air dried for at least 20 minutes. The completely dried membrane stick is immersed into 0.5 ml of latex immunobead suspension, removed after 10 min, rinsed in saline or water, shaken to remove excess liquid,air dried, and the membrane observed for color. The result is scored as negative, no distinct color on the membrane; weakly positive, light bluepurple color on the membrane, with or without a darker band at the meniscus level; or strongly positive, membrane is colored and has more than one dark band at the meniscus level. All fish with results reading weakly positive and higher should not be eaten. Positive control samples at both high and low concentrations of toxins consisted of pooled extracts of implicated HDOH ciguateric fish at7.0 mg/ml and 3.0 mg/ml of methanol containing residual ciguatoxin of 2.24 ng and 0.96 ng ciguatoxin/ml of methanol, respectively. Negative controls were blank membranes used directly or soakedin methanol anddriedthoroughlybeforeimmersingintotheimmunobeadsolution.Thesequential steps of the MIA procedure are presented in Fig. 6. During the development of this method, several synthetic membranes were examined and the hydrophobic membrane proved to be the best, since less than 3% of negative controls showed nonspecific binding of the immunobeads. The data in Table 7 represent an assessment of fish recently involved in clinically documented ciguatera poisoning cases using the MIA. Of 13 fish samples examined, 10 were found to be positive and 2 borderline, containing approximately 60-160 pg ciguatoxin or related toxin per gram of extract. This gave a sensitivity of 92.3%, with 7.7% false negatives. A Sphgmerza bcrrr-clcudcl sample from the island of Hawaii gave an initial negative response in the MIA. However, further examination of the extract of this fish by Membrane stick
I
L
Methanol Tissue sample Step 1
Step 2
Fig. 6 MembraneImmunobeadAssay
Colored bead with MAb -CTX suspension
Negative
Step 3
(MIA) procedure.
Weak Pos. Strong Pos. Step 4
231
Ciguatera Fish Poisoning
Table 7 Membrane lmnunobead Assay of Ciguatera-Implicated Fish Samples, Hawaii Department of Health Species
Remarks
Source Oahu Oahu Kauai Big Island Oahu Kauai Kauai Oahu Maui
unidentified) (species Snapper Barbedos (via Red fish (species unidentified) Barracuda (species unidentified) Crpkalopko1i.s q q u s (mi)
MIA result sample +
+
+ -.I
+
+ + +
+ t
New Jersey) Texas DOH Texas DOH Big Island
+ + ?h
Cooked Cooked sample Cooked samplc Cooked sample Tissue Tissue Tissue Cooked sample Cooked sample sample Cooked Tissue Tissue sample Cooked
-.
Results are shown as +. positlvc; 5 . borderline; and negative. Thls sample was extracted and tested with the MIA and reacted weakly at 7.0 nlglml in laethanol and negative at 3.5 mg/ml. Mouse toxicity results (dead In 7 minutes after intraperltoncal injection) suggest presence of PTX- or MTX-like toxms. The guinea pig atrium analysis suggested the presence of low levels of CTX-like compounds i n this sample (sec Fig. 7). h One patient ate the samespec~es for three consecutive meals: it is uncertain if the same or three different tishcs were consumed. Three others ate only one meal of roi with no toxicity reported. A roi from the same catch was tested and found t o be negative.
mouse toxicity and guinea pig atrium analyses demonstrated that it contained a potent toxin unlike ciguatoxin. I n comparison with a classical ciguatoxin pattern showing strong inhibition by tetrodotoxin (TTX), the S. barrncudcl pattern showed only a slight tetrodotoxin effect (Fig. 7). Nevertheless, the extract at 7.0 mg/ml showed a weak MIA response and a 100 mg dose killed a mouse in 7 minutes. Still, the patient who consumed part of this fish demonstrated classic ciguatera symptoms (141). Taken together, these data suggested the presence of palytoxin, maitotoxin. or as yet undetermined toxins in this barracuda, along with low levels of ciguatoxin-like compounds. The analysis of various fish species with the MIA is presented in Table 8. This table includes the species names, sources, total number of samples, and MIA results directly from fish tissues. The Cmmx sp. (jack or papio) from Hawaii shows a higher percentage of borderline and positive results (23%) than those from Kwajalein and Kosrae (0% and lo%, respectively). In Hawaii, the Crrmnx sp. is one of the major targetsof sportfishermen and is the leading cause of ciguatera poisoning in nearly all of the islands of Hawaii. In summary, of the 154 samples tested with the MIA, 132 (85.7%) were negative, 14 (9.1%) wereborderline,and 8 (5.2%) were positive. The specificity (85.7%) shown iswithin acceptable levels for biological assays of this nature. All negative fishes were consumed without any reports of ciguatera poisoning; in other words, no false negatives were recorded. Figure 8 represents the titration of the standard pooled HDOH-positive extract in the MIA. The data show MIA results of the extracts tested at different concentrations and
232
DOH-poritiw extract 10 rng (b)
Hokama and Yoshikawa-Ebesu
Tetrodotoxin V e r a p d
Rinse
Fig. 7 Inotropic patterns from guinea pig atrium assays for (a) S. burrcrcudn and (h) DOH-positive (containing CTX) extracts with addition of tetrodotoxin, 10 ' M (12.5 p]), verapamil, 10 ' M (12.5 pl), and rinse, respectively. The difference in the TTX block is clearly demonstrated i n the CTX containing extract (h).
Membrane Immunobead Assay of Routine Catches of Fish from Hawaii and Pacific Islands
Table 8
MIA results
Total Species
Ncgative Borderline Positive fish Source
C~YULY sp. (pepiolulua) Curunx sp. (papio) Curmx sp. (papio)
Ctenochaer~rstrigosus (kole) Mugil cephml11.s(mullet) Mulloidichthys aurifluuztnu (weke) Serioln rlumeriti (kahala) Aphureusfitrcn (wahanui) Mu1loirlichthy.s pflugcvi (weke ula) Rodianrts bilinulrrt~rs(a'awa) A p i o r ~virescens (uku) Ccphalopholis arg~ts(roi) S ~ ~ J ~ Nhelleri ~ I I L(barracuda) I Prirrc~or~thu.~~ meeki (aweoweo) A c ~ n r ~ t h u trio.steg~t.s r~s (manini)
Hawaii Kwajalein Kosrae Midway Hawaii Hawaii Hawaii Hawaii Hawaii Hawaii Maui Oahu Oahu Oahu Oahu
Total number (%) Perccntage values arc shown
Other
26 82 20 3 3
3 3
2 (7.6) 4 (5.0) 0 (0.0) 0 0
5
1
4
2 3 2
1
0 1
2 2 1
1 1 1
154 In
parcntheses
20 (77.0) 78 (95.0) 18 (90.0)
2 0
2
2 2
0 0 0 1 0 0
0 0 1
1 132 (86.0)
14 (9.0)
4 (15.4) 0 (0.0)
2 (2.0) 0 0 0 1 0 0
0 0 1
0 0 0
8 (5.0)
233
Ciguatera
3.0+ 2.0+
2.0C
1.6+
1.6+
l.O+
ff
-- -
Fig. 8 Examples of DOH mixedciguatericfishextracts from 0.1 through 10.0 mgextractin 1 ml of methanol (in duplicate), tested in the MIA. The numbers in the parentheses are calculated concentrations of CTX in the crude extracts. The -, t,and numbers represent relative degree of activity.
+
the approximate calculated ciguatoxin concentration. The minimal detection point was 0.08 ng or 80 pg (80 ppt) ciguatoxin in the crude HDOH mixture. This new MIA procedure was compared with previously developed immunological (142). The S-EIA used test procedures which used correction fluid-coated bamboo sticks bamboo skewers with a correction fluid coat and MAb-CTX conjugated to HRP with appropriate substrate as the detection system. The SPIA used a correction fluid-coated bamboo paddle and MAb-CTX adhered to colored polystyrene beads to detect ciguatoxins. The MIA system consists of a solid-phase hydrophobic synthetic membrane laminated onto a solid plastic support. The sensitized membrane is dipped into an aqueous suspension of mixed polystyrene beads of two different colors and diameters bound to MAb-CTX and the intensity of the resulting color on the membrane assessed. This new procedure was used to assess clinically implicated HDOH ciguatera fish samples, a variety of routine fish samples from endemic reef areas of Hawaii, a number and of Caranx sp. from Kwajalein and Kosrae. The results presented in the tables show good detection of HDOH ciguatera-implicated fish, with one exception: direct examinationof the S. barracuda tissue showed no activity in the MIA, but the extract was highly toxic in the mouse assay and showed response in the MIA with the extract at a higher dose (7 mg). Analysis of the extract in the guinea pig assay suggested its major toxic component to be other than ciguatoxin (Fig. 7), as stated earlier. Comparison of Carum sp. from Hawaii, Kwajalein, and Kosrae showed that those from Hawaii had a higher toxicity level (23%) than the samples from Kwajalein (5%) and Kosrae (10%). In part, this may be attributed to the fact that the Caram sp., from Hawaii were obtained from several areas on different islands, while the Kwajalein and Kosrae samples were from one area on a single island. Other groups of fish samples from Hawaii were too small to properly evaluate. Nonetheless, the overall sampling by the MIA shows reasonable ciguatoxin detection levels, with 86% negative, 9% borderline, and 5% positive. The variability dueto nonspecific bindingof the immunobeads to the membrane is minimized by the use of the hydrophobic membrane, with only3% of 179 blank membranes tested showing a borderline reaction. The sensitivity achieved with the small num-
234
Hokama and Yoshikawa-Ebesu
ber of HDOH toxic samples was 92.3%, while the specificity indicated for unknown reef fishes was 85.7%. These values were within the acceptable ranges for a biological test system. The sensitivity of the older procedures (RIA, S-EIA, SPIA) ranged between 95.6% and 97.2% and the specificity was close to 50%. This low specificity suggested nonspecific binding of the antibody conjugate and the antibody bead to the liquid paper on the stick. In part, the antibody bead tended to bind to the bamboo paddles nonspecifically in 22 ( 1 0.6%) of the 207 blank sticks examined. As mentioned, the MIA blanks showed only 2.8% nonspecific binding. The low specificity with liquid paper-coated bamboo paddles needs further studyif these procedures are to be used for commercial ventures. However, the SPIA has been useful in preventing ciguatera in recreational fishermen in Hawaii (132). With the exception of okadaic acid, cross-reaction studies of MAb-CTX with other polyethers are limited (140,143). As indicated by Landsteiner (144), MAb-CTX reacted best with ciguatoxin, its homologous antigen (epitope) at the 1 ng level, while 5 ng of okadaic acid was necessary for comparable activity. Other related polyethers, including brevetoxin, palytoxin, maitotoxin, and congeners of okadaic acid, require levels of 2 5 0 ng to give comparable reactions with MAb-CTX in the S-EIA assay. In the development of the MIA, several factors critical to obtaining accurate and repeatable results were noted. First, the membrane portion of the membrane stick must not be touched, since this can cause false-positive reactions. Second, the membrane stick must be soaked in the methanol/fish sample suspension forat least 20 minutes for optimal results. Third, both the stick and the test tube must be completely dried before adding the latex immunobead suspensionto the test tube; failure to doso can also cause false-positive reactions.Lastly, to preventfalse-positiveresults,themembranestickshouldnotbe soaked in the latex immunobead suspension for longer than I O minutes. If these points are followed, the MIA procedure should prove to be simple and reliable for the detection of ciguatoxin and related polyethers in fish. Preliminary studies suggestedthat the binding sites of ciguatoxin to sodium channel receptors and the monoclonal antibody to ciguatoxin were on opposite sites of the ciguatoxin. Evidence for this interpretation was based on immunological analysis with epithelial extracts of pig small intestine (sodium channels) and MAb-CTX in the guinea pig atrium assay. Ciguatoxin addedto the physiobath containing guineapig atrium induces an inotropic (increase in the atrium contraction, and thus the amplitude of the heartbeat) response. Preaddition of varying concentrations of pig intestine extract or MAb-CTX into the physionot the latter bath inhibits the inotropic effect in the former (pig intestine extract) but (MAb-CTX). Further, immunological studies employingthe S-EIA demonstrated that the west sphere synthetic tricyclic rings A, B, and C did not react with MAb-CTX, but the east sphere synthetic tetracyclic rings K, L, M, and N reacted with MAb-CTX (57). Furthermore, computer molecular modeling for ciguatoxin suggests the reactive siteof ciguatoxin on the fifth domain of the sodium channel is the west sphere, which is similar to brevetoxin in its reaction with the synaptosome of the rat brain tissue (122). Modified immunoassays for polyether toxins have been developed, including radioimmunoassay for brevetoxin (1 18,122). However, to date, no extensive testing or evaluato show sensitivity, but tion of this immunoassay has been established. They appeared specificity has not been evaluated and the procedures were generally not applicable for simple screening of toxic fish.
Ciguatera Fish Poisoning
235
F. Chemical Methods Analyses for polyether marine toxins including palytoxin, tetrodotoxin, okadaic acid,brevetoxin, alld ciguatoxin have useda variety of chemical methods. Thin layer chromatography and high-performance liquid chromatography (HPLC) have been explored. Chemical techniques have been utilized for the analysis of okadaic acid and other polyethers in extracts of fish ( 1 18,145,146). Fluorometric analysis has been developed for assessing tetrodotoxin (okadaic acid, DSP poisoning) and ciguatoxin ( 1 18).
G. Summary Various assays have been developed to identify the toxin of interest in natural marine products, in the purification process, and for screening purposes to maintain wholesome and healthy products for consumption. This has been the case for shellfish and finfish some products. Thus the review and comments addressed in this section were dedicatedto detection.Thebasicgoalsare to achievesimplicity,sensitivity,and specificity i n anassay system to be universally applicable for home, field, and laboratory use in ciguatera poisoning. Most of the bioassays using animals or cell systems tend to be nonspecific. However, for ciguatoxin,use of sodium channel targets suchas synaptosomes and cell bioassay with neuroblastoma indicate some level of specificityandsensitivity. The immunochemical assay appears to be the most promising for practical use cornmercially in screening for ciguatera toxic fish because of its simplicity, practicability, and most of all, its specificity and sensitivity.
VIII. CLINICAL Consumption of toxic fish associated with ciguatera poisoning results in the appearance of clinical symptoms within 10 minutes to 12 hours, but on occasion 24 hours, after consumption (mean 4-6 hours). The gastrointestinal symptoms are characteristic of many other food poisonings, but ciguatera poisoning can be differentiated by unique features affecting the neurologic system. Table I summarizes the many clinical symptoms associated with ciguatera poisoning (15,147). The major toxin implicated has been designated ciguatoxin (23), although the complex symptoms and peculiarities among patients from different tropical and subtropical areas (Caribbean and Pacific) suggest multiple toxins (15,64). For example, Bagnis and Legrand (85) and Bagnis et al. (102), in examining numerous cases from the SouthPacific, showed subtle differences in certain clinical symptoms between Melanesians and Polynesians. Pruritis, ataxia, abdominal pain, and weakness were more commonly reported by Melanesians than by Polynesians. Whether these differences were due to variability in toxins (sourceof fish) or the susceptibility of different ethnicities to ciguatoxin remains unclear (85,102). Diagnosis was based on clinical presentation with paresthesia and dysesthesia, considered a clinical hallmark of ciguatera poisoning, especially in the South Pacific. This essentially differentiates ciguatera from other formsof food poisoning ormild gastroenteritis (64,85,102). In Hawaii, the use of immunological tests including the MIA has aided in the confirmation of ciguatera poisoning by examination of the remaining contaminated fish tissues.
236
Hokama and Yoshikawa-Ebesu
Reports of ciguatera poisoning from the Caribbean (including Florida) and the Pacific (Hawaii, French Polynesia, American Samoa, and Australia) all generally show variations of the clinical symptoms includedin Table 1. These variations can change from area to area. The neurological symptoms, especially dysesthesia and paresthesia, the were major clinical diagnostic hallmarksof ciguatera poisoning followingconsumption of toxic fishes, although these symptoms can also occur in shellfish poisoning and tetrodotoxin intoxication (147). Ciguatera poisoning caused by Scarvs gibbus from the Gatnbier Islands showed initial clinical symptoms characteristic of the ciguatera syndrome. However, after 5-10 days, a second phase appeared, characterized by an ataxic gait, dysmetria, asthenia, loss of static and dynamic equilibrium, and muscle tremor, which was induced by an effect on the cerebellum. This second phase set in for about 7 days and required about 4 weeks for complete recovery (1 5). This variation was probably due to another polyether toxin, isolated and named scaritoxin (148,149). Recently scaritoxin (Table 2) has been identified as a congener of ciguatoxin and named CTX3B (Molecular Weight 1077). Palytoxin has been implicated by Japanese investigators following consumption of a parrotfish (Y/,sisccrru.sovifrom) ( 1 SO). One patient died 4 days after consuming the fish and had symptoms not unlike ciguatera poisoning (150). Earlier (1983), Yasumoto et al. (15 I ) reported the presence of palytoxin in two species of crabs (Loph0zozymrr.s pictor and Detmllicr crlcalcri) associated with lethal poisoning of humans in the Philippines. Palytoxin toxicity has also been reported in Hawaii from smoked Decapterus mac" rosoIm imported from the Philippines ( 152). I n laboratory analysis of toxicity induced by several species, low levels of palytoxin were demonstrated in 4of the 14 fish examined (141). It seems that the term ciguatertr encompasses fish poisoning associated with a group of lipid polyether compounds,of which ciguatoxin appears to be one of the most hazardous and probably the most prominent i n carnivores (23). A study of two outbreaks in Hawaii i n 1985 involving a herbivore (Ctet~ochtretus strigosus) and a carnivore (Seriola d u m r i l i ) showed clinical similarities and differences (64). C. strigosus induced only gastrointestinal and neurological symptoms i n all 16 patients with no hospitalization. Seven of the 14 patients who had eaten S. durnerili were hospitalized and 7 had cardiovascular symptoms in addition to gastrointestinal and neurological problems. A similar observation was reported by Bagnis (15) in Tahiti. This observation of ciguatera poisoning by two fish species of different eating habits suggests the following: (a) there is more than one toxin, (b) changes in the toxin occurred during the passage from herbivore to carnivore, and (c) there were no differences between toxins, but poisoning was a dose-response effect because C. strigosus was small and the viscera (liver, roe, etc.) was not consumed. Other factors worth noting in ciguatera poisoning include (a) multiple poisoning of an individual causes an enhancement of clinical severity and greater sensitivity to the toxin in subsequent poisonings; (b) the poisoning is rarely lethal ( 14,16,85,98,101,102) when the flesh is consumed, and lethality (less than 1%) occurs when the most toxic parts of the fish are eaten (liver, viscera, organs, roe, etc.); and (c) susceptibility to the toxin(s) may vary among individuals. Mammals susceptible to ciguatera poisoning include dogs, cats, dolphins, California sea lions, Hawaiian monk seals, and domestic pigs. Therefore care should be taken in feeding these animals fish from endemic areas. Transmission of toxin via ciguateric mother's milk during breast-feeding has been demonstrated for ciguatera poisoning, along with placental transfer of the toxin, which has resulted in abortions during the first trimesterof pregnancy (85,102). Sexual transmis-
Ciguatera Fish Poisoning
237
sion via seminal fluid from an intoxicated husband to wife has also been reported ( 153). Thereforeciguatoxinanditscongenerscan be transmittedthroughbreast-feeding,the placenta, and body fluids (seminal fluid) from a ciguateric patient to a healthy individual. This is probably made possible by the ability of ciguatera to bind normal serum and tissue proteins, one of which may be albumin. Table 4 summarizes the fish most commonly implicated in ciguatera poisoning i n the Caribbean, including Florida, and the Pacific (Hawaii, French Polynesia, American Samoa, and Australia). Most of the fish listed are carnivores. Part of the variation in toxicity relative to fish between areas may be attributed to ethnic variations in taste for various fish species; another contributing factor could be the variation due to the nature of secondary prevailing toxins. In all studies reported, the cyclic effect of a seasonal pattern of intoxication was inconsistent. I n American Samoa, toxicity has occurred all year round. Toxicityin Florida occurs frequentlyin the spring and silnnner months. In Hawaii, no cyclic pattern of toxicity has been shown. A close examination of 14 fishes consisting of eight species implicated in ciguatera poisoning in 1994 withrespect to clinical symptoms, immunological tests of fish, and guinea pig atrium (sodium channel effect) and mouse bioassays has been reported (141). It was concluded fromthe comparison of the clinical symptomsin the patients and analyses of the fish consumed by the patients i n the immunological test, guinea pig atrium, and mouse toxicity assays that there was a diversity of toxins in the ciguateric fish examined. Potentially, in any given fish there was probably more than one toxin causing the ciguatera symptoms. Diversity of toxins or congeners has been previously suggested by investigators in ciguatera poisoning studies (15,64,85,102,104,14I.l52.154~. Based on the mosquito, mouse, and MIAtests of the consumed ciguatericfish, it has been estimated that illness occurs when man consumes at least23-230 ng ofciguatoxin in a meal. This was referred to as the pathogenic dose (PD) by Bagnis et al. (85,102). The wide range of toxicity suggests variability in individual susceptibility and the effect of previous exposures to very low concentrations of ciguatoxin or congeners (cumulative effect). Others have indicated the minimal level of toxic fish consumption to be 50-100 pg/g of fish (155,156).
IX.
PATHOLOGY
A.
Human
Pathological findings in postmortem studies of humans following death from ciguatera poisoning are relatively scarce. This is due, in part, to the fact that deaths from ciguatera poisoning are very rare (less than 1 % worldwide). Most deaths reported were probably due to palytoxin, particularly to intoxication from eating crustaceans. The crabs Dernclrlitr wqnaudi, D. cdcnltri, and hphozozqr~ru.spicfor from the Philippines and Singapore have been implicated in toxicities and death, but these involved palytoxin as the causative factor. Postmortem examinations of human tissues generally showed little or no histologic tissue changes with routine hematoxylin and eosin (H & E) staining. Evidence of morphologic tissue changes detectable either by light or electron microscopy in human deaths due to ciguatera poisonings is very rare (44). However, three reports in the literature demonstrated these findings: (a) histologic lesions in nerve fibers with striking edemain the vacuoles of Schwann cell cytoplasms and vesicular degeneration of myelin ( 1 57); and (b) muscle biopsy on a ciguatera poisoning victim revealed fiber-
238
Hokama and Yoshikawa-Ebesu
splitting degeneration and necrosisof the liver ( 158,159). No electron microscopy findings have been reported in human fatalities as a consequence of ciguatera poisoning.
B. Animal Studies 1. Ciguatoxin and Brevetoxin Experimental animals administered whole fish tissues, crude organic solvent extracts, and purified ciguatoxin presented with clinical symptoms characterized i n human intoxication with ciguatera poisoning. Wild mongoose given toxic moray eel flesh was noted to have increases in ulcers of the stomach (28). However, i n nature mongoose has been found to develop stomach ulcers due tostressfulenvironments (28). Experimental mice models show few light microscope changes with routine H & E staining. but definite pathologic changes can be seen with the electron microscope i n target organs of mice administered pure ciguatoxin ( 1 60). Repeatedintraperitonealandoraladministrations of ciguatoxin 1B(CTXIB), CTX4C, and PbTx-3 derived from L I I ~ ~ N bI oI hI cI wS and CTX4C purified from G. roxicus to male ICR miceat a dose of 0.1 pglkg for 15 days resulted in marked swelling of cardiac cells and endothelial lining cells of blood capillaries in the heart. A single dose showed no discernible pathologic changes in the heart. Damage to the capillaries was followed by prominent effusion of serum and erythrocytesinto the interstitial spacesof the myocardium. Swelling of the endothelial lining cells of the capillaries caused narrowing of the lumen and accumulation of blood platelets in the capillaries, which caused multiple necroses of single-cell cardiac muscle cells. Myocytes and capillaries appeared normal after 1 month of the treatment with toxins. The effusion in the interstitial spaces resulted i n the formation of bundles of dense collagen, which persisted for 14 months. Diffuse interstitial fibrosis was prominent in the septum and ventricles, with bilateral ventricular hypertrophy. A single dose of 0.1 pg CTXI/kg intraperitoneally resulted in severe acute heart injuries, followed by diffuse myocardial fibrosis ( 160,161 ). It is well established that the west sphere of ciguatoxin acts on the membrane of cells, with the receptor being the fifth domainof the sodium channel, similarto brevetoxin (162). However, ciguatoxin has been shownto have a greater affinity for the receptor than brevetoxin. Both compounds depolarize nerve terminals through their actions on sodium channels, which potentiate the membrane and cause the influx of sodium ions into the cells. It has been suggested that this increase in the sodium influx creates the pathological patterns of the targeted cells when examined by light or electron microscopy ( 160,161 ). Nevertheless, surprisingly, damage by acute levelsof PbTx (200 pg/kg) showed less tissue pathology than CTX at a sample dose of 0.35 pg CTXI /kg. CTX1-treated animals showed marked edema in the heart muscles as well as the nerve tissues. Whether given in shortterm successive or long-term intermittent doses, the effect of CTXl on the heart muscles was similar to the single dose of 0.35 pg CTX 1 /kg. by stimulation of the motor neurons Acute dosesof brevetoxin showed tonic seizures and death with a short opisthotonosis. Pathologic changes in the heart, kidney, and liver by PbTx-3 were less severethan those of ciguatoxicosis. Table 9 summarizesthe comparison of the toxic effects of pure ciguatoxin and PbTx-3 i n ICR mice (161). The effects of CTXI (0.35 pg/kg) on the cardiac tissues at a lethal dose were much more severe than PbTx-3 at a larger lethal dose (200 pglkg), though both toxins are sodium channel ago-
Ciguatera Fish Poisoning
(relative intensity) muscle Cardiac cells Blood capillaries (endothelial lining cells) Thrombosis(heart.liver.penis) Cunlulative effect heart in
239
31 3+ 3+
2+
+ +/-
nists. The affinity for the sodium channel, however, is much greater for CTX I than PbTx3. This physiological difference may account for the variance in the cardiac tissue pathology. Mice given high (death in 65 minutes), moderate (deathin 2 hours 43 minutes), and low doses (death i n 7 hours 15 minutes) of ciguatoxin all demonstrated changes in the small and large intestines. Small intestines showed degeneration of nuclei and swelling (edema) of the lamina propiain the high-dose (CTXI) administration, while the moderatedose CTXl mice showed degeneration of the villi tips. Stripping of brush borders of the villi was observed i n the small intestines with the low-dose CTXl treatments. The lowdose CTXl also caused epithelial surface degeneration, sloughing into the lumen, and increased mucin production. These findings were demonstrated by light microscopy (163). Eighty percent of a mouse unit of semipure CTX 1 B from Gytnt~othormj a w r n i c m s given per mouse induced diarrhea within 10 minutes after intraperitoneal injection and lasted for 30 minutes. The changes were shown i n the large intestines. CTX 1 B induced accelerated mucus secretion and peristalsisi n the colon and stimulated defecation resulting in prominent diarrhea. Large quantities of mucus were secreted in the colon from mature as well as immature goblet cells and epithelial cell damage was observed in the upper portion of the large intestines, but not the lower half. The morphologic changes of the upper large intestine induced by CTX I B were similar to those seen with cholera toxin ( I 64,165). Diarrhea appears to be induced in mice by CTX 1B in doses between I and 7 ng/mu. Mice given less than I ng and greater than 7 ng intraperitoneal injections showed no diarrhea. Mice given oral doses at any levels showed no diarrhea. It appears that variable patterns of diarrhea after intraperitoneal injection of ciguatoxin crude extracts may be attributable to the C T X l B concentration in the crude extract. The pathology induced after administration of ciguatoxin in experimental animals can be summarized as follows: (a) acute poisoning by CTXlB or CTX4C showed damage in the heart, nledulla of the adrenal glands, autonomic nerves, penis, and small and large intestines (163,164);(b) atropine suppresses symptomsof diarrhea, but had no effecton the
240
Hokama and Yoshikawa-Ebesu
injury to cardiac muscle; (c) reserpine aggravatedthe clinical symptoms and pathological findings; and (d) guanethidine and S-hydroxy dopamine and bilateral adrenalectomy had no significant effects on ciguatoxicosis.
2. Dinophysistoxin-l and Pectenotoxin-l Pathological tissue changes occurred in experimental mice administered other related polyether marine toxins. Sequential intrastructural changes were studiedi n the gastrointestinal system of the BALB/c strain of mouse following intraperitoneal administration of dinophysistoxin- 1 (DTX- 1 ) and pectenotoxin- 1 (PTX- 1 ), causative agents of DSP, DTX- 1, a diarrheagenic toxin, produced severe mucosal injuries in the small intestine within an hour after the injectionof the toxin. The injuriesin the small intestines included extravasation of villi vessels, degeneration of absorptive epithelium, and desquamation of the degenerated epithelium from the lamina propia. PTX-I, on the other hand, a nondiarrheagenic toxin from DSP-causing mussels, demonstrated no pathologic changes in the small intestine, but induced pathologic changesin the liver within an hour after intraperitoneal administration. Numerous nonfatty vacuoles appeared in hepatocytes around the periportal regions of the hepatocyte lobules. These vacuoles originatedfrom invaginated plasma membranesof the hepatocyte, as shown by electron microscopy with colloidal iron ( 166).These studies were carried out with suckling BALB/c mice of either gender given various intraperitoneal doses of DTX- 1 and PTX- I .
3. Maitotoxin Maitotoxin (MTX), one of the toxins implicated in ciguatera poisoning and probably the most toxic substance isolated from cultured G. toxicus, induced severe pathomorphologic changes i n the stomach andheart,andatrophy of lymphoidtissueswithreduction of circulating lymphocytes after release of cortisol from the adrenal gland. A reduction of IgM and an increase in calcium in the adrenal glands were also noted in mice. In the gastric nwcosa, multiple lesions were seen with a marked increase in total calcium level 1-24 hours after intraperitoneal administration of 200 or 400 ng/kg of MTX. There was no close temporal association between accumulation of calcium and the morphologic appearance of dead cells in the heart and thymus. After administration of 200-400 ng/kg MTX, marked swelling was seen the in endothelial lining cellsof blood capillaries between cardiac muscle fibers within 30 minutes, followed by cell death of the cardiac muscle cells. MTX at 2200 nglkg also resulted in necrosis of the lymphocytes in the cortex of the thymus at 4 hours and the medulla at 8 hours after administration. Mice pretreated with CoCI,, a calcium channel inhibitor, and/or adrenalectomized mice showed no discernible changes i n the lymphoid tissue after repeated administrations of MTX. It was concluded that MTX initially stimulated calcium influx into the adrenal glands, which induces the release of cortisol into the circulation. The increase of cortisol i n serum results intheacuteinvolution of the thymus and lymphoidtissues,andthus reduction in Ig synthesis ( 167.168).
4. Palytoxin The effects of palytoxin (PTX) on young male ICR mice were examined with ouabain and several cations, including potassium, and the tissues examined by light and electron Illicroscopes for changes. The target organs of PTX were the heart, kidneys, pancreas,
24 1
Ciguatera Fish Poisoning
slnall intestine, and liver. Pathomorphologic changes of the heart induced by PTX were silllilar to those of a lethal dose of KCI. However, no pathologic changes were shown in the kidney for mice treated with KCI. Similar kidney damage in the pro xi ma^ as well as the distal convoluted urinary tubules in mice given 5 mg/kg ouabain were shown, while the heart of Illice given ouabain i n the same dose demonstrated no discernible pathologic changes. Ouabain given together with palytoxin had no effect on the damage induced i n the heart and kidney by palytoxin at 8 mg/kg. It was concluded that palytoxin caused a syt11pto111 col11pIex of hyperkalemia and induced profound inhibition of Na-/K -ATPase ( 169.170). Table 10 presents the LDS,,of mouse toxicity for various known pure nlarine toxins studied. The decreasing order of toxicity i n mouse is as follows: '
MTX
> PTX > CTX > TTX > PbTX > OA > PbTx-B.
5. Toxicity in Fish Generally fish associated with ciguatera poisotlitlg are not affected by consulllption of G. fo.xicll,y, whether in herbivores or in consumption of herbivores by carnivores. For example, toxic fish fed to carnivores caused no toxicity or pathologic changes, but carnivores accumulated the toxin in their tissues ( 1 IO). However, abnormalities occur when fish not associated with ciguateric transmission (freshwater fishes, for example) are fed either G. f o x f u r s or ciguatera-contaminated flesh. Why ciguateric fish are not affected remains to be determined; whether it is metabolic (ability to assimilate toxins into tissues), or a buildup of tolerance with constant exposure to the toxins remains to be studied (44). I n studies prior to availability of pure toxins, pathomorphologic changes induced by administration i n experimental animals were difficult to assess because of the potential mixture of toxins i n crude extract. A case in point is the finding of anticholinesterase activity in fish extracts associated with ciguatera poisoning (52). Present studies with purified ciguatoxin, maitotoxin, palytoxin, and the okadaic acid group of toxins have given a better account of pathomorphologic changes and the clinical symptoms from observations in both the anirnal studies and the symptoms observed in the clinical setting.
6. Summary Thestudies in mice modelswith
pure marinetoxinscarried out by Terao et al. ( 16 1,162,164- 169) probablyreflect the pathology in humans, where studies are very lim-
Table 10 ComparativePotency o f MarineNatural Toxins in Mice
Rcl'erence toxin
L D , , (pg/kg)
Molecular
i n mice (IP)
weight
Source
Hokama and Yoshikawa-Ebesu
242
ited. I n humans, the major target organs affected by marine toxins are the gastrointestinal tract, nervous system, and muscles, explaining the symptomology seen for ciguatera fish poisoning (Table 1 ).
X.
THERAPY
A.
Earlier Treatment
Treatment of ciguatera poisoning can be separated into two periods. I n the first period, limited information about the mode of action, chemistry of the toxins, and definite patterns of s y m p t o m were known for ciguatera poisoning. A thesis by Chanfour ( 17 I ) presents the treatment for ciguatera poisoning in French Polynesia. which was quite similar to the therapy devised by Russel (147). Essentially, treatment was based on the action of the drugs on the s y m p t o m observed. Thus, in humans, drips of atropine at 0.5 nlg every 4 hours were used i n a continuous, multiple-electrolyte solution (147). Atropine appeared to have an effect on the gastrointestinal and cardiovascular symptoms and signs. However, atropine had no significant effect on the skeletal muscle symptoms and showed only a slight improvement i n the neurological findings following injection of atropine. Cyanotic and respiratory diseased patients were placed on oxygen. Patients were maintained on high electrolyte solutions and intravenous daily doses of calcium gluconate, a vitamin B complex, vitamin C, and time-disintegratillg capsules including phenobarbital, hyoscyamine sulfate, atropine sulfate. and hyoscine hydrobromide. These drugs were used i n an attempt to control the presunled pharmacologic action of fish poisoning toxins. These treatments were confined to patients seen within 48 hours of the fish poisoning (147). In cases seen 3-14 days after poisoning, the most severe complaints were sensory disturbances. Atropine had no effect on these patients; that is, no detnonstrable improvements were seen. These patients did best with a high protein diet and administration of intravenous vitamin B complex, calcium gluconate, and oral vitamin C. Extremes i n temperature were avoided because they caused recurrenceof painful pruritus, paresthesia, and nausea ( 147). The use of pyridine-2-aldoxin~e methiodide (2-PAM) based on the anticholinesterase concept of ciguatoxin action produced no improvement i n the patients’ conditions. This was demonstrated in earlier reports ( 172). In 1975, one of the best courses of treatment for ciguatoxin, as recommended by Russel (147) was atropine to control the gastrointestinal corlplaints and cardiovascular disturbances in the early phases of ciguatera poisoning, and intravenous high electrolytes, calcium gluconate, and vitamin B complex, coupled with a high protein diet and vitamin C. Thisappeared togivethemostsatisfactoryresultsfollowingtheacuteperiodand shortened the duration of illness. For pruritus, analgesic cream was used because steroid creams were of no value. For insomnia, diazepam I O mg was rccommcnded because steroids were ineffective.
B.
Recent Treatment
Tocainide has been used with some success i n three patients with long-term, chronic ci( 173). Amitripguatera, i n which neurological symptoms have persisted over several years tyline was also given to these same patients prior to tocainide with no effect (173). At
Ciguatera Fish Poisoning
243
this tilne, 110 significant large-scale studies on the treatment of chronic ciguatera poisoni% with tocainide or aI11itriptyline have been reported. In part, studies of chrOIlic ciguatera a sufficient patient pOpLllapoisoning are limited i n the United States due to the lack of tion. A more recent preliminary study suggested the useof antioxidant therapy for chronic ciguatera poisoning ( I 74). It was suggested that long-term antioxidant therapy might have 21 role i n the nlanagement of chronic ciguatera poisoning. Vitamin E has been shown to provide protection from various chemicals thought to initiate free radical-induced illjury by blocking the deleterious effect of free radicals i n the cellular membrane (175). The suggested therapy for chronic ciguatera poisoning was vitaminE 1000 IU/day for 1 week. followed by vitamin E 2000 IU/day for 2-8 weeks, vitamin C 500 mg/day, and betacarotene 100 mg/day. Amitriptyline (an antisuppressant drug) givenat a low doseo f 2 5 mg to an individual in improvementandeventualrecoveryafterseveral withciguaterapoisoningresulted weeks (176). However, amitriptyline had no significant effect in studies carried out on the Marshall Islands (177). In the western Pacific, traditional remedies prepared from numerous plants have been used both in oral treatment and topical applications for rashes, pruritus, and other skinailmentsassociatedwithciguaterapoisoning.Extracts of leaves.bark.roots,and fish poisoning. Although some fruits of various plants have been traditionally used for plant extracts alleviate someof the symptoms of ciguatera poisoning, none of the hundreds of plants examined was specific for the treatment of ciguatera poisoning ( 178). The most significant clinical discovery in the treatment of ciguatera poisoning occurred serendipitously, when two coma patients in the Marshall Islands (Jaluit Atoll) were treated with mannitol for edema of the brain. Both patients were suspected of ciguatera poisoning.Afewminutesafterintravenousinfusion of mannitol,bothpatientsawoke from their comas (177). Subsequently, all patients with severe ciguatera poisoning have routinely been treated with intravenous infusion of mannitol at the Majuro Hospital in the Marshall Islands. In thefirst JAMA report (177), 24 patientswithacuteciguaterapoisoningwerc treated with intravenous mannitol and each patient's condition improved dramatically.All patients exhibited marked lessening of the neurologic and muscular dysfunction within n1inutes of the mannitol infusion. Relief from gastrointestinal symptoms took longer. Two patients in coma and one i n shock responded within minutes with full recovery after mannitol infusion. These observations were empirical anduncontrolled.Nevertheless,later studies i n Australia and the United States (179,180) have verified and repeated the studies reported in 1988 ( 177). Mannitol is presently used i n severe ciguatera poisoning in most emergency units whereciguaterapoisoningoccurs(Hawaii,Florida,PuertoRico,Australia).Mannitol treatnlent is most effective when given within the first 24-72 hours after ciguatera poisoning, and after 4 weeks following intoxication, mannitol may be of no help ( 177,179,180). The exact mode of mannitcl action i n acute ciguatera poisoning is not clear, but it may be attributable to its pharmacological actions.In addition to being a potent diuretic, mannitolcan causedecreasingcellularedema,reestablishingionicbalanceanddecreasing plasma pH; mannitol is a scavenger of selected chemical structures including hydroxyl group and free radicals ( I 8 1 ). A major effect maybe the interference or removal of bound ciguatoxin at the receptor sites of sodium channels of nerve and muscle tissues ( 1 82). Thus mannitol acts best during the initial period of acute ciguatoxin poisoning.
Hokama and Yoshikawa-Ebesu
244
C. Summary As seen by recent approaches to treatment, nontoxic drugs which would have a protective effect on the sodium channelof the nerves and muscles would probablybe the choice for alleviation of the clinical symptoms associated with ciguatera poisoning.
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Tetrodotoxin
I. 11.
Introduction 254 SourceofToxin 255 A. Distribution 255 B. Tetrodotoxin-bearing organisms 255 C. Transmission o f tetrodotoxinthrough the foodweb D. Bacterial origin tetrodotoxin of 257 E. Major sources of human intoxication 260
257
111. Detection Methods 263 A. Mouse bioassay 263 B. High-pcrformnnce liquid chromatography 263 C. Mass spectrometry 263 spectrometry 264 D. Gas chrornatogmphy-mass E. Thin-layer mass spectrometry (TLC-MS) andliquid 264 chromntography-Inass spectrometry F. Thin-layer chromalography 265 G. Capillary isotachophoresis 266 H. IR spectrolnetry 266 I. ' H-Nuclearmagneticresonance (NMR) spectrometry268 J. Electrophoresis 269 K. Cytotoxicity assays 269 L. Immunoassays 269 IV. Clinical 269 A. Symptoms 269 B. Trentlnent 270 C. Incidences 271 V. Toxicology 276 VI. Pharn1acological Actions 276
253
Yoshikawa-Ebesu et al.
254
Use of Tetrodotoxin as a PharmacologicalTool277 A. Organismal level 277 B. Molecularlevel 277 VIlI. Summary 281 References 282 VII.
1.
INTRODUCTION
Tetrodotoxin (TTX) is one of the most potent nonproteinaceous toxins known, responsible for numerous fish poisonings. This toxin is one of' the oldest known toxins, recorded as early as 2700 B.C. in Chinese literature describing the toxicity of puffers and around 2500 B.C. in Egyptian history in hieroglyphs depicting the fatal poisoning of a pharaoh who had eaten one of these fishes (1,2). In the late 188Os, Yoshizumi Tahara named this toxin after the order Tetraodontiformes,sincemany of its memberscarrythetoxin. In additiontotheTetraodontidae (puffer, balloon fish, globe fish, swell fish, fugu, toad fish, and blowfish), two other families, the Canthigasteridae (sharpback puffers) and Diodontidae (spiny puffers or porcupinefish) also include members associated with tetrodotoxin. Tetrodotoxin poisoning cases due to ingestion of puffer have frequently occurred in the Orient, especially in Japan where these fish are a traditional food. The preferred forms of this delicacy are slices of raw flesh (sashimi) and slightly cooked liver (kimo). Japanese people are aware of the puffers' toxicity and have devised ways to reduce tetrodotoxin in the liver. Judging from the statistics in Japan, the number of deaths due to puffer poisoning has steadily declined, from morethan 100 cases (with morethan 50% mortality) every year before 1959, to between 10 and 100 cases (with more than 25% mortality)
Tetrodotoxin ('IXX)
acidTetrodonic
255
every year between 1960 and 1981,to between 1 and I O cases (with morethan 6% mortality)everyyearsince1982 ( 3 ) . Thisdecline in incidence is probablydue toincreased government regulations. Tetrodotoxin is a water-soluble heterocyclic guanidine whose chemical structure has been characterized (Fig. l ) . Tetrodotoxin only has one guanidium moiety. As can be seen in Fig. 1, many derivatives of tetrodotoxin have been found, although their toxicitiesvary widely. First isolated in 1964 from puffer of the family Tetraodontidae, tetrodotoxin has since been found in diverse organisms, including marine fish and an octopus as well as some terrestrial amphibians such as newts and frogs (see Ref. 4). Tetrodotoxin has been found in several bacteria species, including Shewcmella crlgu and Vibrio spp., and is now believed to be of bacterial origin (5-8).
II. SOURCE OFTOXIN
A.
Distribution
Various toxic tetraodontid species are globally distributed (9,lO). However, thesefish are consumed only in certain parts of the world. Areas that have reported tetrodotoxin poisoning cases due to consumption of toxicpufferfishinclude Japan, Taiwan, China, Hong Kong, Thailand, Korea, Singapore, Malaysia, Australia, the United States, Kiribati, Papua New Guinea, and Fiji. Other organisms found to possess tetrodotoxin are listed in Table 1. Like puffers, these animals are found worldwide: Japan (goby, blue-ringed octopus, various gastropods, starfish,xanthidcrabs),Taiwan(goby,variousgastropods,xanthidcrabs),Philippines (goby), Costa Rica and Panama (Atelopus frogs),Australia(blue-ringedoctopus),the United States (newts), and Thailand (horseshoe crab).
B. Tetrodotoxin-Bearing Organisms Puffers were long believed to be the exclusive source of tetrodotoxin (Fig. 1) (9,lO). The distribution of tetrodotoxin in these fish is mainly in the ovaries (eggs), liver, and skin, though the pattern varies to some degree among species. The origin of tetrodotoxin was often debated to be either endogenous or exogenous. In 1964, however, this toxin was unexpectedly detected in the California newt (Tnrichn torosa) (see Ref. 1 l), thus settling the controversy. In this organism, tetrodotoxin was concentrated in the skin, ovary, muscle, and blood. Since then, tetrodotoxin has been found in a variety of animals (Table l). Other newt species including T. rivularis, T. granulosa, T. vulgcrris, T. cristcrtus, T. nlpestris, T. mmoratus, Notophthalmus viridescens, Cyopsis pyrrhogrrster, and C. erlsicaudcr were also discovered to possess tetrodotoxin in almost the same anatomical distribution as T. torosa. In 197 I , tetrodotoxin was foundin the skin, viscera,and muscle of a goby (Yongeicht l y s crirliger) inhabiting the Amami and Ryukyu Islands, Japan. This goby is also distributed in the Philippines and Taiwan, whereit has been implicated in several human poisoning cases. In addition, tetrodotoxin was detected in the skin of Atelopus frogs inhabiting Costa Rica and Panama. In 1978, tetrodotoxin was isolated from the postsalivary gland of the blue-ringed octopus (Hnl~7aloc~hlerenn mcrcrdoscr) which mainly inhabits northern Australia. Human tetrodotoxin cases are occasionally reported in this area due to envenomation by H. nlucu-
256
Yoshikawa-Ebesuet al.
Table 1 Distribution of Tetrodotoxin inAnimals (9)
Animals
Parts
1. Platyhelminthes:
Turbellaria:
Whole body
2. Nemertinea:
3. Mollusca:
Gastropoda:
Cephalopoda:
Whole body Whole body Whole body Digestive gland Digestive gland Digestive gland Digcslive g h d Digestive gland Whole body Digestive gland Digestive gland Postsalivary g h ~ d
4. Annelida:
Polychaeta: 5 . Arthropoda:
Whole body Whole body Whole body EEE
6. Chactognatha:
Head Head 7. Echinodermata:
X. Vertebrata;
Pisces: Amphibia:
Whole body Whole body Whole body Skin, liver, ovary Skin, viscera, gonad Skin, egg, ovary. muscle, blood Skin, egg, ovary Skin, egg, ovary, muscle, blood Skin, egg, ovary. muscle. blocd Skin Skin
lostr. This octopusalso occasionally appears in middle to south Japan. The octopus secretes tetrodotoxin from the posterior salivary gland to paralyze its prey. In 1979, a serious poisoning incident dueto ingestion of the trumpet shell (Chtrmtria .scmliae),whose muscle is widely consumed, occurred in Shimizu, Shizuoka, Japan. The causative agent was isolated from the digestive gland and identified as tetrodotoxin ( 12). Two similar food poisoning incidents followed in Wakayama and in Miyazaki, Japan, in 1982 and 1987, respectively. This toxic carnivorous trumpet shell is widely distributed in middle to south Japan. Other carnivorous gastropods suchas the frog shell or ootrtrr’utoDor’rr (Tutujcr lissostorncl), hatmnushiro,yc~i(Zeuxis suquijorewsis), and trrcrrepi (Niotha clnthmtcr) in Japan and Niotlrtl lineclttr (13) in Taiwan also possess tetrodotoxin i n their
Tetrodotoxin
257
digestive glands. In addition, in 19S0, tetrodotoxin was detected in the ivory shell(Berbylorritr , j c r p o r ~ i c c z ) collected , i n Fukui, Japan. Mass food poisonings due to ingestion of the eggs of the horseshoe crab sporadically occur i n Thailand, where during early 1996 they caused about 20 deaths. Tetrodotoxin in the eggs was later determined to be the cause of death. Xanthid crabs (Ater;~crfi.s,pnr.inlr.s, Zo.sitr~rr.scrcr~tw),flatworms (Plcrrrot~)nrspp.), and ribbon worms (Nemertinea) have also been shown to possess tetrodotoxin. Presently, distribution of tetrodotoxin is known in only a limited number of organisms (Table l). In many of these animals, including puffers, newts, flatworms, and horseshoe crabs, tetrodotoxin accumulatesi n the ovaries and skin, where it may play a defensive role against predators. In other animals, however, tetrodotoxin maybe used as an offensive substance to immobilize and capture prey by secretion or envenomation of the toxin from the proboscis in ribbon worms or salivary glands in the blue-ringed octopus.
C.
Transmission of Tetrodotoxin Through the Food Web
Tetrodotoxin in puffers seems to come directly from their food, such as toxic gastropods ( / ? c t r ? ~ r r r l r r . s / ? j ~ o ~ ~flatworms, ~ri), and starfish (9,lO). Tetrodotoxin commonly accumulates in the eggs of pufiers, presumably transmitted from the female, since thetoxingenerally disappears from the larvae about 1 week after fertilization. In the wild, the toxin usually reappears in the larvae i n about 2 weeks, probably through ingestion of toxic organisms. Indeed, when larval puffers initially possessing tetrodotoxin during the first week after fertilization are cultured and fed a tetrodotoxin-free diet, they do not become toxic (14). Thus the tetrodotoxin toxification mechanism in puffers is likely to occur through the food web, though intestinal bacteriain some species of these fish reportedly produce negligible amounts of the toxin. Puffers, however, seem inclined to seek tetrodotoxin in their diets. Ingested toxin is initially accumulated in the liver, followed by the skin and other tissues. Fish, except those possessing tetrodotoxin, such as puffers and the tropical goby Y0rrgcit.hrhys t.r.inigc~r.,do not accumulate tetrodotoxin even when toxin-containing diets are fed to them at sublethal doses. Other tetrodotoxin-bearing animals such as gastropods and starfish probably BCCLImulate their toxins through the food web as well. In the trumpet shell (Chttmrricrsaulirrc), tetrodotoxin is ingested through its prey. During elucidation of the tetrodotoxin transmission mechanism for this animal, starfish such as Astwpectetr polyacarrfhus.A. ltrtc'S I ~ / I I O . S t I . S , and A. .sco~~trr.i~r.s on which the gastropod feeds were determined to be links in the food web leading to tetrodotoxin toxitication (IS). These toxic starfish are widely distributed in Japan. Tetrodotoxin-containing starfish probably derive the toxin through their diets. The ivory shell (B~l~ylorricr j ~ r p o r ~ ilikely c ~ ) becomes toxified by ingesting toxic puffers that fishermen discard in the ocean. Tetrodotoxin toxification mechanisms through food web links have already been established by model experiments (15). Fromthesestudies,thetoxificationmethodis hypothesizedasillustrated in Fig. 2. Theinitialsource of tetrodotoxinfound in most animals, however. is belicved to be marine bacteria. '
D. Bacterial Origin of Tetrodotoxin Since tetrodotoxin is distributed i n a variety of invertebrates and vertebrates and there is a wide individual variation in toxin content, even among members of the same species,
258
Yoshikawa-Ebesu et al.
bacter'a 1
Pseudomonas SD., etc. I
-
\
U
absorbed on and precipitated Wlth v
planktonic carcass, etc. Tropical
Ribbonworm Arrowworm Xanthid crab
sediment
1
I Puffetfish
l
Large gastropods
Small gastropods
Fig. 2 Assumedmechanism of toxification of tetrodotoxin-bearing animals (9)
the origin of tetrodotoxin was deduced to be a universal organism such as a microbe. Using instrumental high-performance liquid chromatography (HPLC) and gas chromatography-mass spectrometry (GC-MS) analyses for tetrodotoxin, an intestinal bacteria isolated from a toxic xanthid crab (Atergatis jl0ridu.s) collected from Shimoda, Shizuoka, Japan, was discoveredto produce tetrodotoxin (5). This bacterium was alsonoted as analogous to Vibrio.fischeri. Soon after, V. aigit~oiyticusand several other Vibrio spp. isolated from the intestines of the puffer Tcrkififirgusrlyrleri, the starfish Astropcterr po1~trc~rrrrtilu.s. the blue-ringed octopus H(rpcr1ochl~errrrt ~ r r r ~ ~ d oand s ~ rthe , horseshoe crab Ccrrc~irloscorp i u s rnturrdicaudrr were also demonstrated to produce tetrodotoxins using similar analytical techniques. Simidu etal. (7) showedthat 12 of 24 type culture strainsof marine bacteria tested clearly produced tetrodotoxin or related substances (Table 2). Yasumoto et al. (6) and Matsui et al. (8) ascertained that A1teronroncr.s tetmorlotris isolated from a calcareous alga (Jcmia sp.) and She~vcrwellcrputrefaciem isolated from a puffer (T. rliphokles) produced tetrodotoxin and anhydrotetrodotoxin, respectively. Typical tetrodotoxin production by Vibrio group VI11 ( 16) isolated from the intestines of Atergatisjoridus was demonstrated through instrumental analyses for tetrodotoxin as well as cell toxicity assays. An appreciable amount of tetrodotoxin (30 MU/flask) was produced by Vibrio group VI11 strain (Table 3). The tetrodotoxin fraction showed two peaks (tetrodotoxin and anhydrotetrodotoxin) in HPLC analysis, and in GC-MS analysis for the toxin ( C , base) exhibited Inass fragment ions at t r d : 407 (molecular peak), m/: 392 (base peak),and m / z 376, all of which are specificto trimethylsilylated C, base derived from authentic tetrodotoxin (Figs. 3 and 4). It is recognized fromthe above evidencethat many bacteria can produce tetrodotoxin and/or its derivatives. Tetrodotoxin productivity in vitro, however, seems largely dependent upon culture conditions. The optilnal culture conditions for maximumtoxin yield by tetrodotoxin-producingbacteriaremainunknown.Suchbacteriamayproduceminimal
Table 2
Analysis of Tetrodotoxin in BacterialCells (7) HPLC AnhydroTetrodotoxin tetrodotoxin GC-MS
Bacterial strains used
+
+ + + + t
t
+
t
+
+. clearly detected; =
cultivated
111
2 . dil‘licult t o detect; n fl.eshwatcr medium.
-. not detected
Table 3
Analyses for TetrodotoxinandRelatedSubstancesinthe Extracts of Sevcral Vilwio Groups of Bacteria Isolatcd from Intestines of A. jloridus Specimens Collected from Kojitna in Ishigaki Island, Okinawa (16)
Anhydro-Vihrio group
Lethal potency (MU)
HPLC Tetrodotoxin tetrodotoxin GC-MS
+
260
I
0
Yoshikawa-Ebesu et al.
.
.
-
.
I
.
10
I
20
Retention time (min) Fig. 3 HPLC of tetrodotoxinfractionfrom Vihrio group VI11 isolated from a Kojimaspecimen (upper) and o f authentic tetrodotoxin (lower) (16).
amounts of tetrodotoxin in the intestines of fish and invertebrates. In addition, based on the observationsthat trumpet shells can become highlytoxified by ingesting toxic starfish, and that the debris of small gastropods are often found in the digestive ducts of toxic puffers, and that cultured puffers (nontoxic) can become toxified by feeding on the liver of toxic puffers, it is presumed that tetrodotoxins in carnivorous organisms able to accumulate the toxin could originate from tetrodotoxin-producing bacteria.
E. MajorSources of Human Intoxication 1. Puffers Toxic puffers are limited to the family of Tetraodontidae ( 1 7 ) . The toxicity of Japanese puffer species was extensively surveyedby Tani ( 1 7 ) (Table 4). In this study, wide individual, regional, and seasonal variationsof toxicity were recognized. Especially large regional variations in toxicity were reported in the muscle of hignt~jiig~r (Trrk
26 l
I
1
9
l1
13
Retention time (min)
Fig. 4
15
250 350
400
300 Mass (m/z)
450
100
200 400 300 Mass (mlz)
GC-MS of tetrodotoxin fraction of Vibrio group VI11 from a Kojima specimen
(16). GC,
left; MS, right.
f l ~ r v i c f u , ~mrrshifirgu ), (T. e.\-crcurws),tak$~rgrr (T. ohlongus),firtcrtsuhoshi~~gu (Ltrgocephn/us lrrnuris), serlrlirfugu (L. sceler~tus), rnoyof1grr (Arothrotz stellatus), strzcrr~rrrr~itirg~r (A. hispitlus), and okirltrwtfl&u (Chelorldorr ptrtocLr)were later added to the list of toxic species (3,9). For a more complete list of toxic tetraodontid species, refer to Ref. 18. Tetrodotoxication most commonly occurs in Japan, where puffer or fugu is considered a delicacy. Eating fugu either raw or cooked can be hazardous since tetrodotoxin is heat stable. One of the main reasons connoisseurs eat fugu is to experience the slight oral tingling sensation and even feelings of euphoria that occur with consumption due to trace amounts of tetrodotoxin. In Japan, chefs must be specially trained and licensed in order to prepare and serve fugu. Sake or rice wine infused with fugu testes is also believed to be an aphrodisiac ( 1 1). The toxicity of puffer is related to their spawning season, with the highest toxicity levels occurring between March and June for Japanese puffers. Consequently, i n Japan, fuguiseatenprimarily between October and March. The toxinis concentrated in the ovaries, liver, and often, the skin, making the female puffer more toxic than the males, especially during spawning season. The parts of the puffer considered less toxic are the musculature, fins, andtestes.Althoughthemuscletissuemaybetoxic inat least two puffer species, Fugrr p ~ r r d d i sand L . ~ I ~ O C ~ ~ ~ lunnris ~ I I M S (see Ref. 19), no fatal cases of tetrodotoxication have yet been reported due to eating the meat of the puffer as sashimi, or sliced rawfish, i n Japan (18). Onthe other hand, the dishes known as chiri, fillets partially cooked in a stew of skins, livers, intestines, or testes, and kirno. partially cooked (9,10,18). livers. have been associated with many fatal intoxications
2. Goby Although tetrodotoxin-bearing fish were originally thought to be limited to puffers, tetrodotoxin has also been found i n two gobiid species, the Indo-Pacific goby(Gohius criniger)
Yoshikawa-Ebesu et al.
262
Table 4 ToxicityofJapanesePuffers,Surveyed
Family
Species
Ovary
by T a n i (17)
Testes Blood Intestine Muscle Skin Liver
0 0 0 0 0 0
o e o o o o 0 . o X
0 0 0
0 0 0 0
X
0
8
X
o
x
X
o 0
x 0
X
X
o
x
X
X
X
X
X
X
X
X
x
x
X
X
-
o
o
0
X
-
X
X
X
X
-
X
-
x
x
X
X
-
X
X
-
X
X
-
X
X
x x x
X
X
x x x
X
X
X
X
-
X
X
X
0 0 0
0
0 0 0 8 0 0 0 0 X
0 Strongly toxic, lethal at less than IO g. 0 Moderately toxic, not lethal at less than I O g. 0 Weakly toxic. no^ lethal at less than I O 0 g. X Negative. not lethal at less than 1000 g. - N o data availahle.
( 1 8-20) and a subtropical goby (Yongeichthys cr-itliger.) inhabiting the Amami Islands, theRyukyuIslands, thePhilippines,andTaiwan (21). In thelattergoby,the toxin is distributed in the skin, viscera, and muscle (21). Although the origin of tetrodotoxin i n Y. cr-itfigerremains unknown, these fish are scavengers and are probablytoxified through the food web by feeding on the tissues of dead toxic puffers (Chelonodonpcltoccr), whose habitat is similar to that of the goby.
3. Other Sources of Tetrodotoxin Through human toxication cases, tetrodotoxin has been discovered in several species of gastropodmollusks,including N~rsscrriusccrstus (Gould) and N . cor1oickrli.s (Deshayes) ( 19,22), N . linecrtrr, N. clcrthr-crta,TutufLI lissostonrtr, and Zerr.ris suquijor-er1si.s ( I 3 ) , and Cl~ar-oniasaulicre ( 1 2). Another type of tetrodotoxication can occur through envenomation by the blueringedoctopus (Hcrplochlcrenn mcrculosa) (23). Excessivehandling of the animalhas been the cause behind most of these cases.
Tetrodotoxin
111.
263
DETECTION METHODS
The official method for tetrodotoxin determination has been the mouse assay. With the development of more advanced technology, however, additional methods are being devised that are more rapid, sensitive and specific than this assay (24-27).
A.
Mouse bioassay
Twenty-gram mice are injected intraperitoneally with toxin or unknown samples. Toxicity is expressed i n mouse units (MU), where I MU is the amount of tetrodotoxin that can kill a 20 g male mouse of ddY strain in 30 minutes (28,29). One MU is equivalent to 220 ng of tetrodotoxin (28). Although this bioassay is easy, inexpensive, and relatively quick, its sensitivity and specificity are limited.
B.
High-Performance Liquid Chromatography
This technique will probably soon replace the mouse assay for experimental tetrodotoxin analysis to obtain more consistent toxicity data. Several attempts (24,251 have been made to determine tetrodotoxin type using HPLC. An example of one of these methods follows: Analysis for tetrodotoxin is perfomled using reverse-phase HPLC on a YMC-Pack AM314 column (6 mm inside diameter X 300 mm), two constant flow pumps for eluent and reagent, and a reaction coil (Teflon tube, 3 mm inside diameter X 10 m). The column is developed with a mixture of 2 mM sodium I-heptanesulfonic acid and 1% methanol in 0.05 M potassiunl phosphate buffer (pH 7.0) at a flow rate of 1 ml/min (24). The eluate is mixed with an equal volume of 3 N NaOH and heated in a reaction coil at IOO'C. The intensity of fluorcscence is measured at SOS nm with 380 nm excitation. Retention time o f tetrodotoxin is determined using authentic tetrodotoxin solution in diluted acetic acid (IS pg of tetrodotoxin/ml). A calibration curve of thetoxinis derived fromthe dose relationship between tetrodotoxin at concentrations of 0.1 -1 pg/IO p1 and its resulting fluorescent intensity (peak area). From this curve, tetrodotoxin in unknown samples can be determined. An example of such a curve is illustrated in Fig. S. In this case, more than 0.03 pg of tetrodotoxinll0 p1 can be detected.
C.
Mass Spectrometry
The nlass spectrum of tetrodotoxin can be directly measwed by fast atom bombardment 0. I mg of tetrodosecondary ion mass spectrometry (FAB-SIM). For this technique, about toxin and glycerol as matrix are placed on the sample stage of a mass spectrometer, mixed well, and introduced into the ion chamber of the spectrometer (FAB). Both positive and negative mass spectra of tetrodotoxin are then measured (26,30,31). As shown in Fig.6,tetrodotoxinexhibits (M + H ) ' and(M + H - H,O)' ion peaks at t t d : 320 and tn/: 302, respectively, in the positive mass spectrum, and an (M H ) - peak at W / : 318 in the negative. This technique may beused to identify pure samples of tetrodotoxin from mass number 319.
264
Yoshikawa-Ebesu et al.
-
0
15
30
Retention time (min)
Fig. 5 HPLC chron~atogra~n of authentic tetrodotoxins: ( a ) tetrodotoxin;(h) 4-cpitetrodotoxin:(C) anhydrotetlodotoxin (16).
D. Gas Chromatography-Mass Spectrometry Gas chromatography-mass spectrometry ( 12) is recommended to detect tetrodotoxin in contaminated or very dilute samples that are difficult to purify for tetrodotoxin. In this type of analysis, tetrodotoxin and its derivatives are decomposed to 2-amino-6-hydroxymethyl-8-hydroxyquil~azoline(C, base) withalkalihydrolysis and then converted with to GC-MS trimethylsilane (TMS) to TMS derivatives. These derivatives are submitted analysis as follows: A I O nil flask containing 0.2 m l of tetrodotoxin solution (25 MU, equivalent to about S pg tetrodotoxin) and 0.2 rill of 1 .S N NaOH is heated at 80°C-90°C for 30 minutesto derive a C, base from the toxin. After being cooled to room temperature, the reaction mixture is adjusted to pH 3-5 with 10% HCI and extracted three times with 0.5 1111 of 1-butanol. The CCJbase in the residue is converted to the TMS derivative in the presence of N,O-his (trimethylsilyl) acetamide, trimethylchlorosilane, and pyridine (2:1 : I). Each example of GC-MS selected ion monitoring (SIM) (m/: 407, W / : 392, m/: 376) and the resulting mass spectra are illustrated in Fig. 4. Each peak of SIM ( m / : 407, m/: 392, / ] I / : 376) typical of those of Cq base which appears at a retention time of 14.0 minutes is evidenced by spectrometry. Sharp fragment ions appear at HI/:407 [M'; C, base-(TMS)?], m/: 392 ( M ' - CH,), and / T I / : 376 [ M ' - H - (CH,)?].
E. Thin-Layer Mass Spectrometry (TLC-MS) and Liquid Chromatography-Mass Spectrometry Both TLC-MS (32) and LC-MS (30) are promising detection methods for tetrodotoxin. The latter methodis presently being developed.An example of each procedure is described below.
265
Tetrodotoxin ("10)
100-
-
185(2G+H)'
50. (M+H)' 320
f l
l00
c
200
300
400
500
200
300
400
500 m/z
Fig. 6 Positiveandnegativefastatombombardmcnt Cij) (27).
(FAB) lmss spectra (MS) of tetrodotoxin
For TLC-MS, the TLC plate used is an LHP-K high-performance precoated plate (Whatman, Clifton, NJ). The solvent system is pyridine :ethyl acetate: acetic acid: water ( 15 :5 : 3 :4). For MS, a FAB-MS apparatus, a JEOL DX-303 mass spectrometer with a JEOL DA-S000 data system,is used. An exampleof an analysis of tetrodotoxin, tetrodonic acid, and anhydrotetrodotoxin with this system is shown in Fig. 7 (32). For LC-MS, HPLC and MS are combined to increase measurement accuracy. In this example, the HPLC apparatus used is an Hitachi L-6200 with an ODs-3 column. The solvent system is 50% CHiCN with an ionization level of ESI. Fig. 8 depicts an analysis of tetrodotoxin derivatives using this method (30).
F. Thin-LayerChromatography The TLC technique (26) is conducted on a 5 cm X 20 cmsilica gel precoated plate (Merck) with a solvent system of pyridine: ethyl acetate: acetic acid: water (75 :25 : 15 : 30), 3-butano1 : acetic acid: water(2: 1 : l), or 1-butanol :acetic acid :water (60:1 S : 25). Tetrodotoxin
266
Yoshikawa-Ebesu et al.
100 7
50 302
0
0.2
0.4
0.6
0.8
1.0
100
200
300
1
412
400
Rf
500
m/z
Fig. 7 TLC-MS of tetrodotoxin and derivatives (32): (left) TLC; (right) MS.
is visualized as a pink spot with the Weber reagent or as a yellow fluorescent spot under a UV lamp (365 nm) after spraying with 10% KOH followed by heating at 100°C for 10 minutes. The Rf values of tetrodotoxin are 0.70 with pyridine:ethyl acetate: acetic acid: water (75 : 25 : 15 :30), 0.45 with 3-butanol :acetic acid : water (2 : 1 : 1 ), and 0.2I with 1 -butanol : acetic acid: water (60:15 : 25).
G. Capillary lsotachophoresis When tetrodotoxin (2.1 pg) is applied to an isotachophoresis apparatus, a zone appears at the position with a potential unit value of 0.32. The analytical time is less than 20 minutes. This method enables quantitation of tetrodotoxin content in crude extracts of puffer. The detection sensitivity for tetrodotoxin using capillary isotachophoresis (26) is about 0.2-5 pg, which is equivalent to that of the mouse assay. This procedure, however, eliminates the necessity for using live animals.
H. IR Spectrometry In this method, tetrodotoxin in a KBr pellet is analyzed by a spectrometer (26). The resulting spectrum is complex (3350, 3240, 1670, 1612, 1075lcm) but helpful for identification of the toxin from various sources.
267
Tetrodotoxin
TIME(M1N.)
-
a)
5 .O
G
10.0 l
. 75
TIC
J
G
320 * 5.0 SCAN NO. 200
,
100
: 1250
SAMPLE NO.
SCAN NO. 67-59 TIME(MIN) : 4.2
, -
100
1
276
-
-
60
320
153
249
185 I
I
I
I
I
I
I
1
II
100
1
'
"
l
!
l
,
l
I
l
l
l
l
l
l
l
l
l
l
l
300
200
0
-
400 TIME(M1N.)
1.0
5
3.0
2.0
5.0
4.0
6.0
1633 23
- "IC
f--" 1
I
l
I
I
1
I
50 SAMPLE NO. : 1246
100
304 * 30.0
I
SCAN NO.
SCAN NO. : 78 TIME(M1N) : 4.9
100
-
-
133
100
304
-
158
-
0
-
235
; 100
I
I
I
I
!
~
.
.
.
.
,
.
,
1
'
200
,
,
1
i
l
.
,
,
~
.
l
.
.
.
,
l
0
:
300
400
Yoshikawa-Ebesu et al.
268
H (9.9)
5.49
4.25
3.96 4.02
4 4a 5 7 8 9 11
Tetrodotoxin
2.34 (9.5) 4.08 4.29 4.02 4.04
Newt toxin
5.48 (9.5) 2.33 (9.5) 4.23 4.06 4.27 3.94 4.00
Chemical shifts are expressed as ppm. Coupling constants in parentheses are given as Hz. Fig. 9
1.
'H-NMR spcct~u~n of ncwttoxinanditsdata
on tetrodotoxin.
H-Nuclear Magnetic Resonance (NMR) Spectrometry
This procedure can be used to determine the chemical structure of pure tetrodotoxin and related compounds. One milligram of authentic crystalline tetrodotoxin or purified newt toxin is dissolved in 0.3 1nl of 3% CD,COOD in DzO and submitted to analysis at 400 MHz using a JEOL GSX-400 spectrometer with tetramethylsilane (Me,Si) as the external standard ( 1 2 ) . Fig. 9 shows an example of an analysis using this method.
Tetrodotoxin
J.
269
Electrophoresis
One microliter of tetrodotoxin solution ( I O MU or 2 pg tetrodotoxin) is applied to a 5 cm X 18 cm cellulose strip (Chemetron) with 0.08 M Tris buffer (pH 8.7) and electrophoresed at 0.8 mA/cm for 20 minutes. For detection of tetrodotoxin the strip is treated as described for TLC. In this type of electrophoresis (26), tetrodotoxin moves toward the cathode with a mobility clearly smaller than that of the saxitoxin standard, which exhibits a blue fluorescent spot under UV light after being treated with alkali.
K. Cytotoxicity Assays An assay based on the cytotoxicity of tetrodotoxin was first developed in 1988 (33). In this method the mouse neuroblastoma cell line neuro-2A (ATCC, CCL 13 I ) was used to quantitatively measure sodium channel blocking toxins such as tetrodotoxin and saxitoxin based on their ability to protect the cells from the cytotoxic effects of veratridine and ouabain. Since cytotoxicity was evaluated by microscopic examination of the morphological differences between living and dead cells, this procedure was time consuming and subjective. An improvement to this method was recently made usinga water-soluble tetrazolium salt, 2-(4-iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazoliun~, monosodium salt (WST-I), which is an indicator of living cells (34).
L. Immunoassays Several immunoassays have been developed for tetrodotoxin. These methods(35-37) had low sensitivity or required considerable time to yield results (38). Recently an enzyme immunoassay (EIA) was developed to quantitatively measure tetrodotoxin. This method israpid as well as highlysensitive to tetrodotoxinatconcentrations as low as 2-100 ng/ml (38). Rather than using an antibody raised against tetrodonic acid or tetrodotoxin conjugated to keyhole limpet hemocyanin as in previous assays, this assay uses an antibody against tetrodotoxin conjugated to bovine serum albumin to increase the sensitivity and specificity for this toxin. Moreover, this EIA can yield results in 30 minutes after 60 minutes of preparation (38).
IV. CLINICAL Clinical cases of tetrodotoxin poisoning are diagnosed based on the symptoms and history of exposure to the toxin.
A.
Symptoms
Tetrodotoxication is characterized by a myriad of symptoms, due almost entirely to the sodium conductance blocking actionof the toxin. The type, severity, andvariety of symptoms depend on the amount of toxin ingested. There are four main stages or degrees of tetrodotoxication based on the severity and evolvement of the poisoning (see Table 7, p. 273). Depending upon the amountof toxin ingested, symptoms usually appearwithin 1045 minutes of exposure, though some cases have reported being asymptomatic until as
270
al.
Yoshikawa-Ebesu et
much as 3-6 hours after exposure. Oral paresthesia is usually the initial symptom, and gradually spreads to the extremities and trunk. Other early sylnptorns include taste disturbance, dizziness, headache, diaphoresis, and pupillary constriction. These may or may not be accompanied by gastrointestinal symptomsof salivation, hypersalivation, nausea, vomiting,hyperemesis.hematemesis,hypermotility,diarrhea.andabdominalpain.These symptoms are characteristicof first degree tetrodotoxicationi n a system devisedby Fukuda and Tani (39). Second degree tetrodotoxicationis characterized by additional neuromuscular symptoms, such as advanced general paresthesia, paralysisof phalanges and extremities, pupillarydilation,and loss of pupillaryandcornealreflexes.Respiratorydistressmayalso begin. In third degreetetrodotoxication,patientsexperienceincreasedneuromuscular symptoms. Paresthesia of the larynx may lead to dysphagia and aphagia. Other neuromuscular symptoms include dysarthria. lethargy, muscular incoordination, and ataxia; sensations of floating due to numbness; cranial nerve palsies and muscular fasciculations. Cardiovascularandpulmonarysymptoms of hypotension or, morerarely,hypertension; vasomotor blockade; cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, andatrioventricularnodeconductionabnormalities;cyanosis,pallor,anddyspnea may also occur during this stage of intoxication. Dermatologic symptoms of exfoliative dermatitis, petechiae, and blistering are also often observed. Fourth degree tetrodotoxication involves respiratory failure, extreme hypotension, seizures, and loss of deep tendon and spinal reflexes. Although some patients may exhibit impaired mentalfacultiesandmayevenbecomecomatose,mostpatientsremainfully conscious and alert until inmediately before death occurs. Since death usually occurs in 6-24hours, if thepatientsurvives past 24 hours the prognosis for recovery is good. Otherwise, death is caused by progressive ascending paralysis involving the respiratory nluscles. I n addition to the symptoms just described, several unusual symptoms have been reported in a few cases. These include hypertension (22,40) and cranial diabetes insipidus (4 I ). I n the patients who experienced dramatic increases in blood pressure in response to tetrodotoxin. Deng et al. (40)noted they all had preexisting hypertension and sensitivityto sympathetic stimulation. The case involving cranial diabetes insipidus is described below.
B. Treatment At present, there are no known antidotes or antitoxins to tetrodotoxin, SO treatment of symptolns is supportive. Diagnosis is based on the clinical symptoms and history of consumption of toxic organisms. To reduce exposure to unabsorbed tetrodotoxin, emetics lnay be administered if vomiting has not already occurred. In addition, gastric lavage, especially with 2 % sodium bicarbonate, followed by activated charcoal is recomniended (42). Fluid and electrolyte replacement therapy may be usedto reduce resulting fluid loss. Atropine may also be given to counteract hypotension and bradycardia (42). In cases of respiratory difficulty or failure, oxygen and other ventilatory support, including endotracheal intubation. is often necessary. Several researchers report that administration of the anticholinesterases edrophoIlium and neostigmine enhance the recovery of motor power and markedly reduced paresKao (48). thesia ;llldnL1mbness (43-47). Although these reports are contrary to those of
Tetrodotoxin
271
Chew et al. (46,47) suggest that tetrodotoxin causes a competitive reversible block at the motor end-plate as well as at the motor axon and muscle membrane. The effectiveness of the anticholinesterases can thus be explained by their action in increasing the quantal release of acetylcholine at the neuromuscular junction, thereby reversing the tetrodotoxin blockage (47).
Incidences C. 1. Japan In Japan, many puffer poisoning cases occur every year, resulting in numerous deaths with approximately 7% mortality over the past 10 years (Table 5) (3). Japanese people know that puffer, especially its liver (kirno) is very toxic, but more than a few kirtro fans dare to ingest the liver, believing that the toxin has been eliminated fromit using their own “special” detoxification method. Consequently most puffer poisoning cases are caused by ingestion of toxic livers. Since the Japanese Ministry of Health and Welfare published a guideline of ediblepuffers in 1983,withupdates i n 1993and1995(Table6) (3). the number of puffer poisonings i n Japan seems to have diminished (Table 5). These guidelines only prohibit puffer livers to be served in restaurants. Consequently many tetrodotoxin poisonings still occur due to consumption of homemade puffer liver dishes from fish usually caught by a family member. Tetrodotoxin poisonings due to the ingestion of digestive glands of the edible trumpet shell (Cl~rrro~~irr strulirre) occurred in 1979 i n Shimizu, Shizuoka Prefecture (12). in 1982 in Wakayama, and in 1987 in Miyazaki, Japan. Typical examples of tetrodotoxin poisoning cases are as follows: Poisorlirlg Due t o the Liver of‘ the Plrfler “Korrlorlfirgu” (Trrkifiqu poecilonA 48-year-old man in NagasakiCity,NagasakiPrefecture,atemore thanfour slightly cooked livers (kimo) along with some flesh of wild T. poecilonotus i n the evening in October 1996. The fish had been caught earlier i n the day. Thirty to 60 minutes after in his hands and ingestion of the puffer tissues, the man began to suffer from numbness (1.
orus).
Table 5
PufferPoisoningCases Number
Year
of cases
1987
35
1988 1989
26
1990
31 33
1991
29
1992 l993 1994
33
1995
l996 Total
28 16 30 21 382
Number o f patients
S2 46 45
(1987-1996) inJapan ( 3 ) Mortality Number of deaths 4 S
5
(%)
1.7 10.9 11.1
55
1
I .8
45 57 44
3 4 4
6.7 7.0
23
I
42
2
4.3 4.8
34
3
488
32
9.1
8.8 6.6
272
et
EdiblePufferSpeciesAvailable Welfare) (3)
Table 6
Yoshikawa-Ebesu
a/.
i n Japan(Ministry of Healthand
Edible part Male Family
Species
* Applicable t o this species caught
Muscle gonad Skin
0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0
-
0 0 0 0
-
0 -
0 0 0 0
0 0 0 0 0 0 0 0 0 0 0
-
-
0 0 0 0
0 0 0 0
-
-
0 0 0 -
i n Ar~akcSea and Tachihana Bay.
limbs, followed by cyanosis and respiratory failure during the next 60 minutes. Although he was immediately hospitalized, the patient died during the following hour. Toxicity scores of the puffer livers and flesh were 715-4260 MU/g (equivalent to 0.14-0.85 mg of tetrodotoxin/g)andless than 5 MU/g (equivalentto 0.01 n1g of tetrodotoxin/g), respectively. In this case, the cause of death was detemined to be tetrodotoxin in thewild 7‘. poecilonofus liver, and the victim died due to ingestion of a total toxicity score of more than 10,000 MU (about 2 mg tetrodotoxin), equivalent to the LDS,, of tetrodotoxin for man.
b. Poisotling Due t o the Liver of tlw Pufer “Kurosrrbclfirgu (Lqocepholus gloveri). A 59-year-old woman cooked and consumed 3.5 livers (kimo) (estimated at 300 g) of fresh wild puffer, L. gloveri. Fifty minutes after ingestion she complainedof numbness of her lips and the tip of her tongue, followed by numbness of her fingers. After vomiting once, the victim was immediately hospitalized. Five hours after ingestion the victim devel”
Tetrodotoxin
273
Table 7 The Four Main Degrees of Tetrodotoxication and Associated Symptoms
Degree FirstNrurotnuscularsymptoms(paresthesia of lips,tongueandpharynx;tastedisturbance; dizziness: headache; diaphoresis; pupillary constriction); gastrointestinal symptoms (salivation, hypersalivation, nausea, vomiting, hyperemesis. hematemesis. hypertnotility, diarrhea, abdominal pain) SecondAdditionalncuromuscularsytnptonls(advancedgeneralparesthesia;paralysis of phalanges and cxtremities; pupillary dilatation, reflex changes) ThirdIncreasedneuromuscularsymptoms(dysarthrta;dysphagia,aphagia;lethargy;incoordination, ataxia; floating sensation; cranial nerve palsies; ~nuscularfasciculations). cardiovasculnrlpulmonary symptoms (hypotension or hypertension; vasomotor blockadc: cardiac arrhythmias including sinus bradycardia, asystole, tachycardia, and atriovcntricular node conduction abnormalities; cyanosis: pallor; dyspnea). dermatologic sylnptollls (exfoliativc dermatitis, petechiae, blistering) loss o f FourthRespiratoryfailure,impairedmentalfaculties,extremehypotension,seizures, deep tendon and spinal reflexes
oped dyspnea, mydriasis, and paralysis of the extremities. Artificial respiration through an endotracheal tube was then administered. Nine hours after ingestion, arrhythmia began and was successfully treated with antiarrhythmic drugs; the patient recovered soon after. The toxicity score of the leftover wild puffer livers was 3.5 MU/g (equivalent to 0.0007 mg tetrodotoxin/g). The total amount of toxin ingested by the patient was estimated to be 900-1000 MU (or 0.18-0.2 mg of tetrodotoxin), which may be near the minimum dose necessary to cause tetrodotoxin symptoms. Although L. glover-i isbelieved to be nontoxic, specimens from Taiwan occasionally show weak toxicity (42 MU/g; 0.008 mg of tetrodotoxin/g) in the liver (3 1).
c. Poi.soni~r~g Due tothe Flesh of “Dokrr.snbr!fugu (L. lunaris lunnris). Specimens of L. 1urltrri.s 1uncrri.s (49), a puffer similar to the nontoxic species “sabafugu” (L. luncrris .sprrdicus)were caught in the Vietnam Sea in September 1959 and the heads, skin, and viscera removed. The flesh with tails were quickly frozen. Five men ( 1 7-27 years old) each ate one pieceof the fried L. lunar-is lulraris flesh at noon i n October 1959. All five men developed signs and s y m p t o m typical of tetrodotoxin poisoning 1 hour after ingestion and were hospitalized within 3 hours of ingestion. Three of the men died within 3.5-6.5 hours following ingestion. The causative fish was initially thought to be the nontoxic “sabafugu” (L. lurrrrr-is .s/~rrdicrrs). Since the flesh was toxic, however, classification of the fish was reinvestigated, resulting in the identification of a new species, L. lurttrr-is 1unar.i.s. I ’
cl. Poisorrir~gDue to the Digestive Glarld of n Trumpet Shell (Char-onin sauliae). In December 1979 a 41-year-old man ate a boiled digestive gland (approximately 60 g) of “boshubora” or trumpet shell (C. scrulitre) which he caught in the Shilnizu Sea. Thirty minutes after ingestion, he began to develop paralysisof his lips and limbs, followed by cyanosis and respiratory paralysis. He was immediately hospitalized and received artificial respiration. Although he remained unconscious for 2 days, he recovered fully in 5 days.
274
Yoshikawa-Ebesu et al.
The leftover digestive gland of the C. sarrlicrc. showed a toxicity score of 17.000 in the bioassay for tetrodotoxin. Thus the victim MU (equivalent to 3.7 mg of tetrodotoxin) may have ingested about 10,000 MU (2 mg of tetrodotoxin), the approximate LD5,,of the toxin for man. Tetrodotoxin was also identified as the causative agent i n samples from subsequent similar tetrodotoxin poisoning cases in Wakayama i n 1982 and in Miyazaki in1987using TLC,electrophoresis,andinstrumentalanalyses with IR. 'H-NMR. and GC-MS.
2. United States Incidences of tetrodotoxication are rare i n the United States. As i n Japan, the main Sollrce of this poisoning is puffer. Only 10 known fatalities from the consumption of puffer have occurred in the United States, 3 of them between 1950 aid l990 (50,5 1). Four other deaths occurred in Hawaii between 1903 and 1925 (before statehood), all due to consumption of Awthron sp. (52). An additional case occurred i n Hawaii in 1986 clue to consumption of the liver of Diodoll hystri.x (42). Although the patient did not present t o a hospital until 24 hours after exposure, he recovered within 1 week. More recently, in 1996, there were three cases of tetrodotoxin poisoning in California. Three men consumed 0.25- I .5 oz. of puffer brought from Japan as a prepackaged, ready-to-eat product (53). The two men who had eaten only 0.25 oz. each of the puffer began to experience oral paresthesia and dizziness within 10-20 minutes, while the other man who had eaten 1.5 oz. of puffer began to experience these symptoms within 2-3 minutes. All three men experienced additional typical tetrodotoxin poisoning symptoms, including anxiety, feelings of doom, headache, difficulty speaking, chest tightness. facial flushing, shaking, nausea, vomiting, weakness, and collapse. After hospitalization, all were treated with intravenous hydration, gastric lavage, and activated charcoal. All victims recovered within 24 hours (53). There have been two other cases of tetrodotoxication i n the United States. one of them fatal, due to swallowing the Oregon rough-skinned newt (Ttrrichcr grrrrlulostr) (54). In both cases, the victims may have been under the influence of alcohol, and swallowed the newts as pranks. Consistent with the presence of tetrodotoxin in this species of newt, the two individuals involved experienced the classic signs and symptomsof tetrodotoxicatlon.
3. Elsewhere Although not as common as i n Japan, puffer poisoning occurs in other Asian and Pacific countriesincludingTaiwan,HongKong,Thailand,Singapore.Malaysia,Kiribati,Fiji, Australia, and Papua New Guinea. Unfortunately their records may not always be as complete as those in Japan. In Taiwan, for example, tetrodotoxin poisoning is not a reportable disease, and many physicians are unfamiliarwith the symptomatology of the disease ( 18). Nonetheless, a study of the Taiwan Poison Control Center data from 1988 to 1995 showed a significant number of tetrodotoxin poisonings: a total of 20 incidents involving 52 patients ( 1 8). A detailed listing of these incidents including dates and locations of occurrences, sources of toxin, number of deaths, gender distribution, age of victims. and laboratory confirmation are shown in Table 8. In Singapore, the ingestion of SpAcreroitles rmcrrltrtrrs and Arothrorf stelltrtrrs have resulted in several tetrodotoxin poisonings (41,46,47). Althoughthe patients in these cases experienced typical tetrodotoxication symptoms, no fatalities were reported among them. An unusual symptom was noted in an additional tetrodotoxication case in Singapore
Table 8
Demographic Data of Tetrodotoxin Poisoning in Taiwan, 1988-1995 (18)
Date of occurrence
Location of occurrence:
Mar 1988 Mar 1988 Apr 1988 Jun 1988
Northwest Northwest Midwest Northeast
Whole puffer Flesh of puffer (L. luritrris) Whole puffer Roe of puffer (L. lirriciris)
Jan 1989
Northwest
May 1989 Jul 1989 May 1990 .Tun 1991 Dec 1991 Dec 1992 Jan 1993 Jan 1994 Apr 1994 Apr 1994 May 1994
Northwest Midwest Southeast Midwest Midwest Southeast Midwest Midwest Southwest Midwest Southwest
Decapitated puffer and liver of puffer Roe of puffer Whole puffer Whole puffer Flesh of puffer Whole puffer Whole puffer Whole puffer Roe of puffer Roe of puffer Whole puffer Gastropod mollusks ( N . e m firs and N. corioiddis)
Jul 1994 Dec 1994 Apr 1995 Oct 1995
Northeast Northwest Midwest Midwest
Source of toxin
Whole Whole Whole Whole
No. of patients (deaths) 4 ingested, 1 was poisoned (0) 5 ingested. 2 were poisoned ( 1). 5 (0) 2 (1)
2 P
:
0
.Age (years) 60 36 9, U" (4) 3-60 62
2 (0) l(1) 3 (1) 1 (0)
I(1) 3 (1) 1 (0) 4 (0) 2 (0)'
5 ingested. 3 poisoned ( 0 ) 26 poisoned, I7 reported ( 1 )
fish of Gobirrs criniger puffer puffer puffer
The location of occurrence was dclincd according to different parts of the Taiwan island M/F/U denotes male. female. and unknown sex. respectively. ' Only m e case was reported t o the Poison Control Center-Taiwan with adequate clinical information ,'The symbol "U" denotes unknown age. _I
Gender distribution (M/F/U)
9 im-2 38 53-59. U ( I ) 57 53 5-40 38 25-29. U ( I ) 50 15-71 12-7 I
32-34 19 50 56
Toxin assay None 120 MU/g (inusclej None 1200 MU/g (roe). 45 MU/g (muscle) None I100 MU/g (roe) None None None None 150 MU/g (muscle) None 150 MU/g (roe) None None 150 MU/g ( N . cu.sriis), 13 MU/g ( N . C O I I O I ddS) None None None None
z.3 J
Yoshikawa-Ebesu et al.
276
involving the consumption of Ar-orhr-orz r-eficwlar-is (41). In this case, a 24-year-old man developed nausea and abdominal discomfort after eating this fish. Soon after hospitalization, he became apneic and lost consciousness. After successful cardiopulmonary resuscitation, he remained in a deep c o m and was placed on mechanical ventilation. One noteworthy feature of this case is the first reported occurrence of cranial diabetes insipidus due to tetrodotoxin poisoning. Tambyah et al. (41) suggest that tetrodotoxin may have blockedsodiumchannels i n the axons of thenlagnocellularneurons of the neurohypophysis, thus inhibiting the release of vasopressin and resulting i n diabetes insipidus. Since cranial diabetes insipidus is indicative of extensive irreversible brain damage and impending death, even more unusual was the patient’s complete physical and mental recovery within 50 hours. Reports of tetrodotoxication from other countries are sporadic. These include eight cases (one fatality) from unidentified puffer species in Hong Kong (5556); several cases Ltrgoceph(llu.7 Irrgocephalus (Linnaeus) in Kiribati (for(two fatalities) from the puffer merly the Gilbert Islands) (57); four cases (two fatalities) from Diodorl h y t r - i s in Papua New Guinea (58); five cases (one fatality) from the same puffer species in Fiji (43); four cases (one fatality)in Australia, two from Amhl~r-/?~r~chnre.~ r-ichei,one from Sphcrer-oides gluher- (44,59), and one from Termcferm glaher- (60); seven nonfatal cases i n Thailand, six from Terr-croclorl frrrlgi (61) and one from an unknown puffer species (62); and four cases (one fatality) from an unknown puffer species i n Malaysia (63).
V.
TOXICOLOGY
Many animals as well as humans are sensitive to tetrodotoxin. I n humans, the LD?,,of tetrodotoxin i n man (50 kg) is approximately 10,000 MU, equivalent to 2 mg of tetrodotoxin. Early studies examined the toxic nature of tetrodotoxin in cats, dogs, pigs, chickens, pigeons, sparrows, doves, rabbits, mice, guinea pigs, rats, snakes, frogs, turtles, dragonflies, newts, puffers, and various freshwater and marine fish (for a detailed review, see Refs. 19,481.
VI.
PHARMACOLOGICAL ACTIONS
I n the intact animal, tetrodotoxin has toxic effects on the neuromuscular, cardiovascular, and respiratory systems, probably all due to its voltage-insensitive blocking effect on sodium channels. Tetrodotoxin can induce local anesthetic, emetic, hypothermic, hypotenall of sive, vascular smooth muscle relaxant, and respiratory depressant symptoms (64), which reflect the progressive paralysis of the excitability mechanismof nerve and muscle. Tetrodotoxin also possesses a direct relaxant effect on vascular smooth muscle that may be responsible for producing hypotension i n vivo (65). For an extensive historical review on the pharmacology and toxicology of tetrodotoxin, see Ref. 19. Additional effects of tetrodotoxin have been noted i n various tissues, including pacinian corpuscles, and cardiac and neural tissues. In Pacinian corpuscles, tetrodotoxin may block the sodium channel action potentialas well as reduce the mechanoreceptor potential (see Ref. 66). In rat heart mitochondria, tetrodotoxin decreases in vitro calcium uptake, inhibiting respiration and ATP (adenosine triphosphate) generation capacity at high doses;
Tetrodotoxin
277
this effect is probably due to blockade of the sodium-calcium uniporter by the toxin (67). In embryonic chick heart cell aggregates. tetrodotoxin blocks spontaneous activity in a dose-dependent manner and reduces action potential duration, but has little effect on interbeat interval (see Ref. 68). Neurophysiological studies have been conducted in at least two different human tetrodotoxication cases. I n both cases, although the conduction velocity and amplitude of both muscle and sensory nerve action potentials were decreased, neither temporal dispersion nor focal conduction block occurred. This phenomenon seemed diffuse, occurring in most or all fibers in affected nerve trunks and resulting in synchronous slowingof impulse conduction (58,641. Recent animal studies have shown 4-arninopyridine can reverse saxitoxin and tetrodotoxin-induced cardiorespiratory depression (69). Since this compound is a potent potassium channel blocker, however,it can have serious side effects and even toxicity at higher doses, making its use as a tetrodotoxin antidote unfeasible without further studies.
VII. A.
USE OF TETRODOTOXIN AS A PHARMACOLOGICAL TOOL Organismal Level
Due to its sodium channel blocking effect, tetrodotoxin possesses anticonvulsant and neuroprotective activity in certain situations. In some in vitro studies, low concentrations of tetrodotoxin can have anticonvulsant-like effects similarto those of phenytoin or carbamato duplicate this effect. During pathologizepine (70,71 ); in vivo studies have been unable cal conditions such as seizures or ischemia, sodium influx from voltage-dependent sodium channels may cause a cascade of undesirable effects: opening of calcium channels and calcium-dependent glutamate release, opening of potassium channels, and relief from magi n vitro and in nesium-dependent block of NMDA-type glutamate receptors (72). Both vivo studies have demonstrated that selective sodium channel blockers such as tetrodotoxin can block the initial sodium influx and thus prevent most of these detrimental effects that may contribute to cellular damage during ischemia.In vitro models examined include rat hippocampal slices, optic nerves, neuronal cultures, and spinal explant cultures; in vivo models include gerbil and rat forebrain ischemia, isolatedrat head, and rat c l d’lac < r arrest (see Ref. 72). The toxicityof naturally occurring sodium channel blockers such as tetrodotoxin precludes their therapeutic usefulness (73). Nonetheless, they have been instrumental i n further elucidation of the damaging nature of such phenomenon as ischemia and in the subsequent development of preventive measures against these conditions.
B. Molecular Level Neurotoxins, especially tetrodotoxin. have long been used as tools to study sodium channels in excitable membranes because of their specificity for these channels. The unique action of tetrodotoxin on blocking action potentials by selective inhibition of the membrane sodium conductance upon depolarization wasfirst discovered in lobster giant axons (74). This action was found to be exerted at nanomolar concentrations, to occur without affecting the kinetics of the transient conductance (potassium) increase, to block the tran-
Yoshikawa-Ebesu et al.
278
sient channel without impairing the gating mechanism, and to be effective only when tetrodotoxin was applied to the outside of the squid axon and nodal lnenlbranes (see Ref. 65). Thus tetrodotoxin must act on the outer surface of the sodium channel. In addition, it was noted that action potentials not based on sodium ion fluxcs were insensitive to tetrodotoxin. These included calcium-based action potentialsof various animal nluscles (see Ref. 75). Subsequent studies showed that tetrodotoxin had no effect on neurotranslnitter (both acetylcholine and norepinephrine) release from various nervous tissues (65), contirming the direct effect of tetrodotoxin on sodium channels. The original mechanism of action of tctrodotoxin was thought to be via the guanidinium group on the toxin “plugging” the sodium channel pore and physically preventing the passage of sodium ions through the pore (76). This was based on the fact that the guanidinium moietics were critical for the pharmacological actions of the toxin, and that free guanidinium ions could pass through sodium channels. The elucidationof the chernical structure of tetrodotoxin, however, altered this idea. One of thc first hypotheses of tetrodotoxin binding to sodium channels is illustrated in Fig. 10. In this model, Hille (77) proposed that the tnain binding site on both tetrodotoxin and saxitoxin, a toxin with similar actions to tetrodotoxin, for the sodium channel receptor wasthe unusually electropositive. homologous hydrated ketone group on carbon 1 0 of tetrodotoxin (designatedby the circled X i n Fig. 10). Further hydrogen bonding and electrostatic attraction between the guanidinium group and the negative charge of the selectivity filter were helievcd to stabilize the complex. Moreovcr, this model helped to explain why even minor moclifcations in the structure of either tetrodotoxin or saxitoxin caused drastic reductions in biological activity; for example,the low biological activityof anhydro, alkyl. and deoxy derivatives of tetrodo-
Tetrodotoxin
Tetrodotoxin
279
toxin can be accounted for by the loss of hydrogen bonds between the modified toxin and sodium channels (75,771. Most analogues of tetrodotoxin, including nortetrodotoxin. are relatively inactive against sodium channels. A methoxamine product of nortetrodotoxin, however, was discovered to be about one-third as active as tetrodotoxin (78). Previously the guanidinium moiety and the cationic head at the carbon 2 position were thought to be most important in the binding of tetrodotoxin t o the sodium channel (see Fig. 1). This finding suggested the importance of the 1 I end of the tetrodotoxin molecule i n sodium channel binding. Although all action potentials that are blocked by tetrodotoxin are based on sodium currents, the converse is n o t necessarily true. Certain types of excitable tissues with action potentials due to sodium currents are insensitive to tetrodotoxin. For example, the nerve Iron1 the newt Ttrrichtr torostr is at least 30,000 times more resistant to tetrodotoxin than the frog nerve, and the nerve of the Atlantic puffer (Spheroides nrtrcwltrtrrs) is 1000 times n1ore resistant to tetrodotoxin than frog nerve(6.5).Tetrodotoxin-resistant sodium channels also have much lower c o t l d ~ ~ c t than a ~ ~thcir ~ e ~toxin-sensitive counterparts (79).
I extracellular
intracellular
v
coo-
Fig. l l Sodium chnnncl structure. including regions o f the protein that have been associated with various functions. T, the TTX-binding site: H, the crucial portion of the inactivation gate: +. the and is necessary for activation gating; P. phosphorylacharged portion that sensed menlhrnnc voltage tion sitcs along an extensive cytosolic loop that is absent in skeletal muscle and ecl electroplax: G. sitcs of extracellular glycosylation. (Inset) An expanded view o f the domain IV-S6 region, crucial for the binding o f local anesthetics and phcnyloin. Numbering of amino acid residues begins a t the amino ( N H ? )terminus and continues sequentially through approximately I800 amino acids toward the carhoxyl (COOH) terminus (72).
280
Yoshikawa-Ebesu et al.
Other tetrodotoxin-resistant sodium-based action potentials are found in nerves of puffers, octopus, mollusks, denervated mammalian muscle, etnbryonic and newborn rat by Baker muscle, and rat skeletal muscle L6 Inyotubes (see Refs. 75.80). Experiments and Rubinson (81) with carbodiimide-nucleophile treated nerves showed that the group in the sodium channel involved in the sensitivity of the action potential to tetrodotoxin can, under appropriate conditions, be dissociated from the ability of the channelsto generate action potentials. Thus sensitivity to tetrodotoxin can be abolished while retaining the physiological function of the sodium channel. Tetrodotoxin has been used to demonstrate that sodium channels sensitive and resistant to thistoxin have different physiological and pharmacological properties (see Ref. 80). In rat dorsal root ganglia, both types of channels are present in immature as well as (82,83). adult neurons, suggesting that they both may be essential to sensory integration Indeed, distinct types of sodium channels may be involved i n conveying different types of sensory information (80). For example, tetrodotoxin-resistant sodium channels may be more important in nociception than tetrodotoxin-sensitive ones (84,85), especially under chronic neuropathic pain conditions (86). Further insight into the interactions between tetrodotoxin and sodiunl channels, as well as the differences between tetrodotoxin-sensitive and tetrodotoxin-resistant sodium channels, has been accomplished with the use of molecular biological methods. I n particular, the S5-S6 region of each domain is thought to contain two short segments, SS1 and SS2, that may partly span the membrane as a hairpin (Fig. 1 1 ). Various point mutations in this region have been made in an attempt to map the site of tetrodotoxin and saxitoxin block of the sodium channel. A single point mutation of the rat sodium channel I1 at position 387, replacing glutamic acid with glutamine. was found to reduce its sensitivity to tetrodotoxin and saxitoxin by more than three orders of magnitude (87). This mutation effectively rendered the rat sodium channelI1 insensitive to concentrations of tetrodotoxin up to I O FM, while hardly affecting the macroscopic current properties. The glutamic acid residue at position 387 is conserved in all sodium channels with known sequence and is located between segments SS and S6 of domain I. In the present sodium channel model, this would place glutanlic acid 387 on the extracellular side of the membrane, probably in close proximity to the channel opening (87). The results of this study are consistent with this location, since the binding site for tetrodotoxin is known to be located on the extracellular side (77). Other point mutationsof the rat sodium channel I1 have shown similar results. Mutaat equivations involving two groupsof predominantly negatively charged residue. located lent positions i n the seven-amino acid SS2 segment of the four repeated domains, strongly reduce tetrodotoxin and saxitoxin sensitivity. Mutations of adjacent residues, howevcr, have little or no effect on sensitivity to these toxins (88). These researchers have found that all mutations reducing net negative charge in the amino acid clusters i n segment SS2 markedly diminish single-channel conductance. and have suggested that these two clusters of residues form ring structures which form part of the extracellular pore opening and/ or the pore wall of the sodium channel (88). Molecular techniques have also been used to discover the structural determinants that confer tetrodotoxin sensitivity or resistance of certain sodium channels.AS mentioned, sodill111 channels in brain and innervated skeletal muscles are tetrodotoxin-sensitive and respond to nanomolar concentrations of tetrodotoxin. On the other hand, those which are tetrodotoxin-resistant, such as sodium channels in denervated skeletal tmsclc and RH1
Tetrodotoxin
281
sodium channels in heart, are only blocked by micromolar tetrodotoxin concentrations. RH1 sodium channels are also more sensitive to cadmium than are tetrodotoxin-sensitive sodium channels. Two of the seven amino acidsin segment SS2of domain I differ between RH1 and the tetrodotoxin-sensitive sodium channel isoforms (see Ref. 89). A point mutation of one of these two amino acids, cysteine at position 374 to either phenylalanine or tyrosine, in anRH1 mutant, confers three propertiesof tetrodotoxin-sensitive sodium channels: (a) a 730-fold increase in tetrodotoxin sensitivity; (b) a 28-fold decreased sensitivity to cadmium;and(c)alteredadditionalblock by tetrodotoxinuponrepetitivestimulation (89). Similar homologous mutations have also resulted in reduced tetrodotoxin affinity: an aromatic tyrosine or phenylalanine substituted for cysteine at position 374 i n tetrodotoxinsensitive, voltage-gated sodium channels (90); cysteine substituted for tyrosine at position 401 of the skeletal muscle isoform (p 1) of the rat muscle sodium channel (91 ); and serine changed to phenylalanine at position 376 in sensory neuron specific voltage-gated sodium channels (92). Apparently then, the aromatic residue at position 374 is critical for highaffinity tetrodotoxin binding. The magnitude of the increased tetrodotoxin binding affinity due to a point mutation at this position in RH1 sodium channels suggeststhat this interaction may be through an ionized hydrogen bond (89). Furthermore, the importance of the two amino acid clusters in tetrodotoxin and sodium channel binding proposed by Terlau et al. (88j may be limited to secondary electrostatic interactions (89). The salient features of sodium channels havebeen collectively analyzed to generate a potential model for the tetrodotoxin binding site of the sodium channels of both cardiac and skeletal muscle (93). I n general, this model fits well with most studies. For example, modifications of the hydroxyls at carbons 9 and I O of tetrodotoxin (to tetrodonic acid) result in a totally ineffective molecule (see Ref. 65). Another feature of this model was revealed when the toxin binding site was analyzed with the tetrodotoxin removed: the binding site consisted of a structure resembling a funnel that terminated in a narrowed region. The dimensions and chemical properties of this region werc consistent with those of the hypothesized selectivity filter of Hille (77,94) (Fig. 10). This novel model of thetetrodotoxinandsaxitoxinsodiumchannelbindingsite provides a basis for the differential binding of these toxins to various channel types. This model does not, however, consider interactions of the tetrodotoxin/saxitoxin binding site with other portions of the sodium channel protein. Thus, further experiments and analyses are needed to confirm the actual structure of this macromolecule.
VIII. SUMMARY One of thebest ways to prevent tetrodotoxin poisoning is, of course, not to eat toxic puffer. but many find the delicacy too delicious to resist. Fortunately, both the number of incidences and mortality rate for tetrodotoxication have declined in Japan, where this type of poisoning occurs most often. Key factors i n this reduction are probably increased governmental regulations and the availability of net-cultured nontoxic puffers. Unfortunately, supportive treatment remains the only remedy for tetrodotoxication. Ovcr the past few decades, many tetrodotoxin poisonings have been reported due to the ingestion of seemingly unrelated organisms. Thc discovery of tetrodotoxin in a
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diverse array of animals, both marine and terrestrial, has led to the hypothesis of a bacterial source of the toxin. Presently the toxicity scoreof tetrodotoxin is officially measured by the mouse assay (4). but improved HPLC analysis for the toxin is being developed to replace it due to the increased accuracy, time savings, and ethical concerns of this method. Many instrumental analyses (7,8), including'H-NMR,C'7"NMR. IR, GC-MS, TLC-MS. and LC-MS, are also presently available to identify tetrodotoxin.In addition, cytotoxicity and immunological assays for tetrodotoxin may also be employed for mass screening of samples. Although tetrodotoxin is a potent neurotoxin with an LD,,, of 2 n1g for man, in some areas it is also an important analgesic for the severe pains of rheumatism, neuralgia, and cancer. Furthermore, this toxin is a significant biochemical and pharmacological reagent a s a sodium ion channel blocker (10).
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toxin by thin-layer chrorn~~toglaphy/f~lst atombotnhardmenttnassspectrometry.AnalBiochem 175:258. 1988. 33. K Kogurc, ML Tamplinc. U Sitnitlu. RR Colwcll. Atissue culture assay for tetrodotoxin. saxitoxin and related toxins. Toxicon 26: I9 1. 1988. 34, K Hamasaki. K Kogure. K Ohwada. A biological method for the quantitative measurement of tetrodotoxin (TTX): tissue culturc bioassay i n combination with ;I water-soluble tetrazolium salt. Toxicon 34:490. 1996. 35 S Watabe, Y Sata, M Nakaya. K Hashitnoto, AEnomoto. S Kuninogawa. K Yamnuchi. Monoclonal antihody raised against tctrodonic acid. a derivative of tetrodotoxin. Toxicon 27:265. 1989. 36. K Matsumura, S Fukiya. Indirect competitive enzyme immunoassay for tetrodotoxin using a biotin-avidin system. J AOAC Int 75:883, 1992. 37. K Mntsutnura. A monoclonal antibody against tetrodotoxin that reacts to the active group for toxicity. Eur J Phartnacol 293:41. 1995. 38. KKawatsu.Y Hamano. T Yoda. Y Tcrano. T Shihata. Rapid and highly sensitive cnzylne immunoassayforquantitativedctcrtnination o f tetrodotoxin.Jpn J Med SciBiol S O : 133, 1997, 39. T Fukuda. I Tani. Rccortls o f puffer poisonings, rcpt. 3 [ i n Japonesc]. Nippon Igaku Oyohi Kenko Hoken 3258:7, 1941.
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9 Epidemiology of Seafood Poisoning
I. Introduction 288 11. Epidemiologic Overview 288 Epidemiology A. 288 B. Epidemiology of seafood poisoning 289 Surveillance C. 290 111. EpidemiologyofSeafoodPoisoning291 1V. EpidemiologyofSeafoodPathogens292 Bacteria A. 292 B. Viruses 296 C. Parasites 297 D. Allergies 297 Toxins E. 298 V. Prevention 303 Environmental A. 303 B. Education 304 VI. Conclusions 304 References 306
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INTRODUCTION
Seafood poisoning accounts for a largeandgrowingproportion of all food poisoning incidents. In the United States, fish, shellfish, and other marine organisms are responsible for at least 1 in 6 food poisoning outbreaks with a known etiology, and for 1.5% of the deaths associated with these particular outbreaks. The proportion of foodborne illnesses attributable to seafood, based on reporting for 1988-1992, has markedly increased over the previous decade, when seafood consumption was associated with 10% of foodborne illnesses with an identified etiology (1-4). In other parts of the world, the impact of seafood poisoning is even greater. In the period 1971-1990, seafood was the single most important vehicle in food poisoning outbreaks in Korea (32%) and Japan (22%), where seafoodwasresponsiblefor 43% and 62%, respectively, of outbreak-relatedfatalities (.5,6).As with general food poisoning, seafood poisonings share the following epidemiologic characteristics: ingestion as the primary route of exposure, a wide variety of etiologies (bacterial, viral, parasitic, and toxin), significant underreporting, and an apparently increasing incidence in human populations. This chapter reviews the general epidemiologic principles and issues relatedto seafood-borne disease, explores reasons for the apparently increasing incidence of seafood poisoning, and summarizes the epidemiology of bacterial, viral, parasitic, and toxin-related seafood illnesses.
II.
EPIDEMIOLOGIC OVERVIEW
A.
Epidemiology
Laboratory scientists establish causation by manipulating the laboratory environment. Epidemiologists most often establish causation by observing the natural environment, specifically by assessing naturally occurring patterns of disease in populations. Often, surveillance information provides the first evidence of an unusual pattern. Epidemiologists then design studies to identify differences between ill people and well people that could explain why the disease occurred. T o prove epidemiologic causation, the following are important: an appropriate time to the agent under study), strengthof associasequence (i.e., the disease follows exposure tion (people with the exposure are more likely than those without the exposure to get disease: the greater this increased risk, the greater the evidence for causation). evidence of a dose-response relationship (the probability of illness increases as the exposure increases), biologic plausibility, consistency with other studies, and laboratory evidence (such as toxicologic) that supports the causal relationship (7-1.5). For a seafood poisoning outbreak, it is important to show that the persons who became ill were infected with the suspect organism and had consumed the appropriate seafood vehicle before they became ill: that the attack rate (percentill) was higher among those who consumed the seafood than among those who abstained, and was highest among those who ate the most seafood; and that as the seafood vehicle was contaminated with the specific pathogen or toxin. Of note, with many foodborne diseases, seafood-borne poisonings often appear as disease clusters (i.e., unusual increasesin morbidity or mortality in time and space), since seafoodis often shared among families and friends as well as through commercial venues such as restaurants (13,161.
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B. Epidemiology of Seafood Poisoning Key epidemiologic activities in establishing causation are (a) identifying a target population for study, (h) formulating a case definition, (c) tneasuring exposures of interest, and (d) comparing exposures or outcomesi n subgroups within the target population (see Fig. 1). Ideally the target population is at high risk for both exposure and disease, and thus has a high incidence of disease. In the case of seafood poisonings, a target population would consume seafood contaminated with the agent under investigation. In acute outbreaks, the target population is clear: the group within which the outbreak is occurring. In order to nleasure the amountof disease causedby a specific exposure in the target population, a case definition is required. The case definition may be based on historical a consensus or developed for the purposes of a particular investigation. For example, classic case definition for fish-associated scombroid seafood illness would be the acute onset of gastrointestinal symptoms and upper torso skin rash (both eliminated by antihistamines) within minutes to hours of eating fish. To the extent possible, the case definition should also incorporate some objective measurements such as biomarkers to reduce misdiagnosis. The case definition should also have clinical relevance so that it can be used in the diagnosis and reporting of disease. An exposure isany agent(chemical,biological,etc.)orcharacteristic (a human behavior, a particular genotype) suspected of causing or increasing the risk of disease. In the epidemiology of seafood poisoning, exposures usually are physical agents, such as chemicals or bacteria. Measures of these exposures and their surrogates, physiologic effects, are known as biomarkers (73). Biolnarkers have been divided into markersof exposure and markers of effect. Biotnarkers of exposure are the actual levels of the toxins or their metabolites in body fluids and seafood, such as methyl mercury in the hair of a person who consumes contaminated fish, or identification of a specific virus in the stool of persons suffering from seafood-associated gastroenteritis (17,18). Markers of effect are indicators of subclinical physiologic change, such as conduction changes in the peripheral
Establish the presence o f an outbreak. Confirmthediagnosis. Develop a CLISK definition. Have informal conversations with affected persons to generate hypotheses. S. Develop a questionnaire. 6. Administer the questionnaire to well and ill persons. 7. Look for associations between exposllres and ihless: cakUlatK n lncasure of effect (risk ratio or odds ratio). X. Based on #7. identify a vehicle or transvector. 9. Test the suspected vehicle for the organism.
I. 2. 3. 4.
Tips:
Avoid testing foods a t random before a pathogenic agent is idcntifed in patients and an association between illness and specific food(s) is established. Always collect sanlples of stool/vomitusfromill persons. Keep some stool samples fresh, unpreservcd, unfrozen, so they can be tested for viruses. (Refs. 12-14. 16. 21)
Fig. 1 Steps intheinvesLigation
of n seafood-borneoutbreak
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nerves of persons suffering from the fish toxin disease ciguatcra (19.20). I n addition to biomarkers,epidemiologistsuseotherIneasurements of exposure such as information about food consumption and symptoms associated with a seafood poisoning outbreak collected through questionnaires (21). The final step in an epidemiologic investigation is the calculation of a measure of effect that indicates how much more likely exposed people in the target population are to developdiseasecompared totheunexposedpeople inthetargetpopulation. These measures usually are the risk ratio or the odds ratio, both of which can be interpreted to be the attack rate (or incidence of disease)in the exposed population divided by the attack rate in the unexposed population.
C. Surveillance Surveillance is the ongoing and systematic collection, analysis, interpretation, and dissemination of health-related data. This information isused to plan, implement. and evaluate public health interventions ( 1 1-15,21). With regard to foodborne disease, surveillance permits the early detection of new or newly recognized diseases; early identification of outbreaks so they can be contained (see Figs. 1 and 2): the identification of specific food sources of infection; and the conduct of studies to develop new ways to keep foods clean. Surveillance for seafood poisoning involves two groups of activities: collection of information about human illnesses, and monitoring of marine habitats for evidence of contamination, Data on human illnesses come primarily from traditional passive surveillance programs based on state laws that require clinicians and laboratories to report cases of specific diseases (e.g., paralytic shellfish poisoning, ciguatera, Vibrio infections, tetro-
On July 30, 1987. an outbreak of an acute neurologic illness occurred in Champcrico (population 689 1 ) on the coast of Guatemala ( 2 2 ) . Patients con1plained of headache. numbness o f face and extremities,difficultywalking,dizziness,andother synlptonls. Twenty-sixof 187 cases died o f respiratory arrest within hours of onset. To establish the characteristics and sizeof the outbreak. initially a “case” was broadly defined onset betwccn July 23 as an acute illness in a person with at least two neurologic symptoms with and August 7. Subsequently for the formal epidemiologic study. the case definition was narrowed to an acute illness with headache, numbness o f two or n~oreareas o f the body, and a t least two 0 1 five specific symptoms. Nineteen households, each with sick and well persons in it, were interviewed with a food questionnaire. Colnparison ofCoods eaten at lunch on July 30 showed that clams were associated with the illness. The shellfish were gathered at local beaches by individual families. Fifty-six of S7 patients reported eating clams, compared to S of41 controls. The odds ratio for clam soup was 38.5 [95% confidenceinterval (CI) = 4.6 325.01, signifying that cases werealmost 40 tinm more likely compared with well family members to have eaten clam soup. Soup and clanls were tcstcd using a mouse bioassay and found to contain saxitoxin [the chemical agent of paralytic shcllfish g of clan1 meat; the lethal dose was calculated :IS poisoning (PS€‘)) at levels up to 30,000 1~.U/100 11.000-35,000 pU.Toxin was also isolated from the stomach contents of a fatal casc. at three samplingsitcs along After this epidenlicof PSP, shellfish surveillance was established the Pacific coast. There was also increased awareness anlong thc fishernlen, consuI11ers. and health care providers of the potential seafood consumption. ~
Fig. 2 Seafood-borneoutbreakcasestudy
Epidemiology of Seafood Poisoning
29 1
dotoxin poisoning)to health departments ( 1,23). Health departmentsuse these case reports to monitor long-term trends in disease incidence (i.e., the number of new cases of disease i n a defined population during a specific time period) and to identify disease clusters that may signal outbreaks. Marine habitat surveillance involves monitoring of shellfish beds for paralytic shellfish poisoning (PSP), infectious organisms such as Vibrio species, and toxins (16,241. One example of surveillance techniques at work was the January 1995 closure of oyster beds in Florida’s Apalachicola Bay after studies traced multiple disease clusters of Norwalk viralgastroenteritis to theconsumption of oysters from the bay. Subsequent investigation revealed an earlier outbreak among oyster harvesters, who may have spread the infection to oyster eatersby dumping raw sewage from their boats into the bay, where the oyster beds became infected (25,26). Although the exact temperature and length of time of cooking were uncertain, studies conducted during this investigation also raised doubts about the usefulness of cooking as the sole means of preventing infection (27).
111.
EPIDEMIOLOGY OF SEAFOOD POISONING
One factor in the apparent increasing incidence of seafood poisoning is the worldwide increase in seafood consumption (28). In the United States, the average annual per capita consumption of commercial seafood increased from 5.7 to 6.7 kg from 1980 to 1992. In Canada, total per capita seafood consumption increased from 6.6 to 7. I kg from 1980 to 1990 (29,30). This consumption is not evenly distributed among populations. Degner et al. (28) studied a random sample of 8000 Florida households by telephone survey and found that the avcrage annual seafood consumption was 7.6 kg (with finfish consumption of 6.0 kg) 33% of 1071 peoplesurveyedfromMiami(30). However, in thel-weekrecall,only Dade County consumed marine tinfish (31). Different ethnic subpopulations also have different seafood consumption and preparation patterns. For example, many Southeast Asian and South American groups traditionally eat raw fish, while northern Europeans consume fish preserved by pickling. These cultural differences have important implications for prevention and control. Health warnings that are not culturally targeted and i n the appropriate language may not reach these ethnic subpopulations. For example, locally caught fish contaminated by heavy metals such as mercury are often consumed by ethnic subpopulations that may not have been informed about the dangers (14,lS). In addition, many populations, including ethnic subpopulations, believe that the consumptionof raw seafood is healthier and/or has aphrodisiac properties (as with raw oysters or fugdpufferfish) ( 3 2 3 ) . Seafood poisoning also varies etiologically by vehicle or transvector. In the United States during the period 1983-1992,fish was the transvector for scombroid and ciguatoxin, while shellfish was the transvector for Norwalk-like viruses, paralytic shellfish poisoning, Vibrio species, and hepatitis A virus ( 1 J ) . Seafood poisoning has certain transmission mechanisms in common with all foodborne illness. One is improper food handling. I n 1992, the Centers for Disease Control and Prevention (CDC) recorded 240 foodborne outbreaks witha known etiology (51% of total outbreaks reported). For those outbreaks, the contributing factors included ilnproper holding temperatures (62%), inadequate cooking (29%). contaminated equipment ( 18%), food from unsafe sources (7%), and poor personal hygiene (29%) (1). For seafood, as for other types of food, the relative importance of restaurant and
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other commercial establishments versus private food sourcesin the epidemiology of food poisoning is unclear. Twenty percent of the seafood consumed is derived from recreational and/or subsistence fishingthatisoutside most public health controls (2). Yet the true contribution of this food source to food poisoning is unknown because outbreaks involving these sources are less likely to be reported than restaurant outbreaks. A further global problem with all foodborne illness outbreaks is that,in nonendemic areas, medical personnel often do not recognize the illness or know of reporting requirements, further clouding the true epidemiologic picture due to significant under-reporting (1,2,16,32,33). As with other foodborne pathogens, the internationalization of the food supplymore than 50% of the commercial seafood eaten in the United States is imported-has greatly facilitated the transmission of seafood pathogens. National and international commerce allows forthe rapid and wide geographic spreadof contaminated seafood. Examples range from choleraon international airplanes to Norwalk-like gastroenteritis in five different states associated with the same contaminated collection site (17,27,34-40). The pathogenic organisms themselves can cross national and international boundaries, as seen with the spread of bacterial and dinoflagellate species through the dumping of ballast and bilge 3) (34,39,40). water from international shipping (see Fig. of public These new transmission dynamics have had a major impact on the practice a public health standpoint, the tracing and recall of contamhealth and epidemiology. From inated seafood is much more difficult when the product is widely distributed, and therefore the potential for prolonged outbreaks is much greater (1 7). Epidelniologically it is much more difficult to study human disease when the target populations become diffuse. In addition to the transmission mechanisms it shares with other foodborne illnesses, seafood poisoning has unique modes of transmission related to the marine environment. Discharge of treated and untreated human fecal material, other sewage, and industrial wastes into estuaries and coastal watersis extremely common, even in developed nations. The coastal waters of the United States receive more than 3 X 10" L of municipal sewage each day. Viruses can settle out of the water column and attach to sands, clays, aquatic life forms, and sediments, accumulating in the loose layer over the compact bottom sediment. This layer can be easily resuspended after storms, dredging, or even boating; tidal currents can then transport these resuspended viruses to distant waters (41). Shellfish, especially the filter feeding bivalve mollusks (oysters, scallops, mussels, clams, cockles), live in estuarine areas and obtain their food by filtering large amounts of water. A wide variety of organisms and toxins pathogenic to humans can accutnulate in the shellfish alimentary tract, especially if the filtered water is contaminated by sewage or chemical pollutants. After contamination it can take hours to weeks of filtering in uncontaminated water before these pathogenic organisms are removed. Since the alimentary tract of these bivalves fonns the major edible portion for humans, these mollusks can serve as extremely effective vehicles fora wide range of organisms and toxins pathogenic to humans (30,42,43). The other commonly consumed group of shellfish is the crustaceans (e.g., shrimp, crab, and lobsters). Although not filter feeders, they can acquire contamination from contaminated water.
