Advances in Environmental Research, Volume 13

ADVANCES IN ENVIRONMENTAL RESEARCH

ADVANCES IN ENVIRONMENTAL RESEARCH. VOLUME 13 No part of this digital document may be reproduced, stored in a retrieval system or transmitted in any form or by any means. The publisher has taken reasonable care in the preparation of this digital document, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained herein. This digital document is sold with the clear understanding that the publisher is not engaged in rendering legal, medical or any other professional services.

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ADVANCES IN ENVIRONMENTAL RESEARCH

ADVANCES IN ENVIRONMENTAL RESEARCH. VOLUME 13

JUSTIN A. DANIELS EDITOR

Nova Science Publishers, Inc. New York

Copyright © 2011 by Nova Science Publishers, Inc. All rights reserved. No part of this book may be reproduced, stored in a retrieval system or transmitted in any form or by any means: electronic, electrostatic, magnetic, tape, mechanical photocopying, recording or otherwise without the written permission of the Publisher. For permission to use material from this book please contact us: Telephone 631-231-7269; Fax 631-231-8175 Web Site: http://www.novapublishers.com NOTICE TO THE READER The Publisher has taken reasonable care in the preparation of this book, but makes no expressed or implied warranty of any kind and assumes no responsibility for any errors or omissions. No liability is assumed for incidental or consequential damages in connection with or arising out of information contained in this book. The Publisher shall not be liable for any special, consequential, or exemplary damages resulting, in whole or in part, from the readers‘ use of, or reliance upon, this material. Any parts of this book based on government reports are so indicated and copyright is claimed for those parts to the extent applicable to compilations of such works. Independent verification should be sought for any data, advice or recommendations contained in this book. In addition, no responsibility is assumed by the publisher for any injury and/or damage to persons or property arising from any methods, products, instructions, ideas or otherwise contained in this publication. This publication is designed to provide accurate and authoritative information with regard to the subject matter covered herein. It is sold with the clear understanding that the Publisher is not engaged in rendering legal or any other professional services. If legal or any other expert assistance is required, the services of a competent person should be sought. FROM A DECLARATION OF PARTICIPANTS JOINTLY ADOPTED BY A COMMITTEE OF THE AMERICAN BAR ASSOCIATION AND A COMMITTEE OF PUBLISHERS. Additional color graphics may be available in the e-book version of this book. LIBRARY OF CONGRESS CATALOGING-IN-PUBLICATION DATA

ISBN: 978-1-61209-049-8 (eBook)

ISSN: 2158-5717

Published by Nova Science Publishers, Inc. † New York

CONTENTS Preface

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Short Commentary The Global Extent of Black C in Soils: Is It Everywhere? Evelyn Krull, Johannes Lehmann, Jan Skjemstad, Jeff Baldock and Leonie Spouncer

3

Research and Review Studies Chapter 1

Chapter 2

Chapter 3

Chapter 4

Chapter 5

Current and Emerging Microbiology Issues of Potable Water in Developed Countries William J. Snelling, Catherine D. Carrillo, Colm J. Lowery John E. Moore, John P. Pezacki, James S. G. Dooley and Roy D. Sleator Vermiculture Biotechnology: The Emerging Cost-Effective and Sustainable Technology of the 21st Century for Waste and Land Management to Safe and Sustainable Food Production Rajiv K. Sinha, Sunil Herat, Gokul Bharambe, Swapnil Patil, Uday Chaudhary, Priyadarshan Bapat, Ashish Brahambhatt, David Ryan, Dalsukh Valani, Krunal Chauhan, R. K. Suhane and P. K. Singh

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Human Waste - A Potential Resource: Converting Trash into Treasure by Embracing the 5 R‘s Philosophy for Safe and Sustainable Waste Management Rajiv K. Sinha, Sunil Herat, Gokul Bharambe, Swapnil Patil, Pryadarshan Bapat, Krunal Chauhan and Dalsukh Valani

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Effective Removal of Low Concentrations of Arsenic and Lead and the Monitoring of Molecular Removal Mechanism at Surface Yasuo Izumi

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On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) Loads in Mink Accompanying Parasitic Infection by the Giant Kidney Worm (Dioctophyme Renale) Glenn H. Parker and Liane Capodagli

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Contents

Chapter 6

Aerobically Biodegraded Fish-Meal Wastewater as a Fertilizer Joong Kyun Kim and Geon Lee

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Chapter 7

Equity of Access to Public Parks in Birmingham, England Andrew P. Jones, Julii Brainard, Ian J. Bateman and Andrew A. Lovett

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Chapter 8

An Idea for Phenomenological Theory of Living Systems Svetla E. Teodorova

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Chapter 9

A New Trait of Gentoo Penguin: Possible Relation to Antarctica Environmental State? Roumiana Metcheva, Vladimir Bezrukov, Svetla E.Teodorova and Yordan Yankov

Chapter 10

Assessing Population Viability of Focal Species Targets in the Western Forest Complex, Thailand Yongyut Trisurat and Anak Pattanavibool

Chapter 11

Protection of Riparian Landscapes in Israel Tseira Maruani and Irit Amit-Cohen

Chapter 12

Hydraulic Characterization of Aquifer(s) and Pump Test Data Analysis of Deep Aquifer in the Arsenic Affected Meghna River Floodplain of Bangladesh Anwar Zahid, M. Qumrul Hassan, Jeff L. Imes and David W. Clark

Chapter 13

Application of DNA Microarrays to Microbial Ecology Research: History, Challenges, and Recent Developments John J. Kelly

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285 305

325

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Chapter 14

Food Safety in India: Challenges and Opportunities Wasim Aktar

Chapter 15

Impact of Pesticide Use in Indian Agriculture Their Benefits and Hazards Wasim Aktar

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Ozone Decomposition by Catalysts and its Application in Water Treatment: An Overview J. Rivera-Utrilla, M. Sánchez-Polo and J. D. Méndez-Díaz

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Chapter 16

Chapter 17

Index

Use of Microarrays to Study Environmentally Relevant Organisms: A UK Perspective Michael J. Allen, Andrew R. Cossins, Neil Hall, Mark Blaxter, Terry Burke and Dawn Field

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PREFACE Short Communication - The latest projections of the Intergovernmental Panel on Climate Change (IPCC) estimate a 3°C increase in global temperatures within the next 100 years (IPCC 4th Assessment Report, 2007), and global warming is seen as a major driver in accelerating decomposition of soil organic matter, resulting in increased production of CO2 (e.g. Davidson and Janssens, 2006). The 2007 IPCC report recommended coupling models of terrestrial biogeochemical and atmospheric and oceanic processes in order to improve general circulation models and to recognize the quantitative value of soil organic carbon (SOC) in the global carbon cycle. Estimates of CO2 emissions from soil rely on predictions of the response of different SOC pools to global warming and correct estimation of the size of these pools. In comparison to the pool representing the most stable and biologically unreactive fraction, commonly referred to as passive or inert organic carbon (IOC), the decomposition of labile C is expected to be faster as a response to temperature increase. IOC is a fraction of the SOC pool that is not readily available for microbial decomposition and has turnover times exceeding 100 years (e.g. Krull et al., 2003). Black C (BC) is usually considered the most abundant form of IOC and is defined as the ‗carbonaceous residue of incomplete combustion of biomass and fossil fuels‘ (Schmidt and Noack, 2000). BC is important to several biogeochemical processes; for example, BC potentially modifies climate by acting as a potential carbon sink for greenhouse gases (Kuhlbusch, 1998) and leads to increasing solar reflectance of the Earth‘s atmosphere, but also to a heating of the atmosphere (Crutzen and Andreae, 1990). BC production from fossil fuel combustion contributes to aerosol C, decreasing surface albedo and solar radiation (IPCC 4th Assessment Report). Due to its condensed aromatic structure, BC has a low biochemical reactivity. 14C ages of BC in soils vary between 1160 and 5040 years (e.g. Schmidt et al. 2002). Chapter 1 - Water is vital for life; for commercial and industrial purposes and for leisure activities in the daily lives of the world‘s population. Diarrhoeal disease associated with consumption of poor quality water is one of the leading causes of morbidity and mortality in developing countries (especially in children <5 years old). In developed countries, whilst potable water is not a leading cause of death, it can still pose a significant health risk. Water quality is assessed using a number of criteria, e.g. microbial load and nutrient content which affects microbial survival, as well as aesthetic factors such as odor. In water systems, the presence of disinfectant, low temperatures, flow regimes and low organic carbon sources do not appear to be conducive to microbial persistence. However, frequently this is not the case.

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A variety of human pathogens can be transmitted orally by water and in the developed world water quality regulations require that potable water contains no microbial pathogens. Chlorine dioxide is a safe, relatively effective biocide that has been widely used for drinking water disinfection for 40 years. Providing the water is of low turbidity, standard chlorination procedures are sufficient to prevent the spread of planktonic bacteria along water mains. However, despite this, bacterial contamination of water distribution systems is well documented, with growth typically occuring on surfaces, including pipe walls and sediments. Rivers, streams and lakes are all important sources of drinking water and are used routinely for recreational purposes. However, due to fouling by farm and wild animals, these sources can be contaminated with microbes, e.g. chlorine resistant Cryptosporidium oocysts, no matter how pristine the source or well maintained the water delivery system. The high incidence of Cryptosporidium in surface water sources underlines the need for frequent monitoring of the parasite in drinking water. The use of coliforms as indicator organisms, although considered relevant to most cases, is not without limitations, and is thus not a completely reliable parameter of water safety, e.g. Campylobacter contamination cannot be accurately predicted by coliform enumeration. Furthermore, the presence of biofilms and bacterial interactions with protozoa in water facilitate increased resistance to antimicrobial agents and procedures such as disinfectants and heating, e.g. Legionnaires‘ disease caused by Legionella pneumophila. The high cost of waterborne disease outbreaks should be considered in decisions regarding water utility improvement and treatment plant construction. The control of human illnesses associated with water would be aided by a greater understanding of the interactions between water-borne protozoa and bacterial pathogens, which until relatively recently have been overlooked. Chapter 2 - A revolution is unfolding in vermiculture studies (rearing of useful earthworms species) for multiple uses in environmental management and sustainable development. (Martin, 1976; Satchell, 1983; Bhawalkar and Bhawalkar, 1994; Sinha et.al 2002; Fraser-Quick, 2002). Vermiculture biotechnology promises to provide cheaper solutions to following environmental and social problems plaguing the civilization – Management of municipal and industrial solid wastes (organics) by biodegradation and stabilization and converting them into useful resource (vermicompost) – ‗THE VERMICOMPOSTING TECHNOLOGY‘ (VCT) ; Treatment of municipal and some industrial (food processing industries) wastewater, purification and disinfection - ‗THE VERMI-FILTRATION TECHNOLOGY‘ (VFT); Removing chemical contaminations from soils (land decontamination) and reducing soil salinity while improving soil properties- ‗THE VERMI-REMEDIATION TECHNOLOGY‘ (VRT); Restoring and improving soil fertility and boosting crop productivity by worm activity and use of vermicompost (miracle growth promoter) while eliminating the use of destructive agro-chemicals - ‗THE VERMI-AGRO-PRODUCTION TECHNOLOGY‘ (VAPT); Vermi-composting, vermi-filtration, vermi-remediation and vermi-agro-production are self-promoted, self-regulated, self-improved and self-enhanced, low or no-energy requiring zero-waste technology, easy to construct, operate and maintain. It excels all ‗bio-conversion‘, ‗bio-degradation‘ and ‗bio-production‘ technologies by the fact that it can utilize organics that otherwise cannot be utilized by others. It excels all ‗bio-treatment‘ technologies because it achieves greater utilization than the rate of destruction achieved by other technologies. It

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involves about 100-1000 times higher ‗value addition‘ than other biological technologies. (Appeholf, 1997). About 4,400 different species of earthworms have been identified, and quite a few of them are versatile waste eaters and bio-degraders and several of them are bio-accumulators and bio-transformers of toxic chemicals from contaminated soils rendering the land fit for productive uses. Chapter 3 - Waste is being generated by the human societies since ancient times. Ironically waste was not a problem for the environment when men were primitive and uncivilized. Waste is a problem of the modern civilized society. Materials used and waste generated by the traditional societies were little and ‗simple‘ while those by the modern human societies are large and ‗complex‘. With modernization in development drastic changes came in our consumer habits and life-style and in every activity like education, recreation, traveling, feeding, clothing and housing we are generating lots of wastes. The world today generate about 2.4 billion tones of solid waste every year in which the Western World alone contributes about 620 million tones / year. Discarded products arising from all human activities (cultural and developmental) and those arising from the plants and animals, that are normally solid or semi-solid at room temperature are termed as solid wastes. Municipal solid waste (MSW) is a term used to represent all the garbage created by households, commercial sites (restaurants, grocery and other stores, offices and public places etc.) and institutions (educational establishments, museums etc.). This also includes wastes from small and medium sized cottage industries. We are facing the escalating economic and environmental cost of dealing with current and future generation of mounting municipal solid wastes (MSW), specially the technological (developmental) wastes which comprise the hazardous industrial wastes, and also the health cost to the people suffering from it. Developmental wastes poses serious risk to human health and environment at every stage – from generation to transportation and use, and during treatment for safe disposal. Another serious cause of concern is the emission of greenhouse gases methane and nitrous oxides resulting from the disposal of MSW either in the landfills or from their management by composting. Dealing with solid household waste in more sustainable ways involves changes not only to everyday personal habits, consumerist attitudes and practices, but also to the systems of waste management by local government and local industry and the retailers. This chapter reviews the causes and consequences of escalating human waste, the increasing complexity of the waste generated, and the policies and strategies of safe waste management. It also provides ‗food for thought‘ for future policy decisions that government of nations may have to take to ‗reduce waste‘ and divert them from ending up in the landfills, drawing experiences from both developed nation (Australia) and a developing nation (India). Chapter 4 - New sorbents were investigated for the effective removal of low concentrations of arsenic and lead to adjust to modern worldwide environmental regulation of drinking water (10 ppb). Mesoporous Fe oxyhydroxide synthesized using dodecylsulfate was most effective for initial 200 ppb of As removal, especially for more hazardous arsenite for human's health. Hydrotalcite-like layered double hydroxide consisted of Fe and Mg was most effective for initial 55 ppb of Pb removal. The molecular removal mechanism is critical for environmental problem and protection because valence state change upon removal of e.g. As on sorbent surface from environmental water may detoxify arsenite to less harmful arsenate. It is also because the evaluation of

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desorption rates is important to judge the efficiency of reuse of sorbents. To monitor the low concentrations of arsenic and lead on sorbent surface, selective X-ray absorption fine structure (XAFS) spectroscopy was applied for arsenic and lead species adsorbed, free from the interference of high concentrations of Fe sites contained in the sorbents and to selectively detect toxic AsIII among the mixture of AsIII and AsV species in sample. Oxidative adsorption mechanism was demonstrated on Fe-montmorillonite and mesoporous Fe oxyhydroxide starting from AsIII species in aqueous solution to AsV by making complex with unsaturated FeOx(OH)y sites at sorbent surface. Coagulation mechanism was demonstrated on double hydroxide consisted of Fe and Mg from the initial 1 ppm of Pb2+ aqueous solution whereas the mechanism was simple ion exchange reaction when the initial Pb2+ concentrations were as low as 100 ppb. Chapter 5 - Patterns of metal uptake and accumulation in mink living under conditions of environmental pollution and simultaneously inflicted with the invasive giant kidney worm (Dioctophyme renale) parasite have not been examined, nor is the combined effect of these dual insults on the health and physical condition of the animal known. Using animals collected within the influence of the long-active ore-smelters at Sudbury, Ontario, an examination was made of toxic metal (Cd, Ni and Pb) levels and their tissue distributions within adult male mink bearing different intensities of parasite infection. Higher metal burdens were indicated within infected specimens than those uninfected. Combined renal and hepatic nickel and lead burdens were highest for mink with multiple worm infections, although only lead accumulations reached statistical significance. Cadmium accumulated to the greatest extent in the hypertrophied left kidney and liver, whereas nickel and lead were deposited more readily in the bony spicule of the parasitized right kidney cyst. The relative distribution of cadmium among renal, hepatic and renal cyst tissues (cast, spicule, worms) remained unchanged subsequent to D. renale infection, while the proportions of nickel and lead deposited in hepatic tissue were reduced. Metal burdens in female D. renale were threefold higher than those of male worms, with the difference being attributable to the substantially greater size of the females. Canonical Correlation Analyses of condition measures and body metal burdens failed to indicate a direct relationship between infection intensity and body fat deposits but did confirm a positive association between metal loads and increased fat levels, along with enhanced gonad weights, neck circumference and reduced spleen weights. Such associations may be productive aspects for future investigation into the combined effects of increased metal loads and parasitic infection on the host system. Chapter 6 - Reutilization of fish-meal wastewater (FMW) as a fertilizer was attempted, and aerobic biodegradation of the FMW were successfully achieved by microbial consortium in a 1-ton bioreactor. During the large-scale biodegradation of FMW, the level of DO was maintained over 1.25 mg∙l-1, and a strong unpleasant smell remarkably disappeared in the end. Although the level of total amino acids and the concentrations of N, P and K in the biodegraded FMW were relatively lower than those in two commercial fertilizers, the concentrations of noxious components in the biodegraded FMW were much lower than the standard concentrations. The phytotoxicity of the biodegraded FMW was almost equal to that of the commercial fertilizers. The fastest growth in hydroponic cultures of red bean and barley was achieved at 100-fold and 500-fold dilution, respectively, the growth of which was comparable to those of 1,000-fold diluted commercial-fertilizers. From all above results, it was concluded that a large-scale biodegradation of FMW was successful and the properties of

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FMW were acceptable. This is the first study to demonstrate aerobic production of liquidfertilizer from FMW. Chapter 7 - Provision of public parks has long been advocated as an equalising measure between different elements of society. This study assesses equity of park provision for different ethnic and income-status populations in the urban area of Birmingham in central England. Parks in Birmingham were categorized into a two group typology of green areas suited for more solitary and passive activities (amenity parks) or open spaces designed more for informal sports or other physical and group activities (recreational parks). Using a geographical information system, measures of access to these green spaces were computed for populations of different ethnicities and levels of material deprivation, derived from data from the 2001 UK Census and the 2004 Index of Multiple Deprivation. Distance-weighted access scores were calculated and compared for five population groups ranked by relative deprivation, and for five ethnic groups; Bangladeshis, blacks, Indians, Pakistanis and whites. Statistical analysis found that there were strong disparities in access with respect to deprivation whereby the most income-deprived groups were also the most deprived with regard to access to public parks. There was little evidence of unequal access between ethnic groups. The implications of these findings are discussed. Chapter 8 - An idea for developing of new science field, biodynamics, as biophysical macroscopic theory is propounded. The functioning of living organism as an organized entirety is the main specificity of the life. Hence it is quite reasonable to describe behaviour of biological systems in terms of own theoretical basis. In this article a new state variable vitality as integral characteristic of biological object and measure unit bion are stated. A quantity biological energy is introduced as energy form related to biological selfregulation. Quantity synergy is suggested as measure of selfregulation quality. Biological principle for maximum synergy in healthy living systems is stated. On the basis of variational principle an equation describing recovery process of a biological object after disturbance is obtained. The quantity optimal vitality, related to homeostasis, decreases in lifespan scale and its evolution is described by ordinary differential equation. The potential lifespan maximum of several species at different life conditions may be calculated at different parameters of the equation. A wide range of environmental influences on the living organism could be promptly and easily assessed in the terms of the biodynamics approach. Such an approach could be used for a simple estimation of a patients‘ health status. Chapter 9 - Some morphological traits of Antarctic animals could be considered in the context of a trend to more direct relation between environmental conditions and possible adaptive mechanisms of animal‘s organism. Penguins are excellent object for biomonitoring. Here a preliminarily report is presented regarding a new trait, a spot-like coloration (―yellow spot‖) of the bill, observed on the upper mandible of Gentoo penguin (subspecies Pygoscelis papua ellsworthii). The spot varied in size and colour. It was recorded among chicks over two months old and adult birds (normal and molting). The trait had no significant relationship to the animal' s sex. Among all inspected females 31% and among all inspected males 27% exhibited beak spot. Three breeding colonies were investigated at three different geographical locations at the Antarctic Peninsula – Livingston Island, South Shetlands (6238 S), Wiencke Island (64o52 S), and Petermann Island (6510 S). Yellow spot was found with different frequencies at these three locations: 20%, 36%, and 30%, respectively. All spotted penguins from the three colonies were 32% when compared to all non-spotted. The possible reasons for the spot-like coloration are discussed. The trait could be a phenotypic characteristic. It could

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play some role in the mate choice. A possible connection to carotenoid pigments content does not be eliminated in the context of Gentoo diet. A probable cause for the beak spot appearance seems to be the increasing of ozone depletion above Antarctica. The ultraviolet radiation enhances free radical production. Other studies reported an increase of the flux of transsulfuration pathway as a defense reaction, resulting in elevated level of cysteine. When cysteine concentration is raised, an attaching of cysteine to melanin synthesis pathway occurs and this results in formation of reddish pigments. Chapter 10 - The Western Forest Complex (WEFCOM) in Thailand covers approximately 19,000 km2. This protected area complex comprises 11 national parks and 6 wildlife sanctuaries. During 1999-2004, the Danish Government provided financial support to the Royal Forest Department to manage this forest complex through the ecosystem management approach. The WEFCOM Project employed rapid ecological assessment (REA) to determine the current distribution statuses of wildlife species, develop a Geographic Information System (GIS), and define habitat uses of wildlife. This paper is based upon the achievements of the WEFCOM Project. It aims to define suitable habitats of selected key wildlife species in the WEFCOM and to assess the current and desired statuses under a population viability estimate for those species. The focal wildlife species were sambar (Cervus unicolor), gaur (Bos gaurus), banteng (Bos javanicus), Asian elephant (Elephas maximus), and tiger (Panthera tigris. The authors used logistic multiple regression to determine habitat uses of wildlife and employed minimum dynamic area and landscape matrix surrounding suitable habitats as criteria to assess population viability. The results indicate the current suitable habitat mainly remains in Huai Kha Khaeng and Thung Yai wildlife sanctuaries. In addition, the current viability condition is good for sambar, fair for gaur, elephant and tiger; and poor for banteng. However, landscape matrices outside the suitable habitats for all species range from moderate to high connection of native vegetation. If the project aims to upgrade the viabilities to the next level in the next 10 years, park rangers and multi-stakeholders have to increase the amount of suitable habitats for all species from 12,630 km2 or 67% of the WEFCOM to 16,750 km2 or 89%. By doing this, the number of suitable patches would significantly decrease and the mean patch size would increase substantially, thereby indicating less fragmentation. Chapter 11 - Riparian landscapes are natural habitats of unique ecological and scenic values, which are highly sensitive to human intervention and impact. Yet, due to their qualities, and especially the presence of water, they are also usually attractive for recreation purposes. This is more so in arid and semi-arid zones like Israel. Nevertheless, in the past, the importance of riparian landscapes in Israel did not receive adequate attention in policy and planning. As a result, over the years they were exposed to various negative impacts, including pollution by industrial and agricultural effluents, exploitation of water for agricultural and other purposes, and land use conflicts. Although in recent years with the growing awareness of their ecological and recreational potential, considerable efforts are being invested in the rehabilitation of deteriorated riparian landscapes, their protection is still deficient. This chapter reviews and examines policy tools used for the protection of riparian landscapes in Israel, based mainly on regulations, reports and existing literature. It concludes by offering some lessons for policy-making in general and suggestions for improving the protection of riparian landscapes in Israel in particular. Chapter 12 - To determine the hydraulic characteristics of aquifers and development potential of deep aquifer for sustainable long-term use, study was undertaken by assessing

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water levels of different aquifer formations and conducting pumping test in deep aquifer under Meghna floodplain area of southeastern Bangladesh. Because of arsenic contamination in shallow groundwater, characterization of deeper aquifers and assess their hydraulic connectivity is now an important issue in Bangladesh. Study shows that groundwater pumping for irrigation and other uses cause large seasonal water level fluctuations that is between 2 and 4.5m, 6.5 and 11m and 6.5m in the shallow, main and deep aquifers, respectively. The trend of groundwater level fluctuations supports the hydraulic connectivity of these aquifers. Aquitards separating aquifers are not continuous regionally. This implies that uncontrolled development of deep aquifers may cause leakage of arsenic from contaminated shallow depths to aquifers below. Water levels dropping below sea level for over withdrawal may also cause saline water intrusion as well. However, during the constantdischarge pumping test for deep aquifer, water levels in observation wells open to the shallow and main aquifers showed no noticeable effect from pumping i.e. under conditions of moderate groundwater use for public supply, arsenic-rich groundwater in the shallow aquifer are not likely to be drawn into the deep aquifer. The transmissivity values of the aquifer is generally favorable for groundwater development and ranged from about 1,070 m2/day to 2,948 m2/day at a distance of 44 m from the pumped well. Transmissivity ranged between 1,570 m2/day and 2,956 m2/day at a distance of 120m. Transmissivity was calculated as 2,385 m2/day using recovery data. Estimated storage coefficient values ranged between 0.0000375 and 0.00268, indicates that the aquifer is confined to leaky-confined or semiconfined in nature. Chapter 13 - During the last three decades, molecular methods have dramatically expanded the author view of microbial diversity in natural and engineered systems. A variety of molecular approaches, including both PCR-based and hybridization-based techniques, have been applied extensively to the analysis of complex microbial communities and have yielded new insights. However, the majority of molecular methods that are widely used in microbial ecology are limited in their ability to encompass the incredible diversity of microbial communities. DNA microarrays, which were first introduced in the early 1990s, are one of the fastest growing technologies in biology, and they offer tremendous potential for microbial ecologists. DNA microarrays consist of nucleic acids spotted within a very small area on some solid support, and they enable the immobilization and simultaneous hybridization of hundreds of thousands of nucleic acids. This represents a dramatically higher degree of multiplexing than is possible with other widely used technologies. In addition, microarrays offer the advantages of increased speed of detection, low cost, and the potential for automation. Microarray technology has been used extensively for measuring gene expression in a wide variety of organisms, including human cells, plants, yeast, and bacteria, but its application to microbial ecology has been more limited. There are significant challenges to the use of microarrays in microbial ecology studies, including optimization of specificity and sensitivity and quantification of targets. However, in recent years several research groups have made significant progress in overcoming these challenges, and microarrays are beginning to be applied more frequently to microbial ecology studies in a variety of systems including terrestrial soils, wetland sediments, and freshwater and marine ecosystems. This article will provide a brief history of the use of molecular methods in microbial ecology, and will then review the development of microarray technology, the challenges that exist for application of microarrays to microbial ecology, the available strategies for overcoming these challenges, and some recent applications of microarrays to studies in microbial ecology.

