Organic Bromine and Iodine Compounds Handbook of Environmental Chemistry

Preface Environmental Chemistry is a relatively young science. Interest in this subject, however, is growing very rapi...

Author: Alasdair H. Neilson

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Preface

Environmental Chemistry is a relatively young science. Interest in this subject, however, is growing very rapidly and, although no agreement has been reached as yet about the exact content and limits of this interdisciplinary discipline, there appears to be increasing interest in seeing environmental topics which are based on chemistry embodied in this subject. One of the first objectives of Environmental Chemistry must be the study of the environment and of natural chemical processes which occur in the environment. A major purpose of this series on Environmental Chemistry, therefore, is to present a reasonably uniform view of various aspects of the chemistry of the environment and chemical reactions occurring in the environment. The industrial activities of man have given a new dimension to Environmental Chemistry. We have now synthesized and described over five million chemical compounds and chemical industry produces about hundred and fifty million tons of synthetic chemicals annually. We ship billions of tons of oil per year and through mining operations and other geophysical modifications, large quantities of inorganic and organic materials are released from their natural deposits. Cities and metropolitan areas of up to 15 million inhabitants produce large quantities of waste in relatively small and confined areas. Much of the chemical products and waste products of modern society are released into the environment either during production, storage, transport, use or ultimate disposal. These released materials participate in natural cycles and reactions and frequently lead to interference and disturbance of natural systems. Environmental Chemistry is concerned with reactions in the environment. It is about distribution and equilibria between environmental compartments. It is about reactions, pathways, thermodynamics and kinetics. An important purpose of this Handbook, is to aid understanding of the basic distribution and chemical reaction processes which occur in the environment. Laws regulating toxic substances in various countries are designed to assess and control risk of chemicals to man and his environment. Science can contribute in two areas to this assessment; firstly in the area of toxicology and secondly in the area of chemical exposure. The available concentration (“environmental exposure concentration”) depends on the fate of chemical compounds in the environment and thus their distribution and reaction behaviour in the environment. One very important contribution of Environmental Chemistry to the above mentioned toxic substances laws is to develop laboratory test methods, or mathematical correlations and models that predict the environ-

VIII

Preface

mental fate of new chemical compounds. The third purpose of this Handbook is to help in the basic understanding and development of such test methods and models. The last explicit purpose of the Handbook is to present, in concise form, the most important properties relating to environmental chemistry and hazard assessment for the most important series of chemical compounds. At the moment three volumes of the Handbook are planned. Volume 1 deals with the natural environment and the biogeochemical cycles therein, including some background information such as energetics and ecology. Volume 2 is concerned with reactions and processes in the environment and deals with physical factors such as transport and adsorption, and chemical, photochemical and biochemical reactions in the environment, as well as some aspects of pharmacokinetics and metabolism within organisms.Volume 3 deals with anthropogenic compounds, their chemical backgrounds, production methods and information about their use, their environmental behaviour, analytical methodology and some important aspects of their toxic effects. The material for volume 1, 2 and 3 was each more than could easily be fitted into a single volume, and for this reason, as well as for the purpose of rapid publication of available manuscripts, all three volumes were divided in the parts A and B. Part A of all three volumes is now being published and the second part of each of these volumes should appear about six months thereafter. Publisher and editor hope to keep materials of the volumes one to three up to date and to extend coverage in the subject areas by publishing further parts in the future. Plans also exist for volumes dealing with different subject matter such as analysis, chemical technology and toxicology, and readers are encouraged to offer suggestions and advice as to future editions of “The Handbook of Environmental Chemistry”. Most chapters in the Handbook are written to a fairly advanced level and should be of interest to the graduate student and practising scientist. I also hope that the subject matter treated will be of interest to people outside chemistry and to scientists in industry as well as government and regulatory bodies. It would be very satisfying for me to see the books used as a basis for developing graduate courses in Environmental Chemistry. Due to the breadth of the subject matter, it was not easy to edit this Handbook. Specialists had to be found in quite different areas of science who were willing to contribute a chapter within the prescribed schedule. It is with great satisfaction that I thank all 52 authors from 8 countries for their understanding and for devoting their time to this effort. Special thanks are due to Dr. F. Boschke of Springer for his advice and discussions throughout all stages of preparation of the Handbook. Mrs. A. Heinrich of Springer has significantly contributed to the technical development of the book through her conscientious and efficient work. Finally I like to thank my family, students and colleagues for being so patient with me during several critical phases of preparation for the Handbook, and to some colleagues and the secretaries for technical help. I consider it a privilege to see my chosen subject grow. My interest in Environmental Chemistry dates back to my early college days in Vienna. I received significant impulses during my postdoctoral period at the University of California and my interest slowly developed during my time with the National Research

Preface

IX

Council of Canada, before I could devote my full time of Environmental Chemistry, here in Amsterdam. I hope this Handbook may help deepen the interest of other scientists in this subject. Amsterdam, May 1980

O. Hutzinger

Twentyone years have now passed since the appearance of the first volumes of the Handbook. Although the basic concept has remained the same changes and adjustments were necessary. Some years ago publishers and editors agreed to expand the Handbook by two new open-end volume series: Air Pollution and Water Pollution. These broad topics could not be fitted easily into the headings of the first three volumes. All five volume series are integrated through the choice of topics and by a system of cross referencing. The outline of the Handbook is thus as follows: 1. 2. 3. 4. 5.

The Natural Environment and the Biochemical Cycles, Reaction and Processes, Anthropogenic Compounds, Air Pollution, Water Pollution.

Rapid developments in Environmental Chemistry and the increasing breadth of the subject matter covered made it necessary to establish volume-editors. Each subject is now supervised by specialists in their respective fields. A recent development is the accessibility of all new volumes of the Handbook from 1990 onwards, available via the Springer Homepage http://www.springer. de or http://Link.springer.de/series/hec/ or http://Link.springerny.com/ series/hec/. During the last 5 to 10 years there was a growing tendency to include subject matters of societal relevance into a broad view of Environmental Chemistry. Topics include LCA (Life Cycle Analysis), Environmental Management, Sustainable Development and others.Whilst these topics are of great importance for the development and acceptance of Environmental Chemistry Publishers and Editors have decided to keep the Handbook essentially a source of information on “hard sciences”. With books in press and in preparation we have now well over 40 volumes available.Authors, volume-editors and editor-in-chief are rewarded by the broad acceptance of the “Handbook” in the scientific community. Bayreuth, July 2001

Otto Hutzinger

Introduction

Except for astatine whose chemistry is largely unknown, fluorine and iodine are the first and last of the halogens. This is shown in a number of ways including the successive decrease in the redox potential Hal–/Hal2 and the electronegativity, and increase in the covalent and van der Waals radii. The substitution of hydrogen by fluorine does not greatly alter the structure of organofluorines in contrast to the effect of introducing bulky bromine or iodine substituents. Although fluorides are found abundantly in a range of minerals, the taming of both elemental fluorine and the hydrogen fluorides presented serious experimental difficulties that were solved only after many years of dangerous work and were a prelude to the synthesis of organofluorines. Bromide is present in seawater at a concentration of 65 ppm and iodide at 0.05 ppm although these concentrations are greatly exceeded in hypersaline lakes that are the current source of bromide and iodide. The preparation of both elemental bromine and iodine was accomplished more than 60 years before that of elemental fluorine, bromine in 1826 and iodine in 1811’ and the synthesis of organobromine and organoiodine compounds presented fewer problems. A wide range of organofluorines has achieved industrial importance as refrigerants, surfactants, pharmaceuticals, dyestuffs, whereas the range of organobromine and organoiodine compounds in general use is much more limited. Organofluorine compounds exist only in the monovalent state whereas all the other halogens may exist at oxidation levels up to 7. Organoiodine compounds may exist in the trivalent and pentavalent states that have seen numerous applications: they have been used extensively in organic synthesis as oxidizing agents [Zhdankin and Stang 2002], benziodoxoles have attracted attention as synthetic reagents for the destruction of chemical weapons [Morales-Rojas and Moss 2002] and iodonium salts have been used to develop a silver-free, single-sheet imaging medium [Marshall et al. 2002]. The number of naturally occurring organofluorines is structurally limited and essentially confined to higher plants in contrast to the plethora of organobromine – and to a lesser extent organoiodine – metabolites produced mostly by marine biota. Iodide is essential for many biota including humans, and organic compounds of iodine have long attracted interest as a result of the physiological importance of iodinated tyrosines in thyroid function and the antiseptic property of diiodine released from triiodomethane. More recently they have achieved importance as X-ray contrast agents.

XIV

Introduction

Organic compounds of bromine have a greater diversity of application. Dibromoethane was once used extensively in automobile fuel containing tetraethyl lead to diminish engine corrosion, while methyl bromide has a long history of use as fumigant and has attracted attention as a result of concern with global warming and ozone depletion. In addition, oxidants produced in the bromine cycle in the troposphere have been shown to be important in mobilizing elementary Hg to species that are both accessible to biota and accumulate in Arctic snow [Lindberg et al. 2002]. Polybrominated aromatic compounds, and especially diphenyl ethers, have been used as flame-retardants and are now widely distributed in the environment. A relatively small number of agrochemicals including bromoxynil, bromacil and bromethuron have been used. This volume addresses a broad spectrum of the environmental issues surrounding organic bromine and iodine compounds. In assessing their environmental significance it is important to assess their partition among the environmental compartments and the potential for their long-range dissemination: these issues are discussed by Cousins and Palm. Orlando discusses atmospheric chemistry in the context of ozone depletion and global warming, and the significant difference between the reactions of methyl bromide and methyl iodide are underscored. Mammalian toxicity is discussed by DePierre and the mechanisms of their degradation and transformation by Allard and Neilson. There has been considerable interest in naturally occurring metabolites in the current debate on the fate and partition of methyl bromide that is – or possibly by the time this is published was – important nematocide and is produced in substantial quantities as a metabolite of marine algae. There has also been speculation on the natural occurrence of diphenyl ethers and Neilson discusses plausible mechanisms for the biosynthesis of representative organic bromine and organic iodine metabolites. Once again, it is a particular pleasure to thank the authors who were prepared to sacrifice their valuable time and take on the additional burden of making their contributions. This is particularly appreciated since, in these days of continual stress, potential contributors feel themselves already overburdened with the demands of seeking financial support and producing publications to justify their existence. Any success with this volume is entirely due to the contributors, and I feel sure that their effort has been well rewarded in producing an exciting volume. Lindberg SE, S Brooks, C-J Lin, KJ Scott, MS Landis, RK Stevens, M Goodsite and A Richter (2002) Dynamic oxidation of gaseous mercury in the Arctic troposphere at polar sunrise. Environ Sci Technol 36:1245–1256 Marshall JL, SJ Telfer, MA Young, EP Lindholm, RA Minns, L Takiff (2002) A silver-free, singlesheet imaging medium based on acid amplification. Science 297:1516-1521 Morales-Royas H and RA Moss (2002) Phosphorolytic reactivity of o-iodosylcarboxylates and related nucleophiles. Chem Rev 102:2497–2521 Zdankin VV and PJ Stang (2002) Recent developments in the chemistry of polyvalent iodine compounds. Chem Rev 102:2523–2584

Stockholm, July 2003

Alasdair H. Neilson

The Handbook of Environmental Chemistry Vol. 3, Part R (2003): 1 – 74 DOI 10.1007/b11447HAPTER 1

Degradation and Transformation of Organic Bromine and Iodine Compounds: Comparison with their Chlorinated Analogues Ann-Sofie Allard 1 · Alasdair H. Neilson 2 Swedish Environmental Research Institute Limited IVL, Sweden 1 E-mail: [email protected] 2 E-mail: [email protected]

An overview is given of the pathways for the degradation and transformation of selected brominated and iodinated aliphatic and aromatic compounds. Although greater emphasis is placed on reactions mediated by microorganisms, examples of important abiotic reactions are also given. A mechanistic outline of the enzymology is provided when possible and comparisons are made with the chlorinated analogues which have been more extensively studied. Keywords. Biodegradation and biotransformation, Abiotic transformation, Aliphatic compounds, Aromatic compounds

1

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

2

2

Aliphatic Compounds . . . . . . . . . . . . . . . . . . . . . . . .

4

2.1 2.1.1 2.1.1.1 2.1.1.2 2.1.1.3

Halogenated Methanes . . . . . . . . . . . . . . . . . . . . . . Methyl Halides . . . . . . . . . . . . . . . . . . . . . . . . . . . Methane Monooxygenase Pathway . . . . . . . . . . . . . . . . Methyl Transfer and Corrinoid Pathways . . . . . . . . . . . . . Corrinoid Transmethylations in Aerobic and Anaerobic Metabolism of Methyl Halides . . . . . . . . . . . . . . . . . . . 2.1.2 Di- and Trihalomethanes . . . . . . . . . . . . . . . . . . . . . 2.1.2.1 Aerobic Organisms . . . . . . . . . . . . . . . . . . . . . . . . 2.1.2.2 Anaerobic Organisms . . . . . . . . . . . . . . . . . . . . . . . 2.2 Halogenated Alkanes and Related Compounds with Two or More Carbon Atoms . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Halogenated Ethenes . . . . . . . . . . . . . . . . . . . . . . . 2.4 Haloalkanols . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5 Haloaldehydes . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6 Haloalkanoates . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7 Halogenated Ethers . . . . . . . . . . . . . . . . . . . . . . . . 2.8 Reductive Loss of Halogen . . . . . . . . . . . . . . . . . . . . 2.9 Brominated and Iodinated Alkanes and Related Compounds as Metabolic Inhibitors . . . . . . . . . . . . . . . . . . . . . .

. . . .

6 6 6 8

. 9 . 12 . 12 . 13 . . . . . . .

15 20 22 23 23 26 27

. 30

. . . . . . . . . . . . . . . . . . . . . . . . . . 31

3

Abiotic Reactions

3.1 3.2

Photohydrolytic Reactions . . . . . . . . . . . . . . . . . . . . . 31 Reductive Reactions . . . . . . . . . . . . . . . . . . . . . . . . . 32 © Springer-Verlag Berlin Heidelberg 2003

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A.-S. Allard · A. H. Neilson

4

Aromatic Compounds : Aerobic Reactions . . . . . . . . . . . . . 34

4.1 4.1.1 4.1.2 4.1.3 4.2 4.2.1 4.2.1.1 4.2.1.2 4.2.1.3 4.2.1.4 4.2.1.5 4.2.1.6 4.3 4.3.1 4.3.2 4.4

Hydrocarbons . . . . . . . . . . . . . . . . . . . Degradation and Growth . . . . . . . . . . . . . Metabolism Without Growth . . . . . . . . . . . Biotransformation to Dihydrodiols . . . . . . . . Benzoates . . . . . . . . . . . . . . . . . . . . . Dioxygenation . . . . . . . . . . . . . . . . . . . Dehalogenation of 2-Halogenated Benzoates . . . Loss of Halogen in 4-Halogenated Phenylacetates Halohydrolases . . . . . . . . . . . . . . . . . . . Reductive Dehalogenation . . . . . . . . . . . . . Denitrification . . . . . . . . . . . . . . . . . . . Fungi . . . . . . . . . . . . . . . . . . . . . . . . Phenols . . . . . . . . . . . . . . . . . . . . . . . O-Methylation of Halogenated Phenols . . . . . . Fungal Metabolism . . . . . . . . . . . . . . . . . Amines . . . . . . . . . . . . . . . . . . . . . . .

5

Alternative Mechanisms of Dehalogenation . . . . . . . . . . . . 51

5.1 5.2 5.3

Peroxidase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Dehalogenation by a Polychaete . . . . . . . . . . . . . . . . . . . 51 Dehalogenation by Thymidylate Synthetase . . . . . . . . . . . . 51

6

Anaerobic Reactions . . . . . . . . . . . . . . . . . . . . . . . . . 52

6.1 6.2 6.2.1 6.2.2 6.2.3 6.3 6.3.1 6.3.2 6.4

Introduction . . . . . . . . . . . . . . . . . . . . . . . Halogenated Hydrocarbons . . . . . . . . . . . . . . . Polyhalogenated Benzenes . . . . . . . . . . . . . . . PCBs . . . . . . . . . . . . . . . . . . . . . . . . . . . PBBs and Diphenylmethanes . . . . . . . . . . . . . . Anaerobic Degradation of Benzoates . . . . . . . . . . Dehalogenation . . . . . . . . . . . . . . . . . . . . . Oxidation and Reduction of Aromatic Carboxylates and Aldehydes . . . . . . . . . . . . . . . . . . . . . . . . Phenols . . . . . . . . . . . . . . . . . . . . . . . . . .

7

Concluding Comments

8

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62

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34 34 35 39 40 41 41 43 44 45 45 45 46 48 49 49

52 54 54 55 56 59 59

. . . . . . 60 . . . . . . 60

. . . . . . . . . . . . . . . . . . . . . . . 61

1 Introduction In the course of preparing this chapter it became evident that relatively few studies were directed primarily to brominated compounds. Many were concerned with chlorinated compounds, and some brominated analogues were fortuitously included. It was therefore necessary to glean the literature on chlorinated com-

Degradation and Transformation of Organic Bromine and Iodine Compounds

3

pounds and extract details on their brominated and iodinated analogues that were sometimes included. It was then decided to include results for selected chlorinated compounds for several reasons: (i) for comparison with their brominated analogues; (ii) when studies of the brominated compounds were lacking and had been carried out only with the chlorinated analogues; (iii) when studies only with chlorinated analogues illustrated important principles of metabolism. Attention is drawn to a selection of reviews that cover various aspects of dehalogenation [23, 53, 55, 84, 94, 162, 188, 226]. Three different metabolic situations have been encountered: (i) growth at the expense solely of the brominated compound; (ii) loss of bromide during incubation with cell suspensions or enzymes; (iii) inclusion of a brominated substrate in the course of enzymological studies. It is worth noting that for some halogenated substrates, evidence for diminution of its concentration has been demonstrated in spite of the absence of dehalogenation. This may plausibly be attributed to simple biotransformations such as oxidation or dehydrogenation under aerobic conditions. It is important to note the different experimental procedures that have been used. Experiments under anaerobic conditions have been carried out under a variety of conditions using: (i) pure cultures; (ii) metabolically stable mixtures of organisms; (iii) unselected suspensions of soil or sediment. The last can lead to problems in interpretation since the sample used for assay will generally contain some of the putative degradation products. A cardinal issue that not been addressed here is the accessibility of brominated compounds – especially hydrocarbons and phenolic compounds – to the appropriate organisms in suspended matter or in the sediment phase containing organic carbon. This is only noted parenthetically with references to some representative illustrations from the relevant literature from chlorinated analogues. Attention is drawn in the text to some taxonomic changes. For simplicity, these synonymies are duplicated in the table below. Previous name

Current name

Alcaligenes eutrophus Flavobacterium sp. Hyphomicrobium sp. strain CM2 Methylobacterium sp. strain CM4 Pseudomonas cepacia Pseudomonas paucimobilis Pseudomonas pickettii Rhodococcus chlorophenolicum Clostridium thermoautotrophica Enterobacter agglomerans

Ralstonia eutropha Sphingomonas chlorophenolica Hyphomicrobium chloromethanicum Methylobacterium chloromethanicum Burkolderia cepacia Sphingomonaspaucimobilis Ralstonia pickettii Mycobacterium chlorophenolicum Moorella thermoautotrophica Pantoea agglomerans

For chlorinated compounds, the greatest attention has been given to groups of substances that are considered environmentally unacceptable, for example, low

4

A.-S. Allard · A. H. Neilson

molecular mass chlorinated aliphatic compounds used as solvents, chiral chloropropionates incorporated into agrochemicals, hexachlorocyclohexane, PCBs, and pentachlorophenol used as a wood preservative. Although brominated organic compounds are important as agrochemicals, pharmaceuticals and flame-retardants, and iodinated compounds as X-ray contrast agents, these have been studied less exhaustively than their chlorinated analogues. In addition, the appropriate compounds may not be commercially available as substrates and it may seem unusually academic to synthesize them. Reliance has of necessity been placed on the metabolism of chlorinated compounds, particularly for the structures of those enzymes that have been determined by X-ray analysis. Greatest weight has been placed on studies in which the biochemistry of degradation and transformation has been elucidated, and in which comparison among the halogens is possible. There have been major methodological developments during recent years. These include the success in obtaining crystals of enzymes that enable the application of X-ray analysis to the study of enzyme mechanisms: these provide important details and where available the results of such studies have been included. Although 13C NMR has been used generally with cell suspensions, the availability of on-line LC-NMR opens this to wider application in establishing the structure of transient metabolites. There have been substantial advances in establishing the genetics of degradation, and procedures for comparing amino acid and nucleotide sequences among groups of enzymes. This has made it possible to establish relationships between enzymes from different organisms and encouraged speculation on their evolution. This aspect has not, however, been treated here in the depth that it deserves. It is worth noting that – with the exception of simple bromophenols – virtually no investigations have been directed to the biodegradation of the plethora of naturally occurring brominated organic compounds that are discussed by Neilson.

2 Aliphatic Compounds Introduction

A number of brominated alkanes including methyl bromide, 1,2-dibromoethane, 1,2-dibromoethene, and propargyl bromide have been used in agriculture as fumigants and nematicides. Concern for the adverse effect of these on the destruction of ozone and the long half-life of methyl bromide has resulted in studies on their microbial degradation, as well as attempts to quantify their natural production and the extent to which the ocean serves as a sink. This is discussed in the chapter by Cairns and Palm and, on the basis of the “replacement principle”, attention has been redirected to the use of propargyl bromide [248]. Polychlorinated ethanes and ethenes have been extensively used as solvent and degreasing agents in the metallurgical industry and concern has arisen over their adverse health effects. This has stimulated efforts to study their degradability and

Degradation and Transformation of Organic Bromine and Iodine Compounds

5

to find replacements: indeed this applies equally to all polyhalogenated aliphatic compounds. An outline of the primary C-Br fission reactions encountered during the degradation of brominated alkanes, alkenes, brominated alkanols and bromoalkanoates is given in Fig. 1. Further reactions are, of course, possible, when reactive intermediates such as epoxides or ethenes are formed.

Fig. 1. Outline of primary reactions involved in degradation or transformation of haloalkanes

and related compounds

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A.-S. Allard · A. H. Neilson

2.1 Halogenated Methanes 2.1.1 Methyl Halides

A Taxonomic Note – To avoid possible confusion, taxonomic changes in this area [128] are given: Methylobacterium sp. strain CM4 Æ Methylobacterium chloromethanicum Hyphomicrobium sp. strain CM2 Æ Hyphomicrobium chloromethanicum together with the names of other organisms that are discussed: Leisingeria methylhalidovorans Methylobacterium extorquens Degradation in the Natural Environment. It has been established that the biodegradation of methyl bromide occurs in a number of natural environments. For example, a methylotrophic bacterium IMB-1 isolated from agricultural soil was able to degrade methyl bromide to CO2 [75]. The degradation of methyl bromide in a mixed soil bacterial flora occurred at concentrations several powers of ten lower than have been used in previous experiments (ten parts per billion by volume) [82]. At these low concentrations, neither chemical degradation nor anaerobic degradation occurred. This potential has been examined in freshwater, estuarine, and seawater samples in which non-bacterial degradation was eliminated from the results by lack of inhibition of degradation by methyl fluoride: both methyl bromide and methylene dibromide were examined but only in the freshwater sample was degradation observed [68]. Two strains were able to degrade methyl bromide at concentrations of the mixing ratio of tropospheric methyl bromide with surface water and displayed no evidence of a threshold concentration for uptake [70]. Both strains IMB-1 and Leisingeria methylohalidovorans strain MB2 degrade methyl bromide by respiration that is mediated by a methyltransferase whereas in Methylomonas rubra that is also able to take up methyl bromide at comparably low concentrations degradation of the substrates is mediated by a monooxygenase. Strains Involved in Degradation. There are two distinct mechanisms for the degradation of halomethanes involving (1) methane monooxygenase and (2) corrin-dependent, and the biodegradation of methyl bromide has been demonstrated in both groups. 2.1.1.1 Methane Monooxygenase Pathway

A number of strains with monooxygenase activity have been examined and it is convenient to add some comment on the enzyme since various types of methane monooxygenase have played important roles in the degradation of halo-

7

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 1. Concentrations of bromide (µmol) in cultures in which the substrates hadbeen de-

graded. The theoretical values are 4.5 for methyl bromide and 11 for dibromoethane [196] Organism

Methyl bromide

Dibromoethane

Methylosinus trichosporium OB3b Strain ENV 2041 Mycobacterium vaccae JOB5

4.5 5.3 3.0

9.2 14.1 NA

NA = not available.

genated aliphatic compounds. The enzyme exists in both a soluble and a particulate form of which the former has been more extensively studied. The enzyme consist of three components: a hydroxylase, a regulatory protein that is not directly involved in electron transfer between the hydroxylase, and a third protein that is a reductase containing FAD and an [2Fe-2S] cluster. Details of the structure of the hydroxylase and the mechanism of its action involving the FeIII-O-FeIII at the active site are given in a review [112]. The particulate enzyme contains copper or both copper and iron, and the concentration of copper determines the catalytic activity of the enzyme [194]. For example, trichloroethene is degraded by Methylosinus trichosporium strain OB3b under copper limitation when the soluble monooxygenase is formed, but not during grown with copper sufficiency when the particulate form is synthesized [152]. Examples of methyl bromide-degrading strains include the following: 1. The soluble methane monooxygenase from Methylococcus capsulatus (Bath) is able to oxidize chloro- and bromomethane, but not iodomethane with the presumptive formation of formaldehyde [36]. 2. The methane degrading Methylosinus trichosporium OB3b has been shown to degrade both methyl bromide and dibromomethane [9, 196] and the propane-degrading Mycobacterium vaccae JOB5 methyl bromide (Table 1) [196]. The degradation pathway for Methylosinus trichosporium OB3b was examined in an elegant study using 13C NMR with [13C]CH3Br and [13C]CH2Br2 as substrates. Although the expected formaldehyde from the former could not be demonstrated possibly on account of its rapid further transformation, the formation of CO was shown for CH2Br2 by 13C NMR, and the involvement of methane monooxygenase was supported by inhibition of activity by acetylene [9]. Although it was postulated that the initial reactions were monoxygenation followed by loss of hydrogen bromide, more recent studies on the metabolism of both methyl chloride and methyl bromide show that the reactions are corrinoid-dependent (see below). It is also worth noting the contrasting mechanisms for the degradation of methyl chloride and dichloromethane. One of the fascinating results of the studies with methyl chloride is the similarity of pathways proposed for aerobic and strictly anaerobic bacteria. 3. Suspensions of Nitrosomonas europaea were able to oxidize a number of halogenated alkanes more effectively in the presence of NH4+ that was oxidized to nitrite (Table 2) [221].

8

A.-S. Allard · A. H. Neilson

Table 2. Loss of haloalkanes by Nitrosomonaseuropaea during oxidation of NH4+. Substrate remaining after 24 h as % of the initial amount [222]

Substrate

Remaining substrate %

Substrate

Remaining substrate %

Dichloromethane Dibromomethane Bromoethane cis-Dichloroethene cis-Dibromoethene

0 4 25 9 3

1,2-Dibromoethane trans-Dichloroethene trans-Dibromoethene

16 25 100

2.1.1.2 Methyl Transfer and Corrinoid Pathways

This pathway is used by a number of organisms, and a general outline is given: 1. Strain IMB-1 is able to grow at the expense of methyl bromide [239] and belongs to a group of organisms that are also able to degrade methyl iodide but unable to use formaldehyde or methanol [176]. A single gene cluster contained six open reading frames: cmuC, cmuA, orf 146, paaE, hutI, and part of metF. Although CmuA from this strain had a high homology with the methyl transfer of Methylobacterium chloromethanicum and Hyphomicrobium chloromethanicum, CmuB that has been identified in these strains was not detected. A study with Hyphomicrobium chloromethanicum strain CM2 revealed a cmu gene cluster containing ten open reading frames: folD (partial), pduX, orf153, orf 207, orf 225, cmuB, cmuC, cmuA, fmdB, and paaE (partial). CmuA, CmuB, and CmuC from this strain showed a high similarity to those from Methylobacterium chloromethanicum (Table 3) [239] and it was postulated that the pathway for chloromethane degradation in this strain was similar to that in Methylobacterium chloromethanicum [126]. 2. Methylobacterium sp. CM4 [222, 223] is able to degrade methyl chloride, and details of the metabolism by this strain (now classified as Methylobacterium chloromethanicum) have been resolved and are discussed below. Table 3. Summary of Hyphomicrobium chloromethanicum methyltransferase genes and iden-

tity (%) with representative proteins [239] Gene FolD CmuB CmuC CmuA PaaE

Inferred function Methylene tetrahydrofolate Cyclohydrolase/dehydrogenase Methyltransferase Methyltransferase Methyltransferase and corrinoid protein Reductase

Sequence comparison (% identity)

M. chloromethanicum CM4 cmuB (57) M. chloromethanicum CM4 cmuC (36) and orf414 (34) M. chloromethanicum CM4 cmuA (80) E. coli PaaE (33), P. putida Tdn (32)

Degradation and Transformation of Organic Bromine and Iodine Compounds

9

2.1.1.3 Corrinoid Transmethylations in Aerobic and Anaerobic Metabolism of Methyl Halides

The existence of corrinoids in anaerobic bacteria in substantial concentrations is well established and their metabolic role in acetogenesis and in methanogenesis understood. Their involvement in degradation pathways of aerobic organisms is more recent, and it has emerged that their roles under these different conditions are similar. These issues are explored in the following paragraphs with a view to illustrating the similar metabolic pathways used by both aerobes and anaerobes. Corrinoids are involved in aerobic degradation as noted below for the degradation of methyl chloride by the aerobic Methylobacterium sp. strain CM4, and also C1 degradation by Methylobacterium extorquens [34]. Methyl corrins are key components in transmethylation and examples illustrating the similarity of pathways in aerobic and anaerobic metabolism will be summarized. In the following discussion, tetrahydrofolate or tetrahydromethanopterin (Fig. 2) are implicated in the form of their methyl (CH3), methylene (CH2), methine (CH), and formyl (CHO) derivatives (Fig. 3). The formation of a CH3-Co bond is integral and generally the 5,6-dimethylbenziminazole is displaced by histidine. Aerobic Degradation of Methyl Chloride – Methylotrophic bacteria have been isolated that are able to use methyl chloride aerobically as the sole source of energy and carbon. The substrate is metabolized to formaldehyde and undergoes subsequent oxidation either to formate and CO2 or incorporation via the serine pathway. A study using a strain CC495 that is similar to the strain IMB-1 noted above revealed the complexity of this reaction [38] while details had emerged from a somewhat earlier of methyl chloride degradation by the aerobic Methylobacterium sp. strain CM4 (Methylobacterium chloromethanicum). Cobalamin was necessary for growth with methyl chloride, though not for growth with methylamine, and use of mutants containing a miniTn5 insertion

Fig. 2. Partial structures of tetrahydrofolate (H4F) and tetrahydromethanopterin (H4MPT)

10

A.-S. Allard · A. H. Neilson

Fig. 3. Dehydrogenation of CH3-tetrahydrofolate to CHO-tetrahydrofolate

and enzyme assays revealed the mechanism of the degradation involving initial methyl transfer to a Co(I) corrinoid followed by oxidation via tetrahydrofolates to formyltetrahydrofolate and thence to formate with production of ATP (Fig. 4) [222]. Anaerobic Degradation of Methyl Chloride – The anaerobic methylotrophic homoacetogen Acetobacterium dehalogenans is able to grow with methyl chloride and CO2 and uses a comparable pathway for dehydrogenation of the methyl group involving tetrahydrofolate, a corrinoid coenzyme while the activity of CO dehydrogenase and the methyl tetrahydrofolate produce acetate (Fig. 5) [130]. Some of the gene products are shared with those involved in metabolism of methyl chloride [222] (Table 4). The methyl transfer reactions and those involved in the subsequent formation of acetate have been explored for the demethylase of this strain [101] and also resemble closely those for the aerobic metabolism of methyl chloride by aerobic methylotrophs.

Degradation and Transformation of Organic Bromine and Iodine Compounds

11

Fig. 4. Degradation of methyl chloride by Methylobacterium chloromethanicum (Redrawn from

[222])

Fig. 5. Degradation of methyl chloride by Acetobacterium halogenans (Redrawn from [101,

130])

12

A.-S. Allard · A. H. Neilson

Table 4. Genes, inferred function and identity (%) to representatives [222]

Gene

Inferred function

folD

5,10-Methylene-H4folate dehydrogenase/ 5,10-methenyl-H4folate cyclohydrolase purU 10-Formyl-H4folate hydrolase cmuA Methyltransferase/corrinoid protein metF 5,10-Methylene-H4folate reductase cmuB Methyl transfer

Comparison

Identity

FolD (E. coli)

49

PurU (Corynebacterium sp. MtbA /Methanosarcina barkeri Orf (Saccharomyces cerevisiae) MtrH (Methanobacterium thermoautotrophicum

47 24 24 30

2.1.2 Di- and Trihalomethanes 2.1.2.1 Aerobic Organisms

Several organisms including species of Pseudomonas, Hyphomicrobium, and Methylobacterium [105] have been shown to utilize dichloromethane, and the dehalogenases closely resemble each other. The mechanism for dechlorination involves a glutathione S-transferase that produced dideuteroformaldehyde from dideuterodichloromethane from cell extracts of Hyphomicrobium sp. strain DM2 [72], so that neither elimination-addition nor oxidation-reduction mechanisms are possible. Cell extracts of this strain were able to dehalogenate a number of dihalomethanes (Table 5) [197]. There are two different dichloromethane-dehalogenating glutathione S-transferases, neither of which contain metals, the enzyme from Hyphomicrobium sp. strain DM 4 with an a6 and that from Methylobacterium sp. strain DM 11 with an a2 structure [105]. Glutathione S-transferase is also involved in the degradation of isoprene (2-methyl-buta-1,3-diene) by Rhodococcus sp. strain AD45 [215] and is able to transform cis- and trans-1.2dichlorethene to the epoxides with formation of glyoxal. The enzyme has been purified [216] and has a wide range of substrate specificity (Table 6). Glutathione is also involved in the dechlorination of hexachlorocyclohexane catalyzed by LinD in Sphingomonas paucimobilis strain UT26 [141] and the dehalogenation of tetrachlorohydroquinone by Flavobacterium sp. [246]. The degradation of dibromo- and tribromomethanes has been examined under different conditions: (a) an enrichment culture from seawater was able to de-

Table 5. Rate of dehalogenation (mKat/mg protein) of dihalogenated methanes by cell extracts of Hyphomicrobium sp. strain DM 2 [197]

Substrate

Rate

Substrate

Rate

Dichloromethane Bromochloromethane

3.0 3.3

Dibromomethane Diiodomethane

2.6 0.8

13

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 6. Substrate specificity of S-glutathione transferase from Rhodococcus sp. strain AD45

(isoprene monoepoxide=100%) [216] Substrate

Activity

Substrate

Activity

Epoxyethane 1,2-Epoxybutane 1,2-Epoxyhexane

37 26 25

cis-1,2-Dichloroepoxyethane Epifluorohydrin Epichlorohydrin Epibromohydrin

4 28 21 25

grade 14CH2Br2 to 14CO2; (b) degradation was studied in a marine strain of Methylobacter marinus strain A45 and new type I methanotrophic strains designated KML E-1 and KML E-2: the first was able to degrade methyl bromide and dibromomethane whereas the last was able to degrade tribromomethane but not dibromomethane [69]. It has been established that strains bearing plasmids for monooxygenation of toluene are able to dechlorinate chloroform: for example, the toluene 4-monooxygenase from Pseudomonas mendocina strain KR1 [127], and the generalized toluene 2-, 3-, and 4-monooxygenase from Pseudomonas stutzeri strain OX1[32] (halogenated alkenes are discussed elsewhere in this chapter). 2.1.2.2 Anaerobic Organisms

Acetobacterium dehalogenans is able to degrade dichloromethane and the pathways resemble formally that for the anaerobic degradation of methyl chloride (Fig. 5). A strain of Dehalobacterium formicoaceticum is able to use only dichloromethane as a source of carbon and energy forming formate and acetate [138]. The pathway involves initial synthesis of methylene tetrahydrofolate of which two-thirds is degraded to formate with generation of ATP while the other third is dehydrogenated, transmethylated, and after incorporation of CO forms acetate with production of ATP (Fig. 6). The formation of [13C]formate, [13C]methanol, and [13CH3]CO2H was elegantly confirmed using a cell suspension and [13C]CH2Cl2. It was suggested that a sodium-independent F0F1-type ATP synthase exists in this organism in addition to generation of ATP from formyltetrahydrofolate. A strain of Acetobacterium woodii strain DSM 1930 dehalogenated tetrachloromethane to dichloromethane as the final chlorinated product, while the carbon atom of [14C]tetrachloromethane was recovered as acetate (39%), CO2 (13%), and pyruvate (10%) [49]. Since the transformation of tetrachloromethane to chloroform and CO2 is a non-enzymatic corrinoid-dependent reaction [50, 77] it seems safe to assume operation of the acetyl-CoA synthase reaction and the synthesis of acetate that also takes place during the degradation of dichloromethane by Dehalobacterium formicoaceticum and in which the CO2 originates from the medium [139]. Synthesis of Corrinoid-Dependent Reactions – It is appropriate to bring together a number of related reactions. These resemble those noted above even

14

A.-S. Allard · A. H. Neilson

Fig. 6. Degradation of methylene chloride by Dehalobacterium formicoaceticum (Redrawn

from [138])

though they do not involve halogenated substrates. The metabolism of acetate and lactate by Desulfomaculatum acetoxidans and Archaeoglobus fulgidus respectively and the pathways are given (Fig. 7), and are clearly essentially identical to those used for anaerobic degradation of methyl chloride and dichloromethane. In a wider context methanogenesis by Methanosarcina barkeri is worth noting: methane can be synthesized from CO2 and H2, or acetate, or methanol or methylamine. There are, however, important differences from the reactions noted above: 1. Use of tetrahydromethanopterin in place of tetrahydrofolate 2. The involvement of an aminofuran as acceptor of the formate produced from CO2 3. The different structure of the corrin that transfers the methyl group to coenzyme M and thence to methane Important details on the structures of the corrinoid reductases may be found in a review [117].

Degradation and Transformation of Organic Bromine and Iodine Compounds

15

Fig. 7a,b. Comparison of degradation of: a acetate by Desulfomaculatum acetoxidans; b lactate by Archaeoglobus fulgidus (Redrawn from [202a])

2.2 Halogenated Alkanes and Related Compounds with Two or More Carbon Atoms

Alkanes with a Single Halogen Atom – The monooxygenase in ammonia-oxidizing Nitrosomonas europaea has been examined for oxidation of halogenated ethanes and resulted in production of acetaldehyde presumably by initial terminal hydroxylation (Table 7) [158] while a strain of Nitrosomonas europaea was shown to oxidize a number of substrates including dibromoethane, and cis- and trans-dibromoethene (Table 2) [221]. The rates of dehalogenation of a range of 1-substituted haloalkanes was examined in an Arthrobacter sp. strain HA1 (Table 8) and the enzyme was a halohydrolase that produced the corresponding alkanol and dehalogenated a much wider range of substrates than could be used for growth [179]. Further investigations with the same strain confirmed that the reaction was hydrolytic, showed that there were three dehalogenases, examined the pattern of induction, and extended the range of growth substrates to 1-brominated alkanes C10, C12, C14, and C16 [180]. Pseudomonas sp. strain ES-2 was able to grow with a range of bromi-

16

A.-S. Allard · A. H. Neilson

Table 7. Rate of formation of acetaldehyde [nmol/min/mg protein] by whole cells of Nitro-

somonas europaea from 1-halogenated ethanes in the presence of 10 mmol/l NH4Cl [158] Substrate (µmol)

Rate

Substrate (µmol)

Rate

Fluoroethane (27) Chloroethane (9)

93 221

Bromoethane (4,8) Iodoethane (1.1)

122 19

Table 8. Rates of hydrolysis of 1-haloalkanes in cell extracts of Arthrobacter strain HA1 grown with 1-chlorobutane=100 [179]

Alkane

C1

C2

C3

C4

C5

C6

C7

C8

C9

1-Bromo 1-Iodo

ND 53

159 148

163 186

78 89

40 58

56 28

98 22

116 ND

125 ND

ND = not determined.

nated alkanes that greatly exceeded the range of chlorinated or unsubstituted alkanes: bromoalkanes with chain lengths of C6 to C16, and C18 could all be utilized [187]. A range of chlorinated, brominated, and iodinated alkanes C4 to C16 was incubated with resting cells of Rhodococcus rhodochrous NCIMB 13064 [40], and dehalogenation assessed from the concentration of halide produced (Table 9). The range of substrates is impressive and the yields were approximately equal for chloride and bromide and greater than for iodide. Possibly more remarkable is the metabolic capacity of species of mycobacteria including the human pathogen Mycobacterium tuberculosis strain H37Rv [96]. The specific activities in extracts of M. avium and M. smegmatis to a range of halogenated alkanes is given in Table 10. On the basis of aminoacid and DNA sequences, the strain that was used contained three halohydrolases and the debromination capability of a selected number of other species of mycobacteria is given in Table 11. The haloalkane dehalogenase gene from M. avium has been cloned and partly characterized [97]. Table 9. Dehalogenation of long-chain alkyl halides by Rhodococcus rhodochrous strain

NCIMB 13064 measured as halide release (1-chlorobutane=100). The symbols in parentheses designate growth at the expense of the substrate [40] Substrate 1-Chloropropane butane octane dodecane hexadecane octadecane

Dehalogenation 121 (+) 100 (+) 43 (+) 19 (+) 5 (+) 0 (+)

Substrate 1-Bromopropane butane octane dodecane hexadecane octadecane

Dehalogenation 112 (+) 102 (+) 135 (–) 20 (+) 15 (+) 8 (+)

Substrate

Dehalogenation

1-Iodopropane butane

92 (+) 30 (+)

dodecane

21 (+)

octadecane

0 (+)

17

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 10. Relative specific activity (µmol alkanol produced/mg protein/min) of extracts of

Mycobacterium avium MU1 and M. smegmatis CCM 4622 to halogenated alkanes [96] Substrate

Product

M. avium

M. smegmatis

1-Bromopropane 1,3-Dibromopropane 1-Chlorobutane 1-Bromobutane 1-Chlorohexane 1-Bromohexane 1-Iodohexane

Propan-1-ol 3-Bromo-propan-1-ol Butan-1-ol Butan-1-ol Hexan-1-ol Hexan-1-ol Hexan-1-ol

0.87 1.23 0.11 0.66 1.02 0.77 1.38

3.58 2.50 0.42 1.12 2.62 0.91 3.32

Table 11. Specific activity (µmol bromide produced/mg protein/min) of dehalogenase from

selected species of Mycobacterium towards 1,2-dibromoethane [96] Taxon

Activity

Taxon

Activity

M. bovis BCG MU10 M. fortuitum MU8 M. triviale MU3 M. smegmatis CCM4622

99 76 61 49

M. avium MU1 M. phlei CCM 5639 M. parafortuitun MU2 M. chelonae

36 22 22 20

Alkanes with More than a Single Halogen Atom – Some strains are able to use a,w-dichlorinated alkanes for growth, and the activity of the hydrolase from Rhodococcus erythropolis strain Y2 was high for 1,2-dibromoethane, 1,2-dibrompropane, and the a,w-dichloroalkanes (Table 12) [172] whereas the range of a,w-dichlorinated alkanes that was used for growth of Pseudomonas sp. strain 273 was limited to the C9 and C10 substrates [238]. Dehalogenase activity was demonstrated in a strain of Acinetobacter GJ70 that could degrade some a,w-dichloroalkanes, and 1-bromo- and 1-iodopropane. Although 1,2-dibromoethane could be converted to 2-bromoethanol, this could not be used for growth possibly due to the toxicity of bromoacetaldehyde and the unsuitability of dihydroxyethane as growth substrate [92]. In a later study the enzyme from this strain showed dehalogenase activity towards a wide range of substrates including halogenated alkanes, alkanols and ethers [95] (Table 13). Table 12. Relative substrate activities (1-chlorobutane (=100) of crude halohydrolase from

Rhodococcus erythropolis Y2 towards a,w-dihaloalkanes [172] Substrate

Relative activity

Substrate

Relative activity

1,2-Dichloroethane 1,3-Dichloropropane 1,4-Dichlorobutane 1,6-Dichlorohexane 1,9-Dichlorononane 1,10-Dichlorodecane

6 202 232 168 61 61

1,2-Dibromoethane 1,2-Dibromopropane

802 132

18

A.-S. Allard · A. H. Neilson

Table 13. Rates of dehalogenation with purified dehalogenase from Acinetobacter sp. strain

GJ70 relative to 1-bromopropane (=100) [95] Halomethane

Rate

n-Haloalkane

Rate

Halogenated alkanol

Rate

Methyl bromide Methyl iodine Dibromomethane

143 75

Bromoethane Iodoethane 1-Brompropane 1-Iodopropane

143 93 100 66

2-Bromoethanol 3-Bromopropanol

55 123

It seems valuable to provide a brief summary of the degradation of 1,2dichloroethane by Xanthobacter autotrophicus strain GJ10 which has been used to delineate all stages of the metabolism (Fig. 8). The activity of a haloalkane dehalogenase initiates degradation and is discussed in this section, while the alkanol dehydrogenase, the aldehyde dehydrogenase, and the haloacetate dehalogenase are discussed in subsequent sections. The enzyme responsible for dehalogenase activity has been purified from Xanthobacter autotrophicus strain GJ10, consists of a single polypeptide chain with a molecular mass of 36 kDa, and was able to dehalogenate chlorinated, and both brominated and iodinated alkanes [103]. Details of the mechanism have been explored using an ingenious method of producing crystal at different stages of the reaction [224]. The overall reaction involves a catalytic triad at the active site: Asp124 binds to one of the carbon atoms, and hydrolysis with inversion is accomplished by cooperation of Asp260 and His289 with a molecule of water bound to Glu56 (Fig. 9). In a Mycobacterium sp. strain GP1 that belongs to the group of fast-growing mycobacteria, 1,2dibromoethane could be used as a source of carbon and energy. Although it was converted into the epoxide by a haloalkane dehalogenase that could be used for growth and thereby circumvent the production of toxic bromoacetaldehyde, degradation of the epoxide was unresolved [156]. It is worth noting that, in contrast, 1,2-dichloroethane can be used for growth by Xanthobacter autrotrophicus strain GJ10: the pathway is shown in Fig. 8, and the appropriate enzymes have been demonstrated [93]. The second step in the degradation of g-hexachlorocyclohexane by Sphingomonas paucimobilis UT26 is carried out by the hydrolytic dehalogenation of 1,3,4,6-tetrachlorocyclohexa-1,4-diene to 2,5-dichlorocyclohexa-2,5-diene-1,4-diol [141] and the enzyme is also able to carry out debromination (Table 14) [142]. Pseudomonas putida strain G786) that harbors the CAM plasmid debrominated some polybrominated ethanes under anaerobic conditions [89], for example 1,1,2,2-tetrabromoethane, was reduced to a mixture of cis- and trans-1,2dibromoethene that were also formed from 1,2-dichloro-1,2-dibromoethane (Fig. 10).

Fig. 8. Degradation of 1,2-dichloroethane by Xanthobacter autotrophicus strain GJ10 (Redrawn

from [94])

Degradation and Transformation of Organic Bromine and Iodine Compounds

19

Fig. 9. Active site of haloalkane dehalogenase with 1,2-dichloroethane as substrate (Redrawn from [224])

Table 14. Selected substrates dehalogenated by the dehalogenase (LinB) from Sphingomonas

paucimobilis strain UT26 (chlorobutyrate=100) [142] Substrate

Relative rate

Bromoethane Chlorobutane Bromobutane 1,2-Dibromoethane 1,2-Dibromopropane

257 100 234 355 241

(a)

(b) Fig. 10a, b. Metabolism of: a 1,2-dibromo-1,2-dichloroethane; b tetrabromoethane by Pseudomonas putida G786 (Redrawn from [89])

20

A.-S. Allard · A. H. Neilson

Table 15. Relative time (min) required for degradation of haloepoxides by Xanthobacter sp.

strain Py2 and toxicity (% activity remaining after 2 h incubation with propene) [190] Epoxide

Time required for degradation (min)

Toxicity (% remaining after 2 h)

2,3-Epoxypropane 1-Fluoro-2,3-epoxypropane 1-Chloro-2,3-epoxypropane 1-Bromo-2,3-epoxypropane

3.5 20 30 > 120

105 90 72 8

Haloepoxides – Xanthobacter sp. strain Py2 is able to grow with propene or propene oxide and the pathway is dependent on the presence or absence of CO2 [189]. This strain is also able to degrade 1-fluoro-, 1-chloro-, and 1-bromo-2,3epoxypropane with decreasing ease (Table 15) [190]. The toxicity of the epihalohydrins occurs in the opposite order, and for 1-chloro-2,3-epoxypropane the first detectable metabolite was chloroacetone that is formed by an isomerization and not by a hydrolytic reaction: details of the further metabolism of chloroacetone remained unresolved. 2.3 Halogenated Ethenes

The degradation of chlorinated ethenes has attracted considerable attention (references in [53]) under aerobic conditions by ammonia monooxygenase, by soluble methane monooxygenase, and by toluene 2-, 3-, and 4-monooxygenases (Table 16). The soluble methane monooxygenase from Methylosinus trichosporium OB3b produced a number of products from trihaloethenes [58]: formate from trichloroethene and tribromoethene, and glyoxylate from trifluoroethene, together with the volatile fluoral, chloral, and bromal respectively (Fig. 11). The controlled oxidation of trichloroethene by Methylosinus trichosporium OB3b produced both trichloroethene epoxide and trichloroacetaldehyde, and the last was transformed biologically to trichloroethanol and trichloroacetate by a Cannizarro-like dismutation: the formation of chloroform and formate were abiotic reactions [149]. A strain of a methane-oxidizing bacterium was able to degrade trichloroethene when a growth substrate such as methane or methanol was present [113]. The reactions proceeded through an intermediate epoxide from which formate, Table 16. Toluene monooxygenases and the products from hydroxylation of toluene

Organisms

Cresol produced

Reference

Burkholderia cepacia strain G4 Pseudomonas pickettii strain PKO1 Pseudomonas mendocina strain KR1 Pseudomonas stutzeri strain OX1

ortho meta para o, m, p

[186] [153] [234] [32]

21

Degradation and Transformation of Organic Bromine and Iodine Compounds

Fig. 11. Products from the activity of methane monooxygenase on: a trifluoroethene; b tri-

bromoethene

Fig. 12. Generalized degradation of trihaloethenes by methanotrophs (Redrawn from [113])

CO, and glyoxylic acid were formed, while its spontaneous chemical degradation led to dichloroacetate by chloride migration (Fig. 12) [113]. There are several toluene monooxygenases that can oxidize halogenated ethenes. Comparably with the methane monooxygenases, the purified toluene 2monooxygenase from Burkholderia cepacia strain G4 produced CO, formate, and glycolate from trichloroethene in an approximate ratio of 4:2:1, but neither chloroacetic acids nor chloral were formed [150]. Trichloroethene and 1,1dichloroethene were degraded stoichiometrically to chloride by the toluene monooxygenase in Pseudomonas stutzeri strain OX1 that is able to carry out 2-, 3-, and 4-monooxidation of toluene [32]. The degradation of a selection of halogenated ethenes by the toluene monooxygenases is given in Table 17 [127]. Considerable effort has been devoted to the anaerobic dechlorination of chloroethenes and is discussed elsewhere in this chapter. Table 17. Degradation of halogenated substrates by toluene-oxidizing strains given as concentrations remaining µmol/l after 3 h incubation with20 µmol/l substrate. The dibromoethenes were used as a 1:1 mixture ofthe isomers with a total concentration of 20 µmol/l [127]

Strain

1,2-Dichloroethane

Trichloroethene

Chloroform

cis-1,2-Dibromoethene

trans-1,2-Dibromoethene

T4MO T3MO T2MO

2.7 20 20

0 0 0

4.7 20 20

9.1 2.7 0.2

0.9 1.9 2.6

Monooxygenases were from the following strains: T4O: Pseudomonas mendocina strain KR1 T3O: Pseudomonas pickettii strain PKO1 T2O: Burkholderia cepacia strain G4.

22

A.-S. Allard · A. H. Neilson

2.4 Haloalkanols

The conversion of vicinal haloalkanols to epoxides is of considerable interest and was apparently first recognized in a strain of Flavobacterium [24]. Their formation is stereospecific to produce the trans-epoxide from erythro 3-bromobutan2-ol and the cis- from threo 3-bromobutan-2-ol, and the epihalohydrins are able to react with halide to produce 2-hydroxy-1,3-dihalobutanes [8]. The enzyme has been purified from Arthrobacter strain AD2 that is able to utilize 3-chloro-1,2propandiol for growth, has a molecular mass of 29 Da, and consists of two equal subunits [214]. It is able to form epoxides from vicinal chlorinated and brominated alkanols (Table 18) and carry out transhalogenation between epihalohydrins and halide ions. By contrast, the halohydrin hydrogen-halide lyase B from Corynebacterium sp. strain N-1074 is able to hydrolyze some brominated 2-halogen-substituted alkanols (Table 19) [143] as well as carrying out the transformation of 1,3-dichloro-propan-2-ol to the epichlorohydrin enriched in the R-isomer (Fig. 13). Haloalkanol dehalogenase activity has also been found in a number of different bacteria (Table 20) [188]. The complete degradation of 1,2-dichloroethane by Xanthobacter autotrophicus strain GJ10 requires the synthesis of 2-chloroethanol dehydrogenase. This has been purified and is a quinoprotein showing the typical absorption at 345 nm and a shoulder at 410 nm, has a maximal activity at pH 9–9.4 and a temperature of 40 °C [93]. Table 18. Substrate specificity of haloalkanol dehalogenase from Arthrobacter sp. strain AD2,

and rates relative to that for 1,3-dichloro-propan-2-ol [214] Substrate

Product

Relative rate

1,3-Dichloropropan-2-ol 1,3-Dibromopropan-2-ol 1-Chloropropan-2-ol 1-Bromopropan-2-ol 2-Bromoethanol

Epichlorohydrin Epibromohydrin Propene oxide Propene oxide Ethene oxide

100 31,000 11 3,800 100

Table 19. Relative substrate activities (1,3-dichloropropan-2-ol=100) of halohydrin halide lyase B from Corynebacterium sp. strain N-1074 to various substrates [143]

Substrate

Activity

Substrate

Activity

1,3-Dichloropropan-2-ol 1,3-Dibromopropan-2-ol

100 161

2-Chloroethanol 2-Bromoethanol

0.13 7.9

Fig. 13. Transformation of 1,3-dibromopropan-2-ol of halohydrin hydrogen-halide lyase in Corynebacterium strain N-1074 (Redrawn from [145])

23

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 20. Activity of haloalcohol dehalogenases relative to 1,3-dichloropropanol for selected

strains [188] Substrate

Strain A

Strain B

Strain C

Strain D

Strain E

1,3-Dichloropropanol 1,3-Dibromopropanol 1-Chloropropanol 1-Bromopropanol

100 134,000 NA NA

100 31,000 11 3,780

100 12,500 22.7 NA

100 161 18.5 NA

100 60 27 NA

NA: not available. A = Pseudomonas sp. strain AD1 B = Arthrobacter sp. strain AD2

C = Corynebacterium sp. strain N-1074: dehalogenase Ia D = Corynebacterium sp. strain N-1074: dehalogenase Ib E = Arthrobacter erithii strain H10a.

2.5 Haloaldehydes

In the degradation of haloalkanes, the initially formed aldehyde is dehydrogenated to the carboxylic acid by an NAD-dependent dehydrogenase that was isolated from a 1,2-dichloroethane-degrading bacterium strain DE2 [198]. 2.6 Haloalkanoates

Halogenated alkanoic acids are produced from the aldehydes during the degradation of 1,2-dihaloethanes. The degradation of chlorinated alkanoic acids has been extensively investigated and the dehalogenases have been grouped according to their reactions with 2-chloropropionate to carry out hydrolysis with or without inversion and the effect of sulfhydryl-inhibitors [55]. The enzymology of 2-haloalkanoate dehalogenases is discussed in detail in [188]. By contrast, the brominated analogues have not been so systematically examined and only a summary account can be given. Organisms able to utilize n-undecane were able to debrominate 6-bromohexanoate but not 2-bromohexanoate [154]. Halohydrolases have been isolated from a number of bacteria and displayed activity against 2-haloacetates and, for some, 2-chloropropionate: 1. The halohydrolase H-2 from Delftia acidovorans (Moraxella sp. strain B) produced glycollate from 2-chloro-, 2-bromo-, and 2-iodoacetate [102]. 2. Xanthobacter autotrophicus GJ 10 is able to grow at the expense of short-chain halogenated hydrocarbons and carboxylic acids. The strain produces two constitutive dehalogenases that differ in their heat stability, in their pH optima, and in their substrate specificity (Table 21) [91]. It was proposed that the metabolism of 1,2-dichloroethane is proceeded by hydrolytic dehalogenation, dehydrogenation, and dehalogenation of the resulting chloroacetate to glycolate. The specific activities of these enzymes in this strain are given in Table 22 [93]. 3. The haloacid dehalogenase from a strain of Xanthobacter autotrophicus GJ that was able to utilize 1,2-dichloroethane using a haloalkane hydrolase, a haloalkanol dehydrogenase, an aldehyde dehydrogenase, and lastly a haloacid dehydrogenase has been purified and characterized. It was able to dehalogenate a restricted range of halogenated substrates (Table 23), and produced

24

A.-S. Allard · A. H. Neilson

Table 21. Specificity of dehalogenation in extracts of Xanthobacter autotrophicus strain GJ10, effect of pH and % activity after heat treatment [91]

Substrate

Specific activity (mU/mg protein)

1,2-Dichloroethane Chlorobutane Bromoethane Chloroacetate Dichloroacetate Bromoacetate Dibromoacetate

pH 7.5

pH 9.0

% activity

310 91 45 89 167 94 225

210 54 35 367 471 520 670

0 0 0 102 102 100 110

Table 22. Specific activities of enzymes involved in the degradation of 1,2-dichloroethane by

Xanthobacter autotrophicus strain GJ10 [93] 1,2-Dichloroethane dehalogenase

2-Chloroethanol dehydrogenase

Chloroacetaldehyde dehydrogenase

Chloroacetate dehalogenase

232

330

174

416

Table 23. Relative rates of dehalogenation (chloroacetate=100) for Pseudomonas strain CBS 3

[140] and Xanthobacter autotrophicus strain GJ10 M50 [217]

Chloroacetate Dichloroacetate Bromoacetate Dibromoacetate (S)-2-Chloropropionate (R,S)-2-Bromopropionate

Pseudomonas strain CBS 3

Xanthobacter autotrophicus

100 10 930 ND 15 270

100 134 98 371 92 ND

lactate from L-2-chloropropionate but not from the D-isomer [217].A strain of Pseudomonas sp. strain YL is also able to catalyze hydrolysis not only of 2haloalkanoic acids but also of long-chain halogenated substrates (Table 24) [114]. In both cases, inversion takes place at the halogenated carbon atom. Two 2-haloacid dehalogenases have been isolated from this strain [114] differing in their thermostability and their stereospecificities. One of them (L-DEX) in cells grown at the expense of 2-chloropropionate is thermostable, is inducible by 2-chloropropionate, and catalyzed the dehalogenation of both D- and L-2chloropropionate with inversion and production of D- and L-lactates. The other (L-DEX) is produced in cells grown with 2-chloroacrylic acid, is not thermally stable, and acts on L-2-haloalkanoic acids to produce the D-hydroxyalkanoate. The substrate specificities of the two enzymes are given (Table 25). These results may be compared with the constitutive dehalogenases from Xanthobacter autotrophicus GJ 10, one of which is thermally stable and dehalogenates

25

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 24. Activities of dehalogenase L-DEX from Pseudomonas sp. strain YL for long-chain

halogenated alkanoates (L-2-chloropropionate=100)[114] Substrate

Relative activity

Substrate

Relative activity

L-2-Chloropropionate DL-2-Chloroheptanoate DL-2-Bromooctanoate

100 17 11

DL-2-Bromodecanoate DL-2-Bromododecanoate DL-2-Bromotetradecanoate DL-2-Bromohexadecanoate

24 4 16 19

Table 25. Activities of dehalogenases (relative to L-2-chloropropionate=100) of Pseudomonas sp. strain YL [114]

Substrate

Activity (DL-DEX)

Activity (L-DEX)

Monochloroacetate Monobromoacetate Monoiodoacetate L-2-Chloropropionate D-2-Chloropropionate

105 92 111 100 122

65 90 75 100 0

halogenated alkanoic acids and the other that is thermally unstable dehalogenates halogenated ethanes [91]. The X-ray structures of the enzymes from both Pseudomonas sp. strain YL [83, 111] and Xanthobacter autotrophicus GJ [166] have been established and are broadly similar except for access to the active site [166]. The structure of the haloacid dehalogenase from Xanthobacter autotrophicus GJ is quite different from that of the haloalkane hydrolase from the same strain and the catalytic triad is missing: Asp124 is on another strand and the pair His289 and Asp260 have no counterpart [166]. The following catalytic mechanism was proposed: the substrate is bound to Ser114 via its carboxylate group, Asp8 is available to form an ester intermediate with fission of the C-Cl bond, and there is an unresolved site for an activated water molecule analogous to the His289Asp260 residues in the haloalkane dehalogenase. In the Pseudomonas sp. strain YL structure,Asp10 forms the ester with the substrate and Arg41 is the acceptor of the chloride anion (Fig. 14) [111]. 4. A dehalogenase (II) found when a cosmid gene bank of Pseudomonas strain CB3 constructed in Escherichia coli Hb 101 was screened, was purified and assayed for activity to a number of substrates (Table 23) and removed chloride from (R)-2-chloropropionic acid with inversion of configuration [140]. 5. An apparently unique enzyme has been found in Pseudomonas sp. strain 113 that is able to hydrolyze both D- and L-2-chloropropionate to the hydroxyalkanoic acids with inversion of configuration of both substrates (references in [144]), and it has been proposed [144] that the enzyme has a single common catalytic site for both enantiomers. 6. The purple non-sulfur anaerobic phototrophic bacteria Rhodospirillum rubrum, Rh. photometricum, and Rhodopseudomonas palustris DSM 123 were

26

A.-S. Allard · A. H. Neilson

Fig. 14. Reaction intermediates of L-2-haloacid dehalogenation (Redrawn from [112])

able to grow at the expense of chloroacetate, 2-chloropropionate, 3-chloropropionate, and 2-bromopropionate by dehalogenation to acetate or propionate: low concentrations of bromoacetate were used only by the last strain [129]. It emerged that one limitation to utilization of haloalkanoates was the toxicity of the substrates. This was shown for bromoacetate that inhibited growth of Pseudomonas putida strain PP3 [233] although a mutant of Xanthobacter autotrophicus GJ10 overexpressed the bromoacid dehalogenase and was able to grow with substrate concentrations between 10 mmol/l and 25 mmol/l [218]. A strain of Pseudomonas cepacia MBA4 was able to utilize bromoacetate as sole source of carbon and energy, and the inducible dehalogenase (designated IVa) was isolated and purified [206]. Its activity is given in Table 26. It was subsequently shown that although this dehalogenase (DehIVa) had a 68% identity to the dehalogenase (DehCI) from Pseudomonas sp. strain CB3 the former is dimeric and the latter monomeric [205]. 2.7 Halogenated Ethers

Two strains of an Ancylobacter aquaticus AD25 and AD27 were isolated by enrichment using 2-chloroethylvinyl ether and the dehalogenase displayed activity towards a range of halogenated alkanes (Table 27) [212] and the degradation of

27

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 26. Dehalogenase specific activities (µmol substrate converted/mg protein/min) of Pseudomonas cepacia strain MBA4 under different growth conditions [206]

Growth substrate

Assay substrate CA

BA

2-ClP

2-BrP

DiClA

CA BA 2-BrP

0.46 0.41 0.22

0.67 0.54 0.49

0.08 0.05 0.03

0.27 0.21 0.19

0.08 0.06 0.06

CA = chloroacetate BA = bromoacetate DiCIA = dichloroacetate.