IV. EPIDEMIOLOGY OF SEAFOOD PATHOGENS
A.
Bacteria
Traditionally the most frequently reported seafood poisoning outbreaks (Table I ) have been associated with bacteria. Although increased sophisticationof laboratory testing now
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implicates viral origins, new bacteria-associated seafood poisonings are appearing due to the increasing worldwide consumption of seafood by sensitive subpopulations.
1. Vibrios Vibrios are natural inhabitantsof marine environments. Pathogenic marineVibrio species, and V.wltl$cus, most commonlycause especially V. cholercre, V.parcrktrer~lol~~tic~rs, disease when they are ingested in raw shellfish and, less commonly, i n fish (44,45). Many Vibrio species are not associated with fecal contamination, so the use of fecal colifornl counts as clean water indicators does not ensure that water or seafood beds are free of pathogenic vibrios (3). Cholera, caused by the toxin-producing V. cholerae, can be a severe infection with mortality rates as high as 50%, but is more commonlyan asymptomatic infection. Seafood is the most common vehicle of outbreaks. The infection can be acquired through fecal contamination of food by a person who is ill or an asymptomatic carrier, through seafood harvested in a sewage contaminated area, or through seafood gathered i n natural environmentalreservoirs of V. cholerrre. Nevertheless,epidemiologicstudieshaveshownthat unhygienic food handling in the home or on the street are the most common sources of seafood-borne cholera (34,46). Cholera has the potential for rapid international spread, as evidenced by the seven pandemics since 1817, including the recent Peruvian epidemic. In a particularly dramatic example of the potential for the spread of disease through international travel, 75 people developed cholera after taking an airplane flight from Latin America to California during which they consumed a cold seafood salad plate from Lima, Peru (35-37). In another example of international spread, shellfish beds in the United States have been contaminated of ships arriving from Latin with V. cholercre transported in the bilge and ballast water America (34,40). V. pcrrahaemo1~tieu.sis associated with the consumption of inadequately cooked or refrigerated crustaceans and fish (32). In Japan, upto 50% of reported foodborne outbreaks have been caused by V. ~~crrcrIrhnen~o1~ticu.s; in Guam, the risk of laboratory confirmed V. ptrrrrhtretnol~tie~~.s food poisoning was highly associatedwith recent seafood consumption, especially fish (OR = 37.59, C1 8.30-220.2). In the United States, V. vuln~ficusis rare outside the coastal states of the Gulf of Mexico, but is relatively common in these states; Florida recorded 141 cases during the period 1981- 1993. This organism can be associated with mortality as high as 50% in persons who are immunocompromised or have liver disease; the risk of oyster-related V. ~ulw$cus infection is 80 times greater i n adults with a history of liver disease or who of infection from eating oysters is have chronic alcoholism (2,26,43,45-48). The risk highest in the months April through October, when more than 90% of raw oyster-associated V. vulw$cus infections occur (49). Florida and other states require restaurants that serve raw oysters to post warnings about the dangers of consunling raw oysters. Although not directly seafood consumption,V. vuln$cu.s also can cause wound infectionsin persons who go wading or swimmingin contaminated waters. In Florida, during the period 19811993, one-third of the 141 V. w l n @ x . s infections were wound infections (49). 2. Listeria In New Zealand and elsewhere, Listcrirr ~lowoc~togetles, found on the external surfaces of fresh and frozenfish as well as in the processing plant environment, has been implicated in several seafood-borne outbreaks of listeriosis. These outbreaks have included perinatal infections associated with transmission from infected mothers, who may be asymptomatic
Table 1 Reported Seafood Poisoning Outbreaks by Etiology Etiology
Seafood transvector
Clinical presentation
Reference
Mollusks Mollusks, crustaceans, fish Mollusks Mollusks Shellfish. seafood
Septicemia Gastroenteritis, septicemia (at risk immunocompromised, liver disease) Gastroenteritis Gastroenteritis Gastroenteritis (at risk inimunocompromised)
27.43. 108 43, 108 29. 43. 52
Shellfish Shellfish Shellfish. seafood Seafood
Gastroenteritis Gastroenteritis Gastroenteritis Listeriosis
43 43 42. 43. 55 50, 51
Mollusks Shellfish
Hepatitis Gastroenteritis
42. 53. 62, 63, 108 17. 18, 26. 27. 53. 60. 101, 108
Shellfish Shellfish
Gastroenteritis Gastroenteritis
43 43
Fish (raw) Herring, cod. whiting. haddock. salmon Fish
Abdominal discomfort. eosinophilia. allergy
32. 43. 64-66, 70
Abdominal discomfort, eosinophilia. allergy. eosinophilic meningitis
66
Bacterial Sulnioriellu (typhi. purutyphi) Vihrio fcholerue. purcrhcreniolyticit.s. niimicus, hollisue. j m i a l i s , vultiij?cirs) Sli ig ellu Cuniphylobucter Aeromotius hydrophila, veronii sohriu, cuviue Bacillus cereus EdLcw-dsiellu turdu E. coli (including enterotoxigenic) Listeriu nioriocyrogeries Viral Hepatitis A Small round structured viruses, Nonvalklike viruses (Nonvalk. cockle. snow mountain. calicivirus) Rotavirus Astrovirus Parasitic Airisrikis
Giiut hosfonia
29. 43, 57, I08 35, 42-44, 48, 53. 68. 108
z! 3. 3 0
m
M
22
W
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E
W
m
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(50,Sl). A wide range of seafood transvectors have been associated with these reported listeriosis outbreaks, from raw fish and shellfish to ready-to-eat (but insufficiently processed) cooked shrimp and crabmeat, and smoked mussels.
3. Salmonella Strltnonelltr and Aeromonrrs species are frequent contaminants of raw fish and shellfish, especially if the seafood has had prolonged exposure to elevated temperatures. The risk of transmission is higher for shellfish than for fish because shellfish often are refrigerated for many days without obvious spoilage andthen eaten raw (29,52). As with Vibrio infections, persons who are elderly, immunocompromised, or very young are at greater risk I C I S , enteropathofor more severe gastroenteritis after infection with A ~ ~ O I I I O Iespecially genic strains. 4. Other A wide spectrum of bacterial species has been cultured from shellfish and fish without definitive etiologic connections to seafood poisoning outbreaks ( 5 1-55). Some of these organisms, like Arrornonas, are part of the nonnal flora of the seafood. Others are more likely introduced by unhygienic hutnan handling; enterotoxigenic Esclwrichitl coli, the conmon pathogen of travelers' diarrhea, and Stc~~?ltr'loc.occu.s rwreus have both been detected on seafood (52-56).
5. Future Threats Internationaltradeinwild-harvestedseafood,andincreasinglyinaquacultureseafood products, has facilitated the introduction of pathogens into new geographic areas, as well as into seafood and hunlan communities (30). For example, Strlmonellcr crgoncr was first introduced to Europe following the importation of Peruvian fish meal, with subsequent rapid spread into other food products, producingan increase in human outbreaks. Furthermore, the intensive use of antibiotics in the aquaculture industry, leading to antibiotic accurnulation in seafood tissues, increases the potential for the development of multiply resistant bacteria (57,58).
B. Viruses As discussed above, shellfish harvested in waters contanlinated with raw or inadequately treated sewage are extremely efficient transvectors of seafood pathogens because the shellfish filter the water as they feed, concentrating the pathogens (1,2,29,59). Paradoxically refrigeration increases the pathogens' geographic range, permitting transportof apparently healthy shellfish to many geographic areas, thus extending and prolonging outbreaks. Inadequate procedures for tracing and recallof contaminated seafood also serve to extend outbreaks ( 1 7,18,27).
1. Small Round Structured (Norwalk-Like) Viruses In theperiod1978-1987,approximatelyone-third of seafoodoutbreaks intheUnited States and two-thirds of all cases were associated with consumption of raw molluscan shellfish,accordingto CDC and FDA records (2). In themajority of thesecases,the pathogens were snlall round structured viruses (SRSVs) (26,29,43). SRSVs,orNorwalk-likeviruses,areclassifiedascalicivirusesandarecoInmon causes of outbreaks of gastrointestinal illness in the United States. Because the infectious dose is small, cooking theshellfishdoesnotreliablyeliminatetherisk of contracting
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gastroenteritis. For example,i n an outbreak of Norwalk-like gastroenteritis in North Carolina following consumption of oysters at a church supper, the attack rate (56%) did not differ based on whether the oysters were eaten raw or steamed for at least 12 minutes. Outbreaks of Norwalk-likegastroenteritishavebeenreportedaftergrilling,stewing, steaming, and fryingthe shellfish (26.27.60). Furthermore, routine water quality tests from the areas of contaminated shellfish collection were within normal limits, demonstrating that fecal coliform monitoring is inadequate protection for SRSV contamination (27,61).
2. Hepatitis A The proportion of hepatitis A cases in the United States that are attributable to foodborne or waterborne outbreaks is small (7.5% in the period 1983-1989) (43). Nevertheless, the most common causeof these outbreaksis the consumptionof raw or inadequately prepared shellfish taken from sewage-contaminated waters (32,42,62,63). As with other seafoodrelated pathogens, suchas V. clwlerae, seafood poisoning with hepatitis A has been associated with a significant geographic spread due to seafood export patterns. For example, an outbreak of hepatitis A after consumption of raw oysters from Florida resulted in cases in five different states, including Hawaii (38). C.
Parasites
Consumption of raw and inadequately cooked seafood, especially in certain ethnic subpopulations, is associated with parasitic infections, particularly with anisakids and cestodes (232). Correct food preparation and handling will eliminate the majorityof these diseases.
1. Anasikiasis Anasikiasis is a rare nematode (roundwoml) infection acquired through the consumption of rawfish(especiallycod,herring,mackerel,andsalmon)andcephalopods(such as squid). Traditionally associated with Asian cultures, because of changing consumption patternsanasikiasisisnowreported in Europeand the UnitedStates,although rarely (64,65). Although often asymptomatic, patients can presentwith eosinophilia, abdominal discomfort, invasion, or hemorrhage dueto burrowing of the womi, as well as granulomatous and frank allergic reactions (32,43,65). A similar disease associated with the helminth Gntrrhostomn has been reported in Southeast Asia and the Middle East.
2. Diphyllobothriasis Diphyllobothriasis or fish tapeworm disease was traditionally associated with gefilte fish preparation by Jewish women. Although often asymptomatic. megaloblastic anemia (secondary to vitamin B I Zdeficiency) and eosinophilia are classic findings. Approximately 10% of people in Scandinavia are reportedly infected with Diphyllobothrium. Although usually associated with freshwater fish consumption, diphyllobothriasis has been reported with the ingestion of raw Pacific salmon (32,66).
D. Allergies Seafood allergies include true food allergies (i.e., type I IgE-mediated hypersensitivity) and food intolerance (i.e., nonimmunologic hypersensitivity) (67,68). It is estimated that 3% of 3-year-old Finnish children are allergic to fish, and the prevalence of fish allergy is close to 1 in 1000 in the general Norwegian population (67). In a study in Spain of 3034 persons over 14 years old seen as outpatients in an allergy unit, skin testing and/or
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RAST testing found 30 (0.98%) persons with a possible food allergy, with 14.9% related to seafood (especially shellfish) and 4.2% to fish. Symptoms ranged from acute cutaneous disorders to gastrointestinal complaints to respiratory symptoms (69). In South Africa, among 105 persons with suspected fish allergy, adverse reactions were reported with shellfish [prawns (46.7%), crayfish (43.8%), abalone (35.2%), and black mussels(33.3%)] and withfish [hake(24.8%),yellowtail(21.9%).salmon(15.2%),andmackerel(15.2%)]; these reports were confirmed by double-blind, placebo-controlled food challenge (67). Allergic reactions, ranging from urticaria to anaphylaxis, have been reported after consumption of seafood with parasitic or certain natural toxin (i.e., scombroid) contamination. Allergic reactions after seafood ingestion have been reported even with negative skin or exposure test to fish or shellfish; these are often associated with parasitic infestation and specific allergic reaction rather than seafood-associated allergy. In particular, chronic intermittent allergic reaction type I and/or I11 with eosinophilia have been reported after the consumption of Aniscrkis-parasitizedfish. The ingestion of even safely cooked but to allergicreactionswithIgE-mediatedsensitization Anisakis-parasitizedfishhaslead (64,65,70).
E. Toxins The marine environment contains a variety of chemicals, both natural and man-made,that can be acutely and/or chronically toxic to humans if ingested in large enough quantities. Unfortunately the marine organismsthat humans prefer as food tend to concentrate toxins through a process known as bioaccumulation. Many of the fish that humans eat are at the top of the marine food chain; that is, they are predators (salmon) or even predators of predators (sharks). Toxins from the smaller organisms these predators eat accumulate in the predators’ tissues (particularly if the toxins are lipophilic), and often reach levels high enough to cause acute or chronic disease in humans. The largest and oldest fish of each species contain the most toxin, and also tendto be the most desired by seafood consumers. Molluscan shellfish, also prized as a food source, are filter feeders, concentrating natural and chemical toxins indiscriminately (2,43,7 1,72). Often the toxins that marine organisms accumulatehave no obvioushealtheffects on thefish andshellfish,thustheyremain available for capture for human consumption, and they appear healthy and appetizing to the consumer. Among the diseases caused by natural toxins, only scombroid, botulism, ciguatera, PSP, and tetrodotoxin (fugu) are required to be reported to the CDC (1). Individual states mayrequirereporting of other diseases (26). Therefore, as withallthe seafood-borne diseases, under-reporting is a major epidemiologic issue.
1. Bacteria The seafood diseases caused by bacterial toxins generally are associated with improper food preparation, handling, and storage. Scombroid poisoning is probably the most commonly reported fish-associated illness in the United States (1,3,32,67,68,73). It is often mistakenly diagnosed as “fish allergy” because the symptoms resemble an IgE-mediated reaction and respond rapidly to antihistamines. Scombroid poisoning actually is caused by bacterial overgrowth associated with inadequate fish storage, especially of fish i n the Scotnbroidae family (i.e., tuna, mackerel, and jacks)as well as mahi-mahi or dolphinfish, bluefish, and sardines. The surface bacteria (e.g., halophilic Vibrio spp., Proteus, Klebsiella, Enrerobacter) decarboxylatethehistidinepresent naturallyin dark-meat fish to
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produce high levels of histamine, optimally when the temperature is between 20°C and 30°C. The phenomenon occurs with consumption of fresh and canned fish. The bacteria Clostridium botulinutn type E is prevalent in both freshwater and marine environments. The bacteria produce botulism toxin E on smoked fish, fish eggs, and uneviscerated and salted whitefish. The majority of the cases are associated with inadequate cannillg procedures. Although smoking or light cooking may not kill the bacterial spores, the toxin is heat labile. The toxin produces an acute gastroenteritis followed by cranial nerve dysfunction and symmetrical descending weakness. Although rare, this disease is OccasionalIy seen among ethnic minority communities and with home canned fish (2,32,33,68). Stcll,hy/ococcu.sL I U ~ P U elaborates S an enterotoxin on improperly stored seafood, especially if the fish is garnished with cream sauces or mayonnaise (63).
2. Marine The marine toxin diseases are caused by a myriad of natural toxins produced by minute organisms such as dinoflagellates and diatoms. These phytoplankton are part of the base of the marine food web and are ubiquitous in the marine world (74). Humans are most often exposed through consumption of fish and shellfish that accumulate these toxins. The toxins, small nonpeptides, are some of the most powerful natural substances known; ciguatoxin is toxic to humans in a total body dose of 70 ng. Because these toxins are tasteless, odorless, andheat and acid stable, normal screening and food preparation procedures will not prevent intoxication if the fish or shellfish is contaminated (39,75). The marine toxin diseases are categorized into two groups based on their primary transvectors. Shellfish harbor the toxins that produce paralytic shellfish poisoning (PSP), neurotoxic shellfish poisoning (NSP), diarrheic shellfish poisoning (DSP), and amnesic shellfish poisoning (ASP). Fish carry the toxins responsible for ciguatera poisoning and tetrodotoxin (fugu or puffer) poisoning. The shellfish-associated diseases generally occur in association with algal blooms or “red tides,” which may be characterized by patches of discolored water and dead or dying fish. The fish-associated diseases are more localized to specific reef areas (ciguatera poisoning) and fish (fugu poisoning). Underdiagnosis and underreporting, especially in endemic areas, make it difficult to know the true worldwide incidence of the marine toxin diseases. For example, it is believed that ciguatera affects at least 50,000-100,000 people per year who live in or visit tropical and subtropical areas, but there is significant underreportingof this relatively common marine toxin disease in endemic areas (16,39). The primary target of marine toxins is the neurologic system, although affected individuals usually present witha wide rangeof symptoms, resultingin a confusing clinical picture. Gastrointestinal symptoms begin minutes to hours after eating contaminated seafood. In the case of PSP, fugu, and ciguatera, accompanying acute respiratory distress may be fatal within hours. Ciguatera and ASP may also produce debilitating chronic neurologic symptoms lasting monthsto years. Chronic disease (neurologic, immunologic, etc) associated with the marine toxins is an area of active scientific research. I n the past, these illnesses have been highly localized to endemic island and coastal communities. With increasing worldwide seafood consumption and trade,as well as international tourism, these diseases are expanding beyond their traditional geographic boundof diagnosis and treatment of disease in aries. One side effect has been the high costs traditionally nonendemic areas. In Canada, with an estimated 1000 casedyear related to tourism and food importation, the average medical cost per case of ciguatera was $2470 in 1990 (76,77).
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In addition to increased worldwide seafood consumption, other anthropogenic factors may have helped spread the dinoflagellates and their toxins. Human-assisted transportation of the dinoflagellates or their cysts occursin spat cultivation (young bivalve shellfish sold conimercially to global markets for aquaculture) and dumping of ship ballast water. In response, new international regulations require ships to purge ballast water i n the open ocean prior to docking (40). Human-generated environmental changes, such as reef destruction and eutrophication, also may help explain the apparent increase in human marine toxin disease, as well as the increase in red tides reported worldwide. Global climate change, which some suggest are linked to human activities, also may help explain the apparent global increase in algal blooms as well as the appearance of new marine toxin diseases like Pfiesteria (30,75,78-80). There is even new research connecting red tides to cholera outbreaks since copepods carrying V. cholercre feed on marine algal blooms; thus these algal blooms can lead to cholera disselnination and outbreaks associated with increasingly frequent monsoon flooding (79). cr. Parrrlyic Shel@sh Poisoning. ClassicPSPsymptomsincludeparesthesias of the mouth and extremities, frequently accompanied by gastrointestinal symptoms, within minutes to hours after eating. The disease occurs worldwide. Although often associated with a red tide or algal bloom, significant epidemics of PSP can occur in humans in the absence of a known red tide, as discussed in Fig. 2 with the Guatenlalan PSP epidemic of 1987 ( 2 2 ) . The dinoflagellates associated with PSP produce at least 12 toxins that are heat and acid stable; saxitoxin was the first characterized and the best understood (39). In a population-based study in Alaska after five local outbreaks of PSP, 70% of the 170 people interviewed had eaten shellfish gathered from the same area and 13% reported symptoms consistent with at least one episode of PSP (81). The case-fatality rate was about 8.5-9.5% in two large series; the 1987 Guatemalan outbreak on the Pacific coast had a case fatality rate of 14% overall, but 50% in young children, who may be more sensitive to PSP toxins. Access to emergency medical services in acute cases is crucial to survival. Chronic health effects have not been studied (22).
b. Neurotoxic ShellJsh Poisoning. Red tides that occur offtheFloridacoastare associated with two distinct clinical syndromes, depending on the route of exposure. Ingestion of contaminated shellfish (and less commonly contaminatedfish) causes gastroenteritis and neurologic symptoms similar to but less severe than those associated with PSP. Inhalation of toxins, predominantly brevetoxin, from the sea spray associated with the red tide and accompanying fish kills causes an upper respiratory syndrome in humans and other mammals (39). The classic causative organism, Gytnrlodinium breve, is a dinoflagellate restricted to the Gulf of Mexico and Caribbean waters, although similar species occur throughout the world (82). NSP was first identified by Walker in 1844 on the west coast of Florida. Since then, NSP has been reported from the Gulf of Mexico (including the coasts of Texas, Alabama, and Mississippi), the east coastof Florida, and the North Carolina coast. Recent prolonged NSP red tides in the Gulf of Mexico have been associated with die-offs of endangered manatees and dolphins, as well as respiratory problems in humans (26,83). Fish feed harvested from red tide-contaminated areas also has been blamedfor fish kills in the aquaculture industry. c. Diarrheic ShellJsh Poisottitlg. The first cases of DSP were reported from the Netherlands in the 196Os, followed by similar reports in the late 1970s from Japan. Since with the peak season from then, Inore than 1300 cases have been reported from Japan,
Epidemiology of Seafood Poisoning
30 1
April to September. Other outbreaks have been reported in Europe and South America as well as the Far East (39). i ~ . dinoflagThe causative organisms are the marine dinoflagellates D i t ~ o p h y ~These ellates are widely distributed, but do not always form redtides. They produce atleast nine different toxins consisting of okadaic acid and its derivatives. Because okadaic acid is a potent animal carcinogen, the issue of chronic disease from DSP poisoning needs to be addressed in humans (39,84).
d. Anulesic Shellfish Poisonir~g. First recognized in an outbreak onPrinceEdward Island, Canada, i n 1987, this syndrome produces nausea, vomiting, severe headache, abdominal cramps, and diarrhea within 15 minutes-38 hours of eating mussels, accompanied in about 25% of cases by acute lnemory loss. The 1987 outbreak, associated with consumption of mussels, produced 153 cases and 4 deaths. Some persons with ASP develop apparently permanent neurologic deficits, especially dementia. The toxin responsible is domoic acid, elaboratedby the pennate diatomNit:.schicr prngnc’s. It has been suggested that the index bloomof the diatom may havebeen related to fertilizer runoff from extensive identified as a continuing tobacco farming in the area (39,85). The organism was later problem among seabirds, sea lions, and shellfish in Washington, Oregon, and California. e. Cigrrcrtero Poisoniwg. Ciguatera isthemost conmon foodborne illnesscaused by a marine toxin. Its most distinguishing symptom is temperature reversal (hot coffee tastes cold, ice cream tastes hot). It is caused by the consumption of reef fish that have toxicus and other reef-dwelling dinofed on organisms contaminated with Grrmbic~rdiscrr,s flagellates. Ciguatera has been reported since ancient times and occurs i n tropical and subtropical areas around the world, with epicenters i n the Caribbean and the Indo-Pacific islands. It is not associated with red tides. Ciguatera is another of the seafood-related diseases that have spread geographically because of tourism and national and international commerce (39,75). For example, a single amberjack from a dealer in Key West (Florida), sold to two restaurants and two grocery stores,resultedin at least 20 ciguateracases i n at least two states (86). An estimated 10,000-50,000 people/year who live in or visittropical and subtropical areas develop ciguatera poisoning. The CDC and others estimate that only 2-10% of cases are actually reported i n the United States. Human impact on the environment may also play a role in the spread of ciguatera. Disturbances of coral reefs by military and other human activities stinlulate rapid recolonization by G. to.ricus, which appears to grow better after disturbance than under normal conditions (75). Ciguatera has had a measurable social and economic impact in endemic regions. Because the toxin is so widespread among reef fish, and because it cannot be detected by smell or taste or destroyed by cooking, populations in several endemic areas have abandoned local fish as a food source. Lewis (87) found that ciguatera in the South Pacific caused depression of both the local and export fishing industries and tourism, and had an indirect affect on human health due to avoidance of fresh fish consumption.
J: Pufet:fish PoisorlinR/FuRu. Pufferfish poisoning ortetrodotoxinintoxication (fugu) produces symptoms similarto PSP, with case fatality rates as high as 60%. Tetrodotoxin poisoning is found worldwide, associated predominantly with the ingestion of pufferfish. Fugu poisoning was known to the ancient Chinese as early as 2800 B.C. Unlike diseases caused by marine toxins, fugu poisoning is not due to dinoflagel-
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lates. The toxin, tetrodotoxin, is found i n the order of fish known as Tetraodontiformes, especially in the family Tetraodontidae (puffers). Marine bacteria colonize thegutand skin mucosal layers of the pufferfish following infection, and produce persistent levels of in gonads, liver.and.toalesser extent, muscle tetrodotoxinwhichthefishsequesters (39,75). The most toxic pufferfish are found along the coasts of China and Japan, where they are considered a delicacy and are eaten only after preparation by specially trained chefs. In Japan, where the seafood is known as fugu, 60 poisonings were reported in the period 1974-1979, with 20 deaths. About 50%;of fatalfood poisonings i n Japan each year are due to eating fugu; cases are more conlmon among men than women, probably related to eating habits. Cases have been reported in Europe associated with the mislabeling of imported fish, and elsewhere in the world due to ignorance of the toxic potential of thepuffers.Thus, fuguisavery circumscribedpublic health problem withahigh mortality. Chronic health effects have not been studied (39). g. Otlwr Ncrturd Mtrrinr To.rins. Numerous other types of poisoning are associated with the consumption of a wide variety of fish and shellfish. However, at present these poisonings have very restricted geographic and race-ethnic population distributions. These include a pseudoallergic reaction after eating Japanese callista during the spawning season to hallucinatoly fish poisoning after eating nmllet i n the tropical Pacific and Indian Oceans to hypervitaminosis A and neurologic disorders after consuming shark contaminated with unknown toxins (68,73,75). In addition, consumption of edible red algae, agorlori, i n Japan has been associated with acute foodbome illness and death (88). As with theotherseafooddiseases,internationalseafoodcommerce.aswellastourism,may change the geographic and population profiles of these illnesses in the future.
3. Chemical Toxins Humans, through their industrial activities, liberate mercury and other heavy metals from the earth and send them into the seas. where they bioaccun~ulate throughthe food chain and come back to humans in the form of contaminated seafood. Man-made chemicals and chemical by-products such as polychlorodibenzo-p-dioxins (PCDDs) and polychlorinated biphenyls (PCBs), manyof them lipophilic and nondegradable, follow the same pathways and are consumed by humans. Infants and fetuses are at particular risk because many of (72.89). these substances are incorporated into breast milk and cross the placenta Evaluating the impact of these toxins requires a thorough understanding of the ecotoxicology of pollutants. For example, arsenic bioconcentrates i n shellfish. Nevertheless. years of research have revealed that little of the organic arsenic accumulated by humans from the consumption of seafood is converted into toxic inorganic arsenic. Thereforc, seafood containing arsenic represents a low risk to hurnan consumers (90,91). Traditionally it was believed that many chemical toxins contaminated the marine environment through single-source polluters such as specific industries. One extreme example of single-source pollution was the epidemic of. chronic neurologic disease that occurred when the people of Minimata, Japan, consumed fish contaminated with a methylmercury effluent from a local chloralkali plant (92). Other exanlples include highly visible marine oil spills that can force closure of shellfish beds and fish spawning areas for years. For example, after the Braer oil spill i n theShetlands,thesediment of thetraditional herring spawning beds rose from “background oil levels” o f 5 0 ppb to 100-350 ppb (93).
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However, i n recent years, more global sources of pollution have been recognized. For example. many freshwater and marine fish have become contaminated with methylmercury not from industrial point sources but viaa complex exposure pathway originating withglobalburning of fossilfuelthatproducesinorganicmercury as a by-product (72,94,95). Atmospheric and oceanic currents then spread this mercury to distant sites. Persistent contaminants, such as heavy metals and organochlorines, have been found i n the Arctic food chain and i n biological sampling of Arctic natives (72). Even radioactive materialscanbebioconcentrated in seafood.Radioactivewastedumping.atmospheric fallout, leaks from spent reactors, and runoff from contaminated rivers have lead to measurable radioactivity in seafood found in some Arctic regions (96). Pesticide residues increasingly are being found in seafood. This is probably due to agricultural runoff and the lipophilic properties of many pesticides that cause them to bioaccumulate in both the seafood and the hunlan consumer. In 1993, in the FDA residue monitoring program, 15.8% of 444 imported fish/shellfish samples analyzed for pesticide residues were above regulatory levels; by comparison, only 3.3% of 2261 imported fruits were above regulatory levels during the same sampling time period (97). Long-term exposure to relatively low levels of these chemical contaminants through seafood ingestion may be important in the etiology of immune diseases and cancer. Multiple myeloma, a relatively rare and deadly form of B-cell neoplasm, has been associated with the consumption of fish contaminated with dioxins (possible immunogens) in Baltic Sea fishermen and Alaskan natives (98). Recent research points to the increased sensitivity of children, infants, and fetuses to heavy metals and pesticides, especially to bioaccumuto the dramatic lowering of these allowable residue lated neurotoxins, which may lead levels in seafood and other foods i n the future (89.99).
V.
PREVENTION
Primary disease prevention, the goal of public health, reduces the incidence of disease by preventing human infection ( 1 1 - 13,23). Secondary andtertiary prevention seek to reduce, respectively, the duration of and complications from disease. Of the three approaches, primary prevention is almost always the most effective, from both economic and public health standpoints, although it may not always be possible. Primary prevention of seafood poisoning has two foci: the environment in which the seafood grows and is processed, and the people who prepare and eat the seafood.
A.
Environmental
After a large outbreak of typhoid fever was traced to contaminated shellfish, i n 1925 the U.S. Public Health Service established the Interstate Shellfish Sanitation Program (ISSP) to ensure that contaminated shellfish donot reach the retail market ( 1 7,42). Recommendations to prevent shellfish contamination included sewage control, limiting shellfish harvests to areas with clean water based on fecal coliform counts, and tagging all boxes of shellfish to indicate harvest location and date. Because fecal coliform counts arenot sensitive to the presence of many Vibrio species and Norwalk-like viruses, this has led to a shift from wastewater-associated bacterial outbreaks to outbreaks involving wastewater-
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derived viral pathogens and bacteria of environmental origin (100-102). More recently, in the wake of a 1998 Texas outbreak of V. ~ ~ t r ~ t r k c r e r ~ r o l ~the t i c uISSP s , has adopted new standards that call for the sampling of shellfish meat from areas associated with illnesses caused by V. y~r~.crh~renlol~ticus. The recent passage of the National Food Safety Initiative is a significant primary prevention intervention that includes the following seafood safety initiatives ( 103). Required seafood testing for pathogens and toxins as soon as it has left the hands of the fisherman. Increased personnel, epidemiologic, and laboratory resources devoted to surveillance programs. Implementation of a Hazard Analysis and Critical Control Point (HACCP) approach to seafood processing. HACCP rules require food industries to design and implement preventive measures that identify points where contamination is likely to occur and to implement controls to prevent it. Epidemiologic evaluation will be very important i n the future to determine the implementation and efficacy of these programs.
B. Education In surveys in Florida and elsewhere, from 17 to 25% of theadultpopulationreported eating raw clams and oysters in the preceding year (49,103).These data suggest the need for targeted food safety messages to educate consumers about the dangers of seafoodborne illnesses, especially sensitive subpopulations such as the immunosuppressed, the elderly, and children. The health care providersof these sensitive subpopulations need to be aware of the potential dangers of seafood consumption. I n addition, food processors andfoodservice workers need information about proper food handling procedures for each step of the process from the marine environment to the consumer’s plate. Seafood testing information, as well as food handling and storage procedures. needs to be conlmunicated back tothefishermen and the seafoodindustry to seek out and providesafer seafood. Finally, decreasing global pollutionwill protect not only one seafood supply, but also the crucial habitats.
VI.
CONCLUSIONS
Seafood-borne poisonings, both acute and chronic, are under-reported, but are probably increasing in incidenceandgeographicspread. I n particular,therehasbeenverylittle research into chronic disease associated with seafood poisoning. Furthermore, subpopulations, such as ethnic and the illlmunosuppressed, may be at increased risk for seafood diseases. Although primary prevention is the most desirable, the continual emergence of new seafood-borne diseases signifiesthat active disease surveillance will always be necessary. Finally, although required in the United States by the Food Safety Act, reliance on the testing of food will never be enough, since such testing must always be targeted and it is impossible to test for new or different pathogens and toxins in all seafood prior to human consumption. Therefore, education with regard to seafood safety (particularly issues suchas the consumption of raw seafood andthe importance of sanitation) will remain important (3,38).
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The education of health care providers and public health officials in traditionally nonendemic areas is necessary concerning the diagnosis andthe importance of official reporting of imported "exotic" or emerging seafood diseases (37). Groups that should be targeted include international travelers (both workers and tourists), recent emigrants, certain ethnic subpopulations, and imlllunosuppressed individuals. I n addition, education and cooperation of seafood industry workers concerning the risks of seafood-borne poisonings as wellasprimaryand secondary prevention programs are necessary for these programs to function effectively ( I 1-13.104). There should also be cooperative exploraas seafood tion and education about new and inexpensive protection technologies, such irradiation, between international and national health agencies, and the seafood industry (4,105). Given the high reported prevalence of disease with multiple etiologies associated with the consumption of raw or inadequately cooked seafood, thorough cooking of all seafood is a basic recommendation (53,62). This is particularly true for persons who are immunocompro~nised and/or suffering from liver disease (2,50,106), although not a coniplete panacea (for example, some viruses have high virulence, and many toxins cannot be removed by cooking) (27.60). Furthermore, it is critical that the necessary time and temperature studies be done with seafood to determine the degree of cooking that will denature a virus such as SRSV. It is also important to conmunicate the dangers of raw seafood consumption to ethnic subpopulations using culturally sensitive and appropriate language (33,94,107). In addition. laboratory and epidemiologic evaluation of possibly protective culturally acceptable practices, such as adding acidic substances (i.e., lemon juice in the Latin American marinated seafood dish of ceviche against Vibrio cholera) should alsobe explored (14,15,34.35). Alternative technologies such as the use of naturally occurring bacteria in seafood with antagonistic activity toward pathogens such as Lister-icr are being tested (51). Good sanitation. including facilities for human waste disposal and for proper hand washing with soap, are important to protect food and water supplies. Furthermore. education should be focused on the dangers of human fecal waste near drinking, agricultural, or aquacultural water supplies to prevent the contamination of irrigation and runoff water (34). Educational efforts targeting food handlers, food handling, and preparation to both prevent fecal contamination and maintain food integrity without spoilageor bacterial overgrowth should be undertaken (34.46,53). Even food storage should be emphasized; for example. i n Hong Kong, cholera may have been spread through the consumption of fish kept i n tanks with contaminated water (44). As seen i n several seafood-borne outbreaks, national and international collllnerce can lead to the rapid, wide, and prolonged spread of contaminated seafood with multiple resultant disease outbreaks ( I , 17,27.34,35,37,38). Fecal colifornl counts are agood initial screen for water contamination by fecal material, but are inadequate in the case of some bacteria, and even more for many pathogenic viruses and both natural and chelllical toxins. New laboratory techniques such as polymerase chain reaction (PCR) will lead to better diagnosis, tracing, and ultinmtely prevention of seafood poisonings (3,17,18). Todd and Harwig (29) recommend a risk analysis approach for seafoodthat has the following characteristics: widely consumed, international sources (both feral and aquaculture), associated with emerging diseases, and potentially produced under unsanitary conditions. They emphasize the importance of integrating this risk assessment approach into the HACCP system to more accurately determine the hazards and control of seafood processes. They particularly recommend focusing on potentially unsafe preparation and con-
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sumption practices (such as raw or smoked seafood), since these processes are known to increase the riskof disease. Additional recommendations include economic data collection and presentation to evaluate both the impact and cost-effectiveness of intervention programs, as wellas of present seafood practices (15,29,5 1,57). Thus, epidemiology will necessarily play an active role in evaluating the impact of seafood poisoning and future preventions and interventions.
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96. TG Sayzkina, I1 Kryshev. Current and potential doses from Arctic scafood consutnption. Sci TotalEnviron202:57-65. 1997. 1993. J Assoc 97. Anonymous.FoodandDrugAdministrationpesticideresiduemonitoring: Offic Anal Chcm Int 77(5):161A-185A. 1994. 9X. G Schwartz.Multiplemyeloma:clusters,cluesanddioxins.CanccrEpidetniolBiotnarkcrs Prev6149-56,1997. 99. National Research Council. Pesticides in the dicts of infants and children. Washington. D.C.: NationalAcademyPress,1993. 100. SR Rippey. Infectious diseases associated with molluscan shellfish consumption. Clin Microhiol Rev 7:419-425. 1994. 101. A Stollc, B Sperner. Viral infections transmitted hy food of anitnal origin: the present situationintheEuropeanUnion.ArchVirolSuppl 13:219-228. 1997. 102. CP Gerha, SM Goyal, RI LaBelle, et al. Failure of indicator bacteria to reflect the occurrcncc of enteroviruses in marine waters. Am J Public Health 69: 1 116-1 I 19. 1979. 103. FoodandDrugAdministration.May1997.Wcbsitc: vm.cfsan.frla.gov/-.d~ls/i~sre~)ort.htt~~l. 104. ELBaker,ed.Surveillance i n occupationalhealth and safety.Washington.D.C.:American PublicHealthAssociation,19x9. 105. PLoaharanu.Irradiation as a cold pasteurizationprocess o f I'ood. VetParasitol64:71-82. 1996. 106. D Hellcr. ON Gill. E Raynhatn. et al. An outbreak of gastrointestinal illness associated with consumption of raw depurated oysters. Br Met1 J 292:1726-1727. 19x6. 107. pJ Shubat, KA Raatz, RA Olson. Fishconsutnptionadvisories and outreuch programs for southeast Asian immigrants. Toxicol Ind. Health 12:427-434, 1996. lox. pH Rheinstein. KC Klontz.Shellfish borne illnesses. An1 h111 Physician 47: 1837-1840. 1993. 109. M Wittner. JW Turner. G J q u e t t e . LRAsh. MP Salgo, HB Tanowilz.Eustrongylidinsis. 21 p:ur;lsitic infection acquired hy eating sushi. N Engl J Med 320: 1124-1 126. 1989.
10 The Medical Management of Seafood Poisoning
I. 11.
Introduction 31 I Fish-RelatedPoisonings A. B. C. D.
Botulism
312
312
Scombroid poisoning 312 Tetrodotoxin or pufferfish poisoning Ciguatera 313 Ill. Shcllfiah-Related Poisonings 315 A. Vibrios 315 B. Shellfish toxins 316 IV. Conclusion 3 17 References 3 1 X
1.
3 13
INTRODUCTION
Since seafood is now universally accepted a s a nutritious, high-protein, low-fat red meat substitute, it is being increasingly consumed ( 1 ). Activities associated with tourism and travel,aswellasever-increasinginternationaltrade,facilitateconsumerexposuretoa widearray of seafoodproducts,bothfreshandfrozenfromdifferentmarinesources. Withcontemporaryconsumerhabitsinvolvingmoretwo-incomefamilies,greaterfrequency of eating out in restaurants is also seen. Many patrons who might not routinely cook fish at home, enjoy eating it when it is well-prepared at a restaurant. It is therefore
31 1
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312
unexpected when a seemingly healthy and probably expensive dinner results in food poisoning. In this chapter, the medical management of acute seafood poisoning is approached by separatingseafoodintofish-relatedpoisoningandshellfish-relatedpoisoning.Fishrelated poisonings include botulism, scombroid poisoning, fugu poisoning, and ciguatera. Shellfish-related poisonings include Vibrio, and the four distinct shellfish poisonings (paralytic, neurotoxic, diarrheic, amnesic). Fish-related poisoning, secondary to bacterial toxins or parasites, is well known to be associated with inadequate refrigeration and/or improper preparation. Howcver, toxinmediated seafood poisoning can occur in a perfectly prepared and healthy fish. Shellfishrelated poisoning, especially bacterial and viral,is known to be associated with inappropriate harvesting, handling, or storage. Shellfish poisoning can also be caused by extrinsic toxins produced by algal blooms. Diagnosis of seafood poisoning is, by necessity, associated with a history of recent seafood ingestion. Because most seafood poisonings are self-limited, they are usually dismissed and not diagnosed or reported. Sharingof food oftcn leads to small disease clusters ill and the or epidemics of seafood poisonings. However, if more than one person gets cluster is reported to the public health authorities, a more accurate etiology can then be gleaned through epidemiological investigation. Before asking the epidemiological questions, however, the poisoning sufferer mustbe treated. This chapter dealswith the medical treatment of seafood-borne poisonings.
II. FISH-RELATED POISONINGS A.
Botulism
Type E botulism is associated with air-dried, and both hot and cold smoked fish.Clostricli m b o t d i r r w ? r is responsible for type E botulism (2,3). Pressure cooker temperaturcs are highaltitudeswithlowboilingpoints.Unrequired to killthisbacteriaespeciallyat dercooking can result in acute gastroenteritis, bilateral sixth cranial involvement with diplopia and ptosis, progressive descending symmetrical flaccid paralysis, and respiratory arrest. Treatment is ventilatory support, intravenous hydration. gastric lavage, cathartics, and enemas. Fortunately, a trivalent antitoxin E is available and should be used i n all suspected type E botulism victims. All sinlilarly canned and smoked fish should be safely discarded. Survivors usually recover completely.
B. Scombroid Poisoning Scombroid poisoning is a pseudoallergic intoxication also known as "histaminc food poisoning." It resembles an acute allergic reaction and results from the ingestion of spoiled fish from the Scombridae family (including tuna, mackerel, jack, and bonito). These fish have a high content of dark meat that is rich in histidinc. Unlike thc othcr fish-related toxins, scombroid is caused by inadequate refrigeration, allowing bacterial growth which can transform histidine into histamine.Histamineis not affected by hcat or any other method of preparation. Scombroid poisoning is, however, entirely preventable if the fish is itntnediately and properly refrigerated. Signsandsymptomsbeginminutesafteringestion.Theyincludedramaticskin flushing, throbbing headache, oral burning, abdominal cramps, nausea, and diarrhea. Hives
Medical Management of Seafood Poisoning
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and swelling of the tongue are less common. Victims report that the fish tasted peppery or metallic (4).The disease course is self-limited and symptoms abate i n less than a day if left untreated. Antihistamines and H: blockers, shorten the course and ameliorate the symptoms. Angioedema and bronchospasms require epinephrine.
C. Tetrodotoxin or Pufferfish Poisoning Famed as one of the most potent nonprotein poisons i n nature, tetrodotoxin is naturally found i n pufferfish, from which the coveted “fugu” is prepared. Long considered a delicacy i n Japan, fugu is said to produce exhilaration, perioral tingling, a floating sensation, and a feeling of overall warmth (S). It is also considered to be an aphrodisiac. Fugu chefs are specially trained to take particular care in cutting away the more toxic fish parts such as the viscera and gonads. Tetrodotoxinfunctions by blockingvoltage-dependentsodiutnchannels in nerve cell membranes. This blockage causes perioral paresthesia. nausea, and dizziness. The more rapid the onset of the symptoms, the poorer the prognosis (5,6).More severe symptoms include weakness, generalized numbness,loss of coordination, slurrcd speech, bradycardia, hypotension, and respiratory paralysis. Subsequent death results from respiratory arrest. There is no antitoxin at present. Gastrointestinal decontamination is recommended and supportive respiratory therapy is life saving. Survivors reportedly have spontaneous and complete recovery.
D. Ciguatera Ciguatera was first recorded by Caribbean explorers i n IS 1 1 . It is the most colnnion cause of tropical fish poisonings (4,6-8). It is barely recognized in the medical literature, and remains misdiagnosed, inadequately treated, and underreported. Ciguatera is caused by the ingestion of ciguatoxic fish. These fish are contanlinated with ciguatoxins that arc produced by the marine dinoflagellate Gcrrrrbirr.ili.sc,rr.s to.ric~r4.s(8).Ciguatera has been associated with the consumption of reef fish including barracuda, grouper, snapper, jack, tropical mackerel, morays, parrotfish. surgeonfish, hogfish, sea bass, and wrasse.
1. Acute Phase Ciguatera is classically described as an acute illness. Gastrointestinal symptoms typically begin 3-6 hours after the ingestion of the fish (7,9,lO). The gastrointestinal symptoms are overlapped or followed by a myriad of neurological symptoms. Cardiovascular symptoms are less comnlon and rarely severe. Ciguatera is seldom fatal. The gastrointestinal symptoms of ciguatera are typical of any food poisoning: nausea, vomiting, cramps, abdominal pain, and watery diarrhea. These symptoms are generallyself-limited i n aday or two. The neurological symptoms include intense pruritus, paresthesia, and dyesthesia. They usually begin several hours after the ingestion of the toxic fish and overlap the gastrointestinal phase. Occasionally neurological symptoms do not begin until 3 or 4 days later, well after the gastrointestinal symptoms have subsided. Patients report a myriad of highly unusual complaints, including tap water tasting carbonated, bugs crawling on their skin, a sensation of their teeth falling out, cold feeling hot. and air conditioning as intolerable. Chills are a frequent complaint. but actual temperature elevation is not seen. Urination can burn and sexual intercourse can be painful. Arthralgias, weakness, and easy fatigability are cotnlnon complaints which can last for years. Alcohol
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ingestion is known to aggravate all of these symptoms. Paralysis, especially facial nerve palsies and coma, have been reported ( 7 3 ) . Cardiovascular events include bradycardia and hypovolemic hypotension. Death is rare and usually attributed to hypovolemic shock. Individual variation in symptom response is the rule even when all affected eat the same source of contaminated fish. The diagnosis of ciguatera remains clinical. There are no currently available clinical diagnostic tests for ciguatera in humans.
2. Chronic Phase I n chronic ciguatera, the gastrointestinal and cardiovascular events have subsided, but the neurological symptoms persist. These neurological symptoms are usually diminished in intensity, but can be exacerbated, with patients describing "good days and bad days." Patients can usually identify triggering events such as the ingestion of alcohol, nuts, or fish (1 1 - 15). Physical exercise is exhausting, and libido and joie devivre are diminished. Chronic fatigue, arthralgias, and myalgias are particularly frustrating. Despair can be overwhelming. Reassurance that almost all ciguatera is eventually self-limited is a most welcome prognosis.
3. Treatment The treatment of ciguatera is prilnarily supportive therapy. Severe volume depletion and hypotensive shock require urgent treatment with an intravenous normal saline or lactated ringers solution. Bradycardia is usually mild, with normal sinus rhythm at 50-60 beats/ min, andusually does notrequiretreatmentwithatropine.Gastricdecontamination is probably futile, as symptoms typically begin several hours after the ingested meal. Antiemetics and antidiarrheics should be avoided. Neither antibiotics nor corticosteroids have been shown to help (6,7,12,16). Antioxidants and B-complex vitamins seem harmless,but are of questionable value. Antihistamines and H z blockers may decrease pruritus. Nonsteroidal anti-inflammatory drugs help chronic arthralgias, myalgias, and dental pain. Calcium channel blockers are used for palpitations caused by subsequent tachycardias, which are self-limited. Intravenous (IV) mannitol therapy has been shown to bring a dramatic improvement if administered early (1 l): results have been less impressive if given more than 48-72 hours after ingestion of the toxic fish (1 2- 14,16). In hyperosmolar concentrations, decreased cerebral edema and peripheral nerve edema occur. Clinical reports from controlled studies indicate that a 20% mannitol IV infusion over 30-120 minutes at a dose of 1 g/ kg body weight is safe and well tolerated in the hydrated patient, and seemingly effective if given early enough (8). Less dramatic, symptomatic improvement has been reported when IV mannitol therapy has been administered as late as 1-7 weeks following the onset of ciguatera in uncontrolled studies (1214). The delay in therapy is the result of the typical delay in the diagnosis of ciguatera. Chronicciguatera, in general, does not respond to IV mannitol therapy ( 12.13). Chronic treatment is symptomatic. Antihistamines, H2 blockers, analgesics, and nonsteroidal anti-inflammatory drugs may help to relieve the paresthesia, dysesthesia, and arthralgia ( 1 l,l2). Amitryptiline and fluoxetine have been reported to be beneficial for the chronic fatigue, insomnia, and clinical depression ( 1 7,18). There is no antitoxin for ciguatera, and local folklore and native homeopathic remedies have questionable efficacy. Most ciguatera victims recover spontaneously within a few weeks (19).
Medical Management of Seafood Poisoning
111.
SHELLFISH-RELATED POISONINGS
A.
Vibrios
315
1. Exposure Infectionbythepathogenic Vibrio species generally occurs following theingestion of contaminated water and food, especially raw or insufficiently cooked shellfish. Vibrios are natural inhabitants of tidal rivers and bays that have moderate salinity. Marine shellfish and plankton are the main reservoirs for Vibrio species. Of these Vibrio species. V. cholerne, V. / ) ~ ~ ~ C I I I C I ~ I and ~ I ( ~V.I ~wltriji(~ans ~ ~ ~ ~ ~ ~ . ~have , been implicated most frequently in seafood poisonings (20-28). V. vulrrjjicwrrs can result in septicemia with a high mortality rate. The othcr Vibrio species are associated with gastroenteritis of varying severity (211.
2.
Clinical Symptoms
Vibrio cholor~tr The Vibrio species responsible for “epidemic” cholera are V. cholertr serogroup 01 biotype E l Tor strain and the recently identified V. cholern serogroup 01 39 biotype Bengal. No major outbreaks of this disease have occurred i n the United States since 191 1. Casesreported in theUnitedStates have beenassociatedwithtravelersfromSouth America during the most recent pandemic, or from illegally smuggled crustaceans that were held at improper temperatures (21-24,291. Epidemiologic studies have shown that unhygienic food handling is the most commonsource of seafood-bornecholera CI.
(20,24,25,29,30). Acute diarrheal illness occurring 24-48 hours after the ingestion of contaminated shellfish or fish is conmon to both subgroups. Diarrhea. abdominal pain or cramps, and nausea are generally reported, with fever, vomiting, and headache in about SO% of cases. I n severe cases, stool volume can exceed 250 n N k g in the first 24 hours (31). Coniplications from the effectsof fecal volume and electrolyte depletion include hypovolemic shock and acute renal failure. With early rehydration these complications can be avoided and the illness resolved in a few days. However, untreated, cholera can result in death in a matter of hours. V. cholarrr serogroup non-01 is primarily caused by the consumption of raw, improperly cooked or stored shellfish. Diarrhea is usually less severe than in epidemic cholera. Abdominal cramps, nausea, vomiting, and fever are conmon symptoms whichusually persist for about a week (29,3 1,32). b. Vibrio ~~t~rc~htrerllol~tit.us V. ~ ~ ~ ~ r r r l r c r ~infections ~ r ~ ~ o lare ~ ftypically i ~ ~ ~ ~ linked .~ to the ingestion of undercooked or improperly handled seafood. In Japan, up to SO% of foodborne disease outbreaks have been associated with this Vibrio species (27). Generally, symptoms include watery diarrhea, accompanied by abdominal cramps, nausea, and vomiting. Less colmnon are fever, chills, and dysentery, along with bloody, mucoid stools. The incubation period for this illness is between 4 hours and 4 days, with symptoms present for approximately 3 days.
Vibrio ~ ~ u l r r ~ f i c ~ t r r l s V. w h $ u r r r . s is thc most important cause of severe Vibrio infections in the United States, but is rare outside the Gulf of Mexico coastal states. Typically patients with V. c‘.
316
Blythe et al.
~ w l n i f i c ~ infections ~.s are men over the age of 40 who are immunocon~proll~ised or have cirrhosis, hemochromatosis, or diabetes mellitus. Most patientshave ingested raw oysters from coastal states between May and September within 2 days of onset of symptoms. approaches SO%, with most deaths due touncontrolled Mortality i n theUnitedStates sepsis ( 2 6 , 2 8 3 ) . In a reported case series from Japan, mortality was 68%, and the authors noted that the mortality rate appeared to increase with greater delay between the onset of illness and the initiation of the antibiotic treatment (34). Vibrio can be confirmed by the identification of the causative organism from the diarrheic stool of infected individuals. Laboratory isolation of the organism to differentiate species of Vibrio is possible.
3. Treatment Rapid and adequate replacementof fluids and electrolytes is necessary for allVibrio infections. A fluid, administered orally, containing an Na*content of 90 mmol/L, 20 mmol/L of K*, 80 mmol/L Cl-, 10 mmol/L citrate, and 110 mmol/L glucose has been recommended by the World Health Organization (3 1). This treatment is safe for infants if it is alternated with the consumption of sodium-free fluids such as breastmilk or water. If patients are severely dehydrated, intravenous fluid replacement is required. The use of antibiotics for the treatment of cholera is controversial. It can have the effect of shortening the duration and volume of fluid loss, and clearing the organism from the stool. Multidrug resistance of cholera has been noted in recent studies (30,3 1 3 ) . Tetracycline, ciprofloxacin, chloramphenicol, and erthromycin have been used. Tetracyclineisconsideredthetreatment of choicefor V . \wlniJicu.s infections (30,31,34) in combination with gentamicin. I n addition to antibiotics, surgical debridement and management of shock, coagulopathies, and renal and respiratory failure are critical. Most casesof V. I"/r"/zcrfnrol~ticu.sare self-limited, and require neither antimicrobial treatment nor hospital care. Severe cases should be treated with fluid replacement and antibiotics.
B. Shellfish Toxins There are four distinct shellfish poisoning syndromes caused by the marine toxins: paralytic (PSP), neurotoxic (NSP), diarrheic (DSP), and amnesic (ASP) shellfish poisoning. The toxins responsible for shellfish poisonings are produced by microscopic algae known as diatoms and dinoflagellates (9). Shellfish are filter feeders and they accumulate many local toxins, someof which are heatand acid stable. Toxins implicated in disease outbreaks are often associated with harmful algal blooms, such as red tides (6).
1. Paralytic Shellfish Poisoning Saxitoxin is the major toxin responsible for PSP. It is produced by the dinoflagellates Gorryrrrlas ctrtclltr and G. ttrmtrr.etrsi.s.Saxitoxin manifests toxicity by interfering with the voltage-dependent sodium channels i n nerve cell membranes. This mechanism is similar to that of tetrodotoxin, and quite possibly explains the similar clinical presentations. Of all the shellfish poisonings, PSP and ASP produce the most severe illness. Patients experience paresthesia of the mouth, face, and limbs, frequently associated with nausea, vomiting, and diarrhea. Most deaths occur within the first few hours after ingestion and are attributable to respiratory paralysis. Prompt respiratory therapy is imperative for survival because there isno antidote presently available. Mortality from this disease ranges
Medical Management of Seafood Poisoning
31 7
from 1% to 12% (36-38). PSP usually lasts about 3 days; those surviving past 24 hours have a reportedly excellent prognosis.
2. Amnesic Shellfish Poisoning ASP is caused by domoic acid (DA) which is produced by the diatom Nif:.schitr p r t g e r r s . Domoic acid acts as a potent excitatory neurotransmitter which affects the motor axons. Like the other shellfish poisonings, ASP is associated with gastroenteritis. A unique toxic encephalopathy caused by eating mussels contanlinated with dolnoic acid was reported in Canada. Cognitive dysfunction, loss of short-term memory, confusion, disorientation, seizures, and coma presented in an outbreak of ASP hours after eating mussels harvested off Prince Edward Island (36). For some individuals, the cognitive dysfunction and memory loss were permanent, and deaths were also reported. Treatment is symptomatic.
3. Neurotoxic Shellfish Poisoning The culpable agent i n NSP, brevetoxin. is produced by the dinoflagellate Ptyhotliscw brevis, responsible for the Florida red tides. Brevetoxins open the sodium channels on nerve membranes, which inducesan influx of sodium. NSP presentswith acute gastrointestinalandneurologicalsymptoms.Vomiting and diarrheaoccursil~lultancneously with paresthesia, temperature reversal, vertigo, pupil dilation, throat tightness, and a choking sensation. Symptoms begin about 3 hours after ingestion of toxicshcllfish and lastno more than a few days (6).NSP is milder than PSP and is reportedly self-limited. Treatment is symptomatic.
4. Diarrheic Shellfish Poisoning DSP is caused by okadaic acid (OA), whichis claborated by the dinoflagellates Diwplrysis and Pr.oroccvtrrwu. Okadaic acid induces diarrhea by stimulating the phosphorylation that controls sodium secretion i n the gut (6). This poisoning is common in Japan. An ephemeral illness, gastroenteritis occurs 12 hours after ingestion and persists for about 24 hours. There have bccn no neurological manifestations or deaths reported. Antiemetics and antidiarrheics should be avoidcd. DSP is reportedly self-limited with the elimination of the toxin.
W. CONCLUSION From the medical and public health point of view, the best treatment for seafood poisoning is the primary prevention of illness. Therefore, primary prevention should focus on the harvesting, transport. storagc. and preparation of seafood. Nevertheless, even with proactive measures, seafood poisoning cannot be completely prevented, especially for sensitive subpopulations consuming raw or inadequately cooked seafood. Botulism from undercooked canned or smokedfish, is usually thc result of inexperience. Fortunately, antibotulism toxin E is life saving, provided that emergency medical care is promptly available. Fugu poisoning can turn a thrill-seeking adventure meal into a self-destructive and hardly glamorous near-death experience for those risk takers fortunate enough to survive it. The risk of poisoning from tetrodotoxin is closely related to precautions taken in the preparation of the puffertish. Scombroid poisoning requires surface bacterial growth and is prevented by prompt to only a few days. and continuous refrigeration. Even refrigerator shelf life is limited
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Blythe et al.
Fish should be frozen early if consumption cannot be ensured within 2 days. Scombroid poisoning is easily preventable. Ciguatera is not so easily prevented. Highly prized, usually healthy and delicious coral reef fish can occasionally be toxic. The large predator fish, barracuda, is typically the most toxic and should be avoided. The sale of barracuda is, in fact, banned i n Miami, Florida. Barracuda is still considered a delicacy in most of the Caribbean, especially by the natives. Unfortunately, there is no reliable field test currently available to analyze fish for ciguatoxins before they are sold to the consun1cr. There are also no clinical diagnostic tests for ciguatera. Leftover fish canbe tested for toxicity, however, there is rarely sufficient fish remaining from the meal for toxin analysis. The true incidence of ciguatera is unknown and only speculative. An antitoxin is not available, but if correct diagnosis can be made quickly, IV nwlnitol treatnlent is helpful. Shellfishharvestingiscloselymonitoredtoavoidacute Vibrio gastroenteritis. V. 1v~h(ficm.s canalso cause a toxic septicemia with gangrene and death, especially when in these highassociated with liver disease. Raw shellfish should be avoided altogether risk consutners, and not just in the “month’s without R’s.” Fortunately the four shellfish toxicities-PSP, NSP. DSP, and ASP-are less common. Their descriptivenames-paralytic, neurotoxic, diarrheic, and amnesic-are nlenacing indicators of their potent toxins. Unfortunately no antitoxins are available.
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2.
3. 4.
S. 6.
RL Dcgner. CM Adams. SC Moss. SK Mack. Per capita fish and shellfish consumption in Florida. Industry Report 94-2. Gainesvillc: Florida Agricultural Research Center, 1994. H Badhey, DJ Cleri, RF D’Amato, et al. Two fatal cases of type E adult food borne hotuolism with early symptoms and terminal neurological sign. J Clin Microhiol 23:616-61 X. 1986. TL Bott. JS Deffncr. EMcCoy,EM Foster. C/o.stridiIm b o t c t l i u m r type E i n fish from the Great Lakes. J Bacteriol 91:919-924, 1966. LE Fleming. DC Blythe, DC Baden. Ciguatera fish poisoning. Shoreland‘sTravel Med Month l(6):1-4. 1997. AR Mills, R Passmore, R Pelagic. Paralysis. Lancet 1:161-164. 1988. DC Baden. LE Flcnling, JB Bean. Marine toxins. Handbook Clin NeuroI 2 l(65):14 1 - 175. 1995.
7. LR Juranovic, DL Park. Foodhornc toxins o f marinc origin: ciguatera. Rev Environ Contam Toxicol I 17:s 1-94. 199I. X. R Bngnis. Ciguatern fish poisoning. In: ID Falconer. cd. Algal toxins in seafood and drinking water. New York: Academic Press, 1993, 105- I 15. 9. C Frennette, JD Maclean. TW Gyorkos. A large coInmon-source outhreak of ciguatera fish poisonings. J Infect Dis 158:I 12X- 1 13 1, 19XX. IO. DNLawrence.MBEnriqucz,RMLumish,AMaceo.CiguaterafishpoisoninginMiami. JAMA 244:254-258. 19x0. 11. NA Palafox, LC Jain. AZ Pinano, TM Gulick, RK Williams. IJ Schatz. Successful treatmcllt o f ciguatcra fish poisoning with intravenous mannitol. JAMA 259:2740-7742, 1988. 12. DG Blythc, LE Fleming. RA Ayaar. DP DeSylvn, DC Baden.K Shrank. Mannitol therapy for acute and chronic ciguatera fish poisonings. Me111 Quecllsl MUS34:465-470. 1994. 13. DG Blythe. DP DcSylva. S Cramcr-Castro. Ciguatcra: fish poisoning-the name may he diffcult t o rclnemhcr. hut if you ever get this disease. you’ll ucvcr forget it. Mixmi Med 31 -32. 1992.
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14. DG Blythe, DP DcSylva. LE Fleming, RA Ayaar. DG Baden. K Shrank. Clinical experiences with IV mannitol in the treatment of ciguatera. Bull Soc Pathol Exot 85:425-426, 1992. 15. WR Lange. FR Snyder, PJ Fudala. Travel and ciguatera fish poisoning. Arch InternMed 1 52: 2049-2053,1992. Man 16. JH Pearn. Ciguatera: dilctntnas In clinical recognition, presentation and management. Queens1Mus 34:601-604, 1994. 17. PB Bowman. Amitriptyline and ciguatera. Med J Aust 140:802, 1984. 18. RM Berlin. SL King, DG Blythc. Sytnptotnatic itnprovcmentof chronic fatigue with fluoxetine in ciguatera tish poisoning. Med J Aust 157:567, 1992. McdClinN Am 19. D Mines. S Stanner. SM Sheperd. Poisonings: food. fish, shellfish. Etnerg 15:157-177,1997. 20. SL Lee, ST Lai, JY Lai, NK Leung. Resurgcnce of cholera in Hong Kong. Epidetniol Infect 117:43-49,1996. 21. GR Matte, MH Matte. MIZ Satto. PS Sanchez, IG Rivera, MT Martins. Potentially pathogenic vibrios associated with mussels from a tropical region on the Atlantic coast of Brazil. J Appl Bacteriol 77:28 1-287. 1994. 22. S Rippey. Infectious diseases associated with molluscan shellfish consumption. Clin Microbiol Rev7:419-425. 1994. 23. J Eberhart-Phillips, RE Besser, MI Tormcy. et al. An outbreak of cholcra from food served on an internationalaircraft.EpidetniolInfect 116:9-13, 1996. 24. MJ Albert, M Neira, Y Motarjemi. The role of food in the epidemiology of cholera. World Health Stat Q 50:l 1 1 - 1 18. 1997. 25. M Landgraf. KBPLeme. ML Garcia-Morcno: Occurrence of emerging pathogenic Vihr.io spp. in seafood consumed in Sao Paulo City. Brazil. Rev Microbiol 27: 126-130. 1996. 26. RC Noble. Death on the half shell: the health hazards o C eating shellfish. Perspcct Biol Med 33313-323, 1990. infections 27. RL Haddock. AF Cabanero. The origin of non-outbreak Vihr-io/Jn~trhnc~rrro!\'tic.lrs in Guam. Trop Geogr Med 46:42-43, 1994. Vihrio , ~ r / r ~ j f i c ~from ~ t s rawoysters. J F121 McdAssoc 28. WGHlady.RCMullen.RSHopkins. 80:536-538, 1993.
29. Food and Drug Administration. May1997.Websitc: vm.cfsan.fria.gov/--dtl~s/fsreport.htn~l. 30. JT Weber, ED Mintz. R Canizarcs, et al. Epidemic cholera in Ecuador: multidrug resistance and transmission by water and seafood. Epidetniol Infect 1 12: 1 - 1 I , 1994. 31. AS Fauci, et al.. cds. Harrison's principles o f intcrnal medicine, 14th cd. New York: McGraw Hill. 1998. 32. PH Rhcinstein.KC Klontz. Shellfish borne illnesses.Am Fam Physician 47: 1837-1840, 1993. 33. WG Hlady, KC Klontz. Thc epidemiology of Vihrio infections in Florida, 1981-1993. J Infect Dis173:1176-1183.1996. 34. K Kikawa. K Yamasaki, T Sujiura, H Myose, M Chinen, K Tsursumi, N Iwao, T Dohsozo. A success~ullytreated case of Vihr-io ~ w / r t ( f i t i c . c septicemia ts with shock. Jpn J Med 29313319. 1990. 35. P Maggi. A Carbonam, C Fico, T Santantonio. C Romatlelli, E Sforza, G Pastore. Epidemiological, clinical and therapeutic evaluation of the Italian cholera epidemic in 1994. Eur J Epidcmiol13:95-97.1996. 36. BDGessner, JP Middaugh. Paralytic shellfish poisoning in Alaska: a 20-year retrospective analysis. Ani JEpidetniol141:766.1995. 37. K Hartigan-Go. DN Bateman. Rcdtide in thc Phillipines. Hum Exp Toxicol 13:824, 1994. 38. TM Pcrl, L Bcdard. T Kosatsky, JC Hockin, ED Todd, R Remis. Encephalopathy caused by 1990. contaminated mussels. N Engl Mcd J 322: 1775-1780,
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11 The U.S. National Shellfish Sanitation Program
Introduction 321 A. Interstate shellfish sanitation conference 322 B. Dcvclopment of the model ordinance 323 11. RegulatoryAuthorityfortheNSSP 323 111. Foreign Inspection Procedures 324 A. The Canadian shellfish sanitation program 325 IV. Seafood Safety 326 A. Bacterial contatninants 326 Viral cot1t;uninmts B. 327 V. NSSPGuidefortheControlofMolluscanShellfish327 A. Model ordinance 327 B. Marine biotoxins 330 C. Canadianinterpretationof NSSP growingwaterstandards 33 1 D. Marinas 332 E. Shellstock relaying 332 F. Aquaculture 332 G. Shellfish harvesting and handling practices 332 H.Hazardanalysis and criticalcontrol p a n t (HACCP) 333 Depuration I.335 VI. Conclusions 336 References 337 I.
1.
INTRODUCTION
The need for public health controls for the consumption of shellfish in the United States became a national concern duringthe late 19th and early 20th century when a large number 321
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of illnesses were linked to the consumption of raw oysters, clams, and mussels. I n the winter of 1924, a widespread outbreak of typhoid fever in several major U.S. cities was tracedtothe consumption of sewage-polluted oysters. In response tothisconccrn.the Surgeon General held a conference i n 1925 with local, state, and federal health officials. The outcome of this conference was the publication of eight resolutions capturcd i n a 1925 supplement to Public Hacrlth R q m ? ~titled “Report of the Committee on Sanitary Control of the Shellfish Industry i n the United States“ (17). These resolutions related to the need for sanitary shellfish growing areas, inspection of shellfish processing facilities, a mechanism to ensure thatallshellfish were harvested from a sanitary source, proper procedures to ship shellfish, and the establishment of an acceptable microbiological standard for all shellfish ( I ) . As a result of this conference, a conmittee was established to further dcvclop guidelines to control and improve the sanitary practices of the shellfish industry. Shellfish producing states agreed to issue certificates to approved shellfish proccssors. and the Public Health Service (now the FDA) accepted responsibility for acting as the conduit for inforIllation regarding the effectiveness of the state control programs. As part of this responsibility, the Public Health Service published a list of all shellfish shippers that were certified by those states that maintained “satisfactory” control program. The principles developed i n the 1925 conference formed the basis for the current National Shellfish Sanitation Program (NSSP). Although the basic public health principles of the NSSP have remained unchanged over the years, program procedures have been updated and inlproved at regular intervals. I n the 1940s. the NSSP was updated to reflect growing concern with paralytic shellfish poison (PSP). In 1957, proccdures wcre revised to include public health controls for radionuclide pollutant. In the late 1 C)SOs, the original 1925 “Report of Conunittee on Sanitary Control of the Shellfish Industry i n the United States” was divided into two parts. Part 11, “Sanitation of Harvesting and Processing of Shellfish,” was issued in 1957 and Part I. “Sanitation of Shcllfish Growing Areas.” was published i n 1959. In the 1960s and 1970s, the programwasagainrevised toaddress public health concerns associated with heavy metals and pesticides (2). The NSSP manual of operations is divided into two parts to reflect the two major strategies to control the sanitation of bivalve mollusks. Each of these parts is further divided into a number of chapters; each chapter deals with a particular subject and is organized similarly. An introduction to the topic is provided first, and actual requirements are listed in bold face type. A detailed explanation along with a relevant discussion of the literature on the subject is provided in a section titled “Public Health Explanation.”
A.
Interstate Shellfish Sanitation Conference
In 1983. state and fcderal officials. the academic community, and members of the shellfish industry joined together to form the Interstate Shellfish Sanitation conference (ISSC) (2). The ISSC consists of agencies from shellfish producing and receiving states, the FDA, theNationalMarineFisheriesService of the U S . Departmen1 of Commerce, andthe shellfish industry. The purposes of forming the lSSC were threefold. First, it provided a structure for regulatory authorities to meet on a regular basis to discuss ways to improve shellfish sanitation and safety. Second, it provided a formal structure for participants to provide input to the NSSP’s manual of operations. Third. it was intended to address concerns that some state shellfish control agencies were not adopting rcvisions and enforcing NSSP guidelines in a uniform and timely lnanner.
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B. Development of the Model Ordinance Withitlthe ISSC are three taskforces:growingareas,processinganddistribution,and administration. Committees are often appointed to assist task forces in developing recommendations. Delegates from each state shellfish control agency vote on recomtl~endations submitted by the task forces (2). A ma,jor goal of the ISSC has been the development of a “model ordinance” to rewrite the compliance rcquirements of the NSSP manual of operations parts I and I1 into reglllatory language that can be easily adopted by individual states. A committee of the ISSC began development of the tnodel ordinance in 1987. I n 1992, the initial draft ordinance was presentedanti adopted by the ISSC. I n 1994, the FDA and the ISSC entered into a memorandum of understanding (MOU) in which the FDA agreed to provide technical assistance to the ISSC, and the ISSC agreed to assist the FDA by further development of the Shellfish Sanitation Model Ordinnnce (2). I n 1997, the FDA was asked by the ISSC to adopt the model ordinance. The annual meeting of the ISSC, at earlier meetings, and again at the July 1997 meeting provided an essential forum for the tlevclopment of revisions to the NSSP. The NSSP model ordinance should strengthen the credibilityof the NSSP and the Interstate Certified Shellfish Shippers List (ICSSL). The ICSSL identifies shellfish dealers certitied by the state of residence as being i n compliance with NSSP guidelines (3). Members of the ISSC are expected to implement these guidelines to ensure that harvested shellfish will be acceptable to the other tnelnbers of the ISSC. The ISSC, withthe concurrence of the FDA, established Jan~lary1. 1998 as the implementation date for the model ordinance. DiStefano (4) describes the success of the NSSP as largely dependent on the states adopting nnd implementing the reconmended shellfish control practices for the operation of effective programs. These recommended practices, which traditionally have been incorporatcd into the NSSP manual of operations, have been reconstituted in the form of an “NSSP Guide for the Control of Molluscan Shelllish” (4). The purpose of this handbook is twofold. First. it serves to redraft existing guidelines contained in the NSSP manuals of Operation into an NSSP model ordinance. The language was chosen intentionally so that it could be readily adopted as law or regulation by a state. Second, the handbook sets forth supportive documentation pertinent to the language of the model ordinance. The documentation includes the NSSP’s history, public health reasons and explanations specific to the guidelines contained i n the model ordinance, NSSP guidance documents, suggested NSSP forms, shellfish policy-setting documents, pertinent federal regulations, ancl references.
II. REGULATORY AUTHORITY FOR THE NSSP The FDA is responsible for public protection and seafood regulation i n the United States ( S ) . DiStcfano (3) writes that the agency enforces the federal Food, Drug, and Cosmetic Actandcertainportions of the Public Health Service Act. These laws require that the foods shippedin interstate commercebe prepared, packed, and stored under sanitary conditions, that labeling be accurate and informative, and that the food itself be safe, clean, and sanitary (S).The agency meets this obligation for seafood safety by reviewing state compliance programs for fishery products. The FDA also conducts inspections for donlestic seafood harvesters. growers. wholesalers, warehouses, carriers, and processors. These
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in-plant inspections consider product safety, plant/food hygiene, and economic fraud issues. In addition,theFDAalsohasfunding to conduct analyses for defects including chemicalcontaminants,decomposition, net weight,radionuclides,variousmicrobial pathogens. food and color additives, drugs, pesticides, filth,andInarinetoxins such as PSP and dolnoic acid (3). The FDA is authorized to accept assistance from state and local authorities in the enforcementof laws to prevent and to suppress the spread of communicable disease. This latter authority makesit possible to use the NSSP as a specific regulatory program directed toward the sanitary harvest and production of fresh and frozen bivalve shellfish. The NSSP reflects a tripartite cooperative arrangement between the FDA and voluntary participation by state regulatory agencies and the shellfish industry. The safety of raw molluscan shellfish for human consumption begins with ensuring the quality of the water in which these sedentary organisms are grown and harvested. Because the growing waters are mostly state resources, the NSSP is based on the premise that public health controls forraw molluscan shellfish can best be accomplished under state laws with federal technical support and industry participation (3). The three partners work together to develop and implement a tripartite cooperative program, each with individual responsibilities. First, each participating state adopts adequate laws and regulations for sanitary control of the molluscan shellfish industry, classification of shellfish growing areas, control of harvesting from restricted areas, and certification of shellfish plants. Italso conducts laboratory investigations and implements control measures to ensure lnolluscan shellfish are harvested and processed under sanitary conditions. Second, the FDA conducts an annual review of each state shellfish control program to determine the degree of conformity with the NSSP. Third, the shellfish industry cooperates by obtaining shellfish from safe sources, provides plants that m e t sanitary standards, maintains sanitary operating conditions, tags or labels each shellfish package withthe proper certificate number, and keeps recordsof the origin and disposition of the shellfish.
111.
FOREIGN INSPECTION PROCEDURES
The Sanitary and Phytosanitary (SPS) Agreement under Article 4 of the World Trade Organization (WTO) Agreement on the Application of Sanitary and Phytosanitary Measures states that each member nation of the WTO, including the United States, must accept as equivalent, another country’s food regulatory system as long as it provides the same level of health protection as is provided to consulners by its own system ( 6 ) . Equivalent regulatory systems need not be identical, however,the burden of demonstrating that equivalence exists rests with the exporting country. The importing country has the right to decide for itself whether the regulatory system of the exporting country is equivalent to its own or is inadequate to achievea desired level of protection. The FDA has experience developing and entering into bilateral agreements with trading partners for the purpose of providing assurance that food from those countries will be safe for U.S. conswners (6). In addition to controlling interstate commerce, the FDA uses the NSSP as its mechanism to “certify” foreign shellfish sanitation programs. The FDA establishes international MOUs with official agencies i n those foreign countries that wish to export shellfish to the United States. The foreign government must demonstrate to the FDA that they have laws or regulations equivalent to those published in the manual, and that they to are supported by trained personnel, laboratory facilities, and other resources required
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ensure control over their export shellfish industry. Once the two countries agree to the MOU, the foreign shellfish control authority submits a list of their certifiedshellfish dealers to the FDA. The FDA includes the names of certified foreign shellfish shippers in the ICSSL as evidence of an approved source of shellfish ( 3 ) . The ICSSL covers 23 coastal shellfish-producing states and nine foreign countries including Australia, Chile, England, Iceland, Japan, Republic of Korea, Mexico, New Zealand, and Canada (7). Some countries have expressed an interest i n renegotiating their agreement as an equivalence agreement rather than a compliance agreement. Where differences exist betweenanexporting country’sprogramanddetails in the NSSP manual of operations, the FDA judges the significance of the differences. For example, a country seeking a detern~inationof equivalence with the United States for raw molluscan shellfish needs to demonstrate that its program meets the purposes and objectives of the manual of operations. The whole system must be able to provide assurances beyond those that would be provided solely through end-product testing.
A.
The Canadian Shellfish Sanitation Program
An example of an equivalent regulatory system is that established in Canada. A MOU exists between Canada and the FDA to allow the export of Canadian bivalve products into the United States. The Canadian program, knownas the Canadian Shellfish Sanitation Program (CSSP), began in response to the1925 U.S. outbreak of typhoidfeverfrom contaminated oysters. In 1925, Canada passed regulations under the Fish Inspection Act to require that oysters be a “safe food product.” Canada’s and the United States’ mutual interest i n protecting the public from the consumption of Contaminated bivalves led i n 1948 to thesigning of a formal bilateral agreement. This MOU outlined each party‘s commitment that the harvesting and handling practices used and the administrative procedures followed will ensure adequate sanitary practices in the shellfish industries of the two countries (8). Terms of the MOU require that a mutually acceptable manual be developed to describe the sanitary principles usedto control the certification of shellfish shippers,shellfish handling facilities, and growing water areas. Further, the MOU stipulates that both countries must notify each other of the extent to which their processors meet these sanitary principles. Canada’s response to this requirement was to produce a manual of operations called the “Canadian Shellfish Sanitation Program Manual of Operations.” This document sets out the policies and procedures to be employed when applying the Canadian acts and regulations. Three Canadian federal agencies, the Canadian Food Inspection Agency (CFIA), the Department of the Environment (DOE), and the Department of Fisheries and Oceans (DFO), share administration of this program. The CFIA is responsible for regulating the import and export, processing, packaging, labeling, shipping, certification, storage, and repacking of shellfish. It will suspend operations or decertify shellfish processors when necessary, regulate the depuration (i.e., controlled purification) of shellstock, verify product quality and purification effectiveness, evaluate laboratories performing shellfish analyses, and maintain a biotoxin program for shellfish growing areas. The DOE is responsible for classifying shellfish growing areas accordingto their suitability for shellfish harvesting on the basis of sanitary quality and public health safety. The DFO prevents shellfish harvesting from areas that are classified as closed. Fishery officers patrol the growing areas;
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regulate and supervise relaying. transplanting, and replanting: and restrict harvesting of shellfish trot11 actual and potentially aft‘ectecl growing areas i n a public health emergency.
IV. SEAFOODSAFETY Both the FDA and ISSC issued similar policy statements i n 1993 regarding the consumption of raw molluscan shellfish. The policies recognize the importance of molluscan shellfish in the diet of some consumers and that molluscan shellfish are often consutned raw or partially cookcd. There is a recognized risk of illness when consuming raw shellfish. and the majority of these illnesses are the result of consuming polluted shellfish. Although common illnesses range frommild intestinal disorders to acute gastroenteritis, the risks are much greater for certain medically compromised individuals. For medically compronlised only exposure to sewage-contaminatecl bivalves, but also individuals, risks include not exposure to shellfish infected with common marine vibrio species that are unrelated to pollution. Environmental controls are not effective i n dealing with vibrio-type contamination. The policies point to the NSSP as the FDA’s way of reducing the risk of illness, t o keep risks to a tninimum (9). and states that adhere to NSSP controls help
A.
Bacterial Contaminants
The safety of raw molluscan shellfish is affected by bacterial contaminants present i n the marine environment. Because testing shellfish growing waters or tissue specific for pathogens is very expensive and technically difficult, indicator organistns arc tested for rather than actual pathogens. The indicator organisms currently used by the NSSP to determine t o the coliform group of thesanitaryquality of water and molluscanshellfishbelong bacteria. This groupis considered a good indicator of sewage pollution becauseit contains bacteria found primarily in the intestinal tract of warm-blooded xnirnals. Fecal colitorms and the principal member of this group, E.schc,ricY~irrc d i . are directly associated with the feces of warm-blooded animals and meet thc requirements of a good indicator organism. The presence ofcoliformsi n sanlples is easy to determine andthey are consistently present in large numbers in sewage. Fecal coliforms arc not normally present i n seawater. but are able to survive. They are unable to multiply i n seawater ( I O ) . Some indicators and pathogens are capable of persisting i n terrestrial soil, fresh and marinewaters,andaquaticsediment f’or manydays,whileothers arceven capable of growthexternal t o ahost.Asmallnutnher of shellfish-borneillnesseshave also been associated with bacteria of the genus Vibrio. The vibrios are free-living aquatic microorganisms, generally inhabiting nlarine and estuarine waters. An article written by the Houston Medical School ( I 1) cites 1 1 species of Vihrio thitt have been identilied as pathogenic Vibrio to humans and have the potential to cause extremc illness and sotnetimes death. species are transmitted to humans via infected water or through fecal transmission and have also been detected i n some environnlental samples recoveredIron1 areas free of overt sewage contamination and coliforms. Among the unarine vibrios classified as pathogenic are strains of non-01 Vibrio cholcrtrr, V. p t r , . n / ~ a c ~ , ~ r o I ~ r iand c ~ r ~V.~ , 1 ~ 1 1 1 j f i c 1 1 .All s . three species have been recovered from coastal waters i n the United States and other parts of the world. Both V. chol~rrrand V. I-’trr.c/hrrc.,rlo!\.fic.rl.s can cause severe diarrhea, cramps, nausea, and fever. The greatest concern for shellfish sanitation is V. \ ~ l r f $ f ~ / r . s , which can cause rapid and devastating infection i n humans. Unlike other Vihrio, this species is inva-
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sive and is able to enter the bloodstream through the epithelium of the gut. Fever. vomiting. and chills are the symptoms normally associatcd with infection of this organism. In addition, wound infection may also occur from contaminated seawater. These bacteria cannot be dctected with the fecal coliform test, and therefore present a serious problem.
B. Viral Contaminants There are more than 110 differcut viruses present i n human feces, collectively called the “enteric viruses." Hepatitis A hasbeenidentifiedasasource of seafoodillness,and Norwalk \irus is now considered a major causeof nonbacterial gastroenteritis. From 1976 to 1980, the CDC reported that Norwalk virus caused42% of the outbreaks of nonbacterial gastroenteritis ( I O ) . Viruses can remain viable for long periods of time i n seawater and havebeen shown to survive as long a s 17 months i n marine sediment. It is generally accepted that coliforms do not accurately indicate the presence or absence of viruses. Generally bacteria do not liveas long asviruses i n the marineenvironment.Therefore it ispossibleforenteric virusestobepresent i n water thatisfree of bacteria ( I O ) . Marinesedimentactsasa reservoir for viruses and may be resuspended by any kind of turbulence, such as boating, storms, or clredging. Rainstorms can also increase virnl concentrations i n the water by incrcasing land runoff and by overflow of sewage from treatment plants. Viruses become concentrated i n bivalves at levels higher than the surrounding water; although they do not multiply in bivalves, thcy do accunlulate in the liverlike digestive gland.
V.
NSSP GUIDE FOR THE CONTROL OF MOLLUSCAN SHELLFISH
To understand the administration and application of the NSSP. it is important t o bccome familiar with the components o f the program’s handbook. The handbook is divided into I O major sections. The first section is a brief description of the purpose of the program. The second section isthe nwdcl ordinance, theregulatoryportion of the handbook. It describestheminimumrequirementsnecessarytoregulatetheinterstatecotnmerce of molluscan shellfish and to establish a program to protect the public health of consumers. The third section describes the public health reasons for and explanations of the requirements stated in the model ordinance. I n section four, guidance docutnents are provided which further detail necessary standards and practices. Section five provides examples of suggested forms. Section six contains the NSSP’s policy documents. In the seventh section, interpretations of specific requirements are provided in response to questions posed by ISSC members. Section eight provides the pertinent federal regulations that form the backbone of the NSSP. A briefhistory of the NSSP is described i n sectionnine,and finally, scction ten lists the references from the NSSP manual of operation (9).
A.
Model Ordinance
The model ordinance is made up of 15 chapters. Chapter I sets out theadministrative requirements of the shellfish authority to establish a statewide shellfish safety and sanitation program. All participating states must have regulations i n place to control the classification of shellfish growing areas, the harvesting of shellfish, shellfish processing proce-
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dures and facilities, product labeling, storage, handling and packing, shellfish shipment i n interstatecommerce, shellfishdealers,andbivalveaquaculture.Otheradministrative requirements include keeping adequate records, providing a description of duties where overlapping responsibilities exist between agencies, establishing necessary procedures to regulate the shellfish industry and to investigate incidents of shellfish-borne disease, and developing a management plan to deal with commingling of shellstock. Chapter I also describes the process necessary to certifyshellfish dealers, the number of required inspections, and the need for enforcement action.
1. RiskManagement Chapter 11, “Risk Assessment and Risk Management,” describes the procedures the control agency must follow when outbreaks of shellfish-related illncss occur. upon finding human pathogens in shellfish meat, and upon determinationof toxic substances i n shellfish meat. Chapter 111, “Laboratory,” reviews the requirement for proper quality control of the laboratory procedures and the need for ongoing evaluation of any laboratories used in the context of the NSSP. It also specifies the methods for microbiological, chemical, physical, and biotoxin analyses that must be used. Accepted microbiological and biotoxin assays for different samples are listed in Table 1.
2. Shellfish Growing Areas Chapter IV, “Shellstock Growing Areas,“ encompasses the requirements previously captured in part I of the NSSP nmnual of operations. The provisions set out are of critical Table 1 ApprovedNSSPLaboratoryTests:MicrobiologicalandBiotoxinAnalyticalMethods
shellfish
shellfish Application
plate
Growing area classification: Seawater Controlled relaying: Seawater Shellstock Wct storage: Seawater Shellstock Depuration: Seawater, tank water, uv effluent Shellstock Market shellfish: Shucked Shucked. frozen Shellstock Shellstock, frozen
Total Fecal coliform Standard Paralytic Neurotoxic coliform MPN,’A1 MPN.’ M”poison,’ poison,’.” count,’
d
d
d
d d
d d
d
d d
d d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d
d d d
d d d
d d d
d d d
d d d
d
d
d
d
d
.‘APHA: American Public Hcalth Rccommcndcd Proccdurcs for thc Ex;mination of SC:lWatCr and ShClhh. 4th d . . 1070. MPNand SPC. pp. 28-47; PSP, pp. 57-61; NSP, pp. 61-65. h AOAC: Association of Official Analytical Chcmlsts-Oflic~al Mcthods o f Analysis. 15th Cd.. IW3.PSI’. hiological method. pp. XXI-8x3; AIM tcst for fecal coliforms. pp. 436-437.
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importance because proper growing water classification and restriction of harvesting are two of the most essential points in controlling contamination of molluscan shellfish. Without proper water classification. any market standards would have minimal effect on consumer protection ( 12). Bivalves obtain their food by sieving large quantitiesof water through their meshlike gills. The microscopic particles of food that float i n the sea are caught on the gills and transported to the mouth. The result of this feeding method is that as these animals feed, they extract and concentrate bacteria, viruses, and other contaminants present in the water (9). Fecal contamination of growing waters may come from a number of sources. Feces deposited on land can release pathogens into surface waters via runoff. Most freshwater streams eventually emptyinto an estuary where fecal bacteria and viruses may accumulate in sediment and subsequently can become resuspended. Direct discharge from boats or sewage outfallsalso provides a source of microbial and chemical contaminants.In addition to pathogenic microorganisms, poisonous or deleterious substances may enter shellfish growing areas via industrial or domestic waste discharges or seepage from waste disposal sites or agricultural lands. The potential public health hazard posed by these substances must be considered when assessing the safety of shellfish growing areas. (I. Scrnirnry S u n y ~ . Chapter IV also describesthe“SanitarySurvey.”This isa written evaluation of all environmental factors, including actual and potential pollution sources, that have a bearingon water quality in a shellfish growing area. A sanitary survey is made up of the data and analysis from five components and is the key to the accurate classification of shellfish growing areas. The principal components of a sanitary survey include a shoreline survey, a survey of the bacteriological quality of the water, an evaluaon tion of the effect of any meterologic, hydrodynamic, and geographic characteristics the growing area, an analysis of the data from these surveys, and a determination of the appropriate growing area classification. Once an initial sanitary survey has been completed foran area, portions of the report must be updated annually. A more thorough reevaluation is required every third year. A new, complete sanitary surveyis necessary at least once every 12 years for all areas where shellfish harvesting is permitted. Updatinga sanitary survey consists primarilyof routinely evaluating major pollution sources, collecting water quality data from key stations under adverse conditions, and analyzing the data to ensure that the sanitary survey continues to be representative of current sanitary conditions in the harvest area. When routine monitoring reveals a substantial change in the sanitary condition of the harvest area, intense and comprehensive reevaluation is required. The nianual provides direction on how each of the five components of a sanitary survey must be conducted. A shoreline survey consistsof a review of all actual and potential sources of pollution and how they may affect the growing area. A pollution source survey should be conductedof the shoreline area and watershed to locate direct discharges such as municipal and industrial waste discharges, malfunctioning septic tanks, and nonpoint sources of pollution such as stornlwater runoff and agricultural and wildlife area runoff. Municipal and industrial wastewater treatment facilities should be evaluated in of pollutants terms of design capacity versus actual loading, the type and concentration discharged, and the type and effectiveness of pollution control devices. Bacteriological all pollution sources. Results are interpreted using sampling must be conducted to evaluate either fecal coliform or total coliform and are based on an analysis of at least 15 water samples taken under adverse pollution conditions.It is also permissible to use a systematic
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random sample water quality survey method. Examples of hydrographic and meteorologic characteristics that may affect the distribution of pollutants include tidal amplitude, water circulation patterns, depth, salinity, and rainfall patterns and intensity. b. Class$cwtio~lof the Glowi11,yAretrs. The result of a sanitary survey is a classification of the growing area. Each growing area is classified within defined boundaries as either approved, conditionally approved, restricted, conditionally restricted, or prohibited. Growing areas without a written sanitary survey report must be closed. An “approved” classification is applied to those growing areas with sanitary survey and marine biotoxin data indicating that fecal material, pathogenic microorganisms, and poisonous or deleterious substances are not present in dangerous concentrations. A “conditionally approved” area is a growing area that is subject to intermittent microbiological pollution. Seasonal population, nonpoint source pollution, or sporadic use of a dock or harbor facility Inay affect the sanitary quality of an area. Harvest from a conditionally approved area is contingent upon the development of a written management plan. This plan must include an evaluation of each of the potential sources of pollution that may affect the area and their correlation with predictable environmental conditions or other factors affecting the distribution of pollutants into the area. An area is classified as “restricted” when a sanitary survey indicates a limited degree of pollution. This may arise when levels of fecal pollution or poisonous or deleterious substancesarelowenoughthatrelayingordepuratingwillmaketheshellfishsafeto market. As with conditionally approved areas, the “conditionally restricted” classification is applied to those growing areas that are subject to a greater degree of intermittent microbiological pollution. A growing area isclassified as “prohibited” under two circumstances. The first case is when there is no current sanitary survey on file. The second case occurs when the sanitary survey or other monitoring program data indicate that fecal material, pathogenic microorganisms, poisonous or deleterious substances, marine biotoxins, or radionuclides may reach the area in excessive concentrations. Shellfish may not be harvested for any human food purposes from areas with this classification.
B. Marine Biotoxins The next subject covered in Chapter IV is marine biotoxins caused by blooms of toxic dinoflagellates. In a 1998 publication by the FDA ( 1 3), marine biotoxins were represented as a worldwide problem. The publication stated that blooms of PSP-causing organisms have occurred in New England, Canada, the northwestern United States, England, Norway, Brazil, Argentina, India, Thailand, and Japan. PSP toxins are accumulated and depurated by bivalves during normal feeding. Because the toxins cannot be destroyed by normal cooking, freezing, or smoking, the best way to prevent PSP is by detecting the toxins i n shellfish and discarding them before they reach the market. The detection method used most often is the mouse bioassay. The minimum quantity of PSP toxin that will cause intoxication in the susceptible person is not known. Epidemiological investigationsof PSP in Canada have indicatedthat 200-600 pg of poison will produce symptomsin susceptible persons. One death has been attributed to the ingestion of a probable 480 pg of poison. Investigations indicate that lesser amounts of the poison have no deleterious effects on humans. Growing areasmust be closed at a low enough toxin level to provide an adequate margin of safety, since toxicity levels can change rapidly. For this reason, a regulatory
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standard of 80 pg has been established. Many of the species of toxic phytoplankton that cause PSP are indigenous to the coastal and estuarine waters of the United States. Cases of PSP, including several fatalities, have been reported on both the Atlantic and Pacific coasts ( 14). Pytc.hotli,sc~lr,s is recognized as thecausativeagentforneurotoxicshellfish poisoning (NSP). Blool11s of this dinoflagellate are usually associated with fish kills, but theycanalsomakeshellfishtoxictohutnans. NSP is primarilylimitedtotheGulf of Mexico along the west coast of Florida. Diarrhetic shellfish poisoning (DSP) was recognizedi n Japan within the last decade. In some areas, DSP dinoflagellates have co-occurred with PSP dinoflagellates, making monitoring and management of shellfish harvesting areas difficult. that causes Diatoms such as Nit:schirr pw~,qwsproduce thetoxin,domoicacid, amnesic shellfish poisoning (ASP). This organism is a cotntnon coastal water alga of the Atlantic, Pacific, and Indian Oceans and was considered an innocuous alga u n t i l 1987, when a blootn off the coast of Prince Edward Island produced domoic acid. To date, cases of ASP have only beenassociatedwithmusselsfromPrinceEdwardIsland. The first documented case of ASP occurred i n 1987, and to date hasnot occurred outside of Canada. The blooms of 1987and1988resultedin approximately 130 illnessesand 2 deaths in Canada. During 1991 and 1992, there was a spread of dotnoic acid-producing organisms throughout the world. Dotnoic acid has also been found in shellfish i n Washington and Oregon. Because the consumption of toxic levels of marine biotoxins can cause severe and potentiallyfatalillnesses. it is necessary to have the followingcontrols i n place. It is important to develop a contingency plan that provides for emergency shellfish sampling and assaying, closing of growing areas, harvesting prevention of contaminated species. and product recall. A routine marine biotoxin monitoring program should also be maintained. If the assay results determine unacceptahle levels of marine toxins, the growing area must be closed until data exists to show that the toxin content of the shellfish in the growing area is reduced to acceptable levels. Where heat processing is used t o reduce toxinlevels,propercontrolsareneeded.Finally,theshellfishauthoritymustmaintain proper records of the monitoring data. bw~lis
C. Canadian Interpretation of NSSP Growing Water Standards Environment Canada has hecn assessing the sanitary quality of shellfish growing waters on the west coast of Canada on a regular basis since [he early 1970s (14). Surveys are conducted according to protocols outlinedi n the CSSP. I n addition to bacteriological monitoring surveys, growing areas may be assessed using hydrographic and dye release studies. shoreline investigations of point and nonpoint pollution sources, outfall modeling, and sewage treatment plant evaluations. Previously unsurveyed areas must have a comprehensive survey completed prior to any comnlercial harvesting. If the water quality standard is met and no point or nonpoint pollution sources are identified, the nrea is classified as “approved” for harvesting. Once classified as approved. each area undergoes an annual sanitary review, which may involve additional sampling if deemed necessary, and a completereevaluationevery 3 years.Shellfishgrowingwaterqualityonthewestcoast of Canada is assessed using a network of approximately 2800 marine and IS00 freshwater sampling stations from which 8000 samples are collected annually for fecal coliform anal-
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ysis. Salinity measurements are also taken on the marine samples for comparison with precipitation data obtained from the Atmospheric Environment Service. The survey results are used to assess the adequacy of shellfish closure boundaries, to evaluate water quality at new aquaculture and wild harvest sites, and to quantify pollution levels at point and nonpoint sources.
D. Marinas The use of a body of water as a marina will have a significant impact on its suitability for harvesting shellfish. As a result of the impact on public health, marinas are classified as prohibited, conditionally restricted, or conditionally approved.
E. Shellstock Relaying Chapter V, “Shellstock Relaying.” outlines the need to control thc harvest of shellstock, the initial level of contamination, approved holding containers,and water quality. Speciesspecific water quality parameters must be established and a study is required to determine the effectiveness of the contaminant reduction plan. Additional controls are required to license the shellstock relaying operation, manage the relaying activities, and license the harvesters. Research has shown that shellfish have the ability to purge themselves of certain contaminants when placed in clean saltwater. The rate of purging depends on the specific contaminants. the species of shellfish, and several environmental factors. Relaying is the practice of harvesting shellfish from n1arginally polluted growing areas and placing them i n a clean marine environment for a sufficient time for them to reduce contaminating microorganismsto safe levels. It is important to ensure that adequate controls are in place to provide assurance that the relayed shellfish are all actually relayed to approved or conditionally approved waters and are adequately purified. Strict controls on the operation are necessary to ensure that the shellfish remain in the relay waters long enough to purge contaminating microorganisms. At the same time, they must not be contaminated with poisonous or deleterious substances that would not effectively be purged from the shellfish during the cleansing phase of the relay.
F. Aquaculture The guide also setsout the requirements for aquaculture. TheNSSP describes aquaculture as the culturingof nlolluscan shellfish i n natural and artificial growing areas. Requirements for the authority include a general requirement for records of the aquaculture facility, controls on the source of seed shellstock, and an inspection system for land-based aquaculture. The harvesteris required to operate in accordance with the provisions of their license, to ensure that the water meets a niinimum standard, and that harvesting, processing, storage,shipping,andrecordrequirementsmeetthesamestandardthat is applied to wild shellfish.
G. Shellfish Harvesting and Handling Practices Wet storage is a common practice whereby shellstock is held temporarily in near-shore floats, baskets, or sacks, and onshore i n tanks. The purpose of wet storage is primarily as
The U.S. National Shellfish Sanitation Program
333
a convenience for the harvester and processor. Chapter VI1 of the guide discusses the requirements for wet storage i n approved and conditionally approved growing areas. Gena eral provisions are included to ensure that the shellfish are held, handled, and stored in manner that maintains wholesomeness. “Control of Shellfish Harvesting” (Chapter VIII) requires that a program be established to control the harvestof shellstock through licensingof harvesters, patrol of growing of areas whereshellfish harvest is not permitted, areas, enforceable penalties, identification and a list of all growing areas including their geographic boundaries and classific?t‘ c Ion. Also included in this chapter is the requirement for harvesters to ensure proper operation and maintenance of vessels to prevent contamination of shellfish and proper disposal of human sewage. Harvesters are also required to wash the shellstock as soon after harvesting as is practical, use proper labeling, and maintain temperature control at the harvest site. Chapter IX, “Transportation,” sets out the control requirements that the authority must ensure for all shellfish shipped in interstate commerce. Criteria are set to define an acceptable shipment, includingthat tags and shipping documentsbe in place, the shellstock is alive and cooled to specified temperatures, shucked shellfish must meet minimum temperature requirements, and bacterial samples are taken when required. The harvester or dealer must also follow defined procedures for the use of acceptable transport vehicles, storage bins, temperature controls, shipping times, and sanitation requirements to prevent cross-contamination.
H. Hazard Analysis and Critical Control Point (HACCP)
In Chapter X, “General Requirements for Dealers,” hazard analysis and critical control point (HACCP) requirements arebuilt into the NSSP ( 1 5). In December 1997, a new FDA program went into effect to further ensure the safety of seafood. This program required that foreign exporters and domestic seafood processors, repackers, and warehouses use HACCP requirements as the basis for their quality control program. This system focuses on identifying and preventing hazards that could cause foodborne illnesses rather than relying on spot-checks of rnanufacturing processes and random sampling of finished seafood products to ensure safety. The guidelines for seafood HACCP as set out in the Code of Federrrl Rrgrrkrtior~s (1 6) require that every processor conducta hazard analysis for each kindof fish processed and write and implement an HACCP plan. This plan must list the food safety hazards identified food safety reasonably likely to occur, the critical control points (CCPs) for each hazard, the critical limits that must be met, the procedures and frequency of monitoring each CCP, and any corrective action plansthat have been developed. Each processor must also verify that the HACCP plan is adequate to control food safety hazards, keep adequate records, and set out sanitation control procedures. HACCP requirements apply to all seafood processors; however, molluscanshellfish handlers must follow a few additional rules. For example, part of the HACCP plan must state how the processorwill control the origin of the shellfish, that processors only process shellfish harvested from growing waters apto provedforharvesting,thatshellstockisproperlytagged,andthatrecordsarekept indicate that they have come from an approved source. The model ordinance uses the (16) and then embellishes them by adding detailed regulations as set out by the FDA requirements, particularly for general sanitation requirements. Other requirements for the dealer include the need to maintain certification by the state authority, shellstock identification through proper handling and labeling of shellstock
334
Reid and Durance
and shucked shellfish, and maintenance of shipping documents and records. The practice of wet storage i n tanks is permitted according to specific requirelnents pertainingto proper operation of the facility and water supply. Chapter XI, “Shucking and Packing,” reviews specific operating requirements for dealers who shuck and pack shellfish. A dealer who shucks and packs shellfish is certified a s a shucker-packer (SP), and may also act as a shellstock shipper or reshipper or Inay repack shellfish originating from other certified dealers. Onetopicdiscussedisheatshock.This is amethod of preparingshellfish for shucking that is used in several regions and for several species of shellfish. The process is not intended to open the shellfish but rather to cause the shellfish to relax its adductor muscle(s) so it can be more easily shucked. Short-term heat shock should not inlpair the keeping qualities of packed, refrigerated shellfish meat. Avariety of heat shock processes are currently in use. The state authority is required to approve the scheduled process for heat shock and ensure thatcriticalfactorsaffectingtheprocesshavebeenconsidered. Requirements for the dealer include establishing a CCP and critical limits the for incoming shellstock and during processing. I n addition, the minimum acceptable standards are specified for the plant location and grounds, the source of shellfish, the dry storage facilities, protection of shellstock, andconstruction of floors,walls,andceilings,shuckingbenches,tables,utensils. and equipment. Also outlined are insect and vermin control, minimum lighting levels, necessary heating and ventilation standards. source and sanitation of the water supply, adequate plumbing and sewage disposal, and use and storage of poisonous or toxicnnatcrials. Operational requirements specify the general maintenance and cleanliness of the facility, adequate cleaning and sanitizing of equipment and utensils, minimum time and temperature controls for the shucking, packing, and shipping of shellfish, disposal of shell and waste, labeling of shucked shellfish, and useof ice. The standards for employee health and cleanliness and proper supervision are specified. In response to the dangers of the bacteria Vibrio v u h ~ [ f i c ~ r .postharvest s, processing has become an acceptable way to consistently and reliably reduce V. Iwlrl(/icu.sto nondetectable levels. During the 1991 conference. the ISSC adopted a position that in the absence of definitive information regarding V. wrlnificlrs, the only realistic approach was the education of high-risk groups. This position was reflected in the ISSC’s policy statement regarding the consumption of raw molluscan shellfish. Despite attempts to educate consumers, the use of warning labels, and the imposition of strict temperature controls. the ISSC could not discern a reduction in the cases of illness caused by the organism. During the 1997 ISSC, a resolution was passed regarding postharvest treatment processing. This resolution acknowledged that the use of postharvest treatment processes could reduce V. w r l r l ( f i c u s bacteria in raw oysters to nondetectable levels, and that neglecting available technology that could make products safer could cause potential liabilities for public health authorities and the shellfish industry. The model ordinance requires that an HACCP plan be developed and approved by the state authorities. Components of the plan must include an end-point criterion for the process as nondetectable. a sampling program to demonstrate that the end point is met, proper packaging and labeling, and records. The guide describes requirements for a variety of packing and shipping operations. “Repacking of Shucked Shellfish” follows the same general requirements specified for shucker-packer operations except for the provisions specifically relating to the shucking operation. A repacker (RP) certification is provided to a dealer other than the original
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335
certified shucker-packer who repacks shucked shellfish into other containers. A repacker may repack and ship shellstock but is not permitted to shuck shellfish. Repacking is the process of removing shucked shellfish from one package and placing them in another. Repacking of shucked shellfish exposes them toadditionalhandlingandincreasesthe possibility of contamination. “Shellstock Shipping” applies to those dealers certified as shellstock shippers (SS). These dealers may grow, harvest, buy, or repack and sell shellstock but may not shuck shellfish or repack shucked shellfish. A shellstock shipper mayalso ship shucked shellfish. Critical control points for shellstock shipping are the source of the shellfish and storage conditions. Sanitation requirements are similar tothe SP operation,but are consistent with the more limited nature of the operation. Postharvest processing is permitted where an approved HACCP plan exists. “Reshipping” applies to those dealers certified as reshippers (RS). These dealers may purchase shucked shellfish or shellstock from other certified shippers and sell the product without repacking or relabeling to other certified shippers, wholesalers, or retailers. The reshipper designation is most applicable to those dealers located a considerable distance inland where obtaining shellfish from diggers or harvesters is unlikely. It is intended to provideadditionalcontrols tothestateauthority in case a productrecall is necessary. Critical control points are necessary for receiving incoming product. Sanitation requirements reflect the minimal handling of the product and are limited to controlling the safety of the water for processing and ice production, the condition and cleanliness of food contact surfaces, maintenance of hand washing and toilet facilities, prevention of cross-contamination, protection from adulterants, proper labeling, storage and useof toxic compounds, and pest control. Other standard requirements regardingthe construction and operation of the facility are included.An important aspect for controlis the record-keeping requirements. A reshipper may not shuck or repack shellfish, norshall a reshipper remove or alterany existing label information. Appropriate additional information should be added to indicate the reshipper’s name and certification number.
1.
Depuration
Chapter XV, “Depuration,” applies to those processors certified as depuration processors (DPs). These dealers may receive shellstock from approved or restricted growing areas and submit the shellstock to an approved depuration process. Depuration is intended to reduce the number of pathogenic organisms that may be present in shellfish harvested from moderately polluted (restricted) watersto such levels that the shellfish will be acceptable for human consumption without further processing. The criteria for successful depuration of several species are outlinedin Table 2. The process is not intended for shellfish from heavily polluted (prohibited) waters nor to reduce the levels of poisonous or deleterious substances that the shellfish may have accumulated from their environment. The acceptability of the depuration process is contingent upon the State Shellfish Control Agency (SSCA) exercising very stringent supervision over all phases of the process. Requirements of the SSCA include establishing administrative proceduresto control the harvestof shellfish from restricted areas and their transport to the depuration plant. Also required are approval of plant designand operation, certification ofprocessors, inspection ofdepuration activities and facilities, and prohibitionof interstate shipments in the event that nonconformity is found. The SSCA must ensure that plant location, design, and construction meet certain
Durance 336
and
Reid
Table 2 MPNFecal Coliform Limits for Depurated Shellstock at the End of EachBatch
Process Geometric not
mean
Number of samples
sample Shellfish species
No sample may exceed
to exceed
may exceed
No valuc No value
No valuc No value
170
125
No value
170
75 l10 45
No value
100
No value No value
170
50 20
100
170
45
100
50
130
170
20
70
100
100
100
minimum standards. Dealers must meet a number of stringent requirements i n order to maintain their DP certification number. Verification of the depuration processis an important responsibility. Controls are necessary forthe source of the shellfish, the water supply, equipment construction, the scheduled depuration process, and process verification. Depuration activities involve the requirement for the dealer to have a written depuration plan identifying the scheduled depuration process and the general plan operating instructions accessible to trained employees. Shellstock washing, storage, and commingling must be properly controlled. Depuration tank operation must meet minimum requirements based on the physiology of the species being depurated. A quality assurance program must be developed and maintained to ensure that the depuration activities follow the scheduled process, and that each process batch of shellstock, once depurated, meets the end-point criteria necessary. Finally, specific labeling and record keeping is required.
Vi. CONCLUSIONS The NSSP guide has been developed over some 70 years, from the original eight resolutions described i n the Surgeon General’s “Report of the Committee on Sanitary Control of the Shellfish lndustry in the United States,” to the more than 400 page handbook of today. It evolved from general principles to comprehensive guidelines over that period. In 1992, the introduction of the model ordinance concept marked the beginning of a more standardized approach to shellfish regulation among individual states. The model ordinances were purposely written in a form that allows individual states to readily adopt them into regulation to further improve compliance with this important program. The guide has alwaysbeen a working document, and wecan expect it to continue to change with the changing faceof the U.S. shellfish industry. The introduction of HACCP principles in 1997 reflectscurrenttrendsinfood processing throughout the world, and the guide now supports this. We expect further integration of HACCP into future NSSP guides. As the United States represents one of the world’s premier shellfish markets, U.S.
The US. National Shellfish Sanitation Program
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requirements have effects far beyond national boundaries. Countries who wish to export into the United States must model their shellfish programs according to the NSSP. The shellfish industry has always been uniquely vulnerable to environmental pressures because of its requirement for clean growing waters. This pressure canonly increase with the ever-expanding world population. At the same time, the number of medically compromised individuals, due to disease and aging, will also increase. An effective shellfish sanitation program must continue to adapt to the ever-changing needsof the population as new pathogens emerge and environmental conditions degrade.
REFERENCES 1.
2. 3.
4.
5.
6. 7. 8. 9.
FDA. Part 11-Sanitation of the harvesting processing and distribution of shellfish. National ShellfishSanitationProgramManualofOperations,1995Revision.U.S.Department of Health, Office of Seafood Center for Food Safety and Applied Nutrition, U.S. Food and Drug Administration. Washington, D.C. 1995a. ISSC. The history of the National Shellfish Sanitation Program. Interstate Shcllfish Sanitation Conference,1998a. l~ttp://www.issc.org/NSSP/Backgr~)ut~d/History-NSSP.htt~~. P DiStefano. Application of good guidance practices to revisions to the shellfish sanitation model ordinance. Fcrl Reg 6234480-3448 l , 1997. P DiStefano. NSSP guide for control of molluscan shellfish. Publication no. 98D-0393. Departand Human Services, Food and Drug Administration. 1998. http:// ment of Health libra~y.kcc.hawaii.edu.praise/news/t~ssp.ht~~~l. FDA. FDA Federal Register proposed rule: to establish procedures for safe processing and importing of fish and fishery products. Frvl R q 1995b. FDA.Draftguid:unce on equivalence criteria for food. Fed Keg 62:30593-30600, 1997a. FDA.Intcrstatecertifiedshellfishshippers list. U.S.FoodandDrugAdministrationCenter for Food Safety and Applied Nutrition. 1997b. Fishand Seafood ProductionCanadianShellfishSanitationProgram (CSSP). 1997. http:// www.cfia-acia.agr.ca/et~glish/~~t~itnal/~sh-~~nd-seaf~)od/cssp-index.htn~l. ISSC. National Shellfish Sanitation Program-guide for the control of molluscan shellfish. U.S. Department o l Health and Human Serv~ces,Public Health Service, Food and Drug Ad-
ministration.1997. 10. M Friedman. Statement by Michael Friedman, M.D., Deputy Commissioner for Operations.
1I .
12. 13.
14. 15. 16. 17.
Food and Drug Administration, Department of Health and HumanServices before the Subcommittee on Livestock, Dairy, and Poultry Committee on Agriculture US House of Reprcsentatives.1996.fdahomepage.html. HoustonMedicalSchool. Vibrio species.Univcrsity of Texas. 1995.http://t1ledic.med.uth. t~~~c.edu/path/00001508.ht1n. Microbiological criteria for raw molluscan shellfish. J Food Protect 55:463-480, 1992. FDA. The badbug book-foodborne pathogenic microorganisms and natural toxins handbook. Centre for Food Safety and Applied Nutrition. 1998. http://v~n.cfsan.gda.gov/~now/. Shellfish Water Quality Protection Program on the west coast of Canada. Environmental Protection Branch Pollution Abatement Division. 1995. http://www.doe.ca. P Kurtzweil. Critical steps toward safer seafood. FDA Consumer. 1998. fdahotnepage.html. FDA. Scal‘ood HACCP. Code ~f’FedCr(/l R e ~ ~ ~ l r ~2r1CFR i ~ r ~123:240-248, .~, 1997~. ISSC. History of the Interstate Shellfish Sanitation Conference, Interstate Shellfish Sanitation Conference.1998b. http://www.issc.org/ISSC/Background/History-ISSC.httn.
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12 HACCP, Seafood, and the U S . Food and Drug Administration Kim R. Young, Miguel Rodrigues Kamat, and George Perry Hoskin U.S. Food
tllltl
Orlr
Introduction 339 11. Results 1 -Year Out 340 1.
111.
Lessons 1.eLuned
342
Keep HACCP practical 342 B. Apply fairly and evenly 342 C. Federal/state pnrtncrships are advantageous D. Significant scientific issues will arise 3-14
A.
IV
FurtherInformation References 345
1.
INTRODUCTION
343
345
Seafood is an inlportant component of the American diet. Annual per capita consumption in thc United States is approximately IS pounds. Consumers spent about $46 billion for seafood in 1997. Cannedtuna isthemost commonlyconsumedproduct,followed by shrimp, pollock. and salmon. These products, along with cod, catfish, clams,flatfish, crabs, and scallops, conlprise approximately 82% of the scafood consumed in this country ( I ) . I n recent years, the United States typically has been both the second largest importer and the second largest exporter of seafood. Seafood, as is true for all foods, nlay pose risks for illness (2). After intense public discussion on seafood safety during the late 1980s and early 1990s, the U.S. Food and Drug Administration (FDA) issued final regulations mandating the use of the Hazard Analysis Critical Control Point (HACCP) system for the processing of seafood. Enforcement began on December 18, 1997 (3). The goal of this program isto ensurethesafety of seafood. The confidence of consumers i n the safety of seafood should increase as they learn that potential hazards are under documented control. The HACCP concept consistsof a science-based systemof preventive controls. The process and materials used are analyzed for potential hazards that are reasonably likely tooccur.Criticalcontrolpoints (CCPs) arethenidentified where the hazardsmaybe 339
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Young et a/.
controlledusingmeasuresthatprevent,eliminate,orreducethehazardstoacceptable levels. The controls and CCPs are hazard, product, and process specific. The acceptable range for variation isestablishedforeachcriticallimit. If thereis a deviationfrom a critical limit at a CCP that exceeds the allowable deviation for that parameter, corrective actions should be applied to bring the process back under control. The processor should identify and correct the underlying reasons for the deviation and dispose of or redirect the violative product. HACCP systems are designed and used by industry. Processors regularly monitor the performance of their CCPs and record the results. Records provide the manufacturer with information on how well the plan is working. The firm can use the HACCP records it generates to monitor and control its own processes with regard to food safety. Processors have reported that HACCP provides them with an additional level of control by spotting trends that, if left uncorrected, can lead to safety problems i n the future. When HACCP use is mandated, certain records are required which are available to inspectors from the food safety authority. These inspectors audit the processor’s monitoring, corrective actions, and verification records during an inspection and verify the effectiveness of the HACCP plan and whether it is being followed as designed. Record keeping is the key feature of the HACCP system which allows inspectors to determine that the operation has performed satisfactorily between inspections. Adopting HACCP procedures provides a systematic, preventive, and demonstrable way for the industry to meet its responsibility to produce safe food. This concept is receiving increasing acceptance by the international community in the European Union, Canada, Australia,NewZealand,Japan,Thailand,Mexico,andothercountries.Moreover,the Food and Agricultural Organization (FAO)/World Health Organization (WHO) sponsored Codex Alimentarious Committee has endorsed the HACCP principles. A HACCP-based food inspection system overcomes two shortcomings in the food inspection system that preceded it. Where regulatory inspection is periodic, a review of a firm’s HACCP monitoring records permits the regulatory agency to review the perfornlance of key operations at a plant for the entire period since the last inspection. Rather than a “snapshot” impression of conditions at the plant from which assumptions nlust be made of its overall success in safe food production, an accurate assessment of performance can be made. Second, under a HACCP-based inspection system, processors must demonstrate to the inspector that they understand potential hazards and are taking preventive steps to keep them from happening. Previously a processor’s obligation during an inspection did not extend beyond allowing the inspector intothe processing environment. At that point the inspector was often left on his own to figure out whether or not understanding and control of potential hazards was being accomplished.
II. RESULTS l-YEAR OUT A 2-year interval between publication of the regulations and FDA enforcement of them gave the industry and the agency time to prepare to meet their respective obligations. A high rate of noncompliance, almost 7096, was found once HACCP-based inspections began. An examinationof the causes of noncompliance revealed that more than half of these firms did not have a HACCP plan. Furthermore, the rate was determined based on the initial inspection reports and did not include subsequent reevaluations for plants when disagreements were resolved in the plant’s favor.
HACCP, Seafood, and the U S . Food and Drug Administration
34 l
Conducting a successful hazard analysis requires training and experience. Even after considerable efforts at training of both industry representatives and inspectors, there were differences of opinion between plant operators and inspectors. A hazard was frequently not identified by a firm because the plant had not experienced the problem and did not view it as likely to occur. Typically the plant already had control procedures in place, but did llot recognize them as the CCP procedures and limits expectedto be incorporated into an HACCP plan. In such situations the firm simply needed to recognize these points and control limits and keep monitoring records. In general, if processors identified appropriate hazards for their HACCP plans, they included adequate controls. For example, more than 75% of processors who correctly identifiedagivenhazardalsosetappropriatecriticallimitsanddescribedappropriate monitoring procedures. A noteworthy exception was thatof histamine, the index for scombrotoxin hazard. For operations where inspectors deemed histamine to be a hazard, only 70% of processors described control and monitoring procedures at receiving, 63% described effective control and monitoring procedures during processing, and just over 70% described effective control and monitoring procedures at packing and during storage. During the first year, FDA and state inspections of domestic firms showed that the plants were deficient i n prerequisite sanitation programs or monitoring of them, did not have an adequate plan, had failed to identify appropriate hazards. were deficient in monitoring or records procedures, or failed to take appropriate corrective actions. The types and frequencies of deficiencies seemed to be relatively constant during the first year. For importers the compliance rate was markedly lower, about 20%. Previously importers were not subject to FDA inspections; rather foods were inspected upon arrival at port and if found in violation, the regulatory actions were taken against the foods. Only in rare cases where an importer was found to have deliberately committed a criminal act was the importer held responsible. The new regulations, however, require importers to verify that the products they import have been produced under HACCP systems. Many importers, unlike domestic processors who directly handle the seafoods they produce and process, may have had little first-hand familiarity with the physical nature and potential hazards associated with the foods they have imported and the conditions under which they were produced. Consequently, in spite of considerable efforts to inform importers of this new responsibility, many were initially unprepared for the enforcement phase of these regulations. As a means of discovering systematic difficulties, FDA divides compliance deficiencies into threc categories: critical, significant, and other. Critical deficiencies are those that result i n failure to identify and control hazards. Significant deficiencies include errors in recording appropriate data or adequately documenting corrective actions. The "other" category includes minor and administrative deficiencies. This analysis showed that about 30% of processors did not have critical or significant deficiencies. About 30% of the inspected tirms hadno plan when one was needed. Poor sanitation monitoring, not necessarily poor sanitation. was often reported, and the specific hazard of i n the operation of 40% of firms requiring histamine histamine was inadequately dealt with control. The first year has, as was expected, been a learning process for inspectors and for the industry. What is important, now that HACCP systems are required and are in place, is that firms continue to improve. As firms become more skilled at applying the HACCP of safety, in turn, leads to system, seafood safety becomes more certain. More certainty greater consumer confidence in seafood.
342
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Young et al.
LESSONS LEARNED
Four principles enlcrged from our experience establishing HACCP requirements for the seafood industry. These include that HACCP requirements must be kept practical; these requirements must be applied fairly and evenly throughout the industry; implementation of the requirements is best served by establishing effective partncrships between federal and state regulators; and scientific issues which must be resolved will come to light.
A.Keep
HACCP Practical
Considerable initial concernwasexpressed by many members of theindustryand by governmental oversight authorities that requiring HACCP and its attentlant records could small operations would simply be unable to afford be needlessly burdensome. Further, compliance. HACCP compliance was evaluated to the maximum extent possible i n the context of what the prudent processor already did. There was 110 expectation that processors currently producing safe products would need to undertake significant capital expenditures to come into compliance. In fact, if such expenditures were found necessary, they were doubtless necessary regardless of the formalization of safety control brought to the operation through the application of HACCP principles. The FDA recognized that many seafood producers would have difficulty identifying the hazards reasonably likely to occur from their products and practices. Actually, many within the FDA would find thisa daunting task. TheOffice of Seafood drafted a document to aid in designing correctly targeted HACCP plans, the Fislr m t l I;'i.s/wric~.sHtrrrrdr t r r d Cor~/r.ol.vGuide (4). The availability of this guide was published i n the F c ~ l t ~ t uKoSqistcr l along with the notice of the proposed rule, and comnents were solicited. After evaluating the comments. the draft was revised and the first edition published. The primary intent of the Hazards and Controls Guide wasto support the efforts by processors and importers in conducting their hazard analyses and i n identifying controls and setting critical control limits. The guide also benefited inspectors by providing useful information for evaluating HACCP operations during inspections. The guide included as much information as available on practical control procedures and effective control limits. I t was also intended to be updated as practical control infortuation became available through research and industrial documentation of successful processes and procedures. A revised second edition was published in 1998 which contains modifications based on FDA studies and on information supplied by seafood firms and trade organizations.