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Chapter 14 - Rising incomes and urbanization, an expanding domestic consumer base concerned about food quality and safety, and rapidly growing agricultural exports have been important drivers for the increased attention to food safety in India. But the development of effective food safety systems is hampered by a number of factors, including: restrictive government marketing regulations, weak policy and regulatory framework for food safety, inadequate enforcement of existing standards, a multiplicity of government agencies involved, weak market infrastructure and agricultural support services. The small farm structure further limits farmer capacity to meet increasing domestic and export food safety and SPS requirements. Addressing food safety concerns in India will require adoption of appropriate legislation, strengthening capacity to enforce rules, promoting adoption of good agricultural, manufacturing and hygiene practices, greater collective action, and some targeted investments. Implementing these actions will require joint efforts by the government and the private sector. Developing countries are paying increased attention to food safety, because of growing recognition of its potential impact on public health, food security, and trade competitiveness. Increasing scientific understanding of the public health consequences of unsafe food, amplified by the rapid global transmission of information regarding the public health threats associated with food-borne and zoonotic diseases (e.g. E. coli and salmonella, bovinespongiform encephalopathy (BSE), severe acute respiratory syndrome (SARs) and H5N1 avian flu) through various forms of media and the internet has heightened consumer awareness about food safety risks to new levels globally (Lindsay 1997, Unnevehr 2003, Buzby and Unnevehr 2003, Kafersteing 2003, Ewen et al. 2006, Bramhmbatt 2005). Increased understanding of the impact of mycotoxins, which can contaminate dietary staples such wheat, maize, barley and peanuts, has further raised food security and public health concerns in many developing countries (Dohlman 2003, Bhat and Vasanthi 2003, Unnevehr 2003). As developing countries seek to expand agricultural exports especially to OECD countries, many are receiving a wake-up call on the challenges of meeting both government and private sanitary and phyto-sanitary (SPS) standards in export markets (Otsuki et al. 2001, Henson 2003, Unnevehr 2003, World Bank 2005a). Private standards or supplier protocols have grown in prominence over the past decade as a means to further ensure compliance with official regulations, to fill perceived gaps in such regulations, and/or to facilitate the differentiation of company or industry products from those of competitors. Trends in private standards increasingly tend to blend food safety and quality management concerns (i.e. the recent creation of ISO 22000), or to have protocols which combine food safety, environmental, and social (child labor, labor conditions, animal welfare) parameters (Willems et al. 2005, World Bank 2005). At the same time, increasing globalization of trade introduces greater risks of cross-border transfer of food-borne illnesses. Recent cases of disease episodes in the United States resulting from imported food produce, such as cyclospora from raspberries, hepatitis A from strawberries and salmonella from cantaloupe (Calvin 2003), illustrate to developing countries the potential food safety challenges that can arise in a more globalized market. Weaknesses in food safety systems can have a high cost to society and the global economy. The World Health Organization (WHO) estimates that 2.2 million people worldwide die from diarrheal diseases caused by a host of bacterial, viral and parasitic organisms, which are spread by contaminated water (WHO 2006a). In India, it is estimated

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that 20% of deaths among children under five are caused by diarrheal disease (WHO 2006b). The SARs outbreak in 2003 in East Asia is estimated to have caused an immediate economic loss of about 2% of the Region‘s GDP in the second quarter of that year, even though only 800 people died from the disease (Brahmbatt 2005). The Lowy Institute for International Policy (2006) estimates that a mild global outbreak of the avian flu can cost the world 1.4 million lives and close to 0.8% of GDP (US$330 billion) in lost economic output. At the same time, country reactions to protect its citizens from food safety risks can also have large consequences for exporting countries. Otsuki et al (2001) examined the projected impact of the EU‘s new harmonized aflatoxin standard on the value of trade flows to 15 European countries from 9 African countries and found that it could decrease African exports by 64% (US$670 million). Food safety concerns are getting widespread attention in India. The country‘s rural development strategy, for which a key element is the promotion of increased agricultural exports as a means to foster rural growth and poverty reduction, is coming up against tightening food safety and SPS standards in prospective markets (World Bank 2006a, 2006b). From a domestic perspective, the large national market of 1.2 billion people is undergoing rapid change. Increasing incomes, a growing middle class, increased urbanization and literacy, and a population highly tuned to international trends fueled by the information technology boom are creating a large consumer base giving increasing value to food quality and safety. Improving food safety systems, to meet domestic and export requirements, however, face a number of policy, regulatory, infrastructural and institutional obstacles. Chapter 15 - The term pesticide covers a wide range of compounds including insecticides, fungicides, herbicides, rodenticides, molluscicides, nematicides, plant growth regulators and others. Among these, organochlorine (OC) insecticides, used successfully in controlling a number of diseases, such as malaria and typhus, were banned or restricted after the 1960s in most of the technologically advanced countries. The introduction of other synthetic insecticides – organophosphate (OP) insecticides in the 1960s, carbamates in 1970s and pyrethroids in 1980s and the introduction of herbicides and fungicides in 1970s - 1980s contributed greatly in pest control and agricultural output. Ideally a pesticide must be lethal to the targetted pests, but not to non-target species, including man. Unfortunately, this is not, so the controversy of use and abuse of pesticides has surfaced. The rampant use of these chemicals, under the adage, ―if little is good, a lot more will be better‖ has played havoc with human and other life forms. Chapter 16 - Ozone has recently received much attention in water treatment technology for its high oxidation and disinfection potential. The use of ozone brings several benefits but has a few disadvantages that limit its application in water treatment, including: i) low solubility and stability in water, ii) low reactivity with some organic compounds and iii) failure to produce a complete transformation of organic compounds into CO2, generating degradation by-products that sometimes have higher toxicity than the raw micropollutant. To improve the effectiveness of ozonation process efficiency, advanced oxidation processes (AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on ozone decomposition into hydroxyl radicals (HO·), which are high powerful oxidants. This chapter offers an overview of AOPs, focusing on the role of solid catalysts in enhancing ozone transformation into HO· radicals. Catalytic ozonation is a new way to remove organic micropollutants from drinking water and wastewater. The application of several homo- and heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the

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parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation has largely been limited to laboratory applications, the good results obtained have led to investigations now under way by researchers worldwide. It is therefore timely to provide a summary of achievements to date in the use of solid materials to enhance ozone transformation into HO· radicals. Chapter 17 - Historically, the majority of microarray work has been restricted to welldefined model organisms. This was primarily due to the limited availability of genomic or transcriptomic sequence data and the then high cost involved in developing microarrays. However, recent technological developments have greatly enhanced the speed of generating the underpinning sequence data for non-model species, and have opened up more costeffective approaches for microarray production to make them far more affordable for researchers at the lower end of the budget range. These developments have been seized upon by the environmental genomics community within the UK. The creation of a network of closely integrated facilities for sequencing, microarray printing and bioinformatics has opened the gateway for the study of environmentally relevant organisms. Here, the authors describe the infrastructure for microarray development within the UK, and the diverse applications for which they are currently being used. Versions of these chapters were also published in Environmental Research Journal, Volume 3, Number 1, published by Nova Science Publishers, Inc. They were submitted for appropriate modifications in an effort to encourage wider dissemination of research.

SHORT COMMENTARY

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

THE GLOBAL EXTENT OF BLACK C IN SOILS: IS IT EVERYWHERE? Evelyn Krull, Johannes Lehmann, Jan Skjemstad, Jeff Baldock and Leonie Spouncer THE ROLE OF BLACK CARBON IN GLOBAL CLIMATE MODELS The latest projections of the Intergovernmental Panel on Climate Change (IPCC) estimate a 3°C increase in global temperatures within the next 100 years (IPCC 4 th Assessment Report, 2007), and global warming is seen as a major driver in accelerating decomposition of soil organic matter, resulting in increased production of CO2 (e.g. Davidson and Janssens, 2006). The 2007 IPCC report recommended coupling models of terrestrial biogeochemical and atmospheric and oceanic processes in order to improve general circulation models and to recognize the quantitative value of soil organic carbon (SOC) in the global carbon cycle. Estimates of CO2 emissions from soil rely on predictions of the response of different SOC pools to global warming and correct estimation of the size of these pools. In comparison to the pool representing the most stable and biologically unreactive fraction, commonly referred to as passive or inert organic carbon (IOC), the decomposition of labile C is expected to be faster as a response to temperature increase. IOC is a fraction of the SOC pool that is not readily available for microbial decomposition and has turnover times exceeding 100 years (e.g. Krull et al., 2003). Black C (BC) is usually considered the most abundant form of IOC and is defined as the ‗carbonaceous residue of incomplete combustion of biomass and fossil fuels‘ (Schmidt and Noack, 2000). BC is important to several biogeochemical processes; for example, BC potentially modifies climate by acting as a potential carbon sink for greenhouse gases (Kuhlbusch, 1998) and leads to increasing solar reflectance of the Earth‘s atmosphere, but also to a heating of the atmosphere (Crutzen and Andreae, 1990). BC production from fossil fuel combustion contributes to aerosol C, decreasing surface albedo and solar radiation (IPCC 4th Assessment Report). Due to its condensed aromatic structure, BC has a low biochemical reactivity. 14C ages of BC in soils vary between 1160 and 5040 years (e.g. Schmidt et al. 2002).

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Figure 1. Distribution of soilsused for the prediction of BC in surface soils and global NPP.

Figure 2. Distribution of BC%SOC and %SOC in surfaces soils across latitudes.

Baldock (2007) reported that BC constitutes up to 60% of SOC, indicating that BC can make up a significant part of SOC affecting the response of SOC to temperature changes and the overall turnover time of SOC. Thus, the effect of such a large and unreactive C pool must be effectively integrated into global C cycle and climate models. However, the latest IPCC report regards BC only as an aerosol and not as part of SOC, despite an earlier recommendation to the IPCC to ―… better gauge the influence of BC on the global carbon cycle‖ as the result would ensure a ―more accurate global black C budget and a better understanding of the role of BC as a potential sink in the global C cycle.‖ (2006 IPCC guidelines for national greenhouse gas inventories; appendix 1). The lack of incorporating

The Global Extent of Black C in Soils: Is it Everywhere?

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soil BC in global climate change assessments may be largely due to the lack of global databases on BC in soils and sediments and the understanding of factors and processes that may influence BC contents in soils (climate, soil texture, primary productivity and fire abundance). In order to provide reliable projections of future CO2 emissions from soils, due to global warming, it is important to consider the global distribution of soil BC. Through an initial assessment of the World Soil Archive (http://library.wur.nl/isric/) we demonstrate the variability and trends of global soil BC distribution between different climates and soil types and discuss the implications of this chemically recalcitrant form of C on the global C cycle. By doing this, we also demonstrate that current methods exist to routinely analyse BC and in the future to develop a global BC map.

ARE THERE ROUTINE METHODS TO GENERATE A GLOBAL BC MAP? We have developed a rapid fourier-transform infrared-based technique that provides predictive capability for major soil properties, including BC content. BC was quantified by a novel mid-infrared (MIR) method coupled with partial least squares (PLS) (Janik et al., 2007) that allowed rapid analysis of a large number of samples, not feasible by other published methods (Hammes et al., 2007). This method was originally developed and calibrated on Australian soils (Janik et al., 2007); however, subsequently, we were able to verify that the predictive capacity of the MIR technique for soils from other parts of the world was robust in most cases.

BC IN WORLD SOILS: WHAT DRIVES BC PRODUCTION AND DECOMPOSITION? Our initial assessment took advantage of the large collection of soils that constitute part of the ISRIC – world soil information database (http://library.wur.nl/isric/). We obtained over 400 samples that span all major climate zones and soil types across most parts of the world (Fig. 1). Utilising the MIR/PLS technique, we developed a database that illustrates for the first time the predicted proportion of BC in this large soils dataset. We hope that this initial dataset may become an incentive for the rigorous establishment of a global BC map, data from which may then be incorporated into future IPCC reports and C cycle models. Figure 2 shows the proportion of BC (as % of total SOC: BC%SOC) as well as total SOC contents across northern and southern hemispheres. These data show that BC in surface soils (A and A/B horizons) is ubiquitous in large parts of the world and occurs in the majority of the sampled locations. The variability was large, resulting in BC%SOC varying from over 50% to almost 0%. The soils richest in BC occur in latitudes of 20-30° in both hemispheres, situated mostly in central and South America and southern parts of Africa. These areas correspond to tropical climates with a pronounced dry season in winter (‗Aw‘ in the Koeppen classification). In higher latitudes in the northern hemisphere (60-70°), BC contents were also high and were associated with an increase in total SOC (Fig. 2). These areas are classified as

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Evelyn Krull, Johannes Lehmann, Jan Skjemstad et al.

humid-temperate, constantly moist climates (Cf in Koeppen classification). Higher SOC content at high latitudes results from cool climates and low decomposition rates, which promotes accumulation of organic matter. Despite the high spatial variability of BC%SOC, soils with a high proportion of BC fell largely in the soil group of Vertisols. Vertisols occur worldwide in seasonal climate zones and in lower relief positions (Richardson and Vepraskas, 2000). Such areas of deposition would favour accumulation of BC through erosion and deposition. Another factor contributing to the high amounts of BC%SOC in Vertisols could be the comparably high proportion of clay (known to stabilize soil organic matter) and the type of clay (smectitic). The MIR-predicted data showed that Vertisols were amongst the soil types with the highest clay contents (average 44%). High clay content may promote retention of fine BC. However, clay alone is not a determining factor for high BC%SOC. Oxisols and Ultisols, having the highest clay content (from MIR prediction) of all analysed soil types (45%), had the lowest BC %SOC values. Oxisols, and to a lesser degree Ultisols, are mostly found in high-rainfall tropical climates, lacking seasonal rainfall (Af in Koeppen classification) and having a lower fire activity. While fire activity and BC content are both high in the southern hemisphere this relationship is not consistent and does not concur with findings in parts of the northern hemisphere where fire activity is lower, yet BC%SOC is still high (Carmona-Moreno et al., 2005). Thus, a combination of factors, such as climate (the necessity of a pronounced dry season to ensure fire occurrence), position in the landscape (areas of accumulation) and mineralogy (high amount of expansive clays) may be instrumental in promoting the formation and/or retention of BC%SOC in soils. As illustrated in Figure 1, net primary productivity alone did not appear to be a significant factor globally as it is highest in equatorial regions where BC%SOC was lowest. While we show here that BC contents in surface soils are often significant, BC can also accumulate deeper in the soil. In addition, soil erosion and transport of BC may result in significant accumulation of BC in rivers, estuaries and off-shore sediments (Krull et al., 2006).Thus, the inclusion of BC in global climate models will require a thorough assessment of BC contents not only in surface but in deeper soil horizons as well as aquatic sediments. The analyses conducted in this study indicate that methods exist to accomplish BC measurements for large areas. However, the high variability of the data indicates that broad empirical measurements and extrapolation over large areas are not sufficient for the aim of producing a global BC map. Thus, the next steps in a comprehensive assessment of global BC stocks and distribution have to include a detailed and consistent sampling format as well as a thorough assessment of the processes that control sources and sinks of BC.

ACKNOWLEDGMENTS We thank Janine McGowan for assistance in the analyses of the samples and both her and Ryan Farquharson for review of an earlier version of the manuscript. We thank CSIRO Land and Water for supporting the analyses of this work and ISRIC for access to samples from the World Soils Database.

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REFERENCES Baldock, J.A. Composition and cycling of organic carbon in soils (2007), in Marschner, P. and Rengel, Z. (eds.), Nutrient Cycling in Terrestrial Ecosystems. Springer, Berlin. Carmona-Moreno, C., Belward, A., Malingreau, J.P., Hartley, A., Garcia-Alegre, M., Antonovskiy, M., Buchshtaber, V., Pivovarov, V. (2005), Characterizing Interannual Variations in Global Fire Calendar Using Data From Earth Observing Satellites, Global Change Biology 11, 1537-1555. Crutzen, P.J. and Andreae, M.O. (1990), Biomass burning in the tropics: impact on atmospheric chemistry and biogeochemical cycles, Science 250, 1669-1678. Davidson, E.A. and Janssens, I.A. (2006), Temperature sensitivity of soil carbon decomposition and feedbacks to climate change, Nature 440, 165-173. Hammes, K. et al. Comparison of quantification methods to measure fire-derived (black/elemental) carbon in soils and sediments using reference materials from soil, water, sediment and the atmosphere. Global Biogeochemical Cycles 21, GB3016, doi:10.1029/2006GB002914 (2007). IPCC 4th Assessment report (2007), Working Group I: The physical science basis of climate change, http://ipcc-wg1.ucar.edu/wg1/wg1-report.html 2006 IPCC Guidelines for National Greenhouse Gas Inventories, vol. 4, Agriculture, Forestry and other Land use, Appendix 1, http://www.ipcc-nggip.iges.or.jp/public/2006gl/ vol4.htm Janik, L.J., Skjemstad, J.O., Shepherd, K.D., and Spouncer, L.R. (2007) The Prediction of Soil Carbon Fractions Using Mid-Infrared-Partial Least Square Analysis. Australian Journal of Soil Research 45, 73-81. Krull, E.S., Baldock, J.A., and Skjemstad, J.O. (2003), Importance of Mechanisms and Processes of the Stabilisation of Soil Organic Matter for Modelling Carbon Turnover, Functional Plant Biology 30, 207-222. Kuhlbusch, T.A.J. (1998), Black carbon and the carbon cycle. Science 280, 1903-1904. Richardson, J.L. and Vepraskas, M.J. (2000), Wetland Soils: Genesis, Hydrology, Landscapes, and Classification. CRC Press, Florida. Schmidt, M.W.I. and Noack, A.G. (2000), Black carbon in soils and sediments: Analysis, distribution, implications, and current challenges, Global Biogeochemical Cycles 14, 777-793. Schmidt, M.W.I., Skjemstad, J.O., and Jäger, C. (2002), Carbon isotope geochemistry and nanomorphology of soil black carbon: Black chernozemic soils in central Europe originate from ancient biomass burning, Global Biogeochemical Cycles 16, 1123 doi:10.1029/2002GB001939.

RESEARCH AND REVIEW STUDIES

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 1

CURRENT AND EMERGING MICROBIOLOGY ISSUES OF POTABLE WATER IN DEVELOPED COUNTRIES William J. Snelling1, Catherine D. Carrillo2, Colm J. Lowery1, John E. Moore3, John P. Pezacki4, James S. G. Dooley1 and Roy D. Sleator5 1

Centre for Molecular Biosciences, School of Biomedical Sciences, University of Ulster, Cromore Road, Coleraine, Co., Londonderry, Northern Ireland, United Kingdom, BT52 1SA, 2 The Steacie Institute for Molecular Sciences, National Research Council of Canada, 100 Sussex Drive, Ottawa, Ontario, Canada K1A OR6. 3 Bureau of Microbial Hazards, Health Canada, 251 Sir Frederick Banting Driveway, Ottawa, ON, Canada, K1A 0L2 4 Department of Bacteriology, Northern Ireland Public Health Laboratory, Belfast City Hospital, Belfast, United Kingdom, BT9 7AD 5 Alimentary Pharmabiotic Centre, University College Cork, Cork, Ireland

ABSTRACT Water is vital for life; for commercial and industrial purposes and for leisure activities in the daily lives of the world‘s population. Diarrhoeal disease associated with consumption of poor quality water is one of the leading causes of morbidity and mortality in developing countries (especially in children <5 years old). In developed countries, whilst potable water is not a leading cause of death, it can still pose a significant health risk. Water quality is assessed using a number of criteria, e.g. microbial load and nutrient content which affects microbial survival, as well as aesthetic factors such as odor. In water systems, the presence of disinfectant, low temperatures, flow regimes and low organic carbon sources do not appear to be conducive to microbial persistence. However, frequently this is not the case. A variety of human pathogens can be transmitted orally by 

Correspondence: Dr Roy Sleator, Alimentary Pharmabiotic Centre, University College Cork, Ireland. Phone: 00 353 21 490 1366, Fax: 00 353 21 490 3101, email: [email protected]

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William J. Snelling, Catherine D. Carrillo, Colm J. Lowery et al. water and in the developed world water quality regulations require that potable water contains no microbial pathogens. Chlorine dioxide is a safe, relatively effective biocide that has been widely used for drinking water disinfection for 40 years. Providing the water is of low turbidity, standard chlorination procedures are sufficient to prevent the spread of planktonic bacteria along water mains. However, despite this, bacterial contamination of water distribution systems is well documented, with growth typically occuring on surfaces, including pipe walls and sediments. Rivers, streams and lakes are all important sources of drinking water and are used routinely for recreational purposes. However, due to fouling by farm and wild animals, these sources can be contaminated with microbes, e.g. chlorine resistant Cryptosporidium oocysts, no matter how pristine the source or well maintained the water delivery system. The high incidence of Cryptosporidium in surface water sources underlines the need for frequent monitoring of the parasite in drinking water. The use of coliforms as indicator organisms, although considered relevant to most cases, is not without limitations, and is thus not a completely reliable parameter of water safety, e.g. Campylobacter contamination cannot be accurately predicted by coliform enumeration. Furthermore, the presence of biofilms and bacterial interactions with protozoa in water facilitate increased resistance to antimicrobial agents and procedures such as disinfectants and heating, e.g. Legionnaires‘ disease caused by Legionella pneumophila. The high cost of waterborne disease outbreaks should be considered in decisions regarding water utility improvement and treatment plant construction. The control of human illnesses associated with water would be aided by a greater understanding of the interactions between water-borne protozoa and bacterial pathogens, which until relatively recently have been overlooked.

INTRODUCTION Currently, over 1 billion people worldwide have no access to safe drinking water (CDC, 2007a). In the developed world water quality regulations require that potable water does not contain any microbial pathogens (Percival and Walker, 1999). Residents of affluent nations are remarkably lucky to have high-quality, safe drinking water supplies that most residents of modem cities enjoy, particularly when considered in contrast to the toll of death and misery that unsafe drinking water causes for most of the world's population (Hrudey and Hrudey, 2007). The supply of clean drinking water is a major, and relatively recent, public health milestone (Berry et al., 2006). However, some may presume that drinking-water disease outbreaks are a thing of the past, but complacency can easily arise (Hrudey and Hrudey, 2007). Increasing human populations and urbanisation have placed burdens on water sources, i.e. rivers, streams and lakes, used to provide potable water to most metropolitan areas (Calderon et al., 2006). Excreta from humans, pets, livestock and wildlife, e.g. foxes, present in these source waters have the potential to harbour hundreds of pathogenic microorganisms of public health concern (Leclerc et al., 2002). In developed countries, major outbreaks of waterborne infections have fuelled widespread public concern regarding the microbiological quality of potable water (De Paula et al., 2007; Pankhurst and Coulter, 2007). Potable water can be a source of various potentially infectious microorganisms, which in the majority of cases are contracted by ingestion, but can also be contracted via inhalation of aerosol droplets or by direct dermal exposure (Stojek and Dutkiewicz, 2006). Rivers, streams and lakes, which are not only important sources of drinking water but are also routinely used in the pursuit of human leisure interests, are often contaminated with microbes, e.g. chlorine

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 13 resistant Cryptosporidium oocysts, no matter how pristine the source, or well maintained the water delivery system (Snelling et al., 2006). Fecal contamination of surface waters can occur via wastewater discharge, farming activities and fouling by wild animals. Perhaps the best known potential pathogens are certain strains of Escherichia coli and related Gram-negative species of fecal origin belonging to the Enterobacteriaceae family, usually referred to as ―coliforms‖, which are commonly used as sanitary indicators of potable water quality (Stojek and Dutkiewicz, 2006). Important agents of waterborne infections include various genera of Gram-negative bacteria such as Campylobacter, Escherichia coli O157:H7, Legionella, Salmonella, Shigella, Yersinia, Vibrio cholerae, mycobacteria (Gram positive), enteroviruses and intestinal protozoa (Giardia, Cryptosporidium) (Stojek and Dutkiewicz, 2006). Potable water distribution systems generally present a hostile environment for growth of microorganisms due to low nutrient levels as well as the presence of disinfection residuals (Långmark et al., 2007; Momba et al., 2007). Yet despite this, biofilms form ubiquitously in environments that have been subjected to a range of disinfection processes including chlorination and ultra-violet (UV)-treatment (Momba et al., 1998). Most of the bacteria in drinking water distribution systems, e.g. M. avium, L. pneumophila, and E. coli, are associated with biofilms. In biofilms, their nutrient supply is better than in water, and biofilms can provide shelter against disinfection (Lehtola et al., 2007). Pathogenic bacteria and viruses entering water distribution systems can survive in biofilms for at least several weeks, even under conditions of high-shear turbulent flow, and may be a risk to water consumers (Lehtola et al., 2007). Also, considering the low number of virus particles needed to result in an infection, their extended survival in biofilms must be taken into account as a risk for the consumer (Lehtola et al., 2007). Recent inquiries into the microbial ecology of distribution systems have found that pathogen resistance to chlorination is affected by microbial community diversity and interspecies relationships (Berry, et al., 2006). Research indicates that multispecies biofilms are generally more resistant to disinfection than single-species biofilms (Berry, et al., 2006). Control of microbial growth in drinking water distribution systems, often achieved through the addition of disinfectants, is essential to limiting waterborne illness, particularly in immunocompromised subpopulations (Berry et al., 2006). Whilst disinfection residuals within distribution systems reduce the growth of autochthonous and allochthonous pathogens and reduce the potential re-growth of heterotrophic bacteria, they do not insure the sterility of distribution waters (Payment et al., 1993). The omnipresence of heterotrophs does not in itself constitute a potential health hazard, they can be used to evaluate the microbiological quality of the water and the efficacy of water treatment and distribution processes, as well as the biological stability of distributed waters (Långmark et al., 2007). Waterborne diseases occur worldwide, and outbreaks caused by the contamination of community water systems have the potential to cause disease in large numbers of consumers (Table 1) (Karanis et al., 2007). These cases create a lack of confidence in potable water quality and in the water industry in general; waterborne outbreaks have economic consequences far beyond the cost of health care for affected patients, their families and contacts. In addition to outbreaks caused by contaminated potable water, there is also a risk associated with the accidental ingestion of recreational (or other) waters. National statistics on outbreaks linked to contaminated water have been available in the USA since 1920, and since 1971, the Centers for Disease Control (CDC), the US Environmental Protection Agency (USEPA), and the Council of State and Territorial Epidemiologists have maintained a

Table 1. Summaries of Notifiable Diseases in the United States in 2005 (McNabb et al., 2007)

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collaborative surveillance system for collecting data pertaining to the occurrence and causes of outbreaks of waterborne disease (Karanis et al., 2007). In Europe during 1986–96, 277 outbreaks associated with drinking and recreational water were reported from 16 European countries (Kramer et al. 2001).

VIRUSES Viruses are possibly the most hazardous of all enteric pathogens and have relatively low infectious doses (Santamaría and Toranzos 2003). Viruses are found in very low concentrations in treated water due to the effect of dilution and to the potabilization process (Gutiérrez et al., 2007). For this reason, it has been proposed that large amounts of water need to be collected in order to detect them. A negative result might not be meaningful, while a positive result is important, particularly when viruses are detected in small water samples (Gutiérrez et al., 2007).Thus, the paucity of data regarding waterborne viruses makes it difficult to determine the true risk they represent and precludes the development of plans to prevent viral transmission through contact with environmental water (De Paula et al., 2007). It has been proposed that more than 140 different types of viruses, apparently not eliminated by massive purification treatments, can be found in drinking water (Gutiérrez et al., 2007). The main source for water contamination is human excreta (Gutiérrez et al., 2007). In fact, viruses infiltrate the ground, penetrating to depths greater than 67 m and can remain latent there for several months, as long as the temperature remains low and the environment humid (Gutiérrez et al., 2007). Under these conditions they can easily reach aquifers. Enteric pathologies due to the presence of Rotavirus (RV), Norovirus (NV), Astrovirus (HAstV), Adenovirus (Ad), Hepatitis A (HAV), polio, Coxsakie, and echo type Enteroviruses, among others, have been reported to be associated with consumption of fresh water (Gutiérrez et al., 2007). Enteroviruses are a group of viruses including the polioviruses, coxsackieviruses, echoviruses, and others (CDC, 2007c). In addition to the three different polioviruses, there are 62 non-polio enteroviruses that can cause disease in humans: 23 Coxsackie A viruses, 6 Coxsackie B viruses, 28 echoviruses, and 5 other enteroviruses (CDC, 2007c). Non-polio enteroviruses are second only to rhinoviruses - causative agent of the "common cold" as the most common viral infectious agents in humans (CDC, 2007c). The enteroviruses cause an estimated 10-15 million or more symptomatic infections per year in the United States (CDC, 2007c). All three types of polioviruses have been eliminated from the Western Hemisphere, as well as Western Pacific and European regions, by the widespread use of vaccines (CDC, 2007c). Most people who are infected with an enterovirus exhibit no clinical manifestations of disease. Infected individuals who do exhibit clinical signs of disease usually develop either mild upper respiratory symptoms (a "summer cold"), a flu-like illness with fever and muscle aches, or illness with an associated rash (CDC, 2007c). A less common, though none the less possible, manifestation of the illness is "aseptic" or viral meningitis (CDC, 2007c). Rarely, infected individuals may develop an illness that affects the heart (myocarditis) or the brain (encephalitis) or causes paralysis (CDC, 2007c). Human noroviruses (NoVs) are a significant cause of non-bacterial gastroenteritis worldwide with contaminated drinking water a potential transmission route (Bae and Schwab,

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 17 2008). As members of the Caliciviridae family, NoVs (previously known as ―Norwalk-like viruses‖) are small (27 nm), icosahedral, non-enveloped human enteric viruses that cause acute gastroenteritis (Bae and Schwab, 2008). Due to their non-enveloped structure which is similar to other human enteric viruses such as poliovirus (PV), coxsackievirus, and echovirus, NoVs are presumed to be as resistant to environmental degradation and chemical inactivation as the aforementioned viruses (Bae and Schwab, 2008). The most common vehicles for Hepatitis A virus (HAV) transmission are ingestion of contaminated water, consumption of contaminated foods and contact with infected individuals (De Paula et al., 2007). Hepatitis E virus (HEV) is an emerging food borne pathogen in developing countries, e.g. Asia, Africa and Latin America (Skovgaard, 2007). Transmitted by the fecal–oral route, infection which results in liver inflammation, is linked to the consumption of swine; the primary source of the virus for humans. The incidence of sporadic cases has begun to increase recently in developed countries, but it is uncertain whether these cases were food- or water borne (Skovgaard, 2007). The incidence of hepatitis E infection is highest in adults between the ages of 15 and 40. Children are also succeptable to infection but are less likely to become symptomatic. Mortality rates are generally low as Hepatitis E is a ―self-limiting‖ disease; symptoms generally dissipate by themselves and the patient usually recovers. It is spread mainly through fecal contamination of water supplies or food; person-to-person transmission is uncommon. Outbreaks of epidemic Hepatitis E most commonly occur following disruption to water supplies caused by heavy rainfalls and monsoons. Viruses can be inactivated in the environment by the breakage of their capsid and the liberation of the contained nucleic acid, which can be easily degraded when deprived of the capsid protection (Gutiérrez et al., 2007). Environmental degradation of viruses can result from extremes in pH, thermal inactivation, sunlight and predation or release of virucidial agents from endogenous microorganism in environmental water (Hurst 1988; Yates etal., 1985). Chlorine, the most commonly used drinking water disinfectant, can also inactivate enteric viruses if sufficient dose and contact time are provided (Ellis, 1991). Despite some opinions to the contrary, this means that detection of viral protein (VP) in laboratory assays is not a sufficient criterion as to the infectious nature of the virus (Gassilloud et al., 2003).

TOXIGENICITY Microcystis aeruginosa is a gas vacuolate, bloom-forming cyanobacterium that is of interest from a water quality perspective because of its ability to produce a range of toxic compounds (Saker et al., 2005). The cyclic peptides, also known as microcystins, are produced by several species of cyanobacteria including M. aeruginosa, and have been implicated in the death of humans as well as domestic and wild animals (Saker et al., 2005). These compounds inhibit protein phosphatases 1 and 2A in a similar manner to okadaic acid, and have been linked to liver cancer in humans (Saker et al., 2005). To date, more than 60 microcystin variants have been identified and chemically characterized (Saker et al., 2005). In recognition of their toxic properties, the World Health Organization has adopted a maximum allowable concentration in drinking water of 1µgl-1 (WHO 1998). With appropriate water treatment, maximum exposure to total microcystins is probably less than 1 μgl-1, based on the

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above data. While average exposure is generally well below this level (WHO, 1998), not all water supplies are treated by filtration or adsorption; many are untreated or simply chlorinated (WHO, 1998). In addition to microcystins, many strains of Microcystis are known to produce other peptides including aeruginosins, anabaenopeptilides, cyanopeptolins, anabaenopeptins and microginins, which show a diverse range of bioactivities (Saker et al., 2005).