2-CIP = 2-chloropropionate 2-BrP = 2-brompropionate

Table 27. Relative activities (%) of haloalkane dehalogenase from Ancylobacter aquaticus [212]

Substrate

Relative activity

Products

1,2-Dichloroethane 1,2-Dichloropropane 1,2-Dibromopropane 2-Bromoethanol

100 0.6 133 24

2-Chloroethanol 1-Chloropropan-2-ol, 2-chloropropan-2-ol 1-Bromopropan-2-ol, 2-bromopropan-2-ol 1,2-Dihydroxyethane

Table 28. Specific activity (n mol halide/min/mg protein) of dehalogenase inextracts of strains

AD25 and AD27 of Ancylobacter aquaticus towards halogenated ethers [213] Substrate

Product

Strain AD25

Strain AD27

1,2-Dichloroethane 2-Chlorovinyl ether 2-Chloroethylvinyl ether 2-Bromoethylethyl ether

2-Chloroethanol 2-Hydroxyethylvinyl ether 2-Hydroxyethylmethyl ether 2-Hydroxyethylethyl ether

4710 655 323 697

865 82 46 ND

ND = not determined.

some halogenated ethers has been described (Table 28) [213]. The C-O-C bond is not fissioned enzymatically in the substrate and chemical hydrolysis is involved in two possible pathways (Fig. 15). For the sake of completeness attention is drawn to investigations on the biodegradation of vinyl chloride: in both, elegant use was made of 13C NMR of whole cell suspensions. One used Methylosinus trichosporium OB3b (Fig. 16) [29] and the other a strain of Pseudomonas sp.(Fig. 16) [30]: whereas the first involved chloroethene epoxide, the second was carried out by direct hydrolytic fission of the C-Cl bond. 2.8 Reductive Loss of Halogen

In contrast to all of the above reactions that are not dependent on the presence of oxygen, reductive loss of bromine has been demonstrated in anaerobic bacteria that depend on its absence. The reduction of tetrachloroethene to cis-1,2-

28

A.-S. Allard · A. H. Neilson

Fig. 15. Degradation of 2-chloroethylvinyl ether by Ancylobacter aquaticus (Redrawn from

[213])

Fig. 16a,b. Degradation of vinyl chloride by: a Pseudomonas sp. (Redrawn from [30]); b Me-

thylosinus trichosporium OB-3b (Redrawn from [29])

dichloroethene by the enteric organism Enterobacter (Pantoea) agglomerans may be noted [185] as one of the few examples of the metabolic role of Enterobacteriaceae. Dehalorespiration in which dehalogenation is coupled to the synthesis of ATP has been demonstrated for halogenated ethenes in Dehalospirillum multivorans, Dehalobacter restrictus, Desulfuromonas chloroethenica, and strains of Desulfitobacterium sp. (references in [84]), and in Dehalococcoides ethenogenes that is capable of reductively dehalogenating tetrachloroethene to ethene [121, 125]. The

29

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 29. Substrate range and products of trichloroethene reductase for selected brominated substrates [121]

Substrate

Product

Tribromoethene Vinyl bromide 1,2-Dibromoethene cis- and trans-1-Bromoprop-1-ene 5-Bromopent-1-ene

Dibromoethenes, vinyl bromide, ethene Ethene Ethene Propene Penta-1,4-diene

electron donors were generally H2 or pyruvate, or for Dehalococcoides ethenegenes methanol. The chloroethene reductases contain cobalamine and Fe-S clusters, and the range of brominated substrates for trichloroethene reductase from Dehalococcoides ethenogenes is given in Table 29 [121]. In addition, the TCE reductive dehalogenase from this organism is able to debrominate substrates containing C2, C3, and C4 or C5 carbon atoms, albeit in decreasing ease: an illustrative reaction is the debromination of tribromoethene to dibromoethenes, vinyl bromide, and ethene. Further examples include the debromination methanogenic bacteria of 1,2-dibromoethane to ethene and 1,2-dibromoethene to ethyne by (Fig. 17) [15]. Details of these dehalogenations have emerged from studies with methanogens. The formation of ethene from 1,2-dichloroethane with hydrogen as electron donor has been demonstrated in cell extracts of Methanobacterium thermoautotrophicum DH, and in Methanosarcina barkeri this reaction has been shown to involve cobalamin and F430 using Ti(III) as reductant [85]. Further comments are given in the section on abiotic reactions. It is also worth pointing out that the aerobic Pseudomonas putida G786 carrying the cytochrome P450CAM plasmid is also able to carry out selective reductive debromination [110]. The reductive debromination of BrCCl3 with formation of HCCl3 by cytochrome P450CAM has been shown [28] and examined with a greater range of substrates (Table 30) [110]. Reductive dehalogenation is one of the series of reactions involved in the degradation of g-hexachlorocyclohexane (aaaeee) [141]: (i) initial elimination

Fig. 17. Reductive debromination by methanogenic bacteria (Redrawn from [15]) Table 30. Products from reductive debromination of halogenated alkanes with cytochrome P450CAM [110]

Substrate

Product

Substrate

Product

Br2CCl2 BrCCl3

HBrCCl2 HCCl3

BrHCCl2 HClBrCCF3

H2CCl2 H2ClCCF3

30

A.-S. Allard · A. H. Neilson

Fig. 18. Dehalogenation of hexachlorocyclohexane by Sphingomonas paucimobilis strain UT 26 and reactions catalyzed by LinA, LinB, LinC and LinD (Redrawn from [141])

catalyzed by LinA, (ii) hydrolysis by LinB, and (iii) reductive loss of chloride from 2,5-dichlorohydroquinone catalyzed by glutathione (Fig. 18). The last reaction also occurs during the degradation of pentachlorophenol [246]. Both the active enzyme, the heat-inactivated enzyme from Dehalospirillum multivorans, and cyanocobalamin are capable of dehalogenating haloacetates (Table 31) [147] and the rate of abiotic dehalogenation depends on the catalyst that is used (Table 32). 2.9 Brominated and Iodinated Alkanes and Related Compounds as Metabolic Inhibitors

Most of the reactions of inhibitors depend on the chemical reactivity of brominated and iodinated alkanes and related compounds, and are consistent both with the chemical reactivity of these compounds and their biological reactivity that has been exemplified in hydrolytic or reductive reactions: 1. Methyl iodide and propyl iodide bind to corrinoid coenzymes and thereby inhibit their activity. The reaction is reversible in the presence of tungsten-filament white light, and these reagents have been used to establish the requirement for corrins in biological reactions, for example, (i) the synthesis of acetate from CO2 by Clostridium thermoaceticum [64], (ii) the biosynthesis of Table 31. Rate (sec–1) of dehalogenation by native enzyme from Dehalospirillum multivorans, heat-inactivated enzyme and cyanocobalamin [147]

Substrate

Dichloroacetate Dibromoacetate Bromoacetate Iodoacetate

Product

Chloroacetate Bromoacetate Acetate Acetate

Rate Native enzyme

Inactivated enzyme

Cyanocobalamin

0.67 39 14 42

36 1000 214 103

0.25 12.5 1.5 0.75

31

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 32. Rates (sec–1) of dechlorination of trichloroacetate with various catalysts and reduced methylviologen as electron donor [147]

Catalyst

Lower axial ligand

Rate

Cyanocobalamin Pseudovitamin B12 Cobinamide p-Cresolylcobamide

Dimethylbenzimidazol Adenine None p-Cresol

14 250 452 1472

methionine, and (iii) the formation of methane from acetate by Methanosarcina barkeri [51]. 2. Coenzyme M (HSCH2CH2SO3–) is a key cofactor in methanogenesis and the analogue 2-bromethansulfonate has been suggested as a specific inhibitor of methane formation [71]. It has emerged, however, that it is not specific and inhibits other microbial reactions, for example, anaerobic reductive dechlorination of PCBs [249], and alters the community structure of an anaerobic trichloroethene-degrading consortium [35]. It is worth noting that methanogens may reduce this to ethene [15]. 3. Iodoacetate is a well-established inhibitor of the activity of enzymes containing sulfhydryl groups.

3 Abiotic Reactions It must be admitted at the beginning that very few of these reactions involve comprehensive examination of brominated compounds. The justification for the inclusion of chlorinated compounds lies in the putative similarity of reactions involving their brominated analogues and the expectation that fission of the C-Br bond will be more easily accomplished than that of the C-Cl bond. The literature on chlorinated compounds is enormous so that only some illustrative examples relevant to brominated compounds are given. One recurring issue is best addressed at the beginning. Considerable activity has been given to putative chemical reactions in which, for example, porphyrins or corrins or related compounds are invoked. Enzyme mediated reactions will almost always involve these catalysts, so that the distinction between enzymatic and non-enzymatic reactions seems blurred. 3.1 Photohydrolytic Reactions

The chemical hydrolysis of halogenated alkanes is normally slow but is considerably accelerated (≈105) in the presence of light. For example, dibromoethane is hydrolyzed to 2-bromoethanol that is then converted to the epoxide followed by hydrolysis to dihydroxyethane [25]. The corresponding reaction with 1,2-dibromo3-chloropropane is more complex and produces a number of products although the transformation of 1-chloro-2,3-dihydroxypropane is very much slower (Fig.19) [31].

32

A.-S. Allard · A. H. Neilson

Fig. 19. Photohydrolysis of 1,2-dibromo-3-chloropropane (Redrawn from [31])

3.2 Reductive Reactions

These have been extensively studied partly with a view to simulating biological reactions and their possible role in degrading chlorinated ethanes and ethenes that are used as degreasing solvents. It is, however, worth noting that although this use has diminished and these are sufficiently valuable to justify recovery: 1. In view of concern with the fate of halogenated acetic acids in water distribution systems, their reactions been studied in deoxygenated water containing finely divided Fe0. Successive reductive removal of halogen occurred with formation of chloroacetate from trichloroacetate and acetate from tribromoacetate [86]. 2. Cr(II) has been used to bring about dehalogenation of alkyl halides and results in the production of alkyl radicals. The order of ease of reduction is generally iodides > bromides > chlorides and tertiary halides are most reactive and primary halides least (Table 33) [26, 27]. Rates for selected vicinal halides are given (Table 34) (references in [23]). Vicinal dichlorides such as tetrachloroethene are degraded by zero-valent metals by b-elimination to produce dichloroacetylene and finally acetylene [167]. Table 33. Products and yields (%) from the reduction of alkyl halides by chromous sulfate in

dimethylformamide [26] Substrate

Product

Yield

Dibromomethane Dibromoethene Allyl chloride Allyl bromide Allyl iodide 1,4-Dibromobutane

Methane Ethene Propene Propene Propene n-Butane

101 100 98 98 NA NA

NA = not available. Table 34. Rates (M–1 min–1) of dehalogenation of vicinal alkyl halides with chromous sulfate in aqueous dimethylformamide (references in [23])

Substrate

Rate

Substrate

Rate

2,2-Dibromopropane 2,2-Dichloropropane meso–2,3-Dibromobutane

138 0.49 18

Dibromomethane Chloroform

0.76 15

33

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 35. Rates (M–1 sec–1) of reaction of alkyl halides with Fe(II) deuteroporphyrin IX [227]

Substrate

Reaction

Rate

Bromotrichloromethane Propargyl bromide Allyl chloride Allyl bromide Allyl iodide 1,2-Dibromoethane

Hydrogenolysis Coupling Coupling Coupling Coupling Elimination

310 1.9 1.2¥10–3 0.25 160 1.2¥10–2

3. The stereospecificity of dehalogenation of vicinal dibromides to olefins was examined for reducing agents including Cr(II), iodide and Fe0 [204].Whereas, for dibromostilbene the (E)-stilbene represented >70% of the total olefin produced, for threo-dibromopentane reduction by Cr(II) produced ca. 70% of (E)pent-2-ene, and iodide and Fe0 only <5% of this. 4. The reactions of alkyl halides with Fe(II) deuteroporphyrin IX have been examined and some examples are given in Table 35 [227]. Three classes of reaction were observed: (i) hydrogenolysis, (ii) elimination to alkenes, and (iii) coupling of alkyl free radicals, and further discussion is given in [23]. 5. In the context of model systems for Factor F-430 that is involved in the terminal step in the biosynthesis of methane and that is able to dechlorinate CCl4 successively to CHCl3 and CH2Cl2 [107], nickel (I) isobacteriochlorin anion was generated electrolytically and used to examine the reactions with alkyl halides in dimethylformamide [79]. The three classes of reaction were the same as those observed with Fe(II) deuteroporphyrin IX.A selection of the reactants and products is given in Table 36. 6. Considerable attention has been directed to dehalogenation mediated by corrinoids and porphyrins in the presence of a chemical reductant (references. in [62, 67, 240]). The involvement of corrinoids and porphyrins is consistent with Table 36. Reaction of alkyl halides with Ni(I) octaethylisobacteriochlorin anion in dimethyl

formamide [79] Substrate

Product

Yield

Substrate

Product

Yield

Methyl bromide

Methane Ethane

27 73

Isopropyl chloride

70 30

n-butyl chloride

n-Butane n-Octane

74 26

Isopropyl bromide

n-butyl bromide

n-Butane n-Octane

50 50

Isopropyl iodide

n-butyl iodide

n-Butane n-Octane

42 58

1,2-Dichloroethane

Propane 2,3-Dimethylbutane Propene Propane 2,3-Dimethylbutane Propane 2,3-Dimethylbutane Ethene

5 68 32 70 30 88

34

A.-S. Allard · A. H. Neilson

the occurrence of analogous mechanisms for biological reactions. Illustrations are provided by the dechlorination and elimination reactions carried out by titanium(III) citrate and hydroxocobalamin [19, 67]. For example, in aqueous solution cobalamin with titanium(III) as reductant dehalogenated tetrachloroethene to the trichloroethene radical and thence to the dichlorovinyl radical, chloroacetylene and acetylene [67]. 7. Although not involving brominated compounds, the dechlorination of hexachloroethane to tetrachloroethene by hydrogen sulfide in the presence of 5methylbenzo[1,4]quinone electron acceptor should be noted [155], and has been confirmed in the sulfide-containing anoxic hypolimnion from a lake [132]. 8. The dehalogenation of haloacetates by heat-killed cells of Dehalospirillum multivorans and cyanocobalamin has already been noted above.

4 Aromatic Compounds: Aerobic Reactions Introduction

Generally aromatic bromine and iodine have only specialized application. These are, however, important and range from the use of highly brominated compounds as flame retardants to highly iodinated benzoates as X-ray contrast agents. The first group includes hexabromobenzene, polybrominated diphenyl ethers, polybrominated biphenyls, and tetrabromo-bisphenol-A, and polybrominated diphenyl ethers have achieved some notoriety [42] in view of their distribution in biota in Scandinavia, Japan, the United States and the Eastern North Atlantic [122], and in samples of air from the Great Lakes [195]. In addition a number of agrochemicals contain bromine including 5-bromo-3-sec-butyl-6-methyl uracil (bromacil), 3,5-dibromo-4-hydroxybenzonitrile (bromoxynil), and some phenylurea herbicides (e.g., metobromuron). 4.1 Hydrocarbons

The mechanisms used for the metabolism of halogenated aromatic compounds differ considerably from those for aliphatic substrates, and will be briefly summarized. 4.1.1 Degradation and Growth

Bacterial degradation of hydrocarbons under aerobic conditions involves two consecutive initial reactions before fission of the aromatic ring: dioxygenation with the introduction of both atoms of O2 to produce a cis-dihydrodiol, and dehydrogenation to a catechol. This is followed by ring fission by either of two pathways. The mechanisms have been extensively explored with both halogenated

Degradation and Transformation of Organic Bromine and Iodine Compounds

35

and non-halogenated substrates and it should be noted that there has been increased interest in carrying out the initial stages without the extension to ring fission. Although the putative formation of catechol during degradation had been proposed earlier, it was the paper of Gibson et al. [65] that established dioxygenation of halogenated hydrocarbons. Cells of Pseudomonas putida grown with toluene as sole source of carbon were able to oxidize fluoro-, chloro-, bromo-, and iodobenzene to the corresponding catechols. The key observation was made that cells were able to transform 4-chlorotoluene to two products–4-chloro-2,3-dihydroxytoluene and cis (+)-4-chloro-2,3-dihydroxy-1-methylcyclohexa-4,6-diene. This became the basic mechanism for dioxygenation of aromatic compounds, and has been established for virtually all PAHs and, with extension, to benzoates. Ring fission of substituted catechols is accomplished by dioxygenation with fission of the ring either between the substituents (intradiol or 1,2-fission), or between one hydroxyl group and the adjacent carbon atom (extradiol or 2,3-fission). Although extradiol fission is generally preferred, both may occur and whether extradiol or intradiol fission is followed depends on the substrate. For example, 3-chlorocatechol generally inhibits its further metabolism by catechol 2,3-dioxygenase and the inhibitory effect of 3-chlorocatechol on catechol 2,3dioxygenase may be lifted by the synthesis of catechol 1,2-dioxygenase. One of the key steps in degradation occurs after ring fission and involves maleylreductase. For substrates with a halogen atom at the 2-position, reductive loss of halide takes place using 2 mol of NADH per mol of substrate: otherwise only 1 mol of NADH is required (Fig. 20) [100]. Illustrative results for Pseudomonas sp. strain B13 are given in Table 37). 4.1.2 Metabolism Without Growth

Investigations have been directed to strains in which growth was not demonstrated but in which the substrate was metabolized. Only some examples of growth with unsubstituted brominated arenes exist: 1. The metabolism of 1-chloro- and 1-bromonaphthalene was examined in two strains of bacteria one of which could use the first compound as sole source of carbon: this produced 8-chloro-1,2-dihydrodihydroxynaphthalene from which 3-chlorosalicylate was formed. 3-Bromosalicylate was formed from

Fig. 20. Degradation of halogenated dienelactones, maleylacetate and reduction to b-ketoadipate (Redrawn from [100])

36

A.-S. Allard · A. H. Neilson

Table 37. Reaction of halogenated maleylacetates with maleylacetate reductase from

Pseudomonas sp. strain B13: NADH consumption and halide formation (mol/mol substrate) [100] Halogenated maleylacetate

NADH consumption

Halide formation

Product

2-Fluoro2-Chloro2-Bromo3-Chloro3-Bromo5-Bromo2,5-Dibromo-

2.00 1.93 1.92 0.99 0.91 1.05 1.96

0.94 0.98 0.99 < 0.05 0.08 0.12 1.22

Maleylacetate Maleylacetate Maleylacetate

5-Bromomaleylacetate

1-bromonaphthalene and a pathway for metabolism was proposed, erring only in assignment of a trans configuration to the dihydrodiols [228]. 2. A strain of Sphingomonas sp. SS33 that was adapted to growth with 4,4¢-difluorodiphenyl ether was able to oxidize–but not grow with–4,4¢-dibromodiphenyl ether (Table 38). Analogous to the fluorinated and chlorinated analogues, 4-bromophenol and 4-bromocatechol were produced (Fig. 21) by dioxygenation of one ring [177]. In view of the possible formation of brominated dibenzofurans and benzo[1,4]dioxins, it is worth drawing attention to a study on the degradation of the chlorinated compounds using a dibenzofuran-utilizing Terrabacter sp. strain DBF63 and a carbazole-utilizing Pseudomonas sp. strain CA10. Although angular dioxygenation was readily accomplished, further degradation occurred only with 2-chlorodibenzofuran that formed 5-chlorosalicylate [74]. 3. A strain of Sphingomonas paucimobilis BPSI-3 that was able to grow with biphenyl or benzoate transformed 4-chloro- and 4-bromobiphenyl to products that were assigned as 4- or 5-chloropyridine-2-carboxylate and 4- or 5-broTable 38. Rates of oxygen uptake by cells of Sphingomonas sp. strain SS33 grown with 4,4¢-di-

fluorodiphenyl ether (=100) exposed to various substrates [177] Substrate

Rate

Substrate

Rate

Diphenyl ether 4,4¢-Difluorodiphenyl ether 4,4¢-Dichlorodiphenyl ether 4,4¢-Dibromodiphenyl ether

82 100 35 15

4-Fluorodiphenyl ether 4-Chlorodiphenyl ether 4-Bromodiphenyl ether

123 58 41

Fig. 21. Transformation of 4,4¢-dibromodiphenyl ether by Sphingomonas sp. strain SS33 (Re-

drawn from 177)

Degradation and Transformation of Organic Bromine and Iodine Compounds

37

Fig. 22. Degradation of 4-bromobiphenyl by Sphingomonas paucimobilis strain BPSI-3 (Redrawn from [41])

mopyridine-2-carboxylate (Fig. 22) [41] in which the nitrogen atom of pyridine originated from NH4+ in the medium. 4. Although this is not the place to discuss the extensive investigations into the aerobic degradation of chlorinated biphenyls [2, 202], a short summary seems justified to place investigation of their brominated analogues in a wider perspective. The degradation of PCBs proceeds by dioxygenation to cis-2,3-dihydro-2,3-diols, dehydrogenation to the catechol, extradiol ring cleavage between C1 and C2 positions, and hydrolysis of the vinylogous b-keto acid to benzoate. These reactions are mediated by a suite of enzymes comprising biphenyl-2,3dioxygenase, biphenyl-2,3-dihydrodiol dehydrogenase, 2,3-dihydroxybiphenyl dioxygenase, and the hydrolytic enzymes that produce benzoate encoded by the genes bphA, bphB, bphC, and bphD (references in [2]). PCB congeners in which the less highly substituted ring is almost invariably degraded to a carboxylic acid apparently by extradiol cleavage of the intermediate catechol: for example, in the 2,4,5-, 2,2¢,5¢-, and 2,3,4,5-congeners. As for 1,2,4,5-tetrachlorobenzene [173], elimination of halogen may also take place concomitantly with dioxygenation [3]. Burkholderia sp. strain LB400 was examined for its metabolism of biphenyls 2,2¢-disubstituted with fluorine, bromine, nitro groups [183]. Dioxygenation took place at the 2,3-position with concomitant elimination of the substituents and formation of the catechol by further dehydrogenation. For the sake of completeness, attention is drawn to some complementary issues that are important determinants of the biodegradability of PCBs and are equally relevant to their brominated analogues: a) Metabolites and inhibition – Total degradation of PCBs necessitates degradation of the chlorobenzoates produced by the foregoing reactions. Metabolism of chlorobenzoates produces chlorocatechols and thence muconic acids, and it has been shown with P. testosteroni strain B-356 that the metabolites from 3-chlorobenzoate strongly inhibited the activity of 2,3-dihydroxybiphenyl-1,2-dioxygenase and therefore the degradation of the original substrates [191].An important metabolic situation involves the formation of protoanemonin (4-methylenebut-2-ene-4-olide) as an intermediate in the degradation of 4-chlorobenzoate formed by partial degradation of 4-chlorobiphenyl. This is formed by intradiol fission of 4-chlorocatechol to 3-chloro-cis,cis muconate followed by loss of CO2 and chloride (Fig. 23) [17]. The synthesis of this metabolite adversely affects the survival in soil microcosms of organisms that metabolize 4-chlorobiphenyl, although its formation can be obviated by organisms using a modified pathway that produces the cis-dienelactone [16].

38

A.-S. Allard · A. H. Neilson

Fig. 23. Formation of protoanemonin from 4-chlorobenzoate

b) Alternative metabolites – Although the ultimate products from degradation of PCBs are generally chlorinated muconic acids, the unusual metabolite 2,4,5-trichloroacetophenone has been isolated [14] from the degradation of 2,4,5,2¢,4¢,5¢-hexachlorobiphenyl (Fig. 24) by Alcaligenes eutrophus H850 which has an unusually wide spectrum of degradative activity for PCB congeners. c) An example of the NIH shift occurs during the transformation of 2-chlorobiphenyl to 2-hydroxy-3-chlorobiphenyl by a methanotroph, and is consistent with the formation of an intermediate arene oxide [1]. d) The significance of substrates capable of inducing the metabolism of PCBs was shown in a study with Arthrobacter sp. strain B1B [66]. Effective degradation of a number of congeners in Arochlor 1242 was induced by carvone that could not, however, be used as a growth substrate and was toxic at high concentrations (>500 mg/l). Other structurally related compounds including limonene, p-cymene, and isoprene were also effective.

Fig. 24. Aerobic biodegradation of 2,2¢,4,4¢,5,5¢-hexachlorobiphenyl

39

Degradation and Transformation of Organic Bromine and Iodine Compounds

4.1.3 Biotransformation to Dihydrodiols

Very substantial effort has been directed to studying the transformation of aromatic hydrocarbons to the dihydrodiols on account of possible biotechnological interest: 1. The transformation of 3-chloro-, 3-bromo-, and 3-iodotrifluorobenzene to 3chloro-, 3-bromo-, and 3-iodotrifluoromethylcatechols by cells of Pseudomonas sp. strain T-12 that were grown with toluene [165]. In this example, dioxygenation resulted in preferential concomitant loss of fluoride. 2. A wide range of halogenated benzoate cis-dihydrodiols including the 3- and 5-brominated compounds were prepared by oxidation with Alcaligenes eutrophus strain B9 that is defective in dihydrobenzoate dehydrogenase [163]. Chlorinated benzene and toluenes were transformed by TecA to dihydrodiols, and by TecB to the dehydrogenated products by the gene products of Ralstonia sp. strain PS12 [157]. 3. An important issue is the configuration and enantiomeric excess of the cis-dihydrodiols.A method developed for monosubstituted arenes has been applied to 1,4-disubstituted arenes using the dioxygenase in Pseudomonas putida strain UV4 [21]. The results for some of the brominated and iodinated substrates are given in Table 39. Like toluene and benzene dioxygenase, chlorobenzene dioxygenase in Pseudomonas sp. strain P51 is a three-component enzyme and was used to examine the formation of cis-dihydrodiols from a range of substrates including substituted biphenyls, and heterocyclic compounds [159]. An important issue is the enantiomeric excess of the cis-dihydrodiols. This is given in Table 40 for three strains of Pseudomonas, two using Table 39. Configuration and enantiomeric excess (%) of 2,3-dihydrodiols of 1,4-disubstituted

benzenes formed by Pseudomonas putida stain UV4 [21] Substituent 1

Substituent 4

% enantiomeric excess

Configuration

CF3 I I I I

I F Cl Br Me

> 98 88 15 22 80

2R: 3S 2S: 3R 2S: 3R 2S: 3R 2S: 3S

Table 40. Enantiomeric excess (%) of cis-benzene dihydrodiols formed from (A) Pseudomonas

putida strain UV4 toluene dioxygenase, (B) Pseudomona sputida strain F39/D toluene dioxygenase, and (C) Pseudomonas sp. strain P51 chlorobenzene dioxygenase [159] Substrate

Strain A

Strain B

Strain C

4-Fluorotoluene 4-Chlorotoluene 4-Bromotoluene 4-Iodotoluene

83 15 37 80

90 10 26 80

49 77 77 98

40

A.-S. Allard · A. H. Neilson

toluene dioxygenase and the third chlorotoluene dioxygenase: the data for Pseudomonas putida strain UV4 are from [21]. 4.2 Benzoates

The metabolism of benzoate was studied in a mutant of Alcaligenes eutrophus that was blocked in benzoate degradation. The first reaction was dioxygenation with formation of a 3,5-cyclohexadiene-1,2-dihydrodiol-1-carboxylate–tentatively the cis- isomer [164]. Benzoate dioxygenase is a two-component enzyme of which the reductant contains FAD and [2Fe-2S]. Degradation of benzoates generally involves oxidative decarboxylation to a catechol by a dioxygenase that is distinct from that used for hydrocarbons. The enzymes for 2-halogenated benzoates in which halide is lost simultaneously is discussed later. Hydrolytic loss of halide to produce a phenol that is further degraded by hydroxylation occurs with some carboxylic acids.A complication emerges from the metabolic inhibition imposed by intermediates such as 3- and 4-chlorocatechol. In addition there are hydrolytic and reductive dehalogenations. As introduction examples of growth of brominated benzoates are summarized: 1. The rates of oxidation of halogenated benzoates by cells of Pseudomonas fluorescens grown with benzoate in given in Table 41 [87]. 2. The rates of substrate consumption were measured in three benzoate-degrading bacteria. The results for Pseudomonas putida mt-2 grown with 3methylbenzoate are given in Table 42 [161]. Table 41. Rates of oxidation (µ mol O2 · h–1 · mg dry weight–1) of halogenated benzoates by

whole cells of Pseudomonas fluorescens strain KBI after growth with benzoate [87] Substrate

Rate

Substrate

Rate

Substrate

Rate

Benzoate (10 µmol) 2-Fluorobenzoate 3-Fluorobenzoate 4-Fluorobenzoate

10 6 6 6

Benzoate(10 µmol) 2-Fluorobenzoate 2-Chlorobenzoate 2-Bromobenzoate 2-Iodobenzoate

11 5 0.5 0.2 0.2

Benzoate (2 µmol) 3-Fluorobenzoate 3-Chlorobenzoate 3-Bromobenzoate 3-Iodobenzoate

6 3 2 1 0.2

Table 42. Relative rates of oxygenation of halogenated benzoates (Benzoate=100) by

Pseudomonas putida mt-2 grown with 3-methylbenzoate and Pseudomonas sp. strain B13 grown with 3-chlorobenzoate [161] Substrate

3-Fluorobenzoate 3-Chlorobenzoate 3-Bromobenzoate 3-Iodobenzoate ND = not determined.

Pseudomonas sp. strain B13

Pseudomonas putida strain mt-2

Rate

Rate

67 31 9 1

ND 48 34 39

41

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 43. Relative rates (2-bromobenzoate = 100) of oxygen uptake for halogenated benzoates

by cells of Pseudomonas aeruginosa strain 2-BBZA grown with 2-bromobenzoate [Higson and Focht 1990] [81] Substrate

Relative rate

2-Fluorobenzoate 2-Chlorobenzoate 2-Bromobenzoate 2-Iodobenzoate 3-Fluorobenzoate 3-Chlorobenzoate 3-Bromobenzoate 3-Iodobenzoate 4-Fluorobenzoate 4-Chlorobenzoate 4-Bromobenzoate 4-Iodobenzoate

115 98 100 81 49 41 28 14 30 8 23 14

Substrate

Relative rate

2,5-Dichlorobenzoate 2,5-Dibromobenzoate

61 33

3,5-Dichlorobenzoate 3,5-Dibromobenzoate

17 9

Table 44. Oxygen uptake (nmol O2/mg protein/min; relative to benzoate=100) by cells of Pseudomonas aeruginosa strain JB2 grown with benzoate [80]

Substrate

Rate

Substrate

Rate

2-Fluorobenzoate 2-Chlorobenzoate 2-Bromobenzoate 2-Iodobenzoate

40 30 22 28

3-Chlorobenzoate 3-Bromobenzoate

16 20

3. The results of a range of experiments that included bromobenzoates are summarized. A strain of Pseudomonas aeruginosa was isolated by enrichment with 2-bromobenzoate and was able to degrade a number of other halogenated benzoates (Table 43) [81]. It was suggested that degradation occurred by initial formation of salicylate. Another strain isolated by enrichment with 2-chlorobenzoate was able to degrade di- and trichlorinated benzoates and cells consumed oxygen when incubated with a range of halogenated benzoates (Table 44) [80]. 4.2.1 Dioxygenation

Dioxygenation is involved in two quite different reactions that result in concomitant loss of halogen: (a) with decarboxylation and (b) with elimination of hydrogen halides. In both, catechol is formed directly so that, in contrast to hydrocarbons, no dehydrogenation is necessary. 4.2.1.1 Decarboxylation of 2-Halogenated Benzoates

In these studies, although oxygen consumption was measured, growth did not generally take place. It may therefore be assumed that a critical step after the ini-

42

A.-S. Allard · A. H. Neilson

tial oxidation was limiting. Extensive studies have delineated the mechanism of degradation of halogenated benzoates. Dioxygenation is clearly the first step in degradation with the formation of catechol and concomitant decarboxylation. The relevant enzymes have been purified from different pseudomonads: 1. A two-component 2-halobenzoate 1,2-dioxygenase has been purified from Pseudomonas cepacia strain 2CBS that is able to metabolize 2-fluorobenzoate, 2-chlorobenzoate, 2-bromobenzoate, and 2-iodobenzoate to catechol by concomitant decarboxylation and loss of halide (Table 45) [56]. The inducible 2halobenzoate 1,2-dioxygenase consisted of two components, an oxygenase A that is an iron-sulfur protein and consists of non-identical subunits, a (Mr 52,000) and b (Mr 20,000) in an a3b3 structure. The reductase B is an iron-sulfur flavoprotein (Mr 37,500) containing FAD. It is broadly similar to benzoate dioxygenase though different from the FMN-containing dioxygenases. It has been shown that the gene cluster encoding the enzyme cdbABC is localized on a plasmid [73]. Significant homology existed with the amino acid sequence of benABC that encode benzoate dioxygenase in Acinetobacter calcoaceticus andxylXYZ that encode toluate dioxygenase in Pseudomonas putida mt-2 2. The 2-halobenzoate dioxygenase of Burkholderia sp. strain TH2 was active to a number of 2-halobenzoates (Table 45) and the predicted amino acid sequences of all the gene products cbdABC were very similar to those of Pseudomonas sp. strain 2CBS. It was shown that the effectors of the transcriptional regulatory gene Cbds were 2-chloro-, 2-bromo-, 2-iodo-, and 2-methylbenzoate [201]. 3. Pseudomonas aeruginosa strain 142 was isolated from a PCB-degrading consortium and is able to degrade a range of 2-halogenated benzoates (Fig. 25). The enzyme consists of three components, one of which is a ferredoxin containing a single Rieske-type [2Fe-2S] cluster [169], and whose amino acid sequence is similar to those of other three-component dioxygenases containing ferredoxin such as benzene dioxygenase, toluene-2,3-dioxygenase, biphenyl dioxygenase, and naphthalene dioxygenases. The specificity to selected halogenated benzoates is given in Table 45. Dioxygenation may not, however, necessarily result in dehalogenation. For example, a strain of Arthrobacter keyseri 12B (formerly Micrococcus sp. strain 12B) Table 45. Substrate specificities (relative to 2-chlorobenzoate=100) of halobenzoate dehaloge-

nases from Pseudomonas cepacia 2CBS [56], Pseudomonas aeruginosa 142 [169] and Burkholderia sp. strain TH2[201] Substrate

2-Fluorobenzoate 2-Chlorobenzoate 2-Bromobenzoate 2-Iodobenzoate Benzoate ND = not determined.

Pseudomonas cepacia 2CBS

Pseudomonas aeruginosa 142

Burkholderia sp. TH2

Rate

Rate

Rate

105 100 78 38 45

ND 100 107 130 37

ND 100 66 7 43

43

Degradation and Transformation of Organic Bromine and Iodine Compounds

Fig. 25. Degradation of 2-bromobenzoate by Pseudomonas aeruginosa strain 142 (Redrawn

from [169])

that degrades phthalate by enzymes that are clustered in the pht operon phtBAaAbAcAdCR transforms 2-chloro-, 2-bromo-, and 2-iodobenzoates to the corresponding 3,4-dihydroxybenzoates by dioxygenation and dehydrogenation without dehalogenation [46]. 4.2.1.2 Loss of Halogen in 4-Halogenated Phenylacetates

Pseudomonas sp. strain CBS is able to grow at the expense of 4-chlorophenylacetate, and loss of chloride is mediated by a dioxygenase that produces 3,4-dihydroxyphenylacetate which is degraded by an extradiol catechol dioxygenase. The dioxygenase consists of two components A and B. Component A has been purified and has a molecular weight of 140,000 consisting of three equal subunits, and contains iron and acid-labile sulfur. The substrate specificity is given in Table 46 [124]. Component B which is a reductase that is monomeric with a molecular weight of 35,000 and contains per mol of enzyme 1 mol of FMN, 2.1 mol of Fe, and 1.7 mol of labile sulfur. After reduction with NADH the ESR spectrum showed signals that were attributed to a [2Fe-2S] structure and a flavosemiquinone radical [181]. The molecular and kinetic properties of the enzyme are broadly similar to the Class IB reductases of benzoate 1,2-dioxygenase and 4methoxybenzoate monooxygenase-O-demethylase. Table 46. Substrate specificity of 4-chlorophenylacetate 3,4-dioxygenase in component A of Pseudomonas sp. strain CBS [124]

Substrate

Relative activity (%)

Substrate

Relative activity (%)

4-Fluorophenylacetate 4-Chlorophenylacetate

30 100

4-Bromophenylacetate 3-Chlorophenylacetate

102 10

44

A.-S. Allard · A. H. Neilson

4.2.1.3 Halohydrolases

The metabolism of 4-halobenzoates is different from that of 2-halobenzoates since simultaneous loss of halide and the carboxyl group cannot occur. Instead hydrolytic loss of halogen is involved as an early step in the degradation of 4-halogenated benzoate by a number of strains. It has been shown that, in general, the carboxyl group of strains with this dehalogenating activity is activated initially by a coenzyme A ligase to form benzoyl-coenzyme A (references in [39]). The substrate specificity of the 4-halobenzoate-CoA ligase in Pseudomonas sp. strain CBS3 [119] is shown in Table 47; the kinetic parameters for dehalogenation of 4-halobenzoyl CoA in Acinetobacter sp. 4-CB1 are also given in Table 47 [39]: 1. Alcaligenes denitrificans strain NTB-1 is able to use 4-chloro-, 4-bromo-, and 4-iodobenzoates as sole sources of carbon and energy. Rates of dehalogenation for cells grown on various substrates are given in Table 48. The pathway involves hydrolytic dehalogenation to 4-hydroxybenzoate followed by hydroxylation to 3,4-dihydroxybenzoate [211]. 2. A comparable pathway was used by Arthrobacter sp. strain TM-1 that was able to grow with 4-chlorobenzoate and 4-bromobenzoate [123]. 3. Hydrodehalogenation has been shown in cell extracts of Pseudomonas sp. strain CBS3 [118, 178] and the dehalogenase has been shown to consist of three components, an unstable one with molecular weight of 3000 and two stable components with molecular weights of ≈86,000 and 92,000 [52]. 4. A strain of Acinetobacter sp. 4-CB1 is able to dehalogenate 4-chlorobenzoate with formation of 4-hydroxybenzoate after initial formation of the 4chlorobenzoyl CoA ester [37]. Table 47. Substrate specificity (% relative velocity: 4-chlorobenzoate=100) of 4-halobenzoate-

CoA ligase in Pseudomonas sp. strain CBS3 [119], and values of kcat (s–1 for dehalogenation of 4-halobenzoyl CoA esters in Acinetobacter sp. strain 4-CB1 [39] Substrate

% velocity

Kcat (s–1)

4-Fluorobenzoate 4-Chlorobenzoate 4-Bromobenzoate 4-Iodobenzoate

36 100 105 90

0.003 1.30 2.31 ND

ND = not determined. Table 48. Rates of dehalogenation (nmol substrate consumed/mg protein/min) for cells of

Alcaligenes denitrificans strain NTB-1 grown with various substrates [211] Assay substrate

4-Chlorobenzoate 4-Bromobenzoate 4-Iodobenzoate

Carbon source for growth 4-Chlorobenzoate

4-Bromobenzoate

4-Iodobenzoate

18 15 18

11 11 9

12 12 10

Degradation and Transformation of Organic Bromine and Iodine Compounds

45

Two points are worth noting: (1) hydrolytic dehalogenation is one of the steps in the aerobic degradation of pentachlorophenol and g-hexachlorocyclohexane and (2) the formation of CoA thioesters is well established in the degradation of aromatic carboxylic acids, all of which are discussed in this chapter. 4.2.1.4 Reductive Dehalogenation

This is an important though less common reaction carried out by aerobic bacteria though often only as one stage in complete loss of halogen. Anaerobic dehalogenation with be discussed later: 1. Alcaligenes denitrificans strain NTB-1 (now designated as a coryneform) that was isolated on 4-chlorobenzoate is able to grow with 4-bromo- and 4iodobenzoate, and in addition on 2,4-dichlorobenzoate. The first step in the pathway for 2,4-dichlorobenzoate is reductive loss of the ortho chlorine substituent whereas the second step is, however, carried out by a halohydrolase that forms 3,4-dihydroxybenzoate followed by ring fission [211]. 2. It has been shown that the reductive dechlorination of this strain and of Corynebacterium sepedonicum involves initial synthesis of the coenzyme A thioester and NADPH as the reductant. In addition, hydrolytic 4-chlorobenzoyl-CoA dehalogenase, 4-hydroxybenzoate 3-monooxygenase, and 3,4-dihydroxybenzoate 3,4-dioxygenase activities were found and enable construction of the degradative pathway [170]. 4.2.1.5 Denitrification

Although a number of strains were isolated by enrichment of halogenated benzoates including bromobenzoates, no growth on either 2-bromo- or 4-bromobenzoate was observed under either aerobic or denitrifying conditions. Some were able to grow with 3-bromobenzoate under denitrifying conditions but not aerobically. On the basis of 16S rRNA sequences. these organisms belonged to Thauera aromatica, Pseudomonas stutzeri, or Ochrobactrum anthropi [192]. The extent to which these exhibit anaerobic metabolism is moot. Further discussion on anaerobic reactions is given later. 4.2.1.6 Fungi

Substituted 2,4,6-triiodinated benzoates are incorporated into X-ray contrast agents, and their transformation has been examined in the white-rot fungus Trametes versicolor [168]. Since these compounds are putatively unable to pass the cell walls of the fungus, it is important that although lignin peroxidase activity was not observed, non-specific extracellular manganese-dependent peroxidase and laccase activities were found. There was no introduction of oxygen into the ring and the main reactions were successive deiodination to the monoio-

46

A.-S. Allard · A. H. Neilson

Fig. 26. Transformation of 3,5-diacetamido-2,4,6-triiodobenzoate by Trametes versicolor (Redrawn from [168])

dinated compound (Fig. 26). Attention is drawn to dechlorination of a range of chlorophenols by the laccase from Trametes versicolor [171]. Finally it is worth noting that exposure to halogenated substrates leads to selective enrichment of mutants deficient in catabolic pathways [236]. For example, mutants in the plasmid-encoded pathway for the degradation of pcymene (4-methylisopropylbenzene) in Pseudomonas putida strain JT811 were obtained by exposure to 4-iodobenzoate at a frequency of 68 in 76 total survivors. 4.3 Phenols

A number of quite different mechanisms may be involved depending on the number of substituents and the organism. Loss of halogen may take place before, or after ring fission. Hydroxylation (monooxygenation) to a halogenated catechol and hydrolytic dechlorination to a phenol may be followed by a suite of reactions involving reductive dechlorination. This is an important reaction that will be discussed later for anaerobic bacteria and is also mediated by aerobic bacteria, though often only as one stage in complete loss of halogen: 1. A strain of Azotobacter sp. GP1 is able to degrade 2,4,6-trichlorophenol. The first step in the degradation is dechlorination to 2,6-dichlorohydroquinone that is catalyzed by a monooxygenase requiring NADH, O2, and FAD [235]. 2,4,6-Tribromophenol was able to support growth of this strains and possessed TCP-monooxygenase activity (Table 49) while the dissimilation of 2,4-dichlorophenoxyacetate by Azotobacter chroococcum produces 4-chlorophenoxyacetate, 4-chlorophenol and 4-chlorocatechol [7]. 2. Substantial effort has been directed to the degradation of pentachlorophenol and the details have emerged from studies with three organisms: Flavobac-

47

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 49. Substrate specificity of 2,4,6-trichlorophenol-4-monooxygenase from Azotobacter sp.

strain GP1 for halogenated phenols (2,4,6-trichlorophenol=100) [235] Substrate

Consumption of substrate

Consumption of NADH

Substrate

Consumption of substrate

Consumption of NADH

2,4,6-Trichloro3,4-Dichloro2,3,4,6-TetrachloroPentachloro-

100 50 67

100 41 68

2,4,6-Tribromo2,4,6-Triiodo-

57 50

a

0

8

a

57

Inhibition of NADH consumption in the absence of substrate.

terium sp. ATCC 39723, Rhodococcus chlorophenolicus strain PCP-1, and Mycobacterium fortuitum strain CG2. The changes in classification should be noted to avoid confusion: Rhodococcus chlorophenolicus Æ Mycobacterium chlorophenolicum and Flavobacterium sp. Æ Sphingomonas chlorophenolicus In all three of the strains, the substrate is degraded by a monooxygenase to 2,3,5,6-tetrachlorohydroquinone followed by successive glutathione-dependent reductive dechlorinations to 2,6-dichlorohydroquinone and hydrolysis to 1,2,4-trihydroxybenzene that undergoes ring fission. Cell extracts have a wide specificity for all polyhalogenated phenols and hydroquinones (Table 50) [207]. Flavobacterium sp.ATCC 39723 is able to degrade 2,4,6-trichloro-, 2,4,6tribromo-, and 2,4,6-triiodophenol (Fig. 27) [245] and the pathway is revealed by the wide substrate activity of pentachlorophenol hydroxylase from this strain that produces the 2,6-dihalogenated hydroquinone from pentachlorophenol, 2,4,6-trichlorophenol, 2,4,6-tribromophenol, and 2,4,6-triiodophenol (Table 51) [247], and from 3,5-dibromo-4-hydroxybenzonitrile (Bromoxynil) [203]. 3. Reductive dehalogenation–Apart from the preceding example of pentachlorophenol, partial reductive dehalogenation has been demonstrated during the transformation and degradation of di- and trichlorophenoxyacetates. The transformation of 2,4-dichlorophenoxyacetate by Azotobacter Table 50. Specific activity (nmol substrate/mg protein/h) for halogenated phenols and hydro-

quinones by cell extracts of pentachlorophenol-induced cells of Mycobacterium fortuitum CG-2 [207] Substrate

Activity (cell extract)

Activity (supernatant)

Activity (pellet)

Pentaflurophenol Pentachlorophenol Pentabromophenol Tetrafluorohydroquinone Tetrachlorohydroquinone Tetrabromohydroquinone

2 1.8 1.7 14.3 10.1 7.1

< 0.05 < 0.05 < 0.05 12.5 8.8 6.0

1.1 1.2 0.9 1.4 1.0 0.5

48

A.-S. Allard · A. H. Neilson

Fig. 27. Transformation of 2,4,6-triiodophenol by Flavobacterium sp. ATCC 39723 (Redrawn from [245]) Table 51. Substrate loss by pentachlorophenol hydroxylase in pentachlorophenol-induced cells of Flavobacterium sp. (pentachlorophenol=100) [246]

Substrate

Product

Rate

Pentachlorophenol 2,4,6-Trichlorophenol 2,4,6-Tribromophenol 2,4,6-Triiodophenol

2,3,5,6-Tetrachlorohydroquinone 2,6-Dichlorohydroquinone 2,6-Dibromohydroquinone 2,6-Diiodohydroquinone

100 29 35 172

chroococcum involves an initial reductive dechlorination to 4-chlorophenoxyacetate [7]. It is worth noting, however, that Pseudomonas cepacia strain AC1100 that is able to dehalogenate effectively a wide range of chlorophenols, as well as 2,4-dibromo-, 2,4,6-tribromo-, and pentabromophenol, was unable to dehalogenate 2,4,6-triiodophenol [99]. 4.3.1 O-Methylation of Halogenated Phenols

O-Methylation of halogenated phenols has been shown in both Gram-positive and Gram-negative bacteria assigned tentatively to the genera Rhodococcus, and Acinetobacter and Pseudomonas respectively, and included a number of brominated phenols (Table 52) [6]. Cell extracts mediated O-methylation when supplemented with S-adenosyl methionine that was therefore presumed to be the methyl donor, and for a Gram-positive Rhodococcus sp. strain 1395 were active towards a wide range of chlorinated and brominated phenols (Table 53) [146]. 2,4,6-Tribromoanisole has been identified in samples of fish and mussels (Mytilis edulis) in Japan although its concentration was only ca. 1% of that of the total PCBs [230] and tetrabromobisphenol-A dimethyl ether in samples of mussels from Japan [231]. Halogenated anisoles have in fact a ubiquitous distribution in the environment (references in [61]) and measurements of their concentration in the troposphere over the Atlantic Ocean are consistent with their relative volatility and suggest that the brominated anisoles are largely of biogenic origin in contrast to their chlorinated analogues that are anthropogenic [61]. This is supported by the finding that bromophenols which are widespread in the marine environment may be produced from 4-hydroxybenzoate by bromoperoxidasecatalyzed bromination [57].

49

Degradation and Transformation of Organic Bromine and Iodine Compounds

Table 52. Rates (10–10 µg/h/[cells per ml]) of O-methylation of halogenated phenols by strains

1395 and 1678 [6] Substrate

Rate (1395)

Rate (1678)

Substrate

Rate (1678)

Rate (1678)

2,4-Dibromophenol 2,6-Dibromophenol 2,4,6-Tribromophenol Tetrabromobisphenol-A Tetrabromocresol Tetrabromoguaiacol

22 280 35 4.7 490 110

14 9.6 4 0 12 0

2,4-Dichlorophenol 2,6-Dichlorophenol 2,4,6-Trichlorophenol

16 30 550

1.6 2.4 2.4

Table 53. Specific activities (pmol/min/mg protein) of cell extracts of strain 1395 supplemented

with S-adenosylmethionine (40 µmol/l) towards halogenated phenols [146] Substrate

Specific activity

Substrate

Specific activity

2,6-Dibromophenol 2,4,6-Tribromophenol Tetrabromoguaiacol 4,5,6-Tribromoguaiacol

10 7.6 1.9 7.5

2,6-Dichlorophenol 2,4,6-Trichlorophenol Tetrachloroguaiacol 4,5,6-Trichloroguaiacol

4.4 17 1.5 6.8

4.3.2 Fungal Metabolism

By contrast with these pathways, the degradation of chlorinated phenols by the white-rot fungus Phanerochaete chrysosporium is quite different. Under conditions of nitrogen limitation both lignin and manganese-dependent peroxidases are induced and the pathway for 2,4-dichlorophenol is given (Fig. 28a) [208]. By contrast, the degradation of 2,4,6-trichlorophenol that is initiated by lignin peroxidase and manganese peroxidase involves two reductive step in the formation of 1,2,4-trihydroxybenzene (Fig. 28b) [160]. 4.4 Amines

Although the mechanism for the degradation of anilines is less well established it involves oxidative deamination to a catechol with concomitant deamination. In Pseudomonas putida UCC22(pTDN1) that is able to degrade aniline, conversion to catechol is accomplished by an oxidase encoded by the small subunits tdnA1 and tdn2 and a reductase encoded by a large subunit tdnB, together with the genes tndQ and tdnT that may be involved in amino group transfer [60].A strain of Moraxella sp. strain G is able to utilize as sole sources of carbon and nitrogen 4-fluoro-, 4-chloro-, and 4-bromoaniline but is unable to use 4-iodoaniline [251]. The pathway for 4-chloroaniline involves oxidative deamination to 4-chlorocatechol and loss of halogen by the modified ortho-fission pathway to TCA cycle in-

50

A.-S. Allard · A. H. Neilson

a

b Fig. 28a,b. Dechlorination in Phanerochaete chrysosporium: a 2,4-dichlorophenol (Redrawn

from [210]); b 2,4,6-trichlorophenol by oxidative reduction (Redrawn from [160])

termediates may then occur by the pathways used for halogenated catechols. From the broad specificity of aniline oxygenase (Table 54), it is plausible to assume that the pathway for all 4-halogenated anilines is the same. Despite many further investigation on the degradation of chloroanilines and the isolation of further strains able to carry this out (references in [18]) there seems not to have been any major development in exploring the mechanism except (i) confirmation that the degradation of aniline proceeds by an initial dioxygenation [59] and (ii) that the initial dioxygenase for aniline and 3-chloroaniline are different [18]. Table 54. Aniline oxygenase activity (µmol O2/g protein/min) induced by 4-chloroaniline in Moraxella sp. strain G and recovery of substrate (%) after incubation for 1 h [251]

Substrate

Oxygenase activity

Recovery (%)

Aniline 4-Fluoroaniline 4-Chloroaniline 4-Bromoaniline 4-Iodoaniline

43 44 29 29 9

50 54 54 67 96

Degradation and Transformation of Organic Bromine and Iodine Compounds

51

As may be expected in view of concern about its presence in the environment and possible recalcitrance, a considerable amount of work has been directed to its aerobic degradation. In the course of this, the mechanism has been elucidated in detail and a number of organisms implicated. In addition, the application of white-rot and brown-rot fungi to dechlorination of dichlorophenol is noted since the pathway is entirely different from that used by bacteria.

5 Alternative Mechanisms of Dehalogenation 5.1 Peroxidase Dehalogenation

Peroxidase brings about the oxidation of 4-halogen substituted anilines [88]: 4bromo- and 4-iodoanilines produced the bis(4-bromo- and 4-iodophenyl)-2amino-5-(4-bromo- and iodoanilino)-benzo[1,4]quinonimine, while 4-iodoaniline produced in addition 4,4¢-diiodoazobenzene (Fig. 29). 5.2 Dehalogenation by a Polychaete

It has been shown that the marine terebellid polychaete Amphitrite ornata produces no detectable halogenated metabolites, and that it synthesizes a dehalogenase that is able to remove oxidatively halogens from halogenated phenols, with fluoro, chloro, or bromo substituents [33]. One of the enzymes (DHB 1) has been purified, and consists of two identical subunits (Mr 15,530) each containing heme and histidine as the proximal Fe ligand. In the presence of H2O2, 2,4,6-tribromophenol is oxidized to 2,6-dibromo-benzo-1,4-quinone [109]. It has been proposed that the dehalogenase fulfils an ecological role in protecting sediment organisms from the toxicity of bromophenol metabolites. 5.3 Dehalogenation by Thymidylate Synthetase

Thymidylate synthetase catalyses the reductive methylation of 2¢-deoxyuridylate to thymidylate using 5,10-methylene-5,6,7,8-tetrahydrofolate as both methyl

Fig. 29. Transformation of 4-iodophenol by peroxidase (Redrawn from [88])

52

A.-S. Allard · A. H. Neilson

Fig. 30a, b. Thymidylate synthetase: a the biosynthetic reaction; b dehalogenation of 5-bromo-

and 5-iododeoxyuridylate (Redrawn from [232])

donor and reductant with formation of dihydrofolate (Fig. 30a). Studies of the mechanism have used 5-fluoro-2¢-deoxyuridylate as a pseudo-substrate and revealed that anionoid enzymatic addition at C6 plays an important catalytic role, and it has been shown that thymidylate synthetase mediates release of bromide or iodine from 5-bromo- or 5-iodo-2¢deoxyuridylate (Fig. 30b) [232].An entirely different flavin-dependent pathway has been observed in bacteria including some pathogens and archaea such as species of Pyrococcus.

6 Anaerobic Reactions 6.1 Introduction

There are some salient features of reactions with aromatic halogenated compounds that should be observed. Dehalogenation – especially dechlorination – has been widely observed but growth with the product is not always possible. This depends on the susceptibility to anaerobic metabolism involving cleavage of the ring followed by utilization of the products for incorporation into anaerobic pathways for energy production and biosynthesis. As noted later, however, there is evidence that the chlorinated substrates may themselves be used for generation of energy and this is discussed for the first pure culture that was able to dechlorinate 3-chlorobenzoate anaerobically. Although the greatest weight has been placed on dehalogenation by strictly anaerobic organisms, there has been considerable effort in the study of dehalogenation under nitrate-reducing conditions. The relevance of these reactions in natural ecosystems will probably be minimal except in the rare situations where high concentrations of nitrate are available. In addition, Rhodopseudomonas palustris is able to degrade 3-chlorobenzoate to benzoate that is subsequently degraded to acetate and CO2 [47]. Fairly exten-

Degradation and Transformation of Organic Bromine and Iodine Compounds

53

sive paragraphs are devoted to what we regard as important issues that should always be kept in mind in interpreting the results of studies on anaerobic dehalogenation. It has been shown that dechlorination may be induced by exposure to brominated analogues. This supports the view that fission of the C-Br bond is more easily accomplished than the C-Cl compounds, and justifies the somewhat extensive discussions of anaerobic dechlorination. There are some aspects of anaerobic dechlorination that have been explored in detail in dechlorination that may plausibly be extrapolated to anaerobic debromination: 1. Bioenergetics – The bioenergetics of dechlorination has been most thoroughly examined in a strain of Desulfomonile tiedjei that is able to dechlorinated 3chlorobenzoate to benzoate. It has been shown that reductive dechlorination leads to production of ATP that is synthesized by coupling proton translocation to dechlorination [45, 134]. Cells induced by growth with 3-chlorobenzoate were able partially to dechlorinate polychlorinated phenols specifically at the 3-position whereas the monochlorophenols were apparently resistant to dechlorination [133]. It has been shown in Desulfitobacterium dehalogenans that the yield of ATP in cells using pyruvate as both electron donor (forming acetate) and electron acceptor (forming lactate) is doubled when pyruvate as electron donor is replaced with 3-chloro-4-hydroxybenzoate (forming 4-hydroxybenzoate) [120]. The membrane-bound reductive dehalogenase from Desulfomonile tiedjei has been solubilized and purified [151], is distinct from the glutathione-dependent tetrachlorohydroquinone enzyme from a strain of Flavobacterium sp. [246], and plausibly plays a role in the energy transduction of Desulfomonile tiedjei. A membrane-bound cytochrome c is co-induced with the activity for reductive dechlorination, and has been purified and shown to be a high-spin diheme cytochrome distinct from previously characterized c-type cytochromes [116]. It has been suggested that a chemiosmotic process may be used to rationalize the coupling of energy production with concomitant dechlorination [115]. Anaerobic dechlorination is not therefore a tangential metabolic activity but is directly coupled to the metabolism and bioenergetics of the cell. 2. Partial dechlorination – Although complete dechlorination of polyhalogenated compounds under anaerobic conditions has been observed, the most common situation is that in which only partial dehalogenation occurs: all of these reactions are therefore strictly biotransformations. Illustrative examples include the following: pentachlorophenol [131], hexachlorobenzene [54, 244], 3,4,5trichlorocatechol [5], 2,3,5,6-tetrachlorobiphenyl [220], chloroanilines [108], and 2,4,6-chlorobenzoate [63]. A good example using cell suspensions of a pure culture of Desulfomonile tiedjei is given (Table 55) [133]. This clearly illustrates the specificity for 3-halogenated benzoates. Collectively, these results show that the less highly substituted congeners are more resistant to dechlorination so that they are more recalcitrant. In general this is the reverse of the situation pertaining under aerobic conditions, and this suggests that the com-

54

A.-S. Allard · A. H. Neilson

Table 55. Relative dehalogenation of chlorophenols by cell suspensions of Desulfomonile tied-

jei during 20 h incubation [133] Substrate

% Removal

Product

Pentachlorophenol 2,3,4,6-Tetrachlorophenol 2,3,4-Trichlorophenol 2,4,6-Trichlorophenol 3,4-Dichlorophenol 2,3-Dichlorophenol

100 100 100 1 100 34

2,4,6-Trichlorophenol 2,4,6-Trichlorophenol 2,4-Dichlorophenol None 4-Chlorophenols 2-Chlorophenols

plete degradation of these polyhalogenated compounds will probably involve both kinds of reactions; a similar situation has been suggested for halogenated alkanes and alkenes. 6.2 Halogenated Hydrocarbons 6.2.1 Polyhalogenated Benzenes

Considerable attention has been directed to halogenated benzenes since trichlorobenzenes have been used in wood treatment and as intermediates in the chemical industry and hexachlorobenzene was used as a fungicide. The transformation of hexachlorobenzene has been extensively studied [54, 244] and is consistent with the comments above proceeds with partial removal of chlorine atoms with formation of 1,2- and 1,3-dichlorobenzene as stable end products. In anaerobic columns biological dechlorination of all three trichlorobenzene isomers to chlorobenzene was demonstrated [20]. The anaerobic debromination or deiodination of disubstituted benzenes was examined in anaerobic slurries (Table 56) [200] and clearly illustrates the preferential of bromine over chlorine and iodine over bromine. Partially brominated benzenes have been found in various sediments in Japan, and these were plausibly attributed to anaerobic debromination of the flame retardant hexabromobenzene [229]. Table 56. Rates of dehalogenation (day–1) of halogenated benzenes in anaerobic sediments and

their products [200] Substrate

Rate (day)–1

Product

3-Chlorobromobenzene 3-Chloroiodobenzene 3-Bromoiodobenzene 3-Chlorotrifluoromethylbenzene 3-Bromotrifluoromethylbenzene

0.0065 0.0116 0.0342 0.0016 0.0251

Chlorobenzene Chlorobenzenes Bromobenzene Trifluoromethylbenzene Trifluoromethylbenzene

Degradation and Transformation of Organic Bromine and Iodine Compounds

55

6.2.2 PCBs

PCBs have been used as insulators in high voltage transformers and switching gear and their virtual ubiquity in environmental samples has caused alarm. As a result, there is an enormous literature on their degradation, though it is not intended to cover this in detail. Studies on the degradation of PCBs are exacerbated not only by the number of congeners but by the fact that heavily orthosubstituted congeners occur in enantiomeric forms. Attention is directed to reviews on anaerobic dechlorination [11] and to those that include a summary of aerobic degradation [10, 202]. The last also includes valuable comments on the regulation of aerobic degradation of PCB and the significance of chlorobenzoate degradation. The anaerobic dechlorination of PCBs has been extensively studied both in microcosms and in field samples from heavily contaminated sites in the United States. Three main patterns have been found: N that removed flanked meta chlorines, P that removed para chlorines, and LP that removed unflanked para chlorines [13]. The example of the 2,2¢,3,4,4¢,5,5¢-heptachloro congener is given in Fig. 31. It is important to appreciate that there are important factors that affect the relative effectiveness of these processes: (i) temperature [241] that is discussed in more detail below and (ii) the structure of the PCB congener that primes the dehalogenation [242].