B. Apply Fairly and Evenly The FDA tried to maximize compliance by educating processors, :hereby minimizing the need for regulatory action. For this reason. the first HACCP inspections were treated priInarily as an educational experience for processors. The FDA issued an "untitled letter," that is. an informational letter, advising the firm of HACCP deficiencies if the observcd deficiencies did not pose an imminent health hazard. For these deficiencies, the FDA did not expect a subsequent enforcement action would be taken. The FDA districts were alsoi n a learning phase. To ensure consistencyand accuracy among FDA districts, initially all untitled letters were reviewed by the FDA's Office of Seafood before they were sent. After several months the letters were issued directly by
HACCP, Seafood, and the U.S. Food and Drug Administration
343
the local FDA inspection oflices. The Office of Seafood monitored for consistency by reviewing some of the letters that had been sent. Evenness and fairness by regulators requires uniform training o f inspectors and of the industry. As in earlier experiences with the establishment of low-acid canned food regulations. the FDA ancl the industry recognized that both would benefit from infortnation sharing between the regulators and the regulated. To obtain the best infortnation available, a consortium called the Seafood HACCP Alliance was formed. The alliance consisted of federal and state agency representatives. The active organizations included the Association of Food and Drug Officials (AFDO), FDA, National Marine Fisheries Service, U.S. Department of Agriculture, academia (Sea Grant universities) and industry trade organizations (National Fisheries Institute, National Food Processors Association). A training curriculum was developed and a standard core HACCP training tnanual was produced antl made available for use in these courses(S). The manual provided thernaterial that should be presented in a standard course. Participation in the courses, examinations, on the job training, and on-site audits to demonstrate proliciency have been designed into the training regime for inspectors and will continue to be required and to be refined as experience and information permit. Courses were established antl provided to a l l fetleral and state inspectors. Courses were also held for processors and importers tluring the 2-year period between publication of the regulations and the initiation of inspections for compliance with the new regulations. By the end of the first 321 domestic and 25 international courses resulting in year, the alliance had conducted over 9600 trained studcnts. The training was built around a "train the trainers" structure which provided 600 trainerscertified by the AFDO. These trainers qualified at one or more o f 17 dotnestic and 2 international train-the-trainer courses. Preenforcement-period training offeredto the industry was helpful, but both training and cnforcenlent are neededto create a successful new system. Our experience with implementing the HACCP system showed that additional training would be needed once actual inspections began. After one district found poor compliance among inspected importers, the district held targetcd workshops for then1 and compliance immediately improved. Enforcement may have gotten thcir attention, but education then became the key to achieving compliance. The alliance continues to develop materials i n support of HACCP implementation. It is presently completing another reference. the ConrprrdiwTr of Fish trrrcl Fish Proc1Lrc.t.s Proc~~.s.sr.s, Hcr:trrds, crrrd Corltrols. This compendium will aid producers in establishing processesand i n selectinganalyticalmethodstoverifyplansandmonitorcontrol points (6). Thc fact that the training was designed collaboratively among federal, state, academic experts, and trade experts lends both crcdibility and accuracy to the training effort. Creating this collaboration was a unique and highly effective means of sharing information and of engendering a more cooperative rather than an adversarial relationship between industry and regulators.
C.
Federallstate Partnerships are Advantageous
While this lesson is particular to the United States, there are analogous fcderal/local governmental structures i n many countries which may experience jurisdictional inefficiencies similar to the U.S. experience. Where federal and local s y s t e m both exist and include
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Young et al.
inspection, establishing a partnership between them will increase efficiency because duplicate inspections can be avoided and the inspection procedures can be standardized. Most states inspect seafood processorsthat operate within their boundaries, so some overlap of inspection effort between the federal and state programs has long existed. Although coordination to avoid duplicate inspections has been attempted, differences i n the expectations of the inspection organizations led to differences in inspection results. The complaint that the federal and state inspection efforts resulted i n a “patchwork” program was frequently heard. The federal seafood HACCP regulations and the general climate of interest in HACCP provided an opportunity for federal/state partnering. The adoption of the HACCP approach provided a colntnon base for establishing a collective federal/ state inspection system. The intent of the partnerships is t o promote national consistency, data sharing, and adequate verification that a partnership is workingit as should. Variations among individual states with respect to the industries within them and the structurcs and capabilities of their regulatory infrastructures, including inspection and laboratory capabilities, have indicated a need for flexibility and individually detailed partnerships state by state. The Presidential Food Safety Initiative, announced in January 1997, seeks to reduce foodborne illness by strengthening and improving food safcty practices and policies. The initiative included funding specifically for food safety and made possible federal support to the states to obtain or upgrade computers and inspection equipment, to support training, and to undertake other actionsto facilitate the states’ ability to join in federal/state partnerships. A numberof partnerships have been formed and should increase. These partnerships and the national inspectional database containing federal and state partner inspection results have begun to bring together a more efficient national seafood inspection system.
D. Significant Scientific Issues willArise Producers and processors need additional information in order to develop HACCP plans in order to thatarespecifictotheiroperations. The FDA needs additional information verifythatplansareappropriate to the operation under inspection. For example, Inore of parasites comprehensive epidemiological data are needed to shed light on the prevalence infish,especially i n certainspecies,and to definecontroloptionsfortheseparasites, apart from freezing. Additional options for control measures to restrict the formation of Clostridium horrrlinurn toxin are necessary, i n addition to acidification. smoking, and control of salt and water activity. The time and temperature controlsthat are cited in the Fish m c l Fisltel:\~Proclrrcfs Htrzcrrt1.s m d Controls Grriclo (4) to prevent the formation of histamine i n susceptible species need to be refined as more species and process-specific inforlnation is obtained. The application of chemical and instrumental methods, in addition to sensory analyses, can lead to a better understanding of the conditions that must be controlled to prevent decomposition in general and the formation of hazardous decomposition products that must be controlled under a HACCP plan. Researchis necessary to determine the possiblc hazard from low levels of bacteria on fish with respect to the need for processing control or the suitability of relying upon final cooking preparation. Cooking protocols are needed for many cooked, ready-to-eat products to ensurc destruction of pathogenic organisms. These topics, and others, are being incorporated into theagency’sresearchplans.Hazardanalysis,HACCPcontrols,andcontrollitnitsarc based on the best information available. They are subject to changc as additional scientific information is obtained. Additional information will, in many situations. lead to a better
HACCP, Seafood, and the
US. Food and Drug Administration
345
understanding of hazards, their source, and their controls. The agency hopesthat all interested parties, government, commercial operations and trade organizations, and academia will undertake studies for their resolution.
IV. FURTHER INFORMATION For further information, please visit FDA‘s website at http://www.fda.gov, and then select Fish t r r ~ dFishFoods/Seafood/HACCP. Available information and documents include the eries Products H(alr(1.s r r r d Controls Guide, 2nd edition, and other information such as the acceptable market names for fish in commerce in the United States and related information on identification using electrophoresis with more than 50 species and patterns given. In addition, the Office of Seafood has an e-mail address for seafood HACCP-related questions, which is
[email protected]. The FDA’s Center for Food Safety and Applied Nutrition maintains a “Seafood Hotline” ( 1-800-FDA-4010) which is automated and accessible 24 hours a day to provide answers to consumers’ questions regarding hazards, purchasing, storing, handling, labeling, nutrition, economic fraud, or other matters. Callers who wish to speak with a specialist may do so between the hours of 12 P.M. and 4 P.M.. eastern standard time, from Monday through Friday.
ACKNOWLEDGMENTS The authors thank Philip C. Spiller for his significant suggestions script and Dr. Patricia Schwartz for her helpful review.
i n revising this manu-
REFERENCES I. 2.
3.
4.
5. 6.
NationalMarineFisheriesService.Fisheries oftheUnited States, 1997. Silver Spring, MD: U.S. Department of Commerce, 199X. FE Ahmed, ed. Seafood Safety. National Academy Press, Washington. D.C., 1997. Department of Health and Hunlan Services. 2I CFR Ports 123 and 124: Procedurcs for the Salk andSanitaryProcessingandImportingofFishandFisheryProducts. F d R q 242:6509665202, 1995. and Fish and Fisheries Products Hazards and Controls Guide. 2nd ed. Washington. D.C.: Food DrugAdministration.1998. D Ward. K Hart. eds. HACCP: Hazard Analysis and Critical Control Point Training Curriculum. North Carolina Sea Grant publication UNC-SG-96-02. Compendium of Fish and Fishery Product Processing Methods. Hazards and Controls. National Seafood HACCP Allinnce for Training and Education. I n preparation.
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13 Toxicology and Risk Assessment
1.
INTRODUCTION
Somewherc between the litany from regulatory agencies and othersthat “North America has the safest food supply in the world“ and the alnlost daily media bombardment about some deadly neurotoxicant, teratogen, carcinogen, and so on, of the week in food lies the actual truth of the matter. Mentionof the word “chemical” conjurcs images of devastating diseases, cancers, and birth defects, giving rise to the syndrome c.hc.t~rol,hoDitr. a fear of all chemicals. As stated sotne time ago by David Bradley, “There is nothing like a good dose of media exposure to turn people off a healthy food” ( l ) . He was referring to the controversy of daminozide (AlaYrM)i n apples. The public is well aware that it is exposed involuntorily to a myriad of man-macle chemicals i n air, water, and food that are present accidentally or put there for a purpose. The public is lcss conscious of their involuntary exposure to many natural components in food, which when examincd i n the same rigorous battery of toxicological tests required for drugs. pesticiclcs, and so on, do not fare too well, emphasizing a note of caution to the blind acccptance of the adage “natural is better” ( 2 ) .The pharmacologically active componcnts i n various herbal teas are a case in point (3). People are haunted by the spectcr of possible hazards that this potpourriof synthetic and natural chemicals might
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pose to their health throughout a lifetime of exposure. How much istoo much? How little is too much? How “safe” is safe enough? Since this text deals with foodborne diseases, the following discussion of chemicals, toxicology, andrisk assessment is confined to food. The various chemicals added purposefully to our foods (i.e., food additives) may play one or more of five roles in foods (4). First, they may make foods more attractive or appealing. Second, they may extend the shelf life (preservation) of foods. Third, they may facilitate food processing and mass production. Fourth, they may improve the nutritional quality of foods. Fifth, they may provide the specific requirements of consumer groups with special dietary needs. The functions of some common classes of food additives, with specific examples, are shown i n Table 1. With these chemicals we are presentedwith a dichotomy. New food additives entering the marketplace must undergo toxicological evaluation in all pertinent aspects, including cumulative, synergistic. and potentiating effects. Those judged to be safe at the level of intended use are endorsed, with reevaluationin the lightof new information with regard
Table 1 The FunctionsofSomeCommonFoodAdditives
mples additive Specific Function Class
of foods
Colors
Improvcs appearance
Natural-carotene Synthetic-tartrazitlc
Anticaking agents Antimicrobial agents
For free-running powders Prevent microbial growth
Antioxidants
Prevent rancidity, enzymatic browning
Drying agents
Absorb moisture
Firming agents
Maintain fnnness and texture Prevent food from sticking on surfaces during processing Retention of tnoisture to prevent drying and t o improve shelf life To produce a sweet taste
Magnesium carbonate Sodium benzoate, sodium diacetate Natural-ascorbic acid Synthetic-cthoxyquin, butylated hydroxytoluene Corn starch. anhydrous dcxtrose Calcium chloride, aluminum sulfate Mineral oil, oleic acid, hydrogenated sperm oil. vegetable oils Sorbitol, propylene glycol. sodium tripolyphosphate Sucrose, lactose. corn syrup, honey, molasses Acidifying--ncetic acid. citric acid Alkalizing-sodium bicarbonate Glycerine. c ~ r nsyrup, mono- and diglycerides
Lubricants and releasing agents Humectants
Nutritive sweeteners
pH adjusters
To increase or decrease acidity or alkalinity in foods
Texturizing agents
Produce and ~naintaina desired consistency i n foods
Butter, cheese. orange drink ”c1ystals” Icing sugar Pickles, bread Fruits, pet foods, cooking oils
Canned vegetables and fruits Baked goods Shredded coconut, lnarshmallows Cereals, canned fruit Pickles. soft drinks
Ice cream
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to their use and safety (5).Their toxicological assessment doesnot differ fromthat required for a pesticide, a drug, an industrial chemical, or a home product. I n contrast, many of the food additives that have been in use for many decades have never received adequate toxicological assessment according to the standards required at present, and have been relegatedtothegenerallyrecognizedassafe (GRAS) list of the U.S. Food and Drug Administration (FDA) primarily on the basis of no observed adverse health effects occurring during their long usage. In a 1984 report, a committee of the National Research Council (NRC), National Academy of Sciences (Washington. D.C.), examined the toxicity database of 8627 food additives, reporting that for 46% of the additives there was no toxicity information available and for another 3 4 8 some information was available, but of minimal quality and quantity (6). For 5 % of food additives, cotnplete health hazard assessments could be performed. For the remainder, only partial or inadequate health hazard assessments could be done. This is not encouraging, but think of the monumental task of initiating the studies required to permit complete toxicological and health hazard assessments. Investigators and regulatory agencies have been chipping away at the GRAS list, but it is a slow and tedious task. A select committee was established by the FDA in 1970 to examine the reports of several study groups and committees on GRAS chemicals and to reaffirm the GRAS status of the agents, convert the substances to GRAS status with certain limitations in use, advise additional testing with the continued use of the agent under some interim regulation, or prohibit the substances’ use (7). By 1977 the conmittee had exatnincd and made decisions on 118 reports (chemicals) from a list of more than 1500 GRAS compounds. To be fair to the committee, the chemicals examined were those for which therc was a toxicity database; the information for the remaining chemicals will be much more difficult to obtain. While the public wishes to hear that there are no residues of man-made chemicals in their food,they must appreciate that there isno such thing as absolutely “zero” residue. The level(s)of “detectable residues” changes with technological advances. Modern nnethods of analysishavepushedthezerolevel into thepicogram ( I O g)andfemtogram ( I O g) ranges, almost guaranteeing that if a trace amount of a chcmical is present, it can be quantitated. However, this says nothing about thc biological significance of the low-level residue of the chemical. Contrary to widespread public belief. the results of FDA and state monitoring prograins show that 60-80% of all foods salnpled (fruits, vegetables, grains, meats, dairy products) contain 110 detectable residues (8-10). Residues that are detected are well below the legal tolerance established for that chcmical. Approxinmtely 1% of salnples exceed of the legal tolerances and, invariably, these cxceptions arise from either the illegal use an agent 011 a crop for which it is not registered (e.g., the use of aldicarb in watermelons) or misuse (i.e., noncompliance with the spccified application rate or the recomn1ended interval between treatment and harvesting). Similar results have been reported for Canadian fruits and vegetablcs, with the reasons for exceeding the tolerances usually being well-known examples of product misuse ( 1 I ). However, it is these exceptionsthat receive widespread coverage by the media. I n my opinion, public health suffers more from the scaremongcring than from the ingestion of minute quantities of residues. No amount of legislation and/or reg11lations will prevent the accidental or purposeful misuse of pesticides, but when One contemplates the billions ofpoul~dsof home-grown as well as imported fruits, vegetables, and processed food handled annually, ;I 1 % incidence of noncompliance does not appear to be excessive.
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However. some would consider such an incidence rate to be too high. Industry as well as government agencies a t the local, state/provincc, national, and international levels have been developing intensive monitoring programs to "catch" the homegrown and imported products in violation of tolerances before they reach the market. There will an be exorbitant cost to these programs and, even if successful, the monitoring will be fortunate to catch half of that I % of products exceeding established tolerances, still leaving the problem of assessing health hazards of residues. The public's chemophobia can be reduced by increasing their level of understanding (perception) of what constitutes risk (12). It is hoped that the following discussion will dispel some ofthe mystcry associated with hazard/risk assessmentto the point that making personal hazard assessments about chemicals i n air. water. and food will be less frightening. Yes, despite what you hear and read, there is a rational approach to risk assessment, and the focus of this chapter is to explore the techniques used.
II. TOXICOLOGICAL ASSESSMENT To estimate the potential hazard of any chemical, one nlust begin with the degree or level of toxicity i n conjunction with an estimation of exposure: Hazard
= Toxicity X
(inherent) (level
Exposure X duration)
First, every chemical possesses some degreeof inherent toxicity, a property of the chemical that, i n many cases, is as distinctive as the physical and chemical properties and that guarantees. within reasonable bounds, the chemical always will behave qualitativelyin the same manner regardlessof the species absorbingit. This is a basic tenetof the discipline of toxicology. However, the concentration required to elicit a given biological effect may vary widelyacrossspecies. Second."exposure" intheequationaboverepresentsthe amount of agent necessary to elicit a biological effect; this amount must be ascertained and consists of two components, the concentration of the agent and the duration (time) of exposure. One could encounter situations in which individuals might be exposed t o very high concentrations of a toxicant for a very brief period of time, as could occur i n an intiustrial accident. At the other end of the spectrum, indivicluals might be exposed to very low concentrations of the same toxicant throughout their entire lifetime, with the source(s) arising from drinking water and/or food. While the total dosage (mg/kg body weight) might be of the same nlagnitude i n the two situations, i t is unlikely that the observed biological etfect(s) would he similar, since the dosage per u n i t time of exposure would be vastly different. Another basictenet of toxicology is the concept of a relationship between the amount of toxicant administered and either the number of experimental individuals showing a particular biological effect (dose-response relationship) or an increasing severity in a particular quantifiable, biological effect obscrvcdi n the experimental individuals (dose-effect relationship) (Fig. 1). Idcally this relationship should be signloidal (S-shaped) i n nature, withlowconcentrations o f the agentproducing little or noeffect,progressingovera dosage range i n which there is a reasonable linearity between concentration and effect, with a flattening of the relationship at the highest dosages a t which either a l l animals have responded or the maximum biological effect has been attained. Using appropriate animals as surrogates for the human i n an array of toxicological studies with defined end points,
351
Toxicology and Risk Assessment
0
r
0
z U)
W
K
c z W
o K W
a
DOSAGE
( DmOgSl k AgG E
( mglkg
1
the careful dcvelopment of dose-response (effect) relationships will generate such exposure levels as the no observable adverse effect level (NOAEL) orthe no observable effect level (NOEL). These same experiments also should yield information as to whether or not threshold or nonthreshold conformations can be established for the biological effect(s) and concentrations of the toxicant. Of concern to both the consumer public and regulators is at what levels do adverse biological effects begin to appear. A thre.sl7okf t o x i c m t is presumed to pose no risk below some experimentally determined concentration expressed as milligrams per kilogram of body weight per day (mg/kg/day). In contrast, a r l o r ~ r l l ~ t . s h a lto.uicurlt d is presumed to pose solne risk at all dosages above zero. These two characteristics of a dose-response relationship assume particular importance when considering the problems 111. of risk assessnlent and are reexamined in Sec. No single species of animal is a perfect surrogate for humans since none mimic completely the biochemical and physiological functions of humans. This deficiency is offset by conducting studies in a number of animal species. The general hypothesisis that the more times an agent produces similar biological effects across a number of species of test animals, the greater is the likelihood that at some as yet unknown dosage the same effect might occur in humans. Rodent species used include mice, hamsters, rats, and guinea pigs, whereas rabbits, clogs, and primates are the most-utilized nonrodent species. The objectives of such toxicological studies are ( a ) to reveal any physiological, biochemical, and/or morphological changesi n the test animals and (h) to establish a dose-effect relationof the potential toxicant. ship betweenthese quantitated changes and known concentrations The full range of toxicological assessment must include the entire life span of thc animal, from preconception to the geriatric stage. Since it is impossible to assess all of the toxicity
352
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end points ina single study encompassing all of these stagesof development, the investigation is broken up into segments, each manageable portion addressing specific biological issues in the spectrum of the animal’s life. The exorbitant costs of conducting extensive toxicological assessmentsof chemicals as quickly as possible during make it imperative that any potential toxicity be identified the development of the food additive, pesticide, etc. Generally toxicity studies progress in two “waves” (see Table 2). The first wave consists of a group of studies that can be colnpleted in a relatively short period of time (approximately 2 months) at minimal cost and givea good indicationof what hazards might he expected following acute occupationa exposure (acute toxicity, eye and dermal irritation, dermal sensitization), as well as ascertaining the mutagenic and teratogenic potential of the agent. Positive results in these tests in the range of levels to be found in foods (i.e., acute toxic effectsat low dosage, irritation or dermal delayed hypersensitivity, mutagenicity in vitro tests, or mammalian teratogenicity) obviously would result in the rejection of any commercial interest in the chemical, and the company would proceed to thenext analogue inthe hope of obtaining better results. Obviously, it would be a hazard to have workers exposed to such a chemical during the manufacture or processing of the food. However, if the chemical showed a low order of acute toxicity and was neither mutagenic nor teratogenic, a decision would be made to proceed to the more costly and time-consuming second-wave studies, the proposes of which are to study the long-term effects of administering low levels of the agent on various target organs of the body, including studies of fertility, reproduction, and carcinogenicity. Results from studies in this wave will be in hand only after some 3-4 years and the costs may be as high as $5 million. Appropriately conducted, the results of the second-wave studies will provide a baseline of NOAEL or, more optimistically, NOEL values that can be used by regulatory agencies to develop scenarios for a virtually safe dose (VSD) for humans. If one restricts interests to agents appearing accidentally or by purposeful design in foods, acute toxicity studies mightnot be gennane to the consumers’ concerns. An exception to this would be the incident of aldicarb in watermelons, in which the illegal use of a water-soluble, highly toxic carbamate insecticide resultedi n severe adverse health effects in those consuming the contaminated melons (13). While rare, these accidents can and will happen periodically. A similar misuse of aldicarb on English cucumbers in Canada resulted in several cases of adverse health effects (14). However, hypersensitivity to lowresidue concentrations of pesticides and certain food additives is not unknown, and at least some chemicals causing these allergic-type reactions might be identified in dermal Table 2 Toxicity Database for Chemicals: The Progression of Studies
First Wave Acute(lethality)studiesviaroutes of exposure anticipated for humans Irritationstudics(ocular, dermal) Dermalsensitizationstudies Mutagenicitystudieswith i n vitromicrobialand mamnalian celllines Teratogenicity sludies (nlousc, rat, rabbit) with agent administered to pregnant nninia~s Sccond Wave Subchronic studies. 2 1 -90-day feeding studies t o rodcnt and nonrodent species Chronic/carcinogenicltystudies of 12-monthand24-monthduration,rcspeclively,torodents Reproductivestudies,generally i n rodents,withcffectsstudiedinboth males andfemales
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sensitization studies conducted in guinea pigs and rabbits. More gernlane to the concerns of the consumer should be the results of chronic toxicity, reproduction/fertility, teratogenicity, and carcinogenicity studies reflecting the ingestion of seemingly nonharmful levels of chemicals over considerably longer time periods duringthe reproductive years, during pregnancy, or over a lifetime of 70-90 years.
A.
First-WaveStudies
1. Acute Toxicity The objectives of any acute toxicity studies are to discover and report any adverse health effect(s) that could be attributed to high-level exposure tothe agent, such information being important forthe occupational health and safety of workers involved in nianufacturing, transporting, and/or handling the pure chemical or concentrates prior to or during the mixing or diluting of it in an end-use product. In addition, these data are valid for the general public in the event of exposure to high concentrations as a consequenceof accidental spills, tank car derailments, and so on during transportation or handlingof the chemical. Acute toxicity studies are conducted in surrogate animal models to ascertain the total adverse biological effects caused duringfinite a period of time following the administration of the test agent as a single. usually large, dose by one or more appropriate routes (i.e., ingestion, percutaneous, inhalation)by which the human might acquire the chemical. Since the doses tend to be relatively high, there is usually a good correlation between the amount adlninistercd and sonle end point of toxicity being monitored. Lethality, as determined by thc LD,,, (median lethal dose) value, is frequently one such end point, but it must be emphasized that it is not synonymous with acute toxicity testing but is only one focal point. Valid reasons can be presented for determining the LD5,,for a chemical, mainly i n terms of identifying the hazard potential of an agent in an accidental spill situation and for the transportation of dangerous goods. However. much more attention should be focused on the morbidity of target organ toxicity that does not involve the death of the test animal. The potency of a toxicant frequently is expressed in t e r m of the LD,,,; this index is defined best as a statistical estimate of the acute lethality of an agent administered to a certain sex, age, and strain of a species of animal exposed under the defined conditions of the test (Fig. 2). Standardized protocols for this test have been described (15,16). The value of the LD,,, is in its use as an index of relative toxicity of an unknown chemical compared to the toxicity of known chemicals administered i n the same manner to the same sex, age, and strain of a species. Albino mice and rats, even though of different strain designations, generally are used in determining LD5,, values for chemicals, and a large body of literature has been established for these species. The route (oral, dermal. inhalation) of administration is selected on the basis of anticipated routes of exposure in humans. such as oral and/or dermal routes for food additives or inhalation for occupational exposure. Invariably the dosage administered is high, in keeping with the need to observe toxicity. Unless it is indicated, the LDS,, is assunled to represent the median lethal dose for deaths occurring in the first 24 hours after treatment. Close observation of the appearance and behavior of the animals i n the 24-hour period is crucial to reveal possible clues to the mechanism(s) by which the test agent is causing toxicity. In addition to the onset, intensity, and duration of toxicity, it should be possible to monitor changes i n behavior, respiration, and the nervous (both sensory and motor), gastrointestinal, and cardiovascular
354
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Trealment
D e a d or rnorlbund animals
SURVIVORS Study of
- adverse health effects -duration of toxicity -secondary toxicity -recovery
LDKO
- physiological and biochemical indices
systems. Those animals dying i n the initial 24-hour period. plus any seriously moribund animals that should be euthanized. are subjected to gross anatomical cxamination, with tissues being preserved for microscopic examination. The most ilnportant anitnals in the classical LDillstudy are those surviving the toxic insult, and these are monitored for an additional 14 days. The tests' value lies i n the vast amount of information that can be gleaned by close observation of behavior, growth, and developmcnt. the biochemical analysis of biological (blood. urine) fluid. and so on. The nature of the close-dependent duration of toxic effects and/or secondary toxicity arising from agent-related actions on other target organs days after treatment can be documented. The recovery of the animals from the toxic insult also can be studied. Any animals becoming moribund during the posttreatment period arc cuthanized for subsequent gross cxamination of the organs and the collcction and preservation of tissue samples for microscopic evaluation. Basecl on observations of these LD,,, test survivors, adclitional acute toxicity studies may be warranted at dosages lower than lethal ones, with attention focused on one or Inorc particular biological parameters i n order to develop proper dose-effect rclationships. Given the fact that, with the exception of some pesticides,most anthropogenic agents found a s residues i n foods are not highly toxic, therc is little need to cletermine a precise LD,,,. 'There are valid methods for obtaining an estimate of this index of toxicity using a linlited number ( I I = 6 or 8) of animals rather than the S O or 60 required i n the classical assay ( 17- 19).Results from these limited assays have shown that,for some 86 chemicals. 76 (88%;)of the cstinlated L D , , values were within 30% of the LDi,, value tierermined
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related to concentrations of thetest agents, thereby avoiding the somewhat unreliable, subjective assessments associated withthe in vivo testing criticized by animal rights activists. A full discussion of the merits of these various tests has been published (23).
3. Ocular Irritation The ocular irritation test, no longer in favor because of the trauma to the eyes of test animals, is not likely to be conducted for man-made components in foods, except for the need to assess the accidental occupational exposure to splashes and spills, as this information is an essential component of material safety data sheets (MSDSs). In most cases, a positive dermal irritation test indicates that the test chemical likely would be irritating to the eye. However, given the fact that the corneal membrane and the epidermis are quite different i n structure, there would be obvious exceptions to the above prediction. Unfortunately, while alternative strategies to the eye irritation test have not been validated or accepted by federal regulatory agencies, all home products will continue to be tested i n this manner to assure the consumer that damage (reversible or irreversible) to the eye cannot occur. In vitro tests have been developed to avoid the controversial useof animals in testing ocular irritation. The end points of in vitro toxicity, corneal opacity, inflammation, and of the i n vitro tests, but a selective battery cytotoxicity cannot be addressed by any one of tests can quantify all of these parameters (Table 3) (23). It is possible to identify strong and moderate irritants via these screening techniques, leaving only the weak irritants to be confirmed by testing in animals. A full discussion of these alternative test systems has been published (23).
Table 3 I n VitroOcular Tests ~~~
~~
Toxicity end point Cytotoxicity (using immortalizcd cell lines such as HeLa CHO. V79, MDCK, etc.)
Opacity
Moditicd from Kef. 16
Test system Entry into viable cclls Neutral red assay Fluorcscein diacctate Entry into damaged cclls Propidium iodide Ethidium bromide Trypan blue MTT: cell metabolism EYETEX Ih': nonbiological CEET: chicken enucleated eye test BCO-P: bovine corneal opacitypermeability test Bovine corneal cup Rat vaginal tissuc Chorioallantoic membrane (CAM) or HET-CAM (chicken cmbryo) CAM-VA: vascular changes i n 14-day-old chick embryos
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4. Mutagenicity Testing Chemical lnutagens interact with cellular DNA by alkylation reactions to form adducts that cause a heritable change i n the chromosomes and the genetic information carried therein. If the reaction is severe enough to be irreparable, cellular lethality results, but s1nall defects Inay cause imperfect replicationof the chromosomes during cellrepliCatiO11, resulting in dysfunctions, chromosomal breaks, deletions, fragments, and exchanges,with these mutations being transferred faithfully to descendent cells. While the latency period for autogenic effects is usually very long, extended exposure to very IOW concentrations of mutagens could cause subtle alterations in the chromosomes, with the COnsequellceS reflected in infertility, embryolethality, embryotoxicity, spontaneous abortion, Congenital anomalies, lowered resistance to infections, decreased life span, and possibly carcinogenesis. Such scenarios, fueled by appropriate media attention, have raised the specter in the public’s mind that low levels of chemicals appearing by accident or by design in our food might be responsible for a variety of diseases. They certainly have given credence to the “80 percent of cancers are caused by the oft-quoted but misinterpreted statement that environment” or, more specifically, “environmental factors,” including foods, substance abuse, life styles, occupations, and so on (24). The difficulty (time, impracticality, inconclusiveness of results, cost) of conducting meaningful mutagenicity studies in intact animals led scientists to a search for short-term, inexpensive biological tests sensitive and reproducible enough to permit their use to predict the mutagenic potential of chemicals. To a certain degree, this search has been successful. (1. Microbicrl A.sstrys. A variety of microbial systems using auxotrophic bacteria, yeasts, and fungi were developed partly because the genetics of unique strains of these organisms were well studied and experiments could be conducted rapidly and reproducibly, and partly because the organisms were responsive to the chemical being tested. Cost was also a factor. Many of the assays in use today still bear a striking resemblance to the Scrltrrorlella hphirt~uriwrras the prototype assay, the well-known Ames test, which used indicator organism (Fig. 3) (25,26). Known concentrations ( I X 10 organisms/ml) of a genetically defective strain of S. tlvl,himuriunr (TA98, TA 100, TA 1535, TA 1538) dependent upon the provision of an essential nutrient for growth (histidine in the case of S. fyphimuriun?,but other organisms require sugars, amino acids, purines, pyrimidines, etc.) are incubatedwith a range of concentrations of the suspect mutagen in solution, then spread on agar medium devoid of the essential nutrient and incubated for 18-24 hours, following which the number of growing colonies denotesthe mutagenic reversion to a prototrophic or “wild-type” organism capable of synthesizing the required nutrient itself, with little effects being seen at very low concentrations of the mutagen, while at excessively high concentrations, reversion is obscured because of lethal mutations and the death of cells. With this simple assay, at minimal cost and in a relatively short time, a quantitative relationship can be attained between the concentration(s) of mutagen and the extent of the reverse mutation(s) of the microorganism. The fact that many mutagenic compounds existas promutagens requiring substantial biotransformation to reactive intermediates prior to acting on genetic material necessitated the modification of the assay procedure, since the microorganisms could not perform this role. The “modified” Ames test, now the standard test, requires the incubation of the microorganisms with the promutagen in the presence of an active, metabolizing enzyme
358
Ecobichon Top Agar + Low Nutrlent Concenlratlon Nulrlenl-Free
h
""""_
Organlsms Qrown on Agar
"""
""""""
Blank
Control Plate
S-9
ControlPlato S-0
Fracllon
S-9
+
Chemlcal
Posltlve Plate
system. The usual system is an aliquot of a 9000 X g (S-9 fraction) supernatant fraction obtained from homogenized rodent (mouse, rat, hamster) liver, the donor animal having been pretreated with a suitable enzyme-inducing agent as polychlorinated biphenyl mixture (Aroclor 1254) (25,26).This rodent liver provides maximally induced, hepatic microsomal cytochrome P-450 monoxygennses, the enzymes required to convert promutagens to active mutagenic intermediates. The resulting incubation mixture is applied to an agar
Table 4 Battery of GetlotoxicityTestsAdopted bytheOrganization Economic Cooperation and Development
lor
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Such chemicals, called terntogetls, are a major concern to the public, to industry, and to regulatory agencies because of the low levels required to initiate cellular damage, in some cases at levels on the order of those detected in foods. The fundamental aim of the screening studies used for the evaluation of teratological potential of chemicals is to predict the absence of a teratological hazard for humans. Timing iscriticalinteratogenicstudiesbecauseexposuremustoccurduringthe period of organogenesis, which occurs immediately after the implantationof the fertilized ovum (30). In humans, organogenesis occurs i n the first 8 weeks of pregnancy, during an interval of time when the woman may he unaware of the pregnancy (31). Frequently the critical “toxic window” of time is only a few days duration, with agents acquired before and after the window showing no effect on the particular target organ, hut perhaps causing some adverse effects at another site. The same principles applyto testing surrogate animal models such as the mouse, rat, and rabbit, complicated by thefactsthatthegestation period is not 36 weeks but is condensed to 21 days (mouse, rat) or 32 days (rabbit) and that the toxic window for certain target organs may be as narrow as 24 hours (30,31). To beteratogenic in eitherhumansorsurrogatemodels, a chemical mustbe in the right place at the right time and at an appropriate concentration. With completion of organogenesis, further chemical exposurewill not elicit teratogenicity except i n the central nervous system, which in most species continues to developthroughoutgestation and even well into the postnatal period. However, some observed embryo and fetal toxicities (i.e., mortality and growth retardation) may he related to maternal and nutritional toxicity, especially if high concentrations of a rather unreactive chemical are being administered via the diet. The assessment of teratogenicity is routinely conducted in two species and involves the daily administration of a range of three appropriate dosages to different groups of timed-pregnant rats and rabbits throughout the period of organogenesis, days 6-15 of gestation for the rat and days 6-18 for the rabbit (30). The animals are euthanized 24 hours prior to the calculated day of parturition (day 20 for rats, day 31 for rahhits) and undergo a complete necropsy. The numbers of dead and living fetuses are determined and the uterine muscle is examined for reabsorption sites indicative of embryonic deaths. The position of each pup in the uterine horns is recorded, each is weighed, the sex is determined, and eachis examined for external abnormalities prior to dissection to detect internal malformations. Whole fetuses may he fixed in special fluids for histological examination or for the detection of skeletal anomalies. Evidence has demonstrated that pre- and postnatal exposure to certain metals and other substances can cause covert behavioral and cognitive deficits that are not detected by morphological examination of the brain. Such concerns have resulted in modifications to the above protocol, with some of the treated animals being allowed to give birth and to rear their young for 6 weeks after parturition, at which time the pups are examined by one or more testing strategies designed to assess behavioral and cognitive development. Completion of the battery of testing strategies listed in the first-wave studies (Table2)providesadequateinformationconcerning theacute(high-level,short-duration) toxicity of a test chemical in occupationally exposed individuals, plus a measure of the mutagenic potential and possible mechanism(s) of action, as well as whether or not any identified mutagenicity can be translated into teratogenicity in two nomxdly sensitive animal models. These studies, completed in approximately 3-4 months at a relatively low cost, will be the basis upon which decisions will be made either to proceed to the more-
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B. Second-Wave Studies In contrast to acute toxicity situations in which individuals may be exposed in a short period of time to relatively high levels of a potential toxicant, humans are Inore frequently exposed to agents at relatively low levels over a much longer period. The simulation of such exposures necessitates the developmentof other testing strategies, that is, short-term (subacute, subchronic) and long-term (chronic) studies (32). As is indicated in Table 2, the objectives of these studies are focused toward mechanisms of target organ toxicity. ~ l s grouped o in the second wave are the extensive reproductive studies designed to explore target organ toxicity in the various phases of both male and female reproduction, many of which are extremely susceptible to chemically induced damage and/or dysfunction (30).
1. SubchronicStudies Subchronic studies are designed to explore possible mechanisms of toxic action in animals over a longer period of time and at a range of dosages lower than that reported to be lethal. Male and female animals of the same age, strain. and species are used. At least three dosages (low, intermediate, and high) generally are derivedfrom the results of acute toxicity studies; they frequently are fractional dosages of the LD,,,, such as l%, 5%, and 10% of the LD5,,.The route of administration is usually that by which humans would be expected to acquire the test chemical, for instance, ingestion of the agent in the daily diet or drinking water or administration by oral gavage using a feeding needle. The duration of such studies is usually 21-90 days. A wide range of physiological, biochemical, and morphological parameters are examined quantitatively to detect any adverse biological effect(s) that could be attributed to the level of the agent and the duration of treatment and are used to ascertain the mechanism of toxicity. It is imperative to maintain a degree of flexibility in such studies since one cannot predict if and when toxicity will appear (Fig. 4). I n addition to anticipated sites of action predicted from physiological properties of the test agent and experience with analogues, serendipitous observations are the rule rather than the exception. Therefore it is important to include sufficient numbersof animals at the “front end” of the study in orderto demonstrate a dose-related toxicity without the death of too many test subjects, as well as to ensure that a number of animals in each treatment group survive the exposure and form a nucleus to study the possible permanence or reversibility of the toxicity. For a 90-day study, the investigator should plan to euthanize subgroups of each treatment group at 30,60, and 90 daysafter initiating treatment. In this manner, the various parameters being assessed may detect the appearance and the progressionof a lesion rather than waiting for treated animals to become ill. Parameters used to assess animal wellbeing include such indices as body weight gain, food and water consumption, hematology, clinical chemistry (electrolytes, enzymes), and urinalysis,as well as innovative subjective measurements of organ function and behavioral changes. All dead and severely moribund animals should undergo careful necropsy and examination, with tissues being preserved for embedding, sectioning, staining, and microscopic examination. A number of body organs are removed at necropsy forpriority examination
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ACClimallOII Period
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RecoveryPeriod
Subgroups of anlmalr euthanlxed f o r In-depth examination
of posslbletarget
-10
assoss reversibility O f tOXlCitY
organ l o x i c l t y
Fig. 4 A schematic diagranl illustrating the experimental design of a 90-day subchronic toxiclty study.indicatingtheanimalacclimationperiodandthe plantled intervals for periodic(every 30 days) selection and euthanasia of representative subgroups of treated animals for an assessment of agent-related adverse health effectsduring and after termination of the treatment, thereby permitting an estimation of the tninimunl number of animals required for the study. General practices would require an adequate g r ~ u pof control animals plus groups treated with one of a range of three (low. intermediate, high) dosages of chemical.
or are held for special study (33). Sections of major organs from control animals and all treatment groups are examined by light microscopy as a first priority to establish doseeffect relationships. Second-priority tissues are examined from control and high-dose animals, with additional study of the two intervening dose groups if significant differences are observed between control and high-dose tissues. Other tissues maybe held for special study. With each parameter chosen for measurement. the objectives are to identify the target organ (tissue) affected and to develop a quantitative dose-effect relationship.
2.
Chronic Studies
As with subchronic studies,the objectives of chronic studies are to characterize the mechanisms by which an agent may induce some toxic effect(s) when administered over a considerable portion of the life span of the test animal. As stated by Huff et al. (Ref. 34, p. 6301, “A major challengein designing long-term toxicological experimentsis to calibrate exposure levels to allow a reasonable normal laboratory life (health, appearance, growth and development) for the animals while guaranteeing obvious evidence of chronic toxicity over and above that typically seen in aged animals.” A decade ago, chronic studies were of 2-years duration whether in rodents or in nonrodents (e.g., the dog or primate). There was nothing magical i n this number of years except that i t represented approximately 80% of the life span of a rodent. Studies conducted in longer-living species required an adjustment (increase) in dosage so thatthe animals acquired a “life-span dosage” of 2 years. Immediately one can see difficulties: exposure levels would be so high that the animals would be unable to biotransform and excrete the test agent efficiently. Currently most national and international regulatory organizations require only a 12-month chronic study, but there are discussions to reduce the time period to 6 months. The debate has focused on whether or not chronic toxicity can be assessed adequately in
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this time frame when studies are initiated with young (6-week-old rodents, 8-12-monthold dogs) aninlals at the peak of their physiological/biochelnical functions, yet many of the adverse health effects usllally are observed in older (more than 14 months in rodents, more than 5 years in dogs) animals as functions are beginning to deteriorate. It has been stated that no new chronic toxicity has been detected i n 24-tnonth studies that catmot be seen in “appropriatcly clesigned” 12-month studies. The argument comes back to exposure levels and the ability of animals to cope with the agent. I n the scheme shown i n Fig. 5, a range o f three dosages (low, intermediate, high) is chosen on the basis of results from the subchronic (90-day) studies. the route ofadministration being selected as described i n Sec. I1.A. 1 (32). Usually a tninimum of 50 animals of each sex per treatment group is chosen, with possibly a larger groupof control animals. Flexibility is as important hereas i n the subchronic studies, for the sanle reasons. Sufficient animals shouldbe incorporated into the front endof the study to allow for (a) serendipitous observations: (b) some mortalityat one or more dosage levels; (c) the selection ofrepresenlative subgroups of treated animals for euthanasia and in-depth study at regular intervnls,
~
.
_
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-Acrecrmenl of tumor formallon (Incldencc, rrrllrr apprarance, t y p r ) OR -SubgrOUpS of anlmalr rulhanlzrd Jt Intrrvrlr to arrecs t ~ r r u rhyprrplrrla, prenroplrrllo trrlona, etc. tor apprarancr and prosretrlon of tumors
A schematic diagram forthedesignof a combined12-monthchronictoxicitystudy and a 24-month carcinogenicity study. indicating the animal acclimation period, and the planned intervals for the periodic selection and euthanasia of representative subgroupsof trcatcd animalsfor an assessment o f agent-related adverse health effects during and after the treatment period. thereby permitting an cstimation of the minimum number of animals required for the study. I n general. a range o f three (low, intermediate. high) dosages would be required for the chronic toxicity study. Apart from these dosage groups. additional groups of animals would he exposed to lower dosagcs of thc test agent end point of this aspecl of thestudybeingtheinduction of tumors a s for24months.theonly determined by a morphological assessment of hyperplasia, prcneoplasia, and neoplasia. Adequate numbers of cage-control animals should be carried throughout the study period.
Fig. 5
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say every 2-3 months, in order to detect both the appearance and progression of a lesion; a study of the and (d) sufficientanimals at the end of thetreatmentperiodtopermit reversibility of the toxicity. Similar biochemical, physiological, and morphological parameters will be measured i n an attempt to develop dose-effect relationships for significant biological changes that can be attributed to the test agent. Extensive morphological examination will be conducted, particularly in control animals, since the toxic effects may be masked effectively by normal geriatric changes.
3. Carcinogenicity Studies Carcinogenesis, a multistep process, occurs over a prolonged period of time and progresses through a series of stages that include (a) irlitirrtiorl by agents that elicit the original toxic insult to the nucleic acidsi n the form o f a mutation that causes an aberration in the genome; (b) p m t r w t i o r l by agents that are not carcinogenic per se but trigger events that either increase the number of tumors or reduce the latent periodof appearance; and (c)progrc’.ss i o r r or prdifercrtiorl, the rapid replication of the aberrant cells into foci that will become the tumors (35). Considerable attention has focused on the role of orm)ge/w.y,pieces of retroviral RNA found within the genome of mammalian cells that, when “promoted” by a chance mutation or a chemically induced activation, are converted from inactive protooncogenes to cancer-producing genes (36). The presence of oncogenes in the mammalian cell genome is indicative that initiation already may have taken place and that the cells only await the appropriate chemical or physical insult for promotionto proceed. However, despite many years of study, the exact relationships between chemically induced cell proliferation and carcinogenicity remain unclear. The operational definition of a cc~rc~irzogerlis an agent having the ability to cause (a) an increased incidence of tumors overthe incidence foundin controls, (b) an occurrence of tumors earlier than seen in controls, (c) the development of different tumor types not normally seen, and (d) an increased multiplicity of tumors i n individual animals (37). I n general, prolonged exposure,in excess of I2 months,of animals is required to demonstrate the carcinogenic potential of a chemical. The normal 12-month chronic toxicity study is not of sufficient duration to demonstrate this properly. The high costsof conducting independent chronic toxicity and carcinogenicity studies have resulted in modifications, with the inclusion of additional groups of animals receiving lower dosages in studies that will be carried on through a second 12-month period (Fig. 5). While high exposure levels are usedto identify chronic toxicity, investigators are interested in one end point, carcinogenicity, in the animals studied.In the 2-year portion of the combined study, the highest dosage selected is one that can be given without producing of clinical signs of toxicity and which affects long-term health or the normal longevity the test animal population (38). This dosage level, known as the maximum tolerated dose (MTD), usually is derived from the results of subchronic studies. Exposure levels lower thanthe MTD areused in an attempt to establish a dose-related incidence of tumors, decreased latency period, and so on. As with the chronic toxicity study, sufficient animals should be included so that subgroups can be euthanized at intervals of 3 months to detect any appearance or progression of target organ lesions. I n addition to the regular monitoring parameters, particular attention is focused at necropsy on the detection of “lumps and bumps” and at later microscopic examination, on the detection and identification of cell hyperplasia, preneoplastic nodules, and tumors (benign and malignant) i n order to satisfy the criteria listed above for a carcinogen. One advantage of having the higher-dosage animals i n the 12-month chronic toxicity study is
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the chance that somepreneoplastic or neoplasticchangesmay be discovered, thereby predicting what might take considerably longer to produce at lower dosages. The MTD is controversial, some scientists claiming that it is not high enough to elicit the anticipated effects, while others state that it is equivalent to “chronic wounding,” with some 50% of the chemicals tested chronically being carcinogenic (39). Still other investigators state that if the test chemical is a weak carcinogen, as is seen with many foodadditivesandpesticides,the MTDbecomesexceedinglyhigh,disruptingnormal biotransformation and causing cellular injury, toxic hyperplasia, and toxicity-induced carcinogenesis through abnormal cellproliferation(40). An alternativetotheMTDisto determine a level of exposure, called a pharmacokinetic adjusted dose (PAD) through pharmacokinetic assessment during subchronic studies. This permits the choice of a level of exposure that does not exceed the primary metabolic “break point,” thereby avoiding unnecessary “stress” (metabolic, oxidative, peroxisome proliferation)i n the animals. Ref. 41 provides a good presentation of this controversy. The costs of long-term studies have forced an examination of less time-consuming in vivo assays using special subgroups of rodents having a predisposition toward developing high incidences of certain tumor types. Chenlical exposure for 90- 120 days frequently is sufficient to cause an increased incidence and/or an earlier appearance of tumors. Mouse models for pullnonary tumors and skin cancer are available, as are rat models in which mammary tumors are foundin high incidence. It is assumed that such susceptible strains already are compromised (or initiated) genetically and that subsequent exposure is promotional in nature. An even more rapid technique is the rat liver foci assay in which young adult rats are partially hepatectomized within 24 hours of administering the test chemical in the diet or drinking water, and aftera suitable time period of hepatic regeneration, the foci of altered or abnormal cells that behave differently than the surrounding normal tissue are identified using selective enzyme-detecting stains (27,42).
4. Reproductive Toxicology Studies While the mutagenic and teratogenic potentialsof a toxicant are assessed in the first-wave studies, no exanination of perhaps the most-sensitive portion of the life span of test animals has been camed out. Reproductive toxicologyis the study of the occurrence, causes, manifestations, and sequelae of adverse effects of exogenous agents on reproduction (43). Reproductive “hazards” encompass adverse health effects to the prospective mother and father (loss of libido, sterility, infertility) as well as to the developing offspring (abortion, fetal and/or perinatal death, and mutagenesis and teratogenesis with resulting physiological, biochemical, and dysfunctional changes, as well as behavioral and cognitive anomalies). The gametes (sperm, ova), thepre-andpostimplantationembryo,andallofthe rapidly developing cells that are differentiating into organs in the fetus are exquisitively susceptible to physical and chemical insult primarily because of frequent cell divisions and replication of the cellular DNA. The mandate to study the toxicity of an agent encompasses the entire breadth of the life cycle. Reproductive studies should be designed to detect possible adverse effects on any segment of the reproductive cycle (Table 5) (30). In the past, much emphasis has been placed on studies that examine the effects of agents on the female in her role ;\S a vehicle for the developing embryo; these are essentially the teratogenic studies discussed above (known as segment I1 studies). More recently, considerable attention has been given in order to examine any to pretreating either the nlale or female animals before mating effects on the gametes of the animals, since gamete damage may play an important role
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Table 5 Segments of theMammalian Reproductive and Development Cycles
Fecundity Libido
Gametogenesis Gametes Transport Function Mating behavior Fertilization Zygote transport Implantation
Placenta Formation Function
Embryo fortnation Differcntiatiotl Organogenesis Fetal maturation Parturition Neonatal Viability
Development Lactational nutrition Postnatal maturation Sexual maturation Gatnetogenesis
in theviability of the developing embryo. Paradigms can be developed to explore the mechanisms of toxicity in either or both of the sexes (Fig. 6). In a classical segment I (fertility/reproduction) study, groups of either male or female rodents are treated prior to mating with a range of dosages, usually three levels, for one gametogenic cycle (60 days) for the male rodent or IS days for the female rodent. Following conception, the pregnant female will continue to receive the same daily treatment for (a) the duration of the pregnancy or (b) beyondthe pregnancy if there is interest in the postnatal development period. If the postnatal development period is being studied, the animal is allowed to givebirthandtoreartheyoungpasttheweaningage,thus permitting the study of postnatal viability and the investigation of bellavioral/cognitive development. In contrast, if the male animals have been treated for 60 days, they subsequently are mated with untreated, virgin females, and the focus of the study pertains to whether or not the testagent can producean effect on spermatogenesis, expressed as an alteration in a number of indexes (i.e., fertility, mating, fecundity, gestational, live birth, and survival) calculated at the termination of the pregnancy (30). In this bioassay, it is assumed that the reproductive biologyof the female is normal, with onlythat of the male being affected. The classical reproductive study has been one in which groups of young, healthy female animals (F,)generation) are treated with the test agentat three different "exposure" levels from the time of conception (mated with normal males), throughout the development of the fetuses and the rearing of the postnatal progeny, and at least through one repeated breeding cycle (44,4S). The female progeny, the F, generation, receive the same level of exposure to the test agent in the diet or drinking water upon weaning, with these animals being bred with control males upon reaching maturity to produce an F: generation. A variant of this test exists in which half of the exposed females are euthanized at day 13 of the pregnancy, dissected, and theiruteri examined microscopically for the number and distribution of embryos, embryonic death (placental scars denote resorption), and the number of dead embryos. A number of indexes (litter size,live birth, viability, survival) are determined. The numberof uterine implantations (absoluteand percentage) per female, the number of corpora lutea in the ovaries (indicative of the number of ova released), and the fate of the implantations (live implants, early fetal deaths, late fetal deaths) are deter-
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R1ElMRANlAllOH
B
SEGMENT 11
Fig. 6 A schenlatic diagram illustrating the design of Segment I (fertility/reproduction) and Segment I1 (tcratology)studies. In the former, either male or female animals are treated with the agent for 60 or 15 days, respectively. before mating with control (untreated) animals of the opposite sex. The pregnant anilnals continue to receive the same dosage of test agent throughout gestation and the postpartum period to insure both transplacental and milk acquisition of the agent by the developing fetus and the suckling offspring during thc assessment of adverse health effects. (Additional details are provided in text.) In Segment II studies, healthy, untreated, pregnant animals are treated throughout the pcriod of organogenesis (Days 6 through 15 of gestation for the rat and mouse and Days 6 through 18 of gcstation for the rabbit). with euthanasia at some suitable time point before parturition for an in-depth morphological examination of the developing fctuses for external and internal anomalies. A variant of this test strategy requires the pregnant animal to give birth and rear the young to weanling age, permitting a Inter assessment of behavioral and cognitive skills of the offspring.
mined. All euthanized animals are subjected to an extensive gross and microscopic pathological examination for soft tissue and skeletal anomalies. Such a protocol addresses the physiological problems (a) that the primordial cells forming the ovaries of the developing conceptus would be exposed transplacentally to the test agent if the dam was treated and (b) that this exposed mammalian conceptus carries, in her ovaries, all of the ova that will be used during her lifetime of breeding. As mentionedabove,reproductivestudiesmaybeseparated so thateffects of a potential reproductive toxicant can be examined only in the male animal. The dominant lethal test is restricted to the male since, unlike the female, gamete production (spermatogenesis) is a continuous synthesis from stem cellsin the testes, with these cells vulnerable to damage by agents at any time (30). A dominant lethal mutation is one that kills any offspring heterozygous for the mutation, and usually is indicative of structural chromosomal damage. The main advantage of this assay is the ability to test the sensitivity of mammalian, male germ cellsin vivo at different stages of spermatogenesis. The disadvantage is that, if a chemical is only a weak mutagen, it will not produce a significant amount of chromosomal damage and may "escape" the screening assay. In the assay, young, adult nlale rodents (mice. rats) receive either single doses or daily doses for 5 consecutive days at subtoxic concentrations, say 20% of the LDS,,,fol-
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lowed by sequential mating of each male with untreated virgin females over a period of several weeks, long enough to have encompassed one complete gametogenic cyclei n the males. As the testes will have gametes at premeiotic, meiotic. and postmeiotic stages of development at any point i n time, a short-term treatment may elicit mutations at one or more of the stages (30). The pregnant females are euthanized on day 14 of gestation and assessment of the number of implantations, the number of corpora lutea in the ovaries, and the fate of the implantations (uterine scars indicating early fetal death, late fetal deaths, live implants) are determined. Indexes are calculated to describe whether or not, over several weeks, the test agent had an effect at one stage of spermatogenesis. As an example, agent-induced damage at the meiotic stage of development should result in a significant decrease in the number of pregnancies (sterility if a lethal mutation occurred) or a reduction i n the number of live pups, with an increase in early fetal deaths (if damage permitted fertilization but not the maintenance of a viable fetus) (30).While some investigators consider this to be a crude assay of no predictive value, the end point of lethality does provide a signal that more subtle types of chromosomal damage and gene mutations might occur at lower doses, causing birth anomalies rather than death.
111.
RISK ASSESSMENT
The previous sections have described briefly the various toxicity tests required for the development of a registration “package” for a chemical. These may apply to new agents being introduced into the marketplace; however, rarely does one find the full spectrum of tests for any food additive, pesticide, etc. This deficiency was documented graphically by a 1984 NRC report (6). While risk in the dictionary is associated with the chance of injury, damage, orloss, the term frequently encountered is “acceptable risk””acceptab1e to whom? We have come in a full circle, back to the questions raised in the introduction to this chapter. Risk assessnlent has become a routine component of decision making for setting limits of acceptable exposure [acceptable daily intake (ADI), tolerable daily intake (TDI), and reference dose (RtD)] to chemicals in air, water, and food.Risk assessment is a scientific hypothesis about the most likely range into which the risks from a chemical exposure will fall. Risk assessment is that activity undertaken to assign probabilities to harmful events that occur with frequencies too low to be detected by available methods of toxicology and epidemiology (46). The publication of such influential reports as the National Research Council’s Risk As.se.sswleilt in the Federcrl Goverrlnlerlt: Mmtrgirlg the Process have provided an analytical framework that has gained acceptance and has become dogma (47). It conjures up the concept of a specific nlethodological approach to extrapolate from sets of animal and human data, observed under conditionsof intense exposure, to develop quantitative estimates of risk of human exposure from low levels of agent(s) (46). However, conducting risk assessments does not require a specific methodology, being only a good way to organize knowledge regarding potential hazardous activities or substances, permitting a systemic approach to the question of risks posed under specific conditions (46). Any line of inquiry into the risk assessment for a specific chemical must address four questions:
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I . What type(s) of toxic injury can the chemical cause? (hazard identification) 2. How do the probabilities of occurrence of each of the types of injuries change as exposure changes? (dose-response assessment) 3. What exposures do people experience? (human exposure assessment) 4. What are the probabilities that exposed people will experience the various types of toxic ill-jury (hazards) under their exposure conditions? (risk characterization) Q~rtrrrtirtrtil~~risk tr,sse.s.srtwrIf is defined as the estimation of levels of exposure to a toxic substance that leads to specified increases in lifetime incidence rates or in the probable occurrence of a given undesirable consequence(48). In this context, it is the general population that we are concerned about-individuals characterized by an age range from the very young to the elderly, a range of healthstatusesfrompoor to excellent,inherent susceptibilities to ccrtain chemicals as a consequence of altered biochemical and physiological functions throughout life, and who have been exposed continuously to a broad in food, air, and water. The components of a range of potential toxicants at low levels health-risk assessment have been described many times and the reader is referred to Refs. 49-S2 for a more in-depth review. Research cannot demonstratethat a riskdoes not exist, but it can establish probabilistic bounds on possible risks, and if those bounds are sufficiently “low,” then therisk should be acceptable (53). Research may not show that a hazard exists, but it can demonloss can be inflicted strate that under certain specific circumstances, injury, damage, or in living systems. When does one stop doing additional research that is unlikely to identify any new or different toxic properties that can be usedto make a better assessmentof hazards inherent in a chemical, physical agent, or some form of energy? Some are critical of the costs in comparison to the returns in new data that could be useful. Still others, proponents of the “if i n doubt, cut it out” approach, would deregister the agent or ban its use entirely. All of the concerns distill to one pertinent question: How good are the predictions of risk from existing or developed toxicity databases for chemicals that all of us come into contact with daily? To answer this question, let us examine four aspects of risk assessment (Fig. 7 and Table 6).
A.
HazardIdentification
A potential hazard to human health may come to light in a number of ways, with public concern over ( a ) the presence of certain ingredients in foods that are required i n their processing, preservation, or appearance [e.g., the food color tartrazine, FD&C (Food, Drug and Cosmetic Act) yellow #S]; (b) the chance or purposeful appearance of an adulterant i n a food (a famous example being the chemicaltri-o-tolyl phosphate in Jamaican ginger extract); (c) the detection of pesticide residues i n fresh fruits and vegetables (AlarTM in apples); (d) a natural component in beverages (e.g., toxic components in herbal teas); or (e) an inorganic or organic contaminant in drinking water. A “food allergy,” chemical hypersusceptibility of a subpopulation of individuals, also may focus attentionon a particular synthetic ingredient commonly found in foods. The risk assessor must develop arational approach by conducting a literature review related to each observed adverse effect in animals (and humans) caused bytheagent administered via the same route(s) as that by which the human would acquire the agent and at dosage ranges within the probability of those to which humans would be exposed.
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MEASUREMENTOF
P U W C CONCERNS CHANCE OCCURRENCE
TOXICITY STUDIES
FOOD PROCESSING PURPOSEFULADULTERATION
IDENTIFlCATlON
fmdpdnl malyslsl
-
RESPONSE ASSESSMENT
\ I
A C W EXPOSURES ilood, walal. drl
-
I RISK CHARACTERIZATION
ASSESSMENT
I
RISK MANAGEMENT
REGULATORYRESPONSE
Fig. 7 Asscssnwlt of the potential h;mrd t o health o f exposure t o a toxic chemicnl. The components o f the risk assessment include ( I ) the idcntitication of the potential ha,wd, ( 7 ) the qualitative and quantitative dose-effect assesstlwlt using animal and human toxicity data. (3) the quantitntion of actual exposures from sources of the agent and the identification of susceptible populations. and (4) the characterization of the risk. determining safe levels of exposure based either on the applicat i o n of arbitrary uncertainty factors or mathematical models to the cp:untitative toxicity data. Risk tnatlagemcnt utilizes the results of the risk assesment t o devclop regulatory responses and strategies lor reducing or eliminating exposures.
In particular, the animal studies should address the issues of ( a ) the suitability of the test species as a surrogate for hunlans, ( h ) the breadth of the study (animal numbers, sexes, treatment groups. duration), (c) the types of observations made and quantitative methods o f analysis. (d) thenature o f allpathologicalchangesobserved, and ( e ) alterations i n metabolic responses, both species and sex related (49). All of the evidence is considered in terms of strengths and weaknesses anti i n the confidence that the toxicity observed is applicable across species of test subjects.
B. Dose-Response (Effect) Assessment On the basis of the literature review. the risk asscssor now must proceed to a quantitative evaluation of the dose of the test agent and various biological effects, ~tsually adverse 6). 'Three types of dose-relatedrelationshipsmaybeidentified: healtheffects(Table quantal relationships, graded relationships, and continuous-response relationships (49). I n general, the intensity (or frequency)of response increases with the dosage, giving rise to linear,convex,concave,orsigmoitialdose-responserelationships. Most impor-
371
tantly. does the dose-response relationship appear to be threshold or nonthreshold i n naturc? For systemic (noncarcinogenic, nonmutagenic) toxicity, i t is assumed that there is a t h w s l r o l d t l o s c , a level of exposure below which thereis no significant riskof occurrence of a health effect. 'The threshold concept is important i n the regulatory context because it holds that a range o f exposures trom zero to some tinite value can be tolerated hy an individual with essentially no chance of an adverse health effect occurring. In contrust, t~otrtl~r-e.sho/tl to.\-icvr/rt.s, generally carcinogenic or mutagenic in nature. are presumed to pose sonne risk a t a l l dosesabovezero. As willbe discussedbelow. the nonthreshold concept requires a clit'ferent evaluation, making use of various mathetnatical models to extrapolate t o the subexperilnental range (SO). While the dosage range used in animal studies may not reflect that encountered by exposed humans, this should not be considered tlctrilnental t o the assessnwnt. The nunnber of times that the same biological effect is elicited in different species, the better chance that, at some dosage. the same effect might be encounteredovera 1000-told range i n three species. For risk assessment, the results from the most susceptible species (and sex) would he chosen arbitrarily as a starting point, this being the nnost conservative approach possible.
C.
Exposure Assessment
The exposure assessment componentof risk assessment involves the process of measuring (or estimating) the intensity. frequency, and duration of human cxposures t o m agent in
Ecobichon
372
an “environment” (in this case, food) or estimating hypothetical exposures that might arise from the release of agents intothe environment. It describes the magnitude, duration, schedule, and route of exposure; the size, nature, and classes of human populations exposed; and the uncertainties in all the estimates (47). Toxicity database availability, relevance, completeness, and deficiencies should be addressed. The amounts of the chemical under scrutiny are quantitated in various media (food, water, air) by which the human could acquire it (51,52). All of these parameters will provide a composite history of the chemical in an attempt to reduce the magnitude of the uncertainties and to address strategies by which exposure can be reduced.
D. Risk Characterization The risk characterization component of risk assessment may be defined as the process of estimating the probable incidence of an adverse health effect (hazard) to humans under various conditions of exposure. If at all possible, dose-response relationships from human studies should be used, but barring that, relevant, well-controlled chronic animal studies should be used as substitutes. Recalling the hazard equation, the risk assessor is concerned about the level in the source and the durationof exposure. What is desired, given the facts thatthe chemical may be needed in food processing or preservation or that low-level residues of a pesticide, metal, or other organic contaminant may always be present in a food, is what will constitute an ADI, TDI, RfD, a virtually safe dose (VSD), or an estimated population threshold (EPT-H). How is this accomplished?
1. ArbitraryApproach With a good database and a well-documented dose-response relationship for a threshold chemical that will permit the derivation of a NOEL or a NOAEL, regulatory agencies can apply a series of predetermined, arbitrary uncertainty factors (UFs) by a factor of 100 (a IO-fold uncertainty factor for intraspecies variability i n response to the agent and a 10fold factor to account for interspecies variability).In a routine first analysis, a risk assessor would apply this 100-fold factor to the NOEL (or NOAEL) or the lowest dose producing a biological effect. Where does this derived level of exposure (expressed in milligrams of agent per kilogram of body weight per day) ‘‘tit’’ in relation to that anticipated by worst-case scenario calculations of exposure? It is a first step.
RfD
=
NOEL
UF,
X
UF:
Additional lnodifying factors(MFs) may be applied to the NOEL or NOAEL values based on the confidence of the assessor on the quality and quantity of data and the fact that the most susceptible species have or have not been studied. Given that the toxicity database 5- or IO-fold to yields ollly a reliable NOAEL, the modifying factor could be increased arbitrarily take care of the fact that, while the lowest dose did not produce adverse health effects, there were detectable differences between this dosage group and the controls. If neither of these values could be ascertained from animal studies but a confident lowest observable adverse effect level (LOAEL) was obtained, the assessor would be correct in applying a larger modifying factor to the LOAEL based on reservations about the quality of the study and the lack of information at lower dosages,with the LOAEL/ 1000 becoming a minimum RfD or VSD.
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RfD =
NOAEL UF, X UFZ X M F
(3)
RfD
LOAEL UF, X UFI X MF, X MFI
(4)
=.
2. ExtrapolationApproach Given the difficulties of choosing an appropriate range of dosages for a chronic toxicity study before the study is done, it is easy to see that many chronic toxicological studies for threshold chemicals will not yielda NOEL. Most studies are less thanperfectand require some type of theoretical extrapolation to yield a plausible VSD or RfD. One such example is shown in Fig. 8. In the first method, a line is drawn from the experimental
0.01
0.001
! NOEL11000 NOELllOO
\
0.l
1.0
/ NOEL
5
l0
50
IO0
/ / LOAEL \ NOAEL
DOSAGE (mglkglday) Fig. 8 A theoretical dose-response relationship showing demonstrable dose-related toxicity over range of 1.0 to 100 mg/kg/day. identifying the TD,, (the dosage that produces a response in 50% of the test animals), the lowest observed adverse effect level (LOAEL), the n o observed adverse effect level (NOAEL), and the no observed effect level (NOEL), all dosage reference points that can be used to derive acceptable exposure values. Three different methods of linear extrapolation from this expcrirnental data to the zero dosage are shown. In # l , a straight line is drawn from the lowest cxpcritnental value. I n #2, the line is drawn from the upper limit of the 95% confidence limit of thelowestexperimentalvalue (i.e., m a n value + 2 standard deviations), with this technique accommodating for the highly sensitive subpopulation responding to a low concentration of test agent. The technique in #3 is usedin carcinogenicity risk assessnlcnts to determine risk-specific doses (RSDs), with the line bCgilllling at TD5,,,a point of minimal deviation in the dose-response relationship. (From Ref. 5 8 . ) ;I
374
Ecobichon
threshold dose (or the lowest dose producing an adverse effect)to the zero value. resulting i n an estimation of effects at substantially lower theoretical doses. The second extrapolation technique, which takes into account the upper confidence limit of the response variability for the population i n the test group, consists of drawing astraightlinefromtheupperconfidenceinterval of the NOAEL [mean -C 2 standard deviations (SD)] to the zero value (53). It can be appreciated that the larger the groups of animals that are used at these lower dosages, the better (or narrower) the confidence limits will be. By using this technique, the assessor arbitrarily anticipates the presence of aberrant, hypersensitive individuals i n a population who, if they could be identified, might respond to concentrations of the chemical lower than the threshold level (54). A third approach, utilizing the full complementof test dosages and the linear, curvilinear, or sigmoidal dose-response relationship, consists of drawing a straight line from is minimal the TDr0 (the toxic dose for 50% of thetestsubjects.atwhichpointthere deviation) to the zero value (55). Manywould consider this linear relationship to bea gross overestimation of possible toxicity at lower dosages. However, the overall goal of the risk assessor is not to underestimate human risk. If one used this third approach, one might have serious considerations about the entire toxicity database. Up to this point we have considered the extrapolation tcchniques for threshold-type chemicals for which there is an exposure levelbelowwhich adverse health effects are unlikely to occur. What about nonthreshold-type (zero threshold) chemicals for which, theoretically, there is always some response no matter how small the exposure is (i.e., one molccule might induce an adverse health effect)? Acceptable concentrations of nonthreshold chemicals are determined by various risk analysis techniques (47).
3. Quantitative Risk Estimation The nonthreshold concept hasbeen derived largely from experimentsi n radiation-induced carcinogenesis for which such exposure might induce an irreversible, self-replicating lesion arising from a nonlethal mutation i n a single somatic cell (56).Not surprisingly, this theory was extended to the quantitative risk estimation for chemicals where onc molecule might be sufficient to cause the mutation in the genome to change the future direction of a single cell irreversibly. However, the nonthreshold concept is contrary to evidence. While everyone is exposed to trace amounts of numerous carcinogens daily, n o t everyone develops cancer i n their lifetime. suggesting that, i n most cases. the exposure was low enough to elicit no response. While the nonthreshold concept has been the dogma tor many years, there appear to berecentshifts i n carcinogenesis theories that may accomodate a threshold concept (57). One area to watch closely is the dose-response relationship for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD),other chemicals, and carcinogenicity (58,Sc)). However, for lack o f good experimental data, the nonthreshold concept is stili i n vogue. If acceptable exposure levels t o carcinogenic compounds could be determined by the application of some n1axinlun1 uncertainty factor, it is important t o recognize that this method of determining a population threshold exposure does not take into account the slope of the dose-response curve. This might give an adequate margin o f safety. given a steep dose-response relationship. but might be insuflicicnt if the slope was very shallow. as many arc at thc low-dose exposure (Fig. 8). There is usually a very poor picture of the relatiomhip at the lower end, made more difficult by thefactthatthe dosage range of carcinogenic concern forthe general population lnay be scvcral ordersof magnitude brlow thoseknowntoelicitanycarcinogenic responsc at higher-level occupational exposure.
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Historically the rejection of a population threshold for carcinogens has been based on (a) variablethresholdsbetweenindividuals or populations, (b) theone-hit,one-cancer hypothesis described in Sec. III.B, ( c ) that cancers arise from single mutated cells and with survival, development, and progression cause metastases in other organs. and (d) the original theories of carcinogenesis being derived from radiation-induced cancers. There is an extensiveliteratureon the number of mathematicalmodels usedto achieve VSD values for carcinogens, summarized in Ref. 60. Three types of models have been proposed: tolerance distribution models (probit, logit, Weibull), mechanistic models (one hit, multihit, multistage), and time-to-tumor-occurrence models (lognormal, Weibull) (50). The utility of these various models is for data obtained from appropriate chronic animal studies, the objective beingto determine a slope factor forthe cancer-dose relationship. Working from a preselected, acceptable level of risk, usually 1 extra cancer case i n a population of IO0,OOO or 1 million (mathematical risks based on scientific assumptions used in risk assessment), and having a suitable graphic derivation of the dose-response relationship, the risk assessor can begin to estimate a VSD for the agent. With the aid of several models, a range o f VSD values can be generated and compared. In a review of extrapolatedvaluesfornitrotriaceticacid,sodiumsaccharin,aflatoxin,and2-acetylaminofluorene (2-AAF). it becameevidentthattheWeibull,logit,multihit.andprobit models all had much steeper slopes, thcreby giving much higher VSD values, than did the multistage or simple linear extrapolation models (6 I ) . The multistage model is believed lo be the most biologically plausible since it incorporates contemporary understandingof chemical carcinogenesis, assumes no threshold for cancer initiation, and allows for the use and "best" fit of the full range of experimental data (49). However, atpresentthereis no completelyacceptablemanner in which to reliably determine a threshold for a carcinogen, which is one fact that is agreed upon by all regulatory agencies.
IV.
CONCLUSIONS
This chapter presentsan overview of the development o f a toxicity database and, regardless of the type of chemical or biological actions.the application of these data to theassessment of potential risks to human health. While it is not a perfect system, a good estimation of potential risk can be detemlincd. provided that a good toxicity database and the correct information on exposurc assessment are available. The inability of regulatory agencies to mect the stringent demands of a critical public is based primarily on the absence of adequate toxicity data for agents that have been in use for many decades.
REFERENCES 1 . D Bradley.Pests. problems andthepublic. New Sci, Nov. 23:57-58, 1991. 2. BN Amcs. Dietary corcinogens and anticarcinogens. Science 22 I : 1256- 1263. 1983. 3. PM Riker. Toxic cfrects o f herbal tens. ArchEnvironHealth 42: 133-136. 19x7. 4. JM Concon. Food atlditivcs. In: Food Toxicology. Part B. Contaminants and Additives.New York: Marccl Dekker. 19x8. pp. 1249- 1340.
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Joint Codex Alimcntarius Commission. List of Additives Evaluated for Their Safety-in-Use in Food. Rome: Food and Agriculture Organizntion of the Uniretl Nations, 1973. National Research Council. Toxicity Testing. Strategies to Determine Needs and Priorities. 1984. Washington, D.C.: National Academy Press, SelectiveCommitteeonGRASSubstances.Evaluationaspects ofGRASfoodingredients: lessons learned and questions unanswered. Fed Proc 16:2527-2562,1977. SO Archibald, CK Winter. Pesticides i n our food.Assessingtherisks. I n : CKWinter. JN Sciber, CF Nuckton. cds. Chemicals i n theHumanFood Chain. NewYork:VanNostrand Reinhold, 1990. NewYork:pp. 1-50, CF Wilkinson. Introduction and overview. In: SR Baker and CF Wilkinson, eds. The Effect of Pesticides on Human Health. Advances in Modern Environmental Toxicology Series, vol. 18. Princeton, NJ: Princeton Scientific Publishing, 1990, pp. 5-33. BG Tweedy, HJ Dishburger, LC Ballantine, J McCarthy. Pesticide Residues and Food Safety. A Harvest of Viewpoints. Washington, D.C.: American Chemical Society, 1991. R Frank, HE Braun, BD Ripley. Residues of insecticides, fungicides and herbicides in fruit produced in Ontario, Canada, 1980-1984. Bull Environ Contam Toxicol 39272-279, 1987. P Slovic. Perception of risk. Science 236280-285, 1987. Aldicarb food poisoning from contaminated melons-California. J Food Protect 49573-574,
1986. 14. PRBennett.Aldicarb-human illness. Health Welfare Canada Memo March IO. 1996. 15. PKChan.AWHayes. Principles and methods for acute toxicity and eye irritancy. In: AW Hayes, ed. Principles and Methods of Toxicology. New York: Raven Press, 1989, pp. 169-
220. DJ Ecobichon. Acute toxicity studies. In: The Basis of Toxicity Testing. 2nd etl. Boca Raton, FL: CRC Press, 1997. pp. 43-86. 17. WB Deichmann, TJ LeBlanc. Determination oftheapproximatelethaldosewith about six animals. J Indian Hyg Toxicol 25:415-417, 1945. I 8. RD Bruce. An up-and-down procedure for acute toxicity testing. Fund Appl Toxicol S : 15 1 16.
157,1985. 19. RD Bruce. A confirmatory study of the up-and-down method for acute Fund Appl Toxicol W 7 - 100, 1987.
oral toxicity testing.
t o express the 20. WB Deichmann. EG Mergard. Comparative evaluation of methods employed degree of toxicity of a compound. J Indian Hyg Toxicol 30:373-378. 1948. of irritation and toxicityof 21. JN Draizc, G Woodward. H 0 Calvery. Methods for the study substances applied topically t o the skin and mucous nlenlbranes. J Phannacol Exp Ther 82: 377-389,1944. 22. AH McCrecsh, M Steinberg. Skin irritation testing i n animals. In: FN Marzulli, HI Maibach, eds. Delmatotoxicology, 2nd cd. New York: Hemisphere Publishing, 1983, pp. 147-166. 23. DJ Ecobichon. Acute toxicity studies. In: The Basis of Toxicity Tcsting. 2d etl. Boca Raton. FL: CRC Press, 1997. pp. 43-86. 24. J Higginson. Present trends in cancer epidemiology. Can Cancer C o d 840-75. 1968. 25. BN Amcs, J McCann. E Yamasaki. Methods for detecting carcinogens and mutagens with the Strlrt~or~elltrlmammalian microsome mutagenicity test. Mutat Res 3 1 :347-364, 1975. 26. J McCunn, E Choi, E Yamasaki, BN Ames. Detection of carcinogens and mutagens with the Solrtror~ellrrlmicrosorne test: assayof 300 chemicals.ProcNatAcadSci 72:5 135-5 139, 1975. I n : The Basis of Toxicity Testing. 2 ed. Boca 27. DJ Ecobichon. Mutagenesis-carcinogenesis. Raton,FL:CRCPress, 1997, pp. 157-190. 28. OrganizationforEconomicCooperationandDevelopment.OECDGuidelinesforTesting
1983. Chemicals. Washington, D.C.: OECD Publications and Infornlotion Center. 29. JL Schardcin. Chemically Induced Birth Defects. New York:MarcelDekker, 1985. 30. DJ Ecobichon. Reproductive toxicology.In: Thc Basis of Toxicity Testing.2d cd. Boca Raton, FL:CRCPress. 1997. pp. 117-156.
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57. EK Weisburger. Mechanistic considerations in chemical carcinogenesis. Reg Toxicol Phannacol 12:41-52,1990. 58. L Roberts.Dioxinrisksrevisited.Science251:624-626,1991. 59. KT Kitchin, JL Brown. Dose-response relationship for rat liver DNA damage caused by49 rodent carcinogens. Toxicology 88:31-49, 1994. 60. DJ Ecohichon. Risk assessment. In: The Basis of Toxicity Testing, 2d ed. BocaRaton. FL: CRCPress,1997,pp.191-210. 61. DR Krewski, J VanRyzin.Dose-responsemodelsforquantalresponsetoxicitydata. In: J Csorgo, D Dawson, JNK Rao, and E Shaleh, eds. Statistical and Related Topics. New York: Elsevier/North Holland, 1981, pp. 201-23 l.
14 Nutritional Toxicology
Introduction 380 II. Nutritional Dcliciencies 381 111. Sugars 383 1.
A.Toxicity IV. Lipids 388
of glycationandglyco-oxidationreactions383
Fatty A. acids 388 B. Trtrr1.s fatty acids 392 C. Cholesterol 393 Macrominerals V. 394 Calcium A. 394 B. Magnesium 396 VI. Microminerals 397 A. 397 Iron B. Selenium 399 C. 399 Zinc D. Mercury 400 E. Aluminum 401 F. Catltnium 401 VII. Food Additives 402 salt substitutcs 402 A. Salt and B. Nitrates 403 C. Methylxanthines 404 VIII.FortificationandSupplctnentation41 A. Vitamin D 41 1 B. Vitamin E 414 C. Vitamin C 416 D. Folic acid 417 IX. Naturally Occurring Toxins 417 Aflatoxins A. 417
1
379
380
Kitts
X. Processed Derived Toxins 420
Benzo(a)pyrenc Heterocyclic amines 422 XI. Conclusions 424 References 425 A. B.
1.
420
INTRODUCTION
Food and oxygen areboth essential for survivalof hulnan life and are intrinsically involved in chemical reactions which initiate, regulate, and modify toxicity in the living organism. The exposure of living organisms to the 20.9% oxygen present in air contributes to the dependency on biological oxidation reactionsas a source of energy for growth and mainte2% of the oxygen consumedby mitochonnance. Under normal conditions, approximately dria is incompletely reduced, resulting i n the generation of oxygen radicals ( 1 ). The body’s antioxidant status is comprised of endogenous antioxidant enzymes [e.g., superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px] as the first line of defenseagainstreactiveoxygenspecies.Inaddition,nonenzymaticantioxidantsarealso required to collectively deactivate reactive oxygen species and reduce oxidative stress. These two systems combined, attenuate the accunwlation of toxic oxygen species, thereby protecting the organism against cellular membrane damage and acute or chronic tissue dysfunction-associated pathologies that Inay lead to chronic disease and aging. The rolc of nutrition in maintaining adequate protection against endogenous and exogenous forms of oxidative stress is an important component of nutritional toxicology (2). Foods represent a vast number of chemical compounds, many of which do not directlycontributetoanutritionalfunction i n the organism. Moreover, thepotential for exposing man to toxins present in food is high dueto the different sources of toxic materials that can enter the food system (Table l). Only in a few, but very important cases, is acute toxicity expressed by foodborne chemical toxicants (e.g., food allergics and intolerances). Rather, toxic effects of many food chemicals are most likely expressed following long-term exposure whichcan lead to the development of chronic diseases, such as cancer, atherosclerosis, diabetes, rheumatoid arthritis, and the aging process. The susceptibility of the individual to chronic diseases is determined not only by the hazards of particular food chemicals but also the genetic make-up of the individual with regard to the metabolism and clearance efficiencies of native or generated toxins. The biological implications of these reactions have been shown to contribute not only to the loss of food quality (e.g., sensory, nutritional. and safety parameters) over time, but also increased exposure of the organism to reactive intermediates, capableof initiating genotoxic and cytotoxic responses and thus increased risk of chronic diseases. Both deficiencies and excesses of particular nutrients will modify the disorder either positively or negatively. Another important factor relevant to food chemical toxicity is the interaction between food components and the metabolism of the host. Such activity is the essence of nutritional toxicology and involves both nutrient-nutrient or xenobiotic-nutrient interactions which take place within the host and yield metabolic products with distinct risks for injury. The role of nutrition in protecting the host from a barrage of potential foodborne toxinsisalso vitally important in appreciatingthespecificnlechanismsinvolvedwith detoxifying reactive species present in food or generated on metabolism. Both epidemio-
Nutritional Toxicology
381
Table 1 ToxicChemicalsinFoods
Inadvertent or accidental contaminants toxicants Derived toxicants occurring Naturally
Food additives (food dyes) Food processing practices (irrndiation. hydropcnation) Nutrient-toxicant metabolic interactions (nitrosamines) Cooking (thermal degradation, oxidation. leaching of nutrients)
Food fortification or preparation accidents Food contaminants during production (nntihiotics. pesticides) processing (stabilizing agents). storage (packaging materials)
logical and experimental studies have provided convincing evidence that the human diet contains”extranutrients” whichare capable of affordingprotectionagainstoxidative stress and associated chronic diseases (3.4). Toxicology is a scientific discipline concerned with the detection, occurrence, physiochemical properties, physiological effects, and risk assessment of potentially toxic materials. Nutritional toxicology emphasizcs both the potential deleterious effects of nutrient deficiencics or excesses in enhancing food chemical toxicity, as well as the mechanisms of action of nutrient-nutrient and nutrient-xenobiotic interactions in the manifestation and neutralization of potentially harmful reactions.
II. NUTRITIONALDEFICIENCIES Nutritional deficiencies can influcncc the sensitivity of tissues to oxidative stress. Dietary components such as vitamin E, selenium.sulfur-containingaminoacids,anddifferent minerals, such as iron, copper, and zinc, have a bearing on thc oxidative status of the organism ( 5 ) . Many studies havc classified vitamins A, B-complex, and C, together with of preserving the integrity of various zinc, as antioxidant nutricnts from the standpoint anatomical barriers that ensure cell repair and regeneration in the prevention of disease. Other nutrients, such as a-tocopherol, p-carotene, and ascorbate, also have important roles in preventing free radical propagation and chain elongation. Riboflavin, by maintaining intracellular glutathione (GSH) concentration, is an important cofactor in glutathione reductase (GSSG-Red) activity, an enzyme requircd to regenerate the nonenzymatic antioxidant GSH (2). Increases in the generation of frce radicals derived from oxygen metabolism are accompanied by a predisposition to lipid pcroxidation reactions and cellular damage. Thus
382
Kitts
it follows that a conlplex network of nonenzymatic and enzymatic defense that protects the cell from oxidative stress is closely dependent on the level of nutrient availability. In general, a lowering of dietary assisted tissue antioxidant defense is associated with an increase in free radical reactions that ultimately increase cellular damage and accelerate the aging process. One apparent exception to this is found with food energy restriction, which has been shown in rodents to increase longevity and retard the aging process. The reduction in total calorie intake, by reducing feed intake in mice, decreases spontaneous and chemically induced tumors (6) and increases life span (7). Since i t has been shown that the accumulation of lipid peroxidation products and changes in SOD and glutathione GSH-Px activities are indicators of the aging proccss (8-10). itis of interest that diet in SOD (Cu, Zn-SOD). catalase (CAT), and GSH-Px in restriction leads to an increase most tissues, with the exception of the intestinal nmcosa ( 1 1 ). An increased tissue antioxidant enzyme activity responseto caloric reduction is likely due to decreasesin free radical damage, which assists in the explanation of the corresponding reduced lipid peroxidation ( 12) and protein and DNA oxidation ( 13,14) reactions noted in rodents fed restricted diets. Since food restriction does not seem to reduce metabolic rate( I S), we may conclude that energy restriction does not alter oxygen uptake and therefore the production of free radicals. Protein deficiency, on the other hand, has been shown to cause loss of appetite and resultant loss of weight leading to severe wastage. This condition results i n a reduction in intestinal and hepatic protein concentration and adversely influences antioxidant status, as evidenced by the reduction in glutathione (GSH) and other tissue-specific and enzymespecific changes i n antioxidantenzymedefense in animalsfed proteinrestricteddiets (16,17). Restriction of dietary protein reduces the availability of the cellular tripeptide, GSH, which iscriticalforGSH-Pxactivity.Associated withtherestriction of dietary methionine is the nutrient-nutrient interaction that involves the metabolism of selenium for available selenium-dependent GSH-Px biosynthesis ( 18). These findings correspond to observations made by others that animals fed low-protein diets are predisposed to an accumulation of lipid oxidation products [thiobarbituric acid reactive substances (TBARS)] (19). The effect of proteinmalnutritionmaybefurthercomplicated by the evidence that vitamin E utilization is impaired in protein-restricted diets (30).This predisposes tissues to lipid peroxidation, which Inay occur due toa combination of lowered nonenzymatic antioxidant (e.g., vitamin E ) activity and reduced enzymatic antioxidant activity. For example, supplementing diets with vitamin E has been shown to reduce the toxicity of silver-related selenium deficiency ( 2 I ), thus further exemplifying the complexity of nutrient-nutrient interactions i n the expression of toxicity. These and other examples have clinical importance, with the example of kwashiorkor. ;1 condition manifested by protein deficiency, with or without a delicit in total energy,in children from very underdeveloped countries, occuring as a result of free radical-initiated tissue damage (22). Other studies have demonstrated that a protein deficiency in rats increases the acute toxicity of aflatoxin B 1, an observation which can be reversed if the animals recieve DDT (23). This finding indicates that microsomal hydroxylating enzyme systems detoxify aflatoxins and as a result reduce toxicity by favorably altering the metabolism or clearance of the toxin. Other workers have further demonstrated that protein-deficient rats were no re sensitive to aflatoxin and pesticide toxicity (24,2S) than counterparts fed adequate protein diets. The importance of GSH S-transferase and sulfotransferase activities in conjugntion detoxification reactions involved with removalof xenobiotics further explain the effectof methionine and cysteine deficiencies and low protein intake on increased aflatoxin toxicity intakes (24).
Nutritional Toxicology
111.
383
SUGARS
Sugars represent important functional properties in food systems, in addition to offering a sweet taste and providing a readily available source (e.g., approximately2 1%) of dietary energy. Sugars include the primary monodisaccharides as well as the carbohydrate sweeteners (e.g., sucrose, honey, high-fructose syrups). Although there are many concerns regarding the suggested association between sugar and health concerns, there are no conclusive data to link normal sugar intake with public health hazards, with the exception of the contribution of sugar to dental caries (26). The high sweetness coefficient for fructose makes it a primary monosaccharide for use in beverage and formulated food systems. The structural difference between fructose (2-ketohexose) and glucose (l-aldohexose) is paramount in regard to the unique aspects of fructose metabolism relative to other hexose sugars. In normal individuals, 80-90% of fructose following consumption passes through the intestinal epithelial cells, with the remaining 10-20% transformed by the enzyme fructokinase into fructose 1-phosphate. Only a small quantity of fructose is converted to fructose 6-phosphate by hexokinase in the intestinal mucosa, since the efficiency of metabolizing fructose by hexokinase is very low, and glucose competes for the hexose enzyme. Fructose, in contrast to glucose, does not elicit pancreatic insulin release, which is an important metabolic characteristic when of fructose consumption.In the liver, fructose is phosphorevaluating the risks and benefits ylated by fructokinase to yieldfructosel-phosphate; an intermediatewhichissubsequently split by fructose 1-phosphate aldolase into trioses, dihydroxyacetone phosphate, and D-glyceraldehyde. Since fructose enters metabolism by escaping the limiting step of fructose 6-phosphate to fructose 1,6-diphosphate conversion,the flow of this monosaccharide to the terminal products of hexose dissimulation, such as pyruvate, lactate, mitochondrial acetyl-coA, and tricarboxylic acid metabolites is greatly increased. The excess of metabolites in the tricarboxylic acid cycle enhances metabolic processes such as lipogenesis. Thus hepatic lipogenic capacity is increased and hyperlipidemia can be expressed by a fructose-rich diet (27).
A.
Toxicity of Glycation and Glyco-oxidation Reactions
Schiff bases form in vivo at a rate directly proportional to the glucose concentration, since protein concentrations are relatively constant. The reversible equilibrium nature of early glycosylation products is therefore important because it implies that the total amount generated under physiological conditions is a function of tissue compartmental sugar concentration (28). Schiff bases rearrange to fom1 stable Amadori glycosylation products:a reaction which varies in rate between sugars (29,.30) and proteins (31,321. Due to the slow turnover into Amadori products, theearlyphase of theglycosylationpathwayistime dependent. Moreover, Schiff base products at equilibrium levels are established in hours (32), whereas Amadori rearrangement equilibrium levels are reachedin weeks (28). This reversible equilibrium behavior of early glycosylation products is important, since it implies that the total amount of glycation products generated reaches a steady-state plateau even in long-lived proteins within a short time period. Indeed, glycation of human lens protein and skin collagen remain essentially constant between the ages of 5 and 80(33,34). The production of early glycosylation products, irrespective of whether originating from hemoglobin or basement membrane proteins, increases when blood glucose levels are high (35). and returns to normal after blood glucosc returns to regular levels
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Whereasglycation(glycosylation)involvesthedevelopment of Amadori rearrangement products from the food, referred to as the source of the Maillard reaction, glyco-oxidationistheoxidation of free glucose and intermediates withinthe Maillard 1). It was initially reported reaction to form highly reactive fragmentation products (Fig. that the oxidative fragmentation of the Schiff base prior to Amadori adduct formation was the primary mechanism forthe generation of 2- and 3-carbon browning intermediates(36). The Schiff base therefore undergoes an oxidative retroaldo condensation at both the C2 and C3 positions to eventually form glycoaldehyde and glycoxal, respectively. The significance of this mechanism is that the rates of browning formation by small molecular dicarbonyl compounds are considerably higher than reducing sugars and, moreover, that at neutral pH fragmentationof the Schiff base sugar moiety contributes substantially more to nonenzymatic browning. This mechanism, albeit demonstrated under nonphysiological conditions, showed the potential reactive capabilities ofdicarbonyl compounds which have been established to form in vivo (37). Schiff bases therefore can contribute to the formation of advanced glycation end products (AGE)by way of an intermediate pathway. An alternative mechanism involves an autoxidative glycosylation reaction, whereby a-ketoaldehydes formed by oxidation of glucose react with protein and initiate the advanced stages of the Maillard reaction (38). The enolization of glucose contributesto reduced molecular oxygen under physiological conditions through a transition metal catalyst, yielding a-ketoaldehydes and intermediates (39). Given that the autoxidation of glucose is slow, the quantitative amount of a-ketoaldehydes and oxidizing agents formed with respect to time correlates with protein damage and cross-linking (40). Moreover, the development of reactive oxygen species in the presence of copper ion and oxidation would also be expected to contribute to the collateral damage of proteins and lipids. During the life span of most plasma and cellular proteins. Amadori products are in equilibrium with the glucose concentration and due to transformation rates, which take weeks to months to establish, these products do not develop further in the nonenzymatic browning pathway. However, some early glycosylation products of collagen and other long-lived proteins present in blood vessel walls undergo a slow, complex seriesof chemicalrearrangementreactions to formirreversibleadvancedglycosylationendproducts (AGEs) (35). AGEs are currently believed to include diverse structures. some of which are already cross-linked to aminesor are at the intermediate stageof cross-linking (35,41). One important aspect concerningthe formation of AGEs is that the rate of AGE accumulation is proportional to the time-integrated blood glucose level over long periods of time (42). This implies thatthelevel of AGEs does notreturntonormalwhenglucoseis withdrawn, but rather the products may accumulate over the life span of the proteins. Of equal importanceis the fact that the quantity of cross-linked AGEs increases proportionally with the equilibrium concentrationof Alnadori product. Therefore once proteins have been exposed to excess Anladori product, as is the case with diabetes mellitus, the restoration of glucose control maynot prevent progression of glucose-mediated macromolecule crosslinking. Two of the primary AGE compounds identified are shown in Fig. 2. Pyrraline, derived from the reaction of 3-deoxyglucosone with free lysine residues, under physiological conditions has been reported to be present at twice the concentration in hyperglycemic individuals (43), and is present in elevated amounts in glomerular and vascular wall basement membranes (44). The accumulation of the acid stable product pentosidine has also been reported to be positively correlated with aging and diabetes, with levels increasing exponentially from S to 75 pmol/mg collagen overthe life span and a 3- to IO-fold increase
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-11
t
-
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Pentosidine
Fig. 2 Chemicalstructures of pyrralineandpentosidincadvancctlglycosylation (Refs. 65, 66).
end products.
insubjectswithinsulin-dependentdiabetesmellitus(IDDM) (45). Sincetotal in vivo plasma pentose concentrations are many fold less than glucose, the presence of elevated levels of pentosidine would also suggest abnormalities in pentose nletabolisrn in subjects suffering from IDDM. Alternatively, this AGE product has been shown to be derived not only from pentoses, but also from hexoses and ascorbate (31). Lipid-AGE formation has also been reported from examples including anline-containing lipids such as phosphatidylethanolamine (PE) (46). Many of the health-related effects associated with AGES are presented in Fig. 3. AGES are known for both direct and indirect chemical reactivity and have been linked to numerous pathological manifestations of diabetes and aging (47,48). Other workers
Quenching of nitric oxide action
-
Reduced endothelium derived relaxing factor (EDRF)
.1
Defective vascular relaxation and hypertension 0
0
0
Cross-linking of collagen
P Increasedaging
Cross-linking of soluble plasma proteins m low densitylipoproteins m IgG
+ Reduced 3
LDL receptor clearance Altered immune system
DisturbanceofmacrophageandmonocyteAlterationsinimmune-likegrowth necrosis tumor >(IGF-l), factors proteins surface factor-a (TNF-a), and granulocyte macrophage colony stimulating factor
Fig. 3 Pathologicalsignificance of advancedglycationproducts.
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hypothesize that AGEs are not necessarily directly involved in the pathogenesis of diabetes, but rather are indirectly involvedby being a sourceof reactive oxygen species (38.49). Correlations between AGE formation and diabetic conlplications have been reported in human skin collagen, retinopathy, and arterialand joint stiffness (50-52). AGEs also lead toa decrease in solubilityandsusceptibilitytoenzymaticdigestionaswellasprotein cross-linking. which are particularly relevant to connective tissue and extracellular matrix collagen, since both are associated with increased rigidity, thickness, and breakage, typical of the tissue aging process (51.53). The quenching of nitric oxide, an active component of endothelium-derived relaxing factor, by AGE-modified proteins is also a function of AGE concentration, thereby indicating that AGEs may be involved in diabetic or agerelated defective vascular relaxation and hypertension (54). Other findings indicate that AGE modification impairs LDL receptor-mediated clearance mechanism, thus contributing to elevated absolute and oxidized LDL levels in diabetic patients (55,56). Glycated LDL is immunogenic and may alter platelet function or promote thrombosis (57). Moreover, glycation of HDL-Apo A1 impairs cholesterol efflux from cells (58). Lipid-AGE formation, which has been found to be elevated almost fourfold in diabetic patients, has been positively correlated with LDL oxidation, fatty acid oxidation ( 5 6 ) , and associated with an increased rate of lipid peroxidation (38.59) and glycated hemoglobin levels (60). Collectively these observations have lead to the hypothesis that malondialdehyde (MDA), a secondary product of lipid peroxidation, may be involved in glycosylation reactions and in progression of atherosclerosis in diabetes mellitus (61) may be a contributing factor (Fig. 4).
Malonaldehyde COOH
Enolate
Fig. 4
Schiff Base
Lysillc-malondialtfehydc conjugation Schil'l products
COOH
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Considerable investigation has been dedicated to determine the mechanism(s) by which inhibition of AGE formation could occur for the purpose of preserving the function of collagen basement and other membranes which may play pivotal roles in the long-term complications of diabetes or aging. To date, three possible strategies have been investigated, including Inhibition directly at the site of the glycation pathway Work toward adjusting protein characterization Reduction of secondary oxygen species Aspirin (acetylsalicylic acid) has been shown to prevent nonenzymatic glycosylation at the site of lens cystallins (62).Aminoguanidine, a small hydrazine-like molecule is also thought to prevent advanced glycosylation by reacting with nonprotein-bound derivatives of early glycation products, suchas 3-deoxyglucosone, and prevent developmental complications of diabetes in animal models (63). A potential adverse effect of this drug involves its pro-oxidant activity, which hasbeen shown to involve generation of hydrogen peroxide and inactivation of the antioxidant enzyme catalase in vitro (64). Dietary measures to reduce AGE formation include the water-soluble vitamins B I and B,,, as well as the lipidsoluble vitamin E. Thiamine pyrophosphate and pyridoxamine both exhibit anti-AGE formation properties, with pyridoxamine possessing stronger inhibition by forming Schiffbase linkages with carbonyls of open-chair sugars and Amadori products (65). Further in vivostudiesarerequired to determine theefficacyofthispotential,sincepreviousin vitro investigations were performed at elevated levels (65,66). Other studies performed in diabetic patients and rat models have reported reduced glycated hemoglobin levels with vitamin E supplementation (67). With the generation of glycoxidation products and reacit has been confirmedthat vitamin tive oxygen species occurring during AGE arrangement, E inhibition likely works to block protein glycation by inhibiting MDA formation (68); a noted precursor for primary AGE adduct generation (69).
IV. LIPIDS A.
Fatty Acids
Fat represents an important nutrient for the body, as a primary source of energy and for the presence of linoleic, linolenic, and arachidonic acids, all of which are required for norlnal metabolic and physiological function. These three fatty acids are also referred to as essential fatty acids (EFAs), due to the fact that they are not synthesized efficiently by the anilnal organism and therefore must be received from the diet (70). The partitioning of fats for oxidation or synthesis (e.g., incorporation into adipose triacylglyceride or tissue structural lipids) depends on the fatty acids that comprise the lipid source (71) as well as the metabolic demands of the individual. The biological roles of EFAs entail maintaining growth and reproductive function, skin and hair health, and regulating sterol and cicosanoid metabolism. These roles are greatly important to the metabolic homeostasis of the organism. A list of symptoms associated with EFA deficiency is given in Table 2. Clear symptoms of EFA deficiency occur only after severe withdrawal of EFAs for considerable time periods, since linoleic acid can reside i n body stores and therefore mask an EFA deficiency. Moreover, once the deficiency has been addressed by supplementation or a change of diet to include EFAs, symptomsof EFA deficiency disappear relatively quickly
(72).
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Table 2 DeficiencySymptoms of EssentialFattyAcids
toms
Gross
Reducedgrowthrateand invisualacuity Desquamated dermatitis skin Scaly Kidney abnormalities
loss
DecreasedPUFA in membranephospholipids
Reduced glomerular permeability, filtration and Naexcretion (PGF,.,,,, PGE,, PGH?, PGI,) Decreased capillary resistance Reduced w3-w6 cicosanoids (Pgl,, PG1 \, TXA2, TXA,) Increased erythrocyte fragility Erythrocyte deformation Reduction in immuneresponseReducedcytokineresponse,chemotaxis,andleukocyte receptor exprcssion Abhrcvlattons: PGF,, = prostaglandin F,,. PGI,, = prostaglandin l,,: TXA,, = thrombox;~neA,, (nun1el-i-
cal subscripts rcfcr to cicosanoid subclass).
Epidemiological studies have established a link betweendietarylipidintake and coronary heart disease (CHD).In Japan, the incidence of heart disease is relatively low and the diet is rich in polyunsaturated fats (73). In contrast, Scandinavian and North American countries not only consume an average diet containing traditionally more fat (e.g., 40% of total calories), but also a higher intake of fats rich in saturated fatty acids (74). Risk factors for atherosclerosis include hyperlipidemia, hypertension, insulin resistance, and genetic predispositions, manyof which are influenced by diet. In the case of genetic predisposition to atherosclerosis, familial hypercholesterolemia and family histories of hypertension are noted (75). Clinical studies have also demonstrated that the level of dietary lipid intake can affect the incidence of dyslipidemia (76,77). Individual lipoprotein fractions including very low density (VLDL), low density (LDL), and high density (HDL) lipoproteins are useful indicators of CHD status, and in both human and animal studies have been shown to be sensitive to dietary polyunsaturated fatty acid (PUFA) intake (78,79). The affinity of 0-6 and 0-3 PUFA to bring about changes in plasma lipid concentrations has been related to associated effects on lipoprotein composition and size, as well as tissue lipoprotein metabolism (80,Sl). Hamsters fed PUFA (e.g., linoleic acid) exhibit reduced plasma LDL cholesterol concentrations compared to counterparts fed saturated fats; an observation that was associated with a reduced LDL cholesterol production rate and an increase in hepatic LDL receptor activity. In further studies to investigate the effect of dietary PUFA on lipoprotein kinetics, a reduced plasma LDL cholesterol concentration attributed to a 50% reduction i n LDL apolipoprotein B pool size was reported (82). Moreover, there wasa significant correlation between hepatic apolipoproteinB/E receptor number and in vivo receptor-mediated LDL fractional catabolic rate. These findings indicate that PUFA diets modulate plasma LDL cholesterol levels in part by altering lipoprotein composition and receptor-mediated catabolism. It is noteworthy that the effect of the W3 PUFAonloweringplasmacholesterol isrelated to theratio of linoleic (CIX.2e,.h) to saturates consumed (83). Studies have demonstratedthat the beneficial effects of 0-3 fatty acids were greater in subjects consuming mainly saturated fats, as opposed to vegetable oils. This observation indicates a potential competition between 0-3 and 0-6 fatty acids for specific metabolic enzymes that involve plasma lipid responses to dietary fats. In addition to vegetable-derived W-3 fatty acids, it has also been demonstrated that the W-3PUFAs found in fatty fish have a role(s) in reducing the risk of coronary heart disease. Consider-
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able study has focused on the relationship between0-3 PUFAs and changesi n platelet and vessel wall prostaglandin biosynthesis as wellas platelet function (84,85).Consumption of marine oils has also been linked to reduced levels of plasma cholesterol and triacylglycerides (8637). The fatty acid composition of dietary lipids are not the only faclor influencing serum lipids, as evidenced by the role of cholesterol originating from the saponifiable fraction of animal fat sources in modifying the lipidetnic effect of some saturated fatty acids and liver lipids (88.89). The cholesterol raising effect of saturated fatty acids, palmitic and myristic in particular, is significant when considered in combination with cholesterol intake (90). Alternatively, the presence of phytosterols, abundant i n 18t-soluble fractions of plants, can reduce plasma total and low density lipoprotein cholesterol levels. with no effect on HDL or triacylglyceride levels(9 1 . K ) . The free phytosterols present i n the intestine followinghydrolysisdecrease thesolubility of cholesterol i n theoil andmicellar phases, thus reducing cholesterol absorption. Blood pressure in humans may also be influenced to some extent by dietary lipid composition (93,94), althougha direct cause-effect relationship between hypertension and dyslipidemia has not been clearly established.In a study on the long-term effects of various diets containing different levels of saturated, monounsaturated. and polyunsaturated fatty acids, it was shown that an increased intake of saturated fatty acids raised blood pressure in men, while a diet low in saturated fats and high i n linoleic acid actually lowered blood pressure (95). The theory that unsaturated fatty acid incorporation into membranes will increase cell permeability and the activity of sodium pump and cation exchange transport (96) has not been confirtned by supplementation with linoleicand oleic acids(97). Alternatively, blood clotting factors correlate well with serum triacylglyceride levels (98). Elevated LDL levels have been linked to the inhibition of an endothelium-derived relaxing factor. and low HDL-cholesterol levels were reported to interfere with endothelial production of prostacyclin or contribute to prostacyclin degradation. These mechanisms would be expected to affect vasodilation and thrombotic events. Diabetes mellitus. another risk factor for CHD, occurs i n combination with dyslipidenlia as evidenced by the elevated serum cholesterol and triacylglyceride levels common to diabetics. Moreover, hypertension and insulin resistance are also highly significantly associated with diabetes, as are hyperinsulinenlia, a conitnon pathophysiological feature of obesity, glucose intolerance. and hypertension (99,100). These findings Inay describc interrelatedcontributingfactorsthatcanbemodified by dietaryfoodhabits.including possibly dietary fat intake. Oxidative cellular injury is a comnon source of several diseases, including atherosclerosis,hypertension. anddiabetes,andmaybemodulatedbyspecificdietarylipids (87,101). The peroxidation of PUFAs i n biological membranes causes alterations in fluidity and reduction in membrane potential leading to an increased permeability of H - and other ions and culminating in eventual rupture and release of cell and organelle contents such as lysosolnal hydrolytic enzymes. Fig. 5 illustrates the generation of free radicals by chain process peroxidation reactions that lead to the deterioration of PUFAs and which generatelipidoxidationproductssuchasfattyacidhydroperoxides.Usingtheradical generator AAPH (2,2’-azobis(2-amidi11opropatle) di-hydrochloride) to test peroxyl radical generation fro111 different PUFAs, Tirosh et al. (102) demonstrated that the relative order of susceptibility to lipidperoxidationwas C:: , + C:,, ”+ CIS 2 . The observation that subjects consuming thermally oxidized soybean oil exhibited higher levels of thiobarbitllric reactive substrates (TBARS) i n chylomicrons compared to control individuals, dem-
39 1
Nutritional Toxicology
Chain-H-
Ch. +H*
v=v 1-H*
J Chain-OOH
cyclic Peroxide Fig. 5
Schenlcforlipidperoxidationreactionsgeneratinglipidoxidationproducts.
onstrated that lipid oxidation products are indeed absorbed from the gut (103). Linoleic acid hydroperoxide and unsaturated aliphatic aldehydes are toxic to hunian endothelial cells ( 104). Moreover, endothelial cells, monocyte/nlacrophage and smooth muscle cells can modify LDL by a similar peroxidation reaction, producing a modified apolipoprotein containing LDL that resides with the lysolecithin component of the molecule, and which exhibits cytotoxic and atherogenic properties. Oxidized LDL induces functional changes i n endothelial cells which accelerates the formation of fatty streaks and enhances the production of chemotactic activators. These reactions ultimately lead to the generation of foam cells and the advanced lesions characteristic to atherosclerosis (87). Dietary fats have also been associated with the initiation and/or promotion of mammary carcinogenesis (105). A number of studies conducted in rodents have specifically demonstrated that 0 - 6 PUFA derived from vegetable oils can promote lnamnary tumor development ( I06), where flax seed, cc-linolenic acid ( 107), and marine oils rich in eicosapentenoic and docosahexenoic acids protect against breast cancer (108). The0-3 oil coniponent of the flax seed has beenidentified as having a greater chemoprotective effect when tumors are already established (107). The fact that the ratio of long chain 0-3 fatty acids to total 0-6 fat was found to be inversely associated with the incidence of breast cancer i n four of five different European communities, further indicates that a balance between w-3 and w-6 fat sources is important in protecting against breast cancer (109) and possibly other disease states. The underlying mechanisms that specific fat source differences provide i n promoting or protecting against mammary cancer have included generation of diverse precursors i n prostaglandin products ( 1 IO) and the inhibitory efficacy of
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0-3 PUFA i n reducing the mevalonate pathway ( 1 1 1 ), which is required for activation of growth-regulatory proteins involved in signal transduction pathways. Thermal processing of fats and oils also results i n the production of triacylglyceride degradationproducts(e.g.,hydroperoxides,aldehydes,carbonyls, and polymericcompounds) which possess mutagenic and genotoxic activities( 1 12). Repeat frying of cottonseed oil produces significant mutagenic activity i n S. /?phinruriurn TA 98, 100, and 102 strains that are attributed to lipid oxidation products ( 1 13). Similar mutagenicity could not be detected with more saturated fats, such as coconut or palm oil (1 14). It is also apparent that consumptionof oxidized lipids can deplete a-tocopherol in the gastrointestinal tract, thereby reducing the effectiveness of protecting against free-radical scavenging to activities ( I 15). Ingestion of thermally oxidized sunflower oil has also been shown reduce the synthesis of arachidonic acid from linoleic acid by inhibiting A5 and A" desaturase activity. The overall effect of this modification is the change with membrane lipid composition and fluidity and associated functions ( 1 16). The potential toxicity from thermal processing of fat is not limited to oils, as shownby the extensively thermally oxidized beef tallow which can result in a peroxide value greater than 200 mEq/kg and can elicit a positive response to a number of crypt foci i n rodent colon tissue ( 1 17).
Trans Fatty Acids
B.
Trcrrls fatty acids occur both naturally by fermentation in ruminant animals (e.g., vaccenic acid-l 1 trorrs-C,, I ) or through processing of vegetable oils using hydrogenation procedures. With consumption of hydrogenated vegetable oils, the intake of t r v r r l s fatty acids has been estimated to comprise 5 1 0 % of fat in American diets. The three primary changes that take place with the fatty acid structure during the hydrogenation process include
1.
An increase in thesaturatedandmonounsaturatedfattyacidcomposition,resulting i n a decrease in the PUFA content. 2. The formation of double bonds which take the form of either cis or f r v m configurations due to isomerization. 3. The formation of geometric isomers as the double bonds migrate ontheacyl chain. The chemical changes occurring with the lipid source due to hydrogenation facilitate a transition from the liquid stateof the oil to a plastic vegetable fat with reduced susceptibility to lipid oxidation. Studies that have evaluated the health aspects of frvrrrs fatty acids have remarked on the concerns regarding increasing the proportion of dietary saturated fat and reducing the level of EFAs, such as linoleic and linolenic acids (118). Epidemiological studies have also reported that f r m s fatty acids alter cholesterol and are associated with coronary artery disease in some countries (1 18-120). The primary concern involves the production of tr.crrrs-CIs (elaidic acid), which is absorbed to the same extent as thecisC,, (oleic acid) isomer, but due to a less efficient utilization leads to increased retention i n membrane lipids which may reduce membrane fluidityasa consequence. A similar phenomenon with membrane function is observed with saturated fatty acids. The true significance of this outcome has to be clearly shown to warrant the concerns expressed from studies that have reported nonfatal acute myocardial infarction to be associated with a moderate to high intake of margarine, with an even greater risk associated with older individuals and smokers( 121). An evaluation of the conclusions drawn from epidemiological studies has identified the importance of taking into consideration the timing sequence
,
,
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of exposure to t r m s fatty acid intake and health outcomes (122). It has been shown that while total fat intake in the American diet has decreased 4% (38% to 34%), this change i n dietary habits has not translated into an acceptable decrease in t r ( / m fatty acid consumption ( 1 18). However, a replacement of 2% of the energy derived from t r n m fatty acids by unhydrogenated, unsaturated fats has been estimated to reduce CHD risk by 53% ( 1 19). Thus greater reductionsi n t r m s fatty acids intake will likely only be achieved by changing manufacturing processes rather than altering specific food choices.
C. Cholesterol In elucidating the mechanism of development of cardiovascular disease (CVD), plasma hypercholesterolemia is considered to have a critical role in the etiologyof atherosclerosis ( 1 23). A hallmark of atherogenesis is the presence of lipid-laden foam cells containing cholesteryl esters beneath the arterial endothelium ( 1 24). Similarly, acute responses i n rabbits to the intravenous adlninistration of cholesterol oxidation products (COPS) has indicated the presence of arterial lumen damage similar to that seen in the early stages of atherogenesis (125). Although the progressionof atherosclerosis appears to follow the general stages of lipid peroxidation reactions described in food systems, the nature of the initial injury to the arterial endothelium has yet to be clearly identified. This notwithstanding, cytotoxic, oxidatively modified LDL (mLDL) and COPS (e.g., 25-hydroxycholesterol, cholestane-3P, Sa6P-triol) play key roles in the etiology of atherosclerosis ( I 26). Cholesterol(C,,H,,O)is a tetracyclicstructurecontaining a secondaryhydroxyl group and a double bond. Enzymatic and nonenzymatic reactions are implicated in the formation of numerousoxysterolsboth in food systems and in vivo. The free radical auto-oxidation of cholesterol is initiated by azo compounds, peroxides, hydroperoxides, transition metal ions, and excited molecular oxygen. Photolysis by visible and UV light andionizingradiationcan also generatefreeradicals. The extent of cholesterol autooxidation depends on the physical state of the molecule, pH, temperature, time, and the presence of transition metal ions (e.g., Cu” 10. M) and of metal ion sequestering agents. Fig. 6 shows the mechanism of cholesterol auto-oxidation. The issue of the quantity of cholesterol oxidation products in the human diet is exceedingly important in light of the conclusion that cholesterol oxidation products are far more damaging to vascular health than native cholesterol itself (127). Some of the products of cholesterol oxidation are also weakly mutagenic in the Ames test (1 28). Only recently has there been data generated to identify the different food sources of COPS and their relative concentrations ( 1 12). COPS are present in meat and egg products as a result of native cholesterol and relatively high levels of free iron or other transition elements. In unprocessed food products, levels of COPS are very low; however, following thermal processing (e.g., spray drying, hot air drying) and storage, the concentrations of different 1 to 50 ppm.Absorption of COPS fromthe COPSincrease to levelsthatrangefrom intestine via the chylomicron fractions occurs relatively quickly and similar to standard triacylglyceride (129). Following absorption, COPS reach levels as high as 34% in VLDL, 56% in LDL, and 10% in HDL. It has been postulated that the preferential transport of 25-hydroxycholesterol by VLDL and HDL are the basis for potent atherogenicityof these lipoprotein species. It should be noted that oxysterols are formed enzymatically for physiological purposes, as is the case for the synthesis of 3P,7a-diol, which is a precursor for bile acid synthesis. Oxysterols also may serve as regulatory agents that are implicated in de novo sterol biosynthesis [e.g., inhibition of 3-hydroxy-3 methylglutaryl coenzyme A
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J$y4AH&
H
=H&oo.
Radical Peroxy Radical Cholesterol Allyl
7-Hydroperoxide 5,6-Epoxide Radical Alkoxy
H&OH
H & -
7-Hydroxycholesterol 7-Ketocholesterol Fig. 6 Scheme for cholesterol autoxidation and
generation of cholesterol oxidation products.
reductase (HMG-CoA) activity], or in other in vivo eventsthat influence plasma membrane permeability, function, and stability ( 1 12). For example, a number of cholesterol oxides, including25-hydroxycholesterol,7-ketocholesterol, 7 a - and7P-hydrocholesterol,and cholestane-3P,5a,6P-triol exhibit potent inhibitory effects on cholesterol biosynthesis in a variety of tissues by blocking HMG-CoA reductase, HMG-CoA synthetase, and methyl sterol oxidase activities. In contrast to phospholipid properties within the membrane bilayer, the presence of cholesterol increases stability, decreases the mobility of molecules, and reduces the permeability of the membrane as a result. Moreover, cholesterol requires a higherenergyrequirement to oxidize the molecule compared totheperoxidation of phospholipids. These important facts have led to the suggestion that cholesterol exhibits a protective effect by intercepting oxidants and giving rise to less toxic oxysterols (130). In contrast to the potential toxic effects of cholesterol and the oxidized products of cholesterolaretheinterestingfindingsthatcholesterolinhibitedbothratmammary tumorigenesis ( 131 ) and promotion of aberrant crypt foci in rodent colon (1 32). Oxidized cholesterol has becn shown to enhance ( 1 33) or produce no significant effect ( 1 32) in the development of colon aberrant crypt foci, which represent putative precancerous lesions. An almost identical result concerning products of oxidized cholesterol, including 3P, 5a. 6P-cholestanetriol, 7P-hydroxycholesterol, cholesterol 5a,6a-epoxide, and 7-ketocholesterol, was reported with the inhibition of rat mammary tumorigenesis ( 131 ).
V.
MACROMINERALS
A.
Calcium
Calcium (Ca) makes up 39% of the total human body minerals and 2% of body weight. This mineral has several important functions, including mineralization of bone and teeth, blood clotting, stimulation of secretory activity in endocrine, exocrine, and neurocrine cells, and regulation of muscle contraction. It is an essential nutrient in young children
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for the preventiol1 of rickets, and in adults for preventing osteoporosis, or brittle bones. in later life. More recently, an important interaction between Ca metabolism and EFAs has beell noted in regard to osteoporosis and ectopic calcification ( 134). It is geI1erally accepted thatthemajority of thepopulation does notreceivethe reco111111el1decl dailyallowance(RDA)for Ca (700 mgfor wo111etl; 800 111g for Illen) (135.136). I t is therefore important to know which foods represent good Sources of Ca and have high Ca bioavailability. Dairy products represent a primarysource Of highly available Ca ( 137). Results obtained from fractional absorption studies of radiolabeled den1ol1strated sil11ilar Ca bioavailability from whole milk, cheese, yogurt. imitation milk. and chocolate milk. which was greater than the Ca absorbed from CaCO, ( 137). This finding disproved the perception that oxalate, found i n chocolate milk, contributed to reduced ca absorption from this product. Similarly,no difference in Ca absorption, whether c a is present in ionic (e.g., yogurt) or colloidal (milk) form, was observed, confirming earlierreports by Sl11ith et al. (138) that Cabioavailabilitywas not different between whole Illilk and yogurt. Ca in cheese is also highly absorbable and the length ofthe ripening perio(1 does not affect Ca bioavailability ( 1 39). Lactose, a component of milk atld cheese, is known to facilitate nonsaturable. paracellular C3 uptake in the distal intestine (ileum) (140,141). Earlier studies reported a positive correlation between Ca bioavailability and the ratio of lactose to Ca, to a maximum level to which this effectis observed (142). Ascorbic acid, hypothesizedto form soluble conlplexes withCa in the gastrointestinal tract resulting i n a potential increase i n Ca bioavailability, has been disproved when used as a supplement for both CaCO; and cheese ( 143). Another component derived from milk potentiallyinvolved in Caabsorption are thecaseintrypticdigestionproducts, termed caseinophosphopeptides (CPPsj (144-146). CPPs can form soluble complexes with Ca ions and reduce the formation of insoluble Ca salts i n the distal small intestine. Although intestinal Ca solubility and paracellular absorption can be augmented by CPP supplementation, the net effect of this is minimal on Ca balance and utilizationfor alleviating hypertension or enhancing bone calcification when Ca is not limiting in the diet ( 145- 147). As is the case with conutrient interactions that facilitate Ca absorption, considerable effort has been directed toward food constituents that reduce Ca hioavailability. Oxalic acid, found i n spinach, beet greens, rhubarb, and peanuts, will bind to Ca. thus reducing Ca bioavailability from a food source that is relatively rich in Ca. When conlpared to milk, which has a mean absorption average of 27.6%. Ca absorption from spinach is only 5.1 “C (148). Heaney et al. (149) studied kale, a low-oxalate vegetable from the Rrtrs.sic~rgenus, andreportedthatCafromkalewasactually more availablethanCafrommilk,albeit nutritionally insignificant. Whole wheatflour, known to contain a range of phytate conccntrations as well as various minerals including Ca, Zn, and Se, has a low bioavailability for Ca ( 1 SO). Fiber itself has little effect on the absorption of Ca ( I S 1 ); therefore phytate content could be a limiting factor for calcium bionvailability in whole wheat flour. There is some controversy regarding this; however, wheat flour containing high phytate concentrations lowered intrinsically labeled ‘iCa absorption, but soy flours with low to medium phytate levels were less effective at decreasing Ca bioavailability( 1 52). The contradictory findings reported by many independent laboratories indicate that dietary or intestinal phytase activities may be involved, or more likely the relative Ca:phytate ratio is important in determining the phytate-related inhibition of Ca bioavailability. Moreover, Ca absorption has been reported to be better i n leavened products compared to unleavened products, a fact attributed to the affinity of yeasts t o reduce phytic acid during bread making (153). Other studies have shown that CaCO;, when given with a meal, had a higher absorption
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than without the meal ( 154). Components of the meal, such as fat, helpto decrease gastrointestinal transit time and thus allow for more absorption. There is some evidencethat the development of essential hypertension can be linked in part to low Ca intake. Diets thatarelowin Ca or high in Nahavebeen shown to predispose individuals to hypertension ( 155). Using a dietary recall methodology, McCarron et al. ( 156) reported that hypertensive patients, controlled for age, race, and sex, but not for socioeconomic background, reported a lower Ca intake. Calcium intake by Japanese men from dairy foods has also been inversely associated with blood pressure, whereas Ca from nondairy foods has not (157), thereby inferring that Ca bioavailability as well as intake are important factors. In the spontaneously hypertensive rat nlodel (SHR), Cadepleted diets exacerbate hypertension noticed after I O weeks of age, but there is less agreement as to the protective effect of high-Ca diets in protecting against hypertension (144,158). Similarly, Ca has been linked to reducing certain cancers. such as colon cancer, by inhibiting the initiation effect caused by free fatty acids and bile salts in the colon (159). Some epidemiological studies have also reported a lower incidence of colon cancer in subjects that consumed high-Ca diets (160). The mechanism involved i n protection likely includes the formation of Ca salts with certain fatty acidsor bile salts, thus forming insoluble Ca soaps and removal of precursors involved in promotional cytotoxic reactions. Animal studies involving the chemical induction of colon carcinogenesis havc also reported inhibition by Ca when fed at high dietary levels ( 16 I , 162).