FOODBORNE ‗CONTAMINATING‘ BACTERIA Several foodborne pathogens, e.g. Salmonella typhimurium, Campylobacter jejuni, Yersinia enterocolitica, Mycobacteria, Escherichia. coli O157:H7 and Cryptosporidium, are common intestinal contaminants frequently found in both farmed animals and wildlife (Doyle and Erickson 2006), and in several cases are carried asymptomatically (Snelling et al., 2005; Thompson et al., 2007). These pathogens are generally shed in feces in large populations and can be transmitted to surface water, both directly and indirectly (Doyle and Erickson 2006). Soil as a recipient of solid wastes is able to contain enteric pathogens in high concentrations, which can then be spread to surface water (Figure 1) (Santamaría and Toranzos, 2003). Although most E. coli strains are harmless, E. coli O157:H7 produces a powerful toxin that can cause severe human illness (CDC, 2007b). E. coli O157:H7 has been found in the intestines of livestock (CDC, 2007b), is part of the natural microbiota of the soil, and is therefore also associated with manure, crops, minimally processed ready to eat foods and surface water (Selma et al., 2007; Himathongkham et al., 2007). Infections often lead to bloody diarrhea, and in some individuals, particularly children under 5 years of age and the elderly, the infection can cause haemolytic uremic syndrome (HUS), in which the red blood cells are destroyed and the kidneys fail (CDC, 2007b). E. coli O157:H7 is a major global cause of foodborne illness (CDC, 2007b). Based on a 1999 estimate, 73,000 cases of infection and 61 deaths occur in the United States each year. People can become infected with E.coli O157:H7 in a variety of ways (CDC, 2007b). Though most illness has been associated with eating undercooked, contaminated ground beef, people have also become ill from eating contaminated bean sprouts or fresh leafy vegetables such as lettuce and spinach (CDC, 2007b). In addition, infection can occur after drinking raw unpasturized milk and after swimming in or drinking sewage-contaminated water (CDC, 2007b). Salmonella are enteric bacterial pathogens, of which mainly S. enterica and S. typhimurium cause a variety of food and water-borne diseases ranging from gastroenteritis to typhoid fever (Ly and Casanova 2007). Previous investigations have found that sewage effluent regularly contain Salmonella and Campylobacter (Kinde et al., 1997). Globally, C. jejuni is the major cause of human bacterial diarrhoeal illness and is by far the most common Campylobacter species associated with human illness. The major C. jejuni reservoir is poultry (Snelling et al., 2005). However, C. jejuni has been linked to several drinking water-related epidemics in Finland (Lehtola et al., 2006). C. jejuni is not normally able to multiply in drinking water or in biofilms, although it may survive in biofilms and/or within protozoa (Lehtola et al., 2006; Snelling et al., 2006).

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 19

Figure 1. Diagram showing the different, most important cycles of transmission for maintaining Giardia and Cryptosporidium. As well as direct transmission, water and food may also play a role in transmission. Question marks indicate uncertainty regarding the frequency of interaction between cycles (Hunter and Thompson, 2005).

Shigellosis is a global human diarrhoeal disease leading to dysentery and is caused by S. dysenteriae, S. flexneri, S. boydii and S. sonnei (Niyogi et al., 2005). Shigella dysenteriae type 1 produces severe disease and may be associated with life-threatening complications (Niyogi et al., 2005). The symptoms of shigellosis include diarrhoea and/or dysentery with frequent mucoid bloody stools, abdominal cramps and tenesmus (Niyogi et al., 2005). Transmission usually occurs via contaminated food, e.g. ready to eat salads (Ghosh et al., 2007), water, livestock manure, or through person-to-person contact (Niyogi et al., 2005). Of the estimated 165 million cases of Shigella diarrhoea that occur annually, 99% occur in developing countries with 69% of episodes occuring in children under 5 years of age (Kotloff et al.,1999). Mycobacterium is a genus of Actinobacteria, given its own family. Mycobacteria are aerobic and generally non-motile bacteria, that are characteristically acid-alcohol fast (Ryan and Ray, 2004). Environmental mycobacteria are common heterotrophic bacteria in soils and natural waters, especially in boreal drinking water systems (Torvinen et al., 2004). Mycobacteria are classified as an acid-fast Gram-positive bacterium due to their lack of an outer cell membrane (Ryan and Ray, 2004). All Mycobacterium species share a characteristic cell wall, thicker than in many other bacteria, making a substantial contribution to the hardiness of this genus (Ryan and Ray, 2004). Therefore, compared to most other bacterial species, mycobacteria are exceptionally resistant to chlorination and heating (Torvinen et al., 2004). Some thermotolerant species, including the important mycobacterial pathogens M. avium complex (MAC) and M. xenopi, tolerate heating and may even survive in hot water (>60°) (Torvinen et al., 2004). Thus, they may survive chemical treatments at waterworks and enter the distributed water and finally tap water, where their occurrence has been known since the beginning of the 20th century.

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Tuberculosis (TB) affects one-third of the world's population, claiming a life every 10 seconds and global mortality rates are increasing, especially in developing countries (Sacchettini et al., 2008; Tabbara, 2007). The incidence of tuberculosis is increasing with the increase in the HIV infected population and increased strain drug resistance (Tabbara, 2007). Complex interactions involving humans, domestic animals, and wildlife create environments favorable to the emergence of new diseases (Palmer, 2007). Today, reservoirs of M. bovis subsp. paratuberculosis (MAP), the causative agent of tuberculosis in animals and a serious zoonosis, exist in wildlife (Palmer, 2007). The presence of these wildlife reservoirs is the direct result of spillover from domestic livestock, especially cows, in combination with anthropogenic factors such as translocation of wildlife, supplemental feeding of wildlife and wildlife populations reaching densities beyond normal habitat carrying capacities (Palmer, 2007). Toxigenic Vibrio cholerae, the causative agent of cholera, is a native inhabitant of the aquatic environment (rivers, estuaries, and coastal waters) which is transmitted through drinking water and still remains a leading cause of morbidity and mortality in many developing countries, especially in Asia and Africa (Chomvarin et al., 2007). In aquatic environments V. cholerae associates with the chitinous exoskeletons of copepod molts, which serves as a surface of nutrients thus facilitating biofilm formation and induces competence for natural transformation (Blokesch and Schoolnik, 2007). Despite more than a century of investigation, much remains to be discovered about how pathogenic strains of V. cholerae interact with the human host and how the biology of disseminating stool V. cholerae drives devastating cholera outbreaks (Nelson et al., 2007). The V. cholerae species encompasses more than 200 serogroups (Blokesch and Schoolnik, 2007). Cholera is an ancient secretory diarrheal disease caused by the O1 and O139 serogroups of V. cholerae (Nelson et al., 2007). Despite the dramatic reduction of mortality rates due to the development of oral rehydration solution, the emergence of multiple drug-resistant V. cholerae may reduce the efficacy of antimicrobial treatment and alter the dynamics of outbreaks (Nelson et al., 2007). V. cholerae is a bacterial pathogen of the gastrointestinal tract and the secreted toxin is largely responsible for the massive fluid loss that may reach between 0.5 and 1.0 liter per hour (Nelson et al., 2007). Yersinia (family Enterobacteriaceae) are faculatative anerboic bacteria, of which Y. enterocolitica are foodborne pathogens which are mostly associated with human disease. Y. enterocolitica is responsible for outbreaks of acute human gastroenteritis and chronic sequela, e.g. reactive arthritis, and livestock morbidity, e.g. mastitis (Meusburger et al., 2007; Rudwaleit et al., 2000; Shwimmer et al., 2007). Y. pseudotuberculosis (food-borne route) and Y. pestis (flea-borne zoonotic disease) can also cause disease in humans. Rodents are the major reservoirs of Y. enterocolitica; and while humans are the primary hosts other mammals may also be infected. Infection may occur either through blood (in the case of Y. pestis) or occasionally via consumption of food products (especially vegetables, milk-derived products and meat) contaminated with infected urine or feces. An important property of Yersinia is its ability to multiply at temperatures close to 0 C (Skovgaard, 2007). The minimum infection dose of Yersinia is relatively high (>100,000), and in many countries, e.g. Denmark, Yersinia interest and concern have declined (Skovgaard, 2007).

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 21

PROTOZOAN PARASITES At least 325 water-associated outbreaks of parasitic protozoan disease have been reported (Karanis et al., 2007). North American and European outbreaks accounted for 93% of all reports and nearly two-thirds of outbreaks occurred in North America (Karanis et al., 2007). Over 30% of all outbreaks were documented from Europe, with the UK accounting for 24% of outbreaks, worldwide (Karanis et al., 2007). Giardia duodenalis and C. parvum account for the majority of outbreaks (132; 40.6% and 165; 50.8%, respectively), Entamoeba histolytica and Cyclospora cayetanensis have been the aetiological agents in nine (2.8%) and six (1.8%) outbreaks respectively, while Toxoplasma gondii and Isospora belli have been responsible for three outbreaks each (0.9%) and Blastocystis hominis for two outbreaks (0.6%) (Karanis et al., 2007). Balantidium coli, the microsporidia, Acanthamoeba and Naegleria fowleri were responsible for one outbreak each (0.3%) (Karanis et al., 2007). Waterborne parasites produce transmission stages which are highly resistant to external environmental conditions, and to many physical and chemical disinfection methods routinely used as bacteriocides in drinking water plants, swimming pools or irrigation systems (Gajadhar and Allen, 2004). Resistant stages include cysts of amoebae, Balantidium, and Giardia, spores of Blastocystis and microsporidia, oocysts of Toxoplasma gondii, Isospora, Cyclospora and Cryptosporidium and eggs of nematodes, trematodes, and cestode(Gajadhar and Allen, 2004). These exogenous transmission stages are microscopic in size and of low specific gravity, which facilitate their easy dissemination in fresh water, or seawater (Gajadhar and Allen, 2004). The exogenous stages of waterborne parasites possess outer surfaces capable of withstanding a variety of physical and chemical treatments (Gajadhar and Allen, 2004). The resistant surfaces are comprized of multiple polymeric layers of lipids, polysaccharide, proteins or chitin (Gajadhar and Allen, 2004). Examples of these are the two protein layers of coccidian oocysts derived from the coalescence of wall-forming bodies, the chitinous wall of microsporidian spores, the multi-layered (inner lipid/protein-, middle protein/chitin-, outer protein/mucopolysaccharide) shell of Ascaris eggs, and the impermeable embryophore of the Echinococcus egg which is constructed of polygonal blocks of keratinlike protein held together by a cement substance (Gajadhar and Allen, 2004). The intestinal protozoan parasites Cryptosporidium (Apicomplexan) and Giardia (G. duodenalis) are major global causes of diarrhoeal disease in humans (Smith et al., 2007). Significantly, normal concentrations of chlorine and ozone used in mass water treatment are not adequate to kill these microbes (Smith et al., 2007), which have life cycles suited to both waterborne and foodborne transmission (Smith et al., 2007). Giardia causes intestinal malabsorption and diarrhoea (giardiasis) in humans and other mammals worldwide (Smith et al., 2007). Giardia is one of the most prevalent pathogens that should be removed from drinking water (Smith et al., 2007). In developing countries, the prevalence of human giardiasis is on average 20% (4–43%), compared with 5% (3–7%) in developed countries, where it is associated mainly with travel and waterborne outbreaks (Smith et al., 2007). G. duodenalis assemblages A and B have been found in humans and most mammalian orders (Smith et al., 2007). Giardiasis is a disease with economic ramifications due to the large impact on domestic animals such as cattle and sheep (Smith et al., 2007). The role of animal transmission of human giardiasis is unclear, but the greatest risk of zoonotic transmission seems to be from companion animals such as dogs and cats. Interestingly, the capacity of

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Acanthamoeba to predate Cryptosporidium oocysts has recently been demonstrated (GómezCouso et al., 2007). Free-living-amoeba (FLA) may act as environmentally resistant carriers of Cryptosporidium oocysts and, thus, may play an important role in the transmission of cryptosporidiosis (Gómez-Couso et al., 2007). Zoonotic Cryptosporidium parvum and anthroponotic C. hominis are the major cause of human cryptosporidiosis, although other species including C. meleagridis, C. felis, C. canis, C. suis, C. muris and two corvine genotypes of Cryptosporidium have been associated with human gastroenteritis (Xiao and Ryan 2004; Caccio et al. 2005). Cryptosporidium can survive for months in a latent form outside hosts, as its oocysts retain their infectivity for several months in both salt and fresh water (Fayer et al. 1998; Sunnotel et al., 2006a). Cryptosporidium causes self-limited watery diarrhoea in immunocompetent subjects, but has far more devastating effects in the immunocompromized and in some cases can be lifethreatening due to dehydration caused by chronic diarrhoea (Caccio 2005, Chen et al. 2005). Cryptosporidiosis is responsible for significant neonatal morbidity in farmed livestock and causes weight loss and growth retardation, leading to significant economic losses (McDonald 2000). Over the last two decades, increasing numbers of cryptosporidiosis (in particular water related) outbreaks have been recorded in developed countries (Craun et al. 2005). In 1993, the largest Cryptosporidium outbreak was registered in Milwaukee, Wisconsin, USA where 403,000 people were infected through contaminated drinking water (MacKenzie et al. 1994). This outbreak was caused by C. hominis (Peng et al. 1997), and the total cost of outbreakassociated illness was estimated at >$96 million in both medical costs and productivity losses (Corso et al. 2003). Cryptosporidium has also been associated with treated water in swimming and wading pools (Craun et al. 2005). Shellfish have the ability to filter large amounts of water and concentrate oocysts within their gills (Gomez-Couso et al. 2006). Thus, despite prevention measures, e.g. standard UV depuration treatment, the consumption of raw or undercooked shellfish, may still be a potential health risk (Sunnotel et al., 2007). Also, the use of surface water for irrigation can indirectly cause human infection via the consumption of contaminated fresh produce, e.g. lettuce (Shigematsu et al., 2007). Outbreaks have been reported in healthcare facilities and day-care centres, within households, among bathers and water sports enthusiasts in lakes and swimming pools, and in municipalities with contaminated public water supplies or people served by private water supplies. In 2005 the European Basic Surveillance Network (BSN) recorded 7,960 human cases of cryptosporidiosis from 16 countries. Microsporidial gastroenteritis; a serious disease of immunocompromized people, can have a waterborne etiology (Graczyk et al., 2007). Microsporidia are obligate intracellular eukaryotes parasitizing a wide range of invertebrates and vertebrates with over 1,200 species, of which 14 are opportunistic human pathogens, with Encephalitozoon intestinalis, E. hellem, E. cuniculi, and Enterocytozoon bieneusi being the most common (Graczyk et al., 2007). Currently, Microsporidia are on the Contaminant Candidate List of the U.S. Environmental Protection Agency due to their unknown transmission routes, technologically challenging identification and the difficult treatment of human infections (Graczyk et al., 2007). Considerable evidence indicates involvement of water in the epidemiology of microsporidiosis, however, this link has not been conclusively substantiated (Graczyk et al., 2007). Risk factor analysis for encephalito zoonosis has previously suggested groundwater as a source of infection, and a massive outbreak of microsporidiosis was epidemiologically

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 23 linked to a drinking water distribution system (Cotte, et al., 1999). More accurate Microsporidial epidemiological data is urgently required to accurately assess the importance of this emerging pathogen.

EMERGING AND OPPORTUNISTIC PATHOGENS Opportunistic microbes are a subset of the emerging pathogens and include species of both fecal and environmental origin. Treated potable water contains a variety of microbes that are not well characterized (Stelma et al., 2004). Many of these organisms grow slowly and require nutrient-poor media for culturing (Stelma et al., 2004). Although there is evidence that these microbes are generally not hazardous to the general healthy population, there is a possibility that some of them may be opportunistic pathogens and may be capable of causing adverse health effects in individuals with impaired body defences (Stelma et al., 2004). Examples of opportunistic bacterial pathogens of potable water origin include Aeromonas hydrophila, L. pneumophila, M. avium, and Pseudomonas aeruginosa. There is no reason to assume that the currently known opportunistic pathogens are the only opportunistic pathogens indigenous to potable water (Stelma et al., 2004). Many cases of respiratory infections and digestive system infections are still of unknown etiology and it is possible that some of them could be due to pathogens that are currently unknown (Stelma et al., 2004). Opportunistic bacterial pathogens have the potential to reproduce in the natural environment, particularly in the presence of increased temperatures and nutrients, often when associated with free-living protozoa (Långmark et al., 2007). Free-living protozoa are in most cases non-parasitic, however, some species are known to cause human illness (Långmark et al., 2007). The potential significance of free-living protozoa, e.g. amoeba, as potential environmental reservoirs for aquatic pathogens has been recognized for more than twenty years (King et al., 1988), as has the often complex interspecies relationships between mixed biofilm populations, e.g. bacteria and protozoa (Snelling et al., 2006; Långmark et al., 2007). Yet despite this, with the exception of L. pneumophila, relatively little attention has been payed to studying and understanding interactions between free-living protozoa and other smaller human pathogens (Brown and Barker, 1999; Snelling et al., 2006). Some microorganisms have evolved to become resistant to protozoa digestion and these amoebaresistant microorganisms include established bacterial pathogens, such as Legionella spp., Chlamydophila pneumoniae, M. avium, P. aeruginosa, and C. jejuni, and emerging pathogens such Simkania negevensis, Parachlamydia acanthamoebae, and Legionella-like amoebal pathogens (Snelling et al., 2006). The fate of internalized bacteria can be divided into three main outcomes; i)

Bacteria which multiply and cause lysis of amoebal cells (FLA), e.g. Legionella and Listeria monocytogenes. ii) Bacteria which multiply without causing cell lysis, e.g. Vibrio cholera. iii) Bacteria which survive without multiplication, e.g. mycobacteria (Snelling et al., 2006).

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FLA feed on bacteria, fungi and algae and as such are ubiquitous predators that control microbial communities while at the same time acting as reservoirs for many human pathogens. FLAs are thus often regarded as the ―Trojan horses‖ of the microbial world (Chang and Jung, 2004; Greub and Raoult 2004; Molmeret et al., 2005). Dictyostelium discoideum is an established host model for several pathogens including, P. aeruginosa, Mycobacterium spp., and L. pneumophila (Snelling et al., 2006). Significantly, numerous bacterial species survive disinfection treatments when internalized within protozoa, compared to the exposed and thus more sensitive planktonic state which forms the basis of current disinfection policies (Snelling et al., 2005b; King et al., 1998; Whan et al., 2006). Interactions such as these are unaccounted for in current disinfection models (Berry, et al., 2006). FLA provide habitats for the environmental survival of L. pneumophila, which have been observed undergoing binary fusion within intracellular vacuoles of amoeba (Atlas, 1999; Colbourne et al., 1984). To date it has never been shown that Legionella can multiply in its own natural environment outside protozoa (Atlas, 1999; Colbourne et al., 1984). Legionella pneumophila are fastidious bacteria which develop mostly in water, causing legionnaires‘disease [(LD), legionellosis, or atypical pneumonia/flu-like illness] in humans (Stout et al. 1992; Stojek and Dutkiewicz, 2006). In environmental water bodies L. pneumophila proliferates intracellularly in more than 15 protozoan genera, e.g. Acanthamoeba, Hartmannella, Vahlkamphia and Ehinamoeba (Steinert et al., 1994). Infection of humans, generally the elderly and immunocompromized, occurs after inhalation of aerosolized bacteria from contaminated water sources (Steinert et al., 1994). The infectious particle is unknown, but may be excreted as legionellae-filled vesicles, intact legionellaefilled amoebae, or free legionellae that have lysed their host cell, resulting in L. pneuomphila, not amoeba, persisting within the respiratory tract (Steinert et al., 1994). The processing of L. pneumophila by A. castellanii shows many similarities to monocyte phagocytosis, e.g. the uptake of L. pneumophila by coiling phagocytosis (Steinert et al., 1994). Phagosome–lysosome fusion is prevented based upon L. pneumophila expressing dot/icm genes that code for a putative large membrane complex which forms a type IV secretion system that is used to alter the endocytic pathway (Steinert et al., 1994). Genes such as hfq appear to play a major role in exponential phase regulatory cascades of L. pneumophila (McNealy et al., 2005; Solomon and Isberg, 2000). Globally, potable water supplies which harbour L. pneumophila are important sources of community acquired LD (Stout et al. 1992). LD accounts for an estimated 8,000 to 18,000 cases of hospitalized community-acquired pneumonia in the United States annually (Phares et al., 2007). Individuals are most often infected with Legionella by inhaling bacteria-laden aerosol droplets, e.g. via bathing, but can also become infected by the oral route through drinking water or via traumatized skin or mucous membranes (Stojek and Dutkiewicz, 2006). Approximately 35% of all LD cases reported to the Centers for Disease Control and Prevention (CDC) are acquired in health care facilities (Phares et al., 2007). M. avium is a common cause of systemic bacterial infection in patients with AIDS (Miltner and Bermudez, 2000; Snelling et al., 2006). Infection with M. avium, commonly occurs through the gastrointestinal tract and has been linked to bacterial colonization of domestic water supplies, where A. castellanii may serve as an environmental host for M. avium AIDS (Miltner and Bermudez, 2000). M. avium can also survive within A. polyphaga cyst outer walls and bacterial growth occurs in cocultures (Steinert et al., 1998; Snelling et al., 2006). M. avium enters and replicates in A. castellanii, and similar to mycobacteria within

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 25 human macrophages, inhibits lysosomal fusion and replicates in vacuoles that are tightly juxtaposed to the bacterial surfaces within amoebae (Cirillo et al., 1997). Growing M. avium in amoebae enhances invasion and intracellular replication of the bacterium in human macrophages, the intestinal epithelial cell line HT-29, as well as in mice (Miltner and Bermudez, 2000), and also increases resistance to rifabutin, azithromycin, and clarithromycin, which might have significant implications for prophylaxis of M. avium infection in AIDS patients (Miltner and Bermudez, 2000).

INDICATOR METHODS For more than thirty years the measurement of bacteria of the coliform group has been extensively relied on as an indicator of water quality (Yáñez et al., 2006). Among the coliforms, the specific determination of E. coli contamination can be performed as one of the best means of estimating the degree of recent fecal pollution (Edberg et al., 2000). The European Drinking Water Directive 98/83/EC (Anon, 1998) defines that the isolation of coliforms using Lactose TTC agar with Tergitol (Tergitol-7 agar) by membrane filtration (Anon, 2000) be the reference method for the enumeration of total coliforms, including E. coli in drinking water (Yáñez et al., 2006). However, numerous outbreaks, e.g. Crytosporidium, Campylobacter, and viruses, have made it clear that the presence of bacterial indicators of fecal contamination does not consistently correlate with pathogen levels (De Paula et al., 2007). Alternatively, a number of instrumental methods for the rapid determination of microorganisms based on their metabolic activity have been developed, but not really utilized, for example, impedance (Madden and Gilmour, 1995), conductance (Gibson, 1987), chemiluminescence and fluorescence (Van Poucke and Nelis, 2000). Different chromogenic and/or fluorogenic culture media have also been developed in recent years (Manafi, 2000). One is TBX agar medium, a modification of Tryptone bile agar medium where the substrate 5-bromo-4-chloro-3-indolyl-b-d-glucuronide (BCIG) is added. This substrate is cleaved by the enzyme β-d-glucuronidase (GUD) that is produced by approximately 95% of the E. coli strains investigated (Adams et al., 1990), and the released chromophore produces easy to read blue-green coloured E. coli colonies. Furthermore, the TBX agar method complies with the ISO/DIS Standard 16649 for the enumeration of E. coli in food and animal foodstuffs (Anon, 2001). In addition, different chromogenic media such as Chromocult Coliform agar (CC agar), and coli ID agar have been developed for the simultaneous detection of total coliforms and E. coli, based on the presence of chromogenic substrates for the enzymes β–galactosidase (Lac) and β-D-glucuronidase (GUD) (Geissler et al., 2000). Results from the evaluation of these two media have been reported previously (Finney et al., 2003). In spite of their advantages, the main drawback of chromogenic media is the relatively high cost for routine analysis laboratories where a great number of analysis are performed (Yáñez et al., 2006). The results obtained with the combination of the two media, Tergitol agar and TBX agar, for the enumeration of total coliforms and E. coli have been comparable to those obtained with the individual chromogenic media assayed, CC agar and coli ID (Yáñez et al., 2006). However, the combined method has the advantage of reduced analytical cost since the TBX agar is used only for total coliform-positive samples (Yáñez et al., 2006). In addition, using

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this small modification, the enumeration of E. coli in total coliforms-positive samples can be performed in only two hours, maintaining the advantages of rapid turn-around time and the specificity of the individual chromogenic media (Yáñez et al., 2006). The combination of Tergitol agar with TBX fulfils the European Drinking Water Directive where the ISO Standard 9308 is indicated for the analysis of total coliforms and E. coli (Yáñez et al., 2006). Moreover, this small modification provides a simple and comparatively cost-effective method that is as specific and rapid as the chromogenic media, and which can be used easily by laboratories dedicated to routine analysis of water (Yáñez et al., 2006).

RAPID DETECTION METHODS Many bacterial pathogens are routinely grown on selective/differential agar or broth, e.g. Shigella (Niyogi et al., 2005). Infection with E. coli O157:H7 is diagnosed by detecting the bacterium from stool samples (CDC, 2007b). About one-third of laboratories that culture stool still do not test for E. coli O157:H7, so it is important to request that the stool specimen be tested on sorbitol-MacConkey (SMAC) agar for this organism (CDC, 2007b). All individuals presenting with bloody diarrhoea should have their stool tested for E. coli O157:H7 (CDC, 2007b). However, a major limitation of enrichment methods is the length of time required to complete the testing, since at least two days are needed for the complete identification of bacterial typical colonies (Yáñez et al., 2006). To overcome this problem, new techniques are continually being developed, and in particular, molecular biology methods appear to be interesting alternatives for rapidly detecting pathogens in water samples (Yáñez et al., 2006). To maximise the useful impact of epidemiological data, it is important to be able to rapidly detect and identify low numbers of oocysts from different sample types, including water, and if possible to ascertain if they are viable (Sunnotel et al., 2006a; Sunnotel et al., 2006b). It is important to be able to accurately differentiate between nonpathogenic and pathogenic species of Cryptosporidium, and between different Cryptosporidium isolates of the same species (Sunnotel et al., 2006a; Sunnotel et al., 2006b). Also, the rapid and accurate detection and identification of E. coli O157:H7 and V. cholerae O139 pathogens is crucial for diagnosis, treatment and eventual control of the contagious disease outbreak (Jin et al., 2007). Accurate and reliable epidemiological and molecular typing techniques are crucial for tracing sources of pathogen infection, the recognition of which is important for the implementation of preventive measures (Fendukly et al., 2007). Methods include ribotyping, amplified fragment length polymorphism analysis (AFLP), pulsed field gel electrophoresis (PFGE), restriction fragment length polymorphism analysis (RFLP), restriction endonuclease analysis (REA), and arbitrary primed PCR (Fendukly et al., 2007). Nevertheless, molecular techniques are unfortunately still incompatible with most routine water laboratories because of the expense and the need for trained personnel (Angles d'Auriac et al., 2000). Disappointingly, despite the steady improvement in modern molecular biology techniques, the epidemiology of many infections remains unclear (Snelling et al., 2005b). This confusing epidemiological evidence is partly because of the lack of standard global typing methods and communication between laboratories (Wassenaar and Newell 2000). However, these

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 27 problems are being addressed via initiatives like CAMPYNET (Wassenaar and Newell 2000) and PulseNet for standard molecular typing. Nucleic acid based detection methods such as PCR and real-time PCR are more rapid and sensitive than traditional culturing techniques. However, they are limited by the number of targets that can be detected in a single assay. PCR products can be multiplexed, but typically not more than six targets can be assayed at a single time, and optimizing such a complex reaction can be challenging. For example, primers must not interact with one another to form primer-dimers, and amplification of each target must proceed with equal efficiency. Analysis of the products of the reaction requires size separation by electrophoresis, thus amplicons must be of significantly different size in order to be easily distinguished. Real-time PCR (qPCR) is more rapid as it monitors the amount of PCR products as they are being produced and does not require size separation (Kubista et al. 2006; Zhang and Fang, 2006). In addition qPCR is more sensitive than traditional PCR methods and can estimate the initial concentration of nucleic acids. As with standard PCR methods, multiplexing qPCR is limited by the difficulty in optimizing reactions so that all targets are amplified with similar efficiencies. Moreover, the number of assays that can be multiplexed is limited by the number of fluorescent dyes that can be distinguished in a single reaction (typically no more than 4, depending on the specifications of the instrument being used). Saker et al., (2007) found that PCR can be used to detect inocula for cyanobacterial populations and therefore provides a useful tool for assessing which conditions particular species can grow into bloom populations. PCR methods were particularly useful when the concentration of the target organism was very low compared with other organisms (Saker et al., 2007). The European Working Group for Legionella Infections (EWGLI) has implemented the AFLP and more recently the sequence-based typing (SBT) to define its collection of L. pneumophila serogroup 1 strains (Fendukly et al., 2007). However, the SBT has not been widely used for genotyping non-serogroup 1 L. pneumophila isolates (Fendukly et al., 2007). Of the many phenotypical, immunological and molecular typing methods which have been implemented for the characterization and epidemiological typing of L. pneumophila, only the AFLP and the SBT techniques have gained acceptance by the EWGLI. Both methods were shown to have good reproducibility and accuracy and obviated the need to submit Legionella strains between microbiological laboratories in different countries (Fendukly et al., 2007). The AFLP gel images were, however, more difficult to interpret (Fendukly et al., 2007). This method allows for comparison of the allelic profile of an isolate to previously assigned allele numbers of 6 genes in the following order: flaA, pilE, asd, mip, mompS and proA (Fendukly et al., 2007). Some enteric viruses grow poorly in cell culture, which constitutes a problem when investigating strategies for virus control and prevention (Atmar and Estes, 2001; De Paula et al., 2007). Current methods of detecting such viruses in environmental water samples rely on genome amplification using molecular techniques such as qualitative and quantitative realtime reverse-transcription polymerase chain reaction (RT–PCR) (De Paula et al., 2007). Worldwide, virus detection in environmental and potable water samples is becoming an important strategy for preventing outbreaks of infection with waterborne viruses, e.g. HAV (De Paula et al., 2007). To evaluate the public health threat posed by V. cholerae occurring in water, a rapid and accurate method for detection of toxigenic V. cholerae is essential (Chomvarin et al., 2007).