Fig. 31. Anaerobic dechlorination of 2,2¢,3,4,4¢,5,5¢-heptachlorobiphenyl (Redrawn from [13])

56

A.-S. Allard · A. H. Neilson

Temperature is an important parameter particularly for naturally occurring mixed cultures of organisms in the natural environment: temperature may result in important changes in the composition of the microbial flora as well as the rates for different processes. Some illustrative examples of its importance of temperature situations include the following: 1. An anaerobic sediment sample was incubated with 2,3,4,6-tetrachlorobiphenyl at various temperatures between 4°C and 66°C [241]. The main products were 2,4,6- and 2,3,6-trichlorobiphenyl and 2,6-dichlorobiphenyl: the first was produced maximally and discontinuously at 12°C and 34°C, the second maximally at 18°C, and the third was dominant from 25°C to 30°C. Dechlorination was not observed above 37°C. 2. Sediment samples from a contaminated site were spiked with Arochlor 1242 and incubated at 4°C for several months [237]. Degradation by aerobic organisms in the upper layers of the sediment–but not in those >15 mm from the surface – occurred with the selective production of di- and trichlorobiphenyls. Some congeners were not found including the 2,6- and 4,4¢-dichlorobiphenyls and a wider range of trichlorobiphenyls which were presumably further degraded. There is therefore extensive evidence for the anaerobic dechlorination of PCBs but the extent and specificity of this depends on both the organism and the structure of the congener. 6.2.3 PBBs and Diphenylmethanes

Mixed cultures of organisms isolated from sediments contaminated with PCBs and PBBs were shown to debrominate PBBs under anaerobic conditions [136] and the dominant congener–2,2¢,4,4¢,5,5¢-hexabromobiphenyl–could be successively debrominated to 2,2¢-dibromobiphenyl. On the other hand, in sediments from the most heavily contaminated site containing contaminants in addition to PBBs, very little debromination occurred and the recalcitrance was attributed to the toxicity of the other contaminants [137]. Ingenious experiments have used addition of specific PCB congeners that are more readily dechlorinated to “prime” dechlorination at specific positions [11] and extended to the use of dibrominated biphenyls in the presence of malate to stimulate dechlorination of the hexachloro- to nanochlorobiphenyls [11]. Results from these experiments provide valuable evidence of important differences between anaerobic dechlorination and anaerobic debromination. The use of brominated biphenyls to induce dechlorination of highly chlorinated biphenyls has been examined in detail. Di- and tribromobiphenyls were the most effective in dechlorinating the heptachloro, hexachloro, and pentachloro congeners, and were themselves reduced to biphenyl [13], and 2,6-dibromobiphenyl stimulated the growth of anaerobes that effectively dechlorinated hexa-, hepta-, octa-, and nanochlorobiphenyls over the temperature range from 8°C to 30°C [243]. In anaerobic sediment microcosms, a range of tribrominated

57

Degradation and Transformation of Organic Bromine and Iodine Compounds

biphenyls was successively debrominated to dibromo- and monobromo compounds before complete debromination to biphenyl: of specific interest; both the 2,2¢,4¢,5- and 2,2¢,5,5¢-tetrabrominated congeners were debrominated to the 2,2¢dibromo congener that was slowly completely debrominated to biphenyl within 54 weeks [12]. The pathways for debromination of 2,2¢,4¢,5¢- and 2,2¢,5,5¢-tetrabromobiphenyls are shown in Fig. 32. In comparative microcosm experiments, a number of important features were observed: 1. All of the tribromo congeners were debrominated to products including biphenyl in acclimation times of between <1 and 7 weeks. 2. For the corresponding chloro congeners with comparable acclimation times, only loss of single chlorine was noted and biphenyl was not observed. 3. The mono and dichloro congeners required long periods of acclimation before onset of dechlorination. Other compounds have also been examined as “primers” for dechlorination of hexachloro to nonachloro PCB congeners [44]: a number of substituted brominated monocyclic aromatic compounds were examined and 4-bromobenzoate was effective–though less so that 2,6-dibromobiphenyl (Table 57). The chlorobenzoates that are metabolites of aerobic degradation were ineffective. The positive effect of brominated biphenyls in “priming” the anaerobic dechlorination of CBs has also been encountered in the dechlorination of octachlorodibenzo[1,4]dioxin to the 2,3,7,8 congener induced by 2-bromodibenzo[1,4]dioxin in the presence of H2 [4]. Polybrominated biphenyls are not only debrominated microbially under anaerobic conditions, but are able to induce effective dechlorination of their chlorinated analogues. Debromination may, however, be limited in the presence of other contaminants. Debromination of tetrabromobisphenol-A that is used as a flame retardant has been demonstrated under methanogenic and sulfate-reducing condiTable 57. The effect of selected halogenated benzoates on the dechlorination (%) of hexa- to nonachlorobiphenyls in river sediment slurries [44]

Halogenated benzoate

Halobenzoate dehalogenation

Identified product(s)

% Dehalogenation

2-Bromo3-Bromo4-Bromo2-Iodo3-Iodo4-Iodo2,5-Dibromo-

+ + + + + + +

0–50 0 40–70 10–50 0–30 10–50 10–50

2,5-Diiodo-

+

Benzoate Benzoate Benzoate Benzoate Benzoate Benzoate 3-Bromobenzoate, benzoate 2-Iodobenzoate, benzoate

2,4,6-Tribromo2,3,5-Triiodo-

ND ND

ND = not determined.

10–50 10–20 10–20

Fig. 32. Anaerobic dechlorination of 2,2¢,5,5¢-tetrabromobiphenyl (Redrawn from [12])

58 A.-S. Allard · A. H. Neilson

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Degradation and Transformation of Organic Bromine and Iodine Compounds

tions [225] and in anaerobic slurries [170a], whereas tetrachlorobisphenolA was only partially dechlorinated successively to the trichloro and monmochloro isomers [225]. Under aerobic conditions, bisphenol-A can be degraded [170a] by rearrangement via 4-hydroxybenzoate and 4-hydroxyacetophenone. 6.3 Anaerobic Degradation of Benzoates 6.3.1 Dehalogenation

Although Desulfomonile tiedjei is able to dechlorinate 3-chlorobenzoate – but not 2- or 4-chlorobenzoate – it is capable of dehalogenating not only 3-bromo- and 3-iodobenzoate but also 2- and 4-iodobenzoate (Table 58) [43]. Debromination of bromobenzoates was examined under a variety of anaerobic conditions. Of these, Fe-reducing were negative and varying activities were observed for methanogenic and sulfidogenic conditions [135]. Under denitrifying conditions Thauera aromatica [193] dehalogenated 3-chloro-, 3-bromo-, and 3-iodobenzoate [93]. The anaerobic degradation of benzoate in the light by anaerobic phototrophic bacteria has been known for many years (references in [78, 106]), and the pathway and genetics of its degradation are now well established, and follow substantially those for Thauera aromatica [48, 76]. Degradation of benzoate by the non-sulfur Rhodopseudomonas palustris proceeds via the CoA thioester, ring reduction, and ring fission that results in the formation of glutaryl-CoA before conversion to crotonyl-CoA. Degradation of halogenated benzoates has also been demonstrated in strains of Rps. palustris. For example, strain WS17 is able to degrade 3-chlorobenzoate in the presence of benzoate [98], and strain DCP3 is able to degrade both 3-chloro- and 3-bromobenzoate in the absence of benzoate [219]. In the light of later evidence on the pathways for degradation of 3chlorobenzoate via benzoate, it seems plausible to attribute the first step to dehalogenation of the intact ring. Table 58. Substrate specificity of dehalogenation (nmol substrate dehalogenated/min/mg pro-

tein) in cell extracts of Desulfomonile tiedjei [43] Substrate

Product

Rate

Substrate

Product

Rate

3-Chlorobenzoatea 3-Bromobenzoatea 3-Iodobenzoatea

Benzoate Benzoate Benzoate

0.615 0.68 2.8

2-Iodobenzoateb 3-Iodobenzoateb 4-Iodobenzoateb

Benzoate Benzoate Benzoate

0.31 1.5 0.62

a b

4.42 mg protein: 20 h incubation, 3.5 mg protein: 30 h incubation.

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A.-S. Allard · A. H. Neilson

6.3.2 Oxidation and Reduction of Aromatic Carboxylates and Aldehydes

The reduction of 3-chloro- and 3-bromobenzoate to the corresponding benzyl alcohols has been observed with Desulfomicrobium escambiense that was isolated from a 3-chlorobenzoate dechlorinating coculture. This required pyruvate that was transformed to acetate, lactate, and succinate [184] and no dehalogenation was observed. Metabolically stable enrichment cultures reduced the aldehyde group of 5-bromovanillin and debrominated it to 4-methylcatechol, and debrominated 3-bromo-4-hydroxybenzaldehyde to 4-hydroxybenzoate [145]. One culture produced the corresponding carboxylic acid that was partly degraded to phenol [145]. The converse oxidation of benzaldehydes to benzoates has been observed in Desulfovibrio strains [250]. 6.4 Phenols

It is worth noting that the anaerobic degradation of non-halogenated phenols generally proceeds by ring carboxylation to benzoates, followed by formation of CoA thioesters, dehydroxylation and ring fission and subsequent steps as for benzoates.Although anaerobic dechlorination of phenols has been described in various species of Desulfitobacterium including D. dehalogenans, D. frapperi, D. chlororespirans, and D. hafniense, and in Desulfomonile tiedjei strain DCB-1 and Anaeromyxobacter dehalogenans, only relatively few examples of anaerobic debromination of phenols can be given. As discussed more fully in the chapter by Neilson, brominated phenols are widespread in the marine environment and play many roles including deterrent to animals and algae: 1. The debromination of 2,4-dibromo- and 2,4,6-tribromophenol naturally produced by the hemichordate Saccoglossus kowalwskii has been demonstrated in anoxic slurries and resulted in the production of phenol [104]. 2. Debromination of brominated phenols was examined under the conditions noted for halogenated benzoates. As for bromobenzoates, bromophenols were poor substrates under Fe-reducing conditions, and 2-bromo-, 3bromo-, and 4-bromophenols were degraded with highly variable effectiveness [135]. 3. Growth of Desulfovibrio strain TBP-1 grown with lactate and using sulfate as electron acceptor is able to debrominate 2-bromo-, 2,4-dibromo-, 2,6-dibromo-, and 2,4,6-tribromophenol [22]. 4. Desulfitobacterium chlororespirans strain Co23 is able to couple dehalogenation of halogenated phenols as electron acceptors to oxidation of lactate [175]. Whereas 2,4,6-trichlorophenol and 2,4,6-tribromophenol supported growth with production of 4-chlorophenol and 4-bromophenol, neither 2-bromophenol nor 2-iodophenol supported growth. 5. Desulfitobacterium dehalogenans is able to dehalogenated ortho-substituted phenols (Table 59), the enzyme has been purified and characterized [209], and a model proposed for the energy yield from halorespiration [210]. The mem-

Degradation and Transformation of Organic Bromine and Iodine Compounds

61

Table 59. Specific activity (unit/mg) of ortho-chlorophenol dehalogenase in purified extracts of Desulfitobacterium dehalogenans [209]

Substrate

Specific activity

2-Chloro-4-hydroxyphenylacetate 2-Bromo-4-chlorophenol 2,4-Dichlorophenol 2,3-Dichlorophenol 2,6-Dichlorophenol Pentachlorophenol

12 24 4 15.5 0.8 0.2

Detection limit 0.12 unit/mg.

brane-associated enzyme had pH and temperature optima of 8.2 and 52 °C, and contained one [4Fe-4S] cluster, one [3Fe-4S] cluster, and one cobalamin per monomer. Both of these [Fe-S] proteins are also found in the PceA protein involved in reductive dehalogenation of tetrachloroethene by Dehalospirillum multivorans [148]. 6. Several strains of Anaeromyxobacter dehalogenans are able to dehalogenate a number of ortho-halogenated phenols including 2-chloro- and 2-bromophenol but neither 2-fluoro- nor 2-iodophenol [174]. Acetate served as electron donor and was completely oxidized. By comparison, Desulfovibrio dechloracetivorans that was able to dechlorinate 2-chlorophenol to phenol by oxidizing acetate was unable to debrominate 2-bromophenol [199].

7 Concluding Comments Virtually all the types of reactions that are outlined in Fig. 1 have been illustrated with organic bromine compounds, and it seems reasonable to draw some general conclusions on the degradability of organic bromine compounds in spite of the sometimes limited range of experiments. Data for iodoorganic compounds are sparse. Environmental recalcitrance is an issue that must address in addition the cardinal question of bioavailability: 1. Experimental methods – Care should be exercised in comparing results using different experimental procedures, for example aerobic growth on solid agar medium, experiments in which the sediment phase remains, and those in which metabolically stable cultures have been used. 2. Comparison of halogens – The degradation of organobromine and organoiodine compounds generally proceeds with greater ease than with their chlorinated and fluorinated analogues. 3. Pathways of degradation and transformation – The pathways for dehalogenation of organochlorine, organobromine, and organoiodine compounds are broadly similar. 4. Isomer-specificity – This is well-marked in anaerobic dechlorination of polyhalogenated substrates, and those with the greater number of halogen substituents are more readily dehalogenated.

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8 References 1. Adriaens P (1994) Evidence for chlorine migration during oxidation of 2-chlorobiphenyl by a type II methanotroph. Appl Environ Microbiol 60:1658–1662 2. Ahmad D, Sylvestre M, Sondossi M (1991) Subcloning of bph genes from Pseudomonas testosteroni B-356 in Pseudomonas putida and Escherichi coli: evidence for dehalogenation during initial attack on chlorobiphenyls. Appl Environ Microbiol 57: 2880–2887 3. Ahmad D, Massé R, Sylvestre M (1990) Cloning, physical mapping and expression in Pseudomonas putida of 4-chlorobiphenyl transformation genes from Pseudomonas testosteroni strain B-356 and their homology to the genomic DNA from other PCB-degrading bacteria. Gene 86:53–61 4. Albrecht ID, Barkovskii ALK,Adriaens P (1999) Production and dechlorination of 2,3,7,8tetrachlorodibenzo-p-dioxin in historically-contaminated estuarine sediments. Environ Sci Technol 33:737–744 5. Allard A-S, Lindgren C, Hynning P-Å, Remberger M, Neilson AH (1991) Dechlorination of chlorocatechols by stable enrichment cultures of anaerobic bacteria.Appl Environ Microbiol 57:77–84 6. Allard A-S, Remberger M, Neilson AH (1987) Bacterial O-methylation of halogen-substituted phenols. Appl Environ Microbiol 53:839–845 7. Balajee S, Mahadevan A (1990) Dissimilation of 2,4-dichlorophenoxyacetic acid by Azotobacter chroococccum. Xenobiotica 20:607–617 8. Bartnicki EW, Castro CE (1969) Biodehalogenation. The pathway for transhalogenation and the stereochemistry of epoxide formation from halohydrins. Biochemistry 8:4677–4680 9. Bartnicki EW, Castro CE (1994) Biodehalogenation: rapid oxidative metabolism of monoand polyhalomethanes by Methylosinus trichosporium OB-3b. Environ Toxicol Chem 13:241–245 10. Bedard DL (1990) Bacterial transformation of polychlorinated biphenyls. In: Kamely D, Chakrabarty A, Omenn GS (eds) Biotechnology and biodegradation, vol 4. Gulf Publishing, Houston, USA, pp 369–388 11. Bedard DL, Quensen JF III (1995) Microbial reductive dechlorination of polychlorinated biphenyls. In: Young LY, Cerniglia CE (eds) Microbial transformation and degradation of toxic organic chemicals. Wiley-Liss, New York, USA, pp 127–216 12. Bedard DL, van Dort HM (1998) Complete reductive dehalogenation of brominated biphenyls by anaerobic microorganisms in sediment.Appl Environ Microbiol 64:940–947 13. Bedard DL, van Dort HM, Deweerd KA (1998) Brominated biphenyls prime extensive microbial reductive dehalogenation of Arochlor 1260 in Housatonic River sediment. Appl Environ Microbiol 64:1786–1795 14. Bedard DL, Haberl ML, May RJ, Brennan MJ (1987) Evidence for novel mechanisms of polychlorinated biphenyl metabolism in Alcaligenes eutrophus H 850. Appl Environ Microbiol 53:1103–1112 15. Belay N, Daniels L (1987) Production of ethane, ethylene and acetylene from halogenated hydrocarbons by methanogenic bacteria. Appl Environ Microbiol 53:1604–1610 16. Blasco R, Mallavarapu M, Wittich R-M, Timmis KN, Pieper DH (1997) Evidence that formation of protoanemonin from metabolites of 4-chlorobiphenyl degradation negatively affects the survival of 4-chlorobiphenyl-cometabolizing microorganisms. Appl Environ Microbiol 63:427–434 17. Blasco R,Wittich R-M, Mallavarapu M, Timmis KN, Pieper DH (1995) From xenobiotic to antibiotic, formation of protoanemonin from 4-chlorocatechol by enzymes of the 3oxoadipate pathway. J Biol Chem 270:29,229–29,235 18. Boon N, Goris J, deVos P, Verstraete W, Topp EM (2001) Genetic diversity among 3chloroaniline- and aniline-degrading strains of the Comamonadaceae. Appl Environ Microbiol 67:1107–1115

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The Handbook of Environmental Chemistry Vol. 3, Part R (2003): 75– 204 DOI 10.1007/b11448HAPTER 1

Biological Effects and Biosynthesis of Brominated Metabolites Alasdair H. Neilson Swedish Environmental Research Institute IVL, Stockholm Sweden E-mail: [email protected] In memory of Arthur Lapworth, Robert Robinson, and Christopher Ingold, pioneers of the electronic theory of organic chemistry Abstract. On the basis of halogenated metabolites assembled by Gribble, an attempt is made to provide a hypothesis for the biosynthesis of representative groups. Examples of brominated and iodinated metabolites are drawn from the major classes of terpenoids and acetogenins, and related to the biosynthesis of their putative precursors. Brominated and iodinated carbocyclic and heterocyclic aromatic compounds are discussed as well as mechanisms for the oxidative coupling of phenolic substrates.A few chlorinated metabolites are introduced for additional illustration. Halogenation by cationoid (Hal+) reactions catalyzed by haloperoxidases is discussed, and attention drawn to the alternative role of anionoid (Hal–) halogenation. The presentation is extended to the biosynthesis of isonitriles and dichloroimines by anionoid reaction of precursors with cyanide. The range of biota that produce brominated substrates is briefly indicated, and comments made on the ecological significance of metabolites and associations among biota. Keywords. Brominated metabolites, Iodinated metabolites, Terpenoids,Acetogenins, Carbocyclic

compounds, Heterocyclic compounds, Biological activity, Peroxidase, Metal-free halogenation

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Cationoid Halogenation . . . . . . . . . . . . . . Metabolites Containing the Trichloromethyl Group Cationoid Halogenation . . . . . . . . . . . . . . Anionoid Halogenation . . . . . . . . . . . . . . Synthesis of H2O2 . . . . . . . . . . . . . . . . . .

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Reactions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97 Enzymes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98

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Terpenoids and Steroids . . . . . . . . . . . . . . . . . . . . . . . 109

7.1 7.2 7.2.1 7.2.2 7.2.3 7.2.4 7.2.5 7.2.6 7.3 7.3.1 7.3.2 7.3.3 7.3.4 7.3.5 7.4 7.5 7.6

Monoterpenoids . . . . . . . . . . . . . . . . . . . . . . . Sesquiterpenoids . . . . . . . . . . . . . . . . . . . . . . . Spirochamigrenes . . . . . . . . . . . . . . . . . . . . . . . Bisabolanes . . . . . . . . . . . . . . . . . . . . . . . . . . Eudesmanes and Related Compounds . . . . . . . . . . . . Aromatic Structures – Cuparanes and Related Compounds Rearranged and Degraded Sesquiterpenoids . . . . . . . . Isonitriles, Isothiocyanates, and Dichloroimines . . . . . . Diterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . Laurencia Metabolites . . . . . . . . . . . . . . . . . . . . Epoxide-Derived Diterpenoids . . . . . . . . . . . . . . . . Rearranged Diterpenoids: Sphaerococcus Metabolites . . . Briaranes . . . . . . . . . . . . . . . . . . . . . . . . . . . Isonitriles and Isothiocyanates . . . . . . . . . . . . . . . . Sesterterpenoids . . . . . . . . . . . . . . . . . . . . . . . Triterpenoids . . . . . . . . . . . . . . . . . . . . . . . . . Steroids . . . . . . . . . . . . . . . . . . . . . . . . . . . .

8

Non-Terpenoids: Acetogenins and Other Structures . . . . . . . . 139

8.1 8.2 8.2.1 8.2.2 8.2.3 8.2.4 8.2.5

Laurencia Metabolites . . . . . Other Structures . . . . . . . . Polyhalogenated Higher Alkanes Polyacetylenes . . . . . . . . . Prostanoids . . . . . . . . . . . Furanones . . . . . . . . . . . . Macrolides . . . . . . . . . . .

9

Carbocyclic Aromatic Compounds

9.1 9.2 9.3

Synthesis of the Carbon Skeletons . . . . . . . . . . . . . . . . . . 146 Peroxidase Coupling of Aromatic Compounds . . . . . . . . . . . 148 Cytochrome P450 Coupling . . . . . . . . . . . . . . . . . . . . . 152

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111 114 115 118 118 118 119 123 123 127 130 130 131 134 134 134 136

139 143 143 143 144 144 145

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Biological Effects and Biosynthesis of Brominated Metabolites

9.4 9.5 9.6 9.7 9.7.1 9.7.2 9.7.3 9.7.4

Simple Phenols . . . . . . . . . . . . . . . Quinones and Related Compounds . . . . Prenylated Phenols . . . . . . . . . . . . . Brominated Tyrosines . . . . . . . . . . . Spiranes and bis-Spirooxazolines . . . . . Rearranged Tyrosines . . . . . . . . . . . Metabolites with Several Tyrosine Residues Iodinated Derivatives . . . . . . . . . . . .

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153 155 156 161 162 163 164 166

10

Aromatic Metabolites Containing Several Rings . . . . . . . . . . 169

10.1 10.2 10.3

Biphenyls and Diphenyl Ethers . . . . . . . . . . . . . . . . . . . 169 Diphenylmethanes . . . . . . . . . . . . . . . . . . . . . . . . . . 174 Depsides and Depsidones . . . . . . . . . . . . . . . . . . . . . . 177

11

Heterocyclic Compounds

11.1 11.2 11.2.1 11.2.2 11.2.3 11.3 11.4 11.5 11.5.1 11.5.2 11.5.3 11.5.4

Pyrroles and Simple Indoles . . . . . . . . . . . . . . . . . . . . Complex Indoles . . . . . . . . . . . . . . . . . . . . . . . . . . Tryptamine-Based Metabolites . . . . . . . . . . . . . . . . . . . Dimeric Indoles . . . . . . . . . . . . . . . . . . . . . . . . . . . Prenylated Indoles . . . . . . . . . . . . . . . . . . . . . . . . . Carbazole . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Iminazole, Quinoline, Pyrimidine, Pyrrolopyrimidine and Purine Metabolites . . . . . . . . . . . . . . . . . . . . . . . Oxaarene: Furans, Pyrones, Coumarins and Dibenzo[1,4]Dioxins Furans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Pyrones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Dihydrocoumarins . . . . . . . . . . . . . . . . . . . . . . . . . Dibenzo[1,4]Dioxins . . . . . . . . . . . . . . . . . . . . . . . .

12

References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 193

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178 179 182 185 186 188

. 189 190 . 191 . 191 . 191 . 193

1 Introduction Bacteria, fungi, algae, and marine biota produce a wide range of structurally diverse halogenated metabolites that encompass a plethora of structures ranging from alkanes to terpenoids, acetogenins, complex phenols, and macrolides. Most contain chlorine or bromine, and a smaller number, iodine. Attention has been directed to compounds in marine biota with valuable therapeutic properties while interest in global climate change and the diminishing ozone layer has generated interest in volatile halogenated low-molecular-mass metabolites since very substantial volumes of these are produced in the marine environment. This is discussed in detail in the chapter by Orlando elsewhere in this volume. Chlorine-containing antibiotics – generally from fungi – are well known, and their brominated analogs may be produced during growth on artificial media in the presence of bromide, for example 7-bromotetracycline from Streptomyces au-

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reofaciens, and 7-bromo-4,6,2¢-trimethoxy-6¢-methylgris-2¢-ene-3,4-dione from species of Penicillium. In general, however, with the exception of the brominated polyene xanthomonadin [96] in some species of Xanthobacter that may play a role in photochemical protection [170] and the metabolites from Central Asia that are noted in the section “Source of the Halogen”, bromine-containing metabolites are confined to marine biota. This review attempts to provide a framework of plausible routes for the biosynthesis of brominated and iodinated metabolites. In virtually all of them, the qualifier “hypothetical” should precede the term biosynthesis.Virtually all the examples are taken from the comprehensive surveys of Gribble [68–70] without which this review would never have been possible.With his kind permission, the numbers for the structures used by him are given here in bold face. Besides providing continuity, this makes it possible to avoid duplicating the encyclopedic compilation of references provided by Gribble. It is impossible in a few paragraphs to cover the plethora of structures contained in these volumes, and it would therefore be more appropriate to designate this an essay rather than a review. The references are eclectic and make no attempt to cover the seminal contributions of specific authors. It is hoped, however, that the principles enunciated will be reinforced by the structures of more recently isolated metabolites. This is a reductionist approach, and the goal has been to provide principles of biosynthesis that have a wide application and achieve some degree of unity in the diverse structures. The recurrent theme is halogenation by the cationoid halonium cation that provides an attractive unifying hypothesis. There are, however, metabolites for which a more plausible mechanism involves anionoid bromination with halide, and this is supported by recent studies which have demonstrated the synthesis of 7-chlorotryptophane by anionoid chlorination. The biosynthesis of the carbon skeletons is considered only parenthetically.Although the absolute configurations of many terpenoids has been established unambiguously by X-ray analysis – and are used here – no attempt has been made to rationalize the configurations of enantiomorphic metabolites, many of which occur in the same taxonomic group of biota though differing in the configuration of methyl groups and halogen atoms. The finding of molecular iodine in a range of algae from the West Coast of Sweden by Kylin in 1929 [117] may be seen as cardinal to understanding the production of halogenated metabolites in algae. Iodine was found in representatives of green algae (Cladophora rupestris), brown algae (species of Laminaria and Polysiphonia), and red algae (species of Rhodomela). Although the activity was associated with what was then termed an oxidase, this is consistent with the now established oxidation of iodide to iodine by iodoperoxidase. Important organic iodine compounds include iodinated alkanes, iodinated tyrosines, and – unusually – iodinated sesquiterpenoids. In view of the broad similarity of bromine and iodine, organic iodine compounds may plausibly be synthesized by mechanisms comparable to those for organic bromine compounds. Since, however, iodide is a more effective nucleophile than bromide, it is possible that anionoid iodination may play a relatively greater role than anionoid bromination.Although it is plausible to assume that the biosyntheses of many organic iodine compounds follow

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the pathways used for the bromine analogues, the enzymes may be different and attention is drawn to the activity of lactoperoxidase, myeloperoxidase, and eosinophil peroxidase. Although many metabolites contain both chlorine and bromine, and metabolites with vicinal bromine and chlorine substituents are well represented in red algae of the genus Laurencia, these have not been systematically included. The mechanism for their formation seems to differs from that in which vicinal halogen and hydroxyl groups are present, and a few examples of terpenoids with these structural features are used as illustration. It is, however, clear that generalizations are fraught with danger since even in the same organism metabolites containing vicinal bromine and chlorine substituents in both the cis and the trans orientation may occur, and for trans isomers both diaxial and diequatorial configurations may exist. Only representative examples have been chosen for the vast array of structures. Although for a number of these, closely related compounds may be produced by the same or different organisms, only an illustrative example has been chosen. Metabolites containing chlorine and a group containing isonitrile, isothiocyanate, or dichloroimino groups have been noted parenthetically: 1. For the sake of illustration and to exemplify the range of structures, a number of organic chlorine compounds have been included for which it could be anticipated that the corresponding brominated metabolites might also be found. An illustrative example is the chlorinated bicyclo[8:4:0]tetradecane ring that has been found in gorgonians, sea pens, octocorals, and soft corals (section “Biaranes”), and the dichloroimines that are noted below. 2. The biosynthesis of organochlorine compounds is sometimes mediated by reactions other than conventional peroxidases (section “Anionoid Halogenation”), and it is worth noting the possible involvement of this even in the synthesis of organic bromine compounds. 3. A word of justification is offered for inclusion of isonitriles, isothiocyanates and dichloroimines: the mechanism of their formation by anionoid cyanide is well established [39, 59, 101, 191, 192] and it therefore seemed appropriate to include these metabolites that are synthesized by a reaction that is formally equivalent to anionoid bromination (sections “Isonitriles, Isothiocyanates, and Dichloroimines” and “Diterpenoid Isonitriles”). The alternative mechanism from glycine is noted in sections “Isonitriles, Isothiocyanates, and Dichloroimines” and “Prenylated Indoles”. In this review, the older terms anionoid and cationoid proposed by Lapworth and Robinson are used since they clearly describe the active halogenating agents Hal– and Hal+: the widely accepted modern terms nucleophilic and electrophilic used by Ingold convey this less clearly.

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2 Biological Background 2.1 Geographical Distribution

Greatest attention has probably been devoted to brominated organics metabolites from biota in tropical and sub-tropical environments,particularly the Pacific Ocean, and to a lesser extent the coasts of Spain and Portugal and the Mediterranean.A survey of bromoperoxidase activity in a range of marine algae has been given, and includes samples from Maine, Hawaii, California, and Japan (Table 1) [143]. Attention is drawn to the occurrence of brominated phenols, brominated pyrroles, and chlorinated alkanes among polychaeta and hemichordata and mollusca at two temperate sites on the coast of South Carolina [47]. It should be emphasized that only a restricted range of localities has been examined and that those with harsh climates or that are more inaccessible have been investigated only superficially. Some species such as the brown alga Laminaria sp. have a very wide geographical range, and the green alga Cladophora trichotoma is distributed along the Pacific Coast of North America from Vancouver Island (48°N) to La Paz in Baja California (24°N), while some species apparently occur on both sides of the Pacific Ocean [186]. Although many brominated metabolites have been isolated from red and brown algae, only a very limited number of taxa within these large groups have been systematically examined, and many of these occur in cold-water and even polar areas. Illustrative examples include: 1. Brominated methanes have been found in seaweed beds from Svalbard [40] in the ice diatoms Nitzschia stellata and Porosira pseudodenticulata from the underside of sea ice in McMurdo Sound, Antarctica [199] and in seaweeds from the West Coast of Scotland [153]. Table 1. Activities (µg monochlorodimedone transformed/min/5 g algae) of bromoperoxidase

in selected algae [143] Group (% positive) Rhodophyseae [45] Asparagopis sp. Bonnemaisonia humifera Corallina officinalis Corallina vancouverensis Gracilaria sjoestedtii Plocamium cartaligenium Phaeophyceae [2] Ascophyllum nodosum Dictopteris ubdulata Chlorophyceae [15] Codium cylindricum Enteromorpha linza

Activity

Source

43.5 72 63 19 104 8.4

Hawaii New Hampshire Maine California California California

2.3 0.3

Maine California

7.8 2.6

Japan New Hampshire

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2. Brominated cyclic monoterpenoids have been found in Pantoneura placamioides from the South Shetland Islands, Antarctica [27]. 3. A number of macroalgae from the coast of the Netherlands displayed bromoperoxidase activity assayed by bromination of phenol red by bromide in the presence of H2O2 [229]. 4. A range of organic bromine compounds or bromoperoxidase activity has been found in samples from cold-water localities in Japan and New Zealand [143]. 2.2 Bacteria and Cyanobacteria 2.2.1 Nomenclature Whereas taxonomy has not been taken into account in this essay, the position for cyanobacteria (blue-green algae) merits brief comment. The Botanical Code of Nomenclature was followed for algae and blue-green algae that were ranked as algae in classical algological treatises such as Tilden [212] and Geitler [62]. Appreciation that blue-green algae were in fact prokaryotes means, however, that cyanobacteria – as they are now termed – fall within the Bacteriological Code. This has inevitably resulted in some confusion in nomenclature, the use of unwarranted specific names and probable false identification of genera that have not been finally resolved. Important groups of cyanobacteria that have been shown to produce brominated metabolites include the genera Lyngbya, Oscillatoria, Schizothrix, Calothrix, and Rivularia, while other genera including Fischerella, Hapalosiphon, and Westelliopsis produce chlorinated isonitriles. 2.2.2 Marine or Freshwater Very few of the strains cited appear to be truly marine with a requirement for marine media for cultivation, and the truly marine unicellular genera have seldom been examined for their metabolic capabilities. An exception is provided by the isolation of the chlorinated carbazole (1474) from the cyanobacterium Hyella caespitosa. It should be noted that virtually all samples were obtained from field material and that many cyanobacteria described in algological treatises were epiphytic [212]. Whereas virtually all the higher biota are undoubtedly marine, the position for bacteria (including cyanobacteria) is more complex. Some seem to be truly marine, but many bacteria of terrestrial origin are able to withstand moderate salinity [151], so that even if cyanobacteria were found in marine environments they would not necessarily be classed as marine. This may pragmatically be defined as the obligatory requirement for a saline medium for growth. As a result of the requirement in the Bacteriological Code, growth must be achieved and the need for a saline medium determined independent of the source of the inoculum. For example, strains of Fischerella sp. [98] and Calothrix brevissima [163] have been grown in a freshwater medium without addition of marine salts and this probably applies to others: these should be termed adven-

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tive marine organisms. Other cyanobacteria have been grown in marine media – whether obligatory is not disclosed. 2.3 Associations The term association is used here without any implication of obligate dependence. It is important to appreciate the range and degree of associations that include epiphytes on algae, and sponges with their accompanying biota, while many biota occupy important positions in the marine food chain either as predators or as consumers: 1. Red algae – The sea hare is a major predator on large algae such as species of the red alga Laurencia which are probably the ultimate source of brominated metabolites. For example, species of the sea hare Aplysia sp. accumulate halogenated terpenoids apparently from red algae [46], and it has been shown that polyhalogenated monoterpenoids in Aplysia californica were derived from their diet of algae [195]. 2. Cyanobacteria – It has been shown that a brominated diphenyl ether isolated from the sponge Dysidea herbacea is associated with the filaments of a cyanobacterium [214]. Association between the sponge Dysidea sp. and the cyanobacterium Lyngbya majuscula has also been suggested [79]. This is supported by the isolation from free-living Lyngbya majuscula of the molluscicidal barbamide [158] and the dysidenins [97] that contain both trichloromethyl substituents and a thiazole ring. It is worth noting the number of metabolites produced by cyanobacteria including L. majuscula (references in [140]). In addition, the origin of diketopiperazines containing trichloromethyl substituents that have been isolated from sponges of the genus Dysidea (references in [54]) has been attributed to the biosynthetic activity of Oscillatoria spongeliae [52]. 3. Sponges – The dorid nudibranch Phyllidiella pustulosa acquires isonitrile sesquiterpenoids from their diet of the sponge Acanthella cavernosa that is the source of these secondary metabolites [39]. A marine species of Vibrio sp. strain KMM 9-81-1 was isolated and cultivated from a sponge Dysidea sp. in a complex sea-water medium, and was shown to produce 3,5-dibromo-2(3¢,5¢dibromo-2¢-methoxy)phenol that was isolated from extracts of the sponge [42].An unidentified fungus isolated from the sponge Jaspis aff. johnstoni and cultured on a complex marine medium produced a number of sesquiterpenoid chlorinated metabolites two of which had already been reported from the terrestrial fungus Coriolus consors [23]. 4. Fungi – The fungal endophyte of the marine brown alga Ascophyllum nodosum has been cultivated as an axenic culture in a freshwater synthetic medium containing bromide and, under these conditions, a number of brominated phenolic compounds were produced including 2,3-dibromo-4,5-dihydroxybenzyl alcohol (1803) [162] that is an established metabolite in a number of marine species of Polysiphonia and Rhodomela [164].A number of toxins from marine biota are in fact produced by the accompanying bacteria (references in [196]).

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In the light of these observations, the ultimate source of metabolites isolated from higher organisms may be equivocal. Whereas it seems safe to conclude that biota producing brominated metabolites have an essentially global distribution with prevalence in those from tropical and semi-tropical marine systems, it should be appreciated that knowledge of the distribution of these metabolites is dependent on the choice of sites that have been explored including their accessibility. 2.4 Use of Field Material Virtually all the structures that are discussed have been based on the analysis of field material and a number of factors should be considered: 1. Samples of putatively the same taxon may contain different metabolites. Some illustrative examples include the following: a) Samples of the red alga Plocamium cartilagineum from different geographical localities differed in the types of monoterpenoid components (references in [12]), and samples collected in New Zealand from closely similar localities contained quite different halogenated acyclic monoterpenoids [12]: one contained only a chlorinated bisnor monoterpenoid that had previously been reported, whereas the other contained a series of chlorinated and brominated acyclic monoterpenoids. b) Samples of Laurencia pacifica from the coast of California contain distinct metabolites that are retained in unialgal laboratory cultures, and its was suggested that there might have been a sterility barrier between the populations [86]. c) Laurencia nipponica is widely distributed along the coasts of Japan and on the basis of analysis of natural samples and laboratory cultures it was suggested that there were chemically distinct populations that could be described as chemical races [136]. d) It has been shown that the composition of metabolites in samples of the sea hare Aplysia dactylomela differed among individuals [228]. Attention is drawn to the predation of red algae by sea hares. 2. It may be difficult to detect and exclude the occurrence of more than one species in the material that is collected: it may therefore be difficult to associate unequivocally the structure of metabolites with a specific organism. This problem is exemplified for a collection of the cyanobacterium Symploca sp. used for isolating the anti-tumor agent dolastatin 10 [128]. 3. It is not generally possible to take into account environmental effects on the concentrations of metabolites, and there are unavoidable uncertainties associated with season of collection: a) It has been shown that, in the green alga Ulva lactuca in Eastern Australia, there is a marked yearly variation both in the content of 2,4,6-tribromophenol and in bromoperoxidase activity, and that these are not coincident [50]. The same effect has been observed in the red alga Corallina pilulifera [91].

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b) The seasonal activity of the bromoperoxidase in the red alga Corallina pilulifera varied by a factor of up to 20 although its activity was almost constant and it was shown that the specific activity of the enzyme is determined by the concentration of vanadium [95]. c) Chlorinated, brominated, and iodinated alkanes are produced by the tropical red alga Euchema denticulatum, and it was shown in unialgal cultures that the synthesis of diiodomethane and chloroiodomethane increased during combined stress of an increase in light intensity and in pH from 8.0 to 8.6 [146]. This was not significant for tribromomethane although bromination activity measured by the production of tetrabromophenol from phenol red increased with increasing light intensity or with mechanical stress [204]. Seasonal variations and environmental factors should therefore be taken into consideration in comparing levels of metabolites and enzymatic activity in samples collected at different times of the year. 4. Variations within the organisms may vary and two examples are given as illustration: (i) concentration of lanosol (2,3-dibromo-4,5-dihydroxybenzyl alcohol) in the thallus of the red algae Neorhodomela larix [19] and Rhodomela larix [167] vary within the thallus; (ii) concentrations of brominated furanones in the predator Aplysia arvula that originate in the red alga Delisea pulchra are greatest in the digestive glands that is consistent with their source as food from the alga [33]. 2.5 The Range of Biota The occurrence of organic fluorine compounds has been addressed [71], and they seem to be restricted almost entirely to higher plants. By contrast, the range of biota which produce organic bromine compounds is extremely wide and includes: – Eubacteria and cyanobacteria (blue-green algae) – Rhodophytes (red algae), brown algae (pheophytes), and green algae (chlorophytes) – Sponges and their associated population of for example, crustaceans and cyanobacteria, and green and red algae – Marine organisms including corals, acorn worms, sea-hares, and gastropods. A study from Eastern Australia identified bromophenols in marine algae [230], fish [231], marine polychaetes and bryozoans [232] (Table 2), and in gut samples of both pelagic and benthic carnivorous fish (Table 3). It may reasonably be concluded that algae were the major primary producers of 2,4,6-tribromophenol that proceeded through the food chain to polychaetes, bryozoans, and fish. It was even suggested that the flavor of prawns may be dependent on the bromophenol content. Excellent summaries of the distribution of organic bromine compounds among marine biota has been given by Gribble [68–70]. A valuable review by

85

Biological Effects and Biosynthesis of Brominated Metabolites

Table 2. Concentration (µg/g) of 2,4,6-tribromophenol in Southern Australian biota [230, 231,

232] Group Algae Red Brown Green

Taxon

Amphiroa anceps Pterocladiella capillacea Ecklonia radiata Phyhllospora comosa Enteromorpha intestinalis Ulva lactuca

Polychaeta

Barantolla lepte Cirrifgormia filigera Marphysa sanguinea Lumbreneris latreilli Naphtys australiensis Ceratoneris aeqiseitis Phyllodoce cf novaehollandiae

Bryozoa

Amathia wolsoni Bugula dissimilis Cladostephus ventricosa Orthoscuticella ventricosa

Concentration

1.3 1.3 0.19 0.28 1.4 1.2 8300 130 260 250 860 940 160 1.1 0.61 0.54 0.66

Table 3. Concentration (ng/g) of 2,4,6-tribromophenol in gut samples of groups of Australian ocean fish [231]

Feeding habit

Fish (sample)

Concentration

Pelagic carnivore

Platycephalus caeruleopunctatus (PC 94) Helicolenus percoides (HP 95) Centroberyx affinis (CA 95) Sillago flindersi (SF 94) Nemadactylus douglasii (ND 94) Paristiopterus labiosus (PL 95) Branchiostegus wardii (BW 94) Pseudocaranx dentex (PD 95) Meuschenia trachylepis (MT 94) Girella tricuspidata (GT 94) Kyphosus synneyanus (KS 95)

3.5 2.6 11 54 170 100 100 26 6 32 7

Benthic carnivore

Diverse omnivore Restricted omnivore

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Garson [59] directed to non-halogenated metabolites provides valuable comments on the biology and metabolites of a wide range of marine biota, and one by Coll [26] contains structures of many non-halogenated metabolites from octocorals. 2.6 Natural and Anthropogenic Compounds Although anthropogenic organic bromine compounds have been discussed by Allard and Neilson elsewhere in this volume, a few cautionary comments are given here on analytical procedures: 1. Mass spectrometric analysis of field samples of biota for environmental contaminants of anthropogenic origin may reveal organic bromine compounds of natural origin. Although some of these may resemble those that are undoubtedly anthropogenic, it is possible that they are naturally occurring metabolites of marine biota. Unless full-scale mass spectra are carried out, incorrect sources may readily be attributed. 2. Brominated diphenyl ethers represent an important group of anthropogenic compounds. They have been used as flame retardants, and are widely disseminated in the environment [36, 221]. They are distinguished by the lack of hydroxyl or methoxy substituents in the rings. In addition to these contaminants, a derivative bearing a single methoxy group was found in the herbivorous dugong [222] and corresponds to O-methylation of the naturally occurring brominated diphenyl ether metabolite found in the sponge Desidea herbacea (1869). Indeed, this organism contains a plethora of brominated diphenyl ethers bearing one or two hydroxyl or methoxyl groups (references in [18, 68]). These are putatively produced by oxidative coupling of naturally occurring di- and tribrominated phenols and catechols (section “Simple Phenols”), followed by O-methylation that is well established in brominated phenols from laboratory investigations [3]. 3. There is an additional source of compounds bearing single phenolic hydroxyl groups – and their O-methylated analogues. Although the metabolism of anthropogenic brominated diphenyl ethers by biota is unresolved, it may be mediated by cytochrome P450 hydroxylation of the aromatic ring and these metabolites compounds recovered from environmental samples. Indeed, one example of these has already been detected even though the orientation of the substituents has not been determined [73]. The origin of the methoxy compounds is, however, more simply rationalized on the basis of the arguments produced above. This may also be relevant to the metabolism of brominated dibenzo[1:4]dioxins that have been found during pyrolysis of brominated organic compounds, particularly 2,4,6-tribromophenol [211]. The naturally occurring hydroxylated and methoxylated polybrominated dibenzo[1:4]dioxins are discussed in the section “Oxaarenes – Furans, Pyrones, Coumarins, and Dibenzo[1,4]dioxins”. It should be pointed out, however, that the metabolism of PCBs by higher biota takes place by ultimate formation of methyl sulfones and this may also be the preferred detoxification mechanism for the polybrominated diphenyl ethers.

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The structures of brominated diphenyl ethers that are found during screening of environmental samples should therefore be established by full-scan spectra and not merely by selected-ion-monitoring. It is desirable that the presence of putative phenolic groups should be attempted by derivatization. 2.7 Source of the Halogen Although there is no point at issue for the source of bromide in metabolites produced by marine biota, many halogen-containing metabolites occur in terrestrial systems. For example, depsides and depsidones, and xanthones are widespread lichen metabolites, and an appreciable number of xanthones contain chlorine. Chloride-containing aerosols presumably supply this, and presumably account for the introduction of chlorine into the wide range of other chlorinated terrestrial metabolites given by Gribble [68–70] and into polymeric material from pristine areas [51, 148]. Attention is therefore drawn to the unusual existence of brominated metabolites in the lichen Acorospora gobiensis from Central Asia: (i) brominated fatty acids [175] and bromoallenic acids with terminal C=C=C. Br (R and S) groups [176] in the vicinity of Lake Issyk-Kul and (ii) brominated depsidones from the Tian-Shan Mountains in Uzbekistan [177]. Aerosols from saline or hypersaline sources presumably provide the necessary bromide. 2.8 Biological Activity and Ecological Significance of Metabolites One reason for the upsurge of interest in naturally occurring compounds is the increased realization of their valuable pharmacological activity. This is an extension of folk medicine based on the therapeutic use of higher plants, and this activity has spread to the marine environment. Only a few examples of the types of activity that have been recognized are given as illustration. Biological effects have been widespread, and in non-halogenated metabolites include, for example: (a) among briarane diterpenoids cytotoxic, anti-inflammatory, antiviral, insecticidal and immunomodulatory effects (references in [116]); (b) among staurisporine derivatives inhibition of kinase activity, platelet aggregation, smooth muscle contraction, and reversal of drug resistance (references in [184]). In the course of these investigations many brominated metabolites have been isolated by serendipity, and it has been shown that marine biota produce a plethora of brominated metabolites. A range of biota produces brominated metabolites and attention has been given to the important ecological significance of some of these: 1. Dehalogenation by Amphitrite ornata – It has been shown that the marine terebellid polychaete Amphitrite ornata produces no detectable halogenated metabolites, and that it synthesizes a dehalogenase that is able to oxidatively remove halogens from halogenated phenols, with fluoro-, chloro-, and bromosubstituents [21]. One of the enzymes (DHB 1) has been purified, and consists of two identical subunits (Mr 15,530) each containing heme and histidine as the proximal Fe ligand [119]. In the presence of H2O2, 2,4,6-tribromophenol

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is oxidized to 2,6-dibromobenzo-1,4-quinone. It has been proposed that the dehalogenase fulfils an ecological role in protecting sediment organisms from the toxicity of bromophenolic metabolites. 2. Recruitment of Nereis vexillosa – The polychaetes Thelepus setosus and Th. crispus produce 3,5-dibromo-4-hydroxybenzyl alcohol, and it has been shown in both laboratory experiments (mean concentrations 4.8 mg/l against control values of 0.08 mg/l) and field experiments laboratory (mean surface concentrations 4.0 mg/l against control values of 0.14 mg/l, and 0–8 cm cores 13.4 mg/l against 0.5 mg/l) that the contaminated sediments reduced significantly the recruitment of juvenile Nereis vexillosa larvae [238]. 3. The hemichordate Saccoglossus kowalewskii produces 2,4-dibromophenol and it has been suggested that this adversely affects aerobic bacterial metabolism in the walls of the burrows [107]. From the distribution of brominated phenols among polychaetes in temperate intertidal sand flats [47], it may plausibly be assumed that this is a general phenomenon. 4. The brominated diphenylmethanes avrainvilleol (1853) from the green alga Avrainvillea longicaulis [201] and vidalol A (1857) from the red alga Vidalia obtusaloba [233] (Fig. 1a) are deterrents to herbivorous reef fish. The halogenated norsesquiterpenoid kumepaloxane (946) (Fig. 1b) (section “Degraded Sesquiterpenoids”) that contains both bromine and chlorine substituents is exuded from the mucus of the bubble shell Haminoea cymbalum when they

Fig. 1. a–h. Metabolites displaying biological activity

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Fig. 1. d–h (continued)

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5.

6.

7.

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are disturbed and is a significant deterrent to carnivorous fish in both field and laboratory experiments [169]. A number of brominated pyrroles have been isolated from sponges of the genus Agelas sp. and Stylissa caribica. The metabolites include both monocyclic brominated pyrroles and dimeric analogues, and some are active as fishfeeding deterrents: in laboratory experiments using the bluehead wrasse Thalassoma bifasciatum, the order of activity was: sceptrin (1260) (Fig. 1e) > N-methyldibromidisophakallin > oroidin (Fig. 1d) (1242) > 4,5-dibromopyrrole-2-carboxylate (Fig. 1d) (1243) has been established [7]. It is significant that all these structures incorporate a 2-aminoiminazole structure that is widely distributed in marine sponges, several of which are fish deterrents.Additional examples include stevensine (1274) from Axinella corrugata [235], and the spirotetracyclic N-methyldibromophykellin (1269) from Stylissa caribica [8]. Further comments are given in the section “Pyrroles and Simple Indoles”. It is noticeable that all these structures incorporate a 2-aminoiminazole structure found in oroidin whose biosynthesis from 2-aminoiminazole-3-(2-propenylamine) may provide a plausible biosynthetic route to these brominated pyrroles. Two conclusions are worth noting from a study using the Thalassoma bifasciatum assay to detect fish feeding-deterrent activity in extracts from a range of Caribbean sponges [160]: (a) there was no correlation between this activity and toxicity and (b) although activity was uniformly high among species of Agelas, Aplysina and Verongula it was highly variable among other genera. In order to avoid any misconception, it is pointed out that fish feeding deterrence is not restricted to brominated metabolites based on pyrrole and has been found in diverse non-halogenated terpenoids from a range of biota. Examples include sesquiterpenoids from the gorgonian Heterogorgia uatumani [130], diterpenoids from the brown alga Dilophus okamurae [207], and sesterterpenoids from Glossodoris nudibranchs [53]. The brominated fimbrolides (1044–1046), their hydroxyl and acetoxyl analogues (1047–1060) and the cyclobutanes (1067–1069) (Fig. 1e) (1060) are furanone metabolites (section “Furanones”) in the red alga Delisea pulchra, accumulate in the sea hare Aplysia parvula and one of them functions as a deterrent to fish under field conditions [179]. Their plausible biosynthesis is discussed in the section “Furanones”. The stony coral Tubastraea micrantha produces an unusual 4-bromobenzoate ester (Fig. 1f) (1195) that functions as a deterrent to the destructive Crownof-Thorns seastar Acanthaster planci [2]. It has been stated [182] that T. micrantha contains high concentrations of 3-bromobenzoate whose biosynthesis is readily rationalized by anionoid bromination of prephenate or its precursor chorismate (Fig. 2a).Whereas the origin of the 4-bromobenzoate is less obvious, it would be attractive to rationalize its formation by an NIH-type shift of the 3-brominated chorismate (Fig. 2b). Metabolites of the red alga Odonthalia corymbifera including brominated catechols such as lanosol (1803) (Fig. 1g) and polybrominated diphenylmethanes containing ortho hydroxyl groups in both rings (1851) (Fig. 1g) functioned as potent feeding deterrents to juvenile abalone (Haliotis discus hannai) and sea urchins (Strongylocentrotus intermedius) [113].

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Fig. 2a,b. Biosynthesis from chorismate of: a 3-bromobenzoate; b 4-bromobenzoate

9. The brominated diterpenoids parguerol triacetate (Fig. 1h) (688) and the related deoxyparguerol 16-acetate from Laurencia saitoi were feeding deterrents to juvenile abalone (Haliotis discus hannai) [114]. 10. Tribromomethane – that is among the brominated alkanes produced in Corallina pilulifera by a bromoperoxidase – is important in eliminating epiphytic algae and its seasonal variation paralleled that of superoxide dismutase [154]. 11. The polybrominated dibenzo-p-dioxins from the sponge Dysidea dendyi have been assayed for inhibition of cell division of the fertilized eggs of the sea urchin Strongylocentotrus intermedius and, as would be expected, the hydroxylated metabolites were much more toxic than their methoxylated analogues [216].

3 Biological Bromination and Iodination It is important to emphasize the word “hypothesis” in the title of this essay. Although a great deal has been established on the mechanisms for biological halogenation of organic compounds, the number of examples in which the mechanism for the introduction of bromine into naturally occurring metabolites has only relatively seldom been established. In the absence of this, it seems plausible to extrapolate from mechanisms which have been established for chloroperoxidase and bromoperoxidase that catalyze the reaction of organic substrates with halide and H2O2. Nonetheless, considerable caution should be exercised: 1. Although bromoperoxidases have been isolated from Streptomyces aureofaciens none of them are involved in the synthesis of 7-chlortetracycline from the same organism [29]. 2. Elucidation of the structure of enzymes that are capable of catalyzing chlorination has shown that not all of them contain metals: these systems are explored in greater detail in subsequent paragraphs.

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It has emerged that biohalogenation is a much more complex reaction than it might have seemed some years ago, and that biohalogenation is mediated not only by conventional haloperoxidases. In this essay, although bromination and halogenation have been assumed to occur primarily by reaction of the substrate or its precursor with cationoid bromine (Br+), it has also been argued here that anionoid bromination by Br– is also plausible for substrates with a low electron density. Indeed, it has been shown that these mechanisms differ primarily in the substrate for initial oxidation–peroxidase-oxidation to Br+ or FADH2-catalyzed oxidation of the substrate. A number of issues should be considered in evaluating the hypotheses that are erected here. Although examples of organic chlorine metabolites are used for illustration, the mechanism for their synthesis may be different from that of bromination/iodination; the examples given for antibiotics in the section “Anionoid Halogenation” are illustrative. Although there are two broadly different mechanisms for the biosynthesis of organic bromine compounds, involving formally Br+ or Br–, an attempt is made to show that the differences depend primarily on the stage at which oxidation occurs. The stage at which halogenation occurs may also differ: 1. Most commonly this is the final step, for example, in the synthesis of brominated phenols, pyrroles, and indoles. 2. Halogenation may accompany the synthesis of the carbon skeleton, for example during cyclization of polyepoxides (section “Epoxide-Derived”). It is also possible, however, that halogenation of a simple precursor takes place and that this is carried through the biosynthetic process.A paragraph in the next section is devoted to exploring this the biosynthesis of trichloromethyl-containing metabolites that putatively originate from leucine. 3.1 Cationoid Halogenation In addition to the nature of the halogenating agent, the stage in the biosynthetic pathway at which halogenation takes place is important, and the biosynthesis of trichloromethyl metabolites is used as an illustration. 3.1.1 Metabolites Containing the Trichloromethyl Group Metabolites containing the trihalomethyl group are well represented among aliphatic metabolites and their biosynthesis is readily rationalized on the basis of cationoid halogenation of an enolate precursor. A group of metabolites are produced by the sponge Dysidea or its cyanobacterial associates (sections “Geographical Distribution” and “Bacteria and Cyanobacteria”, and [79]), and contain the trichloromethyl group in positions that are not easily accommodated by production from enolates. There are a number of these metabolites, containing thiazole (1102–1107) and barbamide [158], diketopiperazines (1110–1112), and [54], pyrrolidones (1101; 1113–1115), and the simplest repre-

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Fig. 3. a Trichloromethyl metabolites. b Aeruginosamide from Microcystis aeruginosa

sentative herbacic acid [131] (Fig. 3a). Three hypotheses for their biosynthesis are considered, although there is no experimental evidence to confirm them – or otherwise: 1. Free-radical mechanism – It has been suggested [80] that a free radical mechanism is involved in which abstraction of a hydrogen radical produces a methylene radical that reacts with HOCl – presumably produced by a chloroperoxidase – to produce the product, in the case of trichloromethyl groups by successive cycles. A variant of this has been proposed [131] in which chlorination of leucine itself takes place and this is then funneled through further anabolic reactions. Although there is currently no evidence supporting free radical chlorination, this possibility should be borne in mind in view of the important role of free radicals in a range of enzymatic reactions [198]. 2. Cationoid chlorination of leucine precursors – The biosynthetic pathway for leucine (Fig. 4) that suggests a possible alternative involving cationoid chlorination of an activated methyl group, in, for example, pyruvate – or acetolactate formed from it by acetohydroxyacid synthase. There are attractive features of this hypothesis which could plausibly be extended to the range of metabolites originating in valine, and this seems a simpler and less provoca-

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Fig. 4. Biosynthesis of leucine

tive suggestion than that of the hitherto unsupported role of free radical chlorination as a terminal step in the biosynthesis [80]: a) The ready formation of metabolites containing dichloromethyl or trichloromethyl groups that is consistent with established aliphatic metabolites formed from enolizable keto groups, and the less likely introduction of six chlorine atoms at the same terminal group. b) The stereospecific preference for chlorination of the pro-S-methyl methyl group in barbamide that suggests dissimilarity in the environment of the methyl groups [80]. c) The occurrence of metabolites in which both of the chlorinated leucine entities are equally chlorinated (2 or 3 chlorine substituents). d) The isolation of the simplest trichlorinated leucine metabolite herbacic acid from Dysidea herbacea [131]. It is noteworthy that in aeruginosamide (Fig. 3b) [121] isolated from the unicellular cyanobacterium Microcystis aeruginosa, the terminal part of the leucine structure is replaced by dimethylallyl. It is therefore possible that allylic halogenation would produce the metabolites, even though there appears to be no evidence for polyhalogenation of allyl groups. 3.1.2 Cationoid Halogenation The most widely used mechanism of bromination involves the catalysis by bromoperoxidase of the reactions between H2O2 and bromide with the formation of HOBr – formally Br+. The reactant then undergoes an electrophilic reaction with centers of high electron density, for example those in enols, pyrroles, indoles, and phenolic compounds.Allylic bromination is widely observed and may most simply be rationalized on the basis of rearrangement of the double bond. Cationoid bromination is also involved in the biosynthesis of oxacins and A-ring brominated diterpenoids (sections “Laurencia Metabolites”) in which bromination occurs concomitantly with synthesis of the cyclic structures.

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This mechanism finds support from the structures of bromine-containing metabolites. For example, both 2,4,6-tribromophenol and 2,3,4,5-tetrabromopyrrole are readily produced chemically by electrophilic (cationoid) bromination, and are naturally occurring metabolites. In addition, and consistent with this mechanism, the introduced halogen atoms are located at positions ortho and para to the phenolic groups in virtually all naturally occurring bromophenolic metabolites. Substrates with a high electron density such as ethenes, phenols, and pyrroles are readily brominated chemically and are also found in biota, and are produced by formally comparable mechanisms.Additional examples include indoles from species of the acorn worm Ptychodera that are brominated at the 3and 6-positions (1301) and of the red alga Rhodophyllis membranacea that are brominated at the 2-, 3-, 4-, and 7-positions (1313), and brominated pyrones from the red alga Ptilonia australasica that are noted in the section “Oxaarenes – Furans, Pyrones, Coumarins, and Dibenzo[1,4]dioxins”. The unusual 3-bromo-1,4dimethyl-7-isopropylazulene noted in the section “Synthesis of the Ring” in an undisclosed benthic gorgonian [122] is presumably synthesized by bromination of the intermediate from (E,E)-farnesyl PP by Br+. In addition, Br+ may function as an oxidizing or dehydrogenating agent. 3.2 Anionoid Halogenation Care should be exercised in postulating universal bromination by Br+ since there is an alternative reaction that uses bromide without direct oxidation to Br+. There are three classes of this synthetic route: 1. The biosynthesis of methyl bromide and methyl iodide [5] involves methylation by formally CH3+ in the form of S-adenosyl methionine, but is clearly limited to monobromination: polybrominated methanes that are widely produced by red algae cannot be produced by this mechanism, but rather by cationoid bromination (section “Cationoid Halogenation”). 2. An example of anionoid bromination is provided by 1,4-dihydroxylated benzenoids such as the brominated hydroquinones and brominated benzo-1,4quinones produced by acorn worms belonging to the genus Ptychodera. There are several reasons to support this hypothesis that is discussed further in the section “Quinones and Related Compounds”: a) Species of Ptychodera produces brominated benzo-1, 4-quinones (209–212) together with a series of related brominated cyclohexenones (215–217). b) Quinones are susceptible to attack by nucleophiles and, for benzo-1, 4quinone and bromide,the sequential synthesis of 2-bromo- and 2,6-dibromo-1, 4-dihydroxybenzene by bromination and oxidation is readily visualized. Since iodide is generally more reactive than bromide as a nucleophile, this mechanism might be important for certain classes of iodinated metabolites. These possibilities arise only in compounds such as quinones, and not in other phenols and related compounds. Anionoid bromination involves a series of oxidations and reductions so that the fea-

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sibility of these reactions is dependent on the redox potentials of the quinones. 3. There are unequivocal examples in which halogenation is accomplished in the demonstrable absence of haloperoxidase activity. Important examples include the synthesis of chlortetracycline [29] and pyrrolnitrin that is discussed in the section “FAD2-Dependent Chlorohalogenases”. It has been shown that tryptophan-7-chlorogenase in Pseudomonas fluorescens involves NADH and a flavin reductase and it was postulated that the halogenation proceeded via oxidation of the substrate by monooxygenation involving the flavin hydroperoxide [105]. Further consideration provides a unification of these mechanisms: – For reactions in which peroxidases are involved, the enzyme oxidizes the halide to hal+ (or its equivalent hal2) that is the active halogenating agent. – When haloperoxidase is absent, halogenation involves oxidation of the substrate by an oxygenase followed by its reaction with the halide anion. The two differ therefore only at the stage and the mechanism by which oxidation occurs. In addition, the plethora of halogenated metabolites that could be formed from electron-rich substrates by reaction with hal+ is compatible with facile oxidation followed by reaction with hal–. Examples of this are given for brominated quinones (section “Quinones and Related Compounds”). The two mechanisms are not exclusive and either may be invoked. For example, although the biosynthesis of 3-bromo-4-hydroxyanthraquinone-1-carboxylate and 3-bromobenzoate [182] might involve anionoid bromination, it could also be formed by cationoid bromination by analogy with the Graebe and Liebermann synthesis in 1870 of 2,3-dibromoanthraquinone. In this case, additional care should also be exercised in view of the differing pathways used for the biosynthesis of anthraquinones: acetate-malonate and shikimate-mevalonate. An example is given in the section “Biphenyls and Diphenyl Ethers”. All of these issues have a direct bearing on the biohalogenation of these compounds, and the structures of coupling products including diphenylmethanes, diphenyl ethers, and depsidones. 3.3 Synthesis of H2O2 Hydrogen peroxide is needed for halogenation by haloperoxidases and halide anions, and can be produced by a number of reactions including those of the cells themselves. Since, however, hydrogen peroxide is toxic to cells, any excess is normally destroyed by the action of catalase or peroxidases: indeed these may be synthesized simultaneously with peroxidases [93]. The synthesis of H2O2 can be accomplished by a number of reactions, and in yeasts the enzymes are located in the peroxisome. The examples given illustrate the formation of H2O2 by oxidases that are flavoproteins produced by both prokaryotes and eukaryotes: 1. It is produced during oxidation of primary amines in Klebsiella oxytoca (ATCC 8724) and Escherichia coli (ATCC 9637) by a copper quinoprotein amine oxidase [72].