B. Magnesium Magnesium (Mg) is an essential element for survival, with diverse roles in bone mineralization, lipid metabolism, hypertension, and susceptibility to oxidative stress ( 163- 165). Rich sources of Mg (> 100 mg/ 100 g) are nuts, soybeans, whole grains, and dried beans. Good sources of Mg (50-100 mg/ 100 g) include crustaceans and spinach. Poor sources of Mg (<25 mg/ 100 g) are milk, eggs, and some vegetables. The presence of organic acids, such as phytic and oxalic acid, can modify the extent of Mg mineral absorption (166). The capacityof dietary phytate to impact onMg status may impart broad nutritional implications only among those individuals that have a high phytate intake and are marginally deficient in Mg reserve. Intracellular Mg deficiency has been characterized as a common underlying pathology for a number of risk factors associated with one or more of the clinical complications of CHD. Magnesium deficiency can also contribute to the development of hypertension, heart disease, diabetes,and chronic fatigue syndrome( 167- 169). In Mg depleted animals, there are several linesof evidence showing Mg deficiencyto be associated with hyperlipidemia and atherogenesis ( 170- 172), altered lipid metabolism ( 173, I74), variations in plasma fatty acid composition and platelet aggregation (175), altered erythrocyte membrane fluidity (176). and lipoprotein and tissue peroxidation ( 177). The role of Mg in lipid metabolism is best exemplified by the study reporting that swine fed margarine and butter diets resulted in a Mg-dependent modification in plasma total and LDL cholesterols, independent of the dietary lipid source (177). Moderate Mg deficiency can also result in significant changes in lipid metabolism, as shownby decreased clearanceof triacylglycerides ( 178)andanincreasedlowdensitylipoprotein:highdensitylipoproteinratio(17 I). Marked reductions of plasma phosphatidylcholine-sterol acyl transferase (EC 2.3.1.43) due to Mg deficiency could explain the decreased esterificationof cholesterol contributing
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to impaired transport and disposal of triacylglyceride (178). Considerable evidence also exists that indicates Mg deficiency influences the pathology of vascular lesions (179), associated with pro-oxidant or inflammatory responses, which can lead to freeradical generation. The promotionof free radicals and the subsequent enhancement of lipid peroxidation by these agents predisposes tissues to endothelial cytotoxicity( 1 80), lipid peroxidation ( 1 8 1 ), and fibrogenesis ( 182). These events leadto increased oxidative stress and an accelerated growth response of aortic vessel walls, which is further proof of the link between Mg deficiency and increased risk to atherosclerosis (170) and cardiovascular disease ( 1 64,174). Low Mg potentiates norepinephrine-induced vasoconstriction of mesenteric resistant arteries, not seen with normotensive WKY controls ( 1 83), further demonstrating the potential role of Mg in the modulation of peripheral resistance in hypertension.
VI.
MICROMINERALS
Although dietary macrominerals, such as fats, sugars, and macrominerals, have been the focus of much research in determining possible relationships with the etiology of chronic disease, considerable attention has also been focused on the role of microminerals on health.
A.
Iron
Iron is an essential nutrient, due to its role in the transport of oxygen and carbon dioxide (hemoglobin), transitional storage of oxygen in tissues (myoglobin), and as a functional component for a number of enzymes, including the antioxidant enzyme catalase. An optimal iron balance is important for the healthof the organism, and iron deficiency is considered a serious nutritional concern in populations throughout the world. especially those developing countries where iron deficiency is partly due to a predominately cereal-based diet. Among the factors that contribute to the prevalence of iron deficiency in both developing and industrialized countries is the lack of available iron i n foods and the associated poor bioavailability. Iron bioavailability is influenced by dietary factors such as tannins, oxalates, and phytates which reduce the bioavailability of vitamin C and heme-iron which enhance iron absorption. Supplementation of the diet with iron through fortification strategies is often used to improve iron status. Challenges arise with fortification technologies employing the most easily absorbed iron compounds, causing unacceptable organoleptic changes i n food with alterations in appearance, flavor, and odor. Moreover, iron supplementation may result i n a reduced bioavailability of other trace minerals, such as copper and zinc, by evoking mineral interactions (184). An excess of iron is also potentially harmful, as shown by the rare inherited disease hemochromatosis. This condition causes excessive iron absorption, leading to the deposition of iron in parenchymal cells of critical organ systems. It is suspected that iron fortification may be detrimental to such individuals because it may induce clinical manifestations of the disease at an early age (185). Although iron overload i n susceptible individuals may lead to oxidative damage, the association with increased risk to CHD is at present controversial ( 1 86,187). The reactive natureof iron is attributed to its capacity to catalyze redox reactions between oxygen and biological macromolecules when present in a free, unbound form. Following ingestion, iron is either oxidized and stored i n the iron storage protein, ferritin, or it is transported as a tightly bound complex with the ligand transferrin
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in the plasma pool for subsequent storage as ferritin. Both transferrin and ferritin hold iron in the ferric [Fe”], nonreactive state, which in this form protects against redox reactions. Iron is liberated from ferritin following attack by the superoxide radical ( 0 ’ ), ~ an underlying consequence of high exposure to oxidants or low antioxidant status that is common in many pathologic states such as infectious disease, ischemia, and reperfusion and inflammation.Forexample,transferrinconcentrationsare significantly lower than normal in the condition of kwashiorkor, thus increasing the Fe-dependent susceptibility to oxidative stress (1 88). Complexing iron by soluble chelate such as citrate and ethylenediaminetetraacetic acid (EDTA) willnot preclude iron fromtaking parr in theHaberWeiss cycle ( I 89).
+ 0:- + Fe’+ + 0’ 0:- + 2H’ + H202 + Fe” + H 2 0 2+ Fe” + OH + .OH Fe2- + H 2 0 2-+ FeO’.’ + H 2 0 Fe”
0 2
In the Haber-Weiss cycle, reducing agents such as ascorbic acid. reduced glutathione, and many plant polyphenols reduce ferric ion to ferrous, which in turn reduces oxygen to a superoxide anion for the eventual combination of ferrous ion and hydrogen peroxide [Eqs. (l-3)]. Theseproducts,referred to as Fentonreagents[Eq. (3)] generatehighly reactivehydroxylradicalswhichcandepolymerizepolysaccharides,denatureproteins, as well as produce DNA scissions. inactivate enzymes, and initiate lipid peroxidation, The metal-catalyzed generation of hydroxyl radical from hydrogen peroxide requires iron containing a minimum of one coordination site. While the general consensus is that the interaction between iron, superoxide, and hydrogen peroxide results i n the production of a potent oxidant, there is controversy as to whether it is the hydroxyl radical or a ferry1 ion [Eq. (4)], whichhassimilar butlesspotentoxidationactivity. In addition to free iron, iron-protein complexes such as hemoglobin and oxyhemoglobin have been shown to promote hydroxyl radical formation (190). which in turn promotes lipid auto-oxidation and oxidative stress (191). Many studies have examined the potential relationship between iron overload, as reflected by high plasma ferritin levels in both acute and chronic disease states. The controversies concerning the role of iron in oxidative stress-initiated disease, largely centers around theuse of plasma ferritin, an acute-phase protein which could represent a source of iron or reflect protection based on its sequestering affinity of iron to a nonreactive state. Elevated ferritin levels have been reported i n Nigerian children suffering from malnutrition and infection (192), thus suggesting that iron overload-induced oxidative stress is associated with this aspect of disease pathology. On the other hand, since ferritin levels are elevated during the activation of the immune system, or may reflect a condition of liver damage, caution is needed to use this parameter as an accurate indicator of iron overload. With chronic disease, such as heart disease, some studies have provided direct relevance of an iron paradigm, while others have produced indirect relevancy to this association (193). For example, iron may be associated with heart disease from the standpoint of its role in catalyzing atherogenesis and ischemic reperfusion injury. In a prospective investigation on the association of dietary iron intake and the risk of acute myocardial infarction (AMI) conducted in middle-aged Finnish men, it was reported that subjects with serum ferritin levels2200 pg/lhad a 2.2-fold greater risk of AMI compared (194). This association was adjusted for other known to men with lower serum ferritin risk factors suchas family history, body Inass index, and blood pressure. Ischemic exercise
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was also strongest in men with the highest LDL concentrations. Moreover, dietary iron intake, as assessed using 4-day food records from 50 subjects on an annual basis, was positively associated with risk of AMI. A reported increase in risk of AMI was reported to be 5% for a 1 mg increase in daily iron intake (194). Other studies have also reported that high dietary iron intake can result in elevated serum cholesterol ( 1 86,195). Taken together, these findings support the long-held notion that regular blood loss, and thus iron loss, in cyclic women is a protective mechanism against ischemic heart disease; a protective measure that is lost with the cessation of menses or hysterectomy (196). Arguments that high serum ferritin indicatea large iron storage that is potentially available for promoting LDL oxidation and vascular and myocardial injury have also been given (197). Iron overload has also been identified in the risk of hepatocellular cancer in hemochromatotic patients (198), which can be reduced significantly by phlebotomy therapy (199). Stevens (200) suggested that the positive association between increased body stores of iron and risk of cancer (e.g., colon, bladder, or esophagus) was related not only to the catalytic action of iron in the production of toxic free radicals that promote carcinogenesis, but also the enhanced survival and proliferation of malignant cells.
B. Selenium The recommended dietary allowance of selenium (Se) for adult men (70 pg/day) and women (55 pg/day) was set i n 1989. Selenium (as selenocystein) is a vital component of several essential selenoenzymes, such as glutathione peroxidase (GSH-Px), phospholipid hydroperoxide glutathione peroxidase, thioredoxin reductase, selenoprotein P (plasma), of Se in maintaining a healthy oxidative and selenoprotein W (muscle). The important role status against symptoms of vitamin E deficiency and prevention of dietary liver necrosis in rats was shownby Muth (2011. In animal husbandry studies, Se deficiencies are common and lead to white muscle disease in cattle and sheep, hepatosis dietetica in swine, and exudative diathesis in poultry. In human studies, malnourished Jordanian infants showing symptoms of anemia before dietary Se supplementation exhibited a striking reticulocy response after receiving30-50 pg sodium selenite (202). The link between oxidative stress and Se was further strengthened with the conclusion that Se may be implicatedin reversing symptoms of kwashiorkor, since Se reserves have been reported to be low in children sufferingfromkwashiorkor(203).Seleniumsupplementationstudies incysticfibrosis patients have also shown increases in HDL and reductions in cholesterol and LDL lipid subfractions (204). Selenium also has a role in thedetoxification of xenobiotics,with the example of 1,2-dimethylhydrazine (DMH)-induced colon tumors in Se-deficient rats exceeding those on a Se-replete diet. The generation of reactive oxygen species due to acute DHM exposure included decreased body weight and elevations in serum cholesterol and urea nitrogen, all attributable to liver toxicity and particularly severe in Se-deficient animals (205).
C. Zinc Zinc (Zn) is another trace element widely used in nutritional supplements (206) and required for its rolein appetite regulation (207), bone calcification (2081, regulation of cellular redox potential (209), and prevention of oxidative stress induced by iron (210). The role that Zn plays in maintaining oxidative stress appears to involve a number of mechan i s m that include fatty acid metabolism, generation of cofactor and cytochrome P-450
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enzyme activity, and competitive inhibition of iron-oxygen-enoic complexes. MildZn deficiencies, which are collitnon in populations subsisting on high cereal protein diets, do not necessarily change the plasma Zn concentration or produce clinical symptoms of deficiency. However, dietary Zn deficiencies lead to suppressed cholesterol biosynthesis 3), (21 l ) , lower glutathione concentrationsin tissues (212), increased lipid peroxidation (21 and impaired Cu, Zn-superoxide dismutase activity (214). Zinc also plays a role i n the inhibition of free radical generation reactions by altering cytochrome P-450 activity via the inhibition of NADPH cytochrome-c reductase activity. These findings coincide with clinical studies that have reported lower serum Zn concentrations in breast carcinogenesis in both animal (215) and human (216) subjects. There are examples, however, of Zn eliciting a pro-oxidant effect and resulting in cytotoxicity (217). Zinc also has an indirectroleinprotectingagainstoxidativestressbyinducing metallothioneine synthesis, an essential single-chain polypeptide that functions to detoxify heavy metals such as cadmium and mercury, and protecting against DNA strand scissions which result from oxidative stress (218). In contrast, excessive intakes of Zn can lead to deficiencies in copper (21 9) and iron (2201, as well as impaired calcium metabolism (221) and adversely effecting immune responses in susceptible individuals (222). Copper deficiency not only lowers cupro-enzyme activities, but also the activities of other enzymes such as glutathione peroxidase that do not contain copper (223) and are critical for maintaining oxidative balance. Thus Zn can have a potential indirect effect on oxidative status by interacting with copper availability that is normally required for antioxidant enzyme activity. The balance of monounsaturated and polyunsaturated fatty acid incorporation into phospho!ipids and LDL is modified by Zn (224), and the efficiency of this effect can be of Zn deficiency governed by the fatty acid compositionof the diet. For example, the effect on plasma cholesterol, phospholipid, and LDL is greatest with a diet high in PUFAs (224). Hyperlipoproteinemicsubjectswithclearclinicalsymptoms of atherosclerosisexhibit lower serum Zn concentrations than hyperlipidemic subjects without clinical symptoms of atherosclerosis (225). Moreover, Zn-deficient rats fedlinseed oil (0-3 PUFA) exhibited greater susceptibility to copper-induced LDL oxidation than Zn-adequate counterparts fed the same diet. Thus Zn deficiency may increase the likelihood of lipid peroxidation reactions by increasing the PUFA composition of phospholipid or LDL.
D. Mercury Mercury (Hg) primarily occurs naturally in the environment in inorganic (vapor and salt) and organic (methylmercury) forms. Both organic and inorganic Hg show toxicological effects, which include nephrotoxicity, neurotoxicity, and gastrointestinal toxicity with ulceration and hemorrhage (226). There is a low absorption efficiency of Hg from the gastrointestinal tract in both humans and animals, which corresponds to low Hg transfer into milk i n lactating subjects (227). The consumption of mercury-contaminated fish by fisherman has been associated with increased chromosomal damage, the extent of which was correlated closely to blood Hg levels (228). The formation of reactive oxygen species following exposure to methylmercury has been shown both with iron-mediated oxidative damage and glutathione depletionand lipid peroxidation (229). These events are evidence of the pro-oxidant activity of Hg (230) in critical target organs such as the cerebellum, thusexplainingthenotedneurotoxicity of thismetal.DietaryHgtoxicityleadstoan inactivation of localized alkaline phosphatase activityi n cerebral tissue, which predisposes
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leaky microvessels typical of neuropathological conditions associated with blood-brain barrier damage (231). The suppression of redox cycle enzymes and GSH metabolism by Hg toxicity also facilitates membrane lipid peroxidation and oxidative stress, resulting i n morphological damage i n kidney tissue (232).
E. Aluminum Aluminunl (AI) occurs naturally i n foodandwater as well as parenteralpreparations. Exposure to AI has been shown to reduce the zinc content of soft tissue and bone in rats fed low-calcium diets, denotinga significant mineral interaction potential (233). Excessive intake of AI produces an accumulation and toxicityin critical tissues, leading to neurological diseases such as Alzheimer’s disease, Parkinson’s dementia complex, and both dialysis encephalopathy and osteomalacia (234). The concentration of AI’* in ferritin recovered from patients suffering from Alzheimer’s disease is considerably higher than in healthy patients (235). Although lipid peroxidation reactions are increased i n neurological disease conditions, and the transition metal ions such as copper and iron are noted pro-oxidants that accelerate auto-oxidation of membrane lipids, i t is interesting to note that AI” does not directly facilitate lipid peroxidation reactions (236,237). Aluminum-fed rats display decreased thiolstatus i n brainhomogenate,whichwasassociatedwith a reductionin oxidized glutathione, thus accounting for the noted oxidative stress (237). A mechanism suggested to be attributable to this form of oxidative stress was the noted A13’-induced impairment of NADPH synthesis from glucose-6-phosphate dehydrogenase, a reducing cofactor for GSSG-red activity and regeneration of GSH. Other workers have shown that AI’ ’ , while ineffective at promoting phospholipid peroxidation in liposomes, is effective in accelerating lipid oxidation induced by iron salts (236). The valency of AI being three, however, does not make it an effective promoter of lipid peroxidation (similar to Fe”), and therefore the mechanism for the induced pathological effects attributed AI“-induced toxicity remain to be fully elucidated.
F. Cadmium Cadmium (Cd) has been recognized as a ubiquitous environmental pollutant and metallic toxicant that can be found in leafy vegetables, grains, bivalves, and animal kidney tissue (238). Cd uptake can be influenced by dietary factors, such as calcium restriction, which increases Cd absorption in the small intestine and tissue deposition (239,240). Evidence exists as wellthat a protectiveeffect of both calcium(239)andiron(241)exists in preventing Cd accumulation. Cadmium can disturb the normal metabolic homeostasis of living organisms (242,243) and potentially contribute to prostate carcinogenicity in occupational environments (244). A potential hazard of Cd exposure is also related to its longterm retention in the kidney and resultant tubular damage (245). There is limited evidence from cytogenetic studies to suggesta direct chromosomal damaging potential for Cd (246) and a potential for interaction with mercury. Alterations i n carbohydrate metabolism associated with hyperglycemia have been attributed to Cd exposure (247). It has also been recognized that Cd toxicity, as expressed i n both lipid metabolism and peroxidation reactions (248-250), may involve interactions with other trace minerals, suchas selenium, iron, and copper. Selenium,an essential component of GSH-Px enzymes, active in removing organic hydroperoxides and protecting against oxidative stress, plays an important role against Cd toxicity. Unlike chromium,
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which facilitates cholesterol catabolism and excretion, Cd may enhance cholesterol deposition in aortic tissue (251). Cd toxicity and unetabolism is also interrelated with other minerals, most notably zinc (245). Cd and zinc have been shown to be mutually antagonistic due to similar uptake mechanisms i n the intestinal mucosa. Adequate zinc supply reduces Cd tissue accumulation, while zinc deficiency results i n an enhancement of Cd retention. Cd-exposed rats fed calcium-deficient diets exhibit greater reductions i n body weight and bone mineral content than rats fed calcium-replete diets (252). In bone tissue. Cd-Ca interactions are associated with interferences i n bone remodeling. This is realized by the binding activity of Cd for calcium-binding protein (253): albeit lower, it can bind to the intestinal mucosal protein when calcium intake is restricted and deposit into both kidney and bone tissue.
VII. A.
FOOD ADDITIVES Salt and Salt Substitutes
Salt (NaCI), along with other ingredients such as sodium nitrite. sodium nitrate. sodium ascorbate, and tnonosodium glutamate, has an important role i n food as a tlavor and texture enhancer. as well as a very important preservation aid. The addition of NaCl to storcd food products will control pH, moisture, andtotal electrolyte level and prevent the growth and toxin production of Clostr-idiwtr b o t d i u w t l . Sodium (Na). potassium (K), and chlorine (Cl) are primary elements which represent the electrolytes in body water and play important roles i n body functions. Na ions are requii-ed for the maintenance of blood pressure, blood volume, regulation of external and internal relative cellular fluid volumes, and transmission of nerve impulses. Similarly, Cl ions are required for maintaining the blood acidbase balance, tissue osmolality, and gastric hydrochloric acid formation. Excess NaCl may result in acute and toxic effects. Na and Ca interactions have been investigated to determinetheireffects i n modulatinghypertension(253).Epidemiologicalstudieshave reported minor incidences of hypertcnsion i n societies that have minimal NaCl intake and hypertension i n the samesocieties when NaClintakeisincreased(254). A cause and effect relationship remains controversial (255). High NaCl intake has also been linked to atrophic gastritis and occurrenceof stomach cancer i n countries such as Japan wherehigh NaCl intake is related to a higher incidence of stomach cancer (256). An alternative to Na salt has been the use of potassium (K), which like Na has no established recommended intake, but a safe daily intake 1x1s been established as 1 .c)-5.6 g (255). Potassium has a similar refractive index, specific gravity, and a critical humidity, thus providing functional propertiesi n meat and bakery products similarto Na salts. There is a lower threshold for bitterness in K, which makes its use limited in some food systems: however, blending KC1 with reduced lactose whey or citric acid and tricalcium phosphate will reduce the perception of bitterness. While there is a potential benefit associated with reduced risk of hypocalcemia, lowering of blood pressure. and protection against possible stroke (256), a potential risk from K salts has been found. Swales (257) calculated that the increase i n daily K intake from its use as a conltnon salt substitute results in difficulty in effective excretion of the extra load of K. Those at risk wereidentitiedaselderly patients, patients with renal impainnent.and patients taking K-sparing diuretics, angiotensin-converting enzyme inhibitors, and nonsteroidal allti-infatllmatories (257). Moreover,
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B. Nitrates Froln 170th econolllic and technical considerations, sodium nitrite andsodill111 nitrate represent very important food additives. Nitrite gives cured meats a characteristic color and a range of bacteria,includingpathogenic fl:lvor, whileactingtoillhibitthegrowthof Illicroorganisms such as C'/osfvjdiuvl hofu/irlwn,Srlq'hg/ococ~(~fr.s O I O ' ~ M . S , and Btr~i///i.S ('er'pLi,s arid food spoilage organisms such as corynebacteria, psychotrophs, and pseLIdoII10Ilads. It should benoted,however,thatnitritedoesnotindefinitelypreventsporeoutgrowth, spoilage, or toxin production and prolonged storage or temperature abuse can result i n low levels of ineffective residual nitrite. Nitrite has also bee11 shown to protect against lipid oxidation. thereby reducing rancidity and generated off-flavors i n meat and Ineat products (260). The antioxidant effect of nitrite in heme-catalyzed peroxidation is i11depcl1detlt of pH in the pH range characteristic to meat and meat products. The mechanisn1s of antioxi(iant activity associated with nitrite include stabilization of the lipid fraction i n meats by reaction with the carbon-carbon double bonds and formation of stable complexes by coordination to the iron center of the heme proteins and preventionof catalytic breakdown of hydroperoxides by h e m proteins. The role of nitrite in contributing of nitrite with myoglobin, to the characteristic reddish-pink color is due to the reaction which reduces to nitrosylmyoglobin, and followinga thermal process stabilizesto nitrosylhemochrome. Typical products t o which nitrite is added include bacon, ham, bologna, weiners, sausages, cured turkey, ham, and jerky (with average residual nitrite levels of approximately 20 ppn1). The level of nitrate i n these products is relatively low compared to the nitrite levels found i n nlany vegetables and alcoholic beverages. Nitrite is also recycled i n the salivafollowing intake of nitrite-containingfoods, which provides a prolonged exposure to nitrite. Radiotracer studies etnploying 'iN-labeled sodium nitrate i n rats have estimated that 60% of the label was excreted i n the urine, with a range of 6 to 19% excreted i n theurine. A smallerproportion (3-10%) of labeledcompoundwasretained in the body (26I ). Nitrite is a reactive ion and associates with secondary anlines and amides under acidicconditionstoformnitroso-compoundssuchasnitrosamines and nitrosamides. These compounds following reduction of the nitro- group provide the antimicrobial and potentially toxic expression. The ti,rmation of nitroso- compounds is metabolized further of secondary amines to fully reduced amines. Nitrosamines represent N-nitroso derivatives which show an organ-specific activity relationship with tumor formation, regardless of the route of exposure. In the rat, specifically. symmetrical dialkyls are a source of tumors in hepatic tissue, where unsymmetrical dialkyls are specifically esophageal carcinogens. For example, dimethylnitrosanlinecan be detected throughout the body in rats,but induces tunlors only in the lung, liver, and kidneys. Nitrosanlirles require metabolic activation to carcinogens which is achieved by the matnmalian mixed-function oxidase system (cytochrome P-450) to yield electrophilic products. Dimethylnitrosanlitle is a potential carcinogen in the lung, liver, and kidneys. Nitrosamides, on the other hand. donot require metabolic activation to form tumors at the site of exposure, but since they are relatively less
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stable and unlikely to survive typical cooking temperatures, they provide low toxicological risk. Mutagenic activity has been reported with nitrite in a number of mutagenicity tests (262). which correspond to other studies that have reported an induction of tumors i n rats exposed to N-nitroso- compounds derived from the nitrite precursor (263). Other studies have not reported a positive dose responsein either the induction time for tumor formation or the incidence of tumors in both male and female rats (264). Moreover, there are no studies that have directly associated food-related nitrite use with human cancer. Acute toxicity associated with excessively high intake of nitrites can also occur with methemoglobinemia. This condition is characterized by cyanosis and anoxia and results from a defect in oxygen transport by high levels of methemoglobin in the blood. Nitrate from well water which is converted to nitrite within the body has been associated with this condition due to the affinity of nitrite to oxidize hemoglobin to methemoglobin, thus resulting i n methemoglobinemia. Acute poisoning, resulting i n hepatic necrosis and diffuse bleeding of the lung and gastrointestinal tract, has also been reported from repeated ingestion of or high exposure to dimethylnitrosamine (265). Many nutrients and conutrients are effectivein reducing the toxicological risk associated with chronic exposureto low levels of nitrosamines. Ascorbic acid, erythrobic acid, and a-tocopherol effectively block nitrosamine formation by reducing nitrite to nitric oxide (266), thus inhibiting adenonla induction by NaNO? (267). Plant polyphenols, such as gallic and ferulic acids from wheat bran (268) and beetle nut extracts (269). as well as caffeine (267) have been shown to reduce nitrite toxicity. Soluble fibers derived from pectin and carrageenan have also been shown to provide substrate for microbial, nonspecific reduction of nitro- groups as a result of dietary adaptation (270). The importance of a balanced diet including fruits and vegetables, combined with advances in food technologies to reduce the nitrosamine content of human foods, provides relatively little risk to human health from nitrite use.
C. Methylxanthines Methylxanthines are naturally present in many plant products and in the case of caffeine is used as a food additive for taste enhancement of sweetness. Methylxanthines are widely consumed by humans of all ages in a variety of beverages, foods, and medications, including coffee, tea, cola, chocolate, and analgesics. The average consumer ingests 2-3 mg caffeine/kg of body weight ora minimum of 10 mg/kg/day from coffee, tea, or soft drinks. to physiological Methylxanthines have no direct nutritional significance; however, due and metabolic activities, these compounds can influence the nutritional status of the individual. For example, caffeine is a diuretic which promotes mineral excretion (271,272) and stimulates the sympathoadrenal medullary system, the latter resulting in mobilization of free fatty acids (273). A sparing of glycogen due to increased fatty acid oxidation and reduced muscle fatigue follows. Moreover, a stimulation of lipolysis inhibits glycolysis and glucose uptake in muscle tissue, thereby acting to spare carbohydrate. Caffeine is efficiently absorbed following oral consumption, with 99% absorption occurring within 45 minutes (274). Caffeine is a powerful central nervous system stimulant that affects all portions of the cerebral cortex, resultingin reduced fatigue and drowsiness and clearer thought processes. Dosages of caffeine contained in a 12-ounce serving of soft drink or one or two cups of coffee or tea will improve auditory and visual vigilance. Peak plasma concentrations are reached in 15-20 minutes (2751, and the plasma half-life
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after oral ingestion is 2.5-4.5 hours (276). Due to the small molecular weight and partial hydrophobic character of caffeine, distribution into all body fluids occurs with transport across both theblood-brain and placental barriers. In humans, 80% of caffeine is metabolized to paraxanthine in the liver by the cytochrome P-450 mixed-function oxidase system. Most of the remaining 20% is converted into theophylline, theobromine, trimethyluric acid, and numerous other metabolites. The biotransformation of caffeine to dimethylxanthines, dimethyl and monmethyl uric acids, trimethyl- and dimethylallantoin and uracil derivatives occursi n liver microsomes. The oxidationof 1 methylxanthine to l-methyluric acid is mediated by xanthine oxidases (277). The major clearance pathway for paraxanthine occurs via transformationto 5-acetylamino-6-fonnylamino-3 methyluracil (AFMU), which accounts for 67% of paraxanthine elimination. Metabolism of paraxanthine to 1methylxanthine, and then by xanthine oxidase to l-methyluric acid, can account for 25% of caffeine transformation. The conversionof caffeine directly to theophylline by demethylation is a reversible reaction, however, only 6% of theophylline is converted back to caffeine (278). The metabolites of caffeine are primarily excreted in the urine, with 25% excreted in feces. The physiological effects of caffeine and metabolites are based on three primary mechanisms of action which include the following: (a) Antagonism of adenosine receptors: Methylxanthine antagonize adenosine receptors (AI and A2) which are present in large numbers in the brain, trachea, heart, kidney, and fat cells. The blocking of adenosine-receptor activity by caffeine at concentrations that produce behavioral signs of stimulation is explained by the otherwise characteristic inhibitory neurophysiological actions of adenosine, resulting in notable physiological effects such as sedation, bradycardia, hypotension, and hypothermia. With the competitive antagonism at extracellular adenosine receptors by caffeine, the result is generally the opposite to adenosine and excitatory behavioral effects are observed. Adenosine A 1 receptors increase reversal frequencies of action potentials, resulting in decreased excitability. There are AI receptors that also inhibit phospholipase C, which yields inositol and diglycerides. High-affinity A2 adenosine receptors stimulate adenylate cyclase via guanyl binding protein and occur i n the brain, platelets, and liver, whereas low-affinity A2 receptors are chiefly found in thebrain and fibroblasts. Methylxanthine-sensitive receptorshave been found to trigger the relaxation of smooth muscle, with caffeine having equipotent activity at AI and A2 receptors. Dimethylxanthine derivatives, theophylline, and paraxanthine are more potent at A2 than at AI receptors. Conversely, theobromine is more potent at AI receptors. Activation and release of serotonin can be caused by stimulation of xanthine-sensitive receptors. Studies on selectivity of methylxanthines toward certain types of receptors have shown that the caffeine derivatives theophylline and paraxanthine are more potent and selective antagonists than caffeine (279). (h) Inhibition of phosphodiesterase: Caffeine actsto inhibit phosphodiesterase activity since adenosine is a potent stimulator of adenylate cyclase in many tissues, including the brain. Methylxanthines stimulate the cardiovascular system by inhibition of phosphodiesterase, resulting i n increased arterial blood flow. Xanthines stimulate beta-adrenergic receptors which also contribute to vasodilation and bronchodilation. Caffeine and theophylline have been found to cause hippocampal excitation and enhanced long-term hippocampalpotentiation,respectively (280,28I ) . Chroniccaffeinetreatmentmaycause an increase in norepinephrine concentrations resulting in a slight decreased density of betaadrenergic receptors. Different parts of the brain respond distinctively to methylxanthine stimulation. Caffeine increases norepinephrine i n the frontal cortex and cerebellum, while
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increasingserotonin in thestriatumandcerebcllunl.anddopamineinthc striatuI11. 111 the renal system. xanthines increase diuresis and blood flow (282). likely causecl by a11 antagonistic effect on inhibition of renin release, vasoconstriction of specific arterioles, and changes in glomerular filtration. I n general. the significance of caffeine consulnption 011development of chronic disease remains controversial. despite the noted physiological activities o f this compound.
1. HeartDisease Foremost i n the issue of relationships between diet and CHD disease h;ls beet1 the debate over the association between coffee (caffcine consumption) and CHD (283-286). The consumption of coffee at levels exceeding S cups/day have been reported to be associated with elevated plasma cholesterol levels (287). Acute cuffeinc consumption i n subjects that normally do not ingest caffeine causes increases i n blood pressure and heartrate,with near-complcte tolerance after 1 --4 days for both humoral and hemodynamic paranleters (288). The observation that a lower sensitivity to the pressure-elevating effect of caffeine that the caffeine-induced pressor occurs i n subjects with a high coffee intake suggests effect is related to the amount and frequency of caffeine intake and on the rate o f caffeine metabolism (283). Thisconclusion is controversialsincesubsequentstudiescould not show a correlationbetweenbloodpressureresponse or heartrateandrate of caffeine metabolism (2x9). The association between coffec consumption and plasma cholesterol in North America has been confirmed by some investigators (290) but refuted by others (791). Moreover.clinicalstudieshavedemonstratetl that caffeinewas not effective at raising cholesterol (292). Further examination of a number of studies has indicated that the consumption of caffeine-containitlg beverages such as coffee and tca is not correlated with the incidence of hypertension, gastrointestinal ulcers, cancers o f the colon. rectum, and bladder, and cardiac infarctions (293). Mnnn and Thorogood (294) reported anincreased risk of myocardial infarction in women consuming 6 or more cups of coffee per day; however, this study failed to directly associate this event with caffeine. Variations i n the volume of coffee consumed and the concentration of caffeine present i n the beverage i n many studies has made possible relationships between coffee consumption and various health risks problematic (295). It may be concluded that there arc confounding effects associated with the diet that preclude m ; accurate assessment o f risk of hyperlipidemia associated with caffeine intake. For example. studies conducted i n hypercholcstct-olelnic rats fed caffeine-cholesterol supplemented diets. showed no difference i n aorta or peripheral tissue cholesterol content to control animals. From these findings, i t may be concluded that caffeine alone is not involved i n initiating atherosclerotic lesions (296).The disparity of lindings between different studies strongly suggeststhat there are possibly other factors which play ;I n1orc important role i n thc relationship between coffec beverage consuniption and elevated cholesterol than the caffeine content. Coffee isa complex mixture. composed ofapproxin1;ltcly 1000 different chen-rical constituents(297), sotne of which have bioactivc properties (298.299). Support for this hypothesis is obtained from results o f studics condllcted with tea, which also contains caffeine, but which failed to clcv;~te seruni cholesterol (300). Moreover. van Dusselclorp levels to the same extent as that observed with coffee et al. (301 ) reported 110 differences i n blood lipids between sub.jccts consuming regular coffee al1dtI1osc consuming decafl'cinated coffee. Although it nlay he argued that there is 110 sillglc compound unique to coffee, two potential constituents present i n coffee i n addition to caffeine have been shown t o produce a cholesterol-raising effect. CoI11l11otl to
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coffcc arc the Maillard reaction products (MRPs) or brown pigments of nonenzymatic browning. High molecular weight MRPs have been shown to chelate trace elements and minerals (298.302). and although metal chelation of coffee is generally associated with phytic acid, thiol, and other phenolic compounds (303), MRPs are also present i n coffee and vary considerably depending on the processing conditions used in the preparation of the coffee. I n particular, copper, zinc, and iron are sequestered by the chelating action of brown pigments. It is important to note that both copper and zinc deficiency i n rats can produce significant hypcrcholesterolemia (304). The mechanism for this proposed effect is largely associated with a reduced activity in plasma lecithin cholesterol acyltransferase (LCAT) activity,resulting in anassociatedmarkedincrease i n p l a s m cholesteroland apolipoprotcin concentration. I t is therefore possible that the association between coffee rtnd serum cholesterol may be related to the amount or the strength of the coffee. Other coffee constituents that have been attributed to the cholesterol-raising effect include those present i n the coffee lipid fraction (305).The compounds have been identified as being the rliterpcnoitl alcohols cafestol and kahweol (Fig. 7a) found i n boiled coffee but not filtered coffee (306).Both compounds are associated with the nontriacylglyceride fraction of coffee lipids and are likely retained by the filter paper used for filtering coffee, thus explaining thc absence of a cholesterol-raising effect in subjects that switchetl fron1 consutning boiled to filtered coffee. Ratnayake et a l . (307) reported from analyzing nun1crous different coffee brews that both the total lipid content and the diterpene content were greatest i n boiled coffee; the source of processed coffee reported earlier to be associated (308). I t is noteworthy that thesediterpenoidalcohols withclcvatcdserumcholesterol have d s o bee11 shown t o possess weak estrogenic activity (309), as well 21s depressing serum creatinine and ~-glutamyl-tr~~nsferase (306).
A
B
0
II
CH= CHCOH
OH
Caffeic acid
0 II CH= CHCOH
HOOC
0
O O C e CH-
1 \ OH QH
OH
Ferulic acid
Chlorogenic acid
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Kitts
2. Cancer The issue of whether caffeine-containing beverages induce or promote cancer has bee11 a controversial debate over the years (295). Epidemiology studies have reported clear associations between caffeine or coffee consumption and an increased incide11ce of cancer. Studies by Aeschbacher et al. (310,31 l ) , however, showed 110 evidence of Inutagenic activity from coffee when in the presence of metabolic enzymes, nor any evidence of mutagenic metabolites in the urine of coffee drinkers. Other workers have reported on average 20,000 revertants from mutagens derived from one cup instant of coffee, compared r to 2500 revertants present in one cup of drip coffee using the TA 100 S d / ~ n e l l t test strain (312). Although ~nethylglyoxal was identified to account for an appreciable amount of mutagenicity in coffee (313), it is not known to what degree metabolic activities germane to liver and intestinal xenobiotic enzymes have in reducing the carcinogenic potential of this or other potential componentsin coffee. Results of a 2-year chronic exposure study inof mice to caffeine using an instant coffee vehicle reported reduced growth, despite creased energy expenditure by the animal, but no increased incidence of tumor formation (314). I n fact, caffeine itself has been proposed to possess antioxidant activity (315), as does chlorogenic acid and other plant phenolics (Fig. 7b) which are primary constituents of coffee (299). There is sufficient evidence from both chemical and biological studies to conclude that caffeine does not possess carcinogenic potential, especially at the dosages comn~onin human consumption.
3. ReproductivePerformance Most women consume less caffeine during pregnancy, the primary source of this reduction i n caffeine intake is in the decreased consumption of coffee (3 16). Howcver. a large percentage of pregnant women (approximately 95%) still consume caffeine in some form of beverage. A marked decrease in birth weight hasbeen reported in childrenof heavy maternal caffeine consumers (>300 mg/day), following correction for nicotine use (317). This response was independent of changes in pregnancy weight gain, length of labor, or birth length. Reproductive studies conducted i n rodents during the last trimester have shown that especially high exposure levels of caffeine (equivalent to 10-40 cups of coffee/day) resulted i n embryo resorption and fetal growth suppression (318). The fact that a similar effect was observed for both caffeinated and decaffeinated coffee suggests that the notcd effects were also partially attributed to some component in coffee other than caffeine. A primary sourceof concern regarding caffeine exposure during pregnancy is the characteristically longhalf-life of caffeine. I n humanstudies, thehalf-life of caffeineincreases throughout gestation to a maximum of 18 hours in late pregnancy before returning to nonpregnant values of about 5.5 hours during the postpartum period (319). Since the hepatic mixed-function oxidase system is involved in caffeine metabolism (320) as well as the oxidative metabolism of steroid hormones (32 1 ), the longer half-life of caffeine in late pregnancy is due in part to the reduced metabolism of caffeine and the greater volume of distribution of theconstituent.Radioactivemeasurementsafter 2 hours of ratfetal tissues from dams administered[ 1 Me"C]-caffeine reveals a majorityof fetal and placental tissue radioactivity is attributed to caffeine, with theophylline being a principal metabolite of caffeine in rodents (Fig. 8). It was not determined if the presence of this theophylline represented fetal or placental caffeine metabolism, or whether it was derived from maternal metabolism prior to placental transfer to the fetus. Regardless, i t is noteworthy that the deposition of maternal caffeine reaches the fetus quickly and caffeine concentrations between maternal and fetal tissues reach unity very quickly (Fig. 9a,b).
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Nutritional Toxicology A
1
Carbon -14 Spectrum 'a Violet Spectrum
(254nm)
Fig. 8 Representativeclutionpattcms of methylxanthine(a-thcohrominc.b-thcophyllincand cthcohromine standards) and [ l -methyl "C] caffeine radioactivity from reverse-phase C- 18 HPLC column and radioactive Row detection apparatus. A = methylxanthinc standards;B = 2-hour maternal plasma sample; C = 2-hour maternal urine sample: D = ?-hour placental tlssue extract; E = 2-hour fetal liver tissue extract.
The teratologic effectsof caffeinc were first reported by Nishimura and Nakai (322) in pregnant mice receiving a large. single, intraperitoneal dose (250 mg/kg) of caffeine between days 9 and 14 of gestation. This study demonstrated a cause and effect response between caffeine exposure and notable defects i n fetal development that were manifested by digital and cleft palate malformations, and prompted other workers to investigate the relationship between caffeine-induced maternal or placental catecholamine release and teratogenic effects (323).Observations from various sources have indicated that caffeine can stimulate an adrenal-associated increase i n plasma catecholamine levels (288), and that fetal catecholamine exposure corresponds to a higher incidence of limb malformations as evidenced by the prevention of caffeine-induced teratogenicity by maternal adrenalec-
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Brain Lung
Heart
Liver
Kidney
Organ
Lung
Heart
Braln
Liver
Kidney
Organ
Fig. 9 Caffeine disposition in fetal and maternal organs 2 hours after [ I-Me "C-caffeine injcction. (A) Activity expressed per organ weight; (B) activity cxprcsscd per mg/kg tissuc.
Nutritional Toxicology
41 1
tomy (324). Other workers have demonstrated that both the mandible and femur bone of caffeine-exposed animals weighed less and were smaller in volume and softer thanin control animals (325). The femur, being an endochondral bone, would be particularly sensitive to caffeine exposure if calcification processes that rely on readily available calcium pools are affected. This indeed was shown to be true in mice administered a range of sustained-release caffeine dosages (50-250 mg/kg), resulting in a decreased weight gain by dams and lower fetal weights and associated reduced ossification; the latter being a good indicator of disrupted calcium metabolism (326). Sincc caffeine consumption is conmonly associated with smoking and/or alcohol intake (327,328), it is often difficult to predict the true risk associated with caffeine in fetal growth and development. In rats the threshold for caffeine-induced teratogenic effects has been set at 80 mg/kg body weight in one dose (329). Similarly, the threshold for which there may appear to be a teratogenic effect i n humans has been estimated to range from I O to 14 cups of coffee taken at one sitting (330). Obviously these threshold concentrations for teratogenicity represent unlikely scenarios, thus suggesting that caffeine consumption itself is not a prinmy risk to fetal growth abnormalities. Exposureof both pregnantrats andrabbits to cocoapowder,whichcontainstheobromineas the main methylxanthanine,failed to show the teratogenicity observed earlier for caffeine ( 3 3 1,332). Many of the more serious concerns regarding caffeine intake during pregnancy and lactation are related to the long-term consequences that relate to the development of the fetus and neonate. Tanaka and Nakazawa (333) studied the effects of caffeine exposure of dams on neonatal brain parameters from days1 -10 postpartum. The inclusionof 0.0470 caffeine in the drinking water of the dams resulted in reduced brain weight and higher tyrosine content in related pups. It was suggested that caffeine, which increases CAMP, promotes the activation of tyrosine hydroxylase through CAMP protein kinase, which in turn results in a disturbance of catecholamine metabolism in fetal and neonatal cerebrum. Another study that exposed rat pups to caffeine from days 2-6 postpartum reported an up-regulation of adenosine AI receptors that persisted to 90 days of age (334). It was concluded from examining brain tissue of experimental animals that various regions of the brain were distinctively sensitive to caffeine, depending on the stage of maturation and the time of exposure. However, compensatory growthof rat pups fed 122 mg/kg/day during prcgnancy has been reported(335), thus indicating that the effect of caffeine exposure during pregnancy on neonatal growth may be overstated.
VIII. A.
FORTIFICATIONAND SUPPLEMENTATION Vitamin D
Vitamin D, along with calcitonin and parathyroid hormone (PTH), function to maintain calcium and phosphorous homeostasis. In response to a decline in serum calcium levels. vitamin D is metabolized to a biological form [ 1,25-(OH)? cholecalciferol], whichin turn acts on the bone to promote Ca” resorption and induction of Ca” transcellular absorption within the intestine. It is relatively difficult to define the exact requirement for vitamin D, since vitamin D; can be produced in vivo upon exposure to the UV rays of sunlight. The FAO/WHO recommended intake of vitamin D in children under 6 years old is 400 IUlday: however, with maturity the daily intake drops to 100 IU/day. Other workers have
412
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estimated that the brief, casual exposure of the face, arms, and hands to sunlight is equivalent to ingesting 200 IU ( 5 pg) of vitamin D (336). Vitamin D deficiency results i n inadequate transcellular intestinal absorption and renal reabsorption of calcium and phosphate. Consequently serum calcium and phosphate are lowered and serum phosphatase activity increases. The actionof parathyroid hormone with reduced 1,25-dihydroxyvita1nin-D~ due to the deficiency results in skeletal demineralization and the onsetof rickets in children and osteomalaciain adults. Air pollution or low exposure to sunlight may necessitate the consumption of vitamin D by food fortification or through supplementation. Moon et al. (337) provided epidemiological evidenceto suggest that lower vitamin D biosynthesis upon exposure to UV rays was a causative factor for the greater susceptibility of developing rickets in dark-skinned infants. Vitamin D supplements have also been recommended for elderly individuals who are institutionalized or have limited access to the outdoors because of limited mobility or confining conditions or diseases. Low circulating concentrationsof 25-hydroxyvitamin D or 1.25-dihydroxyvitamin D intheelderlyarecharacteristic of vitamin D insufficiencyratherthandirect deficiency, and are largely attributed to reduced capabilities of skin biosynthesis and vitamin D conversion. Typical recommended dosages of vitamin D supplements for elderly subjects i n the United States are 10 pg/day (338). It is noteworthy that of all vitamins, vitamin D has the greatest potential for toxicity if ingestion is excessive. A minimum toxic dose for vitamin D has been estimated to be 50.000 IU for adults and 1000-2000 IU for infants (339). For example, a daily intake of 3000 IU (75 pg) vitaminD has been shown to result in a rapid increase in 1,25-dihydroxyvitamin D to extreme physiological levels, while 2000 IU/day vitamin D produces hypercalcemia and 800 IU/day has been associated with development of renal calculi (337). Hypervitaminosis D results in a 15-fold increase in plasma 25-hydroxyvitamin D (340). The hypercalcemia induced by vitamin D can also persist for weeks to months depending on the half-life of the vitamin D (341). Since excessive amounts of vitamin D are not easily obtained from dietary sources, fortification and supplementation strategies designed to combat osteoporosis, parathyroidism, or just daily vitamin supplements creates a requirement for assessing the health risks/benefits of fortifying foods with vitamin D. The toxic manifestations of vitamin D represent serious complicationsthat are associated with calcificationof soft tissues in the heart, lung, and kidney especially, and demineralization of bone.Intoxicationsymptomsincludehypercalcemia,hypercalciuria,anorexia, nausea, thirst, polyuria, and muscular weakness. Although rickets in experimental animalscan be cured by vitamin D treatment, it is also possibletoinducerickets by excessive vitamin D consumption, where bone Ca” mobilization representing the source of hypercalcemia causes bone Ca?- wasting. An endemic of hypercalcemia occurring in Britain during the 1950s due to supplementation of milk with 2000 IU vitamin D and vitamin D enriched cereals resulted in numerous cases of hypercalcemia, with symptoms of growth failure, mental deficiencies,and supravalvular aortic stenosis (341). A risk associated with hypervitaminosis D and hypercalcemia has also been reported in Japan with infants administered vitamin D supplemented formula, which required monitoring of infant serum calcium and phosphorus levels (342). Hypercalcemia dueto vitamin D intoxification can be described in two categories: hyperabsorption of calcium from the intestine. and increased mobilization of calcium from the bone. Whereas the former will potentially result in soft tissue calcium deposition that is a concern in atherosclerosis, the latter has particular relevance from the osteoporosis standpoint. For example, chronic vitamin D excesshas beenrelated to an elevated1,25-dihydroxyvitamin D concentration(337),
Nutritional Toxicology
413
which in turnhasbeenassociated with reduced bone mass in Aleutian islanders (ages 40-75) (343). It is likely that the increased levels of circulating 1,25-dihydroxyvitamin D increases osteoclast activity and thus decreases both the calcium and magnesium content of bone. The effectsof extreme hypercalcemia(> 15 mg/dl) dueto vitamin D intoxification are listed in Table 3. It has been known for some time that vitamin D may represent a potential concern in cardiovascular health (344,345). Epidemiology studies have reported a lower incidence of atherosclerosis in underdeveloped countries, where food is not fortified with vitamin D (337). These workers indicated that dark-skinned individuals were less susceptible to the toxic effects of vitamin D compared to their fair-skinned counterparts, as evidenced by the existence of large differences i n the average incidence of atherosclerosis among Caucasian and Negro populations. The rate of decline in age-adjusted heart disease deaths recorded from 1968 to 1975 among women and blacks was half that of Caucasian men, which is consistent with the attenuated effect of vitamin D in blacks and premenopausal women. In a community-based study, Scragg et al. (346) showed that the increased exposure to sunlight was protective against CHD. A photometabolic interaction between cholesterol and vitamin D is based on the fact that both vitamin D and cholesterol are derived from squalene, andthe conversion of squalene to 7-dehydrocholesterol could be enhanced bysunlight,whereasintheabsence of sunlight,cholesterolisformed(347).Supplementing the dietof 19 1 volunteers, ages 63-76 years, with vitaminD to evaluate a relationship between cardiovascular health and vitamin D indicated that serum 25-hydroxyvitamin D waspositivelycorrelatedwithserumLDLandnegativelycorrelatedwithHDL (348,349). Other workers have shown a strong relationship between artery calcification and an increase in total serumcholesterol(350).Aorticcalcification is asrelevant in predisposing animals to atherosclerosis as is lipid infiltration and plaque formation (351). There are case studies describing vitamin D toxicity that were attributed to both supplementation and fortification practices. Gross hypercalcemia attributed to vitamin D toxicitywasreported ina 6-month-old boy who had beenadministered ancxcessive amount of a vitamin mixture composedof vitamins A, C , and D since the ageof 4 months. The child exhibited symptomsof vomiting, constipation, and increasing apathy, as well as an undetectable parathyroid hormone plasma concentration (352). Milk and infant formula preparations can be either underfortified or overfortified as well. In 1990 a reported eight cases of hypervitaminosis D resulting in hypercalcemia, anorexia, and other symptoms in weredetectedcaused by amassivefortification of milkwithvitamin D atadairy Massachusetts (353). More than 500 times the amount of vitamin D listed on the label was present i n the product, occurring as a result of equipment malfunction. A follow-up study conducted to determine the variance in vitamin D levels i n milk and infant formula
Table 3 AdverseEffects of Vitamin D Toxicity
Organ Skeletal Cardiovascular
reduced bone nuss Hypcrcalcifcation of bone, Ahnormal contraction of vnscular smooth muscle (hypcrtension), calcification of arteries
Calcium-phosphateprecipitation within renal tubules. urinary tract stones in skin, artery, and gastric mucosa Soft tissue Calcification Renal
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from several dairies i n five states produced results that showed only 62% of the 42 milk samples contained less than 80% of the amount claimed on the label (354). No vitamin D was detected i n 3 of the 14 samples of skim milk tested, i n which one milk sample labeled as containing vitamin D (ergocalciferol) actually contained vitamin D (cholecalciferol). Seven of the I O infant formulas contained tnore than ZOO%, of the amount stated on the label; one containing more than 400%)of the stated amount. A similar study conducted in the United States reported 80% of milk samples contained either 20% less or 20% more vitamin D than the amount stated on the label (355). Of particular concern was of whole milk thereportedfindingthat one sample of chocolate milk and one sample contained more than 900% and 300% vitamin D. respectively, of the amount stated on the label. These studies identified the need for monitoring fortified products for vitamin D content and possibly improving the technologies used to fortify milk with vitamin D. There is concern that abolishing the current directive on vitamin D supplementation would cause a resurgence of rickets. which was a health issue i n parts of Canada even in the late 19SOs, prior to when vitamin D was added to milk (356). Despite the potential risks of vitamin D toxicity due to inaccuraciesin fortification and supplementation technologies or practices, it may be unrealistic to expect that oral vitamin D supplements could reach all populations atrisk (e.g., pregnant women, infants, disabled persons) who are presentlyprotected by vitamin D fortifiedfootls. Thesituations of chronically ill and elderly persons warrant additional attention dueto the fact that they tend toconsume lower levels of vitamin D-containing foods and beverages and have less access to the outdoors and sunlight.
B. Vitamin E Vilamin E, which appears to be the least toxic of the f a t soluble vitamins, includes more than 30 different tocopherols (a-.p-, r-, and 6-tocopherols) witha common homolog. Excellent sources o f tocopherols are soybean. cottonseed, canola, and wheat germ oils. The accepted daily intakeof a-tocopherol is 0.15-2 mg/kg of body weight. and generally a clinical deficiency of vitamin E is rare. Although tocopherols are found naturally in all high plants, fortification is still required where food products arc easily exposed to thermal oxidation or decomposed by exposure of light. The limit of uses in olive oil is 200 mg/ kg. in coconut oil, palm kernel oil, and tallow is S00 mg/kg, and i n infant food it is 300 mg/kg fat (Codex Alimentarius). The antioxidant activity of tocopherols is i n the following descending order: a- > p- > z- > F- at 37°C and 6- > r- > p- > a- at 5O0C-l00"C. It is importanttonote thatwhenpresent i n highconcentrations in foodsystems.prooxidant effects of vitamin E may also result (357,358). Vitamin E deficiency i n humans is rare, but it canoccur in prematurcneonates and i n people suffering from cystic librosis anti biliary atresia. I n most cases, vitamin E deficiencies occur becauseof lipid malabsorption syndromesand the dependence of enteric absorption of vitamin E on adequate lipid absorption. Low dietary intake of vitamin E, coupled with high physiological demands can result in oxidative stress symptoms such as erythrocyte hemolysis and hemolytic anemia. For example. an increase i n the requirement for vitamin E has been associated with oxidative stress generated by excessive fishoil consumption (359). Moreover, a low body content of tocopherols has been shown to result i n an increased tissue (360) and HDL (361) cholestcrol content. This observation o f vitamin E in protecting may be attributed to the important and often overlooked role against depletion of important membl.ane PUFAs which are required for preserving both
415
Nutritional Toxicology
physical properties and enzymatic activities involved in cholesterol metabolism. Further support for this idea comes from the importance of vitamin E in preventing a cholesterolinduced increase in aortic catalase and glutathione peroxidase activity (362). There has been strong support for a positive role for vitamin E in the prevention of ischemic heart disease by induction of antiatherogenic properties (363). Inhibition of lipid oxidation by vitamin E occurs by donating a hydrogen atom from one of the hydroxyl groups i n the vitamin peroxyl (LOO) and alkoxyl (LO') radicals or tocopheryl free radical [Toc'; Eq. ( S ) ] . The inhibition reactions are as follows:
+ TocH H LOOH + Toc' + Tot' + LOO TOC + TocH + LOH + Toc' + TOC'-+ LO TOC
LOO'
LOO' LO' LO'
-
-
Inhibition of peroxyl and alkoxyl free radicals by tocopherol (TocH) can prevent lipid oxidation and decomposition reactions. The rates of reaction Eqs. (S)-@) are important in determining the anti- and pro-oxidant activities of tocopherols. The faster the forward reaction (e.g., the larger the k value). and the slower the backward reaction (e.g., the smaller the - k value), the greater the antioxidant activitiesof tocopherols. The antioxidant activity of vitamin E has alsobeen shown to enhance antioxidant defense mechanisms and prevention of chronic disease. In vitro studies have demonstratedan efficacy for vitamin E to delay the oxidation of low density lipoproteins (363,364). Studies conducted in vitro with rabbits fed D,L a-tocopherol reported a resistance to in vitro forced peroxidation reactions (36S), but no effect i n preventing atherosclerosis. Tocopherol (TocH) can also be converted to a tocopheryl radical (Toc'), which in turn can promote lipid oxidation by abstracting PUFA (LH) to produce an alkyl free radical (L'). In addition, a tocoquinoperoxy1 radical (QOO') can be produced by the oxidation of tocopherol radical (Q'), which also abstracts hydrogen from lipids, thereby promoting lipid oxidation. The pro-oxidant reactions are as follows: TOCH+ 0: + HOO' Toc'
+ TOC'
+ LH + TocH + L'
Toc' -+ Q'
+ 0 2 + QOO. QOO' + LH + QOOH + L' QOOH + LH -+ QOH + L' + OH
Q'
The rates of these reactions dictate the anti- or pro-oxidant activity of tocopherols under different conditions. For example, the reaction rate constant in Eq. ( S ) is many-fold relatively higher than the rate constants in Eqs.(5') and ( I O ) at high tocopherol concentrations, resulting in the large quantities of tocopheryl radicals produced i n Eq. (10). The accumulation of excessive tocopheryl radicals results i n the production of more peroxyl radicals, which promotes Eq.( 1 l ) . These reactions generate free radicals for further propagation reactions i n lipid oxidation. A pro-oxidant effect of vitamin E attributed to photosensitization has also been shown with an intravenous lipid emulsion product usedto treat neonatal jaundice in premature infants (366). Following a 24-hour exposure to phototherapy, thelevel of hydroperoxides increased almost sixfold. The potential physiological significance of these reactions, or the relation between the concentrations of anti- and pro-
Kitfs
416
oxidant activities of tocopherols is unclear (367). However. high concentrations of vitamin E can reduce transition metals and produce radical species from redox reactions (368). The radical center in the aqueous phase is transferred to the lipid phase and a tocopheryl radical (Toc’) is generated when an aqueous peroxyl radical (LOO’) comes into contact with the surface of the LDL, where endogenous a-tocopherol is present to donate a hydrogen to the peroxyl radical according to
The a-tocopherol radical formedin LDL, being water-soluble, is trapped in the lipid phase of LDL, and a chain reaction propagating additional tocopheryl radicals (Toc’) occurs according to the following scheme:
The exposure of LDL to pro-oxidant depletes a-tocopherol prior to generation of lipid peroxidation products, which is consistent with the free radical scavenging property of vitamin E (369). Halliwell(370) demonstrated that the reduction of Cu” to Cu‘ by vitamin E also promotes lipid oxidation. The pro-oxidant activity of vitamin E in the presence of Cu” prevented a characteristic antioxidant effect when tested in a detergent dispersion model micellar solution containing Cu” and a-tocopherol (37 I). The tocopheryl radical (Toc’) generatedby abstraction of hydrogen from tocopherol can be efficiently regenerated in the presence of ascorbic acid (AscoH) according to the scheme
+ TocH + Toc’ + LOOH L‘ + TocH(slowreaction,inefficient) Toc’ + LH Toc’ + AscoH + Asco’ + TocH (fast reaction, efficient) LOO’
Asco’
+ ROO’ -+
Asco-OOR (stable, and inactive product)
The synergistic antioxidant activity between ascorbic acid and a-tocopherol (372) is characterized by the return of tocopherol to antioxidant status and the generation of ascorbic radical that can further stabilize the peroxyl radical by forming a stable and inactive product. In addition to regeneration of tocopheryl radicals, the peroxyl radical stabilizing effect makes ascorbic acid an effective synergistic antioxidant.
C. Vitamin C Vitamin C (ascorbic acid) is a water-soluble reducing agent that has important roles in the regulation of antioxidant and pro-oxidant functions, xenobiotic detoxification, and iron metabolism. Ascorbic acidis found in both fruits (e.g., papaya, oranges, cantaloupe, strawberry) and vegetables (e.g., broccoli, green peppers, cauliflower, kale); however,it is generally accepted that extreme conditions in food processing and storage will reduce the ascorbic acid content dramatically. Exposure of food to extremes of heat, light, oxygen, and pH result in losses associated with thermal destruction, photo- and enzymatic oxidation, and leaching of ascorbic acid. A deficiency of vitamin C is well documented to result in hemorrhage, hyperkeratosis, hypochondriasis, and blood abnormalities, the symptoms
Nutritional Toxicology
41 7
of the condition scurvy. Epidemiological evidence indicatesthat ascorbic acid is a significant variable in modulating the incidence of gastric cancer (373). Other workers have reported a significant inverse relationship between vitamin C status and both diastolic and systolic blood pressure (374,375). Plasma vitamin C concentrations have also been found to be significantly lower in individuals with CHD; however, unlike vitamins A and E, plasma vitamin C levels are not related to a risk of coronary artery disease (376). These findings are potentially related to the affinity of vitamin C to protect against oxidative stress, as evidenced by a strong reducing power both in vitro (377) and in vivo (378), and an affinity to delay LDL lipid peroxidation (379). Vitamin C has been referred to as thefirst defense andmost importantantioxidantinplasma(380).Asisthecasewith vitamin E, ascorbic acid can delay LDL lipid peroxidation and may provide longer protection by inhibiting aqueous peroxyl radicals(38 1) and sequestering transition metals (382). The significance of vitanlin C antioxidant activity may also be the underlying cause for the observed prolonged survival of patients with terminal cancers (383). Reactions specificto enhanced intestinal iron absorption attributable to ascorbic acid and associated transition metal-induced redox cycling or mixed-function cosubstrate activity has resultedin a potential pro-oxidant toxicityrisk associated with vitamin C (384,385). Both mutagenic and genotoxic effects of ascorbic acid have been demonstrated in vitro (386,387). Although these studies are often conducted in the absence of catalase, they denlonstrate the importance of reducing transition metal ions (e.g.,Fe‘+, Cu”) in generating Fenton reaction-induced hydroxyl radicals from the Habcr-Weiss cycle in vitro. The interpretation of these findings to an in vivo condition requires caution, however, since it should not be overlooked that the interactions between antioxidant enzymes and other nonenzymatic antioxidants provide both a diverse and synergistic function in protecting cells from oxidative stress.
D. Folic Acid Folate represents an important vitamin for coenzyme activity with single-carbon transfer in biochemical reactions and has important roles in nucleic acid synthesis, erythrocyte function, and hair health. Fortification of cereal grain food systems with folic acid has been accepted asa necessary strategy for preventingboth lnacrocytic anetnia and debilitating neural tube birth defects (388). This practice is based on the understanding that the folate requirement of 400 pg/day is not obtainable from an average diet consumed by women of childbearing age.In general, folate deficiency is conmot1 in the North American diet and has been regarded as a risk factor for many chronic diseases, including anemia, cancer, and cardiovascular disease. Current fortification levels for folate are listed at 140 pg folate/100 g cereal grain (389). This notwithstanding, concern hasbeen expressed that high folate levels could also mask vitamin B deficiency in some populations, including the elderly (390).
IX. NATURALLYOCCURRING TOXINS A.
Aflatoxins
Aflatoxins are secondary metabolites produced by the molds A.spr~yillusj f m u s and A. prrrmiticus i n agricultural commodities such as corn, peanuts, figs, tree nuts. rice, dried fruits, cassava, and various seeds. Moreover, aflatoxin residues can also occur in egg or
418 Table 4
KittS
ExposureRoutesfor Aflatoxin
Routc DirectconsumptionDietary
intake of AFB, from cereal, grain. and nut products IndirectconsumptionMetabolites of allatoxin (AFM, in milk) Inhalation Occupational risk: aflatoxincontaining dust
milk products as a result of ingestion of aflatoxin-contaminated feed by poultry or lactating dairy cows. Toxic components isolated from peanut meal used as an ingredient in poultry feed were resolved into four fluorescent spots when applied to silica gel and eluted using chloroform-methanol as the tnobile phasei n a thin layer chromatographic procedure (391 ). The two fluorescent blue and two fluorescent green spots identified under a UV light were named aflatoxins B1, B2, G I , and G2, respectively (392). The structures of aflatoxin are derived from a condensed bisfuradcoumarin ring, with isomers B? and B I representing dehydro- derivatives of aflatoxins B1 and G I . respectively. Aflatoxins M1 and M2 are typicallytnammalianmetabolites of B1 andB2.respectively,althoughtheymayalso occur in fungal cultures. Special research intcrest has been placedon aflatoxin B I (AFB 11, since it is the most prevalent and the most toxic secondary metabolite. Food and feeds are susceptible to invasion by Aspergillus at all stages of production (production, preharvest, harvest, processing, transportation, and storage). Relative humidities of 88 to 95% and storage temperatures in the 25"C-3OoC range are optimal for growth of Aspet.gi//rts species from air, soil, or insect vectors (393). Essential of oilscloves, cinnamon, and onion and garlic extracts, and eugenol all have inhibitory effects on aflatoxin production and growth of A. ,flmws. In addition, caffeine is effective in inhibiting growth and mycotoxin production of A.spct;qillus and Pmicilliwn species (394); a finding which appears to be specific to caffeine since other mcthylxanthines such as theobromine are less effective (395). The possible routes of human exposure (Table 4) and subsequent health risks are presented in Table S . Direct consumption of aflatoxins is related to the intake of principle aflatoxins from dietary sources, which are distinct from the indirect consumption of aflatoxin metabolites from edible animal tissues. Inhalation of aflatoxins poses an occupational risk of allatoxicosis for workers in grain mills, oil presses, and employees of livestock Table 5 TargetOrgansforAllatoxin
Organ systctn Respiratory Gastrointestinal Hepatic Reproductive
B,
Characterization of discasc Lung, trachea, and bronchus tumors Colon carcinotna, small intestine tumors Hcpatocellular carcinotna, xute hcpatitis, preneoplastic lesion Etnbryotoxicity. reduced fertility. inhibition of lymphocyte function, reduced cell mediated immunity
Nutritional Toxicology
419
feed-processing facilities. Although acute toxicity of AFB 1 results in hepatitis and death in countries such as India and Kenya (396). it is the chronic exposure to low levels of aflatoxins that represents themost c o m m n risk associated with carcinogenesis. Aflatoxins are relatively low molecular weight. lipophilic compounds, suggesting efficient absorption from the small intestine into the mesenteric circulation prior to entering the liver through the portal circulation. Allatoxins act predominantly at the liver and biliary tract. AFB 1 is a procarcinogen that once metabolically activated to the 8,9-epoxide intermediate, a putative and ultimatecarcinogen,formsadducts(N7-guaninederivative2,3-dihydro-2-(N7puanyl)-3-dehdroxya~atoxinB 1) that are primarily found i n GC-rich regions of DNA (Fig. IO). Thcse adducts result in a GD-TA nucleic acid transversion and DNA mutation (397). Biliary excretion of AFB 1 has been reported to involve an AFB l-glutathione complex as a major metabolite excreted at 6 to 8% of administered dose in I O minutes, with the peak rate of excretion occurring at 30 minutes (39X). Elimination of AFB 1 is relatively slow, with an apparent plasma half-life of 92 hours for cumulative AFB 1 fecal excretion com-
Procarcmogen (without polar groups) Aflatoxin B1 Phase I Enzyme (Cytochrome P450)
Polar groups introduced Electrophilic Intermediate (AFBI 8.9 oxide)
Conjugate
Reaction
I
Phae II Enzyme (Glutathione S-transferase) Aflatoxin Glutathlone
Carclnogenic Adducts
I
Excretion 8,9dihydro-B,9dihydroxy Aflatoxin B1
9-Hydroxyaflatoxin B1
420
Kitts
pared to IS% urinary excretion 23 daysafter dosing (399). The three major urinary metabolites recovered by radiotracer methods are AFM 1 (41-SO%), AFPl (
X.
PROCESSED DERIVED TOXINS
A.
Benzo(a)pyrene
Polycyclic aromatic hydrocarbons (PAHs), of which there are more than 100 different compounds,areformedduring theincolnpletecombustion of organicmatterandare widely distributed in our environment (Table 6). Benzo(a)pyrenc, a primary isomer and potent carcinogen prototypeof PAH, is a highly lipophilic compound which through inhalation, dermal, and oral exposure represents a high risk of tumor formation at several organ sites, including lung, liver, and skin. The removal of B(a)P from the body is facilitated by a number of metabolic steps that convert B(a)P into hydrophilic metabolites for ready removal (Fig. 11). B(a)P is oxidized by cytochrome P-450-dependent mono-oxygenases, epoxide hydroxylase, and prostaglandin H-synthetase pathways to initially form phase I metabolites, which include epoxide phenol, dihydrodiols, and secondary metabolites, such as diolepoxides, tetrahydrotetrols, and phenol-epoxides. The moderately polar phaseI metabolites are subsequently conjugated with glutathione, glucuronic acid, and sulfates to
421
Nutritional Toxicology Table 6 Sources o f B(a)P
Source Cigarette smoke Gasoline engine exhaust Charcoal broiled steaks Fats (margarine, butter) Roasted coffee Tea Fruits, vegetables, cereals
20-40 pg/cigarctte 2- 170 pg/mg extract 8 pg/kg processed food 62 W k g 0.1 - 1 1.5 pg/kg 3.9-21.3 pg/kg 0-48 pglkg
form phase I1 polar metabolites which are more easily excreted than parent hydrocarbons by urinary and biliary routes (402). As a result of the metabolic biotransformation process, internlediate B(a)P metabolites become reactive intermediates which can induce mutations, transform cells, and form covalent linkages with nucleophilic sites on tissue macro(1 12). The balance between the rate of molecules, including proteins and nucleic acids formation and the rate of detoxification of xenobiotic products govern the potential for cellular injury (403,404). Regional blood flow along with tissue enzyme substrate affinity and detoxification activity are primary determinantsof B(a)P clearance. While hepatic tissue playsa primary role in the metabolic clearance of circulating B(a)P in vivo (405), the lung tissue, by receivingtheentirefraction of cardiac output, has a relatively higher blood flow and
I PHASE II I
1 PHASE1 I I
I
Introduction of polar groups Polycyclic aromatic hydrocarbons(PAH)
Conjugation anddetoxification Epoxides
MetaboIic Activafion
IBenzo(alpyrene1
P-450
hydrolase
-Cytochrome rnonooxygenase system -Prostaglandin H-synthase pathway -Epoxide Glutathione
Carcinogenic reactions in target tissues
-
Secondary metabolites epoxides Diol Tetrahydrotetrols Phenol epoxides
Fig. 11 Enzymatic biotransformation of benzo(a)pyrene by phase I (activation) and phase I1 (detoxification) reactions.
422
Kitfs
therefore exposure of toxicant. Physiologically based pharmacokinetic (PBMK) modeling for B(a)P describes B(a)P metabolic clearance to follow the exprcssion:
where CL = organ clearance; Q = blood flow to the organ: E = extraction. or the fraction of xenobioticremovedfrom the bloodas it passesthrough the organ;Cl’ = intrinsic clearance (the amount and character of the enzyme(s) of disposition); f,, = fraction of the drug not bound to blood elements (406,407). PBPK modeling has shown that tissue enzyme activity is critical, along with organ perfusion,for the metabolicclearanceofB(a)P.Usingthisapproach,B(a)Pintestinal absorption has been estimated to be 40%i n the rat, with a first-pass metabolism extraction rate of 0.4 at doses IO-l000 times higher than the estimated daily intake of B(a)P by man (SO ng/kg) (406). Approximately 50% of total body clearance of B(a)P proceeds via the bile, however, biliary excretion may not necessarily indicate final detoxification since B(a)P can be reabsorbed and reactivatedby hydrolases present i n the intestinal tract (408). The significance of nutritional status on the toxicity expression of B(a)P and other polycyclic aromatic hydrocarbons is relevant to the specific target organ changes i n enzymatic activation of the parent cornpound by cytochronle P-450 and epoxide hydroxylase leading ultimately to generation of principle mutagens and carcinogens, such as the bayregion diol-epoxide moiety of the hydrocarbon. Natural plant phenolics present i n vegetables (ellagic acids) (409) and tea [( -)epigallocatechin-3-gall~1te] (410) block the adverse biological effects of phase I enzymes. By forlning covalent adducts with diol-epoxides. plant phenolics modulate biotransformation and facilitate elimination of the xenobiotic metabolite. The chemoprotective effects of phenols also influence B(a)P-induced tumorigenesis by enhancing phase I1 antioxidant enzyme s y s t e m such as glutathione-S-transf e m e and NADP(H):quinone reductase.
B. Heterocyclic Amines Heterocyclic amines (HCAs) are formed as pyrolysis products during the cooking (frying. roasting, baking, or broiling) of protein-rich foods. These products represent a source of food-derived mutagens and carcinogens and thus are a potential risk to public health in thc etiology of human cancer (41 1.412). The foodborne HCAs are subdivided into several classes, including indoles and imidazoles, which are derived from amino acids and proteins, and quinoline, quinoxalines, and pyridines. which are derived from Maillard-type reactions involving creatine, creatinine, free amino acids, and monosaccharides produced during cooking at temperatures of 150°C-300°C. Predominant i n Western foods are the quinoline structured HCAs, which have been identified as 2-amino1 -methyl-6-phenylitnidazo [4,5-61 pyridine (PHIP), 2-amino-9H-pyrido [2,3-b] indole, 2-amino-3, 8-dimethylimidazo [4,S-fl quinoxaline (MeIQx).2-amino-3,4.8-trimethylimidazo [4,5-t] quinoxaline (DiMeIQx), and 2-amino-3-n1ethylinidazo [4,S-f] qunoline (IQ) (Fig. 12). IQ and MeIQ havebeenreported to induce indirect frameshift bacterial tnutagens (413) to a greater extent than aflatoxin B , and yield positive responses i n the Srrlrrrorlellcr microsomal bioassay (414). IQ hasbeen found to be carcinogenic in rodent models (415,416) and hepatocarthe potential for both IQ cinogenic i n monkeys (417). Other studies have demonstrated and MeIQ to be converted to direct-acting mutagens following short-term (e.g., 1 hour)
423
Nutritional Toxicology
PhlP
MelQx
IQ
MelQ
F3
F4
GmNH2 bq
GIu-P-1
GIu-P-2
Fig. 12 Chemical structures of quinoline (IQ, MeIQ), quinoxaline (MeIQX),pyridine (PhIP),and amino acid pyrolysis products (Glu-P- I , Glu-P-2, TIT-P-], Trp-P-l,Trp-P-2).
treatment with SO mM sodium nitrite (pH3) at 37°C (418). IQ can also be converted to the direct-acting mutagen, 2-nitro IQ (3-methyl-2 nitroimidazo-[4,5-f] quinoline) following treatmentwithnitrite(419).Proteinpyrolysisproductsderivedfromcharredparts of broiled fish and beef and expressing mutagenic activity have also been identified as 3amino- 1.4-dimethyl-SH-pyrido [4,3-b]indole (Trp-P- 1 ) and 3-amino- 1 methyl-SH-pyrido[4,3-h]indole(Trp-P”),fromtryptophanpyrolysate and 2-amino-6-methyl-dipyr1 ), and 2-aminodipyrido[ 1,2-a: 3,2-d]imidazole (Gluido[ 1,2-a: 3,2-d] imidazole (Glu-PP-2), from glutamic acid pyrolysates. Both Trp-l and Trp-2, found in broiled foods, are hepatocarcinogenic in mice, with females being more susceptible than males (420). In addition to representing a carcinogenic risk, HCA dietary exposure has been associated with risk factors for cardiovascular disease i n humans (421,422). Heart tissue collected from rats exposed to dietary MeIQx has been shown to contain damaged DNA in proportional quantities to the exposure level of MeIQx (423), thus providing further evidence for an impact on cardiovascular health. Following rapid absorption from the gastrointestinal tract, metabolic activation of HCA is required in order to formDNA adducts and exert mutagenic, cytotoxic, or carcinogenic activities. The major routeof activation is a two-step process where the cytochrome P-450-mediated mixed-function oxidase system acts to oxidize the amino moiety to Nhydroxylated intermediates, which are then converted by phase I1 enzymes to reactive electrophilic species that are capable of forming covalent linkages with DNA or protein (424). Alternatively, these products can be transformed to sulfate or P-glucuronic acid
424
Kitts
conjugation products with detoxification reactions and are ultimately excreted i n the urine or feces. Excretion elimination of HCA is less in humans than in rodents (425). As is the case with other foodborne xenobiotics,a balance between phaseI activation/detoxification and phase I1 activation/detoxification enzyme activities are required to reduce the risk of HCA-associated chronic disease. Differences in enzymatic biotransformation of parent HCA compounds to carcinogenic metabolites i n specific target organs may explain the different rates of DNA adduct formation observed in these organs. Thus dietary factors that regulate enzymatic biotransformation activities are noteworthy modulators of natural chemoprevention. For example, the conjugated double-bond isomeric derivatives of linoleic acid (CLA) present in fried beef and dairy products has efficacy to block IQ DNAadduct formation in liver, lung, large intestine, and kidneys (426). Plant flavonoids and polyphenolic acids (427), as well as retinol (vitamin A) (428), inhibit HCA mutagenicity. Dietary fiber has been shown to be effective at reducing the mutagenic activity of foodborne HCA in vivo as well (429). The chronic feeding of HCA in rodent studies has been performed using dosages ranging from0.02 to 0.08% of the diet (430). These maximum tolerable dosages far exceed the average intakes (e.g., 5 ng/day equivalent to 83 pg/day/kg body weight of a 60 kg individual) consumed in food cooked under normal conditions. Four primary HCAs identi> MeIQx > DiMeIQx > IQ (431). PhlP fied in the diet in decreasing order were PhIP is present in the greatest amounts in fried beef and represents the greatest exposure to HCA i n the North American diet. It is noteworthy that the carcinogenic potencies of these HCAarealmost in reverse order (IQ > DiMeIQx > MeIQx > PhlP), whichmaybe explained by the fact that PhIP, while undergoing phase I activation, is cleared efficiently by extrahepatic tissues. From dietary records collected from more than 3500 individuals on intakes of HCA, an upper bound estimate of incremental cancer risk was estimated at 1.1 X 10", when using cancer potencies based on body surface area. Nearly half (46%) of the incremental risk was due to the ingestion of PhIP, with the consumption of meat and fish products contributing most (80%) of the total risk.
XI. CONCLUSIONS Epidemiological, experimental, and metabolic studies have provided convincing evidence that the human diet contains both nutrients and bioactive nonnutrients which provide protection against degenerative diseases including atherosclerosis and cancer (Table 7). Xenobiotic compounds are metabolized by enzyme systems that have evolved in response to selection pressure and are dependent on the nutritional status and the macro- and micronutrient composition of the diet. The role of dietary constituents in influencing the toxicity of xenobiotic agents needs to be considered along with nutrient-nutrient interactions in evaluating risks to dietary-induced environmental carcinogenesis, mutagenesis, teratogenesis, or other chronic diseases such as CHD. Phase I reactions that are necessary for the detoxification of xenobiotic materials will be suppressed by fasting or induced by highprotein diets or the availability of key nutrients such as niacin or riboflavin. Phase I1 enzymes, located in the cytoplasm and endoplasmic reticulum, are active in conjugation reactions that produce more polar metabolites from GSH conjugates, sulfates, and glucuronides in removal of electrophilic xenobiotics derived by endogenous phaseI enzyme activity, or from exogenous sources. Phase I1 enzymes are also regulated by nutritional status concerning various specific nutrients, as well as other food constituents that include iso-
425
Nutritional Toxicology
Table 7 NonnutritiveChctnoprevcntiveAgents
in Foods
Agent Resveratrol
Grape.mulberry
I3C.I Phenyl isothiocyanate Sulphoraphone Crambene
Cabbage, broccoli Cabbage, broccoli Broccoli Cabbage, broccoli, mustard Spice (Cucltttrrr longcl)
Turmeric
Eugenol
CLA-conjugated linoleic acid
Oil extracts from clove, cinnamon. basil, nutmeg Beef, cheese
Inhibition of cyclooxygenase and hydroperoxidase activity, anti-inflammatory activity, induction of phase I1 NADPHquinone reductase activity Inhibition of flavin tnonooxygcnase Inhibition of P-450 cnzytncs Induction of phase 11 enzymes Induction of GST activity Antioxidant activity, inhibition o f cyclolipoxygenase, scavenging of diolepoxides Anti-inflammatory activity, induction of Phase I1 enzymes, modulation of immune system Antioxidant activity, modulation of cyclolipoxygenase activity
thiocyanate and that are present in many cruciferous plants (broccoli, cabbage, kale, and cauliflower) (3,432). The result is dietary nutrients as well as nonnutrients that work in concert with the general nutritional status of the organism to minimize the toxicity of xenobiotic agents. Attempts to relate the chenloprotective properties of various dietary components to the development of chronic disease states will continue to be an active area of study in nutritional toxicology.
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Chemical,BiologicalandMedicalAspects. NewYork:PergamonPress,1991,pp.5764. 373. M Cohen, HN Bhagavan. Ascorbic acid and gastrointestinal cancer. J Am Col1 Nutr 14:565578, 1995. 374. DA McCarron, CD Morris, HJ Henry, JL Stanton. Blood pressure and nutrient intake in the United States. Science 224:1392-1398, 1984. 375. JP Moran, L Cohen, JM Greene, X Guifa, EBFeldman, CC Hamcs, DS Feldman. Plasma ascorbic acid concentrations relate inversely to blood pressurei n human subjects. Am J Clin Nutr 57:213-217, 1993. 376. R Delport, JB Ubbink, JA Human, PJ Beckcr, DP Myburgh, WJ Hayward Vcrmaak. Antioxidant vitamins and coronary heart disease risk in South African males. Clin Chim Acta 278: 55-60,1998. 377. PA Glascott, MTsyganskaya,EGilfor,MAZern, JL Farber.Theantioxidantfunctionof the physiological content of vitamin C. Mol Pharmacol 50:994-999, 1996. 378. I Jialal, SM Grundy. Preservation of the endogenous antioxidants in low density lipoprotein by ascorbate but not probucol during oxidative modification. J Clin Invest 87597-601, 1991. 379. K Kunert, AL Tappel. The effect of vitamin C on in vivo lipid peroxidation in guinea pigs as measured by pentane and ethane production. Lipids 18:271-274, 1983. 380. BFrei,LEngland, BN Ames.Ascorbateis an outstandingantioxidant in humanblood plasma. Proc Natl Acad Sci USA 86:6377-6381, 1989. 381. KL Retsky,MWFreeman, B Frci.Ascorbicacidoxidationproduct(s)protecthumanlow density lipoprotein against atherogenic modification. Anti- rather than prooxidant activity of vitamin C in the presence of transition ions. J Biol Chem 268:1304-1309, 1993. of 382.KLRetsky,BFrei.VitaminCpreventsmetalion-depcndentinitiationandpropagation lipid peroxidation in human low density lipoprotein. Biochim Biophys Acta 1257:279-287, 1995. 383. E Cameron, L Pauling. Supplemental ascorbatei n the supportive treatment of cancer: reevaluation of prolongation of survival times in terminal cancer patients. Proc Natl Acad Sci USA 75:4538-4542,1978. 384. A Samumi, J Aronovitch, D Godinger, M Chevion, G Czapski. On thc cytotoxicity of vitamin D and metal ions. A site-specific Fenton mechanism. Eur J Biochem 137: 119-124, 1983. 385. V Herber. Viewpoint: Does mega-C do more good than harm or more harm than good? Nutr Today28:28-30,1993. 386. HFStich,JKarim, J Koropatnick,LLo.Mutagenicaction of ascorbicacid.Nature260: 722-724,1976. 387. MP Rosen, RHC San. HF Stich. Mutagenic activity of ascorbate in mammalian cell cultures. Cancer Lett 8:299-305, 1080. 388. GS Ranhotra, PM Keagy. Adding folic acid to cereal-grain products. Cereal Food World 40: 73-76.1995. 389. S Daly, JL Mills, AM Molloy, M Conley, YJ Lee, PN Kirke, DC Weir. JM Scott. Minimum cffective dose of folic acid for food fortification to prevent neural-tube defects. Lancet 350: 1666-1669,1997. 390. LB Bailey.Folateintakerecommendationsfrom a nutritionalscienccperspective.Cereal Food World 40:63-66, 1995. Nature 195:1062391. BF Ncsbitt, J O’Kelly, A Sheridan. Toxic metabolite ofA.s/~e,.Xi/lfts~~ffkl,ft.s. 1063,1962. 392. LL Zaika, RL Buchanan. Review of compounds affecting the biosynthesis and bioregulation of aflatoxins. J Food Protect 50:691-708, 1978. 393. DL Park, B Liang. Perspectives on aflatoxin control of human food and animal feed. Trends Food Sci Techno1 4365-381, 1993. 394. RL Buchanan, DC Hoover, DB Jones. Caffeine inhibition of aflatoxin production: mode of action. Appl Environ Microbiol 46: I 193-1 200, 1983.
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395. RL Buchanan, RS Applebaum, P Conway. Effect of theobromine on growth and aflatoxin production by Aspergillus porrrsiticus. J Food Safety 1:211-216, 1978. 396. JM Cullen, PM Newbeme. Acute hepatotoxicity of aflatoxins. In: DL Eaton, JG Groopman, pp. 3-26. eds. The Toxicology of Aflatoxins. New York: Academic Press, 1994, in the content of the multistage nature of 397. YP Dragan, HC Pitot. Aflatoxin carcinogenesis cancer. In: DL Eaton, JG Groopman, eds. The Toxicology of Aflatoxins. New York: AcademicPress,1994,pp.179-206. 398. CJ Holeski, DL Eaton, DH Monroe, GM Bellamy. Effect of phenobarbitol on the biliary excretion of aflatoxin PI-glucorunide and aflatoxin Bl-S-glutathione in the rat. Xenobiotica 17:139-153,1987. 399. RA Coulombe, RP Sharma. Clearance and excretion of intratracheal and oral administration of aflatoxin B1 in the rat. Food Chem Toxicol 23:827-830, 1985. 400. CD Groopman, LG Cain, TW Kensler. Aflatoxin exposure in human populations: measurements and relationship to cancer. CRC Clin Rev Toxicol 19:113-145, 1988. 401. JP Whitty, LF Bjeldanes. The effectsof dietary cabbage on xenobiotic metabolizing enzymes and the binding of aflatoxin B1 to hepatic DNA in rats. Food Chem Toxicol 25:581-587, 1987. 402. DH Phillips. Fifty years of benzo(a)pyrene. Nature 303:468-472, 1983. 403. AW Wood, RL Chang, W Levin, RE Lehr, M Schaeffer-Ridder, JM Karle, DM Jerina, AH Conney. Mutagenicity and cytotoxicity of benzo(a)anthracene diol epoxides and tetrahydro epoxides: exceptional activity of the bay region 1,2 epoxides. Proc Natl Acad Sci USA 74: 2746-2750,1977. 404. 0 Plekonen, DW Nebert. Metabolism of polycyclic aromatic hydrocarbons: etiologic role in carcinogenesis. Pharmacol Rev 34: 189-222, 1982. 405. BR Smith, WR Brian. The role of metabolism in chemical-induced pulmonary toxicity. Toxicol Pathol 19:470-48 I , 199l. 406. DA Wiersma, RA Roth. Clearance of benzo(a)pyrene by isolated rat liver and lung: alterations in perfusion and metabolic capacity. J Pharmacol Exp Ther 225: 121-125, 1983. 407. RA Roth, A Vinegar. Actionby the lungs on circulating xenobiotic agents, witha case study of physiologically based pharmacokinetic modeling of benzo(a)pyrene disposition. Pharmacol Ther 48:143-155, 1990. 408. SG Bowes, AG Renwick. The intestinal metabolism and DNA binding ofbenzo(a)pyrenein guinea-pigs fed normal, high-fat and high cholesterol diets. Xenobiotica 16:543-552, 1986. 409. P Lesca. Protective effects of ellagic acid and other plant phenols on benzo(a)pyrene-induced neoplasia in mice. Carcinogenesis 4: 1651-1653, 1983. 410. SK Katiyar,RAganval,MTZaim, H Mukhtar.ProtectionagainstN-nitrosodiethylamine and benzo(a)pyrene induced forestomach and lung tumorigenesis in A/J mice by green tea. Carcinogenesis14:849-855,1993. 41 1. T Sigimura. Carcinogenicity of mutagenic heterocyclic amines formed during the cooking process.MutatRes 15033-41, 1980. 412. JS Felton, MC Knize. Occurrence, identification and bacterial mutagenicity of heterocyclic amines in cooked food. Mutagen Res 259205-218, 1991. 413. T Sigimura, S Sato. Mutagen-carcinogen in food. Cancer Res 43:S2415-S2421, 1983. 414. T Sigimura, K Wakabayashi. Mutagens and carcinogens in the diet. In: MW Pariza, JS Felton. HU Aeschbacaher, S Sato, ed. Mutagens and Carcinogens in the Diet. New York: WileyLiss,1990,pp. 1-18. 415. H Ohgaki, K Kusama, N Matsukura, K Morino, H Hasegawa, S Sato, S Takayama, T Sugimura. Carcinogenicity in mice of a mutagenic compound, 2-amin0-3-methylimidazo[4,5-f] quinoline from broiled sardine, cooked beef and beef extract. Carcinogenesis 5921-924, 1984. 416. S Takayama, Y Nakatsuru, M Masuda, S Sato, T Sugimura. Demonstration of carcinogenicity in F344 ratsof IQ from broiled sardine, fried beef and beef extract. Gann 75:460-470, 1984.
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417. RS Adamson. UP Thorgeirsson. EG Snyderwine, SS Thorgeirrson. J Reeves. DW Dalgard. S Takayama. T Sugimura. Carcinogenicity of 3-amino-3-n~ethyli1nida~o[4,5-f]quinoline in nonhuman primates: induction of tunwurs in three macques. Jpn J Cancer Res 81:10-14, 1990. 418. JK Lin, JT Cheng. SY Lin-Shiau. Enhancement ofthemutagenicityofIQandMeIQby nitrite in Salmonella system. Mutat Res 278977-287, 1992. 419. C Sasagawa,M Muramatsu. T Matsushima. Formationof direct mutagens from amino-imidazoazaarcnes by nitrite treatment. Mutat Res 203386, 1988. 420. T Matsushima, T Sugimura. Mutagen-carcinogens i n amino acid and protein pyrolysates and in cooked food. Prog Mutat Res 2:49-56. 198 1. 421. RH Adamson, UP Thorgeirsson. Carcinogens in foods: heterocyclic amines and cancer and 369:211-220, 1995. heart disease. Adv Exp Mcd Biol 422. JW Gaubatz, S Rooks.Dietary-linked DNAdamagein cardiaccells.FASEBJ10:A967. 1995.
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EOvcrvik,MOchiani.MHirose,TSugimura,MNagao.Theformation of heartDNA adducts in F344 rats following dietary administrationof heterocyclic amines. Mutat Res256: 37-43, 1991. CD Davis, HAJ Schut, EG Snyderwine. Enzymatic phase I1 activation of the N-hydroxyl14: amines of IQ, MeIQx and PhIP by various organs of monkeys and rats. Carcinogenesis 207 1-2096, 1993. AR Boobis, J Segura, NJ Gooderham, DS Davies. CYPIA2-catalyzed conversion of dietary heterocyclic amines to their proximal carcinogens is their major route of metabolism in humans. Cancer Res 54:89-94. 1994. HX Zu, HAJ Shutt. Inhibition of 2-a~nino-31nethylin~idazo[4,5-f]quinoline-DNA adduct formation in CDF, mice by heat altered derivatives of linoleic acid. Food Chem Toxicol 30: 9-16, 1992. AJ Alldrick, J Flynn,IR Rowland. Effects of plant-derived flavonoids and polyphenolic acids on the activity of mutagens from cooked food. Mutat Res 163:225-232. 1986. L Busk, UG Ahlborg. L Albanus. Inhibition of protein hydrolysate mutagenicity by retinol (vitamin A). Food Chern Toxicol 20:535-539. 1982. P Lindeskog, E Overnik, L Nilsson. C-E Nord, J-A Gustafsson. Influence of fried meat and fibre on cytochrome P-450 mediated activity and excretmn of mutagens in rats. Mutat Res 202:553-563, 1988. DW Layton. KT Bogen. MC Knize. FT Hatch, VM Johnson, JS Fclton. Cancer risk of heterocyclic amines in cooked foods: an analysis and implications for research. Carcinogenesis 16: 39-52. 1995. Y Zhang. TW Kenslcr,CC Cho, CH Posner, P Talalay. Anticarcinogenic activities of sulforaphane and structurally related synthetic norbornyl isothiocyanatcs. Proc Natl Acad Sci USA 91:3147-3150. 1994. M Jang, L Cai.GO Udeani, KV Slowing, CF Thomas, CWW Beechcr. HH Fong.NR Farnsworth, AD Kinghorn, RG Mehta, RC Moon, JM Pczzuto. Cancer chemopreventive activity of resveratrol, a natural product derived from grapes. Science 275218-220, 1997. EN Frcnkel, AL Waterhous. JE IOnsella. Inhibition of human LDL oxidatlon by resvcratrol. Lancet 34111 10.3-1 104, 1993. MTHuang,TLysz. T Ferraro, AF Abitli, JD Laskin, AH Conney.Inhibitoryeffects of curcumin on in vitro lipooxygcnase and cyclooxygenase activities in mouse epidermis. Cancer Res 51:813-819, 1991. K Polasa, B Sesikaran,TP Krishna. K Krishnaswamy. Turmeric-induced reduction in urinary 1991. mutagens. Food Chem Toxicol 29699-706, AC Reddy, BR Lokesh. Effect of dietary turmeric on iron-induced lipid peroxidation in the rat liver. Food Chcm Toxicol 32279-283, 1994. CJM Rompelberg. SJC Evertz, GCDM BruijnlJesrozier. PD Van Den Heuvel, H Vcrhagen.
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Effect of eugenol on the genotoxicity of established mutagens in the liver. Food Chem Toxicol34:33-42,1996. CJM Rompelberg. M-J ST Steenwinkel, JG Van Asten, JHM Van Dalft, RABaan, H Verhagen. Effect of eugenol on the mutagenicityof benzo(a)pyrene and the formationof benzo(a)pyrene-DNA adducts i n the -1acZ-transgenic mouse. MutatRes369:89-96, 1996. TH March, EH Jeffery, MA Wallig. The cruciferous nitrile. crambene, induces rat hepatic and pancreatic glutathione-S-transferase. Toxicol Sci 42:82-90, 1998. Y Li, EJ Wang, L Chen, AP Stein. KR Rcuhl, CS Yang. Effects of phenethyl isothiocyanate on acetaminophen metabolism and hepatotoxicity. Toxicol Appl Pharnmol 144:306-314, 1997. S Larsen-Su, DE Williams. Dietary indole-3-carbinol inhibits F M 0 activity and the expression of flavin-containing nmnooxygenase form 1 i n rat liver and intestine. Drug Metab Dispos 24:927-93 l , 1996. YL Ha, NK Grimm, MW Pariza. Newly recognised anticarcinogenic fatty acids: identification and quantification i n natural and processed cheese. J Agric Food Chem 73:75-81, 1989. C Ip. JA Scinleca. Conjugated linoleic acid and linoleic acid arc distinctive modulators of manunary carcinogenesis. Nutr Cancer 27:13 1-1 35, 1997.
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15 Food Additives
I. Introduction 448 11. Functions of FoodAdditives 448 Preservation A. 448 448 Processing B. C. Appeal and convenience 449 Nutrition D. 449 111. Food Additive Categories 451 N . FoodAdditiveSupplyIndustry454 V.ResearchandDevelopment455
VI. Manufacturing 458 VII. Government Regulations 459 United A.States 459 B. European Union (EU) 463 C. Japan 466 VIII.Trends and Issues 466 1X. Description of MajorFoodAdditives 468 Sweeteners A. 468 B. Thickeners and stabilizers 474 C. Colors 480 substitutes Fat D. 485 Enzymes E. 489 Vitamins 490 F. G . Antioxidants 495 H. Preservatives 499 I. Emulsifiers 502 J. Flavors 504 X. AdverseEffectsofFoodAdditives508 A. Food additives banned from use 509 Industrial B. chemicals 510 C. Foodallergiesandotheradversefoodreactionstofoodadditives D. Food additives derived from allergenic food 515 Bibliography 5 15
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1.
INTRODUCTION
The broadest definition of a food additive is any substance that becomes part of a food product, either directly or indirectly, during some phase of processing, storage, or packaging. The universe of food additives encompasses Direct food additives, those that are intentionally added to food for a functional purpose, in controlled amounts, usually at low levels (from parts per million to 1-2%, by weight), and Indirect or incidental food additives, those entering into food products in small quantities as a result of growing, processing, or packaging. The difference between food ingredients and additives is mainly in the quantity used in as food (e.g., sucrose), any given formulation. Food ingredients can be consumed alone while food additives areused in small quantities (usually less than 2%) relative to the total of desirable and food compositionbut which nonetheless playa large part in the production safe food products. as minor ingredients incorporated into foods Food additives may be looked upon to affect their properties in some desired way. Most commonly, the effects desired relate to color, flavor, texture, nutritive value, or stability in storage. There is no rigorous definition that meets all needs. The Codex Alime~mrius,which dominates actions in international circles, considers as a food by itself and normally an additive as an ingredient “not normally consumed by the used as a typical ingredient.” This obviously leaves great latitude for judgment committee. The U.S. Food, Drug, and Cosmetic Act has a complex definition of food additives that comes closeto any component of food introduced into U.S. commerce after 1957 and it will be addressed in the section dealing with government regulations.
11.
FUNCTIONS OF FOOD ADDITIVES
Direct food additives serve several major functions. Many additives, in fact, are multifunctional (Table 1). The basic functions of direct food additives include the following:
A.
Preservation
Food preservation techniques have advanced in the past 100 years and now include thermal processing,concentrationanddrying,refrigerationandfreezing,modifiedatmosphere, and irradiation. However, the use of chemical preservatives frequently augments these basic preservation techniques and represents the most economical way for food manufacturers to ensure a reasonable shelf life for their product. Antioxidants and antimicrobial agents perform some of these functions as well.
B. Processing Food processors are increasingly using food additives to ensure the integrity and appeal of their finished products. Emulsifiers maintain mixtures and improve texture in breads, dressings, and other foods. They are used in ice cream when snloothness is desired, in breads to increase shelf life and volume and to distribute the shortening, and in cake mixes
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Table 1 FoodAdditivesandTheirFunctions
Food additive Anticalilng agents Antioxidants
Appeal Preservation improvement modification Nutrition
Process X
X
Colors
Emulsifiers Enzymes Fat substitutes Flavors Humectants Leavening agents agents pH control Preservativcs Processing aids Sweeteners (sugars only) Sweeteners. high intensity Thickeners stabilizers and Vitamins and minerals
X X
X X X X X
X X X
X X X X
X X X X
X X X
X X
X X X
to achieve batter consistency. Stabilizers and thickeners assist in presenting an appealing product with consistent texture. Sorbitol, a humectant and sweetener, is used to retain moisture and enhance flavor. With the removal of sugar from many foods for dietetic reasons, a substitute bulking agent is needed.
C. Appeal and Convenience The changing eating habits of consumers, partly brought about by the large increase in the number of women who work outside the home, is creating a growing need for convenience foods. In many of these types of foods, it is essential that a variety of additives be used to provide the taste, color, texture, body, and general acceptability that are required. This need for convenience, while maintaining aesthetic appeal and taste, is becoming extremely important. Most food additives such as gums, flavoring agents, colorants, and sweeteners are includedby food processors because consumers demandthat food look and taste good as well as be easy to serve.
D. Nutrition There have been tremendous advancesin the knowledge of human nutrition, and consurners are increasingly awareof the value of good nutrition. Vitamins, antioxidants, proteins, and minerals are added to foods and beverages as supplements in an attempt to ensure proper nutrition for those whodo not eat a well-balanced diet.In addition, additives suchas antioxidants are often used to prevent deterioration of natural nutrients during processing. Recently more importance has been attributed to disease prevention through proper nutrition, as well as to increasing performance through sport nutrition products. On the other hand, the desire for good nutrition througha balanced diet may adversely affect consumer demand for some food additives such as fat substitutes.
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Adopted from the National Academy of Sciences/National Research Council national survey of food industries, the following ternls describe the physical and technical effects of various food additives: Anticaking agent orfree-flow agent: substance added to finely powdered or crystalline food products to prevent caking, lumping, or agglomeration. Antimicrobial agent: substance used to preserve food that prevents the growth of microorganisms and subsequent spoilage, including fungistats, mold and rope inhibitors, antimicrobial agents, antimyotic agents, preservatives, and mold preventing agents (indirect additives). Antioxidant: substance used to preserve food by retarding deterioration, rancidity, or discoloration due to oxidation. Boiler water additive: substance used in a steam or boiler water system as an anticorrosion agent to prevent scale or to effect steam purity. to preserve or enhance the color or Color or coloring adjunct: substance used shading of a food including color fixatives and color-retention agents. a unique flavor and/or color to Curing or pickling agent: substance imparting food, usually producing an increase in shelf-life. Dough strengthener: substance usedto modify starch and gluten, thereby producing more stable dough. Dryingagent:substancewithmoisture-absorbingabilityusedtomaintainan environment of low moisture. Emulsifier or emulsifier salt: substance which modifies surface tension in the component phase of an emulsion to establish a uniform dispersion or emulsion. Enzyme: Used to improve food processing and the quality of finished food. Firming agent: substance addedto precipitate residual pectin, thus strengthening the supporting tissue and preventing its collapse during processing. Flavor enhancer: substance added to supplement, enhance, or modify the taste and/or aroma of a food without imparting a characteristic taste or aroma of its own. Flavoring agent or adjuvant: substance added to impart or help impart a taste or aroma in food. Flour treating agent: substance added to milled flour to improve its color and/ or baking qualities, including bleaching and maturing agents. Formulation aid: substance used to promote or to produce a desired physical state or texture in food, including carriers, binders, fillers, plasticizers, film-formers, and tableting aids, etc. Freezing or cooling agent: substance that reduces the temperatureof food materials through direct contact. Fumigant: volatile substance used for controlling insects and pests. Humectant: hygroscopic substance incorporated in food to promote retention of moisture. Leavening agent: substance used to produce or stimulate production of carbon dioxide in baked goods in order to impart a light texture, including yeast, yeast foods, and calcium salts. Lubricant or release agent: substance added to food contact surfaces to prevent ingredients and finished products from sticking to them (direct additives), including release agents, lubricants, surface lubricants, waxes, and antiblocking agents (indirect additives).
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Malting or fermenting aid: substance used to control the rate or nature of the malting or fermenting process, including microbial nutrients and suppressants and excluding acids and alkalis. Masticatory substance: substance that is responsible for the long-lasting and pliable property of chewing gum. Nonnutritive sweetener: substance having less than 2% of the caloric value of sucrose per equivalent unit of sweetener. Nutrient supplement: substance necessary for the body’s nutritional and metabolic processes. Nutritive sweetener: substance having greater than 2% sucrose per equivalent unit of sweetening capacity. Oxidizing or reducing agent: substance which chemically oxidizes or reduces another food ingredient, thereby producing a more stable product. pH control agent: substance addedto change or maintain active acidity or basicity, including buffers, acids, alkalis, and neutralizing agents. to enhance the appeal Processing aid: substances used as a manufacturing aid or utility of a food or component, including clarifiers, clouding agents, catalysts, flocculents, filter aids, crystallization inhibitors, etc. Propellant: gas used to supply force to expel a product or to reduce the amount of oxygen in contact with the food in packaging. Sequesterant: substance which combines with polyvalent metal ions to form a soluble metal complex to improve the quality and stability of products. Solvent or vehicle: substance used to extract or dissolve another substance. Stabilizer or thickener: substance used to produce viscous solutions or dispersions, impart body, improve consistency, or stabilize emulsions, including suspending and bodying agents, setting agents, and bulking agents. Surface-active agent: substance used to modify surface properties of liquid food components for a variety of effects, other than emulsifiers. Includes solubilizing agents, dispersants, detergents, wetting agents, rehydrating enhancers, foaming agents, defoaming agents, etc. Surface finishing agents: substance used to increase palatability, preserve gloss, and inhibit discoloration of foods, including glazes, polishes, waxes, and protective coatings. Synergist: substance used to act or react with another food ingredientto produce a total effect different from or greater than the sum of the effects produced by the individual ingredients. Texturizer: substance which affects the appearance or feel of the food. Tracer: substance added as a food constituent (as requiredby regulation) SO that levels of this constituent can be detected after subsequent processing and/or combination with other food materials. Washing or surface removal agent: substance used to wash or assist in the removal of unwanted surface layers from plant or animal tissues.
111.
FOODADDITIVECATEGORIES
Substances that come under the general definition of direct food additive number in the thousands and include
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Inorganic chemicals (e.g., phosphates, sulfites, calcium chloride, etc.) Syntheticorganicchemicals(e.g.,dyes,benzoates,aromachemicals,vitamin A, etc.) Extraction products from and derivatives of natural sources (e.g., pectin, essential oils, vitamin E, etc.) Fermentation-derived products (e.g., enzymes, citric acid, xanthan gum, etc.) Most food additives have a long history of use; others are the result of recent research and development to fill particularrequirements of modcrnfoodprocessing.Someare common chemicals of industry that are upgraded in t e r m of purity to allow their use i n food. Major categories of foodadditivesincludepreservatives,colorants,antioxidants, flavors, thickeners and stabilizers, emulsifiers, acidifiers and buffers, enzymes, and sweeteners. Examples of major products in each category are shown in Table 2. Within this same category, products may belong to several chemical classes and offer specialized functionality (e.g., water- and oil-soluble antioxidants that include ascorbic acid and hindered phenols, respectively, and water-soluble azo dyes and water-dispersible carotenoids as food colors). Basic foodstuffs are excluded from the definition, although ingredients added to foods (e.g., high fructose corn syrup, MSG, and protein concentrates) are often included among food additives. Certain food additives, such as colors, flavors, gums, emulsifiers, and preservatives may find use also in pharmaceutical products and in toiletries and cosmetics (e.g., toothpaste, lipstick, etc). The same Food Chemical Codex (FCC) grade as in food is typically used in these applications, however, the combined value of the additive for these other applications does not exceed 10% of food use. Indirect food additives have no purposeful function in food and may be divided into the following categories: Components of adhesives (e.g., calcium ethyl acetoacetate 1,4-butanediol modified with adipic acid) Components of coatings (e.g., acrylate ester copolymer coatings and polyvinyl fluoride resins) Components of paper and paperboard (e.g., slimicides, sodium nitratehrea complex, and alkyl ketone dimers) Basic components of single- and repeated-use food contact surfaces (e.g., cellophane,ethylene-acrylicacidcopolymers,isobutylenecopolymers,andnylon resins) Components of articles intended for repeated use (e.g., ultrafiltration menlbrancs and textiles and textile fibers) Compounds controlling growth of microorganisms (e.g., sanitizing solutions) Antioxidants and stabilizers (e.g., octyltin stabilizers in vinyl chloride plastics) Certain adjuvants and production aids (e.g., animal glue, hydrogenated castor oil, synthetic fatty alcohols, and petrolatum)
as contaminants. In the United In many countries, these materials are defined and regulated States, these materials are food additives under the law. They are commonly classed as indirect food additives, but the FDA handles them in the same way as direct additives. Just as with direct additives, they may be generally recognized as safe (GRAS) substances and thereby escape explicit regulation because that status makes them, in fact, not food
453
Food Additives Table 2 SelectedMajorFoodAdditives
Thickeners and stabilizers Agar Alginates Carageenan Carboxymethyl ccllulose (CMC) Casein Gelatin Gellan gum Guar gum Gum Arabic Locust bean gum Modified starches Pectin Xanthan gum
Sweeteners Acesulfame-K Aspartame Dextrose Lactitol Mannitol Sorbitol Saccharin Xylitol Colors Certified food colors Dyes Lakes Noncertified colors Caramel Plant extracts Synthetic carotenoids Fat substitutes Partially or nonmetabolizable Sucrose polyester (Olestra) Caprenin Fat tnimetics Carbohydrate based products Protein based products Emulsifiers Flavors Aroma chemicals Vanillin Essential oils/natural extracts Menthol Flavor compositions Strawberry flavor Enzymes Amylases (alpha-amylase, etc.) Glucose isomerase Pectinases Proteases Rennin
Vitamins VitaminA Vitamin A acetate Vitamin B I Thiamin hydrochloride Vitamin B. Thiamin mononitrate Vitamin B,, Pyridoxtne hydrochloride VitaminB Cyanocobalamin Vitamin C Ascorbic acid VitaminD Ergocalciferol, cholecalciferol VitaminK Menadione Antioxidants Ascorbic acidlsodium ascorbate Erythorbic acidlsodium erythorbate BHA (butylated hydroxyquinone) BHT (butylated hydroxytoluene) PG (propyl gallate) TBHQ (tert-butyl hydroquinone) Tocopherols Sulfur dioxide/sulfite salts Preservatives Benzoic acidlbenzoates Propionic acidlpropionates Parabenes Sorbic acidlsorbates Sulfites Emulsifiers Mono- and diglycerides Lactylated esters Lecithin Polysorbates Propylene glycol esters Sorbitan esters Sucrose csters Anticaking agents Aluminum calcium silicate Calcium silicate Salts o f fatty acids (stearates) Silicon dioxide Tricalcium silicate Yellow prussiate of soda pH control agents Citric acid Malic acid Phosphoric acidlphosphatcs Sodium citrate Sodium hydroxide
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additives. In general, however, regulation of these materials is more extensive and more rigorous in the United States than in other counties. As might be expected, packaging materials which have been used for a long time, such as glass, receive less close scrutiny than more newly introduced materials and those materials just being proposed for introduction.
W. FOOD ADDITIVESUPPLY INDUSTRY Food additive suppliers are an important part of the food manufacturing system, supplying products to both commodity processors and food processors (Figure 1). Practically every food manufacturing operation depends to some degree on the use of food additives, but the range of additives necessary for the formulation varies (Table 3). Overall, the food additive industry appears to be highly fragmented, consisting of more than 500 companies supplying a variety of chemically and functionally different products that serve a common end-use market-the food industry. However, suppliers tend to beeitherhighlyspecializedparticipantsinthemajorproductcategories(e.g., Novo with enzymes, Warner-Jenkinson Company with certified food colors, etc.) or large chemical companiesthat offer food-grade versionsof a few industrial products (e.g., Lonza's emulsifiers, FMC Corp.'s cellulose derivatives). Manufacturers are typically involved in supplying additives in a limited number of product categories (e.g., colors, vitamins, or enzymes) or servicing selected food sectors (e.g., processed meats, dairy-based products, or bakery products). While a company or group of companies may tend to dominate sales in each of the specific categories (e.g., Novo with enzymes, Rhodia with vanillin, or NutraSweet/KelcoCo. with aspartame and biogums), no single company or small group of companies dominates the entire food additive industry. Forty years ago it was relatively easy and lucrative for chemical companies to stumble into the role of food additive supplier and reap profits by upgrading the purity and quality of chemicals originally developed for other industrial markets. Today, however, the long time and high costs associated with gaining regulatory approval (estimated 510 years and $1.5-$40 million) have taken away the incentive to commercialize products
Fig. 1 Integrated view of the U.S. food manufacturing system.
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from basic research. As a result, food additives represent only a minor portion of large chemical companies’ overall business. Most large chemical companies that supply food additives, such as FMC, Monsanto, Hercules, Lonza, Hoffman-La Roche, Huls, Rh6nePoulenc, and Eastman Chemicals, have diversified chemical operations, with perhaps only 5% or less of total sales generated by food additives. Figure 2 depicts the food additive industry structure and the flow of its products. Some 60-70% of food additives are used in the manufacture of food; about 20-30% are usedin commodity processing operations such as flourmilling,meatpacking,oilseed crushing and refining, vegetable packaging, animal feeds, and fruit juice processing; and the remaining 5-10% are used for things such as pharmaceuticals and cosmetics.In addition to basic additive producers, the food additives industry includes companies that specialize in compounding of specialty product mixes, and national and local distributors (Figure 2). Specialty compounders formulate mixed products for the food industry such as dairy ingredients, baker’s mixes, curing blends, thickener and emulsifier blends, cheese aids, ethnic flavors, total seasoning packages, and spice blends. They are generally very knowledgeable about additive and ingredient properties and are experiencedin food technology overall. Compounding companies are often relatively small, sell directly to a food processor, are highly service oriented, and market product lines that have a high level of perceived differentiation. Their formulations offer convenience and enjoy higher gross profit margins than single food additive sales. Distributors also playan important rolein the distribution of food additives. Additive producers typically use distributors to service their smaller accounts or for warehousing and servicing of accounts that geographically the producers cannot cover effectively or economically.
V.
RESEARCH AND DEVELOPMENT
Because there are so many dissimilar and unconnected segments of the food additives industry, the participating companies exhibit different approaches to research and development (R&D). Many stress applications research to uncover new niches for existing additives or modifications of currently FDA-approved additives. Some emphasize innovative research or new, high-value products, but these are very few because of the cost and time for basic research, development, regulatory approval, and market acceptance of a new food additive product. For example, NutraSweet’s aspartame product took more than 11 years to gain FDA approval; acesulfame K took 6 years for FDA approval and a total of 21 years since development. The total costs of research, development, and approvals for aspartame were close to $25 million. Procter & Gamble’s OlestraTM was in research and development for 20 years, yet in developwasn’t submittedto the FDA until June 1987. After spending over $200 million ment costs and waiting more than 8 years for FDA approval, the fat substitute received approval in January 1996. Approval of thefoodadditive islimited to snacks such as potato chips and tortilla chips. Moreover, it has a special regulatory constraint: OlestraTMcontaining products require fortification with vitamins A, D, E, and K to compensate for the limited absorption of these fat-soluble vitamins, and the products must be labeled with the statement, “This product contains Olestra. Olestra may cause abdominal cramping and loose stools. Olestra inhibits the absorption of some vitamins and other nutrients. Vitamins A, D, E, and K have been added.”
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COMMODITY ADDITIVES PROCESSORS Sugar refining Grain milling 9 Oil seed processing Fruit and vegetables processing
4
FOOD MANUFACTURERS Acidulanls Emulsiliers Flavors, colors, etc. High-Intensity sweeteners
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OTHER USES * Pharmaceutical
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Daily products Bread. c m k i e s , etc.
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Fig. 2 Food additives, pattern of use in food, and other applications.
In general, only large, well-financed companies can afford the
R&D efforts neces-
sary to bring a new food additive product to market. Small companies simply are unable
to deal with the complexity, costs, and required time. Personnel staffing requirements for R&D of food additives vary significantly. Because statistics for the food additives business of most producing companies are not reported separately, only estimates can be made. R&D expenditures as a percentage of sales typically range from 1% or less for products such as preservatives, to 5-6% for more technically oriented products such as fat substitutes and certain natural colors, and 5-1076 for flavors.
VI.
MANUFACTURING
Manufacturing processes for food additives vary widely in their nature and technological sophistication. Some of the specific processes for the more important food additives are described in later sections of this report. A common characteristic of all food additives manufacturing, however, is that the products must be made to a high degree of purity and under sanitary conditions similarto those of food processing plants. Production equipment must be dedicated to food additive products and cannot be used forother industrial production. Plants producing food additives are subject to periodic inspection by the regulatory agencies. Typically chemical additives made by synthesis (e.g., BHT, saccharin) or by fermentation(e.g.,aspartame,microbialenzymes,xanthanandgellangums)require a high level of capital investment. The former additives have industrial uses and are likely to share their basic production costs with the industrial-grade material; a small portion of
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the total production is then upgraded and purified to food-grade quality in separate dedicated plant units. Accurate long-term market forecasts for products are essential in order to minimize the risk associated with capital investment decisions in single-purpose plants. Other participants in the food additives business that are not involved in chemical production on a large scale for the extraction and purification of natural products, small-scale synthesis of aromatic chemicals, and for flavor and ingredient compounding have much lower capital requirements.
VII. A.
GOVERNMENT REGULATIONS
United States
The application of food additives is highly regulated worldwide, although regulatory philosophy, the approval of specific products, and the level of enforcement differ from country to country. Basic regulations in the United States, Western Europe, and Japan are described below. These three major industrial regions are the largest consumers of food additives. With only 13% of the world’s population, these countries account for more than twothirds of the food additive market. The U.S. Food and DrugAdministration(FDA) istheprincipal U.S. regulatory body controlling the use of food additives. It does so through the 1958 Food Additives Amendment to the Food, Drug & Cosmetic (FD&C) Act of 1938. The amendment was enacted with the threefold purpose of 1.
2. 3.
Protecting public health by requiring proof of safety before a substance can be added to food. Advancingfoodtechnology. Improving the food supply by permitting the use of substances in food that are safe at the levels of intended use.
According to the legal definition, food additives that are subject to the amendment include “any substance the intended useof which results or may reasonably be expected to result directly or indirectly in its becoming a component or otherwise affecting the characteristics of any food.” This definition includes any substance used in the production, processing, treatment, packaging, transportation, or storage of food. If a substance is added to a food for a specific purpose it is referred to as a direct additive. For example, the low-calorie sweetener aspartame, which is used in beverages, puddings, yogurt, chewing gum, and other foods, is considered a direct additive. Indirect food additives are those that become part of the food in trace amounts due to its packaging, storage, or other handling. This class includes all materials that would not usually become part of food if man could completely control food production. In practice, indirect food additives are found in agricultural produce in quantities well within acceptable and legal tolerances. For example, minute amounts of packaging substances may find their way into foods during storage. A variety of chemicals, including plastic monomers,plasticizers,stabilizers,printingink,andothersubstances,migrate at extremely low levels into foods. Lead andtin are perhaps the main concerns associated with packaging materials. The storage of acidic foods in inappropriate containers can result in the leaching of toxic heavy metals, such as zinc and copper, into the food. Food packaging manufacturers therefore must prove to the FDA that all materials coming in contact with food are safe before they are permitted for use in such a manner.