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The culture method (CM), which is routinely used for assessing water quality, has not proven as efficient as molecular methods because this notorious pathogen survives in water mostly in the viable but non-culturable state (VBNC) (Chomvarin et al., 2007). The isolation and identification of V. cholerae by CM is expensive, time-consuming, labour intensive, and unable to precisely distinguish between toxigenic V. cholerae and non-toxigenic V. cholerae (Chomvarin et al., 2007). Fluorescent monoclonal antibody combined with molecular genetic based methods can demonstrate the presence of toxigenic V. cholerae in the aquatic environment (Chomvarin et al., 2007). The last three decades have witnessed exponential progress in our understanding of the biology of pathogens. The complete sequencing of the human genome and of many microbial pathogens associated with potable water have rapidly fuelled the development of gene array technology, improved pathogen detection systems and analysis methods. A host of different molecular techniques for studying the transmission dynamics and detection of drug resistant pathogens have been developed (Katoch et al., 2007). DNA microarrays (or gene arrays) consist of segments of DNA called probes or reporters arrayed on a solid surface (Call, 2005; Lemarchand, 2004). DNA spots are typically very small, on the micrometer length scale, and can be arrayed in high density with hundreds to thousands of spots present on a single array. Detection using microarrays is based on the hybridization of fluorescently labelled nucleic acids (RNA or DNA) isolated from the test matrix to these probes, and subsequent measurement of fluorescence at each spot. Microarrays allow the simultaneous detection of thousands of targets in a single assay. Thus, a single array could be used for the detection of a wide range of pathogens, virulent strains of a particular pathogen, antimicrobial resistant strains, and even molecular typing. Microarrays have been applied to microbial quality monitoring of water (Lee et al., 2006; Lemarchand et al., 2004; Maynard et al., 2005). However, methods involving direct hybridization onto microarrays are inherently lower in sensitivity relative to PCR because of the lack of oligonucleotide amplification. For example, Lee et al. (2006) were only able to detect pathogens in wastewater at levels of 2 x 108 copies of the target gene, using direct hybridization of DNA isolated from wastewater. Straub et al. (2005) have developed a microarray-based system for analysis of RNA isolated from microbial communities. This system shows great promise, as detection of RNA could be linked to the presence of viable cells; however, the sensitivity of this system is currently too low to be useful for pathogen detection. Sensitivity can be greatly improved by PCR amplification of the target, before hybridization to the array. However, production of multiple PCR products can be labour intensive and slow. Amplification of universal genes (i.e. rRNA genes) gets around this problem as a single primer set can be used to amplify a segment from a gene that is conserved among the pathogens of interest. Species-specific sequence variability within the amplicon is detected by hybridization of the PCR products to a microarray. Using this method, with the 23s rRNA gene, Lee et al. (2006) demonstrated an exponential increase in the sensitivity of their array to 20 copies of the target gene. Maynard et al. (2005) used a similar strategy, but improved discriminatory power of their array by the inclusion of multiple genes (cpn60, 16s rRNA and wecE ). They were able to detect 100 copies of a target gene, but this detection level was decreased when the genomic DNA of interest was in a background of complex DNA mixtures. PCR amplification favors the amplification of the most abundant bacterial species and low-level targets may be underrepresented, or undetectable on the array. This is

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 29 an important problem, as pathogens are likely to represent a small proportion of the microbial communities in water supplies. Kostic et al. (2007) were able to overcome this problem to a degree by using a unique labelling method (sequence-specific end-labelling of oligonucleotides) and inclusion of competitive oligonucleotides during labelling. They were able to detect pathogens which comprised 0.1% of the total microbial community analyzed. Wu et al. (2006) used a method of total DNA amplification (multiple displacement amplification) to non-specifically amplify total genomic DNA isolated from contaminated groundwater prior to microarray hybridization. This method seems promising, as it appears to eliminate the bias of PCR based amplification, while increasing the sensitivity of the microarray-based assay. Microarrays have proven to be useful tools for monitoring microbial communities in water and environmental samples. However, there are practical limitations in the use of microarray-based approaches including the requirement of specialized equipment and trained personnel. Automation of array systems would contribute significantly to the ease of deployment of these systems. Suspension arrays, such as the Luminex 100 system, are particularly amenable to automation (Baums et al., 2007; Gilbride et al., 2006). These arrays work on similar principles to planar microarrays, but they are rapid, cost effective, flexible and provide high reproducibility. In these systems, probes are immobilized onto solid surfaces of microspheres that are labelled with fluorophores (up to 100 colours are available) to facilitate their identification. After hybridization, the beads are analyzed in a flow cytometer. A red laser identifies the bead, and a green laser registers if a target has been captured. This system was found to be effective for detecting six different fecal indicating bacteria in environmental samples (Baums et al., 2007). Straub et al. (2005) have developed a comprehensive system for sample preparation and pathogen detection called ―BEADS‖ (biodetection enabling analyte delivery system). This system incorporates immunomagnetic separation to capture cells for concentration and purification of the analytes, a flow through thermal cycler and detection of PCR products on a suspension array. Using this system with river water, Straub and colleagues were able to detect 10 cfu of E. coli. When multiplexed with Shigella and Salmonella, sensitivity was reduced, but 100 cfu of each organism could be detected. Biosensor technologies offer the promise of portable devices delivering reliable results within short periods of time. Biosensors can be defined as analytical devices incorporating a biological material such as specific antibodies or DNA probes to confer specificity, integrated within a transducing microsystem (optical, electrochemical, thermometric, piezoelectric, magnetic or micromechanical) (Lazcka et al., 2007; Gilbride et al., 2006). Biosensors can be incorporated into miniaturized microfluidic devices commonly referred to as ―lab-on-a-chip‖ devices. Typical ―Lab-on-a-chip‖ devices contain wells for samples and storage of reagents, and microchannels for distribution of the samples and reagents to reaction chambers. They are connected to an instrument for control and detection of reactions by biosensors and to a computer for data analysis. The use of such devices reduces reagent costs, increases speed of analysis, minimizes handling steps, and provides portability, capability for parallel operations and the automation of complex assays. Several biological assays have been miniaturized and automated, including PCR, qPCR, DNA electrophoresis and microarrays, and immunoassays (Chen et al., 2007). A hand held device comprising electrochemical biosensors was successfully used to screen environmental water samples for 8 pathogens, within 3-5 h (LaGier et al., 2007). The detection limit of the device was equivalent to 10 cells of Karenia

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brevis when purified genomic DNA was assayed with the device. The scale of such devices, in terms of the number of targets that can be analyzed at a time, is still limited. The incorporation of high-density microarrays into integrated microfluidic devices containing biosensors has the potential to address the important issues of sensitivity and assay automation while maintaining the high information content of a microarray. Such systems may enable the reliable real-time monitoring of microbial communities, and detection of pathogens in water systems. While current devices show promise, there is a need to improve sensitivity, to improve specificity when dealing with complex samples, to provide portability and low cost in order to gain widespread use at water treatment facilities.

CONCLUSION In most developed countries, the removal or inactivation of the majority of pathogens, e.g. Cryptosporidium oocysts, can be accomplished by conventional water treatment technology, which generally includes flocculation, coagulation, sedimentation, filtration and chlorination (Betancourt and Rose 2004). Conventional treatment consists of coagulation/flocculation, sedimentation, and filtration (Sunnotel et al., 2006a). Implementation of multiple barriers to safeguard drinking water is recommended as pathogen loads vary during the season, with peak loads during early spring and late autumn (Ferguson et al. 2003). The high occurrence of many important human pathogens in surface water sources underlines the need for frequent monitoring of the parasite in drinking water (Frost et al. 2002). The high cost of waterborne disease outbreaks should be considered in decisions regarding water utility improvements and the construction of treatment plants (Morgan-Ryan et al. 2002). The integration of watershed and source water management and protection, scientific management of agricultural discharge and run-off, pathogen or indicator organism monitoring for source and treated water, outbreak and waterborne disease surveillance is needed to reduce waterborne transmission of human diseases (Ferguson et al. 2003). Water treatment facilities employing second-line treatment practices such as UV irradiation and ozone treatment can alleviate the danger imposed by chlorine resistant pathogens, e.g. Cryptosporidium (Keegan et al. 2003). However, outbreaks of waterborne illness can still occur in developed countries, because of malfunction or mismanagement of water treatment facilities (Ferguson et al. 2003). In these cases, hazard analyses protocols (microbiological hazards based on fecal coliforms (FC) and turbidity (TBY) as indicators) for critical control points (CCPs) within each facility may help to minimise the risk of contaminated water distribution in cases of system component failure, where CCPs include raw resource water, sedimentation, filtration and chlorinedisinfection (Jagals and Jagals 2004). Without knowing the occurrence of pathogens in water it is difficult to determine what risk they present to consumers of contaminated potable water (Karanis et al., 2007). Standardized methods are required to determine the occurrence of both established and emerging pathogens in raw untreated water abstracted for potable water, water treatment systems, potable water, and also recreational waters (Karanis et al., 2007). Also, because of the potential for pathogens to interact within protozoa and/or biofilms, the total populations of potable water must be assessed and monitored, e.g. how do populations vary with seasonality

Current and Emerging Microbiology Issues of Potable Water in Developed Countries 31 and from one country to another? An improved understanding of the microbial ecology of distribution systems is necessary to design innovative and effective control strategies that will ensure safe and high-quality drinking water (Berry, et al., 2006). Better education and increased awareness by the general public and pool operators could potentially reduce the number and impact of swimming pool and other recreational waterrelated outbreaks (Robertson et al. 2002a). Improved detection methods, with the ability to differentiate species, can be useful in the assessment of infection and identification of contamination sources. These will also provide vital data on the levels of disease burden due to zoonotic transmission (Fayer 2004; Sunnotel et al., 2006a). The roles of humans, livestock and wildlife in the transmission of microbial pathogens remain largely unclear for many different areas. The continued and improved monitoring (using appropriate molecular methods) of pathogens in surface water, livestock, wild life and humans will increase our knowledge of infection patterns and transmission of pathogens in potable water (Fayer 2004; Sunnotel et al., 2006a). For zoonotic pathogens which are spread both directly and indirectly to potable water, a wide array of interventions have been developed to reduce the carriage of foodborne pathogens in poultry and livestock, including genetic selection of animals resistant to colonisation, treatments to prevent vertical transmission of enteric pathogens, sanitation practices to prevent contamination on the farm and during transportation, elimination of pathogens from feed and water, additives that create an adverse environment for colonization by the pathogen, and biological treatments that directly or indirectly inactivate the pathogen within the host (Doyle and Erickson, 2006; Sunnotel et al., 2006a). To successfully reduce the carriage of foodborne pathogens, it is likely that a combination of intervention strategies will be required (Doyle and Erickson, 2006). Collaborative efforts are needed to decrease environmental contamination and improve the safety of produce. There is a very real need for studies that monitor the quality of the water as well as for policies and investments that focus on sanitation (De Paula et al., 2007). Unfortunately, in developed countries, the currently adopted short-term and blinkered strategy of simply detecting pathogens, e.g. a positive/negative Cryptosporidium result, without further species and isolate information impedes any significant or meaningful epidemiological analysis and/or effective implementation of intervention strategies. Because of the current necessity to minimise costs, current molecular methods (outlined previously) and future nanotechnology based methods are not being routinely employed, and thus may never fulfil their maximum potential. This situation demands significant and immediate attention and collaborative input from governments and water industries world-wide – resulting in a more concerted effort from both scientists and policy makers to standardise the global epidemiological response. Currently, due to the significant amount of work involved in sample collection and statistical analysis, there is a time lag of approximately 2-3 years in attaining up to date pathogen data (see CDC table 1). A global data base and collective goals for biological contaminant loading are vital for achieving safe potable water. More funding is needed to speed up this process, and to better educate the general public of the advantages of effective intervention strategies. Globally, rapid and accurate monitoring and species typing must be carried out routinely, not just when outbreaks occur. There is also a need for much more accurate and conclusive information on the overall microbial populations present in water and their relationships to one another symbiotic, parasitic or otherwise (Snelling et al., 2006). Once successfully

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introduced in developed countries, then and only then, can this template be effectively applied to the developing world. If successful the substantial economic cost (clinical costs and lost working hours) of water borne pathogens might, in time, be reimbursed through improved epidemiological data collection which, with proactive intervention, will dramatically reduce the health and economic burden of waterborne disease.

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In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 2

VERMICULTURE BIOTECHNOLOGY: THE EMERGING COST-EFFECTIVE AND SUSTAINABLE TECHNOLOGY OF THE 21ST CENTURY FOR WASTE AND LAND MANAGEMENT TO SAFE AND SUSTAINABLE FOOD PRODUCTION Rajiv K. Sinha1, Sunil Herat 2 , Gokul Bharambe 3, Swapnil Patil3, Uday Chaudhary3, Priyadarshan Bapat3, Ashish Brahambhatt3 David Ryan3, Dalsukh Valani3, Krunal Chauhan3, R. K. Suhane4 and P. K. Singh4 1

Visiting Senior Lecturer, School of Engineering (Environment), Griffith University, Nathan, Campus, Brisbane, QLD-4111, Australia 2 Senior Lecturer, School of Engineering (Environment), Griffith University 3 School of Engineering (Environment), Griffith University 4 Scientists, Rajendra Agriculture University, Bihar, India Keywords: Vermicomposting; Vermifiltration; Vermiremediation; Vermi-agro-production; Vermitreatment-Self-Promoted Odor-Free Process; Earthworm Gut – A Bioreactor; Earthworms – Reduce Greenhouse Gases; Earthworms Stimulate Microbial Degradation; Vermicompost – Rich in Minerals and Hormones; Vermifiltered Water - Chemicals and Pathogen Free and Nutritive.

1. INTRODUCTION A revolution is unfolding in vermiculture studies (rearing of useful earthworms species) for multiple uses in environmental management and sustainable development. (Martin, 1976; 

Principal and Corresponding Author: [email protected]

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Satchell, 1983; Bhawalkar and Bhawalkar, 1994; Sinha et.al 2002; Fraser-Quick, 2002). Vermiculture biotechnology promises to provide cheaper solutions to following environmental and social problems plaguing the civilization – 1) Management of municipal and industrial solid wastes (organics) by biodegradation and stabilization and converting them into useful resource (vermicompost) – ‗THE VERMI-COMPOSTING TECHNOLOGY‘ (VCT) ; 2) Treatment of municipal and some industrial (food processing industries) wastewater, purification and disinfection - ‗THE VERMI-FILTRATION TECHNOLOGY‘ (VFT); 3) Removing chemical contaminations from soils (land decontamination) and reducing soil salinity while improving soil properties- ‗THE VERMI-REMEDIATION TECHNOLOGY‘ (VRT); 4) Restoring and improving soil fertility and boosting crop productivity by worm activity and use of vermicompost (miracle growth promoter) while eliminating the use of destructive agro-chemicals - ‗THE VERMI-AGRO-PRODUCTION TECHNOLOGY‘ (VAPT); Vermi-composting, vermi-filtration, vermi-remediation and vermi-agro-production are self-promoted, self-regulated, self-improved and self-enhanced, low or no-energy requiring zero-waste technology, easy to construct, operate and maintain. It excels all ‗bio-conversion‘, ‗bio-degradation‘ and ‗bio-production‘ technologies by the fact that it can utilize organics that otherwise cannot be utilized by others. It excels all ‗bio-treatment‘ technologies because it achieves greater utilization than the rate of destruction achieved by other technologies. It involves about 100-1000 times higher ‗value addition‘ than other biological technologies. (Appeholf, 1997). About 4,400 different species of earthworms have been identified, and quite a few of them are versatile waste eaters and bio-degraders and several of them are bio-accumulators and bio-transformers of toxic chemicals from contaminated soils rendering the land fit for productive uses.

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2. EARTHWORMS: THE GREAT WASTE AND LAND MANAGERS ON EARTH The earthworms have over 600 million years of experience in waste and land management. No wonder then, Charles Darwin called them as the ‗unheralded soldiers of mankind‘ and ‗friends of farmers‘; and the Greek philosopher Aristotle called them as the ‗intestine of earth‘, meaning digesting a wide variety of organic materials including the waste organics, from earth. (Darwin and Seward, 1903).

Versatile Waste Eaters and Decomposers Earthworms are versatile waste eaters and decomposers. They feed lavishly on the organic waste, and also on the microorganisms (bacteria, fungi and the actinomycetes) that invade and colonize the waste biomass. Most earthworms consume, at the best, half their body weight of organics in the waste in a day. Eisenia fetida is reported to consume organic matter at the rate equal to their body weight every day.(ARRPET, 2005). Earthworm participation enhances natural biodegradation and decomposition of organic waste from 60 to 80 %. Study indicates that given the optimum conditions of temperature (20-30 C) and moisture (60-70 %), about 5 kg of worms (numbering approx.10,000) can vermiprocess 1 ton of waste into vermi-compost in just 30 days (ARRPET, 2005). Upon vermi-composting the volume of solid waste is significantly reduced from approximately 1 cum to 0.5 cum of vermi-compost. Earthworms can also treat and purify municipal wastewater (sewage) and also some industrial wastewater from the food processing industries significantly reducing the BOD and COD loads, the total dissolved and suspended solids (TDSS). It does this by the general mechanism of ‗biodegradation‘ of organics in the wastewater, ‗ingestion‘ of heavy metals and solids from wastewater and also by their ‗absorption‘ through body walls. What is more significant is that, there is no ‗sludge‘ formation in the process and the resulting vermifiltered water is clean, nutritive and disinfected enough to be reused for irrigation in farms, parks and gardens.

Bio-Accumulators of Toxic Soil Chemicals and Contaminants Earthworms have been found to bioaccumulate heavy metals, pesticides and lipophilic organic micropollutants like the polycyclic aromatic hydrocarbons (PAH) from the soil (Contreras-Ramos et. al, 2006). After the Seveso chemical plant explosion in 1976 in Italy, when vast inhabited area was contaminated with certain chemicals including the extremely toxic TCDD (2,3,7,8-tetrachlorodibenzo-p-dioxin) several fauna perished but for the earthworms that were alone able to survive. Earthworms which ingested TCDD contaminated soils were shown to bio-accumulate dioxin in their tissues and concentrate it on average 14.5 fold. (Satchell, 1983). E. fetida was used as the test organisms for different soil contaminants and several reports indicated that E. fetida tolerated 1.5 % crude oil (containing several toxic organic pollutants) and survived in this environment. (OECD, 2000; Safwat et al., 2002).

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Earthworms also tolerate high concentrations of heavy metals in the environment. The species Lumbricus terrestris was found to bio-accumulate in their tissues 90-180 mg lead (Pb) / gm of dry weight, while L. rubellus and D. rubida it was 2600 mg /gm and 7600 mg /gm of dry weight respectively.(Ireland, 1983).

Earthworm Species Suitable for Waste Management (Waste Biodegradation) Long-term researches into vermiculture have indicated that the Tiger Worm (Eisenia fetida), Red Tiger Worm (E. andrei), the Indian Blue Worm (Perionyx excavatus),the African Night Crawler (Eudrilus euginae),and the Red Worm (Lumbricus rubellus) are best suited for vermi-composting of variety of organic wastes and vermifiltration of both municipal and industrial wastewater under all climatic conditions. (Graff, 1981). E. fetida and E. andrei are closely related. Our study has indicated that E. fetida is most versatile waste eater and degrader and an army of the above 5 species combined together works meticulously.

Earthworm Species Suitable for Land Remediation (Soil Decontamination) Certain species of earthworms such as Eisenia fetida, Aporrectodea tuberculata, Lumbricus terrestris, L. rubellus, Dendrobaena rubida, D. veneta, Eiseniella tetraedra, Allobophora chlorotica have been found to tolerate and remove wide range of chemicals from soil. Our study also indicate that E. fetida is most versatile chemical bio-accumulators. (Ireland, 1983). Several of above species are common to India and Australia, both nations being biogeographically very close. (Sinha et. al,.2002).

3. THE BIOLOGY AND ECOLOGY OF EARTHWORMS Earthworms are long, narrow, cylindrical, bilaterally symmetrical, segmented animals without bones. The body is dark brown, glistening and covered with delicate cuticle. They weigh over 1400-1500 mg after 8-10 weeks. On an average, 2000 adult worms weigh 1 kg and one million worms weigh approximately 1 ton. Usually the life span of an earthworm is about 3 to 7 years depending upon the type of species and the ecological situation. Earthworms harbor millions of ‗nitrogen-fixing‘ and ‗decomposer microbes‘ in their gut. They have ‗chemoreceptors‘ which aid in search of food. Their body contains 65 % protein (70-80 % high quality ‗lysine rich protein‘ on a dry weight basis), 14 % fats, 14 % carbohydrates and 3 % ash. (Gerard, 1960; ARRPET, 2005).

Enormous Power of Reproduction and Rapid Rate of Multiplication Earthworms multiply very rapidly. They are bisexual animals and cross-fertilization occurs as a rule. After copulation the clitellum (a prominent band) of each worm eject lemon-

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shaped ‗cocoon‘ where sperms enter to fertilize the eggs. Up to 3 cocoons per worm per week are produced. From each cocoon about 10-12 tiny worms emerge. Studies indicate that they double their number at least every 60 days. Given the optimal conditions of moisture, temperature and feeding materials earthworms can multiply by 28 i.e. 256 worms every 6 months from a single individual. Each of the 256 worms multiplies in the same proportion to produce a huge biomass of worms in a short time. The total life-cycle of the worms is about 220 days. They produce 300-400 young ones within this life period. (Hand, 1988). A mature adult can attain reproductive capability within 8 – 12 weeks of hatching from the cocoon. Red worms takes only 4-6 weeks to become sexually mature. (ARREPT, 2005). Earthworms continue to grow throughout their life and the number of segments continuously proliferates from a growing zone just in front of the anus. Table 1. Reproductive Capacity of Some Environmentally Supportive Worms Species

E. fetida E. eugeniae P. excavatus D. veneta

Sexual maturity time (days) 53-76 32-95 28-56 57-86

No. of cocoon. 3.8 3.6 19.5 1.6

Cocoons hatching time (days) 32-73 13-27 16-21 40-126

Egg maturity days 85-149 43-122 44-71 97-214

% hatching 83.2 81. 90.7 81.2

No. of hatchlings 3.3 2.3 1.1 1.1

Net reproduction rate/week 10.4 6.7 19.4 1.4

Source : Edwards (1988).

Sensitive to Light, Touch and Dryness Earthworms are very sensitive to touch, light and dryness. They tend to migrate away from light. Cold (low temperature) is not a big problem for them as the heat (high temperature). Their activity is significantly slowed down in winter, but heat can kill them instantly. They temporarily migrate into deeper layers when subjected to too cold or too hot situations. It seems worms are not very sensitive to offensive smell as they love to live and feed on cattle dung and even sewage sludge. However, offensive smell can persist only for a short while in any environment where worms are active. They arrest all odour problems by killing anaerobes and pathogens that create foul odour.

4. VERMICULTURE : A GLOBAL MOVEMENT The movement was started in the middle of 20th century and the first serious experiments for management of municipal / industrial organic wastes were established in Holland in 1970, and subsequently in England, and Canada. Later vermiculture were followed in USA, Italy, Philippines, Thailand, China, Korea, Japan, Brazil, France, Australia and Israel (Edward,1988). However, the farmers all over the world have been using worms for composting their farm waste and improving farm soil fertility since long time. In UK, large 1000 mt vermi-composting plants have been erected in Wales (Frederickson, 2000). The American Earthworm Technology Company started a 'vermi-

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composting farm' in 1978-79 with 500 t /month of vermicompost production (Edward, 2000). Collier (1978) and Hartenstein and Bisesi (1989) reported on the management of sewage sludge and effluents from intensively housed livestock by vermiculture in USA. Japan imported 3000 mt of earthworms from the USA during the period 1985-87 for cellulose waste degradation (Kale,1998). The Aoka Sangyo Co. Ltd., has three 1000 t /month plants processing waste from paper pulp and the food industry (Kale,1998). This produces 400 ton of vermicompost and 10 ton of live earthworms per month. The Toyhira Seiden Kogyo Co. of Japan is using rice straw, municipal sludge, sawdust and paper waste for vermicomposting involving 20 plants which in total produces 2-3 thousands tons of vermicompost per month (Edward, 2000). In Italy, vermiculture is used to biodegrade municipal and paper mill sludge. Aerobic and anaerobic sludge are mixed and aerated for more than 15 days and in 5000 cum of sludge 5 kg of earthworms are added. In about 8 months the hazardous sludge is converted into nutritive vermicompost (Ceccanti and Masciandaro, 1999). In France, 20 tons of mixed household wastes are being vermi-composted everyday using 1000 to 2000 million red tiger worms (Elsenia andrei) in earthworm tanks. (ARRPET, 2005). Rideau Regional Hospital in Ontario, Canada, vermi-compost 375 - 400 kg of wet organics mainly food waste everyday. The worm feed is prepared by mixing shredded newspaper with the food waste. (ARRPET, 2005). In Wilson, North Carolina, U.S., more than 5 tons of pig manure (excreta) is being vermi-composted every week. (NCSU, 1997). In New Zealand, Envirofert is a large vermicomposting company operating in over 70 acre site in Auckland converting thousands of tons of green organic waste every year into high quality compost. (www.envirofert.co.nz). Almost all agricultural universities in India are now involved in vermiculture and a movement is going across the sub-continent especially involving the poor rural women with dual objectives of ‗making wealth (food and fertilizer) from waste and combating poverty‘ while ‗cleaning the environment and combating pollution of land and soil‘. Earthworms have enhanced the lives of poor in India. It has become good source of livelihood for many. (White, 1994; Hati, 2001). Vermiculture is being practiced and propagated on large scale in Australia too as a part of the 'Urban Agriculture Development Program' (to convert all the municipal urban wastes into compost for local food production) and ‗Diverting Waste from Landfills Program‘ (for reducing landfills in Australia).

5. THE VERMICOMPOSTING TECHNOLOGY (VCT) Vermicomposting is a rapid biological degradation process (aided by earthworms and soil microbes) in which several kinds of organic materials are converted from ‗unstable product‘ (which is likely to decompose further, creating objectionable odors, generating greenhouse gas methane and producing environmental insanitation) to an increasingly more ‗stable product‘ whose value is upgraded as nutritive materials for the soil and that can remain in the environment without creating any environmental problem. All organic wastes by its very nature (chemical composition) is bound to disintegrate anaerobically in environment and generate greenhouse gas methane (CH4). Only if they are allowed to degrade under aerobic conditions (which is readily facilitated by earthworms) that this can be prevented.

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5.1. Wastes Suitable for Vermicomposting Earthworms can physically handle a wide variety of organic wastes from both municipal (domestic and commercial) and industrial (livestock, food processing and paper industries) streams. Earthworms are highly adaptable to different types of organic wastes (even of industrial origin), provided, the physical structure, pH and the salt concentrations are not above the tolerance level. Another matter of considerable significance is that the earthworms also partially ‗detoxify‘ (by bio-accumulating any heavy metals and toxic chemicals) and ‗disinfect‘ (by devouring on pathogens and killing them by anti-bacterial coelomic fluid) the waste biomass while degrading them into vermi-compost which is nearly sterile and odorless. (Pierre et al., 1982).

Some Industrial Wastes Suitable for Vermi-Composting Solid waste including the ‗wastewater sludge‘ from paper pulp and cardboard industry, brewery and distillery, sericulture industry, vegetable oil factory, potato and corn chips manufacturing industry, sugarcane industry, aromatic oil extraction industry, logging and carpentry industry offers excellent feed material for vermi-composting by earthworms. (Kale, et al., 1993; Kale and Sunitha, 1995; Seenappa et al.,1995; Gunathilagraj and Ravignanam, 1996; Lakshmi and Vijayalakshmi, 2000; ARRPET, 2005.) Worms Can Even Vermicompost Human Excreta (Feces) Bajsa et al. (unpublished data) studied vermicomposting of human excreta (feces). It was completed in six months, with good physical texture meeting ARMANZ (1995) requirements, odourless and safe pathogen quality. Sawdust appeared to be the best covering material that can be used in vermicomposting toilets to produce compost with a good earthy smell, a crumbly texture and dark brown colour. There was no re-growth of pathogens on storing the compost for longer period of time and the initial pathogen load did not interfere in the die off process as the composting process itself seemed to stabilize the pathogen level in the system. Waste Materials Preferably to be Avoided for Vermi-composting 1) Heavily salted products unless soaked in water for 24 hours; 2) Excess citrus (orange and lemon peels and crushes) and onions wastes (might reduce pH and impair worm activity); 3) Feces of pets (may carry viral or bacterial toxins); 4) Fresh green grasses and foliages (green waste) creates high temperature and can harm worms; 5) All non-biodegradable wastes; 6) Meat and dairy products and slaughterhouse waste are to be avoided in the initial stage till the number of earthworms become high enough in the composting bed (it may invite ‗maggots‘ and also create bad odor for sometimes). However, Nair et al (2006) studied that thermophilic aerobic composting of ‗food waste‘ for 7 days followed by vermicomposting can eliminate the need for screening off of ‗citrus‘ and other acidic wastes like ‗onion peels‘ from vermicomposting. It comprises a short period

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of high temperature (around 55 C) followed by a period of lower temperature. This also leads to reduction of pathogens in much shorter time. (See table 9 below). The Envirofert, New Zealand, which is vermicomposting thousands of tons of green waste every year, also practice putting the green waste first to a lengthy thermophilic cooking, and then to vermicomposting by worms after cooling. Cooking of green waste also help destroy the weeds and pathogens which may come from the feces of pets in grasses. They claim that each worm eat the cooked green waste at least 8 times leaving an end product which is rich in key minerals, plant growth hormones, enzymes, and beneficial soil microbes. (www.envirofert.co.nz).