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2. Oxidation of methanethiol to formaldehyde and sulfide is catalyzed by an oxidase in Hyphomicrobium sp. strain EG and is accompanied by the synthesis of H2O2. 3. Oxidation of cholesterol to cholest-4-ene-3-one with the production of H2O2 is carried out by an oxidase in Brevibacterium sterolicum and Streptomyces sp. strain SA-COO [156]. 4. D-Aminoacid oxidase has been isolated from a number of yeasts and produces H2O2. 5. Yeasts belonging to the genera Candida and Endomycopsis are able to degrade alkanes via alkanoates. The degradation of their CoA esters is accomplished by an oxidase with the production of H2O2. 6. Extracellular H2O2 is required for the activity of peroxidases in white-rot fungi, and is produced in Phanerochaete chrysosporium by glyoxal oxidase or by an aryl-alcohol oxidase in Bjerkandera adusta.

4 Haloperoxidases 4.1 Reactions In this essay, the term haloperoxidase will be used to cover not only classical haloperoxidases but also lactoperoxidase, and eosinophil peroxidase that are endowed with comparable biosynthetic activity – and by extension myeloperoxidase. The halogenation of organic substrates by peroxidases in the presence of halides and H2O2 is complex and it has been shown that enzymes not generally considered as halogenating enzymes are also able to carry out this reaction (section “Halogenation by Metal-Free Enzymes”). Attention will be directed to all these systems. In the presence of halide anion and hydrogen peroxide these enzymes accomplish halogenation through the reaction of electrophilic Hal+ with the substrate Sin the presence of hydrogen peroxide: S–H + H Æ H2O2 + HalH Æ S–Hal + 2 H2O The spectrum of active products may, at least for myeloperoxidase, be extended to nitrite that is discussed later. The overall reaction is supported by the fact that, in the absence of a substrate, chloroperoxidase from the fungus Caldaromyces fumago catalyzes the formation of Cl2 and Br2 from chloride and bromide respectively [123, 144]. It will therefore be assumed that Br+ (or OBr–) is the active agent in the bromination of the compounds discussed in this essay. The extent to which the substrate is bound to the active site of the enzyme determines whether halogenation is carried out at the active site or after release of the halogenating agent.

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4.2 Enzymes Assay of haloperoxidase activity is generally carried out using a surrogate substrate: 1. Most frequently using 2-chloro-5, 5-dimethylcyclohexan-1,3-dione (monochlorodimedone) that is brominated at the 2-position. 2. Phenol red which at pH 7 produces the dichloro compound from chloroperoxidase or the tetrabromo compound from bromoperoxidase [127] and references in [149]. 3. Aniline that is brominated at positions 2,4 and 6 positions.An exception is the direct assay of iodoperoxidase activity by reaction with I– to produce I3– [4]. Enzyme activity is not, however, necessarily correlated with the presence of brominated metabolites, and may differ between specimens collected at different times of year and location [50]. Haloperoxidases have been isolated from a range of organisms including bacteria, fungi, and red and brown algae, annelids and acorn worms (Table 4). The structures of the enzymes differ considerably, and may contain heme (bacteria, fungi, and algae), vanadium (V) (fungi and algae) or contain no metal prosthetic groups (bacteria) (Table 5). Since eosinophil peroxidase from mammalian sources is capable of halogenation in the presence of halide and H2O2, mammalian peroxidases such as eosinophil peroxidase and myeloperoxidase have potentially important pathological functions through formation of reactive halogenating or oxygenating

Table 4. Distribution of bromoperoxidase activity

Bacteria

Fungi Algae

Rhodophyta

Pheophyta

Annelids Acorn worms

Chlorophyta Polychaeta Hemichordata

Streptomyces lividans Str. phaeochromogenes Str. aureofaciens Str. griseus Pseudomonas aureofaciens Ps. pyrrocinia Ps. pyrrolnitrica Curvularia inaequalis Cystoclonium purpureum Rhodomela larix Corallina (several species) Cystoclonium purpureum Ascophyllum nodosum Laminaria (several species) Macrocystis pyrifera Ecklonia stolonifera Penicillus capitatus Thelepus setosus Ptychodera flava

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Type of enzyme

Group

Organism

Halogen

Reference

Heme

Bacterium

Streptomyces phaeochromogenes Cystoclonium purpureum Penicillus capitatus Penicillus capitatus Caldariomyces fumago Notomastus lobatus Ascophyllum nodosum Ecklonia stolonifera Laminaria digitata Laminaria saccharina Macrocystis pyrifera Corallina pilulifera Corallina pilulifera Curvularia inequalis Pseudomonas fluorescens Pseudomonas putida

Br

[218]

Br

[161]

Br Br Cl Cl Br Br Br Br Br Br Br Cl Cl

[9] [134] [144] [22] [223] [76] [100] [32] [194] [111] [94] [190] [105]

Br

[92]

Pseudomonas pyrrociniacus Streptomyces aureofaciens Acinetobacter calcoaceti

Cl

[234]

Br

[227]

Br

[102]

Rhodophyta

Flavin Vanadium

Non-heme/flavin Non-heme/ nonflavin Non-heme/ nonmetal

Chlorophyta Chlorophyta Fungus Polychaete Phaeophyta Phaeophyta Phaeophyta Phaeophyta Phaeophyta Rhodophyta Rhodophyta Fungus Bacterium Bacterium Bacterium

species (references in [57, 240]). Examples of reactions mediated by mammalian lactoperoxidase are given later in this section, and of the eosinophil peroxidase in the sections “Simple Phenols” and “Iminazoles, Quinolines, Pyrimidines, Pyrrolopyrimidines, and Purines”. There has been an upsurge of interest in myeloperoxidase in neutrophils as a result of its role in mediating the physiological effect of nitric oxide [41] produced from L-arginine by nitric oxide synthetase [109]. Nitrated aromatic metabolites including 3-nitrotyrosine [41, 217] and 8-nitro-2¢-deoxyguanosine [17] that are produced have been used as markers for the physiological production of nitric-oxide derived oxidants although the nature of the active species has not been unequivocally established. Suggestions have included NO2, peroxynitrite [181], or NO2+ and, although the last is not favored [217], it is conceptually attractive by analogy to halogenation with Hal+. For the sake of completeness, mention is made of the dehaloperoxidase from the polychaete Amphitrite ornata that carries out the replacement of halogen substituents (fluorine, chlorine, or bromine) in halogenated phenols in the presence of H2O2 with production of benzo-1,4-quinones and halide ion. The crystal struc-

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ture has been determined and suggests the mechanism by which the enzyme functions [119]. It is worth drawing attention to a number of additional features of haloperoxidases: 1. The enzymes may, or may not, display specificity for chloride or bromide or iodide: for example, bromination and iodination activity may be displayed by chloroperoxidase as noted below, and conversely bromoperoxidases may not be able to accomplish chlorination. 2. Lactoperoxidase is limited to reactions involving bromide or iodide in the presence of H2O2. Illustrative examples of synthesis mediated by these include: (a) E-prelaureatin (825) from 3Z,6S,7S laurediol [56], (b) deacetylalurencin (822) from 3E,6R,7R laurediol [55], (c) laurallene (829) from E-prelaureatin, (d) laureatin (823) from Z-prelaureatin [90] (Fig. 5), and (e) iodolactones (1019, 1020) from polyenoic acids produced though iodonium cations [14] (Fig. 6a,f) and cyclic lactones (1708, 1709) from arachidonic acid (C20 ∆5,8,11,14) [13], Fig. 6a,b)

Fig. 5a–d. Brominations with lactoperoxidase, H2O2, and bromide: a E-prelauretin from 3Z,6S,7S-laurediol; b deacetyllaurencin from 3E,6R,7R-laurediol; c laurallene from E-prelaureatin; d laureatin from Z-prelaureatin

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Fig. 6a,b. Biosynthesis of iodinated lactones by lactoperoxidase, H2O2, and iodide: a 1019;

b macrolide lactone 1708

3. Interest has emerged in the use of haloperoxidase to accomplish potentially valuable reactions with synthetic substrates: a) For isolated double bonds reaction with bromide, H2O2, and chloroperoxidase produces stereospecific formation of trans-bromohydroxy compounds by reaction of the initially formed bromonium ions (Fig. 7a) [150]. b) Ring opening of cyclopropanes may yield 1-halogen-3-hydroxylated metabolites [61] (Fig. 7b). c) Neighboring carboxyl group may facilitate bromination at a sterically accessible position (Fig. 7c) [171]. Although bromination was quantitatively observed for 2-exo-methylbicyclo[2.2.1]hept-5-ene-2-endo-carboxylic acid

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Fig. 7a–c. Chloroperoxidase-catalyzed synthesis using synthetic substrates: a 9-dehydroprogesterone; b methylcyclopropane; c bicyclo[2:2:1]cyclohexene carboxylate

and for bicyclo[3.2.0]hept-2-ene-6-one there was no evidence for chlorination. It is not known whether this selectivity is limited to synthetic substrates. 4. Even when enzymatic activity is present, there may be no evidence for the presence of halogenated metabolites in the organism, for example, the chloroperoxidase from Streptomyces lividans [11]. 5. Chloroperoxidase is capable of carrying out a number of stereospecific oxidations using tert-butyl hydroperoxide as oxygen source, for example, epoxidation of 3-methylbutenols [120], hydroxylation of alkynes [88], oxidation of cyclopropylmethanols [87], and the vanadium bromoperoxidase oxidation of 1,3-ditert-butylindole [135]. Of greater relevance to this essay are the dimerization of phenols to biphenyls that are discussed in the section “Simple Phenols”. Consideration is first given to haloperoxidases that contain either Fe or V. Enzymes that lack these metals such as the bromoperoxidase from Pseudomonas putida that contains neither heme nor vanadium and whose activity is stimulated by Co2+ [92] are discussed in the section “Halogenation by Metal-Free Systems”: 1. The chloroperoxidase from the fungus Caldaromyces fumago (generally referred to as CPO without qualification) has been isolated in pure form. It contains ferroprotoporphyrin IX, and displays, additionally, peroxidase and catalase activities. In the absence of organic substrates it catalyzes the formation of Cl2 and Br2 from chloride and bromide respectively [123, 144]. X-ray analysis shows the presence of a thiol proximal ligand (Cys29) [202] that is found also in cytochrome P450 so that both halogenation and oxygen insertion re-

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Fig. 8a,b. Bromination by extracellular lignin peroxidase from Phanerochaete chrysosporium:

a ring bromination of 3,4-dimethoxybenzyl alcohol; b side-chain bromination of 3,4-dimethoxycinnamate

actions can be carried out each involving Fe4+=O [203]. In addition the acidbase catalytic group that participates in fission of the peroxide O-O bond is glutamate (Glu183) rather than histidine in conventional peroxidases. Chloroperoxidase in the presence of H2O2 is also capable of oxidizing styrene stereospecifically to the epoxide [159]. A purely schematic representation of its mode of action is given by [202, 203]: Fe3+ – OH2 + H2O2 Æ Fe3+ – OOH + H3O+ Fe3+ – OOH + H+ Æ Fe4+ = O + H2O Fe4+ = O + Cl– + H2O Æ Fe3+ – OCl(H) + H3O+ Fe3+ = OCl(H) + H2O Æ Fe3+ – OH2 + HPCl H2O2 + Cl–+H+ Æ HOCl + H2O 2. The major isozyme of extracellular lignin peroxidase (LipII) contains heme Fe and in the presence of H2O2 and Br– or I– is able to bring about bromination and iodination; both fluoride and chloride inhibit bromination [172]. It is capable of mediating both bromination of the aromatic ring of 3,4-dimethoxybenzyl alcohol followed by oxidation (Fig. 8a) as well as side-chain bromination of 3,4-dimethoxycinnamic acid (Fig. 8b). 3. The structure of the vanadium enzyme in the terrestrial fungus Curvularia inaequalis has been determined by X-ray analysis [139]. The trigonal bipyramidal structure consists of four oxygen atoms hydrogen bonded to aminoacids and one N from histidine496. The oxygen atoms are hydrogen-bonded to pairs of aminoacids: arg360 and argN1490; gly403 and lys353; and ser402 and argN2490. A schematic representation is given and the mechanism may be deduced from the structures of the native enzyme and the peroxide (Fig. 9) that makes it possible to present details of the mechanism of the enzymatic activity. A review of vanadium peroxidases has been given [16]:

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Fig. 9. Structure of vanadium chloroperoxidase from Curvularia inaequalis

H2O2 + VOH – enz Æ V.O2H – enz + H2O V.O2H – enz + HO– Æ V.[O – O] – enz + H2O V.[O – O] – enz + HCL Æ V.[O – O].Cl – enz V.[O – O].CL – enz + H2O Æ VOH – enz + HOCl+HO– H2O2 + HCl Æ HOCl + H2O 4. A number of bromoperoxidases have been isolated and these are briefly summarized: a) Bromoperoxidase, which has been isolated from Pseudomonas aureofaciens ATCC 15926, also displays peroxidase and catalase activities, and contains ferriprotoporphyrin IX [219]. b) A particulate enzyme has been isolated from the red alga Cystoclonium purpureum [161] and displays bromoperoxidase or peroxidase activity depending on the pH of the assay: in the presence of 2,5-dihydroxyphenylacetic acid with 4-hydroxybenzyl alcohol as substrate, 3-bromo-4-hydroxybenzyl alcohol was produced at pH 4.7, but at pH 5.4 to 6.7 increasing amounts of 4,5-dihydroxybenzyl alcohol were produced at the expense of bromination. c) Four different bromoperoxidases have been isolated from Streptomyces griseus [243]. Only one of them, however, contains ferriprotoporphyrin IX and displays peroxidase and catalase activities. This illustrates that there are two different groups of enzymes of which one lacks heme prosthetic groups as discussed in the section “Halogenation by Metal-Free Enzymes”. d) The iodoperoxidase from the brown seaweed Saccorhiza polyschides contains three isoforms and vanadium (V) is essential for activity [4]. e) Search for homologies with the vanadium-dependent bromoperoxidase from the red alga Corallina pilulifera [189] revealed highly conserved sequences with the bromoperoxidase from the marine brown alga Ascophillum nodosum and the chloroperoxidase from the fungus Curvularia inaequalis.

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5. The chloroperoxidase from the polychaete Notomastus lobatus [22] consists of a flavoprotein and heme protein in a 1:1 ratio and is able to oxidize chloride, bromide and iodine in the presence of H2O2: it produces 2-bromo-, 2,4dibromo-, and 2,4,6-tribromophenol from phenol in the presence of bromide and H2O2.

5 Halogenation by Metal-Free Enzymes 5.1 FADH2-Dependent Chlorohalogenases The biosynthesis of halogenated metabolites has revealed that not all of them are mediated by conventional haloperoxidases and that some contain neither a prosthetic group nor metals such as Fe and V that have been discussed above. For example, the chloroperoxidase from Streptomyces lividans does not contain metal ions and in the absence of absorption in the Soret band of the spectrum is assumed not to contain protoporphyrin [11]. In addition, it is not associated with the synthesis of chlorinated metabolites. Both these features [15] also occur in Serratia marcescens.Although bromoperoxidase activity in Pseudomonas putida and P. aeruginosa is shown by bromination of monochlorodimedone, aniline, and phenol red, none of them had a heme-type peroxidase [93]. Pyrrolnitrin is a secondary metabolite of Pseudomonas fluorescens with strong antifungal activity and the genes involved in its synthesis have been designated prnA, prnB, prnC, and prnD [75]. The biosynthetic pathway of pyrrolnitrin is shown in Fig. 10 together with the appropriate designation for each of the enzymes [108]. The activity of prnA also carried out chlorination of tryptophane at C7, and the enzyme has been partly purified [105]. It consists of two components and requires NADH and FAD for activity. The mechanism of chlorination

Fig. 10. Biosynthesis of pyrrolnitrin

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Fig. 11. Biosynthesis of 7-chlorotryptophane

is given in Fig. 11 and involves an anionoid reaction between chloride and a putative indole-6,7-epoxide followed by aromatization. 5.2 Peroxyacid-Mediated Synthesis of Active Halogen In other studies, chloroperoxidase activity has been found in degradative enzymes: 1. The enzyme in the bacterium Rhodococcus erythropolis NI86/21 that is involved in the degradation of thiocarbamate herbicides is a non-heme haloperoxidase that does not occur in other strains of rhodococci that can degrade thiocarbamates [35]. 2. The 3,4-dihydrocoumarin hydrolase in Acinetobacter calcoaceticus strain F46 is able to brominate monochlorodimedone in the presence of H2O2 and 3,4dihydrocoumarin or acetate or butyrate [102]. It was proposed that acyl peroxides be produced from an acylated serine site on the enzyme by the action of H2O2 and oxidized bromide to the active brominating agent BrO–. This is analogous to the mechanism proposed earlier [168]. 3. The gene bearing the esterase from Pseudomonas fluorescens was expressed in Escherichia coli and the enzyme displayed both hydrolytic and bromoperoxidase activity [165]. In addition to haloperoxidases containing heme or V, there are a number of bacterial halogenating enzymes that do not contain metals. The X-ray analysis of that from Streptomyces aureofaciens (ATCC 10762) revealed a catalytic triad ser98, asp228, and his257 that is normally associated with hydrolytic activity [81]. Bromination in the presence of H2O2 takes place only in buffer containing acetate or propionate, or when peracetic acid was used in place of H2O2. It was therefore proposed that the halogenation occurred by peracetic acid oxidation of Br– to Br2

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that produced racemic bromohydrins by reaction with activated double bonds [168]. In addition, the enzyme possesses esterase activity towards 4-nitrophenyl acetate, and the bromoperoxidase has been characterized as an a/b hydrolase. This proposal is consistent with a number of other non-halogenation reactions that have been observed with enzymes possessing peroxidase activity.

6 Halogenated Alkanes In this section only halogenated methanes will be considered. Other polyhalogenated alkanes are considered in the section “Other Structures”. 6.1 Monohalogenated Methanes Many simple chloroalkanes and bromoalkanes are produced biologically, and it has been speculated that their production is globally significant. It is convenient to begin with the biosynthesis of methyl chloride and methyl bromide that differs from that of virtually all other organic bromine compounds. Although CH3Br may plausibly be formed by methylation of bromide by analogy with the synthesis of methyl chloride from chlorideby the fungus Phellinus pomaceus [78], this is not a plausible route for the more highly brominated methanes including CH2Br2 and CHBr3 that are produced during laboratory storage of brown, red and green seaweeds [153]. Methyl iodide also has a global distribution [224]. There has been enormous concern over ozone destruction in the troposphere, and attention has been directed to reactive halogen compounds including methyl chloride and methyl bromide. It has been unambiguously established that methyl chloride is produced by a range of fungi [226] and methyl bromide by the marine brown algae Macrocystis pyrifera [132] and by the diatom Nitzschia stellata [199]. A number of bacteria have been isolated that are able to methylate iodine with production of methyl iodide [5]: these include both terrestrial (Rhizobium sp. strain MRCD 19, Variovorans sp. strain MRCD 30) and marine (Alteromonas macleodii strain IAM 12920, Photobacterium leiognathi strain NCIB 2193, and Vibrio splendidus strain NCIMB 1) organisms. S-Adenosyl methionine transferase activity has been demonstrated in the white-rot fungus Phellinus pomaceus and the red alga Endocladia muricata [241], so that the source of the methyl group has been established: the methyl halides are formed by nucleophilic attack of halide ion at the electrophilic CH3S+ group. It is worth noting, however, that most red and brown algae produce the more highly brominated methanes that cannot be produced by this mechanism. They are plausibly biosynthesized by successive bromination of the enols of C2 or C3 units containing C=O groups followed by loss of C1 (CH2O from CH3CHO and CH3CHO from CH3.CO.CH3). This noted again in the following section. The cardinal environmental issue is whether their biological production is consistent with global measurements of the concentrations of these compounds

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in the oceans and the atmosphere. The issue has become much less transparent in recent years with better measurements, and there are two major uncertainties for both methyl chloride [106] and methyl bromide [193] resulting largely from more extensive and better measurements of the fluxes of these compounds: (1) are the quantities of biogenically produced compounds significant on a global basis and (2) is the ocean is a sink or a source of these compounds? It therefore seems wise to accept a degree of uncertainty in the interpretation of the measurements, and their significance. In an assessment of fluxes and tropospheric transport a number of issues should be addressed. In a broader perspective, it is convenient to arrange these according to principles that are applicable to all compounds that are volatile. 6.2 Polyhalogenated Methanes Most red and brown algae produce, however, the more highly brominated methanes that are unlikely to be produced by this mechanism. These are plausibly biosynthesized by successive bromination of the enols of C-2 or C-3 units containing C=O groups followed by loss of C1 (CH2O from CH3CHO and CH3CHO from CH3.CO.CH3) with concomitant formation of CH2Hal2, and CHHal3. This mechanism is supported by the biosynthesis of CH2Br2 and CHBr3 when the bromoperoxidase from the green macro alga Ulvella lens was incubated with oxalacetate, bromide and H2O2 [155]: the eightfold stimulation in enzymatic activity by added Co2+ is noteworthy. It has been suggested [186] that in California this alga is an epiphyte on Laurencia that is well established as a source of brominated metabolites. Other reactions could produce tribromomethanes: 1. Trichloromethane is formed during aqueous chlorination of a range of phenols including 2,4,6-trichlorophenol [58], and tribromomethane during chlorination of water containing natural organic matter in the presence of bromide. Since 2,4,6-tribromophenol is formed from phenol by the action of the chloroperoxidase from Notomastus lobatus (section “Enzymes”) in the presence of bromide and H2O2 [22] it is plausible to suggest that this could be an alternative source of tribromomethane, brominated acetones (e.g., 54) that are found in the alga Asparagopsis taxiformis and dibrominated acetate that has been found in archival firn from Antarctica [220] (Fig. 12). 2. The biosynthesis of long-chain ketones from acetate is noted in the section “Polyhalogenated Higher Alkanes” and bromination of these followed by fission could produce tribromomethane.

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Fig. 12. Ring fission of phenol during cationoid bromination

7 Terpenoids and Steroids Terpenoids encompass a wide range of structures. The group comprises C10 monoterpenoids, C15 sesquiterpenoids, C20 diterpenoids, C25 sesterterpenoids, C30 triterpenoids, and C40 tetraterpenoids formed by cyclization of linear unsaturated precursors and often with rearrangements. The basic structures are derived from mevalonic acid–monoterpenoids from geranyl pyrophosphate (GPP), sesquiterpenoids from farnesyl pyrophosphate (FPP), diterpenoids from geranyl geraniol pyrophosphate (GGPP), triterpenoids by condensation of FPP groups, and tetraterpenoids by condensation of two GGPP groups. The less common sesterterpenoids are formed from diterpenoids by addition of dimethylallyl pyrophosphate (Fig. 13). C15 oxacins are not terpenoid and are derived from fatty acids synthesized from acetate. Virtually all of the brominated compounds are produced by cationoid bromination with Br+, and for di- and triterpenoids by bromination during construction of the rings. This is the preferred mechanism for diterpenoids that do not usually have an oxygen function at C3. On the other hand, for triterpenoids that are oxygenated at C3, bromination could also take place via the corresponding ketone. Since there are a large number of terpenoids of marine origin that contain bromine, only some representatives of each of the major classes have been used as illustration, and some of these contain both chlorine and bromine. The synthesis of these is discussed below, and if only chlorinated metabolite have so far been identified these are given as illustrations of cationoid halogenation.

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Fig. 13a–d. Schematic biosynthesis of a: isopentenyl PP and dimethylallyl PP; b monoter-

penoids; c sesquiterpenoids; d diterpenoids

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7.1 Monoterpenoids

These are widely distributed among red algae in species of Plocamium, Portieria (syn. Chondrococcus, Desmia) hornemanni, and Ochtodes and are represented by both acyclic and monocyclic structures. Acyclic monoterpenoids containing both halogens are numerous, and include those isolated from Plocamium hamatum (287) (Fig. 14a) and P. cartilagenium (257 and 259) differing in the configurations at C5 and C6) (Fig. 14b). Although the positions of halogenation at the 2,2a, 5,6 and 8 positions are related to reactive sites in geraniol pyrophosphate (GPP), halogenation also takes place at other sites. Many acyclic monoterpenoids are highly halogenated with up to six halogens (both chlorine and bromine) so that halogenated octatrienes in, for example, P. cartilagineum (257/259) must involve dehydrogenation steps in their biosynthesis (Fig. 14b) (205c).

Fig. 14a,b. Biosynthesis of acyclic monoterpenoids: a in Plocamium humatum (287); b in P. car-

tilagenium (257/259)

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The basic structural types of cyclic halogenated monoterpenoid metabolites are given in Fig. 15a, and examples in Fig. 15b–d. They are highly halogenated with both chlorine and bromine and illustrative examples are summarized in Table 6. A few halogenated monoterpenoids contain oxygenated rings, for example in Plocamium cartilagineum (346) (Fig. 15e), and tetrahydrofuran (Fig. 16a) and tetrahydropyrans (Fig. 16b) in the taxonomically distinct Pantoneura placamioides from the South Shetland Islands, Antarctica [27, 30]. By analogy with epoxide intermediates in the biosynthesis of diterpenoid metabolites (section “Epoxide-Derived”), it is attractive to involve epoxide intermediates in the biosynthesis of these highly oxygenated metabolites. Aromatic structures including phenols and indoles are C-alkylkated – prenylated – by terpenoids and examples given in sections “Prenylated Phenols” and “Prenylated Indoles”.

Fig. 15a – e. a Outline of structural groups of cyclic monoterpenoids. b 324. c 349. d 325. e 346

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Fig. 15d– e (continued)

Table 6. Cyclic monoterpenoids, their structural group (Fig. 15a), source and figure illustrat-

ing biosynthesis Structural group

Source and structure

Figure

1 2 3 Group 2 Tetrahydrofuran Pantoisofuranoid Pantopyranoid

Plocamium cartilagineum 324 Portieria hornemannii 349 Plocamium cartilagineum 325

15b 15c 15d

Portieria hornemannii 346 Pantoneura plocamioides Pantoneura plocamioides

15e 16a 16b

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Fig. 16a,b. Monoterpenoids with oxygenated rings from Pantoneura plocamioides: a pan-

toisofuranoid; b pantopyranoid

7.2 Sesquiterpenoids

These are widely distributed in marine biota, particularly the red algal species of Laurencia, and the green alga Neomeris annulata and they encompass a wide range of structures. The number of acetogenins from species of Laurencia is noted in the section “Laurencia Metabolites”. There are four major structural groups that are discussed: spirochamigrenes (Fig. 17a) (section “Spirochamigrenes”), bisabolanes (Fig. 17b) (section “Bisabolanes”) eudesmanes (Fig. 17c) (section “Eudesmanes and Related Compounds”) and aromatic cuparenes and related compounds (Fig. 17d) (section “Aromatic Structures – Cupranes and Related Compounds”). In addition, there are a number of metabolites that contain rearranged or degraded sesqui-

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Fig. 17a–d. Major structural groups of sesquiterpenoids

terpenoid structures (section “Degraded Sesquiterpenoids”) for which the term degraded is used here in place of transformed since significant alterations in the structure may have occurred. In the section “Isonitriles, Isothiocyanates, and Dichloroimines” a group of metabolites containing isonitrile, isothiocyanate and dichloroimine substituents are given. The structural classes of sesquiterpenoids, the relation among them, their biosynthesis and sources are shown in Table 7 and Fig. 18a–e including the algaone (Fig. 18f) [138]. As noted in the section “Associations”, it is probable that the original sources of all of those in Aplysia are species of Laurencia that serve as food for sea-hares. 7.2.1 Spirochamigrenes

Many halogenated spirochamigrenes have been isolated from species of Laurencia including L. glandulifera, L. obtusa, L. nipponica, and L. rigida. The chamigrenes are a major alicyclic group, many of them also contain both chlorine and bromine, and a few brief comments are made on those with vicinal substituents: 1. Many of the spirocyclohexane metabolites from species of Laurencia have a double bond at C3 or C4 in the B-ring. Metabolites with both chlorine and bromine in the B ring are formed by formation of the halonium+ from a C=C bond followed by reaction with a halide anion. The vicinal chlorobromo compounds have therefore have the trans configuration, either diequatorial (obtusol) (513) (Fig. 19a) or diaxial (isoobtusol) (516) (Fig. 19b) and this seems always to be true for ring-B metabolites in Laurencia. This is also true for metabolites with vicinal halogen and hydroxyl for which a mechanism involving synthesis of a cyclic bromonium ion and further reaction with water is most likely. 2. Metabolites with vicinal halogen substituents seem uncommon in ring A, and the situation for vicinal bromohydroxy metabolites is more complex than in ring B. In virtually all structures, the bromine substituent at C10 is equatorial, and in many structures – if not most – the substituents have the cis configuration, either in obtusol (513) or in isoobtusol (516). There are, however, exceptions where they are trans, and this is revealed by the structure of the

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Table 7. Illustrativese squiterpenoids: structure, biosynthsis and source

Structure

Example

Figure

Source

Monocyclic Spirobicyclic Cuparene

Preintricaterol (431) Obtusol (519) Laurenterol (589) Cupalaurenol (616) (612)

18a 18b 18c 18d 18e 18f

L. intricata L. obtusa L okamurai Aplysia dactylomela Laurencia majuscula Aplysia dactylomela

Cyclohexadiene Algoane a a

McPhail et al. (1999) [138].

Fig. 18a–f. Biosynthesis from farnesyl PP: a preintricatol (431); b obtusol (519); c lautenterol (589); d cupalaurenol (616); e cyclohexadiene (612); f the algoane

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Fig. 19a–f. Chamigrene sesquiterpenoids: a obtusol (513); b isoobtusol (516); c metabolite (490); d metabolite from L. rigida; e metabolite (507); f rearranged pannosanol from L. pannosa

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Table 8. Configuration of vicinal-substituted spirochamigrenes from species of Laurencia

Ring A: configuration

Ring B: configuration

Substituent and source

Figure

cis ae cis ae None trans ee cis ea

trans ee trans aa trans ee none trans ee

Bromochloro (513) L. obtusa Bromochloro (516) L. obtusa Bromohydroxy (490) L. nipponica Bromohydroxy (a) L. rigida Bromohydroxya (507) L. rigida

19a 19b 19c 19d 19e

a

König and Wright 1997 [118].

diequatorial metabolite from L. rigida [118]. In view of the contrasting situation for ring B, it may plausibly be assumed that the gem-dimethyl group at C11 and the spirocyclohexane at C6 (513) contribute to the tendency to form bromohydroxy metabolites with the cis- (C9-a or C9-e, and C10-e or C10-a) configuration, with the frequent – though not universal – equatorial conformation for bromine substituents at C10. The synthesis of the trans metabolites is consistent with reaction of a cyclic bromonium ion with water. Illustrative examples of the structural possibilities are given in Fig. 19a–e including the rearranged pannosanol from L. pannosa (Fig. 19f) [206] and are summarized in Table 8. 7.2.2 Bisabolanes

Bisabolanes occur widely in species of Laurencia and a scheme for the biosynthesis of one of the basic structures from L. caespitosa (577) is given (Fig. 20). 7.2.3 Eudesmanes and Related Compounds

There is a plethora of these structures in sesquiterpenoids, and therefore also in their brominated analogues. Eudesmane and related metabolites are frequently found in species of Laurencia and they differ primarily in the configuration of the bromine substituent at C7. Related structures are found in the green alga Neomeris annulata and are formed by a range of alternative cyclizations of farnesyl PP (Fig. 21).Although many of them contain the bromine and hydroxyl substituents at the same positions and with the cis configuration, the absolute configuration of the cis angular methyl group and the adjacent bromine substituents may be different. 7.2.4 Aromatic Structures – Cuparanes and Related Compounds

These metabolites contain bromine in the aromatic rings at positions para to a hydroxyl group and cationoid bromination provides a plausible mechanism for their

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Fig. 20. Biosynthesis of the bisabolane sesquiterpenoid caespitane (577) in Laurencia caespitosa

Fig. 21. Bromination of farnesyl pyrophosphate by alternative cyclizations in Neomeris annulata to eudesmane-related metabolites (557, 559)

biosynthesis. Two ring structures are involved: cuparene and the closely related laurene, and examples of aromatic brominated metabolites may be produced by alternative cyclizations (Fig. 22a,b). The iodinated metabolites from Laurencia nana (caraibica) 600 and 601 may be produced from laurinterol (589) (Fig. 23). 7.2.5 Rearranged and Degraded Sesquiterpenoids

Rearranged Metabolites – There is a wide range of rearranged sesquiterpenoids, and illustrative examples are given for representatives from species of Laurencia and from the green alga Neomeris annulata in Table 9 and Fig. 24a–e.

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Fig. 22a,b. Brominated aromatic sesquiterpenoids produced by alternative cyclizations

Fig. 23. Biosynthesis of iodinated aromatic metabolites 600 and 601 from laurinterol 589

Table 9. Rearranged sesquiterpenoid metabolites

Metabolite

Source

Figure

Oppositol (566) Neomeranol (557) Cycloeudesmane (559) Perforatone (569) Perforenol (571)

Laurencia subopposita Neomeris annulata Neomeris annulata Laurencia perforata Laurencia perforata

24a 24b 24c 24d 24e

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Fig. 24a–e. Illustration of rearranged brominated metabolites derived from farnesyl PP: a oppo-

sitol (566); b neomeranol (557); c cycloeudesmane (559); d perforatone (569); e perforenol (571)

Degraded metabolites – A number of metabolites originate in sesquiterpenoids that have been degraded, and examples containing tetrahydrofuran or tetrahydropyran rings are given: 1. The C12 furocaespitane and the related furanone (458, 459) (Fig. 25) in Laurencia caespitosa are formed from degraded caespitane by loss of a C3 unit, and the biosynthetic pathway [44] is supported by the structure of the metabolite 461 from the same alga. This is comparable to the loss of a C3 unit during biosynthesis of the furanocoumarin psoralen and the furanoquinoline dictamnine. 2. Some tetrahydropyrans are formed from sesquiterpenoids by rearrangement and loss of the original methyl group at C10, for example in the C14 (944) metabolite (Fig. 26a) from the sponge Haliclona sp. and the C12 (946)metabolite kumepaloxane (946) (Fig. 26b) from the bubble shell Haminoea cymbalum [169]. Loss of the methyl group may plausibly be accomplished by oxidation mechanisms analogous to those suggested for the biosynthesis of norterpenoids [152]. The non-halogenated norsesquiterpenoid clavukerin from Alcyonium molle is derived not from mevalonate but from acetate that is a degradation product of mevalonate [28].

Fig. 25. Origin of the furocaespitane (458) and the related furanone (460) by degradation of

caespitol (574)

Fig. 26a,b. Biosynthesis of tetrahydropyrans through degradation of sesquiterpenoid inter-

mediates: a C14 (944) from the sponge Haliclona sp.; b the C12 (946) kumepaloxane from Haminoea cymbalum

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7.2.6 Isonitriles, Isothiocyanates, and Dichloroimines

A group of sesquiterpenoid metabolites contain isonitrile, isothiocyanate, or dichloroimine substituents.Although none of these metabolites contain bromine, the analogous dichloroimines are widely distributed and their probable biosynthesis takes place by anionoid reactions from pyrophosphates analogous to anionoid bromination. Further details are given in reviews [39, 59, 101, 191, 192]. Dichloroimines (carbonimidic chlorides) have been isolated from sponges (562–565) and nudibranchs [210] and are represented by linear (Fig. 27a), monocyclic (Fig. 27b), bicyclic (Fig. 27c), spiro (Fig. 27e), and tricyclic (Fig. 27f) structures (Table 10). The formation of these metabolites may plausibly occur by reactions in which both the initially produced isonitrile or isothiocyanate undergo cationoid chlorination to the dichloroimines [192] (Fig. 28). Cyanide is the precursor of the isonitrile group and this has been confirmed for the linear sesquiterpenoids from the sponge Stylotella aurantium using 14C-labeled cyanide [192]. Formation of isonitriles and thiocyanates plausibly involves reaction of the anionoid cyanide or thiocyanate with the pyrophosphate of the terpenoid precursor [39, 191, 192]. In addition to the bicyclic dichloroimines, the corresponding aldehydes that may be considered degradation products have also been isolated from the sponge Stylotella aurantium (Fig. 27d) [147]. Although the isonitrile group in hapalindoles from Hapalosiphon fontinalis may be formed by incorporation of cyanide, by contrast the isocyanides isolated from other cyanobacteria that have similar structures – fischerindole (1432) (Fig. 29a) and N-methyl welwitindolinones [98] (Fig. 29b) – are formed by a completely different reaction: the incorporation of glycine and not by reaction of pyrophosphates with cyanide (1417–1427, 1432, 1433) (Table 11). 7.3 Diterpenoids

A wide range of structures is found including those in species of the sea hare Aplysia, in red algal species of Laurencia that may serve as food for sea hares, in species of Sphaerococcus, and in gorgonians and sea pens. Table 10. Sesquiterpenoid dichloroimines

Structure

Biosynthesis

Source

Reference

Linear

Figure 27a

Monocyclic Bicyclic

Figure 27b Figure 27c

Spiro Tricyclic

Figure 27e Figure 27f

Pseudaxinyssa pitys Styletella aurantium Pseudaxinyssa pitys Retulidia fungia Stylotella aurantium Axinella cannabina Ciocalalypta sp.

[239] [192] [239] [210] [147] [37] [101]

Fig. 27a–f. Dichloroimine sesquiterpenoids: a linear; b monocyclic; c bicyclic reticulidins A and

B in Reticulidia fungia; d related aldehydes; e spiro[4,5]decane axisonitrile-3; f tricyclic pupukeanane-2-isonitrile

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Fig. 27d–f (continued)

Fig. 28. Biosynthesis of isonitriles, dichloroimines and isothiocyanates from pyrophosphate precursors of terpenoids

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Fig. 29a,b. Biosynthesis of isonitrile indoles in Fischerella sp. containing a geranyl structure:

a fischerindole (1432); b N-methyl welwitindolinone isonitrile

The group is pragmatically divided into five main groups: (1) metabolites from Laurencia (section “Laurencia Metabolites”); (2) those derived from epoxides by cyclization (section “Epoxide-Derived”); (3) rearranged diterpenoids (section “Rearranged Diterpenoids”); (4) briaranes and related structures (section “Briaranes”); and (5) diterpenoids containing isonitrile substituents (section “Diterpenoid Isonitriles”).

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Biological Effects and Biosynthesis of Brominated Metabolites Table 11. Indole and oxindole isonitriles in cyanobacteria

Metabolite

Organism

Attachment of geranyl PP to indole

Attachment of glycine to indole

Attachment of isopentyl PP to indole

Ambiguine isonitrile A (1433) Fischerindole L (1432) N-methylwelwitindolone (2424) [3,3]-Spiro oxindole a

Fischerella ambigua Fischerella muscicola Hapalosiphon welwitschii Hapalosiphon welwitschii

C-4

C-3

C-2

C-2

C-3

None

29a

C-3

C-4

None

29b

C-3

C-3

a

Figure

Stratmann et al. 1994 [197].

Whereas brominated Laurencia metabolites may be produced by direct cyclization reactions, an attractive mechanism of biosynthesis for other brominated diterpenoids is based on cyclization of epoxide precursors. This is analogous to the occurrence of triterpenoids brominated at C3 (section “Triterpenoids”) and is applicable also to sesterterpenoid scalaranes. 7.3.1 Laurencia Metabolites

Diterpenoids in species of Laurencia may be divided into four main structural groups (Table 12): 1. Monocyclic metabolites from L obtusa exemplified by obtusadiol (685) (Fig. 30a) and laurencianol ( 686) (Fig. 30b).

Table 12. Groups of Laurencia diterpenoid metabolites

Group

Metabolite

Origin

Figure

Monocyclic

Obtusadiol (685) Laurencianol (686) Pinnatol A (663) Parguerane (693) 3-Bromobarekoxide a Neoirieone (684) Prepinnaterpene (683) Pinnaterpene A (680) Kahukuene B (697)

L. obtusa L. obtusa L. obtusa L. pinnata L. luzonensis L. irieii L. pinnata L. pinnata L. obtusa

30a 30b 30c 30d 30e 30f 30g 30h 30i

Polycyclic

Bicyclo[8:4:0]tetradecane Kahukuene a

Kuniyoshi et al. 2001 [112].

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Fig. 30a–i. Biosynthesis of Laurencia metabolites: a obtusadiol (685); b laurencianol (686);

c pinnatol A (663); d parguerane (693); e 3-bromobarekoxide; f neoireone (684); g prepinnaterpene (683); h pinnaterpene A (680); i kahukuene B (697)

Fig. 30e–i (continued)

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Fig. 31. Ring A rearranged diterpenoids

2. A series of bi- and tricyclic metabolites: in metabolites from L obtusa the gemdimethyl cyclohexane of ring A is transformed into a methylcyclopropane in paragueranes (687–693), a cyclobutane (694) or enlarged to a methylcycloheptane (695) (Fig. 31). a) Pinnatols (663) (Fig. 30c) from L. pinnata resembling the metabolites found in sea hares which may in fact originate in species of Laurencia b) Pargueranes from L. obtusa (693) (Fig. 30d) c) 3-Bromobarekoxide from L. luzonensis (Fig. 30e) [112] d) Neoirieone (684) from L. irieii (Fig. 30f) and the related neoirietetraol from L. yonaguniensis [209] 3. The group of bicyclo[8:4:0]tetradecane prepinnaterpenoids (683) (Fig. 30g) and pinnaterpene A (680) (Fig. 30h) in Laurencia pinnata. 4. The kahukuenes (697) from L obtusa (Fig. 30i). Clearly the range of structures synthesized by species of Laurencia is truly remarkable. 7.3.2 Epoxide-Derived Diterpenoids

A number of structures may be derived by cyclization reactions of epoxides and include aplysin-20 from the sea-hare Aplysia kurodai that was the first marine diterpenoid that was isolated. The diterpenoids such as aplysin-20 (647) and isoaplysin-20 (648) from species of the sea hare Aplysia have a b-bromine substituent at C3 and may plausibly be produced by Br+ catalyzed cyclization of GGPP (Fig. 32). More complex structures include dactylomelol (653) from Aplysia dactylomela (Fig. 33a), the unusual structure in rotalin B (715) from the sponge Mycale rotalis (Fig. 33b), and the dactylopyranoid in A. dactylomela (Fig. 33c) [228]. 7.3.3 Rearranged Diterpenoids: Sphaerococcus Metabolites

Substantially rearranged metabolites occur in species of the red alga Sphaerococcus and their biosynthesis from GGP (Fig. 34) may be rationalized in three stages:

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Fig. 32. Cyclization of geranyl geraniol PP with Br+ to form the 3b-brominated diterpenoid aplysin-20 (647) and isoaplysin-20 (648) from Aplysia kurodai

1. Formation of bromosphaerene-A (705) by migration of methyl groups in allylic positions from C-9 to C-8, C-13 to C-12, and transannular migration of the methyl at C-5 to C-15. 2. Cyclization between C-2 and C-15 with loss of pyrophosphate, and between C-7 and C-12. 3. Cyclization between C-6 and C-15 with formation of a double bond at C-4 in bromosphaerol (699). 7.3.4 Briaranes

Chlorinated briarane diterpenoids that are based on bicyclo[8:4:0]tetradecanes are widespread in diverse taxa of gorgonians, sea pens, and soft corals although their brominated analogues have not so far been found. All of them are highly oxygenated and bear an allylic chlorine substituent at the same position.A plausible route for the biosynthesis of briarein A (727) from the gorgonian Briareum asbestinum whose structure is typical of these norditerpenoids is given in (Fig. 35a). In addition, contraction of ring A may occur (Fig. 35b) and has been shown to occur during treatment with BF3 etherate of the epoxide stylatulide (735) found in the sea pen Stylatula sp.

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Fig. 33a–c. Biosynthesis of halogenated diterpenoids from geranyl geraniol PP epoxide in-

termediates: a dactylomelol (653) in Aplysia dactylomela; b rotalin-B (715) in Mycale rotalis; c dactylopyranoid in Aplysia dactylomela

Fig. 34. Biosynthesis from GGPP of rearranged diterpenoids in Sphaerococcus: bromocorodienol (707) and bromosphaerol (699)

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Fig. 35a,b. Biosynthesis of: a chlorinated briarane norditerpenoids briarein (727); b ring A-contracted chlorinated briarane norditerpenoids

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7.3.5 Isonitriles and Isothiocyanates

The biosynthesis of sesquiterpenoid isonitriles, isothiocyanates, and dichloroimines has been discussed in the section “Isonitriles, Isothiocyanates, and Dichloroimines”, and the analogous diterpenoid isonitriles and isothiocyanates are discussed here. It may be assumed that their biosynthesis occurs by reaction between anionoid cyanide or thiocyanate and geranyl geraniol pyrophosphate (GGPP) by the reactions shown in Fig. 36. Isonitriles and related metabolites are widely distributed in sponges. Those of the genus Acanthella contain a series of kalihinols with isonitrile (716) or isothiocyanate groups (724) (Fig. 36a), and an extensive series of isonitriles and isothiocyanates occurs in Phakellia pulcherrima [235]. The cycloamphilectene bisisonitrile diisocyanodociane found in the genus Amphimedon presents an unusual structure in which both isonitrile groups originate in cyanide [59] (Fig. 36b). It is closely related to the 7-isonitrile in Halichondria sp.(Fig. 36c) [142] and the N-formyl-7-aminoamphilectene (Fig. 36d) [25] in Axinella sp. that is presumably formed from the nitrile by hydrolysis. On the other hand, the nitrile found in an unidentified sponge [74] is probably formed by a 1,6 addition to a quinone methide and is therefore analogous to bromopuupehenone that is formed by 1,4 addition (section “Prenylated Phenols”). 7.4 Sesterterpenoids

These terpenoids contain a C25 skeleton formed from C20 diterpenoids and C5 isopropenyl pyrophosphate units and the greatest number hitherto isolated are fungal metabolites or in a series of furanoids that have been isolated from marine sponges of the genus Ircinia. A number of scalarane sesquiterpenoids occur in nudibranchs of the genus Glossodoris and, although those that have been isolated [53] do not contain halogen, their biosynthesis follows that of di- and triterpenoids and could be predicted to encompass synthesis of the 3-bromo compounds. Although the brominated sesterterpenoid bromodesidein has been isolated from Desidea pallescens (802) (Fig. 37) this could most plausibly be rationalized by cationoid bromination of a prenylated 1,2,4-trihydroxybenzene. Sesterterpenolids are typically marine, and the highly rearranged neomangicol B was found in a marine species of Fusarium sp. from the Bahamas [173]. The related mangicols do not contain halogen and have a structure in which the subterminal cyclohexane ring is replaced by a cyclopentane ring [174]. 7.5 Triterpenoids

Triterpenoids (C30) are formed by head-to-head-coupling of farnesyl pyrophosphate (FPP, C15) and they encompass a wide range of structures. In terrestrial plants they are well represented by pentacyclic structures and their rearrangement products, and in the marine environment an important group of polyoxy-

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Fig. 36a–d. Biosynthesis of diterpenoid isonitriles and isothiocyanates: a kalihinol-A (716);

b diisocyanodociane; c 7-isonitrilo-11-cycloamphilectene; d N-formyl-7-amino-11-cycloamphilectene

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Fig. 37. Biosynthesis of bromodesidein from Desidea pallescens

genated metabolites. Brominated triterpenoids occur in a number of red algae including Laurencia and, by analogy with brominated diterpenoids (section “Epoxide-Derived Diterpenoids”), may plausibly be produced from polyepoxides. This hypothesis is analogous to the cascade synthesis for polyene polyepoxides of the polyether brevitoxins in Gymnodymium breve and ciguuatoxin in Gambierdiscus toxicus (references in [188]) and the biosynthesis of steroids and triterpenoids from oxidosqualene [1]. Examples include callicladol from Laurencia calliclada [205] (Fig. 38a), aurilol from the sea hare Dolabella auricularis (Fig. 38b) [200], intricatetraol (799) (Fig. 38c) from L. intricata (799), and thyrsiferol (796) (Fig. 38d) from L. viridis. It is also to be expected that nortriterpenoids (C27–30) analogous to those in the terrestrial environment may be found. 7.6 Steroids

Although a few examples of chlorinated steroids have been found, for example, in the sponges Strongylacidon sp. (815–817) and Xestospongia sp. (818), the brominated analogues seem not to have been isolated. Steroids are produced by demethylation of lanosterol, and the 3-hydroxyl group is a remnant of the initial squalene epoxide. The metabolites from Strongylacidon have chlorine substituents at C-4 and their biosynthesis is readily rationalized by cationoid chlorination of the 3-keto enolate. Other possibilities are given as conceivable biosynthetic routes involving either cationoid or anionoid bromine: 1. A number of polyoxygenated sterols have been isolated from Dysidea sp. [31]. One of these contains a ∆7,8 sterol and a C9,11-epoxide, while others are oxy-

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Fig. 38a–d. Biosynthesis of brominated triterpenoids from squalene epoxides: a callicladol

from L. calliclada; b aurilol from the sea-hare Dolabella auricularis; c intricatetraol from L. intricata (799); d thyrsiferol (796) from L. thyrsifera

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Fig. 38d (continued)

Fig. 39a,b. Hypothetical biosynthesis of brominated sterols from precursors in: a Dysidea sp.;

b Dendronephthya gigantea

genated at C-11. Anionoid bromination of the first at C-7 could produce 7bromo-11-hydroxy-8-enes (Fig. 39a). 2. Hydroxylation at C-5 and C-6 is also found in some structures both from Dysidea sp. and from the octocoral Dendronephthya gigantea [242], and it is tempting to suggest that these originate either from an epoxide or from the corresponding ∆5,6 compound. In the first, anionoid bromination could be hypothesized and in the second cationoid bromination (Fig. 39b).

139

Biological Effects and Biosynthesis of Brominated Metabolites

8 Non-Terpenoids: Acetogenins and Other Structures 8.1 Laurencia Metabolites

Red algae of the genus Laurencia produce a wide variety of brominated acetogenins. Most of them have a -C=C-CCH or BrC=C=C- entity, many contain an oxacyclooctane (oxacin), a bicyclo[2:2:1]oxaheptane ring, and a number of other structures to which the 6- and 7-hydroxyl groups contribute to form tetrahydrofuran rings. They are derived from laurediol and the impressive range of structures is provided by Gribble [68]: a selection of structures is summarized in Table 13. A range of oxocins has been isolated from species of the red alga Laurencia. These are derivatives of fatty acids and the biosynthesis of deacetyllaurencin (822) (Fig. 40a) and notoryne (840) (Fig. 40g) in L. nipponica from trans-laurediol is supported by the synthesis of deacetyllaurencin by bromination of translaurediol with bromide, H2O2 and lactoperoxidase [55, 56]. Further metabolic possibilities (Table 13) are illustrated by prelaureatin (825) and laurallene (829) (Fig. 40b), obtusenyne (851) (Fig. 40c), trans-maneonene B (843) (Fig. 40d), obtusin (852) (Fig. 40e), ocellenyne (940) (Fig. 40f), notoryne (840 (Fig. 40g), the tetrahydropyran (947) in the encrusting sponge Mycale rotalis (Fig. 40h), and kasallene (859) (Fig. 40i). The ocellenyne (940) (Fig. 40f) in the sea hare Aplysia oculifera may plausibly be derived from species of Laurencia. Table 13. Acetogenins in species of Laurencia and Aplysia and the sponge Mycale rotalis [69]

Structure

Representative

Origin

Figure

Oxocins

Deacetyllaurencin (822) Prelaureatin (825) and laurallene (829) Obtusenyne (851) trans-maneonene B (843) Obtusin (852) Ocellenyne (940) Notoryne (840) (947) Kasallene (859)

L. nipponica L. nipponica

40a 40b

L. obtusa L. nidifica L obtusa Aplysia oculifera L. nipponica 40g Mycale rotalis L. obtusa

40c 40d 40e 40f

Oxononane Bis-tetrahydrofuran Spiro bis-tetrahydrofuran Bicyclic tetrahydrofuran Tetrahydropyran 1:4 Dioxan

40h 40i

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Fig. 40a–i. Biosynthesis by bromination of laurediol: a deacetyllaurencin (822); b prelaureatin

(825) and laurallene (829); c obtusenyne (851); d trans-maneonene B (843); e obtusin (852); f ocellenyne (940); g notoryne (840); h tetrahydropyran (947); i kasallene (859)

Biological Effects and Biosynthesis of Brominated Metabolites

Fig. 40d–f (continued)

141

142

Fig. 40g–i (continued)

A. H. Neilson

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Fig. 41. Biosynthesis of polybrominated metabolites in species of Bonnemaisonia

8.2 Other Structures 8.2.1 Polyhalogenated Higher Alkanes

Polybrominated alkanes with chain lengths of C7 in Bonnemaisonia humifera (135), C9 in B. nootkana (163), and C8 in species of Delisea are presumably formed by biosynthesis from acetate with a carbonyl functions at C-5 and C-8 followed by decarboxylation. Cationoid bromination may therefore occur at C-5 and C-7, and C-6 and C-8 respectively (Fig. 41) while C8 metabolites with ketone groups at C-6 are presumably the products of Favorskii rearrangements [137]. These are discussed further in the section “Furanones” in the context of furanones and in the section “Oxaarenes – Furans, Pyrones, Coumarins, and Dibenzo[1,4]dioxins” in the context of pyrones. Tribromomethanes could be produced by brominated fission of the ketones (section “Polyhalogenated Methanes”). The brominated furanones in species of Delisea that are noted in the section “Furanones” may be presumed to have a similar origin of the carbon skeleton. 8.2.2 Polyacetylenes

Fatty acids are derived from acetate, and a wide range of C16 and C18 brominated derivatives exist, mostly unsaturated containing double and triple bonds. Most of the brominated acetylenes of marine origin are produced by sponges, and they share a number of characteristic features: – Proximal carboxyl groups – Distal vinyl bromide groups conjugated with both a triple and a doubl bond: Br.CHCH.CC.CHCH— – Conjugated unsaturation (..; .; .) at C5 – Chain lengths of typically C18 (1637–1656) It is worth noting the isolation of 18-bromooctadeca-5,7,17-triynoic acid and lipids containing the allene 15-hydroxy-18-bromo-12,16,17-octadecatrienoic acid from the lichen Acorospora gobiensis collected in Central Asia [175, 176].

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Naturally occurring polyacetylenic acids are widely distributed and are plausibly formed by desaturation of C16 or C18 fatty acids. Although the distal methyl group may be transformed by oxidation and decarboxylation, this cannot be the case for C16 or C18 compounds, and only the first stage of the oxidation may occur, followed by cationoid bromination. The polyacetylenes from Petrosia sp. [124] are unusual in several ways: two central triple bonds are non-conjugated connected by a carbonyl group, and they contain a terminal HCCHOH.C group that might plausibly undergo cationoid bromination. 8.2.3 Prostanoids

A range of related compounds differing in the halogen substituents and the stereochemistry of the side chains has been found in the soft coral Clavularia sp. Bromovulone (1083). Iodovulene (1039) from Clavularia viridis is representative of the range of metabolites that are related to the chlorinated punaglandins (references in [224]), and the putative biosynthesis of bromovulone is shown (Fig. 42). Useful background on the non-halogenated analogues in a range of marine biota has been given for both prostanoids [60] and the wider group of oxylipins [63]. 8.2.4 Furanones

The brominated fimbrolides and acetoxyfimbrolides are furanone metabolites from the red alga Delisea pulchra, they accumulate in the sea hare Aplysia parvula, and one of them functions as a deterrent to fish under field conditions [179]. Polybrominated furanones (1046, 1048) in the red alga Delisea elegans are

Fig. 42. Biosynthesis of bromovulone I (1038)

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145

derived from a C9 brominated precursor followed by a Favorskii rearrangement [137] and a series of dimeric metabolites (1067–1069) has also been isolated from this organism (Fig. 43). 8.2.5 Macrolides

Attention is directed only to those with phenolic rings whose bromination follows the expected pattern, for example, aplysiatoxin (1711) in the sea hare Stylocheilus longicauda and the tribrominated structural analogue (1716) in cyanobacteria that are plausibly the source of the metabolite found in the seahare. In view of the complex and often uncertainly in the biosynthesis of macrolides that involve both C2 (acetate) and C3 (propionate) components, no attempt has been made to rationalize the structures of others containing bromine in branches of the cyclic structure, for example, the BrCH=C in oscillariolide (1710) in Oscillatoria sp., lyngbyaloside in Lyngbya boullonii, aurisides in the seahare Dolabella auricularia, and phorboxazole in the sponge Phorbas sp. [70].

Fig. 43. Biosynthesis of brominated furanones in the red alga Delisea fimbricata (1046, 1065, 1067, and 1068)

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9 Carbocyclic Aromatic Compounds Two broad groups of aromatic compounds will be considered – phenolics and heterocyclics. Their common feature is the high electron density on the rings, and it is this that facilitates cationoid halogenation. Two large structural groups will be considered: (1) those containing only a singe aromatic ring and (2) those formed by coupling reactions to form biphenyls, diphenyl ethers, depsidones and dioxins. 9.1 Synthesis of the Carbon Skeletons

The pathways for synthesis of the carbon skeletons have a direct bearing on the possibilities for bromination and the positions at which bromination is most likely to take place. Brief attention is therefore given to the main routes for biosynthesis of the structures that are discussed in this essay. A good general summary is available [133] and only a brief outline will be given here. The main precursors are acetate for acetogenins, mevalonate for terpenoids and sterols, and shikimate for aromatic compounds.A brief account is also given of products formed by coupling of aromatic compounds since many of these occur naturally. Aromatic compounds are synthesized biologically by three pathways: (1) the acetate pathway, (2) the mevalonate pathway, and (3) the shikimate pathway. The shikimate pathway is initiated by the reaction of erythrose phosphate with phosphoenol pyruvate to produce successively dehydroquinate, dehydroshikimate, shikimate acid-3-phosphate and arogenate (Fig. 44) followed by reactions resulting in the final production of phenol (Fig. 44). It has been shown that crude bromoperoxidase from Ulva lactuca was able to produce 2,4,6-tribromophenol from a range of precursors including phenol and 4-hydroxybenzoate that was suggested as the most likely [48, 49]. These are the precursors of most of the simple brominated phenols that are discussed in this essay.

Fig. 44. Intermediates in aromatic biosynthesis and further transformation to phenol

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147

Some structures such as complex phloroglucinols, depsides, and anthraquinones are, however, polyketides with hydroxyl groups at the meta positions derived from acetate. It should be noted that many of the principles have evolved from schemes to rationalize the biosynthesis of a plethora of natural products. These studies have most frequently tracked the positions of the carbon atoms using labeled precursors taking advantage of plausible chemical reactions, but with the exception of investigations on the biosynthesis of vitamin B12 the enzymology has not generally been systematically examined. Some unusual metabolites are worth noting: 1. The brominated dihydrofulvene 190 [103] is an unusual metabolite isolated from the red alga Vidalia spiralis, and a plausible route for its biosynthesis by ring-contraction of 3-dehydroshikimic acid is given in (Fig. 45). 2. The pseudo-aromatic 3-bromo-1,4-dimethyl-7-isopropylazulene (Fig. 46) in an undisclosed benthic gorgonian [122] is presumably synthesized by bromi-

Fig. 45. Biosynthesis of a brominated cyclopentene (dihydrofulvene) (190) from dehydroshikimic acid

Fig. 46. Biosynthesis of 3-bromo-1,4-dimethyl-7-isopropylazulene

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nation of the intermediate from (E,E)-farnesyl PP by Br+ and is clearly related to neomeranol (557) (Fig. 24b) from the green alga Neomeris annulata and the non-halogenated trisnorsesquiterpenoid clavukerin. 9.2 Peroxidase Coupling of Aromatic Compounds

The mechanism of the reaction for coupling of phenolic compounds is supported by evidence from a number of different directions: 1. Models for the biogenesis of a wide range of natural products – especially alkaloids – from phenolic precursors by one-electron in vitro chemical oxidation, generally with ferricyanide. 2. Metabolites whose structure reveals the mechanism of coupling involving the synthesis of cyclohexadienones. 3. Intermediates isolated from peroxidase oxidations. In the horseradish peroxidase oxidation of pentachlorophenol, NMR and EPR have confirmed the formation of the cyclohexadienone [104] (Fig. 47). Tetrachlorobenzo[1,4]quinone was an artifact produced by subsequent reactions in organic solvents. The classic example was the synthesis by Barton of usnic acid that involved the rearrangement of the phenol O-radical to a ring C-radical that was then involved in the C-C coupling reaction resulting in the synthesis of the bicyclic ring system of the lichen metabolite usnic acid. Examples are given of reactions involving OC coupling (Fig. 48a) or C-C coupling (Fig. 48b). Phenolic coupling of phenolic diterpenoids is illustrated by the metabolite isolated from the gorgonian octocoral Pseudopterogorgonia elisabethae (Fig. 49) [178]. The plausibility of these reactions is supported by the reactions of biphenyl and diphenyl ether with the white rot fungus Pycnoporus cinnabarinus: hydroxylation takes place at positions ortho to the ring junction followed by dimerization to products containing C-C linkages [99], or from chlorohydroxybiphenyls of products in which dechlorination has also taken place [183]. Polymerization of aromatic compounds containing single aromatic rings is accomplished by free radical reactions catalyzed by peroxidase. The products include, for example, biphenyls, diphenyl ethers, bis-indoles, dibenzofurans, and dibenzo[1,4]dioxins. The same principle is applicable to polyketides including the gymnochromes 1,6,8,10,11,13-hexahydroxy-2,5,9,12-tetrabromobisanthene7,14-quinone (phenanthro[1,8,9,10-fghij]perylene-7-14-quinone) (2199) derivatives (Fig. 50) produced by the crinoid Gymnocrinus richeri [34].