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A wide variety of chemicals are used in modern agricultural practice. Residues of these chemicals can linger in raw and processed foods, although federal regulatory agencies evaluate the safety of such chemicals, and regulate and monitor their use on food products. The major categories of agricultural chemicals include insecticides, herbicides, fungicides. fertilizers, and veterinary drugs, including antibiotics. Industrial and/or environmental pollutants may migrate into foods i n small amounts. On rare occasions, hazardous levelsof polychlorinated biphenyls (PBCs) and polybrominated biphenyls (PBBs) have been found in foods. For regulatory purposes, all food additives fall into one of three categories: Generally recognized as safe (GRAS) substances Prior sanctioned substances Regulated direct/indirect additives. GRAS substances (approximately 700 compounds) are a group of additives regarded by qualified experts as “generally recognized as safe.” These substances are considered safe because their past extensive use has not shown any harmful effects. Prior sanctioned substances (approximately 1400 compounds) are products that were already in use in foods prior to the 1958 Food Additives Amendment and are therefore considered exempt from the approval process. Some prior sanctioned substances also appear on the GRAS list. This is the grandfather clause of the amendment. The FDA is involved in an ongoing review of the GRAS and prior sanctioned substance lists to ensure thatthesesubstances are tested by means of thelatestscientific methods. Likewise, the FDA also reviews substances that are not currently included on the GRAS list to determine whether they should be added. All other additives are regulated-that is, a specific food additive petition must be filedwiththe FDA requesting approval for use of theadditive in anyapplicationnot previously approved. A food or color additive petition must provide convincing evidence that the proposed additives perform as intended. Animal studies using large doses of the to show that the substance will not cause additive for long periods are often necessary harmful effects at expected levels of human consumption. In deciding whether an additive should be approved, the agency considers the composition and properties of the substance, the amount likely to be consumed, its probable long-term effects, and various other safety factors. Absolute safety of any substance can never be proven. Therefore the FDA must determine if theadditiveissafeunderthe proposed conditions of use, based on the best scientific knowledge available. I n addition, the FDA operates an Adverse Reaction Monitoring System (ARMS) to help serve as an ongoing safety checkof all additives. The system monitors and investigatesall complaints by individuals or their physicians that are believed to be related to specific foods, food additives, or nutrient supplements. Color additives for food represent a unique and special category of food additives. They have historicallybeen so considered in legislation and regulation. The current legislation governing the regulation and use of color additives in the United States is the Food, Drug & Cosmetic Act of 1938, as a mended by the Color Additive Amendment of 1960. Colors permitted foruse in foods are classified eitheras certified or exempt from certification.Certifiedcolorsare man-made, with each batch being tested bythe manufacturer and the FDA (certified) to ensure that they meet strict specifications for purity. Color additives that are exempt from certification include pigments derived from natural sources. However, color additives exempt from certification also must meet certain
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legalcriteriaforspecificationsandpurity.One of thefeatures of theColorAdditive Amendment of 1960 was the equal treatmentof synthetic colors and the so-called natural colors in respect to pretesting requirements. Moreover, currently all color additives, certified and uncertified (“natural”), are designated on the label as artificial color. Flavor substances are regulated somewhat differently, and the rules are less restrictive. However, the use of aroma chemicals as flavor ingredients is regulated under laws that may differ from country to country. Following the lead of the United States, inclusion on a positive list that spells out which chemicals are permitted for food use has become the prevalent legislation for regulating flavor chemicals worldwide. The United States has a list of flavor substances that are deemed GRAS based on the history of use, review of of experts. These GRAS lists (through GRAS 18) available toxicology, and the opinion have been compiled since 1977 by the expert panel of the Flavor Extracts Manufacturers Association of the United States (FEMA). Over the years, more than 1800 materials appeared on FEMA lists. Formed in 1909. FEMA is an industry association that originally started pursuing voluntary self-regulation and later was granted quasi-official status on regulatory matters regarding flavor chemicals by the FDA. The FEMA expert panel was formed in1960. Thisindependentpanel,composed of eminentlyqualifiedexpertsrecruitedfromoutsidetheflavorindustry,hasexpertiseinhumannutrition,physiology, metabolism,toxicology,andchemicalstructure-activityrelationships.Mostindustrial countries more or less follow the U.S. system. Although the FDA has primary jurisdiction over food additives, clearance for use of additives in certain products must be obtained from other government agencies as well. For example, the U.S. Department of Agriculture (USDA) through the Meat Inspection Division (MID) exercises jurisdiction over additives and ingredients for meat and poultry; the Bureau of Alcohol, Tobacco, and Firearms (BATF) of the U.S. Department of the Treasury controls the ingredients used in alcoholic beverages. The standards of identity specify in detail what can and cannot be packaged under a given product name. Standards of identity exist for milk, cream, cheese, frozen dessert. bologna products, cereal products, cereal flours, pasta, canned and frozen fruits and vegetables, juices, eggs, fish,nuts, nonalcoholic beverages, margarine, sweeteners, dressings, and flavorings. An approved food additivein the United States may be precluded from use in certain foods characterized by the standards of identity unless the additive is specifically required by or is listed as an optional ingredient in the standards. The standardsof identity establish the ingredient composition of a given food, which can then be labeled by its common name. If the manufacturer does not adhere to the standard composition, the food must be labeled “imitation.” The Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), which was issued in 1972 and amended in 1988, covers pesticides used on raw agricultural products. The FDA, however, is responsible for enforcing tolerances for pesticide residues that end up in food products. In the United States, label disclosure of food additives is mandated with few exceptions. Under FDA, USDA, and BATF regulations, the ingredients of a food or beverage must be stated on the product label i n decreasing order of predominance. For many direct additive categories, chemical constituents must be identified by their common names and the purpose for which they were added. One of the recent regulations involving the food industry, as well as food additive manufacturers, came with the passing of the Nutrition Labeling and Education Act of 1990 (NLEA), which anlends the Federal Food, Drug & Cosmetic Act, to make nutrition
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labeling mandatory for most FDA-regulated foods. The nutrition labeling regulations issued by the FDA and the USDA Food Safety and Inspection Service (FSIS) required compliance by August 8, 1994. The FDA’s nutrition labeling regulations focus on nutrients currently accepted as significantly affecting consumer health. The serving size is the basis for reporting each food’s nutrient content. Serving sizes are defined for most foods reflecting the amount people actually eat and are shown in both common household and metric measures. The amount per serving of the following nutrients are required to be included on labels: total calories, calories from total fat, total fat, saturated fat, cholesterol, total carbohydrates, complex carbohydrates, sugars, dietary fiber, protein, sodium, vitamin A, vitamin C, calcium, and iron. Listing other essential vitamins and minerals such as thiamin, riboflavin, and niacin, among other nutrients, is optional. A simplified nutrition label format is allowed for foods containinginsignificant amounts of more than half the required nutrients. The minimum label includes total calories, total fat, total carbohydrates, protein, and sodium. The FDAregulation requires the nutrition content be based on amounts of the product customarily consumed, and expressedin both common household and metric measures (e.g., 1 cup and 240 ml). Serving size reference amounts are based on food consumption survey data on amounts of food commonly consumed per eating occasion by persons 4 years of age and older. Manufacturers must follow the procedures to convert the reference amounts to serving sizes appropriate for their specific products. Any package containing less than two servings is considered a single-serving container. Nine terms are presently allowed by the FDA to describe a food, including free, low, high, source of, reduced, light/lite, less (or, for calories, fewer), more, and fresh. Claims for cholesterol are tied to levels of saturated fat in the food. Meal-type products are not allowed to use the term reduced. Health claims are allowed for only the following nutrient/disease relationships: a a a a
a a
a a a a
a
Calcium and osteoporosis Sodium and hypertension Unsaturated fats, low cholesterol intake, and cardiovascular disease Dietary lipids and cancer Fiber-containing grain products, fruits and vegetables, and cancer Fruits, vegetables, and grain products that contain fiber and risk of coronary heart disease Dietary fiber from fruits and vegetables and cancer Folic acid and neural tube defect Sugar alcohols and tooth decay Psyllium-containing foods and the riskof heart disease (when consumed as part of a diet low in saturated fat and cholesterol Soy protein and reduced risk of coronary heart disease (FDA proposal as of January 1999, not finalized yet)
Changing dietary recommendations and labeling requirements impact food additive producers both positively and negatively. Products used for fat-sparing/substitution (e.g., hydrocolloids, starches, other fat substitutes) and low-calorie sweeteners fare well, as food manufacturers striveto lower the caloric and saturated fat content of their products. Natural colors (provided they can be substituted), as well as other natural or seemingly natural
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products will also be in higher demand so as to provide consumers with a more healthy and nutritious product (or at least one with a more healthy-sounding label). The Food Additives Amendmentalso contains what is knownas the Delaney clause, which mandatesthe FDA toban any food additive found to cause cancer in manor animals, regardless of dose level or intended use. The clause applies not only to new food additives but also to those in use prior to 1958. The Delaney clause is totally inflexible in that it does not recognize any threshold level below which the additive might not present a health hazard. Thus it has caused a number of problems for the food industry and for food additives. Certain additives (e.g., the sweetener cyclamate, etc.) have been banned after they were found to be potential carcinogens-even though feeding tests in animals at massive dose levels may not bear any correlation to the potential risk to man of chronic ingestion at very low levels. Were it not for a moratorium mandated by Congress, saccharin would also have been banned in the United States several years ago by the FDA in compliance with current U.S. food laws. Although congressional sentiment has been running for some time in favor of repealing the Delaney clause, to date, attempts to replace it with a more practical and realistic law have been unsuccessful.
1. Approval Process A new substance gains approval for fooduse through the successful submission of a food additive petition that must document the following: Safety, including chronic feeding studies in two species of animals. Intended use. Efficiency data at specific levels in the specified food system. Manufacturingdetailsandproductspecifications. Methods for analysis of the substance in food. Environmentalimpactstatement. Quite frequently, this process can be lengthy-up to I O years in the case of aspartame and OlestraTM-and costly in terms of man-hours and dollars. There is little doubt that every level of the U.S. food additives business is affected by regulations, and operates with a constant awarenessof the importanceof FDA decisions.Not only is the introduction of a new food additive impossible without FDA approval, but the additives in use are under constant scrutiny by the regulatory agency and remain vulnerable to new unfavorable toxicology findings. While the barring of an additive may create opportunities for suppliers to develop new or substitute materials, the potential market is often too small loss of the ingredient may cause havoc within afto create sufficient incentive, and the fected sectors of the food industry. For example, the ban on cyclamates, followed by the close call on saccharin, almost caused the demise of the diet soft-drink industry. The wellrecognized need for alternative safe sweeteners undoubtedly was a stimulus for G . D. to engage in a IO-year effort to have Searle (now Monsanto’s NutraSweet Kelco division) aspartame cleared for food use.
B.
European Union (EU)
Food additives intended for human consumption are regulated by the member states as described in Directive 89/107/EEC of December 21, 1988. The EU food additive law recognizes 106 food additives. Later, several amendments and adaptationsof the directive were introduced or proposed including
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A list of additives the use of which is authorized to the exclusion of all others. The list of foodstuffstowhichtheseadditivesmaybeadded,theconditions under which they may be added and, where appropriate, a limit on the purpose of their use. The rules on additives used as carrier substances and solvents, including their purity criteria. In 1990 the commission proposed a first specific directive relating to sweeteners and food additives other than colors and sweeteners. The Sweetener Directive took effect on July 30, 1994. Member countries were asked to adopt the directive by December 3 1, 1995, which did not occur. The sweetener guidelines are expected to open up new markets for low-calorie food products and will simplify logistical matters. Efforts have been toward a uniform registration process so that a registration obtained in one country would be valid in all EU member countries. The new EU food additive law, however, will not prevent individual countries from asking for additional or country-specific requirements for new product registrations. At the EU level, several institutions and groups are involved in the development of food additives law, illcluding of the institutions of the European Commisthe Scientific Committee for Food (SCF), one sion which deals with safety issues, representatives from different national professional organizations, representatives from the food industry, retailers, etc. The Standing Committee on Foodstuffs ensures close cooperation between the commission and the member states. and labeling of novel food such as The EU rules for theevaluation,marketing, genetically modified foods are also being developed. The new nlarketing rules wouldalso oblige manufacturers to obtain permission before placing new foods or ingredientson the market, with the exception of products that are substantially equivalent to existing foods. The new rules have still to be cleared by the European Parliament, which has the power to veto under the new co-decision procedure introduced in 1995. In many countries, additives must be declared in the labeling. Within the EU, some additive groups have been uniformly codified with “E” numbers for the orientation of consumers. Some countries, suchas Germany, have gone further, adopting regulations on an acceptable daily intake (ADI) basis that build on the latest toxicological knowledge. Some examples of “E” numbers are presented in Table 4. Under EU food law, any claim that a food has the property of preventing, treating, or curing a human disease or condition, or any implication of such properties, is prohibited. This aspect of the law has been strictly enforced in all member states of the EU. As early as 1980, the European Commission recognized that the area of food claims required harmonization and circulated the first proposal for a directive. By the end of 1998, this approach had not succeeded. Recently the introduction of genetically modified (GM) corn and soy into Europe has caused considerable activity within governments and consumer organizations. European Parliament and Council Regulation no. 258/97 on novel foods and novel food ingredients requires prior approval of foods and food ingredients containing or consisting of a GM organism, and food and food ingredients produced from, but not containing GM organisms. More recently, Council Regulation1 139/98 came into force, requiring that any product containing GM soy or corn, or derivatives of GM soy or corn containing protein or DNA, must be labeled with the statement “produced from genetically modified soy” or
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Food Additives Table 4
Selected EUFoodAdditivcsandCodes
Colorants El00 El01 El02 El 10 E120 E150 El60 E 160a El 60c E162 E163 Preservatives
-
Curcumin Riboflavin Tartrazin Yellow no. 6 Carmin Caralnels Annatto Beta-carotene Paprika Beetroot red (betanin, betanidin) Enocyanin (grape-skin cxtract)
Sorbic acid Sodium sorbate E20 1 Potassium sorbate E202 Calcium sorbate E203 Benzoic acid E210 Sodium benzoate E21 1 Potassium benzoate E212 Sodium propionate E28 1 Potassium propionate E282 Calcium propionate E283 Antioxidants L-ascorbic acid (vitamin C) E300 Synthetic alpha-tocopherol (vitamin E) E307 Propyl gallate (PG) E31 1 Butylhydroxyanisole (BHA) E320 Thickcners and stabilizers alginate Sodium E40 l E415 E420 E440a Emulsifiers E322 E47 1 Mono- diglycerides and of fatty acids Polyglycerol E475 acids fatty esters of Sodium steroyl-2 lactilate E48 I E200
"produced from genetically modified maize." However, refined oils or lecithin that are very unlikely to contain GM protein or DNA are exempt from such labeling statement requirements. A further labeling change that comes into force in February 2000 is the quantitative declaration of ingredients (QUID). This applies to foods and beverages with more than oneingredient,withvery fewexceptions. The quantity of ingredients,expressed as a percentage of the food or drink, must appear in or immediately next to the name of the food or i n the list of ingredients next to the ingredient concerned.
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C. Japan In Japan, the Food Chemistry Division of the Ministry of Health and Welfare (MHW) has jurisdiction over food additives through the Food Sanitation Law. It was in 1948 that the term “food additive” appeared in the law (in the Food Sanitation Law) and a positive list of food additives was created in Japan.It was the first positive list created in the world, and it did not distinguish between syntheticor natural additives. Several amendments were adopted later. Amendments to the regulations, as well as additions or deletions to Kohetish0 (the Japanese Codexof Food Additives), were mostlyinfluenced by two major objectives: protection of food sanitation and customer safety, and harmonization with international regulatory requirements. In the Food Sanitation Law, the term “additive” means anything added to, mixed into, permeating, or otherwise put in or upon food forthe purpose of processing or preservto defining ing it. Most discussions on regulating food additives in Japan have been related what food additives should be under legal restriction and on labeling requirements. Very often in these discussions, differentiating “synthetic” and “natural” food additives had been at issue. In Japan, those two generally used terms have often misled customers into a blind belief in natural food additives. However, regulatory bodies, as well as the food additive industry, no longer distinguish additives with these terms. The latest amendment of the law (May 24, 1995) includes deletion of the term “chemically synthesized substances.”Thus“natural”foodadditivesareregulatedunder the amended law (being enacted from May 24, 1996), unless theyarelisted as “existingfoodadditives.” The MHW then disclosed the list of “existing food additives” on August IO, 1995. Today, when new natural food additives are used in Japan, suppliers also need to report them to the MHW. However, in general, data requirements for natural additives are still not as strict as those for chemical substances. Natural food additives reported to the MHW are listedin a table separate from the conventional positive list for chemical food additives. There are about 1200 items in the natural additive list, while the conventional list contained 349 compounds as of 1992. In 1983 the MHW addeda new regulation that requires labeling by the name of the compound used as a food additiveas well as by the purpose of its use. Today both synthetic and natural food additives need to be labeled.
VIII. TRENDS AND ISSUES While there are many differences in food tastes and preferences among consumers, the major trends driving the food additives industry appear to be very similar: Concern for health and nutrition. Food safety/health consciousness. Desire for convenience. The concept of value added. High costs associated with R&D and product commercialization. Growing awareness of the connection between diet and diseases suchas cancer and heart disease has caused consumersto reexamine their diets and lifestyles and seek healthier alternatives. Consumer desire for healthier, more nutritious foods favors natural addi-
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tives and ingredients (and those that are perceived as natural), as well as those that reduce calories, sodium, cholesterol, and the overall fat in foods. Fortification withthe “right level” of vitamins, amino acids, and trace minerals is important, and additives that sound natural(e.g.,gelatin,pectin,vitamins,etc.)versuschemical(e.g.,potassiumbenzoate, butylated hydroxyanisole, etc.) have a more favorable consumer image. The shiftawayfromcommodity to moreprocessed,higher-valuefoodproducts favors an increased use of additives in processing. Additives that are perceived favorably by consumers as healthy or natural foods are likely to grow faster. Finally, demands are high for fat replacers, high-intensity sweeteners, low-calorie bulking agents, certain gums, freezehhaw stabilizers, and natural flavors. Sales of ingredient and additive blends will dominate in the future. The synergistic effects that enhancethefunctionality of thesematerials,whilereducingthequantity needed, will play an ever-more significant role in formulated foods. Information on these blends will be scarce, because they will be developed in house by food additive suppliers and food manufacturers wishingto maintain confidentiality in order to optimize exclusive commercial benefit. Other issues affecting the growth and broadening of the food additives industryincludeincreasinggovernmentregulatoryactivity;increasing R&D andlegal expenses; and the great length of time needed to perfect, gain approval for, and market a new food additive product. In addition to traditional processed food products, a variety of health-related products known as “functional foods” and “nutraceuticals” have appeared on the market. Functional foods are food products that improve performance or provide a health benefit beyond meeting the basic nutritional needs of humans. Although functional foods are consumed for their taste, aroma, or nutritional value, they are also consumed by health conscious adults for their perceived benefits in preventing the onset of degenerative diseases such as arthritis, cancer, or heart disease. Nutraceuticals are specific vitamins, minerals, amino acids, herbs and other botanicals, or constituent parts thereof that are taken in oral form to promote natural ways of preventing or treating various degenerative disease conditions. Nutraceuticals differ from functional foods in that they are only consumed for their health benefits rather than for taste, aroma, or nutritive value. In the United States nutraceuticals will have to overcome regulatoryconstrainedbeforetheycangainalargemarket. In constrast,inJapanand several countries in Europe the conceptof nutraceuticals is well established,both in terms of regulations and consumer acceptance. The market for fat replacers is the number one concern of customers in the United States, and following the recent approval of Olestra, the first truly heat-stable fat substitute will increase new product activity and interest in reduced or no-fat products. The fastest growth for fat substitute-containing products is expected to be in the United States, where diets have historically been higher in fat and sugar and consumers appear to have more problems with obesity/weight control and associated diseases. Europe may be the next window of marketing opportunity for light products. Especially in the UK, France, and Germany, the popularity of foods and beverages with less fat, sugar, and calories appears to be moving toward US.levels. In Japan the market for fat substitutes is currently very small, because the problem of excessive consumption of fat is not as serious as in the United States or Western Europe. However, with the growing influx of Western culture and along with it “fast food,” demand for low-calorie/low-fat foods is likely to grow. The success of these products will depend on the success/acceptance of low-calorie/lowfat products in the United States and Western Europe.
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The safety of the food supply continues to receive a great deal of attention from the press, the public, and the governments. In 1993, an outbreak of food poisoning i n the United States, eventually traced to undercooked beef, caused fundamental changes in regulatory policies and demonstrated to food processors the need for increased caution against food pathogens. The recent European outbreaksof bovine spongiform encephalopathy (BSE), known as “mad cow disease,” have created more serious and worldwide concerns about cattle-derived food products, including some dietary supplements (e.g.. gelatin capsules) and personal care products. Fast-paced lifestyles will continue to drive the demand for savory, high-quality convenience foods. Microwaveable and shelf-stable products that are tasty and healthy require additives such as specialized flavors, colors, and stabilizers to enhancelmaintain quality and will result i n continuing growth of the market for these additives. The concept of value-addedproducts is also ofgreatinterest tofoodprocessors as foods with added value, or at least perceived added value (e.g., low-fat, low-calorie, vitamin fortified, more convenient form/package, perceived prevention against particular diseases), garner higher margins. Therefore consumption of additives that can aid in adding value to processed foods will continue to increase. Following trends in the United States,the European market shows increased interest in ethnic foods and vegetarianism. The motivation in both cases is to promote health. Also, more products are being introduced which are aimed at very specific groups. These include children, teenagers, women, and in particular the growing population of elderly persons.
IX.
DESCRIPTION OF MAJOR FOOD ADDITIVES
Direct food additives comprise more than 30 types. With about 3000 food additives, including more than 1800 flavoring substances currently approved for use i n the United States (and more petitioning for approval), it would be difficult in a chapter such as this to discuss each and every substance. Ten major food additives were selected for discussion in this chapter.
A.
Sweeteners
Sweeteners are used in formulated foods to impart sweetness and to perform several other functions. They render certain foods palatableand mask bitterness; add flavor, body, bulk, and texture; change the freezing point and control crystallization; control viscosity, which contributes to body and texture; and prevent spoilage. Certain sweeteners act as preservatives by binding moisture in food that is required by detrimental microorganisms. Alternatively, some sweeteners can serve as food for fermenting organisms that produce acids that preserve the food, thus extending shelf life by retaining moisture. These auxiliary flmctions must be kept in mind when considering applications for artificial sweeteners. Sweeteners can be classified in a variety of ways: 0
0
Nutritive or nonnutritive. Materials either are metabolized and provide calories, or are not metabolized and thus are noncaloric. Natural or synthetic.Commercialproductsthataremodifications of anatural product, for example, honey or crystalline fructose, are considered natural.
Food Additives 0
0
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Regularor low-calorie/dietetic/high-intensity. Althoughtwosweetenersmay have the same number of calories per gram, one may be considered low-calorie or high-intensity if less material is used for equivalent sweetness. As foods. For example, fruit juice concentrates can impart substantial sweetness.
Sweetness is measured via sensory methodsby taste panels. It is a subjective perception influenced by a multitude of variables including the temperature of the food being tasted, pH, other flavors and ingredients in the food, physical characteristics of the food sweetener, concentration, rate of sweetness development, and permanence of sweetness and flavor. Also, results can vary depending on the foods consumed priorto testing (even several hours before testing), the flavors to which the taster is accustomed, tasting experience of the panelist, time of day, and the physical surroundings in the test room. Sucrose, commonly known as table sugar (orrefined sugar), is the standard against which all sweetenersaremeasured in t e r m of quality of tasteandtaste profile.It is consumed in the greatest volume of all sweeteners. Sucrose, high-fructose corn syrup (HFCS), and other natural sweeteners (e.g., molasses, honey, maple syrup, and lactose) are food commodities and are not considered as food additives, therefore they will not be covered here. The discussion that follows is limited to the polyol alternative sweeteners and the high-intensity sweeteners.
1. Polyols Polyols(sugaralcoholsorpolyalcohols)arechemicallyreducedcarbohydrates.These compounds are important sugar substitutes that are utilized where their different sensory, special dietary, and functional properties make them desirable. Also polyols are utilized in low-calorie food formulations. The sweetness of polyols relative to sucrose and their caloric values are shown in Table 5. Moreover, because polyols are absorbed more slowly from the digestive tractthanis sucrose, they are useful in certain special diets. When consumed in large quantities (in excess of 25-50 g/day), however, they have a laxative effect, apparently becauseof the comparatively slow intestinal absorption.In the EU countries, if a food product contains morethan 10% by weight of a polyol, a warning statement must be added to the label stating that excessiveconsumption may induce a laxative effect. I n the United Statesfood products sweetenedwith polyols and containingno sucrose can be labeled as “sugarless.” “sugar free,” or “no sugar” but must also bear the statement “Not a reduced calorie food,” “Not a low calorie food,” or “Useful only for not promoting tooth decay.” Table 5 RclativcSwcctncss and Caloric Value of Polyols
Relative swectncss Caloric valuc (sucrosc = 100)
(U.S. allowance: kcal/g)
Erythritol Hydrogenated starch Hydrolysates
60-70 25-50
0.4 3.0
lsolllalt
45-65 40 90 70 50-70 100
2.0 2.0 3.0
Polyol
Lactitol Maltitol Mannitol Sorbitol Xylitol
I .6 2.6
2.4
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Sorbitol occurs naturally in many edible fruits and berries including pears, apples, 1974 the cherries, prunes, and peaches. Its nontoxic nature has long been recognized. In FDA included sorbitol as one of the first four chemicals in its revised list of GRAS substances. Sorbitol is only70% as sweet as sucrose. However, it has many functional properties to body desirable in a sweetener, such as bulking agent ability, high viscosity (contributing and texture), hygroscopicity (resulting in its humectant as well as its softening nature), cool taste, sequestering ability, and crystallization modification (retardation). Because sorbitol can be digested without insulin and is also noncarinogenic, it is used as a sugar substitute in diabetic and sugarless foods and candies. In general, sorbitol is used in foods to aid retention of product quality during aging, or to provide texture or other product characteristicsto the formulation. In its major applications-sugarless chewing gum, candies, and mints-liquid sorbitol is used primarily as a bulking agent and not for its sweetness. Sorbitol’s noncariogenic nature and the fact that it does not promote tooth decay may account for its wide use in these applications. Mannitol is only about 70% as sweet as sucrose and is also noncariogenic. Because of its nonhygroscopic nature, mannitol is used as a dusting powder and anticaking agent, besides its special dietary food application. The highest demand for mannitol is in sugarless chewing gum and sugar-free chocolates. However, mannitol has a more serious laxative effect than sorbitol and a warning label is required when consumption is likely to exceed 20 gtday. Xylitol is a five-carbon poly01 with sweetness similarto sucrose. It is found in small amounts in a variety of fruits and vegetables, and is formed as a normal intermediate in the human body during glucose metabolism. Xylitol has good solubility, blends well with foods, and has a lower melting point than sucrose, an advantage in the manufacture of confectionery products. There is also evidence that xylitol is not only noncariogenic but reduces tooth decay when used as a replacement for sucrose.It is mainly used in compressed candies, chewing gum. and overthe-counter pharmaceutical products. Xylitol is expensive, therefore it is usually used in small amounts in combination with other sweeteners. In a blend with aspartame, the two compounds havean excellent synergistic effect. Also, xylitol is blended with other polyols to minimize undesirable properties, such as hygroscopicity or the laxative effect of sorbitol, or to improve the solubility of mannitol. Lactitolmonohydrate,asugaralcohol,hasphysicochemicalpropertiesdifferent from those of sugars. It has a sweetness value approximately one-third that of sucrose and is therefore suitable where bulking with low sweetness is required. To increase the sweetness it can be blended with high-intensity sweeteners. It is derived from milk sugar and used as a sweetener in Japan, Israel, and Switzerland. In the United States a selfaffirmation GRAS statement petition has been submitted to the FDA for its use in chocolate, confections, and baked goods.
2. High-Intensity Sweeteners High-intensity sweeteners, once used mainly for dietetic purposes, are now used as food additives in a wide variety of products. They are termed high-intensity because they are many times sweeter than sucrose. But because of their very low use levels, high-intensity sweeteners cannot perform other key auxiliary functions in food and often must be used in conjunction with other additives such as low-calorie bulking agents. High-intensity sweeteners are also used in pharmaceuticals, cosmetics, animal feed, and biocides. The
471
Food Additives Table 6
Rcgulatory Status andSweetnessRelative to Sugar" Sweetness (sucrose = 1)
Cyclamate, Na salt Aspartam Accsulfarne K Saccharin Sucralose" Thaumatin (talin) Alitarne' Neohesperidin DC Stevioside Glycyrrhizin
30 200 200 300 600 3000 2000 2000 300 50
U.S.
Canada
Europc
Japan
P A A A A N P N N
A A A N' A N
A
P
P
N N
N"
N
A N N
N A N A N A P N A A
A A A P N
" A = approved: P = petitiontiled; N = not approved. "Sucralose is approved in U.S.A. Australia. Russia. Brazil. New Zealand. Quasar. Romania. and Mcxico. ' Alitam IS approved 111 Australia. New Zcsland. Pcoplc's Republican o l Chlna. Indonesia, Colombia. and Mexico. "Glycyrrhizin is approved as a llavoring. but not as a sweetener i n thc United States. "Sacchurm i n Canada is limited for use In personal care products and pharmaceutical. but it is banned In Ibods and beverages.
regulatory status and sweetness relative to sugar of high-intensity sweeteners are showr! in Table 6. Aspartame was approved in the United States i n 1981 for use in prepared foods, dry beverage mixes, and as a tabletop sweetener, and in 1983 for use in liquid soft drinks. It gained instant popularity and has become the sweetener in virtually all diet soft drinks in the United States. Aspartame has impacted not just the dietetic soft drink market but also many other sweetener markets. Its success has encouraged R&D, and FDA approval is currently being sought for its use in baked products, since aspartame can now be made heat-stable through an encapsulation process. Aspartame first appeared inthe U.S. dietsoft drink market in combination with saccharin (30% aspartam and 70% saccharin). Presently about 98-99% of canned or bottled diet sodas contain 100% aspartame. However, aspartame may be replaced in many products because i n 1998 other high-intensity sweeteners were approved for beverages. Aspartame can be used in many diverse applications. It is approved for use in more than 100 countries worldwide, and more than 5000 products cor.tain aspartame. In the United States, FDA approval is being given to more and more applications. In 1981 it was approved for use in prepared foods, dry beverage mixes, and as a tabletop sweetener and for carbonated liquid products in 1983. More recently, in 1993, FDA approval was extended to many other products, and thelist of approved products now includes the following categories:
0
Nonalcoholicbeveragesandready-to-servenonrefrigerated,pasteurized,aseptically packaged fruit juice beverages, including sport drinks Frozendesserts(dairyandnondairy) Refrigerated,flavoredmilkbeverages Fruit and wine beverages containing less than 7% alcohol
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Yogurt-type products in which aspartame is added after pasteurization and culturing Refrigerated,ready-to-servegelatindesserts Confectionaries(hardandsoftcandies) Bakedgoods,includingbakingmixes Low-alcoholbeer(containing less than 3% alcohol) Aspartame is about 200 times sweeter than sucrose. Unlike many other low-calorie sweeteners, aspartame isdigestedbythebody to amino acids, which are metabolized normally.However,because of itsintensesweetness,theamountsingestedaresmall enough that aspartame is generally considered noncaloric. Aspartame has a sugarlike taste, and enhances some flavors. Before aspartame was approvedby the FDA, it underwent the most rigorous review the agency ever gave a food additive. The process took approximately 10 years to complete. In early 1984. aspartame’s safety in beverages was again brought into question by researchers at the University of Arizona and the Community Nutrition Institute. The FDA, however, rejected a request for further hearings, saying it was satisfied that aspartame is safe in soft drinks. More recently, some research reports show that artificial sweeteners have had no effect on lowering weight levels and suggest that artificial sweeteners may actually increase appetite and thirst. To date, these findings have not appeared to affect the American consumer’s perceived benefit of low-calorie sweeteners. A few cases relating aspartame consumption to severe medical reactions have been reported in medical journals. About 4000 consumer health complaints of headaches and other reactions havebeen received by the FDA, allegedly due to the consumption of aspartame. The clinical validity and resultant outcome of these claims are not known at the present time. Saccharin was discovered in 1879 and has been used as a food additive since the early 1900s. Saccharin is approximately 300 times as sweet as sucrose. Because it is acidic and not very soluble in water, it is used primarily as its sodium salt. Saccharin combines well with other sweeteners and has an excellent shelf life. Its main disadvantages are a bitter, metallic aftertaste and concern over its safety. In the United States, a warning label regarding its safety must be attached to all food products containing saccharin. Saccharin is the most widely used nonnutritive sweetencr worldwide and is the least expensive on a swcetness basis. The FDA took saccharin off the GRAS list in the early 1970s as a result of a study in the United States was suggesting it caused cancer in rats. A ban on saccharin used proposed by the FDA but was stayed by Congress i n 1977 because of the ensuing public uproar fueled by the fact that there was then no noncaloric sweetener to replace it. However, saccharin has now been cleared of the possibility of causing bladder cancer by n number of studies. It is banned in Canada. Saccharin has been used primarily i n soft drinks, but also as a tabletop sweetener and in a wide range of other beverages and foods. A drop in the demand for saccharin for use in soft drinks occurred in early 1985 after Coca-Cola and Pepsi-Cola substituted a major portion of their saccharin use with aspartame. However, it is still used in other products in the rapidly growing dietetic soft drink market. In July 1988, the FDA approved the use of Hoechst AG’s acesulfameK (SunetteTM) for use in chewing gum, dry beverage mixes, instant coffee and tea, gelatins, puddings, and nondairy creamers. In 1998, the FDA approved its use i n nonalcoholic beverages. 200 times that of sucrose. It has a Acesulfame K has a rapidly perceptible sweet taste
Food Additives
473
good shelf life and is relatively stable across temperature and pH ranges associated with the preparation and processing of foods. Baked goods, candies, and dry mixes are believed to be the most viable markets for this low-calorie sweetener. A limitation is an unusual taste detected at levels required for adequate sweetness, which will no doubt prevent its widespread use in diet soft drinks. No toxicity problems have been reported in a multitude of studies to date. Sucralose is the onlylow-caloriesweetenermadefromsugar.Since 1991 it has been authorized for use in foods and beverages in more than 30 countries worldwide, including Canada, Mexico, Brazil, Australia, New Zealand, Argentina, Lebanon, Russia, and Romania, and it received FDA approval in April 1998. Developed by Tate & Lyle (UK), sucralose is a chloroderivative of sucrose, 600 times sweeter than sucrose, made by altering the sucrose molecule. Unlike sugar, sucralose is not converted into energy by the body, and therefore containsno calories. In addition, sucralose does not promote tooth decay, and is stable in a wide range of pH and thermal process conditions. Its uses include soft drinks, dairy products, baked and extruded products, puddings, breakfast cereals, jams and jellies, canned fruit, and chewing gum. Other high-potency sweeteners not approved for use in the United States but used elsewhere include the following compounds: Cyclamate is 30 times sweeter than sucrose. It has a sugarlike taste, a good shelf life, and a synergistic effect when combined with saccharin or aspartame. Cyclamate was introduced as a food sweetener in the 1950s, but was banned in 1970 because of its suspected carcinogenic potential. Since then, Abbott Laboratories, the developer and main producer of cyclamate, has undertaken further studies and submitted petitions to the FDA that demonstrate its safety. In June 1985, the National Academy of Sciences concluded that cyclamate was not a carcinogen. The FDA, however, has not reapproved use of the sweetener. Cyclatnate use is currently permitted in more than 40 countries, including Canada and the EU (excluding the United Kingdom). Cyclamate is used as a tabletop sweetener, in beverages, and in low-calorie foods, particularly in combination with saccharin. The use of cyclamate with saccharin givesa better taste to beverages than saccharin alone. Thus saccharin producers would welcome reintroduction of cyclamate inview of competition from aspartame. Developed by Pfizer in 1979 (prior to selling its food business), alitame isa dipeptide made of two amino acids, L-aspartic and D-alanine. It is 2000 times as sweet as sugar, with the same taste as sugar; thus its use level would be 25-400 ppm. Composition and use patents had been issued in 32 countries. The U.S. patent expires in 2000. Alitame is approved in Australia, New Zealand, the People’s Republic of China, Indonesia, and Mexico for use in food, beverage, and tabletop applications. Approval is still pending in the United States, Japan, Canada, and the EU. Potential market applications for alitatne include bakery products, snack foods, candies and confectionaries, ice cream, and frozen dairy products. A reported advantage of alitame over aspartame is lower loss during cooking and heating, since it is heat-stable. Thaumatin, a mixture of sweet-tasting proteins from the seeds of T l ~ ~ u t m cotro cmceus dcrniellii, a West African fruit, is about 2000-2500 times sweeter than sucrose. Its taste develops slowly and leaves a licorice aftertaste. Thaumatin acts synergistically with saccharin, acesulfame K, and stevioside. Potential applications include beverages and desserts; it cannot be used in baked products. Thaumatin is generally recognized in the United States as safe for chewing gum, and the supplier, Tate & Lyle, is seeking GRAS extensions for other foods. Thaumatin has been permittedin Japan as a natural food addi-
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tive since 1979. Although it is approved for use as a sweetener in the United Kingdom and Australia, it is used primarily as a flavor enhancer. Stevia rebaudinnrr, a plant native to South America, is the source of the stevia extract, which is a natural sweetener. Stevia can beused in food products that require baking or cooking because of its stability in high temperatures. This product is approved for use as a sweetener in Japan,but is not approved for use in the United States. Thereis currently a proposal in Brazil that the sweetener be included in any sugar-free soft drink in that country. Brazil is believed to be the third-largest soft drink market in the world, after the United States and Mexico. Dihydrochalcones (DHCs) are derived from bioflavonoids of citrus fruits and are 300-2000 times sweeter than sucrose. They leave a licorice aftertaste and give a delayed 2000 times sweeter than sucrose is properception of sweetness. Currently DHC that is duced from bitter Seville oranges by hydrogenation of natural neohesperidin (the main flavone of some oranges). In low concentration in combination with other sweeteners, it has potential usesin chewing gum, candies, some fruit juices, mouthwash, toothpaste, and pharnlaceuticals. It is approved for use in Spain, the Netherlands, Germany, Belgium, and Zimbabwe. Several other high-potency plant constituents (in addition to stevia and thaumatin) that have been considered as food sweeteners include monellin from the African “serendipity berry”; glycyrrhizin, also discussed as a flavor enhancer and extracted from the licorice root; and hernandulcin, an oil extracted from a Mexican plant. Such sweeteners could potentially be used in addition to or as substitutes for synthetic sweeteners that are now used to sweeten low-calorie or dietetic foods and beverages.
B. Thickeners and Stabilizers Thickeners and stabilizers (also called hydrocolloids, gums, or water-soluble polymers) provide a number of useful effects in food products. The technical base for these effects results from the ability of these materials to modify the physical properties of water. Most food and beverage products largely consist of water. Water-soluble materials function as rheology modifiers, affecting the flow and feel (mouth) of food and beverage products; act as suspensionagentsforfoodproductscontainingparticulatematter;stabilizeoil/ water mixtures; act as binders in dry and semidry food products; and create both hard and soft gels in food products that require this physical form. During the 1990s, fat replacement (discussed in detail in a later section) became a major application for modified starches and gumsas these additives provide unique texturizing, bulking, and emulsifying properties. Moreover, natural gums have been preposed as good sources of dietary fiber. Thickeners and stabilizers are generally used in very small anlounts in most food products (e.g., 0.15% in jam, 0.35% in ice cream, and 1-2% in salad dressings). Table 7 indicates the primary functions of many food thickeners. Two principal classes of these materials are recognized: natural materials obtained from plants and animals, and semisynthetic nlaterials that are manufactured by chemical derivatization of natural organic materials, generally based on a polysaccharide on microbial fermentation-based substances. A third class known as “synthetic polymers,” obtained from petroleum or natural gas precursors, is not used as a food additive. Figure 3 shows the sources and the various hydrocolloids used by the food processing industries. Unmodified or natural corn starch, produced by the wet millingof tield corn. supplies the majority of thickening material for the American food and beverage market. Other
Table 7 Major Food Thickeners and Stabilizers and Their Functions Emulsion Thickening stabilization Unmodified starches Modified starches Casein Gelatin Carboxymethylcellulose Methylcellulose Guar gum Alginatcs Xanthan gum Pectin Locust hean gum Gum arabic Carageenan Agar
X X X X X X X X X X X
X X X X X X X X
Suspending properties Gclation
X X
Crystallization control
X X
Water binding
Mouth feel
X X
X
X
X
X X
X X
X
X
X X
X X X
Flavor fixation
Protective film forming
Synergistic Fat cffect substitution X
X X
X X
Foam stabilization
X
X X X X
X
X
X X
X X X X X X X
X
X X X
X X
X X
X X
X X
X
X
X
X
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Food Additives
4 77
natural starches of significance include potato, tapioca, arrowroot, and sago starches. Unmodified cornstarch, commonly called pearl starch, is used in the food processing industry in the preparation of sauces, gravies, and fillings. It is the choice thickening ingredient in many formulated food products because of its modest price. It is modestly priced in comparison to modified starch, but even more so compared with additives such as cellulose ethers, guar, xanthan, and alginates. Major use categories for unmodified starch include 0 0
0
0 0
Meat gravies Cookedpuddings Cream-stylesauces Pie fillings Barbecue sauces Saladdressings Bakedgood fillings
Modified starches used in food products or food processing have been modified to Extend the bodying or gelling effect of norlnal starches Improvc resistance to acid or heat degradability and to low temperature and frecze/ thaw (eliminating aggregation) Improve texture Modify gelling tendencies as desired Increase viscosities at high temperature without gelling on cooling Provide instant solubility and gelling i n cold water Modified or derivatized starches are generally designed for more selective food applications than unmodified starch. Modified starches are used in a wide variety of products, including baby foods, purees, candy (e.g., bonbons and butter creams), jellies, cake mixes, dough, various soup powdcrs and liquids, instant noodles, puddings, pie fillings, batter mixes, sauces, salad dressings, dairy desserts, snack foods, and canned foods. In meat products such as sausage, ham, and luncheon meat. modified starches serve as a binder as well as a thickener. Recently, modified starchcs have been used as fat substitutes in margarine-like spreads, salad dressings, cookies, and baked products. Casein is a protein occurring naturally in, and obtained from, milk: it is thc main ingredient in cheese. Casein is marketed as sodium, calcium, potassium, or magnesium caseinatc and is uscd in confections, puddings, bakery fillings and frostings, coffee whiteners, and whipped toppings. I n 1970 only five protein products were available from milk. Since then, nunlerous advances have been made used in the methods to isolate and modify proteins. As a result, most suppliers now offer multitudes of specialty casein products. Gelatin is obtained from pork skin and bones (type A), or beef skin and bones (type B). Type A is mostly used for confectionery products and type B for dairy applications. Gelatinisabout97%protein,but it has no beneficialvalueto humannutrition.Food applications for gelatin includes dairy products such as yogurts, confectionary products such as g u n m y animal chcwables, meats suchas canned hams, and gelatin desserts. Gelatin is hygroscopic, capable of absorbing up to 10 times its weight in water. Under refrigeration i t forms a thermally reversible gel of high strength. Gelatin seems to exhibit little synergy with other thickeners and stabilizers. Therefore it appears to be of little benefit to blend gelatin with other gums to produce custom
formulations.
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Carboxymethylcellulose (CMC) is the primary cellulose ether consumed in food and beverage applications, mainly in pet foods, frozen dairy products, beverages, bakers’ goods, dry drink mixes, syrups, glazes, icings, and toppings. The current search for microwave-compatible food additives makes CMC a candidate for this rapidly growing formulated food market. CMC, a nonnutritive substance, is also popularin diet food formulations requiring thickeners and stabilizers. Methylcellulose (MC) and hydroxypropylcellulose (HPC) are also used in specialized food and beverage applications, but their relatively high market prices preclude them from large volume applications. Guar gum is the galactomannan derived from the endospenn of guar seeds (Cynrnoposis tetmgonolobus) grown in India and Pakistan since ancient times. It is one of the most economical and widely used gums, with extensive use in a variety of food applications since the 1950s. In spite of several attempts by major domestic guar concerns to encourage domestic production of guar in Texas and other arid agricultural areas of the American Southwest, mostof the guar gum consumedin the United States is derived from imported degermed guar beans (splits), mostly from India. Becauseof changing supplies due to weather and harvest conditions, as well as the demand for guar gum for industrial applications, the price of guar gum tends to shift dramatically. However, guar is expected to remain one of the most cost-effective thickeners on the market. Major uses for guar gum include ice cream, dessert toppings (e.g., Cool Whip@), frozen and refrigerated prepared meals, cheese, imitation bakery jellies and dry-mix bakery formulations, fruit drinks, soups, gravy and sauce mixes, water-based frozen desserts, salad dressings, and instant hot cereals. Alginates are extracted from different types of seaweeds, mainly from brown seaweed, Mcrcrocytis pyrifern and Lroninuria sp. The alginates include the various salts of alginic acid and propylene glycol alginate (PGA). Sodium alginate is used primarily as a binder in frozen desserts, reconstituted onion rings, crab and shrimp analogs, instant pudding mixes, fabricated puddings, sauces and gravies (particularly those containing milk or requiring the low “weep” property of alginates), and re-formed meats. Propylene glycol alginate (PGA) is used in the United States as a foam stabilizer in beers and ales. In addition, PGA is used by major food manufacturers in salad dressing formulations. The nonsodium light metal salts of alginic acid are used as sodium alginate alternatives in low-sodium and dietary food specialties. Xanthan gum, a fermentation product, is used in salad dressings, relishes, syrups, sauces, bakery fillings, prepared puddings, glazes and toppings, processed cheese products, dry cake and beverage mixes, and fruit and carbonated beverages. A significant use is in dairy products, where it prevents the separation of the contained whey from the rest of the food product. Xanthan doesnot exhibit any reactivity with milk proteins and therefore is often used in combination with other hydrocolloids, particularly carrageenan. Moreover the stability of xanthan gum to acid and high salt content makes it very useful for many types of foods. Gellan gum isthe latest hydrocolloid approved for food use, produced with a fermentation process likethat used for the fermentationof xanthan gumby the organismAur-omonas elodecr. The FDA approved gellan gum for use in icings, frostings, bakery fillings, and low-solids jams and jellies and confections.It is also approved for food usein Japan. Gellan gum can be used at levels substantially below those required by conventional hydrocolloids. There are two forms of gellan gum. The first is a high-acetyl gum, which is partlyacetylatedandprovidesthermoreversiblegels.Thesecondisalow-acetyl gum forming a firmer and more brittle gel.
Food Additives
479
Pectin is a fruit extract from the peel of citrus fruits and apple pomace. The main commercial types used in foods are pectin itself and potassium pectinate, sodium pectinate, of esterification and amidated pectin. Commercial products include high ester [degree (DE) of 501 or low ester (DE of less than 50) pectins. Traditional food uses of pectins are in jellies and jams. Newer applications include gummy candies and fruit-flavored juices and carbonated drinks.In gummy candies and jellies, it is replacing starch for improvement of fruity flavor.In fruit-flavored drinks,it stabilizes the constituents and makes the product more appealing. A constraint on the supply of pectin is the approval required by the EPA to start up a new plant. Because of the high costs of compliance to dispose of the large volume of waste generated during pectin production, the last North American pectin production plant was relocated from Florida to Mexico. Locust bean gum is obtained from the carob tree. The major source of locust bean gum is the Mediterranean countries. The size and quality of the crop is directly related to climatic conditions, producing periodic shortages of supply and great fluctuations of price. Chemically locust bean gumis similar to guar gum. Anionic, cationic, and hydroxyalkyl derivatives are also produced commercially. Locust bean gum swells in cold water, but heating is necessary for maximum solubility. Locust bean gum is widely used in frozen dairy products, in conjunction with guar gum and carrageenan, and is used for preventing syneresis in cream cheese. In addition, locust bean gum is used in many nonemulsified sauces and dressings as a thickener, in prepared meals, and in bakery products as a moisture retention aid. Much of the locust bean gum is supplied in a blended form to the dairy industry. Gum arabic is obtained from various trees of the genus Acacia, primarily from A. senegd. It is highly soluble in water (up to 50%), and its solutions are of relatively low viscosity. Other advantages of gum arabic as a food additive are its nontoxicity and lack of odor, color, and taste. These properties are especially useful in systems requiring emulsifying properties, such as high solid suspensions. It is used as an emulsifier in beverages for citrus oil and flavors,a foam stabilizer in beer, as a crystallization retarder and emulsifier in confectionaries, and as a stabilizer in dairy and bakery products. Since the source of supply has sometimes been unreliable because of political and social events in the Middle East, many U.S. users have turned to substitutes, including starch derivatives. Carrageenan is extracted from Irish moss (Chondrusand Gignrfincr species) that is harvested off the Atlantic shores of New England, the Canadian Maritime Provinces, and several European countries. Carrageenan is readily soluble i n water to form an inelastic gel and is commonly used with other gums. Its most unique property is a high degree of reactivity with certain proteins, such as casein. The largest application of carrageenan in food use is in dairy products (e.g., frozen desserts,flavored milk powder, nondairy creamers). For example, cocoa can be suspended in milk with the use of about 0.025% carrageenan. One of the most significant recent developments for carrageenan suppliers has been its widespread use in poultry applications for moisture retention. The product Serves to retain moisture before and during cooking and allows the poultry to be pumped with large amounts of water. In 1990, the FDA decidedto allow an unrefined seaweed extract knownas Philippine Natural Grade (PNG) to be sold under the carrageenan heading. Traditionally, refined carrageenan is made in a 10-step process in which carrageenan is extracted from the seaweed and then filtered to remove the cell walls, or cellulose, and other substances from the seaweed. PNG carrageenanis prepared in a five-step process that extracts the unwanted
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substances from the seaweed, leaving both carrageenan and about 10-14% cellulose, as opposed to the less than 1% found in traditionally refined product. PNG is currently being used in the meat, cheese, and pharmaceutical markets. Agar (also called agar-agar) is obtained from various red-purple seaweeds of the class Rhodophycae. Agar is used primarily in baked foods (icings, toppings, meringues) and in confectionary products. Because agar is the most expensive of the seaweed extracts, there have been efforts to substitute with other gums such as carrageenan. A number of other thickening agents are used by the food industry, but these represent a very minor portion of the food additives market. Most are higher priced, in erratic supply, and face increasing competition from the principally used thickeners. Such other thickening and stabilizing agents and their principal uses include Ghatti gum. Obtained from India and Sri Lanka; no other functional properties are known than thickening and emulsion stabilization. Tragacanthgum.ObtainedfromtheMiddleEastandusedinsaladdressings and sauces. Karaya gum. Obtained from India and used for extreme thickening to pastelike gels.
C. Colors Colors are additives used to improve the overall appearance of foods and influence the perception of texture and taste. Products are derived from either natural origins or producedsynthetically. IntheUnitedStates,colorsaredividedintotwotypes:certified (FD&C) and natural (exempt from certification) colors. Food colors are listedi n the Code of Federnl Regultrtions (CFR) Title 21 parts 70-82. If an additive is not specifically included in these sections, it may not be used for coloring food, drug, or cosmetic products that will be sold in the United States. There is some confusion about the term “natural” colors. The definition of “natural” varies between the United States, Europe, and Japan. This section will concentrate on the U.S. regulations, with occasional reference to others. In the United States, from a regulatory point of view, there is no definition for natural colors, only “certified dyes” and “color additives exempt from certification.” Certified colors are synthetic materials whose purityis checked by theFDA.Colorsobtainedfromanimal,plant, or mineral origins are not certified because they often contain complex mixtures of many components. The exact composition of natural color varies from plant variety to plant variety, from region to region, and from season to season. Users depend on the integrity of their suppliers to ensure product quality. The certification process concerns only batch purity, it does not guarantee the safety of the color molecule. There is no inherent reason why certified colors are either more safe or less safe than natural colors (colors exempt from certification).In order to market their products, U.S. producers must submit product samples from each batch of material in an FDA laboratory to ensure and pay a certification fee. The materials are analyzed that they meet specific purity specifications. In other parts of the world, only self-certification exists, except in Japan where certification of synthetic colors has been required since 1994. Certified food colors, both primary and blends, are produced in a variety of forms including powder, liquid, granules, plating blends, nonflashing blends, pastes, and dispersion; the least expensive form is powder.
Food Additives
481
A number of formerly certified FD&C colors have been banned under the provisions of the Delaney clause of the Food, Drug & Cosmetic Act, either because they were found to be carcinogenic or because there was no assurance that they could be made free from carcinogenic impurities. These actions have steadily reduced the number of certified dye in 1950 to 7 in 1998. (An colors available to the U.S. food industry from more than 22 additional color, FD&C citrus red no. 2, is permitted for coloring the skins of oranges that are not intended or used for processing, but it has not been produced in the United States in recent years.) Certified food colorants can be divided into dyes and lakes. Dyes are chemical compoundsthat exhibit their coloring power or tinctorial strength when dissolved in a solvent. Lakes are insoluble colored materials that color by dispersion. Table 8 shows the physical properties of these seven certified food colorants. Color regulations specify a legal minimum of 85% pure dye for primary colors,but most dye lots contain from 90-93% pure dye. Certified dyes fall into several chemical classes: azo-dyes (yellowno. 5 , yellow no. 6, red no. 40, citrusred no. 2),triphenylmethane dyes (blue no. 1, green no. 3), xanthine type (red no.3), and sulfonated indigo (blue no. 2). FD&C dyes are also used in the production of lakes, which are pigments prepared by combining a certified dye with an insoluble alumina hydrate substratum. Lakes are both water and oil insoluble and impart color through dispersion in food. Thus they are suitable for coloring foods that cannot tolerate water and products in which the presence of water is undesirable.Examplesinclude bakery products(icings,fillings,cakeand doughnutmixes),confections,dairyproducts(hardfatcoatingsforicecreamnovelties, wax coatings for cheese, yogurtwith fruit syrups), drypet foods, dry beverage bases, and dessert powders. The FD&C lakes do not have a legally specified minimum dye content; manufacturers use formulations of from 1 1 % (standard) to 42% pure dye (concentrated). Noncertified colors can be from either natural origins (primary sources), such as vegetables and fruits, or produced synthetically. Traditional markets for noncertified food as butter, margarine, shortening, colors include lipid-based, high-fat food systems such popcorn oil, processed cheeses and spreads, salad dressing, and snack foods. Water-soluble forms are also available and are used in beverages, baked goods, confections, and dairy products. Food color additives exempt from certification, their colors and sources listed are in Table 9 and described in more detail below. Annatto extract (Bixin, Norbixin, etc.) is an extract of a seed from a shrub called Bixa or-ellrrrw L. that grows in South America, East Africa, and the Caribbean. Oil- and water-soluble fonns exist depending on the method of extraction. Annatto extracts exhibit various yellow shades, and are commonly used in cheddar cheese and bakery products, often in combination with turmeric or paprika oleoresin. Beet juice/powder (betanin, beet-root red, etc.) is a water-soluble color found as the predominant pigment in red beets (Betcl v ~ d g m i ~Several ). forms exist, including dried ground beets, or dehydrated beet powder; beet juice concentrate, the liquid obtained by concentrating the expressed juice from mature beets; and beet powder, made by spray drying beet juice concentrate onto a carrier of maltodextrin. Canthaxanthin (Roxanthin) is a synthetically prepared carotenoid that is conm1ercially available as a water-dispersible powder. It exhibits reddish orange to dull violet shades. Caramel (burnt sugar) color results from the controlled heat treatment of food-grade carbohydrates. Often catalysts are added to drive the reaction to the desired color end point. Caramel colors exhibita colloidal charge anda variety of shades from yellow brown
Table 8 Physical and Chemical Properties of Certified U.S. Food Colorants Stability to FD&C name
Common name
Light
Red no. 3 Red no. 40 Yellow no. 6 Yellow no. 5 Blue no. 1
Erythrosine Allura red AC Sunset yellow FCF Tartrazine Brilliant blue FCF
Fair Very good Fair Good Fair
Fair Fair Fair Fair Poor
Blue no. 2
Sodium indigo disulfonates Fast green FCF
Very poor Fair
Green no. 3
Oxidation
pH change
Compatibility with food components
Tinctorial strength
Hue
Poor Very good Good Good Good
Very good Very good Good Good Excellent
Blue Yellow Red Lemon yellow Green- blue
Poor
Poor Good Good Good Good (unstable in alkali) Poor
Very poor
poor
Deep blue
Poor
Good
Good
Excellent
Blue
Water solubility 9 25 19 20 20 1.6
20
483
Food Additives
Table 9 Food Color Additives Exempt from Certification"
Additive
Color
Source
Annatto extract Beet juice Dehydrated beets Canthaxanthin" Caramel Apocarotenal' Beta-carotene Carrot oil Cochineal extract (carmine) Fruit juice (grape and cranberry) Grape skin extract" (enocianina) Paprika Paprika oleoresin Riboflavin Saffron Titanium dioxide" Turmeric Turmeric oleoresin Vegetable juice
Yellow Red Purple Red Brown Orange Yellow Yellow Red
Vegetable Vegetable Vegetable Synthetic Semi-synthetic Synthetic Synthetic Vegetable Insect
Red
Fruit
Red
Fruit
Red Red Yellow Yellow White Yellow Yellow Red
Vegetable Vegetable Synthetic Vegetable Synthetic Vegetable Vegetable Beet and red cabbage juice
"Under the Code of Federal Regulations, Title 21. No color additive may be used in foods for which standardsof idcntity have been promulgated under Section401 of the Federal Food.Drug & Cosmetic Act, unless the use of added color I S authorized by such standards. "May not exceed 66 mg/kg of solid. o r pint of liquld, food. ' May not exceed 33 mg/kg of solid, or pint of liquid, food. "Used only in beverages. "May not exceed l % by weight of the food.
to reddish brown, and is available in powder and liquid forms. Caramel has a very large market in cola beverages. It is also used in bakery products and confectionaries. Apocarotenal (beta-apo-8'-carotenal) is a red-orange synthetically prepared carotenoid that is oil soluble. The pigment is found in oranges and tangerines and is commonly used in products such as cheese spreads and snack foods. In the United States, a usage restriction of 15 mg/lb of semisolid or solid food exists. Commercial products of natural beta-carotene exist from several sources, including the alga Dunaliella salina and palm oil. Beta-carotene can be also synthesized. It is oil soluble and exhibits a characteristic butter to egg-yolk shade. It is commonly used in baked goods, beverages, and confections. Cochineal extract, or carmine, the lake pigment of cochineal extract, is an extract of a female cochineal insect Dacrylopius coccus, or Coccus cacti. It is a stable colorant used since antiquity. At pH 4 and below it is orange, at pH 4-6 it is magenta-red color, and at pH greater than 6, it has a blue-red shade. The insect is commonly cultivated in
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Peru, Ecuador, and the Canary Islands. Approximately 70,000 insects are requircd to produce one lb of SO% carminic acid lake. It is cotnmonly used in beverages, sausage products, aperitifs, and confections. Cochineal extract is not kosher. Fruit juicesthat typically contain carotenoid- or anthocyanin-type pigments are often usedin concentrated or single-strength forms as coloring agents. In the United States, fruit juices mustbe expressed from mature varietiesof edible fruits or a water infusionof the dried fruit. Fruit juices that are used for coloring include cranberry, cherry, raspberry, elderberry, grape, orange, and tomato. Grapeskinextract(enocianina) is obtained by an aqueousextraction of fresh, deseeded marc remaining after grapes have been pressed to produce grape juice or wine. It contains the common componentsof grape juicebut not in the same proportion. During the steeping process, sulfur dioxide is added, and most of the extracted sugars are fermented to alcohol. The extract is concentratedby vacuum evaporation, during which practically all of the alcohols are removed. A small amount of sulfur dioxide may be present. I n the United States, grape skin extract is permitted only for use in coloring beverages. Paprika is the ground form of sweet red peppers (Cqxicurw ~ I I U ~ HPaprika ) . oleoresin is a solvent extract of the coloring principles of sweet red peppers. Extraction of the peppers is cauied out withseveralpermittedsolvents,includinghexane,ethylene dichloride, and various alcohols. Oil is commonly added to the extract to standardize the strength, with typical designations in American Spice Trade Association (ASTA) units and color value units (CVU). Paprika oleoresins are oil soluble, reddish orange shades. Typical applications include coloring salad dressings, snack foods, cheese product, baked goods, breading, and crackers. lo as lactoflavin and vitamin B'. Riboflavin, a bright yellow color, is also referred It is a naturally occurring yellow pigment isolated from milk, and it can also be synthesized. It has limited solubility, a bitter taste, and is light sensitive, therefore it has limited USC.
Saffron is the dried stigmas or extractof Crocus .wtivu.s. The predominant pigments arecrocinandcrocetin.Saffronislimitedinitsapplicationduetoitsveryhighcost; approximately 165,000blossoms are required to produce 1 kg of colorant. Saffron is commonly used as a spice and colorant i n rice products. Its bright lemon-yellow color is also used in applications such as soups, baked goods, and certain dairy products. Titanium dioxide is a white pigment that is reacted product from a mineral oxide called ilmenite, a type of iron ore. The crystal form, anatase, is the form of choice as a colorant for food. In the United States, purity of 99% or greater is required. Titanium oxide isthe onlywhitepigmentcurrentlypermittedasacoloradditive intheUnited States. It is often used to opacify systems such as low-fat/no-fat salad dressings and dairy products, pet foods, baked goods, sugar-coated candies, and other confections. It colors by dispersion, as it is not water or oil soluble. Turmeric and turmeric oleoresin is a bright yellow pigment from the rhizome CurL ' I I I I I ~ ~lotzgcr, which is grown predominantly in India. The principle coloring agent is curcumin. The oleoresin form is extracted by solvents, such as alcohol and acetone. It is available with or without flavor components. Some vegetable juices, typicallyin a concentrated form, are used as coloring agents. In the United States, vegetable juices must meet the criteria of the federal regulation, which describes juice expressed from mature varieties of ediblc vegetables. An example of a commercially available vegetable juice colorant isred cabbage juice, which contains anthocyanins. Most other vegetable juice concentrates contain chlorophyll pigments and
Food Additives
485
are often not of sufficient color concentration nor stable enough to be used commercially. In addition, the flavor impact is often undesirable. Eight synthetic dyes are permitted for use in the EU countries. Twelve water-soluble dyes and eight of their lake colors are allowedi n Japan for foods. Compared to American consumers, European and Japanese consumers are more prone to demand that their food products contain natural colorants. There is a growing body of evidence that many natural colorants perform additional functions. They also act as vitamins, antioxidants, and antimicrobial and antiviral agents. Natural colorants also may have anticancer properties and can be used to treat vascular diseases and improve night vision. This information was discovered in recent years.
D.
Fat Substitutes
Although fats are essential for a healthy diet, excessive consumptionof fat has been related to health problems. Fat replacers are those ingredients that can help to reduce a food’s fat and calorie levels while maintaining some of the desirable qualities fat brings to food, such as “mouth feel,” texture, and flavor. Fat replacers can be carbohydrate, protein, or fat based. Three alternative approaches are being pursued in this area: Fatsubstitutes(Table IO). Thesearepartiallyor fully nonmetabolizablecompounds that possess fatlike properties and can replace fats on a one-for-one basis. Most fat substitutes are synthetic compounds that possess fatlike properties and can replace fat in a broad range of applications. Fat mimetics (Table 1 l ) . These are nonfat ingredients that mimic the mouth feel and other functional properties providedby fat, but have fewer caloriesthan fat. I n recent years, numerous approaches have been undertaken to partially replace or to eliminate fatsin food by using FDA-approved traditional nonfat food ingredients suchas novel carbohydrates and gums,as well as other innovative ingredients, including microparticulated milk and egg proteins, and modified oat fibers. These products are capable of duplicating many of the functional properties of fats, such as lubricity, tenderization, opacity, flavor release, slipperiness, melt. and plasticity. These products cannot substitute for fatson a one-to-one replacement basis. Moreover, these ingredients are suitable as fat replacements only in foods that do not require extensive heat processing (e.g., salad dressings, frozen desserts, margarine-like spreads, etc.). Emulsifiers are fat- or fatty acid-derived compounds that have the ability to modify the surface properties of solids or liquids and possess many of the properties Table 10 FatSubstituteApproved
in theUnitedStatcs
Composition
Name
Olcstra (OleanTM) Caprenin“ Salatrim (BenefatTM) Medium-chain triglycerides (MCT)”
Sucrose hexa-, hepta-, and octaesters Monobehenin esterified with C, ,, and C,,,l, acids Monostcarin esterified with C2 C , ,I, and C, I, acids Esters of fractionatcd coconut oil fatty acids
“Application is limited t o soft candy and confectionary coatings ”Because o f ~ t shigh prlce, ~t IS used in medical foods only.
(,,
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486 Table 11 SelectedProducersandDevelopers
Producer Carbohydrate-based products AVEBE America Inc. Cerestar International Conagra Inc. Crompton & Knowles, Inc. Cultor Food Science, Inc. FMC Corporation
Grain Processing Corporation Hercules, Incorporated National Strach and Chemical Co. The Quaker Oats Company Remy Industries N.V. Rhodia Food Ingredients A. E. Staley Manufacturing Co.
Zumbro, Inc. Protein-based substitutes Cultor Food Science, Inc. Nutrasweet/Kelco Opta Food Ingredients Emulsifiers American Lecithin Co. Central Soya Co., Inc. Cultor Food Science, Inc. Lonza Inc. Lucas Meyer Inc. Gumslhydrocolloids Alko Ltd. (Finland) Asahi Kasei Kogyo K.K. (Japan) Dow Chemical Co. FMC Corporation Hercules Incorporated Nutrasweet/Kelco
of FatMimetics
Compound
Trade name
Potato maltodextrine Modified high-amylose corn Oat maltodextrine Cellulose gel Polydextrose Fatlcarbohydrate emulsion Microcrystalline cellulose Mixtures of cellulose, maltodextrine, etc. Corn maltodextrine Pectin Modified waxy corn, tapioca starches
Paselli SA2 Amalean I and I1 oatrim Miracle Middles Litesse Very-low Novagel 200 Avicel
Oat maltodextrine Rice starch Oat maltodextrine Modified corn starch Modified potato, tapioca or waxy corn starch Hydrolized oat flour Rice maltodextrine
Maltrin Spledid N-Oil, N-Lite, Slenderlean Oatrim Remygel Oatrim Instant Stellar Sta-Slim Trimchoice Ricetrin 3
Whey protein Concentrate Microparticulated egg white and milk protein Zein, a corn gluten derivative
Dairy-Low Simplesse
Phospholipid fraction derivative of soy lecithin Lecithin Emulsion of Soybean Oil or milk fat Emulsifier Modified soy lecithin
N.A.
Enzyme modified cellulose Cellulose (>l pm) Methylcellulose, hydroxypropyl methylcellulose Cellulose Pectin Xanthan gum Gellan gum
N.A. Cellucream Methocel
Lita
Centrolex VeryLo N.A. M-C-Thin HL66
Avicel Splenda Dricoid 280
food Additives
487
of a fat or an oil. The caloric value of most emulsifiers is similar to that of triglycerides. However, depending on the degree of esterification and polymerization, some emulsifiers such as polyglycerol esters may have a lower calorie content. Polyglycerol esters contribute only 6 kcal/g. Typically 2% fat in a formula can be replaced with1% emulsifier with no loss of functionality. However, due to regulatory constraints and flavor considerations, emulsifiers are usually used at 1% concentration or less in formulated foods.
1. Low andNoncaloricLipids Currentlyonlyfournewlystructuredlipidsareapprovedforuse as fatsubstitutesin theUnitedStates(Table 10). Thesearemade bytheinteresterification of natural oils. Olestra (brand name OleanTM), developed by Procter & Gamble Co. (P&G), was approved by the FDA in January 1996 for use in preparing potato chips, tortilla chips, and other savory snacks. Olestra is a sucrose polyester made from sucrose backbone and six to eight fatty acids. The number and type of fatty acids vary depending on the performance characteristics desired. The fatty acids are derived from vegetable oils found in soy, corn, and cottonseed oils. Olestra molecules are much larger than those of ordinary fats, so the body’s digestive enzymes cannot break it down. Thus Olestra is neither digested nor absorbed, passing straight through the body. It is noncaloric and nonsweet. Olestra was discovered about 25 years ago. Its submission to the FDA was withdrawn numerous times as the information on it was refined. At the time of its approval, more than 300 volumes covering more than 100 laboratory studies on seven species and 98 clinical investigations involving 2500 humans comprised the body of knowledge on this compound. P&G spent more than $200 million for the development and regulatory approval process of olestra. There are some concerns about olestra because it blocks the absorptionof fat-soluble vitamins consumed with it. Therefore the FDA requires that fat-soluble vitamins A, D, E, and K be added to foods made with olestra. Also it is reported to cause abdominal discomfort and may act as a laxative in some cases. Therefore products with olestra have to carry a warning label that these effects are possible. Approval of olestra brought a range of responses from scientists and consumer advocates who disapprove of its use, as well as endorsements by groups such as the American Dietetic Association, which identifies olestra as “one more choice for consumers in the war against fat.” Caprenin, or caprocaprylobehenin, is a reduced-calorie alternative to cocoa butter and other confectionary fats. Caprenin is a fat, a triglyceride composed of naturally occurring fatty acids-caprylic (C8,(,),capric (C,,,(,), and behenic (C??(,) acids. The mediumchain fatty acids are derived from coconut and palm kernel oils. The long-chain behenic acid, which comes from hydrogenated rapeseed oil, is mostly unmetabolized in the gastrointestinal track. Therefore caprenin hasa caloric density of 5 kcal/g, instead of the 9 kcal/ g in conventional fats. According to P&G’s patent, this material can substitute for about 70% of the fat in confectionary products, which contain usually 25-45% fat. Instead of the traditional FDA clearance processthat requires several years to complete, Caprenin has been cleared by a “self-affirmation process.” That process was based on the recommendationof an expert panel (convened by the Life Sciences Research Office of the Federation of American Societies for Experimental Biology), which reviewed published data and P&G’s research and concluded that caprenin is safe as a confectionary fat.
488
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Salatrim (BenefatTM),an esterified monostearin, is a family of reduced-calorie fat products consisting of short- and long-chain fatty acids. These fats are derived from ingredients found in nature and provide only 5 kcal/g instead of the 9 kcal/g provided by traditional fats. This calorie reductionis achieved because the short-chain fatty acids (acetic, propionic, or butyric) used are inherently low in calories, and the long-chainfatty acid (stearic acid) used is not fully metabolized. Because BenefatTMis fat, it delivers the taste and performance of conventional fats. Medium-chain triglycerides (MCT) are estersof fractionated coconut oil fatty acids. These compounds provide 8.3 kcal/g, only slightly less than conventional fats. However, recent physiological studies suggest that MCTs are burned readily for energy and have little tendency to be incorporated into tissue lipids that are not deposited as fat. MCTs are GRAS compounds, have been used in medical and infant feeding products for more than 30 years, but have not been used in consumer food products because of their high cost. More recently, their use has been expanded into sport/nutrition foods.
2. Fat Mimetics Table 1 1 lists some conventional food ingredients that have been used as fat replacers. Many new ingredients are being recommended, therefore this list should not be considered exhaustive. However, it provides an overview of the various types of replacers and fat extenders presently available.
N. Cnrboh~dmte-BasedSubstitutes. Nearly 40 different products based on starch have been recommended for fat replacement. Some of these exist as products with other uses, although several havebeen developed specifically as fat mimetics. Mostof the materials in this category are used to form a gel containing modified starch and water. The gel is then substituted for fat in the formula on an equal-weight basis. Starch-based fat mimetics have many different properties dependingon the parent starch and on the degree of cross-linking,substitution,andacidmodification. In manyinstances,two ormore starches must be used together to give the desired effect, or they can be combined with other polymers and emulsifiers. Maltodextrins are productsof the acid hydrolysisof starch, and actas bulking agents, giving the mouth-feel qualities of fat. One of the first on the market, in 1984, was N-Oil, a hydrolyzed tapioca maltodextrin. The substance forms a thermoreversible gel in aqueous foods, and therefore creates the mouth feel of fat. N-oil and several similar products are used in frozen desserts, salad dressings, margarine-type spreads, dips, baked products, and snacks. Several other starch-based maltodextrin fat replacers are available. One of the most widely used fat mimetics is AvicelTM,a cellulose derivative used in frozen desserts, salad dressings, and baked goods. Methocel, a food gum, is made of cellulose ethers for use in bakery products, fried foods, and salad dressings. Pectin is a gum that forms a gel. It has a large water-holding capacity and therefore helps to overcome some of the dry impression of fat-free foods. A gum is a soluble fiber, so it must be countedas a carbohydrate (4 kcal/g) in the calculationof calories for labeling purposes. In 1991 Splenda, a specialty pectin, was introduced as a fat replacer. Another gum fat replacer, carrageenan, that was used in low-fat hamburger, failed to achieve wide consumer acceptance. Other hydrocolloids and gumsthat are not listed in Table 1 1 are frequently promoted as fat-sparing agents. Xanthan, gelatin, carrageenan, algin, guar konjak, locust bean gums, etc., can be utilized as well for their fat-sparing function.
Food Additives
489
Polydextrose-“LitesseTM” and “Veri-LoTM”-is recommended for replacement of fats. Polydextrose is a water-soluble, reduced-calorie polymer of dextrose that contains small amounts of sorbitol and citric acid. It provides 1 cal/g as it is only partially metabolized by the human body. Oatrim, developed in the USDA laboratory in Peoria, Illinois, is an amylodextrin with 5% P-glucan extracted from oat flour. It is used as a fat replacer in baked products.
b. Protein-Bused Substitutes. SimplesseTM is a mixtureof eggwhite and milk proteins, water, sugar, pectin, and citric acid subjected to high shear (e.g., microparticulation) that f o r m a gel of protein spheroids. The small spheres produced by microparticulation provide the mouth feel of fat. This product is used in frozen desserts. A similar version, Simplesse 100, is made from whey protein and is approved for use in baked products. With its 1-2 kcal/g, 1 g of microencapsulated protein can replace I g of fat. Several other protein-based fat replacers are on the market and are used in both cooked and uncooked products. Lita is based on zein, a microencapsulated protein from corn. Like other proteinbased fat replacers, it contributes less than 2 kcal/g. It is used in frozen desserts, whipped toppings, and mayonnaise. c. En~u/s~)?er.~. Emulsifiersarefat-basedsubstances thatareused withwater to replace all or part of the shortening content in cake mixes, cookies, icings, and vegetabledairy substitute. Most emulsifiers provide the same calories as fat, but less is usell, resulting in fat and calorie reductions. Many emulsifiers simply play a “fat-sparing’’ role. However, polyglycerol esters may have a lower caloric content than triglycerides, depending on the degree of esterification and polymerization. The commonly used food emulsifiers that have applications as fat replacers include lecithin, mono- and diglycerides, and derivatives such as acetylated, succinylated, and diacetyl tartaric esters of distilled monoglycerides, polyglycerol esters, polysorbates, and sucrose esters. More information on specific compounds can be found in the emulsifier section of this chapter.
E.
Enzymes
Enzymes are catalysts used during food processing to make chemical changes to the food. They are biological catalysts that make possible or greatly speed up chemical reactions by combining with the reacting chemicals, bringing them into the proper configuration for thereaction to take place. They are notaffectedbythereaction. All enzymes are proteins and become inactive at temperatures greater than about 40°C or in unfavorable conditions of acidity or alkalinity. Some of the specific functions food enzymes perform include Speed up reactions Reduce viscosity Improve extractions Carry out bioconversions Enhance separations Develop functionality
Somogyi
490
Create/intensify flavor Synthesize chemicals Food enzymes are usually classified into the following categories: Carbohydrasesandamylasesarecommercially the mostimportantsubgroup, hydrolyzing 1,4-glycosidic bonds in carbohydrates Proteases,hydrolyzepeptidebondsinproteins Lipases,splithydrocarbonsfromlipids Pectic enzymes and cellulases, hydrolyze plant cellwallmaterial Specialtyenzymes These enzyme categories can be divided further into 15-20 subgroups. The traditional roles of enzymes in the food industry have been in the processing of bakery goods, alcoholic beverages, and starch conversion. But interest is now focused on newer and more varied applications, such as hydrolysis of lactose, the preparation of modified fats and oils, the processing of fruit juices, and other processes where newer enzymes are being identified. Today many food processes utilize enzymes. Food-grade enzymes encompass a wide variety of commercial products that are employed in the production, conversion, and modification of foods because of their highly efficient and selective catalytic functions. Table 12 lists many of the major food enzymes and gives some applications in foods and food processing. The largest application of enzymes in the food industry is the use of alphaamylase, glucoamylase, and glucose isomerase for starch conversion and production of high fructose corn syrup. Rennin, a protease enzyme used in cheese making, is also of significant value, followed by a host of other enzymes, including pectinases, invertase, lactase, and maltase (used for the modification of starches and sugars), catalase, pepsin, glucose oxidase (an antioxidant for canned foods), and bromelin, ficin, and papain (plant proteases used for tenderizing meat and producing easily digestible foods). Enzymes are highly specific and can act only on a single classof chemicals, such as proteins, carbohydrates, or fats. These same enzymes are also used in nonfood applications such as pharmaceuticals, textiles, detergents, and waste treatment. Enzymes are produced from animal tissues (e.g., pancreatin, tripsin, lipase), plant tissues (e.g., ficin, bromelin), and most frequently by microorganisms (e.g., pectic, starch enzymes). Microbial production from a variety of species of molds, yeasts, and bacteria is increasingly becoming the predominant source of enzymes. Application of genetic engineeringto the developmentof enzymes has already made a significant impact. The first food additive produced by genetic engineering was chymosin, “Chy-MaxTM”, a microbial rennet that has been approved by regulatory agencies intheUnitedStates,Canada, theUnited Kingdom,Australia,Italy,andseveralother countries. Advantages of the bioengineering product are increased yields, relative ease of manufacture, lower price, and the ability to label the product as kosher.
F. Vitamins Vitamins are nutritive substances required for norrnal growth and maintenance of life. They play an essential role in regulating metabolism, converting fat and carbohydrates into energy, and forming tissues and bones. Vitamins can used be as functional ingredients
49 1
food Additives Table 12 Applications for Enzymes intheFoodIndustry
Food Alcohol production Baking
Amylases Fungal proteases
Brewing
Microbial proteases, papain, pectinase Invertase Pectinases, cellulase
Confections Coffee
Dairy
Rennins, lactase, lipase
Fats and oils Flavors
Lipase, phospholipase Protease, lipase
Fruits and vegetables Fruit juice and wine
Cellulase Pectinases
Protein
Bromelin, papain pepsin, pancreatin
Sugar processing
Amylases, cellulase
Starch conversion
Glucose isomerase
Other
Proteases
Starch liquefaction Dough conditioning, flour bleaching, malting, and antistaling Low-calorie beer, chill proofing, barley, altemative adjunct liquefaction, and saccharification Cream candy centers Removal of burnt flavor in UHT (ultra-heattreated) milk Separation of bean, viscosity control of extracts Cheese making, accelerated cheese ripening, natural cheese flavor concentrates, whey utilization, lactase intolerance Coca-butter substitutes, flavor-ester synthesis Synthesis of savory flavors, natural flavor esters Breakdown of cellulose structure Mash treatment, depectinization, starch/araban haze removal, citrus pulp wash viscosity reduction, natural cloud production Rendering, soy milk production, egg white replacement emulsifier production, functional hydrolysates Removal of undesirable starches and polysaccharides in the processing of cane sugar High fructose corn syrup, maltose, and dextrin syrups Meat tenderizing, coffee soluble-extract viscosity reduction
in foods. Vitamin E (tocopherol) and vitamin C (ascorbic acid) protect foods by serving as antioxidants to inhibit the destructive effects of oxygen. This helps protect the nutritive value, flavor, and color of food products. In addition, ascorbic acid enhances the baking quality of breads, increases the clarity of wine and beer, and aids color development and inhibition of nitrosamine formation in cured meat products. Beta-carotene and beta-apo8”carotenal are vitamin A precursors, which are brightly pigmented and may be added to foods such as margarine and cheese to enhance their appearance. The roles of these substances outside their nutritional functions are discussed elsewhere in this chapter (see Antioxidants, Preservatives, and Color). Thirteen vitamins are recognized as essential for human health, and deficiency diseases occur if any one is lacking. Because the human body cannot synthesize most vitamins, they must be addedto the diet. Most vitamins are currently consumed as pharmaceutical preparations or over-the-counter vitamin supplements. Some, like vitamins B, C, D, and E are added directlyto food products. Ready-to-eat breakfast cereals are a good example of fortification. Because the primary use of these cereals is as a complete breakfast entree, they are commonly formulatedto provide 25% or more of the daily value (% DV) per serving of the 10-12 important vitamins and minerals common to cereals. Another
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important example is the fortification of fruit drinks with vitamin C. Other foods that typically have added vitamins include margarine. infant formula, meal replacements, and breakfast bars (Table 13). Vitamins are added to processed foods for several reasons: T o restorevitalnutrientslostduringprocessing-thisisimportantwithdried milk, dehydrated vegetables, canned foods, and refined and processed foods. To standardize nutrient levels in foods when these fluctuate because of seasonal variations, soil differences, and methods of preparation. To fortify fabricated foods that are low in nutrients and promoted as substitutes for traditional products; this includes complete breakfasts, breakfast drinks, meat extenders, and imitation products such as eggs, milk, cheese, and ice cream. To fortify a major staple, such as bread, with a nutrient known to be i n short supply. For the preparation of designer foods (nutraceuticals) containing vitamins that are shown to be useful in preventing chronic diseases. Vitamins are typically divided into two groups: fat soluble and water soluble. Fatsoluble vitamins are usually measured i n international units (IUS) and consist of vitamins A, D, E, and K. The water-soluble group, usually measured in units of weight, consists of vitamin C (ascorbic acid) as well as the B vitamins. Humans need eight nutritive Bcomplexvitamins:niacin,riboflavin,panthotenicacid,pyridoxine,folicacid,thiamin, biotin, and vitamin B ,?.Table 14 outlines synonyms for andthe most commonly marketed forms of the major vitamins consumed in the United States as food additives. I n 1993 the FDA introduced reference daily intakes (RDI) [formerly recommended daily allowance (RDA)] for the labeling of foods and vitamin supplements. The RDI was designed to spell out the nutritional requirements of an average American. Those with greater than average needs (young woman, the elderly, and cigarette smokers, for example) are responsible for knowing their additional requirements and supplementing their diet. RDI values for vitamins established by FDA regulations are listed in Table 14. Table 13 FortifiedFoodGroups
Vitamin
Food Milk
Vitamin D
420 IUA
Beverages (noncarbonated) Cereals
Vitamin C
15-100% of U.S. RDI
Most essential vitamins Thiamin, Riboflavin, Niacin Vitamin A
25-100% of U.S. RDI
Flout Margarine Miscellaneous foods (e.g.. instant breakfast, energy bars. etc.)
per serving
Most essential vitamins
per serving S - W k of U.S. RDI per 2 oz. serving 33,100 lU/kg
Optional, but generally added Optional; also added as an antioxidant Optional; added to 90% of cold cereals Mandatory Optional, hut gcncrally added Added to position food as complete meal replacement
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Table 14 VitaminsConsumcd as FoodAdditivesand U.S. RDIValues
U.S. RDI
ajor synonyms Principal Vitamin Vitamin A A, Az Vitamin B Niacin Thiamin Riboflavin Pantothenic acid Pyridoxine Cyanocobalamin Folic acid Biotin Vitamin C
Vitamin D D1 D, Vitamin E
Vitamin K K, K,
5000 IU
Retinol
Vitamin A acetate Vitamin A palmitate
Dchydroretinol Vitamin B? Vitamin B , Vitamin B2 VitaminB, Vitamin B,) Vitamin B Vitamin B,
Nicotinic acid Thiamin hydrochloride Riboflavin Calcium pantothenate Pyridoxine hydrochloride
Ascorbic acid
Ascorbic acid Sodium ascorbate Calcium ascorbate
Folate
20 mg 1.5 mg 1.7 mg 10 mg
2 mg 6 Pg 0.4 mg 0.3 mg 60 mg
400 IU
Ergocalciferol Cholecalciferol Tocopherols
Phytonadione Menadione
DL-alpha tocopherol acetate D-alpha tocopherol D-alpha tocopheryl acid succinate
30 IU
Phylloquinone
65 Pg
Vitamin A is generally added to margarine and milk. Muchof the vitamin A content of milk is obtained by feeding cows supplements of the vitamin. In addition, vitamin A is frequently added to instant breakfast foods, granola bars, and quick preparation or energy bar food products to better position those foods as complete meal replacements. Most natural vitamin A is derived from fish oils or carotenoid pigments found in chlorophyll-containing plants. These carotenoid pigments are the source of several provitamins, of which alpha- and beta-carotene are the most important. Important commercial forms include beta-carotene, retinol, retinol acetate, and retinol palmitate. Practically all the vitamin A used today is obtained by synthesis from the chemical intermediate, betaionone. Thiamin (vitamin B , ) is found in all plants, but cereal grains, milk, legumes, nuts, eggs, and pork contain large amounts. Thiamin is essential for the proper functioning of the central nervous system. Important commercial products include thiamin hydrochloride and thiamin mononitrate. Thiamin is obtained synthetically by several different routes, including linking chlorate-thylpyrimidine with 4-methyl-5-(hydroxy-ethyl)thiazole. Another method is the conversion of 4-amino-5-cyano-pyrimidine into a thioformylaminomethyl derivative via catalytic hydrogenation and reaction with sodium dithioformate.
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Riboflavin (vitamin B:) occurs in plant and animal cells. Important dietary sources include organ meats, yeast, and dairy products. Riboflavin is produced synthetically from D-ribose and fermentation processes. Pantothenic acid (vitamin B,) occurs i n all animals and plants and i n some microorganisms. Natural sources of pantothenic acid include liver, eggs, broccoli, cauliflower, tomatoes, and molasses. Commercially available forms include the liquid D-pantothenyl alcohol (panthenol), as well as calcium D-pantothenate and racemic calcium pantothenate. It is produced commercially by condensation of D-pantolactone with beta-alanine. Niacin (vitamin B,) is a generic term that includes both niacin (nicotinic acid) and niacinamide (nicotinamide). Poultry, meats, and fish are the most important sources of niacin. Both niacin and niacinamide are important commercial forms. Niacin is produced synthetically by the oxidation of quinoline, or 2-methyl-5-ethyl-pyridine. Niacinamide is produced by amidation of niacin. Pyridoxine (vitamin B,) refers to naturally occurring pyridine derivatives that have vitamin B, activity. Most forms of the vitamin occur in plants and animals, but chemical synthesis is a far more efficient and economical method of production than natural isolation. Pyridoxine hydrochloride is produced by the condensation of ethoxyacetylacetone with cyanoacetamide. Cyanocobalamin (vitamin B,:) is found in dairy and meat products. Cyanocobalamin and hydroxocobalamin are the important commercial forms, produced by fermentation using either Streptomycetes griseus or S. crureofkciens. Vitamin B is essential for bone as well as fornormal marrowcells,thenervoussystem,andthegastrointestinaltract, blood function. Folic acid, a member of the vitamin B complex, is a yellow-orange crystalline powder found in brewer’s yeast, wheat, nuts, legumes, and liver tissues. Folic acid and the calcium salt of folic acid can be obtained synthetically by a number of routes from triaminohydroxypyridine and para-aminobenzoylglutamic acid. Folic acid functions as a coenzyme in the synthesis of nucleic acid, purine-pyrimidine metabolism, and other systems. Recently folic acid gained importance because of its role in reducing the chances of neural tube birth defects and its role of controlling homocysteine, a risk factor in atherosclerosis. Medical studies indicate that folic acid and pyridoxine (vitamin B,) can reduce high levels of homocysteine, an amino acid,in the blood. A high levelof blood homocysteine was found to be an independent factor from cholesterol leading to increased risk of heart attack and stroke. Therefore folic acid and possiblyvitamin B , use in nutraceuticals and other fortified food products (e.g., breakfast cereals, cereal bars, calorie control and fitness food products, etc.) will increase substantially in the next few years. Vitamin C (ascorbic acid) is the most important vitamin used as a food additive in terms of volume. Primary applications include fruit juices, still beverages, juice-added sodas, and dry cocktail or beverage powder mixes. Ascorbic acid is also used as a food to preserve and preservative. Fruit juice makers, in particular, are applying the vitamin protect against color change in fruit ingredients. By doing so, they can also promote the high vitamin content of juices. Vitamin D: (ergocalciferol) and vitaminD l (cholecalciferol) are produced synthetically by the irradiation of the provitamins ergosterol and 7-dehydrocholesterol. respectively. Vitamin D! can also be isolated from fish liver oils. Although vitamins D: and Dl areboth important commercial products, most of thevitamin D issynthesized by the photochemical conversion of 7-dehydrocholesterol to D,.
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495
Vitamin E is found widely throughout nature, but the main dietary sources include vegetable fats and oils, dairy products and meat, eggs, cereals, nuts, and leafy green and yellow vegetables. The role of tocopherol as an antioxidant that can reverse the damaging effects of free radicals and therefore prevent certain chronic diseases has received increasingly wide attention in recent years. Naturally occurring vitamin E can be obtained from vegetable oil sources by distillation, and recently two major vegetable oil processors initiated the commercial production of natural tocopherols. However, large quantities of tocopherols are synthetically derived. Vitamin K, found i n green leafy vegetables, tomatoes, cauliflower, egg yolks, soybean oil, and liver, is essential for the formation of prothrombin and other blood-clotting factors in the liver. Menadione and its sodium bisulfite and diphosphoric acid ester derivatives are the lnost common commercial forms of the vitamin K group of compounds. Menadione (vitamin K \ ) is produced synthetically by treating 2-methylnaphthalene with chromic acid in the presence of sulfuric acid.
G. Antioxidants Antioxidants are food additives that retard atmospheric oxidation and its degrading effects, thus extending the shelf life of foods. Examples of food oxidative degradation include products that contain fats and oils in which the oxidation would produce objectionable also rancid odors and flavors, some of which might even be harmful. Antioxidants are used to scavenge oxygen and prevent color, flavor, and nutrient deterioration of cut or bruised fruits and vegetables. Recently, definitive studies have shown and been widely publicized in the news media that antioxidant nutrients suchas ascorbic acid (vitaminC) and tocopherols (vitamin E) can protect against harmful cell damage and thus prevent certain human diseases. Foods formulated with antioxidants and other vitamins are now recommended to prevent and cure cancer, cardiovascular diseases, and cataracts. The same antioxidants that are used to prevent oxidative deterioration of food may be used in functional foods (nutraceuticals, designer foods, etc.)to create products that prevent or cure certain chronic diseases.In this section, however, only the food preservation function of antioxidants will be discussed. To improve the performance of antioxidants,twoothertypes of foodadditives, sequestrants (e.g., EDTA, citric acid) and synergists (e.g., mixtures of antioxidants and lecithin), are frequentlyused with them. Antioxidants may also be presentin food packaging as indirect food additives, but such use is not covered in this chapter. Food antioxidants can be divided into water-soluble and oil-soluble compounds and also classified as natural or synthetic, as shown in Table 15. The most frequently used natural antioxidants are ascorbic acid (vitamin C), its stereo isomer erythorbic acid, and their sodium salts, plus the mixed delta and gamma tocopherols. While ascorbic acid finds its major use as a nutritive supplement orin pharmaceutical preparations, smaller amounts are intentionally used for antioxidant purposes. Erythorbic acid (iso-ascorbic acid) is virtually devoid of vitamin C activity (only 5% that of ascorbic acid). Citric acid and tartaric acid are also natural antioxidants (and antioxidant synergists), but are predominantly added to foods as acidulants. Synthetic antioxidants used as direct food antioxidants include butylated hydroxyanisole(BHA),butylatedhydroxytoluene(BHT),tert-butylhydroquinone(TBHQ), and propyl gallate (PG). These antioxidants are effective in very low concentrations (0.01%
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Table 15 FoodAntioxidantsandTheirManufacturingProcesses
Manufactured compound Antioxidant Oil-soluble products Butylated hydroxyanisole (BHA) Butylated hydroxytoluene (BHT) Tert-butyl-hydroquinone (TBHQ) Propyl gallate (PG) Tocopherols Thiodipropionic acid Dilauryl thiodipropionate Ascorbyl palmitate Ethoxyquin Water-soluble products Ascorbic acid Sodium ascorbate Erythorbic acid Sodium erythorbate Glucose oxidase/catalasc enzymes Gum guaiac Sulfites
Rosemary extract
by Synthesis Synthesis Synthesis Synthesis Extraction or synthesis Synthesis Synthesis Synthesis Synthesis Fermentation or synthesis Fermentation or synthesis Fermentation or synthesis Fermentation or synthesis Fermentation Extraction Synthesis Extraction
or less in animal fat) and not only retard rancidity but also protect the nutritional value of the food by minimizing the breakdown of vitamins and essential fatty acids. At one time, safety questions were raised about several synthetic antioxidants. These have largely been resolved, although the impact on the market for synthetic antioxidants still exists. The major applications of antioxidants in foods are listed in Table 16. The fats and oils industry and the snack/fast food/convenience food industries are the major users of food antioxidants. While the growth of fat-containing foods is declining because of consumer concerns related to the adverse effectsof fat and high caloric intake to health, the increasing preference for “healthier” unsaturated fats will increase demands for oil-soluble antioxidants because these fats require more protection against rancidity. Butylated Hydroxyanisole (BHA) was introduced commercially in foods in 1948. The major applications for BHA are in frying fats and oils, salad oils, and shortenings. BHA can beused in blends with BHT, TBHQ, or PGto optimize performance. For example, blends of BHA and propyl PG are used as stabilizers in edible lard and tallow. BHA is approved by the FDA as a GRAS substance, but the use is limited to a maximum 0.02% of the total fat and oil content of the product. Past developments have had a detrimental effect on the demand for BHA. In 1982, the Japanese government reported that, according to feeding studies done in Japan with BHA at 2% of the entire diet, BHA was found to be carcinogenic. Consequently BHA would not be allowed in food products sold in Japan after July 1982. Various governments, including the United States, Canada, and the UnitedKingdom,requestedadelay intheimplementationdate until further studies could be done. The date was then deferred to February 1, 1983, and the ban was never implemented. Subsequently the World Health Organization’s Food and Agriculture Organization studies showed that BHA dosage levels would have to be high
497
Food Additives Table 16 FoodApplications for Antioxidants
Oil-soluble antioxidant applications (to retard onset the rancidity) of
Water-soluble antioxidant applications (to prevent oxidative deterioration of color, flavor, and nutrients)
Edible fats vegetables and fruits Fresh Vegetable oils Dried fruits uit Nuts ruits Frozen Shortenings Margarine Salad oils productsConfectionery frying foodFast oils Flavoring compositions products Bakery Processed 111eat spreads poultryand Meat Cheeses fruits processed Thermally chicken Processed Snack foods, nuts Canned meat and poultry Pancnke/cake mixes Breakfast cereals Dehydrated potatoes Chewing gums
(e.g., about2% of the oil or fat content of the food) beforeany carcinogenic effects would become apparent. (The normal BHA content level is 200 ppm of the fat or oil content of the food.) However, because of Japan’s announcement of its initial study, BHA was removed from some of the food and food packaging sold in the United States and Japan. The findings of the Japanese study relative to BHA were surprising since several other studies conducted worldwide had found BHA to be noncarcinogenic. The original Japanese researcher has now agreed that BHA is not carcinogenic: however, irreparable damage to BHA as a food antioxidant has occurred, and the product’s unhealthy image is unlikely to be reversed in the future. Butylated hydroxytoluene (BHT) was approved for use as a food antioxidant in 1954. BHT is often used in blends with BHA or BHA/PG in vegetable oils and in edible animal fats to take advantage of the synergism obtained. Although BHT was never removed from the FDA’s GRAS list, demand for BHT as a direct food additive dropped significantly because of an FDA proposal to restrict the use of BHT as a food additive throughout the 1980s. That proposal, as well as the general trend toward the use of allnatural ingredients in foods, has negatively impacted BHT use in foods. Producers of both BHA and BHT have petitions filed with the FDA to recognize the existence of “prior sanctions” for the use of the chemicals as food antioxidants at levels not to exceed 0.02%. Such recognition would eliminate the necessityof classifying the chemicals as food additives. Tertiary-butyl hydroquinone (TBHQ) is relatedto BHA and has good heat stability. It was first introduced for food applications in 1972. TBHQ shows exceptional ability i n protecting unsaturated vegetable oils and animal fats from rancidity. One of its largest applications is in soybean oils. Although mostly used by itself, TBHQ canbeused in combination with BHT and BHA. TBHQ is often used as a replacement product for PG.
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Propyl gallate (PG) has been used as a food antioxidant since the 1950s. Its current primary use is more as a synergist in combination with BHA and BHT. The active part gallic acid, can be extracted from natural sources and can be synthesized. Propyl gallate is effective in vegetable oils as well as animal fats, but it is not heat stable, even at cooking temperatures. Total consumption of PG is very small because of its relatively high price and competition from TBHQ. Ascorbic acid (vitamin C) and sodium ascorbate are widely used as natural antioxidants and vitamin supplements. As an antioxidant, ascorbic acid is used primarily in prepared foods (canned fruits and vegetables, juice drinks, applesauce, potatoes) and in processed meats (sausages). Manufacturers use it if for its protective function in soft drinks, for example, but declare it as “added vitamin C.’’ Ascorbic acid is insoluble in fats and oils, and its almitoyl ester is synthesized to impart some lipid solubility. Used alone, the ester is not very effective in fats and oils, and it is normally used in combination with tocopherol. Erythorbic acid (iso-ascorbic acid) and sodium erythorbate are used primarily as antioxidants in cured meats (e.g., bacon, sausages) and by salad bars as an oxygen scavenger. They arealso used in frozen fruits, vegetablefats and oils, and frozen fish and seafood. Erythorbic acid (and its salts) has benefited significantly from the FDA’s ban on the use of sulfites for fresh or uncooked vegetables i n salads. Approximately 80% of the total U.S. consumption is estimated to be in the form of sodium erythorbate. The greatest use is i n cured meat to minimize the formation of nitrosamines during the curing or cooking process. USDA regulations governing the maximum level of nitrite permitted for curing uses of erythorbacon require the useof 500 ppln of ascorbates or erythorbates. Other food bates are in fresh cut meat, frozen fruits and vegetables, and raw fresh cut vegetables. In Europe, erythorbic acid use has been permitted since 1995. Tocopherols are the fastest growing antioxidants used in the United States. Because the United States trades heavily with Japan, where synthetic antioxidants are banned,U.S. food exporters are reformulating products using natural antioxidants. Mixed tocopherols appear to be the product of choice. Although all isomeric forms (alpha, gamma. and delta) of tocopherol show antioxidant activity, the 80% garnma/20% delta mixture of natural tocopherols has the best antioxidant activity. Mixed natural tocopherol products can be used to protect a variety of food products, including dehydrated and processed vegetables, pasta and noodles, animal fats, salad dressings and oils, snacks, meats, and baked foods. Residues from vegetablerefining contain a small but significant level of tocopherols. Using techniques such as molecular distillation, these can be concentrated to give a brown oily product with goodantioxidantproperties. Thecompositionvaries withtheorigin (type of vegetable oil), and both gamma-rich and delta-rich versions are used. Sulfites serve multiple functions in foods: ( 1 ) inhibition of enzymatic and nonenzymatic browning, and (2) control of microbial growth. For years, sulfur dioxide and sulfite to help preserve the color of dried fruits and vegetables. salts have been widely used Sulfites areused in wine making and the wet milling of corn to prevent undesirable microbial growth.Restaurantsandotherfoodserviceoutletspreventthebrowningoffresh produce with the use of sulfites. But because of the allergic reactions of some consumers (especially asthmatics) to sulfites, regulations were issued and alternatives sought. In July 1986, six sulfiting agents-sulfur dioxide, sodium sulfite, sodium bisulfite, potassium bisulfite, sodium metabisulfite, and potassiunl metabisulfite-were banned by the FDA for use i n raw vegetables and fruits on salad bars. In July 1987, the FDA ruled that all pack-
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499
aged foods containing I O ppm or more of sulfur dioxide equivalents must disclose on the label that sulfiting agents are present. In 1990. the use of sulfites on fresh potatoes was banned. Treatment of fruits and vegetables with sulfitesis the most effective means available today to control browning. However, because sulfites have been banned in certain food categories and their regulatory status i n other categories is in question, alternative treatments to retard enzymatic browning and other oxidative reactions havebeen investigated. To date, however, alternatives to sulfites are not equivalentto sulfites in their effectiveness, cost, or functionality. The promising antioxidantlpreservativealternatives generally contain ascorbic acid or erythorbic acid in combination with one or more adjuncts, such as citric acid or some other acidulant, a calcium salt. a phosphate, sodium chloride, cysteine, or a preservative such as potassium benzoate or sorbate. The ascorbic acid derivatives, ascorbic acid 2phosphate and ascorbic acid-6-fatty acid esters, are also reportedly effective. Another suggested substitute (which functions in water but not with fats and oils) is the sequesterant and chelating agent ethylenediaminetetraacetic acid (EDTA), which hasbeen widely used i n processed potatoes, salad dressings, sauces, and beverages. Cyclodextrin is another sulfitealternativethatcanbeusedtopreventbrowning. Finding a good substitute for sulfites, however, has not yet been realized. This is because sulfites not only act as antioxidants to prevent browning, but also perform preservative functions i n preventing unwanted microbial spoilage. The above chemicals are ineffective against microbes. Ethoxyquin is included in the FDA regulation but limited to specific applications only. It is cleared for retarding oxidation of carotene, xanthophylls, and vitamins A and E in animal feed and fish food, and as an aid in preventing the development of organic peroxides i n canned pet food. Gum guaiac is an approved antioxidant for natural flavoring substances and other natural substances used in conjunction withflavors. It is also approved for addition to aninlal feed and food-packaging materials.
H. Preservatives Preservatives (antimicrobial agents) are capable of retarding or preventing the growth of microorganisms such as yeast, bacteria, molds, or fungi and subsequent spoilage of food. The principal mechanisms are reduced water availability and increased acidity. Sometimes these additives also preserve other important food characteristics, such as flavor, color, texture, and nutritional value. Important food preservatives used include sorbic acid and its potassium salt, calcium and sodium propionates, sodiumand potassium benzoates, and parabens. Sulfur dioxide and sulfites are also used extensively for controlling undesirable microorganisms in soft drinks, juices, wine, beer, and other products. Salt, organic acids, sugar, alcohol, spices, essential oils, and herbsalso inhibit the growth of microorganisms, but usually their primary function is different when added to food. Chemical preservatives play a very important role i n the food industry, from manufacture through distribution to the ultimate consumer. The choice of a preservative takes into consideration the product to be preserved, the type of spoilage organism endemic to it, the pH of the product, the shelf life, and the ease of application. No one preservative all organisms, and therefore combinations are canbe used in every producttocontrol
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oftenused. In certainfoods,specificpreservativeshaveverylittlecompetition.Inthe concentrations used in practice, noneof the preservatives discussed here is lethal to microorganisms in foods. Rather, their actionis inhibitory. Major uses for preservativesby food industry sector are listed in Table 17. In general, increased demand by consumers for lightly processed, lightly prepared foods (as people tend to do less cooking at home but at the same time are looking for products that are fresh, suchas prepared salads) has stimulated use of antimicrobial preservatives over the past several years. At the same time, however, media and consumer reacof several preservatives in tion to chemical preservatives has stymied or limited the growth favor of “all natural” and “no preservatives added” food products. However, significant displacement of traditional preservatives with naturally derived new products is not expected in the near future. Important areas for preservatives are in fruit beverages and convenience foods. For example, low fat/low calorie salad dressings require a preservative, while the traditional high oil-containing products had lower water activity and therefore an acceptable shelf life without chemical preservatives. Potassium sorbate and sorbic acid are used as preservatives in a great variety of foods and can be used as directadditives, as spraysordipbaths,and as coatingson wrapping materials, inhibiting yeasts, molds, and bacteria. Potassium sorbate is used where high water solubility is desired. Because sorbates have no effect on the microorganisms that produce lactic acid, they can be utilized to prevent yeast and mold spoilage of foods, such as mostcultureddairyproductsandpickles,withoutinterferingwiththedesired bacterial cure. Potassium sorbate solutions may also be used for spray and dip bath applicat i m s on cheese, dried fruits, smoked fish, and similar products. The effectiveness of potassium sorbate is based on its ability to depress fatty acid metabolism in the microorganisms. Useof sorbic acid is limited because of its low solubility in water. Therefore potassium sorbate is the primary form used in foods. It is effective against microbes at pH 6.5 or less. As the pH decreases, the antimicrobial activity of this preservative increases. Onan equal weight basis, potassium sorbate has 74% of the activity of sorbic acid. Sorbic acid and potassium sorbate are GRAS additives. Normal use levels are in the range of 0.05-0.10/0. Sorbates are used in cheeses, baked goods, spreads, margarine, dried fruits, jams, and jellies. Because of its corrosive nature, propionic acid, a liquid, is rarely used in the food industry. Its sodium and calcium salts are used in its place, yielding the free acid within
Table 17 MajorUSCSofPreservatives by Use Sector
Preservative SorbatesMoldandyeastinhibition in proccssedcheeseandspreads, other low-acid foods, and dried fruits. Effective in the acidic pH range up to pH 6.5 BenzoatesBeverages,fruit juice, pickles.Effective in pHrange 2.5-4.0 PropionatesMoldandropeinhibitors inbreadandbakedgoods ParabensAntibacterialforuseinlow-acidfoods(pHgreaterthan 5.0) such as meat and poultry products
Food Additives
50 1
the food at low pH. They are highly effective mold inhibitors. but have essentially no effect against yeast. They have negligible activity against bacteria, except for their effectiveness against the rope-causing Bacillus rtleset1tericu.s. Propionic acid occurs naturally in Swiss cheeses at levels as high as 1 %. Its calcium salt, and to a lesser extent its sodium salt, have been used for more than 30 years as an inhibitor of mold growth in bread. The main market for propionate salts isinbakery products, chiefly because these salts do not inhibit yeast action (they also have almost no activity against bacteria). The propionates have GRAS status for use i n foods and have no upper limits imposed except for breads, rolls, and cheese, which come under the Standards of Identity. They can be used up to 0.3% in cheese products and to 0.32% by weight of the flour in white bread and rolls. Benzoic acid is one of the oldest chemical preservatives used in foods, having been described as a preservative in the 1800s. It has been used in foods since the early 1900s. Benzoic acid occurs naturallyin some fruits and spices, suchas cranberries, prunes, cinnamon, and cloves. Sodium or potassiunl benzoates are most effective in the pH range of 2.5-4.0. Benzoates have activity against yeasts, molds, and bacteria. However, benzoates are not recommendedfor bacterialcontrol becausetheirantimicrobialactivity is poor in above pH 4, where bacteria are the greatest problem. As benzoates are very efficient controlling yeasts, they cannot be used i n dough or in other yeast-raised bakery products. The most important uses for benzoates are in fruit juices and carbonated beverages, jams and jellies, and condiments. In carbonated drinks, 0.03-0.05% is used; in noncarbonated drinks, up to0.1 % is used. Benzoates arealso used for fats and oils, gravies, frostings, puddings, and gelatins. Potassium benzoate became commercially available in 1984 and can be used as a substitute for sodium benzoate in many of the above food products. It is also useful in margarine, potato salad, fresh fruit cocktails, and pickles. Although the amount of sodium added with the benzoate salt is nutritionally insignificant, the potassium salt was developed specifically for use in reduced or low-sodium food products to avoid sodium declaration on the label. The potassiunl or sodium salts of benzoic acid are more soluble in water than is benzoic acid and consequently are preferred for use in many food products. They do not destroy yeasts or molds but instead retard further growth of organisms already present, provided the degree of contamination is not too high. Benzoic acid and benzoates are GRAS substances and are pernlitted for usei n foods up to a maximum of 0.1% concentration. Parabens are esters of para-hydroxybenzoic acid. A combinationof methyl and propyl esters and sodium benzoate ismostoftenused,buttheethyl and butyl esters also have utility. Parabens are the only phenols approved for microbiological preservation of foods. Parabens are effective against molds and yeasts and are relatively ineffective against bacteria, especially the gram-negative bacteria. Their antimicrobial activity extends up to pH 7.0, making the parabens the only antimicrobial agents effective at higher pH values. The methyl and propyl parabens are GRAS ingredients, but their use is limited to 0.1% (combined). tz-Heptyl paraben is permitted in beer at a maximum concentration of 12 ppm. Parabens are used in baked goods, beverages, fruits, jams and jellies, and olives and pickles, but not in dairy products. Because parabens are the most expensive of the availablepreservativesandhavesometechnicalproblemsassociatedwiththeirusein foods, use by the food industry remains limited.
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About I O years ago, sodium nitrite and sodium nitrate were used i n curing bacon and other meats to prevent the growth of bacteria that cause botulism. The nitrate and nitrite were linked to the formation of nitrosamines in the meat, which were considered carcinogenic in experimental animals. Though the public outcry has largely subsided and nitrites continue to be used in smaller amounts, the continued use of these preservatives probably s t e m from the absence of suitable alternatives. Sodium ascorbate and sodium erythorbate are effective catalysts in the curing process, and the addition of one of these antioxidants to bacon makes it possible to reduce the quantity of sodium nitrite used. Most chemical preservativesin use today have specialized uses and established niche markets in the food industry. A great amountof interchangeability does not exist because of specific inhibitory actions toward bacteria, molds, or yeasts. Blendsof antioxidants and preservatives (some natural and some synthetic chemicals) can provide multiple functions for multiple food products. One such combination of ingredients is a blend of erythorbic acid, citric acid, and potassium sorbate as an antioxidant and antimicrobial substitute for sulfites on fresh vegetables.
1.
Emulsifiers
Emulsifiers are additives that allow normally immiscible liquids, such as oil and water, to form a stable mixture. They are widely used in foods in order to achieve the texture, taste, appearance, fat reduction,and shelf life desired in foods. Bread and bakery products is the largest food segment utilizing emulsifiers. In this application, they soften the bread and strengthen the dough by distributing the fat within the product so less fat (shortening) needs to be added. Emulsifiers are utilized as fat-sparing agents i n salad dressings and bakery and dairy products. Visible fats and oils routinely need emulsifiers for food-product processing, appearance, maintenance of shelf life, texture, and taste uniformity. They are also included i n low-fat formulations (e.g., frozen desserts, bakery products), often more so than in formulations with normal fat levels. In addition, food emulsifiers are widely used in convenience, snack, and microwaveable food products. The multiple applications and functions of food emulsifiers are shown in Table18, and several of the more prominent food uses of emulsifiers are shown in the following listing:
Breads Frozen desserts Icings Cream fillings Chocolate milk Whipped toppings Coffee creamers Instant breakfasts Infant formula Dessert mixes Rolls
Cakemixes Fresh cakes Donuts Cereals
Food coatings Instant potatoes Pastas Snack foods Ice cream Dips
Shortenings Margarine and diet spreads Peanut butter Candy Caramels Chewing gum base Chocolate Toffees
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Table 18 Functions of Emulsifiers
Function
Antistaling
Margarine, creamy salad dressing. coffee whiteners, frozen desserts Most baked goods
Modifying texture
Bread, cakes, macaroni
Wetting
Coffee whiteners. drink mixes, instant breakfasts Color and flavor systems Shortenings, margarine, peanut butter Ice cream, frozen desserts
Emulsifying
Solubilizing Crystal modification Preventing agglomeration Foaming
Defoaming Reducing tackiness Fat sparing
Improving palatability
Whipped toppings, icings. cakes. convenience desserts, ice cream Processing of syrups, yeast Candies, chewing gums Baked products, frozen desserts, whipped toppings, margarine, spreads, imitation sour cream Icings, confectionary coatings
Disperses small droplets of immiscible substances Complexing action on starch reduces firming of crumbs Complexing action 011 starch reduces clumping, improves consistency and uniformity Reduces interfacial tension between liquid and solid Improves solubility Modifies mode and ratc of crystal formation Controls coagulation of fat particles Controls dispersion of a gas in a liquid Breaks emulsions Assures texture Reduces size of fat globules, resulting a wider dispersion and reduced fat levels Improves mouth feel
The most common and commercially important emulsifiers are monoglyceridesand diglycerides of fatty acids and their esters (e.g., glyceryl monostearate), lactylated esters (e.g.,sodiumstearoyllactylate),propyleneglycolmono-anddiesters(e.g.,propylene glycol monostexate), lecithin, sorbitan esters (e.g., sorbitan monostearate), and polysorbates (e.g., polyoxyethylene 80 sorbitan monolaurate). With the exception of lecithin, few emulsifiersareused as a single additive. Most food emulsifiers are used as blends of emulsifiers, water, fats, and other classes of food additives such as gums. These products are formulated for specific applications (or specific customers) so that the combination provides both enhanced performance and ease of use. Emulsifiers are regulated as food additives in most countries. The FDA classifies lecithin, monoglycerides and diglycerides, diacetyl tartaric acid ester (DATEM), and triethyl citrate as GRAS substances. The other emulsifiers have specific regulations that permit their use in specific products at set levels. Monoglycerides and diglycerides are used in the largest amounts (more than 50% of the total volume for emulsifiers), mainly because of their low cost. Important applications are in the preparation of shortenings, in bread and other bakery products, and in ice cream. Lecithin. Commercial lecithin is usually a by-product from the refining of crude soybean oil. The term lecithin describesa complex mixtureof phospholipids, triglycerides, fatty acids, and other componentsthat occur naturally in soybean oil. The major phospholipids of lecithin are phosphatidylcholine(PC), phosphatidylinositol (PI), phosphatidylethanolamine (PE), and phosphatidic acid (PA). The unique structureof these phospholipids
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and other minor constituents gives lecithin its emulsification properties. Lecithin is often modified to improve its effectiveness as an emulsifier. The common modified lecithins thatare commercially available are the hydroxylated, acetylated, and enzyme-modified lecithins. The result is more hydrophilic, water-dispersible lecithin with enhanced oil-inwater emulsion properties. The primary applications for lecithin are in baked products, dairy blends, baby foods, nutritional drinks, margarines, chocolates, chewing gunis, and confectionaries. Polysorbates are a group of emulsifiers that contain sorbitans, various types and amounts of fatty acids, and polyoxyethylene chains. Heating sorbitol with stearic acid in the presence of a catalyst cyclizes sorbitol andf o r m an ester to produce sorbitan monostearate and tristearate. Other sorbitan esters of importance are monooleate and tristearate. Any of the three esters maybe reacted with ethylene oxide to give polyoxyethylene derivatives, which are much more hydrophilic than sorbitan esters. The monostearate derivative is known as polysorbate 60, the tristearate is polysorbate 65, and the monooleate is polysorbate 80. Polysorbates and sorbitol esters are used chiefly in ice cream. imitation dairy products, and in baking applications. Polyglycerol esters contain polymerized glycerol and various types and amounts of fatty acids. The polyglycerol portion is synthesized by heating glycerol in the presence of an alkaline catalyst. The polyglycerol backbone is then esterified either by direct reaction with a fatty acid or by interesterification with triglyceride fat. Sucrose esters are manufacturedby adding fatty acids to a sucrose molecule. Sucrose has eight free hydroxyl groups that are potential sites for esterification with fatty acids. Derivatives containing one to three fatty acid esters are emulsifiers and are approved for food use. There are a large number of other emulsifiers used in the food industry. but their volumes are negligible. Examples include lactylated esters, used in direct baking (notthe shortening) and in imitation dairy products, and propylene glycol esters, used i n various prepared mixes, shortening. and baking.
J.
Flavors
Flavors consist of concentrated preparations, with or without solvents and carriers, used to impart a specific taste to food. Flavor ingredients are the largest single group of direct food additives utilized by the food industry. They also represent the highest value among the food additives segments. Flavoring substances are classified as Naturalflavoringsubstance-obtained by physical separation, enzymatic processes, or microbial processes from vegetable or animal sources, either in the raw state or after processing (including drying, torrefaction, and fennentation). Nature-identicalflavoringsubstance-obtained by synthesisorisolated by chemical processes and chemically identical to substances naturally present in the vegetable or animal sources (this classification is used in Europe butnot allowed in the United States). Artificial flavoring substance-obtained by chemical synthesis and not found in nature. Flavoring preparation-products other than natural substances, whether concentrated or not, with flavoring properties, obtained by physical separation or enzymatic or microbial processes from material of vegetable or animal origin, either
Food
Additives
0
505
i n the raw state or after processing (including drying, torrefaction, and fernlentation). Process flavorings-products obtained by heating to a temperature not exceeding 180°C for a period not exceeding 15 minutes using a mixture of ingredients, not necessarily having flavoring properties themselves,of which at least one contains nitrogen (amino) and another is a reducing sugar. Smoke flavorings-smokeextractsusedintraditionalfoodstuff smokingprocesses. Flavor enhancers-some amino acids and nucleotides, as well as sodium salts (such as monosodium glutamate, sodium inositate, and sodium guanylate), have only a weak taste by themselves but have the power to considerably enhance the taste sensation caused by other ingredients in savory flavors.
The flavor industry is not a single homogeneous entity, but a composite of closely interrelated and somewhat overlapping sectors including essential oils and natural extracts, aroma chemicals, and compounded flavors. The first two sectors providethe raw materials used for compounding flavors. Essential oils are usually defined as the volatile aromatic material obtained from botanical or animal sources by the process of distillation, expression, solvent extraction, or maceration. The most common physical process used for removal of essential oils is steam or water distillation. to a material that has been removed from a plant by a The term “extract” refers solvent, after which the solvent is evaporated to concentrate the oil. Absolutes, which are alcohol-soluble liquids, and concentrates, which are usually waxy solids, are both extracts. Oleoresins are thick, viscous products obtained by extraction of plant material with a nonaqueous solvent (e.g., hydrocarbon)that is subsequently removed. Extractsof vanilla beans and other fruit extracts are the most important product examples of this class. Essential oils and natural extracts represent complex aroma mixtures containing hunto condreds of chemical constituents. They may be used for imparting scent or aroma sumer products or lnay be used as raw materials for compounding flavor and fragrance compositions, or they may be the source of isolated aroma chemicals, also used in compounding. Essential oils can be classified into three chemical groups: straight hydrocarbons, oxygenated compounds, and benzene derivatives. Aroma chemicals comprise organic compounds with a defined chemical structure thatareisolated from microbial fermentation, plant or animal sources, or produced by of organic synthesis. Isolation consists of the physical removal of the flavor compound interest from a natural source that contains it (e.g., L-menthol isolated from cornmintoil). Isolates may be further chemically modified. Aroma chemicals used to compound flavors are of two types: ( l ) isolates, which have been physically removed from natural sources that contain them and which may be further chemically modified; and(2) synthetic aroma chemicalsthat duplicate the structure and aroma characteristicsof their counterparts found in nature. Synthetic aroma chemicals that duplicate the structure and aroma characteristics of their counterparts found in nature are known as “nature identical.” Those that are not known to occur in nature but display an aroma reminiscent of known natural products with unrelated chemical structure are defined as “artificial.” However. the legal definition of natural and artificial varies, depending on each country’s legislation. Aroma chemicals are used as raw materials for flavor compositions. While technical merits are not at issue, naturally occurring aroma
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chemicals may enjoy preferential status for theiruse i n certain countries because of labeling regulations. More than 80% of aroma chemicals in use contain only carbon, hydrogen, and oxygen in their structure, the large majority being esters, ketones, aldehydes, and alcohols. A few contain nitrogen (nitro and nitrile groups, pyrazines) and/or sulfur (mercaptane, thiazoles). About 4% of the chemicals are unsaturated hydrocarbons, primarily with cyclic and acyclic terpene structures (e.g., limonene, pinenes, etc.). Mostof the aroma chemicals are oil-soluble, water-insoluble liquids. Aroma chemicalsof commerce can be broadly classified according to their chemical structure and are grouped into three categories as follows: Benzenoids (including naphtalenoids): chemicals containing a benzene or naphthalene ring, including alcohols, acids. esters, aldehydes, ketones, phenols, phenol esters, and lactones. Terpene and terpenoids: chemicals with (or closely related to) characteristic terpene structures, both acyclic and cyclic, having two or more isoprene (CiH,) moieties and oxygenated derivatives of the terpene hydrocarbons, including alcohols, aldehydes, ketones, and esters. Other aroma chemicals: includes aliphatic, alicyclic, and heterocyclic compounds and esters of lower fatty acids. Of the thousands of aroma chemicals includedi n compounded flavors. the following compounds are used in very large quantities: 3-phenetyl alcohol and esters, vanillin, ethyl vanillin, esters of lower fatty acids, benzyl acetate. alpha-hexyl-cinnamaldehyde, 1m e n thol (synthetic), geranioUnerol and esters, and anethol. The universally applicable definition of flavor compositions is that of mixtures of aromatic materials that are added to foods and beverages in order to improve palatability. Flavor compositions consist of complex mixtures of various aromatic materials from few to 100 or more constituents. Compounded flavors may contain aroma chemicals, natural extracts, essential oils, solvents, andin some cases other functional additives (e.g.. antioxidants,acidulants.emulsifiers,etc.).Certainrawmaterialsthatcanbeuseddirectly as flavors without compounding (e.g., vanilla, peppermint) and those products with a taste of theirown,such as sweeteners,acidulants,andsalts,arenotincluded i n theabove definition. Flavors serve all sectors of the food processing industry, including carbonated and still beverages, processed foods, confectionary, and dairy foods, and are added to foods and beverages for the following reasons: T o create a totally new taste To enhance, extend, round out, or increase the potency of flavors already present To supplement or replace flavors to compensate for losses during processing To simulate other more expensive flavors or replace unavailable flavors To mask less desirable flavors-to cover harsh or undesirable tastes naturally present in some foods Thetypes of flavor compositions,theirmanufacturingprocess andthestarting materialsformanufacturingthem,andtheircommonproductformaresummarized in Table 19.
Food Additives
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X.