5.2. Vermicomposting of Municipal Solid Waste Millions of tons of municipal solid wastes (MSW) from homes and commercial institutions, cattle and farm wastes are generated in the human society and are mostly ending up in the landfills everyday, creating extraordinary economic and environmental problems for the local government to manage and monitor them (may be up to 30 years) for environmental safety (emission of greenhouse and toxic gases). Construction of engineered landfills incurs 20-25 million US dollars before the first load of waste is dumped.

5.3. Mechanism of Worm Action in Vermicomposting Worms Reinforce Microbial Population and Act Synergistically Promoting Rapid Waste Degradation Earthworms promotes the growth of ‗beneficial decomposer aerobic bacteria‘ in waste biomass and this they do by improving aeration by burrowing actions. They also act as an aerator, grinder, crusher, chemical degrader and a biological stimulator. (Dash, 1978; Binet et al., 1998 and Sinha et al., 2002). Earthworms hosts millions of decomposer (biodegrader) microbes in their gut (as they devour on them) and excrete them in soil along with nutrients nitrogen (N) and phosphorus (P) in their excreta (Singleton et al., 2003). The nutrients N and P are further used by the microbes for multiplication and vigorous action. Edward and Fletcher (1988) showed that the number of bacteria and ‗actinomycetes‘ contained in the ingested material increased up to 1000 fold while passing through the gut. A population of worms numbering about 15,000 will in turn foster a microbial population of billions of millions. (Morgan and Burrows, 1982). Singleton et al. (2003) studied the bacterial flora associated with the intestine and vermicasts of the earthworms and found species like Pseudomonas, Mucor, Paenibacillus, Azoarcus, Burkholderia, Spiroplasm, Acaligenes, and Acidobacterium which has potential to degrade several categories of organics. Acaligenes can even degrade PCBs and Mucor can degrade dieldrin. Under favorable conditions, earthworms and microorganisms act ‗symbiotically and synergistically‘ to accelerate and enhance the decomposition of the organic matter in the waste. It is the microorganisms which breaks down the cellulose in the food waste, grass clippings and the leaves from garden wastes. (Morgan and Burrows, 1982; Xing et al. 2005) Vermicomposting of organic waste involves following actions of worms –

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1) The waste feed materials ingested is finely ground (with the aid of stones in their muscular gizzard) into small particles to a size of 2-4 microns and passed on to the intestine for enzymatic actions. The gizzard and the intestine work as a ‗bioreactor‘; 2) The worms secrete enzymes proteases, lipases, amylases, cellulases and chitinases in their gizzard and intestine which bring about rapid biochemical conversion of the cellulosic and the proteinaceous materials in the waste organics. Earthworms convert cellulose into its food value faster than proteins and other carbohydrates. They ingest the cellulose, pass it through its intestine, adjust the pH of the digested (degraded) materials, cull the unwanted microorganisms, and then deposit the processed cellulosic materials mixed with minerals and microbes as aggregates called ‗vermicasts‘ in the soil. (Dash, 1978). 3) Only 5-10 percent of the chemically digested and ingested material is absorbed into the body and the rest is excreted out in the form of fine mucus coated granular aggregates - the ‗vermicasts‘ rich in nitrates, phosphates and potash. 4) The final process in vermi-processing and degradation of organic matter is the ‗humification‘ in which the large organic particles are converted into a complex amorphous colloid containing ‗phenolic‘ materials. Only about one-fourth of the organic matter is converted into humus. The colloidal humus acts as ‗slow release fertilizer‘. (ARRPET, 2005).

5.4. Experimental Studies on Vermicomposting a). Study of Biodegradation Abilities of Individual Worm Species This study was underataken by Sinha et al. (2002) at University of Rajasthan, Jaipur, India. About 150 mixed species of composting worms Eisinia fetida, Eudrilus euginae, and Perionyx excavatus were added to 1 kg each of buffalo dung, garden and kitchen wastes in different containers. In another three containers, each of these wastes and the three individual species of worms were used separately to assess their individual feeding and biodegradation abilities. (See table 2). Table 2. Vermicomposting of Cattle Dung, Kitchen Wastes and Garden Wastes by Individual Composting Worms Waste Materials 1 kg of each Cattle dung (Week old & semi-dry) Raw food wastes (Spinach stems, cauliflower & eggplant rejects) Garden wastes (Grasses & dry leaves)

Time Taken in Vermicomposting & Complete Degradation (in days) E. fetida E. euginee P. excavatus Mixed Species 59 44 62 45

78

61

83

70

89

69

91

80

Source: Sinha et al., (2002): The Environmentalist, UK.

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Findings and Results Results clearly showed that cattle dung is accepted quickly and composted rapidly by earthworms. Dung is partially degraded materials and the worms love to feed on them as they are rich in microbes. Raw food waste and garden wastes which contain cellulosic materials are accepted slowly as they are hard to degrade. Performance of Eudrilus euginae was best among the three species followed by Eisinia fetida. b). Study of Composting with Worms (Vermicomposting) and without Worms (Conventional Aerobic Composting) to Assess the Efficiency of Two Processes This study was made by Sinha et al. at University of Rajasthan, Jaipur, India on different cooked and raw kitchen wastes. In each container, 100 mixed species of composting worms (Eisinia fetida, Eudrilus euginiae and Perionyx excavatus) were added. A control pot was also organized for conventional aerobic composting without worms. Experiments were continued throughout the year in both summer, rainy and winter seasons to assess the effect of temperature variation, humidity and climate change on worm activity. (See table 3). Table 3. Biodegradation of Raw and Cooked Kitchen Wastes With and Without Worms

Source: Sinha et al., (2002) : The Environmentalist, UK. (Data is for warmer climate in India - MayJuly 1998: Temperature Min. 20C ; Max. 42C; Values in brackets are for cold periods Dec.1998 – Feb. 1999 : Temperature Min. 8 C; Max. 18C) Key: EP = Earthworms Present (Vermicomposting); EA = Earthworms Absent (Aerobic Composting) ; NA= Never Achieved Within the Period of Study.

Findings and Results Study confirmed beyond doubt that vermicomposting by worms is most efficient process (nearly 4-6 times faster) over aerobic composting which involves only microbial decomposition. Worms act both ways – carry out enzymatic degradation and also enhance microbial decomposition. Worm activity is at still greater threshold during warmer periods than in cold. c). Study of Vermicomposting of Raw and Cooked Food Wastes to Assess the Food Preferences of Earthworms and the Time Taken in Degradation of Each Food Component The study was made by Sinha et al. (2005) and Patil (2005) at Griffith University, Brisbane, Australia. Vermi-composting was carried out in a specially fabricated plastic ‗vermiculture bin‘ (Figure 1) sold in major hardware stores all over Australia. There is adequate provisions for aeration from side walls and top is open with cover lid on it. The

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vermicomposting bed was prepared with a thick layer of wet newspaper and about 4 inches of garden soil spread over it. About 250 numbers of earthworms were released in this soil bed. Food wastes were collected from homes and restaurants. About 100 gm of each of the food waste components were used. Mixed species of Elsenia fetida, Eudrilus eugeniae and Perionyx excavatus were used. Worms ranged between 3-6 cm in length and 0.1-0.2 cm in thickness. Moisture content was maintained at around 60 % by regular spray of water. Temperature within the bin was maintained at around 22-24 C as in the glass-house.

No of Days taken for complete Biodegradation

100 90 80 70 60 50 40 30 20 10 0 E. fetida,

E. euginae,

P. excavatus

Mixed Species

Earthworm Species Used Cattle dung

Kitchen w astes

Garden w astes

Figure 1. Time Taken To Complete Biodegradation of Different Food Waste by Various Earthworm Species.

A control VC bin was organized with all above features except for the earthworms to compare the rate of waste biodegradation without worms (but by aerobic process), odor problems etc., and to exactly determine the role and efficiency of earthworms in waste biodegradation. Results of vermicomposting have been given in tables 4 and 5. 120

16

% Reduction

12 80

10

60

8 6

40

4 20

2

Raw potato cuts & peels

Crushed egg shells

Cooked chicken, lamb & beef (curry)

Indian chapatti

Cooked potato, cauliflower etc. Raw rejects of cauliflower etc.

Baked potato, tomato etc.

Apple, pear, kiwi fruit etc.

Raw cabbage, lettuce etc.

Rice, noodles & pasta

0

Bread, buns & naans

0

Food Waste components % Biodegradation With Worms

% Biodegradation WithoutWorms

Figure 2. Rate of Biodegradation of Food Waste Components.

Weeks

Duration (Weeks)

14

100

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Findings and Results Worms can eat and degrade all food components including the cooked meat wastes (except the bones) conveniently according to their food preferences. They prefer softer ones first. But they are not comfortable with egg-shells, raw onions and raw potato rejects. Table 4. Rate of Biodegradation of Raw and Cooked Food Wastes With and Without Earthworms Under Identical Climatic Conditions (Food waste components about 100 gm each with 250 worms)

Source: Sinha et al. (2005) Keys: * 100 % degradation was achieved within the study period (Week 14) though with foul odor and heavy invasion of fungus; ** 100 % degradation could never be achieved, had odor problem and badly invaded by fungus (blue, black and green moulds) and had to be terminated; *** Terminated in week 4 because of maggots, heavy fungus and offensive smell;

Table 5. Rate of Biodegradation of Fried and Oily Food Wastes With and Without Earthworms Under Identical Climatic Conditions (Food waste used was about 9.5 kg with 500 worms)

Source: Patil (2005) Key: ND = Not detectable * Mild foul odor ** Highly offensive foul odor T= Terminated.

% Biodegradation

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100 90 80 70 60 50 40 30 20 10 0 2

4

6

8

10

12

14

Time (Weeks) % Biodegradation With Worms of Crushed Food

% Biodegradation With Worms of Intact Food

% Biodegradation Without Worms of Crushed Food

% Biodegradation Without Worms of Intact Food

Figure 3. Rate of Biodegradation of Fried and Oily Food Wastes With and Without Earthworms Under Identical Climatic Conditions

Figure 4. The Vermiculture Bin

Figure 5. Fried chicken nuggets and fish, calamari rings, potato fries.

Figure 6. Worm size before vermicomposting.

Figure 7. Worm size and health after vermicomposting.

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Figure 8. Food Waste in VC Bin (Half Crushed and Half Intact) on Day 1 (30.8.05).

Figure 9. Biodegraded (Vermicomposted) food waste on Day 29 after week 8 (26.10.05).

Findings and Results Worms slowly accept the fried greasy and oily foods although reluctantly in the beginning, and then more faster, after adapting with new food products. They prefer crushed food materials.

5.4. Vermicomposting of Sewage Sludge : Some Preliminary Studies Sludge is an inevitable hazardous and odorous byproduct from the conventional water and wastewater treatment plants which eventually require safe disposal either in landfills or by incineration incurring heavy cost. When the sludge is dewatered and dried they are termed as ‗biosolids‘. Management of the biosolids remains problematic due to the high cost of installing sewage sludge stabilization reactors and dehydration systems. Vermicomposting has been successfully used for treating and stabilizing municipal (water and wastewater treatment plants) as well as industrial (paper mill, dairy and textile industry) sludge, diverting them from ending up in the landfills. (Elvira et al. 1998; Lotzof, 1999; Ndegwa and Thompson 2001; Fraser-Quick, 2002; Contreras-Ramos et al., 2005). The quality of compost is significantly improved in terms of nutritional and storage value by worms. (Klein et al., 2005). The chemical and biological composition of sewage sludge is unpredictable as they may contain chemicals and pathogens from industry and agriculture. They are potential health hazard as they have been found to contain high numbers of cysts of protozoa, parasitic ova, fecal pathogens like Salmonella, Shigella and E. coli and also heavy metals such as zinc (Zn), cadmium (Cd), mercury (Hg) and copper (Cu). However, they also contains organics and essential plant nutrients like nitrogen (N), phosphorus (P), potassium (K) and various trace elements. Sludge is stabilized to reduce or eliminate pathogens and heavy metals, eliminate offensive odor, and reduce or eliminate the potential for putrefaction. Stabilized sewage sludge can become good source of organic fertilizer and soil additive for beneficial reuse in farms. (McCarthy, 2002). Earthworms feed readily upon the sludge components, rapidly convert them into vermicompost, reduce the pathogens to safe levels and ingest the heavy

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metals. Volume is significantly reduced from 1 cum of wet sludge (80 % moisture) to 0.5 cum of vermicompost (30 % moisture). (Eastman, 1999). Contreras-Ramos et al. (2005) studied the vermicomposting of biosolids (dried sewage sludge) from various industries but mainly from textile industries and some households (municipal) mixed with cow manure and oat straw. 1,800, 1,400 and 1000 gms of aerobically digested biosolids were mixed with 800, 500 and 200 gms of cow manure and 200, 100 and 0 (zero) gms of oat straw in triplicate set up. A control was also kept with only biosolids. Cow manure was added to provide additional nutrients and the oat straw to provide bulk. 50 earthworms (weighing 40 gm live weight) were added in each sample and the species used was Eisenia fetida. They were vermicomposted at three different moisture contents – 60 %, 70 % and 80 % for two months (60 days). The best results were obtained with 1,800 g biosolids mixed with 800 g of cow manure and no (0) straw at 70 % moisture content. Volatile solids of the vermicompost decreased by 5 times, heavy metals concentrations and pathogens (with no coliforms) were below the limits set by USEPA (1995) for an excellently stabilized biosolid. Carbon content decreased significantly due to mineralization of organic matter, and the number of earthworms increased by 1.2 fold. Vermiprocessing of sludge (biosolids) from the sewage and water treatment plants is being increasingly practiced in Australia and as a result it is saving large landfill spaces every year in Australia. The Redland Shire in Queensland started vermi-composting of sludge (biosolids) from sewage and water treatment plants with the aid of Vermitech Pty. Ltd. in 1997. The facility receives 400-500 tons of sludge every week with 17 % average solid contents and over 200 tons of vermicast is produced every week by vermicomposting. (Vermitech, 1998). The Hobart City Council in Tasmania, Australia, vermicompost and stabilize about 66 cum of municipal biosolids (from sewage sludge) every week, along with green mulch diverting them from landfills. Zeolite mixed with the biosolids helps balance the pH and also in absorbing ammonia and odor. About two-third of this volume (44 cum) becomes ‗vermicompost‘ which is sold to public. The City Council is saving AU $ 56,000 per year just from avoiding landfill disposal (transport and tipping fees etc.). They are earning an equal amount as revenue from the sale of vermi-compost. (Datar et al., 1997).

5.4.1. Mechanism of Worm Action in Vermicomposting of Sludge : Worms Act as Sludge Digester The basic mechanism of worm action is same as it is in the degradation and composting of general organic waste components of MSW. However, vermistabilization of sludge involves very complex mechanical, chemical and biological transformation processes and the resultant product has higher stabilization and soil supplement value than traditional composting that relies on mechanical incorporation of sludge with green waste in large compost heaps. It decompose the organics in the sludge, mineralise the nutrients, ingest the heavy metals and devour the pathogens (bacteria, fungus, nematodes and protozoa) found in sludge making them chemicals and pathogen free ready to be reused as soil additive and organic fertilizer. Essentially, it works as a ‗sludge digester‘ which is accomplished in the following steps1) The sludge is softened by the grume excreted in the ‗mouth‘ of the earthworms and from there it goes to the ‗esophagus‘;

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2) In the esophagus the softened sludge components is neutralized by calcium (Ca) (excreted by the inner walls of esophagus) and passed on to the gizzard and the intestine for further enzymatic action.

5.4.2. Experimental Study of Vermicomposting of Sewage Sludge Brahambhatt (2006) studied the vermicomposting of sewage sludge. The sludge was obtained from the Oxley Wastewater Treatment Plant in South Brisbane and earthworms were obtained from Bunnings hardware. It contained mixed species of Eisenia fetida, Perionyx excavatus and Eudrillus eugeniae. Cow dung was obtained from cattle farm in Ipswich. Both sewage sludge and the cow manure was partially air dried for 5 days to prevent any methane and hydrogen sulfide generation that might harm the worms. Vermicompost was obtained from our vermiculture lab in the School of Environmental Engineering. Five sets of experimental bins (40 litre HDPE containers) with biosolids were prepared with one as CONTROL and were studied for morphological (color and texture), biological (pathogens) and chemical (heavy metals) changes over the 12 weeks period. Table 6. Status of Coliforms in the Untreated (Control), Vermicomposted and Microbiologically Composted Biosolids After 12 Weeks

Source: Brahambhatt (2006). Key: VC = Vermicomposting; * Microbial Composting. % of stabilisation

% of stabilisation

100.00% 80.00% 60.00% 40.00% 20.00% 0.00% Biosolids (CONTROL)

VC Biosolids by Earthw orms

VC Biosolids *Composted by Biosolids by Earthw orms + Cow Dung Cow Dung

*Composted Biosolids by Organic Soil

Nature of samples tested

Figure 10. Status of Coliforms in the Untreated (Control), Vermicomposted and Conventionally Composted (By Microbial Degradation) Biosolids after 12 Weeks.

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Findings and Results Results clearly shows that the earthworms significantly reduce or almost eliminates the pathogens from the digested (composted) sludge. Sludge treated with earthworms (with or without feed materials) only showed ‗negative results‘ by the Colilert test under the UV lamp. And this was achieved in just 12 weeks. It also infers that under the microbial composting systems the pathogens remains in the sludge for longer time even after treatment until it is completely dry with all food and moisture exhausted making them difficult to survive. Table 7. Status of Heavy Metals Cadmium (Cd) and Lead (Pb) in the Untreated (Control), Vermicomposted and Microbiologically Composted Biosolids (mg/kg of soil) in 12 Weeks

Source: Brahambhatt (2006). Key: * Conventional Composting by Microbial Degradation; NC = No Change in Value.

90 80

2.5

70

2

60 50

1.5

40

1

30 20

0.5

Lead (mg/kg of soil)

Cadmium (mg/kg of soil)

3

10

0

0 Biosolids (CONTROL)

VC Biosolids VC Biosolids *Composted *Composted by by Biosolids by Biosolids by Earthw orms Earthw orms + Cow Dung Organic Soil Cow Dung

Nature of sample tested

Cadmium Lead

Figure 11. Status of Heavy Metals Cadmium (Cd) and Lead (Pb) in the Untreated (Control), Vermicomposted and Conventionally Composted Biosolids after 12 Weeks.

Findings and Results Results clearly show that the earthworms reduce the heavy metals cadmium (Cd) and lead (Pb) from the digested sludge. There was no change in the values of heavy metals between the untreated sludge (Bin 1) and those treated by adding cow dung and by organic soil (Bins 4 and 5) to enhance microbial composting. Although biosolids can be slowly

Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al.

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stabilized by microbial degradation over a period of time, the heavy metals will remain in the system for quite sometimes after which it may leach into soil or get bound with soil organics by chemical reactions occurring in the soil. % Reduction in Cadmium

% Reduction in lead

70%

% Reduction

60% 50% 40% 30% 20% 10% 0% Biosolids (CONTROL)

VC Biosolids VC Biosolids *Composted by Earthw orms by Earthw orms Biosolids by + Cow Dung Cow Dung

*Composted Biosolids by Organic Soil

Nature of sample te ste d

Figure 12. Percentage Reduction in Cadmium and Lead in the Untreated (Control), Vermicomposted and Conventionally Composted Biosolids after 12 Weeks.

Though there was not very significant removal of heavy metals by earthworms in the 12 weeks of experiment yet their role in heavy metal removal cannot be undermined. Providing additional feed materials (Bin 3) enhanced worm activity and also their number (stimulating reproduction) and led to greater removal of heavy metals. It infers that over a period of time and with enhanced worm activity the heavy metals can be completely removed from the biosolids.

5.5. Critical Factors Affecting Optimal Worm Activity and VermiComposting 1. Moderate Temperature: In general earthworms prefers and tolerates cold and moist conditions far better than the hot and dry ones. Most worms involved in vermicomposting require moderate temperature between 20 – 30  C. Heat causes more problems in vermi-composting than the cold. Red worms are reported to become inactive above 29 C. They are at the highest levels of both waste degradation and reproduction activity as the weather cools in the fall and warms in the spring. 2. Adequate Moisture: Earthworms requires plenty of moisture for growth and survival ranging from 60 to 70 %. The bed should not be too wet as it may create anaerobic condition adversely affecting worm activity. 3. Adequate Aeration: Vermi-composting is an aerobic process and adequate flow of air in the waste biomass is essential for worm function. Worms breath through their skin and need plenty of oxygen in the surrounding areas. Although worms constantly aerate their habitat by burrowing actions, periodical turning of waste biomass can improve aeration and biodegradation.

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4. Neutral pH (7.0): Earthworms are sensitive to pH. The decomposition of organic matter produces ‗organic acids‘ that lower the pH of the bedding soil and impair worm activity. Although the worms can survive in a pH range of 4.5 to 9 but functions best at neutral pH of 7.0. Although worms can lower pH of its medium by secreting calcium (Ca), it is suggestive to add ground limestone (calcium carbonate) powder to the bedding periodically. This would serve two purposes- maintain neutral pH and also supply the much needed calcium (Ca) to the worms for its metabolism. 5. Carbon / Nitrogen (C/N) Ratio of the Feed Material: High C/N ratio above 30:1 in waste biomass can impair worm activity and vermi-composting. Although earthworms help to lower the C/N ratio of fresh organic waste, it is advisable to add nitrogen supplements such as cattle dung or pig and goat manure or even kitchen waste (which are rich in nitrogen contents) when waste materials of higher C/N ratio exceeding 40:1 such as the green cellulosic wastes (grass clippings) are used for vermi-composting. 6. Adequate Supply of Calcium (Ca): Calcium appears to be important mineral in worm biology (as calcarious tissues) and biodegradation activity. Although most organic waste contains calcium, it is important to add some additional sources of calcium for good vermi-composting. Egg shells are good source of natural calcium. Occasionally limestone powder should be added.

5.6. Advantages of Vermi-Composting Earthworms have real potential to both increase the rate of aerobic decomposition and composting of organic matter and also to stabilize the organic residues in the MSW and sludge – removing the harmful pathogens and heavy metals from the compost. The quality of compost is significantly better, rich in key minerals and beneficial soil microbes. In fact in the conventional aerobic composting process which is thermophilic (temperature rising up to 55 C) many beneficial microbes are killed and nutrient especially nitrogen is lost (due to gassing off of nitrogen). Vermicomposting by earthworms excels all other ‗bio-degradation‘ and ‗bio-conversion‘ technologies by the fact that - it can utilize waste organics that otherwise cannot be utilized by others; achieve greater utilization (rather than the destruction) of materials that cannot be achieved by others; and by the fact that it does all with ‗enzymatic actions‘ and enzymes are biological catalysts giving pace and rapidity to all biochemical reactions even in minute amounts. It also keeps the system fully aerated with plenty of oxygen available to aerobic decomposer microbes. Aerobic processes are about 10 times faster than the anaerobic.

1. Nearly Odor-free Process Earthworms create aerobic conditions in the waste materials by their burrowing actions, inhibiting the action of anaerobic micro-organisms which release foul-smelling hydrogen sulfide and mercaptans.

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2. Destroy Pathogens in the End Product (Compost) Making Them Pathogen Free The earthworms release coelomic fluids that have anti-bacterial properties and destroy all pathogens in the waste biomass. (Pierre et al.,1982). They also devour the protozoa, bacteria and fungus as food. They seems to realize instinctively that anaerobic bacteria and fungi are undesirable and so feed upon them preferentially, thus arresting their proliferation. More recently, Dr. Elaine Ingham has found in her research that worms living in pathogen-rich materials (e.g. sewage and sludge), when dissected, show no evidence of pathogens beyond 5 mm of their gut. This confirms that something inside the worms destroys the pathogens, and excreta (vermicast) becomes pathogen-free (Appelhof, 2003). In the intestine of earthworms some bacteria and fungus (Pencillium and Aspergillus) have also been found (Singelton et. al, 2003). They produce ‗antibiotics‘ and kills the pathogenic organisms in the sewage sludge making it virtually sterile. The removal of pathogens, faecal coliforms (E.coli), Salmonella spp., enteric viruses and helminth ova from sewage and sludge appear to be much more rapid when they are processed by E. fetida. Of all E.coli and Salmonella are greatly reduced. (Bajsa et al., 2003). Bajsa et al. (2004 and 2005) studied the pathogen die-off in vermicomposting of sewage sludge spiked with E.coli, S.typhimurium and E.faecalis at the 1.6-5.4 x 106 CFU/g , 7.25 x 105 CFU/g and3-4 x 10 4 CFU/g respectively. The composting was done with different bulking materials such as lawn clippings, sawdust, sand and sludge alone for a total period of 9 months to test the pathogen safety of the product for handling. It was observed that a safe product was achieved in 4-5 months of vermicomposting and the product remained the same quality without much reappearance of pathogens after in the remaining months of the test. Eastman (1999) and Eastman et. al., (2001) also studied significant human pathogen reduction in biosolids vermicomposted by earthworms. Lotzof (2000) also revealed that the pathogens like enteric viruses, parasitic eggs and E.coli were reduced to safe levels in sludge vermicast. Cardoso and Remirez (2002) reported a 90 % removal of fecal coliforms and 100 % removal of heliminths from sewage sludge and water hyacinth after vermicomposting. Contreras-Ramos et, al., (2005) also confirmed that the earthworms reduced the population of Salmonella spp. to less than 3 CFU/gm of vermicomposted sludge. There were no fecal coliforms and Shigella spp. and no eggs of helminths in the treated sludge. (Eastman et al.,2001; Kumar and Sekaran, 2004). Our studies, Brahmbhatt (2006) has also confirmed complete removal of coliforms by earthworms. In conventional aerobic composting system, which is ‗thermophilic‘ process, pathogens are killed due to high temperature (beyond 55 C). Wu and Smith (1999) studied that for efficient composting and pathogen reduction a temperature of 55 C must be maintained for 15 consecutive days. But this also kills several beneficial microbes. If this high temperature is not achieved which could be the case in small scale composting pathogen die off will not be effective. Vermicomposting is a ‗mesophilic composting‘ system where temperature does not increase beyond 30 C and the harmful microbes (pathogens) are killed selectively by the worms through biological (physiological), microbial and enzymatic actions. Nair et al. (2006) studied a combination of thermophilic followed by vermicomposting and observed that the combination of the process leads to faster reduction of pathogens than the same period of thermophilic composting (21 days) and after 2 and 3 months (Table 9). It was also noticed that 21 days of a combination of thermocomposting and vermicomposting produced compost

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with acceptable C:N ratio and good homogenous consistency of a fertilizer. However the E. coli and E. faecalis was greater than 110 MPN/g) while S. typhimurium was undetectable. The optimum period to obtain pathogen-free compost was 9 days of thermophilic composting followed by 2.5 months of vermicomposting (Nair et. al., 2006). The study also indicated that vermicomposting leads to greater reduction of pathogens after 3 months upon storage. Whereas, the samples which were subjected to only thermofilic composting, retained higher levels of pathogens even after 3 months. Table 8. Removal of Pathogens (E. coli and E. faecalis) in Thermophilic Composting Vis-a-Vis Vermicomposting Processes

Source: Nair et. al., (2006) Key: dT = days of Thermophilic composting; dV = days of Vermicomposting N.B. E.coli and E. faecalis was tested using the Most Probable Number (MPN) per gram of compost (Standards Australia, 1995 a and b respectively).

3. Vermicompost Is Free of Toxic Chemicals Several studies have found that earthworms effectively bio-accumulate or biodegrade several organic and inorganic chemicals including ‗heavy metals‘, ‗organochlorine pesticide‘ and ‗polycyclic aromatic hydrocarbons‘ (PAHs) residues in the medium in which it feeds. (Nelson et al., 1982; Ireland, 1983). 4. Low Greenhouse Gas Emissions by Vermi-composting of Waste Emission of greenhouse gases carbon dioxide (CO2), methane (CH4) and nitrous oxide (N2O) in waste management programs of both garbage and sewage has become a major global issue today in the wake of increasing visible impacts of global warming. Biodegradation of organic waste has long been known to generate methane (CH4). Studies have also indicated high emissions of nitrous oxide (N2O) in proportion to the amount of food waste used, and methane (CH4) is also emitted if the composting piles contain cattle manure. N2O emission is relatively high at the beginning of the conventional aerobic composting process, but declines after two days. N2O is mainly formed under moderate oxygen (O2) concentration. High emission of N2O at the beginning might be due to the metabolism of the microbial community coming from food waste, as food waste have been found to generate N2O even stored at 40 C. (Toms et, al. 1995; Wu et, al. 1995; Wang et, al., 1997; and Yaowu et. al., 2000). In theory, vermicomposting by worms should provide some potentially significant advantages over conventional composting with respect to GHG emissions. Worms significantly increase the proportion of ‗aerobic to anaerobic decomposition‘ in the compost

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pile by burrowing and aerating actions leaving very few anaerobic areas in the pile, and thus resulting in a significant decrease in methane (CH4) and also volatile sulfur compounds which are readily emitted from the conventional (microbial) composting process.(Mitchell et al., 1980). Molecule to molecule, methane is 20-25 times more powerful GHG than the CO2. Earthworms can play a good part in the strategy of greenhouse gas reduction and mitigation in the disposal of global organic wastes as landfills also emit methane resulting from the slow anaerobic decomposition of waste organics over several years. Recent researches done in Germany has found that earthworms produce a third of nitrous oxide (N2O) gases when used for vermicomposting (Frederickson, 2007). Molecule to molecule N2O is 296 to 310 times more powerful GHG than carbon dioxide (CO2). However, analysis of vermicompost samples has shown generally higher levels of available nitrogen (N) as compared to the conventional compost samples made from similar feedstock. This implies that the vermicomposting process by worms is more efficient at retaining nitrogen (N) rather than releasing it as N2O. This needs further studies and is being done at Griffith University, Brisbane, Australia (Middleditch, 2008).