Fig. 47. Product from the action of peroxidase on pentachlorophenol

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Fig. 48a,b. One-electron oxidation of phenol and formation of: a O-C; b C-C coupling products

Fig. 49. 7-Hydroxyerogorgiaene and its dimer in Pseudopterogorgonia elisabethae

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Fig. 50. Biosynthesis of a gymnochrome (2199)

The coupling reactions that produce dimeric products from phenols are initiated by one-electron oxidation by peroxidases in the presence of hydrogen peroxide. The phenoxy radical is mesomeric with the radical at the ortho and para positions, and it is the further reactions of these that produce the final products. There are three broad classes of structure that may be formed by dimerization of a monohydric phenol: 1. A biphenyl retaining two hydroxyl groups. 2. A diphenyl ether or a dibenzofuran each retaining one hydroxyl group. 3. A dibenzo[1,4]dioxin in which both of the original hydroxyl groups are involved in the heterocyclic ring. An interesting example of a structure containing three of these structural elements is provided by the heptaacetate of the phloroglucinol-derived fucofureckol C (Fig. 51) [64] isolated from the brown alga Eisenia arborea. The coupling of 4-propioguaiacone to the biphenyl and the diphenyl ether [166] by peroxidase in the presence of H2O2 (Fig. 52) are the archetypal reactions discussed in this section, and it is important to underscore that the alternatives are

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Fig. 51. Biosynthesis of a gymnochrome (2199)

Fig. 52. Dimerization products from 4-propioguaiacone

Fig. 53. Dimerization of 4-methylphenol with chloroperoxidase

determined by the structures of the precursors: this is illustrated for halogenated tyrosines in the section “Biphenyls and Diphenyl Ethers”. Although these coupling reactions are characteristically catalyzed by peroxidases in the presence of H2O2, they have also been observed with chloroperoxidase even though there are differences at the active sites of the enzymes [20].A mixture of a dibenzofuran and a biphenyl (Fig. 53) was isolated from the reaction with 4methylphenol in the presence of H2O2 and provide support for the reactions discussed in this essay. Coupling of 2-hydroxy-5-chlorobiphenyl and 3-chloro-4-hydroxybiphenyl is mediated by the laccase from Pycnoporus cinnabarinus [183] with formation of dimeric products that may or may not contain chlorine. A number of other coupling reactions have been hypothesized in this essay. These involve the formation of radicals from the methylene groups in halogenated tyrosine and indole. Two illustrative examples are given, prepolycitrine A that is the precursor of polycitone A (Fig. 54a) in the ascidian Polycitor

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africanus [180] and the epimeric gelliusines from the coral Gellius/Orina sp. (2417) (Fig. 54b). 9.3 Cytochrome P450 Coupling

Although the oxidative coupling of phenolic compounds is well established and is generally carried out by one-electron oxidation catalyzed by peroxidases, coupling may also be accomplished by cytochrome P-450 systems, for example, by the cytochrome P450 in the higher plant Berberis stolonifera that is able to carry out the synthesis of diphenyl ether alkaloids (Fig. 55) [110]. In spite of broad similarities, however, NMR studies of the reduced states of chloroperoxidase from Caldariomyces fumago and of the bacterial cytochrome P450cam exhibit significant differences at the active sites [129]. Although this is an oxidase and differs from the bacterial P450cam in the replacement of the glycine residue by proline at

Fig. 54. a Prepolycitrin A from Polycitor africanus. b Gelliusine (2417) from Gellius/Orina sp.

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Fig. 55. Cytochrome P450 oxidative coupling

the oxygen-binding pocket, cytochrome P-450 enzymes are distributed in both eukaryotic and prokaryotic microorganisms. 9.4 Simple Phenols

Brominated phenols are widely distributed in marine biota including algae, worms, polychaetes, and sponges (Tables 14 and 15), and may contain additional substituents such as aldehyde, hydroxymethyl, and chlorine. All the structures are consistent with cationoid bromination. Some examples are given in Table 16 including a simple phenol (1829) (Fig. 56a), a hydroquinone (1830a) (Fig. 56b), a structure with a hydroxymethyl substituent (1803) (Fig. 56c), a phloroglucinol (1820) (Fig. 56d), and dibenzyl ether (1895) from red algae Symphocladia latiuscula (Fig. 56e) that is formed from two molecules of 2,3-dibromo-4,5-dihydroxyphenol. The tunicate Ritterella rubra contains the biologically active rubrolide G (1082) (Fig. 56f) formed by combination with a C5 residue. The closely related quinones are discussed in the section “Quinones and Table 14. Biota producing brominated phenolic metabolites

Cyanobacteria Algae

Chlorophyta Rhodophyta Pheophyta

Marine worms

Polychaete worms Sponges

Hemichordata Hemichordata Phoronida Polychaeta Porifera

Calothrix brevissima Avrainvillea nigricans Polysiphonia (several species) Rhodomela confervoides Ascophyllum nodosum Eisenia arborea Balanoglossus biminiensis Ptychodera sp. Phoronopsis viridis Lanice conchilega Aplysina aerophoba

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Table 15. Biota producing brominated diphenyl ether metabolites

Algae Sponges

Marine chlorophyta Porifera

Cladophora fascicularis Dysidea (many species)

Table 16. Examples of brominated phenols

Source

Structure

Figure

Balanoglossus carnosus Numerous red algae Rhabdonia verticillata Symphocladia latiuscula

1829, 1830a 1803 (lanosol) 1820 1895

56a,b 56c 56d 56e

Fig. 56a–f. Examples of brominated phenols

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Related Compounds”, prenylated phenols in the section “Prenylated Phenols”, and brominated tyrosines and their derivatives in the section “Brominated Tyrosines”. Metabolites formed by coupling of phenols to produce diphenyl ethers, biphenyls, diphenyl methanes, depsidones, and dibenzo[1,4]dioxins are discussed in the section “Metabolites Containing Several Rings”. 9.5 Quinones and Related Compounds

Cationoid bromination by Br+ is not the only possible mechanism for bromination. Whereas the biosynthesis of brominated phenols plausibly involves cationoid bromination, the possibility of anionoid bromination occurs for phenols with hydroxyl groups in the para position.An example of this is provided by 1,4-dihydroxylated benzenoids such as the brominated hydroquinones (Fig. 57a) produced by acorn worms belonging to the genera Ptychodera and Glossobalanus [84]. These may be produced by anionoid bromination of the quinones. There are two supporting reasons for this hypothesis: (a) species of Ptychodera also produce brominated benzo-1,4-quinones together with a series of related brominated cyclohexenones (Fig. 57b) [83] and (b) quinones are susceptible to attack by nucleophiles and, in the case of benzo-1,4-quinone and bromide, the sequential synthesis of 2-bromo- and 2,6-dibromo-1,4-dihydroxybenzene by bromination and oxidation is readily visualized (Fig. 58a). This mechanism is clearly relevant only for 1,4-dihydroxyphenols. Brominated cyclohexenones (Fig. 58b) are presumably formed from an epoxyquinone. Additional examples of anionoid bromination include the following. 1. The biosynthesis of the polycyclic brominated quinones such as those belonging to the gymnochrome group, for example, the gymnochromes 1,6,8,10,11,13-hexahydroxy-2,5,9,12-tetrabromobisanthene-7,14-quinone

Fig. 57. a Brominated hydroquinones (1830, 1830a). b Brominated benzoquinone (212) and related cyclohexenones (215, 216)

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Fig. 58. a Biosynthesis of brominated hydroquinones (1830, 1830a) by reaction of Br– with benzoquinone. b Reaction of quinone epoxide with Br– (215, 216)

(phenanthro[1,8,9,10-fghij]perylene-7-14-quinone) derivatives (Fig. 50) produced by the crinoid Gymnocrinus richeri [34]. 2. Structures containing quinone-methides or double bonds conjugated with more than a single ketonic group exist and these may be susceptible to anionoid bromination at the conjugated double bond. Illustration is provided by the metabolite (637) in an encrusting sponge (Fig. 59a) and by the 1:6 addition of anionoid cyanide to puupehenone isolated from Hyrtios sp. (Fig. 59b) [244] to form the naturally occurring antiviral metabolite cyanopuupehenol that occurs in a sponge of the order Verongida [74]. 3. Bromide may react with epoxides to produce bromohydrins (Fig. 58b) that are widely distributed terpenoid metabolites. 9.6 Prenylated Phenols

Metabolites formed by the alkylation of phenols or indoles (section “Prenylated Indoles”) by pyrophosphates of terpenoid precursors are widely distributed, and they are obviously susceptible to cationoid bromination. Among them are metabolites formed by condensation with hydroquinones that have already been noted in the sections “Anionoid Halogenation”, “Monoterpenoids”, and “Quinones and Related Compounds”. Examples from a wide range of biota including a bacterium, algae, a fungus, and sponges including Cacospongia sp. [10] are given in Table 17 and Table 18 and their biosynthesis in Fig. 60a–h.

Fig. 59. Biosynthesis of involving anionoid addition to quinone methides: 1,4 addition to bromopuupehenone (637) and 1,6 addition to the nitrile in Hyrtios sp.

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Table 17. Biosynthesis of prenylated metabolites from monoterpenoid precursors

Terpenoid precursor

Prenylated metabolite and source

Figure

C10 geranyl PP

Cymopol (621), cyclocymopol (623), cymopochromenol (627) in the green alga Cymopolia barbata Tribromocacoxanthene (635) in the sponge Cacspongia sp. Spiro metabolite a in the sponge Cacspongia sp. Napyradiomycin B3 (1743) from the fungus Chainia rubra

60a

C10 geranyl PP

C10 geranyl PP + C5 isopentenyl PP a

60b 60c 60h

Bali et al. 1990 [10].

Table 18. Biosynthesis of prenylated metabolites from sesquiterpenoid and sesterterpenoid precursors

Terpenoid precursor

Prenylated metabolite and source

Figure

C15 farnesyl PP C15 farnesyl PP C15 farnesyl PP C25

Bromopuupenone (637) in an undetermined sponge Bromomarinone (1748) in a marine bacterium Peyssonol A (560) in the alga Peysonnelia sp. Bromodesidein in Desidea pallescens (802)

60d 60e 60f 60g

Fig. 60a–h. Biosynthesis of prenylated hydroquinones and 1:4-quinones: a cymopol (621), cy-

clocymopol (623), and cymopochromenol (627); b tribromocacoxanthene (635); c the spiro metabolite; d bromopuupenone (637); e bromomarinone (1748); f peyssonol A (560); g C25 in bromodesidein (802); h napyradiomycin B3 (1743)

Biological Effects and Biosynthesis of Brominated Metabolites

Fig. 60b–d (continued)

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Fig. 60e-g (continued)

A. H. Neilson

161

Biological Effects and Biosynthesis of Brominated Metabolites

Fig. 60h (continued)

A note of caution should be entered since in some cases both a quinone and the corresponding hydroquinone may be present so that either cationoid bromination of the phenol or anionoid bromination of the quinone may take place: an illustrative example is bromopuupenone (637) that could also be produced by anionoid bromination of the quinone (Fig. 60d). 9.7 Brominated Tyrosines

A wide variety of brominated structures are derived from tyrosine (Table 19). The major ones include simple spiranes and bis-spirooxazolines (section “Spirotyrosines and bis-Spirooxazolines”), rearranged products (section “Rearranged Tyrosines”), metabolites containing several tyrosine entities (section “Metabolites Table 19. Examples of structurally modified tyrosines [68]

Structure

Organism

Figure

4-Hydroxycyclohexadienone 1914 3,4-Dihydroxycyclohexadiene 1925 Bis-spiroisoxazole at C4 1993 Spirolactone 1929 OH/alkyl groups 1935 Alkyl/bromine rearrangement 2276 Alkyl/alkyl rearrangement 2270 Oxepin 2012 Tetrameric diphenyl ethers 2033 Biphenyl 1908 Diphenyl ether 1898

Aplysina fistularis Aplysina aerophoba Aplysina aerophoba Aplysina aerophoba Aplysina aurea Amathia wilsoni a Aplysia kurodai Psammaplysilla purpurea Ianthella basta Cancer pagurus b

61a 61b 61b 61c 63a 63b 63c 64 65 70a 70b

a

In scleroprotein, b Bryozoan.

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with Several Tyrosine Residues”), and metabolites containing iodine (section “Iodinated Derivatives”). In some, the decarboxylated tyramine or its hydroxyimine are present. Halogenated biphenyls and diphenyl ethers based on tyrosine are discussed in the section “Biphenyls and Diphenyl Ethers”. It is worth noting that eosinophil peroxidase produces 3-bromo- and 3,5-dibromotyrosine from tyrosine in the presence of bromide and H2O2 [240] and it was suggested that these metabolites could serve as molecular markers for oxidative damage of proteins by reactive bromine species. 9.7.1 Spiranes and bis-Spirooxazolines

Two broad groups of spiranes have been found: (i) simple spiranes (1945) derived by angular oxidations at C4 followed by cyclization (Fig. 61a) and (ii) a more complex series from sponges of species of Apysina in which the spirooxazoline ring is formed from two 3,5-dibromotyrosines, for example in aerothionin (1993) (Fig. 61b). In some of the metabolites an additional hydroxyl group

Fig. 61a–d. Biosynthesis of: a simple spirane (1914) from 3,5-dibromotyrosine (1903) by oxidation and further cyclization to (1945); b bis-spirooxazoline aerothionin (1993); c 3-hydroxylated epimers 1925 and 1926 and cyclization of the former to 1929; d the bisepoxide calafianin

Biological Effects and Biosynthesis of Brominated Metabolites

163

Fig. 61c, d (continued)

may be introduced at C3 with alternative configurations, for example (1925, 1926), in Ailochroia crassa: the hydroxyl group introduced at C3 may form spirolactones in aeroplysinin 2 (1929 from Ianthella sp. (Fig. 61c), or the epoxide in calafianin from Aplysina gerardogreeni (Fig. 61d) [43] by reaction with the vicinal bromine atoms. Discorabdins from the sponge Latrunculia sp. represent structures in which a spiro tyrosine is annellated to a metabolite formed by cyclization of tryptamine, for example, in discorabdin C (1438) from the sponge Latrunculia sp. that is derived from 3,5-dibromotyramine and 7-hydroxytryptamine (Fig. 62). 9.7.2 Rearranged Tyrosines

There are several types of rearrangements that are encountered in tyrosine metabolites and they may be rationalized as dienone-phenol rearrangements involve different groups at the C-4 position:

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1. OH/alkyl groups (1935) during the transformation of 3,5-dibromo-4-hydroxyphenylacetamide (1931) in Aplysia archeri to the cyclohexadienone (1914) in Aplysia fistulans followed by rearrangement in Aplysina aurea (Fig. 63a). 2. Br/alkyl groups in amathamide (2276) Fig. 63b) and the related amathaspiramides (Fig. 63c) [145] both from the bryozoan Amathia wilson. 3. Alkyl/alkyl groups in aplaminone (2270) from the mollusk Aplysia kurodai (Fig. 63d). More complex rearrangements are illustrated by the biosynthesis of psammoplysin B (2012) in the Red Sea sponge Psammaplysilla purpurea (Fig. 64) that is one member of a family of psammaplysins [126]. 9.7.3 Metabolites with Several Tyrosine Residues

There is an important group of metabolites that contain several tyrosine groups in addition to the spiro-oxazolines (1993) that have been noted above. The bas-

Fig. 62. Biosynthesis of the spirane discorhabdin C (1438) in Latrunculia sp. from 3,5-dibromotyrosine

Biological Effects and Biosynthesis of Brominated Metabolites

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Fig. 63a–d. Dienone-phenol rearrangements: a (1935); b amathamide D (2276); c amathaspi-

ramide; d aplaminone (2270)

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Fig. 63d (continued)

tadins (2031–2047) are a group of related sponge metabolites produced by the Queensland sponge Ianthella basta and the Pohnpeian sponge Psammaplysilla purpurea. They contain one or two diphenyl ether structures formed from 3-bromotryptamine or 2-bromo-6-hydroxyryptamine in which the NH2 group has been oxidized to =NOH (Fig. 65). Polycitone A (1233) from a species of the ascidian Polycitor may be produced by dimerization of 3,5-dibromotyrosine (Fig. 66). Metabolites containing biphenyl or diphenyl ether structures based on other halogenated tyrosines are discussed in the section “Biphenyls and Diphenyl Ethers”. 9.7.4 Iodinated Derivatives

Iodinated derivatives of tyrosine are widely distributed (Fig. 67a), and thyroxine in the mammalian thyroid fulfills a physiologically important function (Fig. 67b). These compounds have also been isolated from a number of marine organisms

Biological Effects and Biosynthesis of Brominated Metabolites

Fig. 64. Rearrangement to the oxapin psammaplysin-B (2012)

Fig. 65. Brominated tyrosine structural elements in bastadin-6 (2033)

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Fig. 66. Polycitone A (1233) derived from 3,5-dibromotyrosine

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Fig. 67. a 4-Hydroxy-3,5-diiodophenylalanine (1896). b Thyroxine (1897)

including algae, sponges, ascidians, and gorgonians. Attention is directed to the iodinated tyramine derivative from the Senegalese sponge Ptilocaulis spiculifer [38] in which the hydroxyl group is alkylated by a [CH2]3-NH2 group. The isolation [4] of a vanadium-dependent iodoperoxidase from the brown seaweed Saccorhiza polyschides is worth noting.

10 Aromatic Metabolites Containing Several Rings 10.1 Biphenyls and Diphenyl Ethers

Diphenyl ethers are produced by dimerization of simple phenols whose halogenation has been discussed in the section “Simple Phenols” and attention is directed here to their formation by the coupling of phenolic precursors. Illustrative structures from various sources include the sponge Dysidea herbacea (1868) (Fig. 68a), from the green alga Cladophora fascicularis (1880) (Fig. 68b), from D. fragilis (1870) (Fig. 68c), and the octabromopolyether (1894) (Fig. 68d) from the acorn worm Ptychodera flava.A summary is given in Table 15.A number of products formed from brominated tyrosines have been discussed in the section “Iodinated Derivatives”, and further examples are given here including the formation of biphenyls. Examination of the structures of brominated diphenyl ethers suggests a number of pathways for their biosynthesis including unsymmetrical coupling to give products with (a) a single OH group (1870) (Fig. 69a), (b) with hydroxyl groups in both rings (1875) (Fig. 69b), or (c) from brominated catechol followed by reduction and dehydration to a product with hydroxyl groups in both rings (Fig. 69c). In addition, either biphenyls or diphenyl ether may be formed. The alternatives may be illustrated with the products from dimerization of halogenated tyrosines: whether a biphenyl or a diphenyl ether is formed depends on the halogen substituent. Two examples are given: 1. The brominated biphenyl (1908) (Fig. 70a) in the cuticular tissue of Cancer pagurus is produced from 3-bromotyrosine that is widely distributed in the scleroprotein of sponges, gorgonians, and mollusks, while the analogous chlo-

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Fig. 68a–d. Brominated diphenyl ethers: a 1868; b 1880; c 1870; d octabromopolyether 1894

rinated biphenyl (3-chlorodifucol) is formed from 2-chlorophloroglucinol (Fig. 71a) [65] in the brown alga Carpophyllum angustifolium. 2. The iodinated diphenyl ether (1898) (Fig. 70b) that is widely distributed in sponges, ascidians, gorgonians, and marine algae is produced from 3-iodotyrosine, and the analogous iodinated diphenyl ether (2-iodophlorethol) from 2-iodophloroglucinol (Fig. 71b) [65]. For the sake of completeness, the occurrence of brominated terphenyls, quaterphenyls, and quinquephenyls (Fig. 72) in the brown alga Analipus japonicus is noted [66]. Attention has already been drawn in the section “Natural and Anthropogenic Compounds” to the problem of natural and anthropogenic diphenyl ethers and the issue of the origin of methoxylated metabolites. For the sake of completeness, brief note is made of chlorinated diphenyl ethers that are metabolites from terrestrial and not marine biota. Three groups are illustrated: 1. From the freshwater fungus Kirschsteiniotelia sp. (1882) (Fig. 73a). 2. The biologically active ambigol B (1884) [45] (Fig. 73b) from the cyanobacterium Fischerella ambigua. 3. The cytotoxic russuphelins (Fig. 73c) from the toxic fungus Russula subnigricans [157] that are optically active due to steric interference from two ortho-chlorine substituents and the hydroxyl and methoxyl substituents of the central ring. It is worth noting that a fungus of the genus Aspergillus produces chlorinated diphenyl ethers and that the biosynthesis of its non-halogenated analogue asteric

Biological Effects and Biosynthesis of Brominated Metabolites

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Fig. 69a–c. Biosynthesis of brominated diphenyl ethers by unsymmetrical coupling of bromophemnols: a 1870; b 1875; c coupling of a bromocatechol followed by reduction and dehydration

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Fig. 70a,b. Dimerization products: a biphenyl (1908) from 3-bromotyrosine; b diphenyl ether (1898) from 3-iodotyrosine

Biological Effects and Biosynthesis of Brominated Metabolites

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Fig. 71a,b. Dimerization products: a chlorinated biphenyl (3-chlorodifucol) from 2-chlorophloro-

glucinol; b iodinated diphenyl ether (2-iodophlorethol) from 2-iodophloroglucinol

Fig. 72. Brominated polyphenyl (1893)

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Fig. 73a–c. Chlorinated diphenyl ethers from: a (1882); b ambigol B (1884); c russuphelin from

Russula subnigricans

acid is accomplished via an anthraquinone, the corresponding benzophenone and grisandiene rather than by unsymmetrical coupling of chlorophenolic precursors [77]. 10.2 Diphenylmethanes

These metabolites are quite widespread in algae, and they retain both hydroxyl groups from the phenolic precursors in contrast to diphenyl ethers in which one of them is retained as hydroxyl groups and the other provides the ether linkage. One of the hydroxymethyl groups is lost as “CH2O” during aromatization. For example, dibromodihydroxybenzyl alcohols are found in species of the red algae Polysiphonia and Rhodomela and the corresponding diphenyl methane occurs in the red alga Rhodomela larix (Fig. 74). It is worth noting that the postulated intermediate quinone methide structure is analogous to that formed during metabolism of 4-methylphenol by Pseudomonas putida [85], and is represented in non-halogenated triterpenoids such as dispermoquinone and celastrol. There are a number of variants: 1. Rearrangement and retention of the hydroxylmethyl group in 2-bromo-3,4dihydroxybenzyl alcohol (1810) occurs, for example, for 5¢-hydroxyisoavrainvilleol (1854) (Fig. 75) in the green alga Avrainvillea longicaulis. 2. Two identical dibromodihydroxybenzyl alcohols may form an ether group by dehydration of the hydroxybenzyl alcohols in the dibenzyl ether (1895) iso-

Biological Effects and Biosynthesis of Brominated Metabolites

175

Fig. 74. Formation of the brominated diphenylmethane (1851) from the brominated hydroxybenzyl alcohols (1803)

Fig. 75. Rearrangement of hydroxymethyl group in 1810 to form 5¢-hydroxyisoavrainvilleol (1854) by a dienone/phenol rearrangement

lated from the red algae Odonthalia corymbifera and Symphocladia latiuscula (Fig. 76) [115]. 3. The grisan thelepin (2215) (Fig. 77) in the annelid Thelepus setosus is formed by reaction of the phenolic hydroxyl group in one ring with a dienone formed by oxidation of the other ring. 4. Rawsonol (1855) from the green alga Avrainvillea rawsoni contains four orthobromophenol rings linked by three methylene groups leaving one free hydroxymethyl group (Fig. 78a) and is clearly produced by extension of the reactions used for biosynthesis of diphenylmethanes.

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Fig. 76. Dibenzyl ether (1895) from the red algae Symphocladia latiuscula and Odonthalia

corymbifera

Fig. 77. Formation of the grisan thelepin (2215) from Thelepus setosus

Biological Effects and Biosynthesis of Brominated Metabolites

177

5. Cyclotribromoveratrylene (2283) in the red alga Halopytis pinastroides is derived from the brominated catechol (1810) that occurs in Polysiphonia sp. (Fig. 78b). 10.3 Depsides and Depsidones

In contrast to the metabolites already discussed, both of these groups are lichen metabolites [6].Which of the symbionts is involved in biosynthesis is unresolved, but it may reasonably be assumed that their biosynthesis parallels that for biphenyls, diphenyl ethers, and diphenylmethanes. Depsides and depsidones are polyketides, retain oxygen functions at the meta positions, and their structures closely resemble each other. Many of them contain chlorine [68] and the isolated example of the brominated depsidone from the lichen Acarospora gobiensis has already been noted [177]. Its biosynthesis is suggested in Fig. 79).

11 Heterocyclic Compounds Pyrroles and pyridine together with their benzo analogues comprise the two main groups of nitrogen-containing heterocyclic aromatic compounds that contain halogen-substituted metabolites. A simplistic discussion will be given of

Fig. 78. a Tetrameric phenylmethane rawsonol (1855) in the green alga Avrainvillea rawsoni.

b Cyclotribromoveratrylene (2283) in the red alga Halopytis pinastroides

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Fig. 79. Biosynthesis of dibrominated depsidone from Acarospora gobiensis

their electronic structures that are central in determining their susceptibility to cationoid halogenation – both chemical and biological. On the basis of these considerations, it may plausibly be suggested that the biosynthesis of halogenated derivatives of these compounds involves either Hal+ generated from halide and H2O2 catalyzed by haloperoxidases, or directly by Hal–. Two additional comments are inserted: (a) compounds containing hydroxyl or amino substituents are generally more accurately described as cyclic amides or amidines, and (b) with increasing numbers of nitrogen substituents in iminazoles, pyrimidines and purines, it becomes increasingly difficult to predict positions at which halogenation may take place. 11.1 Pyrroles and Simple Indoles

These are based on the azacyclopentadiene pyrrole and two electrons from the N atom contribute to the aromatic sextet while the third is involved in forming an N-H bond that is weakly acidic. These compounds are therefore anionoid, and the range of halogenated derivative parallels those expected on the basis of chemical reactions with Hal+. Pyrrole is highly reactive to cationoid reactants so that polybrominated pyrroles are widely distributed among bacteria, algae, and higher biota: representatives from bacteria include those from Chromobacterium sp. (1228) (80a) and the antibiotic pyrrolonitrin (1210) from Pseudomonas fluorescens (Fig. 80b) whose biosynthesis has been discussed in the section “FADH2Dependent Chlorohalogenases”. More complex pyrroles have been isolated and display important biological properties. For example, 4,5-dibromopyrrole-2-car-

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Fig. 80a,b. Halogenated pyrroles from: a Chromobacterium sp. (1228); b) pyrrolonitrin (1210) from Pseudomonas fluorescens

boxylate occurs in sponges of the genus Agelas that also produce oroidin in which this is linked to 2-aminoiminazole and dimeric products containing a central cyclobutane (sceptrins) or a cyclohexane ring (ageliferins) (Fig. 81a,c,d): their role as fish-feeding deterrents has been noted in the section “Biological Activity and Ecological Significance of Metabolites”. There are also a number of more complex tetracyclic metabolites in sponges that include the fish-feeding deterrent Nmethyldibromoisophakellin (1270) from Stylissa caribica [8] and the structurally related cytotoxic metabolite from Axinella brevistyla [213]. They contain 2,3-dibromopyrrole rings bearing a substituted carboxamide group at C5 and a 2aminoiminazole at C3 (references in [67, 213]). The plausible biosynthesis of one of them from girolline that also occurs in Axinella brevistyla is given (Fig. 82) [213]. Although the putative involvement of lysine was supported by the chemical synthesis from lysine of N-a-(4-bromopyrrolyl-2-carbonyl)homoarginine that occurs in Agelas wiedenmayeri [125], there have been alternative suggestions for the ultimate source of the 2-aminoimidazole including histidine and ornithine (references in [125]). Brominated indoles are widely distributed (Table 20), and naive considerations (Fig. 83) suggest that halogenation of indole may take place at any position and, like the corresponding pyrroles, bromination frequently takes place at several positions (Fig. 84a–d) (Table 21). Pseudomonas aureofaciens that produces pyrrolnitrin also produces 7chloroindoleacetic acid, and it has been shown [105] that the chlorination of tryptophane at the 7-position is catalyzed not by a chloroperoxidase but by an alternative mechanism–NADH-dependent flavin induced halogenation that involves the oxidation of the substrate and subsequent reaction with Cl– (section “Anionoid Halogenation”). Care should therefore be exercised in assuming that cationoid bromination is necessarily involved in the synthesis of brominated indoles that are frequent metabolites in marine biota. 11.2 Complex Indoles

The range of brominated metabolites based on indole is wide and includes condensation products from tryptamine (phytostigmines) (section “TryptamineBased Metabolites”), dimeric indoles (section “Dimeric Indoles”), and prenylated derivatives (section “Prenylated Indoles”). These are summarized in Table 22.

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Fig. 81a–d. Brominated pyrrole metabolites in sponges of the genus Agelas: a oroidin (1242); c sceptrin (1260); d ageliferin (1265)

Fig. 82. Formation of tetracyclic metabolite by cyclization between 2,3-dibromopyrrole carboxamide and girolline in Axinella brevistyla

Biological Effects and Biosynthesis of Brominated Metabolites Table 20. Distribution of simple brominated indoles

Group

Taxon

Cyanobacteria Algae (Rhodophyceae)

Rivularia firma Laurencia brongniartii L. nipponica L. rigida Rhodophyllis membranacea Corallistes undulatus Dercitus sp. Dysidea herbacea Hexadella sp. Iotrocha sp. Oceanapia bartschi Penares sp. Pleroma menoui Plocamissa igzo Spongosorites sp. Topsentia genetrix Dendrophylla sp. Didemnum candidum Eudistoma fragum Mancinella sp. Murex sp. Chartella papyracea Flustra foliacea Zoobotryon verticillum Balanoglossus carnosus Ptychodera sp. Didemnum candidum Eudistoma fragum

Sponges

Corals Tunicates Molluscs Bryozoa

Marine worms Tunicates

Fig. 83. Positions in indole susceptible to cationoid halogenation

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Fig. 84a–d. Brominated indoles: a 1297), (1302) from the acorn worm Ptychodera flava laysan-

ica; b (1303) from Balanoglossus carnosus; c (1318) and (1313) from Laurencia brongniartii; d 6-bromotryptamine (1357) from Didemnum candidum Table 21. Positions of bromine substituents in polybrominated indole metabolites [68]

Position of bromine

Example

Source

33,64,63,5,72,3,4,72,3,5,6-

1299 1302 1304 1297 1312 1318

Acorn worm Ptychodera flava laysanica Acorn worm Ptychodera flava laysanica Acorn worm Glossobalanus sp. Acorn worm Ptychodera flava laysanica Red alga Rhodophyllis membranacea Red alga Laurencia brongniartii

11.2.1 Tryptamine-Based Metabolites

A substantial group of brominated metabolites contain several rings, and their structures may readily be rationalized on the basis of reactions between tryptophanes or tryptamines formed by decarboxylation, for example 6-bromotryptamine (1357) from Didemnum candidum (Fig. 84d).

183

Biological Effects and Biosynthesis of Brominated Metabolites Table 22. Examples of metabolites containing halogenated indole entities [68]

Structure

Example

Source

bis-indole

1346–1352

bis-tryptamine

Dragmacidon B (1381)

bis-indolymethane

(1384)

Phytostigmine

Flustramine A (1393)

Carboline

Eudistomin G (1498)

Indolo[2,3-a]carbazole

Tipanazole D (1490)

Cyanobacterium Rivularia firma Tunicate Didemnum candidum Tunicate Didemnum candidum Bryozoan Flustra foliacea Tunicate Eudistoma olivaceum Cyanobacterium Tolypothrix tjipanasensis

Figure

91

85

90

A group of metabolites (flustramines and relatives 1393–1407) from the marine Flustra foliacea are cyclization products of 6-bromotryptamine prenylated at N(1), 2, 3, or 8 with C5 units derived from isopentenyl PP: their biosynthesis is illustrated for flustramine A (Fig. 85a). The complex chartelline A (1412) belongs to a group of structurally related metabolites from the marine bryozoan Chartella papyracea and is plausibly derived from 4,5,6-tribromotryptophane, histamine and isopentenyl PP (Fig. 85b) [24]. The bisindolylmethanes containing the aminoethyl group at the bridging position include metabolites from the tunicate Didemnum candidum from the reaction of 6-bromoindole and 6-bro-

Fig. 85a. Biosynthesis of flustramine A (1393) from Flustria foliacea

Fig. 85b. Chartelline A (1412) from Chartella papyracea

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motryptamine (1384) (Fig. 86) and the metabolite (2417, 2418) from Gellius/ Orina sp. with three indole rings (Fig. 87). A number of brominated carbolines, the eudistomins (1493–1507), are produced by the Caribbean tunicate Eudistoma olivaceum and the related eudistomidins (1509–1515) by E. glaucus from Okinawa. These metabolites are considered here since their biosynthesis involves cyclization of tryptamines with a C1 or more complex units, while bromination in the indole ring is consistent with patterns already noted. A single illustrative example is given in Fig. 88. 11.2.2 Dimeric Indoles

The bis-indoles (1346–1352) formed by radical-coupling contain bromine at C2, C3, C4, C5, are widespread in the cyanobacterium Rivularia forma, and contain two indole rings coupled though C3 of one ring to the N of the other, or the C4

Fig. 86. Formation of bisindolymethane (1384) by free-radical coupling of tryptophane in Didemnum candidum

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Fig. 87. Gelliusines (2417, 2418) from Gellius/Orina sp.

Fig. 88. Brominated carboline eudistomin-J (1500) from the tunicate Eudistoma olivaceum

of one ring to the C3 of the other (Table 23). These reactions may plausibly be rationalized on the basis of free radical oxidative coupling initiated by formation of the N radical by analogy with that of phenol (both phenol and indole are weak acids) (Fig. 89). A similar origin accounts for the synthesis of the chlorinated metabolites from the cyanobacterium Tolypothrix tjipanensis such as tjipanazole D (1490) (Fig. 90) and the dimeric tryptamine dragamicidon B (1381) in Hexadella sp. may be formed by the reactions shown in Fig. 91. 11.2.3 Prenylated Indoles

A large number of metabolites contain anionoid structures including phenols and indoles that are amenable to alkylation – prenylation with the pyrophosphate precursor of terpenoids. The simplest group containing a C5 unit has been noted in the flustramines (section “Tryptamine-Based Metabolites”), and others are discussed here since skeletal rearrangements may occur. In addition some contain isonitrile substituents that are derived from glycine, and not from cyanide that occurs in sesquiterpenoid (section “Isonitriles, Isothiocyanates, and Dichloroimines”) and diterpenoid isonitriles (section “Isonitriles and Isothiocyanates”). Complex indoles containing chlorine are formed from geranyl py-

Biological Effects and Biosynthesis of Brominated Metabolites Table 23. Examples of bis-indoles from the cyanobacterium Rivulariafirma

Structure

Examples

Bromine substitution

C3 Æ C3¢ C3 Æ N C4 Æ N C4 Æ C3

1351 1350 1349 1346

2,2¢,5,5¢ 2,2¢,3¢,4,5,5¢ 3,3¢,5,5¢,7-methoxy 2¢,3,5,5¢,7-methoxy

Fig. 89. Formation of dimeric indoles by free-radical coupling

Fig. 90. Biosynthesis of tjipanazole D (1490) from Tolypothrix tijpanasensis

Fig. 91. Dimeric tryptamine dragamicidon B (1381) in Hexadella sp.

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rophosphate (GPP) and glycine that contributes its nitrogen atom to form isonitriles (Fig. 29). These are found in cyanobacteria, for example, N-methyl weltwitindolinone isonitrile in Hapalosiphon welwitschii [98] and the more complex ambiguines from both Fischerella ambigua and Westelliopsis hibernicus. As already noted in the section “Use of Field Material”, the isonitrile group in hapalindole A (1417) from Hapalosiphon fontinalis may originate in cyanide.As already noted, however, there seem to be ambiguities about the biosynthesis of this group of metabolites. Representatives of the major structural types have been isolated from Hapalosiphon welwitschii and Westiella intricata and a biosynthetic scheme has been proposed [197]. At the risk of perversity, it is suggested that it would be attractive to assume a common origin of the isonitrile group in all these cyanobacterial indoles: glycine would provide not only the nitrogen but also the additional carbon putatively originating in the reaction between tetrahydrofolate and serine: the alternative of isocyanoethylindole [197] seems less attractive except for those metabolites in which this group is attached to the C-3 of indole. In contrast to the formation of isonitriles in sesquiterpenoids and diterpenoids where no additional carbon is required, reaction of activated cyanide with a terpenoid pyrophosphate would leave unresolved the origin of this additional carbon unit. The various structural types formed by condensations of geranyl PP and glycine with indole are given in Table 11. In all of them, the carbonyl of glycine forms bonds at GP C-2. Illustrative structures include fischerindole (1432) in Fischerella sp. (Fig. 29a), N-methyl weltwitindolinone isonitrile (2424) [98] in Hapalosiphon welwitschii (Fig. 29b), and ambiguine A in Hapalosiphon hibernicus (1433) that is formed by alkylation of indole at C2 by isopentenyl pyrophosphate. 11.3 Carbazole

In the apparent absence of brominated carbazoles and phenazines, chlorinated metabolites are given as illustration including 1474 from the unicellular cyanobacterium Hyella caespitosus and chlorinated phenazine (1533) from Streptosporangium sp. (Fig. 92).

Fig. 92. Chlorinated carbazole (1474) from the cyanobacterium Hyella caespitosa and chlori-

nated phenazine (1533) from Streptosporangium sp.

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11.4 Iminazole, Quinoline, Pyrimidine, Pyrrolopyrimidine, and Purine Metabolites

By way of introduction on the biosynthesis of halogenated iminazole, quinoline, pyrimidine, pyrrolopyrimidine, and purines, brief comments are given on the electronic structures of these compounds. Their electronic structures are quite different from those of indoles and pyrroles since only one electron from the N participates in the aromatic sextet and the other two lone-pair electrons make these compounds weakly basic. Since the N atom withdraws electrons from the ring these compounds display cationoid chemical reactivity but only low anionoid activity that is clearly illustrated by (a) the reactivity of pyridine to cationoid reagents (e.g., Hal+, NO2+) at C3, and the greater ease of the reaction of pyridine towards anionoid reagents (e.g. OH–, NH2–) at C2 and C4. Comparably pyrimidine undergoes reaction with cationoid regents at C-5 and with anionoid reagents presumptively at C2 and C4.Although anionoid bromination at C2 in the pyrimidine ring might occur via the pyrophosphate in the biosynthesis of 2-bromo-4-aminopyrimidine in psammopemmin-B (Fig. 93) from Psammopemma sp., this mechanism seems unlikely to be generally applicable. For example, whereas the purine 2-chloro-5¢-deoxyadenine (kumisine) in Theonella sp. [89] and the 2¢-C-methyl analogue in Tachycladus laevispirulifer [185] are halogenated in the pyrimidine ring (Fig. 94a), pyrrolo[2,3-d]pyrimidines that are

Fig. 93. Biosynthesis of 2-bromopyrimidine in psammopemmin B

Fig. 94a–c. a 2-Chloro-5¢-deoxyadenine (kumisine in Theonella sp. b 4-Amino-5-bromopy-

rrolo[2,3-d]pyrimidine) from Echinodictyum sp. (1549). c 4-Amino-5-iodopyrrolo[2,3d]pyrimidine-7-b-5-deoxyfuroriboside (1550) from the red alga Hypnea valendiae

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Fig. 95a–d. Brominated quinolines and derivatives: a a 7-bromoquinoline (1528); b 7-bromoquinazoline-2,4-dione (1531); c 2-bromoleptoclinidinone (1196); d pantherinine (1198)

known from a variety of biota including marine algae, sponges and ascidians (references in [141]) have bromine or iodine substituents at C7 (purine numbering), for example, in 4-amino-5-bromopyrrolo[2,3-d]pyrimidine (1549) from the sponge Echinodictyum sp. (Fig. 94b) and in 4-amino-5-iodopyrrolo[2,3d]pyrimidine-7-b-5-deoxyfuroriboside (1550) from the red alga Hypnea valendiae (Fig. 94c). It is worth noting, however, that bromination of deoxyguanosine or deoxyadenosine with eosinophil peroxidase (EPO) in the presence of bromide and H2O2 or HOBr yields the 8-bromo compounds [187] and of uridine the 5bromo compound [82]. The low activities of quinolines to attack by cationoid reagents well established on chemical evidence and halogenated quinolines and phenazines are apparently less common than pyrrole-based compounds. Illustrative examples include 7bromoquinoline (1528) from the marine bryozoan Flustra foliacea (Fig. 95a) and 7-bromoquinazolinedione (1531) from the tunicate Pyura sacciformis (Fig. 95b) as well as the complex 2-bromoleptoclinidinone (1196) from the ascidian Leptoclinides sp. (Fig. 95c), and pantherinine (1198) from the ascidian Aplidium pantherinum (Fig. 95d). 11.5 Oxaarenes: Furans, Pyrones, Coumarins, and Dibenzo[1,4]Dioxins

Oxo and thia analogues of pyridine are unusual natural compounds. Although the non-halogenated analogues of pyrrole such as furans are not uncommon among natural products, ring-halogenated derivatives are unusual: an example is the 2-bromofuran ring (714) in the sponge Chelonaplysilla sp. The electronic structures of furan and thiophene follow broadly that of pyrrole: the hetero atoms contribute two electrons to the aromatic sextet, and major similarities include the ease of reactions with cationoid reagents and the well-established

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similarity of thiophene and benzene. Although thiophene – that is the sulfur analogue of pyrrole – is found in terrestrial polyacetylenes formed by reaction of a central 1,3-diyne with sulfide, it is not halogenated, and the analogous reaction with brominated polyacetylenes from marine biota seems not to be formed. 11.5.1 Furans

Although furanones (section “Furanones”) and tetrahydrofurans are components of a number of metabolites, ring-brominated furan occurs only seldom and a single example is illustrated by the 2-bromofuran ring (714) in the sponge Chelonaplysilla sp. 11.5.2 Pyrones

Brominated pyrones have been isolated from a few red algae. The formation of the brominated g-pyrone (1560) from the red alga Ptilonia australasica may be rationalized on the formal basis of the cationoid bromination of precursors synthesized from acetate via C9 precursors followed by a Favorskii rearrangement (Fig. 96) [137]. 11.5.3 Dihydrocoumarins

The brominated dihydroisocoumarin hiburipyranone 1573 in the sponge Mycale adhaerens and its chlorinated relatives is plausibly produced from the phloroglucinol that is formed from 6 acetyl CoA units (Fig. 97).

Fig. 96. Biosynthesis of brominated g-pyrone (1560)

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Fig. 97. Biosynthesis of the brominated dihydroisocoumarin hiburipyranone (1573) from acetyl CoA

Fig. 98a–c. Halogenated dibenzo[1,4]dioxins from: a Eisenia arborea; b Tedania ignis; c Dysidea

dendyi

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11.5.4 Dibenzo[1,4]Dioxins

Dibenzo[1,4]dioxins are unusual natural products that may be produced by coupling of phenol radicals. The biosynthesis of halogenated representatives may be expected to follow routes already suggested for diphenyl ethers followed by nucleophilic replacement of halogen and formation of the second ether bond in dioxin. The final products often contain hydroxyl or methoxyl groups that suggest that they were formed from phenols containing an additional hydroxyl group. The structure of a non-halogenated example containing both a dibenzo1,4-dioxin and a dibenzofuran has already been given (Fig. 51), and the parent dioxin with hydroxyl and aldehyde groups in each ring has been identified in the ascidian Aplidiopsis ocellata [237]. Illustrative examples of halogenated dibenzo[1,4]dioxins include: 1. Chlorinated products from the peroxidase dimerization of chlorophenols [208]. 2. 4¢-Bromo- and 4¢-iodoeckol (isolated as hexaacetates) (Fig. 98a) in the brown alga Eisenia arborea [64]. 3. 1-Hydroxy-2,3,6,8-tetrabromodibenzo[1,4]dioxin and 1-hydroxy-2,6,8-tribromo-dibenzo[1,4]-dioxin (Fig. 98b) from the sponge Tedania ignis (Dillman, R.L. quoted by Gribble [68]). 4. The spongiadioxins 1-hydroxy-3,4,6,8-tetrabromo- and 1-hydroxy-2,3,6,8tetrabromodibenzo[1,4]dioxin from Dysidea dendyi [215] (Fig. 98c). One of the congeners was synthesized from the relevant diphenyl ether that has been isolated from the sponge D. herbacea.Additional congeners have been isolated and many in the group also occur as the O-methyl ethers [216]. Acknowledgement. I thank Ann-Sofie Allard for her skill and patience in producing the figures from my sketches, and Östen Ekengren for providing library facilities.

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The Handbook of Environmental Chemistry Vol. 3, Part R (2003): 253 – 299 DOI 10.1007/b11446HAPTER 1

Atmospheric Chemistry of Organic Bromine and Iodine Compounds John J. Orlando National Center for Atmospheric ResearchAtmospheric Chemistry Division, Table Mesa Drive, 1850 Boulder CO 80305, USA. E-mail: [email protected]

In this chapter, the atmospheric sources, sinks, distributions, trends, and impacts of organic bromine and iodine compounds are reviewed. Most studies of bromine in the atmosphere have been driven by its well-characterized contribution to stratospheric ozone depletion. Most organic bromine can be grouped into three classes–methyl bromide, the man-made Halons, and a group of shorter-lived, naturally occurring species (e.g., CH2Br2, CHBr3, etc.). Methyl bromide, which originates from an array of natural and anthropogenic sources, constitutes the major source of bromine to the stratosphere, contributing about half of the 20 ppt Br believed to be present there. The Halons, a group of long-lived compounds of strictly anthropogenic origin, are believed to contribute currently about 35% to this present-day stratospheric bromine burden, while the shorter-lived species (which emanate primarily from the oceans) contribute about 15%. Due to their link to ozone depletion, regulations are now in place (in the case of the Halons) or are soon to be in place (in the case of methyl bromide) to eliminate the production and sales of these species. Thus, the ensuing decades should see a reduction in the stratospheric burden of organic bromine. Most organic iodine in the atmosphere appears to originate from the ocean, though anthropogenic sources (rice paddies, biomass burning) also appear to contribute. Methyl iodide appears to be the largest contributor to the overall budget, though other iodinated methanes and higher alkanes (e.g., CH2I2, CH2ICl, CH2IBr, CH3CH2I, CH3CHICH3, CH3CH2CH2I) also play a role. The lifetimes of iodinated species are short (of the order of a few days or less) due to their rapid photolysis in the troposphere. Thus, the impact of these species is largely restricted to the boundary layer, though a contribution to ozone depletion in the lower stratosphere cannot be entirely ruled out. Keywords. Bromine, Chlorine, Halogens, Ozone depletion, Methyl bromide

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Sources and Sinks of Methyl Bromide Anthropogenic Sources . . . . . . . . Fumigation . . . . . . . . . . . . . . . Biomass Burning . . . . . . . . . . . . Leaded Gasoline . . . . . . . . . . . . Natural Sources . . . . . . . . . . . . The Ocean: A Natural Source and Sink Other Sinks . . . . . . . . . . . . . . . Atmospheric Reactions . . . . . . . . Soil Sink . . . . . . . . . . . . . . . . Methyl Bromide Budget and Lifetime . Sources and Sinks of Halons . . . . .

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2.2.1 2.2.2 2.3 2.3.1 2.3.2 2.4 2.5 2.5.1 2.5.2 2.5.3 2.6 2.6.1 2.6.1.1 2.6.2 2.6.3

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1 Introduction In this chapter, the atmospheric chemistry of brominated and iodinated organic compounds (both natural and anthropogenic) will be summarized, including consideration of their sources and sinks to and from the atmosphere, their atmospheric distributions and temporal trends, and their contribution to key issues in atmospheric chemistry (e.g., destruction of stratospheric ozone, climate change). The total flux of organic bromine to the atmosphere is about 500 Gg/year [155]. Though most of this flux arises from natural sources (predominantly the ocean), there does exist an important anthropogenic component to the emissions, in the form of methyl bromide (used as a fumigant in agriculture) and the Halons (which have found use as fire extinguishants). Because both of these anthropogenic emissions have been categorically linked to stratospheric ozone depletion, their production and sales are now (in the case of the Halons) or will be (in the case of methyl bromide) restricted as part of the Montreal Protocol and its

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subsequent amendments. Concurrently, other shorter-lived organic bromine compounds are being proposed for use (e.g., n-propyl bromide as an industrial solvent), though their reduced atmospheric lifetimes should preclude any significant transport of these species to the stratosphere. Thus, the next few decades will likely see changes in both the overall amount and the speciation of organic bromine in the troposphere and stratosphere. The flux of organic iodine to the atmosphere is not yet firmly established, but is probably comparable to that of bromine.At present, most of these emissions are thought to be of natural, oceanic origin, though emissions from rice paddies [137] and biomass burning [3, 23] are also believed to be significant. Future anthropogenic contributions (e.g., methyl iodide as a substitute for methyl bromide in agricultural practices, and CF3I and related compounds as possible Halon substitutes) have also been proposed. All iodinated organic species have very short atmospheric lifetimes (of the order of a few days or less, owing to their rapid photolysis), thus reducing their atmospheric burden, restricting their transport to the stratosphere, and hence limiting their capacity to contribute to ozone depletion.

2 Organic Bromine Compounds Though bromine is not particularly abundant in the atmosphere (the total Br in organic forms in the lower atmosphere is only about 20 ppt), it can have profound effects, particularly on ozone levels in the stratosphere [163]. Most organic bromine in the atmosphere can conveniently be divided into three classes of compounds–methyl bromide (which originates from both natural and anthropogenic sources), the Halons (which are strictly anthropogenic), and the so-called shortlived brominated species (CH2Br2, CHBr3, CH2BrCl, CHBrCl2, CHBr2Cl, etc., which arise primarily from natural sources). A fourth class of compounds, classified as semi-volatile organic pollutants, and including such anthropogenic compounds as polybrominated biphenyls (PBBs) and polybrominated diphenyl ethers (PBDEs), make up only a small fraction of the total atmospheric Br burden, but may have important health effects. This portion of the chapter will first cover the sources and sinks of each of these classes of compounds, followed by a summary of the current knowledge regarding their distributions and trends in tropospheric air. This will be followed by a discussion of stratospheric Br chemistry, including an account of the organic bromine compounds reaching the stratosphere, the subsequent destruction of these compounds in the stratosphere (which leads to release of free Br-atoms), and the ozone depletion chemistry that ensues. Springtime boundary layer ozone depletion in the Arctic which is definitely linked to inorganic bromine chemistry, but which may or may not involve organic forms of bromine, will also be briefly discussed. 2.1 Sources and Sinks of Methyl Bromide

No single compound has generated as much scientific and political fervor in recent years as methyl bromide; see [154, 155, 163] and references therein. The in-

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clusion of this species among the substances regulated by the Montreal Protocol and its amendments, the complex nature of its atmospheric sources and sinks, and a persistent problem in balancing its atmospheric budget have combined to stimulate a substantial amount of research over the last decade. While the tropospheric mixing ratio (≈10 ppt) of methyl bromide seems to have been constant over the last couple of decades, known sinks for this species (≈200 Gg/yr) currently outweigh known sources (≈125 Gg/yr) by a significant amount [155]. This sub-section will summarize the current knowledge of the various sources and sinks involved in determining the atmospheric budget of methyl bromide, highlighting this perplexing “missing sink” problem that has been evident for much of the last decade. 2.1.1 Anthropogenic Sources 2.1.1.1 Fumigation

Almost all production and sales of CH3Br are related to its use as a fumigant, predominantly for soils but also for durables, perishables, and structures. Production of CH3Br for these purposes increased through the 1980s from 45 Gg in 1984 to 71.6 Gg in 1992 [154, 163]. However, it is important to realize that not all methyl bromide used for these purposes finds its way into the atmosphere, particularly in the case of soil fumigation where chemical and biological consumption occur [155]. Extensive studies [180, 181, 183, 186, 188–190] have shown that about 25–75% of the methyl bromide applied to soil is eventually released to the atmosphere, depending upon the method and depth of injection, the type of plastic covering employed after application, and the soil temperature, pH, and other chemical/ physical characteristics [183]. Other fumigation practices result in larger atmospheric emission factors, ranging from 70% for durables to 100% for structure fumigation [155]. Total fumigation-related emissions to the atmosphere, based on 1992 production and an emission factor of 50% for soil fumigation, are estimated at 41 Gg/year [155]. 2.1.1.2 Biomass Burning

Biomass burning was first proposed as a source of methyl bromide by Singh and Kanakidou [158] and Khalil et al. [89]. Though no measurements were available at the time, this hypothesis was based on the recognition of biomass burning as a source of methyl chloride. Later, Mano and Andreae [105] derived CH3Br/CO2 and CH3Br/CH3Cl emission ratios from laboratory and field burns of savanna grasses, chaparral, and boreal forests, and estimated a global biomass burning source for methyl bromide of 10–50 Gg/year, with a best estimate of 30 Gg/year. Further studies by Andreae et al. [3] and Blake et al. [23] have led to reduced range, 10–40 Gg/yr, and best estimate of 20 Gg/year was put forward [155]. However, a recent evaluation of available data [4] suggests a somewhat larger source strength from biomass burning, closer to the original 30 Gg/year value. The remaining un-

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certainty in this source strength results from the fact that potentially important ecosystems, tropical forests in particular, have yet to be studied [155]. 2.1.1.3 Leaded Gasoline

Brominated organic compounds, most notably ethylene dibromide, have been employed in leaded gasoline as lead scavengers since the 1920s [10, 171], and various studies have shown that methyl bromide emissions from automobile exhaust occur as a result [15, 74, 76]. Thomas et al. [171] combined a calculation of the total bromine content in the leaded gasoline consumed worldwide (on a yearly basis) with published [15, 74, 76] emission factors for methyl bromide (i.e., fraction of total bromine mass emitted as methyl bromide), to obtain the methyl bromide source strength. They found that the total bromine consumption in leaded gasoline peaked at about 170 Gg/year in the 1970s, but had decreased to a value of about 23 Gg/year by 1995. They then combined these data with methyl bromide emission factors [15] to obtain global automobile emission strengths of 43 Gg/year methyl bromide in the early 1970s, decreasing to about 5 Gg/year in 1995. A huge uncertainty exists in this approach, however, since methyl bromide emission factors obtained by various workers [15, 74, 76] vary by as much as two orders of magnitude. A different approach [10, 37] to quantifying CH3Br emissions from mobile sources is based on the determination of a correlation in urban areas of CH3Br concentrations with those of CO (whose concentration above background is dominated by mobile source emissions). Data from two studies of this sort (conducted in the UK [10] and in Santiago, Chile [37]), when scaled to CO mobile emissions worldwide, yielded global methyl bromide source strengths of 1.5 Gg/year [10] and 4 Gg/year [37], respectively. The current best estimate for automobile exhaust emissions of methyl bromide, based on the studies just described, is 5 Gg/year [155], indicating that the contribution of this source to the overall budget is minor. Note, however, that the decrease in this source over the last few decades may have counteracted, at least in part, the increased anthropogenic use of methyl bromide for agricultural purposes [171]. 2.1.2 Natural Sources

Because it is quite well established that the oceans are currently a net sink for methyl bromide (see below) and because anthropogenic methyl bromide production numbers are seemingly well characterized, recent studies have focused on the quantification of naturally-occurring, terrestrially based methyl bromide sources. Lee-Taylor and Holland [98] proposed that CH3Br could be produced from the decomposition of litter by wood-rotting fungi. Their study indicates that this source is likely minor, however, with a best-estimate flux of 1.7 Gg/year, and an uncertainty of roughly a factor of three.Various wetlands (and related) ecosystems have also now been recognized as possible sources of methyl bromide to the atmosphere, including rice paddies (global source strength 1.3 Gg/year [137]), peatlands (0.9 Gg/year [53]), and global wetlands (about 5 Gg/year [53, 175]). In

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addition, Rhew et al. [143] have shown that coastal salt marshes represent a potentially large source, with an estimated global flux of 14 Gg/year. Certain higher plant species have also been shown to generate methyl bromide; global emissions estimates of 7 Gg/year have been obtained for rapeseed and cabbage alone [61]. Rhew et al. [142] have also obtained evidence for methyl bromide emissions from shrubland vegetation, which are strong enough to counterbalance roughly the soil sink in these ecosystems. However, a wide range of herbaceous, deciduous, and coniferous plants have also been recognized as sinks of methyl bromide [81]; data from this study indicate that the vegetative sink term may be of similar magnitude to the soil sink term described below. 2.1.3 The Ocean: A Natural Source and Sink

The oceans act simultaneously as both a major source and sink of methyl bromide. The complexities involved were outlined by Butler [28], who described a two-box model (the surface ocean being the lower box, and the atmosphere the upper box) approach to understanding the system. Processes included in the model were: emission into the atmosphere (from the above-described anthropogenic and terrestrially-based natural sources); transfer from the atmosphere to ocean and the reverse process, evasion of methyl bromide from the ocean to atmosphere; production in the ocean (probably mostly due to phytoplankton, though a quantitative understanding of these processes has not yet been achieved [148, 149, 155]); destruction in the ocean by chemical reaction (hydrolysis and an exchange reaction with Cl– ion) and by biological destruction; and transport of methyl bromide from the surface of the ocean to deeper waters. The net ocean/atmosphere flux of methyl bromide can be obtained from a consideration of the so-called saturation anomaly. That is, waters that are supersaturated in methyl bromide (positive saturation anomaly) represent a net source to the atmosphere, while undersaturated waters will act as a sink. Early measurements in various regions of the Pacific Ocean [89, 158, 161] showed these waters to be supersaturated in CH3Br, thus implying a net source to the atmosphere. Singh and Kanakidou [158] estimated a global net source of 60 Gg/year on the basis of a re-analysis of supersaturations observed [161] in mid-latitude waters. Khalil et al. [89] measured lower supersaturations in the Pacific, which led to a lower estimate for the net oceanic source, roughly 35 Gg/year. However, the entire picture changed following the study of Lobert et al. [100], which showed most of the open eastern Pacific Ocean to be undersaturated in methyl bromide (though some coastal and upwelling regions were supersaturated). Their data thus implied that the oceans were in fact a net sink, with the global sink strength estimated at –13 Gg/year. At this point, some authors [1, 128] suggested that the available measurements were biased to mid- and lower latitudes, and that the colder high-latitude oceans regions could still be a large methyl bromide source, since chemical loss is known to be much slower in colder waters. Subsequent higher latitude measurements made by Moore and Webb (in the Labrador Sea) [114], and Lobert et al. (in the Southern Ocean) [101] showed that these regions were also predominantly undersaturated as well, and the strength of the net

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oceanic sink was refined to 21 Gg/year. Furthermore, the large undersaturations observed in these colder high latitude waters implied a large biological (bacterial) contribution to the oceanic destruction rate, a result confirmed by various workers [44, 71, 91, 173, 174]. Further refinements to the global net flux to the ocean were made on the basis of studies by Groszko and Moore [72] and King et al. [90]. King et al. [90] found that temperate regions of the NE Atlantic were supersaturated in spring, suggesting a seasonal cycle to the overall process, and a revised estimate to the net sink strength, 16 Gg/year, was suggested. Groszko and Moore [72] found a complex, but robust correlation of the methyl bromide saturation anomaly with the sea surface temperature (SST), with the ocean acting as a sink for methyl bromide at both high (> 24°C) and low temperature (< 12°C), but as a net source at intermediate temperatures; this correlation was confirmed by King et al. [90]. On the basis of their saturation anomaly/SST correlation, Groszko and Moore [72] suggested a global sink strength of 10 Gg/year. In addition to net flux determinations based on observed saturation anomalies, parallel efforts have been directed at understanding the gross oceanic CH3Br sink term, and hence its partial lifetime with respect to oceanic removal [28, 196, 197]. As described by Butler [28], this gross oceanic sink term can be obtained from a parameterization of the air-sea exchange rate, and from measurements of the solubility, diffusivity, and oceanic destruction rate of methyl bromide. The initial effort was based only on known chemical loss rates [80] for methyl bromide in the ocean, and implied a partial lifetime of 3.7 yr with respect to oceanic removal. Refinements to the calculation [196, 197], including better spatial resolution and inclusion of biological destruction in the ocean, have led to a calculation of the gross oceanic uptake of methyl bromide of 77 Gg, and a new partial lifetime for oceanic loss of 1.8–1.9 years. Coupling these data with the 16 Gg/year net flux estimate above then implies a gross oceanic source term of 61 Gg/year. Finally, it is worth noting that the ocean, with its large reservoir of methyl bromide, will act to compensate partially for any future changes in anthropogenic (or other terrestrial) source strengths [28]. With the atmospheric and oceanic destruction time constants now believed to be roughly similar in magnitude (about 1.8 years.) and the soil loss term slower in comparison, the decrease in atmospheric mixing ratio may be only half that expected in the absence of the oceanic buffering effect. 2.1.4 Other Sinks 2.1.4.1 Atmospheric Reactions

For all the organic bromine and iodine compounds considered in this chapter, potential loss mechanisms in the atmosphere include reaction with OH, reaction with other atmospheric oxidants (e.g., Cl, O(1D), O3 and NO3), or photolysis. Reaction with OH can occur throughout the troposphere and stratosphere, and will generally occur via abstraction of a hydrogen atom, e.g. OH + CH3Br Æ CH2Br + H2O

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Atmospheric photolysis rate coefficients (j, in units of s–1) can be calculated from the product of the absorption cross section for the molecule of interest (s, in cm2 molecule–1), its photolysis quantum yield (f, unitless), and the atmospheric photon flux (I, photons cm–2 s–1), integrated over all relevant wavelengths [25]: l

j = Ú s(l) f(l) I(l) dl

(1)

For photolysis to occur in the troposphere and lower stratosphere, the molecule of interest must absorb at wavelengths longer than 290 nm, while photolysis at higher altitudes can occur at shorter wavelengths (often in the 200–220 nm “window” between the strong absorption bands of ozone and O2). The major loss process for methyl bromide in the atmosphere is its reaction with OH in the troposphere, reaction as at Eq. (1) above. The rate coefficient for this reaction has been studied by four different groups [38, 79, 107, 200].All available data are in good accord, and a rate coefficient k1 = 4.0¥10–12 exp (–1470/T) is currently recommended (k1 = 1.9¥10–14 at 275 K, the approximate average temperature in the troposphere). Based on a scaling procedure involving methyl chloroform, the accepted partial lifetime for methyl bromide with respect to this process is 1.7 years [155]. Absorption cross sections for methyl bromide [51, 68, 109, 144] in the ultraviolet are shown in Fig. 1. Absorption is weak in the actinic region (> 290 nm), hence the lifetime with respect to this process is long, about 35 years. Reaction of methyl bromide with Cl-atoms has also been measured [63, 123], and a rate coefficient of about 4.3¥10–13 cm3 molecule–1 s–1 has been obtained at 298 K. With globally averaged Cl atom concentrations over the entire troposphere likely no more than 103 cm–3 [146], this reaction is unlikely to be of any significance as a methyl bromide sink (lifetime ≈ 80 years). Similarly, stratospheric removal of methyl bromide via reaction with O(1D) is insignificant when compared to OH reaction and photolysis, though the rate coefficient for this process is large (1.8¥10–10 cm3 molecule–1 s–1) [172]. 2.1.4.2 Soil Sink

Loss of methyl bromide to soil bacteria was first quantified by Shorter et al. [157], who estimated a total global sink of 42(±32) Gg/year, with only a small portion of this loss occurring on cultivated soils. A similar study by Serça et al. [156] led to a larger estimated sink term, 140(±70) Gg/year. The most notable distinction between the two studies was the large difference in the estimated contribution of cultivated soils (2.7 Gg/year [157] vs 66 Gg/year [156]). Shorter and co-workers [176] then studied in more detail the loss of methyl bromide to cultivated soil and revised their previous value upward to 7.5 Gg/year, still considerably smaller than the Serça estimate. Thus, the overall methyl bromide flux to soils remains uncertain. A sink term of 42 Gg/year was adopted by [155], which was the best estimate from the Shorter et al. study. If the new cultivated soil estimate [176] is used, the sink strength would increase to ≈ 50 Gg/yr. However, the data of Serça et al. [156] would still imply a larger sink, even if the cultivated soil term of Varner

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Fig. 1. Ultraviolet spectra for a series of brominated organic species. Data sources are given in text. Methyl bromide (CH3Br), solid circles; Halon-1301 (CF3Br), solid line; Bromoform (CHBr3), solid squares; Chlorodibromomethane (CHClBr2), dashed line; Halon-1202 (CF2Br2), solid triangles; Dibromomethane (CH2Br2), dotted line; Halon-1211 (CF2ClBr), solid diamonds; Halon2402 (C2F4Br2), dash-dotted line

et al. [176] is adopted. Note also that the California shrubland study of Rhew et al. [143], discussed above with regards to possible plant emissions of methyl bromide, suggests a smaller soil sink term for this ecosystem than is inferred from the previous studies. Further discussion of the biodegradation of methyl bromide is provided in another chapter of this volume (Allard). 2.1.5 Methyl Bromide Budget and Lifetime

The most recent budget for methyl bromide was published in the WMO [155] report; see Table 1. This tabulation clearly shows a significant imbalance, with known sinks outweighing known sources by 83 Gg/year. Given the research that has been carried out in the years since that report was published, some refinements to the budget can be made, as were described above and as are outlined in

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Table 1. Sources, sinks, and atmospheric lifetime for methyl bromide (adapted from [155])

Source or sink type

Source or sink strength (Gg/year) Best estimate from [155]

Sources Fumigation Gasoline Ocean Biomass burning Natural terrestrial Subtotal Sinks Ocean Atmospheric reactions Soils Plants Subtotal Total

40.8 5 56 20 122 –77 –86 –42 –205 –83

Atmospheric lifetime (year)

Updated (see text) 40.8 5 61 20 20 147 –77 –86 –50 ? –213 –66

1.9 1.7 3.4 ? 0.7

Table 1. First, the net ocean flux has been revised to –16 Gg/year, from the previous estimate of –21 Gg/year.With best estimate for gross oceanic sink term fixed at 77 Gg/year, the oceanic source term must then be increased to 61 Gg/year. New natural, terrestrial sources of about 20 Gg/year from coastal marsh areas, wetlands, and litter decay can also be added [53, 98, 137, 143, 175]. These adjustments would improve the source/sink discrepancy to 58 Gg/year, from 83 Gg/year. However, the soil sink term is still very uncertain, and is likely larger than the 42 Gg/year estimated in the WMO [155] report (due to the increase in the estimated [176] sink to cultivated soil discussed above). The complex role of vegetation, which may act as both a source and sink of methyl bromide, would appear to require further study before any assessment of its contribution can be estimated. Also presented in Table 1 are partial lifetimes for methyl bromide loss to soil, to the ocean, and in the atmosphere, which combine to give an overall lifetime estimate of 0.7 years. 2.2 Sources and Sinks of Halons 2.2.1 Sources

The Halons, including CF3Br (Halon-1301), CF2ClBr (Halon-1211), and to a lesser extent CF2BrCF2Br (Halon-2402) and CF2Br2 (Halon-1202), have been in use as fire extinguishing agents since the 1960s [60]. Due to their ability to destroy stratospheric ozone, production and sales of these compounds was stopped in de-

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veloped countries as of January, 1994, as part of the Montreal Protocol and its amendments [155]. Production in developing nations is continuing, though a freeze in production at 1995–97 levels is scheduled for 2002 and production is scheduled to stop in 2010 [155]. Reliable production data are only available for the two most abundant species, Halon-1211 and Halon-1301 [60]. Production of these species peaked in the late 1980s at levels of about 20 Gg/year for Halon-1211 and 12 Gg/year for Halon-1301, but have decreased throughout the 1990s. However, production data do not provide the whole picture, as emissions to the atmosphere typically lag production, the result of which has been the build-up of a substantial reservoir for both Halon-1211 and Halon-1301 [60]; see Fig. 2. Thus, emissions have not declined nearly as much as production has in recent years. As shown in Fig. 2, emissions from the existing reservoirs could continue at levels of about

Fig. 2. Predicted emission and reservoir data for Halon-1301 and Halon-1211. Data from [60]. Halon-1211 emissions, solid line; Halon-1211 reservoir, dotted line; Halon-1301 emissions, dashed line; Halon-1301 reservoir, dash-dot-dot line. Reservoir data have been divided by ten for display purposes

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2 Gg/year until 2020, and measurable emissions may persist to the end of the current century [60]. No reliable production data are available for the two less abundant Halon species, Halon-2402 and Halon-1202. However, Fraser et al. [60], in a 2-D global modeling study, estimated emissions of these two species on the basis of observed mixing ratios and known atmospheric lifetimes. They found that Halon2402 emissions probably peaked near 1.7 Gg/year around 1990, but have since declined to levels of less than 1 Gg/year. An analogous study on Halon-1202 revealed that source strength has increased throughout the entire period from 1963 to the late 1990s, with emissions in the late 1990s reaching about 0.8 Gg/year [60]. 2.2.2 Sinks

Because the Halons possess no hydrogen atoms, reaction with OH (or other oxidants, such as NO3 and Cl) does not occur to any measurable extent. Thus, atmospheric destruction of these species can only occur by photolysis (or possibly reaction with O(1D) in the stratosphere), and atmospheric lifetimes of these species are quite long. Ultraviolet spectra of the four Halon species [27, 51, 68, 69, 121] are shown in Fig. 1. Replicate measurements for various species are available and, though good agreement exists in the 200–230 nm atmospheric window (where most stratospheric photolysis occurs), discrepancies at longer wavelengths (where tropospheric photolysis occurs) do exist. Halon-1211, Halon-2402, and Halon-1202 absorb appreciably at wavelengths above 290 nm, and thus these three species photolyze to a certain extent in the troposphere. Tropospheric photolysis of Halon-1301 is extremely slow, however, and destruction of this species occurs entirely in the stratosphere. Atmospheric lifetimes for the Halons, controlled exclusively by their photolysis rate, are 3 years for Halon-1202, about 20 years for Halon-1211 and Halon-2402, and about 65 years for Halon-1301 [27]. 2.3 Sources and Sinks of Short-Lived Species 2.3.1 Sources

A number of relatively reactive, short-lived brominated organic compounds, including CH2Br2, CHBr3, CH2BrCl, CHBrCl2, and CHBr2Cl have been detected in significant quantities in ambient air [6, 7, 16, 33, 34, 40–42, 88, 93, 124, 126, 134, 138, 150, 152, 169, 191, 192, 194]. Though the predominant source of these species is natural, some anthropogenic sources do exist. Bromoform, CHBr3, in particular and other brominated compounds are used to treat and disinfect drinking and cooling waters. The total source strength for this use has been estimated [73] at 5 Gg/year, with the majority of the use in the treatment of freshwater.