ADVERSE EFFECTS OF FOOD ADDITIVES
The practice of adding chemicals (e.g., salt, spices, herbs, vinegar, and smoke) to food dates back many centuries. In recent years, however, the ubiquitous presence of chemical additives in processed foods has attracted much attention and public concern over the long-term safety of additives to man. Although the safety issue is far from subsiding, there is scientific consensus that food additives are indispensable in the production, processing, and marketing of many food products. Moreover, the judicious use of chemical additives-typically in the range of a few parts per million (ppm)to less than 1 % by weight of the finished food-contributes to the abundance, variety, stability, microbiological safety, flavor, and appearance of food. While food additives offer a major contribution to the palatability and appeal of a wide variety of foods, their level of use is relatively insignificantinthetotalhumandiet.Forthemostpart,thepermittedfoodadditivesaresafe, highly effective, and have been in continuous use for a long time. There is much discussion about whether a food additive or food product is natural or synthetic. The fact is that this classification, in many instances, has become somewhat arbitrary. Many food additives synthesized in chemical laboratories are also naturally occurring in normal food. Monosodium glutamate, a flavor-enhancing food additive, is the sodium salt of glutamic acid, an amino acid found i n many foods such as mushrooms and tomatoes and metabolized by the human body using the same biochemical pathways of digestion. Synthetic vitamin C (ascorbic acid) and its isomer, erythorbic acid, are the same chemicals that are found in citrus fruits. Similarly, citric acid, which is today produced commercially by enzymatic fermentation of sugars, is the same chemicallyas the naturally occurring chemical that has been found to make lemons and limes tart. Much of the worldwide public concern about the use of food additives relates to fearsaboutsafetyandhasgeneratedsomesort of regulatorystructure in every major country, as well as in international bodies, to monitor this aspect of the field. There is a Joint Experts Committeeon Food Additives, set up by the Food and Agriculture Organization and the World Health Organization, to consider the safety of additives and set specifications and limits for them. These limits take the form of an acceptable daily intake (ADI). The Codex Committee on Food Additives is required to follow the safety guidelines of the Joint Experts Committee. Its safety criteria are generallynot very different from those used in the United States, although they are not codified. In the United States, criteria for food additives are stated in 21 CFR 9170.22, “Safety factors considered,” and 21 CFR 9 170.20, “General principles for evaluating the safety of food additives.” The key sentence, which also runs through the decisions in other countries, says, “A food additive for use by man will not be granted a tolerance that will exceed I / 100‘” of the maximum amount demonstrated to be without harm to experimental animals.” apply It should be remembered, however, that in the United States, these criteria only to substances that are legally food or color additives and, by interpreting regulation, to thosesubstancesthatare GRAS on thebasis of “scientific procedures.” For those substances that are GRAS because of “experience based on common use in food,” there are no rules. Decisions of safety depend on the knowledge and judgment of the “experts qualified by scientific training and experience to evaluate its safety.” One other aspect of the safety question deserves discussion. In the United States only, there is a special provision, known as the Delaney clause, that says “no additive shall be deemed to be safe if it is found to induce cancer when ingestedby man or animal.”
Food Additives
509
This ~neansthat an additive is not to be permitted at any level, no matter how low, if it induces cancer at any level, no matter how high. The risks posed to the consumer by the food supply are rated in decreasing order of severity as follows:
1.
Microbiological hazards (food poisoning from bacteria or bacterial toxins salmonellosis, botulism, etc.). 2. Nutritional hazards (excessive consumption of sodium, saturated fat, etc.). in fish,lead from car exhaust, etc.). 3. Environmental pollutants (mercury 4. Natural toxicants (mushroom poisoning, solanine in potatoes and other solanaceous plants, shellfish toxins, etc.). 5. Pesticide residues (maximum residue levels are enforced in the United States but may exceed federal limits i n imported produce). 6. Food additives (documented cases of poisoning due to food additives are rare and were due to noncompliance with federal regulations). Although the risk to human health from food additives ranks the lowest anlong food hazards, some potential risks from food additives do exist.
A.
FoodAdditivesBanned from Use
In the United States, the FDA prohibited the use of a number of chemicals in foods for human consumption because they either present a risk to public health or have not been shown tobesafe by adequate scientific data. Table 20 lists the food additives that are presently prohibited from addition to food. Use of any of these substances causes the food to be in violation of FDA regulations. In the years since 1970, food colors, especially the synthetic dyes, have received tremendous publicity-nearly all of it bad. Color additives for food represent a unique and special category of food additives. They have historically been so considered in legislation and regulation. The current legislation governing the regulation and use of color additives i n the United States is the Food, Drug & Cosmetic Act of 1938, as amended by the Color Additives Amendment of 1960. This amendment allowed forthe provisional of scientific studies determining or temporarylisting of food colorants, pending completion the suitability of these colorants for permanent listing. Pharmacological testing of synthetic “certified” colors was initiated in 1957. Many of the synthetic colors that had been approved for use at some time i n the past have been removed from the approved list as a result of new toxicological test results. This has steadily reduced the number of certified dye colors available to the U.S. food industry from more than 22 in 1950 to 7 in 1999 (an additional color, FD&C citrus red no. 2, is permitted for coloring the skins of oranges that are not intended or used for processing, but it has not been produced in the United States i n recent years). Table 21 provides a history of the status of synthetic colorants in the United States. Another eight dyes are permitted in the EU, but are not permitted in foods in the United States. The EU works on a positive list system using EU Directive no. 95/2/EC, which is the general directive on food additives (other than colors and sweeteners that are covered in separate directives). Thislaw recognizes 106 food additives. If the additive is mentioned in the doctrine then it is allowed, if not it is forbidden. However, the directive includes a list of substances that cannot be used i n flavorings (Table 22).
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Table 20 FoodAdditivesProhibitedfromUseinHumanFood
Food additive
21CFR section Date ruling of
Calamus and its derivatives Cinnamyl anthranilate Cobaltous salts and its derivatives Coumarin Cyclamate and its derivatives Diethylpyrocarbonate (DEPC)
189.110 189.113 189.120 189.130 189.135 189.140
May 9, 1968 Oct.23,1985 Aug.12,1966 March 5 , 1954 Oct.21,1969 Aug. 2, 1972
Dulcin Monochloroacetic acid" Nordihydroguaiaretic acid (NDGA) P-4000 (5-nitro-2-n-propoxyaniline) Safrole Thiourea (thiocarbamide) Chlorofluorocarbon Flectol H ( I ,2-dihydro-2,2,4-trimethylquinoline Lead solders Mercaptoimidazoline and 2-mercaptoimidazoline 4,4'-methylenebis (2-chloroanaline)
189.145 189.155 189.165 189.175 189.180 189.190 189.191 189.220
Jan.19, 19.50 Dec.29,1941 Apr.11,1968 Jan.19,1950 Dec.3,1960 Mar. 17, 1978 Mar. 15, 1977
Flavoring compound Flavoring compound Foam stabilizer Flavoring compound High-intensity sweetener Ferment inhibitor in beverages High-intensity sweetener Preservative in beverages Antioxidant High-intensity sweetener Flavoring compound Antimycotic preservative Propellant Food packaging adhesive
189.240 189.250
June 27, 1995 Nov.30,1969
Can solder Packaging material
189.280
Dec,2,1969
Hydrogenated 4,4'-isopropylidenediphenolphosphite ester resins Tin-coated lead foil capsules for wine bottles
189.300
Feb.17,1989
189.301
Feb. 8, 1996
Packaging adhesive and polyurethane resin Antioxidant and stabilizer in vinyl chloride resins Capsule for wine cork
Functionality
-
"Pernutted i n food package adheslves wlth an accepted m~grat~on levcl up to 1 0 ppb under
4
175.105.
B. Industrial Chemicals Polychlorinated biphenyls (PCBs) and polybrominated biphenyls (PBBs) are toxic industhey trialchemicals.Because of their widespread, uncontrolled industrial applications, have become a persistent and ubiquitous contaminant in the environment. As a result, certain foods, principally those of animal and marine origin, contain PBCs and PBBs as environmental contaminants. PCBs are transmitted to the food portion (meat, milk, and eggs) of food-producing animals ingesting PCB-contaminated animal feed. In addition, a significant percentage of paper food-packaging materials contain PCBs, which may migrate to the packaged food. Therefore temporary tolerances for residues of PCBs as unavoidable contaminants are established by the FDA (21 CFR 3 109.15 and $109.30). The temporary tolerances for residues of PCBs are as follows: 1.5 ppm in milk (fat basis) 1.5 ppm in manufactured dairy products (fat basis) 3 ppm in poultry (fat basis) 0.3 ppm in eggs 0.3 ppnl in finished animal feed for food-producing animals
51 1
Food Additives Table 21 ChronologicalHistory of Certified Food Colors i n the United States Year listed for food
additive 1907 1907 1907 I907 I907 1907 I907
Nanw of certified food color
Red no. 1 Redno.2 Red no. 3
Orange no. 1 Yellow no. I Green no. 2 Blue no. 2
I959
Yellow 110. 5 Yellow no. 3 Yellow no. 4 Green 110. 1 Green no. 3 Red 110. 4 Yellow no. 6 Blue no. 1 Yellow no. 2 Orange no. 2 Red no. 32 Violct no. I Citrus red no. 2
1966 l97 I
Orange B Recl no. 40
1916 1918 1918
1922
1927 l929 I929
1929 1939
1929 1939 1950
Y car delistcd
1961 1976 .4-
I956
I959 I966 b I
1959 1959
1966 E
1976 E E
1969 1956 1956
I973
*
:%
:v :E E
2 ppm in animal-feed conlponents of animal origin, including fish meal and other by-products of marine origin and i n finished animal feed concentrates, supplements. and premixes intcnded for food-producing animals 2 ppm i n fish and shellfish (edible portion) 0.3 ppm in infant and junior foods 10 ppm in paper food-packaging material intended for use with human food or finished animal feed
C.
Food Allergies and Other Adverse Reactions to Food Additives
Although food allergy rarely constitutes a serious, life-threatening concern, it may result in chronic illness. As complete avoidance of the incriminated food is the best defense against adverse reactions. informationis of foremost importance. Food allergensidentified to date are prcdominantly proteins, although some may be polysaccharides. Despite the n d t i t u d e of additives used in foods, only a small number have been associated with advcrse reactions. Table 1.3 provides a list of the food additives that have been associated with adverse reactions.
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Table 22 EuropeanUnionList of Food Additives Prohibited for Usc in Flavorings
(Directive 95/2/EC) EU number E230 E23 1 E232 E233 E234 E235 E239 E242 E249 E250 E25 1 E252 E280 E28 I E282 E283 E284 E31 I E312 E315 E316 E579 E620 E62 1 E622 E623 E624 E625 E626 E627 E628 E629 E630 E63 1 E632 E63 3 E634 E635 E912 E9 14 E927b E950 E957 El 105
Compound Biphenyl, diphcnyl Orthophenyl phenol Sodium orthophenyl phenol Thiabendazole Nisin Nntamycin Hexamethylene tctramine Dimethyl dicarbonate Potassium nitrite Sodium nitrlte Sodium nitrate Potassium nitrate Propionic acid Sodium proprionatc Calcium proprionate Potassium proprionate Sodium tetraborate (Borax) Octyl gallate Dodecyl gallate Erythorbic acid Sodium erythorbate Ferrous gluconate Glutamic acid Monosodium glutamate Monopotassiurn glutamate Calcium diglutarnate Monoammonium glutamate Magnesium diglutalnate Guanylic acid Disodium guanylate Dipotassium guanyhte Calcium guanylate Inosinic acid Disodium inosinate Dipotassium inosinate Calcium inosinate Calcium 5’-ribonucleotides Disodium 5’-ribonucleotides Montan acid esters Oxidized polyethylene wax Caramide Acesulfame K Thaumatin Lysozy1ne
Food Additives
513
Burning of sulfur-containing coal has been used for centuries to preserve food. In addition, sulfite salts (sodium and potassium sulfite, bisulfite, or metabisulfite) are used as a sanitizing agent for fermentation containers and are added to a wide variety of food products, including dried fruits and vegetables, wine, shrimp and other seafood, and citrus beverages. Because of complaints about severe allergic reactions from asthmatic consuniers, in 1986 the FDA banned the use of sulfites in fresh cut fruits and vegetables, and sulfites must be listed on the label if a food product contains i n excess of I O ppm sulfite. The FDA estimates that about 1% of the U.S. population may be sulfite sensitive. However, among the asthmatic patient population, the sensitivity to sulfites is more prevalent, ranging from 2% to 5%. The antimicrobial food preservatives benzoate and paraben are believed to cause adverse reactions, such as asthmatic reactions i n some individuals. Benzoates occur naturally in certain berries and are usedin beverages. and their use is limited 0.1% to concentra-
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514 Table 24 SelectedFoodAdditives Derived from Allergenic Food Staples Milk proteirl rlc,ri\wti\vs
Casein Caseinates Lactose Lactitol Whey Egg protcirl &riw~fi\v.s Albumin Globulin Livetin Lysozyme Ovalbumin Soyhem deriIdw.7
Hydrolyzed soy protein Hydrolyzed vegetable protein Natural flavoring Meat flavoring (natural) Lecithin Soy protein Soy concentrate Soy isolates WllcYrf &;\~rlti\~es
Gluten Vital gluten Starch Vital gluten Vegetable gum Vital gluten Cor11 dc.ri,uti\~es
Caramel coloring Corn sweetener Citric acid Dextrin Dextran Erythritol Food starch Gellan gum Lactic acid Maltodextrine Mannitol Modified food starch, vegetable gum Sorbitol
Xanthan gum
Food Additives
515
tion. Parabens are effective antioxidantsi n low-acid products. However, they are primarily used in cosmetic and pharmaceutical products and rarely in food. They have been implicated as a cause of eczematous or contact dermatitis reactions. Butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) have been linked to adverse reactions in a small number of individuals. These antioxidants are frequently used in fats and oils and in cereal products to retard rancidity. Aspartame (L-aspartyl-L-phenylalanine methyl ester), a widely used artificial sweetener, is a dipeptide. Humans metabolizeit. Aspartame has been citedas the most frequently complained-about product. Soft drinks havebeen mentioned most often as the aspartamecontaining product, and headaches are the common reaction reported.In the United States. the FDA requires that aspartame-containing products include the following label declaration: “Phenylketonurics:containsphenylalinine.”Also, in the EU countries, thelabel declaration “contains a source of phenylaniline” is required. Monosodium glutamate (MSG) isused as a foodadditivebecause of itsflavorenhancing properties. The most commonly reported adverse reaction associated with MSG consumption is Chinese restaurant syndrome. Symptoms of the Chinese restaurant syndromeincludenausea,headache,sweating,thirst,facialflushing, and abdominalpain. These symptoms typically occur 15-30 minutes after consuming food containing a large amount of MSG. A Chinese food meal may contain from S to I O g of MSG. Among the coloring agents used in thefood industry, tartrazine(FD&C yellowno. S ) has most often been implicated as a cause of allergies, especially urticaria and asthma. Respiratory problems subsequent to tartrazine ingestion have been reported by several sources. Tartrazine produces a bright yellow color and it is used in a variety of beverages, bakedproducts,confectionaries,dessertmixes,etc.Tartrazineisalsoused to produce other food colors such as green, maroon, and rust. Lack of yellow color, therefore, is not a guarantee of tartrazine’s absence. Thus the FDA requires that FD&C yellow no. 5 be specifically stated by name on food ingredient labels. Food colorings other than tartrazine (listed in Table 23) have alsobeen implicated as causing adverse reactions in some individuals.
D. Food Additives Derived from Allergenic Food of allergic reactions. In adults, these foods include nuts, peanuts, fish, and shellfish. In children, the main culprits include eggs, milk, peanuts, soy, wheat, and fish. Elimination of these foods from the allergic individual’s diet is essential. However, many food additives are derived from these basic food items, and the allergen compound may be carried over even into highly refined derivatives. Recognition of the presence of such potentially allergenic compounds is sometimes difficult. In Table 24, selected food additives derived from allergenic natural food sources are listed. Recognition of these additives is crucial to avoid potential health hazards to consumers sensitive to certain foods.
A few foods are responsible for the majority
BIBLIOGRAPHY AT Brannen. Food Additives. New York: Marcel Dekker, 1980. Code of Federal Regulations, “Title 21“Food and Drugs. Subchapter B: Food for Human Consumption. Parts 100-199. Washington. DC: U.S. Government Printing Office, 1998.
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MDrozenandTHarrison.Structure/Function Claim forFunctionalFoodsandNutraceuticals. Nutraceut World 1 : 18-20, 1998. FJFrancis. Colorants. St. Paul, MN: American Association of Cereal Chemists, 1998. TE Furia. Handbook of Food Additives, 2nd cd. Boca Raton, FL: CRC Press. 1980. M Clicksman. Food Hydrocolloids. Boca Raton. FL: CRC Press. 1982. YH Hui. Principles nndIssuesin Nutrition. Monterey, CA: Wadworth Health Scienccs Division, 1985. Institute of Medicine. Food Chemicals Codex, 4th cd. Lancaster, PA: Tcchnomic Publishing. 1996. 1989. RJ Lewis, Sr. Food Additives Handbook. New York: Van Nostrand Rcinhold, DD M e t ~ ~ l fHA e . Sampson, RA Simon. Food Allcrgy: Adverse Reactions to Foods and Food Additives, 2nd cd. Cambridge, MA: Blackwell Science, 1996. TNagodawithanaandGReed.Enzymes i n FoodProcessing.SanDiego,CA:AcademicPress, 1993. P Newberne. GRAS flavoring substances, 18th list. Food Techno1 52(9):65-92, 1998. L O’Brien Nabors. RC Geraldi. Alternative Sweeteners. 2nd cd. New York: Marccl Dekkcr, 1991. JE Perkin. Food Allergies and Adverse Reactions. Gnithcrsburg, MD: Aspen Publishers. 1990. G Reineccious. Source Book of Flavors, 2nd ed. New York: Chapman & Hall, 1994. LD Roscnberg, LP Somogyi. The U.S. FoodIndustry in thc1990s.BIPreportD96-2033.Menlo Park, CA: SRI Consulting, 1996. LP Somogyi. Direct food additives in fruit processing. In: LP Sotnogyi. HS Rarnaswamy, YH Hui, eds. Processing Fruits, Biology, Principles. and Applications. Lanccster, PA: Tcchnornic Publishing,1996. LP Somogyi. The flavour and Fragrance industry. Chetn Ind March 4:170-173, 1996. LP Somogyi, H Janshekar. Y Ishikawa, S Bizzari. Food additives.In: Spccialty Chemicals forStratcgies and Success. Specialty Chemicals Handbook. Mcnlo Park. CA: SRI Consulting. 1996.
16 Analysis of Aquatic Contaminants
Introduction S 17 General Consitlelations 5 1 X A. Sampling 518 B. S m p l e storage 518 C. Quality :wurancc 5 18 Ill. Organics 519 A. Introduction 5 19 B. Extraction 520 C. Cleanup 522 D. Analysis of particular organics 524 E. Determination of tainting 526 IV. Inorganics and Organomctallics S26 I.
11.
Introduction 526 B. Matrix dcstruction 527 C. Analysis 527 Rel'erenccs S29
A.
1.
INTRODUCTION
A single chapter cannot provide a complete guide to the analysis of all of the comnlon contaminants of seafood; this chapter is therefore intended as an introduction to the enornlously complex matter of analyzing contaminants in food and to provide some direction for performing these analyses. The considerationsof contaminants in seafood derive from real orperceivedpublichealthconcerns.Knownpublichealthconcernsaregenerally regulated by government agencies whoset allowable limits. Such regulations are generally based on the toxicity and the safety margin of the contaminant and on the expected daily consumption of the particular food. Analysis of contaminants in seafoods is done for two reasons: (a) routine monitoring for compliance purposes (food safety), and (h) for research to determine the effects on various organisms and the expected daily dietary intakeof contaminants by humans. Routine monitoring of seafood safety is meant to identify excessive amounts of contaminants 51 7
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i n batches of seafood and requires analytical methods capable of quantitation at the regulated levels. The analytical methods used for research need to have lower detection limits than those used for monitoring of food safety, since it is necessary to quantitate contaminants not only near the regulated levels but also to quantitate existing levels of contaminants in samples in order to do statistical analysis of the data or for the calculation of expected daily intakes of contaminants.
II. GENERAL CONSIDERATIONS A.
Sampling
One of the first considerations after deciding to analyze a seafood for contaminants is to in individual organisms can vary take the samples for analysis. The contaminant levels considerably (1). Season, food availability, temperature, and other conditions contribute to the variability. It is essential to consider the purpose of the undertaking. If it is desirable to understand sources of individual variability, it is necessary to take many (20-30) Samples of individuals; if onthe other hand thedrivingreasonfortheanalysisisone of consumer health, pooled samples will provide the required answers. However, since seafoods are either from individual organisms or from groups of individuals, precautions must be taken to ensure that the samples are representative of the particular food in question. This can be done by taking pieces of individuals selected at random from a batch of the seafood (in the case of large organisms) or whole organisms (if small), grinding or otherwise homogenizing the resulting pooled sample, and retnoving subsamples of the homogenate for analysis.
B. Sample Storage Having decided on an appropriate sampling strategy, the samples must be stored so as to prevent deterioration of the food or the contaminant, or contamination of the sample during storage. Freezing is the preferred method to prevent spoilage of samples and is generally the most common storage method, but it is best to check with the analyst for conditions for the specific contaminants of interest and for suitable storage containers to prevent contamination of samples during storage (2).
C. Quality Assurance Assuring the quality of measurements is a critical concern, as it is central in determining the validity of conclusions drawn fromthe data. All aspects of analysis must be evaluated, not only the measurement of the analyte. This chapter is not concerned with the levels of contaminants in living organisms, but rather the levelsof contaminants in the organism as it is consumed or presentedto the consumer. The distinction may be trivial for persistent contaminants, but can be very important for any contaminants that are sensitive to pH or redox changes, or subject to enzymatic alteration or to redistribution postmortem. The number of samples, the method of sampling, and the handling of the samples can have considerable impact on the resulting data quality. Many laboratories are now acredited, meaning that they have proved their ability to controlthe analytical processes within their laboratory. The processes outside their facility may, however, compromise the data quality if due consideration is not given to all aspects of the analysis. A detailed account of all
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aspects of quality assurance can be foundin the text of Taylor (3) and many works dealing with detailed application of the principles (2,4-6). There are two aspects of analysis that are often confused; these are accuracy and precision. Consider, for a moment, an individual shooting at a target. The individual in question attempts to hit the bulls-eye repeatedly.If after say 10 shots the target isexamined and all of the shots fall in a small area, the shooter can be said to be precise. Notice that no mention has been made as to what part of the target was hit. If the (small) pattern of shots matches the intended mark, the bulls-eye i n this case, the shooter can be said to be accurate as well as precise. The two aspects are quite different, as are solutions to problems with either. In the case of a lack of precision, it is necessary to find the source of the variability. A problem with accuracy can be corrected by appropriate calibration of the method. Accuracy can be assured by the analysis of appropriate certified reference materials.
111. ORGANICS A.
Introduction
This is a very diverse group of compounds. Those compounds considered in this chapter include: highly volatile compounds that can impart off-flavors or taint seafoods, aromatic hydrocarbons, a number of groups of persistent industrial chemicals, of which there are thousands of individual congeners, as well as pesticides and their persistent metabolites. Some examples of groups of compounds of interest include: aromatic hydrocarbons, also referredto as polycyclicaromatichydrocarbons(PAHs),polychlorinatedbiphenyls (PCBs), polychlorinated terphenyls (PCTs), polychlorinated naphthalenes (PCNs), chlorinated dibenzo-p-dioxins (PCDDs or “dioxins”), chlorinated dibenzofurans (PCDFs or “furans”), chlorinated pesticides (e.g., DDT, mirex, toxaphene, hexachlorocyclohexane, As is evident from cyclodienes), chlorinated cthers, chlorophenols, and n-nitrosamines. this list, chlorinated compounds are the largest group; this is due to the tendencyof highly chlorinated compounds to persist in the environment and to accumulate in organisms. Brominated analogues of many persistent chlorinated compounds werealso manufactured andarefoundintheenvironmentand i n marine organisms (7-16), although inlesser amounts than the chlorinated counterparts. Those organic compounds that tend to persist in the environment, althoughthey are a very diverse group, share some common properties. They have low polarity and thus tend to be much more soluble in the lipidsof organisms than in water; as a result they tend to bioaccumulate. Although these compounds are not highly volatile, they are sufficiently volatile to vaporize from soils, particularly in warm climates, and be transported by air currents over very long distances (17-20). For example, toxaphene was reportedly the most abundant organochlorine in lake trout in the Great Lakes (21) and in Arctic fish (22), even though it was never used in the Arctic or the Great Lakes watershed. Organisms have considerable capabilityto metabolize foreign compounds and either recyclethecarbonorexcretethemetabolites.Many of thechlorinatedenvironmental contaminants are metabolizedvery slowly due to the presence of halogens or other groups that interfere with the binding of degrading enzymes with the particular contaminant. In the environment, abiotic processes such as photodegradation also play an important role in the degradation of many compounds. The compoundsthat tend to persist in the environment do so because of their chemical structure, which enables them to resist degradation
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by biological and environmental processes. As a result they accumulate in organisms and circulate through the food chain and the environment. The total complement of halogenated compounds that are extractable with organic solvent are called extractable organohalogens (EOX). These compounds canbe those containing chlorine (EOCI), bromine (EOBr), or iodine (EOI). The quantitatively predominant ones are the EOC1. Although EOCl is the group about which the most is known, 85-95% of the chlorine that is extractable is on compounds that are as yet unidentified (23,24). The 5-15% of the EOCl that is known constitutes the pesticide and industrial chemicals commonly referredto as organochlorines. Some pesticides and industrial chemicals are known to be metabolized to an oxygenated form (hydroxyl or carboxylic acid). These, being more polar than the original compound, are mostly excreted by organisms. Some of these compounds can. however, enter lipid synthesis pathways andcan be conjugated to lipids. Such compounds are more lipophilic than the parent compound; they are of larger size and are likely to be retained in body fat (25-29). only The reported accumulation of such a conjugate in humans is that of pentachlorophenol-palmitate in human body fat (30). However, since these larger compounds are of the same size as lipids (and are, in fact, lipids) theywouldberoutinelydiscarded with other troublesome lipids during sample clean-up, and it is therefore likely that they are undocumented for lack of looking rather than because of lack of presence. As a group, EOCl is no doubt the largest group of organic contaminants in marine organisms, as well as being the least understood. Although there are about 2500 documented naturally produced halogenated compounds (3l ) , there are no naturally produced organohalogens that bioacculnulated or are knownto be persistent environmental contaminants. Some fractions of lipids containing high levels of EOCl are known to be toxic in animal assays (32), and as a result it seems prudent that these materials should be investigated further. N-nitroso compounds are a major group of chemical carcinogens (33). Although they are not found in living fish or shellfish, they can be formed during processing, storage, or cooking and may therefore be present in seafood as it is consumed.
B. Extraction Most seafood samples contain organic contaminants, at microgram per gram or less levels, in a complex matrixof proteins, lipids, water, and minerals. Modern instruments generally havesufficientsensitivitytodetectevenlowerlevelsofcontaminants,butthematrix components cannotbe introduced into analytical instruments and as a result it is necessary to extract the organic contaminants from the matrix and to further remove any undesirable components (cleanup) prior to analysis in order to prevent interference with the analysis. Highly volatile contaminants, such as the odoriferous compounds that can cause tainting, can be removed from seafood by steam distillation (34,35), or by purging a macerated sample with aninertgasandtrapping the contaminantsforanalysis (36,37). Another approach is that of headspace analysis (38) in which a macerated sample is sealed in a vial, heated to volatilize the contaminants of interest, and vapor in the headspacc gas is analyzed by gas chromatography or other means. For all other types of organic contaminants, an extraction with solvent (generally a nonpolar one) must be used. Many different solvent systems havebeen reported, ranging from supercritical carbon dioxide to the more common petroleum ether, hexane and hexane-acetone, and hexane dichloromethane mixtures (39-41). Different solvent systems are favored for different groups of contaminants
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due not only to the need to extract the particular contaminant reliably but also to prevent breakdown of the contaminants (41-43). Whatever the solvent, extraction can be accomplished only if the sample is suitably macerated. This is generally accomplishedby grindingthe sample first or by blending the sampledirectly in theextractionsolvent.The blending not only macerates the samplebut also provides energyto speed up the extraction process. An alternative procedure is cold column extraction in which a macerated sample is mixed with anhydrous sodium sulfate (to remove water), packedin a glass column, and extracted with solvent passed down the column. Methods of solvent extraction of aromatic hydrocarbons havebeen reviewed by Dunn (44), Griest and Caton (45), Kiceniuk and Holoubek (41), Vassilaros et al. (46), and Zitko (47). Themost commonly used procedure for the extraction of these compounds from seafood is alkaline hydrolysis of the sample followed by extraction with a nonpolar solvent such as hexane, pentane, isooctane, cyclohexane, diethyl ether, benzene, or a mixture of solvent (41). Dichloromethane is a good solvent for aromatic hydrocarbons and has the added advantage that it is less prone to the formation of stable emulsions than solvents such as diethyl ether (41). Emulsion formation causes analytes to be lost during subsequent steps, thus increasing variability in the analysis. The formation of emulsions can be reduced by acidification (48) and phase separation can be improved by addition of salt (46). The sample can be ground with anhydrous sodium sulfate and column extractedwithmethyl-t-butylether/dichloromethane(49)orwithdichloromethane in a blender or tumbler (50,51). Other methods such as Soxhlet extraction and various combinations of methods are also used (41). Phenols and halogenated phenols are found in tissues in the free form as well as conjugates of sulfate, glucuronide, and fatty acids (30). The sulfate esters and glucuronides are water soluble, whereas the fatty acid conjugates are lipophilic. Therefore in order to extract all of the phenols it is first necessary to hydrolyze the conjugates prior to solvent extraction or steam distillation. The procedures for the removal and analysis of phenols are reviewed by Benvenue and Beckman (52), and Dougherty (53). The most conmon methods for hydrolysis of phenolic conjugates reported in recent literature are those using enzymes (53-55). Acid hydrolysis using mineral acids, such as 10% sulfuric acid, has been found to be equally successful (56.57). After deconjugation the phenolic compounds are removed equally effectively by either steam distillation or solvent extraction (53,56). Numerous solvent systems have been used for the extraction of chlorinated ethers (58). The methods used are similar to those for the extraction of polychlorinated dibenzop-dioxins and polychlorinated dibenzofurans (58). The tissue is first homogenized with sodium sulfate followed by Soxhlet extraction with a mixture of hexane/acetone/diethyl ether/petroleum ether (59-62) or, alternatively, the homogenate is packed in a glass column and extracted with dichloromethane (63-65). Alkaline hydrolysis and other methods are also used (58). There are about 750 pesticides, metabolites, and organic impurities that are commonly monitored in food (40). Dueto the similar (lipophilic) natureof these residues there has been a trend to thedevelopment of so-called multiresidue methods for the extractionof groups of similar compounds. Residues can be extracted from tissue by any of the methods previouslydiscussedforremoval of other lipophilic materials. Allextractionmethods coextract lipids which must be removed prior to analysis of most residues (see cleanup). One recently developed method, called matrix solid-phase dispersion (MSPD), combines extraction and clean-up into one procedure. In the MSPD procedure the tissue sample is blended with octadecyl derivatized silica (C,J, transferred to a column prepackcd with
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Florid, and the residues are eluted with methanol or acetonitrile (66-68). There are presently about five methods in use for the isolation and subsequent detection of 200 residues (40). Dibenzofurans are unstable at high pH, therefore alkaline hydrolysis of samples to break down the matrix is of limited use in the extraction of chlorinated furans and dioxins (43). Acid digestion with hydrochloric or sulfuric acid is preferred for matrix breakdown. Often the acid digestion is combined with a solvent such as hexane, pentane, or toluene to extract the dioxins and furans at the same time as doing the digestion (69). Seafood by mixing with having a low fat content can be freeze dried and subsequently extracted ether/acetone/hexane/diethyl dichloromethane(70).Soxhletextractionwithpetroleum ether has been used to extract dehydrated fish holnogenate as well (7 1 ). Homogenate mixed with sodium sulfate can also be packed in a column on top of material used for cleanup, thus combining theextractionandfirststage of cleanup (72). For a detailed review of procedures for the extraction of dioxins and furans see Clement et al. (69). Polychlorinated terphenyls and naphthalenes can be extracted from homogenized tissue with a variety of cold solvents (73-75). Steam distillation and extraction of condensate with hexane (76,77), Soxhlet extraction(78,791, and acid (80) or base (80-82) digestion followed by solvent extraction are also used (75). The extraction of polychlorinated biphenyls (PCBs), has been reviewed by Metcalfe (83). Cold column extraction methods have lower recoveries of PCBs than the more popular Soxhlet methods (84). Saponification followed by extraction with isooctane was used for isolation of PCBs from lobster tissue (85). Extraction by blending with a solvent is also used (83). Supercritical fluid extraction methods have been developed (86,871 and will probably become more prevalent. Volatile nitrosamines are extracted from homogenized food by a multistage extraction using ammonium sulfamate in water, followed by dichlorolnethane (33). For extraction of nonvolatilenitrosaminesthehomogenizedfoodsarnpleismixedwithCelite, packed in a glass column, and extracted with pentane followed by ethyl acetate. The first 225 ml of ethyl acetate eluate is discarded and the last 300 nd is used for analysis ( 3 3 ) . Extractable organohalogens are extracted from tissue by blending with combinations of solvent suchas hexane/isopropanol(50/50)(32,88), acetonelhexane (2/1) (24) acetone/ cyclohexane (2/1) (89). The extraction is repeated, the solvent layers pooled and evaporated, and the solvent exchangedto hexane (24) or cyclohexane(89). The resulting extract is then washed with distilled water (24,88,89) acid, or salt solutions (32,90) to remove halides prior to neutron activation analysis of the organic phase for chlorine, bromine, and iodine.
C. Cleanup Methods for the cleanup of extracts prior to analysis of organic residues can be grouped according to the following mechanisms: Separation by molecular size Gel permeation Dialysis Absorption Liquid-liquid partition Chemical degradation of lipids
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Often a number of these lnechanisrns are required to remove all of the interfering materials. Since lipids form the bulk of the material extracted from tissue, they are usually removed first. Most lipids have molecular weights of 500-1500, whereas the contaminants con.1Inonly analyzed are smaller. Gel permeation chromatography and dialysis methods separate moleculesby size. Bothof these methods can handle the large amount of lipids present in Some seafood and provide nearly complete recovery of the contaminants. Gel permeation can by automated readily and is a common method in analytical laboratories. Dialysis, using polyethylene membrane in organic solvent, is slower than gel permeation chromatography but uses much less solventto fractionate the same amount of material. Dialysis has been used for the preparation of large amounts of COlltaminant fraction for tOXiCOlOgical work (91) as well as for analysis (92-94). Sterols present in many seafood samples will pass through the membrane and mustbe removed prior to most analytical procedures (91). Adsorption chromatography is a commonly used method for cleanup of more than 400 contaminants in food (95), including organochlorine compounds (75) and aromatic hydrocarbons (41). Chemical degradation of lipids by the addition of concentrated sulfuric acid to extracts in hexane is a simple and time-proven method of cleanup of extracts for the analysis of organochlorine compounds such as PCBs and DDTs. The recoveryof compounds after sulfuric acid cleanup has been examined in detail for 39 organochlorine contaminants a Florisil cleanup to that of the sulfuric (96). The same study compared recoveries for acid method fora variety of contaminantsin certified reference materials, including lyophilized fish, and reported comparable results for organochlorines (96). Dioxins and furans are amongthe most toxic contaminants and are foundin seafood at several orders of magnitude lower levels than other organic contaminants. The very low levels of these contaminants, together with the fact that many compounds interfere with the detection and quantitation of dioxins and furans, requires that not only lipids but also the interferents be removed priorto analysis. Lipids constitute the bulkof the material in the extract and are removed by gel permeation chromatography, adsorption on silica gel, or by washing the extract with concentrated sulfuric acid (69). The rigorous removal of interferents prior to analysis is a time-consuming but essential process. Such a cleanup requires from three to five columns (69), depending on the procedure used. For a detailed comparison of methods and a complete review of dioxin and furan analysis see Clement et al. (69). After lipid removal by gel permeation or other means, polychlorinated naphthalenes can be further cleanedup on charcoal (78) or charcoal on polyurethane foam (75) columns to remove compounds such as nonplanar PCBs and other compounds that interfere with the analysis of polychlorinated naphthalenes. Extracts for the analysis of polychlorinated terphenyls can be cleaned up on a silica cartridge with n-hexane (97). Methods for removal of lipids and organochlorine interferents from extracts prior to analysis of PCBs have been reviewed by Erickson (73) and Metcalfe (83). Gel permeation is perhaps the most widely used initial cleanup procedure. Sulfuric acid treatment following GPC has been reported for congener-specific analysis of PCBs in lobster hepatopancreas tissue (85). Methods for the cleanupof samples for analysisof chlorinated pesticides have been reviewed (40,98). Because of the very large number of compounds in this category there are a wide variety of methods for cleanup of samples prior to analysis. Since, i n most cases, a number of pesticides are of interest (multiresidue methods), sample cleanup and initial fractionation are combined to provide fractions such as PCBs and DDE, toxaphene
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and DDT, etc. The initial fractionation is done on columns such as silicic acid, alumina. or Florisil (40,98). Depending on the particular application, the initial fractions may require further cleanup stepsby different columns or other separation methods. For example, nitration has been used to remove compounds other than toxaphene in the analysis of samples for the toxaphene group of compounds (98-100).
D. Analysis of Particular Organics Nitrosamine compounds were first determined (in the 1960s) by thin-layer chromatography (101,102), and by gas chromatography (GC) usingan electrolytic collductivity detector (103). Alkali flame ionization and the more selective Coulson electrolytic conductivity detectors were used in the early to mid-1970s (104). The thennal energy analyzer (TEA), which was developed specifically forthe analysis of N-nitroso compounds, provides high sensitivityandselectivityforthedeterminationofnitrosaminesand is thedetector of choice. In the TEA breaks, N-NO bonds to produce a nitrosyl radical, which is then reacted with ozone to produce exited nitrogen dioxide. Nitrogen dioxide emits therlnal energy (33) which can be detected and measured. This detector canbeused for GC or high performance liquid chromatography (33). Extractableorganohalogensareanalyzed by neutronactivationanalysis.This method detects all chlorine, bromine, and iodine regardless of the chemical bonding state of these elements and, sincethe analysis is done on organic extracts from which all halides have been removed, it measures all of the organohalogen present i n the extract. The sanv ples, typically about 750 p1 of extract or comparator standards, are irradiatedin a neutron fluxto produceradionuclides of the halogens (activation). The irradiated samples are opened in a fume hood and a 500 p1 portion of the contents is transferred to a fresh 1.2 1111 polyethylene vial to eliminate theneed for a vial blank. The new sample vial and contents are then decayed for 5 min and counted for 30 min. The 443 keV, 617 keV, and 1642 keVpeaks of ‘?‘I, ‘“Br, and 3xC1areusedtoquantitatetheiodine, bromine, and chlorine content of the samples. The absolute detection limits are typically 3 ng for iodine, 6 ng for bromine, and 30 ng for chlorine (89). In order to determine individual organic contaminant compounds it is necessary to either use a detection and quantitation method that is highly specific and not subject to interference from other materials or to first separate the individual compounds and then quantitate them. The only detection methods that can be used to identify and quantitate individualcompounds in mixturesareShpol’skiifluorometryfor the determination of some of the aromatic hydrocarbons (41,105,106) and immunoassay. Immunoassay uses antibodies that are specific for a particular compound to quantitate the compound in question. Methods of immunoassay are available for a variety of contaminants, and in some cases provide sensitivity comparableto mass spectrometric methods (107). This methodology has considerable potential for automation and more widespread use particularly for initial screening of large numbers of samples. For all other detection methods, the components must first be separated. The separation process is generally started during cleanup with silica, Florisil, or various combinations of columns. Each fraction from such a column or combination of columns contains a large number of contaminants of similar properties. The chromatographic method that provides the highest resolution of organic compounds is gas-liquid chromatography using wall-coated open tubular columns. The “capillary” columns used for separation of chlorinated pesticides and chemically similar industrial chemicals are
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typically 10-60 In long and 0.2-0.32 mm inside diameter coated with a moderately polar material to a film thickness of about 0.25 pm. The specific column types used for PCBs have been reviewedby Metcalfe (83). Clementet al. (69) have reviewed the gas chromato(58) reviewed the analygraphic separation of dioxins and furans. Paasivirta and Koistinen sis of chlorinated ethers. Columns for the separation of aromatic hydrocarbons have also been reviewed (41), as have those for the analysis of phenols and chlorophenols (53), n-nitrosamines (33), polychlorinated terphenyls and chlorinated naphthalenes (75), and chlorinated pesticides (40). The detector types used for the detection and quantitation of chlorinated pesticides and other compounds are electron capture detectors, electrolytic conductivity detectors, atomic emission detectors, mass spectrometry, and tandem mass spectrometry (mshns). Electron capture and electrolytic conductivity detectors are sensitive to contamination and fouling. They have linearity and overload problems as well, and are unable to discriminate among the halogens (40). These detectors are, however, cheaper than mass spectrometers and remain the methods of choice for routine monitoringof chlorinated pesticides i n large numbers of samples. Atomic emission detectors can detect various elements of interest, but largely due to their expense, havenot gained wide use.Mass spectrometry has become the method of choice for analysis of organic contaminants. This is due to the ability of mass spectrometry to not only quantitate the individual compounds after separation by high-resolution gas chromatography but also to confirm the identification of the compound. When usedinthenegativeion chemical ionization mode, mass spectrometry provides extremely high sensitivity for polychlorinated aromatic compounds while being insensitive to other potentially interfering compounds (74,78). The useof mass spectrometry for the determination of dioxins and furans has been reviewed (108,109). Low-resolution mass spectrometers are often used for screening of samples for high levels of dioxins and furans. When more critical measurements have to be made, however, the more expensive highto resolution machines must be used to obtain the lower detection limits necessary, and provide more freedom from interferences(75). Tandem mass spectrometry provides about I O times higher detection limits than high-resolution mass spectrometry but is capable of quantitation of dioxins and furansin samples with less stringent cleanup than that required forhigh-resolutionmachines (75). Iontrap mass spectrometryhas also beenused for analysis of chlorinated pesticides ( 1 IO, 1 1 1) and polycyclic aromatic hydrocarbons (1 12). High-performance liquid chromatography (HPLC) as well as high-resolution gasliquid chromatography are used for the separation and analysis of aromatic hydrocarbons (41).The detectors used for determination of aromatics by HPLC are ultraviolet absorption detectors or fluorescence detectors. With gas chromatography, flame ionization detectors may be used for routine screening of samples. Most determinations of aromatics are now done by gas chromatography (GC) with some type of mass spectrometric detection (41). Single-ion monitoring (SIM) can be used to filter out interfering spectra and focus on the determination of specific compounds, thus increasing the peak response ( 1 13). The GCSIM parameters for a large number of aromatic hydrocarbons and heterocyclic aromatic hydrocarbons have been published ( 1 13,114) and together with retention indices can be used for the routine identification of these compounds (41). Toxaphene is the trade name for an insecticide that was produced starting in the mid-1940s by the chlorination of camphene. This process results in a mixture consisting of some 200 congeners, many of which have enantiomers (1 15). About half a million metric tons of toxaphene were produced ( 1 16) and presumably used and released to the environment. Although its use was banned in the United States in 1982 (98). its use has
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continued in some countries. Many of the compounds in toxaphene are highly persistent, and even in remote parts of the northern hemisphere, such as the Arctic and the Atlantic, toxaphene is the most or second most common organochlorine contaminant (22,116). The of the analysis of individual compounds in toxaphene is so complex thattheanalysis enantiomers of some of the chiral forms was first reported in 1994 ( 1 1 S).
E. Determination of Tainting Since tainting is by definition “the presence of an objectionable odor or flavor in a food,” it is based on human perception (of what is objectionable). Tainting in the strict sense must therefore be determined by human senses. This is doneby presenting known (clean) samples as well as suspected samples to groups of individuals in one of a number of presentation formats and asking the individuals to evaluate them (taste, smell, or both). The results are then tallied, evaluated, and compared to statistical tables. For a detailed examination of the determination of tainting see Botta ( 1 17). Tainting substances canbe accumulated by organisms and remain in subsequent products or they may originate from packaging materials or other process-related sources ( 1 18). Many types of compounds have been shown to impart undesirable odors or flavors to seafood ( 1 18- 12 1 ). In those cases where a particular compound is identified as a cause of tainting it can be analyzed by instrumental methods. Since these are rather volatile compounds, they can be isolated by steam distillation, purging, or direct headspace analysis. Separation is generally by gas chromatography, with detection by flame ionization or sniffing of the components as they come off the column. For a detailed account of the determination of oderiferous materials, see the review by Karahadian and Lindsay (35). A semiconductor sensor array instrument is now on the market for the characterization of volatile molecules (122,123). Although this instrument is primarily sensitive to polar compounds like the spoilage products of seafood, other sensor arrays maybe developed that will be capable of quantitating other materials, such as tainting compounds.
IV.
INORGANICS AND ORGANOMETALLICS
A.
Introduction
The elements of general interest as contaminants are cadmium, selenium, lead, mercury, and arsenic. Selenium is an essential trace element, but is toxic at high doses. The margin between nutritional levels and toxicity is small( 1 24,125). In seafood, selenium is of interest not only for its potential toxicity but also for its protective role against arsenic, cadmium, and mercury toxicity ( 124,126). Four oxidation statesof selenium have been documented in nature, as well as dimethyl and diethyl organic forms (124). The species of selenium differ in their absorptive ability ( 127) and in their ability to protect against mercury toxicity ( 1 24). It is therefore necessary to analyze not only the total selenium but to also measure the individual species in some cases. All aspects of the analysis of selenium are reviewed by Cappon (124). Mercury is a relatively rare element on earth, but is ubiquitous in the environment due to the volatility of elemental mercury and to pollution. Since mercury is present in seafood mostly in organic form, and mostly as methylmercury (the most toxic form), many investigations of mercury in seafood determine total mercury and assume that all mercury is methylmercury (the worst-case scenario). Theidentification of the specific form (specia-
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B. Matrix Destruction Most analytical methods for the determination of metals require that the sample be in liquid form; therefore the matrix of seafood must be destroyed or otherwise solubilized prior to analysis. Methods such as neutron activation analysis of freeze-dried ( I 34,135) or homogenized samples are done routinely, and energy dispersive X-ray fluorescence spectrometry can be done on freeze-dried samples ( 1 36). Some high-temperature analytical methods such as inductively coupled plasma or flame atomic absorbance spectrometry Hocan tolerate incomplete sample digestion (as long as the material is homogeneous). mogenized slurries of food material can be analyzed by atomic absorbance spectrometry ( 137). For the other methods of analysis the matrix is destroyed by acid digestion or ashing to provide a soluble material. Voltometry or spectrophotometry methods require more complete digestion of samples ( 1 38). Dry ashing consists of heating the sample in the presence of air to first dry it out and then to oxidize the dried material. The remaining ash is then dissolved and analyzed. This method cannot be used for the preparation of samples for mercury analysis, as most forms of mercury are volatile enough to be lost. Cadmium, aluminum, selenium, and lead chloride are also volatile and require precautions to prevent the formation and loss of halides (2). For a review of digestion procedures see Taylor et al. ( 137) and Novozamsky et al. (138).
C.
Analysis
All aspects of methods for the analysis of mercury ( 1 39), cadmium ( 1 28), lead (140), arsenic (141 ), selenium ( I 24), and tin (142) have been reviewed (2). Recently, atomic spectrometry methods for the analysis of all metals and elements of interest have been reviewed ( 137). 1. Total Content Most of the samples analyzed for metals are analyzed for the total metal content of the particular metal. The analytical methods for total metal content can be grouped into two catagories: direct analysis and analysis of a digested sample. The predominant method of direct analysis (homogenized freeze-dried sample)of elements is neutron activation analysis (NAA). NAA has a number of advantages over other methods: (a) analysis can be done without physical destruction of the sample; (b) reagent blanks are not needed; (c) the potential for contamination can be reduced by minimal preirradiation handling; (d) the process is virtually free from matrix interferences; (e) solids, liquids, or gasses can be analyzed; ( f ) qualitative information is obtained as well as quantitative analysis; (g)
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there is multielement specificity; (h) almost all of the elements can be determined; (i) excellentsensitivityanddetectionlimitsareattainable; ( j ) thereishighprecisionand accuracy, with (k) extensive linear range;(l) the cost is reasonable; (m)total analysis time is short, and (n) fully automated systems are available. The most important advantage is that the chemical state of the element does not influence the analysis (143; Chatt, personal communication). Because of these advantages, NAA has been used for the analysis of foods ( 1 34,144) and for the certification of trace elements in reference materials ( 1 34,135). If the element of interest is present at less than the detection limits or there is interference fromotherelements,theelement of interestcanbeseparatedoutbyradiochemical ( 134,145) or preconcentration methods. There are a variety of methods for the analysis of contaminant elements that normally require digested samples. Mostof the methods involve atomic spectrometry, electrochemical methods, or a combination of gas or HPLC with a detector for metals such as atomicabsorbancespectrometry,inductivelycoupledplasma,ormicrowave-induced plasma emission spectrometry.It has been argued that among the presently available methods for the analysis of trace elements (e.g., contaminants) in biological material, the most sensitive, fast, selective, and precise multielement method is inductively coupled plasma mass spectrometry (ICP-MS) (146). However, the special consideration required during sample preparation for the analysis for a particular element can effectively reduce the capability to analyze multiple elements using the same sample preparation. This may account for the few articles using ICP-MS for the analysisof seafoods ( 137). The less expensivegraphitefurnace(GFAAS)orelectrothermalatomicabsorptionspectrometers (ETAAS) are among the most popular methods for analyzing total metal contaminants in seafood, particularly selenium ( 124), cadmium ( 1281, and lead ( 140).
2. Speciation Organometallic compounds tend topartitionintolipids of organisms andthustendto accumulate in organisms i n a manner similar to other organic contaminants. Analytically speaking, organometallics are often detected and analyzed by the metals they contain. Some knowledge of speciation of metals and arsenic is necessary in order to interpret the toxicologic risk of levels of these elements in a particular seafoc-id (141). For example, mercury is often present mostly as the most toxic form (methylmercury) ( 1 39). while arsenic is often i n the form of arsenobetaine, which is relatively nontoxic (141). In addition, although selenium counteracts the toxicity of mercury, the species of selenium have different absorption and protective abilities (124). Speciation analyses are done by extraction, separation, or a combination of both, followed by detection with an element-specific detector. The most prevalent detection methods for contaminant elements are atomic absorbance spectrometry (AAS) and inductively coupled plasma (ICP). Selenium species have been separated by both ion exchange chromatography and ion pairing chromatography followed by ICP-MS ( 137) or flame atomic absorption spectrometry ( 125). Selective hydride generation has also been used to determine selenium species i n biological material ( I 24). For detailed reviews of methods for the analysis of selenium species, see Cappon ( 124) and Taylor et al. ( 137). Arsenic compounds rangei n polarity from water soluble to lipid soluble; as a result, a number of solvent combinations must be used to recover the various arsenic species. Separation of arsenic compounds can be done usingthinlayer chromatography, highperformance ion exchange chromatography, or a combination of methods (141). Detection
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of the species can be by atomic absorption spectrometry, atonic emission spectrometry, 01' ICP-MS (141). Methods for extraction of organomercurials have been reviewed (139,147-150). The earlier procedures generally used dilute acidto liberate protein-bound organic mercury compounds, a halide to promote the formation of halide derivatives, and extraction with an organic solvent such as benzene or toluene. The organomercurials werethen converted to water-soluble thiosulfate complexes and back-extracted from the organic phase with water,leavingnonmecurialorganicsbehind i n theorganicphase.Thecoextraction of lipids tends to form emulsions which are difficult to break and lead to losses of analyte. Extraction of lipids with acetone before the extraction of organomercurials was reported by Hight and Cocoran (15 I ) . A simplified method for the extraction of methyl- and ethylnlercury from tissues has been reported (152) and Inay be applicable to seafood samples. This method uses thiosulfate complexationwith no prior acidification, bromide treatment, and solvent extraction. Another method that has been used for the analysis of methylmercury i n seafood uses sulfuric acid and iodoacetic acid to release the mercury and convert it to a volatile form which can be determined by gas chromatography ( 153). This method is carried out with the sanlples contained in headspace vials and the procedure can be semiautomated to reduce labor costs. A supercritical fluid extraction method hasalso been reported ( 1 54).
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126. ARuiter.Contaminants in fish.In: A Ruiter, ed. FishandFisheryProducts.Wallingford: CAB International, 1995. p. 26 1. ;ind in vitro bioavailability of selenium 127. LH Shen, H van Nieuwenhuizen. JB Luten. Speciation in fishery products. In: JB Luten, T B~rresen,J Oehlenschliiger. eds. Seafood from Producer to Consumer, Integrated Approach to Quality. Atnsterdam: Elsevier. 1995, p. 653. 128. S Ray. Cadmium. In: JW Kicenium,S Ray, eds. Analysis of Contaminants in Edible Aquatic 1994, p. 91. Resources. New York: VCH Publishers, Inc.. 129. W Salomons, U Fiirstner. Metals i n the Hydrocycle. Berlin: Springer-Verlag. 1984. pp. 212287. 130. JF Uthe, DP Scott. CL Chou. Cadmiutn contaminationin American lobster, Hotnarus americanus, near a coastal lend smelter: use of multiple linear regression for management. Bull Environ Contam Toxicol 38:687, 1987. 131. JF Uthe, CL Chou. Cadmium in sea scallop (Placopecten magcllanicus) tissues from clean I , 1987. and contaminated areas. Can J Fish Aquat Sci 44:9 132. JF Uthe, CL Chou. DH Loring, RTT Rantala, JM Bewers, J Dalxiel, PA Yeats. R Levaque Charron. Effect of waste treatment at a lead smelter on cadmium levels i n American lobster (Hotnarus americanus). sediments and seawater in the adjacent coastal zone. Mar Pollut Bull 17:l 18, 1986. 133. DA Wright, PM Welbourn. Cadmium in the aquatic environment: a review of ecological, physiological, and toxicological effects on biota. Environ Rev 2: 187. 1994. 134. SJ Parry. Biomedical applications. In: Activation Spectrometry in Chemical Analysis. New York: John Wiley & Sons. 1991, p. 163. 135. RM Parr. On the role of neutron activation analysis in the certification of a new reference material for trace-elenlent studies, mixedhuman diet, H-9. J Radioanal Nucl Chetn 123:259, 1988. 136. TH Nguyen, J Boman, M Leermakers, W Bacyens. Mercury analysis in environmental samJ Anal Chern 360:199, 1998. ples by EDXRF and CV-AAS. Frcsenius
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137. A Taylor, S Branch, H Crews, DJ Halls, LMW Owen, M White. Atomic spectrometry update-clinical and biological materials, food and beverages. J Anal Atom Spectrom 12:1 19R. 1997. 138. I Novozamsky. HJ van der Lee, VJG Houba. Sample digestion procedures for trace element determination. Mikrochim Acta 1 19: 183, 1995. 139. CJ Cappon. Mercury and organomercurials. In: JW Kiceniuk, S Ray, eds. Analysis of Conp . 175. taminants in Edible Aquatic Resources. New York: VCH Publishers. Inc.. 1994, 140.RLobinski,FCAdams.Leadandorganoleadcompounds.In: JW Kiccniuk, S Ray. eds. Analysis of Contaminants in Edible Aquatic Resources. NewYork:VCH Publishers. Inc., 1994, p. 1 15. 141. Y Shibata. M Morita. Arsenic and organoarsenicals. In: JW Kiccniuk, S Ray, eds. Analysis of Contaminants in Edible Aquatic Resources. New York: VCH Publishers. Inc.. 1993, p. 159. 142. RE Sturgeon. KWM Siu. Tin and organotin. In: JW Kiccniuk, S Ray. eds. Analysis of Contaminants in Aquatic Resources. New York: VCH Publishers, Inc.. 1994. p. 225. 143. WD Ehtnann, DE Vance.Nuclearactivationanalysis.In:WDEhmann,DEVance,cds. & Sons. 1991. Radiochemistry and Nuclear Methods of Analysis. New York: John Wiley p. 253. 144. WC Cunningham, WB Stroube Jr. Application of an instrumental neutron activation analysis procedure to analysis of food. Sci Total Environ 63:29. 1987. 145. I Bayat, ND Raufi. M Nejat. Determination of mercury and other toxic elements i n fish and of foodstuffs using destructive neutron activation analysis. In: Health-Related Monitoring Trace Element Pollutants Using Nuclear Techniques. Vienna: International Atomic Energy Agency. 1985. p.141. 146. CK Mathews Trace toxic elements in nutritional health. In: Proceedings of the International Conference of Health and Diseases: Effects of Essential and Toxic Trace Elements, 4th ed. New Delhi: Wiley Eastern, 1995. p. 43 I . 147.JARodriguez-Vasqucz.Agaschromatographicdctcrtnination of organomercury (11) compounds. A mini review. Talanta 25:299, 1978. 148. AK Shirvastran, SG Tandon. The determination of mercury: a mini-review. Toxicol Environ Chem 5 3 1 I . 1982. In: DFSNatusch, PK Hopke.eds.AnalyticalAspectsof 149.RSBraman.Chemicalspeciation. & Sons. 1983, p. I . AnalyticalChemistry.NewYork:JohnWiley 150. CJCappon.GLCspeciation of selectedtraceelements.LC-GC5:400.1987. 151. S Hight.MCocoran.Rapiddetermination of methylmercury infishandshellfish:method development. J Assoc Anal Chetn 70:24. 1987. 152. AGFBrooks,EBailey,RT Snowden. Determinationofmethyl-andethylmercury in rat blood and tissue samples by capillary gas chromatography with electron-capture detection. J Chromatogr 374:289, 1993. 153. P Larsen, W Baeyens. Improvement of the semiautotnatated headspacc analysis method for the determination of methylmercury i n biological samples. Anal Chitn Acta 228:93, 1990. 154. W Holak. Determination of methylmercury after supercritical fluid extraction. J AOAC Int 78:l 124. 1995.
17 Agricultural Chemicals Debra L. Browning and Carl K. Winter U t ~ i w r s i hof CtrlIfi)r-r~itr-Da\~is, Davis. Califontin
Introduction 537 History A. 538 TI. PesticideTypes539 A. Insecticides 539 B. Herbicides 540 C. Fungicides 541 111. PesticideUsageLevels541 Regulations 543 IV. Pesticide 544 V. PesticideResidueLimits of 1996546 VI. FoodQualityProtectionAct VII. International Regulations 547 VIII. MonitoringandEnforcement547 A. U.S. Department of Agrlculture 549 B. State programs 550 Risks 551 IX. Dietary X. NondietaryRisks 553 XI. Conclusions 554 References 554 I.
1.
INTRODUCTION
“Pesticide” is a generic term for a variety of agents that control pests. Insecticides, herbicides, and fungicides are all commonly known pesticides, but less well known types includemolluscicides,bacteriocides,nematicides,scabicides,pheromones(attractants), plant growth regulators, acaricides, and repellants. Traditionally, pesticides have been used i n agriculture to maximize yield and minimize crop loss to pests. Pesticides can be applied before planting a crop to protect seeds/ seedlings, sterilize soil, protect roots, and kill competing weeds; during growth to mini-
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mize unwanted pests; and after harvesting to minimize damage from rodent, insect, and fungal populations (1 ). It has been estimated that the average time i t takes a farmer to tend and harvest I acre of cropland has decreased from 56 hoursin I830 to 2 hours today ( 1 ). Lower maintenance and increased crop yields have encouraged farmers to apply nlore pesticides. It has been estimated that 40% of the world's food supply would be at risk without pesticides. In developed countries, economic benefits may range from $3.50to $5.00 for every $ 1 .OO spent on pesticides (2). The United States accounts for nearly one-third of the pesticide user expenditures worldwide and one-fifth of the pesticide usage worldwide. The United States uses 4.5 billion pounds of pesticides i n a typical year (of which I .2 billion pounds are used in agriculture) at a user cost of $1 1.3 billion ($7.9 billion on agricultural pesticides) (3).
A.
History
Humans have used pesticides to control invertebrate, vertebrate, and microorganism infestations since as early as 1000 B.c., when the Chinese used sulfur as a fungicide against powdery mildew on fruit ( l ) . Sulfur remains an important pesticide today (79-89 million pounds used in the United States in 1995) (3).Arsenical compounds were popular insecticides in the 16th century, while nicotine, rotenone, and chrysanthemum extract have all been used as insecticides since the 17th century and are still used today ( l). AstheUnitedStatestransitionedfromhorsepower to mechanizedpower i n the 1920s, farm production increased and the use of farm labor decreased. The small and medium-sized farms of the Midwest could not typically afford nor get capital to exploit the advantagesof size; subsequently onlythe large farms survived. The large farms became moreheavilydependent on pesticidesandmechanizedpower to maintainproduction. Heavy use of arsenic pesticides led to the beginnings of consumer concern in this country regarding the use of pesticides on agricultural crops (4). The 1938 creationof the Federal Food, Drug and Cosmetic Act (FFDCA) and subsequent amendments established the requirement for pesticide tolerances to be established when pesticide use could result in residues on food or feed crops. In 1947 the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA) was passed by Congress. I t grouped all pesticide products under one law and mandated labeling and registration requirements. The law was amended in 1975, 1978, 1980, 1984, 1988, and 1996. Additional requirements under these amendments included Appropriate chemical, toxicological. and environmental i n p c t studies Label specification Use restrictions Responsibility to monitor pesticide residue levels i n foods Pesticide reevaluation and reregistration Special consideration for the sensitivity and exposure of infants and children Consideration of aggregate risk from food, water, and domestic exposure Cumulative risks from pesticides possessing a common mechanism of toxicity Despite government intervention and regulation of pesticide use, pesticide residues
in food continue to generate significant societal concern. I n a national consunler attitude survey conducted annually over the past decade, between 72% and 82% of U.S. consumers considered pesticide residuesto be a serious hazard ( S ) . Contributing to the public's aware-
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ness and concern over pesticide residues are several widely publicized incidents and reports. These include an incident in which the insecticide aldicarb was used illegally on watermelons in California in 1985 and resulted in morethan 1000 suspected cases of human illness (6). Other notable events include areport released by the Natural Resources Defense Council and widely covered by the national media alleging that U.S. children faced “intolerable” risks from exposure to a number of pesticides, including the plant growth regulator daminozide (Alar) (7,8), and a 1993 report from the National Research Council that recommended changes in the risk assessment and regulation practices to more appropriately consider the differences in susceptibility and exposure of infants and children to pesticide residues relative to adults (9).
II. PESTICIDE TYPES It is important to appreciate that all pesticides possess some degree of toxicity to some living organisms, otherwise they would have no practical use. It is equally important to recognize that pesticides are usually developed for a target species but physiological and biochemical system similarities in nontarget organisms can lead to undesired responses. Adherence to labelinginstructionsandsafeusepracticescanminimizehumanhealth hazards (1). There are several types of pesticides:
Pest
Type of pesticide
Insect Weeds Fungi Nematodes Mites Leaves Bacteria Rodents Snails Algae
Insecticide Herbicide Fungicide Nematicide Acaracide Defoliant Bacteriocide Rodenticide Molluscicide Algacide
Primarydamage to agriculturalcommodities isattributedtoweeds,insects,and fungi. The U.S. Environmental Protection Agency (EPA) reported that of the estimated 1 billion pounds of conventional pesticides (measured on the basis of active ingredients) used in the United States in 1995, 55% were herbicides, 32% insecticides, 7% fungicides, and 6% other (3).
A.
Insecticides
Insecticides are pesticides designed to control crop-damaging insects. They can be nerve poisons, muscle poisons, dessicants, or sterilants. Chlorinated hydrocarbons, organophosphates, and carbamates are the most common chemical classes of insecticides. Chlorinated hydrocarbons such as DDT, aldrin, dieldrin, and chlordane were developed during the
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1930s and 1940s and ledto rapid improvements in insect pest control throughout the world because of their high insect toxicity and their significant environmental persistence. This same persistence led to problems of environmental build-up and biological magnification, and was manifested in significant ecological and environmental upset that ultimately resulted in the elimination of the majority of the uses of chlorinated hydrocarbon insecticides in the United States. Chlorinated hydrocarbons have more recently been associated with estrogenic and enzyme-inducing properties that could possibly interfere with fertility and reproduction i n human and other nontarget organisms ( l ) . Organophosphate and carbamate insecticides have replacedmost chlorinated hydrocarbons. From two very different chemical classes (esters of phosphoric or phosphorothioicacidandcarbamicacid,respectively), they share a conmlonmechanism of action involving inhibition of cholinesterase enzymes in both insects and in mammals. Organophosphate and carbamate insecticides are less persistentthan the chlorinated hydrocarbon to mammals insecticides that they largely replaced, but are generally more acutely toxic and other nontarget organisms ( I , 10). There are as many as 200 different organophosphate insecticides, of which 39 cornpounds are currently registered for U.S. food use. Examples of organophosphate insecticides include parathion, malathion, diazinon, chlorpyrifos, and azinphos-methyl. About 25 carbamate insecticides are in the marketplace and 14 are registered for U.S. food use. Carbaryl, aldicarb, and carbofuran are examples of carbamate insecticides. One of the newer classes of synthetic insecticides is the pyrethroids. Derived from pyrethrins (the natural extracts of chrysanthemum flowers), pyrethroids are more stable inlightthantheirnatural predecessors and are therefore nlore effective as agricultural insecticides. Pyrethroids are considered excellent broad-spectrum insecticides, cause rapid “knockdown” and mortality in insects at low doses, are of lower toxicity to mamn1als by oral, dermal, and inhalation routes of exposure than the organophosphates and carbamates, and break down fairly rapidly i n the environment. They are in significant demand worldwide and synthesisof new analogs continuesto represent an important research area. Though more selective to target species, the pyrethroids suffer from significant environmental lability that reduces their effectiveness and are more costly than other classes of insecticides ( 1 l ) .
B. Herbicides As indicated previously, herbicides contributeto the majority of U.S. agricultural pesticide use in terms of pounds applied. There are a large number of different types of herbicides availableontheU.S.andworldwidemarkets;examplesincludetriazine,sulfonylurea, phenoxy, and quaternary ammonium herbicides. The wide variety of herbicides also results i n a wide variety of toxicological actions on plant material. Preplant herbicides are applied before crop seeding has begun, preemergents are applied (to the soil) before the appearance of unwanted weeds, and postemergents are used after germination of the crop and/or weeds. Some herbicides, such as glyphosate, are toxic to virtually all types of plant material (although glyphosate resistance is being incorporated into genetically modified plant varieties such as soybeans), while others may be more selective, such as phenoxy herbicides that are toxic to broadleaf plants but do not harm narrow-leaf plants such as grasses. Some herbicides exert their toxicity through direct plant contact, while others are applied to the soil or foliage and are translocated throughout the plant following absorption into the plant (I).
Agricu/fura/ Chemicals
C.
541
Fungicides
Fungicides are chemicals derived from compounds such as inorganic metals and sulfur, aryl, and alkyl-mercurial compounds and chlorinated phenols (1). They are used to control molds and other plant diseases by inhibiting the metabolic processes of growing fungal organisms. Many storage conditions (increaseddecreases in heat, moisture) can lead to massive microbial growth, especially on grain products. Many types of fungi can stress or damage crops and stimulate plant toxin development, and some fungi Aspergilsuch as lus jlavus and Fusarium monilifomze produce aflatoxins and fumonisins, respectively, which represent mycotoxins of significant human and animal health concern (12,13).
111.
PESTICIDE USAGE LEVELS
The EPA provides estimates of pesticide use and expenditures for the United States and the world. It bases estimates on surveys, U.S. Department of Agriculture (USDA) reports, other published reports, and proprietary data when it is available. Not all proprietary data is made available, therefore the EPA can only estimate use data. Pesticides have both agricultural and nonagricultural uses, including household and garden pest control, mosquito abatement, sanitation, and wood preservation. Fifty-one percent (Fig. 1)of the pesticide use in the United States in 1995 involved chlorine and sodium hypochlorite. This is not surprising when one considers the large quantities required for disinfecting potable and wastewater. Large volumes are also used as bleaching disinfectants and in swimming pools (3). Of the 4.5 billion pounds of pesticides used in 1995, only 27%or 1.2 billion pounds were devoted to conventional pesticide usage, which excludes biocides, wood preservatives, and disinfectants. Of that 1.2 billion pounds, 939 million pounds were used in the production of food and fiber products (Fig. 2) and the remainder went to home, govern(3). The average amount ment, industrial, and commercial facility, site, and land uses spent on pesticides is$4200 per farm and$20 per homeowner, though it is estimated that only three-fourths of farms and homes use pesticides (3). The types and amounts of pesticides used in food and fiber production are shown in Fig. 3. Herbicides and plant growth regulators were responsible for nearly halfof the pounds of pesticides applied, followed by fumigantshematicides, insecticideshiticides, and fungicides.
(720 Million Pounds)
6%
Conventtonal Pestlclder (1.22 Billion Pounds) Includes *othef pesticide chemicals not developedlproduced
use as oesticides. Thlsincludes: -sulfur -petroleurn -chlorine
pecialty Blocidea 60 Million Pounds)
Pounds)
Chlorlne#ypochlorltea (2.32Billion
Usage Total U.S. (4.5 Billion Pounds)
Fig. 1 U.S.pesticide use, 1995. (Adapted from EPA (3).)
Browning and Winter
542 IndustrlaUGowmmenWHom~er
I
Production of Food and Fiber Products
Fig. 2 Conventional pesticide use in the United States, 1995. (Adapted from EPA (3).)
Fig. 4 suggests an overall decrease in pesticide use since 1979,though agricultural use of pesticides fluctuates yearly depending on many factors such as floods/unseasonable weather, pest outbreaks, and increases in planted acreage of pesticide intensive crops. Flooding conditions in 1993 led to higher weed infestation problems in 1994,resulting in greater pesticide use(3). Care should be exercised when developing definitive conclusions of pesticide use trends based on comparisons of total pounds applied. The effectiveness of various pesticides may differ dramatically; some may be active at rates of pounds of an ounce per acre.A more effective per acre, while others may be effective at fractions method to determine pesticide use trends is to compare the number of pesticide applicationsratherthantheamountsapplied.Unfortunatelythistypeofdataisfrequently lacking. California has the most comprehensive pesticide use reporting system in the nation, having established the nation’s first full-use reporting system in 1990 (14). Growers are required to report to the county agricultural commissioners all site-specific information related to use of each pesticide, including the total amount of product applied and the specific number of acres treated, as well as the precise location of application. The Calif
Fig. 3 Types of pesticides used (by volume) in the United States in 1995. (Adapted (3)J
from EPA
Agricultural Chemicals
543
O I0 0
800 600
400
200 0 1979 1981 1983 1985 1987 1989 1991 1993 1995
Fig. 4 Annual volume of pesticides used in food and fiber production in the UnitedStates, 19791995. (Adapted from EPA (3.)
nia pesticide reporting system generates realistic pesticide use data which can be used to more accurately guide dietary risk assessment (15).
IV. PESTICIDE REGULATIONS The EPA, the U.S. Food and Drug Administration (FDA), and the USDA are responsible for regulating and monitoring pesticide use in the United States. The EPA registers pesticides for use and establishes allowable levels of pesticides (tolerances) on food and feed crops. The FDA monitors domestic and imported foods, and the USDA monitors meat and poultry. The USDA also has responsibility for the Pesticide Data Program, which, in contrast to the FDA's tolerance enforcement focus, more randomly samples primarily fruits and vegetables in ready-to-eat form to aid EPA's risk assessment activities (16). The USDA was given the initial responsibility in 1947 to administer FIFRA. The original statute was intended to group all pest control products under one law and provide one agency with jurisdiction to deny, suspend, or cancel registrationsof pesticide products. The newly created EPA assumed responsibility forFIFRA administration in 1972, while the FDA and USDA retained basic responsibility for both monitoring residue levels and seizure of foods not in compliance with the regulations. Since 1972 the EPA has been authorized to grant pesticide registration and to establish pesticide tolerances and regulate pesticide residues in food and feed under FIFRA. FIFRA is primarily a risk/balancing statute;if the benefits of the useof a pesticide (such as increased productivity, lower food cost, or public health protection) are deemed by EPA to outweigh competing risks (such as adverse effects to humans, including consumers and agricultural workers) or environmental damage, the EPA has the authority to register the pesticide for specified uses (17). For food use pesticides, the EPA requires a full battery of toxicological tests typically performed on rats, rabbits, and nonhuman primates. Such studies include, but are not limited to, acute, subchronic, and chronic exposure, carcinogenicity, metabolic fate, teratogenicity, and mutagenicity studies. The EPA also requires that the manufacturer provide studies on pesticide effects on nontarget organisms, the environmental offate the
Browning and Winter
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pesticide and its breakdown products, and residue studies. It is estimated that it may take 10 years to fulfill the EPA’s testing requirements for a new pesticide at a cost of approximately $30 million (1). Once collected, the EPA reviews this data to estimate the risks associated with the specified uses of the pesticide and to determine if all appropriate tests have been completed.If such risks are consideredto be lower than the benefits anticipated from the pesticide’s use, the EPA will grant a registration and will specify the pest/comrnodity combinations for which the pesticide may be used and appropriate conditions for use anddisposal,whichincludeconsumer,occupational,andenvironmentalconsiderations. Failure to obey such legal requirements, which are printed on the pesticide label, constitutes a federaloffenseandmayresultinfinancialpenaltiesand/orimprisonment. After registration with the EPA, individual states can restrict or denyofuse a particular pesticide within that state. California is one such state with stringent use restrictions.
V.
PESTICIDE RESIDUE LIMITS
As described previously, when the use of a pesticide on a food or feed crop may have the potential to leavea residue, the EPA typically requires thata “tolerance” or maximum allowable level be established for that pesticide/commodity combination. Residues deto be illegal and may result in tected in excess of the established tolerance are deemed seizure and possibly removal of the commodity from the market andfines to the producer. Illegal residues also result when residues are detected, regardlessof level, on commodities for which the pesticide is not registered for use. The process EPA uses to establish tolerances is rather confusing and often misunderstood; this process is described in much more detail by Winter (17). Briefly, tolerances are established to represent the maximum expected residues of a pesticide on a particular commodity resulting from legal applications of the pesticide under conditions specified for use. As such, tolerances are usefulas enforcement tools to indicate whether application conditions were followed, since it is highly unlikely that residues in excess of tolerances will be encountered under the specified use conditions. In addition, the presence of a pesticide on a commodity for which the pesticide does not have an established tolerance could indicate that the pesticide was used on the wrong commodity or could illustratethat care was not exercised to prevent contamination of other commodities through factors such as drift or residual soil uptake. Tolerances, unfortunately, are often considered as “safety standards,” which is a misnomer, since illegal residues rarely meet toxicological criteria as “unsafe” residues (17). Before granting a tolerance. EPA makes assessments of potential human exposure resulting from all registered (and proposed) uses of the pesticide. Typically the EPA initially calculates the theoretical maximum residue contribution (TMRC), which represents the maximum legal exposure to the pesticide and assumes that (a) the pesticide is always used on all commodities for which it is registered, (b) residues are always present at thc tolerance level, and (c) there is no reduction in residue levels resulting from postharvest factorssuch as transportation,washing,peeling,cooking,processing,etc.The TMRC value is compared with toxicological criteria such as the reference dose (RfD), which represents, based upon the results of animal toxicology studies and extrapolations to humans, the typical daily exposure level not considered to represent any appreciable level of risk. If the TMRC is below the RfD, the risk from the use of the pesticide is deemed
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negligible and the tolerance petition is typically approved, provided that the oncogenic (cancer) risks posed at the TMRC are below the “negligible risk” of level 1 excess cancer per million. The calculation of oncogenic risks uses conservative (risk magnifying) assumptions concerning low-dose extrapolations from the results of animal cancer studies performed at moderate and high doses (18). In cases where the TMRC exceeds the RfD or the oncogenic risk exceeds the negligible risk level, the EPA may use refinements in its risk assessment practices to more accurately assess “anticipated” levels of exposure by considering more realistic pesticide use, residue, and/or postharvest data. Some studies have indicated that the TMRC may often exaggerate exposure levels by offactors 10,000100,000 times (10). Since the tolerance values represent, by definition, the maximum residues anticipated from the proper use of a pesticide, is it clear that assuming all residues As an example, Fig.5 compares tolerance are present at the tolerance level is not realistic. levels, anticipated residues using controlled field trials, and actual regulatory monitoring findings for the fungicide captan on apricots, cherries, and peaches. Both the highest observed regulatory monitoring levels and the anticipated residues from legal worst-case field trials represent only a small fraction of the established tolerance level. In many cases, the artificially high exposures estimated using the TMRC cause the RfD to be exceeded or the oncogenic to exceed a negligible risk level, necessitating the need for refinements in the exposure calculations. Such refinements may include adjustments of actual pesticide use (in contrast to the assumption that 100% of the acreage is treated), the useof more realistic residue data, and/or considerationof potential postharvest effects such as processing that could significantly reduce actual consumer exposure to the pesticide. These refined exposure estimates commonly represent the anticipated residue contribution (ARC) (18). If the ARC is below the RfD, and the oncogenic risk at such an exposure is less than 1 excess cancer per million, the tolerance is usually established.
Apricots
m
level
m
Peaches Cherries Anticipated
observed Maximum
residue’
Fig. 5 Comparison of captan tolerances and residues from controlled field studies and monitoring programs. (Adapted from Winter (17).) “Average residue from controlled field studies using most severe legal applicahon of pesticide. hCalifomia Department of Food and Agriculture Monitoring Programs, 1981-1984.
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I n summary, while dietary health risks from exposure to residues are considered before a tolerance is approved, the actual tolerance level established is not based on safety, but rather represents the maximum residue anticipated from the legal use of the pesticide.
VI.
FOOD QUALITY PROTECTION ACT OF 1996
In August 1996, the Food Quality Protection Act (FQPA) was signed into law and promised to significantly alter the way pesticides would be regulated in the United States. The act was intended to end a paradox in the statutes governing pesticide residues found in FFDCA and FIFRA. The Delaney clause, a 1958 FFDCA amendment. stipulatedthat any food additive shown to cause cancer in humans or laboratory animals could not be used. By definition, pesticide residues were considered to be food additives only in cases where a pesticide concentrated to greater levels i n the processed food than in the raw commodity a processed food form. Inconsistencies werc or when a pesticide was added directly to developed in the regulation of raw versus processed foods, and additional inconsistencies surrounded the two major federal laws; the Delaney clause, under FFDCA, was a “risk only” statute, while FIFRA was a “risk balancing” law that allowed consideration of bothrisksandbenefits.Afteralengthystudy,theNationalResearchCouncilin1987 recommended that the EPA adopt a uniform “negligible risk” policy for the regulation of potentially carcinogenic pesticides in all foods as a reasonable substitute for the zerorisk Delaney clause that applied to processed food forms. The EPA adopted this policy in 1988 but wasimmediatelychallenged in court.After the U S . 9thCircuitCourt of Appeals ruled that EPA’s “negligible risk” policy didnot comply with the Delaney clause ( 19) and subsequent consent decree agreements were developed that specified a time table forwhichpesticideregistrationssubject tothe Delaneyclause wouldbe revoked,the FQPA emerged as a legislative tool to eliminate the Delaney clause from pesticide regulation. on processed The new act establishes one law for all pesticide residue tolerances food and raw agricultural commodities, aswell as standards that applyto all risks, whether oncogenic or nononcogenic. The EPAmust determine that tolerances are “safe,” defined as “a reasonable certainty that no harm will result from aggregate exposure” IO the pesticide (20). “A reasonable certainty of no harm” has historically been interpreted as a one i n one million additional risk of cancer over a lifetime and exposure below the reference dose. TheFQPAalsoadoptsseveral oftherecommendationsfrom the 1993National Research Council report on pesticides in the diets of infants and children. Important new provisions of the FQPA include thepotentialuse of anadditional IO-fold uncertainty factor when extrapolating the results of animal toxicology data of possible human effects to provide additional protection for infants and children, consideration of “aggregate” risk from water and domestic exposure to pesticides in addition to dietary exposure, and consideration of “cumulative“ exposure to familiesof pesticides (suchas the organophosphate insecticides) that possess common mechanisms of toxicological action. The FQPA also expedites approval of safer pesticides, creates incentives for the development and maintenancc of effective crop protection tools for American farmers, requires periodic reevaluation of pesticide registrations and tolerances, requires the EPA to account for possible endocrine disruption, and provides consumer right-to-know provisions (20).
Agricultural Chemicals
VII.
547
INTERNATIONAL REGULATIONS
Pesticide residues on foods entering the United States from other countries must comply with U S . tolerances. Throughout the world, however, pesticide residue standards may vary from country to country. Some pesticides used on commodities in foreign countries may not be allowed on the same commodities in the United States, while other pesticides permitted in the United States may not be allowed in other countries ( 18). The Codex Committee on Pesticide Residues (CCPR) was created to provide international guidance on pesticide residue issues. The CCPR reconmends international pesticide tolerance standards to facilitate international food trade. The Food and Agriculture Organization (FAO) and the World Health Organization (WHO) established the CCPR i n 1961-1962 (21 ). Membership is open to all members and associate members of FAO and/or WHO. Scientific experts from the Joint Expert Committee on Food Additives and the Joint Meeting on Pesticide Residues make independent recommendations to WHO and FAO on pesticide residue limits known as maximum residue limits (MRLs) (22). These MRLs are not recommended unless an acceptable daily intake (ADI), analogousto the U S . reference dose, has been established from relevant data. Any question of the pesticide’s safety will result in reevaluation, and all nations participating may comment on the limits set by the Codex (21). Thcre are currently several pesticides subject to Codex MRLs for one or more food conmodities common to international trade. The United States is one of Inany countries participating in Codex thathasnot accepted theseinternationalpesticidelimitsunless they match those already established by the EPA. When comparing Codex Alimentarius equivalent MRLs to U.S. tolerances, the standards were equal47% of the time, the Codex MRLs were lower 34% of the time, and U.S. standards were lower 19% of the time (23). Because differing data sets are used, pesticide metabolites are regulated differently, and agricultural production/pest control practices differ, it is difficult to reach a consensus on the best international regulatory numbers (23).
VIII. MONITORING AND ENFORCEMENT Although the EPA, USDA, and FDA all have roles in the regulation of pesticides, the FDA is charged withenforcingtolcrances in domestic and imported foods shipped in interstate commerce (24). The objective of the FDA monitoring program is to monitor foods and feed for illegal pesticide residues andto take regulatory action when tolerances are exceeded or when residues are detected on commodities for which the pesticide is not registered. The FDA primarily performs commodity monitoring from which fruits. vegetables, and other commodities are sampled and analyzed for residues of more than 200 possible pesticides using multiresidue screening techniques (24). Commodity monitoring programs encompass both surveillance monitoring and compliance monitoring. I n the surveillance monitoring program, the typesof conmodities chosen for sampling and the origins of the samples are targeted to improve FDA’s ability to identify violative residues ( 2 5 ) . While targeted, samples collected in this program are far more random than those analyzed in the compliance monitoring program i n which samples are usually drawn as a follow-up to illegalresiduedetection or similar problems (typically related to n spccific shipper,
Browning and Winter
548 1.6%
1,2% 8%
66.0
88.1
Domestic
Import
4429 samples
5223 samples
Residue Found Residue Found 0No Residue Found Violative Violative Not
Fig. 6 Summary of FDA's surveillance monitoring program, 1997. (Adapted from FDA (24).)
grower, geographic area, or country). Most samples gathered by the FDA are from surveillance sampling. Domestic samples are usually collected near the source of production or at the wholesale level, whereas imported foods are sampled at the pointof entry into the United States. Raw agricultural commodities are preferable for commodity samples, whic are then analyzed before washing or peeling. The FDA also tests some processed foods (24). In 1997 the FDA analyzed 9652 surveillance and 191 compliance samples. Fig. 6 compares results from domestic and import monitoring and Table 1 provides a more detailed breakdown by commodity. The FDA notes that the majority of the illegal residues from foodof imported origin occurred when residues were detected for which no tolerance was established on the commodity, not from residues in excess of legal tolerances (24). In the vast majority of these cases, the violative pesticide is registered for use (by the
Table 1 FDASurveillanceMonitoring Program1997 byCommodity Number samples violative limits found within legal of Domestic surveillance Samples (by commodity) Fruits Vegetables Graindgrain products Milk/dairy productdeggs Fish/shellfish/other Baby foods/formula Import surveillance Samples (by commodity) Fruits Vegetables Graindgrain products Milk/dairy products/eggs Fish/shellfish/other Baby foods/formula Source: Adapted from FDA (24).
No residue Residue found Residue found
1171 1707 397 628 369 51
44.1% 69.1% 59.5% 97.0% 68.0% 82.8%
54.7% 28.5% 40.6% 3.0% 32.0% 17.2%
1.2% 2.4% 0.0% 0.0% 0.0%
2034 2356 322 85 158 268
60.6% 63.0% 86.0% 89.4% 93.7% 86.6%
38.2% 34.9% 13.0% 10.6% 6.3% 10.8%
1.2% 2.1% 0.9% 0.0% 0.0% 2.6%
0.0%
549
Agricultural
EPA) on other commodities. Results clearly indicate that residue levels rarely approach the tolerance levels and that illegal residues are infrequently encountered. The FDA also performs its Total Diet Study annually to acquire incidence/level data on particular commodity/pesticide combinations. This study uses a market basket approach, with 261 foods comprising each market basket which are gathered once a year in each of the four geographical regions of the United States from three different cities in each region. Each collectionof foods is prepared for table-ready consumption and then analyzed for pesticide residues. As an example, FDA inspectors may purchase apples, flour, eggs, and sugar to use in the baking of an apple pie and the pie is analyzed for pesticide residues at the time it is ready to be consumed (24). By combining analytical results with estimates of typical consumption rates of the various components of food analyzed in the Total Diet Study, it is possible to estimate typical daily toexposure individual pesticides for the general population as well as for specific population subgroups defined by such factors as age, gender, and geographical location.
U.S. Department of Agriculture
A.
The USDA is another federal agency responsiblefor analyzing foods for pesticide residues. Specifically, its National Residue Program analyzes meat, poultry, and raw egg products for pesticide residues, animal drugs, and environmental contaminants. As with the FDA, samples include both domestic and imported food products. The USDA uses multiresidue analysis methods that detect each major insecticide class (chlorinated hydrocarbons, chlorinated organophosphates, organophosphates, and carbamates) and also detect 40 individual pesticides (10). The USDA’s residue program was expanded to include collection of data on pesti(AMs)was appointed cide residues in food. The USDA’s Agricultural Marketing Service to undertake the creation and implementation of such a program, currently known as the Pesticide Data Program (PDP). PDP has been in operation since May 1991 and has published findings for calendar years 1991 through 1995. ofTen the 50 states currently participate: California, Florida, Michigan, NewYork, Maryland, Ohio, Texas, Wisconsin, Colorado, and Washington (26). In 1996 the PDP collected and analyzed 5771 samples originating in 35 states and 10 foreign countries (26). Fig. 7 summarizes the 1996 PDP results in terms of origin of sample (domestic versus import) and residue findings. Eight fresh fruit and vegetable
24.8% 12%
87.8%
Domestic
5771 Samples
I
No doteetablo rerlduea
\
3.4%
Vlolathre Reslduea
71.8%
Detectable residues
Pesticide Residue
Fig. 7 SummaryofUSDA’spesticidedataprogrammonitoring, (261.)
Results 1996. (AdaptedfromUSDA
Browning and Wfnter
550
commodities were collected, including apples, carrots, grapes, oranges, peaches, spinach, sweet potatoes, and tomatoes. In addition, four processed fruit and vegetable commodities were collected, including apple juice, canned and frozen green beans, sweet corn, and 3.4% sweet peas. Remaining samples were drawn from wheat and whole milk. Of the violative residues detected, most (96.3%)of the violations occurred when a residue was found not licensed for that product(16). The PDP monitoring program shows a higher percentageof detected residues than does the FDA’s surveillance monitoring program. The major reason for the difference is that analytical methods used in the PDP are generally far more sensitive than the FDA’s methods. By incorporating the PDP data with the Total Diet Study data, the EPA can more accurately assess the dietary risks posed by pesticides. The PDP’s sampling procedures are designed to provide more realistic estimates of pesticide residues as close as possible to the point of consumption, thereby improving the reliability and extent of information to the actual percentage available for risk assessment. This gives numerical representation of crop treated with a pesticide and considerations such as washing, cooking, processing, and storage of commodities. Most recent changes in the PDP have led to a greater focus on monitoring foods consumed most frequently by infants and children in response to recommendations of the National Research Council’s 1993 report on pesticides in the diets of.infants and children.
B. State Programs In addition to the FDA’s monitoring system, individual monitoring programs exist in 38 states; such state programs vary considerably with respect to focus and sampling rates. California currently has the largest state monitoring program and typically spends more that $40 million each year to regulate pesticide use. Results of California’s Routine Marketplace Surveillance program for 1995 are shown in Fig.8; of the5502 samples analyzed, the vast majority (64.7%)showed no detectable residues, while illegal residues were de-
Residue less than 10% of tolerance 24.5%
Residue between 10% and 5 0 % of tolerance
Residue between 50% and 100% of tolerance 1 .O%
\
\No Illegal residue
Resldue Detected
64.7%
1.6Yo
Fig. 8 Summary of California’sroutinemarketplacesurveillancemonitoringprogram, (Adapted from California Department of Pesticide Regulation (15).)
1995.
Agricultural Chemicals
551
tected in 1.6% of the samples ( 15). Approximately 75%)of illegal residues occurred when pesticides were detected on commodities for which they were not registered for use; only about 25% of the illegal residues represented tolerance violations. Results also indicated that legal residues. when detected, typically existed at a small fraction of established tolerances, with the majority of detected residues present at less than 10% of tolerance (IS).
IX.
DIETARY RISKS
Calculating the dietary risks I‘aced by consumers from pesticide residues is a challenging task that requires a variety of assumptions to be made (18,27). The types of assumptions made may dramatically influence the nnagnitutles of estimated risks; the use of conservative assumptions Inay produce much higher risk estimates than those obtained using less conservative assumptions. As an example, the NRC estimated oncogenic risks for a number of pesticides by assuming exposure at the TMRC level, which resulted in the finding that most of the pesticides studied generated oncogenic risks far in excess of the negligible (28). The use of more accurate human risk standard of one excess cancer per million exposure data reduced the oncogenic risks for most of the pesticides to levels far below the same negligible risk standard (10). A common method used to address the magnitude of dietary pesticide risks is to present the resultsof regulatory monitoring programs by providing information concerning the percentages of samples found to have either no detectable residues, detectable but legal residues, and violative residues. Unfortunately such findings are of little use in the risk assessment arena since tolerances are not directly related to safety levels, as has been described prcviously, and illegal residues should not be construed as “unsafe” residues in most cases. A Inore accurate approach to estimate risks from pesticides is to use the exposure data developed from market basket surveys such as the FDA’s Total Diet Study and relate that data to reference doses and/or calculate oncogenic risks. While several assumptions relating to food consumption patterns and toxicological potencies still must be made, this type of approach precludes the need to estimate pesticide use patterns, residue levels on raw commodities, or postharvest effects on residue levels. 1991 TotalDietStudyarecompared Exposure estimates developed from FDA’s with EPA’s established reference doses and the analogous acceptable daily intakes estabi n allthree lished by WHO in Table 2 (29). Resultsindicatethatformostpesticides population subgroups, exposure estimates represent only a small fraction (often less than 1%) of the corresponding reference doses or acceptable daily intakes. I n the typical case where reference doses are derived using a 100-fold uncertainty factor when extrapolating from the highest level that does not produce a noticcable effect in animal studies to estimate an appropriate human daily exposure level, exposure at the reference dose would constitute a level 100 times lower than that of the no observed effect level i n animals. Exposure at a level of 1% of the reference dose corresponds to an exposure 10,000 times below the level that does not produce noticeable effects in the animals. Such findings provide an illustration of why the majority of health professionals consider the risksof pesticide residues i n foods tobe far lowerthan a number of other food safety risks such as those posed by nlicrobiological contamination, nutritional imbalance, environmental contaminants, and naturally occurring toxins (30). Even so. these findings
552 Table 2
Browning and Winter
Pesticide Intakes Estimatcd From FDA's Total Diet Estimated exposure (Kglkglday)
Pesticide
0.0002
alpha
Acrphate Azinphos-methyl BHC, BHC, gamma (Lindane) Captan Carbaryl Carbofuran, total Chlordanc. total Chlorpyrifos Chlorpyrifos-methyl DCPA DDT, t o t a l DEF Dcmeton Diazinon Dichlorvos Dicloran, total Dicofol, total Dieldrin Dimethoate Endosulfan, total Endrin Ethion Fenitrothion Fcnuron' Fonofos
Heptachlor, total Hexachlorobcnzene Iprodionc, total Linuron Malathion Mcthamidophos Methomyl Methoxychlor, p,p' Metobromuron" Mcvinphos. total Ncburon' Omthoate Parathion Paration-methyl Pentachlorophenol Permethrin, total Phosalonc Phoslnct Pirimiphos-methyl
6- I 1 months 0.0089 0.0028
14- l6 yearold male 0.01 l 3 0.0033
0.0004 0.0004 0.0478 0.1801 0.0002 0.0001
0.0082 0.0 104 0.0002 0.0095 <0.0001 0.000s
0.0049 <0.0001 0.1926 0.0218 0.0027 0.034 0.0 173 <0.000 1 0.0 I28 <0.0001
0.0004
0.0008
0.0209 0.09 0.000 1 0.0003
0.0034 0.0 126 0.000 1
0.0056 0.000 1 0.0003
0.0022 <0.0001 0.044 0.0077 0.0021
0.0022 0.0 I S8 <0.0001 0.0034 <0.0001 0.0002
Study, 1991 -
60-65 yearold felnalc 0.0 165 0.0029 0.0002 0.0003 0.0595 0.08 1 1 0.0004
0.0002 0.0024 0.0066 0.0002 0.0043 <0.0001
0.0006 0.0022
FAO/WHO ADI.' (pg/kg/day) 30 5 -h
8 I00
0.5 10 1 -
0.06 3
20' -
0.1175
30'l
0.023s 0.002 l
25
0.0035
10
0.1'
0.5
5 -
-
-
0.2 0.5"
0.0002
-
0.00 I9
300'l
0.0008 0.0487
0.001 0.0275 0.019
20 4
0.0066
0.0097 0.0007 0.0016 0.02s 1
0.0073 0.0043 0.0007
<0.0001 0.0013 0.00 16
0.000 I 0.0004 0.0338 <0.0001
0.0009 0.00 1
0.2
2
0.0035
0.0008
<0.000 1 0.0 144
-
0.5 0.05
<0.0001 0.0003
0.0004
-t
-
0.04
0.05" 0.3
0.0026 0.002 1 0.0779
0.0006
500 0.5"
6 0.2
<0.0001 0.0003
0.0037 0.0007 <0.0001 0.0026
0.3
130 -
<0.0001 0.0005
0.0102
-
S'
<0.0001 0.0005 0.0003
0.0 12 0.0053
-l1
10'
2 4
<0.0001
4
10
0.000 1
0.0242
EPA RtD.' (pg/kg/day)
0.0068
0.0002 0.000 1 0.008 1 <0.0001 0.0019
0.0042 0.0001
0.0008 0.0495 <0.0001 0.0027 0.0006
0.1'
-
30 I00 -
l .S' -
0.3 5
20 -
0.8
40 2 20 0.05
25 50 -
30
SO 5
SO
20'
20
l0
10
-
553
Agricultural Chemicals
Table 2 Continued
Estimated exposure (pg/kg/day) 6- 1 I months Pesticide ~~
~
14-16 yearold male
60-65 ycarold female
FAO/WHO ADI.' (pg/kg/day)
EPA RID,' (pglkglday)
~~
Propargite Quintozene, total 0.395Thiabendazole Toxaphene Vinclozolin
0.099 1 0.0004
0.0495 0.0009
0.049 1 0.0003
0.0033 0.0052
0.0059 0.00 I8
0.0024 0.0061
150
7' -
70
20 3 -
25
cannot prove the absence of long-term risks from pesticidesin the diet, asthis is a scientific impossibility. It is also clear that pesticide misuse has historically resulted in incidents of acute poisoning of people consuming tainted foods. The illegal application of aldicarb resulted in more than 1000 cases of human poisoning in the United States and Canada in 1985 (6), and several other documented casesof human poisoning from consumption of excessive levels of pesticide residues are reported by Ferrer and Cabral (3 1). The health benefits from the use of pesticides also require some discussion. It is clear that pesticide use allows for increases in production of a wide variety of food crops; into greater availability and lower consumer costs. A such increases may be translated substantial body of human epidemiological evidence has indicated that diets rich in cow of heart sutnption of fruits, vegetables, and grains may significantly decrease one's risk disease and certain types of cancers, and the National Research Council concluded that the theoretical increased pesticide risks from consuming greater amountsof fruits. vegetables, and grains were greatly outweighed by the health benefits provided by these types of foods (32).
X.
NONDIETARY RISKS
Pesticide concerns are not centered solely on the safety of residues in foods. Other concerns, such as potential acute toxicity to humans from occupational exposure (manufacturof crops) and bystander exposure to ing, loading, application, harvesting, and handling off-target drift from spraying operations, are additional risks associated with pesticides. Other indirect risks associated with pesticide use include Human occupational and domestic exposure Reduction of fish and wildlife populations
Browning and Winter
554
Livestock losses Destruction of susceptible crops and natural vegetation Honeybee losses Destruction of natural enemies Evolved pesticide resistance and creation of secondary pest probletns The health costs associated with pesticide use have been estimated be to $786 nlillion annually based on treatment of pesticide-related illnesses due to poisonings (2). State and federal governments spend i n excess of $240 million for regulatory pesticide pollution controlprograms,training(forpesticideapplicators).registrationandreregistration of pesticides, and other programs geared toward residue detection. These figures do not include the indirect costs such as fish. wildlife, tree, and crop losses, or chronic health costs (such as cancer) which could raise the total cost to as much as $4 billion annually (2).
XI.
CONCLUSIONS
The widespread use of pesticides throughout the United States and the world undoubtedly results in consunler exposure to a large number of pesticides i n the hutnan diet. Specific cases of consumer poisoning resulting from pesticide misuse have been documented. A variety of opinions have surfaced concerning the magnitude of the potential dietary risks from continuous exposure to lowlevels of pesticidesinthediet;somearguethatthe large amount of regulatory scrutiny given pesticides coupled with the resultsof regulatory tnonitoring progranu and market basket surveys indicate exceedingly low levels of risk, while others form different conclusions. In many cases, such differences of opinion may have less to do with the significant amount and quality of scientific data generated for pesticides and more to do with other less quantitative and nonbiological attributes (33,34). It has been argued that the public pays too little attention to levels of calculated risk, while the scientific community pays little attention to such “outrage” factors as the voluntariness, equity distribution. origin. or familiarity of the risks. Because of such differences in the acceptability of pesticide residue risks among various segments of thepopulation, it is clear that debate concerning pesticide issues should continue for some time. In the meantime, significant research into integrated pest management and sustainable agriculture approaches and recombinant DNA technologies. such as the development of pest-resistant varieties of food crops, is being conducted in an effort to reduce the world’s current level of pesticide dependency.
REFERENCES DJ Ecobichon. Toxic effects of pesticides. In: CD Klaassen. cd. Casarctt CG Doull’s Toxicology: The Basic Science of Poisons. 5th ed. New York: McGrnw-Hill, 1996. pp. 64348.5. 2. D Pirnentel. H Acquay, M Biltonen,PRice.MSilva. J Nelson, V Lipner. S Giordano, A Horowitz. M D’Amore. Environmental and economic costs of pesticide use. BioScicnce 42: 750-760, 1992. 3. EPA. Office of Prevention. Pesticides and toxic substances (7503W).Pesticides Industry Sales andUsage. 1994 and 1995 Market Estimlte. 733-R-97-00?, Washington, DC: U S . Governmcnt Printing Office, August 1997. 1,
Agricultural Chemicals
555
oftheAssessment 4. CR Curtis.AgriculturalBenefitsDerivedFromPesticideUse:AStudy Process. Columbus: Ohio State University Press, 1988. CA Beall, S Brown,JOHarwood, CL Lamp, G Stanford, YJ 5 . CM Bruhn,CKWinter. Steinbring. B Turner. Consumer response to pesticide/food safety risk statements: implications for consumer education. Dairy Food Environ Sanit: 278-287. 1998. 6. LR Goldman, M Bcllcr, R Jackson. Aldicarb food poisoningsin California. 1985- 1988: toxicity estimated for humans. Arch Environ Health 45: 141- 147, 1990. 7. JV Aidala. Apple Alarm: Public Concern About Pesticide Residues in Fruits and Vegetables. CRS Report to Congress. Congressional Research Service,89-l66ENR. Washington. DC:U.S. Governmcnt Printing Office. March 10, 1989. in Our Children’sFood. X. NaturalResourcesDefenseCouncil.IntolerableRisk:Pesticides Washington, DC: National Resources Defense Council, 1989. 9. National Research Council. Pesticides in the Diets of Infants and Children. Washington, DC: NationalAcademyPress, 1993. 10. SO Archibald,CKWinter.Pesticides i n our food: assessingtherisks.In:CKWinter.JN Seiber, CF Nuckton, eds. Chemicals intheHumanFood Chain. NewYork:VanNostrand Reinhold. 1990, pp. 1-50. insecticides. In: 11. CJ Marshall. Effects and mechanisms of action of pyrethrin and pyrethroid 1995. LW Chang. RS Dyer. eds. Handbook of Neurotoxicology. New York: Marcel Dekker, pp. 5 I 1-545. 12. PR Cheekc. LR Shull. Natural Toxicants in Feed and Poisonous Plants. Westport. CT: Avi Publishing, 1985, pp. 393-467. 13. LB Bullerman. Occurrence of fusariu~nand fumonisins on food grains and in foods. I n : LS Jackson,JWDeVries. LBBullernxm,cds.FumonisinsinFood.NewYork:PlenumPress. 1996, pp. 27-38. 14. CaliforniaDeparttnent of PesticideRegulation.PesticideUseReporting.Sacranlento, CA: California Dcpartmcnt of Pesticide Regulation, 1995. http://www.cdpr.ca.gov/docs/dprdocs/ userptng/pruhtm.htlll. i n Fresh Produce. Executive Sum15. California Department of Pesticide Regulation. Residues nary of 1995 Monitoring Program. Sacramento. CA: California Department of Pesticide Rcgulation,1997. 16. USDA. Pesticide Data Program, Progress Report. Washington, DC.U.S. Government Printing Office,1998. 17. CK Winter. Pesticide tolerances and their relevance as safety standards.Reg Toxicol Phurmacol15:137-150,1992. 18. CK Winter. Dietary pesticide risk assessment. Rev Environ Contam Toxicol 127:23-65,1992. 19. CK Winter. Pesticide residues and the Delaney clause. Food Technology 47(7):81-86, 1993. 20. EPA. Major issues in the Food Quality Protection Act of 1996. Washington, DC: Office of Pcsticide Programs, 1998, http://www.epa.gov/oppfeadl/fqpa/fq~~a-iss.ht~~~. 21. JR Wesscl, NJ Yess. Pesticide residues in foods imported into the United States. Rev Environ ContamToxicol120:83-104. 1991. 22. FAO. Risk Management and Food Safety. FAO Food and Nutrition Paper 65, Rome: FAO/ WHO, pp. 27. 23. General Accounting Office. International Food Safety: Conqxlrison of U.S. and Codex Pestil99 I . cide Standards. GAO/PEMD-91-22. Washington. DC: U.S. Government Printing Office, 24. FDA. Pesticide Program, Residue Monitoring 1997. Washington, DC: U.S. Government Printing Office. 1998, http://www.cfsan.fdda.gov. 25. DV Reed, P Lombardo, JR Wessel, JA Burke. B McMahon. The FDA pesticides monitoring program. J Assoc Anal Chcm 70:591-595. 1987. 26. USDA.PesticideDataProgram,Annual Sumnary CalendarYear 1996. Washington,DC: U.S. Government Printing Office. 1997. http://www.ams.usda.gov/science/pdp/i~~dex.ht~~~. 27. CK Winter. Lawnlakers should recognize uncertainties in risk assessment. Calif Agric 48:2 l 29. 1994.
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National Research Council. Regulating Pesticides in Food: The Delancy Paradox. Washington, DC:NationalAcademyPress.1987. 29.FDA.PesticideProgram,1991.JAssocAnalChem75:156A,1992. 30. CK Winter. Pesticide residucs in foods: recent events and emerging issues. Weed Techno1 10: 969-973. 3 1. A Ferrer, JPR Cabral. Epidemics due to pesticide contamination of food. Food Addit Contam 6:S95-S98,1989. 32. National Research Council. Dict and Health: lmplications for Reducing Chronic Disease Risk. Washington, DC: National Academy Press, 1989. 33. PM Sandman.Riskcommunication:facingpublicoutrage.EPAJ13(9):21-22,1987. 34. CK Winter, FJ Francis. Assessing, managing, and communicating chemical food risks. Food Techno1 51185-92. 1997.
28.
18 Radioactivity in Food and Water
Introduction 557 A. Radioactivity 557 B. Natural and anthropogenic radioactivity 558 C. Radiation interaction with matter 559 D. Biological effects of radiation 560 E. Measurement radiation of 560 Risks F.radiation of 562 11. Uptake of Radioactivity by Humans562 A. Naturalradioactivity in foodandwater562 B. Anthropogenic sources in food and water 564 C. Food chain concentration of radioactivity 564 111. AccidentalContamination of FoodandWater565 565 A. Sourcesofcontamination B. Interdiction techniques 565 IV. Conclusions 567 A. Necessity for surveillance 567 Consistency B. guidelines of 568 C. Necessity for low-level effects research 568 D. Public education 568 References 569 I.
1.
A.
INTRODUCTION Radioactivity
Radioactivity is the physical phenomenon by which unstable atoms spontaneously change into other atoms and, in the process, emit radiation. The resulting atom can be stable (nonradioactive) orit may be subject to further change (radioactive). If the resulting atom If a parent is also unstable, it is termed the “progeny” of the original, “parent” atom. atom changes or “decays” to a radioactive progeny, a decay chain can occur with one or more radioactive progeny, resulting finally in a stable atom. One familiar decay chain
557
558
Kocol
involves uranium atoms decaying through several steps to thorium, radium, radon, and eventually to lead. I n order to understand radioactivity, it is important to understand something about atoms. Atoms are differentiated by the numbersof protons and neutronswithin their nuclei. The number of protons determines the chemical element,that is, a hydrogen atom contains I proton, while a uranium atom contains 92. The sum of the particles (protons plus neutrons) in the nucleus is the rrtonric nlms of the atom. i.sotope.s, and are distinDifferent types of atoms of the same element are called guished from each other by their different atomic masses. The differences in the atomic masses of the isotopes are due to differences in the numbers of neutrons in the nuclei of the atoms. The atomic masses are written as a superscript before the chemical symbol of the element. As examples, 'H is an aton1 of normal, stable hydrogen containing one proton in the nucleus; 'H (deuterium) is a rare stable atom of hydrogen containing one proton and one neutron i n the nucleus; 'H (tritium), containing one proton and two neutrons in its nucleus, is a radioactive form of hydrogen which will undergo decay to 'He. Note that all the hydrogen isotopes contain one proton in the nucleus; the different isotopes have different numbers of neutrons i n their nuclei. It is thought that radioactivity is the result of some imbalance in the numbers of protons and neutrons in the nucleus which then emits radiation to achieve a more stable condition.Radioactivityisevidencedbytheradiationemittedfromthe a t o m as they decay. It is that radiation, not the radioactivity itself, that is measured and which can cause biological damage to organisms exposed to it. Each radioactive decay is characterized by three parameters: the rate of decay, the type of radiation emitted. and the energy of that radiation. a particular rate of decay, usually designated as the Each radioactive isotope has "half-life" of that isotope. The half-life specifies the time it takes for half the atoms in any given sample of the isotope to decay, irrespective of the amount of atoms present. Since one-half of the atoms remain after one half-life, one-fourth are left after two halflives, one-eighth after three half-lives, and so on. The number of isotope a t o m present is directly related to the rate of emission of the radiation. Some isotopes have extremely short half-lives, on the order of fractions of a millisecond, while other half-lives are measured in billions of years. The half-life of 'H is approximately 12.5 years ( I ) .
B. Natural and Anthropogenic Radioactivity By far the great bulk of radioactivity on earth is due to naturally occurring radioactive materials.Most of it is from"primordial"isotopesandtheirprogeny; theprimordial isotopes are long-lived enough so that they have not had time to decay totally since the formation of the earth 5 billion years ago. Important to this discussion are 'jXU (half-life of 4.5 billion years), "?Th (half-life of 14 billion years), and ?"U (half-life of 710 million years). Important progeny of the primordial isotopes that are found in food and water are ""Ra, "'Rn, '"'Po, and '"'Pb. Otherprimordialisotopes, notparts of decay chains but important i n this discussion, are "'K (half-life of 1.3 billion years) and "Rh (half-life of 47 billion years). In addition, there are several naturally occurring nonprimordial isotopes in the environment and, therefore, in food and water; these have shorter half-lives and are not part
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of decay chains. This group includes 'H and "C (half-life of 5730 years), both of which are produced i n the atmosphere by cosmic ray bombardment. Also i n this category is '7"Pu (half-life of 24,400 years), produced as a result of neutrons from spontaneous fission i n uranium deposits (2). Anthropogenic isotopes are produced through the intervention of people. Some of these materials are produced i n nuclear reactors, such as '"CS (half-life of 30 years), and ""CO(half-life of 5 years), '"'Sr (half-life of 28 years), various radioactive iodine isotopes (radioiodines), and several plutonium isotopes (3). Others are produced by bombardment of materials in accelerators and include tritium (jH) and "C (4). Anthropogenic activities also can concentrate naturally occurring isotopes, making them more readily available to the food chain. When uranium ore is extracted from the earth and made into fuel rods that then are used for electric power, the naturally occurring uranium is concentrated; of course, i n this instance, other isotopes are produced as products of the fission reaction ( 3 ) . Radioisotopesare usedinmanydifferentapplications other thanfornuclear power, including nwlical diagnosis and treatment ( I "CS, ""CO, radioiodines, and technetium), stabilization of airliners and ships (uranium), safety lights on airliners (tritium), watch dials (tritium). smoke detectors ('J'Am), and gas mantles (thorium). Isotopes also are used industrially for package fill and thickness control and in a myriad of research work (S).
C. Radiation Interaction with Matter Radiation can be defined as the transfer of energy through space. The energy transfer can take place via particles or via waves as the energy-carrying medium. Several types of radiation are emitted by most radioactive isotopes; three of the most common are named after the first three letters of the Greek alphabet: alpha, beta, and gamma. Alpha particles are large, very energetic nuclei of normal 'He atoms (such atoms are stripped of their orbital electrons) and thus are packets of two protons (i.e., helium) and two neutrons (i.e., 'He); they are emitted primarily by very heavy radioactive isotopes such as '''U, '"'Pu, and ""Ani. Although alpha particles are emitted at relatively high energies, because they are so massive, alpha particles do not penetrate very deeply into matter: a sheet of paper would beasufficientshieldagainstmostpurealphasources.Fromaradiationsafety standpoint, alpha emitters pose relatively few problems if the sources are external to the body. However, if ingested or inhaled, alpha emitters can be very damaging to internal organs because of the large amount of energy the alpha particles will dissipate to a very small volume of the body. Beta particles are high-speed electrons emitted from the nuclei of some radioactive atoms. They are much smaller than alpha particles and therefore are more penetrating, but they can be stopped by a few millimeters of aluminum. Beta particles can pose a skin radiation problem if they are from an external source and, of course, an internal problem if the source is ingested or inhaled, although somewhat less of a problem than an equally active alpha source. Gamma rays are pure energy, not particles at all, and therefore are very penetrating. To stop most gamma rays, several centimeters of lead is required. Because they are so penetrating, many gamma rays will pass through the body without reacting and therefore have less capability of causing biological damage than alpha or beta emitters.
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With a few exceptions, whenever alpha or beta particles are emitted in radioactive decay, gamma rays will accompany that emission. Other types of radiation that can be emitted through the process of radioactive decay are positrons (positively charged electrons) and X-rays. A positron will disintegrate immediately when coming into contact with an electron, resulting in two gamma rays; thus further interaction with matter will be by those gamma rays. X-rays interact with matter in the same way as gamma rays of the same energy. Besides thegrossdifferences in penetratingpowersfortheemissionsdiscussed above, each radiation emitted can be distinguished by its energy relative to others of the same type. Thus, alpha, beta, and gamma radiations are each found i n large spectra of energies; however, each particular decay emits its characteristic radiation at particular energies. A measurementof the type of radiation, its energy, andthe half-life of the decay can determine the particular radioactive isotope present i n the sample (1).
D. Biological Effects of Radiation The radiations discussed above are all “ionizing radiation” i n that they all contain sufficient energy to remove electrons from atoms of the materials through which the radiation passes. Removal of an electron from an atom results in the formation of two charged particles:theremovedelectron,whichisthenfreefromitsoriginalatom,andtheremaining atom without that one electron, which is therefore electrically charged. Since by convention the electron is said to have a negative charge, the remaining atom is positively charged. Charged particles are called ions; hence the term ionizing radiation. Some electrons in atoms are associated with chemical bonding of the atoms into molecules. Thusthe removal of those electrons can have important effects on the molecular structures of the substance being irradiated. If the atoms being irradiated are in biological tissues, specifically the DNA, there are three possibilities: The DNA can correct the damage, resulting in a DNA molecule of exactly the same type as prior to the ionizing event. The resulting DNA molecule can be so detrimental to the cell that the cell perishes without reproduction. The resultingDNAmolecule can changetheoperation of thecellinsuch a way that its life is impaired (perhaps leading to early death via compromised metabolism) or it reproduces uncontrollably (cancer). Some of the changes canbe detrimental to the cell whilenot affecting the organism; others can lead to the death of the organism ( I ) .
E. Measurement of Radiation The ionizing property is usedto measure ionizing radiation. The familiar Geiger-Mueller counter consists of a gas-filled tube with a center wire that is charged electrically positive with respect to the outer wall of the tube. An ionizing event causes electrical charges to be created i n the tube. Negatively charged particles travel toward the attracting positive center wire where, when the charges arrive, a voltage pulse is created and then detected. of energy, as long as the energy is Each ionizing event results in one pulse irrespective sufficienttocause theionization.Thus theoutput of themeter is a total number of
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“counts” or, more usually, counts per minute. The counter is generally useful for beta and gamma emitters, since most such counters have a wall thickness too great for alpha particles to penetrate. Gamma energies can be determined in the laboratory using gamma spectroscopy with some detecting device (e.g., a crystal of sodium iodide or germanium) that can emit different voltages or current pulses dependingon the energy dissipated within the crystal. Computer analysis of the pulses results in a spectrumof energies so that particular gamma energies can be determined. For alpha emitters or for low-energy beta emitters, the sample must be placed within a counter so as to minimize the absorption of the particles before they have a chance to initiate an ionizing event. Proportional counters can be used that are similar i n construction to a Geiger-Mueller tube but which operate at voltages at which the size of the output pulse is proportional to the energy transfer of the original ionizing event. Thus an energy spectrum of the beta or alpha particles can be obtained and the energy of the radioactive decay determined. In any case, lor isotopes of reasonable half-lives (minutes to years), the decay in terms of count rate can be followed over time to determine the half-life of the isotope being measured. With knowledge of the energy, type of radiation, and the half-life, an identification of the isotope involved can be made. Photographic film reacts to ionizing radiation in a fashion similar to its reaction with light. Exposure of film to radiation causes a darkening, or fogging, of the developed film. Attempts can be made to differentiate energies using selective filtration (aluminum or copper of various thicknesses) with the film for personnel monitoring, but film is not used generally for determination of radioactivity in a sample. I n any determination of radioactivity at low levels, the natural background radiation can interfere with sample analysis. To compensate for the background radiation, a count is obtained without a radioactive sample in place, and that count is subtracted from the background count. Combinations of isotopes, whether occurring naturally in the background or in a complex sample, complicate the analysis of the isotopes present. Computer programs have been written to aid in the analysis of samples containing several isotopes. In many cases, radiations from combinations of isotopes may not be discriminated by the counting methods described above. Theni t may be necessary to separate some elements fromthe sample chemically so that their radiations can be measured without interference. Chemical separations complicate the analysis and increase the time until a result is obtained (1). An international commission redefined radiation units in1980 to relate them to basic physical quantities. Because of that change, some confusion exists in comparing data in recent reports with those published before 1980. The classical unit of the amount of radioactivity had been the curie (Ci), approximately equal to the decay rate of 1 g of radium. The unit in officialusetodayisthe becquerel(Bq),onedisintegration(ordecay) per second. The classical unit of radiation dose had been the rad (radiation absorbed dose), a measure of energy deposited by any ionizing radiation in any matter. The current unit is the gray (Gy), equivalent to 100 rads. It was discovered earlythat different radiations and energies have different effectiveness for producing biological effects. Therefore, when discussing biological effects,a new unit of dose equivalence, the rem (radiation equivalent mammal), was invented. The current unit of dose equivalence is the sievert (Sv), equal to 100 rem (6).
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F. Risks of Radiation Determination of the risks of radiation exposure has been of importance from the first discovery that radiation exposure may not be innocuous. Very early in the 20th century, i t was discovered that skin bums, presumed similarto sunburns, could be caused by ionizing radiation. Later studies with animals determined that various genetic (reproductive) and teratogenic (influencing development of the embryolfetus) effects could be caused by radiation. Hulnan studies, mostly from the effects of the atomic bombings of Japan i n World War 11, and further animal studies determined that cancer also could be a longterm effect of ionizing radiation. All studies of radiation effects have been criticized dueto various problems. Animal studies indicate variability among species, so that exact extrapolation to humans is suspect. Some studies havebeen done with groups containing confounding variables, suchas populations undergoing radiation medical treatments for conditions that may predispose them to the effects observed, or with uranium miners who had a long history of smoking. The current risk hypothesis is that extrapolation can be done from high doses to low doses at some unspecified level for long-term effects, even though some studies indicate that such extrapolation to doses at ordinary background levels may not be valid. The tradition in radiation protection worldwide has been to be very careful, some say overly so, such that any error made will be in the direction of safer doses and fewer effects. With all the above in mind, i t isuseful to recognize that no studies have shown effects, statistically, in large hunlan populations at less than I O rem (0.1 Sv) for a onetime dose or 1 rem (0.01 Sv) per year for a working lifetime. The effects may be present, but they are masked by normal incidences of the diseases that may be linked to radiation exposure (7). Risks differ depending upon the method of exposure. If the radiation source is outside the body, shielding or increasing the distance from the source will greatly reduce exposure. If the source is ingested or inhaled, however, i t may be with the individual for a lifetime, causing internal exposureas the radioactive atoms decay. The ingestion pathway is of prime concern for those who must planforpublicprotection i n the event of an accidental release of radioactive material into the environment (8). Another important determinant of risk is the portion of the body exposed. Different organs exhibit different sensitivities to radiation damage. The female breast and thyroid are particularly susceptible to radiation carcinogenesis, while the central nervous system is relatively unaffected by similar doses. Thus when determining the risksof radioactivity in foods and water, it is important to determine the “target organ” in which the radioactivity will concentrate (7).
II. UPTAKE OF RADIOACTIVITY BY HUMANS A.
Natural Radioactivity in Food and Water
Foodandwater, as most substances in nature,containnaturallyoccurringradioactive materials. Much of the radioactivity in food and wateris composed of primordial radioisotopes, primarily “‘K and the uranium and thorium decay chains. The public health importance of these materials depends on the particular isotopes in question, the amounts of those isotopes i n food and water, the chemical natures of the isotopes (especially water solubility), the organs of the body in which the isotopes are concentrated, and the types
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and energies of the emissions (7). Of course, the amounts of the isotopes found in food and water will depend on the transport mechanisms by which the isotopes contaminate the products (9). Potassium is an important element metabolically. Most plants and animals we use "'K. Potassium compounds are generally for food contain some potassium, which contains water soluble and therefore are easily absorbed into the body. Because of that solubility, excess potassium is also discharged easily from the body so that a dynamic equilibrium exists between uptake and elimination, maintaininga certain concentration. It is estimated that the average adult male contains approximately 60 Bq/kg body weight '"K, mostly from food, leading to a dose equivalent of 0.6 mSv/year. No authority is suggesting the elimination of bananas or low-sodium salts from the diet because of the relatively high amounts of "'K in those foods. Most of the elements in the uranium and thorium decay series are heavy metals and therefore are not particularly water soluble. Thus incorporation into body tissues is much less likely than for potassium. However, the membersof these decay chainsin many cases are alpha emitters, so that the health effects from internal absorption of the isotopes are enhanced. Most insoluble forms of these materials will pass through the gastrointestinal tract without absorption so that the dose achieved is that collected during passage. Some small fraction of the isotopes, of course, will be absorbed, leading to a dose equivalent of approximately 1.3 lnSv/year for the average adult. A majority of this dose is due to the '"'Pb/'"'Po pair (10). One aspect of natural radioactivity that has received much attention is the possibility of radon and radium in drinking water. Bothof these radioisotopes are partsof the uranium decay series. Since uranium is a rather common element on earth, groundwater has the opportunity to dissolve some of these uranium progeny while traveling through mineral deposits. Ofparticularimportanceisradon, a gaseous element which is chemicallyinert. Since gases dissolve readily in water, any radon in minerals through which groundwater flows can be carried away. However, dissolved gases are exchanged readily for other gases so that the radon concentration will drop as the water becomes exposed to the surface and therefore to atmospheric gases, primarily oxygen and nitrogen. A roiling stream or babbling brook probably is not a radon carrier; however, groundwater, such as found i n wells, has been discovered to contain substantial amounts of radon. The problem is especially acute in areas in which known uranium deposits occur, such as the "Reading Prong" in Pennsylvania, New York, and New Jersey, and on the Colorado plateau. As is suggested above, radon in water can be removed in large measure through aeration or boiling. When radon is found i n groundwater, it probably also will be found in thelocalsoil, so that seepage occurs, leading tothe indoor radon problem thathas received much attention ( 1 l ) . Cosmogenicisotopes of importanceare 'H (tritium) and "C. Since all food and drink contain hydrogen andall food contains carbon, these isotopes are of great importance when discussing radioactivity i n food and water. Many formsof these isotopes, of course, are water soluble and easily incorporated into the body. The natural body burden of )H is approximately 26 Bq; however, since the isotope is also a product of thermonuclear reactions, the present body burden has risen to approximately 1000 Bq, leading to a dose of 0.03 mSv/year per person (2). It should be emphasized that, because tritium has such a low-energy beta emission with no associated gamma rays, the health consequences of tritium at these levels generally are not considered important (7).