5. More Homogenous End Products Rich in Nutrient The advantages for employing vermi-composting to process and stabilize organic waste including sewage sludge over conventional composting by microbial action are that the end product are more homogenous, rich in plant nutrients and the levels of contaminants are significantly reduced. Lotzof, 1999). McCarthy (2002) asserts that vermicomposting of sewage sludge has converted them into an end product that is safe for agricultural use. Vermicompost has very ‗high porosity‘, ‗aeration‘, ‗drainage‘ and ‗water holding capacity‘ and also contains ‗plant-available nutrients‘. The resulting product appears to retain more nutrients for longer period of time and also greatly increases the water holding capacity of the farm soil (Hartenstein and Hartenstein, 1981; Appelhof, 1997). Table 9. Initial and Final Nutritional Quality of the Vermicomposted Sludge

Source: Bajsa et al., 2005 Key: SL –Sludge; LC – Lawn clippings; SD – Sawdust; S – Sand

The chemical analyses of casts showed two times more available magnesium, 15 times more available nitrogen and seven times more available potassium compared to the surrounding soil (Kaviraj and Sharma, 2003). This is an excellent biofertilizer and soil conditioner (Jensen, 1998).

6. No or Low Energy Use in Vermi-composting Process Conventional microbial composting requires energy for aeration (constant turning of waste biomass and even for mechanical airflow) and sometimes for mechanical crushing of

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waste to achieve uniform particle size. Vermi-composting do not involve such use of energy. Occasionally turning may be required for aeration which can be done manually.

7. Production of Earthworm Biomass : A Nutritive Earthworm Meal Large scale vermi-composting of sludges would result into tons of earthworms biomass every year as under favorable conditions earthworms can ‗double‘ their number at least every 60 – 70 days. Potentially large quantities of worm biomass will be available as ‗pro-biotic‘ food for the cattle and fish farming, after the first year of composting. This can be a good source of nutritive ‗worm meal‘ rich in essential amino acids ‗lysine‘ and ‗methionine‘. It is superior to even ‗fish meal‘. 8. Mineralization of Nutrients from the Sludge Earthworms accelerates the decomposition of the sludge and mineralization of the organic compounds in it. Most important is that earthworms mineralize the nitrogen (N) and phosphorus (P) in the sludge to make it bio-available to plants as nutrients. They ingest nitrogen from the sludge and excrete it in the mineral form as ammonium and muco-proteins. The ammonium in the soil is bio-transformed into nitrates. Phosphorus (P) contents increased in the vermicomposted sludge treated with earthworms but decreased in the samples without earthworms, while the nitrogen (N) content did not show much difference (Parvaresh, et. al., 2004). Elvira et, al,. (1998) reported increase in the potassium (K) content of the sludge vermicompost. 9. Decrease in Total Organic Carbon (TOC) and Lowering of C/N Ratio of Sludge This has significance when the composted sludge is added to soil as fertilizer. Plants cannot absorb and assimilate mineral nitrogen unless the carbon to nitrogen (C/N) ratio is about 20:1 or lower. Mineralization of organic matter in the sewage sludge by earthworms lead to significant decrease in total organic carbon (TOC) content thus lowering the C/N ratio. This they do by consuming and breaking carbon compounds during respiration. Elvira et al., (1998) found that vermicomposting of paper-pulp-mill sewage sludge for 40 days decreased carbon (C) content by 1.7 fold. Contreras-Ramos et al., (2005) found that carbon content decreased by 1.1 to 1.4 fold in two months. Our work (Brahambhatt, 2006) also indicated that earthworms reduce TOC from composted sludge. 10. Reduction in Volatile Solids (VS) from Sludge Maximum reduction of the volatile solids (VS) is the goal of any sludge stabilization system and reduced VS is an indicator of stabilized sludge. Study found that Elsenia fetida increases the rate of volatile solid sludge (VSS) destruction when present in aerobic sludge and this reduces the probability of putrefaction occurring in the sludge due to anaerobic conditions. (Loehr, et al,1998). Earthworms creates and maintains good aerobic conditions in the sludge due to its burrowing actions and this enhances the process of VSS destruction. Hartenstein and Hartenstein (1981) reported a 9 % reduction in volatile solids over 4 weeks of sludge vermicomposting by earthworms, which was higher than that of control by almost one-third. Fredrickson et al., (1997) found a VS reduction of 30 % in compost after 4 months of conventional composting, whereas, the reduction was 37 % after only 2 months of

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vermicomposting. Higher decrease means more stable product and earthworms plays important role.

6. THE VERMIFILTRATION TECHNOLOGY (VFT) Vermifiltration of wastewater using waste eater earthworms is a newly conceived novel technology with several advantages over the conventional systems. Earthworms body work as a ‗biofilter‘ and they have been found to remove the 5 days BOD (BOD5) by over 90 %, COD by 80-90 %, total dissolved solids (TDS) by 90-92 % and the total suspended solids (TSS) by 90-95 % from wastewater by the general mechanism of ‗ingestion‘ and biodegradation of organic wastes, heavy metals and solids from wastewater and also by their ‗absorption‘ through body walls. Suspended solids are trapped on top of the vermifilter and processed by earthworms and fed to the soil microbes immobilized in the vermifilter. The most significant part is that there is ‗no sludge formation‘ in the process as the worms eat the solids simultaneously and excrete them as vermicast. (Huges et. al., 2005). This plagues most councils in world as the sludge being a biohazard requires additional expenditure on safe landfill disposal. This is also an odor-free process and the resulting vermi-filtered water is clean enough to be reused in industries as cooling water and also highly nutritive to be reused for farm irrigation.

6.1. Vermifiltration of Municipal Wastewater (Sewage) Sewage is the cloudy fluid of human fecal matter and urine, rich in minerals and organic substances. Water is the major component (99 %) and solid suspension amounts to only 1 %. The biochemical oxygen demand (BOD) and oxygen consumption (OC) values are extremely high demanding more oxygen by aerobic microbes for biodegradation of organic matter. Dissolved oxygen (DO) is greatly depleted. Low oxygen in water leads to anaerobic decomposition of organic contents and emission of obnoxious gases like hydrogen sulfide, methane and carbon monoxide. The nitrogen (N) and phosphorus (P) contents are very high and there are heavy metals like cadmium (Cd) and significant amount of coliform bacteria. The total suspended solids (TSS) is also very high like the BOD and nutrients and this often leads to a high anaerobic microenvironment in the sediments.

BOD, COD and SS Values of Raw Sewage and the Acceptable Values of Treated Sewage The average BOD value of the raw sewage ranged between 200 – 400 mg/L, COD ranged between 116 -285 mg/L, the TSS ranged between 300 – 350 mg/L and the pH ranged between 6.9 – 7.3. There is great fluctuation in these values depending upon catchment area, flow rate and season. Sewage from industrial areas can have high COD values, very low or high pH, due to accidental mixing of industrial wastewater. The normal acceptable values for BOD in treated wastewater is 1-15 mg/L, COD is 60-70 mg/L, TSS 20-30 mg/L and pH around 7.0.

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6.2. The Mechanism of Worm Action in Vermifiltration 1) The twin processes of microbial stimulation and biodegradation, and the enzymatic degradation of waste solids by worms as discussed above simultaneously work in the vermifiltration system too. 2) Vermifilters provide a high specific area – up to 800 sq m/g and voidage up to 60 %. Suspended solids are trapped on top of the vermifilter and processed by earthworms and fed to the soil microbes immobilized in the vermifilter. 3) Dissolved and suspended organic and inorganic solids are trapped by adsorption and stabilized through complex biodegradation processes that take place in the ‗living soil‘ inhabited by earthworms and the aerobic microbes. Intensification of soil processes and aeration by the earthworms enable the soil stabilization and filtration system to become effective and smaller in size. 4) Earthworms intensify the organic loadings of wastewater in the vermifilter soil bed by the fact that it granulates the clay particles thus increasing the ‗hydraulic conductivity‘ of the system. They also grind the silt and sand particles, thus giving high total specific surface area, which enhances the ability to ‗adsorb‘ the organics and inorganic from the wastewater passing through it. The vermicast produced on the soil bed also offers excellent hydraulic conductivity of sand (being porous like sand) and also high adsorption power of clay. This is ideal for diluted wastewater like sewage. (Bhawalkar, 1995). 5) Earthworms also grazes on the surplus harmful and ineffective microbes in the wastewater selectively, prevent choking of the medium and maintain a culture of effective biodegrader microbes to function.

6.3. Critical Factors Affecting Vermifiltration of Wastewater 1. Worm Population and Density (Biomass) in Vermifilter Bed (Soil) As the earthworms play the critical role in wastewater purification their number and population density (biomass) in soil, maturity and health are important factors. This may range from several hundreds to several thousands. There are reports about 8-10,000 numbers of worms per square meter of the worm bed and in quantity (biomass) as 10 kg per cubic meter (cum) of soil for optimal function. 2. Hydraulic Retention Time (HRT) Hydraulic retention time is the time taken by the wastewater to flow through the soil profile (vermifilter bed) in which earthworms inhabits. It is very essential for the wastewater to remain in the vermifiltration (VF) system and be in contact with the worms for certain period of time. HRT depends on the flow rate of wastewater to the vermifiltration unit, volume of soil profile and quality of soil used. HRT is very critical, because this is the actual time spent by earthworms with wastewater to retrieve organic mater from it as food. During this earthworms carry out the physical and biochemical process to remove nutrients, ultimately reducing BOD, COD and the TDSS. The longer wastewater remains in the system in contact with earthworms, the greater will be the efficiency of vermi-processing and

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retention of nutrients. Hence the flow of wastewater in the system is an important consideration as it determines the retention of suspended organic matter and solids (along with the chemicals adsorbed to sediment particles). Maximum HRT can results from ‗slower rate of wastewater discharge‘ on the soil profile (vermifilter bed) and hence slower percolation into the bed. Increasing the volume of soil profile can also increase the HRT. The number of live adult worms, functioning per unit area in the vermifilter (VF) bed can also influence HRT. HRT of vermifiltration system can be calculated as – HRT = (ρ x Vs) / Q wastewater where; HRT = theoretical hydraulic retention time (hours) Vs = volume of the soil profile (vermifilter bed), through which the wastewater flow and which have live earthworms (cum) ρ = porosity of the entire medium (gravel, sand and soil) through which wastewater flows Q wastewater = flow rate of wastewater through the vermifilter bed (cum / hr) Thus the hydraulic retention time (HRT) is directly proportion to the volume of soil profile and inversely proportion to the rate of flow of wastewater in the vermifilter bed.

3. Hydraulic Loading Rate (HLR) The volume and amount of wastewater that a given vermifiltration (VF) system (measured in area and depth of the soil medium in the vermifilter bed in which the earthworms live) can reasonably treat in a given time is the hydraulic loading rate of the vermifilter (VF) system. HLR can thus be defined as the volume of wastewater applied, per unit area of soil profile (vermifilter bed) per unit time. It critically depends upon the number of live adult earthworms functioning per unit area in the vermifilter bed. The size and health of the worms is also critical for determining the HLR. HLR of vermi-filtration system can be calculated as – HLR = V wastewater / (A X t) whereLR = Hydraulic Loading Rate (m / hr) V wastewater = volumetric flow rate of wastewater (cum). A = Area of soil profile exposed (sqm). t = Time taken by the wastewater to flow through the soil profile (hr). High hydraulic loading rate leads to reduced hydraulic retention time (HRT) in soil and could reduce the treatment efficiency. Hydraulic loading rates will vary from soil to soil. The infiltration rates depend upon the soil characteristics defining pore sizes and pore size

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distribution, soil morphological characteristics, including texture, structure, bulk density, and clay mineralogy.

6.4. Some Preliminary Studies on Vermifiltration of Sewage A pilot study on vermifiltration of sewage was made by Xing et al,. (2005) at Shanghai Quyang Wastewater Treatment Facility in China. The earthworm bed which was 1m (long) x 1m (wide) x 1.6m (high), was composed of granular materials and earthworms. The worm‘s number was kept at about 8000 worms/sqm. The average chemical oxygen demand (COD) value of raw sewage used was 408.8 mg/L that of 5 days biological oxygen demand (BOD5) was 297 mg/L that of suspended solids (SS) was 186.5 mg/L. The hydraulic retention time varied from 6 to 9 hours and the hydraulic loading from 2.0 to 3.0 m3 / (m2.d) of sewage. The removal efficiency of COD ranged between 81-86 %, the BOD5 between 91-98 %, and the SS between 97-98 %. Gardner et. al.,(1997) studied on-site effluent treatment by earthworms and showed that it can reduce the BOD and COD loads significantly. Taylor et. al., (2003) studied the treatment of domestic wastewater using vermifilter beds and concluded that worms can reduce BOD and COD loads as well as the TDSS (total dissolved and suspended solids) significantly by more than 70-80 %. Hartenstein and Bisesi (1989) studied the use of earthworm for the management of effluents from intensively housed livestock which contain very heavy loads of BOD, TDSS and nutrients nitrogen (N) and phosphorus (P). The worms produced clean effluents and also nutrient rich vermicompost. Bajsa et. al., (2003) also studied the vermifiltraion of domestic wastewater using vermicomposting worms with significant results.

6.5. Experimental Study of Vermifiltration of Sewage Chaudhry (2006) studied the vermifiltration of sewage using earthworms. The raw sewage was obtained from the Oxley Wastewater Treatment Plant in South Brisbane. The study was carried out in the same vermicomposting (VC) bin which was made to work as the vermifiltration (VF) kit. This was located in PC2 lab of Griffith University, as a hazardous material (raw sewage) was to be used. The temperature in lab was maintained at 21.50C, with 50% humidity. The Vermifiltartion kit contained about 30-40 kg of gravels with a layer of garden soil on top. This formed the vermifilter bed. It has provisions to collect the filtered water at the bottom in a chamber which opens out through a pipe fitted with tap. Above the chamber lies the net of wire mesh to allow only water to trickle down while holding the gravels above. The bottommost layer is made of gravel aggregates of size 7.5 cm and it fills up to the depth of 25 cm. Above this lies the aggregates of 3.5 to 4.5 cm sizes filling up to another 25 cm. On the top of this is the 20 cm layer of aggregates of 10-12 mm sizes mixed with sand. The topmost layer of about 10 cm consists of pure soil in which the earthworms were released. The worms were given around one week settling time in the soil bed to acclimatize in the new environment. As the earthworms play the critical role in wastewater purification their number and population density (biomass) in soil, maturity and health are important factors. This may range from several hundreds to several thousands. There are reports about 8-10,000

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numbers of worms per cubic meter of the worm bed and in quantity (biomass) as 10 kg per cubic meter (cum) of soil for optimal function. (Komarowski, 2001).However, in this experiment we started with 500 worms in 0.025 cum of soil bed, which comes to about 20,000 worms per cum of soil. This means even lesser number of worms could have done the job.

The Control Kit without Earthworms: Assessing the Precise Role of Worms A control kit (exact replica of vermifilter kit but devoid of earthworms) was also organised for reference and comparison. It is important to note that the soil and sand particles and the gravels in the kit also contribute in the filtration and cleaning of wastewater by ‗adsorption‘ of the impurities on their surface. They provide ideal sites for colonization by decomposer microbes which works to reduce BOD, COD and the TDSS from the wastewater. As the wastewater passes through, a layer of microbial film is produced around them and together they constitute the ‗geological‘ and the ‗microbial‘ (geo-microbial) system of wastewater filtration. With more wastewater passing through the gravels there is more formation of ‗biofilms‘ of decomposer microbes. Hence it is important to have a control kit in order to determine the precise role of earthworms in the removal of BOD, COD and the TDSS. Experiences, however, have shown that the geo-microbial system gets ‗choked‘ after sometimes due to slow deposition of wastewater solids as ‗sludge‘ and becomes unoperational whereas, the vermifiltration system with earthworms continues to operate. The Experimental Procedures Around 5 – 6 litre of municipal wastewater (sewage) was kept in calibrated 10 litre capacity PVC drum. These drums were kept on an elevated platform just near the vermifilter kit, as shown in figure 1. The PVC drums had tap at the bottom to which an irrigation system was attached. The irrigation system consisted of simple 0.5 inches polypropylene pipe with holes for trickling water that allowed uniform distribution of wastewater on the soil surface (vermifilter bed).

Figure 13. Vermifilter Kits : Regulated flow of wastewater from storage drums determining 1–2 hrs HRT.

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Figure 14.

Figure 15.

Figure 16.

Figure 17.

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Figures 14, 15, 16. Different sizes of gravels used in preparation of the vermifilter bed. Figure 17. Soil bed with earthworms and the device of wastewater discharge and distribution through perforated PVC pipes.

Wastewater from the drums flowed through the irrigation pipes by gravity. The wastewater percolated down through various layers in the vermifilter bed passing through the soil layer inhabited by earthworms, the sandy layer and the gravels and at the end was collected in a chamber at the bottom of the kit. Next day this treated wastewater from both kits were collected and analysed for BOD5 , COD and the TSS. The hydraulic retention time (HRT) was kept uniformly between 1-2 hours in all experiments.

Key Parameters Studied in Sewage Vermifiltration a) Biochemical Oxygen Demand (BOD) The biodegradable organic matter in wastewater is expressed as BOD (Biochemical Oxygen Demand). It is the amount of oxygen needed in a specified volume of wastewater to decompose organic material by the aerobic microbes. The unit of BOD is ppm or mg / L. b) Chemical Oxygen Demand (COD) Many organic substances, which are difficult to oxidize biologically by aerobic microbes, or are toxic to micro-organisms, such as lignin, can be oxidized chemically by using strong oxidizing agent like dichromate (Cr2O7) in acidic media. The unit of COD is ppm or mg / L.

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Earthworms can ingest and remove several organic substances from the wastewater which otherwise cannot be oxidized by microbes and thus bring down the COD values significantly.

c) Total Suspended Solid (TSS) Solids in wastewater consist of organic and inorganic particles and they can either be ‗suspended‘ or ‗dissolved‘. They provide adsorption sites for chemical and biological contaminants. As suspended solids degrade biologically, they can create toxic by-products. TSS can be represented as mg/L or ppm d) Turbidity Turbidity is measured as the cloudiness of water and this is caused by suspended and colloidal particles, such as clay, slit, finely divided organic matter. Turbidity is measured in NTU ( Number of Transfer Units). e) pH Value pH is a symbol that represents negative base -10 logarithm of the effective concentration of hydrogen ions (H+) in moles per Lit. The Analytical Methods Used in Laboratory Study The untreated sewage that was fed to the vermifilter kit and treated sewage which was collected at the bottom of the kit in a chamber were analyzed to study the biological oxygen demand (BOD), chemical oxygen demand (COD), (TSS), turbidity and the pH value. Following standard methods of analysis were adopted, Table 10. Standard Methods adopted to study specified parameters SR. No. 1 2 3 4 5

Description Biological Oxygen Demand (BOD) Chemical Oxygen Demand (COD) Total Suspended Solids (TSS) Turbidity PH

Standard Methods 405.1 5220C 160.2 180.1 D1293-99

The Experimental Results and Discussion a) Removal of BOD5 Results shows that the earthworms can remove BOD (BOD5) loads by over 98 % or nearly complete at hydraulic retention time (HRT) of 1-2 hours (Chowdhary, 2006). BOD removal in the control kit (where only the geological and microbial system works) is just around 77 %. (Table 11).

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Table 11. BOD5 Removal of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT: 1- 2 hrs) Expt No

Untreated Raw Sewage BOD5 (mg/L)

Treated Sewage BOD5 (mg/L) With Worms Without Worms (Vermifiltered) (CONTROL)

1 2 3 4 5

309 260 316 328 275

1.97 4.00 6.02 2.06 4.25

86.3 45.8 63.3 83.2 63.5

% Reduction in BOD5 by Earthworms (Vermifiltered) 99.4 98.5 98.1 99.4 98.5

% Reduction in BOD5 without Earthworms (CONTROL) 72.1 82.4 80.0 74.6 76.9

% Reduction in BOD5

Av. = 98.78 % Av. = 77.2 %. Source: Chowdhary (2006). 100 90 80 70 60 50 40 30 20 10 0 1

2

3

4

5

Experim ent No

% Reduction in BOD5 by Earthworms (Vermifiltered) % Reduction in BOD5 without Earthworms (CONTROL)

Figure 18. % Reduction in BOD5 of swage Water Treated With and Without Earthworms.

b) Removal of COD Results shows that the average COD removed from the sewage by earthworms is over 45 % while that without earthworms (only the geological and microbial system in the control kit) is just over 18 %. (Chowdhary, 2006). (Table 12). COD removal by earthworms is not as significant as the BOD, but at least much higher than the microbial system. Table 12. COD Removal of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT : 1 - 2 hrs) Expt No

Untreated Raw Sewage COD (mg/L)

Treated Sewage COD (mg/L) With Worms Without Worms (Vermifiltered) (CONTROL)

1 2 3 4 5

293 280 280 254 260

132 153 128 112 139

Av. = 45.7 % Av. = 18.4 %. Source: Chowdhary (2006).

245 235 201 217 227

% Reduction in COD by Earthworms (Vermifiltered) 54.9 45.4 54.3 55.9 46.5

% Reduction in COD without Earthworms (CONTROL) 16.4 16.1 28.2 14.6 12.7

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% Reduction in COD

100 90 80 70 60 50 40 30 20 10 0 1

2

3

4

5

Experim ent No

% Reduction in COD by Earthworms (Vermifiltered) % Reduction in COD without Earthworms (CONTROL)

Figure 19. % Reduction in COD of Sewage Wastewater Treated With and Without Earthworms.

c) Removal of TSS Results shows that the earthworms can significantly remove the suspended solids from the sewage by over 90 %, which in the control kit (where geological and microbial system works together) is over 58 % only. (Chowdhary, 2006). (Table 13). Table 13. TSS Removal Efficiency of Sewage Wastewater Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT 1 - 2 hrs) Expt No

Raw Untreated Wastewater TSS (mg/L)

Treated Wastewater TSS (mg/L) With Worms Without Worms (Control)

1 2 3 4 5

390 374 438 379 407

28 24 22 27 25

116 190 184 179 168

% Reduction in TSS by Earthworms (Vermifiltered) 92.82 93.58 94.97 92.87 93.85

% Reduction in TSS without Earthworms (CONTROL) 70.25 49.19 57.99 52.77 58.72

Av. = 92.97 % Av. = 58.34 %. Source: Chowdhary (2006).

Table 14. Turbidity Removal of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) (HRT : 1- 2 hrs) Expt No

1 2 3 4 5

Untreated Raw Sewage Turbidity (NTU) 112 120 74 70 100

Treated Sewage Turbidity (NTU) With Worms (Vermifiltered) 1.5 0.6 1.1 1.2 1.1

Without Worms (CONTROL) 3.6 1.5 1.2 1.8 2.0

Av.= 98.78 % Av.= 97.88 %. Source: Chowdhary (2006).

% Reduction in Turbidity by Earthworms (Vermifiltered) 98.7 99.5 98.5 98.3 98.9

% Reduction in Turbidity Without Earthworms (CONTROL) 96.8 98.8 98.4 97.4 98.0

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% Reduction in BOD5

d) Turbidity Removal Results indicates that the average reduction in turbidity by earthworms is over 98 % while that without earthworms in the control kit is also significantly high and over 97 %. (Chowdhary, 2006). (Table 14).

100 90 80 70 60 50 40 30 20 10 0 1

2

3

4

5

Experim ent No

% Reduction in TSS by Earthworms (Vermifiltered) % Reduction in TSS without Earthworms (CONTROL) Figure 20. % Reduction in TSS of Sewage Wastewater Treated With and Without Earthworms.

% Reduction in Turbidity

100 80

% Reduction in Turbidity by Earthworms (Vermifiltered)

60 40 20 0 1

2

3

4

5

% Reduction in Turbidity Without Earthworms (CONTROL)

Experiment No

Figure 21. % Reduction in Turbidity of sewage treated with and without Earthworms.

e) Improvement in pH Value of Treated Sewage Results indicates that the pH value of raw sewage is almost neutralized by the earthworms in the vermifilter kit. pH value of treated sewage without earthworms also improved but it was not consistent in all experiments. (Table 15).

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Table 15. Improvement in pH Value of Municipal Wastewater (Sewage) Treated by Earthworms (Vermifiltered) and Without Earthworms (Control) Expt. No

Untreated Raw Sewage pH

1 2 3 4 5

6.58 5.76 6.65 6.67 6.05

Treated Sewage pH With Worms Without Worms (Vermifiltered) (CONTROL) 7.05 6.62 7.35 6.05 7.00 7.47 7.25 6.95 7.06 7.15

Source: Chowdhary (2006).

A

B

C

Figure 22. Appearance of Sewage After and Before Treatment. Bottle A : The clear vermifiltered sewage water; Bottle B: The hazy water from the controlled kit; Bottle C : The turbid and cloudy sewage water.

6.5. Significance and Advantages of Vermi-filtration Technology Over Conventional Wastewater Treatment Systems Vermi-filtration system is low energy dependent and has distinct advantage over all the conventional biological wastewater treatment systems- the ‗Activated Sludge Process‘, ‗Trickling Filters‘ and ‗Rotating Biological Contactors‘ which are highly energy intensive, costly to install and operate and do not generate any income. Since the conventional technologies are mostly the flow-processes and have finite hydraulic retention time (HRT) it always results into a ‗residual stream‘ of complex organics and heavy metals (while only the simple organics are consumed) in the ‗sludge‘ that needs further treatment (requiring more energy) before landfill disposal. This becomes unproductive. In the vermifilter process there

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is 100 % capture of organic materials, the capital and operating costs are less, and there is high value added end product (vermicompost).

1) Vermifilter Treatment of Wastewater is an Odorless System There is no foul odor as the earthworms arrests rotting and decay of all putrescible matters in the wastewater and the sludge. They also create aerobic conditions in the soil bed and the waste materials by their burrowing actions, inhibiting the action of anaerobic microorganisms which release foul odor. 2) Vermi-filtration is Low Energy System Some energy may be required in pumping the wastewater to the vermi-filtration unit if gravity flow is not adequate. 3) Synchronous Treatment of Wastewater and the Solids : End Products Become Useful Resource for Agriculture and Horticulture Earthworms decompose the organics in the wastewater and also devour the solids (which forms the sludge) synchronously and ingest the heavy metals from both mediums. This stabilized solids is discharged in the vermifilter bed as ‗excreta‘ (vermicompost) which is useful soil additive for agriculture and horticulture. 4) Vermifiltered Wastewater is Free of Pathogens As the earthworms devour on all the pathogens (bacteria, fungus, protozoa and nematodes) in the medium in which they inhabit the resulting filtered water becomes free of pathogens. The celeomic fluid secreted by worms which has ‗anti-bacterial‘ properties (Pierre et, al., 1982) further eliminate the pathogens. 5) Vermifiltered Wastewater is Free of Toxic Chemicals (Heavy Metals and Endocrine Disrupting Chemicals) As earthworms have the capacity to bio-accumulate high concentrations of toxic chemicals including heavy metals in their tissues the resulting wastewater becomes almost chemical-free. Earthworms have also been reported to bio-accumulate ‗endocrine disrupting chemicals‘ (EDCs) from sewage. Markman et al. (2007) have reported significantly high concentrations of EDCs (dibutylphthalate, dioctylphthalate, bisphenol-A and 17  - estrdiol) in tissues of earthworms (E.fetida) living in sewage percolating filter beds and also in garden soil.

6.6. Decentralized Sewage Treatment by Vermifiltration at Source of Generation All above results were obtained with approximately 500 worms in the vermifilter bed made in about 0.032 cubic meter (cum) of soil. This comes to approximately 16,000 worms per cum of soil. The initial number of worms must have increased substantially over the period of 7-8 weeks of experiment.

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If a vermifilter bed of 0.3 cum soil is prepared with approximately 5000 worms (over 2.5 kg) to start with, it can easily treat 950 - 1000 L of domestic wastewater / sewage generated by (on an average) a family of 4 people with average BOD value ranging between 300 - 400 mg/L, COD 100 – 300 mg/L, TSS, 300 – 350 mg/L everyday with hydraulic retention time (HRT) of the wastewater in the vermifilter bed being approximately 1 - 2 hours. Given that the worms multiply and double its number in at least every 60 days under ideal conditions of temperature and moisture, even starting with this number of earthworms a huge population (biomass) of worms with robust vermi-filtration system can be established quickly within few months which will be able to treat greater amount of wastewater generated in the family. An important consideration is the peak hour wastewater generation which is usually very high and may not comply with the required HRT (1 - 2 hrs) which is very critical for sewage treatment by vermi-filtration system. To allow 1 - 2 hrs HRT in the vermifilter bed an onsite domestic wastewater storage facility will be required from where the discharge of wastewater to the vermifilter tank can be slowly regulated through flow control.