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A much larger source of bromoform and the other short-lived species is from the ocean. Though this source strength is difficult to quantify directly, the most recent study [33] gives a total source for bromoform of 220 Gg/year, with the majority arising from macroalgae. Source strengths for the short-lived brominated organics can also be derived from a consideration of their average tropospheric concentrations and lifetimes. For example, in the WMO report [155], a flux of 200 Gg/year was derived for bromoform, while the fluxes of other species are found to be considerably lower (CH2Br2, 54 Gg/year; CHBr2Cl, 12 Gg/year; CHBrCl2, 6 Gg/year; CH2BrCl, 3 Gg/year).While CHBr2Cl, and CHBrCl2 are likely direct products of biological activity, they may also arise in the oceans from reaction of Cl– ions with bromoform [41]. Another potential future source of short-lived brominated organics may result from their proposed use as industrial solvents. The most likely compound to be put into future use in this context is n-propyl bromide.At present, however, there do not appear to be any reports of ambient measurements of this species nor any estimates of the proposed source strength. 2.3.2 Sinks

Much like CH3Br, other partially halogenated methanes (like CH2Br2, CHBr3, etc.) are destroyed in the troposphere by reaction with OH.As seen in Table 2, rate coefficients for the entire suite of brominated methane species are very similar, [20, 38, 50, 51, 122, 123, 199] falling in the range (1–1.5)¥10–13 cm3 molecule–1 s–1 at 298 K.With global OH concentrations of 106 molecule cm–3, partial lifetimes with respect to OH reaction for these species are then about 100–150 days. For n-propyl bromide, reaction with OH occurs with a rate coefficient of about 1¥10–12 cm3 molecule–1 s–1 at 298 K [54, 65, 78, 118, 170], thus giving this species a tropospheric lifetime of about ten days. Photolysis also can play a role in the atmospheric degradation of these shortlived brominated species (see representative UV spectra in Fig. 1) [20, 67, 116, 122].As seen in the Figure, increasing halogen substitution leads to a red-shift in the observed spectrum, with Br having a greater effect than Cl. Lifetimes for the short-lived species against photolysis are as follows: CH2BrCl, about 50 years; Table 2. OH rate coefficients (at 298 K, in units of 10–13 cm3 molecule–1 s–1), photolysis rates, and

atmospheric lifetimes for short-lived brominated organics. Adapted from [155]. See text (Sect. 2.3.2) for data sources Molecule

kOH

Lifetime vs OH reaction (days)

Photolysis lifetime (days)

Atmospheric lifetime (days)

CH2BrCl CH2Br2 CHBrCl2 CHBr2Cl CHBr3

1.0 1.2 1.2 1.2 1.5

150 130 130 130 100

15000 5000 2220 160 36

150 127 82 72 26

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CH2Br2, 15 years; CHBrCl2, 7–8 months; CHBr2Cl, 5 months; CHBr3, 36 days. Thus, as a general rule, photolysis only plays a substantial role in the case of the triply halogenated methanes, while tropospheric degradation of the less heavily halogenated species is controlled predominantly by OH [20]. 2.4 Semi-Volatile Organic Bromine Compounds

A class of brominated organics that deserve special consideration are a group of semi-volatile, environmentally persistent, and potentially bioaccumulative compounds used as fire retardants [48, 52, 133, 140]. This class of compounds includes polybrominated biphenyls (PBBs), hexabromocyclododecane (HBCD), polybrominated diphenyl ethers (PBDEs), and tetrabromobisphenol-A (TBBP-A), and are used to reduce fire hazard in plastics, textiles, upholstered furniture, and electronic circuit boards [48, 52, 133, 140]. The first type of compound manufactured for this purpose were the PBBs, which have now been phased out due to environmental concerns and replaced by the PBDEs. These compounds have themselves now been replaced in part by TBBP-A [140]. Global production of brominated flame retardants is estimated to be about 150 Gg/year [48, 49], about 67 Gg/year of which is in the form of the PBDEs [141]. Because of their low vapor pressures (10–6 to 10–9 Torr), PBDEs will partition themselves between the gas- and aerosol phase [103, 167], as discussed in more detail in the chapter of this volume by Cousins et al. While in the gas phase, loss processes for PBDEs (and PBBs as well) might include reaction with OH or photolysis, though these processes have not been studied to date. Reaction of OH with the structurally similar polychlorinated biphenyls (PCBs) have been shown [2] to be quite rapid, about (0.5–5)¥10–12 cm3 molecule–1 s–1 (decreasing with increasing chlorine content), and to represent a major atmospheric loss process for these species. Ruzo et al. [147] have reported UV spectra for PBBs in hexane solution which extend beyond 290 nm, thus making photolytic loss viable for these species and likely the PBDEs as well. Measurements of PBDEs in ambient air have now been reported at a few different sites; see [52, 167] and references therein. Concentrations in remote locations (Siberia; Alert, northern Canada) typically fall in the range 1–15 pg/m3 (about 103–104 molecule cm–3, or 10–4 to 10–3 ppt). Somewhat higher concentrations (tens of pg/m3) have been identified in urban, semi-rural, and rural settings in the UK and in the USA [52].While these concentrations are exceedingly small compared to other halogenated species in the atmosphere, the potential for these compounds to travel great distances (as evidenced by their detection at remote high latitude sites), their potential for bioaccumulation [48], their increasing abundance in the environment [52, 141], and their potential health risks have attracted considerable recent attention.

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2.5 Tropospheric Distributions and Trends for Organic Bromine 2.5.1 Methyl Bromide

Measurements of methyl bromide in ambient air were first made in the late 1970s by Singh et al. [159]. Many such measurements have been made in the ensuing 25 years or so, [3, 16, 21–23, 34, 40, 56, 72, 88–90, 94, 96, 100, 101, 105, 114, 124, 126, 134, 150–152, 158, 161, 185, 193] and a survey of data obtained near the earth’s surface is provided in Table 3. Clearly, a general consensus has been reached; the Table 3. Summary of measurements of methyl bromide at the earth’s surface

Reference

Location of measurement

Measurement date

Concentration (pptv)

Singh et al. 1977 [159] Singh et al. 1983 [160] Rasmussen and Khalil 1984 [134] Penkett et al. 1985 [124] Cicerone et al. 1988 [40]

Continental USA

1976

4.7

Eastern Pacific

1979–1981

25 NH, 20 SH

1.25

Barrow, Alaska

1983

UK to Antarctica

1982–83

11.0 (seasonal cycle noted) 15 NH, 11 SH

1.36

11 NH, 9.8 SH

1.12

10.7 NH, 8.0 SH

1.34

11.1 NH, 8.5 SH

1.31

Khalil et al. 1993 [89] Lobert et al. 1995 [100] D Blake et al. 1996 [21] Lobert et al. 1997 [101] NJ Blake et al. 1996 [23]

Alaska, Hawaii, 1985–87 Samoa, New Zealand Oregon, Hawaii, 1978–92 Samoa, Tasmania E Pacific 1994 NE Atlantic

1992

11.4 NH

Southern Ocean

1994

8.4 SH

S Africa, S Atlantic, S. America Groszko and Moore NW Atlantic and 1998 [72] Pacific Wingenter et al. N and S Pacific 1998 [185] King et al. 2000 [90] N Atlantic, N Pacific Yokouchi et al. Alert, N Pacific, 2000 [193] Southern Ocean, E Indian Ocean

1992

10–15

1995

11.4 NH, 10.0 SH

1994–95

NH/SH gradient

1.14 1.21, varied with season

1998

11.9 NH

1996–98

11.4 NH, 9.1 SH

1.25

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global average concentration is of order 10.0 ppt, with the northern hemisphere average (about 11.5 ppt) exceeding the southern hemisphere average (about 9 ppt) by 15–35%. The existence of a hemispheric gradient likely implies a greater source strength in the northern hemisphere relative to the south, information that can prove useful in assessing the location of the missing sink. The many measurements of a hemispheric gradient [16, 40, 72, 100, 152, 161], varying in magnitude from roughly 1.1 to 1.4, have recently been put into context by Wingenter et al. [185] Their methyl bromide concentration data from sites throughout the remote Pacific (northern and southern hemisphere) showed clear evidence for a seasonal cycle in the northern hemisphere, with mixing ratios peaking in March-April (10.5–11 ppt) and reaching a minimum in September (9.5–10 ppt).A similar seasonal cycle in methyl bromide mixing ratios has also recently been noted at Alert, in northern Canada [193]. The existence of this seasonal cycle is consistent with the presence of stronger loss terms (reaction with OH, and possibly soil and oceanic uptake) in summer. However, no such seasonal cycle is evident in data from the southern hemisphere [185]. Thus, the observed [185] inter-hemispheric ratio varies with season, from a maximum of 1.35 in March-April, to a minimum of 1.1 in September, a fact which seems to explain much of the variability in previous datasets. Wingenter et al. [185] reported an average inter-hemispheric ratio of 1.21, and calculated that for total methyl bromide emissions of 185 Gg/year and an assumption of equal loss rates in both hemispheres, northern hemisphere emissions must exceed those from the southern hemisphere by about 40 Gg/year. There is no strong evidence for any temporal trend in recent tropospheric methyl bromide mixing ratios. Khalil et al. [89] noted a small increase in observed mixing ratios in the early 1990s, and suggested an increase of 0.3 ppt/year for that time period, though that trend apparently has not continued over the ensuing decade. On a more long term basis, trend information for most of the twentieth century has recently been obtained from the analysis of polar firn air from various sites in Antarctica [29, 168]. The data of [29] are consistent with a mixing ratio near 5–6 ppt for 1900, with a slow rise over the early part of the century and a more rapid rise (0.05–0.06 ppt/yr) during the 1970s and 1980s (consistent with the onset of methyl bromide use in agricultural fumigation practices). Sturges et al. [168] also noted clear evidence for at least a 2 ppt increase in methyl bromide abundance over the latter half of the century, consistent with the Butler et al. [29] analysis. As noted earlier, decreases in emissions of methyl bromide from the consumption of leaded gasoline may have acted to partially offset increased agricultural use during the 1970s and 1980s. Though the source/sink discrepancies discussed earlier preclude accurate predictions, the early- to midtwentieth century data provide at least an indication of possible future methyl bromide levels following the anticipated ban of its use in fumigation [29]. Altitude gradients for CH3Br within the troposphere are fairly weak, consistent with the relatively long lifetime of this species (0.7 years) [22, 56, 94, 96, 150–152]. Early balloon-based measurements in both mid- and low latitudes, showed relatively constant mixing ratios up to the tropopause (and even beyond) followed by sharp decreases in the lower stratosphere [56, 96]. More recent studies [22, 94, 150, 151] are consistent with this picture. For example, Schauffler et al. [151] re-

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port tropical tropopause values of 9.4 ppt during the STRAT campaign, only slightly below their average free tropospheric mixing ratio for the same time period, 9.95 ppt. The mixing ratios of methyl bromide (and other brominated organics) clearly decline with altitude in the stratosphere, as will be discussed in more detail below. 2.5.2 Halons

As was shown above, the Halon species are fairly long-lived (three years in the case of Halon-1202 to 65 years for Halon-1301) and therefore are rather well mixed in the troposphere. Atmospheric measurements of the two most abundant Halons (1211 and 1301) first became available in the early 1980s [56, 95, 125, 134, 160]; mixing ratios of these species at that time were on the order of 1 ppt. Growth in the tropospheric mixing ratios for these species was rapid through the 1980s and early 1990s [30, 60, 88, 95, 164]. While this growth rate has declined recently in the case of Halon-1301 [30, 31, 60, 110] (growth rate about 0.06 ppt/year for 1995–1996, and 0.03 ppt/year in 1998, compared with values in excess of 0.1 ppt/year in the late 1980s and early 1990s [60]), declines in the growth rate of Halon-1211 have been less evident [31, 60, 110]. As of the late 1990s, mixing ratios of Halon-1211 had reached values of 4.0 ppt, while those of H-1301 were 2.1 ppt. [31, 60, 110]. Extrapolations into the future have been performed by Fraser et al. [60], using estimated future production in underdeveloped countries (based on allowances under the Montreal Protocol) and estimated emission rates from the global bank. Their model indicates that Halon-1211 mixing ratios are likely near their peak at present, while the longerlived Halon-1301 is not expected to reach its maximum atmospheric burden (3.2 ppt) until around 2030. Firn air data for Halon-1211 and Halon-1301 have also been reported [29]. The data show essentially zero concentrations of these species in the first half of the twentieth century, thus confirming the negligible contribution of any natural sources of these species. Concentrations of the less abundant Halons, Halon-2402 and Halon-1202, have also grown since their initial detection [129] in ambient air in the 1980s [60]. The most recent data on Halon-2402 suggest a leveling off of observed mixing ratios near about 0.5 ppt, while data on Halon-1202 suggest that its concentration was still increasing as of the late 1990s, approaching a value of about 0.05 ppt in the southern hemisphere [60]. The lack of production and reservoir data for these species precludes prediction of future atmospheric abundances. As expected for compounds of anthropogenic origin (and hence mostly northern hemisphere emissions), interhemispheric gradients have been observed for the three most abundant Halons, Halon-1211, Halon-1301, and Halon-2402 [31, 111, 160]. Because the tropospheric lifetimes of these species are quite long, these gradients are not large, however (approximately 1.05–1.10 [125]). A seasonal cycle for Halon-1211 has also been reported at high northern latitudes [134], with winter mixing ratios exceeding summer ones. This observation was attributed to either the existence of a stronger sink in summer, or to changes in transport pat-

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terns from industrialized regions to the south. Mixing ratios of the Halons show essentially no variation with altitude throughout the troposphere [150, 151], as expected for such long-lived species. 2.5.3 Short-Lived Species

As noted earlier, numerous measurements [6, 7, 16, 33, 34, 40–42, 88, 93, 124, 126, 134, 138, 150–152, 169, 191, 192, 194] of the short-lived organic bromine compounds have been reported. A survey of some of these data (near the surface) is presented in Table 4. The data display a high degree of variability, due to the spatial variability of the oceanic source (which appears to be related to productivity) and the reasonably short lifetimes of these species. Some features of this composite data set are summarized below [155]: 1. Bromoform is the most abundant of the short-lived brominated organics in the marine boundary layer (MBL), typically 1–2 ppt, followed by CH2Br2 (about 1 ppt), and CH2BrCl, CHCl2Br, and CHClBr2 (all a few of tenths of a ppt). Away from strong source regions, the total Br concentration in these compounds is usually in the range 5–10 ppt, similar in magnitude to the contribution of CH3Br (10 ppt) and the Halons (total 6 ppt) to the overall organic bromine budget. Note, however, that much higher values are often observed for the short-lived species in regions of high productivity. 2. Concentrations of these short-lived species are often correlated, suggesting common (presumably biological) sources [7, 33, 192]. 3. Seasonal cycles (highest mixing ratios in winter, lowest in summer) have been observed for most of the short-lived species in the northern hemisphere [5, 40, 192]. The cycle is strongest for bromoform (ratio of winter/summer concentrations ≈ 3), and the strength of the seasonal cycle appears to be roughly independent of latitude. 4. No temporal trends in the atmospheric mixing ratios of these short-lived species are obvious, though these would be hard to discern given the nature of the available data, the relatively short lifetimes of the species involved, and the spatial inhomogeneity of the sources [155]. Firn air data [168] from Antarctica also show no discernible temporal trend in the mixing ratios of CH2BrCl, CH2Br2, CHBrCl2, CHBr2Cl, or CHBr3. There is now ample evidence which shows that the mixing ratios of the shortlived species display significant vertical gradients in the troposphere (see CH2Br2 and CHBr3 data of Fig. 3, for example) [150, 155]. Free tropospheric mixing ratios are typically lower than those found in the MBL; typical values are about 0.8 ppt for CH2Br2, 0.1–0.2 ppt for CH2BrCl, CHBrCl2, and CHBr2Cl, and 0.3–0.4 ppt for CHBr3 [155]. As shown by Schauffler et al. [150, 151], mixing ratios for the short-lived species are further reduced at the tropical tropopause compared to average free tropospheric values. Typical tropopause mixing ratios are 0.5 ppt for CH2Br2, 0.12 ppt for CH2ClBr, 0.02 ppt for CHCl2Br, and less than 0.1 ppt for CHClBr2 and CHBr3. As expected, the falloff in mixing ratios with altitude is most precipitous for the species with the shortest lifetimes (for exam-

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Table 4. Summary of some representative measurements of short-lived brominated organics at the earth’s surface

Reference

Location of measurement

Compounds measured

Avg. concentration (ppt)

Rasmussen and Khalil 1984 [134] Berg et al. 1984 [16]

Barrow, Alaska Arctic

Penkett et al. 1985 [124]

UK to Antarctica

Khalil and Rasmussen 1985 [88]

South Pole

Class and Ballschmiter 1988 [41] Cicerone et al. 1988 [40]

E. Atlantic Ocean

CH2BrCl 2 CH2Br2 CHBr3 CH2Br2 CH2Br2 CHBr3 CH2BrCl CHBr2Cl CHBr3 CHBr3, CH2Br2 CHBr2Cl, CHBrCl2 CHBr3

2.6 5.0 (3–36) (3–60) 2.7 NH, 1.6 SH 0,85 NH, 0.58 SH 2.5 0.7 1 2.0 0.4 Seasonal cycle in NH

CHBr3

0.26 MBL, 0.20 free troposphere 3.7 3.8 6.3 1.5 2.6 Jan, 1.6 April 0.8 Jan., 0.8 April 0.22 April 0.26 April 0.3 summer, 0.3 winter 0.1 summer, 0.3 winter no seasonal cycle 0.8 Pacific, 1.2 SE Asia 0.7 Pacific, 0.8 SE Asia Seasonal cycles seen in free troposphere 7 1.5 0.5 6.27 0.83

Atlas et al. 1992 [6] Reifenhäuser and Heumann 1992 [138] Atlas et al. 1993 [7] Yokouchi et al. 1994 [191]

Yokouchi et al. 1996 [192]

Alaska, Hawaii, Samoa, New Zealand Mauna Loa, Hawaii Antarctic peninsula

CH2Br2 CHBrCl2 CHBr3 tropical Pacific Ocean CHBr3 Alert, Canada CHBr3 CH2Br2 CH2BrCl CHBrCl2 Alert, Canada CHBr3 CHBr2Cl CH2BrCl

Yokouchi et al. 1997 [194]

W. Pacific Ocean, SE Asia

CHBr3 CH2Br2

Atlas and Ridley 1996 [5] Carpenter and Liss 1999 [34]

Mauna Loa, Hawaii

Many

Coastal Ireland

Carpenter et al. 2000 [33]

Coastal Ireland

CHBr3 CH2Br2 CHBr2Cl CHBr3 CHBr2Cl

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Fig. 3 A – L. Vertical gradients for some organic bromine compounds, as reported by Schauffler

et al. [150]. Crosses represent data from the 1996 NASA Global Tropospheric Experiment Pacific Exploratory Mission-Tropics (PEM-Tropics), and circles are data from the 1996 NASA (continued next site)

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Fig. 3 A – L (continued). Stratospheric Tracers of Atmospheric Transport (STRAT) campaign. Reproduced with permission from the American Geophysical Union. From SM Schauffler, EL Atlas, DR Blake, F Flocke, RA Leub, JM Lee-Taylor, V Stroud, W. Travnicek, Journal of Geophysical Research, Volume 104, No. D17, pages 21,513–21,535, 1999, copyright [1999] by the American Geophysical Union

ple, bromoform is the most abundant of the short-lived brominated species in the MBL, but is often below detection limits at the tropical tropopause). 2.6 Atmospheric Impacts of Organic Bromine Compounds 2.6.1 Stratospheric Ozone Depletion

There is now abundant evidence [22, 56, 93–96, 126, 129, 150–152, 164, 179] that significant quantities of organic bromine are present at the tropical tropopause, the major point of entry of tropospheric air into the stratosphere. The total Br content in organic forms has been measured a number of times through the 1990s.Values ranging from about 15 to 20 ppt have been reported [93, 94, 126, 129, 150–152, 179], with methyl bromide contributing about half of the bromine, the Halons about 35%, and the short-lived species (mostly CH2Br2) contributing

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about 15%. Increasing tropospheric levels of the Halons throughout this time period have led to an increase in the total amount of Br present at the tropopause [179]. Measurements of organic bromine compounds made in the stratosphere, both from balloons and from high-altitude aircraft, clearly show that destruction of these species occurs readily in the lower stratosphere (see Fig. 3 for example) [56, 93–96, 126, 129, 150, 179]. Within the stratosphere, the fractional conversion of each individual organic bromine compound (i.e., the fraction of the compound destroyed at a given point in the stratosphere, relative to its tropical tropopause mixing ratio) has been shown to correlate well with the fractional loss of other stratospheric tracer compounds, for example CFC-11 and N2O [150, 179]; see Fig. 4. As seen from the Figure, at relatively low conversions, the fractional conversions of CH3Br, Halon-1211, and Halon-2402 are all larger than those of CFC11, reflecting their shorter lifetimes in the lower stratosphere. On the other hand, the fractional conversion of the longer-lived Halon-1301 is less than that of CFC11. Using SF6 to determine the age of stratospheric air (where “age” refers to the time since entry into the stratosphere), Wamsley et al. [179] showed that destruction of organic forms of bromine in the stratosphere is essentially complete within five years.

Fig. 4 A – D. Correlations of the fractional dissociation of organic bromine compounds with CFC-11 in the stratosphere, from [150]. Reproduced with permission from the American Geophysical Union. From SM Schauffler, EL Atlas, DR Blake, F. Flocke, RA Leub, JM Lee-Taylor, V Stroud, W. Travnicek, Journal of Geophysical Research, Volume 104, No. D17, pages 21,513–21,535, 1999, copyright [1999] by the American Geophysical Union

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Recent attention has focused on a possible hitherto unrecognized role of bromoform as a source of stratospheric bromine. For example, Sturges et al. [169] recently reported mid-latitude tropopause bromoform mixing ratios of about 0.2 ppt, about 3% of the total organic Br present. They also reported measurable levels of bromoform in mid-latitude stratospheric air with a mean age of up to 1.5 yr. Since the bromoform could not have survived tropical upwelling and transport to the mid- and high latitude stratosphere, the most likely mechanism responsible for its presence there is mid-latitude stratosphere-troposphere exchange. Recent modeling studies [55, 119] have also focused on the possible contribution of bromoform to lower stratospheric bromine loading. While there is some disagreement on the overall magnitude of this source (depending on model transport parameters), it could be that about 1/3 to 2/3 of the inorganic Br just above the mid-latitude tropopause is due to bromoform destruction there. Another potential source of Br to the stratosphere is in the form of inorganic species, generated from the destruction of the brominated organics in the troposphere. While these inorganic species (HBr, HOBr, BrONO2, BrO) are likely removed from the troposphere via precipitative scavenging (on a timescale of about ten days), the potential does exist for transport across the tropopause. Some evidence for a contribution of inorganic bromine to stratospheric Br-loading comes from the balloon measurements of Pfeilsticker and co-workers [126], who have shown that their measurements of stratospheric BrO are higher by 3±3 ppt than could be accounted for from the destruction of the full suite of organic bromine compounds, which were measured essentially simultaneously. This suggestion of transport of inorganic forms of Br to the stratosphere is supported by measurements from the same group of a significant tropospheric column of BrO [59]. The above discussion has served to demonstrate conclusively that approximately 20 ppt of Br is carried into the stratosphere, mostly in organic forms, and that the conversion of organic to inorganic bromine is rapid in the lower stratosphere. It is well established that the ensuing inorganic bromine chemistry is an important contributor to ozone depletion chemistry, particularly in the lower stratosphere [25]. Though a full discussion of this chemistry is beyond the scope of this chapter, a brief summary will be given. Bromine is much more efficient at ozone depletion than is chlorine (by a factor of 40–100 depending on latitude and altitude) [154, 155], due to the lack of stability of potential reservoir compounds, such as HBr, BrONO2, and HOBr. HBr is the longest lived inorganic bromine species, with a lifetime of about one day, but its effectiveness as a reservoir is limited due to a lack of a strong source term (for example, reaction of Cl-atoms with methane provides a large source of the stable reservoir HCl, but the analogous reaction of Br-atoms with methane does not occur to any measurable extent due to the thermodynamics of the system). Photolysis of BrONO2 and HOBr is rapid in comparison to the analogous chlorinated compounds; the lifetimes of these species are less than 1 h in the sunlit mid-latitude stratosphere [25]. Thus, as shown by both measurements and models, e.g., [8, 97, 126], about 40–50% of the available inorganic bromine is present in the form of BrO in the sunlit stratosphere (HOBr and BrONO2 contribute about 10–20% each), and catalytic cycles involving reaction of BrO with ClO, HO2, and NO2 result in ozone depletion, e.g.,

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Br + O3 BrO + HO2 HOBr + hn OH + O3 net: 2 O3

Æ Æ Æ Æ Æ

BrO + O2 HOBr + O2 OH + Br HO2 + O2 3 O2

Br + O3 BrO + ClO Cl + O3

Æ Æ Æ

BrO + O2 Br + Cl + O2 ClO + O2

Æ

3 O2

Æ Æ Æ Æ Æ

BrO + O2 BrONO2 + M Br + NO3 NO + O2 NO2 + O2

Æ

3 O2

net: 2 O3 Br + O3 BrO + NO2 + M BrONO2 + hn NO3 + hn NO + O3 net: 2 O3

These bromine-driven chemical cycles probably contribute on the order of 10–20% to the total ozone loss rate in the mid- to low-latitude, lower stratosphere [25]. Key stratospheric reactions involving inorganic forms of bromine are summarized in Fig. 5.

Fig. 5. Schematic representation of the key stratospheric reactions involving inorganic

bromine compounds

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2.6.1.1 Ozone Depletion Potentials for Organic Bromine Compounds

The Ozone Depletion Potential (ODP) of a particular source gas provides a measure of its ability to deplete ozone relative to an equivalent mass of a reference compound, usually CFC-11 [154, 155]. Thus, by definition, the ODP of CFC-11 is unity. Typical values for other CFC compounds are also near unity (e.g., CFC-12, CFC-113, CFC-114 have ODPs of 0.9), while HCFCs (which have shorter atmospheric lifetimes) typically possess ODPs on the order of 0.1 or less [154]. Both steady-state ODPs (a cumulative measure over an infinite time period) or a timedependent ODP (integrated over a specified time horizon) may be defined. ODPs can be evaluated using atmospheric models or from semi-empirical numerical methods (for details see [154]). For example, in Schauffler et al. [150], the ODP of the brominated source gases at specific locations in the stratosphere was obtained as follows: ODP(q,z) = (FCX/FCCFC-11) (a) (tX/tCFC-11) (MCFC-11/MX)*(nX/3) where (FCX/FCCFC-11) is the fractional conversion of compound X relative to that of CFC-11, a is the ozone depletion efficiency of bromine relative to chlorine, tX is the lifetime of compound X, MX is the mass of compound X, and nX the number of bromines in compound X. Using a value for a of 50, typical ODP values for the mid-latitude lower stratosphere (16–20 km) were found to be 0.4 for CH3Br, 12 for Halon-1301, 9.5 for Halon-2402, and 7 for Halon-1211. By comparison, ODPs for the shorter-lived brominated organics are considerably smaller. For example, Wuebbles et al. [187] have determined an ODP for CH2ClBr of about 0.1 and an ODP for n-propyl bromide of 0.03. 2.6.2 Global Warming Potentials of Organic Bromine Compounds

As radiatively active, fairly long-lived atmospheric constituents, organic bromine compounds have the potential to alter the earth’s climate system through the absorption of long-wave radiation. The concept of a global warming potential (GWP) has been introduced [154] to provide a measure of the integrated radiative forcing of a particular gas relative to that of a reference compound, usually CO2: t¢

t¢

GWPX(t¢) = { Ú FX exp(–t/tX) dt}/{ Ú FCO2 R(t) dt} where GWPX (t¢) is the global warming potential of compound X for a time horizon, t¢, FX is the radiative forcing for compound X, tX is the lifetime of compound X, and R(t) is a representation of the decay of an instantaneous pulse of CO2 injected in the atmosphere. Radiative forcings, lifetimes, and GWPs over various time horizons, as given in [155], are summarized in Table 5. Clearly, the GWPs of the Halons are quite high, owing to their stronger absorption in the infrared window (800–1200 cm–1) and their longer lifetimes compared to methyl bromide.

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Table 5. Radiative forcings, lifetimes and GWPs for selected (longer-lived) organic bromine compounds (from [154, 155])

Compound

CH3Br CH2Br2 Halon-1211 Halon-1301

Radiative forcing (W m–2 ppbv–1)

Lifetime (yrs)

0.01 0.01 0.30 0.32

0.7 0.4 11 65

GWP for various time horizons 20 years

50 years

100 years

16 5 3600 7900

5 1 1300 6900

1 <1 390 2700

2.6.3 Springtime Arctic Boundary Layer Ozone Depletion

One of the more interesting atmospheric phenomena that has come to light in recent years is the observation of sporadic, near-complete ozone depletion in the Arctic MBL upon the return of solar insolation in spring. Numerous field campaigns have been conducted to study these phenomena over the years (see overview papers [11, 12, 24] and references therein). While the evidence is now overwhelming that bromine-based inorganic chemistry (i.e., cycles involving Br, BrO, HOBr, BrCl, Br2, BrONO2) is responsible for the observed ozone depletion, the extent (if any) of the involvement of organic bromine is still uncertain. A full discussion of the chemistry involved in these depletion events is beyond the scope of this chapter, but it is generally accepted that heterogeneous processes are central to the cycling of inorganic bromine. For example, uptake of gas-phase HOBr by aerosol, followed by its reaction with Br– (or Cl–) ion in solution to generate Br2 (or BrCl), which is released to the gas phase and rapidly photolyzed, results in an amplification of the total gas-phase halogen content [108], a so-called “bromine explosion”: HOBr(g) + Br– Br2(g) + hn Br + O3 BrO + HO2

Æ Æ Æ Æ

Br2(g) + OH– Br + Br BrO + O2 HOBr + O2

The role of the organic bromine compounds (most notably bromoform, which is the most abundant and has the shortest lifetime against photolysis), may be in the early phases of ozone depletion events, where photolysis of these species may provide a (small) source of inorganic bromine required to initialize cycles such as the one shown above [108].

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3 Organic Iodine Compounds 3.1 General Overview

Research conducted over the last three decades, spurred by the discovery of CH3I (methyliodide) in the atmosphere by Lovelock et al. [102], has shown that the most of the organic iodine in the atmosphere originates from the ocean. In addition to CH3I, other iodine-containing organics (e.g., larger alkyl iodides, and multiply halogenated methanes such as CH2I2 and CH2ICl) have also been identified in ambient air and/or in ocean water.While the exact nature of the oceanic production of these species is not fully understood, biological and photochemical processes are both likely involved. Total fluxes of iodine to the atmosphere are uncertain but are thought to be on the order of hundreds of Gg per year. The only non-negligible anthropogenic sources appear to be emissions from rice paddies [137], and possibly biomass burning [3, 4, 23, 34]. Future anthropogenic use of organic iodides may come in the form of replacement compounds for Halons (e.g., CF3I) and possibly methyl bromide (methyl iodide). In this portion of the chapter, atmospheric sources of organic iodine will first be reviewed and total fluxes to the atmosphere estimated. The next section will provide a summary of kinetic data related to the atmospheric destruction of the organic iodine species, which will be used to estimate their atmospheric lifetimes. The distribution of organic iodine in the atmosphere will then be discussed, including a summary of any discernible temporal, seasonal, latitudinal, or altitudinal trends in observed mixing ratios. Finally, a brief overview of inorganic iodine chemistry and its influences on the MBL, free/upper troposphere, and lower stratosphere will be provided. 3.2 Sources of Organic Iodine

Most of the sources of organic iodine compounds to the atmosphere are natural and result from oceanic emissions (see for example reviews by Vogt [177], Cicerone [39], and Chameides and Davis [35]) though, as discussed below, anthropogenic sources cannot be ignored. The most abundant organic iodine species found in the atmosphere is methyl iodide [3, 7, 13, 21–23, 43, 47, 75, 99, 102, 112, 113, 120, 135, 139, 159, 161, 194, 195], though other compounds with oceanic sources, including CH2I2 [34, 41, 42, 92], CH2ICl [34, 41, 42, 92, 113], CH2IBr [34], CH3CH2I [194], CH3CHICH3 [92], CH3CH2CH2I [92], and CH3CH2CH2CH2I [92] have also been identified in MBL air and/or in surface sea water. The mechanisms involved in the production of these species in the ocean are not fully understood. Early studies [35, 92, 120, 135] focused on the production of methyl iodide by macroalgae and phytoplankton, though the total production from these sources appears to be small compared to the global source [195]. More recent studies have focused on the possibility of photochemical production in ocean water [75, 115, 195], and this source is now thought to dominate. Regardless of the nature of the

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Table 6. Summary of some representative measurements of methyl iodide near the earth’s

surface Reference

Location of measurement

Measurement date

Concentration (ppt) mean (min–max)

Flux estimate (Gg/yr)

Lovelock 1973 [102] Singh et al. 1977 [159] Rasmussen et al. 1982 [135] Singh et al. 1983 [160] Reifenhäuser and Heumann 1992 [139] Atlas et al. 1993 [7] Oram and Penkett 1994 [120] Davis et al. 1996 [47] N. Blake et al. 1996 [23]

UK to Antarctica

1971–1972

1.2

40,000

California, other USA locations Global

1976

9.2 (1–120)

1981

2.2 (22 max)

1300

Happell and Wallace 1996 [75] Yokouchi et al. 1997 [194] Carpenter et al. 1999 [34] Moore and Groszko 1999 [112] Bassford et al. 1999 [13] Cohan et al. 1999 [43] Yokouchi et al. 2001 [195]

California, E. Pacific Antarctica

1979–1981

2.0

400

1987

2.4 (0.6–7.9)

800

Tropical Pacific

1990

1.1 (0.7–1.6)

Coastal UK

1989–1990

(43 maximum)

W. Pacific

1.0

S. Atlantic, S. Africa, S. America Norwegian Sea

0.5

W. Pacific, E and SE Asia Coastal Ireland

1994

2.4

1991–1994

0.87 (0.05–5) 0.63 (0.24–2) 0.43 (0.12–1.27)

1997

N Atlantic, Pacific 1995–1996

0.7

Coastal Ireland

3.4 (1.9–8.7)

1996

W. Pacific

0.36

NW Pacific, 1996–1999 Northern Canada, Japan

0.74 (0.08–1.94) 0.31 (0.05–0.64) 0.82 (0.30–1.69)

150–350

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281

actual production mechanisms, estimates have been made of the flux of methyl iodide from the ocean to the atmosphere [35, 112, 135, 139, 161]. As is summarized in Table 6, a consensus value has yet to be reached, with flux estimates ranging between ≈250 and 1500 Gg/year (though more recent investigations support values at the lower end of this range). In any event, it is important to realize that, though observed levels of CH3I and other organic iodine compounds are low compared to their brominated analogs, the actual flux of iodine to the atmosphere is roughly comparable to that of bromine. Less is known about the oceanic production and distribution of the multihalogenated methanes CH2I2, CH2ICl, and CH2IBr, or their flux to the atmosphere. Various measurements have shown sea water concentrations of CH2I2 [41, 42] and CH2ICl [41, 42, 113] to be comparable to or greater than those of CH3I, implying significant fluxes of these species to the atmosphere. Sources of these species may be from phytoplanktonic production and/or from Cl– substitution reactions [41], e.g., Cl– + CH2I2 Æ CH2ICl + I–

(2)

Br– + CH2I2 Æ CH2IBr + I–

(3)

The potential importance of these less abundant species in coastal regions was illustrated by Carpenter et al. [34]. Their atmospheric measurements of CH3I, CH3CH2I, CH2ICl, CH2IBr, and CH2I2 at Mace Head, Ireland showed that, while CH3I accounted for about 60% of the organic iodine in MBL air, the contribution of CH3I to the total flux was only 3% or less, and was completely overwhelmed by the (shorter-lived) CH2I2 and CH2IBr species.Macroalgae appear to constitute only a minor source of the multi-halogenated species [64], and of organic iodine in general. Anthropogenic sources of organic iodine to the atmosphere are thought to be minor compared with the oceanic sources just described [35, 39, 166, 177], though these sources are not insignificant. Early reports of elevated CH3I in urban areas [99] are not supported by subsequent measurements [135, 161]. Fossil fuel burning contributes only a negligible (≈0.02 Tg/year) amount to the total atmospheric iodine budget [35]. On the other hand, biomass burning has clearly been identified as a source of atmospheric methyl iodide [3, 4, 23, 34]. The total source strength, most recently estimated to be about 14 Gg/year [4], is small compared to the natural source strength, however. The most significant anthropogenic component to the organic iodine budget appears to be from rice paddies, with an estimated global source strength of 70 Gg/year [137]. Given that the oceanic source of methyl iodide could be as low as about 200 Gg/year, the total anthropogenic contribution to the total methyl iodide source may be as high 30% or so. One possible anthropogenic iodine emission which has received some attention is CF3I [165], a potential replacement for the now-regulated Halon fire suppressants. Although this species is not currently in use, and there have been no reports of its detection in the atmosphere, its possible role in the destruction of stratospheric ozone has been explored [165]. However, even if this species were to be produced in quantities comparable to the pre-Montreal Protocol Halon production rates (about 20 Gg/year), the source strength would still be relatively minor compared to the natural iodine sources just described [165].

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3.3 Loss Mechanisms

All iodine-containing organic compounds possess fairly strong absorption features in the near ultraviolet and thus these species are subject to rapid photolysis, with tropospheric lifetimes of order a few days or less. Other destruction pathways involve the bimolecular reaction of the organic iodine compounds with OH radicals and Cl atoms. These reactions are of minimal importance for smaller species such as CH3I, CF3I, CH2I2, etc., but play an increasingly important role for the larger alkyl halides (e.g., the various isomers of propyl and butyl iodide). Data for relevant kinetic processes and atmospheric lifetimes for the individual compounds are summarized below. 3.3.1 Photolysis

Due to the importance of photolysis in determining the atmospheric lifetime of the organic iodides, a comprehensive set of absorption cross section data has now been obtained for these species over the wavelength and temperature ranges of importance for atmospheric photolysis rate determinations. In most cases, multiple cross-section determinations exist in the literature, and agreement between studies is good in almost all cases (typically within ±10%).Absorption cross section data at 298 K for a series of organic iodine compounds are displayed in Fig. 6. The spectrum of methyl iodide [14, 57, 77, 84, 130, 136, 145] possesses a maximum near 260 nm, with measurable absorption extending to at least 350 nm. Cross sections in this long wavelength tail of the spectrum (shown in Fig. 6) decrease with decreasing temperature [57, 136, 145], as expected on the basis of changes in the relative population of ground state rotational levels with temperature. Spectra for larger alkyl halides (e.g., ethyl iodide, n-propyl iodide, isopropyl iodide, tert-butyl iodide) [77, 127, 130, 136, 145] all possess spectra similar in shape and magnitude to CH3I (see Fig. 6). Full temperature dependence studies have been conducted for ethyl iodide, and isopropyl and n-propyl iodides [136, 145]; like CH3I, lower temperature leads to lower cross sections in the long wavelength region of interest for tropospheric photolysis. Spectra for iodinated organic compounds containing more than one halogen atom, CF3I (absorption maximum near 267 nm [58, 130, 165]), CH2ICl (maximum near 270 nm [136, 145]), CH2IBr (maximum near 280 nm [117]), and CH2I2 (maximum near 290 nm [145]) are all red-shifted compared to the simple alkyl halides (see Fig. 6).As will be explored in more detail below, this shift in the spectra leads to stronger absorption in the actinic region, and thus shorter lifetimes for these compounds relative to methyl iodide. As seen from Eq. (2), in addition to accurate absorption cross section data, photolysis quantum yields are also required as a function of wavelength before atmospheric photolysis rates can be computed. On the basis of the nature of the excited state involved in the absorption process (a s* ¨ n transition localized on the C-I bond), it is universally accepted that quantum yields are unity for photodissociation of all organic iodides at all wavelengths. However, while it is clear

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283

Fig. 6. Ultraviolet spectra for a series or iodinated organic species. Data sources are given in the text. Diiodomethane (CH2I2), solid squares; bromoiodomethane (CH2BrI), dotted line; chloroiodomethane (CH2ClI), solid triangles; trifluoroiodomethane (CF3I), dashed line; n-propyl iodide (CH3CH2CH2I), solid circles; methyl iodide (CH3I), solid line

that photolysis of alkyl iodides does indeed generate I atoms (see, for example, [14, 82, 87, 153] and references therein), little in the way of quantitative quantum yield data actually exist. Estimates of the photolysis lifetimes of organic iodine compounds have been presented by a number of groups [57, 58, 117, 136, 145, 165], under the assumption of a photolysis quantum yield of unity. Though direct comparisons are difficult due to different conditions employed in the calculation (e.g., season, latitude, overhead ozone column, instantaneous vs diurnally averaged loss rates), a general consensus has been reached on the lifetimes of the various iodinated organics (see Table 7 for a summary). CH3I and the other alkyl iodides have the longest lifetimes against photolysis, about 3–5 days at the earth’s surface. Photolysis lifetimes for other compounds are shorter, 1–2 days for CF3I, a few hours for CH2ICl, an hour or less for CH2IBr, and a few minutes for CH2I2. For CH3I and

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Table 7. Atmospheric loss processes and lifetimes (days) for organic iodine compounds

Compound

kOHa, d, e

kCl a, d, f

tOH

tCl b (global)

tCl (MBL) c

tPhot g

CH3I CH3CH2I CH3CH2CH2I CH3CHICH3 CH2I2 CH2ICl CH2IBr CF3I

0.74 (3) 14.7 12.1 (<1) (<1) (<1) 0.25

15 160 660 460 (850) 850 (850) 4–9

150 40 9 10 >100 >100 >100 460

7700 720 175 250 135 135 135 20000

150 15 3.5 5 2.7 2.7 2.7 385

3–5 3–5 3–5 3–5 0.0035 0.12 0.04 1–2

a

Units of 10–13 cm3 molecule–1 s–1. Assuming global average [Cl] = 103 atoms cm–3. c Marine boundary layer, assuming [Cl] = 5 ¥ 104 atoms cm–3. d Brackets indicate estimate value (this work and [45]). e See Section 3.3.2 for data sources. f See Section 3.3.3 for data sources. g See Section 3.3.1 for data sources. b

the other alkyl iodides, photolysis rates are expected to be almost constant with altitude throughout the troposphere (the decrease in the absorption cross sections with decreasing temperature is offset by the increasing actinic flux at altitude) [145]. For the more heavily halogenated species, photolysis rates increase with altitude due to a weaker temperature dependence to the cross section data in the key 300–340-nm region [145]. 3.3.2 Reaction with OH

The reaction of OH radicals with methyl iodide, OH + CH3I

Æ CH2I + H2O

(3a)

Æ CH3 + HOI

(3b)

has been the subject of only two studies [26, 62]. Though the level of agreement between the two measurements is not particularly satisfactory, the rate coefficient seems to be too slow to be of major atmospheric importance. The preferred value appears to be that of Brown et al. [26], k3 = 7.4¥10–14 cm3 molecule–1 s–1. With globally averaged OH concentrations of about 106 molecule cm–3, the lifetime for methyl iodide against OH reaction is about 0.5 years, negligible in comparison with loss via photolysis. Rate coefficient data for reaction of OH with larger alkyl halides are relatively scarce. However, the available data [32] suggest the expected trend, an increase in the rate coefficient with increasing size of the alkyl iodide (see Table 7). Thus, lifetimes for the propyl iodides with respect to OH reaction are about ten days, and OH reaction is thus competitive with photolysis. Extrapolation of the available kinetic data to the butyl iodides indicates that OH reaction may in fact be the most important loss mechanism for these species.

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Atmospheric Chemistry of Organic Bromine and Iodine Compounds

Four measurements of the rate coefficient for reaction of OH with CF3I have been reported [17, 26, 62, 66]. Much like the CH3I case, the overall agreement is poor, with rate coefficient values at 298 K ranging from (2.5 to 12)¥10–14 cm3 molecule–1 s–1. Nonetheless, reaction is slow and thus of negligible atmospheric importance (lifetime > 1 year). Rate coefficients for the other multiply halogenated species (CH2I2, CH2ICl, CH2IBr) are not currently available, but are also likely to be too small to be of atmospheric significance. 3.3.3 Reaction with Cl-Atoms

Another possible destruction pathway for alkyl iodides is their reaction with Clatoms. The reaction of Cl with methyl iodide has been studied in some detail, and has been shown [9, 19, 45, 70, 86] to proceed via a complex mechanism, involving both an abstraction pathway (reaction at Eq. 4) and an adduct formation pathway (reaction at Eq. 5): Cl + CH3I

Æ CH2I + HCl

Cl + CH3I

´

(4)

CH3I∑Cl

(5) 1¥10–12

The abstraction reaction occurs with a rate coefficient of order cm3 –1 molecule near room temperature, while the adduct formation process has a rate coefficient of about 2¥10–11 cm3 molecule–1 s–1. However, adduct formation via the reaction at Eq. (5) is to a large extent reversible, and thus does not represent a permanent sink for CH3I. Methyl chloride has been observed as a minor (≈ 9%) product of the reaction [70], and likely arises from rearrangement of the adduct: CH3I∑Cl

Æ CH3Cl + I

(6)

Other possible fates of the adduct have not been fully explored. However, reaction with O2 does not seem to occur to any measurable extent [19], and the short lifetime (≈ 100 ms at 298 K) would seem to preclude reaction with trace atmospheric species (e.g., NOx). An effective value for k5 at 298 K, 1 atm pressure of 1.5¥10–12 cm3 molecule–1 s–1 has been obtained [19]. Though the reactions of Cl with larger alkyl iodides have not been extensively studied as yet, the available data [45] suggest that Cl-atom rate coefficients increase with increasing size of the alkyl iodide; see Table 7. Product studies [46] indicate that abstraction reactions and alkyl chloride formation (presumably via adduct formation) are both important processes for ethyl iodide, n-propyl iodide, and isopropyl iodide. The reaction of Cl-atoms with CF3I has been shown [45, 85, 104] to be slow, with values of k7 in the range (4–9)¥10–13 cm3 molecule–1 having been reported. The reaction has been shown to occur via I-atom abstraction: Cl + CF3I

Æ CF3 + ICl

(7)

However, reaction of Cl with CH2ICl is extremely rapid [18], with k8 = 8.5¥10–11 cm3 molecule–1 s–1, and also occurs via I-atom abstraction: Cl + CH2ICl Æ CH2Cl + ICl

(8)

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J. J. Orlando

The reason for the rapidity of the reaction at Eq. (8) appears to lie in the thermodynamics [18]; the reaction at Eq. (8) is essentially thermoneutral whereas the reaction at Eq. (7) and analogous reactions involving transfer of an I atom from alkyl iodides to Cl are endothermic (by as much as 20–25 kcal/mol). Thermodynamics estimates indicate that reaction of Cl with CH2I2 and CH2IBr are also likely to be rapid. At present, atmospheric levels of Cl atoms are uncertain and thus the atmospheric lifetime of the iodinated organics with respect to reaction with Cl is difficult to assess.As mentioned earlier, average Cl atom concentrations over the entire troposphere are probably no more than 103 cm–3 [146] but values in the MBL (the region where the majority of the organic iodine is emitted) could be considerably higher (≈ 104–105 cm–3), particularly in the more polluted northern hemisphere [131, 162, 184]. Lifetimes for the organic iodine compounds with respect to Cl-atom reaction are summarized in Table 7. The reactions may be significant sinks in the MBL for CH2ICl, ethyl iodide, n- and iso-propyl iodide, and by logical extension, the larger alkyl iodides [18, 45] as well. 3.3.4 Product Formation in the Oxidation of Organic Iodine

A full accounting of the atmospheric impacts of the organic iodine compounds requires not only that their atmospheric lifetimes be established, but also that the products of their oxidation be determined. Of particular interest would be the formation of any long-lived iodine-containing intermediates, which might be subject to transport to regions away from the initial source. However, this does not seem to be the case. Photolysis, the dominant loss process for all species, of course leads to the immediate release of free I-atoms, e.g., CH3I + hn Æ CH3 + I

(9)

Even CH2I2 photolysis will lead to rapid liberation of both I atoms [46]: CH2I2 + hn

Æ CH2I + I

(10)

CH2I + O2

Æ CH2IO2

(11)

CH2IO2 + NO Æ CH2IO + NO2

(12)

CH2IO

(13)

Æ CH2O + I

As was shown above, reaction of Cl or OH may play a role in the destruction of the ethyl, propyl, and butyl iodides. These process also lead to rapid I-atom release, via reaction pathways analogous to Eqs. (11), (12), and (13). Thus, it seems that, in all cases, the initial organic iodine destruction process results in essentially instantaneous production of free I atoms. As shown below, the ensuing inorganic iodine chemistry likely plays a role in ozone destruction in the MBL, and (to a lesser extent) may also play a role in the free/upper troposphere and stratosphere as well.

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3.4 Atmospheric Distributions of Organic Iodine Compounds

Since the first detection of methyl iodide in the atmosphere by Lovelock et al. [102], numerous measurements of its concentration, particularly in MBL air, have been reported [3, 7, 13, 21–23, 34, 43, 47, 75, 99, 102, 112, 113, 120, 135, 139, 159, 161, 194, 195]. Some representative data sets are summarized in Table 6. This large body of data, which includes measurements extending essentially from pole to pole, shows typical values in the MBL of order 0.1–5 ppt.Values tend to be higher in the tropics than at higher latitudes [194], while mid-latitude measurements [22, 135, 155, 195] show maximum mixing ratios in summer, observations which both support a photochemical source of oceanic methyl iodide. However, a higher northern latitude study [195] revealed a reversed seasonal cycle, with higher values observed in winter. Though CH3I mixing ratios on the order of a few ppt are typical over open ocean water, observations of elevated levels (tens of ppt) are not uncommon. These are normally associated with regions of high ocean productivity [120] (e.g., photoplankton blooms, coastal waters) or with biomass burning plumes [3, 23, 43]. No reports of any temporal trends of CH3I have appeared to date in the literature, though no large trend in a predominantly oceanic source would be expected. Sturges et al. [168] were able to model successfully CH3I firn air data from Antarctica from an assumption of a constant yearly source strength, which probably indicates the lack of any strong temporal trend in CH3I emissions over the last several decades. As mentioned earlier, the presence of organic iodine species other than CH3I has also become apparent over the last 15 years or so. Among the species identified either in MBL air or in ocean surface water are alkyl iodides (CH3CH2I, CH3CHICH3, CH3CH2CH2I, various butyl iodides) [92, 194], and a series of multihalogenated methanes (CH2I2, CH2ICl, CH2IBr) [34, 41, 42, 92, 113]. A summary of some of these observations is provided in Table 8.When observed, typical mixing ratios of these species are on the order of a few tenths of a ppt, less than average methyl iodide mixing ratios. Nonetheless, these species may make a significant contribution to the overall atmospheric organic iodine burden in some locations. As shown above, the lifetimes of organic iodine compounds in the atmosphere are in all cases on the order of a few days or less, and in the case of CH2I2 only a few minutes. Thus, transport over great distances from the source regions is not possible, and it is not surprising that highest concentrations are observed in the MBL near the ocean source. Only methyl iodide has been detected above the boundary layer [3, 23, 43, 47, 135], and its concentration there is considerably lower than is found in the MBL. For example, altitude profiles for methyl iodide were obtained as part of the PEM-Tropics A campaign [43]. Average mixing ratios were observed to decrease from surface values near 0.4–0.5 ppt to values of about 0.1–0.2 ppt near 4 km. Essentially constant mixing ratios observed between 4 and 12 km were interpreted as evidence for a major contribution from deep convection to vertical mixing. Furthermore, the presence of CH3I in the free and

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Table 8. Summary of some representative measurements of organic iodides (other than methyl iodide) near the earth’s surface

Reference

Location of measurement

Compound measured

Concentration (ppt)

Reifenhäuser and Heumann 1992 [139] Moore and Tokarczyk 1992 [113] Klick and Abrahamsson 1992 [92]

Antarctica

CH2ClI

NW Atlantic

CH2ClI

S Atlantic and Swedish coast

Yokouchi et al. 1997 [194] Carpenter et al. 1999 [34]

W. Pacific, SE Asia

CH3CH2CH2I CH3CHICH3 CH2ClI CH2I2 CH3CH2I

Seawater measurements only Seawater measurements only Seawater measurements only

Coastal Ireland

CH3CH2I CH2ICl CH2IBr CH2I2

0.1 (SE Asia only) 0.06 0.11 0.08 0.05

upper tropospheric air was used as a tracer for marine convection, and used to assess the role of convective processes on HOX sources in the upper troposphere [43]. To date, there have been no reports of the detection of any organic iodine compounds in the stratosphere. 3.5 Atmospheric Impacts of Iodine Compounds: Inorganic Iodine Chemistry

Many overviews of the tropospheric chemistry of inorganic iodine, particularly as it relates to the destruction of ozone in the MBL, have appeared in the literature, e.g., [35, 36, 39, 83, 106, 177, 178, 198]. The key chemical reactions involved are shown in Fig. 7. The main fate of I in the troposphere is reaction with ozone to generate IO: I + O3 Æ IO + O2

(14)

The subsequent (rapid) photolysis of IO completes a null cycle, reaction Eqs. (14)–(15), (15) IO + hn (+O2) Æ I + O3 and establishes a steady-state between I and IO. Competitive processes for IO also exist, including its reaction with HO2, NO, NO2, and with itself and other halogen oxides (ClO and BrO). These reactions generate a suite of temporary reservoir compounds, including HOI, IONO2, and OIO. These reservoir species may then photolyze to regenerate I or IO, or may participate in heterogeneous processes that lead either to the removal of iodine from the system (via deposition to the surface) or to a return of iodine to the gas phase, mostly in the form of I2, IBr, or ICl [106]. Many reaction sequences involving the formation and subsequent

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Fig. 7. Schematic representation of the key reactions involving inorganic iodine compounds in the marine boundary layer

(gas- or aqueous-phase) destruction of the reservoirs, result in net ozone loss, for example, I + O3 Æ IO + O2 IO + HO2 Æ HOI + O2 HOI + hn Æ OH + I Æ HO2 + O2 OH + O3 net: 2 O3

Æ

3 O2

As summarized by McFiggans et al. [106], the kinetic database for inorganic iodine chemistry is not as well established as is the case for chlorine and bromine; uncertainties exist, for example, in the rate and mechanism of the photolysis of OIO and IONO2. Nonetheless, their model [106], which satisfactorily reproduced

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observed IO levels in the MBL, shows that iodine-based chemistry could lead to ozone loss rates of a few ppb per day, losses that are comparable in magnitude to those attributed to HOx-based cycles. Observation of measurable CH3I mixing ratios in the free and upper portion of the tropical and sub-tropical troposphere prompted Davis et al. [47] to consider the potential impacts of iodine chemistry there. For moderate iodine loading, effects were modest; iodine-based catalytic cycles (mainly involving HOI formation) contributed on the order of 5–10% to ozone loss rates over the total tropospheric column, and led to < 5% variation in the HO2/OH and NO2/NO ratios. The potential impact of iodine chemistry in the stratosphere was pointed out by Solomon et al. [166] They postulated that, though species like CH3I are shortlived, their transport to the upper troposphere/lower stratosphere could occur rapidly via deep convection, particularly in the tropics. Once in the stratosphere, destruction of the organic species would be rapid, and catalytic cycles involving I-atoms and IO radicals would be very efficient (perhaps as much as 1000 times more efficient than Cl) at removing ozone in lower stratosphere. Key cycles involve reaction of IO with HO2 (shown above), ClO, and BrO: I + O3 IO + ClO Cl + O3 net: 2 O3 I + O3 IO + BrO Br + O3 net: 2 O3

Æ Æ Æ

IO + O2 I + Cl + O2 ClO + O2

Æ

3 O2

Æ Æ Æ

IO + O2 I + Br + O2 BrO + O2

Æ

3 O2

Their modeling studies showed that a supply of about 1 ppt of CH3I to the stratosphere could make iodine-based cycles the dominant loss process for ozone in the lower stratosphere. While recent kinetic studies [50] have shown that the cycles involving ClO and BrO are less efficient than the original Solomon et al. estimates, and atmospheric measurements [132, 182] have shown that IO levels in the stratosphere (at least at mid-latitudes) are considerably less than 1 ppt, (probably no more than 0.2 ppt), the contribution of iodine to lower stratospheric ozone depletion cannot be entirely ruled out.