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All living things incorporate "C into their cells during life. This principle isthe basis for "C dating in archeology, anthropology, and geology. The ratio of "C to total carbon for a relic is determined and compared to the current ratio. Any lessening of "C i n the relic is due to the fact that, after death, no more carbon is incorporated into the cells; therefore the loss of "C is due to its radioactive decay since that time. Correcting for the half-life of "C (5730 years) yields the age of the relic since death (1). For naturally produced "C, the body burden is about 46 Bq/kg for a dose equivalent of about 0.013 mSv/year, rising to about 0.023 mSv/year due to weapons testing (2). Future archaeologists will not be able to use I'C dating for relics from our time.
B. Anthropogenic Sources inFood and Water From the discussion above, it becomes obvious that there are amounts of radioactivity in food and water produced by people (anthropogenic sources) in addition to those that are naturally occurring. Other isotopes of importance inthisfieldarethefissionproducts '"CS, I3'I, and ""Sr, other radioiodines, and the transuranics, mostly plutonium isotopes. Cesium compounds are generally very water soluble and therefore are available for absorption into the body, concentrating in muscle tissue. Because of this absorption i n human and food animal muscle tissue, its relatively high fission yield, its half-life of 30 years, and the high energyof its beta emission,'"CS is considered a very important isotope i n public health considerations i n the event of an accident involving fission materials. "'CS is also considered an indicator of fission product contamination since it is so easily recognized during gamma spectroscopy. Iodine is concentrated in the thyroid and in mammalian milk; therefore radioiodines are considered important public health problems in the event of environmental contamination. Iodine compounds are generally water soluble and thus are available for absorption into the body. The fission product l3II has a half-life of 8 days and a high-energy beta emission with associated gamma rays that can cause biological damage in the thyroid and nearby tissue. Other radioiodines also can be a food and water problemif released into the environment. '"I (half-life of 13 hours) and (half-life of 60 days) are used medically, primarily for diagnosis and treatmentof thyroid conditions. Hospital, patient care, and medical clinic effluents can cause contamination of food and water with these isotopes. Strontium is water soluble in many of its chemical forms and is chemically similar to calcium. Therefore it can concentrate in bone. "'Sr, with its progeny ""Y, is a fission product which, because of its easy incorporation into the body, its half-life of 28 years, and its difficulty of removal from the bone, is considered an important isotope of public health interest in the event of release into the environment. The transuranics, mostly plutonium, generally are insoluble in most of their chemical forms.Thusincorporationinto the body isdifficult.Ingestionprobablywouldleadto some exposure in the gastrointestinal tract as the material passes through, with no longterm uptake. The public health problem with these isotopes, in less than extraordinary amounts, generally is thought to be due to inhalation as particles into the lungs, where the alpha emissions can cause biological damage to the tissue near the site of deposition (1).
C.
Food Chain Concentration of Radioactivity
When radioactive materials are freed into the environment, just as with their nonradioactive counterparts, concentration of the material may occur within certain plants and ani-
ater
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mals. Thus a fallout of fission products onto grazing land may lead to concentration of specific isotopes in certain parts of plants, which can then be ingested by foraging animals and concentrated in the muscle tissue we eat or in milk we drink. In that way, concentrations of materials may be higher i n food animals, and therefore i n humans, than in the original contaminated environment. The concentrationof materials throughoutthe food chain dependson various factors. In some way, the material is released into the environment, making it available to the food chain. Naturally occurring materials are available for sorption by some plant or animal. The material may be found in water that is used for irrigation and then absorbed by plants. The water may be used for drink by food animals, thus concentrating the material in organs that are then eaten by humans. The material may be present i n the soil and then incorporated into a plant that is eaten directly by humans or fed to food animals (9). In the case of anthropogenic sources, similar scenarios apply. Effluent containing radioiodines from a patient study maybe released from a hospital into the sewage system, which then flows into a river from which food animals drink or humans harvest fish. The iodines concentrate in the thyroids and the milk of the animals; some iodines will then be ingested by humans eating the fish or drinking the milk from the animals. Since milk is a primary food source for infants and young children, the problem of radioiodines in milk can be a real concern in the event of milk contamination (12). It is easy to envision other scenarios, as with accidents involving transportof radioactive materials in which a breech of the containment leads to environmental contamination and then to contamination of the food or water supply. To cope with potential public health problems, nuclear power plants are required to have emergency plans in the event that an accident may release any radioactive materials into the environment.
111.
ACCIDENTAL CONTAMINATION OF FOOD AND WATER
A.
Sources of Contamination
Food and water may become contaminated with radioactive materials from natural sources, of natural uranium in the soil, or such as plants grown in an area with a large amount from an accident involving natural or anthropogenic radioactive materials, such as from a power plant. One of the most famous instances of contamination of food and water from anthropogenic sources was that associated with the nuclear power plant accident at Chernobyl in Ukraine in the former Soviet Union. The accident, in April 1986, resulted in a spread of radioactive contamination worldwide. The former Soviet Union reported 3 1 deaths due to burns and radiation dosesin the short term following the accident. Public health estimates of the worldwide impact range into the tens of thousands of early cancer deaths due to population exposures. Even at the highest estimates, except for populations very near the accident site, the total deaths are expected to be statistically unobservable ( 1 3). The major point is that the accident at Chernobyl, in fact, did result in contamination of foodstuffs around the world, and several governments outside the Soviet Union did take steps to protect their populations from unnecessary exposures ( 14,15).
B. InterdictionTechniques 1. Guidelines for Interdiction Guidelinesexist,internationallyand within mostcountries,concerning the maximum must be amounts of radioactive materials allowed in food and water before something
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done to protect public health and safety. Traditionally, maxinlum levels have been set to ensure that no member of the public receives nlore than S mSv from a combination of short-lived and long-lived isotopes. The levels have been set by such international bodies as the International Council on Radiation Protection (ICRP). while i n the United States the advisory levels have been set by the National Council on Radiation Protection and Measurements (NCRP). In the United States, federal and state regulatory agencies generally have followed the advisory guidelines, but often other considerations have ledto stricter regulations. Specifically, in the United States, the Environmental Protection Agency (EPA) has set limits on the amounts of naturally occurring radioactive materials that are allowed in drinking water, and various states have set their own standards, generally following those of the EPA. Limits have beenset i n picocuries per liter for radium (S pCi/L), tritium (20,000 pCi/L), ""Sr (8 pCi/L), and uranium (20 pCi/L) i n public water supplies. The levels have been set at an approximate lifetime cancer risk of 1 in 10,000, corresponding to an exposure of 0.04 mSv dose equivalents per year ( 16). Similarly the EPA has set standards for radiological contamination of foods. The standards in the United States are enforced by the Food and Drug Administration (FDA). The Protective Action Guide (PAC), the level for '"I at which steps must be taken to avoid ingestion of the foods, has been set at IS00 pCi/kg for infant food, including milk, and at 8000 pCi/kg for other foods. For radiocesium, the PAC level has been set at 10,000 pCi/kg irrespective of the type of food (17). The concern about radioiodines has led to one type of radioprotective drug that can be made available in the case of population exposure. Potassium iodide (KI) can be taken to load the thyroid with iodine, making it less likely to absorb more iodine. If the K1 is taken prior to or within 2 hours of exposure to radioiodines. reduced absorption of radioiodines is possible. Because K1 can block the thyroid, some members of the public have concluded that it is a protective drug forany radiation exposure, and some public demands were heard for its distribution in the United States after the Chcrnobyl accident. It must be emphasized that K1 is to be used only for radioiodines, as it is useless for other radioit is to be used only when isotopes, that it is a drug with possible side effects, and that its riskhenefit considerations have been determined to be positive by public health authorities. Most state authorities do not stockpile K1 for public use in emergencies: evacuation is considered a better protective measure. However, most states have K1 available for use by emergency response personnel who would not be evacuated in some emergencies and who, indeed, would be expected to enter areas of some contamination i n order to carry out duties related to public protection ( 12,18). The Nuclear Regulatory Commission (NRC) in the United States currently is reexamining its policy on the use of K1 in emergencies ( 19). If food and water do become contaminated, interdiction techniques maybe considered. The ultimate choice of a selected technique depends on various factors. The potentially exposed population must be considered. A s n d l population is protected nmch more easily than a large one. The interdiction techniques selectedmust do less harnl than would the exposure to the contaminated food and water. Thus the risk to the population of removing food must be balanced against the statistical health risk of the population consuming contaminated foods. Consideration must be given to the problems of food distribution, availability of replacement foods, familiar foods versus unfanliliar replacements, ethnic and religious food preferences, and a nutritious, balanced diet.
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2. Measurements The steps to be taken are dependent upon the level of contamination; therefore analytical techniques and dependable laboratory facilities must be available to determine the mount of that contamination. In the early phasesof an accidental contamination,a reliable estimation of the problem may not be available (20). In some accidents (e.g., Chernobyl) from which the greatest amount of contamination is due to fission products, gamma spectroscopy can give a very reliable indication of the level of contamination. For some isotopes (e.g., tritium or “C), the analytical method of choice may be liquid scintillation counting. Other isotopes may require low-level alpha or beta counting instrumentation. Chemical separation techniques may be necessary for some analyses, thus delaying the time at which data will be available for decision making. In the early stages of a release of radioactivity into the environment, prevention of contamination will be important. Taking meat- and milk-producing animals off pasture and onto stored feed is a fairly simple preventive step. Some fresh fruits and vegetables can be cleaned simply by washing and peeling to remove surface contamination prior to preparation or eating. 3. Storage,Diversion,Dilution, or Embargo If food and water become contaminated, several options are available to regulatory agencies: storage, diversion, dilution, or embargo. Food may be stored for a period of time until the short-lived contaminants decay to a level below accepted guidelines. Making cheese from milk and storing the cheese until radioiodines decay to acceptable levels is an example of the storage option. Food and water may be diverted from human consumption to other uses. There may be industrial applications of the food and water in which the material is processed into nonfood items. Alternately the food and water may be processed into feed for animals that will not be used for human food. Processing into pet foods is an example of such a diversion ( 17). Sometimes food and/or water that is too highly contaminated for human consumption can be diluted with “clean” food so that the contamination in the resulting dilution is less than the action level. In the United States, the FDA generally does not approve such “dilution as a solution to pollution” (17). It is unknown whether countries i n which the food supply is not as diverse and widely available as in the United States would have such qualms. Embargo is a regulatory function by which food is declared unfit for human consumption and its marketing forbidden. This is usually an intermediate step to control the product so that it is held prior to a final disposition decision. The steps taken depend on a variety of factors in addition to those listed above: the particular food or water contaminated, the level of contamination relative to guidelines, andthesocial and economic costs of the steps to betaken. At Chernobyl, the Soviet government used all these techniques to reduce the exposureof the population to contaminated food and water ( 15).
IV.
CONCLUSIONS
A.
Necessity for Surveillance
Real information concerning the amount of radioactivity in food and water is lacking. The U.S. Public Health Service used to performsurveillanceanalyses of “market basket”
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food items collected throughout the United States, especially during the days of aboveground nuclear testing; later that function was performed by the EPA. Unfortunately that program has been cut drastically in recent years due to general budget problems in the federal government. However, without continuing sample collection and analysis, it is impossible to say what the “normal” levels of various radioisotopes may be in our food supply: such information is necessary in order to determine whether low-level contamination has in fact occurred and to determine actions to be taken i n the event of an accident. Some release scenarios may not include notification of public health authorities, either because the release was unknown or because responsible parties do not wish to make a release known. At a minimum, the market basket surveillance program should be reinstated.
B. Consistency of Guidelines Guidelines among agencies must be consistent. It is true that the EPA and the FDA have published guidelines or action levels to be used in emergencies. However, many stateand local agencies set their own guidelinesthat may be quite different from those set by federal authorities. Separate guidelines, leading to different advice to the public in case of emergency, can result in a perception that the authorities do not know what is happening, what a safe level might be, and what the public can do to protect itself. Guidelines, once set, should be changed during an emergency situation only under very strict conditions. One of the sources of confusion at the Three Mile Island nuclear accident in 1979 was that preset guidelines were not followed after the accident occurred (21).
C. Necessityfor Low-Level Effects Research There isverylittleresearch beingdoneconcerninglow-levelradiationeffects.Even though most authoritieshold that such research at near-background levels will not succeed, the public perception is that the scientific “establishment” does not care enough or is not open enough to consider the results to perform that research. Routine monitoring of food and water supplies, mentioned above, can be part of the data collection for such a study. There will be much more data on low-level effects as a result of the Chernobyl accident, but other scientific studies need to be performed with appropriate experimental design; we should not wait for another accidental event to bring to us a population to study.
D. Public Education Finally, it is very important that a real public educational effort be pursuedby the technical community into what is known about radiation, radioactivity, measurement, biological effects, and risk. This field is not so difficult that it is impossible for the public to have some knowledge of its results so as to make societal decisions. When we hear representatives of public interest groups proclaiming that no radiation at all is a standard to be achieved, it is obvious that we have done a poor job, indeed, of passing our knowledge on to the average consumer.
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REFERENCES I . H Cember. Introduction to Health Physics. New York: Pergamon Press, 1980. 2. Exposure of the Population of the United States and Canada from Natural Background Radiation. Report no. 94. Bethesda, MD: National Council on Radiation Protection and Measurements.1987. 3. Public Radiation Exposure from Nuclear Power Generation in the United States. Report no. 92. Bethesda, MD: National Council on Radiation Protection and Measurements, 1987. 4. CM Lederer, JM Hollander, I Perlman. Table of Isotopes. New York: John Wiley I% Sons, 1967. 5. RadiationExposure intheU.S.PopulationfromConsumerProductsandMiscellaneous Sources. Report no.95. Bethesda, MD: National Council on Radiation Protection and Measurements,1987. 33. Washington, DC: International Commission 6. Radiation Quantities and Units. Report no. on Radiation Units and Measurements, 1980. 7. National Research Council. Health Effects of Exposure to Low Levels of Ionizing Radiation. Washington, DC: National Academy Press, 1980. 84. 8. General Concepts for the Dosimetry of Internally Deposited Radionuclides. Report no. Bethesda, MD: National Council on Radiation Protection and Measurements, 1985. 9. Radiological Assessment: Predicting the Transport, Bioaccumulation, and Uptake by Man of Radionuclides Released into the Environment. Report no. 76. Bethesda, MD: National Council on Radiation Protection and Measurements. 1985. 10. Ionizing Radiation Exposure to the Population of the United States. Report no. 93. Bethesda, MD: National Council on Radiation Protection and Measurements, 1987. Its Daughters. Report no. 11. Exposures from the Uranium Series with Emphasis on Radon and 77. Bethesda. MD: National Council on Radiation Protection and Measurements, 1987. of Radioiodine.Reportno.55. intheEventofReleases 12. Protection of the Thyroid Gland Bethesda, MD: National Council on Radiation Protection and Measurements, 1977. 13. LR Anspaugh, RJ Catlin, M Goldman. The global impact of the Chernobyl reactor accident. Science242:1513-1519,1988. 14. IAEA technical assistance in support of contamination monitoring following the Chernobyl accident. Int Atomic Energy Agency Bull 33(2):15-19, 1991. 15. The international Chernobyl project. Int Atomic Energy AgencyBull 33(2):4-13, 1991. 16. DC Kocher. Perspective on the historical development of radiation standards. Health Physics 611519-527,1991. 17. Food and Drug Administration. Accidental radioactive contamination of human food and animal feeds: recommendations for state and local agencies. Fed Reg 47:47073-47083, 1982. 18. Conference of radiation control program directors, Inc. Reevaluation of policy on KI. Newsbrief 4 November, 199 1. 19. Nuclear Regulatory Commission. Potassium iodide. Fed Reg 55:39768-39769, 1990. 20. BW Emmerson. Intervening for the protection of the public following a nuclear accident. Int Atomic Energy Agency Bull 30(3):12- 18, 1988. 21 M Rogovin, GT Frampton Jr. Three Mile Island, A Report to the Commissioners and to the Public. Washington, DC: Nuclear Regulatory Commission, 1980.
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19 Food Irradiation
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Introduction57 1 Purposes o f FoodIrradiation572 History of Food Irradiation572 RadiationConditions572 Changes in IrradiatedFoods572 PanaceaorPanic?S74 FoodIrradiationMyths574 Regulation oftheIrradiationProcess575 SupportfortheIrradiationProcess576 References 577
INTRODUCTION
Food irradiation is the process of treating food and food products with radiation. The process consists of exposing the food to some source of ionizing radiation for a sufficient time that is necessary for the desired effects to occur. Sources of radiation may include particle accelerators, essentially high-energy and high-dose X-ray machines, or radioisotopes, such as ""CO or '"CS ( l ) . In a typical food irradiation facility, the packaged food is transported via a conveyor belt into the shielded irradiation area. The packages are kept in proximity to the radiation source fora time governedby the effect desired.The conveyor belt then leads the irradiated food outside for further processing, storage, or shipment ( 2 ) . (See the Chapter 18 on radioactivity i n food and water for a discussion of radiation, its effects, and its measurement.) The same sources of radiation and techniques have been used for many years to sterilize medical supplies such as bandages, instruments, tubing, and implants (3). At least 39 nations (including Belgium, France, Hungary, the Netherlands, Argentina, Brazil, Denmark, Finland, India, Indonesia, Israel, Norway, the United States, Yugoslavia, and Russia) have approved the process for at least some food products ( 1 ).
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II. PURPOSES OF FOOD IRRADIATION Radiation is used in food processing for several reasons (in order
of increasing dose):
1.
Reduce insectinfestation.Theprocesskillsinsectswithoutleavingpesticide residues. 2. Increase of theshelflife of the food so that it willbeavailablefor a longer time and throughout a wider geographic area. Specifically, sprouting canbe reduced in potatoes and onions; blackening can be reduced in shrimp. 3. Removal of spoilage organisms so that the food can be stored under wider environmental conditions. Milk, milk products, and meats can be stored hermetically sealed for long periods at room temperatures. 4. Control of disease-causing organisms so that the food is more hygienic for use. Trichina, salmonella, and E. coli can be reduced in meats and poultry. 5. Sterilization of the food so that it may be consumedby people who are especially susceptibletovariousorganisms.Theprocesshas beenused forpreserving foods for astronauts and to supply a more varied diet to those with suppressed immune systems, such as cancer and AIDS patients (4). Because of the effects required in food, the radiation doses used in the processing of food are necessarily much higher than normal environmental levels. Increasing shelf life and insect deinfestation require the least radiation dose; sterilization requires the highest doses.
111.
HISTORY OF FOOD IRRADIATION
Research on food irradiation techniques and effects began inthe 1940s whenthe U.S. Army began testing the application of the process for field rations. After years of testing, the process was abandoned because the techniques attempted at the time reduced the food to unpalatable states. Later, research was continued to determine optimum environmental conditions for irradiation by changing the doses delivered and the temperatures and ambient gases used in the process. Today the process produces foods that are difficult to distinguish from unitradiated foods (4).
IV. RADIATION CONDITIONS Radiation conditions must be developed for each particular food item. Even different varieties of the same food, different speciesof cherries, for example,need specific irradiation conditions to deliver an acceptable product. Many foods are also very sensitive to the dose levels delivered; a particularfoodmayverywelltoleratethedosesnecessaryto increase shelf life, but be degraded to unacceptable states when sterilization is attempted.
V.
CHANGES IN IRRADIATED FOODS
If the process is performed properly, chemical and physical tests reveal little hint as to whether most fooditemshadbeenirradiated.Alimitednumber of foodscanexhibit
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distinguishableanalyticalresultsfromelectronspinresonanceorthermoluminescence after irradiation. However, biological testing may yielddata which raise suspicions of irradiation. The product may be much more clean biologicallythan its unirradiated counterpart. Therefore the only generally applicable test that has thus far been developed for irradiation exhibits the result that the food itself is more hygienic than other examples of the same food item (5). Radiation does, in fact, produce changes in food; that is the reason it is used. At the lowest levels, used for insect deinfestation, the radiation kills insects which may be present naturally in the food. Food irradiation has been used for this purpose worldwide for years. Currently almost all spices imported into the United States have been irradiated for this purpose. Spices generally are grown in tropical areas of the world where insect infestation is a problem. Thus irradiation results in a much cleaner product. The same technique can be applied to grains. It has been estimated that the use of food irradiation in this way can reduce the 25% of the world food supply which is lost to insects, bacteria, and rodents every year ( l ) . Food irradiation is very beneficial for increasing the shelf life of many foods. Potatoes, onions, and garlic can be irradiated to prevent sprouting of the stored product. Thus they can be shipped over long distances, supplying people with these food items over a longer period of the year (6). The technique can be used to reduce spoilage organisms in certain foods such as meats and milk, thus allowing storage at room temperature for longer periods. Therefore these foods can become availableto more people, especially in nonindustrialized countries where refrigeration is expensive or nonexistent (6). I n the United States we have problems with trichina i n pork, E. coli in beef, salmonella in chicken, and campylobacter in various foods. These disease-causing organisms can be destroyed in those food products with the use of food irradiation. Currently 9100 people in this country die and 6.5 million cases of foodborne illnesses occur each year, numbers which could bc greatly reduced by using the technique (4). In hospitals caring for patients with compromised immune systems, food preparation is extremely important since even the normal bacterial contamination in foods served to such patients can be lethal. For many years, these hospitals have used irradiated foods at the sterilization level for their patients. Thus the patients are allowed a diet containing items which could not be sterilized by other means, such as icc cream (4). Studies on the effects of irradiation on foods have been done overthe past 50 years. The results of those studies include the following: Irradiation does changethe nutritional valueof some foods,but the change is compaas cooking or rable to those observed in other preservation techniques, such canning. With current techniques, foods have no distinguishable changes in taste or texture. Any changes noted are within the normal parameters for that food item. Foodirradiationdoes not cause any immunity in microorganisnls as is observed with chemicals and drugs. Since the process is a physical one, at energies far higher than those of chemical bonds, no development of resistance by the microorganisms has been observed or is expected. Food irradiation does not induce radioactivity into the treated foods. In the same way that having a medical X-ray docs not cause the irradiated patient to become radioactive, food does not become radioactive through this process.
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There are no increased health risks causedby the degradation of appropriate packaging materials during treatment. There is no increased risk caused by irradiation of food additives or residues, such as pesticides, which may be present during irradiation ( 1). It should be observed that several of these effects could,if they were of real concern, be observed intheresults of irradiation of medicalsupplies;rememberthatthesame As aresult of that techniques,sources, andradiationlevelsareusedinthatindustry. radiation, no radiation-resistant organisms have been produced, no radioactivity is induced in the medical supplies, no degradation of appropriate packaging occurs, and no harmful radiolytic products are produced due to irradiation of minor components of the medical supplies.
VI.
PANACEA OR PANIC?
As with any process, food irradiation is not a panacea. include.
Some problems with the process
Doses to the foods must be carefully controlled so as not to dcgrade the quality of the food. Irradiation conditions must be individually determined and delivered for each food item. Theeffects of theirradiationareonlyfortheirradiatcdconditions. If grainhas beenirradiatedforinsectdeinfestation, it mustbekept from further insect infestation; if food has been sterilized, it must be kept from further contamination to maintain that sterilization. If the food becomes recontaminated, it should not be reirradiated since radiation effects are cumulative and the resulting food may have undesirable qualities. It is conceivable that the irradiation dose could be large enough to eliminate spoilage organisms, yet small enough so that pathogenic organisms survive. Thus an individual could be led, through lack of off odors and flavors of contaminated foods, into believing thata given food item is fit for consumption when it may not be so. Strict regulations concerning radiation doses must be followed to prevent this effect ( I ) .
VII. FOOD IRRADIATION MYTHS
I n the United States, the public has several misconceptions concerning this technique. TO correct these misconceptions, the following facts are important to remember: lrradiation does NOT make the food radioactive. The energies of radiation used are much too low to induce radioactivity in the food. The machines used function such that a lnalfunction will not result i n higher energies, but simply too little radiation for the desired effect. of nutrients. Foods which have been irradiated do NOT lose substantial amounts Such losses are comparable to those observed in other food preservation techniques, such as canning and cooking.
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The technique is NOT being promoted so that we will become so dependent on the ""COand '"CSthat we will need nuclear power simply to produce these isotopes. In industrialized countries, the preferred technique is particle acceleration, a type of high-energyX-ray machine. In nonindustrialized countries, isotopes are probably used because of the instability of electric power in those areas. The argument that there has not been a general test developed for irradiated foods is telling to some people. However,that argument seems to be a proirradiation one; with ourgreatanalyticalcapabilities,wecannotseeanydifferences, chemicallyorphysically,betweenirradiated andnonirradiatedsamples of most food items. Thus perhaps any difference is really irrelevant. Because no general test exists for irradiated foods, and some people are fearful of the process, the FDA has prolnulgated rulesthat all irradiated foods be labeled as such. Some people arguethat the widespread use of these techniques will require transport of radioactive materials with the inherent danger of that shipment. However, particle accelerators are NOT radioactive, and do not pose this problem. If isotopes are used, they are the very same isotopes and quantities which have been used for years medically, with the same transportation problems, and the same lack of public health impact. Some people are concerned about radioactive waste being produced andits inherent danger associated with transport and disposal. However, again, these are the same isotopes that are used medically with the same disposal problems and history of safe transport and disposal. Also, accelerators donot pose this same problem. Finally, some people are concerned aboutthe workers at plants where the irradiation is conducted. The radiation industry has a long, successful history of control of exposures to workers at various sites. As one example, workers are not allowed to occupy the area where the irradiation process is taking place. Before entry, the source needs to be secured. Also,all employees needto be monitored for radiation exposure in the same ways employees are in medical facilities that use radiation techniques (1).
VIII. REGULATION OF THE IRRADIATION PROCESS
I n the United States, the FDA has promulgated rules and regulations for control
of the irradiation process. For its part, the USDA has issued regulations based on the FDA rules and regulations. The exposure conditions must be controlled; records must be kept of the lots, exposures, and types of food; and the records must be made available to government inspectors. Food items must be labeled at sale if the process is used; for irradiated foods which will undergo further processing prior to sale, shipping papers must indicate that irradiation was used and the levelof radiation to which the foods were subjected. Reirradiation of the same batch of food is not allowed. Exposure is to be the minimum necessary for the expected result (7). Inspection procedures are the sameas for any other food process, exceptthat irradiation conditions must also be monitored; this requirement is directly analogous to the requirement that temperatures nlust be controlled and recorded i n food cannery processing.
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Table 1 ApprovedIrradiationUses
Food
in the United States
Approved use
Dose
~
Spices and dried vegetables Seasonings Dry or dehydrated enzyme Preparations All foods Fresh foods Poultry Rcd meat (such as beef,
lamb, pork)
Decontamination and control of insects and microorganisms Control of insects and microorganisms Control of insects Delay of maturation Control of disease-causing organisms Control of spoilage and disease-causing organisms
30 kGy 10 kGy
1 kGy 1 kGy 3 kGy
4.5 kGy (fresh) 7 kGy (frozen)
Thus good manufacturing practices (GMPs) and good radiation practices must be followed throughout the process. Quality control records must be kept; irradiation units must be inspected regularly for proper operation; and shipping documents must indicate that the lot has been irradiated to prevent reirradiation of the same items later in processing. Only foods meeting microbiological criteria and other quality standards may be accepted for irradiation; thus the process cannot be used to “clean up” dirty food items ( 2 ) . Table 1 indicates the uses for food irradiation as approved in the United States by the FDA (3).
IX. SUPPORT FOR THE IRRADIATION PROCESS Several professional public health associations have issued policy statements in support of the food irradiation process. The American Medical Association (AMA) has adopted the following recommendations put forth by its Council on Scientific Affairs: The AMA affirms food irradiation as a safe and effective process that increases the safety of food when applied according to governing regulations. The AMA considers the value of food irradiation to be diminished unless it is incorporated into a comprehensive food safety program based on good manufacturing practices and proper food handling, processing, storage, and preparation techniques. The AMA encourages the FDA and the USDAto continue the requirement that all irradiated fruits, vegetables, meats, and seafood carry the international logo that has become recognized as indicating that the food has been subjected to gamma irradiation. The AMA affirms the principle that the demonstration of safety requires evidence of a reasonable certainty that no harm will result but does not require proof beyond a reasonable doubt (i.e., “zero” risk does not exist) (8). TheAmericanDieteticAssociation(ADA)haspublished statement (9):
the followingposition
Itis the position of thcAmericanDieteticAssociationthatfoodirradiationisonc way to enhance the safety and quality of the food supply. The ADA encourages the
Food Irradiation
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government, food manufacturers, food commodity groups, and qualified dietetics professionals to continue working together in educating consumers about this technology. The Health Physics Society (HPS), a professional scientific organization concerned with protecting people and the environment from radiation, has concluded that ( I O ) Food preservation by irradiation offers great potential benefit with no radiation risk to the consumer. is firmly The technical feasibility of safely preserving certain foods by irradiation established by experimental evidence and experience. Federal regulatory bodies responsible for such matters are proceeding cautiously i n approving new applications of this technology and are basing their approvals/ disapprovals on the best scientific and technical infomation available. Foods preserved by FDA reconmended irradiation procedures d o not become radioactive or toxic as a result of irradiation. The application of this technology to the betterment of mankind should neither be permitted nor precluded on the basis of misinformation.
REFERENCES I.
2.
3. 4. 5. 6.
7. X. 9.
IO.
Facts About Food Irradiation. Vienna, Austria: Intcrnational Consultative Group0 1 1 Food Irradiation, Joint FAO/IAEA Division of Nuclear Techniques in Food and Agriculture. l99 1. H Kocol. Food irradiation issues, technical and public, in the United States. I n : Seventh InternationalCongressoftheInternationalRadiationProtectionAssociation.Sidney.Australia, 1988, pp. 18-21. J Henkcl. Irradiation: ;I safe measure for safer food. FDA Consunwr (May-Junc): 1998. BL Cohen. Irradiated food: Is thcrc a need? Nuclear News (June): 35(8):60-68. 1992. P Loahamnu. Food irradiation: fact or fiction? Int Atomic Energy Bull 32(2):44-48. 1990. J Van Kooij. Food irradiation makes progress. I n t Atomic Energy Bull 26(2): 17-21, 1984. General Accounting Office, Food Irradiation, Federal Requirements and Monitormg. GAOl HRD-90-1 18. Washington. DC: U.S. Government Printing Office, May 1990. AMA House of Delegates. Irradiation of food. Procecdings, Dcccmber 1993. p. 364. Anmican Dietetic Association. Position of the American Dietetic Association: food irradiation. J AmDietAssoc 96:69-92, 1991. Health Physics Society. Position Statement on Food Irradiation. McLean, VA: Health Physics Society, 1988.
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20 Drug Residues in Foods of Animal Origin
Historical Perspective 579 A. Modern animal production and practices 579 B. Toxicological significance o f residues 581 C. Risklbenelit assessment 583 D. Responsibility, compliance, and enforcement 583 E. Ilnportance o f analytical methodology 584 11. Signilicnnt Veterinary Drugs S86 1.
A. B. C. D.
Antiprotozoal drugs
S86
Anthelmintics (dcwormers) 587 Antibiotics, antibacterials. and antimicrobials Growth promotants 593 111. Future Developments 594 Refcrences 595
1.
A.
5x9
HISTORICAL PERSPECTIVE Modern Animal Production and Practices
I n our quest for sustenance i n the form of protein food, we have evolved from hunter to producer, through individual to communal farmer, and come to rely upon complex massproduction animal husbandry practicesin order to supply a large percentageof our protein foods. Sophisticated animal husbandry practices are necessary for modern-day civilizations to prosper and flourish. Such operations involve large numbers of animals raised simultaneously in high-density populations under carefully controlled conditions. Ideal environmental conditions that include scientifically formulated feeds and strict medical regimes serve to produce high-quality, unifornl products. In addition, high-density populations, or confined animals. minimizes the ability of the animals to move about, resulting in improved feed conversion. The most totally integrated mass-production operations are 579
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evidenced inthepoultryandaquacultureindustries,with swine and bovine production being less integrated. Young animals or hatchlings may be supplied as feeder stock to individual operations in which they are fattened for market or used for egg production. Although the individual animals may come from nmltiple local producers, they are fattened and maintained under exacting conditions. Mass-production animal husbandry practices requirea strict medical regime in order to ensure the opti1nunl health and welfare of the animals. In small farming units, animal care can be designed for individual animals as the need arises. The general well-being and health of small herds or flocks can be monitored continuously, and appropriate medications or treatment administered when a condition requiring them is noted. As herd and/ or [lock size increases, the ability to monitor the health of individual animals becomes more difficult. High-density populations prevalent in present-day mass-production operations are susceptible to infections, infestations, parasitism, and stress. Such high-density populationsrequirecarefulmanagement in order to maintain the health and the feedconversion efficiency of the animals. In this regard, drug use in modern animal production enhances producers’ abilities to produce high-quality products at a reasonable cost. Drug use has become an integral part of modern mass-production animal husbandry. For purposes of this chapter, the termv c ? e r i m y drlrgs is defined as any substance applied oradministered to any food-producinganimal.Regulationsgoverning the use, dosage form, and withdrawal times for veterinary drugs can be found in the Code of’ Federd R q d t r t i o r l s ( l ) , Title 21, and other published sollrces (2-4). Veterinary drugs approved for use in animal production and treatment have been thoroughly tested and shown to be effective for their intended use. Guidelines for their intended use are specific in terms of dosage form. formulation, and intended species. The vast array of drugs approved foruse have many applications and can be divided into four broad usage categories: (a) therapeutic. (b) prophylactic, (c) diagnostic, and (d) for modification of physiological function or behavior. Therapeutic treatments are used to cure a diagnosed disease or to prevent one from occurring. Preventive treatment in a mass-production operation takes the form of prophylactic administrations that require subtherapeutic quantities of drug being administered routinely along with feed or water. Prophylactic treatment is practiced in order to minimize the potential for rapid transference of disease throughout a herd or flock. Such treatments also are used as an aid in promoting growth. The growth-promoting aspect of prophylactic treatments can be a direct consequence of treatment with a growth promotant or, alternatively, of treatment with antibiotics that will aid growth by promoting the general well-being of the animal, thus enhancing feed conversion. Drug use for diagnostic purposes is implicit and is not discussed further in this chapter. Animal stress associated with high-density populations or transportation can be modified with drug treatment, and drug treatment for behavior modification commonly is administered to animals just prior to shipment to slaughterhouses. When drugs are used in animal husbandry, the potential exists for drug residues to be present i n consumed animal products; this may have human health implications (5). The animals may be exposed intentionally or unintentionally to drugs. Drug residues in food that exceed the tolerance level may result from misuse, illegal use, improper use of over-the-counter drugs, and/or negligencein observing withdrawal times. These veterinary drug residues include the parent compounds and/or their metabolites that may be present i n human foods originating fromtreated animals. At present, the use of these drugs cannot be eliminated completely from modern animal husbandry practices and still enable societies to provide sufficient protein food to feed their citizens.
Drug Residues in Foods of Animal Origin
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I n general, the types of drugs used in animal husbandry do not vary greatly worldwide. There are, however, differences relative to “safe levels,” controls, and permissible usage, depending upon cultural and political forces. The international market in animal products introduces an additional burden upon regulatory bodies to ensure safe, wholeif we are to ensure absolutely safe, some import and export products. Philosophically, drug-free products, the use of all drugs would have to be banned, which is not realistic. In addition, it can be difficult to determine clearly the exact concentration of certain drugs and/or metabolites in animal products. Present analytical capabilities cannot confirm unuse of such terms as “free of equivocally the absence of a given drug. Therefore the drug residues” or “drug free” may be misleading. The elimination of drug use in animal husbandry practices for food animals is unlikely to occur in the foreseeable future. Thus we must rely upon toxicological and metabolic studies to establish safe acceptable levels. In addition, we must utilize advances in analytical capability to assay and monitor for drug residues in foods of animal origin. B. ToxicologicalSignificance of Residues The toxicological significance of drug residues relative to humans depends on several factors: type and amount of drug present, exposure rate and duration, and the sex, age, and health of individuals consuming the drug. Before the toxicological significance of a drug can be ascertained, metabolic studies are performed on animals in order to evaluate the fate of a given drug. Metabolic studies help to characterize the disposition and elimination of the drug and/or its metabolites in any given animal species. Metabolites of the parent drug are then isolated and identified. Metabolic studies are essential in establishing if there is a predisposition of the drug toward a specific tissue, and whether the parent drug or a metabolite should be selected for toxicological testing. The Food and Drug Administration (FDA) is responsible for considering the safety of any substance formed in or on food by a sponsored compound before approving its use under the Food, Drug and Cosmetic Act(6).Safety evaluations are designed to characterize the parent drug and its metabolites and/or degradation productsthat may be present in consumed animal products. The total drug residue must be considered. Initially, however, it is the responsibility of the drug sponsor to determine the metabolic profile of the drug and to develop toxicological data relative to the amount, persistence, and chemical nature of the total drug residue that may be present in edible tissue. Information obtained from metabolic studies for specific drugs in production-class and laboratory animals is used to evaluate the drugs’ toxicities and/or carcinogenicities. If a chronic bioassay demonstrates a compound to be carcinogenic, then additional testing will be required to include structural elucidation or in vitro genetic toxicity testing of the suspect metabolites. Depending upon the drug, the animal being tested, and the experimental data desired, metabolic studies generally involve the following: Total residue depletion studies Metabolic studies in target animal species (production animals) Metabolism studies in laboratory animals Selection of metabolites for toxicological testing Identification of target tissue and marker residue Data from each of these items is necessary for establishing risk.
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Total drug residue depletion studies involve the measurement of the depletion of total drug-related residues from edible tissues of target animals. In general, total drug residue depletion studies involve the use of radiolabeled compound(s) with label type, location,andspecificactivityappropriateforthestudy.Radioactivity-measuringtechniques will allow the investigator to calculate residue concentration levels at appropriate depletion intervals and give an indication of the amount of drug/metabolite remaining in specific tissues over time. The study itself must take into account such factors as species variation, age, dose, sex, and treatment duration, which may affect the type and rate of metabolite formation as well as the rateof parent/mctabolite excretion. The residue depletion study helps to establish withdrawal times and should be performed on market class animals using a treatment dose and at an interval appropriate for the particular drug. Metabolic studies are necessary to determine the metabolic fate and persistence of drugs and/or metabolitesin edible animal tissues. Such studies generate data relativeto the extraction. fractionation, separation, isolation, and identificationof compound metabolites. Chemical characterization of major metabolites provides useful information in drug evaluations. Drugs and/or metabolites that demonstrate a propensity to be retained i n edible animal tissues for a prolonged time may be toxicologically significant. and may require additional evaluation and testing. Furthermore, metabolic studies may indicate the presence of nonextractable radiolabeled compounds and/or metabolites otherwise known as hound residues. Present technology makes it difficult to assess the toxicological importance or unimportance of bound residues accurately. Such residues may or may not be released when animal-derived foods are consumed. It is unknown whether these released residues pose any health risk because scientists have been unable to characterize or define fully the parameters needed to extract these residues in their native form. Rigorous chemical techniques utilized to free suspected bound residues generally destroy or chemically alter the compound(s), making characterization difficult or impossible. Additional testing of theseboundresiduesmayberequiredtofurthcr cnhance an understanding of their potential risk. An important adjunct to nletabolic studies i n target production animals is metabolic studies of drugs and/or metabolites in laboratory animals. Drugs and/or metabolites that mayexist in consumed animal products may be toxicologicallysignificantinhumans. Laboratory animals are used to evaluate the toxicological aspects of such drugs and/or metabolites, and such studies involve similar procedures a s evaluations of conlpounds in target production animals. Toxicological testing takes into account the proposed drug usage i n animal husbandry and probable exposure of humans to the compounds and/or metabolites. possible biological effects on humans, and the effects on an array of biological testing systems. Toxicological testing in laboratory animals establishes a dose that produces an adverse biological effect in the test animals. It also should establish a dose that does not produce a toxicological or pharmacological effect or no observed effect level (NOEL). Such tests can encompass chronic bioassay, long-term animal studies. teratology studies, and specializedstudies to assess effects onneurologic,immunologic,hormonal,andreproductive systems. Thc metabolic and toxicological studies establishthe last tissue from which residues deplete to ;In acceptable toxicological tolerance level. These tissues are designated as the target tissues. A marker residue also is determined during metabolic studics: this is the residue (parent or metabolite) with a concentrationthat correlates to the total residue ~011cel1tration after depletion. Withdrawal times are established; these are the times required
Drug Residues in Foods of Animal Origin
583
for the drug or metabolites to deplete to a safe level in the target tissue. A tolerance level is determined; this is defined as the maximum residue of a drug that is allowed legally in food. The tolerance level is determined based upon the toxicological data relative to thenature of theresidue,level,toxicity,andexposure.Allappropriatemetabolicand toxicological data are used in establishing the toxicological significance of drugs and metabolites. Risk assessment protocols have a safety margin factored in and are variable and depend on many factors not discussed in this chapter.
C. Risk/Benefit Assessment Data obtained from metabolic studiesand toxicological testing are used in risk assessment in order to establish “reasonable” risk with due consideration to the Delaney clause of the Food, Drug and Cosmetic (FD&C)Act (409(C)(3))(6). Reasonable risk is a theoretical calculation based on toxicological data.It applies a safety factorto the toxic dose observed in laboratory animals. Because of this safety factor, an overtolerance residue usually does not constitute an immediate health threat. Some drug residues can produce chronic adverse effects and maypose a health hazard if consumed by individuals sensitive to that particular drug. Residues that are suspected of causing cancer in laboratory animals are of special concern becausethey may pose an unacceptable health hazardto humans through repeated low-level exposure. Unfortunately the effects of long-term exposure to low-level residues are difficult, if not impossible, to determine. Also, the transfer of toxicological data obtained from animal studies to humans is inherently flawed, however. there is no better way to evaluate drugs. By factoring in a considerable safety margin, we can reasonably assure drug safety. Routine reporting of observed adverse effects in treated animals by animal drug sponsors is required by New Animal Drug Application (NADA) guidelines and acts as an indicator of potential problems ( 1 ). The FDA, underthe Food, Drug and Cosmetic (FD&C) Act, has the sole responsibility for determining the safety and efficacy of animal drugs (6). Metabolic studies and toxicological testing carried out under the FD&C Act provisions provide considerable data relating to drug and/or metabolite disposition, depletion, and elimination. Such information provides a database by which compounds can be evaluated and risk assessments made relative to human consumption and exposure. Such documents as the U.S. Department of Agriculture/Food Safety and Inspection Service’s (USDAIFSIS) Compound Evaluation and Analytical National Residue Program Plan(3) use such datain setting priorities for a compound’s importance in monitoring programs. By taking into account data from metabolic studies, toxicological aspectsof compounds and their metabolites, consumption rates, and age and sex of consumers, the relative importance of compounds can be tabulated and priorities can be set. While the overall process is complex, it does allow for a working model by which to allocate resources. It is important thatinternationalregulatorybodiesmakeaneffort to standardize protocols used to assess the toxicological significance of veterinary drugs. International trade in the global marketplace dictates that the world community set about the task of to preserve a free flow of trade in standardizing drug usage and residue levels in order animal products while protecting the public health.
D.
Responsibility, Compliance, and Enforcement
The FDA, Environmental Protection Agency (EPA), and USDA share responsibility for ensuring that only safe levels of drugs will be present in raw meat and poultry. as well
584
Long and Roybal
as in aquaculture species. The FDA, under the FD&C Act (6), is responsible for (a) ensuring the safety of drugs given to food-producing animals, (b) setting a limit or tolerance on the amount of an animal drug allowed in food, and (c) preventing the marketing of raw meat and poultry containing residues above tolerance levels. The FDA may establish a withdrawal period priorto slaughter for treatedaninuls from whichfood will be derived. The EPA is responsible for(a) determining the safety and effectivenessof pesticides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7), (h) setting tolerances forpesticideresidues in foodunder the FD&C Act (6), and (c) regulating,underthe Toxic Subslances ControlAct (g), the introduction into the environment of 1nost chemical substances not regulated as drugs, pesticides, or food additives. The USDA, under the Federal Meat Inspection Act (9) and the Poultry Products Inspection Act ( I O ) , has the responsibility of preventing the marketing of adulterated raw meat and poultry, including those containing residues in excess of tolerance. The system is rather complex, and the interactions among the regulatory bodies can be explained best with a couple of examples. Simply, the USDA identifies illegal residues of drugs or pesticides i n raw meat or poultry, informs the FDA or EPA of the illegality, sends samples to the FDA (drugs and pesticides) or the EPA (pesticides) for follow-up, and determines what corrective actions may be necessary. The FDA is responsible for enforcing the regulatory statutes under the FD&C Act. The FDA may analyze the samples received from the USDA to determine if a violation has occurred. The FDA determines if a violation has occulred, the chain of evidence is secure, and the case development for the illegal residue has been complete and timely. If there is a violation, then the FDA has theoptionto (a) issueinformationletters tothe growersrelativetotheviolation, (h) prosecute or issue an injunction preventing the sale of the product, or (c) issue a seizure order for products with serious violations involving the deliberate misuse of drugs by owners. However, the Inisuse of animal drugs, in itself, does not violate the FD&C Act. The FDA must prove that the misuse resulted in the marketing of adulterated product. The EPA acts on the USDA’s notification, relative to pesticides, in their own manner: this is not discussed here. The FDA also monitors for pesticides in foods. The interaction betweenthe USDA and the FDA requires close coordination in order to develop a case properly within a reasonable time frame. If the case development has not been complete, it can be difficult to hold responsible parties accountable. The USDA is limited in its ability to implement strategies aimed at nlininlizing potential infractions. The USDA can request a pretest of a small lot of animals representative of the entire lot prior to shipment and slaughter. Herds from certain producers may be certified as drug free after considerable documentation and testing and withthe submission of such documentation. Monitoring of drug residues in animal products destined for hulnan consumption is the responsibility of federal as well as state regulatory bodies. Each state has a department or division of agriculture with the responsibility of ensuring that animal products destined for human consumption meet certain standards. States work closely with the USDA and FDA to ensure that animal products are wholesome.
E. Importance of Analytical Methodology Animal-derived human food products that are found to contain drug residues exceeding the tolerance, or safe, level are covered under the Delaney clause. If a food product is found to contain a drug residue exceeding the tolerance levelor to contain an illegal drug, then the food is considered adulterated andunfit for human consumption. Concerns relative
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to drug residues that may be present in animal-derived foods have resulted in intensified testing of both the milk supply and animal products in general. An increasing burden has been placed upon private, state, and federal testing laboratories engaged in analytical testof the ing activities. Analytical capabilities now available have been able to meet many challenges, but not in all cases. Modernfood-animalproductionand intensifiedtesting of animalproductshave placed an extraordinary burden on the analytical chemist. The analytical chemist is challenged by the ever-increasing requirement of testing multiple matrices for multiple tracelevel drug residues. In many cases, drug residue testingin the low ( 1 - 10) parts per billion (ppb) range is extremely difficult to accomplish.Furthermore, the drugresiduelevels being tested are at the very limit of sensitivity of most present-day analytical technology, and there do not appear to be major advances in analytical technology on the horizon. Ultimately analytical capability is governedby the sample preparation and extraction steps, which should be designed to provide “clean” samples for analysis. Sample extracts should be such that interferences are eliminated or minimized to allow for optimum delectability by present-day analytical techniques. Sample preparation is critical in order for analytical methods to be reliable. Future animal drug residue analytical techniques will require precise selectivity, extreme sensitivity, efficiency in terms of both reagents and analytical time, and cost-effectiveness. Analytical methods can be grossly divided into two basic categories: field tests or drug screens, and laboratory assays. First, drug screening methods destined for field use in modern mass-production operations should be such thattheyarerapid, easy to use, easily interpreted, and reasonably specific. These tests should be accurate and sensitive enough to ensure that the drug residue level present in the animal product is within guidelines. The screening test should be such that a specialist is not required to run or interpret theresults,andsuchtestsshouldberapidenough so as notto impede theproducer’s ability to deliver wholesome products to the market for a fair and reasonable cost. A recent review of the various animal drug screening methods was published by Long and Barker ( 1 1). First, immunoassay screening techniques appear to holdthegreatest promise for usable, on-site drug screening. The introduction of the radioiminunoassay technique for the detection of insulin in human plasma under scoredthe utility of the classical antibodyantigen interaction that is the basis for many modern drug screens ( 12). The analytical chemist quickly recognized the potential of this technique for drug and chemical testing. In recent years there has been a dramatic increase in the development and use of competitive binding techniques, or microbial receptor assays (13), for monitoring drug residues. Kits have been developed to detect a variety of veterinary drug and chemical residues, including antibiotics, pesticides, and mycotoxins. These kits are excellent for rapid screening of classes or groups of compounds. These kits can be utilized best i n several ways: to assist producers and field investigators in identifying potential problems, for qualityto improve laboratory efficiency by control requirements, for follow-up investigations, increasing sample throughput, and as a tool i n regulatory actions. The drug screens available currently have limited direct utility in the field. Ultimately, however, such screens will be developed and used i n the field for positive indications as part of an integrated drug residue control strategy. Such screens will compliment and enhance present analytical capabilities. Second, laboratory assays suchas thin-layer chromatography (TLC), gas chromatography (GC), and liquid chromatography (LC) techniques generally are not amenable to
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fielduse, except for TLC. Such analytical techniques require specialized facilities and equipmentand highlyskilledprofessionalsfortheiruse.Analyticalchromatographic methods are used to confirm positive screens. Various analytical techniques used in animal drug residue testing are reviewed by Long and Barker ( 1 l). The development of analytical methods must take into account such intended end uses as whether the method will be used as a screen, for quantification, or for identification purposes. In addition, one should know whether the technique will be used for single- or multiple-component detection, quality control, or regulatory purposes. The method must answer the need. No single technique can satisfy all of the requirements for drug residue testing. The analytical procedure must result in a reasonable identification based on the criteria mentioned above. Advances i n miniaturizedextractiontechniques,field-usabledrugscreeningkits, and analytical hardware will allow us to keep pace with the ever-increasing demand for an integrated drug testing protocol for ensuring the safety and wholesomeness of the food supply.
II. SIGNIFICANT VETERINARY DRUGS The following sections describe various animal drugs that are used, have been used, or have the potential to be misused i n food-animal production. Each drug has specific withdrawal times associated with its use. These withdrawal times are variable, depending on the drug and the animal species to which it is administered. Exact withdrawal times for specific applications have been published ( 1 -4). There are a variety of hunlan health of these drugs, but wehavenot concerns associated with excessive exposure to some attempted to cover all of these. Also. the list of drugs described is not meant to be all inclusive, but rather it is a list of commonly used drugs or drugsthat arc of special interest. We attempt to present the most pertinent information relevant to each drug in a concise manner and provide the reader with relevant references. Also, references pertaining to analytical methodology are included.
A.
Antiprotozoal Drugs
1. Coccidiostats Dimetridazole (DMZ), ipronidazole (IPR), metronidazole (MTR), and ronidazole (RNZ) belong to a class of coccidiostatsknownasnitroimidazoles. DMZ and IPR had been approved for use in turkeys for the treatment of blackhead disease (histomoniasis). MTR is usedagainstbovineurogenitaltrichomoniasis.RNZisactiveagainstenterohepatitis and histomoniasis i n turkeys and for swine enteritis. Nitroinlidazoles have growth-promoting action when administered i n low doses. Based on available data, a common metabolic pathway for the nitroimidazoles appears to involve the conversion to the 2-hydroxymethyl derivative followed by conjugation through hydroxy with either sulfate or glucuronate, and elimination via the urine. Studies have raised safety concerns over the continued use of nitroimidazoles because data indicate that these compounds are mutagenic, tumorigenic, and possibly carcinogenic. These concerns, combined with the potential for other than intended use of these compounds, have caused several countries to withdraw approval and/or ban their use in food-producing animals ( 14- 18).
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2. Antiprotozoals Nitrofuran antimicrobials arc a class of compounds having a common S-nitrofuran skeleton. Since their introduction in 1950, nitrofurans have been used extensively in animal husbandry. They are activei n vivo against gram-negative and gram-positive bacteria. This antibacterial property is derived from their abilityto disrupt bacterial dehydrogenase activity. The S-nitromoiety of these antiprotozoal drugs is the key structural component responsible for their activity. Two nitrofuran derivativesused commonly in veterinary medicine are nitrofurazone, 3-[[(5-nitr0-2-furanyl)methyS-nitro-2-furaldehyde semicarbazone, and furazohdone, lene]-amin0]-2-oxazolidinone. Botharetype B category I1 animal drugs. Nitrofurazone is approved for use in poultry as an aid i n the prevention of coccidiosis and for controlling secondarybacterialinfections. It also isused i n swineproductionasatreatment for necrotic enteritis caused by S t r l r w n d k r c.ho/er~/rsiu.s. It is effective for the treatment of uterine infections i n lactating cattle. Furazolidone is approved for use in poultry production for the prevention of fowl typhoid, paratyphoid, and pullorum, as well as for an aid in the prevention of coccidiosis caused by E. telrellu, E. mw1tri.v. and E. trc.prv/rlirrtrin chickens. It also is effective against infections due to S. t!phinru/.hnr. Furazolidone helps in the treatment of turkey blackhead disease (histomoniasis, enterohepatitis). When used i n swinefeed. i t aids in the prevention of bacterialenteritisandbloody dysentery (19-22). Nitrofurans are absorbed rapidly and extensively metabolized with little of the parent drug being found i n the urine. Furazolidone demonstrates a propensity to be bound covalently to tissue protein and/or other biological macromolecules, making quantitative extractions difficult. Common metabolites of nitrofurans are formed as a result of the oxidation of thefuranring,leadingto a 4-hydroxyderivativefollowedbyconjugationthat forms the glucuronide for elimination via the urine. Alternatively, metabolites are formed as a result of the reduction of the S-nitro group to form a hydroxyl amine that is reduced further to produce an open-chain cyano derivative of the furan ring. This cyano derivative is capable of binding to tissue via a protein thiol attachment or of rapid elimination via the urine (23-25). Data suggest that nitrofuran colnpounds are mutagenic and possibly carcinogenic. Additional coccidiostatic compoundsnot discussed here include nicarbazin and such ionophores a s nionensin. salinomycin, narasin, and lasalocid.
B. Anthelmintics(Dewormers) 1. Benzimidazoles Benzimidazoles, introduced i n 1960, are used widely in cattle, swine, horses, and sheep for the prevention and elimination of parasitic intestinal worms. Some examples of the more conmionly used benzimidazoles are thiabendazole. fenbendazole, oxfendazole. albendazole, and mebendazole. All have the basic I .2-diatninobenzene backbone structurc. which is responsible for their activity. Except for mebendazole, these vermifuges interfere with the metabolism of w o r m by reacting with fumarate reductase, rendering it inactive. This enzyme is essential in the production of adenosine triphosphate (ATP). Without ATP, the worms lack the energy source for sustenance and ultimately die. Mebendazole functions by inhibiting glucose transport, which is necessary for ATP production. Some benzimidazoles may exhibit both modes of action.
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Thiabendazole undergoes detoxification throughthe formation of S-hydroxythiabenof dazole, followed by conjugation with either sulfate or glucuronate. The metabolism fenbendazole appearsto be extensive, resultingin major metabolites suchas fenbendazolesulfoxide(oxfendazole),fenbendazole-sulfone,p-hydroxy-fenbendazole, and5-phenylthio-2-a1nino-benzirnidazole (26). Since oxfendazole is one of fenbendazole’s initial and major metabolites, we would expect that oxfendazole would have a sinlilar metabolic route of elimination. The majoridentified metabolite of albendazole is its sulfoxide, which is eliminated via urine. Mebendazole is predominantly excreted unchanged. Human subjects have complainedof loss of appetite, nausea, vomiting, and dizziness when exposed to benzimidazoles. Diarrhea, epigastric distress, drowsiness, and headache to this class of comalso have been reported when human subjects have been exposed pounds (3).
2. Other Antinematodals Examples of other antinematodals used extensively in the veterinary arena are phenothiazine,levamisole,pyranteltartrate,morentaltartrate,andivennectin,all of whichare classified as type B category I1 drugs. Phenothiazine, first synthesized by the reaction between diphenylamine with sulfur, in 1883 (27), was one of the first drugs recognized as having antinematodal activity. It has been used extensively since 1938. Although its mode of action is not known, it has proven effective against cecal worms normally associated with poultry. It is used for the control and eliminationof nodular worms in swine. For sheep, goats, and cattle, its application is against stomach w o r m , large-mouth bowel worms, and hookworms. Additional uses in cattle are as an aid in preventing the breeding of horn flies and face flies (4). (As of November 1991, phenothiazine no longer was approved as a single drug additive to animal feeds. Manufacture of type B and C drugs containing phenothiazine as the only additive ceased in September 1991) (28). While not completely understood, the metabolism of administered phenothiazine appears to generate several products. First, the rapidly absorbed phenothiazine is oxidized in the intestine by cellular enzymes to sulfoxide (SO), which may be further oxidized in the liver to the sulfone (SO,). The second, and probably the major metabolic pathway involves the hydroxylation at the 3 and/or 7 position, creating thionol derivatives that thenare conjugated with glucuronateforelimination viatheurine. Phenothiazine and the thionols also may exist i n their leuco forms as a result of reduction in acidic urine (19,20,29,30). Levamisole is the levo isomer of the racemic mixture of d, 1-tetramisole. Levamisole is the active agent in this d,l-mixture. The effectiveness of levamisole is based on its ability to cause sustained muscle contractions in nematodes, which results i n paralysis and death of the organism. Levamisole hydrochloride is approved for use in cattle for the treatment of gastrointestinal and lung wornls. In swine, it is used against infections of large round worms, nodular worms, lung worms, intestinal thread worms, and swine kidney w o r m (4). Pyrantel, 1,4,5,6-tetrahydro- 1-1nethyl-2-[2-(2-thienyl)ethenyl]pyrin~idine, a thiophene/ pyrimidine conjugate, is a broad spectrum anthelmintic. It is approved for use in swine against infections of large round wornls and nodular worms. As is typical with most antinematodal drugs, its activity is associated with the modification of muscular contraction; they are anticholinergic, impeding the physiologic action of acetylcholine and inhibiting cholinesterase. Pyrantel is rapidly metabolized to several metabolites and eliminated via
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theurine.Verylittle of the intact drug survives the metabolic process. Most of these metabolites contain the N-methyl- 1,3-propanediamine as their common moiety; they result from the hydrolysis of the tetrahydropyrimidine portion of the molecule. Morantel is an analog of pyrantel having a methyl group at the 3 position on the thiophene ring. It is approved for use in cattle against infections associated with stomach worms and worms of the slnall and large intestine. The metabolism and elimination of morantel is similar to that ofpyrantel. A comparisonof the anthelmintic properties associated with pyrantel and morantel suggests that morantel has greater anthelmintic activity. Ivertnectin is becoming a very popular anthelmintic used in the animal-producing industry. It belongs to a group of isolates of S. trvertnictilis known as avemlectins. It consists of a mixture of 22.23-dihydroavermectin B,,, and 22,23-dihydroavermectin B,,,at a 4 : 1 ratio. Its popularity stems from its very potent activity compared to other dewormers. As little as 1 mg/kg body weight is required for ivermectin to show anthelmintic activity. As a potent parasiticide, it is effective and active against all major gastrointestinal and lung womls (e.g., ascarids, hairworms, large-mouth stomach worms, stomach bots, threadworms, and heartworms).It is approved foruse in cattle, sheep, and swine for the treatment and control of a wide variety of nematodes of the Strongylidae family, a parasitic gastrointestinal tract roundwortn. In poultry, ivermectin has shown to be effective againstC q d l a ria obsignatrr and Asctlritlitr g d l i . Ivemlectin is not approved for use in lactating animals. Ivermectin iseffectivebecause it inhibitsspontaneousmotion of thenematodes through the increased excretion of ganlma amino butyric acid (GABA), which interferes with neurotransmission and stimuli of muscles, resulting in worm paralysis, death, and expulsion. Ivermectin is eliminated very slowly via the bile and feces with a half-life of 5-7 days. Metabolic studies have shownthe major metabolic residue to be the intact drug. The highest level of residues were detected in the liver and fat (19,31-33).
C. Antibiotics,Antibacterials,andAntimicrobials
1. Aminoglycosides Aminoglycosides, as the name implies, are large antibiotics consisting of highlybasic amino sugars linked through glycoside functional groups;they are effective against gramnegative and gram-positive organisms. The most commonly used aminoglycosides are sulfate salts of streptomycin, neomycin, and gentamycin. All arninoglycoside antibacterial action occurs by a similar pathway. Aminoglycoside interferes with amino acid polymerization, leading to an inhibition of protein biosynthesis. Aminoglycosides attach themselves to the 30s site of the ribosomal subunit, thereby inhibiting codification between transmitter RNA and messenger RNA. Streptomycin was first isolated from Streptomyces griseus by Schatz et al. in 1944 (34). It may be converted to dihydrostreptomycin by the reduction of the streptose moiety in water at atmospheric pressure with a platinum catalyst. This reduced form has similar antibacterial properties as streptomycin and both are effective for treating enteric infections in various animal species. Streptomycin/dihydrostreptomycin are effective against Pcrsteurelltr, Brucella, Hetnophilus, Saltnotudla, Klebsiellcr, Shigella, and Mycobacterium organisms. In cattle, they are used to control outbreaks of leptospirosis. Both have been used in poultry production against infections due to Salmonella arizonae, chronic respiratory disease, infectious sinusitis, and synovitis.Streptomycin/dihydrostreptomycin are excreted primarily via the urine and feces as the intact drug (50-60%). Absorption is minimal
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with oral dosing, while parenteral administration allows for better diffusion, with most of the antibiotic found in plasma (20-30% bound to protein). Neomycin, isolated from the fermentation products of S. fitrelitre in 1949 by Waksman and Lechevalier ( X ) , is a mixture of several constituents, designated as A. B, and C. Neomycin B is the main component of the commercially available drug. Neomycin is as an effective agent forthe treatment used in several species (bovine, porcine, and poultry) of bacterial enteritis. It is active against such gram-negative organisms as E. coli, Etlterobercter treroL~etle.s, Pcrsterrrelltr, Sdtnonellcr, and Vibrio choleroe, and gram-positive organisms like Brrcillus ernthrtrcis, Stcrl’lty1ococcu.s m l r m y , and Listeritr m o t r o c ~ y t o ~ ~ e nWhen cs. administered orally, neomycin is absorbed poorly and rapidly excreted via the feces and urine. Parenteral administration allows for better distribution and increased plasma concentrations of the drug. Gentamycin, also known as gentiomycin, is an aminoglycoside antibiotic that was first isolated from the actinomycetes Mic)vrrrorrosl,or~r echinosporcr and M . purprecr by J. P. Rosselot et al. (36) in 1964. Commercial gentamycin is composed of four closely related constituents varying only i n the placement of a hydrogen and methyl group on the 6’ carbon of the purpurosamine ring. Several minor components of gentamycin also have been identified. Gentanlycin has in vitro activity against E. coli, Klehsiello, Elltrohtrcter, Scrlrnonellcr, Listeria, Brucelltr, andvariousstreptococciinfections.Aswiththeother anlinoglycosidicantibiotics,gentatnycin is absorbedpoorly when administeredorally; therefore intramuscular injections are the preferred route of administration. Parenterally it diffuses into plasma and is distributed to other body fluids. Its main application is in poultry and swine production, primarily underthe care of a veterinarian. Aminoglycosides have been shown to be acutely toxic and cause damage to the kidneys (nephrotoxic) and to the auditory sense (ototoxic) (2J0.29).
2. Chloramphenicol Chloramphenicol (CAP), 2,2-dichloro-N-[2-hydroxyl-(hydroxyn1ethyl)-2(4-nitrophenyl)ethyl] acetamide,abroad-spectrumantibiotichavingactivityagainst both gram-positiveand gram-negative organisms, is derived from Streptomyces vene~r4elcre.It is effective against gram-negative infections in cattle (37). Its antibiotic activity occurs through in vivo reduction of the nitro groupto a nitroso group, which in turn binds irreversibly to the mitochondrial cell wall, inhibiting protein synthesis necessary for bacterial cell wall formation.The para-positioning of the nitro group on the phenyl ring appears to be responsible for its antibacterial activity. CAP has responded favorably against experimental infectionsin animals, including. It butnotlimited to, rickettsial pox, scrub typhus, and Rocky Mountain spotted fever. also has been shown to be effective against typhoid fever, bacterial pneumonias, brucellosis, and salmonellosisin various animal species. It is used for therapeutic and prophylactic treatments in veterinary medicine, and is absorbed rapidly when given orally. The main avenue of elimination or detoxification for chloramphenicol is as the glucuronide conjugate. In addition to the parent drug and conjugate, several other metabolites have been reported in goats, including CAP-oxamic acid, CAP-alcohol, CAP-base, and CAP-acetyl aryl amine (38). Some sensitive human subjects can acquire aplastic anemia, a potentially deadly blood disorder, when exposed to CAP. In addition, human intestinal flora may develop resistance to CAP, thereby making the treatment of S. typhi and S. IxrrtrtydIi infections
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difficult (39). These potential human health hazards have createdneed a to reevaluate and lnonitor for CAP residues in animal-derived foods destined for human consumption.
3. Macrolides Macrolides normally are distinguishedby sugar molecules attachedto a lactone-ring backbone. They have been used as an alternative to penicillin therapy when penicillin-resistant strains have been encountered. Their main mode of action is to interfere with in vitro protein synthesis. These compoundsare effective against gram-positive bacteriaand some strains of Listc.ritr. Examples of some familiar and commonly administered macrolides are tylosin, erythromycin, and oleandomycin. Tylosin is derived from Streptornyws frrrdioc and is used mainly for the treatment of chronic respiratory disease in poultry. It can be administered singly, but normally is administered i n combination with other veterinary drugs such aspyrantel tartrate. ErythroIts antimicrobial activity is against grammycin is isolated from Streptorrrycc’s ~t-ytkrc~us. positive organisms and it functions by interfering with in vitro protein synthesis. It is used primarily as a bacteriostaticagentandappearstohaveitsgreatestactivityatapH of approximately 8. Metabolism of erythromycin leads to its concentration i n the liver and its elimination via bilein high levels. Oleandomycinis produced fromStreptomyvs mltibioticus andisespeciallyeffectiveagainstgram-positivestaphylococciandstreptococci organisms ( 19). As with all antibiotics, the concern relativc to the development of antibiotic-resistant strains being transmitted to foods of animal origin dictates vigilance over Its use.
4. Penicillins Penicillinsbelongtothelargerclass of antibioticsknownasthep-lactams.Sincethe discovery by Fleming in 1929 of the antibacterial action of Pewicillirml colonies and the isolation of penicillin in 1940, these antibiotics have been studied, derivatized, and used extensively for their broad-spectrum therapeutic action. Penicillin G, the first of the penicillins, is effective against gram-positive organisms. With the advent of the new derivatives of penicillin (e.g., ampicillin, amoxicillin, and cloxacillin), treatment against infections caused by gram-negativebacteriabecamecommonplace.Theirability toinhibitthe growth of invading microorganisms i n the host organism with apparently nontoxic side effects led them to be considered the “wonder drugs” of the 1940s. p-lactams are very selective cell wall synthesis inhibitors, attacking transpeptidases and carboxypeptidases, which are essential enzymes in cell wall formation of p-lactam-sensitive microorganisms. The major elimination pathway of penicillins appears to be through the kidney. p-lactam penicillins all share a similar metabolic pathway i n humans. Metabolism normally occursvia a two-step process by which the p-lactam ring is first hydrolyzed, opening the ring to form the major metabolite, penicilloic acid. Second, penicilloic acid is transformed with attack at the thiazolidine ring, leading to the formation of penamaldic acid (40-42). Although the mechanistic details have not been elucidated completely, it is known that approximately 90% is excreted i n the urine unchanged (19). Penicillin is known to be eliminated in the milk of lactating animals (19). Unfortunately the liberal use of the penicillins has created bacterias resistant to the original penicillin. To address this phenomenon, pharmaceutical companies have produced a nlultiple of different penicillin derivatives. Unfortunately some human subjects can become sensitized to this class of drugs. Immunopathologic responses such as this can be serious, which dictates the need to monitor this class of drugs closely in the food supply.
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5. Sulfonamides Sulfonamides (sulfa drugs) have been used as antibacterials for more than 60 years. Their veterinary use has been confined mainly to swine and cattle, but their emergence in the aquaculture industry has broadened their scope greatly. As antibacterial agents, sulfonamides are active against both gram-negative and gram-positive organisms. Examples of some organisms affected by sulfonamides drugs are Streptococcus pyogenes, Streptococcus pneumonirre. some strains of Bacillus mrthr~rcis,Brucella, V. cholercle, and Chlrrtnydia trc~homcrtis.In addition, some strains of E. coli associated with urinary tract infections and Nocarclicr osteroides and vulnerable to sulfonamides. The basic building blockof the sulfa drugs is sulfanilamide (para-aminobenzenesulof sulfonamides is dueto their ability to bind toa critical fonamide). The therapeutic effect enzyme by mimicking para-aminobenzoic acid (PABA), which reduces the production of folic acid, an essential component for bacterial growth in animal cells. The R group on the amido ( N ' ) governs antibacterial activity of sulfas and depends on the R functionality attached to theN' nitrogen. By substituting different R groups, the pharmaceutical industry has generated a wide variety of sulfa agents for a multitude of uses. The degreeof toxicity of theparent, as well as themetabolites,variestremendouslyfrom low toveryhigh, depending on the NI substitution. Two pathways for the metabolism and elimination of sulfonamides are expected. First, acetylation of the sulfonamide at the N' position occurs i n the liver, and metabolism to be more increases with residence time in the blood. These acetylated metabolites appear toxic than the parent drug. The second metabolic route involves the oxidation of the aromatic hydrocarbons (benzene or other heterocyclic ring), leadingto the formation of phenolic constituents ultimately forming conjugates with glucuronate or sulfate conjugates that then are eliminated via the urine (20.29). Sulfamethazine has been implicated as a possible carcinogen (43). The emergence of bacterial resistance is a concern, and could make the treatment of human subjects infected with sulfonamide-resistant strains difficult. Additionalsulfonamidedrugsincludesulfathiazole,sulfadimethoxine,sulfachloropyrazine, sulfaquinoxaline, and others.
6. Tetracyclines Tetracyclines are a group of broad-spectrum antibioticsthat have their origin inStreptomyces. Three of the major tetracyclines used routinelyin the veterinary arena are chlortetracycline (CTC), oxytetracycline (OTC), and tetracycline (TC). The isolation of CTC from S. mueofnciens was first reported in 1948 (see Ref. 19). Two years later, OTC was derived from S. rimosus. Elucidation of the structure of CTC and OTC led to the synthesis of TC by the catalytic reduction of CTC. Tetracyclines are effective against a wide variety of gram-negativeandgram-positivebacteria,includingcertainstrains of beta-hemolytic streptococci, rickettsiae, mycoplasma, chlamydia, and amoebas. In poultry, tetracyclines are effective in the treatment of chronic respiratory disease, nonspecific infectious enteritis, infectious sinusitis, hexamitiasis, and in the control and prevention of synovitis. In swine and cattle, theyareusedtoaidintheprevention of bacterial enteritis. I n addition, they are effective against anthrax, brucellosis, and urinary tract infections caused by E. coli. Tetracyclines act by inhibiting protein synthesisin bacterial cell walls. They perform as a blocking agent by binding with RNA, preventing the interaction of the aminoacyl transfer and messenger RNA ribosome attachment vital to bacterial cellular metabolism.
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The tetracyclines are metabolized and concentratedin the liver and released into the intestine in bile. Excretion occurs throughthe kidney, with a large percentage of the unchanged drug found in the urine (25-50%). Their persistence in blood (plasma) facilitates their passage across the blood-milk barrier, and traces of TC residues can be found in milk up to 48 hours postdosing( 19,20,29). The development of tetracycline-resistant organisms is a concern, and hypersensitive individuals can develop a reaction to low-level tetracycline exposure.
7 . Others Carbadox, or hydrazinecarboxylic acid (2-quinoxalinyl-methylene-,methyl ester,N’,N‘dioxide), trade name Mecadox (Pfizer Inc., Animal Health Division, New York, NY), is an antibacterial approved for use only in swine. It improves feed efficiency and increases the rate of weight gain by effectively controlling swine dysentery and bacterial swine enteritis (scours). Carbadox is active primarily against gram-negative organisms such as E. coli, S d m o t d l a hpl-7hinru1-iun1,and Snltnor-7hellN ckolewesuis. Its activity appears to be attributable to the formation of the dihydroxy quinoxaline metabolite, which inhibits DNA synthesis ( 1 9). Metabolismof carbadox occurs rapidly, with hydrolysis to the major metabbeen detected olite quinoxaline-2-carboxylic acid (QCA) (44). While desoxycarbadox has in the liver and kidney (43, QCA appears to be the more persistent metabolite in liver. Concerns relativeto the developmentof carbadox-resistant bacterial strains(46) and possible carcinogenicity (47,48) have raised questions over carbadox’s control and use. Dyes such as gentian violet, malachite green, and methylene blue are antimicrobial/ antifungal agents that have been used i n food-animal production, including aquaculture. Suchdyesgenerallyare usedtocontrolfungal growth in animalfeedsandthesedye as a residueintheanimals that consumethefeed. residuesmaymanifestthemselves Malachite green may be usedto reduce or eliminate external fungal growthin aquaculture species.
D. Growth Promotants 1. Anabolics Diethylstilbestrol (DES), 4,4-( 1,2-diethyl1.2-ethenediyl) bisphenol, is a nonsteroidal synthetic estrogen. Its very potent estrogenic properties are responsible for its popularity in animal husbandry. It is used primarily as a growth promotant in cattle production. DES is composed of two isomers, the cis (30%) and the tram (70%) isomers. Studies have shown thatthetrans isomer istheactive componentresponsibleforDES’shormonal activity. DES causes the retention of salt and water and enhances the animal’s ability to fix nitrogen. Its mode of action lies in its ability to stimulate the production of RNA and DNA, as well as increasing protein synthesis. DES is eliminated as a conjugate (glucuronate and/or sulfate) and is excreted in the urine. In 1979, after years of controversy, the FDA banned the use of DES as a growth promotant in food-producing livestock. It has been classified as a carcinogen by the EPA (20,27,29). Zeranol, a benzoxacyclotetradecin derivative, is a nonsteroidal anabolic veterinary drug. Zeranol is produced from zearalenone, a mycotoxin produced by Fusnrium roseum, which is found commonly in grain products. It is used commercially in cattle and sheep for increasing the rate of weight gain by improving feed efficiency. Metabolic studies indicate the main pathway of elimination to be excretion of both free compound and the
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glucoronide or sulfate conjugate in the urine. The two metabolites of zeranol are zearalanone (major metabolite) and taleranol (minor metabolite) (49,50). Trenbolone acetate, 17P-acetoxy-estra-4,9, I 1 -trien-3-one, was first synthesized and introduced in 1967 (51). It is an anabolic steroid used in cattle, sheep, goats, and swine for improving feed efficiency to increase the rate of weight gain. Its activity is related to its ability to increase nitrogen retention in heifers. It is therapeutically effective against pathological accumulation of ketone bodies (ketosis) in cattle, sheep, goats, and swine. This appears to be due to its effect on improving the efficiency of the Krebs cycle in the liver. In cattle, it is metabolized rapidly in the blood. Hydrolysis of the acetate leads to the major metabolite, epi-trenbolone (17a-hydroxy-estra-4,9,1 I -trien-3-one), which can be found in the bile, kidney, and liver. Conjugation of trenbolone acetate to form glucuronides or sulfates occurs, and these metabolites are eliminated quickly via the bile and urine. The principal metabolite found in muscle is trenbolone, 17P-hydroxy-estra-4,9,1 I trien-3-one. Milk does not appear to be an avenue of excretion for trenbolone (52-56). Anabolics demonstrate a hyperstrogenic character, which is a concern when children are exposed to this class of colnpounds because excess exposure can result i n a precocious puberty effect. In addition, these compounds have been implicated as cancer promotants. Examples of other anabolic drugs are hexaestrol, ornestrol, estradiol, estriol, and estrone.
2. Synergistic Growth-Promotant Enhancers Progesterone is a honnone secreted by the corpus luteum that is involved in the control of pregnancy. The 6-methylationof progesterone and the 17a-acetylation of progesterone leads to enhanced progestational activity. The introduction of a 16-methylene group into 6-methyl- 17a,acetyl-progesterone likewise is accompanied by an increase i n biological potency. The synthesis of melengestrol acetate (MGA) (6-methyl- 16-methylene- 17a-actoxy-4, 6-pregnadiene-3,20-dione)wasaccomplishedsuccessfully in theearly1960s. MGA is a progestational steroid incorporated into cattle feed for the purposes of growth promotion, improved feed utilization, and estrus suppression. Activity is associated with the inhibition of ovulation and an increase in the frequency and size of the palpable follicles. Metabolic studies show that the normal route of excretion is via the feces and urine. MGA appears to be highly resistant to the metabolic process, with the highest residues of intact MGA (86%of dose administered) foundin fat. Liver contained 29%of administered radioactivity, which consisted of both intact and metabolized MGA. Because of the similarity between MGA and trenbolone acetate, it would be expected that metabolized MGA willcontainseveralhydroxylatedentities. In someinstances. as many as 22different metabolites have been determined, including conjugated and hydroxylated MGA (57-60). Additional examples of this class of drugs include dexamethasone, medroxy progesterone, megestrol acetate, and norgestrel.
111.
FUTURE DEVELOPMENTS
Excess exposure to animal drug residues, aswith any chemical compounds, hasthe potential to be a human health hazard. Toxicological, microbiological, and immunopathological responses can be manifested in some sensitive human subjects, depending on the drug to which they are exposed and the conditions of exposure. Potential and/or observed toxic effects of some drugs have prevented their marketing and/or resulted in previously ap-
Drug Residues in Foods of Animal Origin
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proved drugs being removed fromthemarket,thusrequiringthe development of new drugs. Unfortunately. new-generation drugs are becoming more exotic and complex in order to meet the needs of the food-animal industry. However, the costof developing and marketing these new animal drugs to replace ineffective or prohibited ones is becoming excessive. In addition, the time required to deliver a drug to market can be lengthy. Drug use i n veterinary medicine has intensified in recent years; however, alternatives to drug use should be explored and utilized if economically feasible. In recent years there has been renewed interest in raising food animals in drug-free environments. Closed specific-pathogen-free (SPF) operations require strict adherence to good animal practices i n an attempt to limit the occurrence of infections or disease, thus minimizing the need to use medications.Such operations maynot be completely successful or applicable to all food-animal production, but it is becoming more evident that drug use is not a replacement for sound animal husbandry practices. A more complete understanding of virus/host and/or bacteria/host interactions relativeto infections and immunity will better equip scientists to develop moreefficient drugs. Furthermore, suchan understanding Inay allow future farmers to operate i n closed pathogen-free production systems. Such closed systems may not be realized for decades, if ever, thus new-generation drugs will be needed in the foreseeable future. Future new-generation animal drugs in all likelihood will rely more on combination drugs,thatis,onedrugpotentiatestheaction of another drug through linked enzyme systems. Such synergistic drug combinations have an advantage over single drug administrations i n that they can be more efficient and reduce the potential for the emergence of resistant organisms. The biochemical pathways and mechanistic details associated with these linked enzyme systems, relative to specific drug activities, will require innovative and creative thinking within the pharmaceutical industry. Genetic engineering Inay provide future farmers with more efficient new-generation animal drugs.
REFERENCES 1.
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3. 4. 5.
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Department of Health and Human Services and Food and Drug Administration. New Animal Drugs (21 U.S.C. 360 and 510), Code ofFederalRegulations.1991. SF Sundlof, JE Rivierc, AL Claigmill. FoodAnimalResidueAvoidanceDatabank.Trade Name File. A Comprehensive Compendium of Food Animal Dmgs, 3rd ed. Gainesville. FL: Institute of Food and Agricultural Sciences. University of Florida, 1989. J Brown, ed. Compound Evaluation and Analytical National Residue Program Plan. Washington, DC: USDA. FSIS Science and Technology Program, 1991. S Muirhead, ed. Feed Additive Compendium. Minnetonka, MN: Miller Publishing. 1990. Food and Agriculture Organization. Residues of Veterinary Drugs in Foods. FAO Food and of the United Nations, 1985. Nutrition Paper 32. Rome: Food and Agriculture Organization Department ofHcalthandHuman ServicesandFoodandDrugAdministration. 21 U.S.C. 301, Food. Drug and Cosmetic Act as amended. Washington, DC: U.S. Government Printing Office.1989. a s amended by the Federal Environmental Environmental Protection Agency. 7 U.S.C. 135. Pesticide Control Act of 1972 (7 U.S.C. 136). Federal Insecticide, Fungicide and Rodenticide Act of 1947. Washington, DC: US. Government Printing Office, 1999. U.S. Department of Commerce. 15 U.S.C. 2601. Toxic Substances Control Actof 1976. Washington. DC: US. Government Printing Office, 1999. U.S. Department of Agriculture. 21 U.S.C. 601, US. Department of Agriculture Federal Meat Inspection Act o f 1906. Washington, DC: U.S. Government Printing Office, 1999.
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U.S. Department of Agriculture. 21 U.S.C. 451, U.S. Departmentof Agriculture Poultry Products Inspection Act of 1957. Washington, DC: U.S. Government Printing Office, 1999. AR Long, SA Barker. Antibiotics in foods of animal origin. In: YHHui, ed. Encyclopedia 1. New York: John Wiley & Sons, 1991, p. 59. of Food Science and Technology. Vol. CD Hawker. Radioimmunoassay and related methods. Anal Chem 45:878A, 1973. SE Charm, RChi.Microbialreceptorassayforrapiddetectionandidentificationofseven families of antimicrobial drugsin milk: collaborative study. J Assoc Anal Chem 71:304, 1988. MR Parkhie. Hazards of extra-labeluseofdimetridazole.JAmVetMedAssoc,189:270, 1986. S Sved, G Carignan. The kineticsof elimination of dirnetridazole and its hydroxylated metabolite in food animals. AOAC Spring Workshop, Drug Residue Section. Ottawa, Ontario. Canada, April 29, 1987. Dimetridazole Turkey Clearances Revoked, Food Chem News July 13:47-48, 1987. Approval withdrawn for ipronidazole. Feedstuffs, January 23:9, 1989. F Ramos, M Filipe. C Castilho, I Silvcira. Liquid chromatographic determination of dimetridazole and ronidazole in feeds. J Liquid Chromatogr 14:2131, 1991. NH Booth, LE McDonald. Veterinary Pharmacology and Therapeutics, 5th ed. Ames: Iowa State University Press, 1982. Its Applications to Therapeutics and Toxicology, T Sollmann. A Manual of Pharmacology and 8th ed. Philadelphia: WB Saunders, 1957. T Suortti, K Heinonen. Simple and rapid analysis of nitrofurazone from blood, milk, urine and meat samples. Chromatographia 24:344, 1987. JJ Ryan, YC Lee, JA Dupont. CF Charbonneau. A screening method for determining nitrofuran drug residues i n animal tissues. J Assoc Anal Chem 58:1227, 1975. LHM Vroomen, MCJ Berghmans,JP Groten, JH Koeman,PJ Vanbladeren. Reversible interaction of a reactive intermediate derived from furazolidone with glutathione and protein. Toxicol ApplPharmacol 9553. 1988. LHM Vroomen, MCJ Berghmans, P Hekman, LAP Hoogenboom, HA Kuiper. The elimination of furazolidone and its open-chain cyano-derivative from adult swine. Xenobiotica 17: 1427, 1987. JFM Nouws, TB Vree, MML Aerts, M Degen. F Driessens. Some pharmacokinetic data about furaltone and nitrofurazone administered orally to preruminant calves. Vet Q 9:208, 1987. SA Barker, T McDowell, B Charkhian, LC Hsieh, CR Short. Methodology for the analysis of benzimidazole anthelmintics as drug residues in animal tissues. J Assoc Anal Chem 73: 22,1990. M Windholz, ed. Merck Index, 10th ed. Rahway, NJ: Merck and Co., 1983. S Muirhead, ed. Feed Additive Compendium, vol. 30. Minnetonka, MN: Miller Publishing, 1992. of Therapeutics. 5th ed. LS Goodman. AG Gilman, GB Koelle. The Pharmacological Basis NewYork:Macmillan,1975. J Chromatogr Sci IO: GJ Cimbura. Review of methods of analysis for phenothiazine Drugs. 287,1972. SV Prabhu, TA Wehner, PC Tway. Determination of ivermectin levels in swine tissue at the parts per billion level by liquid chromatography with fluorescence detection. J Agric Food Chem 39: 1468, 1991. PC Tway, JS Wood Jr. CV Downing. Determination of ivermectin in cattle and swine tissues usinghighperformanceliquidchromatographywithfluorescencedetection.JAgricFood Chem 29: 1059, 1981. M Alvinerie, JF Sutra, P Galtier, PL Toutain. Determination of ivermectin in milk by high performance liquid chromatography. Ann Rech Vet 18:269, 1987. ASchatz, S Bugie,SAWaksman.Streptomycin,asubstanceexhibitingantibioticactivity against gram-positive and gram-negative bacteria. Proc Soc Exp Biol Med 57:244-248, 1944.
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35. SA Waksman, HA Lechavalier. Neomycin,a new antibiotic active against strcptomycin-resist a n t bacteria, including tuberculosis organisms. Sci Wash 109:305-307. 1949. A Hamdan, C Joyner, R Schmidt. D Migliore. 36. JP Rosselot, J Marquez, E Mcscck, A Murawski, HL Herzog. Isolation, purification, and characterization of gentamicin. In: JC Sylvester. cd. Antimicrobial Agents and Chemotherapy--1963. Ann Arbor. MI: American Society for Microbiology.1964, pp. 14-16. 37. JM Wal. JC Peleran, GF Borics. High performance liquid chrornatographic determination of chloramphenicol in milk. J Assoc Anal Chem 63:1044, 1980. of metabolic conjugation of 38. JM Wal, JC Peleran. E Perdu. D Rao. G Bories. New aspccls 1988. chloramphenicol.DrugMctabDisposit16:635, 39. JJ Van Der Lee, WP Van Bennekom. HJ DeJong. Dctermination of chloramphenicol at ultratracelevels by high-performancedifferentialpulsepolarography.Application to milkand meat. Anal Chin1 Acta l 17:17 I , 1980. of Action, New Developments 40. M Salton, GD Shockman, cds. P-Lactam Antibiotics. Mode and Future Prospects. New York: Academic Press. 198 I . 41. T Uno, M Masada,K Yakaoka, T Nakagawa. High performancc liquid chrotnatographic detcrtnination and pharmacokinetic investigation of amino-pencillins and their tnctabolitcs i n man. ChetnPharmBull29:1957. 1981. 42. Y Murai. T Nakagawa. K Yalaoka, T Uno. High performance liquid chromatographic analysis and pharmacokinetic investigation of oxacillin and its tnetabolitcs in tnan. Chem Pharm Bull 293290. 198 I . 43. NLittlefield.ChronicToxicityandCarcinogenicityStudies of Sulfamethazine in B6C3FI. Mice. Technical Report41 8. Jefferson. AK: National Center for Toxicological Research. 1988. 4 4 . MJ Lynch. SR Bartolucci. Confirmatory identification of carbadox-related residues i n swine livcr by gas-liquid chrotnatography/mass spectrotnetty with selected ion monitoring. J Assoc Anal Chetn 65:66, 1982. 45. AI MacIntosh, G Lauriault, GA Neville. Liquid chromatographic monitoring of thc depletion of carbadox and its metabolite desoxycarbadox in swine tissues. J Assoc Anal Chetn 68:665, 1985. 46. K Ohmae, S Yonezawa, N Terakado. R plasmid with carbadox resistance from Escherichia coli of porcine origin. Antimicrob Agents Chetnother 1936. 1981. ScrviccsandFoodandDrugAdministration. 21 U.S.C. 47. DepartmentofHealthandHuman 556. Code of Federal Regulations. Washington, DC: U.S. Government Printing Office, 1997. of carhadoxandolaquinox-growth 48. H Yoshimura, M Nakamura, T Kocda. Mutagenicities promoters for pigs. Mutat Res 90:49, 1981. 49. JE Roybal, RK Munns, WJ Morris, JA Hurlbut. W Shitnoda. Determination of zeranolIzcaralenone and their metabolites in edible animal tissue by liquid chromatography with clectroJ Assoc chemical detection and confirmation by gas chromatography/tnass chromatography. Anal Chem 71:263, 1988. 50. RS Baldwin, RD Williams, MK Tcrty. Zeranol: a review of the metabolism, toxicology and analytical methods for detection of tissue residues. Regul Toxicol Pharmacol 3:9, 1983. 51. L Velluz, G Nomine. J Mathieu, R Bucourt, L Nidelcc. M Vignau, JC Gasc. Agencements steroide trienique ct activities anabolisantes. CR Acad Sci (Paris) 264(C):1396, 1967. 52. J Pottier, M Busigny. JA Grandadam. Plasma kinetics, excretion in milk and tissue levels in the cow following itnplantation of trenbolone acetate. J Anim Sci 41:962, 1975. 53. JA Grandadam, JP Scheid, A Jobard, H Drcux, JM Boisson. Results ohtained with ternbolone acetate in conjunction with estradiol 17P in veal calvcs, feedlot bulls, lambs and pigs. J Anim Sci 41:969, 1975. 54. SS Hsu. TR Covey, JD Hcnion. Determination of trenbolone in bovine liver and tnuscle by 10:3033, 1987. HPLC and LCIMSIMS. J Liquid Chronlatogr 55. J Pottier. C Cousty, RJ Heitzman. IP Reynolds. Differencesi n the biotransformationof a 17Phydroxylated steroid, trenbolone acetate, in rat and cow. Xcnobiotics 1 1:489, 1981.
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56. MWAPhillips, DJ Harwood.Assayfor 17a-OH trenholonc.themainbiliarymetaboliteof trcnholonc acetate in the cow. J Vet Pharmacol Ther 5:285, 1982. 57. RC Zimhclman, LW Smith. Control of ovulation i n cattle with tnelengestrol acetate. I. Effcct of dosage and route of administration. J Reprod Fertil 1 I : 185, 1966. 58. RC Zimhelman, LW Smith. Control of ovulation in cattle with mclengcstrol acetate. 11. Effect on follicle size and activity. J ReprodFertil 11:193-201, 1966. 59. LF Krzeminski, BL Cox, RE Gosline. Fate of radioactive melcngcstrol acetate in the bovine. J Agric Food Chem 29387, 1981. 60. JS Cooper, JS Eke, AEKellic.Themetabolism of melengestrolacetate.Biochcm J 104(3): 57P. 1967.
21 Migratory Chemicals from Food Containers and Preparation Utensils
I.
11. 111.
IV. V.
VI. VII.
VIII. IX. X.
1.
Introduction 599 600 Migration Theory Migration of PackagingMaterials601 ConcernswithMigration of PackagingMaterials 603 SourcesofFoodPackagingMigrantChemicals 603 A. Polymers 603 B. Plasticizers 607 Effects of IonizingRadiation on PackagingMaterials 609 MetabolismofPlasticizers 610 Toxicity of Plasticizers 61 1 AlternativeFoodPackagingMaterials 612 Conclusion 612 Rcferenccs6 13
INTRODUCTION
The use of plastic organic polymers and functional additives such as plasticizing agents or antioxidants in the packaging, manufacture, and serving of common foodstuffs such as meat, cheese, margarine, bacon, vegetables, and beverages may result in these packaging materials potentially being considered as indirect food additives ( I ) . This phenomenon occurs because of the potential for contact and migration or transfer of polymer components from containers or packaging films to foods during processing, packaging, and storage. Plastics (i.e., polystyrene) are also extremely prevalent as food service utensils and (23). Polymer additives can fallinto two containers both inside and outside the home functional categories: those that modify the physical properties of polymers, namely plasticizers, lubricants, and coloring agents, etc., and those that have a stabilizing or protective effect on polymer degradation (antiaging additives), such as antioxidants and ultraviolet light protecting agents (4,5).Plasticizers are synthetic organic additives characterized by 599
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low molecular weight with low melting points or low volatility with high boiling points and high miscibility with the parent polymeric matrix [e.g., phthalic acid esters (PAEs)]. They are used to improve the plasticity (i.e.. flow and workability) of polymers such as polyvinylchloride (PVC), polyvinylacetate (PVA), andpolyvinylalcohol(PVAL). In this way plastic formulations can contain up to 60% of plasticizer materials to transform an otherwise rigid polymer into a plastic with improved physical and mechanical properties such as flexibility, durability, and impact resistancc ( 1,6). Plastics have revolutionized the packaging and transportation of foods, allowing foodsto be packaged in lightweight materials while offering excellent physical protection against mechanical damage, water vapor transmission, and light oxidation. However, due to the relatively low molecular weight of residual polymeric monomers and certain additives (i.e., plasticizers and antioxidants), the migration to and absorption of these compounds by both solid and liquid foods is known to occur (7-9). Thismay result i n off-odorsor-flavorsbeingimparted to the packaged food (10,l l ) , resulting i n the loss of product and sales. As well, the toxicology of these substances has been the subject of intense investigation i n animal models (1216) and human subjects ( 18-20). These issues gain relevance given the increased interest in irradiating packaged foods for increased microbiological safety(2 1-23). Gamma-irradiation of packaged foods is known to result i n alterations such as the cross-linking and scission of polymeric materials and component additives which may in turn be adsorbed by the packaged food contained therein. This chapter discusses these issues as well as examining potential alternatives to the traditional polymeric packaging materials.
II. MIGRATION THEORY The migration and sorption of materials fronl food packaging materials into food or from the food into the packaging material itself obey Fick's law of diffusion [see Eq. ( l ) ] and to the physic a1 Interaccan be explained on the basisof the adhesion theory, which refers tion of two materials. '
where
J
=
flux (moles/sec cm')
D = diffusion coefficient (cm'/sec)
C = concentration of substance A (moles/cm') X = distance (cm) There are two mechanisms by which adhesion plays an important role in the migration of packaging material components. The first is through the adhesion and loss of food constituents(i.e.,volatiles)tothepackagingmaterialresulting in areduction of food sensory qualities and thus, acceptability. The second is through the adhesion ofthe packaging material to the foodstuff, which may introduce toxicants, unwanted flavors, or odoractive substances, again resulting i n the loss of food quality as well as safety (4,24). The
Migratory Chemicals from Food Containers and Preparation Utensils
Table 1 General Tenets toAdhesionTheory Materials,'
601
Involving Migration o f FoodPackaging
~~
Molecular interaction
Type of adhesion intcraction Mechanical interlocking Macroand microscale Wetting
Llquid dispersion and surface tension
Electrostatic
Ionized substrates f o r m electrical double layer
Chemical
Covalent bonding betwecn surfaces and polymer componellts Polymer and additive migration
Diffusion
Description of interaction Rugosity of surface increases locking of film components. Electrodynamic intermolecular forces create dispersive and attractive forces at surfaces. Double layer is positively charged while outer interface is negatively charged. Adhesive forces act by attraction through electrical double layer in films. Contact time and processing dependent. Contact time, temperature, tnolecular weight, polymer type, and viscosity dependent.
adhesionphenomenacan be explainedaccording to theconditionslisted in Table 1 (4,24,25). The t e m migration typically refers to the movement of chemical compounds from the packaging material into the food or beverage contained therein. This migration activity includes the movement of gases, low molecular weight compounds, and water vapor from the package into thefood (4). This phenomenon is distinct from sorption, which involves the movement and uptake of food constituents into packaging materials (24). These processes arc dynamic and both can occur simultaneously by diffusion (26). At steady state, flux can be described as a function of the concentration difference of the migrating chemical across the distance of diffusion when referring to Eq. ( I ) (27). In general, migration and sorption are similar processes which can ultimately affect food quality. Both phcnomena are influenced by the chemical and physical properties of the substrate and the receiving medium.
111.
MIGRATION OF PACKAGING MATERIALS
Of primary concern to the food scientist and consumer are the migration and sorption processes which may impact on the organoleptic quality, nutrition, and safety of packaged foods. Migration of plastic packaging components may result in the introduction of undesirable and/or unsafe substances into food, thus rendering the food unsuitable for consumption. Similarly, sorption processes may result in nutrient, flavor, or odor loss from the packaged food into the packaging material, giving rise to a poor quality product. Thus, sorption is a phenomenon involving packaged food, where concern for product sensory qualities and nutrient retention are generally the primary focus. Packaging materials can
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adsorb flavor components from food systems, rendering the food product unacceptable over time (i.e., during the distribution and delivery process) due to "flavor scalping," as in the case of terpenoids in juices (28). In addition, the use of heat treatment or other adhesive phenomena will contribute to weight loss or reduced volumeand altered nutrient retention in packaged foods (29). Migration of packaging materials into food isa function of the chemical composition and concentration of the packaging materials and the solubility of the migrating chemical i n the polymer or its partition coefficient between the polymer and the contact surface of the food. Thefat content and more specifically the fat-releasing propertiesof contact foods can greatly influence the migration of low molecular weight compounds (30). For example, migration of the plasticizer stearyl 3-( 3,5-di-rcrt-butyl-4 hydroxyphenyl) propionate into mayonnaise (an oil-in-water emulsion with moderate fat-releasing properties) resulted i n a mean concentration of 69 pg/dm' compared to 792 pg/dm' into a tcst fat (30).Migration efficiency will also be affected by the storage or thermal processing temperatureused (i.e., heating the food inside the packaging material, as i n microwave cooking), as well as the amount of time the food is in contact with the package material(2). Thus migratory chenlicals derived from packaging niaterials can be transferred into foods according to ( l ) ] andthereforecanbemathematicallymodeled Fick's law of diffusion[seeEq. (3,31,321. Any toxicological hazard associated with the migrationof food packaging materials into foods would therefore be a functionof the amount of migratory material originating from the polymer whichentersthefoodandtheintrinsictoxicity of thismaterial
(15,16,33). Studies evaluating the migration of chemicals into foods from packaging materials 3% NaCI, 3% NaHCO,, 3% acetic use established food silnulants (i.e., distilled water, acid, 3% lactic acid, 20% sucrose, 15-5056 ethanol, and olive oil or isooctane) as model systems to estimate the migration of chemicals into foodstuffs (27,30-32). Frequently, elevated temperatures are used to simulate accelerated storage conditions. Standard test conditions include I O days at 40"C, 2 hours at 70"C, and I hour at 100°C (30). These data, used i n conjunction with toxicological testing, have resulted in the establishment of regulatory allowances for food packaging materials migrating into foodstuffs set at a dietary concentration of 0.5 pg/kg in the United Statesby the Food and Drug Administration (FDA) (32,34,35) and maximum total migration limits set at 10 mg/dm' by the European Union (6). Packaging design and construction that incorporate the concept of functional barriers (i.e., laminate films) to limit the migration of noncarcinogenic chemical components into food to a level less than the threshold of 0.5 pg/kg are regarded as acceptable in theUnitedStates (32). Functional barriers represent a concept whereby the specific package design and construction limit the migration of packaging niaterials into food in amounts regarded below this threshold level (32,361. This is especially pertinellt given the trend to using recycled materials i n packaging. Quite often a contaminated material will be coextruded with virgin materials acting as a functional barrier against taint migration from the inner layerof recycled polymer material (32). Tripartite polyethylene terephthalate (PET) films made from core material consistingof PET contaminated with tohene and chlorobenzene sandwiched between two virgin PET layers demonstrated significant migration of the contaminants into water, 3% acetic acid, andisooctane.Migration of contalninants into 3% acetic acid (50°C over 10-131 days) from the contaminated PET ranged from 4.1 to 33.3 pg/dm? and< 0. I to 20.7 pg/dm' without and with barrier layers, respectively,fortoluene and < 0.1 to17.0 p g / d d and < 0.1 to12.3pg/dlll' without and with barrier layers, respectively, for chlorobenzene (32). Thus, the effects of the diffu-
Migratory Chemicals from Food Containers and Preparation Utensils
603
sion of core layer contaminants into plastic extruded recycled materials.
CO-
films must be considered when using
W. CONCERNS WITH MIGRATION OF PACKAGING MATERIALS The classes of packaging material migrants that fall into the category of food safety concerns include the amine precursors and nitrosamines (37), plasticizers (1,38), and polymer monomers such as vinyl chloride ( 1 6,19). Amine precursors and nitrosamine formation have been reported from the interaction of the rubber netting used to package nitritecontaining cured meats and from other foods contained in paperboard containers (4,37). This chapter will now focus on the polyolefin and vinyl derivative packaging materials and additives. For example, the most commonly used plasticizers to improve the working properties of PVC are the esters of dibasic or tribasic organic acids known as phthalates, which comprise as much as 60% of the market (39). Phthalic acid esters are the subject of concern due to the structural similarity of these compounds to the known teratogen thalidomide(phthalidomide) (1). Indeed,di(2-ethylhexyl)phthalate(DEHP)hasbeen demonstrated to have fetotoxic, embryolethal, and teratogenic effects in rats at a dose of 1 glkglday ( 1 3). Finally, vinyl chloride polymers (e.g., PVC) are widely used in many types of packaging materials and have also been shown to migrate into foods (1,7). PVC contains allylic chlorine atoms, which can be released from the polymer upon exposure to light or heat treatment of the packaged food. The primary concern with PVC, and the monomer vinyl chloride used in the manufacture of this plastic, is due to animal and human epidemiological data from workers exposed to vinyl chloride indicating that exposure to vinyl chloride is strongly linked to hepatic carcinoma ( 1 6,18,19).
V.
SOURCES OF FOOD PACKAGING MIGRANT CHEMICALS
A.
Polymers
Numerous polymers are used in food packaging to store and protect food from deterioration due to chemical or physical damage from radiant or heat energy, while at the same time enabling the consumer toview and evaluate the product. Examplesof common polymers used in food packaging materials and the migratory components of concern are presented in Table 2 , and the chemical formulas of plastic resin monomers and polymers are provided in Tables 3 and 4, respectively.
1. Vinyl Derivatives Polyvinyl chloride film is manufactured by the low-pressure free radical polymerization of vinyl chloride (Table 3) at temperatures ranging from 50°C to 160°C (40). PVC film is a versatile, thin, cling film ( c 2 5 p m or 1 mil thick) that is transparent and has a high oxygen permeability but is naturally brittle and therefore requires plasticizing agents to of vinyl chloride and vinylidene beincorporated into the polymer (40). A copolymer chloride (Table 3), more commonly known by its trade name, SaranTM,is a high quality cling-film wrap used commercially and in the home. As PVC is not polymerized directly into the plastic matrix and has a high mobility due to the low molecular weight of the
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Table 2 Conmon PolymersandMigrationChemicalsinFoodPackagingMaterials,'
of
Example Polymer Polyvinylchloride Beverage water and bottles (PVC) Polyethylene (PE) Dairy products and water Polyethylenetercphthal-Beverageandwaterbottles ate (PET) Polystyrene (PS) Microwave meals, cookie bags and syrup containers PaperandpaperboardMilkand juice cartons
Melamine-fonnaldchyde formaldehyde and Melamine Tableware
Allylic chlorine Acetaldehyde, allyl alcohol. acrolein Acetaldehyde Styrene, polystyrene Polychlorinated biphenyls (PCBs), alkyl and a r y l aldehydes residues
,'See Refs. I . 3. 4. 7. 1 0 , 11. 27. 31, and 40.
branched polymers, migration from the plastic into the food product can occur with extended use. Migration isinfluenced by thefatcontent, pH, and alcohol content of the foodstuff ( l ) . Vinyl chloride (VC) monomer is a residue of PVC film manufacturing that is removed following the polymerization reaction. However, VC has received considerable attention due to its link to angiosarcoma of the liver (ASL) (16,18,19). An Italian study indicated that significant quantities of VC (13-83 ppt) could be detected in PVC-bottled drinking water (7). Moreover, the concentration of VC in bottled water was observed to increase linearly with storage time (approximate rate 1 ng/ 1/day), resulting in the recommendation that storage dates be placed on the labels of PVC-bottled water (7). While migration of VC into vinegar (9.4 mg/l), cookingoil (14.8 mg/kg), butter, and margarine (approximately 50 pg/kg) have been reported ( l ) , the greatest risk of health effects from VC is from occupational exposure in manufacturing plants (17,lS). Workers exposed to VC from manufacturing plants have been reported to suffer from primary nonangiosarcoma liver tumors aswell as ASL (1 7,18). Moreover, mutationsto the p53 tumor suppressor gene have been observedin ASL patients in association with serum anti-p53 antibodies (19). Anti-p53 antibodies could also bedetected i n serum from individuals who were
Table 3
MonomersandDegradationProducts from Food Packaging Polymers Compound Chemical structure name Propylene Ethyl acetate Vinyl acctate Vinyl chloride Vinylidene chloride Styrene
CHCH3 H2C CH3COOCH2CH3 CH3COOCH =CH2 CH2 =CHCl CH2 =CC12
e C H = C H 2
ame on
Migratory Chemicals from Food Containers and Preparation Utensils Table 4
605
CommonFoodPackagingPolymerMaterials
class Compound Polyolefins
Polyethylene Polypropylcne
PE PP
Vinyl derivatives
Polyvinylchloride Polyvinylidene chloride Polyvinylalcohol Polyvinylacctate
PVC PVDC PVAL PV A
Polystyrene
PS
Polyethylene terephthalate
PET
Polyesters
R = OH, MylarTM R = OCH,, Terylene
Polyfluorocarbons
Polytctralluoroethylene
Polyamides
Polyalnidc
PTFE TcflonTM PA
-(W-
W n -
R = (CH?),. i.e., Polyamide 6
subject to occupational exposure to VC, leading these workers to suggest that serum antip53 antibodies may be a biomarker of individuals at risk for development of ASL. Another vinyl derivative that is used commonly in food service utensils and dishes is the mononler styrene (Table 3) in the manufacture of polystyrene (PS; Table 4) foam articles (2,3,8,27,31.41). General purpose and high-impact PS are used in numerous food packaging and preparation containers including thermoset cookware, plates, cups, bowls, egg cartons, meat trays, and hinged take-out containers.In most cases, foods would only a short period of time at relatively mild be in contact with the packaging material for temperatures (approximately 55°C) in a food service situation or for a longer period at refrigeration temperatures (approximately 4°C) (3). Migration of residual styrene monomer from therrnoformed PS foam food-contact articles into CriscoTM brand cooking oil has been reported to be proportional to the square root of the time of exposure (3). The diffusion coefficients of styrene migration into cooking oil ranged from 4.5 X IO"' cm'/ sec at 21°C to 3.4 X I O " cm'/sec at 65.6"C (3). Migration of styrene into 8% ethanol from egg cartons incubated over3 1 days at 4°C was negligible (below the detection limit
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of 0.01 pg/cm') (3). More recently, Philo et al.(31) evaluated the concentration and migration of styrene-7,8-oxideintofood-contactarticles.Styrene-7,8-oxideistheoxidation product of styrene and is thought to be formed during the peroxide-initiated polymerization of styrene resin at 200°C. It is this styrene derivative that has been hypothesized to be as well as responsibleforthetoxicity of styrene,includingrenalandhepaticdamage pulmonary and cardiac abnormalities(31,42). Findingthe oxide i n PS packaging materials butnotinthe base resin pellets indicates that the epoxides are not formed during the polymerization process but rather are formed during injection molding or thermoforming of the plastic. However, the oxide decomposed rapidly in aqueous media at 40°C and 1OO"C, potentially making the exposure levels in foods less of a risk (3 I ) .
2. Polyolefins Polyethylene (PE) is one of the most widespread food packaging materials today. This hydrocarbon polymer (Table 4) can be manufactured with varying amounts of branching within the linear backbone with high-density PE (HDPE) beingthe least branched and lowdensity PE (LDPE) containing the most branching (27). Associated with these structural differences are the differing usesof these polymers: HDPE has great thermal stability and as such is used not only for films but also rigid food containers (i.e., yogurt pots), while LDPE isusedfor bags and cling-films because of its excellent flexibility. The related polymer, polypropylene (PP) has similar characteristics and uses in food packaging as PE. Conditions which favor the migration of chemicals from polyolefin packaging materials such as PE include elevated storage temperatures and exposure to oxygen (10,ll). The resultant reaction products (Table 2) can migrate into foods and cause waxlike odors that contribute to easily detected off-flavors. VitaminE has been shown to reduce the migration of off-odor aldehydes and ketones, and hexadec- l-ene into water storedin HDPE bottles, thus reducing the development of off-flavors in the stored product (IO). Phenolic antioxidants were also demonstrated to reduce the release of off-odors and -tastes from HDPE bottles associated with aldehydes and ketones (low odor thresholds), although a total of 47 volatile components released from bottles could be identified by gas chromatography mass spectrometry ( G U M S ) ( I I ) .
3. Polyesters The polyester polyethylene terephthalate (PET) is synthesized from the condensation of ethylene glycol and terephthalic acid and is more commonly known in North America by its trade name MylarTM (Table 4). PET is often usedin cola-type beverage and water bottles as well as in laminated films to provide excellent strength and abrasion resistance (27). Similar to PE and PVC, PET is also sensitive to thermal and oxidative degradation, resultingintherelease of acetaldehyde as theprinciplevolatilemigratorycompound. Acetaldehyde has also been associated with color changes in PET as the packaging material ages.
4. Other Packaging Materials Paper and cardboard are also commonly used as packagingmaterials.Polychlorinated biphenyls (PCBs) havebeen reported in foods packaged in paperboard made from recycled paper as a consequence of therecyclingprocessorfromtheprintinginkusedinthe packaging (1). In glassine packages, a pine oil hydrogenation product is used in cereal box liners which can contribute to a pineflavor upon migration of chemical constituents (4).
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B. Plasticizers 1. FunctionalityinPolymers There are more than 450 different plasticizers used in polymer formulations, of which approximately 100 are available for commercial use(4). Some common plasticizing agents in use to decrease the rigidity and brittleness of vinyl derivative polymers such as PVC are listed along with chemical formulasi n Table S. Important factors in choosing an appropriate plasticizer include low volatility, miscibility with the plastic polymer, and the avoidance of undesirable properties, such as a predisposition to induce color alterations in the packaging material as well as adverse organoleptic or toxicological properties. Plasticizers are used primarily in polylners that are characterizedby a glassy structure at room temperature (S). The rigid polymer interacts with the plasticizer moleculesto reduce the brittleness (brittleness temperature, Th)as well as the glass transition temperature (T&, allowing an increase in the temperature range of viscoelasticity for the polymer (5). The commonly used phthalic acid esters(PAEs) are low molecular weight esters that consist ofa cyclohexatriene ring (benzene dicarboxylic acid) core esterified to aliphatic substituents (Table 5).
Table 5 PlasticizerAdditives
111
Plastics
Compound name
Abbreviation Chemical structure
Phthalic acid
a
COOH
Dibutyl phthalate
DBP
aCooC COOCqHg
Terephthalic acid
Dioctyl phthalate
DOP
Di(2-ethylhexyl) phthalate
DEHP
Terephthalic acid
esters
Adipic acid esters
Citrates Oils and fatty acids
D
HOOC
Di(2-cthylhexyl) tcrephthalate Adipicacid Dioctyladipnte Di(2-rthylhexyl) adipntc Acetyl tributyl citrate Epoxidizcd soyhean oil Epoxidized linseed (flax) oil N-butyl stearate
DEHT
HOOC(CH 2)qCOOH
DOA DEHA ATBC ESBO EL0
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The PAEs are used in PVC polymers and include compounds such as DOP and DEHP which are characterized by affinity for these polymrs, low volatility, water resistance, and good flexibility of products at low temperatures (5). On the other hand, DBP has a relatively high volatility and is used mainly in cellulose-based varnishes and in adhesives (5). Several other organic acids, such as terephthalic acid (an isomer of phthalic acid), adipic acid, and citric acids, are the parent compounds of other plasticizers such as DOA, DEHA, and ATBC. Other plasticizers include stearyl 3-(3,5-di-fcrf-butyl-4 hydroxyphenyl) propionate foundin both high- and low-density PE and PP as well as rl-butyl stearate, which is used in high-impact polystyrene and PVC (5,30). Polyepoxide plasticizers such as ESBO have a good affinity for the vinylic polymers (5). Frequently, combinations of plasticizers will be used in formulations because of differences in the plasticizing, solvation, lubrication, and creep properties of the finished polylner and plasticizer blends.
2. Factors Influencing the Migration Kinetics of Plasticizers Over time, plastics containing low molecular weight plasticizers will lose plasticizer due to the evaporation of volatiles or migration of the plasticizer during food contact( 5 ) . This results in a change i n the polymer physical properties (i.e., becoming hard and brittle) accompanied by a decrease in weight due to the loss of the plasticizer. The migration of plasticizer from PVC films into foods is thought to follow Fickian behavior occurring via a two-stage process involving,first, the diffusion of plasticizer from the bulk phase within the polymer to the surface, followed by the subsequent diffusionof the migrant compound from the polymer into the food. Transfer of the plasticizer to the surface of the film depends on the nature and properties of the film (i.e., functional barrier in a laminate) as well as the barrier layer thickness (32). The migration of the plasticizer into the food is facilitated by the relatively lower resistance to mass transfer within the food phase compared to that of the polymer. As a result of the differences i n diffusion kinetics between the polymer and food source, both the composition (fat content and fat-releasing properties)and phase of the food, as well as the type and concentration of plasticizer used, are important variables in migration kinetics (30,32). For example, the transport of plasticizer by diffusion is expected within an immobile or solid food phase as opposed to convection kinetics if the food is in the liquid phase. In both of these situations, the migrant transportation rate will be reduced as the system approaches an equilibrium. The physicochemical properties of the plasticizer and the food simulant extraction medium will also influence the rate at which equilibrium is established (30,32). For example, the migrationof stearyl 3-(3,5-difur-butyl-4 hydroxyphenyl) propionate from LDPE into distilled water after incubation at 20°C for 60 days resulted in the transfer of 2.53 pg plasticizer/dm', whereas transfer into olive oil was 720 pg plasticizer/dm' (32). These variables will also be influenced by the duration and temperatureof exposure of films in contact with foods(30,32). Moreover, a lower concentration of the plasticizer within a polymer results ultimately in less migration into the foodstuff (39). Diffusion of plasticizers from a polymer will also occur more rapidly when the temperature is significantly higher than the Ts of the polymer material. However, if the external temperature is approximately equal to the T?, the diffusivity of the plasticizer can decrease by several-fold with a change in the incubation temperature in food simulant model studies. Bieber et al. (30) demonstrated this phenomenon with high-impact PS containing n-butyl stearate incubated with atest fat, resulting in the transfer of plasticizer at a rate of 10.2 pg/dm' at -20°C compared to 41.7 pg/dm' at 20°C after 120 days incubation (30).
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3. Migration of Plasticizer from PVC Films Migration of plasticizers from PVC films has been studied i n a variety of foods and food simulants modeling aqueous and fatty food systems (9.43-45). Several international studies (Canadian and Danish) confirm that DEHA is present in food-contact films and as a migrant chemical in store-wrapped meat, poultry, fish, cheese, and ready-to-eat prepared foods (9,44,45). Levelsof DEHA in store-wrapped cheese were observed to reach a level of 45 mg/kg after only 2 hours at 5"C, increasing to 150 mg/kg after I O days of storage (44). Other workers have reported DEHA levels as high 3asI O and 429 mglkgin cheeses (9,45), which means that consumer intakes close to the tolerable daily intake of 0.3 mg/ kg body weight reconmended by the European Economic Community (EEC) Scientific Committee for Food are entirely possible. More recently a majority of PVC cling-film samples (77%)used at the various stages of food distribution (importers, wholesalers,and retailers) exhibited greater rates of migration of DEHA into fatty foods as modeled by isooctane ( i n place of olive oil) than perrnissiblc by the EEC migration limit of 3 mg DEHA/dtnL (43). Migration of DEHA from PVC films into nonfatty foods as modeled by water was at or below the EEC limit of 0.1 mg/dm? i n this same study. It is noteworthy that while some fresh meats packaged in PVC films have contained 49- 15 1 nlg DEHA/ kg, frozen chicken showed no detectable migration of DEHA (45). Migration of other plasticizers including DEHP have been reported in beverages (mean 0.065 mg/kg) and foodstuffs (0.29 mglkg) packaged in glass containers with DEHP-containing cap or lid seals (9).
VI.
EFFECTS OF IONIZING RADIATION ON PACKAGING MATERIALS
Treatment of foods that represent a microbiological health risk using ionizing irradiation to improve the safety of susceptible foods such as chicken (Scrlmo/wl/cr sp.) and ground cwli 0157:H7) is increasingly recommended given the beef or hatnburger (Es~~IIEI~I'cIII'LI potential severity of these foodborne diseases (21,22). Irradiation must be combined with other safe food handling and prescrvation techniques (i.e.. refrigeration) to prevent the cross- or recontamination of the irradiated product. One way to achieve this is to irradiate prepackaged meat and poultry, however, the irradiation process should neither alter the physicochelnical properties of the packaging films nor result in the transfer of components or residues from the packaging material to contaminate the food in contact with the plastic film (46). Irradiation of plastic films may result in a combination of two basic phenomena: (a) chemicalcross-linkingbetweenpolymerstructuralunits,ultimatelyresulting in a potential increase i n film tensile strength, or (h) fragmentation of polymeric structures resulting in the decreased strength and increased permeability of packaging films (47,48). Polymer additives such as plasticizers and alltioxidants (added a s stabilizers) are also affected by theionizingirradiation of plasticfilms (47-49).Polymerradiation-induced changes are influenced by several factors, including the chemical structure and conlposition (i.e., additives) of the polymer, the processing history, and the irradiation conditions, namely the dose rate. In addition to the scission and cross-linking of polymeric chains, the formation of volatile radiolysis products. which can be intluenced by the presence of 02,occurs in irradiated plastics. As reviewed by Buchalla et al. (47), under vacuum, the
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main radiolysis volatiles produced are hydrogen, methane, and hydrogen chloride from polymers containing chlorine molecules. In the presence of 02,the gaseous products will also contain CO?, CO, as well as Hz, CH,, and other hydrocarbons. The formation of reactive oxygen species as free radicals can result in the oxidation of the polymer forming peroxide, alcohol, and carbonyl compounds (47). The various classes of polymers are noted to vary in their susceptibility to radiolysis. The most stable polymers include the vinyl derivative PS and the polyester PET, with the polyamides (nylons) having intermediatestabilityandthepolyolefins(HDPE,LDPE,PP) as theleaststablepolymers.For example, the exposure of PP to heat, light, or ionizing radiation is known to result in the generation of alkyl radicals (PP.) which can subsequently interact with molecular oxygen to form peroxyl radicals(PP-00.) and ultimately products containing hydroxyl, carbonyl, or carboxyl groups (SO). These volatiles can result in adverse flavor changes to irradiated foods packaged in these polymers. The intensityof off-odors is much greater upon irradiation of LDPE compared to HDPE, which in turn is greater than polystyrene and various polyamides and polyesters (S 1). Moreover, the intensity of off-odors is noted to increase with the availability of oxygen in the atmosphere. Taints from PVC and PS have been reported with doses as low as 2.6-3.9 kGy through sensory testing (5 l). The addition of antioxidants such as BHT is noted to reduce the formation of carboxylic acid derivatives in LDPEfilms(47).However,phenolicantioxidantssuch as Irganox1076, 1010, and 1330 and the arylphosphite antioxidant Irgafos 168 which are added to PVC, PE, and PP polymers are susceptibleto degradation following gamma irradiation (49).It is noteworthy that some antioxidant degradation products may become covalently linked to the matrix polymer, thereby decreasing the potential for migration into foodstuffs. This hypothesis has been validated by studies with “C-labeled packaging antioxidants, indicating that following irradiation, the amount of extractable “C-labeled native antioxidant declined in irradiated polymers. This observation was associated with increases in the nonextractable radioactivity within the polymeric matrix (49). At present, little is known about the toxicity of the radiolysis products from polymers and additives given the variability in migration rates due to potential polymeric entrapment of volatiles from the different polymers into food simulants.