7. THE VERMIREMEDIATION TECHNOLOGY (VRT) Large tract of arable land is being chemically polluted due to mining activities, heavy use of agro-chemicals in farmlands, landfill disposal of toxic wastes and other developmental activities like oil and gas drilling. No farmland of world especially in the developing nations are free of toxic pesticides, mainly aldrin, chlordane, dieldrin, endrin, heptachlor, mirex and toxaphene. There are over 80,000 contaminated sites in Australia, over 40,000 in the US; 55,000 in just six European nations, over 7,000 in New Zealand, and about 3 million contaminated sites in the Asia-Pacific. They mostly contain heavy metals cadmium (Cd), lead (Pb), mercury (Hg), zinc (Zn) etc. and chlorinated compounds like the PCBs and DDT. Cleaning them up mechanically by excavating the huge mass of contaminated soils and disposing them in secured landfills will require billions of dollars. Earthworms in general are highly resistant to many chemical contaminants including heavy metals and organic pollutants in soil and have been reported to bio-accumulate them in their tissues. Vermiremediation (using chemical tolerant earthworm species) is emerging as a low-cost and convenient technology for cleaning up the chemically polluted / contaminated sites / lands in world. Earthworms Efficiently Remove Toxic Organic and Inorganic Chemicals from Soil: Several species of earthworms but more particularly Eisenia fetida, Lumbricus terrestris and L. rubellus, have been found to remove significant amounts of heavy metals, pesticides and lipophilic organic micropollutants like the polycyclic aromatic hydrocarbons (PAH) from the soil (Contreras-Ramos et. al, 2006). Several studies have found definite relationship between ‗organochlorine pesticide‘ residues in the soil and their amount in earthworms, with an average concentration factor (in earthworm tissues) of about 9 for all compounds and doses tested. (Ireland, 1983). Studies indicate that the earthworms bio-accumulate or biodegrade ‗organochlorine pesticide‘ and ‗polycyclic aromatic hydrocarbons‘ (PAHs) residues in the medium in which it lives. (Nelson et al., 1982; Ireland, 1983). Earthworms especially E. fetida can bio-accumulate high concentrations of metals including heavy metals in their tissues without affecting their physiology and this particularly

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when the metals are mostly non-bioavailable. Studies indicate that they can take up and bioaccumulate in their tissues heavy metals such as cadmium (Cd), mercury (Hg), lead (Pb), copper (Cu), manganese (Mn), calcium (Ca), iron (Fe) and zinc (Zn). They can ingest and accumulate in their tissues extremely high amounts of zinc (Zn) and cadmium (Cd). Of all the metals Cd and Pb appears to bio-accumulate in most species of earthworms at greater level. They can particularly ingest and bio-accumulate extremely high amounts of cadmium (Cd) which is very mobile and may be readily incorporated into soft and non-calcareous tissues of earthworms. Cadmium levels up to 100 mg per kg dry weight have been found in tissues. Contreras-Ramos et, al.,(2005) also confirmed that the earthworms reduced the concentrations of chromium (Cr), copper (Cu), zinc (Zn) and lead (Pb) in the vermicomposted sludge (biosolids) below the limits set by the USEPA in 60 days. Malley et. al., (2006) also studied bioaccumulation of copper (Cu) and zinc (Zn) in E. fetida after 10 weeks of experiment. (Table 16). Table 16. Concentration of Cu and Zn in E. fetida Tissues Initially and After 10 Weeks

Initial sample Control Dosage 1 Dosage 2 Dosage 3

Cu (mg/Kg) ±SD 17.29±2 20.34 ±4 104.58 ± 47 158.95 ± 10 213.07 ± 22

Zn (mg/Kg) ±SD 108.22±4 127.54±8 137.52±8 132.03±16 138.51±18

Source: Malley et al., (2006).

Some metals are bound by a protein called ‗metallothioneins‘ found in earthworms which has very high capacity to bind metals. The chloragogen cells in earthworms appears to mainly accumulate heavy metals absorbed by the gut and their immobilization in the small spheroidal chloragosomes and debris vesicles that the cells contain.

7.1. Mechanism of Worm Action in Vermiremediation : The Uptake of Chemicals from Soil and Immobilization by Earthworms Earthworms uptake chemicals from the soil through passive ‗absorption‘ of the dissolved fraction through the moist ‗body wall‘ in the interstitial water and also by mouth and ‗intestinal uptake‘ while the soil passes through the gut. Earthworms eat large volume of soil with microbes and organic matter during the course of their life in soil. Earthworms apparently possess a number of mechanisms for uptake, immobilization and excretion of heavy metals and other chemicals. They either ‗bio-transform‘ or ‗biodegrade‘ the chemical contaminants rendering them harmless in their bodies.

a) Biotransformation of Chemical Contaminants in Soil Heavy metals in earthworms are bound by a special protein called ‗metallothioneins‘ which has very high capacity to bind metals. Ireland (1983) found that cadmium (Cd) and lead (Pb) are particularly concentrated in chloragogen cells in L. terrestris and D. rubidus, where it is bound in the form of Cd-metallothioneins and Pb-metallothioneins respectively

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(i.e. bio-transformed) with small amounts deposited in waste nodules. The chloragogen cells in earthworms appears to mainly accumulate heavy metals absorbed by the gut and their immobilization in the small spheroidal chloragosomes and debris vesicles that the cells contain.

b) Biodegradation of Chemical Contaminants in Soil: Earthworms Promote Microbial Activity for Biodegradation Ma et al., (1995) found that earthworms biodegrade organic contaminants like phthalate, phenanthrene and fluoranthene. It may be noted that several soil microorganisms especially bacteria and fungi also biodegrade several categories of chemicals including hydrocarbons in soil. However, when earthworms are added to the soil they further stimulate and accelerate microbial activity by increasing the population of soil microorganisms and also through improving aeration (by burrowing actions) in the soil, and in totality enhance the rate of biodegradation. The earthworms excrete the decomposer (biodegrader) microbes from their gut into soil along with nutrients nitrogen (N) and phosphorus (P). These nutrients are used by the microbes for multiplication and enhanced action. Reinforcement of microbial activity by earthworms for biodegradation of chemical contaminants in soil have been reported by Binet et al., (1998). Edward and Lofty (1972) showed that the number of bacteria and ‗actinomycetes‘ contained in the ingested material by earthworms increased up to 1000 fold while passing through the gut. A population of worms numbering about 15,000 can in turn foster a microbial population of billions of millions.

7.2. Experimental Study of Vermiremediation of PAHs Contaminated Soils Ryan (2006) studied the vermiremediation of PAHs contaminated soils by earthworms. The soil was obtained from a former gas works site in Brisbane where gas was being produced from coal. The initial concentration of total PAHs compounds in the soil at site was greater than 11,820 mg/kg of soil. (Ryan, 2006). The legislative requirements for soil PAHs concentration is only 100 mg/kg for industrial sites and 20 mg/kg for residential sites. 10 Kg of contaminated soil was taken in each of the four 40 litre black HDPE containers and made into Treatments 1,2, 3 and 4. The first remained as control and no treatment was done. In Treatment 2 approximately 500 earthworms (mixed species of E. fetida, Perionyx excavatus and Eudrillus euginae) of varying ages and sizes were added to the soil. (Worms were contained in about 2 kg of primary feed materials bought from Bunning Hardware). To this was added 5 kg of semi-dried cow dung as secondary feed material. In Treatment 3, about 5 kg of kitchen waste organics were added as secondary feed material to the 500 worms. In Treatment 4, only 5 kg of organic compost was added to the contaminated soil and no worms. This was set up to assess the effect of only microbial action on the contaminated soil as any organic compost is known to contain enormous amount of decomposer microbes. In all the four treatments enough water was added time to time, to maintain the moisture content between 70-80 % and were allowed to stand for 12 weeks. They were kept under shade thoroughly covered with thick and moist newspapers to prevent any volatilization or photolysis of the PAH compounds in the soil.

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Thus, Treatments 2 and 3 had total of 17 kg materials (10 kg contaminated soil + 2 kg of primary feed materials with worms + 5 kg of secondary feed material added additionally). Treatment 4 had 15 kg (10 kg soil + 5 kg compost). Due to addition of feed materials (cow dung and kitchen waste) and compost in the contaminated soil significant dilution of PAH compounds are expected to be made and was taken into consideration while determining the impact of earthworms and the microbes in the removal of PAH compounds. The results have been shown in tables 17 and 18 and figures 23 and 24. Mauli Table 17. Removal of Some PAH Compounds from Contaminated Soil by Earthworms Provided With Different Feed Materials (10 Kg Contaminated Soil + 500 Worms* in 2 kg Feed Materials With Additional Feed Materials Cow Dung (5kg) and Kitchen Waste (5kg) for 12 Wks) and compost (5Kg)

Source: Ryan (2006). *Mixed species of worms (E.fetida, Perionyx excavatus and Eudrillus eugeniae) were used.

Table 18. Percent Removal of Some PAH Compounds from Contaminated Soil by Earthworms Provided With Different Feed Materials (10 Kg Contaminated Soil + 500 Worms* in 2 kg Feed Materials With Additional Feed Materials Cow Dung (5kg) and Kitchen Waste (5kg) for 12 Wks)

Source: Ryan (2006). (%) Values within bracket are those after taking the dilution factor (due to mixing of feed materials) into account. This is just in 12 weeks period.

Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al.

80

100.00% Set 2 Soil + Worms + Cow Dung

% Removal

80.00% 60.00%

Set 3 Soil + Worms + Kitchen w aste

40.00% 20.00%

A ve ra ge

hr ys en F B e lo en u zo ra n (k th ) e F ne lo ur a B n en th en zo e (a B en ) P zo yr en (g e ,h ,i) py re ne

C

(b )

Set 4 Soil + Compost (No Worms)

B en

B en

zo

zo

(a )

an th r

ac en e

0.00%

Extracte d PAH Com pounds

100.00% 80.00%

Set 2 Soil + Worms + Cow Dung

60.00% 40.00% 20.00%

(a )a nt hr ac en e Be C nz hr o ys (b en )F e Be lo ur nz a o nt (k he )F ne lo ur D ib an en th zo en e (a , h Be )P nz yr o en (g e ,h ,i ) py re ne Av er ag e

0.00%

Be nz o

% Removal (Considering dilution factor)

Figure 23. Percent removal of PAH from contaminated soil by earthworms, provided with different feed materials.

Set 3 Soil + Worms + Kitchen waste Set 4 Soil + Compost (No Worms)

PAH Com pounds

Figure 24. Percent removal of some PAH compounds from contaminated soil by earthworms provided with different feed materials, after taking the dilution factor (due to mixing of feed materials) into account.

Findings and Results Results confirm the decisive role of earthworms in PAHs removal which can be by both activities – bioaccumulation, and by promoting microbial activities. When microbial activity in the soil is enhanced alone (without aided by earthworms) by adding microbe rich organic compost to the soil (Treatment 4) the removal rate of PAHs are not very significant. This indicates that earthworms acts in a different manner and contributes decomposer microbes for hydrocarbons which otherwise is normally not available in soil or in ordinary compost. Singleton et al. (2003) has reported some ‗uncultured‘ bacterial flora tightly associated with

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the intestine of the earthworms. Some of them, such as Pseudomonas, Acaligenes and Acidobacterium are known to degrade hydrocarbons. Providing the earthworms with additional feed materials in the form of cow dung and kitchen waste (Treatments 2 and 3) must have played important role in raising worm activity and in its reproductive behavior, and also in stimulating the soil microbial activity. There was not much significant difference in the impact of two types of feeds. Ma et al. (1995) showed an increase in PAH loss from polluted soil when the worms were denied of any additional source of food. They concluded that earthworms increase oral intake of soil particles when driven by ‗hunger stress‘ and consequently ingested more PAHs polluted soil. While this may be a temporary phenomenon in short time (9 weeks of study by authors) it can never be a long-term strategy because any bioremediation treatment of polluted soil is time-taking – in months and years. Worms would starve and die by then. And, adding organic feed materials to the polluted soil has several other advantages. It promotes microbial activities in soil and when the worms ingest them and excrete – the excreted products are nutrient rich organic fertilizers.

7.3. Advantages of Vermiremediation Technology: Earthworms Improves Total Quality of Soil in Terms of Physical, Chemical and Biological Properties Significantly, vermiremediation leads to total improvement in the quality of soil and land where the worms inhabit. Earthworms significantly contribute as soil conditioner to improve the physical, chemical as well as the biological properties of the soil and its nutritive value. They swallow large amount of soil everyday, grind them in their gizzard and digest them in their intestine with aid of enzymes. Only 5-10 percent of the chemically digested and ingested material is absorbed into the body and the rest is excreted out in the form of fine mucus coated granular aggregates called ‗vermicastings‘ which are rich in NKP (nitrates, phosphates and potash), micronutrients and beneficial soil microbes including the ‗nitrogen fixers‘ and ‗mycorrhizal fungus‘. The organic matter in the soil undergo ‗humification‘ in the worm intestine in which the large organic particles are converted into a complex amorphous colloid containing ‗phenolic‘ materials. About one-fourth of the organic matter is converted into humus. The colloidal humus acts as ‗slow release fertilizer‘ in the soil. During the vermi-remediation process of soil, the population of earthworms increases significantly benefiting the soil in several ways. A ‗wasteland‘ is transformed into ‗wonderland‘. Earthworms are in fact regarded as ‗biological indicator‘ of good fertile soil (Neilson, 1951). One acre of wasteland when transformed into fertile land may contain more than 50,000 worms of diverse species. Bhawalkar and Bhawalkar (1994) experimented and concluded that an earthworm population of 0.2 – 1.0 million per hectare of polluted land / wasteland can be established within a short period of three (3) months.

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8. THE VERMI-AGRO-PRODUCTION TECHNOLOGY (VAPT) Vermiculture biotechnology promises to usher in the ‗Second Green Revolution‘ through ‗Organic Farming‘ doing away with the destructive agro-chemicals which did more harm than good to both the farmers and their farmland during the ‗First Green Revolution‘ of the 1960‘s. Earthworms restore and improve soil fertility and boost crop productivity by the use of their metabolic product - ‗vermicast / vermicompost‘ (Tomati and Galli, 1995). They promote soil fragmentation and aeration, and brings about soil turning and dispersion in farmlands. On an average 12 tons / hectare / year of soil or organic matter is ingested by earthworms, leading to upturning of 18 tons of soil / year, and the world over at this rate it may mean a 2 inches of fertile humus layer over the globe (Bhawalkar and Bhawalkar, 1994). Earthworms excrete beneficial soil microbes, and secrete polysaccharides, proteins and other nitrogenous compounds into the soil from their body to improve the physical, chemical and the biological properties of soil.

8.1. Earthworms Vermicast / Vermicompost : A Miracle Growth Promoter Vermicompost produced by biodegradation of MSW by earthworms is a nutritive plant food rich in NKP (2 - 3 % nitrogen, 1.85 – 2.25 % potassium and 1.55 – 2.25 % phosphorus), micronutrients, beneficial soil microbes like ‗nitrogen-fixing bacteria‘ and ‗mycorrhizal fungi‘ and are wonderful growth promoters (Buckerfield, et al.,1999). Kale and Bano (1986) reports as high as 7.37 % of nitrogen (N) and 19.58 % phosphorus as P2O5 in worms vermicast). Moreover, vermicompost contain enzymes like amylase, lipase, cellulase and chitinase, which continue to break down organic matter in the soil (to release the nutrients and make it available to the plant roots) even after they have been excreted. (Chaoui et al., 2003). Vermicompost has very ‗high porosity‘, ‗aeration‘, ‗drainage‘ and ‗water holding capacity‘ and also contains ‗plant-available nutrients‘. Vermicompost appears to retain more nutrients for longer period of time and also greatly increases the water holding capacity of the farm soil (Hartenstein and Hartenstein, 1981; Appelhof, 1997).

a) High Level of Plant-Available Nutrients Atiyeh et al. (2000) found that the conventional compost was higher in ‗ammonium‘, while the vermicompost tended to be higher in ‗nitrates‘, which is the more available form of nitrogen. The nitrogenous waste excreted by the nephridia of the worms is mostly urea and ammonia. The ammonium in the soil is bio-transformed into nitrates. Patil (1993) found that earthworm recycle nitrogen in the soil in very short time. The quantity of nitrogen recycled is significant ranging from 20 to 200 kg N/ha/year. Barley and Jennings (1959) reported that worms significantly improve soil fertility by increasing nitrogen contents. Hammermeister et al. (2004) also found that vermicompost has higher N availability than the conventional compost on a weight basis. They also found that the supply of several other plant nutrients e.g. phosphorus (P), potassium (K), sulfur (S) and magnesium (Mg), were significantly increased by adding vermicompost as compared to conventional compost to soil.

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b) High Level of Beneficial and Biologically Active Soil Microorganisms Earthworms hosts millions of beneficial microbes (including the nitrogen fixers) in their gut and excrete them in soil along with nutrients nitrogen (N) and phosphorus (P) in their excreta i.e. vermicast (Singleton et al., 2003). The nutrients N and P are further used by the microbes for multiplication and vigorous soil remediation action. The mycorrhizal fungi encouraged by the earthworms transfer phosphorus by increasing solubilisation of mineral phosphate by the enzyme phosphatase. Edward and Fletcher (1988) and Edwards (1999) showed that the number of beneficial bacteria and ‗actinomycetes‘ contained in the ingested material increased up to 1000 fold while passing through the gut. A population of worms numbering about 15,000 will in turn foster a microbial population in billions. (Morgan and Burrows, 1982). c). Enhance Seed Germination Worms castings used as a seed germinator produce rapid results with a superior strike rate of ‗unusually healthy seedlings‘

d) Rich in Growth Hormones: Ability to Stimulate Plant Growth Researches show that vermicompost further stimulates plant growth even when plants are already receiving ‗optimal nutrition‘. Vermicompost has consistently improved seed germination, enhanced seedling growth and development, and increased plant productivity much more than would be possible from the mere conversion of mineral nutrients into plantavailable forms (Edwards and Burrows, 1988). Arancon (2004) found that maximum benefit from vermicompost is obtained when it constitutes between 10 to 40 % of the growing medium. Atiyeh et al. (2000) speculates that the growth responses of plants from vermicompost appears more like ‗hormone-induced activity‘ associated with the high levels of humic acids and humates in vermicompost rather than boosted by high levels of plantavailable nutrients. This was also indicated by Canellas et al. (2002) who found that humic acids isolated from vermicompost enhanced root elongation and formation of lateral roots in maize roots. Tomati et al. (1985) had also reported that vermicompost contained growth promoting hormone ‗auxins‘ and flowering hormone ‗gibberlins‘ secreted by earthworms.

8.2. Earthworms Reduce Soil Salinity, Renew Soil Fertility and Improve Crop Productivity Earthworms not only help renew the natural soil fertility but also improve the soil pH and reduce ‗soil salinity‘. Hota and Rao (1985) reported that three tropical earthworm species viz. Perionyx millardi, Octocheaetona surensis and Drawida calebi survived in saline solutions of 9.5 gm, 8.5 gm and 7 gm NaCl per liter (L) respectively. In a study made by Kerr and Stewart (2006) at the US Department of Energy it was found that E. fetida can tolerate soils nearly half as salty as seawater i.e. 15 gm / kg of soil. (Average seawater salinity is around 35 g/L). Farmers at Phaltan in Satara district of Maharashtra, India, applied vermiculture (live earthworms) on his sugarcane crop grown on saline soils irrigated by saline ground water. The yield was 125 tones / hectare of sugarcane and there was marked improvement in soil chemistry. Within a year there was 37 % more nitrogen, 66 % more phosphates and 10 %

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more potash. The chloride content was less by 46 %. Farmer in Sangli district of Maharashtra, India, grew grapes on eroded wastelands and applied vermicasting @ 5 tones / hectare. The grape harvest was normal with improvement in quality, taste and shelf life. Soil analysis showed that within one year pH came down from 8.3 to 6.9 and the value of potash increased from 62.5 kg/ha to 800 kg/ha. There was also marked improvement in the nutritional quality of the grape fruits (Bhawalkar and Bhawalkar, 1994).

8.3. Vermicompost Protects Plants Against Various Pests and Diseases There has been considerable anecdotal evidence in recent years regarding the ability of vermicompost to protect plants against various pests and diseases either by suppressing or repelling them or by inducing biological resistance in plants to fight them or by killing them through pesticidal action. Agarwal (1999),also reported less incidence of diseases in vegetable crops grown on vermicompost. Vermicompost also contains some antibiotics and actinomycetes which help in increasing the power of biological resistance among the crop plants against pest and diseases. Pesticide spray was reduced by 75 per cent where earthworms and vermicompost were used in agriculture. The actinomycetes fungus excreted by the earthworms in their vermicast produce chemicals that kill parasitic fungi such as Pythium and Fusarium.

a) Ability to Repel Pests There seems to be strong evidence that worms varmicastings sometimes repel hardbodied pests (Anonymous, 2001; Arancon, 2004). Edwards and Arancon, (2004) reports statistically significant decrease in arthropods (aphids, buds, mealy bug, spider mite) populations, and subsequent reduction in plant damage, in tomato, pepper, and cabbage trials with 20 % and 40 % vermicompost additions. George Hahn, doing commercial vermicomposting in California, U.S., claims that his product repels many different insects pests. His explanation is that this is due to production of enzymes ‗chitinase‘ by worms which breaks down the chitin in the insect‘s exoskelton (Munroe, 2007). b) Ability to Suppress Disease Edwards and Arancon (2004), also found statistically significant suppression of plantparasitic nematodes in field trials with pepper, tomatoes, strawberries and grapes. The scientific explanation behind this concept is that high levels of agronomically beneficial microbial population in vermicompost protects plants by out-competing plant pathogens for available food resources i.e. by starving them and also by blocking their excess to plant roots by occupying all the available sites. This concept is based on ‗soil-foodweb‘ studies pioneered by Dr. Elaine Ingham of Corvallis, Oregon, U.S.(http://www.soilfoodweb.com). Edwards and Arancon (2004) reported the agronomic effects of small applications of commercially produced vermicompost, on attacks by fungus Pythium on cucumber, Rhizoctonia on radishes in the greenhouse, by Verticillium on strawberries and by Phomposis and Sphaerotheca fulginae on grapes in the field. In all these experiments vermicompost applications suppressed the incidence of the disease significantly. They also found that the ability of pathogen suppression disappeared when the vermicompost was sterilized,

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convincingly indicating that the biological mechanism of disease suppression involved was ‗microbial antagonism. More studies is required to develop this potential of vermicompost as a sustainable, non-toxic and environmentally friendly alternative to chemical pest control or at least its application in farming practices can also lead to significant reduction in use of chemical pesticides.

8.4. Vermiwash: The Nutritive Liquid Byproduct of Vermicomposting Rich in Growth Promoting and Pesticidal Properties The brownish-red liquid which collects at the base of vermcomposting unit due to high moisture content in the compost pile should be collected. This liquid partially comes from the body of earthworms too (as worm‘s body contain plenty of water) and is rich in amino acids, vitamins, nutrients like nitrogen, potassium, magnesium, zinc, calcium, iron and copper and some growth hormones like ‗auxins‘, ‗cytokinins‘. It also contains plenty of nitrogen fixing and phosphate solubilising bacteria (nitrosomonas, nitrobacter and actinomycetes). Farmers from villages near Rajendra Agricultural University in Bihar reported growth promoting and pesticidal properties of this liquid. They used it on brinjal and tomato with excellent results. The plants were healthy and bore bigger fruits with unique shine over it. Spray of vermiwash effectively controlled all incidences of pests and diseases, significantly reduced the use of chemical pesticides and insecticides on vegetable crops and the products were significantly different from others with high market value. These farmers are using vermicompost and vermiwash in all their crops since last 2 years completely giving up the use of chemical fertilizers. (Personal Communication With Farmers in Pusa, December 2006).

8.5. Experimental Studies on Agronomic Impacts of Earthworms and Vermicompost on Crop Plants 1) Potted Wheat Crops Bhatia (1998) and Bhatia et al. (2000) studied the agronomic impact of ‗earthworms‘ on potted wheat crops at University of Rajasthan, Jaipur, India. Wheat seeds (Triticum aestivum Linn) and earthworms were obtained from Rajasthan Agricultural Research Institute, Jaipur. Three identical sets of pots with ten replicates of each were prepared from uniform soil of the same stock, which was assumed to be near neutral, i.e., without any organic matter. Three sets of 10 pots each were prepared. 1) Treatment 1 was kept as a control (without any input); 2) In Treatment 2, fifty (50) worms (mixed species of Eisinia fetida, Perionyx excavatus and Eudrillus euginae) were added to each pot and 250 gm of one week old cattle dung (allowing release of methane from fresh dung) was added as feed material for the worms. 3) In Treatment 3, chemical fertilizers (104 gms urea, 0.2 gms potash and 0.75 gms single super phosphate) were added;

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4) In Treatment 4, about 250 gm of week old cattle dung was added to precisely determine the role of earthworms in growth promotion as the dung also contain nutrients and support growth after degradation and conversion into compost. Ten seeds were sown in each pot (10 x 10 = 100 in ten pots) after mixing the soil with the respective fertilizers (Urea was added in two identical doses, one at the time of sowing and another 21 days). Results are given in table 19. Table 19. Agronomic Impacts of Earthworms Compared With Chemical Fertilizers on Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn.)

Parameters

(1) (2) (3) (4) (5) (6) (7) (8) (9) (10)

Number of seed germinated out of 100 Root length (Av. cm) Shoot length (Av. cm) Ear length (Av. cm) Total height of plant (Av. cm) Leaf length (Av. cm) Dry weight of ears (Av. cm) Number of seed grains per ear (Average) Chlorophyll content (mg/l) Number of tillers per plant

Treatment 1 Control

Treatment 2 Soil Containing Earthworms (50 Nos.) and Cattle Dung (As feed material)

Treatment 3 Soil With Chemical Fertilizers (N=104 gm; K=0.2 gm; P=0.75 gm)

Treatment 4 Soil Containing Cattle Dung (250 gm) only

50

90

60

56

7.13 22.1 4.82

16.46 59.99 8.77

9.32 25.2 5.45

8.23 23.1 5.1

34.16

85.22

39.97

37.30

12.73

26.37

14.19

13.45

0.135

0.466

0.171

0.16

11.8

31.1

19.9

17.4

0.783 1

3.486 2-3

1.947 1-2

1.824 1-2

Scale

Source: Bhatia (1998) and Bhatia et al. (2000): Also in Sustainable Agriculture (Sinha, 2004).; Key: Av. = Average. 100 90 80 70 60 50 40 30 20 10 0 Percentage of seed germination

Root length (cm)

Shoot length Ear length (cm) Total height of (cm) plant (cm)

Leaf length (cm)

Dry w eight of ears (gm)

Number of seed grains per ear

Chlorophyll Number of content (mg/l) tillers per plant

Parameter Set Set Set Set

1 2 3 4

Control Soil with Chemical Fertilizers Soil Containing Live Earthworms & Cow Dung (as feed material) Soil Containing Cattle Dung

Figure 25. Agronomic Impacts of Earthworms Compared With Chemical Fertilizers on Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn.).

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Findings and Results The potted wheat crops with earthworms and added cattle dung as feed materials (Treatment 2) made excellent progress from the very beginning of seed germination up to maturation. They were most healthy and green, leaves were broader, shoots were thicker and the fruiting ears were much broader and longer with average greater number of seed grains per year. Significantly, they were much better even as compared to those grown on chemical fertilizers (Treatment 3). Worms fed on cattle dung and excreted them as vermicast (vermicompost) which worked as the miracle growth promoter. Wheat crops added with cattle dung only (Treatment 4) were almost close to those grown on chemical fertilizers. The dung was eventually converted into compost by soil microbes which worked as growth promoter. 2) Potted and Farm Wheat Crops Reena Sharma (2001) studied the agronomic impacts of vermiculture on the potted as well as on the farm wheat crops at University of Rajasthan, Jaipur, India. In this experiment live population of earthworms and vermicompost were applied separately to pot and farm soil. Same mixed species of worms e.g. E. fetida, P. excavatus and E. euginae were used and vermicompost was also prepared by using same species by composting kitchen waste and cattle dung. The farm was divided into eight plots of 25 x 25 sqm size. Three treatments were prepared: 1) Vermicompost : In the pots 30 gm of vermicompost was used while in the farm it was used @ 2.5 tonnes / ha. 2) Chemical Fertilizers (NPK) : As urea (N), single super phosphate (P) and potash (K), in one full dose and two reducing doses for the pots and one for farm. Vermicompost (30 gm) were applied with both reduced doses of chemical fertilizers; 3) Earthworms : In each pot 50 numbers of earthworms were used, while 1000 worms were released in the farm plot of 25 x 25 sq. mt. ; and 4) The fourth farm plot was kept as control (no inputs). All the treatments were replicated twice. The wheat seed was grown @ 100 kg/ha. Irrigation schedule was maintained as recommended for wheat. Chemical nitrogen (urea) was applied in two split doses (first half at the time of sowing and second half dose after 21 days of sowing) whereas the phosphate and potash fertilizers were applied as single dose at the time of sowing. Results are given in tables 20 and 21.

Findings and Results In the pot experiments the best growth performance in respect of root, shoot and ear length and their weight, weight of 1000 grains were observed where a combination of reduced dose (3/4) of chemical fertilizer (NPK-90:75:60) and normal amount of vermicompost (30 gm) were applied. It was significantly much better than even where full doses of chemical fertilizers were used. More significantly, both vermicompost and earthworms positively influenced the total yield of the grains which is 19.2 and 19.1 grains / ear respectively.

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Table 20. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Potted Wheat Crops Treatments

1. 2. 3. 4. 5. 6.

Vermicompost Earthworms (50 Nos.) NPK (120:100:80) Full Dose NPK (90:75:60) Reduced Dose + VC NPK (60:50:40) Reduced Dose + VC CONTROL

Shoot Length (cm) 41.11 39.27 42.14 47.89 45.96 37.01

Ear Length (cm) 7.65 6.74 7.67 8.91 8.59 5.83

Root Length (cm) 15.5 8.35 16.35 20.2 18.3 7.18

Wt. Of 1000 grains (gm)

Grains/ Ear

33.38 32.41 30.2 40.58 39.29 27.28

19.2 19.1 17.1 18.67 18.91 15.4

Source : Sharma (2001); In Sustainable Agriculture (Sinha, 2004); Key : VC= Vermicompost.