4 Summary The organic bromine content of the present-day atmosphere consists of a mix of anthropogenic (Halons and methyl bromide) and naturally-occurring (methyl bromide, and other short-lived species) compounds. With regulations now in place to eliminate the anthropogenic component of this mixture, due to the deleterious effects these compounds have on stratospheric ozone, the next century should see a reduction in the stratospheric bromine burden. Organic iodine, on

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the other hand, consists mostly of naturally-occurring species. The lifetimes of these compounds are very short (a few days at most), and thus mixing ratios are small and transport to the stratosphere is curtailed. While there is some indication that the future may see the further addition to the anthropogenic component to the atmospheric organic iodine budget (as iodine-containing species are being proposed as Halon and methyl bromide replacements), this burden is likely to be small compared to natural sources and is unlikely to be of any major environmental concern. Acknowledgements. The National Center for Atmospheric Research is operated by the Univer-

sity Corporation for Atmospheric Research under the sponsorship of the National Science Foundation. The author is indebted to Sue Schauffler, Elliot Atlas, and Geoffrey Tyndall (all of NCAR) for many helpful discussions, and for their comments on the manuscript.

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The Handbook of Environmental Chemistry Vol. 3, Part R (2003): 301 – 334 DOI 10.1007/b11992HAPTER 1

Physical-Chemical Properties and Estimated Environmental Fate of Brominated and Iodinated Organic Compounds Ian T. Cousins 1 and Anna Palm 2 1 2

ITM, Institute of Applied Environmental Research, SE 106 91 Stockholm, Sweden. E-mail: [email protected] IVL, Swedish Environmental Research Institute, Box 21060, SE 100 31 Stockholm, Sweden. E-mail: [email protected]

This chapter reviews the environmentally relevant physical-chemical properties, partitioning and reactivity properties of a selection of organobromine and organoiodine compounds. Substitution of hydrogens with bromine or iodine is shown to cause significant changes in vapor pressure, solubility in water, and hydrophobicity (octanol-water partition coefficient). These property changes are similar to those caused by substitution of hydrogen with chlorine, although there are quantitative differences attributable to the size and mass of the halogen atoms and to the effects on the strength of intermolecular interactions. The environmental implications of these changes are illustrated using simple multimedia partitioning models for volatile brominated and iodinated compounds, brominated flame retardants, and an iodinated X-ray contrast agent. The organobromine and organoiodine compounds are predicted to be less mobile in the environment than their organochlorine counterparts due to their generally higher reactivity and hydrophobicity, and lower volatility and solubility in water. Keywords. Physical-chemical, Properties, Bromine, Iodine, Organic, Partition, Model

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Volatile Brominated Organic Compounds . . . . . . . . . . . . Brominated Flame Retardants . . . . . . . . . . . . . . . . . . Volatile Iodinated Organic Compounds . . . . . . . . . . . . . Iodinated Organic Compounds Used in X-Ray Contrast Media

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Halomethanes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 307 PBDEs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 309 Reactivity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 316

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Estimated Fate and Behavior of Soil Fumigants: Methyl Bromide and Methyl Iodide . . . . . . . . . . . . . . . . . . . . . . . . . . 320 Estimated Fate and Behavior of PCBs and PBDEs . . . . . . . . . 324

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Estimated Fate and Behavior of Other Flame Retardants: HBCD, TBBPA, PBBs . . . . . . . . . . . . . . . . . . . . . . . . . 326 Estimated Fate and Behavior of Iopromide, an X-Ray Contrast Agent 328

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List of Abbreviations BFRs DDT EPIWIN EQC HBCD LRT PBBs PBDEs PCBs PCDD/Fs POPs QSPRs TBBPA UV

Brominated flame retardants 1,1,1-Trichloro-2,2-bis(p-chlorophenyl)ethane Estimations Programs Interface for Windows Equilibrium Quality Criterion Model Hexabromocyclododecane Long range transport Polybrominated biphenyls Polybrominated diphenyl ethers Polychlorinated biphenyls Polychlorinated dibenzo-p-dioxins and furans Persistent organic pollutants Quantitative Structure-Property Relationships Tetrabromobisphenol A Ultraviolet light

1 Introduction Since the 1960s a large amount of research has focused on the effects and environmental fate of chlorinated organic compounds. The physical-chemical properties and environmental partitioning of these compounds have been extensively measured and thus they are now reasonably well understood. Substitution of chlorine for hydrogen causes a marked increase in molar mass and volume and results in reduced vapor pressure and lower solubility in water and other solvents [9]. An increase in hydrophobicity also occurs, which leads to increased partitioning to organic components of soils, sediments, and biota while there is increased recalcitrance to degradation by several mechanisms. All of these undesirable properties have led to restrictions and bans on many commercial chlorinated organic compounds including PCBs and DDT. A separate volume of this handbook edited by Paasivirta [50] includes chapters discussing the sources, fate and effects of several groups of organochlorine compounds. In recent years there has been increasing concern about the environmental release of other halogenated organic compounds, in particular fluorinated and brominated organic compounds which have important commercial applications. The environmental fate and effects of iodinated organic compounds have also

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been studied, although less extensively. To understand the partitioning and reactivity of these compounds it is necessary to appreciate the physical-chemical consequences of substituting hydrogens on organic molecules by different halogens. The goals of this review are therefore to (1) compile selected available data on the environmentally relevant physical-chemical properties of brominated and iodinated organic compounds, (2) discuss the nature of the physical-chemical properties induced in the molecule resulting from bromination and iodination, and compare these with chlorination and fluorination, and (3) apply an evaluative model using the physical-chemical properties of selected brominated and iodinated organic compounds. The following sections describe some brominated and iodinated organic compounds that are known to be of concern. There is obviously a much greater range of brominated and iodinated compounds in commercial use that may be present in the environment. However, the aim of this chapter is not to review all of the brominated and iodinated organic compounds present in the environment, but rather to point out the effects of bromination and iodination on the physicalchemical properties and partitioning of a few key compound classes.

2 Bromine and Iodine It is useful to discuss briefly the properties of the elements themselves before discussing the properties and partitioning of brominated and iodinated organic compounds. There are large differences in the physical-chemical properties of the halogens, as summarized in Table 1. Fluorine has an electronegativity of 4.0 on the Pauling scale, which is the highest of all elements, whereas bromine and iodine have lower, but still relatively high electronegativities of 2.8 and 2.7, respectively. Conversely, polarizability increases in the order F < Cl < Br < I. Fluorine atoms show a very low polarizability because their electrons are tightly held (i.e., they are close to the nucleus), whereas iodine atoms are easily polarized (i.e., their electrons are far from the nucleus). Fluorine is the strongest known oxidizing agent, but oxidizing strength decreases as one proceeds from fluorine to iodine. Fluorine is so reactive it even forms compounds with Kr, Xe, and Rn, elements that were once believed to be inert. Iodine is the least reactive halogen, but is still reactive enough to form compounds with most metallic and non-metallic elements. Bromine is used in the manufacture of fire retardants (40%, US figures), drilling fluids (24%), brominated pesticides (12%), water treatment chemicals (7%), and other products (17%), including dyes, photographic chemicals, pharmaceuticals, and rubber additives [62]. Iodine is used in sanitation (45%, US figures), animal feed (27%), pharmaceuticals (10%), catalysts (8%), heat stabilizers (5%), and other uses (5%) including inks and colorants, photographic equipment and industrial disinfectants [63].

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Table 1. Selected properties of fluorine, chlorine, bromine, and iodine atoms where “X” is halo-

gen (properties from [35]) Property

Fluorine

Chlorine

Bromine

Iodine

Atomic mass Ionic radius (nm) Bond length in CX4 (nm) C-X bond energy (kJ/mol) Electronegativity

18.998 0.133 0.132 552 4.0

35.453 0.181 0.177 397±29 3.0

79.904 0.196 0.194 280±21 2.8

126.904 0.198 0.213 209±21 2.7

3 Some Important Brominated and Iodinated Organic Compounds in the Environment 3.1 Volatile Brominated Organic Compounds

Volatile brominated organic compounds have long tropospheric lifetimes and consequently some of the compounds released to the troposphere diffuse to the stratosphere. In the stratosphere the compounds are degraded by UV-light, resulting in the formation of bromine atoms, which, similarly to chlorine atoms, can catalyze the depletion of the ozone layer, the Earth’s primary shield against UV radiation. Free bromine is believed to be more efficient, on an atom per atom basis, than chlorine in removing stratospheric ozone. Indeed, it is suggested that bromine may be 40–100 times more destructive [43]. The major bromine-containing organics found in the troposphere that contribute to stratospheric ozone depletion are bromomethane or methyl bromide (CH3Br), dibromomethane (CH2Br2), and the halons, 1301 (CBrF3), 1211 (CBrClF2), and 1242 (C2Br2F4) [5]. Methyl bromide was used as an effective soil fumigant for control of soil-borne plant pathogens, although it is now being phased out in most countries (e.g., the US Clean Air Act schedules phase-out by 2001) because of concerns over its role in destroying stratospheric ozone [71]. Estimates published in 1992 suggested that agricultural fumigation contributed between 15 and 35% of bromine gas in the atmosphere [1]. The halons, which are bromine- or chlorine-containing fluorocarbons, were used extensively for fire mitigation applications. The Montreal Protocol has mandated a ban on the production of halons in industrialized countries, although their use is still permitted in certain applications [61]. Australia, particularly sensitive to stratospheric ozone depletion, has not only phased out the production of halons, but also totally banned their use. A number of other brominated organic species have also been detected including CH2BrCl, CHBrCl2, CHBr2Cl, and CHBr3 (bromoform), all of which have natural oceanic sources, and CF3CHBrCl, a man-made compound used as an anesthetic [5]. Many organobromine compounds are produced naturally by marine biota (sponges, corals, sea slugs, tunicates, sea fans) and seaweed, plants, fungi, lichen, algae, bacteria, microbes, and some mammals [22–24]. Macroalgae

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[20, 40] and phytoplankton in the oceans [46, 54] are believed to be responsible for the majority of natural production.Anthropogenic sources of volatile brominated organic compounds are considered minor in comparison to natural sources, except for a few specific compounds such as the halons and methyl bromide. 3.2 Brominated Flame Retardants

The manufacture of flame retardants is the most important use of bromine, accounting for about 40% of total use. The use of bromine in fire retardants relates to the fact that it is one of the few chemical elements with fire-resistance properties. The brominated flame retardants (BFRs) are a group of persistent organic pollutants (POPs) that are becoming increasingly studied (see recent reviews [13, 14, 51, 52]). The similarity in structure of PBDEs and PBBs to other well-known classes of persistent organic pollutants (POPs) such as PCBs and PCDD/Fs has prompted concern over possible similar environmental behavior and effects, i.e., similar persistence, bioaccumulation potential, and toxic actions. The BFRs are used to protect against the risk of accidental fire in such as computers, televisions, radios, stereo systems, video players, and in products made of textiles and plastics. BFRs are also used to improve the fire safety of foam padding used in upholstered furniture, in the plastic coating on electrical wire, and even in carpeting. Commercial BFRs are usually produced as technical mixtures that contain a range of brominated organic compounds. Some of the notable compounds of concern that are contained in these technical mixtures include polybrominated diphenyl ethers (PBDEs) (209 separate congeners), hexabromocyclododecane (HBCD), tetrabromobisphenol A (TBBPA), and polybrominated biphenyls (PBBs) (also 209 congeners). BFRs are classified as semi-volatile and hydrophobic.Although the properties of individual BFR compounds vary, they all tend to accumulate in organic-rich media such as soils and sediments, and lipid-rich biotic tissues. Similar to the organochlorines, they are expected to biomagnify in food chains. Much of the research to date has focused on the PBDEs, for which there is evidence of weak dioxin-like toxicity and some endocrine modulating effects [14, 51, 52]. Polybrominated dibenzo-p-dioxins and furans (PBDDs/Fs) and mixed halogenated dioxins and furans are not used as flame retardants, but are unintentionally produced during incineration of, e.g., municipal waste incineration and in internal combustion engines. They may be especially formed if the incinerated waste contains BFRs [67]. 3.3 Volatile Iodinated Organic Compounds

Organoiodine compounds are more reactive than the corresponding chlorine and bromine compounds and are thus not expected to diffuse significantly to the stratosphere. Instead, iodinated organic compounds are expected to play an important role in tropospheric chemistry. Due to their lower photostability, they

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may photodissociate and form free iodine in the troposphere, for example, by dissociation of iodomethane (CH3I) [19]. These iodine radicals are very reactive and can initiate several catalytic cycles, e.g., the destruction of tropospheric ozone. Iodomethane or methyl iodide (CH3I) is believed to be the largest contributor of tropospheric iodine, but other species have been identified including iodoethane (CH3CH2I), 1-iodopropane (CH3CH2CH2I), 2-iodopropane (CH3CHICH3), 1iodobutane (CH3CH2CH2CH2I), 2-iodobutane (CH3CH2CHICH3), diiodomethane (CH2I2), and chloroiodomethane (CH2ClI) (Giese et al. 1999) [19]. For a more complete description of the atmospheric chemistry of the brominated and iodinated organics we refer readers to the chapter of this volume by John Orlando. Similarly to the volatile brominated organics, the vast majority of organoiodine compounds are of biogenic origin [22], predominantly from marine macroalgae [33] and ice algae [16]. Methyl iodide has been proposed as a replacement for methyl bromide by the agricultural industry because it is still an effective soil fumigant, but is considered to be less damaging to stratospheric ozone [17]. 3.4 Iodinated Organic Compounds Used in X-Ray Contrast Media

A major application of iodinated organic compounds is in X-ray contrast media, which are used for X-ray examination of soft tissues in humans. Due to the high oral doses used and the absence of metabolism prior to excretion in urine, iodinated X-ray media are discharged in considerable quantities into the aquatic environment. For example, it has been shown that they are the main contributors to the burden of total adsorbable organic halogens (AOX) in clinical wastewater [18]. Common X-ray media are the derivatives of 2,4,6-triiodinated benzoates such as Diatrozoate (CAS: 131-49-7), Iopamidol (CAS: 60166-93-0), Iopromide (CAS: 73334-07-3) Iothalamic acid (CAS: 2276-90-6), Ioxithalamic acid (CAS: 28179-444), and Desmethoxy isobromide (DMI) (CAS: 76350-28-2) [58]. These compounds have high molecular mass (600–800 g/mol) and possess polar hydroxyl groups in their side chains. They are highly hydrophilic, fairly water soluble due to the many polar groups in their structure, non-volatile and are not readily biodegradable [57]. Their environmental fate and effects have not been extensively studied.

4 Physical-Chemical Properties and Reactivity In this section the physical-chemical properties of brominated and iodinated organics are reviewed and compared to chlorinated and fluorinated organics. A volatile class of organics believed to contribute to stratospheric ozone depletion, the halomethanes, and a semi-volatile class of persistent, hydrophobic organics, the PBDEs, are chosen for detailed examination with particular attention focused on the influence to the molecule of different halogen substitution.

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4.1 Halomethanes

Figure 1 shows the effect of adding multiple halogens to the vapor pressure of halomethanes. The substitution of hydrogen in methane with chlorine, bromine, or iodine causes a consistent decrease in the vapor pressure. Substitution with iodine decreases the vapor pressure more than for bromine, which in turn decreases the vapor pressure more than for chlorine. Interesting, fluorine displays anomalous behavior. The addition of one or two fluorines to methane causes a decrease in the vapor pressure of the compound. However, the addition of a third and fourth fluorine causes an increase in vapor pressure. The main point to consider in understanding these trends is the effect of molecular mass and volume. Heavier molecules require greater thermal energy in order to acquire the velocities sufficiently great to escape the surface of the condensed phase, and because their surface areas are usually much larger, intermolecular forces between molecules in the pure state are also greater. The more strongly a particular molecule attracts like molecules, the lower its corresponding abundance in the gas phase at equilibrium (i.e., vapor pressure) at a particular temperature. A description of the role of intermolecular forces on vapor pressure is found in [56]. Multiple halogenation in the chloro-, bromo-, and iodomethanes results in a consistent decrease in the vapor pressure. The increasing molar volume that results from addition of Cl, Br, and I appears to control the vapor pressure of these compounds. The addition of fluorine causes a relatively smaller increase in molecular volume, which is enhanced by the very low polarizability of fluorine. This may cause trifluoro- and tetrafluoromethane to be less susceptible to inter-

Fig. 1. The vapor pressures of the fluorinated, chlorinated, brominated and iodinated methanes. Measured values were taken from [6, 12, 32, 72]

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Fig. 2. The solubilities in water of the fluorinated, chlorinated, brominated and iodinated methanes. Measured values were taken from [25, 29, 37, 69]

molecular interactions and results in volatility more similar to that of methane [15]. Figure 2 shows the effect of adding multiple halogens to the solubility in water of halomethanes. The two main factors affecting the ability of organic solute to dissolve in water are (1) its molecular size and (2) the specific interactions between the organic solute and the water molecules. It is generally observed that within a specific class of organic compounds there is often a linear relationship between molecular mass or volume and solubility in water (e.g., see PBDEs section later). However, for the halomethanes molecular interactions between the organic solute and water molecules play an important role. In order for the halomethanes to dissolve in water they must disrupt the hydrogen bonding between water molecules. The only attractions possible between methane and water molecules are the much weaker van der Waals forces. The polarity of the halomethanes means that they can also interact with water molecules through the stronger dipole-induced dipole and dipole-dipole attraction forces, which accounts for the much higher solubility of the mono- di- and tri-halogenated methanes relative to methane. The reduction in solubility from monoto trihalomethanes is probably a result of increasing molar volume. However, the larger decrease from tri- to tetrahalomethanes may result from the weaker dipole-dipole interactions between water and the symmetrical tetrahalomethanes, which have no net dipoles. Figure 3 shows the effect of adding multiple halogens to the magnitude of the Henry’s Law constant of the halomethanes. The Henry’s Law constant (Pa·m3/ mol) provides measure of equilibrium distribution of a compound between the pure water and air phases. It is often estimated as the quotient of the vapor pressure (Pa) and solubility in water (mol/m3). The trends in Henry’s Law constants

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Fig. 3. The Henry’s law constants of the fluorinated, chlorinated, brominated and iodinated methanes. Measured values were taken from [21, 28, 30, 34, 45, 47, 70]. If measured values were unavailable for a compound the Henry’s law constant was calculated from the ratio of the vapor pressure (Pa) and solubility in water (mol/m3)

in Fig. 3 are therefore likely to be reflective of the trends in these two other properties. The large drop in the Henry’s Law constant from methane to the monohalomethanes is reflective of the large increase in water solubility of these compounds. The Henry’s Law constant decreases slightly as one progresses from monohalo- to trihalomethanes and then jumps up again for the tetrahalomethanes (especially for tetrafluoromethane tetraiodomethane), reflecting the large decrease in water solubility of these compounds. Figure 4 shows the effect of adding multiple halogens to the hydrophobicity (as expressed by the octanol-water partition coefficient (KOW)) of the halomethanes. The addition of chlorine, bromine or iodine to methane causes a consistent increase in KOW. Substitution with iodine increases the KOW more than substitution by bromine, which in turn increases the KOW more than the substitution by chlorine. Iodomethanes are the most hydrophobic, followed by bromo-, chloro-, and fluoromethanes. This trend was not so clear in the solubility relationship in Fig. 2 and may rather be a reflection of these compounds’ different solubilities in octanol, which will be largely related to molar volume. 4.2 PBDEs

Commercial PBDE mixtures include a large number of PBDE congeners. Each congener has unique physical-chemical characteristics that are dependent on the structure. When released into the environment, the components of the technical mixtures are likely to become dispersed and diluted so that interaction between

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Fig. 4. Log octanol-water partition coefficients (log KOW) of the fluorinated, chlorinated, brominated and iodinated methanes. Measured values were taken from [8, 26, 42]

them is unlikely. For this reason it is more common to measure properties of each of the components of a technical mixture and estimate their environmental fate, exposure and risk for each congener separately. As the interest in brominated flame retardants is increasing, the number of measurements of physical-chemical properties of individual congeners is steadily rising.Watanabe and Tatsukawa [65] performed one of the first studies of the properties of PBDEs, and measured vapor pressures and octanol-water partition coefficients (KOW) of PBDEs with various degrees of bromination. Since then, additional measurements have been performed [7, 27, 41, 49, 59, 60, 68]. Table 2 includes a compilation of physicalchemical property measurements for PBDE congeners. A major concern when interpreting the results from different experimental studies is that the numbers obtained sometimes vary by several orders of magnitude: this complicates the task of assigning relevant property data in, e.g., fate assessments involving environmental modeling. This is the case for many chemical groups [39] and depends on the different methodologies used, the analytical methods, and other, often unknown parameters. In order to achieve “best-estimates” for properties of chemicals, a common approach is to apply so-called quantitative structure-property relationships (QSPRs). The basic concept of QSPR is to select an appropriate molecular descriptor such as, e.g., molecular mass or volume, number of substitutes, or relative retention time and to correlate the properties of well-studied compounds against this descriptor, e.g., using linear regression. The relationship obtained between the molecular descriptor(s) and properties can then be used to derive the properties of unknown compounds. This kind of approach is particularly advantageous for chemicals belonging to a group with similar structures and behavior, such as, e.g., the PCBs, the halogenated benzenes, or the PBDEs.

5.47–5.58 c 5.74±0.22 (# 17), 5.94±0.15 (# 28) m

5.87–6.16 c 6.19 g 6.81±0.08 (# 47) m

7.0±1.0¥10 –2 (# 28) h

1.09¥10 –2 b 1.53¥10 –2 g 1.5±0.2¥10–2 (# 47), 1.8±0.3¥10–2 (# 66), 6.0±0.1¥10–3 (# 77) h

3

4

10.53 (# 47), 10.82 (# 66), 10.87 (# 77) d

9.3 (# 17), 9.5 (# 28) d

log KOA 2.99 a PSL: 0.163 (# 1), 0.128 (# 2) e PSL: 0.259 (# 3) h 1.27–1.89¥10 –2 c PSL: 1.68¥10 –2 (# 7), 1.37¥10–2 (# 8), 2.77¥10–2 (# 10), 1.19¥10–2 (# 12), 1.13¥10–2 (# 13), 9.84¥10–3 (# 15) e PSL: 1.73¥10–2 (# 15) 1.5–2.7¥10–3 c PSL: 2.19¥10–3 (# 17), 2.01¥10–3 (# 25) 1.6¥10–3 (# 28), 4.56¥10–3 (# 30), 2.25¥10–3 (# 32), 1.78¥10–3 (# 33), 1.39¥10–3 (# 35), 1.02¥10–3 (# 37) e PSL: 2.19¥10–3 (# 28) h 2.6–3.3¥10–4c PSL: 1.86¥10–4 (# 47), 1.22¥10–4 (# 66), 6.79¥10–5 (# 77) h PSL: 3.19¥10–4 (# 47), 2.38¥10–4 (# 66), 4.0¥10–4 (# 69), 4.10¥10–4 (# 71), 4.92¥10–4 (# 75), 1.56¥10–4 (# 77) e

PS (Pa)

[56a], b [15a], c [65], d [27], e [68], f [41], g [60a], h [60], i [49], j [68a], k [57a], l [32a], m [7].

5.03 c

0.13±0.02 (# 15) h

2

a

4.21 a

18 a 21 b

log KOW

0 1

No. of Wsol (mg/l) bromines

Table 2. Summary of measured physical-chemical properties of individual PBDE congeners

78.5–79 (# 47), 96–97 (# 51) 134–135 (# 71) 134.5–135.5 (# 75) 94–95 (# 77) f 83.5–84.5 (# 47), 104–108 (# 66) 96.5–98 (# 77) h, 82–82.5 (# 47) i

64–64.5 (# 28), 85–86 (# 30) 77–77.5 (# 32), 48–49 (# 37) f 64–64.5 (# 28) h

56–56.5 (#15) f 57–58 (#15) h

27.45 a

MP (°C)

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8.27±0.26 (# 183) m

1.5±0.3¥10–3 (# 183) h

7

9.97 c

8.35–8.9 c

6.86–7.92c 7.90±0.14 (# 153) 7.82±0.16 (# 154) m

8.7±0.6¥10–4 (# 153) 8.7±0.9¥10–4 (# 154) h

6

8 9 10

6.46–6.97 c 6.57 g 7.37±0.12 (# 85) 7.32±0.14 (# 99) 7.24±0.16 (# 100) m

6.0±0.1¥10–3 (# 85), 9.4±0.8¥10–3 (# 99), 4.0±1.0¥10–2 (# 100) h 9.0¥10–7 j 2.4¥10–3(# 99) k

log KOW

5

No. of Wsol (mg/l) bromines

Table 2 (continued)

11.96(# 183) d

11.82 (# 153), 11.92(# 154), 11.97(# 156) d

11.66(# 85), 11.31 (# 99), 11.13 (# 100), 11.97(# 126) d

log KOA

PSL: 4.68¥10–7 (# 183) 2.82¥10–7 (# 190)h PSL: 9.05¥10–7 (# 190) e 1.2–2.2¥10–7 c

2.9–7.3¥10–5 c PSL: 9.86¥10–6 (# 85), 1.76¥10–5 (# 99), 2.86¥10–5 (# 100) h PSL: 2.81¥10–5 (# 85), 6.82¥10–5 (# 99), 4.99¥10–5 (# 116), 8.07¥10–5 (# 119) e 4.2–9.4¥10–6 c PSL: 1.58¥10–6 (# 138), 2.09¥10–6 (# 153), 3.80¥10–6 (# 154) h PSL: 8.43¥10–6 (# 153) e

PS (Pa)

180–181 (# 140) 142–143 (# 154) 183.5–184.5(# 166) f 160–163 (# 153) 131–132.5 (# 154) h 182.3–182.8 (# 128) 134.2 (# 138), 157.6 (# 153) i 156–157 (# 181), 197–197.5 (# 190) f 171–173 (# 183) h 206 l

123.3(# 85), 92.3 (# 99) i 97–98 (# 100), 199.5–200 (# 116) 86–87 (# 119) f 119–121 (# 85) 90.5–94.5 (# 99) 100–101 (# 100) h

MP (°C)

312 I.T. Cousins · A. Palm

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Cole and Mackay [9] illustrated the QSPR method, “the three solubility approach”, and applied it to the chlorobenzenes, Cousins and Mackay [11] applied it to the phthalate esters, and Palm et al. [51] applied it to the PBDEs. Here the work of Palm et al. is repeated using the wider selection of experimental physical-chemical data that have recently become available (Table 2). Correlations were sought between the chosen molecular descriptor, which was the Le Bas molar volume [53], and the “solubilities” (mol/m3) of the liquid state of the compounds in air, water and octanol (SA, SW, SO), where SO represents the organic carbon- or lipid-containing media in the environment, i.e., soil, sediment and biota (biota include animal and vegetable components in both the aquatic and sediment phases) (Fig. 5). The three solubilities – SA (mol/m3) in air, SW in water and SO in octanol – were calculated from data in Table 2 using the following equations: SA = PSL/RT,

(1)

SW = WS/MF

(2)

SO = KOWSW

(3)

where PS is the vapor pressure of the subcooled liquid at 298 K (Pa), R (8.314 Pa/K.mol) is the gas constant, T (298.15 K) is the temperature, WS (g/m3) is the solubility of the solid in water at 298 K, M (g/mol) is molar mass, F is the fugacity ratio (log F = – 6.79(TM – T)/(2.303T)), and KOW is the octanol-water partition coefficient at 298 K. The correlations obtained for PBDEs were compared to similar correlations for the chlorobenzenes and PCBs that were calculated using experimental physical-chemical from [39] (Fig. 5). The linear regression equations obtained for the PBDEs were as follows: Log(SA) = 3.58 – 0.036 V

r2 = 0.87

SE = 0.55

(4)

Log(SW) = 2.42 – 0.021 V

r2

= 0.75

SE = 0.50

(5)

Log(SO) = 2.95 – 0.001 V

r2

= 0.02

SE = 0.38

(6)

From the solubility correlations (Eqs. 4–6), it is possible to deduce the relationships between molar volume and the partition coefficients KAW (air-water), KOW (octanol-water) and KOA (octanol-air): Log(KOW) = 0.53 + 0.02 V

(7)

Log(KAW) = 1.16 – 0.0015 V

(8)

Log(KOA) = –0.63 + 0.035 V

(9)

Equations (4–9) can be used to estimate properties of congeners for which no measurements currently exist. Figure 5a shows that the solubility in air falls steadily with increasing bromination with a slope slightly steeper than for the chlorobenzenes and PCBs. The absolute values are about a factor of 102 to 104 lower at the same molar volume. The decrease in SA is 0.84 log units per bromine added, i.e., a factor of 7.0. Although the solubilities, both in water (Fig. 5b) and in octanol (Fig. 5c), decrease with increasing bromination, the former (0.49 log units or factor of 3.11)

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Fig. 5. Plots of the log of solubility in air (i.e., vapor pressure/RT), water and octanol (i.e., KOW¥SW) (mol/m3) for the liquid state at 25 °C for PBDEs, chlorobenzenes and PCBs. Physicalchemical data were taken from Table 2

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Fig. 6. Plots of Log KOW, Log KAW, and Log KOA vs Le Bas molar volume. Experimental and estimated values are also shown in the graphs. Physical-chemical data are taken from Table 2

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are greater (0.03 log units or factor of 1.1). This accounts for the increasing octanol-water partition coefficient with increasing molar volume (Fig 6a), 0.46 log units (factor of 2.9) per bromine substituted. The standard errors (S.E.) of the regression lines are less than one log unit. Plots of the three partition coefficients are given in Fig. 6. The reported values for KOW for one substance sometimes vary by one log unit (factor of 10), which is important since KOW is used to estimate sorption and bioaccumulation. The experimental KAW data (as calculated from experimental data of water solubility and vapor pressure), which are essential for estimating air-water exchange, show considerable scatter, and this underscores the need to determine KAW by direct measurement. Indeed, different estimates may cover a range of 102.2. The KAW is estimated to decrease by 0.35 log units (factor of 2.2) per bromine added, which implies a decreasing preference for air relative to water with increasing bromination. Figure 2c indicates that KOA increases by 0.8 log units (factor of 6.5) per bromine added. The calculated values are generally between 0.1 and 1.5 log units lower than the corresponding measured values [27]. Although this difference is less than that reported by Palm et al. [51], it is still significant. Beyer et al. [4] suggested that the observed difference might be a result of the different measurement techniques when determining KOW and KOA, which may imply that the assumption KOA = KOW/KAW is inaccurate. It is therefore desirable to measure melting point, entropy of fusion, KOW, KAW, KOA, and SO for a few selected congeners to ascertain the trend with bromination more accurately. Analyses such as that attempted above would be greatly improved if more high quality physical-chemical property data were available for individual PBDE congeners. Further, there is a general lack of physical-chemical property measurements for other BFRs such as HBCD, PBB congeners, and TBBPA, although an attempt is made to estimate them later in this chapter. Further, these measurements should be undertaken at a range of environmentally relevant temperatures. 4.3 Reactivity

It is revealing to consider the relative strengths of the carbon-halogen bond for different halogens. The order of bond strength is C-F > C-C > C-B > C-I (see Table 1 for bond energies and bond lengths). The carbon-fluorine bond is one of the strongest in nature and thus carbon-fluorine bonds are extremely difficult to break during chemical reactions. Consequently, organofluorine compounds are amongst the most environmentally persistent man-made organic chemicals [15]. The stability of chlorofluorocarbons and some volatile organochlorines enables them to be transported to the stratosphere, where they can breakdown with the help of UV light to form reactive chlorine. The atmospheric chemistry of reactive chlorine compounds in the atmosphere is discussed in [31] and of hydrofluorocarbons and hydrofluoroethers in [64]. Volatile organobromine molecules are also stable enough to reach the stratosphere and have damaging effects, although their higher reactivity and lower abundance in the troposphere determines that free bromine in the stratosphere is less abundant than free chlorine. Hydroxyl radical reactions are a key removal

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317

mechanism for the organobromine compounds, except for the halons that do not contain a hydrogen atom (e.g., CF3Br, CF2Br2, CF2ClBr). Direct photolysis is also important for some volatile brominated compounds. Due to their relatively lower photostability, direct photolysis of volatile iodinated organics is believed to be the dominant removal mechanism in the troposphere [19]. Methyl iodide has a tropospheric residence time of less than ten days as a result of photodissociation and subsequent formation of iodine atoms. Increasing iodination probably makes this compound class even less photolytically stable. John Orlando discusses the atmospheric chemistry of brominated and iodinated organic compounds in more detail in another chapter of this volume. For the semi-volatile organochlorines (e.g., PCBs) increasing the chlorination level increases the stability of the compound in the atmosphere, surface waters, soils and sediments [39]. The principal removal mechanism in the atmosphere is oxidation by hydroxyl radicals and the main removal mechanism in water, soil and sediments is aerobic biodegradation. Increased chlorination imparts stability to hydroxyl radical attack in the atmosphere which can be explained by (1) there being fewer hydrogens to attack and (2) the halogens are strong electron withdrawing groups which stabilize the aromatic ring to electrophilic attack. Increasing chlorination also imparts stability to aerobic biodegradation, which can be explained by (1) reduced solubility in water and thus bioavailability to microorganisms and (2) increased halogenation stabilizes the aromatic ring to attack by enzymes. It is expected that increasing the bromination level of PBDEs or PBBs will have a similar effect on stability to hydroxyl radical attack in the atmosphere and biodegradation in other media. The environmental degradation rates of the PBDEs and PBBs are unknown, but we speculate that the relative weakness of the carbon-bromine bond and lower electronegativity of bromine causes them to be less persistent than structurally similar organochlorine compounds.

5 Estimated Environmental Fate and Behavior A full evaluation of the likely environmental fate of the brominated and iodinated organic chemicals is beyond the scope of this chapter. It is, however, useful to outline how the physical-chemical properties of a few selected brominated and iodinated compounds translate into environmental fate, which can be achieved by conducting evaluative environmental fate modeling. The aim is to establish the general features of chemical behavior, the media into which the chemical will tend to partition, the primary loss mechanisms, the tendency for intercompartmental transport and bioaccumulation, and the potential for long-range transport. The EQuilibrium Criterion or EQC model [38] is a widely used evaluative model that treats an area of 105 km2 with about 10% of the area being covered by water. The temperature in the EQC environment is set at 25 °C, the common temperature at which physical-chemical properties are measured. Evaluative modeling assessments usually progress through three stages of complexity; Levels I, II, and III. Each subsequent level requires more detailed information or includes additional processes providing a step-wise increase in understanding of the behavior of a chemical in the environment. In this chapter only Level III outputs are

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provided, because these are the most environmental realistic of the three “levels” of model output. The Level III calculation shows the dependence of overall environmental fate on mode of entry, highlights the dominant intercompartmental transport pathways (e.g., air-water or air-soil exchange), and calculates the overall environmental persistence in the model environment [66]. Mackay [36, 38] provides a complete description of fugacity calculations with examples at each level of complexity. For the Level III calculation, 1000 kg/h of each chemical was emitted to the air, water, and soil compartments individually. These are evaluative emission scenarios designed to illustrate the effects of different extreme emission scenarios on fate and behavior. Finally, 1000 kg/h of chemical was emitted all three compartments simultaneously. The compounds selected for the modeling exercise are (1) methyl bromide and methyl iodide, volatile compounds that have both natural and anthropogenic sources, (2) PBDE congener 47 (2,2¢, 4,4¢-tetrabromodiphenyl ether), a semivolatile compound contained in some BFRs, which is compared to the structural similar PCB congener 52 (2,2¢, 5,5¢-tetrachlorobiphenyl), (3) other important BFRs, HBCD, TBBPA and PBB congener 52 (2,2¢, 5,5¢-tetrabromobiphenyl), and (4) a non-ionic X-ray contrast agent, Iopromide (CAS number: 73334-07-3). The structures of these compounds are contained in Fig. 7. Table 3 contains a compilation of the key physical-chemical properties and degradation half-life estimates for the selected compounds. The Estimations Programs Interface for Windows (EPIWIN) [44] software was used to estimate properties where measured values were not available (see the Syracuse Research Corporation Inc. web site at http://esc.syrres.com/interkow/epi.htm for a description

Fig. 7. Structures of some brominated and iodinated organic compounds selected for fate modeling

94.94 141.94 485.8 292 641.7 469.8 543.88 791.12

1.52¥104 1.38¥104 3.0¥10–2 3.0¥10–2 3.4¥10–3 6.3¥10–4 1.0¥10–2 23.8

Molar mass Water (g/mol) solubility (g/m3) 2.16¥105 5.40¥104 9.6¥10–5 4.9¥10–3 6.3¥10–5 3.13¥10–5 2.35¥10–9 <10–12

Vapor pressure (pa) 1.19 1.51 6.3 6.1 5.6 7.32 7.20 –2.05

Log KOW

–93.7 –66.4 81.6 87 185 158 181 350

Melting point (°C) 6650 166 256 352 51.2 400 86.8 3.95

Air 360 360 3600 3600 14400 3600 3600 1440

Water

360 360 3600 3600 14400 3600 3600 1440

Soil

1440 1440 14400 14400 57600 14400 14400 5760

Sediment

Assumed 1st order reaction half lives (h)

Vapor pressure from [12], water solubility data from [29], log KOW from [26], atmospheric half-life calculated from atmospheric residence time of 400 days (half-life) [5], (h) = 0.693 ¥ 400 ¥ 24). Degradation half-lives for water, soil and sediment estimated using EPIWIN [44]. b Vapor pressure from [6], water solubility data from [29], log KOW from [26], atmospheric half-life calculated from atmospheric residence time of 10 days (half-life) [19], (t1/2 = 0.693 ¥ 10d ¥ 24 h). Other degradation half-lives estimated using EPIWIN [44]. c Physical-chemical data from the “three solubility approach” (Section 4.2). Degradation rates estimated using EPIWIN [44]. d Physical-chemical data from [39], degradation rates estimated using EPIWIN [44]. e Physical-chemical property data taken from EU risk assesment [48], degradation rates estimated using EPIWIN [44]. f Log KOW from [26] and all other data estimated using EPIWIN [44].

a

Methylbromide a Methyliodide b PBDE-47 c PCB-52 d HBCD e PBB-52 f TBBPA f Iopromide f

Compound

Table 3. Physical-property property data for compounds used in the evaluative modelling

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of the estimation software). The EPIWIN software can estimate physical-chemical properties and reaction rates from structure alone and is now widely used by chemical regulators, particularly in the US. For the purposes of consistency, all first order biodegradation rate constants were estimated using EPIWIN. It is noted that the half-life values assigned are extremely uncertain; however, the ranking of reactivity should be more reliable. Table 4 provides a summary of the model output and the following sections add some interpretation. In addition to calculating the overall environmental persistence (total inventory (kg)/total losses (kg/h)) due to losses by reaction and from advection (transport of chemical in air and water out of the model environment), the reaction residence time, which is the total inventory (kg)/losses by reaction (kg/h), is also calculated. The reaction residence time gives a better indication of the removal of chemical from the environment as a whole. The absolute values for overall persistence that have been estimated may not accurately reflect the actual persistence times in the environments, as there are large uncertainties in the reaction half-lives assigned. However, it is our opinion that the ranking of chemicals for persistence is useful exercise that could be used to support chemical regulation. For example, it is interesting to examine if structurally similar organobromine and organoiodine compounds are more or less persistent than organochlorine compounds. Another criterion used to rank organic compounds for regulation is their potential for long-range transport (LRT). There are two main factors which control a chemical’s LRT potential: persistence in the atmosphere which can be characterized by a half-life, and “stickiness”, the propensity for a chemical to partition to the terrestrial surface if transported in air, or its propensity to partition to sediment if transported in water. Characteristic travel distances (km) in air and water have been calculated for each of the selected compounds using the method suggested by Beyer et al. [3]. Similar to environmental persistence, it is more appropriate to compare the ranking of chemicals rather than pay too much attention to the absolute travel distances estimated. Thus it is useful to compare travel distances for the organobromine and organoiodine compounds with the wellstudied organochlorine compounds. 5.1 Estimated Fate and Behavior of Soil Fumigants: Methyl Bromide and Methyl Iodide

Methyl bromide is used as a soil fumigant and is also produced biogenically in the world’s oceans. Its use as a soil fumigant has been recently restricted since it was believed to contribute to stratospheric ozone depletion. Methyl iodide has been considered as a replacement chemical by the agricultural industry because its higher photoreactivity in the troposphere and thus lower residence time are believed to render it unlikely to be involved in stratospheric ozone depletion. However, the environmental behavior of methyl iodide is essentially unknown [17]. It is therefore interesting to predict and contrast the environmental behavior of these two compounds using evaluative modeling techniques. Figure 8 illustrates the Level III model output for methyl bromide and methyl iodide for 1000 kg/h emission to soil and Table 4 summarizes the model results

Air Water Soil “Total” Air Water Soil “Total” Air Water Soil “Total” Air Water Soil “Total” Air Water Soil “Total”

Methylbromide

HBCD

PCB-52

PBDE-47

Methyliodide

Emission medium

Compound

98,917 (99.8) 45,129 (19.5) 96,752 (89.7) 241,000 (54.9) 70,484 (99.5) 31,711 (14.4) 66,312 (68.8) 169,000 (43.5) 49,458 (2.07) 952 (0.01) 2.02 (<0.01) 50,413 (0.30) 81,406 (31.1) 19,620 (0.29) 79.2 (<0.01) 101,000 (0.83) 39,709 (3.10) 11,160 (0.13) 148 (<0.01) 51,017 (0.17)

Air 168 (0.17) 186,000 (80.3) 1,000 (0.93) 187,000 (42.7) 286 (0.40) 188,000 (85.4) 2,237 (2.32) 195,000 (49.2) 17,040 (0.71) 304,000 (3.22) 178 (<0.01) 321,000 (1.89) 4,128 (1.58) 270,000 (4.00) 172 (<0.01) 274,000 (2.25) 8,197 (0.64) 441,000 (4.96) 1,514 (<0.01) 451,000 (1.46)

Water 18.1 (0.02) 8.25 (<0.01) 10,114 (9.38) 10,140 (2.31) 35.9 (0.05) 16.2 (<0.01) 27,791 (28.8) 27,844 (7.19) 1,810,000 (75.9) 34,844 (0.37) 5,190,000 (99.9) 7,040,000 (41.4) 77,316 (29.6) 18,634 (0.28) 5,190,000 (99.9) 5,280,000 (43.3) 1,080,000 (84.4) 304,000 (3.42) 20,700,000 (99.8) 22,000,000 (71.4)

Soil

Amount at steady state (kg) (percent in brackets)

Table 4. Level III model results for selected halogenated compounds

0.317 (<0.01) 351 (0.15) 1.89 (<0.01) 353 (0.08) 0.608 (<0.01) 399 (0.18) 4.75 (<0.01) 405 (0.10) 510,000 (21.4) 9,090,000 (96.4) 5,335 (0.10) 9,610,000 (56.5) 98,615 (37.7) 6,450,000 (95.4) 4,104 (0.08) 6,560,000 (53.7) 151,000 (11.8) 8,140,000 (91.5) 27,944 (0.14) 8,320,000 (26.9)

Sediment 4.13 (387) 9.64 (26.6) 4.49 (143) 6.09 (45.1) 2.95 (10) 9.18 (18.5) 4.01 (12.0) 5.38 (14.4) 99.4 (208) 393 (779) 217 (217) 236 (358) 10.9 (60.6) 282 (696) 216 (217) 170 (321) 53.3 (90.1) 371 (1,301) 862 (865) 429 (686)

Residence time (d) (Reaction residence time in brackets) a

Air: 1,000 Water: 5,000

Air: 6,400 Water: 1,400

Air: 1,400 Water: 1,700

Air: 3,400 Water: 550

Air: 130,000 Water: 860

Characteristic travel distance in air and water (km) b

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Air Water Soil “Total” Air Water Soil “Total” Air Water Soil “Total”

PBB-52

a

60,041 (3.28) 1,061 (<0.01) 2,05 (<0.01) 61,104 (0.34) 19,873 (0.53) 0.01 (<0.01) <0.01 (<0.01) 19,873 (0.10) 9.82 (<0.01) <0.01 (<0.01) <0.01 (<0.01) 9.82 (<0.01)

Air 8,985 (0.49) 217,000 (2.00) 114 (<0.01) 226,000 (1.26) 14,474 (0.39) 224,000 (2.04) 118 (<0.01) 239,000 (1.20) 453,000 (39.8) 675,000 (99.8) 428,000 (36.0) 1,560,000 (51.8)

Water 1,320,000 (72.2) 23,390 (0.22) 5,190,000 (99.9) 6,540,000 (36.5) 3,000,000 (80.5) 0.765 (<0.01) 5,190,000 (99.9) 8,190,000 (41.2) 685,000 (60.2) <0.01 (<0.01) 761,000 (64.0) 1,450,000 (48.1)

Soil

Amount at steady state (kg) (percent in brackets)

440,000 (24.0) 10,600,000 (97.8) 5,597 (0.11) 11,100,000 (61.9) 694,000 (18.6) 10,800,000 (98.0) 5,672 (<0.11) 11,500,000 (57.5) 857 (0.08) 1,278 (0.19) 810 (0.07) 2,946 (0.10)

Sediment

Estimated residence time in the Level III environment. b Estimated using the method of Beyer et al. (2000).

Iopromide

TBBPA

Emission medium

Compound

Table 4 (continued)

76.4 (200) 453 (809) 217 (217) 249 (384) 155 (201) 457 (816) 217 (217) 276 (355) 47.4 (86.6) 28.2 (86.7) 49.6 (86.6) 41.7 (86.6)

Residence time (d) (Reaction residence time in brackets) a

Air: 0.14 Water: 7,500

Air: 440 Water: 1,100

Air: 2,300 Water: 1,100

Characteristic travel distance in air and water (km) b

322 I.T. Cousins · A. Palm

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Fig. 8. Diagrams showing Level III model outputs for methylbromide and methyliodide for 1000 kg/h emission to soil

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for all evaluative emission scenarios. The most relevant emission scenario to consider is emission to soil, which represents the chemicals’ use as a soil fumigant. When 1000 kg/h of chemical is emitted to soil, the vast majority of both chemicals volatilize (978 kg/h for methyl bromide and 937 kg/h for methyl iodide). To determine which of the two compounds is most likely to reach the stratosphere and contribute to ozone depletion it is necessary to compare their atmospheric reactivity. Methyl bromide is more stable in the atmosphere than methyl iodide, largely because it is more photolytically stable. The tropospheric residence times of methyl bromide and methyl iodide are 400 and 10 days, respectively, as previously discussed. Of the 1000 kg/h that is emitted to the soil, 10.1 kg/h of methyl bromide is removed by reaction in the atmosphere, whereas 277 kg/h of methyl iodide is removed. The characteristic travel distance of methyl bromide in air (130,000 km) is much larger than that of methyl iodide (3400 km), largely a reflection of the differences in atmospheric residence times. In conclusion, the presence of the heavier iodine rather than bromine in the molecule only slightly reduces the volatilization potential of the two compounds and thus almost identical amounts of the two compounds are estimated to enter the atmosphere as a result of their use as soil fumigants. However, as a result of its longer reaction residence time in the atmosphere, more methyl bromide is expected to reach the stratosphere and thus be a more significant contributor to stratospheric ozone depletion than methyl iodide. In general, the higher reactivity of the volatile iodinated organic compounds, and their slightly lower volatility relative to chlorinated and brominated compounds, will ensure that they have a minor impact on stratospheric ozone depletion. 5.2 Estimated Fate and Behavior of PCBs and PBDEs

Figure 9 illustrates the Level III model output for PBDE-47 and PCB-52, and Table 4 summarizes the model results. These compounds were selected because PBDE-47 and PCB-52 are both tetra-halogenated compounds, thus facilitating direct comparison of the effects of chlorination and bromination on environmental fate and behavior. As previously discussed, the substitution of bromine for hydrogen reduces the vapor pressure and solubility in water more markedly than the substitution with chlorine. This results in much higher propensity for the PBDEs relative to the PCBs for atmospheric aerosols, soils, and sediments.As bromination level increases the net rate of transfer to organic carbon and lipid-rich media increases. When emitted to all three media simultaneously, there is a net transfer of PBDE-47 from water to sediment of 848 kg/h, from air to soil of 355 kg/h, and from air to water of 57 kg/h. Soil reaction is the main removal process for this compound (1354 kg/h). The chemical accumulates in soil and sediment and there are relatively small proportions in air and water. Of the chemical present in the air, 26% is estimated to be associated with atmospheric aerosols. For PCB-52, there is a net transfer from water to sediment of 579 kg/h, from air to soil of 18.5 kg/h and from air to water of 19.0 kg/h. There is also a significant movement of chemical from water to air of 246 kg/h, compared to 23.7 kg/h for PBDE-47. As for PBDE-47, soil and sediment are the most important media

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Fig. 9. Diagrams showing Level III model outputs for PBDE-47 and PCB-52 for 1000 kg/h emis-

sion to each of air, water and soil

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of accumulation and soil reaction is the main removal process (1017 kg/h). Of the chemical present in the air, only 0.60% is estimated to be associated with atmospheric aerosols. The characteristic travel distance for PCB-52 (6350 km) is about 4–5 times larger than for PBDE-47 (1400 km), which is a reflection of the higher “stickiness” of PBDE-47 for atmospheric aerosols. Increased aerosol partitioning also increases atmospheric deposition processes and thus reduces atmospheric travel distance. In summary, the behavior of these two classes of compounds is broadly similar in that they are both persistent and bioaccumulative and due to their hydrophobicity are likely to accumulate in organic rich phases such as soils and sediments. The PBDEs, however, are less volatile and less water-soluble at the same level of halogenation and thus partition more strongly to atmospheric aerosols and soil and sediment particles. The result is that they will be less bioavailable and are less mobile in the environment than PCBs.A small proportion of the PBDEs released will travel long distances, evidenced by the fact that they have been detected in the Arctic [2]. This is not unexpected given the widespread usage of these chemicals and their high environmental persistence. However, in our opinion, the vast majority of BFRs released to the environment will remain close to their sources in populated regions, where they will be predominantly stored in the soils and sediments. Transport to remote regions could be increased under special atmospheric conditions or through long-range transport of particles in the atmosphere and water bodies. 5.3 Estimated Fate and Behavior of Other Flame Retardants: HBCD, TBBPA, PBBs

The PBDEs are the best studied of the BFRs. There are fewer studies, however, reporting the fate and behavior of some of other commercially important BFRs such as HBCD, TBBPA, and PBBs. It is therefore interesting to estimate their fate and behavior and identify the key fate processes, which will help researchers target experimental work. Figure 10 illustrates the Level III model output for HBCD, TBBPA, and PBB-52, respectively, and Table 4 summarizes the model results. When emitted to all three media simultaneously, there is a net transfer of HBCD from water to sediment of 458 kg/h, from air to soil of 69.7 kg/h and from air to water of 24.7 kg/h. Soil reaction is the main removal process for this compound (1063 kg/h) although air reaction is competitive at 682 kg/h. The chemical accumulates in soils and bottom sediments and there are relatively small proportions in air and water. Of the chemical present in the air, 0.05% is estimated to be associated with atmospheric aerosols. It is estimated to be a highly persistent chemical with a reaction residence time of more than two years. For TBBPA, there is a net transfer from water to sediment of 1021 kg/h, from air to soil of 684 kg/h and from air to water of 76.0 kg/h. There is negligible transport from water to air (< 0.01 kg/h) and soil to air (< 0.01 kg/h), as this is an extremely non-volatile molecule. It accumulates in soils and in particular in bottom sediments. Of the chemical present in the air, 99.9% is associated with atmospheric aerosols. It has a very low characteristic travel distance in air (440 km) as it is rapidly deposited from the atmosphere to terrestrial and water surfaces.

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Fig. 10. Diagrams showing Level III model outputs for HBCD, TBBPA, and PBB-52 for 1000 kg/h emission to each of air, water and soil

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For PBB-52, there is a net transfer from water to sediment of 977 kg/h, from air to soil of 263 kg/h, and from air to water of 42.6 kg/h. There is only a little movement of chemical from water to air of 18.4 kg/h and soil to air of 0.03 kg/h. Soil and sediment are the most important media of accumulation and soil reaction is the main removal process (1263 kg/h). Of the chemical present in the air, 0.16% is estimated to be associated with atmospheric aerosols. Not surprisingly, given its structural similarity, the behavior of PBB-52 is close to that of BDE-47. It has, however, a higher association to atmospheric aerosols. It is generally expected therefore that the PBBs will have similar fate and behavior to the PBDEs. The PBBs and PBDEs are expected to be less environmental mobile and slightly less persistent than the PCBs. There are broad similarities between the environmental fate and behavior of the various BFRs. They all tend to accumulate in soils and sediments and have a high environmental persistence and low environmental mobility. They have a higher potential to bioaccumulate and biomagnify than the chlorinated compounds due to their higher hydrophobicity, although they are likely to be less bioavailable. TBBPA and the higher brominated BFRs are particularly immobile and should be expected to be limited to areas close to sources. The lower brominated BDEs (tetra- and lower), and to some extent HBCD, will be transported longer distances away from sources, but are not expected to be global pollutants to the same extent as the more mobile organochlorine compounds such as PCBs, hexachlorobenzene and hexachlorocyclohexane. 5.4 Estimated Fate and Behavior of Iopromide, an X-Ray Contrast Agent

Iopromide is the most widely used X-ray contrast agent used and has been detected in wastewaters, river water, and groundwater. Although the usual range of emission scenarios have been run for this compound, the most environmental realistic is discharge to water. Since X-ray contrast media are normally applied in human medicine, polluted municipal sewage treatment effluents and clinical wastewaters are presumably the main source of these chemicals to the environment. Figure 11 illustrates the Level III model output for 1000 kg/h emission to water, and Table 4 summarizes the model results for all other emission scenarios. If 1000 kg/h of Iopromide is discharged to water 675 kg/h is advected out of the water box in out-flowing water, 3.4 kg/h is deposited to bottom sediments, a negligible amount is volatilized, and 325 kg/h is removed by reaction in the water column. The application of the X-ray media to humans requires that they are stable and hydrophilic to guarantee that they are rapidly eliminated via urine without being metabolized. Due to Iopromide’s stability, it will be transported many kilometers downstream of the source as testified by its estimated characteristic travel distance in water of 7500 km. The chemical will almost exclusively remain dissolved in the water column and will thus remain bioavailable. However, as it has a low estimated hydrophobicity (Log KOW of –2.05), it is not expected to bioaccumulate significantly in organisms. Further, it has also been shown to have an extremely low toxicity with no effects on any of various aquatic species observed

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Fig. 11. Diagram showing Level III model output for Iopromide for 1000 kg/h emission

to water

at concentrations as high as 1 g/l [57]. Given that it is detected in the environment at only a few µg/l [58], its risk to aquatic organisms appears to be very low. It may bioaccumulate in plants as its high solubility in water facilitates its uptake by plant roots and accumulation in watery tissues. Its risk to plant life cannot be determined as its phytotoxicity is unknown. As X-ray contrast agents tend to contain ionic groups or polar groups that make them highly water-soluble the environmental behavior of Iopromide is believed to be representative of the behavior of other compounds in this class.

6 Conclusions Substitution of hydrogen with bromine or iodine is shown to cause significant changes in properties such as vapor pressure, solubility in water, and hydrophobicity, as expressed by the octanol-water partition coefficient. These property changes are generally comparable to those that occur as a result of substitution of hydrogen with chlorine although there are quantitative differences attributable to the size and mass of the different halogen atoms and also to the effects that different halogens have on the strength of intermolecular interactions. The substitution of bromine or iodine for hydrogen usually causes a reduction in solubil-

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ity in water and vapor pressure, which is largely attributable to the higher mass and volume of these halogens relative to hydrogen. Therefore, iodine causes a bigger physical-chemical effect than bromine, which in turn causes a bigger effect than chlorine. Exceptions occur as a result of strong molecular interactions occurring between the molecules in their pure state or between the solute and solvent molecules. For example, there is a rise in water solubility caused by substitution of one hydrogen for a halogen in methane caused by the relatively strong interactions between water molecules and the more polar halogenated methanes. Evaluative modeling of several selected compounds is useful in determining the implications of the changes in physical-chemical results resulting from different halogen substitution on organic molecules. The relative potency of the various halomethanes for stratospheric ozone depletion is mainly determined by their reactivity in the troposphere. Brominated methanes can be removed from the troposphere both by photolysis and oxidation with hydroxyl radicals. Increasing bromination reduces the photolytic stability and thus atmospheric residence time of the brominated methanes.Volatile iodinated compounds are photolytically unstable in the troposphere and thus have a negligible contribution to stratospheric ozone depletion. Semi-volatile organobromines such as the BFRs tend to be less volatile, less water soluble and more hydrophobic than their chlorinated analogs. These properties ensure that organobromines accumulate more readily in organic/lipid-rich phases such as biota, atmospheric particles, soils and sediments and are less mobile and less bioavailable than their organochlorine analogues. Although their rates of degradation in the environment are unknown, we speculate that they are more reactive than their organochlorine analogues. Low bioavailability, however, may restrict their biodegradation by microorganisms. In summery, their lower volatility and lower solubility in water would result in lower mobility, and thus lower environmental exposures and reduced potential to pollute remote areas (e.g. Arctic regions) than their organochlorine analogues. There are no iodinated analogs of PCBs or structurally similar compounds to facilitate direct comparison with PCBs and PBDEs. A major application of iodinated organic compounds is in X-ray contrast media. Unlike most of the other halogenated compounds discussed in this chapter, these compounds contain polar groups, which impart hydrophilic properties. They are consequently fairly water soluble, and have low volatility and hydrophobicity. Therefore, they are expected to be present almost entirely in the dissolved phase of surface waters into which they are discharged. Their fairly high stability is expected to facilitate their transport many thousands of kilometers from sources. However, their low bioaccumulation potential and low measured toxicities suggest that they are unlikely to pose a risk to aquatic animal life. Acknowledgements. The authors would like to thank Stockholm University for providing the

Visiting Researcher Fellowship to Ian Cousins, and Thomas Cahill of the University of California at Davis, US and Donald Mackay of Trent University, Canada for providing useful input to the chapter.

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64. Wallington TJ, Nielsen OJ (2002) Atmospheric chemistry and environmental impact of hydrofluorocarbons (HFCs) and hydrofluoroethers (HFEs). Handb Environ Chem 3N:85–102 65. Watanabe I, Tatsukawa R (1989) Anthropogenic brominated aromatics in the Japanese environment. Proceedings.Workshop on brominated aromatic flame retardants. Swedish National Chemicals Inspectorate, Solna, Sweden 24–26 October 1989, pp 63–71 66. Webster E, Mackay D, Wania F (1998) Evaluating environmental persistence. Environ Toxicol Chem 17:2148–2158 67. Wichmann H, Dettmer FT, Bahadir M (2002) Thermal formation of PBDD/F from tetrabromobisphenol A – a comparison of polymer linked TBBP A with its additive incorporation in thermoplastics. Chemosphere 47:349–355 68. Wong A, Lei YD, Alaee M, Wania F (2001) Vapor pressures of the polybrominated diphenyl ethers. J Chem Eng Data 46:239–242 68a. World Health Organization (1994) “Environmental Health Criteria 162: Brominated Diphenyl Ethers”. Geneva, Switzerland 69. Yalkowsky SH, Dannenfelser RM (1992) Aquasol database of aqueous solubility.Version 5. College of Pharmacy, University of Arizona, Tucson, AZ. PC Version 70. Yates SR, Gan J (1998) Volatility, adsorption, and degradation of propargyl bromide as a soil fumigant. J Agric Food Chem 46:755–761 71. Yates SR,Wang D, Ernst FF, Gan J (1997) Methyl bromide emissions from agricultural fields: bare-soil, deep injection. Environ Sci Technol 31:1136–1143 72. Yaws CL (1994) In: Handbook of vapor pressure, vol 1. C1 to C4 compounds. Gulf, Houston, TX, p 347

The Handbook of Environmental Chemistry Vol. 3, Part R (2003): 205 – 251 DOI 10.1007/b11992HAPTER 1

Mammalian Toxicity of Organic Compounds of Bromine and Iodine Joseph W. DePierre Unit for Biochemical Toxicology, Department of Biochemistry and Biophysics, Arrhenius Laboratory, Stockholm University, 106 91 Stockholm, Sweden E-mail: [email protected]

Human beings are exposed to an increasing number and increasing amounts of organobromine and organochlorine compounds, both of man-made and natural origin. The commercial products of this nature present at highest levels in the general environment include alkyl halides (in particular, methyl bromide), aryl halides and polybrominated diphenyl ethers, all of which exert well-documented toxic effects on mammalian cells. The major systems affected include the points of contact with the environment (i.e., the pulmonary and gastrointestinal tracts and the skin), the central nervous system and the liver. In addition, some of these substances disrupt the hormonal status with respect to thyroid hormones and estrogens. If not easily predictable at present, many of these forms of toxicity are not surprising in light of the reactivity, hydrophobicity, and/or structural properties of the compounds in question. Keywords. Toxicity, Organic compounds, Bromine, Iodine

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2.1 2.2 2.2.1 2.2.2 2.2.3 2.2.4

Consequences of the Hydrophobicity of Xenobiotics . . . . . . . . Toxic Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . Chemical Reaction with Cellular Components . . . . . . . . . . . Specific Binding to Proteins . . . . . . . . . . . . . . . . . . . . . Interference with the Metabolism of Other Xenobiotics and of Endogenous Hydrophobic Substances . . . . . . . . . . . . Non-Specific Binding to Proteins and Accumulation in Membranes

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Brominated and Iodinated Alkanes and Related Substances

3.1 3.1.1 3.1.2 3.1.3 3.1.4 3.1.5 3.2 3.2.1 3.2.2 3.2.3 3.2.4

Methyl Bromide . . . . . . . . . . . General Comments . . . . . . . . . Properties . . . . . . . . . . . . . . Human Exposure . . . . . . . . . . Pharmacokinetics and Metabolism Toxicity and Genotoxicity . . . . . Methyl Iodide . . . . . . . . . . . . General Comments . . . . . . . . . Properties . . . . . . . . . . . . . . Pharmacokinetics and Metabolism Toxicity and Genotoxicity . . . . .

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3.3 3.4

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Bromobenzenes and Related Compounds

4.1 4.1.1 4.1.2 4.1.3 4.2

Bromobenzene . . . . . . . . . . . . . . . . . . . . . General Comments, Properties, and Human Exposure Pharmacokinetics and Metabolism . . . . . . . . . . Toxicity and Genotoxicity . . . . . . . . . . . . . . . Polybrominated Benzenes and Related Substances . .