VII.
METABOLISM OF PLASTICIZERS
Plasticizers in food are absorbed from the diet over a wide concentration range and can be found in several tissues, with the most concentrated amounts being found in the kidney and liver and the metabolites excreted in the urine (52,53). Studies to estimate the dietary intake and excretionof DEHA reported a median intake of 23.7 mg in the United Kingdom (53). Urinary excretion of the metabolite 2-ethylhexanoic acid (EHA) acted as a useful biomarker to assess DEHA intake. There doesnot appear to be any significant bioaccumulation of phthalates such as DEHP (20,52). Studies with primates indicate that DEHP is rapidly and extensively metabolized to be excreted in the urine as glucuronide conjugation products of mono(2-ethylhexyl) phthalate (MEHP). DEHP is metabolized to MEHP by the intestinal lipases and esterases. The alkyl side chains are further oxidized and shortchaindialkylphthalatescanbeexcretedunchangedorarecompletelyhydrolyzed to phthalic acid (52). Longer-chain DEHP is converted to polar derivativcs of the monoester by oxidative metabolism prior to excretion. MEHP metabolism occurs in part by a rate-
Migratory Chemicals from Food Containers and Preparation Utensils Table 6 SomeDetrimentalEffects
61 1
of Phthalatc Esters,'
In vitro findings:
Chromosomal aberrations in nlammalian cells Basc pair mutagenicity with/without S-9 mix (MEHP) Genotoxicity to B. srthtilis In vivo findings: Decreased body weight gains (rats) Hepatic and pituitary hypertrophy Neuromuscular and skeletal deformities Decreased testicular weight Hypolipidelnia Hepatic tumorigenesis Embryotoxicity ' S c c Rcfs. I?-. 13, IS. and 53.
limiting peroxisomal P-oxidative mechanism prior to excretion (20). For both DEHP and MEHP, excretion follows a time- and dose-dependent metabolic profile in primates and man. Lipoprotein-bound DEHP metabolites are efficiently eliminated (e.g., peak elimination is 6 hours after ingestion), as demonstrated by the 90% urinary excretion of DEHP and metabolites and 10% fecal excretion (20).
VIII. TOXICITY OF PLASTICIZERS The potential toxicity of phthalate esters is of concern not only from packaging materials but also from the fact that PAEs are noted to leach into the environment (54). A Taiwanese study demonstrated that PAEs such as DOP can be detected in the soil and groundwater as well as in vegetables grown in these environments (54). The LD5,, for DEHP in rats has been reported to be 34 g/kg (53). Phthalate esters have been shown to have a number of potential deleterious effects (Table6). Decreases in testicular weight recorded in rodents receiving MEHP at 2% of their diet have been attributed to the loss of gonadal zinc. The intravenousadministration of 11 mg/kgMEHPdailyfor 13 days to rabbitshasbeen shown to result in33% maternal mortality. Enlargement of the liver and the accompanying hypolipidemia and hepatic peroxisomal proliferation have also been reported in rats administered DEHP (15). Chronic feeding studies of DEHP at dietary intakes of 0.27-0.9 g/kg body weight for 2 years show a significant increase in hepatic tumors. It should be noted, however, that there is a 6000-fold or more margin between the estimated human intake of PAE and the dose that produced liver tumors in rodents. Thus, while phthalate esters have been reported to have teratogenic and fetotoxic effects in animal studies when by hepatofed at levels of 1 g DEHP/kg/day. and impaired lipid metabolism accompanied megaly at 2% DEHP in the diet, the rapid metabolism and excretion of DEHP in primates and humans suggests that this plasticizer is only a minor health risk (13,15,20). Indeed, the Institute of Food Technologists' Expert Panel on Food Safety and Nutrition concluded that phthalates do not represent a significant hazard to human health given the relatively low rates of migration into foods (4).
(PLA)
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IX. ALTERNATIVE FOOD PACKAGING MATERIALS Safety concerns about the environment and the food supply with regard to the disposal of packaging materials in the environment and the leaching of plasticizers into the soil and groundwater (54) have been the impetus behind research into novel food packaging materials. Edible f i l m based on carbohydrate (i.e., starches), lipids (i.e., monoglycerides), and milk protein (i.e., caseinates, whey proteins) have been investigated extensively by food technologists (55-57). However, these f i l m have very high water vapor transmission rates, albeit moderate oxygen permeability. ultimately severely limiting the utility of these biodegradable or edible films i n the food industry in any meaningful way (55,56). Conn a food packaging polymer. Polylacet al. (58) investigated the use ofpolylactide (PLA) as tide is a polymer resulting from the polymerization of lactic acid and its dimer lactide as shown below:
Polylactide
Lactide
Lactic acid has a solid history of use as a food ingredient and has had GRAS status in the United States since 1984. The main physical characteristic of concern with PLA is the Ts value of 60"C, limiting its use to foods which are not extensively heated. Migrant chemicals from PLA may include lactic acid, lactoyl-lactic acid (the linear dimer of lactic acid), small oligomers of PLA, and lactide (58). These chemical species are expected to be hydrolyzed to lactic acid in the aqueous or acidic environment of foodstuffs or ultimately within the gastric contents. Potential applications of PLA include disposable food service items such as dishes, cutlery. and packagingfor fast-food applications at or below room temperature or at elevated temperatures less than 60°C ( S S ) .
X.
CONCLUSION
In summary, the migration of plastic polymer residues (i.e., VC, styrene), plasticizers, and contaminants may adversely influence the taste, odor, and safety of foods and beverages either stored or heated i n specific types of storage containers ( i t . , PE, PVC, paperboard, etc.). Factorswhich influence the degree of severity of these effects include contact surface and time, the aqueous or lipophilic nature of the foodstuff, the fat-releasing properties of the food, the temperature of processing or storage, and the method of processing ( i t . , ionizing radiation). Off-odors and -flavors in foods packaged or stored in polymers, or conversely "flavor-scalping" of food volatiles into packaging materials may reduce the quality and palatability of foods without affecting the health and safety of the consunw. The toxicology of plastics and plasticizers may be associated with occupational exposurc, as in the caseof ASL i n those exposedto VC, or consumption ofphthalate ester plasticizers in animal studies. Exposure of consumers to the amount of phthalate esters which nlay migrateintofoods is not consideredto beahealthrisk.Foodscientistsarecurrently
Migratory Chemicals from Food Containers and Preparation Utensils
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evaluating the utility of alternate packaging materials such as edible films derived fro111 as polylactide, a polymer of the dimer of lactic acid, lipids and dairy proteins as well lactide. Lillliting factors for the application of these novel packaging f i l m include such proble1ns as high ratesof water vapor transmission as well as glass transition temperatures close to the serving temperature of foods. However, new formulations as well as the use of lanlinate films may enhance the physicochemical propertiesof existing f i l m and newer edible f i l m .
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39. E Kondyli, PG Demetrius, MC Kontominas. Migration of dioctylphthalate and dioctyladipate plasticizers from food-grade PVC filnls into ground meat products. Food Chem 45:163-168, 1992. 40. SS Schwartz, SH Goodman. Structure and characteristics of polymers. In: Plastics Materials and Processes. Toronto: Van Nostrand Reinhold, 1982, pp. 130-136. of styrenefrom 41. KM Lehr,GCWelsh, CD Bell, TD Lickly.The'vapour-phase'migration general purpose polystyrene and high impact polystyrene into cooking oil. Food Chem Toxicol 31~793-798, 1993. 42. MMT Janssen. Contaminants. In: J de Vries. ed. Food Safety and Toxicity. New York: CRC Press, 1997, pp. 53-62. 43. JH Petersen, L Lillemark, Lund L. Migration from PVC cling films compared with their field of application. Food Addit Contam 14:345-353. 1997. i n contactwithcheese:health 44. JHPetersen, ETNaamasnscn, PA Nielsen.PVCclingfilm aspects related to global migration and specific migration of DEHA. Food Addit Contam 12: 245-253, 1995. 45. RP Kozyrod, J Ziaziaris. A survey of plasticizer migration into foods. J Food Protect52578580. 1989. 46. CH McMurray. Food irradiation-the challenge. In: DE Johnston, MH Stevenson, eds. Food Irradiation and the Chemist. cambridge: Royal Society of Chemistry, 1990, pp. 1-12. 47. R Buchalla, C Schuttler, KW Bogl. Effects of ionizing radiation on plastic food packaging materials: a review. Part I . Chemical and physical changes. J Food Protect S6:99 1-997, 1993. 48. D Kilcast. Irradiation of packaged food. In: DE Johnston, MH Stevenson,cds. Food lrradiation and the Chemist. Cambridge: Royal Society of Chemistry, 1990, pp. 140-152. 49. DW Allen, A Crowson, DA Leathard, C Smith. The effects of ionising radiation on additives present in food-contact polymers. In: DE Johnston, MH Stevenson, eds. Food Irradiation and the Chemist. Cambridge: Royal Society of Chemistry, 1990, pp. 124-130. so. A Feigenbaum, D Marqui, A-M Riquet. Compatibility of plastic materials with foodstuffs: mechanistic and safety aspects of ionized polypropylene. In: P Ackcrmann, M Jigerstad, T Ohlsson, eds. Foods and Packaging Material-Chemical Interactions. Cambridge: Royal Society of Chemistry, 1995, pp. 87-94. 51. R Buchalla. C Schuttler. KW Bogl. Effects of ionizing radiation on plastic food packaging materials:areview.Part 2. Globalmigration,sensorychangesandthefate of additives. J Food Protect 56:998-1005, 1993. 52. WM Kluwe. Overview of phthalate ester pharmacokinetics in mammalian species. Environ Health Perspect 453-10, 1982. 53. NJ Loftus, BH Woollen, GT Steel, MF Wilks, L Castlc. An assessment of the dietary uptake of di-2-(ethylhcxyl) adipate (DEHA) in a limited population study. Food Chem Toxicol 32: 1-5,1994. 54. M-CYin.K-H Su. Investigation on risk of phthalate ester in drinking water and marketed foods. J Food Drug Anal 4:313-318, 1996. 55. RJ Avena-Bustillos, JM Krochta. Water vapor permeability of caseinatc-based edible films as affected by pH, calcium crosslinking and lipid content. J Food Sci 58:904-907, 1993. 56. JM Krochta, C De Mulder-Johnston. Edible and biodegradable polymer films: challenges and opportunities. Food Techno1 5 1 :61-73, 1997. 57. JM Krochta, JR Maynes. Properties of edible films from total milk protein. J Food Sci 59: 909-9 1 I , 1994. 58. RE Conn, JJ Kolstad. JF Borzclleca, DS Dixler, LJ Filer Jr, BN LaDu Jr, MW Pariza, Safety assessment of polylactide (PLA) for use as a food-contact polymer. Food Chem Toxicol 33: 273-283,1995.
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22 Food and Hard Foreign Objects: A Review
I. Introduction 617 A. Consumerexperiencewithhardobjects in food617 B. Literature onhardobjects in food 618 Product C. tampering 618 D. Filth 618 E. HardobjectsabscntfromtheFood,DrugandCosmeticAct F. Defect action lcvcls 618 11. TheProcessors'Goal6 19 A. Quality assurance procedures and devices 619 B. The returnable bottle 619 111. Poisonous Plants 620 1V. Harvey W. Wiley 620 V. Glass inFoodandDrink 620 VI. IngestionandInhalation of ForeignObjects621 A. Food-related items 622 B. Hard foreign objects i n food 622 C. Rcvicw of case reports 623 Drug Administrationcomplaintreportingsystem624 D.TheFoodand VII.HACCPandHardForeignObjects 624 References 624
618
1. INTRODUCTION A.
Consumer Experience with Hard Objects in Food
Most consumers are awareof the fact that hard objects thatdo not belong in food nevertheless sometimes are present. This is because most consumers have personally found such objects. Furthermore, most such encounters are innocuous in nature. Certain foods are so frequently endowed with physical objects (e.g., grit in spinach) that we are surprised if such objects are not found. In other instances, the encounter between the consumer and 61 7
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a physical object in food or drink may result in an illjury, the severity of which nay vary widely.
B. Literature on Hard Objects In Food In view of the frequency with which consumers encounter hard objects in food during chewing or swallowing, it is surprising that the scientific literature on this subject is remarkablyscarce.Threeauthorshavetouched onthesubjecttangentially (l-3), anda fourthhasdealtwith it somewhatmoresubstantively (4), but only onecontemporary article addresses the issue completely ( 5 ) .
C. Product Tampering Excluded from this review are acts of fraud, sabotage, and product tampering. Hiding a big rock inside a bale of bay leaves to boost the weight would be considered fraud. The deliberate addition of a foreign object to a food or beverage product by an employee of the product’s manufacturing company would be considered an act of sabotage. When, in an effort to cause harm to some other person or to extort money from a manufacturer, someone deliberately adds a physical object such as a needle or a straight pin to a product offered for sale, the act would be considered product tampering (6,7). The most connnon type of product tampering, rarely prosecuted under the Federal Anti-Tampering Act (8) or any other statute, occurs when a consumer deliberately puts a piece of glass, a mouse, or a cockroach-to mention just a few of the possibilitiesinto a food or beverage, and then reports the “find” to the manufacturer, the motive being to get a free quantity of the product or money from the manufacturer.
D. Filth Also excluded from this chapter are those physical objects usually categorized as filth ( 1 ): rodent hairs and feces; bird feathers and feces; insects, mites, and their fragments; and molds and rots associated with fruits, seeds, nuts, and many other foods. The tiny metal fragments sometimes generatedby opening cans with a can opener in the home or restaurant kitchen also are excluded from this discussion.
E. Hard Objects Absent from the Food, Drug and Cosmetic Act The Federal Food, Drug and CosmeticAct (FD&C Act)(8) says nothing about such things as paint chips, ball bearings, or glass shards in food. When such items happen to be found in food or drink by regulatory officials, each incident is evaluated on its own merits and, if deemed actionable under the FD&C Act, it is because the foreign object has rendered the food “unfit” (402[a][3]) for human consumption due to the offensive “mouth-feel” of the adulterated food, or has rendered it “injurious to health” (402[a][4]).
F. Defect Action Levels The defect action levels (DALs) give intofoodsafter theagriculturalphase
no ground to physical objects that find their way of production (9). Somemodestforbearance
is
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619
II. THE PROCESSORS’ GOAL Reputable food and beverage processors prefer to produce products that are freeof foreign objects. Their zeal is driven perhaps in part by the fact that there is little defense against an obvious hard foreign object, especially one that has caused injury to a consumer. No doubt, however, the overriding motive for keeping objects out of foods and beverages is to protect and preserve the company’s reputation. Of course, the economic consequences of downtime on the production line and recalls of shipped products probably also play a role in a company’s pursuit of zero defects in the product line, whatever that might be.
A.
QualityAssurance Procedures and Devices
Many companies have invested heavily in quality assurance devices and procedures to prevent the occurrence of objects in food or to discover them before the foods leave the factory (1 1). Glass, one of the early mass-produced commercial packaging materials, continues to be widely used. Unfortunately glass packaging readily breaks during both the manufacturing of the container and the washing,filling, capping, labeling, and boxing of it on the production line. The problem of glass shards in foods and beverages has been reviewed in the technical literature over a span of many years from early in this century to the present (12-18). Reputable companies that use glass packaging strictly follow the applicable good manufacturing practice (GMP) statements and carefully keep records of problems so that processing equipment can be fine-tuned to reduce breakage to a minimum. Virtually all detection systems for foreign objects involvea human observerto some to degree, but most production line detection systems also involve specialized machines supplement visual observations. There are two different lines of approach to the goal of preventing hard foreign objects from getting into food and drink. One involves various to remove foreign objects screens, shakers, air blasts, filters, magnets, or other devices present in the production stream. The second involves machines that “look” for foreign objects in the finished product before or after packaging. These machines may be very sophisticated technically and capable of detecting many kinds of foreign objects-glass, metal, bone, rocks, shell fragments, and other items (19-26). One recent book appears at first glance to treat this subject, but in fact it deals only with solutions of elemental metals and metallic compounds in food (27).
B. The Returnable Bottle The returnable glass beverage bottle is a special problem for bottlers. Although it is an ecologically sound conservation practice to reuse glass bottles, the bottler cannot always be certain that the containers are restored to their original pristine condition before being refilled. Consumers may subject the bottles to a wide variety of uses other than that for which they were manufactured-storage of insecticides, motor oil, and paint, to mention
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a few. Bottles carelessly discarded along roadsides may become homes to snails, insects (especially the pupal casesof flies) (28), worms, shrews, and mice, or serve as depositories for amphibian eggs. When such bottles are collected and returned to the bottler, the remains of the previous inhabitants or the residues of materials previously stored in the bottle Inay resist the washing process and become an unwelcome surprise in the consumer’s favorite beverage.
111.
POISONOUS PLANTS
A special class of hard foreign objects consists of certain plant parts that are in themselves a toxic. One of these is the seed of the rosary pea or jequirty bean (Abrus preccrforir~~), tropical legume. The seed generallyis known to be inedible, but it is brightly colored (red and black) and apparently attractive to children and domestic animals. The seeds often are used in jewelry, but occasionally, in defiance of all common sense, they may be used to decorate foods, thus increasing their chance of being ingested. The seed ingested whole reportedly is innocuous, but a broken, mashed, or chewed seed releases a toxic protein that may cause profound damage to the intestinal tract of people and animals. Ingestion of a single, well-chewed seed can be fatal (29,30). The shell of the cashew nut, Anacardium occiderltcrle (Anacardiaceae), contains an irritating oil well known as a blistering agent of the skin (29,30). Shell fragments rarely get into the marketed product, but if a piece of shell is swallowed, the oil in it can irritate the buccal and gastrointestinal mucosa. The various kinds of rattlebox (Crotalcrriu species, Fabaceae) include bothwild and cultivatedforms. C. spectcrbilis, C. scrgittalis, andotherspeciesarecommon field and roadside weeds, especially in the southern and central United States. Poisoning occurs in two ways: one is as an accidental contaminant of herbal teas (all parts of the plant are toxic); the other occurs as seeds mixed in (during harvesting) with food and feed grains (29-31).
IV. HARVEY W.WILEY Concern for the possible adverse health effects of hard foreign objects in food and drink was evident early in this century. One might have guessed that Harvey W. Wiley, principal protagonist for the first federal pure food law (32), would have been interested in this subject, and perhaps he was. Butin the record of that era, he makes only passing reference to a study of “cocoa shells in cocoa products” i n his 191 1 report (33). His concern then was probably with the shells as an “economic adulterant” (in the same sense that water was an economic adulterant of milk) rather than with the shell fragments as hard foreign objects in cocoa powder.
V.
GLASS IN FOOD ANDDRINK
Glass shards in foods and beverages were an early cause for concern, but this concern was tempered by observations of professional glass eatersin action. Hancock (12) referred toobservationsmade by Haines (34, p. 889), whowatched a professionalglasseater
Food and Hard Foreign Objects
62 1
consume six test tubes, two good-size lamp chimneys, a 4 ounce medicine bottle, two pieces of window glass (each 4 inches square), and pieces of colored glass (each 1 inch X 3 inches). “He was kept under observation for several hours after eating the articles mentioned, but he at no time showed the least unfavourable symptom.” Hancock failed to note Dr. Haines’ further observation about this same glass eater who “died two or three years subsequently from a subacute gastro-enteritis, presumably from the irritation produced by his long-continued glass-eating” (34, p. 889). Perhaps this last note explains why Dr. Haines (34, pp. 888-889) retained some ambivalence on the matter: There can be no question that the ingestion of particles of glass of a certain size Inay lead to such injury of the lnucous membrane of the stomach and intestines as t o cause death. . . . Still there can be no doubt that the dangers of broken glass and of similar objects have been greatly overestimated.The results of experiments and thc experience of professional glass-eaters indicate that they are far less dangerous than is commonly supposed. Based on the evidence available to him, Hancock ( 1 2, p. 4) came to his own conclusions: Herein doubtless lies the ttuth of the matter. The effects produced by swallowing glass probably depend upon the size and form of the particles swallowed, sharp edged and angular fragments of appreciable size being much more likely to produce injury than powdered glass, which i n this form is readily enveloped by the food taken with it and by the secretions of thc stomach and intestine. That this is the case finds confirmation in the relatively few recordedcases of injury from swallowing glass.for. . . its presence is by no means infrequent in foods. When present, however, it is usually in powdered form, the particles fewin number and identifiable only by the most careful microscopic examination. I n this form it is probably rendered innocuous in the ways mentioned, and would account for the large measure of immunity from injury enjoyed by those (and they can be numbered by the millions) who consume daily one or other of the immense variety of foods and beverages which are put up in glass containers. Hancock had at hand the results of experiments by Simmons and von Glahn (35) to support his conclusions ( 1 2). They showed that the ingestion of ground or powdered glass had no ill effect on dogs.A similar experiment, conducted40 years later, gave similar results. Dogs and rabbits fed glass particles in three size categories (0-3 mm, 3-7 m m , 7-12 mm) and metal filings of the sort produced by a kitchen can opener suffered no gross or microscopic injuries to the gastrointestinal tract (14). Commenting on cases observed in a mental hospital, Eldridge noted that “some patients seemed to prefer glass or china objects, which they would first shatter into sizes that could be swallowed. They would ingest such materials time aftertotime, the exclusion of other items, but, again, all such patients that came to my attention would eventually pass these materials without trouble” (36, p. 666). Cohen agreed with this evaluation(37).
VI.
INGESTIONAND INHALATION OF FOREIGN OBJECTS
Human injury or death associated with the ingestion or inhalation of foreign objects has been reported widely in the medical literature. Many such cases have nothing to do with
622
Gorham
food. The offending object could be a coin, a thumbtack, an earring, a marble, a balloonin other words, a vast arrayof small objects (38).
A. Food-RelatedItems
Many other cases of injury or death have been attributed to various items of food. Hot dogs are notorious offenders, as are grapes, hard candy, nuts, chunks of meat, popcorn, beans, and many other such objects. Liquid foods also may cause problems, especially for infants. Another categoryof objects reported to cause injury or death consists of things that are associated intrinsically with foods: fruit pits and seeds (as in dates, grapes, apples, Spanish limes, and other citrus fruits), bones (beef, pork, chicken, fish), shells (nuts, shrimp, clams, oysters), stems (raisins, string beans), pig's teeth in sausage, and fish scale in canned tuna. Objectsin this category sometimes are considered to be less objectionable than those in the foreign object category because they occasionally may filter through the normal safeguards of good manufacturing practice.
B. Hard Foreign Objects in Food This category consists of foreign objects in foods and beverages. Such objects are described as foreign because they are not intrinsically associated with a food product. Some clearly are associated with harvesting. The small stones or clumps of soil found in dried beans and peas are typical, as are the sand and gritin peanut butter and spinach (Fig.1). Other objects, clearly associated with manufacturing, occur twoincases. The first is associated with the manufacturingof the food container per se. Glass and metal fragments predominate here. The second occurs during food processing. Glass and metal fragments
Fig. 1 Clumps of soil from a commercial package of dried beans.
Food and Hard Foreign Objects
623
Fig. 2 Metalfragmentaccidentallypackedwithsardines.
also predominate in this phase (Fig.2). In earlier days, wood splinters were more common in manufactured foods because then wood was used more commonly in processing equipment. Today, plastic is frequently seen on the processing lines. With wear and tear on the equipment, bits of plastic may find their way into foods and beverages. Plastic shards in a four-ton batch of chicken nuggets prompted a major chicken processor to initiate a recall effort. All but about2500 pounds of the adulterated chicken were recovered. The plastic shards were discovered by children eating lunch served at school. This kindof hard foreign object may cause physical injury or choking; fortunately no injuries were associated with this incident (Duyron Daily News, 9 September 1997). C.
Review of Case Reports
Case reports or reviews of collections of cases involving foreign objectsin the digestive or respiratory tracts typically referto the more severe, the more unusual, or the fatal cases. The degree of severity ranges from the relatively “minor” esophageal tears caused by sharp-edged, solid objects (38,39), through partial blockages of the esophagus (40) or trachea (41), to complete and fatal obstruction of the airwayA(42). review of the medical literature readily available, covering a span of the years 1961-1990, yielded 679 cases of injury or death associated with ingestion or inhalation of foreign objects (43-53). Many of these cases (221) had nothing to do with food, but in a relatively large number (437), of cases (28), some some kindof food material was involved. In a relatively small number food-associated, hard foreign object-pit, shell, bone, stem, seed-was the culprit. In this admittedly incomplete collection of cases from the medical literature, there was no unequivocal report of injury, illness,or obstruction causedby a hard foreign object ingested with food or drink that was not intrinsically associated with food or drink. In other words, ball bearings, wood splinters, conveyor-belt nuts and cleats (staple-like wires
Gorham
624
used to hold together sections of conveyor belt), glass shards, bits of metal or plastic, i n no case and similar extraneous, hard foreign objects ingested with food or drink were unequivocally implicated in obstruction of or injury to the digestive or respiratory tracts. There was one report of intestinal injury caused by a wood splinter that apparently had been swallowed with food, but the patient could recall no such event (54).
D. The Food and Drug Administration Complaint Reporting System The situation in the preceding section standsin marked contrast to that reported by H y m n et al. (5). In a careful analysis of just one 12-month period (October 1988-September 1989). they compiled data on 10,923 complaints about food registered with the U.S. Food and Drug Administration (FDA). Of these complaints, 25% (2726 cases) involved foreign objects in food or drink,and 14% (387 cases)of these involved illness or injury associated with foreign objects ingested in beverages or food.Most of the injuries/illnesses, as might be expected, involved cuts or abrasions in the mouth and throat, damage to teeth or dental prostheses, or gastrointestinal distress. The foreign objects were rank ordered from most to leastcommon:glass,slime or scum,metal,plastic,stonedrocks,crystaldcapsules, shells/pits, wood, and paper. Foreign object complaints involving injury or illness were associated most often with soft drinks, followed in descending order by baby foods, bakery products, cocoa/chocolate products, fruits, cereals, vegetables, and seafoods. The study by Hyrnan et al. (5) revealed that health professionals rarely report cases of injury or illness attributed to foreign objects in beverages and foods. Most often (in 8 2 8 of the cases), it was the consumer who registered the complaint. Hyman et al. (5) also note that the FDA Complaint Reporting System is a much underused early warning of system that, if properly utilized, could greatly benefit both consumers and producers foods and beverages in the United States.
VII. HACCP AND HARD FOREIGN OBJECTS The worldwide food industry is gradually coming under a food safety program called Hazard Analysis Critical Control Points (HACCP). Since HACCP focuses on microbial hazards and their reduction or elimination,the overall safety of food should be enhanced. In spite of the fact that hard foreign objects have little to do with microbial pathogens, hard foreign objects are nevertheless considered a critical control point. This is because hard foreign objects may cause injury if the consumer attempts to chew or swallow them (55).
REFERENCES JR Gorham. Filth and extraneous matteri n food. In: YH Hui, ed. Encyclopedia of Food Science and Technology. New York: Wiley-Interscience, 1991, pp.847-868. 2. OP Snydcr Jr. HACCP-an industry food safety self-control program, part 1V. Dairy, Food EnvironSanit 12230-232, 1992. 3. DA Corlett Jr. RF Stier. Risk assessmcnt within the HACCP system. Food Control 271-72, 1.
1991.
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4. EJ Rhodchamel. Overview of biological, chemical, and physical hazards. In: MD Picrson and DA Corlctt Jr, e&. HACCP: Principles and Applications. New York: AVI/Van Nostrand Reinhold, 1992, pp. 8-28. S . FN Hyman, KC Klontz, L Tollefson. Food and Drug Administration surveillance of the role of foreign objects in foodborne injurtes. Public Health Reports 108:54-59, 1993. 11: 6. KC Deignan.Producttampering:packagingandprevention.DairyFoodEnvironSanit 636-639, l99 1. 7. M McCue. Foil the malicious tamperer. Food Drug Packag 56(9):14-16,18-19, 1992. as Amended,and 8. FoodandDrugAdministration.FederalFood.Drug,andCosmeticAct, Related Laws. HHS publication no. FDA 89-1051. Rockville, MD: U.S. Department of Health and Human Scrvices. 9. Industry Activities Section. The Food Defect Action Levels. Washington, DC: Food and Drug Administration.1989. IO. Meat and Poultry Inspection Operations. Meat and Poultry Inspection Manual. Washington, DC: Food SafetyandInspectionService, US. Department of Agriculture,1987, pp. 129130a. 1 1 . F Jacobson. The detection and elimination of foreign materials. Manufact Confect 61(1 ):4l44, 1981. i n FoodsPacked in Glass 12. GC Hancock.AReportontheOccurrenceofGlassFragments Containers. Reports on Public Health and Medical Subjects no. 37. London: Her Majesty’s Stationary Office, 1927, pp. 3, 4. 13. WF Janssen. Breakage, an index of production efficiency. Glass Packer 19:226-229,254-255, 1940. 14. AJ Lehman. Glass and metal fragments in foods and beverages. Assoc Food Drug Oflic US Q Bull 22:24-26,1958. 15. WV Eisenberg. Inorganic particle contentof foods and drugs. Environ Health Perspect9: 183191. 1974. 48(4):80-83, 1976. 16. LE Slater. Product probing X-rays become on-line sleuths. Food Eng 17. A Wollen. New ART form in bottle inspection. Soft Drinks 36:473,475. 1982. 18. JS Gecan, SM Cichowicz, PM Brickey. Analytical techniques for glass contamination of food: a guide for adtninistrators and analysts. J Food Protect 53:895-899, 1990. 19. Anon. X-ray sorter detects and rejects rocks and foreign matterin almonds. Food Eng 55( IO): 131, 1983. 20. G Mayo. New methods make advances in foreign body detection, Food Eng
21.
22. 23. 24. 25.
26. 27. 28.
29. 30. 31.
60( 11):136,138, 1988. Anon. Automatic detection of foreign matter in food. Br Food J 89(938):52,53.55, 1987. Anon. A Lock. The Guide to Reducing Metal Contamination in the Food Processing Industry. Tampa,FL:Safeline,1990. Anon. Metal detection on processing and packaging lines, Food Processing 53( 1):54 (1992). J Mans. Smart systems for error-free metal dctection. Prepared Foods 161(3):90-92, 1992. M Broderick. Contaminant management systems within the confectionery industry. Proceedings of the 46th Annual Production Conference Pennsylvania Manufacturing Confectioners’ Association, 1992, pp. 95-97. A Lock. Remove failure factors from your metal detection program. Food Drug Packag 56( 12): 20-2 l , 1992. C Reilly. Metal Contamination of Food. London: Elscvier Applied Science, 1991. WA Riley. Fly pupae in bottled milk. J Dairy Sci 2:183-188, 1919. KF Lampe, MA McCann. AMA Handbookof Poisonous and Injurious Plants. Chicago: AmericanMedicalAssociation,1985. NJ Turner, AF Szczawinski. Common Poisonous Plants and Mushrooms of North America. Portland, OR: Timber Press, 1991. DJ Humphreys. Veterinary Toxicology. London: BailliZre Tindall, 1988.
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32. JHYoung.PureFood-SecuringtheFederalFoodandDrugsActof 1906. Princeton, NJ: PrincetonUniversityPress, 1989. 33. HW Wiley. 1911 Report of the Bureau of Chemistry. Food Law Institute Series, Federal Food, Drug and Cosmetic Law Administrative Reports, 1907-1 949. Chicago: Commerce Clearing House, 1951, p. 2 17. 34. WSHaines.Deathfrompoundedglassandothermechanicalirritants. I n : F Peterson, WS vol. 2. Philadelphia: WB Saunders, Haines, RW Webster, eds. Legal Medicine and Toxicology, 1923, pp. 888-897. 35. JS Simmons, WC von Glahn. The effect of "ground glass" on the gastrointestinal tract of dogs. JAMA 71:2127-2128, 1918. 36. WW Eldridge. Foreign bodies i n the gastrointestinal tract. JAMA 178:665-667, 1961. 37. H Cohen. Glass gluttony and gastrointestinal gouging. JAMA 206:1582, 1968. 38. FL Rimell, A Thorne Jr. S Stool, JS Reilly, G Rider, D Stool, CL Wilson. Characteristics of objects that cause choking in children. JAMA 274: 1763-1766, 1995. 39. GF Longstreth. Esophageal tear caused by a tortilla chip. N Engl J Med 322: 1399- 1400, 1990. 40. WA Webb. Management of foreign bodies of the upper gastrointestinal tract. Gastroenterology 94~204-216, 1988. 41. DWVane, J Pritchard, CW Colville. KW West,HEigen, JL Grosfeld.Bronchoscopyfor aspirated foreign bodies in children. Arch Surg 123:885-888,1988. 42. RE Mittleman. Fatal choking in infants and children. Am J Forensic Med Pathol 5:201-210, 1984. 43. TO Honaas, EA Shaffer. Endoscopic removal of a foreign body perforating the duodenum. Can Med Assoc J 116: 164,169, 1977. 44. FM Keith, EJP Charette. RB Lynn, TA Salerno. Inhalation of foreign bodies by children: a continuing challenge inmanagement.CanMedAssoc J 122:52-57,1980. 45. SP Baker, RS Fisher. Childhood asphyxiation by choking or suffocation. JAMA 244: 13431346, 1980. 46. BF Rothmann, DR Boeckman. Foreign bodies in the larynx and tracheobronchial tree in children. Ann Otolaryngol 89:434-436, 1980. 47. RE Mittleman, CV Wetli. The fatal cafe coronary. JAMA 247:1285-1288, 1982. 48. H Meislin. M Kobernick. Corn chip laceration of the esophagus and evaluation of suspected esophageal perforation. AnnEmergMed 12:433-457,1983. 251: 49. CS Harris, SP Baker, GA Smith. RM Harris. Childhood asphyxiation by food. JAMA 223 1-2235, 1984. 50. RM Esclamado, MA Richardson. Laryngotracheal foreign bodies in children. Am J Dis Child 1411259-262, 19x7.
M Melzer-Lange, R Van Howe, JD Losek. Esophageal foreign body presenting with altered consciousness. Am J Dis Child 142:915-916,1988. 66:804-X1 I . 52. PLBhatia.Hypopharyngealandoesophagialforeignbodies.EastAfrMedJ
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1989. 53. JA Lima. Laryngeal foreign bodies in children: a persistent, life-threatening problem. Laryngoscope 99:4 15-420, 1989. 1(4U3):315, 54. KEE Read. Ulceration of a foreign body through the small intestine. Br Med J 1946. I. Reviewof 55. AR Olsen. Regulatory action criteria for filth and other extraneous materials. hard or sharp foreign objects as physical hazards in food. Regul Toxicol Pharmacol 28: 1X 1189, 1998.
23 Food, Filth, and Disease: A Review
I. TheFood,DrugandCosmeticAct627 11. CompetitionforFood 628 111. EarlyIdeasandDiscoveries 628 IV. Transmission Cycles 628
Mechanical Transmission 629 A. Pathogenic microorganisms on pests 629 B. Fate of pathogens on pests 630 in mechanicaltransmission C. Roleofpcsthabits D. Correlationofpestpopulationsanddiseascincidence E. Sampling pathogensfromfoodsandpests631 F. Role of vectors in mechanical transmission 631 G. Role of pantrypestsinmechanicaltransmission VI. NewRisksandRemedies 632 A. New risks: emerging pathogens 632 B. Mechanical transmission (again) 632 C. Mitesasdirectagentsofdisease 632 New D. remedies: HACCP 633 Appendix 633 References 634 V.
1.
THE FOOD, DRUG AND
630 630
632
COSMETIC ACT
The language of theFederal Food, Drug and Cosmetic Act (FD&C Act) seems rather unequivocalwheneveracts of adulteration by filthare mentioned (1). Oneparagraph, 301(k) (21 USC 331(k)}, prohibits anyact done to a food after shipment i n interstate (3) { 21 USC commerce thatresultsinthefoodbeingadulterated.Another,402(a) 342(a)(3)}, statesthat a food shall be deemed to be adulterated if it consists in whole or in part of any filthy, putrid, or decomposed substance or if it otherwise is unfit for food. The final example, from paragraph 402(a)(4)(21 USC 342(a)(4)},states that a food shall be deemed to be adulterated if it has been prepared, packed, or held under unsanitary 627
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conditions by which it Inay have been contaminated with filth, or by which it may have been rendered injurious to health. The federal courts have never insisted that there must always be a cause-and-effect relationship between “filth” and “injurious to health.” Since 1942, when District Court JudgeChestnut (also spelledChesnut)read,during a trial,thedefinition of~filthfrom Welwtcr’s I~~ter.~~cltior~rrl D i c t i 0 1 1 q ~the . ordinary sense of the word has been used in judicial interpretations of the FD&C Act (2). Judges and juries have rendered verdicts both for and against the government‘s allegationsof filth i n food, always based on the practical application of this dictionary definition (3). As used in this chapter, Jilt11 is defined as insect, bird. rodent, or other objectionable matter of animal origin found in or associated with food (4). The more common kinds of filth found during food inspectionsand analyses are whole insects and mites and fragments thereof; rodent and other mammalian hairs; feathers and feather barbules; urine, uric acid, and feces-usually from rodents or birds.
II. COMPETITION FOR FOOD Human foods and animal feeds, as well as the places where these materials are stored and processed, are attractive to a wide variety of animals. Competition between ‘‘LIS’’ and “them” for nutritive substances is keen and continuous. Pests of the food industry may be counted upon to take full advantage of every opportunity to convert foods and feeds to their own use. To prevent this, nothing less than constant vigilance is required of the foodindustry. But evenunder thebest of circumstances, it isdifficulttoconsistently provide the consumer with foods and feeds that are completely free from contamination by food pests ( 5 ) .
111.
EARLY IDEAS AND DISCOVERIES
The idea that pests might be vectors was very slow to dawn upon the human mind. The germ theory itself had to be established thoroughly first, and that was another idea that was slow to take hold. But once the germ theory was accepted, people began to ask about the ways that germs make their way from a person who is sick to a person who is well. By the dawn of the twentieth century, it was well known that the germs that cause some diseases could be transmitted directly from one person to another. This method of transmission,seemingly so simple andlogical,becametheoverridingtheorythatwas applied, correctly or not, to a wide variety of diseases. In some cases, the theory worked perfectly; in other cases, it seemed to work well most of the time, but not all of the time; and in still other cases, it did not work at all.
IV. TRANSMISSION CYCLES Classic studies of malaria, yellow fever, and Texas cattle fever firmly established the fact that somediseasetransmissioncyclesincludeanobligatoryintermediary-thevector. Also, the disease agents themselves were identified as belonging to a wide rangeof categories in the living world-bacteria, protozoa, helminths, viruses, and fungi. The transmisto the very complex. sion cycles of these disease agents varied from the very simple
Food, Filth, and Disease
629
Before the first half of the twentieth century came to a close, a large number of vector-borne diseases had been studied to the point where their mechanisms of transmission were well understood. Two basic cycles of pathogen transmission by vectors had become well known: biological and mechanical. In the simplest form of biological transmission, the pathogen must enter the vector, undergo at least one mandatory phase of multiplication and/or morphologic transformation, and be carried by the vector to a susceptible host. Biological transmission is not discussed in this chapter. In mechanical transmission, the pathogen remains on the external surface of the vector, in its gut. or i n its excretory system. The vector merely transports the pathogen from an infectious source to a susceptible host or to something that a susceptible host eats or drinks.
V.
MECHANICAL TRANSMISSION
What of foodborne pathogens and the waysthey are transmitted? Biological tranSllliSSiOn and the role of food pests as direct agents of disease (that is, in the production of toxins, allergens,orphysicalinjury,andintheinvasion of thedigestivetract by insectsand mites) are not considered here. The focus here is on mechanical transmission of foodborne pathogens. The mechanical transmission of foodborne pathogens is another idea that has been very slow to take hold. It seems very easy for professional health care providers to accept the concept that a diarrheic food handler emerging froma bathroom without hand washing and immediately beginning to prepare a shrimp salad could contaminate the food with pathogenic bacteria that could cause illness in susceptible people who eat that food. This is the classic fecal-oral route of transmission that every clinician and epidemiologist remembers when confronted with a case or an outbreak of probable foodborne illness. When an entomologist puts forth the idea that a house fly or a cockroach also might pick up pathogens from feces and carrythem mechanically to human food and that people might become ill from eating that food, the traditional medical person expresses incredulity and looks for another (logical) explanation. A good exampleof this kind of tunnel-vision approach can be found in the literature on the epidemiology of hospital infections i n which the notion that hospital pests might be vectors of pathogens rarely is considered (6) (see of Appendix A). There is, however, a growing literature on this subject, some examples which are given i n the references cited here. Before the twentieth century was even a decade old, Harvey Wiley, that great champion of food purity, already had made plans to study the role of house flies as mechanical carriers of bacteria (7). In the early years of the twentieth century, L. 0. Howard (8), an entomologist, and Samuel Crumbine ( g ) , a public health administrator (to mention just two among many), castigated the house fly as a menace to public health ( I O ) . Since that time, other scientists have sought to build the case for the roleof vectors in food contamination and foodborne illnesses. There have beenso many such studies that only a few can be mentioned here. Several linesof thought that researchers and reviewers have applied to this question are listed here, each followed by a few examples from the literature.
A.
Pathogenic Microorganisms on Pests
A common approach in evaluating the role of pests as vectors in the epidemiology of foodborne illnesses is to capture a pest from its natural environment and find out what
Gorham
630
kinds of microorganisms it carries on its body surface or in its gut. Hospitals have been a common target for this kind of investigation, with the focus on ants, cockroaches, and other pests ( 1 1- 15). Early in the twentieth century,flies and cockroaches were surveyed for their bacterial loads (16,17). Sincethat time investigators have surveyed flies and various other potential vectors for their bacterial burdens (18-27). Other potential vectors have been surveyed for pathogenic fungi (28,29), protozoans(30-321, helminths (33-35), and viruses (36,37).
B. Fate of Pathogens on Pests Another common technique is to take a clean pest, expose it (externally or internally) to some known pathogen population (usually monospecific), and then see what happens to the pathogens. Root (38) foundthat cysts of Entamoeba kistolytictr, ingested by flies that then were drowned in water, remained viable for 7 days. Strltnonelk~r orrrnienburg fed experimentally to American cockroaches (Periplanctcr crtnericcrntr) could be isolated from the excretal pellets of those roaches over a period of 140 days (39). Griffits showed that ants (Solenopsis gernintrto) can carry bacteria (Shigellu Jexncri) for 24 hours after being contaminated (40).
C.
Role of Pest Habits in Mechanical Transmission
A third approach in evaluating the role of pests as vectors is to study the morphology, physiology, and behavior of a pest to discover what attributes it possesses that make it especially accommodating to the acquisition and carriage of pathogens. Some pests make especially suitable mechanical vectors because they have structural features that facilitate carriage of pathogens (41). Commensal rodents (42), cockroaches (43), ants( 4 4 ) , and flies (8) have habitsthat often take themto both contaminated and clean substrates (e.g., human food or food-contact surfaces). The Asian blow fly ( C h t y s o t n y a tnegcrcephcrla),for example, oviposits on human feces (omnipresent in the Orient) and feeds on confections and fruits in the markets (45). The status of physiological resistance mechanisms of insects to the presence of microorganisms pathogenic to humans is largely unknown. The roaches Periplmetn mstral r r s i m and P. ameticrrncr apparently lack antibacterial substances that deter gut infections by Strlrnonelltr typhi (46). Greenberg found that, more often than not, larval house flies (Musca domestica) lose their salmonellas during ecdysis (47). Vector behavior may be naturally conduciveto mechanical transmission-as by the regurgitation habit of flies, for example (48)"or the behavior sometimes may change as a result of infection. Rats infected with Soltnonellu typhitnuri~tntendto wander more widely than healthy rats (49).
D. Correlation of Pest Populations and Disease Incidence Another line of investigation is largely statistical but still requires intensive work in the field and in the laboratory. This involves correlation of pathogen occurrence in a human population with that same pathogen in the population of a suspected disease vector. Sometimes a marked increase in a probable vector population is followed by an epidemic in the human population. Microbiological samples must be collected from both people and
Food,
Disease
63 1
vectors. A corollary approach, after an epidemic has gotten under way, is to drastically reduce the suspected vector population by some control measure. If the epidemic curve subsequently falls away, taking into account incubation periods, then the implication follows that there was some cause-and-effect relationship between the disease course and the vector population. Polish investigators found that the kinds of pathogenic bacteria collected from patients i n Rzesz6w provincial hospital also were being carried by cockroaches (Blcrttellrr gertnernica, Blcrttcr orienterlis) infesting the hospital (50). In a similar situation, Indian scientists found a certain antibiotic-resistant strain of Klebsiella pt~eurnonicrein patients and in German roaches in a New Delhi hospital (51). An outbreak of shigellosis in Northern Shigelln dysenteriae Ireland was linked to an infestation by German roaches from which wasisolated (52). Scrlmonelln typ/?irnuriurn wasisolatedfromsparrowsinfestingthe kitchen of a mental hospital in which patients were suffering from gastroenteritis caused by the same strain of Scrl~nonella(53). An outbreak of infant diarrhea, attributed to Saltnonellcr bo~~ismorb$cans, inan Australian hospital was traced to improper sanitary practices. The same pathogen was isolated from cockroaches and mice infesting the hospital (54). The case rate during an outbreak of infant diarrhea in a Belgian hospital dropped off shortly after the cockroaches (Blcrttdl~rgermcrrriccr) infesting the ward were killed by an application of dichloro-diphenyl-trichloro ethane (DDT). Scrlmonelk~typhinrurium was isolated from the patients and the cockroaches ( 5 5 ) . Levine and Levine(56),i n a reviewof the literature on the epidemiologyof shigellosis, concluded that fly control measures tend to reduce theincidence of shigellosisin human populations. This conclusion was borne out by an experimental study in Israel. Rates of shigellosis were lower in cornmunities in which fly suppression measures were carried out than in communities without fly control programs (57).
E. Sampling Pathogens from Foods and Pests Another technique i n the investigation of foodborne illness is to sample suspect foods to see if there is a match between the microorganisms found in the food and those taken from people who are ill. A further step is to collect specimens of pests that have had access to the food to see if they are carrying the incriminated pathogen. This approach, too, has a corollary. Inoculate the pest (vector) with a pathogen and letthepest have access to human food. Then, let a volunteer eat the food and see what happens. This corollary approach has few champions currently. In 1935, Staff and Grover (58) did a classic investigation of a foodborne disease outbreak, linking the patients (208, with 3 fatalities), the food product (a filled pastry), and the vector (Rattus norvegicus) to the etiologic agent, Scrlmonellrr enteritidis.)
F.
Role of Vectors in Mechanical Transmission
The last and,so far, least-productive effort is totry to establish a cause-and-effect relationship between human foodborne disease and the activity of a probable vector. The Staff and Grover (58) investigation was exceptional; too often. the evidence is largely circumstantial. But there is at least one disease in which the routeof transmission clearly involves vector contamination of food and drink: Bolivian hemorrhagic fever andthe rodent C&NIyS callos1rs (59).
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632
G. Role of Pantry Pests in Mechanical Transmission Left to themselves, the pantry pests-insects and mites that make their home in raw or processed foods-have little opportunity to pick up pathogens that can be carried on their bodies. Unfortunately they are not left to themselves under most circumstances. The foods i n which the pantry pests live also are visited by ants, flies, mice, rats, birds, cockroaches, and other vermin, all well known for their habit of indiscriminately foraging on filthy substrates andon foods and food-contact surfaces. Thus the more mobilepests bring pathoi n turn, pick up the pathogens gens to the home territory of the pantry pests, and they, and disseminate them wherever they chance to travel within the food material. Experimental work by Klowden and Greenberg (60) demonstrated unequivocally the potential for mechanical transmission to be involved in a dangerous situation for coni n food sumers.When the mechanicaltransmissionoccursafterthefinalbiocidalstep processing, consumers may ingestviable pathogenic organisms andrun the risk of becoming ill. When bacteria (Srrlnrorwlla species) were placed onthe bodies of cockroaches that subsequently were killed, the bacteria survived on the dead bodies for as long as 60 days. If the cockroaches had diedin a food material that was destined to be eaten without further biocidal treatment. then the consumer would have been at risk of foodborne illness.
VI.
NEW RISKS AND REMEDIES
A.
NewRisks: Emerging Pathogens
There may have been a time when one could be nonchalant aboutfilth in food, but now we live i n a much different world.In the past decadeor so we have witnessedthe emergence of foodborne pathogens possessing unparalleled virulence. These pathogens can make even normally healthy people sick: sometimes they have killed otherwise healthy individuals. This means that they are an even greater threat to the immunocompromised population. That population includes those who are infected with HIV, of course; but there are other large segments of the immunocomprolnised population-the elderly, the very young, the traumatized by accident or surgery, and recipients of radiation therapy or chemotherapy.
B.
Mechanical Transmission (Again)
As was demonstrated earlier in this chapter, pathogens may be carried about by insect, mite, rodent, and bird vectors. The pathogen load borne by such vectors has traditionally (but not necessarily accurately) been thought of as very small i n relation to the infective dose, that is, the minimum number of pathogenic units required to generate symptoms i n a normal human host. Now we have a much different situation in the world. Even if the pathogen load carried by a vector is small, it may be enough to produce illness in an ilnlnunocompromised host. Further, the pathogen may be one of the newly emergent “super” microbes; in that case, the “too small” pathogen load may be quite heavy enough to make even a normally healthy person ill.
C. Mites as Direct Agents of Disease The idea that eating insects and mitesin our food or eating food that has been contaminated by insectsandmites is harmless is becoming so pervasivethatsomefoodregulatory
Food, Filth, and Disease
633
agencies have simply stopped doing filth inspections and filth analyses. Of course, it is true: Most of us will not suffer any ill effects from ingesting a few dead insectsand mites with our food. Buttheoccasionalseriousillness,suchasanaphylaxisassociatedwith ingesting mites (61,62),jars the complacency usually associated with such an apparently benign phenomenon and justifies the establishment of regulatory action criteria (63).
D. New Remedies: HACCP The Hazard Analysis Critical Control Points (HACCP) program for food safety, when properly implemented and maintained, has the ability to protect everyone from both the traditionalfoodbornepathogensandparasitesandfromthenewlyemergingones. Although hard foreign objects are treated as one of thecriticalcontrolpoints,theother adulterantsgenerallyreferred to as "filth i n food"receiveverylittleattention in the HACCP protocols. However, this is not to say that filth is considered to be unimportant. HACCP does not deal specifically with the sources of filthin food because the whole edifice of HACCP is built (and must be built) on a foundation of thorough, persistent, and dependable sanitary practices in the food industry. A substantial part of preventing pathogens from getting into our food supply is preventing pests from getting into our food-and this must be done at all stages of production, harvest, storage, manufacture, distribution, retailing, and even to the home kitchen, the latter being the theater where the majority of foodborne infections occur. of mite,insect, Althoughrelativelylittleisknownaboutthehealthsignificance rodent, and bird filth i n food, the information on hand indicates that food pests must be excluded from all situations in the food industry. More laboratory research is needed to determine the pathogenic significance and risk to the consumer of filth i n food. Cases of filth-associated illness must be investigated thoroughly and reported in the scientific literature. Regulatory and quality assurance personnel in the food industry should be alert for opportunities to fill in the gaps in our knowledge of foodborne filth and its relation to human disease (64,651.
APPENDIX: HOSPITAL PESTS AND HOSPITAL PATHOGENSA MISSING LINK* Rats,' flies,! and cockroaches' are famous for their alleged ability to harbor and transport human pathogens. Ants' and beetlesJ are suspect, too. A knowledge of the habits of these creatures makes it easy to imagine how pests might contaminate presumably clean, even sterile, surfaces without leaving any visible traceof their visits. Add to the proved capacity of pests as carriersof pathogens the fact that these pests are frequent guests in our hospitals, and you have a convincing indictment based on circumstantial evidence. The paradox is that circumstantial evidence isallwehave.Investigators have traced hospital-acquired infections to many different sources," but rodents and insects have rarely been definitely incriminated, to my knowledge, i n published reports. The closest thing to it was an epi-
:b
A guest editorial by J. R. Gorhm with literature cited, reprinted fromSonscripr, thc official publication of the Georgia Society of Registered Professional Sanitarians,Vol. 3, No. 1. February 1960, p. 17.
Gorham
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demic of salmonellosis among infantsi n the nursery of a European hospital. The epidemic persisted in spite of traditional control measures. but stopped suddenly after cockroaches infesting the incubators were eradicated.' Leaving aside the minor problem of protecting allergic patients,s is hospital pest control done only for esthetic and economic reasons'? Or are there valid medical reasons too? Sanitarians, housekeeping and food-service supervisors, and physicians need to give some thought to these questions (feedback is welcome!).
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60. MJ Klowden, B Greenberg. Effects of antibiotics on the survival of S r r l r r l o r d l u i n the Alncrican cockroach. J Hyg 79339-345. 61. AM Erben, JL Rodriguez. J McCullough, DR Ownby. Anaphylaxis after ingestion of bcignets contaminated with Derr,rcctol,ltrrRoice.s firrirwc. J Allergy Immunol 92:846-849, 1993. 62. T Matsumoto, T Hisano, M Hamaguchi, T Miikc. Systemic anaphylaxis after eating storagemite-contaminated food. Int Arch Allergy Immunol 109: 197-200, 1996. 63. AR Olsen.Regulatoryactioncriteria for filthandotherextraneousmaterials. 11. Allergenic mites: an emerging food safety issue. Regul Toxicol Pharrnacol 28:190-198. 1998. 64. JR Gorham. Food pests as discasc vectors. In: JR Gorham, ed. Ecology and Management of Food-industry Pests. Arlington, VA: AOAC International, 199 1. 65. AR Olsen. Rcgulatory action criteria for filth and other extraneous materials. 111. Reviewof flies and foodbornc enteric disease. Regul Toxicol Pharnmacol 28:199-211. 1998.
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24 Food Filth and Analytical Methodology: A Synopsis
1. Introduction 639 A. B. C. D.
11.
The practice o f f o o d analysis
Filth. Decomposiiion, and Foreign Matier 641 A. Heavy filth 641 B. Decomposition 04 I C. Light filh 641 D. Soluhlc filth
Ill.
639
Why a n n l y ~food'? 640 Some kinds o f :un:llysis 640 History of filth unalysis 640
Quality Assurancc
642
642
A. Methods o f quality assurance 642 B. Analyst quality assurance 643 C. Benefits t o the public 642 References
643
1. INTRODUCTION This short review should i n no way be construed as a technical guide to filth analysis; it is meant merely to put the practice of filth analysis in its proper place in the overall arena of food safety. Reliable sources of technical infonnation are readily available ( 1-4).
A.
The Practice of Food Analysis
The practice of food analysis occurs at many different levels of sophistication; moreover, i n the vast Imjority of instances, there has to be a good reason to do the analysis. The more informal the analysis, that is, on the lower end of the sophistication scale, the less expensive it is. The more formalthe analysis, that is, higher on the scaleof sophistication, the more expensive it is. This expenditure of money and effort hasto be justified; in other 639
640
Gorham
words, there has to be some compelling reason to carry out the analysis. Generally the more sophisticated procedures take place ina laboratory setting; the less formal procedures may occur in a wide variety of settings, including the laboratory.
B. WhyAnalyzeFood? The reasons for carryingout an analysis vary widely, but the basic motive of food manufacturers is to present to the consumer foodthat tastes good, smells good, and is good. Achieving this maintains or enhances one's reputation, whether individual or corporate. The productionandmarketing of onlygoodandwholesome food also means thatthe food establishment is complying with the pertinent federal, state, and local laws pertaining to foodpurity,thusavoidingtheunpleasantconsequences of violatingtheselaws:fines, prison time, and civil lawsuits, to mention a few (5-7). Other reasons for conducting food analyses arise from the law enforcement side of the equation. Voluntary compliance with the laws and with good manufacturing practices is very beneficial to the consumer, but the only way to know for sure the purity status, as defined by the pertinent laws and regulations, of any given food at any given time is to analyze it. Here, with regard to the Federal Food, Drug and Cosmetic Act (FD&C Act) (S), at least, analysts are guided by the two major filth provisions in the act, namely, that filth has no place being in food or being around food.
C. SomeKinds of Analysis The quick and casual observations by sight and smell that happen whenever a can or package of food is opened in the home kitchen constitutes food analysis-inforl71a1, but often quite telling. Similar things happen in the groccry; cans and packages arc usually not opened unless in connection with a deli operation, but cans and packages are obscrved for defects, and substandard items of all kinds are culled before being presented to the public. Visual inspection (i.e., analysis) takes place in the warehousing and distribution sectors of the food chain. Items that look bad or smell bad are discarded. In the food manufacturing sector, food analysis is an integral part of the operation's quality assurance protocols. Similar analyses, usually informal, but some involving technias ingredients cal expertise, happen at the places where raw materials are collected for sale in the manufacture of foods. Analyses occur, too,at the many places where food materials are harvested. on the wharf or in the warehousc, or at Food materials may be visually inspected the granary or flour mill. It is common for samples to be collected in all of these places and sent to a government food laboratory for further examination.In the laboratory setting a whole range of analytical techniques may be employed, from the simplest to the most technically complex.
D. History of Filth Analysis In the early days of the 20th century, Harvey Wiley andhis group of intrepid food testers focused most of their attention on toxic chemical adulterants (9). However, even in those early days, around the time of the passage of the first federal food purity law ( i n 1906), some effort was made to detect filth in food. Since the detection process required some form of analysis of each kind of food, analytical methods were creatcd out of necessity.
Food Filth and Analytical Methodology
64 1
II. FILTH, DECOMPOSITION, AND FOREIGN MATTER A.
Heavy Filth
Although the word filth is i n the title of this chapter, it is really just the first word in a triad of contaminants: filth, decomposition, and extraneous materials or foreign matter (4). This last term really covers all the others, too. but in conmot1 practice it refers to sand, gravel, paint chips, tnetal fragments, pieces of glass, fruit pits, and a whole host of other hard foreign objects (these kindsof extraneous materials are often collectively called “heavy lilth”). The food defect action levels (DALs) ( I O ) allow very small quantities of fruit pits to be present in food: at these low levels they are considered to be natural and unavoidable constituents of food. Other hard foreign objects are considered adulterants under the provisions of the FD&C Act if they may cause injury to the mouth and digestive organs ( 1 1; see also Chapter 22) or if they simply make the food feel gritty in the mouth ( I O ) . These hard foreign objects can be sieved out of food, or they settle out of the food materials upon agitation. Hard foreign objects are now included in Hazard Analysis Critical Control Points (HACCP) programs because they may cause injury to the consun1er (12).
B.
Decomposition
The detection of decomposition is a specialized kind of analysis. The processof decomposition often leads to the production of characteristic odors that the analyst can detect by organoleptic methods (smell, in this case,but taste is also employed in some cases). Organoleptic methods ( 1 3 ) have been widely used i n the past to detectdecomposition in seafoods and i n the carcasses of meat animals and poultry. With the beginningof HACCP program in the meat industry, organoleptic analyses have become less common. Molds often occur on fruits, nuts, berries, seeds, and nmly other food materials. These defects may be detected by visual examination-techniques that are referred to a s macroanalytical methods ( 14,151. On occasion, to get an approximation of the level of moldiness, the product i n questionissubjectedto a specialsamplingprogram that is designed to make the sample representative of the entire lot. These macroanalytical techniques are often used when unprocessed foods are presented to the food nmnufacturer for use as ingredients. In other techniques. such as the Howard mold count, the sample of food is prepared for analysis i n such a way that the mold fragments become visible under light microscopy and that the mold count in the sample accurately reflects the mold count i n the entire lot that was sampled ( 1 6).
C.
Light Filth
The detection of insect fragments, feather barbules, and rat hairs-all properly called filth, butsolnetitnesreferrcdtoaslight filth-has been a majorfocus of foodanalystsfor more than half a century. Basically these kinds of analytical methods, often referred to as lnicroanalytical methods, involve dispersing extraction oil into an aqueous or alcoholic mixture of the food sample. Since the bits of insect cuticle, feather barbules, and hairs are oleophilic, they tend to rise to the surface. The food materials, being largely hydrophilic, tend to settle to the bottom. The analyst then simply separates the two polar opposites and examines the extractiotl oil fraction under a microscope (17).
Gorham
642
This oil/water technique is often employed when itis necessary to establish (or reexamine) a DAL for a particular product (18). In one instance in which questions were raised about filth adulterants in canned sardines, canned tuna, and canned crabmeat, Samples were collected, using a statistically valid sampling protocol, from many localities around the United States (19). Other products are sampled and analyzedin a similar manner. These sample collection programs produce such huge quantities of product that it is usually not feasible for a single government laboratory to do all the analytical work. Most of the work is performed by contract laboratories in the private sector.
D. Soluble Filth In recent years there has been a trend to go beyond trying to detect just particulatc filth. These efforts have focusedon identifying and quantifying the biochemical signatures left behind by food-infesting animals (birds, rodents, insects, mites) (20-26).
111.
QUALITYASSURANCE
A.
Methods of Quality Assurance
One very important aspect of filth analysis is perhaps little known and little appreciated by the general public. I refer to the great emphasis on quality assurance on the part of the analyst population. For example, when an analyst develops a new analytical technique (a phenomenon that is quite common), that technique does not simply and immediately become a standard method. The technique has to be evaluated by a panel of expert analysts to determine its reliability in the hands of different analytical technicians. The new technique will most likely get wide exposure by its publicationi n the J o u m d ofAOAC h f e r r m f i o r d . If it survives the examination occasioned by thc journal publication, the technique may eventually make it into the Qffcirrl Mefhorls of A11crlysi.sof AOAC Itlremafiolrtrl ( I ) .
B.Analyst
Quality Assurance
Most filth analysts are tested periodically to evaluate their analytical skills. Test samples are sent out to the analysts and the analytical results they derive are then evaluated. In this way ahigh level of consistent expertise is maintained, and any deficiencies in analyst’s performance can be remedied. The achievement and maintenance of a high and consistent level of skill comes by way of unending practice and the pursuit of perfection in carrying out analytical techniques. A huge body of literature (see, e.g., Ref. 27-3 1 , as well as all of the references at the end of this chapter) and a wide array of analytical techniques ( I ) must be mastered by the analyst.
C. Benefits to the Public This emphasis on quality assurance directly benefits the general public. When any givcn food is found to be adulterated, the public can have confidence in that finding and reassurance in knowing that such adulterated foodwill not reach the dinner table. Silnilarlywhen analysts find no filth in food andit has been certified that all applicable HACCP protocols
Food Filth and Analytical Methodology
643
have been observed, the public can be confident that these findings reflect the actual state of the food-that it is wholesome and safe to consume.
REFERENCES 1.
2.
3.
4. S.
6.
7. 8.
9.
IO. I I.
12. 13.
14.
1s.
PA Cun11iff,ed. Official Methodsof Analysis of AOAC International, 16th ed., 3rd rev. Arlington. VA:AOACInternational.1997. JR Gorham. Filthand extraneous matter in food I n : YH Hui, ed. Encyclopedia of Food Science and Technology. New York: Wiley-Interscience, 1991, pp. 847-868. AR()[sen, TH Sidebottom, SA Knight. eds. Fundamentals of Microanalytical Entomology. A Practical Guide to Detecting and Identifying Filth in Foods. Boca Raton, FL: CRC Press, 1996. JR Gorham, cd. Principles of Food Analysis for Filth, Decomposition, and Foreign Matter. FDA Technical Bulletin 1. Arlington, VA: Association of Official Analytical Chemists.198 1. PM Brickey Jr. Concepts of food protection. In: JR Gorham, ed. Principles o f Food Analysis for Filth. Decomposition, and Foreign Matter. FDA Technical Bulletin1. Arlington. VA: Association o f Official Analytical Chemists, 198 1, pp. 3-5. PM Brickey Jr. The Food and Drug Administration and the regulation o f food sanitation. I n : JR Gorham,ed. Ecology and Management of Food-Industry Pests. Arlington, VA: Association of Official Analytical Chemists, 199 I , pp. 49 1-495. JS Kahan. Criminal liability under the Federal Food. Drug, and Cosmetic Act-the large corporation perspective. Food Drug Cosmet Law J 36:314-331. 1981. Food and DrugAdministration.FederalFood,Drug, and CosmeticAct, as Amended, and Related Laws. HHS Publicationn o . FDA 89- 105 1. Rockville, MD: U.S. Department of Health and Human Services, 1989. JHYoung.PureFood-SecuringtheFederalFood and Drugs Act o f 1906. Princeton, NJ: PrincetonUniversityPress, 1989. Industry Activities Staff. Thc Food Defect Action Levels. Washington, DC: Food and Drug Administration,1998. FN Hyman, KC Klontz, L Tollefson. Food and Drug Administration surveillance o f the role o f foreign objects i n foodborne injuries. Public Health Rep 108:54-59, 1993. JR Gorham. Filth and extraneous matter in food. In: Encyclopedia of Food Science and Technology, 2nd ed. Ncw York: Wiley-Interscience. 1999. MD Rosen. The art and science of organolcptics. Lamp 80(2):12-15, 1998. PMBrickey Jr. Macroscopicanalysis. In: JRGorham, ed. TrainingManual for Analytical Entomology in the Food Industry. FDA Technical Bulletin 2. Arlington, VA: Associatioll o f Official Analytical Chemists. 1978, pp. 91-1 1 1. AR Olsen, SA Knight, GC Ziobro, cds. MacroanalyticalProceduresManual. ht[p:// vm.cfsan.fda.gov/-dlns/~np~1~-toc.htll71.
16.
17.
18.
19.
20.
SM Cichowlcz. Analytical mycology. In: JR Gorham, cd. Principles of Food Analysisfor Filth, Decomposition, and Foreign Matter. FDA Technical Bulletin1. Arlington, VA: Associationof Official Analytical Chemists, 198 I . EC Washbon. KR Halcrow. Laboratory procedures.In: AR Olsen, TH Sidcbotto111,SA Knight, eds. Fundamentals of Microanalytical Entomology. A Practical Guide to Detecting and Identifying Filth i n Foods. Boca Raton, FL: CRC Press, 1996. JM Taylor. Establishment and use o f defect action levels. In: Association of Food Industries 82. Matawan, NJ: Association of Food Industries, 1982. RG Dent, JR Gorharn. Collaborative study of extraction of light filth from canned crab Incat, J Assoc Official Anal Chcm S9:825-826. 1976. JJ Thrasher. Detection of metabolic products. In: JR Gorham, cd. Principles of Food Analysis
644
21.
”.3 7 23. 24. 25.
26.
37. 28. 29. 30.
31.
Gorham
for Filth. Decomposition, and Foreign Matter. FDA Technical Bulletin 1. Arlington, VA: Association of Official Analytical Chemists. l98 1 . HR Gerberg.Chemicaltestfor manmalian feccs in grainproducts:collaborativestudy. J Assoc Official Anal Chem 72:766-769, 1989. PEKauffman,DBShah. Enzyme imnlunoassayfordetection o f D r o s q d ~ i /,,re/trrloRtr.slcr ~ antigens in the juice of various foods. J Assoc Official Anal Cheln 71:636-642, 1988. FA Quinn, W Burkholder. GB Kitto. Ilnlnunological technique for measuring insect contamination of grain. J Econ Entomol 85:1463-1370, 1992. B Bradcr. Are food sanitation assays nlcanlngful’? Cereal Foods World 42:759-760, 1997. PA Valdcs. GC Ziobro, RS Ferrera. Use o f urease-bromothymol bluc-agar method for largescale testing of urine on grain and seeds. J AOAC Int 79866-873, 1996. P Valdes-Bilcs and GC Ziobro. The identification of the sourcc of reagent variability in the xanthydrolhrea method. J AOAC Int 81:1155-1161. 1998. G Domenichini. ed. Atlantc delle Impurith Solide negli Alimcnti-Manuale per il Riconoscimento dci Materiali Estranci. Pinerolo, Italy: Chiriotti Editori, 1997. in Food:AnIllustratedKey.AgricultureHandbook JRGorham, ed. InsectandMitePests 655. Washington, DC: U S . Department of Agriculture, 1991. D McClymont-Pcace. Key for Identification of Mandibles of Stored-food Insects. Arlington, VA: Association of Official Analytical Chemists, 1985. DH Ludwig. JR Bryce. Hairs and feathers. In: AR Olsen, TH Sidebottom. SA Knight, eds. Fundanncntals of Microanalytical Entomology. A Practical Guidc10 Detecting and Identifying Filth in Foods. Boca Raton, FL: CRC Press, 1996. JW Gentry, KL Harris. JW Gentry Jr. Microanalytical Entomology for Food Sanitation Control. Melbourne, FL: JW Gentry and KL Harris, 1991.
lndex
AAPCC. 2 Abalone. 56 poisoning, S7 Ahr-us prec~rtorir4.s.620
Acanthuridae. 29 Acmrt\rrrrrr.s 1irlecrtrr.s. 29
Acaricides. 537. S39 Acceptable daily intake (ADI). 547 Acetylsalicylic acid, 388 Acipenseridac, 40 Actrrcdcs t o r r r c v r t o s r r s ,
64
Actiniidac. S 3 Actinodisc~dae,53 Active Foodborne Disease Surveillancc Systeln, 1 15
Acule myocardial infarction (AMT), 398 Additives, food ( S W Food additives) Adenosine receptors, antagonism of, 405
Adenoviridae, 120 Adulteration o f food. 627, 633. 641-642 Advanced glycation end product (AGE) formation: (AGE) formation. 384-388. 39 1-392 pyridoxamine, 3XX thiaminc pyrophosphate, 388 vitamin B,, 388 vitamin B,>,388 Acrotrrntrrr.7. l 18. 152, 153. 154 153 h~r/rophi/rr,111, 113. 118. 123. 125, 153. 154, 155 l~r,rollii,153- I S4 Aflatoxins, 382. 417-420. 541 Africa, 38. 56, 84. 117 Aging, glycation and. 386-388 ctnk..
Agnntha, 25 Agricultural chemicals, 537-556 Agricultural Marketing Service (AMS). S49 Alaska. 70 Altrthyricr corldo/(r. 80 Albacore, 42 Albulidae, 33 Alcyonarians, S 3 Alepocephalidae. 33 Al~~smtdriurrr: c ~ r r t c l l e l l r l ,80 l l l i l l l ~ t r ~ l l 80, r, 95 o.stcr!fildii. 80 l~zlll~rrl~Il.YC. 80 Algacide, 539 Algae. 63 toxic. 28 Alocrrsitr rtrrrcrorr1li:tr. 65 Alsitlirrrrr corrr//irrrrr, 194 Alternatives and mignunt chemicals. 6 1 1 Aluminum. 401 Alrrtcwr sc'riptn, 20
Alzheimer's discase, 401 Amadori glycosylation products, 383-385, 388
American Association of Poison Control Centers (APPCC), 1 ( . v w also Poison control centers) toxic exposure surveill;~ncc system (TESS), S Aminoguanidinc. 388 Anmsic shellfish poisoning, 5 I , 56. 3 17 Amphidinium carterae. 28, 52 Amphineura, 56 Amphipods, 68 Arltrhcrcwcr
circinnlis. 80 645
646
Index
Anacardiaceae, 620 Arltrccrrtlilrnr occitlerlttrle, 620
Analysis. drug residues, 584 Anaphylaxis. 633 Anchovies, 33. 34, 35. 194 Angrtilltr m g r ~ i I / t I 41 . Anguillidae, 41 Anguilliformcs, 41 Anilnal production, 579 Anthozoas, 53 Anthropogenic sources of radioactivity in food and water, 558, 564 Antibacterials. drug residues, 588 Antibiotics. drug residues, 588 Antihclmintics. drug residue$. 587 Antihistamines. 60 Antimicrobials, drug residues, 588 Antioxidants, 495 Antiprotozoal drugs, drug residues, 586 Ants. 630-631, 633 Ap/ysirr, 96 Apodes, 41 Arachidonic acid. 388. 392 Archtrc~btrc~teritr, 1 17 Arctic, 70 Atlantic Ocean, 27 Occan, 70 Arothrorl hispitlrrs, 38 Arotl~ror~ rrrclrtrcyris. 38 Arteries. stiffness. 387 Arthropoda. 63 Arthropods, 65 poisonous. 63 Ascorbic acid. 404, 416 (sec, trlso Vitamin C) Ascorbic acid. calcium absorption and. 395 Asia. 40, 41, 63, 64, 66, 70, 1 16. 127, 128, 130
Asian blow fly, 630 Asia-Pacific region, 78 Asiatic horseshoe crab. 63-64 poisoning. 63 Asiatic porpoise. 68, 69, 70 ASP, 78-79, 90-94, 185,194-201 testingmcthods.194-201 A.sper.gil1~r.s.4 I8 j / ~ / l ' / / .4~ 17-4 . I 8, 54 1 /Jtl~(l.Viti('ll.Y,4 17 Aspirin. 388 Associate o f American Poison Control Center (AAPCC), 2 Asteroidca. 54
Astro1iricim>, 120 Astrolirrts, 119, 120.154.1.56. 159 Atelgotis joridrrs. 64 Atherosclerosis, 387, 390. 393. 397
risk factors for, 389 Atlantic hagfish, 26 Atlantic Ocean, 25, 27, 29, 34. 35. 38, 42, 55, 69, 194 Attractants, 537 Australia,27, 56, 70, 84, 87, 154, 155. 158 Autooxidative glycosylation reaction, 384 Azores, 55 Bd?\./orlicr jlq'oizic~n,58 Brrc~ill1t.s. 1 I8 cercus. 137, 403
Bacteria.121-126 Bacterial toxigcncsis, 24-25, 28. 36 Bactcriocides. 537, 539 Btrlrrerwpter(c horerrlis. 68. 69 Balaenopteridac, 68 Baleen whales. 68 Balistidac, 29 B~rli.sr0itle.s~ ~ o l ~ . s / ' i ~ ~ ~29 /llliil. Barbel, 40 B(li6lr.s. 4 1 h n r l ~ ~ t40 s, Barracuda, 32 Bay of Bengal, 63, 128 Beaked narwhal. 68, 69 Bean, jcquirty, 620 Beche-de-mer. 55 Beetles. 633 Bclonifonncs. 35 Benedryl, 36 Benzo(a)pyrene (B(a)P).420-422 Bering sea, 69 Bioassay methods: for doluoic acid, 195-196 for paralytic shellfish poisoning, 185-189 miscellaneous. 188- 189 II1c)Usc, 185-188,195-196 Biotoxicntions, 61, 64. 65 marine, 25, 37. 43, 51, 53 Biotoxins, in fish. 23 Birds. food-infccting. 631. 642 Birgrts lotro, 6.5 Bivalves, 56 (.sc,c trlso Bivalve mollusks) Bivalve mollusks, 59. 78, 94-95, 1 1 1, 112, 121, 152. 1.57. 158, 185 poisonous, 59, 60. 61 Black Sea, 40
647
Index
Black-tip reef sharks. 27 Blooms, 52, 78, 95-96, 184- 185, 189 dinoflagellate, 52 phytoplankton. 52 Blowfish, 38 Bluefish, 35 Blue-green algae, 94 Blue whales, 68 Bolivian hemorrhagic fever. 630 Bonefish, 33, 40 Bottles, retumahle beverage. 619-620 Botulinum toxin, 160- 16 I Botulism. 159,169, 312 Brcr.s.sic~c1.395 Brevetoxins, 87-90, 96
Brcvitoxic shellfish poisoning, 5 I . 52 British Columbia, 60 Brittle stars. 54 Buccinidae. 58 Bull-head sharks, 27
hi/'/'O", 29 Cerrrrrrs l l l e l t l l l l / ~ ~ < 29 ~f~.s, Carcharhinidae, 27 CorcherrI1irllr.s trrllhoirlc,rl.si.s. 27 Qrrchtrrkirlu.s lelrcas, 27 Corchrhinus rrrc,lrrrrol,terrt.s. 27 Cctrcharotlorr ctrrcktrritrs, 26 C N ~ ~ ~ I I O63 S ~ ~ O ~ ~ J ~ ~ ~ S , roturrrlicod[r, 63 Cardiovascular disease (CVD). 393, 397. 417, 423 Crrrc,ttcr cerretto gigcrs, 66 Carnivora, 67 Curpi1irr.s r r r a c w l n t l r s , 64 Carrihean, 28 Cascillophosphopeptides (CPPs). 395 Cashew nut. 620 Caspian Sea. 40. 160 Castor oil fish, 35 Catalase. 397 Catfish, 1 12 Cclr(I1l.Y
Caahezon, 40-41 Cadaverine. 36 Cadmium. 400. 40 1. 402 Caffeine, 404-4 I 1
Centers for Disease Control and Prevention (CDC), 113, 114, 115, 135,169 CentralAlnerica, 38,127
C'trlici1~iriclert~, 1 19,120,157 Calicivirus. 154.156.157 California, 38,60,129, 148
Ccphalopoda, 56, 62 poisoning, 62 poisonous, 62 Cestodiasis, I 1 l Cetacea. 67 Cetaceans. poisonous. 6X. 69
Callistin shellfish. 59 poison. 60 poisoning. 59 Calls, poison center, 4, 5 Co1onry.s cctl1o.s~t.s.630 Crrrry,ylohtrcrr.f~~r-, 1 16. 1 18. 145- 147 coli, 145, 146 , f i m s , 1 1x /lyoirltesfirlctlis, 145 , j c j r r r r i , 118,122,125. 145. 146 h r i , 145 Canada, 58,91,137, 185. 186. 194, 195, 553
Cancer, caffeine and, 408 calcium and, 396 food additives and. 402, 404 iron and. 399 vitamin C and. 417 C~lrlthi~rr.ster r i ~ ~ l ~ l ( r ~38 (1.s.
Canthigasteridae, 37, 38 Cape Cod, 34 Capillary electrophoresis. for DA, 197 Capillary isotachophoresis. 268 Carangidae, 29
Ceotlos Irrlrhrcrcfrlifercl, 65
Cllelonitr rrryticrs, 66
Chelonidae. 66 Chelonitoxication, 66-67 Chelonitoxin. 67 C'helorl ~wigicr~.si.s, 29
Chemicalmethods for PSP, 186,190-197 Chemicals, agriculture, 537-556 (sec, t r l s o Pesticides) Chesapeake Bay. 138 Chimaeras. poisoning. 26. 28 China, 29, 38. 69. 1 1 I . 157 Chitins, 56,130 Chlorine, 402 Choking, 623 Cholera, 127- 130, 169 Cholesterol, 390, 392-394, 396, 399, 400. 402. 406-407,
413,415
Cholesterol oxidation products, 393 Cl1or1tlrirr ~lrl11tltcl,9 I . 194
Chondrichthycs, 26 Chordata, 25
648
Index
Chronic fatigue syndrome, magnesium and, 396 C I I I ? . S O ~~rrc:errcc~~~hrrlrr, ~YI 630 Ciguatera, 28, 210 animal studies, 238 bioassay. 22 1 biology, 2 1 1 chemical methods, 235 chemistry, 2 13 clinical. 235 detection methods. 221 earlier treatment, 242 epidemiology, 2 18 fish associated in poisoning. 219 geographic distribution, 2 18 history, 2 10 human studies. 237 immunoassay. 226 incidence, 218 in vitro bioassay. 223 in vitro cell assay. 225 memhrane immunobead assay. 227 pathology. 237 pharmacology. 2 16 recent treatment, 242 rcfcrences. 244 therapy, 242 Ciguatcra fish, 28, 29. 32, 58 bioassays for, 32-33 immunoassays for, 33 poisoning. 27, 28, 29. 30, 32-33, 52, 61 toxicity. 29-30, 32-33 Ciguatoxin, 30, 31. 32, 33. 37, 57-58, 115 Ciguatoxin complex, 25,37.52.6 I , 31 3.3 14 Clams. 56, 113, 129, 167, 185, 188, 189, 194 C/orlordli.s, 1 I I Clostridiwl. 36 h ) t ~ t i / ~11 ~ I~, ~118, ~ ~ 125, t , 159-161,166, 170. 3 12. 402. 403
33
~ ~ 1 ~ ~ ~ t lt/fl~i.SStl, l ~ ~ J ~ ~ ~ I l l
Cllrptr: .sl’rclttlr.s. 33 t h r i s s t r , 34 Clupeidae. 33 Clupeiformes, 33 Clupeotoxic fish poisoning, 33-34 Cnidarians. poisonous, 53 Cockroaches, 629-634 Coconut crabs. poisonous, 65 Codex Colnmittee on Pesticide Residue (CCPR), 547 Coelenterata (sec, Cnidarians)
Coenobitidae, 65 Collagen, 387 cross-linking of, 386 Cololtrbis sctircr, 35, 36 Colors, 480 Coniplaint Reporting System, 624 Cur~gercorl,ger, 42 Congridac. 4 1-42 Copepods,68,130 Copper, 38 I , 384, 397. 400, 401. 407. 416 Corals. 53 Coronary heart diseasc (CHD). 389. 390. 392, 393, 397, 413. 417 caffeine and, 406-407 Cottidae. 40 Cow sharks. 27 Crabs, 63. 64, 126,129.137, 150, 166-167, 170, 185. 189 Crmso.stren g i p s . 60 Crayfish,63,64. 138 Crevalle, 29 Crumbine, Samuel, 629 Crustaceans, 63, 64, 65. 1 1 1, 128 zooplankton.129 Cryptosporidiosis.169 Cured meats. 403 Cuttlefish, 56 Cyclostome fish, 25 poisoning, 26 Cyprinidae. 40. 41 Cytotoxicity assays, 268 Dairy products. calcium bioavailability in, 395 Dalatidae, 27 DALs (see Defect action Icvcls) Decomposition. 64 I Defect action levels (DALs), 618-619, 642 Defoliant, 539 ~ C ~ / J ~ l ~ l l I ~c wl c~m ~, 68. f ~ ~69 l t . ~ Delphinidae. 68, 69 Ucw(1rritt t r ) . ~ - i ( n 64 , Dermochelidae, 66 Uertrroche1y.s coritrccw, 66 Detection methods for ciguaterata, 22 1 bioassay, 221 chemical, 235 immunoassay, 226 in vitro bioassay, 223 in vitro cell assay, 225 membrane immunobead assay, 227 summary, 235
Index
649
Detection methods for tetrodotoxin, 263 capillary isotachophoresis, 268 clinical symptoms, 270 cytotoxicity assays. 268 electrophoresis, 268 gas chromatography-mass spectrometry, 264 high-performance liquid chromatography. 263 immunoassays, 268 IR spectrometry, 268 mass spectrometry, 263 n~ousebioassay, 263 'H-nuclear magnetic resonance spectrometry, 268 pharn~acologicalactions. 276 pharmacological tool, 277 references. 282 thin-layer chromatography, 266 thin-layer mass spectrometry and liquid chromatography-mass spectrometry. 265 toxicology. 276 Desrrttrrestir~,57 Diabetes mellitus, 384,386,387,388,390.396 Diarrhctic shellfish poisoning (DSP), 51, 52. 56. 78-79, 84-87, 94 Diarrhetic shellfish toxins (DSTs), 84-86, 87, 95 Diatoms. 9 I , 96, 185, 194 Dinoflagellates, 51, 52. 60. 80. 85, 95. 183185, 189, 192 toxicity. 28, 33, 34, 57
Dosirm j o p o l t i c n . 60
Drug residues. 579 analysis, 584 ani~nalproduction, 579 antibacterials, 588 antibiotics, 588 antihelmintics, 587 antimicrobials, 588 antiprotozoal drugs, 586 foods of animal origin, 579 growth-promotion drugs, 593 historical perspectives, 579 rKfKrCllcCS, 595 regulation, 583 risk/bencfit assessment, 583 toxicology, 58 1 veterinary drugs, 586 DSP (see Diarrhetic shellfish poisoning) Dyslipidcmia. 389. 390 Ehenaceae, 65 Echinidae, 55 Echinodcnnata, 54 Echinoderms, poisonous, 54 Echinoidea, 54 Ecotoxicology, 24 Ecuador. 1 15.129 Erln*crrclsicll~ttrrrdrc. 1 13 Eels, 41 -42, 1 12
x5
Eicosapcntenoic acid, 39 1 Elaidic acid, 392 Elasmobranch fish, 26, 27 poisoning, 26 Electrophoresis. 268 Elephant fish, 26, 28 ELISA assay: for donloic acid, 197-201 for paralytic shellfish poisoning, 192-194 Ellcsmere Island, 70
rotll~trlrltll,
85
Elapidae, 33
tripo.s, 85
Emetics, 70 Emulsifiers. 489. 502 Engraulidae, 33, 34
Dtrtopkysis: r l c ~ l l l ~ l i ~ i ( l t85 f7, O('llt0,
85
Jortii, 6 I , X5 hrt.strrte,
x5
m i m , 85 ilot-lv<~ic~rl,
Dinophysistoxins (DTXs), 61, 84 Diorlo~rhystri.r, 38 Diodontidae. 37. 38 Diospyros mctritiltw, 65 Diphcnhydraminc,36 Disease, vectorborne. 628-629 Docosahexenoic acid, 39 I , 392 Dolphins, 67. 68, 69 Dolnoic acid (DA), 91 -94, 96-97 LDq, of, 92, 185, 194-201 Dose-response (effect) assessment, 370
Engrctlr/i.s c,rtc.rrr.sic./tl,/lrs, 34 Elttttrlroebn /ti.sto/ytic.c~.630 Entericadenovirus, 154, 156, 159
Enterobactcriacecac.36.139,142. 144 Enterovirus, 120, 167 Environmental Protection Agency (EPA). 116, 539, 541, 543-545, 547. 550-551 Enzymes, 4x9 Epidemiologicalstudies, 1
Index
Epidemiology, 1-3 Epidemiology of ciguatcra poisoning, 2 18 fish association with. 219 geographic distribution of, 2 18 incidence of. 2 18 Epinephrine. 37 Eretrlroche/y.s irrrhrictrtcc. 66
70
~~i~~1ltJ~ htir/ltrfLr.s, hll.s
Eriphitr
64 E.sc~hcric~/lict c d i , I I 1. I 17, 1 18. 122. 124. 139, 142-144, 155, 165 diffuse-adhering (DAEC). 142 enteroaggrcgative ( EaggEC), 142 cntcrohemorrhagic (EHEC), 142 cnteroinvasive (EIEC), 142, 145 enteropathogenic (EPEC). 142. 145 enterotoxigenic (ETEC). 142, l45 01S7:H7, 115, 116. 142, 145. 166, 169, scttbricw/rc,
I70 shigatoxin-producing, Esocidac, 40 ESOS
/llc.ilr.s,
145
140
Essential fatty acids (EFAs). 388. 392, 395 deficiency sylnptoms, 389 Estonia. I28 Ethylencdiaminetetrancetic acid (EDTA). 398 Eukaryotes. 1 17 Europe. 24. 40. SS. 58. 70. 78. 127, 130. 150. 154. 160. 188, 391 E L l r h ~ r f l l r l . /sW / t / l l f i . Y . 36
Excitatory amino acid tramporter (EAAT). 92 Exposure assessnwnt. 37 1 Extraneous materials, 64 1 Fabaceac, 620
Fat substitutes. 485 Fatty acids, 388-393 dietary intake of, 389-393 essential (EFAs). 388 mctaholisn1 of, 388 trlllls, 392-393 FDA ( s e c Food and Drug Administration) Fecalcoliform. 142, 143, 144 Feccs. 630 Federal Food. Drug and Cosmetic Act (FFDCA), 538. 546 Federal Insecticide, Fungicide. and Rodcnticidc Act (FIFRA). 538-543 Ferritin, 398 Filefish, 29. 37 Finback whales, 68 Finfish, 1 1 1, 162. 1x5 First-wave studies. 353
Fish carnivorous. 28 ciguatoxic. 32-33 clupeotoxic, 33 genlpylotoxic. 34-35 hallucinogenic. 43-44 ichthyoallyeillotoxic. 43-44 ichthyohcmotoxic. 41 -42 ichthyohepatotoxic. 32-43 ichthyootoxic, 30-41 ichthysarcotoxic, 25-28 sconlhrotoxic, 35-37 tetrodotoxic, 37-40 Fish liver poisoning. 32-43 Fish roe poisoning. 25, 40-41 Flatworms. X I F / r r ~ ~ o / ~ r r t ~ t r l ~~i il rl lr rl ~ r i l l t ; t / ~ ~403 rlll~,
Flavors. 504 Flies. 630-63 1. 633 Fluoronwtric mcthod. for paialytic shellfish poisoning.190 Folic acid. 417 Food additives. 402-4 I I . 448 adverse effects of. 508 adverse food reactions t o . 5 1 1 ;lllcrgics to, 51 1 antioxidants. 495 background. 448 banned from use. S09 categories, 45 I colors. 4x0
derived from allergenic food. 5 I5 description, 468 cmulsiliers, 489. 502 enzymes. 489 European Union ( E U ) rcgulations. 459
suhstitutcs. 4x5 flavors. S04 Functions. 448 industrial chemicals. 5 I O Japanese rcgulations for. 466 manuFacturing, 458 preservatives, 499 rcfCreIlcKS, 5 15 regulations. 459 research and development. 455 supply industry, 454 sweeteners. 368 thickeners and stabilizers, 474 trends and issues, 466 U.S. regulations lor. 450 vitamins. 490 fat
651
Index
Food allergies. 51 1 Food and Agriculture Organization, S47 Food and Drug Administration (FDA). 113. 115, 116. 135, 154. 169, 543, 547-552, 624
Bureau of Chemistry, 1 13 Northeast Technical Services Unit (NETSU), 113-1 14 Foodborne marine intoxicants. 24 Food containers. migrant chemicals and, 599 Food irradiation, S72 background. S72 changes in food and. 572 conditions, 572 controversy, 574 history. 572 myths, 574 purposes, S72 references 577 regulation. S75 support, S76 Food, Drug and Cosmetic Act (FD&C Act). 538, 546, 618, 627. 640-641 Foodnet, 115. 116, 169 Food Quality Protection Act (FQPA), 546 Food radioactivity, 558, S62 Foods of animal origin, drug residues, S79 Foreign matter, 64 I Fortification, 4 I 1-4 17 France, 1 1 1 Frec radicals, 382, 397, 398, 400, 415-416 Fructose, 383 hyperlipidemia and, 383 metabolisln of, 383 Fugu /mrddi.s, 38 Fumigants. 541 Fumonisins, S41 Fungicides, 537. 539, 541 Fungii, 38, 95 FIr.wrim rrrordr'forrrw, S41 Galapagos Islands. 38 Gtrrd>iertli.scus to.uicu.s, 28. S2
Gars. 40 Gas Chromatography-Mass Spectrometry. 264 Gastropoda. 56 Gastropod mollusks. 37. 38 Gastropods, 95, 185 poisonous. 56-59 Gempylids, 3.1-3.5 Gcmpylotoxic fish. 34-45 Germany,128
Germ theory, 628 GilbertIslands,129 Glucose, 383. 384, 386 ~nacro~nolecule cross-linking and. 384 Glutathione peroxidase (GSH-Px). 382, 399 cnzymc. 401 Glycooxidation reactions. 383 Glycosylation reactions, 383-388 GMP (sec Good manufacturing practice) Goby fish, 37, 38 Goldlish, 29 Gonyautoxins (GTXs). 64, 183,189. 190192 Good Inanufacturing practice (GMP), 619, 640 Grains, food and feed, 620 Granulocyte-macrophage colony-stimulating factor. 386 Great white sharks. 26 Greenland. 70 Greenland shark, 27 Group A rotavirus. I19 Grouper, 32. 42 Growth factors, immune-like (IGF- I ), 386 Growth-promotion drugs. drug residues. 593 Guam. 1 IS. 129 Guinna. 38 Guidelines lor radioactivity contamination. 568 Guillain-Barr6syndrome,IS9 Gulf Coast Vibrio Surveillance System. 135 Gulf of Aden, 27 Gulf of Mexico, 40, 69. 87. 134-135. 170 Gulf of St. Lawrence, 27 Gy/r~r~otlit~i~rrr~ h r a r ~(SCC Phc1~oclist~~r.s hrc,1*i.s) Gyrrirwdirliwrl c t r t o t w t u r r l . 80
'H-Nuclcar Magnetic Resonance Spcctrometry, 268 Haber-Weiss cycle, 398, 4 I7 HACCP ( s c ~Hazard Analysis Critical Control Point) Hagfish, 25-26 Htrlithorltlrirr oktrtloi. 84
Haliotidae. S7 Hrrliotis: discus, 57 .sil~lloldi,57 Hallucinogenic fish poisoning. 43-44 Hamnerhead sharks, 27 Hand washing. 629
H n r c w g h o w l i s , 34
Hawaii. 38
652
Hazard Analysis Critical Control Point (HACCP).116,169. 170, 624, 633. 64 1-642 Hazard identification, 369 Heart disease, caffeine and, 406-407 Hemochromatosis, 397 Hemoglobin. 397, 398, 404 Hemolysin,138-139 Hemotoxins, 4 1 Hepatitis, 119 type A, 1 1 I , 119. 120,123,169 type E, 119,120 HepatitisAvirus,156,157,167 Hepatovims,120 Hcptovirus A (see Hepatitis A virus) Hc~ptrtrrlc1lin.s p r l o . 27 Herbicides. 537, 539, 540, 541 Herbivores, toxic, 28 Hernandiaceae. 65 H c m m d i l r s o n o m , 65 Herrings, 33-35 Heterocyclic amines, 422-424 Hexanchidac, 27 High-density lipoprotein (HDL), 389, 390, 393. 396. 399. 413.414 High-Performance Liquid Chromatography (HPLC), 196-197,263 Histaminc, 36, 37 Histamine poisoning (sec. Scombroid poisoning) Histidine, 36 Historical perspectives, drug rcsiducs, 579 Holotkurio trrgus, 55 Holotlllrritr tlt/>Itlo.stI, 55 Holothurins, 55 Holothuroidea, 54, 55 Homaridae, 65 Hong Kong. 128, I30 Horseshoe crabs, poisonous, 63 House fly, 629 Howard, L. 0.. 629 Howard mold count. 641 Human adenovirus, 1 19,120 Human caliciviruses, 119 Human immunodeficiency virus (HIV), 632 Humanpoliovirus. I 19, 120 Hrrso h l r s o , 40 Hydrogenation, 392 Hydroids, 53 Hydrozoans. 53 Hypercalccmia. 4 12-4 13 Hypercholcsterolemia, 389, 393, 406, 407
Index
Hyperglycemia. 401 Hyperinsulinemia, 390 Hyperlipidemia. 383, 389, 396, 406 Hyperlipoproteinernia. 400 Hypertension, 386, 389, 390, 396, 397, 402, 406 calcium and, 396 Hypervitaminosis A, 42 Hypervitaminosis D, 412-413 lchthyoallyeinotoxic fish, 43-44 lchthynhematoxic fish, 41 -42 lchthyohepatotoxic fish. 42-43 lchthyootoxic fish, 25, 40-41 Ichthyosarcotoxic fish, 25-28, 35-37, 40 Immune system, altered. 386, 399 Immunoassays, 268 Immunological assays: for dornoic acid, 197, 198 for paralytic shellfish poisoning, 192- 194 India. 111, 128,170,419 Indian Ocean, 27, 28, 43, 66, 194 Indoncsia. 63. 127 Indo-Pacific region. 27, 29, 34. 35, 38, 43, 53-57, 62, 64-66 Industrial chemicals, 5 10 Infections, hospital. 629, 633-634 Infectious hepatitis (see Hepatitis A vims) Infrared (IR) spectrometry, 268 Insecticides. 537, 539-540, 541 Insects. 628, 63 I , 642 Insulin-dependent diabetes mellitus (IDDM). 386 Insulin resistance. 389, 390 Interdiction techniques, radioactivity contamination, 566 Interstate Shellfish Sanitation Conference (ISSC). 113 Ionizing radiation, migrant chcmicals. 609 Ionotropic glutamate receptors (IgluRs). 92-93 Ireland, 61 Irradiation, food, 572 Ischemic heart disease, 399 Israel,161 Isuridac, 26 Italy,129 Ivory shells. 58 Jacks, 29, 32, 35 JapaI1, 24, 27, 29. 36, 38. 40, 4 1-42, 56-63, 84.95.97, 116-117,137.140.171, 389. 402, 412
Index
653
Japanese dosinia, 60 Japanese littleneck, 60 Jellyfish, 53, 81 Jequirty bean, 620 Joint stiffness, 387 Journal
of AOAC I ~ l t e ~ r l c r t i o n r r642 l,
Kanagawaphenomenon,138,139 Kapchunka, 160- 161 Kenya, 419 King crabs, 63 Klebsiella prmmloniae, 1 I3 Korea, 40,41. 42. 1 1 1 , 116,117.128 Kwashiorkor.398 Kynurenic acid, 93-94 Kyphosidae, 43 Kvpho.s~r.scirlera.scelr.s.43 L m t o h c r r i l l ~ r . s ,36. 1 18
Lactose, 395 38 Lampreys. 25-26 Laxatives. 70 LDq,, 30, 92, 185-187,194-201,197 median lethal dose, 30 Leatherjackets, 29 Lepisosteidae, 40 Lepisosterrs frisfoech~rs, 40 Linoleic acid, 388-392 Linolenic acid, 391, 392. 399 Lipid, 388-394 advanced glycosylation end products (AGES) and, 384, 386 thermal processing, 392 Lipid oxidation, 392, 398, 401, 403, 415 glycation reactions, 382, 391, 392 Lipid peroxidation, 38 I , 386, 387, 390, 39 I , 393, 397.400, 403, 415-417 Lipoproteins (see High-density lipoprotein, Low-density lipoprotein, and Very lowdensity lipoprotein) Lipogenesis, 383 Lisrerilr, 113, 1 16.148 twrwcyfogenes, 113,118,123,146,148, 149,166,170 Listeriosis,146 Lobsters,63. 64, 126,137 poisonous, 65-66, shovel, 65 slipper, 65 spiny, 65 [Ne, 65 Ltr,qocl~/’hn/lr.sIlrrlrrri,s,
Low-density lipoprotein (LDL), 386. 389. 390, 393. 396, 399.400, 413, 415416 glycated, 387 modified,393 receptor-mediated clearance mechanism, 387 Lumanc, 53, 54 Luljanidae. 29 L1rtjalllr.s:
holrtrr, 29
, q i l h r s . 29 wiCqierlsi.s,29
Mackerel. 34-42, 35, 36 Mackerel sharks. 26 Macrominerals, 394-397 Macrophage, surface proteins. 386 Madagascar, 27, 29 Magnesium, 396-397 Mahi-mahi, 35 Maillard reaction, 384 products, 401 Maitotoxin, 30, 37 Malaysia, 63 Malondialdehyde (MDA), glycosylation, 386, 387. 388 Mammalia. 70 Marcus Island. 57 Marianas Islands, 57 Marine food biotoxications. 25, 37 Marine mammals, poisonous, 67 Marine turtles, poisoning, 66-67 Marinka, 40 Mass spectrometry, 263 Mcl.strrrl~~rlol~ir-lrs, 120 Mastigophora, 5 1 Matalelei, 53, 54 Matamala Samasama. 53. 54 Maximum residue limits (MRLs), 547 Measurement of radiation, 560 Mediterranean region, 26, 27, 28, 34, 130 Mediterranean Sea, 40, 55 Mercury, 400-401 Merostomata, 63 Metazoans, 53 Methylxanthines, 404-41 1 Mice, 631 Microbial assays, for PSP, 188 Microcystins, 94 Microminerals, 397-402
654
MiddleEast.127 Migratory chemicals, 599 alternatives to, hl 1 concerns about. 603 i n food containers, 599 history of, 600 ionizing radiation and, 609 in packaging materials, 601 plasticizers, 603. 610, 61 1 polymers, 603 in preparation utensils, 599 references, 61 3 theory. 600 Minimum infectious dose (MID), 632 Minnows, 40, 41 Mites, 628, 631-633, 642 Miticides. 541 Modified atmospheric packaging (MAP), I60 Molds, 641 Molidae. 37 Mollusca.56.143 Molluscan shellfish. 78 Molluscicides. 537, 539 Mollusks. 52. 62. 63, 81, 96. 128. 140 poisonous. 56 Monitoring o f pesticide usage, 547-557 Monocyte, surface proteins, 386 M o r l o r l o r ~rrlorlocc~ros,68 Monodontidae, 68, 69 Monosodium glutamate, 402 Monounsaturated fatty acids (MUFAs). 390, 392,400 Morerelkr, 25 Moray eels, 32, 42 Mor-irtclcr citr-ifolirc. 32, 39-40 Mouse bioassay, 185- 188, 189, 190- 192, 195- 196, 263 Mouse lethality technique, 185- l88 (.sec r d s o Mouse bioassay) Mlrgil w/d1cllu.s, 29, 43 Mugilidae, 29, 43 Mullets, 29 Mullidae.29 Mlrr-crerler lrclenrr. 42 Muracnidne, 42 M l t s c u clorrresticw, 630 Mussels, 90, 94.113, 145, 155. 166,170. 184. 188, 194. 195, 197 toxic. 61 Myoglobin, 397. 403 Myristic acid, 390
Index
Nccssrrr-ius: (‘O.S1II.S,
95
corloicldis, 95
National Clearinghouse for Poison Control Centers, 2 National Marine Fisheries Service (NMFS), I13 National Research Council (NRC), 539, 546, 550-55 I , 553 National Residue Program, 549 National Shellfish Sanitation Program (NSSP), 122,113,135 Natural Resources Defense Council, 539 Nautilus. 62 Nematicides, 537. 539. 541 Nematodiasis. 1 1 1 Neophocrr cirter-er/,70 Neopltoccrerto pllocctertoidc~,68, 69 Neosaxitoxin, 64 Neo.rorltl1irr.s ~ I I I / J I Y S . S I ~64 S, Ne/mrrlcw: rlrltigllcl,
58 58
ill~~~l’.S~’U//J~Cl,
decenlcostertu, 58
Netherlands,145 Neurotoxins, 51, 52, 159-160. 317 New Guinea, 53 New Zealand, 78, X7 Nitrates, 403-404 Nitric oxide, quenching of, 386, 387 Nitrites. 403-404 Nitroso-compounds, 403 Nit:schirr, 9 1
Nonenzymatic browning, 384. 407 Nono tree, 32, 39-40 North America, 24, 40. 41, 70, 87, 170, 188, 195, 389, 417, 424 North Atlantic Ocean. 26, 27, 28 North Pole, 70 North Sea. 27 Nonvalk-likevirus, I 19, 120, 123.157 Norwalkvirus, 1 I I , 1 19, 120, 154. 156, 157, 158, 167 Norway. 1 1 1 Nut, cashew, 620 Nutritional deficiencies. 38 1-382
index
655
Pea, rosary, 620 Pcctcnotoxin (PTX), 61, 62, 84 Pectinidae, 6 I Pelccypoda. 56 Pe/oche/ys hihrorli. 66 I’cwicillilfrr~, 4 18 Pcntosidine, 384. 386 Percworr plarrissirrrrrrrr, 64
Periwmklcs,185 Peroxtdc values, 392 Pcsticidcs, 537-556 dietary risks, 55 1-554 historical uses, 538-539 nondietary risks. 553-554 regulations. 543-544, 546-55 1 residue limits, 544-546, 557 types of, 539-541 usage levels. 541 -543 Pesticide Data Program(PDP),543,547-552 Petromysonidae, 26 Petrorrrysorr f r l t ~ r . i r l l f . s26 , I’jestc~ritl:
PacificIslands,27, l27 Pacific Ocean, 25, 77-29, 43, 57, 62, 66, 69. 160. 185, l94 Packaging materials. migrant chemicals i n , 60 1
Palinuridac, 65-66 Palmitic acid, 390 P l t l y t l l o t r , 53 Palytoxin, 25, 37, 53 Ptrr-trc~c~rltr.ofl1.s /;1*;t/rr.s, Prrr.trgorlirrllrs,
55
lI1
Paralytic shellfish poisoning, 5 I . 56, 60, 64. 78-84. 94, I 15.183-196, 201. 316 testing methods for. 185- 194 Paralytic shellfish toxins (PSTs), 80-83, 87. 95-97, 183- 196 Parathyroid hormone (PTH), 4 1 1 Palhogcns. foodbornc. 629, 632 Pathology. ciguatenta 237 anitnal studies. 238 human,237 P c r f i r w p e c m yc~.s.socvui.s,6 I , 95
poisoning. 52-53 toxin. 52-53 Ptiesteriaceae, 52 Phallosthoids, 29 Phcromones. 537 Philippines,53. l 1 I . 140 Phocidae. 70 Phosphate, vitamin D and, 4 12 Phosphodiesterase inhibition, 405-406 Phospholipid hydroperoxide glutalhione peroxidase. 399 Phy.setcJr c ~ c r t o d o r r ,68. 69 Physeteridae. 68 Physohrcrchitr clorrglasi. 53, 54 Phytates, 395-397 Phytoflagellates. toxic, 25 Phytoplankton, 52, 94 Phytosterols, 390 Pic.onltr,,iritItrc, 1 19, 120,157 Pikes, 40 Pilrtrrrrurs ~~espc~rtillo, 64 Pinnipcdia, 67, 70 Piratea, 65 Pits, fruit, 621. 623, 641 Pltrc~o/>et~terl tlrtrgello1liclt.s. 96 Plankton, I38 Plant growth regulators. 537 Plants: poisonous, 620 toxic, 65
656
Index
Plasnla protcins. cross-linking of, 386 PI, ,. 'l\tlLlzers. : migrant chemicals, 603, 610, 61 1 Plnt!por/itc grnrdorrrtr, 64 Plectognathi.37 P/~~,siorlrorrcl.s .shigel/oides. 113, 1 18, 123, 125, 152- 153 POISINDEX, 3, 6, 7, I O Poison center websites. 10 Poison control centers: American Association of Poison Control Centers, (AAPCC). 1-2 calls handled, 5 calls received, 4 education, professlonal and public, 7 internationalaffiliations,10 quality monitoring, 7 references standards, 6 staffs, 3 toxicology organizations, related to, 7 in United States, I O websitcs, 10 Poison information centers. U.S., 10 Poisonous plants, 620 Poison Prevention Packaging Act, 2 Polar bears. 67, 70-71 Poliovirus, 155, 156.159,167 Pollution, 52 Polychaete worms, 63 Polycyclic aromatic hydrocarbons (PAHs), 420-42 1 Polymerase chain reaction (PCR) technologies. 164- 165 Polymers, migrant chemicals, 603 Polynesia, 29, 65 Polyunsaturated fatty acids (PUFAs), 389392, 400,414-415 0-3, 389-392 0-6. 389, 391 Pompanos, 29 Porcupincfish, 37, 38 Polpoises, 67. 68 Portugal,129 Potassium. 402 Potentiators, 36 Prawns, 1 12 Preparation utensils, migrant chemicals, 599 Preservatives, 499 Prickleback. 40 Processors, food, 6 19 Product tampering. 61 8 '
Prokaryotes archaen, 1 17- 1 19 bacteria, 1 17- I19 Pr~lr~l~~elltrlllll:
~ ~ ~ ~ ~ c u28, t w52, r n 85 , elegnrls, 85 hr!ffiltcrllrtictnlrrrr. 85 lirrrrr. 28, 52, 85 ~Iror-i~~e-leholr~icre, 60 I l l e . Y i ~ ~ t l I I 1 1 1 l 128, . S2
Proteins: advanced glycation end product-modified, 387 cross-linking of, 387 Proteobtrcterio, subdivision of, 1 18, 126 Proteus, 36,154 Protistans, 5 I P r v t n ~ o r r ~ c r u l r ttc~r~mrer~.si.si.s, s~.~ 25 Protothnc~lstrrrrlirletr, 96 Protozoans, 5 I , IS0 P . s e l l ~ / ~ ~ r l r o r l r25 ls. aerllgirlo.srr, 1 1 P.st~lltlo-r~ir,.st~/lia:
r11r.strtrli.s, 9 I
r/e/~ctlti.ssirlrct, 91 rurrltiseries, 9 I , 96 p " " l r t ~ ~ ~ i e / i c ~ c l t ~ . ~9 . s1; l l ~ ~ l ,
prmqerls,
91, 96, 194
serirrtn, 9 I t1rrgidrr/o, 9 1
PSP (see Paralytic shellfish poisoning) Pterothrissidae. 33 Ptychodiscus hrcvis, 87, 96 Pufferfish, 37-38, 40, 117 poisoning, 95 toxic, 25, 37, 38, 39 Puffer poisoning, 37-40 P m n hi.s/1ida, 70 Putrescine, 36 Pyrodir~iwr~ hohurrletw, 80
Pyrraline, 384, 386 Rrltlimthus p"urrrotclrr.si.s, 53, 54 Radiation, biological effects of, 560
Radioactivity. S57 accidental contamination of food and water, 565 anthropogenic sources in food and water, 558. 564 background, 557 biological effects of radiation, 560
Index
[Radioactivity] consistency of guidelines, 568 contamination of food and water, food chain concentration. 564 hutnan uptake of, 562 interaction with matter, 559 interdiction techniques, 565 matter interaction with, 559 measurement of, 560 natural in food and water. 558, 562 necessity for low-level effects research, 568 necessity for surveillance, 567 public education, 568 references, 569 risks, 562 sources of contamination, 565 uptake of by humans, 562 in water, 558. 562 Radioimmunoassay for paralytic shellfishpoisoning, 192- 193 Ratfish, 26. 28 Rats, 630-63 I , 633 Rattlebox, 620 Recommended Dietary Allowance, 399, 41 1 Red algae. 9 1, 194 Red Sea. 34, 38 Red tide, 184- 185 Reef crabs, 37, 38, 64 Reference dose (RtD),544-545 Reo\iridacJ. 120,158 Repellents, 537 Reproduction, caffeine and, 408-41 1 Requiem sharks, 27 Retinopathy, 387 Rhorirctis horcvsi, 53, 54 Ribosomal Database Project (RDP), 117 Risk assessment, 347, 368 background, 347 dose-response (effect) assessment, 370 exposure assessment, 37 1 first-wave studies, 353 hazard identification, 369 references, 375 sccond-wave studies, 361 toxicological assessment, 350 Risk/benefit assessment, 583 Risk characterization, 372 Risks of radiation, 562 Robber crabs, 65 Rodenticide, 539 Rodents, 628, 630, 633, 642
657
Rorquals. 68 Rosary pea, 620 Russia, 24 Rrwettu.7 ~)rc~ionr.s, 35
Ryukyu Islands, 28 Sabotage, 618 Safety standards and pesticides, 544 Salmon,148,162 So/rnorw/la, 1 1 1 - 1 13, 1 16- 1 18, 122, 124, 137,139-141,145,166,170 cholertremis, I37 enderitidis, 139,140 ptrrcJt!phi. 137 typhi, 123, 137, 139-140,166 Salmonellosis,140,169, 634 Salt, 402-403 Salt substitutes, 402-403 Samoa. 53 Saponins, 55 Sardines, 33, 34, 35 Saturated fatty acids, 389, 392, 392 Strsidonllrs gigonteus, 185,186 Saxitoxin (STX), 25, 57-58, 64. 80-83, 96. 183- 1 84, 186- I94 Second-wave studies. 36 1 Scabicides, 537 Scalloped hammerhead shark, 27 Scallops, 56, 61, 1 85 poisoning, 61-62 Scandinavia,150,389 Scaphopoda, 56 Schiff bases, 383-385, 387-388 Schi:ophys crspero, 64 Schizotl~or.ax,41 intermedilrs, 40 Scomberesocidae, 35, 36 Scornber joponiclcs, 36 St.or~rheror~ronrs niphonius, 42 Scombroid poisoning, 35-37 management of, 3 12 Scombroids, 35, 36, 42 Scomhrotoxism, 35-37, 1 15 Scor./~oeIlic~hrhqs. 41 lNtIr~t10rutIlS,40
Sculpins, 40 Scyllaridae, 65 Scyphozoa, 53 Sea anemones, 53 poisoning, 53-54 Sea bass, 42 Sea chubs, 43
658
Index
Sea cucumbers, 54 poisonous, 54-55 Seafoodborne disease, 109- 172 Sea lamprey, 26 Sea lion, 70 Seals, 67. 70 Sea of Azov. 40 Sea urchins, 54 poisonous, 55-56 Sei whale, 68. 69 Selenium. 399 Selenoenzymes, 399 Selenoprotein P, 399 Selenoprotcin W. 399 Serine/threonine phosphatase. 85. 94 Seven-gilled sharks, 27 Sharks.26-27 Shellfish,112,114,117,128,126,132, 146,148.152,154,155.157-160.167, 169-171 sanitation regulations, I 12 Shellfish toxins, 77-108 minor. 94-95 Slrigeller, 1 16,145 clyrlserltericre, 1 13 jie.nwri. 630
Shigellosis,169 Shrimp, 68 129,137.140,167 Sibbuldus wiu.sci~Iit,s,68 Siheria, 70 Singaporc, 128, 129 Skates. 26 Skijack, 36 Sleeper sharks, 27 Slickheads, 33 Slugs, 56 Snails, 56 Snappers, 29, 32 Snow trout, 40 Sodium ascorbate. 402 Sodium nitrate, 402 Sodium nitrite, 402 SO/C'll(lj).Si.Y ~ e l l l i l I f i t A 630 , S o r ~ t ~ t i ~ r. ~ ~r itc. ~s ~ o c ~ e ~ ~27 hcll~ts, South Africa, 27, 140 SouthAmerica, 87, 127 Spain, 78, 95 Sperm whale, 68. h9 s/l/lcier~l~~l~~.s: cllllllllOtilS.
moc'ulcltll.s,
38 38
SphyrrIcl :ygcwrw, 27
Sphyrnidae. 27 Spikefish, 37 Sponge, 84 Sprat, 34 Squids. 56, 62 Stei/'/l!loeocc.il.s, 1 17. I 18 cl~lrcli.s,1 1 1, 113, 122, 125, 137,
161, 403
Starfish. 37, 54 Sterc~11epi.sischirtcrgi,
42
Stichaeidae, 40, 41 Stic~hclerl.s,4 1 grigorjewi, 40 Stoichactiidae, 53 Stre/,foc.oec.its. 1 18, 162.164 inioe, 1 18, 125.162-164
134.
Striped surgeonfish, 29 Sturgeons, 40 Sugars, 383 dental caries and, 383 glycation products. 383-388 Sulfur-bottom whales, 68 Sunfish, 37 Supcroxidc radical. 398 Surgeonfish, 29 Surmullets, 29 Surveillance of pesticide usage, 547-55 1 Sweden.157 Sweeteners, 468 Switzerland.128 ?idlypicw.s, 63
Tahiti, 18 Taiwan,24, 95. 137,170 Tannins, 397 7upe.s .ser~liclec~lt.sseitct.60 Tarpons, 33 Taxonomy, 1 17- 1 1 8. 139, 153 Testudinata, 66 Tetruodorl lirwatlts, 38
Tetraodontidae, 37, 38 Tetraodontiformcs. 37 Tetramine. 58 intoxication, 58-59 Tetrodon poisoning. 37-40, 3 I3 Tetrodotoxic fish, 37-40 Tetrodotoxin, 25, 37, 39. 64, 95 background, 254 bacterial origin. 257 detection methods, 263 distribution, 255 food, 257
659
Index [Tetrodotoxin] human intoxication, 260 incidence, 27 1 major sources, 260 as a phamacological tool, 277 source of toxin, 255 symptoms.270 -bearing organisms, 255 treatment, 270 Teuch, 40 Thailand. 63, 1 I I , 128 T/lolrrn/itcl, 64 Thrr1rlrcto.s tttrlritittt11.s. 70 Theoretical maximum residue contribution (TMRC). 544-545, 55 1 Thermostable direct hemolysin (TDH). 138, 139 -related hemolysin (trh), 138 Thickeners and stabilizers. 474 Thiobarbituric reactive substrates (TBARS), 382, 390 Thioredoxin reductase, 399 Thin-layer chromatography, 266 Thin-layer mass spectrometry and liquid chromatography-mass spectrometry, 265 Three-toothed fish, 37 Thrissina baelama, 34 Thrombosis, 387 T / l m t n t s ~ / I ~ I I I / L 36 S, 7’iIqio, 1 12,162 7ir1c.u.41 tirlctr. 40 Tissue biosensors for paralytic shellfish poisoning,I88 Tocopherols, 414, 416 Tooth shells, 56 Torres Straits, 63 Total Diet Study, 549-553 Toxicological assessment, 350 Toxicology organizations. 7 Toxicologywebsitcs.10 Toxins biogenesis. 24 biosynthesis, 24 dystrophication. 24 eutrophication, 24 in fish, 23 naturally occurring, 417-420 ocean pollution, 24-25 process derived. 420 toxigenesis, 24 Transferring, 398
Travelers’diseasc.142 Trematodiasis, I 1 1 Trepang. 55 Tritlmxrr : g i g ~ s .6 1 ttIr/.vittw, 6 I Tridacna clams. 61 Tridacna shellfish poisoning, 6 I Tridacnidae. 6 1 Triggerfish, 29, 37 Trimethyl oxide, 27 Trionychidae, 66 Tropical reef crabs, 65 poisoning, 64 Trout,162.197 Trunkfish,37 Tumor necrosis factor CI (TNF-a),386 Tunas, 35, 36. 42, 166 Turbinidae, 57 Turban shells, 28, 57 poisoning. 57-58
T1rr.h: r/,a~r.o.storrll/.s,57
57
lllt/r.lflort/fl/.s,
pic‘o, 28
Turtles, 66 Typhoidfever.139,169 U p m t w s nrge, 43 UnitedKingdom,160 United States, 52, 1 11-1 13, 116, 128, 129, 132,134,137,138,140,145,150.152, 154,157-162,168, 169,412, 414 Urchins, 54 sun dollars, 54 Ursidae. 70 U.S. agricultural chemicals, 537-556 U.S. Consumer Product Safety Commission (CPSC), 2 US. Department of Agriculture (USDA), 115. 116, 541, 543, 547. 549-550, 619 Ccntcr for Food Safety and Applied Nutrition (CFSAN). 116 Food Safety and Inspection Service (FSIS), 116 U S . Food and Drug Administration (FDA), 38 U.S. Public Health Service, I 13 USDA (see U.S. Department of Agriculturc)
Vector, 629-630 Veneridac, 59, 60
660
Vcncrupin shellfish, poisoning, 60 Venoms, 23 Vertebrates, 66 fish poisoning, 66 Very low-density lipoprotein (VLDL), 389, 393 modified LDL, 393 Vctcrinwy drugs. 586 Vi/~rio, 25, 36, I 13. I 16- I 18, 126. 129. 130. 135, 138, 152, 169 u l ~ y i r w l y t i m s ,126.139 c w d ~ t r r i t r c ,126 choler-(re, 1 1 I , 113, 1 15. 1 18. 122, 126130, 134-135,167.170 c~ir~c.irtrlntierlsi.s, 126 tlorrrselcr, I26 disease. 126- 127. ,flro~inlis,I26 ,filr-Ili.SSii, 126 hollisrre, 126 infections.134 management. 3 15-3 16 rlret.sc~ltrlikol'ii. 126 r r r i r r r i c x s , 116 /rtrr-crlttrerr~o!\tic~~rs. 1 1 I , 1 15. 1 18, 122, 124,126. 130, 137-139,167,169-170 w 1 r ~ ~ f i e ~ r r . 11 s . I . 115, 1 18. 123,124,126, 130, 132- 138. 167- 169
Vietnam. 63 Viruses. 1 19- 120, 154- 159 fomites.119 morphologies, 1 19- 120 vectors, 1 19 vehicles,I19 Vitamin A, 381, 413, 417, 424 Vitamin B,,388 Vitamin B,?, 417 Vitamin B,,, 388 Vitamin C, 381, 413, 416-417
Index
Vitamin D, 41 1-414 Vitamin E. 381, 382, 388. 399. 414-416, 417 Vitamins. 490 Voltage-sensitive sodium channels (VSSC). 8 1-82, 87-89, 95. 96 Walruses, 67, 70 poisoning.70 Water,polluted,129 Wd?ster-'.sh~tc~r-rlcrtiorltrll>ic,tiorwr;y, 628 West Africa. 55 West Indies, 34 Whales, 67. 68 Whelks.58-59.185 Whitefish,160 White Sea. 27 White whales, 68, 69 Wiley, Harvey W.. 620, 629, 640 World Health Organization (WHO), 1 IO. I I I , 547. 551
Xanthidac, 64 Xcnobiotic compounds. 424-425 Xiphosuridae, 63 Yellowtail,162 Ycr-sinirr, l 16, I 18 er~tcr-oc.olitic.rr, 118, 122, 125, 149,152. 170 /'e.sti.s, 149 J ~ " ' " r c l o t l r b c ~ r - c I t l o s i s , 149 Yersiniosis,149, 150 Yessotoxin (YTX). 61-62, 84 Zinc, 397, 399-402, 407. 381 Zooplankton. 68, 138 Z ~ Z ~ I I rrer~e~rs, I L I S 64 tetrodotoxin, 64