5

Polybrominated Biphenyls (PBBs) and Polybrominated Diphenyl Ethers (PBDEs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 222

5.1 5.1.1 5.1.2 5.1.3 5.1.4 5.1.5 5.2 5.3

PBDEs . . . . . . . . . . . . . . . . General Comments . . . . . . . . . Structures and Properties . . . . . Human Exposure . . . . . . . . . . Pharmacokinetics and Metabolism Toxicity and Genotoxicity . . . . . Polybrominated Biphenyls . . . . . Tetrabromobisphenol-A . . . . . .

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1 Introduction Organobromine compounds are finding greater and greater use for an increasing number of purposes. At present, bromine is used commercially in flame retardants (38%), drilling fluids (22%), sodium bromide solutions (10%), brominated pesticides (mostly methyl bromide) (8%), water treatment chemicals (7%) and others, including photographic chemicals and rubber additives (15%) [20]. In 1998 the total annual world production of bromine-containing compounds was 514 million kg [20, 21]. It seems surprising how many different organobromine compounds we may potentially be exposed to. The National Library of Medicine Specialized Information Services of the United States (http://toxnet.nlm.nih.gov) lists more than 100 such compounds, some of which are shown in Table 1. As can be seen, many of these compounds are used primarily in factories and laboratories and should not be allowed to enter the general environment; while some are no longer produced. The number of organoiodine compounds to which we can potentially be

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Table 1. Organobromine compounds to which humans are exposed (from the National Library of MedicineSpecialized Information Services of the United States(http://toxnet.nlm.nih.gov), search in the Hazardous Substances Data Bank).Pharmaceutical preparations are not included

Compound

Major uses

Methyl bromide Ethyl bromide 1-Bromopropane Isopropyl bromide n-Butyl bromide sec-Butyl bromide t-Butyl bromide Vinyl bromide Allyl bromide Propargyl bromide Chlorobromomethane Bromodichloromethane

Insecticide, fungicide, herbicide Industrial ethylating agent and solvent Solvent, chemical synthesis Industrial reagent Chemical synthesis Chemical synthesis Chemical synthesis Flame retardant, synthesis of plastics Insecticide, chemical synthesis Industrial reagent, fumigant Fire extinguishers, chemical synthesis Fire extinguishers, industrial reagent and solvent, mineral and salt separation Flame retardant, chemical synthesis Refrigerators, air conditioning, fire extinguishers Chemical synthesis, fire extinguishers, refrigerant Industrial solvent and reagent Fire extinguishers Chemical synthesis Refrigerant, fire extinguisher Reagent in the pharmaceutical industry, fire extinguishers, industrial solvent Industrial reagent Fire extinguishers Solvent PVC production Flotation agent Insecticide, chemical synthesis Chemical synthesis Synthesis in the pharmaceutical industry Nematocide Industrial reagent Modifies polymerization of vinyl chloride Flame retardant

Bromotrichloromethane Bromochlorodifluoromethane Bromotrifluoromethane Dibromomethane Dibromofluoromethane Chlorodibromomethane 1,1-Dibromotetrafluoroethane Tribromomethane Tetrabromomethane 1-Bromo-2-fluoroethane 1-Bromo-2-chloroethane 1,2-Dibromo-1,1-dichloroethane 1,1,2,2-Tetrabromoethane Ethylene bromide 1,2-Dibromoethylene 1-Bromo-2-chloropropane 1,2-Dibromo-3-chloropropane 1,2-Dibromopropanol 1,1,1-Tribromo-2-methyl propanol 2,2-Bis(bromomethyl)-1,3propanediol Acetyl bromide Ethyl 2-bromoacetate Bromoacetone 3-Bromopropanoic acid 2,2-Dibromo-3-nitrilopropionamide 1,2,5,6,9,10-Hexabromocyclododecane 1,2-Dibromo-4-(1,2-dibromoethyl)cyclohexane 3-Bromo-1-chloro-5,5dimethylhydrantoin Bromobenzene p-Dibromobenzene Hexabromobenzene

Chemical synthesis Industrial reagent, tear gas War gas, chemical synthesis Chemical synthesis Algicide, bactericide, fungicide, preservative Flame retardant Flame retardant Water disinfectant (germicide, fungicide) Lubricating oil Industrial reagent Flame retardant

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Table 1 (continued)

Compound

Major uses

(2-Bromoethyl)benzene 2,4,6-Tribromophenol Pentabromophenol Benzyl bromide alpha-Bromobenzyl chloride 2-, 3-, and 4-Bromotoluene 2,3,4,5,6-Pentabromotoluene o-,m-, and p-Xylyl bromide beta-Bromostyrene p-Bromoaniline 2-Bromo-4,6-dintroaniline Acetylpyridinium bromide 3,4,5,6-Tetrabromophthalic anhydride 2,4,5,2¢,4¢,5¢-Hexabromobiphenyl 2,2¢,6,6¢-Tetrabromobisphenol A p-Bromophenyl phenyl ether Decabromodiphenyl ether Acarol Bantrol Brodifacoum Bromacil Bromadiolone Bromphos Bromophos ethyl Bromoxynil Chlorbromuron Diaquat dibromide alpha-Epibromohydrin Metabromuron Naled Profenofos

Industrial reagent Antiseptic, wood preservative, industrial reagent Industrial reagent, molluscicide Chemical synthesis War gas Industrial reagent and solvent Flame retardant Tear gas, war gas, chemical synthesis Food additive, fragrance Chemical synthesis Industrial reagent Deodorant, surfactant, topical antiseptic Flame retardant, industrial reagent Flame retardant (formerly) Flame retardant Flame retardant Flame retardant Acaricide Mulluscicide, herbicide Rodenticide (anticoagulant) Herbicide Rodenticide (anticoagulant) Insecticide, acaricide Acaricide, insecticide, larvicide Herbicide Herbicide Herbicide Sporicide, flame retardant Herbicide Fungicide, bactericide Insecticide, acaricide

exposed is considerably smaller (Table 2). However, the number of iodinated pharmaceutical preparations (which have been purposely excluded from Tables 1 and 2; see also below) is actually larger than the number of brominated drugs. In addition to these products of human activities, it is now clear that organobromine and organoiodine compounds, including alkaloids, are produced naturally by bacteria, microalgae, sponges and other marine organisms [137–139, 218, 274, 366]. Human exposure to these natural substances appears to be limited and, to date, virtually nothing is known about how they might affect us. We may be in for some surprises in this connection. Finally, our bodies produce a few iodinated compounds for regulatory purposes, i.e., the thyroid hormones (concerning which much more will be said below) and iodolactones and iodoaldehydes, which are apparently involved in autoregulation of the thyroid gland [93, 129]. Furthermore, 2-octyl-gammabromoacetoacetate has been suggested to be an endogenous inducer of rapid-

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Table 2. Organoiodine compounds to which humans are exposed (from the National Library

of Medicine Specialized Information Services of the United States(http://toxnet.nlm.nih.gov), search in the Hazardous Substances Data Bank). Pharmaceutical preparations are not included Compound

Major uses

Methyl iodide Triiodomethane Iodofenphos Iodoacetic acid Bantrol Dithiazanine iodide

Industrial reagent, fire extinguishers, etching of electronic circuits Wound dressing, adhesives, polymerization control Insecticide, acaricide Food additive (wheat flour), industrial reagent Molluscicide, herbicide Sensitizer of photographic emulsions

eye-movement (i.e., dream) sleep [35]. As far as I am aware, no other organobromine or organoiodine compounds are produced endogenously by mammals. In a chapter such as this, it is neither possible nor advisable to attempt to summarize the known effects of all known organobromine and organoiodine compounds on mammals. I have chosen to focus here on a limited number of compounds/groups of compounds, i.e., alkyl halides, bromobenzenes and polybrominated biphenyls and diphenyl ethers. (Comments on other bromo- and iodosubstances are also included.) This choice was designed to include compounds which are widely spread in the general environment in relatively large amounts (methyl bromide and the polybrominated flame retardants); compounds with relatively similar (various alkyl halides, different bromobenzenes, a variety of brominated diphenyl compounds) and very different (alkyl and aryl halides and brominated diphenyl compounds) structures and chemical reactivities; and substances about which a reasonable amount of information is available. For information concerning other organobromine and organoiodine compounds, I refer the reader to the National Library of Medicine web site described above and the numerous references provided at the end of this chapter. This web site allows searches in a number of different data bases, including HSDB (Hazardous Substances Data Bank), IRIS (Integrated Risk Information System), GENE-TOX (Genetic Toxicology (Mutagenicity)), CCRIS (Chemical Carcinogenesis Research Info System), TOXLINE (Toxicology Bibliographic Info), DART/ETIC (Developmental and Reproductive Toxicology) and TRI (Toxic Release Inventory). I found the HSDB most useful: for each of the bromo- and iodocompounds included, this data base provides information concerning (in this order) human health effects, emergency medical treatment, animal toxicity studies, metabolism/pharmacokinetics, pharmacology, chemical/physical properties, chemical safety and handling, manufacturing and use, and synonyms and identifiers. This web site is updated regularly. In short, this is a valuable source of information for anyone interested in either the basic and/or practical toxicology, as well as other aspects of organobromine and organoiodine compounds.

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2 Some Basic Principles of Biochemical Toxicology On the basis of our fundamental and rapidly expanding knowledge within the field of biochemical toxicology, organobromine and organoiodine compounds, like all xenobiotics, can be expected to be taken up by and exert toxic effects on living organisms by different mechanisms. 2.1 Consequences of the Hydrophobicity of Xenobiotics

The vast majority of xenobiotics to which we are exposed, including organobromine and organoiodine compounds, are moderately to extremely hydrophobic. This simple common chemical characteristic has a number of toxicological consequences: – The permeability barrier of biological membranes consists of long-chain acyl groups attached to glycerol or sphinosine in phospholipids, i.e., this barrier is highly hydrophobic. Consequently, hydrophobic xenobiotics can generally diffuse through the permeability barrier of biological membranes and thus will usually be taken up readily through virtually all our contact surfaces with the environment (the gastrointestinal tract, lungs, skin, etc.). Furthermore, once inside the body, most of these substances can readily diffuse into all cells with which they come into contact. – The liquids which we employ to excrete unwanted substances from our body, primarily urine and bile, are basically aqueous solutions in which xenobiotics are poorly soluble. The whole purpose of xenobiotic metabolism is to render these compounds water soluble for excretion. In the vast majority of cases this process begins with activation of the xenobiotic in question by the cytochromes P-450 system, often to extremely electrophilic and reactive intermediates, e.g., epoxides, free radicals, and carbonium ions. Such reactive intermediates are potentially dangerous to the cell (see below) and must be inactivated by, for the most part, epoxide hydrolase and/or the glutathione transferases. The glutathione transferases, together with the UDP-glucuronyltransferases and sulfotransferases, attach water-soluble endogenous molecules to xenobiotic metabolites, which can then be excreted. – Unmetabolized and, thus, still hydrophobic xenobiotics accumulate in hydrophobic compartments of the body, including the adipose tissue, membranes and hydrophobic pockets on proteins. Such accumulation in membranes and proteins can disturb the structures of these cellular components, thereby preventing them from performing their functions.

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2.2 Toxic Mechanisms 2.2.1 Chemical Reaction with Cellular Components

Organobromine and organoiodine compounds which are themselves reactive and/or reactive metabolites of such substances (see above) can react chemically with different components of mammalian cells. Such reactions often result in covalent binding of the xenobiotic to the endogenous molecule or oxidation of this molecule. The endogenous components which have been most extensively investigated in this context are proteins and DNA. Chemical reactions of xenobiotics and/or their metabolites with proteins can potentially lead to cell death by necrosis or apoptosis; while such reaction with DNA can result in genotoxicity, including mutations, cancer and teratogenicity. 2.2.2 Specific Binding to Proteins

If the structure of a particular xenobiotic is sufficiently similar to that of an endogenous ligand which binds to a specific protein, this xenobiotic might also bind to the same protein with high affinity. Upon binding, the xenobiotic may either act as an agonist (activating the protein) or antagonist (inhibiting the protein’s activity directly and/or by preventing the binding of its natural ligand). The protein targets for such xenobiotic binding which have been investigated most extensively to date are receptors involved in regulating gene expression, e.g., the thyroid hormone receptor, the estrogen receptors, the Ah receptor and the peroxisome proliferator-activated receptors. Thus, a growing number of xenobiotics are being found to exert hormonal or anti-hormonal effects. It is interesting in this context to note that the receptors mentioned above, all of which have endogenous ligands which are highly hydrophobic, bind a much wider range of ligands than do proteins whose ligands are hydrophilic (e.g., transport proteins for sugars and amino acids, receptors for peptide hormones, etc.). One might speculate that it is more difficult to form a highly specific binding site for a hydrophobic ligand on a protein than to form such a binding site for hydrophilic substances, which contain one or more functional groups. 2.2.3 Interference with the Metabolism of Other Xenobiotics and of Endogenous Hydrophobic Substances

The enzyme families involved in the metabolism of xenobiotics to hydrophilic products which can be excreted from the body (see above) all exhibit much broader substrate specificities than do the vast majority of other enzyme systems in mammalian cells. This ability of these enzymes to metabolize virtually anything that is hydrophobic is highly advantageous, allowing us to detoxify virtually all new xenobiotics which appear in our environment.

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At the same time, the broad substrate specificities demonstrated by most xenobiotic-metabolizing enzymes set the scene for extensive interactions between different xenobiotics. For example, one xenobiotic might competitively inhibit the metabolism of another by binding with higher affinity to cytochrome P-450. In addition, many xenobiotics up-regulate (increase the amounts and activities of) the enzymes involved in detoxification, thus accelerating their own metabolism, but also the metabolism of other xenobiotics. Although very little is presently known about such interactions, synergism and antagonism between xenobiotics at the metabolic level can be expected to be the rule, rather than the exception. Furthermore, although much remains to be elucidated in this area, the same enzymes which metabolize xenobiotics appear to be involved in the metabolism of endogenous hydrophobic substances, including steroid hormones, fatty acids, prostaglandins, leukotrienes, vitamin A, melatonin, and bilirubin. Thus, a xenobiotic might interfere with the metabolism of such endogenous molecules in the same way it interferes with the metabolism of other xenobiotics. 2.2.4 Non-Specific Binding to Proteins and Accumulation in Membranes

As mentioned above, non-specific binding of xenobiotics to proteins or their accumulation in membranes may have toxic consequences. For instance, enrichment of xenobiotics in the membranes of mitochondria can inhibit oxidative phosphorylation while such enrichment in nerve cells may interfere with propagation of nerve impulses. Binding of xenobiotics to a hydrophobic region(s) on a protein may prevent this protein from carrying out its function. For example, most xenobiotics bind quite extensively to serum albumin, whose function is to transport hydrophobic endogenous substances such as fatty acids and steroids in the circulation. These basic principles provide a conceptual framework for understanding many of the phenomena associated with the toxicology of organobromine and organoiodine compounds. It is fair to say that the uptake and distribution of these compounds will be determined to a large extent by their hydrophobicity. Furthermore, most of them can be expected to exert a wide and dose-dependent spectrum of toxic effects on mammalian cells via one or a combination of the mechanisms described above.

3 Brominated and Iodinated Alkanes and Related Substances 3.1 Methyl Bromide 3.1.1 General Comments

Methyl bromide is widely used as a gaseous fumigant/pesticide against insects, termites, rodents, weeds, nematodes, and soil-borne pathogens. This compound

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is employed primarily to fumigate soil (85%) in connection with cultivation of, e.g., tomatoes, strawberries, melons, tobacco, flowers, and bell peppers; 8% is used to spray grains, fruits, and vegetables after harvesting; and 5% for structural fumigation, e.g., protecting homes from termites. This simple alkyl halide is also used as a methylating agent, refrigerant, herbicide and solvent; in fire extinguishers; for degreasing wool; and for extracting oils from nuts, seeds and flowers. Annual world-wide consumption is approximately 72,000 tons [3, 99, 100]. Methyl bromide was listed as a Class I ozone-depleting substance in the US Clean Air Act of 1990 [289] and is therefore now being gradually phased out. Use of this compound will be banned by 2005 in industrialized nations and by 2015 in all nations, under the international ozone protection treaty called the Montreal Protocol. It will, however, not be easy to replace, because of its low cost and effectiveness against a wide variety of agricultural pests. 3.1.2 Properties

Methyl bromide is a colorless gas, with a boiling point of 3.5 °C [226].At low concentrations it is practically odorless and thus hard to detect (chloropicrin, a tear gas, is sometimes added as a warning agent), whereas at higher concentrations it has a sweetish, chloroform-like odor and produces a burning taste [47]. The log10 of the octanol-water partition coefficient for methyl bromide is 1.19 [72, 155] and this compound is accordingly freely soluble in organic solvents, but also reasonably soluble in water (approximately 14 g/l at 25 °C) [47, 226, 358]. 3.1.3 Human Exposure

When used as a soil fumigant, as much as 33–80% of methyl bromide may enter the atmosphere. Thus, workers fumigating fields or homes are potentially exposed to high levels. To a lesser extent, drinking water may also be contaminated with this compound. Significant quantities of methyl bromide are also produced by algae and kelp. 3.1.4 Pharmacokinetics and Metabolism

As expected on the basis of its hydrophobicity (see above), methyl bromide enters the human body easily through points of contact (primarily the lungs and skin) and is thereafter rapidly distributed to many tissues, including the lung, adrenal gland, kidney, liver, brain, testicles, and adipose tissue [227, 241]. The fate of methyl bromide in mammal cells is determined by its electrophilic reactivity, which allows methylation primarily of nucleophilic sulfhydryl groups in the tripeptide glutathione (gamma-glutamyl-cysteinyl-glycine) and proteins, releasing a bromide ion at the same time. This reaction with glutathione is both enzymatic, apparently being catalyzed most efficiently by glutathione transferase T1, and non-enzymatic (in contrast to the case with methyl chloride) [46, 152,

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153]. About 25% of the population in Western countries are so-called “non-conjugators”, i.e., possess little or none of this enzyme which catalyzes the reaction between methyl bromide and glutathione [46, 152, 153]. In such non-conjugators a considerably larger portion of the entire dose would be expected to react nonenzymatically with sulfhydryl groups on proteins, as well as with other nucleophilic groups (see also below). Such methylation of hemoglobin (which has a life-time of approximately four months in humans) can be detected at least ten weeks after acute exposure to methyl bromide and is thus useful for monitoring exposure [46, 141, 189, 236, 319]. Exposure can also be monitored by following serum bromide levels [179, 263]. The methyl moiety of this aryl halide is eliminated from rats primarily as carbon dioxide, with a half-life of 10–16 h [37, 240, 241].Apparently nothing is known about how this carbon dioxide is formed, although metabolism via the cytochrome P-450 system does not seem to be involved. The half-life of the associated bromide ion in rats is considerably longer, i.e., between five and nine days [173, 388]. 3.1.5 Toxicity and Genotoxicity

As would be expected from its reactivity and consequent covalent binding to cellular nucleophiles, methyl bromide is highly toxic, causing a large variety of undesirable effects in both humans and experimental animals (Table 3). As would be expected, the most severe acute injury caused by inhalation is on the lung, and the toxic effects on skin and eyes are also easily understandable. However, it is unexpected that the primary organ affected by both chronic and subchronic exposure is the central nervous system [389], even after dermal uptake [168, 227]. The reason for this organ specificity is at present unknown. Abdominal pain, vomiting and nausea may also occur. Symptoms may not appear for 3–12 h or even longer after exposure, making it more difficult to link these to exposure to methyl bromide. Especially in the case of chronic exposure, there may also be damage to the kidneys, liver (humans) and heart (mice and rats) [205]. Such exposure in rats also leads to degenerative and proliferative lesions in the nasal cavity [88]. In addition, long-term studies in experimental animals show cellular damage in the brain, kidney, nasal mucosa, heart, liver, adrenal cortex, and testicles [88]. Finally, a number of workers have died as a result of exposure to methyl bromide [7, 112, 320, 388]. The mechanism(s) underlying this wide spectrum of toxic effects is more-orless totally unknown. Naturally, methylation of sulfhydryl groups on proteins (which ones?) and of glutathione, leading to depletion of this important regulator of cellular redox homeostasis, are attractive, but at present rather vague hypotheses [388]. Concerning the neurotoxicity of methyl bromide, a number of intriguing observations are presently available. This compound decreases neuronal excitability [392]. It has also been demonstrated to inhibit tyrosine hydroxylase in rat brain with a time-course similar to that observed for accompanying decreases in catecholamine concentrations, locomotor activity. and body temperature [166, 167, 169]. Methyl bromide also causes depletion of glutathione and inhibits glu-

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Table 3. Reported acute and chronic toxic effects in humans and experimental animals caused by exposure to methyl bromide

Organ system

Symptoms

References

The lungs and upper respiratory tract

Edema Congestion Damage to mucous membranes Hemorrhage Breathing difficulties Tingling Itching Burning sensation Redness Blisters Pain Redness Pain Blurred vision Temporary loss of vision Dizziness Headache Fatigue, despondency, lethargy Weakness of the arms and legs Numbness Loss of coordination Lack of balance Tremor, muscle spasms Paralysis Convulsions Coma Psychological disturbances: extreme agitation loss of memory and concentration hallucinations loss of speech mood changes insomnia nightmares

[7, 112, 205, 320, 388]

The skin

The eyes

The central nervous system

[7, 112, 205, 320, 388, 397]

[7, 112, 205, 320, 388]

[2, 7, 88, 112, 166–169, 205, 227, 320, 388, 389]

tathione transferase activity in rat brain [80]. The highly interesting, albeit limited observation has been made that neurotoxicity was actually greater in one worker who could conjugate methyl bromide enzymatically with glutathione than in a non-conjugator with the same exposure. This finding suggests that such conjugation does not protect against neurotoxicity, which may actually be caused by formation of methanethiol and formaldehyde from methylglutathione [128]. With regards to genotoxicity, methyl bromide appears to be weakly mutagenic (Table 4), but not carcinogenic in animals [389]. The Environmental Protection Agency of the United States concludes that this xenobiotic is not classifiable as a human carcinogen because of inadequate data on humans and animals. Interestingly, methyl chloride does cause renal tumors in male rats, whereas inhala-

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Table 4. Experimental indications that methyl bromide may cause mutations in mammals

Experimental phenomenon

References

Cause mutations in short-term bacterial tests, e.g., the Ames test, without metabolic activation Mutagenic in Drosophila Mutagenic in mouse lymphoma cells in vitro Leads to unscheduled DNA synthesis (= DNA repair) in rat liver cells in vitro Methylates DNA in vitro Methylates DNA in vivo following inhalation by or oral administration to rats and mice

[36, 92, 216] [92, 216] [92, 216] [92, 216] [339] [36, 298, 299]

tion of methyl bromide causes no tumor formation in rats and mice [36, 299]. One possible explanation for this marked difference between these two alkyl halides may be the prevalence of N-methylation to 3-methyl adenine and 7-methyl-guanine, that are more easily repaired by mammalian cells than the O-methylated 6-O-methyl-guanine of DNA in the case of the bromide [36, 299]. Observations made with benzo(a)pyrene some time ago may be of relevance in this context. It was found that administration of epoxides of this xenobiotic, known to be the immediate carcinogens, to the skin of mice actually gave rise to significantly fewer skin tumors than did application of benzo[a]pyrene itself. Possibly, a strong electrophile may react with proteins to such a great extent that little of the substance reaches the nucleus, where it can alkylate DNA. Suggestive in this regards is the report that in mice exposed to methyl bromide, hemoglobin is methylated to a considerably greater extent than is DNA [92]. One problem associated here with extrapolating mutagenic/carcinogenic studies on rodents to humans is that rats and mice metabolize methyl bromide very rapidly, whereas a rather large subpopulation of humans are “non-conjugators” (see above) [36, 125, 126]. One molecular epidemiological study of workers exposed to methyl bromide in connection with greenhouse sterilization detected no increase in the degree of methylation of their blood leukocyte DNA [298]. In contrast, another such investigation revealed a certain level of genotoxicity (mutations in lymphocytes, micronuclei in oropharyngeal cells) in exposed workers [50]. In addition, a third study found that methyl bromide caused chromosomal aberrations (sister chromatid exchange) in the lymphocytes of nonconjugators, but not of conjugators [153]. On the basis of these findings, it seems likely that methyl bromide will prove to be at least weakly carcinogenic in humans. Reported effects of methyl bromide on reproductive processes are scarce and somewhat contradictory [7, 112, 180, 320, 388]. This compound has been reported to interfere with the reproduction of experimental animals only at very high doses and no lasting effects on sperm quality or spermatogensis in rats could be detected. In one study inhalation by rabbits and rats for 7–8 h each day throughout gestation caused no birth defects. In contrast, other investigators have found

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that methyl bromide does cause birth defects in experimental animals, including missing lobes of the lung, missing gall bladder, and skeletal deformities. 3.2 Methyl Iodide 3.2.1 General Comments

Methyl iodide is employed in microscopy as a light-sensitive etching agent for electron circuits and as a component in fire extinguishers in much smaller total amounts than the usage of methyl bromide [47]. It is thus not surprising that this compound has been much less studied than the bromide. 3.2.2 Properties

Methyl iodide is a colorless, transparent liquid with a boiling point of 42.5 °C [47]. It has a pungent odor resembling that of ethyl ether. The log10 of the octanol-water partition coefficient for methyl bromide is 1.19 [72, 155] and this compound is freely soluble not only in organic solvents but also appreciably so in water (approximately 14 g/l at 25 °C) [47, 226, 358]. 3.2.3 Pharmacokinetics and Metabolism

As expected from its hydrophobicity, methyl iodide is easily absorbed through the gastrointestinal tract and lungs, after which it is distributed to numerous organs (primarily the brain, liver and kidney in the case of rabbits) [55–57, 152]. It further resembles methyl bromide in that it methylates both proteins and glutathione (the reaction with glutathione occurring both enzymatically (with genetic polymorphism) and non-enzymatically), with no significant involvement of cytochrome P-450 in its metabolism [55–57, 152]. 3.2.4 Toxicity and Genotoxicity

Like methyl bromide, methyl iodide exerts similar toxic effects primarily on the central nervous system [38, 39, 57], lungs and upper respiratory tract [55, 56, 305], and skin [207] of both experimental animals and humans [14, 22, 27]. Interestingly, methyl iodide is approximately six times as acutely toxic towards mice as methyl bromide. In this case also, the toxic mechanism(s) is thought to involve protein methylation and depletion of free glutathione (mitochondrial glutathione may be more important in this context than the cytosolic tripeptide) [38, 39, 55–57, 91, 124]. Again, even though methyl iodide is not classified as carcinogenic towards man due to lack of sufficient information [186], this compound is weakly muta-

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genic in the Ames and other bacterial tests, as well as in mammalian cells in vitro [9, 10]. Furthermore, methyl iodide methylates DNA with the same pattern as that observed with the bromide [36, 126, 163, 349, 350, 367, 387]. Interestingly, in the case of methyl iodide, dated animal tests – although not based on modern technology – do suggest carcinogenicity. 3.3 Other Alkyl Bromides

Although in most cases much less information is available, the toxicology of other alkyl bromides including n-butyl bromide [1, 6, 20, 28, 101, 107, 108, 155, 192, 199, 213–215, 225, 234, 237, 266–269, 285, 287, 297, 301, 303, 327], dibromomethane [67, 188], tribromomethane [13, 106, 187], tetrabromomethane [12, 225], and dibromoethane [13, 103, 187] is similar but not identical to that described above. For instance, in some cases the cytochrome P-450 system plays a much more prominent role in the metabolism of the compound and for other of these substances the carcinogenicity in experimental animals is more firmly established. 3.4 Related Compounds

(See Table 5) Table 5. Information concerning organobromine compounds related to alkyl bromides

Compound

Use/occurrence

Toxicity

References

Acetyl bromide

Organic synthesis, dye manufacture

[135, 225, 330, 364]

Bromoacetone

Formerly: chemical war gas, tear gas, synthesis organic manufacture

Bromoacetates

By-products of drinking water disinfection

Irritation of the respiratory tract: Chest pains Pulmonary edema Hypoxemia Bronchospasm Symptoms similar to asthma Persistent abnormalities in lung function Severe skin burns Irritation of the eye: Pain Swelling Corneal erosion Blindness Intensely irritating to the skin and the mucous membranes of the repiratory tract Irritation of the eye: Violent lacrimator Serious, but reversible damage to the cornea Genotoxic both in vitro and in vivo Adverse effects on sperm in rodents

[135, 188]

[131, 228, 292]

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4 Bromobenzenes and Related Compounds 4.1 Bromobenzene 4.1.1 General Comments, Properties, and Human Exposure

Bromobenzene is employed primarily as an additive to motor oil, a reagent for producing phenyl magnesium bromide, a solvent for crystallization, and in various processes in which a dense liquid is desirable. In occupational situations and upon exposure of individuals to motor oil, contact is primarily with the lungs and skin. Waste release of bromobenzene has led to environmental contamination in food, ambient air, and water, resulting in potential uptake by ingestion and inhalation. Bromobenzene is a heavy, mobile, colorless liquid with a pungent, aromatic odor [47, 225] and a boiling point of 156.2 °C [47]. The log10 of the octanol/water partition coefficient of this compound is 2.99 [72, 155], i.e., it is miscible with most organic solvents and very poorly soluble in water [225]. 4.1.2 Pharmacokinetics and Metabolism

As would be predicted from its pronounced hydrophobicity, bromobenzene is readily adsorbed through the lungs, gastrointestinal tract, and intact skin [134]. As expected, this compound has been detected in human adipose tissue. The metabolism of bromobenzene is much more complex than that of the alkyl halides, giving rise to phenols and epoxides (produced by the cytochrome P-450 system and accelerated by exposure to xenobiotics which increase the amount of, i.e., up-regulate (induce), this system), quinones (formed non-enzymatically and, perhaps, enzymatically as well from dihydroxylated products), catechols (arising from intramolecular hydrolysis of epoxides by epoxide hydrolase), glutathione adducts of the epoxides and quinones and secondary products, and sulfated derivatives of the phenols and catechols [26, 89, 156, 165, 197, 223, 224, 248, 275, 308, 312, 352–355, 375, 393, 394]. The primary excretion products are catechol derivatives, both free and conjugated with sulfate, and mercapturic acid derivatives of glutathione conjugates [134, 270], much leaving the human body in bile [235]. Both epoxides and quinones formed by metabolism of bromobenzene bind covalently to nucleophilic atoms in many different proteins [265, 333]. The major epoxide metabolite, i.e., the 3,4-epoxide [11, 94], is highly reactive and binds directly to proteins close to where it is formed (e.g., the cytochrome P-450 system itself in the endoplasmic reticulum); while, in contrast, the 2,3-epoxide is more stable and can diffuse relatively far from its site of formation and bind to other proteins, e.g., hemoglobin [19, 165, 208, 209, 219, 312, 391]. These epoxides bind both to cysteinyl sulfhydryl groups [312] and amino groups on lysine and histidine residues in proteins [26].

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4.1.3 Toxicity and Genotoxicity

As can be seen from Table 6, the toxic profile for bromobenzene in experimental animals exhibits some similarities with that of the alkyl halides. Not surprisingly, the points of entry, i.e., respiratory tract, skin, and gastrointestinal tract, are all irritated, although much less seriously than by, e.g., methyl bromide. The central nervous system can also be functionally impaired to a less pronounced degree than in the case of the alkyl halides. (A speculation: membranes, including myelin, and membrane components such as ion channels play particularly important roles in the functions of the central nervous system. Thus, it may be that many hydrophobic xenobiotics accumulate in these membranes and thereby impair, e.g., the development and propagation of nerve impulses.) The major difference between the toxic effects of bromobenzene and alkyl halides is the severe liver necrosis and milder necrosis of the kidney caused by the aromatic compound (compare Tables 3 and 6). This hepatic necrosis is proposed to be caused by the formation of reactive metabolites, i.e., epoxides and quinones of bromobenzene and subsequent covalent binding of these electrophiles to cellular proteins; and, indeed, such protein adducts are localized in the necrotic areas [60, 347, 352–355, 381]. There is some evidence that bromobenzene can up-regulate its own metabolic activation via the cytochrome P450 system [238], as well as that epoxide hydrolase and glutathione transferase protect against hepatonecrosis by inactivating epoxides and quinones [54]. It has been suggested that epoxide adducts constitute only a small portion of the total covalent binding compared to quinones, but are more important for the toxicological effect [26, 258]. Thus, it is important to identify the reactive intermediates formed and determine to what extent each of these binds to proteins. On the other hand, covalent binding is unlikely to be limited to any particular protein(s), so that there are probably a multitude of different pathways of this nature all of which terminate in cell death. An alternative or, perhaps, complementary hypothesis concerning the mechanism underlying bromobenzene-induced liver necrosis involves glutathione deTable 6. Reported toxic effects in experimental animals exposed to bromobenzene

Organ

Effects

References

Liver

Severe centrilobular necrosis

Lung and upper respiratory tract Skin Gastrointestinal tract Central nervous system

Irritation

[60, 115, 117, 130, 136, 188, 222–224, 248, 343–347, 368, 375, 381] [42, 81, 286]

Kidney

Irritation Vomiting, diarrhea Various types of dysfunction, anesthesia Necrosis (mild)

[134] [134] [188] [45]

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pletion (by covalent binding to the reactive intermediates) and consequent lipid peroxidation [115, 136, 222–224]. Indirect evidence for this hypothesis is provided by observations that cysteine and methionine (directly or by stimulating glutathione synthesis) and their derivatives [53, 87, 352–355], as well as antioxidants [254, 294] can attenuate the necrotic effect. Clearly, these two hypotheses are not mutually exclusive, since depletion of glutathione in isolated rat hepatocytes significantly increases the level of protein adducts formed. At present there is no evidence that bromobenzene is carcinogenic in man.Although this compound is not mutagenic in bacteria, even with metabolic activation (the Ames test) [270], it does cause oxidative damage to DNA in rodent and human hepatocytes in vitro, an effect against which glutathione can protect [371, 386]. Furthermore, the extent of DNA strand breakage is increased in Hep G2 cells treated with bromobenzene [386]. In light of the carcinogenicity of other epoxides and quinones, it appears likely that bromobenzene will prove to be at least somewhat carcinogenic in mammals. The developmental toxicity observed in Xenopus sp exposed to bromobenzene was concluded to be due to the formation of a highly toxic epoxide intermediate(s), which could be detoxified by epoxide hydrolase and glutathione transferase [118]. 4.2 Polybrominated Benzene and Related Substances

To date, very few structure-activity relationships involving bromobenzene, polybrominated benzene, and related compounds have been reported.After intraperitoneal administration of 1,4-dibromobenzene to mice, metabolites produced with the involvement of both the cytochrome P-450 system and glutathione transferase were found to bind covalently to DNA in the liver, kidney, lung, and stomach [69]. In a comparison of the hepatoxicities of bromobenzene; 1,2-, 1,3-, and 1,4-dibromobenzenes; 1,2,4- and 1,3,5-tribromobenzenes; 1,2,4,5-tetratbromobenzene; and hexabromobenzene, it was concluded that increased lipophilicity and molecular size are associated with decreased hepatotoxicity [343–347]. In another study involving 2-bromobenzonitrile, 1,2-dibromobenzene, bromobenzene, 2-bromoanisole, and 3-bromotoluene (listed in order of decreasing hepatotoxicity and increasing rate of in vitro metabolism), good correlation between the degree of toxicity and relative extent of binding to protein was observed [375]. Pentabromophenol has been reported to be only mildly hepatotoxic in mice, while exerting more severe nephrotoxicity in both rats and mice [345], i.e., a pattern opposite to that exhibited by bromobenzene (see Table 6). This phenol also binds to human transthyretin (the protein responsible for transporting thyroid hormone in the circulation) with a higher affinity than does thyroid hormone itself [243]. Accordingly, this compound might well cause hypothyroidism. Finally, hexabromobenzene has been demonstrated to up-regulate cytochrome P-450 in the livers of male and female rats [121] and is not teratogenic in rodents [71, 200]. Even these few observations suggest that there can be dramatic differences in the toxicity exerted by benzenes substituted with different numbers of bromine

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atoms, by different congeners of benzenes brominated to the same extent, and by closely related compounds. This is also obviously the case for polybrominated biphenyls and polybrominated diphenyl ethers (see below). Much closer investigation of such considerations is warranted.

5 Polybrominated Biphenyls (PBBs) and Polybrominated Diphenyl Ethers (PBDEs) 5.1 PBDEs 5.1.1 General Comments

The term brominated flame retardants covers a wide range of different materials, the major ones (historically and/or in terms of quantity) being polybrominated biphenyls (PBBs), polybrominated diphenylethers (or diphenyloxides) (PBDEs), hexabromocyclododecane, and tetrabromobisphenol A [30, 84]. Since the 1960s, PBDEs have been added as non-covalently-bound flame retardants (5–30% by weight) to thermoplastics (i.e., high-impact polystyrene) in electrical appliances, TV sets, computer circuit boards and casings, and building materials. These compounds are also found in foams and upholstery in home and business furnishings, in the interiors of cars, buses, trucks, and airplanes, as well as in rug and drapery textiles [171, 342, 382]. By 1997, PBDEs were the major bromine compounds used in plastics solely as flame retardants, comprising an estimated one-third of all brominated flame retardants in use [243, 383]. Annual world-wide production has increased dramatically since the 1970s and was approximately 67,000 tons in 1999 [98, 158, 159, 171, 243, 325, 382, 383]. For instance, use of BFRs in the United States has increased at least 15-fold since the mid-1960s. Because of their persistence and increasing levels in the environment, as well as the numerous toxic effects they appear to exert (see below), PBDEs have received and are still receiving enormous attention from both the scientific community and the general public. Without exaggeration, one could say that PBDEs are the PCBs (polychlorinated biphenyls) of the present decade [40, 90]. 5.1.2 Structures and Properties

As can be seen from Fig. 1, there are a large number of different congeners of PBDEs. Three major commercial mixtures are employed: deca-BDEs (containing some nona- and octa-congeners); octa-BDEs (also containing hepta-), and pentaBDEs (also containing tetra-). Fully brominated deca-BDEs accounts for 75% of the total production. Less than ten congeners are present in commercial preparations, compared to roughly 80 PCBs in the Arochlor 1254 and 1260 mixtures [171, 382]. Since most toxicological investigations are performed with mixtures

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Fig. 1. Structures of polychlorinated and -brominated biphenyls, polychlorinated dibenzo-[p]-

dioxins and dibenzofurans, polybrominated diphenyl ethers, thyroxine, estradiol, and tetrabrombisphenol-A

of congeners, the characteristics discussed here refer to such mixtures, unless otherwise stated. However, as will be pointed out further below, different congeners may well elicit quite different biological responses. As also depicted in Fig. 1, the structures of PBDEs demonstrate certain similarities with those of PCBs, dioxins, benzofurans, and thyroid hormones (see further below). The log10s of the octanol/water partition coefficients for PBDEs are 5–10 [72, 84, 158], reflecting their extreme hydrophobicity. PBDEs act as flame retardants by releasing, at elevated temperature, bromine radicals which quench the fire-spreading process [171].

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5.1.3 Human Exposure

Since PBDEs are not covalently bound to the products to which they are added, these substances are released more-or-less slowly throughout the lifetime of the products. PBDEs are relatively stable to both chemical and biodegradation, as a result of which they are persistent in the environment, some having half-lives there of two to ten years. This persistence, together with their high degree of hydrophobicity, has led to the widespread occurrence of these compounds in the general environment, including their bioaccumulation in the food chain and enrichment in hydrophobic microenvironments, such as the adipose tissue and fatty breast milk of human beings. Tetra- and pentabrominated congeners exhibit bioconcentration factors of 5000–35,000, while the corresponding values for octaand deca-congeners is 5–<50 because of limited uptake (see below) [111]. As a consequence, tests in Europe, North America, Japan, and the oceans reveal PBDEs in air, water, sediment, shellfish, fresh and saltwater fish, birds, moose, reindeer, seals, dolphins, whales, cow’s milk and human hair, fat, blood, milk, and placenta [15, 24, 66, 77, 78, 82, 83, 85, 86, 88, 144, 146, 171, 193, 245, 279, 280, 283, 323–326, 338, 373, 382], including whales who feed far from shore [85, 229, 351]. Thus, the general population is exposed to PBDEs via breast milk and in the womb, in cow’s milk, fish, chicken [175], and maybe other foodstuffs [105]. Drinking water is probably not so important in this connection. Although still only 1/10–1/100 the levels of PCBs, concentrations of PBDEs in inhabitants of Europe and North America have increased 50–600-fold since 1972 and continue to increase. PBDE concentrations in Swedish breast milk doubled every five years during the period 1972–1997, from 0.07 parts per billion to 4 pbb, while the corresponding levels of organochlorine compounds decreased [77, 245, 246, 279, 280]. Similar increases have been seen in the plasma of Swedish and German citizens and breast milk and placenta of Finnish women [77, 245, 246, 279, 280]. In North America this value has risen from 0.21 ppb in 1982 to 16–25 ppb a few years ago [326, 328]. In addition to exposure via the environment, occupational exposure is known to occur.Workers at shredders in plastic recycling plants and workers who install and repair computers have been reported to exhibit significantly elevated levels of PBDEs in their blood [147, 148, 331, 332]. There is even some evidence, based on a small number of workers, that suggests possible exposure to PBDEs in connection with computer use at work [331] and thus, presumably, also at home. Indeed, PBDEs have been shown to be present in the particulate fraction of air in a number of computerized offices in Stockholm [31, 148]. Combustion of PBDEs produces brominated and chlorinated dioxins [32, 48, 221, 336, 383, 384]. Consequently, workers at incinerators burning mixed waste can be over-exposed to all of these halogenated compounds, as can fire fighters and residents. Indeed, residents may be exposed for many years after a fire [158]. The main congeners in human tissues are the three ortho/para (2,4-) substituted, i.e., 2,2¢,4,4¢-tetra-BDE (PBDE-47), 2,2¢,4,4¢,5-penta-BDE (PBDE-99), and 2,2¢,4,4¢,5,5¢-hexa-BDE (PBDE-153), which are found at similar levels (0.3–98.2 ng/g lipid) in adipose tissue from Sweden [157], Spain [247], Israel [85], and the United States [338]. Such congener patterns may provide information on

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the manner of exposure; relatively large amounts of the deca congener suggest direct, recent and/or occupational exposure to the parent compound, while relatively more tetra may indicate environmental exposure [171]. Breast milk contains higher tetra/deca levels than serum from dismantlers at an electronics-recycling plant, which were also significantly higher than the corresponding values for clerks in the same plant or a control group of hospital cleaners [331]. 5.1.4 Pharmacokinetics and Metabolism

As indicated above, and as expected from their high degree of hydrophobicity, PBDEs may be taken up through all contact points with our surroundings, i.e., primarily the gastrointestinal tract, lungs and skin. Decabromodiphenyl ether is expected to be one of least active congeners, because this compound is poorly absorbed from the gastrointestinal tract (approximately 0.3% of the total dose) and rapidly eliminated (half-life <1 day), at least in rodents [272]. The half-life of decabromodiphenyl ether in human blood has been determined to be 6.8 days [148]. In contrast, lower congeners are completely absorbed and have much longer half-lives in rats, e.g., 20–30 days for a tetra and 45–119 days for two hexa congeners [51, 369]. It would be of considerable interest and importance to know why decabromodiphenyl ether apparently diffuses through biological membranes more slowly than the lower congeners. Except that it appears to be very slow, amazingly little is known about the metabolism of PBDEs in mammalian cells. Hydroxylated products, presumably produced via the cytochrome P-450 system, have been identified and covalent binding of metabolites of tetrabromodiphenyl ethers to cellular macromolecules in various tissues of the rat and mice has been taken as evidence for the formation of a reactive epoxide intermediate [96, 145, 290]. 5.1.5 Toxicity and Genotoxicity

It is hardly surprising (see above) that, as is the case for alkyl halides and bromobenzenes, exposure to PBDEs has been associated with a wide variety of toxic effects (Table 7) [78, 296]. Of course, some of these effects may be interrelated, e.g., antiestrogenic activity and/or hypothyroidism may exert detrimental effects on fetal development or the central nervous system. Most of these toxic effects are, indeed, presently believed to be mediated by relatively specific binding of PBDEs and/or their metabolites to well-characterized proteins, i.e., transthyretin (involved in thyroid hormone transport in the blood), estrogen receptors, and the Ah receptor (which also binds PCBs and dioxins and acts to regulate the expression of numerous cellular proteins in various organs). However, much research remains to be carried out before a firm causal relationship between such binding and the toxicology of PBDEs can be established. Clearly, exposure to PBDEs disrupts the thyroid hormone balance in experimental animals, resulting in hypothyroidism [149–151, 171, 242–244, 304]. Meerts and coworkers [242–244] demonstrated that that PBDEs could compete with thy-

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Table 7. Reported toxic and genotoxic effects caused by exposure of mammalian cells to PBDEs

System

Effect

References

Thyroid hormone balance

Hypothyroidism Tumors Toxicity Dysfunction Abnormalities in development Increased activities of a number of enzymes, including cytochrome P-450 Reduction in vitamin A levels Tumors Estrogenic effects Increased embryo mortality Delayed skeletal formation

[25, 105, 149, 151, 170, 171, 243, 244, 304] [109, 171]

Central nervous system

Hepatic functions

Estrogen balance Development in utero

[51, 52, 65, 105, 149, 151, 170, 171, 244, 369]

[198, 242, 304] [110]

roid hormone (see Fig. 1) for binding to human transthyretin only after metabolism by induced rat liver microsomes, strongly suggesting the involvement of hydroxylated metabolites. Hallgren and coworkers [149–151] administered Bromkal 70–5DE (a commercial mixture of PBDEs) and DE-47 (2,2¢,4,4¢-tetrabromodiphenyl ether) to rats and mice orally (18 mg/kg body weight) once daily for 14 days, after which both plasma free and total thyroid hormone levels were significantly decreased. Finally, 4 of 35 production workers involved in manufacturing decabromodiphenyl ethers and decabromobiphenyls exhibited clinical signs of hypothyroidism, with no such cases among 89 age- and sex-matched controls [25]. As a result of such effects on thyroid hormone and its regulatory functions, the brain may develop abnormally, especially in children exposed in utero or through breast feeding [43, 171, 243]. In rats PBDEs also bind to the Ah receptor, thereby up-regulating several enzymes, including the cytochrome P-450 system [61, 171, 244]. Lower congeners are stronger inducers [51, 52], due to their higher bioavailability and/or affinity. Unfortunately, the affinities with which these various compounds bind to the Ah receptor have not yet been determined. In addition, brominated naphthalenes and iodobenzenes have been found to be excellent ligands for the Ah receptor in rat liver cytosol [65]. Finally, using a cell line transfected with a reporter gene (luciferase) under the control of either the alpha or beta form of the estrogen receptor, several PBDE congeners, and especially hydroxylated metabolites, were found to be relatively potent activators of this receptor, although not nearly as potent as estrogen itself [242]. Possibly, PBDEs and, especially, hydroxylated metabolites might also exert estrogenic effects by more potently inhibiting human estrogen sulfotransferase, an important pathway for inactivation of this hormone [198]. Although the potency of PBDEs in causing such effects is not yet known [171, 365], they seem to be equally or more potent than some PCBs [171, 244, 369],

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while being less potent than other PCBs and dioxins [149, 171, 244]. There is much discussion of the structural similarities between PBDEs and PCBs and PBBs, the DDT family, the herbicide nitrofen, the PCDDs/PCDFs and thyroxine (T4) (Fig. 1). Indeed, some of these substances share toxicological properties as well. From this viewpoint, available data suggest that tetrabromo- to hexabromodiphenyl ethers are likely to be carcinogens, endocrine disrupters and/or neurodevelopmental toxicants, in the same manner as PCBs and dioxins [149, 151, 171, 369]. Dioxins are known to exert an additional plethora of health effects, ranging from chloracne to altered sex ratio [33, 63, 176, 177, 184, 185, 230, 259]. Furthermore, existing evidence indicates that at least certain PBDEs can act synergistically with PCBs and dioxins to increase their toxicity. For instance, exposure of rats to certain PBDEs potentiates the effects of PCBs on thyroid hormones and related enzymes [149, 171]. This is potentially a serious problem, since the highest levels of present exposure of children in utero and through breast feeding to PCBs and dioxins appears already to have detrimental effects on learning and reduce resistance to infections [171, 191, 212, 293, 300, 311, 374]. Furthermore, exposure of the general public to dioxin appears to be close to levels that may cause diabetes, endometriosis, increased risk of cancer, and other health problems [102, 104, 182, 184]. Another aspect of this problem is that pyrolysis of FR 300 BA, Bromkal 70–5 DE, Bromkal 70-DE and Bromkal GI, and commercial mixtures of PBDEs gives rise to relatively high levels of polybrominated dibenzofurans and polybrominated dibenzo-p-dioxins [184, 185, 390]. At present, there is simply too little data available to conclude whether PBDEs, in particular at the doses to which we are exposed, cause cancer in humans [105, 382]. Commercial mixtures of deca-, octa-, and pentabromodiphenyl ethers are not mutagenic in Salmonella tymphimurium [272, 382]. On the other hand, mono-, di-, and tetrabromodiphenyl ethers induce genetic recombination in mammalian cell lines [160]. One study did conclude that there exists an association between adipose tissue levels of PBDE and risk for non-Hodgkin lymphoma among Swedish hospital patients [157]. 5.2 Polybrominated Biphenyls

Production of the non-covalently-bound PBB flame retardants ceased in the United States more than two decades ago, in 1979 [187]. Accordingly, the toxicology of these compounds is becoming increasingly of historical and academic interest. The PBBs were not as extensively studied during the 1970s as the PBDEs are today, presumably because of the lower level of environmental awareness at this earlier time. Only three commercial PBB products named Firemasters and containing limited numbers of hexabrominated, octabrominated, and decabrominated congeners were manufactured [158]. These were used as an additive flame retardant (= not covalently bound) almost exclusively in a particular thermoplastic (i.e., arylonitrile-butadiene-styrene) employed in electronic equipment housings [158], as well as, to a limited extent, to prime the anaerobic dechlorination of

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PCBs [29], i.e., much less extensive usage than for the polybrominated diphenyl ethers.As in the case of PBDEs, the log10 values of the octanol/water partition coefficients of these substances are 5–10 [72, 158]. Many of the other relevant physical and chemical properties of the PBBs are not available. Formerly, PBBs were released to the environment during manufacture and disposal of commercial and consumer products containing these compounds [164, 276]. Monitoring data on the ambient air levels of PBBs is very limited.Although more is known about their levels in water and soil, aquatic life-forms, birds and terrestrial organisms, these data are relatively old now. It is clear that, like PBDEs, PBBs are persistent in the environment [190, 196] and present in the serum, adipose tissue, breast milk, placenta, and cord serum of exposed humans [16, 17, 113, 217, 340, 341, 379, 380]. Congeners with six or less bromine atoms bioconcentrate in aquatic organism [132, 281, 288, 395, 396]. Absorption occurs by inhalation and dermal contact, leading to high levels in the adipose tissue and serum of exposed workers [44, 217, 341], as well as in individuals exposed to contaminated food [113, 178, 217, 250, 380]. Initially after exposure, PBBs accumulate in the liver, lungs, and muscle, but are later redistributed to adipose tissue. The metabolism of PBBs in animals is slow and limited, consisting of hydroxylation and debromination [127, 210, 211, 318]. Dihydrodiol formation has also been demonstrated, suggesting the intermediate formation of an epoxide [211, 337]. A free para position facilitates metabolism and bromine content has a less pronounced influence than the positions of the bromine atoms [74–76]. Inducers of cytochrome P-450 (e.g., phenobarbital, 3-methylcholanthrene) accelerate PBB metabolism, indicating the involvement of this xenobiotic-metabolizing system [74–76, 253]. The toxicological profile of PBBs is in many respects similar to that of the PBDEs (Table 8), although the PBBs have been shown to exert a wider range of effects. In humans, an association has been reported between levels of PBB in adipose tissue and risk for lymphoma and breast cancer [162, 172], but this remains controversial and not at present conclusive. Extensive attempts have been made to elucidate the toxic mechanism(s) underlying these responses to PBB exposure, but with little real success [4, 5, 18, 73–76, 133, 291, 306, 309, 310, 316]. The major hypotheses are similar to those proposed for PBDE toxicity, namely, binding to the Ah receptor and, possibly, other receptors as well [73–76, 95, 251, 261, 262, 291, 310, 317, 321]. 5.3 Tetrabromobisphenol-A

It is important to consider briefly this derivative of polybrominated biphenyls (see Fig. 1), since while PBBs are being phased out in Europe [342], the production of tetrabromobisphenol-A continues to increase. The little information available indicates that tetrabromobisphenol-A is present in the air surrounding computers [30, 147], has a half-life of two days in human blood [148], and exerts both thyroid hormone and estrogenic activity in mammalian cells [206, 242, 243]. This emerging pattern looks like a variation on an all-too-familiar theme.

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Table 8. Reported toxic effects associated with exposure of mammals and mammalian cells to

PBBs Function

Effect

References

Reproduction

Inhibition of in vitro fertiliation Increased incidence of abnormal embryos Oocyte degeneration Decreased fertility Cleft palate in offspring Neurological impairment in offspring Increased hepatic neoplasms and mortality in offspring Up-regulation of the same form of cytochrome P-450 up-regulated by dioxins Increased DNA repair Gene mutations Chromosomal aberrations Promotion of tumor formation initiated by other xenobiotcs Cancer Abnormal morphology Hypothyroidism

[140, 161, 202, 273, 313, 356, 376]

Hepatic functions

Thyroid hormone balance Skin Eyes Immunological defenses Bone marrow cells

Bromacne Irritation Impairment Chromosomal aberrations

[64, 70.122, 142, 194, 202, 203, 220, 249, 255, 256, 264, 271, 314, 372, 377]

[4, 5, 8, 25, 49, 143, 271, 281, 282] [59] [252] [119, 120, 143, 174, 231–233, 370] [252, 281, 282]

6 Organobromine and Organoiodine Compounds Employed as Pharmaceutical Agents and/or Research Tools Since a large and growing number of organobromine and organoiodine compounds are finding use both as experimental research tools (Table 9) and clinical drugs (Table 10), it is appropriate to make some short mention of these. Many such halogenated drugs were discovered serendipitously during derivatization of established drugs in an attempt to improve their efficacy. In other cases, the halogen atom has been added to a molecule simply to make it detectable (e.g., radioactive iodination or iodinated contrast media) or to increase its hydrophobicity (e.g., dibromo cyclic adenosine monophosphate). In some instances specific steric features have been aimed for. In most cases the significance of the bromine or iodine atom has been elucidated in at least some detail. Iodinated contrast media, which are widely employed in visualization of different human organs, elicit adverse reactions in as many as 50% of the patients receiving these preparations [348]. The most common components of such media include salts of benzenoic acid iodinated at the 2, 4 and 6 positions and with other substituents at positions 3 and 5, e.g., the sodium salt of 3,5-diacetamido-

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Table 9. Examples of organobromine and organochlorine compounds employed as experi-

mental research tools Compound

Use

References

8-Bromo analogues of the key second messengers cyclic AMP and cyclic GMP 16-Delta-iodo-17-b-estradiol

Biochemical and physiological investion of signal transduction pathways

[62, 322]

Selective ligand for the alpha form of the estrogen receptor Agonist of the alpha2 form of the adrenergic receptor Inhibitor of choline acetyltransferase Assaying DNA synthesis

[359]

Iodoclonidine Bromoacetylcholine Bromo- and iododeoxyuridine (analogues of thymidine) 1-Delta-25-dihydroxyvitamin D-3-bromoacetate Radiolabeled iodomelatonin Iodoacetamine

Affinity-labeled ligand for the vitamin D receptor Ligand for melatonin receptors Acylation of thiol groups, depletion of glutathione

[58] [204] [79, 307] [260] [328] [154]

2,4,6-triodobenzoic acid. The numerous adverse reactions observed to iodinated contrast media are summarized in Table 11. Interestingly, at least one organobromine compound, namely, 4-bromo-2,5dimethoxy-phenethylamine (2C-B), is known to be psychoactive and addictive [385].

7 Concluding Remarks The three classes of organobromine and organoiodine compounds of central interest here appear to exert toxic effects on mammalian cells by somewhat different mechanisms: the reactivity of alkyl halides causes these compounds to react directly with nucleophilic groups on proteins and nucleic acids. Bromobenzenes can be metabolized to reactive intermediates which can also bind to cellular nucleophiles, and at the same time these compounds can bind more or less specifically to thyroid hormone-binding proteins and to the Ah receptor. In contrast, polybrominated biphenyls and diphenyl ethers do not appear to bind covalently to cellular components, but rather exert their toxicities primarily by reversible association with thyroid hormone-binding proteins, estrogen receptors, and the Ah receptor. It remains to be seen to what extent non-specific accumulation in, e.g., membranes and hydrophobic proteins contributes to the toxicities of these compounds. In hindsight it is clear that many of these toxic effects are, if not entirely predictable, at least not surprising. It seems remarkable to me that toxicology remains a phenomenological area of research to such a large extent (i.e., when this dose of that xenobiotic is administered, these effects are seen). Surprisingly little information is available con-

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Table 10. Examples of organobromine and organochlorine compounds employed or being developed as pharmaceutical preparations

Compound 2-Bromo-5,6-dichloro-1-b-Dribofuranosylbenzimid-azole Bicyclic furano 5-iodopyrmidine nucleosides 3-Bromo- and 3-iodo-2(4-aminophenyl)benzothiazoles 4¢-Iodo-4¢-deoxydoxorubicin

Use

Inhibits the replication of human cytomegalovirus Highly potent and selective inhibitors of varicella zoster virus Potent antitumor activity towards human breast, ovarian, lung, renal and colon carcinoma cell lines Inhibits formation of amyloid beta-peptide polymers and may be useful in treating Alzheimer’s disease p-Iodo-2,6-diisopropylphenol Anticonvulsant and anticonflict activities Ebrotidine/N-{[{E)-[[2-[[[2A new antiulcer agent combining the [(diaminomethylene) amino]- properties of an H2-receptor antagonist 4-thiazolyl]methyl] thio]ethyl] with those of a cytoprotective agent amino]methylene]-4-bromobenzenesulfon-amide Radiolabeled iodobenzamide Ligand for postsynaptic D2 dopamine receptors in diagnosis of Parkinson’s disease Radiolabeled 2-b-carbomethoxy- Ligand for presynaptic dopamine 3-b-(4-iodophenyl)tropane) transporters in diagnosis of Parkinson’s disease Radiolabeled 5-iodo-2Cancer diagnosis and therapy deoxyuridine Radiolabeled 5-iodo-2-fluoro“Reporter substrate” for the herpes 2¢-deoxy-1-b-D-arabinosimplex virus thymidine kinase for furanosyl-5-iodouracil monitoring gene therapy Radiolabeled 6-b-iodomethylFunctional imaging of the adrenal gland norcholesterol

References [97] [239] [41]

[116|

[360] [334, 335]

[357]

[357]

[195] [34, 123]

[315]

cerning questions of key significance to the toxicities of alkyl halides, bromobenzenes, polybrominated biphenyls, and diphenyl ethers, and, indeed, most xenobiotics: – How are these substances distributed in the human body and why (pharmacokinetics)? – How, exactly, are they metabolized? – Are reactive intermediates formed and, if so, at different levels in different organs and cell types? – Are there genetic polymorphisms in this metabolism and, thus, our susceptibility to the toxic effects of organobromine and organoiodine compounds? – What cellular components do the xenobiotics and/or their metabolites actually bind to in vivo and in what manner, i.e., covalently or non-covalently, how specifically?

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Table 11. Adverse reactions in patients administered iodinated contrast media

Principle toxic mechanism Symptoms observed Hypertonicity

Ionic charge

Chemical toxicity

Allergic/anaphylactic reactions

Endothelial lesions in blood vessels Damage to the blood-brain barrier Deformation and rigidity of erythrocytes, resulting in thrombosis and ischemia Cardiovascular effects (local and generalized): vasodilatation, flushing, sensation of warmth, discomfort, generalized hypotension Impaired renal function, sometimes leading to renal failure Experience of nausea, vomiting, fever, chills, faintness, headache, sneezing, metallic taste Altered electrolyte balance Impaired nerve conduction Impaired cardiac function Effects of increased sodium levels (e.g., peripheral vasodilation, deterioration of the blood-brain barrier) Effects of increased iodine levels binding to proteins and inhibition of their functions (e.g., acetylcholinesterase) Acute tubular necrosis cardiotoxicity Sneezing Pharyngeal, cerebral and/or pulmonary edema Bronchospasm Fatal cardiovascular collapse

See References 68, 114, 181, 257, 284, 302, 348, 361–363, 378.

– How significant are subtle differences in chemical structure (e.g., different congeners of dibromobenzene or tetrabrominated biphenyls or diphenyl ethers) to the uptake, pharmacokinetics, metabolism, and toxicity of such compounds, and why? Answers to such questions are sorely needed if biochemical toxicology is to become a predictive science, a change which is highly desirable in light of the tens of thousands of different xenobiotics to which we are exposed and which exert various effects on our bodies, both individually and synergistically. Nonetheless, the persistence and toxic effects of certain organobromine compounds appear to be serious enough that alternatives should be looked for. As mentioned above, methyl iodide is now being phased out. Furthermore, a number of European countries are considering a ban on polybrominated biphenyl ethers and, perhaps, all halogenated flame retardants [342]. Hundreds of flame retardant chemicals are known [295] and some plastics that resist fire without containing polybrominated diphenyl ethers are now available [383]. Some computer companies already offer PBDE-free products [23, 171, 183, 329]. Furthermore, plastic can be replaced by other materials, e.g., in furniture and textiles. And last, but not least, more care should obviously be taken in recycling products containing potentially toxic organobromine, organoiodine and other xenobiotics. However, we should not continue to repeat the common mistake of replacing a commercial compound which has proven to be dangerous with another xenobiotic

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about which nothing is known, but which eventually proves to be at least as toxic. Perhaps the field of biochemical toxicology is already advanced enough to allow us to turn hindsight into at least limited foresight. It seems highly irrational that governments demand careful toxicological testing of clinical drugs before these can be administered to, often, a relatively small number of patients; whereas commercial and industrial xenobiotics which may contaminate the general environment, so that the entire population is exposed, are subject to much less control. It is also highly undesirable that commercial products containing a large number of different compounds, most of which are chemically similar and thus useful for the same purpose, but many of which have quite different effects on living organisms are allowed to seep into the environment. At the same time, it should be remembered that any hydrophobic xenobiotic at a high enough dose will be toxic, if for no other reason than that it accumulates in biological membranes and interacts with hydrophobic sites on proteins, thereby interfering with the functions for which these structures are responsible. The question must always be posed as to whether the doses to which humans are exposed are actually high enough to be toxic, a highly controversial question in the case of, e.g., polychlorinated dibenzo-p-dioxins. Obviously, improvements in the area of quantitative risk assessment are sorely needed. Finally, it will be of considerable interest to continue expanding our knowledge concerning organisms that naturally produce organobromine and organoiodine compounds. For what purposes? To what extent are humans exposed to these natural xenobiotics? And, perhaps most fascinating from a basic biochemical point of view, are there unknown endogenous organobromine and organoiodine compounds of functional importance to human physiology?

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