Table 21. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Farm Wheat Crops Treatments

1. 3 4 5.

Shoot Length (cm) Vermicompost (@ 2.5 t / ha) 83.71 NPK (90:75:60) (Reduced Dose) + VC 88.05 (Full Dose) NPK (120:100:80) (Full Dose) 84.42 CONTROL 59.79

Ear Length (cm) 13.14 13.82

Root Length (cm) 23.51 29.71

Wt. Of 1000 grains (In grams) 39.28 48.02

Grains / Ear

14.31 8.91

24.12 12.11

40.42 34.16

31.2 27.7

32.5 34.4

Source : Sharma (2001): In Sustainable Agriculture (Sinha, 2004); Key: VC = Vermicompost. Key: VC= Vermicompost; N = Urea; P = Phosphate; K = Potash. 60 50

Scale

40 30 20 10 0 Vermicompost

Live Earthw orms NPK (120:100:80) NPK ( 90:75:60 ) + NPK (60:50:40) + (50 Nos.) VC VC

CONTROL

Parameters Shoot Length (cm) Root Length (cm) Grains/Ear

Ear Length (cm) Wt. of 1000 grains (gm)

Figure 26. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Potted Wheat Crops.

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100 90 80

Scale

70 60 50 40 30 20 10 0 Shoot Length

Ear Length

Root Length

Wt. of 1000 grains

Grains / Ear

Parameters Vermicompost

Earthworms (1000 Nos.)

NPK (90:75:60) + VC

CONTROL

Figure 27. Agronomic Impact of Earthworms, Vermicompost and Chemical Fertilizers on Farm Wheat Crops.

Findings and Results In the farm experiment the highest growth and yield was achieved where reduced doses (3/4) of chemical fertilizer (NPK- 90:75:60) were applied with full dose of vermicompost (@ 2.5 tons/ha). However, the total yield of the grain (grain / ear) as well as the ear length and the weight of 1,000 grains of crops grown on vermicompost were as good as with those grown on full doses of chemical fertilizers (NPK). 3). Farmed Wheat Crops This facility was provided by Rajendra Agriculture University, Pusa, Bihar, India under a collaborative research program with Griffith University, Brisbane, Australia. We studied the agronomic impacts of vermicompost and compared it with cattle dung compost & chemical fertilizers in exclusive application and also in combinations on farmed wheat crops. Cattle dung compost was applied four (4) times more than that of vermicompost as it has much less NPK values as compared to vermicompost. Results are given in table - 22 Table – 22: Agronomic Impacts of Vermicompost, Cattle Dung Compost & Chemical Fertilizers in Exclusive Applications & In Combinations on Farmed Wheat Crops Treatment Input / Hectare Yield / Hectare 1). CONTROL (No Input) 15.2 Q / ha 2). Vemicompost (VC) 25 Quintal VC / ha 40.1 Q / ha 3). Cattle Dung Compost (CDC) 100 Quintal CDC / ha 33.2 Q / ha 4). Chemical Fertilizers (CF) NPK (120:60:40) kg / ha 34.2 Q / ha 5). CF + VC NPK (120:60:40) kg / ha + 25 Q VC / ha 43.8 Q / ha 6). CF + CDC NPK (120:60:40) kg / ha + 100 Q CDC / ha 41.3 Q / ha ----------------------------------------------------------------------------------------------------------------------------

Suhane et. al., (2008): Communication of RAU, Pusa, Bihar, India Key: N = Urea; P = Phosphate; K = Potash (In Kg / ha)

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90

50

Control

45 40

Vermicompost (25 Q / ha)

35 30

Cattle Dung Compost (100 Q / ha)

25 20

Chemical Fertilizer (NPK 120: 60:40)

15 10

CF (Full Dose) + VC (25 Q / ha)

5 0 Yield (Quintal / hectare)

CF (Full Dose) + CDC (100 Q / ha)

Figure 28: Agronomic Impacts of Vermicompost, Cattle Dung Compost & Chemical Fertilizers in Exclusive Applications & In Combinations on Farmed Wheat Crops

Findings & Discussion Exclusive application of vermicompost supported yield comparable to rather better than chemical fertilizers. And when same amount of agrochemicals were supplemented with vermicompost @ 25 quintal / ha the yield increased to about 44 Q / ha which is over 28 % and nearly 3 times over control. On cattle dung compost applied @ 100 Q / ha (4 times of vermicompost) the yield was just over 33 Q / ha. Application of vermicompost had other agronomic benefits. It significantly reduced the demand for irrigation by nearly 30-40 %. Test results indicated better availability of essential micronutrients and useful microbes in vermicompost applied soils. Most remarkable observation was significantly less incidence of pests and disease attacks in vermicompost applied crops. 4). Potted Corn Crops Sinha & Bharambe (2007) studied the agronomic impacts of earthworms & its vermicompost on corn plants at Griffith University, Brisbane, Australia. It had two parts A & B. Part A was designed to compare the growth promoting abilities of vermicompost with chemical fertilizers and also the earthworms when significantly present in the growth medium. It had three (3) treatments with three replicas of each and a control. Treatment 1 with 25 number of adult worms only; Treatment 2, with chemical fertilizers; and Treatment 3, with vermicompost and also containing same number of worms. Soluble chemical fertilizers ‗Thrive‘ was used. Approx. 8 gm of chemicals was dissolved in 4.5 L of water. It had total nitrogen (N) 15 %, total phosphorus (P) 4 %, total potassium (K) 26 % and a combination of essential micronutrients. Three applications were made during entire growth period while the worms and vermicompost was applied only onetime. Results are given in table 23 (a).

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Table 23 (a). Agronomic Impacts of Earthworms, Worms With Vermicompost and Chemical Fertilizers on Corn Plants (Added to 4 kg of soil; Av.Growth in cm)

Parameters Studied

CONTROL (No Input)

Treatment – 1 EARTHWORMS Only (25 Nos.) (Without Feed)

Treatment – 2 Soluble CHEMICAL FERTILIZERS

Treatment – 3 EARTHWORMS + VERMICOMPOST (200 gm)

Seed Sowing Seed Germination

29th July 2007 9th Day

Do 7th Day

Do 7th Day

Do 7th Day

Avg. Growth in 4 wks

31

40

43

43

Avg. Growth in 6 wks

44

47

61

58

App. Of Male Rep. Organ (In wk 12)

None

None

46

53

87

None

None

None

48

53 (App. Of Male Rep. Organ)

88

95

None

None

None

New Corn

53

56

92

105

Pale & thin leaves

Green & thin

Avg. Growth In 12 wks App. of Female Rep. Organ (In wk 14) Avg. Growth in 15 wks App. Of New Corn (in wk 16 ) Avg. Growth in 19 wks Color & Texture of Leaves

Male Rep. Organ

Green & stout leaves

Male Rep. Organ

90 Female Rep. Organ

Green, stout & broad leaves

Source: Sinha & Bharambe (2007)

Findings and Results Corn plants in pot soil with earthworms and vermicompost (Treatment 3 – Pot D) achieved good growth after week 4, and those with chemical fertilizers (Treatment 2 – Pot C) after week 6. After that, both maintained good growth over the other two (Control & Treatment 1 – Pots A & B) and attained maturity in 11 weeks (appearance of male spike). Female reproductive organ, however, appeared in corn plants grown on vermicompost only in 14 weeks. The ‗new corn‘ appeared after 111 days (in week 16). Until week 4, corn plants in all four treatments showed almost identical growth. Ironically, the corn plants in pot soil with ‗worms only‘ (Treatment 1) could not make any significant progress. Soil being completely devoid of organics could not be used by worms to produce any vermicast. However, they were all greener and healthier than control. Another significant finding was that pot soil with vermicompost demanded less water for irrigation as compared to others.

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120

100 CONTROL

Height in cm

80 EARTHWORMS (25) Without Feed

60

Soluble CHEMICAL FERTILIZERS (NPK) 40

EARTHWORMS (25) + VERMICOMPOST (200 gm)

20

0 Avg. Growth Avg. Growth in 4 wks in 6 wks

Avg. Growth Avg. Growth Avg. Growth in 12 wks in 15 wks in 19 wks

Figure 29 : Agronomic Impacts of Earthworms, Worms With Vermicompost and Chemical Fertilizers on Corn Plants (In 19 Weeks Period)

(1). (Growth until 4 weeks)

(2). (Growth until 6 weeks)

(3). (Growth until 12 weeks) Plants with chemical fertilizers (CF) and vermicompost (VC) grew well; Male spikes appeared in both plants in week 11. Keys:

(4). (Growth until 15 weeks) Plants with CF and VC maintained excellent growth; Female reproductive organs appeared in VC only in week 14 and ‗new corn‘ in week 16.

(A) Pot soil without any input (CONTROL) (B) Pot soil with worms only (C) Pot soil with chemical fertilizers (D) Pot soil with worms and vermicompost (200gm)

Figure 6 (a). Photo showing growth of corn plants under the influence of earthworms, worms with vermicompost and chemical fertilizers.

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Part – B This study was designed to test the growth promoting capabilities of earthworms added with feed materials and ‗vermicompost‘, as compared to ‗conventional compost‘. It had three (3) treatments with three (3) replicas of each. The dose of vermicompost was ‗doubled‘ (400 gm) from previous study and same amount of conventional compost was used. Only one application of each was made. Crushed dry leaves were used as feed materials (400 gm). Results are given in table 23 (b). Table 23 (b). Agronomic Impacts of Earthworms (With Feed), Vermicompost and Conventional Compost on Corn Plants (Added to 4 kg of soil ; Av. Growth in cm)

Parameters Studied Seed Sowing Seed Germination Avg. Growth in 3 wks Avg. Growth in 4 wks App. of Male Rep. Organ (in wk 6) Avg. Growth in 6 wks Avg. Growth in 9 wks App. Of Female Rep. Organ (in wk 10) App. of New Corn (in wk 11) Avg. Growth in 14 wks Color & Texture of Leaves

Treatment – 1 Earthworms (25) with Feed (400 gm) 9th Sept. 2007 5th Day 41 49 None 57 64

Treatment–2 Conventional COMPOST (400 gm) Do 6th Day 42 57 None 70 72.5

Treatment – 3

None

None

Female Rep. Organ

None 82

None 78

Green & thick

Light green & thin

New Corn 135 Deep green, stout, thick & broad leaves

VERMICOMPOST (400 gm) Do 5th Day 53 76 Male Rep. Organ 104 120

Source : Sinha & Bharambe (2007)

160

Growth in cm

140 120

Earthworms (25) With Feed (400 gm)

100 80

Conventional COMPOST (400 gm)

60

VERMICOMPOST(400 gm)

40 20 0 Avg. Avg. Avg. Avg. Avg. Growth in Growth In Growth In Growth Growth 3 wks 4 wks 6 wks In 9 wks in 14 wks

Figure 30: Agronomic impacts of Earthworms (with feed), Vermicompost and Conventional Compost on Corn Plants (In 14 Weeks Period)

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Findings and Results Corn plants in pot soil mixed with vermicompost (Treatment 3) achieved rapid and excellent growth after week 4 and attained maturity very fast. Between weeks 6 to 11, there was massive vegetative growth with broad green leaves. Male spike appeared in week 6 while the female reproductive organs appeared in week 10 which bored the ‗new corn‘ in 11th week. Corn plants in pot soil with worms only (Treatment 1) and conventional compost (Treatment 2) could not keep pace with vermicompost. Corn plants with worms only, however, were more green and healthy and took over those plants grown on conventional compost finally in the 14th week. Female reproductive organs and ‗new corn‘ could not appear until the completion of the study by 21st November 2007.

Keys:

(A) Pot soil with earthworms only (B) Pot soil mixed with conventional compost (400 gm) (C) Pot soil mixed with vermicompost (400 gm)

Figure 6 (b). Photo showing growth of corn plants under the influence of earthworms, conventional compost and vermicompost.

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Results of both studies (Part A & B) established beyond doubt that the ‗vermicompost‘ (metabolic products of worms) works like ‗miracle growth promoter‘ and is biologically (nutritionally) much superior to the conventional compost. This has also been found by other researchers (Subler et al., 1998; Pajon (Undated); and Bogdanov, 2004). And what is most significant is that when the dose of vermicompost is doubled (from 200 grams in Part A study to 400 grams in Part B, all other conditions remaining same) it simply enhanced total plant growth to almost two-fold (from average 58 cm on 200 gm VC to average 104 cm on 400 gm VC) within the same period of study i.e. 6 weeks. Corn plants with double dose of vermicompost (400 gm) achieved maturity in much shorter time. Male reproductive organs (spike) appeared after 81 days (in week 12) in plants grown on 200 gm of vermicompost, while in those grown on 400 grams, it appeared just after 39 days (in nearly half of the time in week 6). Similarly, the female reproductive organs and eventually the ‗new corn‘ appeared after 96 days (in week 14) and 111 days (in week 16 ) respectively in plants grown on 200 grams of vermicompost, while it appeared only after 69 days (in week 10) and 75 days (in week 11) respectively, in plants grown on 400 grams of vermicompost. It is also significant to note that the corn plants with earthworms only, performed better over those grown on conventional compost, once again establishing the role of earthworms as ―growth promoters‖. Study also confirmed that ‗vermicompost‘ is superior over ‗conventional compost‘ as growth promoter & in retaining soil moisture while also helping the plants to attain maturity and reproduce faster, thus reducing the ‗life-cycle‘ of crops and also shortening the ‗harvesting time‘. Conventional compost fails to deliver the required amount of macro and micronutrients including the vital NKP (nitrogen, potassium & phosphorus) to plants in shorter time. The leaves and stems of corn plants grown on vermicompost was much greener, broader and stouter than those grown on conventional compost (Sinha and Bharambe, 2007).

8.5.1 Current Experimental Study on Potted Wheat, Corn and Tomato Plantys Under Progress: Some Striking Observations of Plants Under Vermicompost Valani, Dalsukh and Chauhan, Krunal (2008) is currently studying the agronomic impacts of vermicompost vis-à-vis conventional cow dung compost and chemical fertilizers on wheat, corn and tomato plants. Some very exciting results have started appearing in the very beginning of the study.

1). High Strike Rates of Wheat Seed Germination The striking rates of wheat seeds were very high under vermicompost. They germinated nearly 48 hours (2 days) ahead of others and the number of seeds germinated were also high by nearly 20 %. The seedlings are also much greener and healthier and have more offshoots.. 2). Rapid Growth of Corn Seedlings There was not much difference in the striking rates of seed germination but once germinated the seedlings grew at a much faster rate almost doubling in size (from 22 cm to 42 cm) in just two days. Seedlings are more greener, stouter and healthier than others.

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3). More Flowering and Rapid Growth of Tomato Fruits Flowering in tomato plants under vermicompost was delayed by 5-6 days but once started there were greater numbers of flowers (average 7-8 per plant as compared to 5-6 in chemical fertilizers) and subsequently fruit developments. Significantly, the fruits grew in size very rapidly overtaking all others and are much greener. One more striking observation is that the tomato plants on vermicompost are showing greater adaptability to higher temperature (about 30º - 35º C) in the glasshouse.

CONCLUSIONS AND REMARKS Vermiculture practices for waste and land management and for improving soil fertility to boost crop productivity has grown considerably in recent years all over the world. Vermiculture is in fact a technological revival of the traditional age-old methods practiced by the ancient farmers for disposal of farm wastes and improvement of farm fertility by earthworms. It is like getting ‗gold from garbage (solid waste) by vermicomposting‘, ‗silver from sewage (wastewater) by vermifiltration‘; ‗converting a wasteland (chemically contaminated and saline lands) into wonderland (fertile land) by vermiremediation‘; ‗harvesting green gold (crops) by using brown gold (vermicompost) – and all with the help of earthworms which are abundant in soil all over the world but most prominently in the tropical nations. The three versatile species E. fetida, E. euginae and P. excavatus performing wide environmental functions occur almost everywhere. Earthworms are justifying the beliefs and fulfilling the dreams of Sir Charles Darwin as ‗unheralded soldiers‘ of mankind. (Sinha and Sinha, 2007).

9.1. The Vermicomposting Technology (VCT) Earthworms have real potential to both increase the rate of aerobic decomposition and composting of organic matter, and also to stabilize the organic residues in them, while removing the harmful pathogens and heavy metals from the end products. Waste composting by earthworms is proving to be environmentally preferred technology over the conventional microbial composting technology and much more over the landfill disposal of wastes as it is rapid and nearly odorless process, can reduce composting time significantly and above all, there is no emission of ‗greenhouse gas methane‘ which plagues both these solid waste management options. Vermicomposting of organic waste to divert massive domestic waste from the landfills are gaining importance and many municipal council in Australia are adopting this rapid and odorless vermicomposting technology (VCT) using waste eater earthworms.(UNSW ROU, 2002(a) and 2002(b)). Poor nations cannot even afford to construct and maintain a truly engineered landfill. Developing countries needs more options for safe waste management, and also low-cost, as they have several other social and developmental priorities with limited resources. Moreover, in the rural communities of both developed and developing world, centralized waste management system is never a good option. Individual households or a cluster of homes can

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treat their wastes better and also recover some useful resources (compost). This will prove more useful in the rural areas where farms are located. The worm number and quantity (biomass) is a ‗critical factor‘ for vermi-composting of organic wastes besides the optimal temperature and moisture which determines worm activity. A minimum of about 100-150 adult worms per kg of waste would be ideal to start with for rapid biodegradation and also odor-free process. Vermicomposting process driven by the earthworms tends to become more robust and efficient with time as the army of degrader worms grows and invade the waste biomass and further proliferating several battalions of aerobic decomposer microbial army. What is of greater significance is that earthworms accept and adapt to all kinds of new food products (even the fried foods) of modern civilization (except a few) to which their ancestors were never used to in history and readily degrade them converting into vermicompost.

9.2. The Vermifiltration Technology (VFT) Vermifiltration of wastewater is a logical extension of ‗soil filtration‘ which has been used for ‗sewage silviculture‘ (growing trees) since ancient days. Healthy soil is a biogeological medium acting as an ‗adsorbent‘ of organics, inorganic, pathogens and parasites. Vermifiltration technology (VFT) can be a most cost-effective and odor-free process for sewage treatment with efficiency, economy and convenience. Any non-toxic wastewater from the households, commercial organizations or industry can be successfully treated by the earthworms and the technology can also be designed to suit a particular wastewater. It can be used in a de-centralized manner in individual industry or cluster of similar industries so as to reduce the burden on the wastewater treatment plants down the sewer system. It can treat dilute (less than 0.1 % solids) as well as concentrated wastewater. It has an in-built pH buffering ability and hence can accept wastewater within a pH range of 4 to 9 without any pH adjustment. Though significant removal of BOD, COD and the TDSS is achieved by the geomicrobial system unaided by earthworms (as shown from our study in the control kit) the system fails to work after some time as it is frequently choked due to the formation of sludge and also colonies of bacteria and fungi (in the vermifilter bed) in the absence of the earthworms which constantly keep devouring on them. Presence of earthworms in the system also improve the ‗adsorption‘ properties of the geological system (sands and soils) by grinding them in their gizzard. Vermifiltration process driven by the earthworms also tends to become more robust and efficient with time as the army of degrader worms grows, further proliferating microbial population (army of aerobic decomposers). It is also a compact biological wastewater treatment system as compared to other non-conventional system such as the ‗constructed wetland system‘ which often suffer from limitations of oxygen for the decomposer aerobic microbes to act efficiently. Wetland based technologies involve mainly treatment and low utilization of waste materials and hence can be wasteful. BOD, COD, TSS and turbidity removal efficiency generally increases with increase in HRT up to a certain limit and is positively affected by the number of earthworms (earthworms biomass) per cubic meter (cum) in the vermifilter bed (soil profile). It can reduce the small BOD loadings of sewage (200-400 mg/L) within 30 – 40 minutes of HRT. Worms

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have been found to remove very high BOD loads (10,000 – 1,00,000 mg/L often found in wastewater from food processing industries) within 4 to 10 hours of HRT. If the population of worms were to be higher, the same efficiency of BOD, COD, TSS and turbidity removal could have been achieved at lower HRT. However, in case of municipal wastewater (sewage) treatment the objectives are not only to remove BOD, COD and TDSS, but also to remove the toxic chemicals including the heavy metals and pathogens from the wastewater. The presence of ‗endocrine disrupting chemicals‘ (EDCs) in sewage which was discovered recently with the development of new instruments (Markman et. al., 2007) is causing great concerns these days. And what is the matter of more serious concern is that they cannot be removed by the conventional sewage treatment methods. They can only be removed by the reverse osmosis methods of ‗membrane filtration technology‘ (MFT) which is cost-prohibitive at present and all nations cannot afford it. The cost-effective vermifiltration technology (VFT) assumes great significance in the removal of EDCs from wastewater. Hence greater hydraulic retention time (1-2 hours) is allowed so that the worms can ingest (bio-accumulate) the toxic chemicals and also devour upon the pathogens completely. Greater interaction with wastewater components also provides better opportunity for the worms to eat all the solids and prevent any sludge formation. Vermi-filtration of wastewater must be started with higher number of earthworms, at least over 15,000-20,000 worms per cubic meter (cum) of soil in the vermifilter bed for good results. It is also important that they are mostly adult and healthy worms. In vermicomposting of solid waste, which is a continuous process (in days and weeks) the worms have to act ‗gradually‘ in phases while their population (new army of bio-degraders) keeps on building up to intensify the biodegradation process. In vermifiltration of wastewater, the worms have to act ‗instantly‘ as the wastewater flow past their body (degrading the organics, ingesting the solids and the heavy metals). That is why the wastewater has to be ‗retained‘ (HRT) in the vermifilter bed for some appropriate period time (which has to be in hours and not in days) while the worms act on the wastewater.

9.3. The Vermiremediation Technology (VRT) Earthworms have great potential in removing hydrocarbons and many other chemicals from contaminated soil, even the PAHs like benzo(a)pyrene which is very resistant to degradation. They are extremely resistant to toxic PAHs and tolerate concentrations normally not encountered in the soil. It is important to mention here that nearly 80 % removal (60-65 % if the dilution factor is taken into account) of seven important PAH compounds was achieved in just 12 weeks period and that too with only about 500 worms (of both mature and juvenile population) in 10 kg of soil (50 worms/kg of soil). And this was during the winter season in Brisbane (March – May 2006) when the biological activities of worms are the lowest ebb. Increasing the number of worms per kg of contaminated soil to about 100 mature adult worm /kg of soil, and the time of remediation up to 16 weeks could have completely (100 %) removed the PAH compounds. Contreras-Ramos et. al, (2006) studied with 10 worms / 50 gms of contaminated soil (which is equivalent to 200 worms / kg of soil) in about 11 weeks and got 50-100 % removal of some PAHs. Many soils contain abundance of pores with diameters of 20 nm or less. Such pores are too small to allow the smallest bacterium (1 um), protozoa (10 um) or root hairs (7 um) to

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penetrate and attack the chemicals. A chemical contaminant residing in such fine pores in soil is thus completely protected from attack by a microbe in the soil for biodegradation action. In other words such chemical contaminants are not ‗bio-available‘ for any biological action. Earthworms play a very important and critical role here by enlarging the pores through continuous ‗burrowing actions‘ in the soil, thus allowing the microbes to enter into the pores and act on the contaminants. It also stimulate the population of decomposer microbes to several folds for enhanced biodegradation action. The ‗gizzard‘ in the earthworms helps to grind the food very thoroughly with the help of tiny stones swallowed by the worms into smaller particles 2-4 m in size. This grinding action may serve to make PAHs or any other chemical contaminants sequestered in the soil ‗bio-available‘ to decomposer microbes for degradation. Vermi-remediation may prove very cost-effective and environmentally sustainable way to treat polluted soils and sites contaminated with hydrocarbons in just few weeks to months. With the passage of time, the remedial action is greatly intensified. As the worms multiply at an enormous rate it can quickly achieve a huge arsenal for enhanced degradation of PAHs in much shorter time. Comparing the cost incurred in mechanical treatment by excavation of contaminated soils and their safe dumping in secured landfills (as hazardous wastes), this technology is most economic. More study will be needed on the PAH removal activities of earthworms with and without additional feed materials and upon different categories and doses of organic feed.

9.4.The Vermi-Agro-Production Technology (VAPT) Earthworms when present in soil inevitably work as soil conditioner to improve the physical, chemical as well as the biological properties of the soil and its nutritive value for healthy plant growth. This they do by soil fragmentation and aeration, breakdown of organic matter in soil and release of nutrients, secretion of plant growth hormones, proliferation of nitrogen-fixing bacteria, increasing biological resistance in crop plants, and all these worm activities contribute to improved crop productivity. (Barley, 1959). Studies have found that if 100 kg of organic waste with say, 2 kg of plant nutrients (NPK) are processed through the earthworms, there is a production of about 300 kg of ‗fresh living soil‘ with 6 % of NPK and several trace elements that are equally essential for healthy plant growth. This magnification of plant nutrients is possible because earthworms produce extra nutrients from grinding rock particles with organics and by enhancing atmospheric nitrogen fixation. Earthworms activate this ground mix in a short time of just one hour. When 100 kg of the same organic wastes are composted conventionally unaided by earthworms, about 30 kg compost is derived with 3 % NPK. This usual compost thus has a total NPK of only about 1 kg. Rest one kg nutrient might have been leached or volatilized during the process of composting. (Bhawalker and Bhawalkar, 1993 and 1994). The metabolic products (vermicast / vermicompost) of worm activities (feeding and excretion) works like a ‗miracle growth promoter‘. Vermicompost is agronomically much superior to conventional microbial compost sold in the market and can play very important role in promoting growth in crop plants, even competing with chemical fertilizers as our studies have shown. It is mainly due to the growth promoting factors (hormones and enzymes) that is present in the vermicompost. Study shows that doubling the dose of

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vermicompost almost double the growth of corn plants within the same period and can help attain maturity much earlier. Worms and its vermicompost have tremendous crop growth promoting potential while maintaining soil health and fertility and significantly reducing (by over 80 %) the use of chemical fertilizers and pesticides, and in some crops can even replace them completely. This is what being termed as ‗sustainable agriculture‘. Use of vermicompost in farm soil eventually leads to increase in the number of earthworm population in the farmland over a period of time, as the baby worms grow out from their cocoons contained in the vermicast. In Argentina, farmers who use vermicompost consider it to be seven (7) times richer than conventional composts in nutrients and growth promoting values (Pajon (Undated); Munroe, 2007). No wonder, Sir Charles Darwin, the great visionary scientist of 19th Century, called the earthworms as ‗friends of farmers‘ and ‗unheralded soldiers of mankind‘ working day and night under the soil.

Tribute to the Earthworms Earthworms are justifying the beliefs and fulfilling the dreams of Charles Darwin. He wrote a book in which he emphasized that there may not be any other creature in world that has played so important a role in the history of life on earth (Bogdanov, 2004). One of the leading authorities on earthworms and vermiculture studies Dr. Anatoly Igonin of Russia has said – ‗Nobody and nothing can be compared with earthworms and their positive influence on the whole living Nature. They create soil and everything that lives in it. They are the most numerous animals on Earth and the main creatures converting all organic matter into soil humus providing soil‘s fertility and biosphere‘s functions: disinfecting, neutralizing, protective and productive‘. (Appelhof, 2003). There can be little doubt that mankind‘s relationship with worms is vital and needs to be nurtured and further expanded.

REFERENCES AND MORE READINGS Anonymous (2001): Vermicompost as Insect Repellent; Biocycle, Jan. 01: p. 19. Agarwal, Sunita (1999): Study of Vermicomposting of Domestic Waste and the Effects of Vermicompost on Growth of Some Vegetable Crops; Ph.D Thesis Awarded by University of Rajasthan, Jaipur, India. (Supervisor: Rajiv K. Sinha) ARRPET (2005): Vermicomposting as an Eco-tool in Sustainable Solid Waste Management; ; Asian Institute of Technology, Anna University, India. Appelhof, M. (1997): Worms Eat My Garbage; 2nd (ed); Flower Press, Kalamazoo, Michigan, U.S. Appelhof, Mary (2003): Notable Bits; In WormEzine, Vol. 2 (5): May 2003 (Available at (http://www.wormwoman.com) Achazi, R.K., Fleener, C., Livingstone, D.R., Peters, L.D., Schaub, K.,Schiwe, E. (1998): Cytochrome P 450 and dependent activity in unexposed and PAH-exposed terrestrial annelids; J. of Comparative Biochemistry and Physiology; Part C, Vol. 121: pp. 339-350. Arancon, Norman (2004): An Interview with Dr. Norman Arancon; In Casting Call, Vol. 9 (2); August 2004. (http://www.vermico.com)

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Atiyeh, R.M., Subler, S., Edwards, C.A., Bachman, G., Metzger, J.D. Shuster, W. (2000): Effects of Vermicomposts and Composts on Plant Growth in Horticultural Container Media and Soil; In Pedobiologia, Vol. 44: pp. 579-590. Barley, K.P. (1959): The Influence of Earthworm on Soil Fertility II : Consumption of Soil and Organic Matter by the Earthworms; Australian Journal of Agricultural Research; 10: pp. 179-185. Barley, K.P. and Jennings A.C. (1959): Earthworms and Soil Fertility III; The Influence of Earthworms on the Availability of Nitrogen; Australian Journal of Agricultural Research; Vol. 10: pp. 364-370. Belfroid, A., Meiling, J., Drenth, H.J., Hermens, J., Seinen, W., Gestel, K.V. (1995): Dietary Uptake of Superlipophilic Compounds by Earthworms (Eisenia andrei); J. of Ecotoxicology and Environmental Safety; Vol. 31: pp. 185-191. Butt, K.R. (1999): Inoculation of Earthworms into Reclaimed Soils: The UK Experience; Journal of Land Degradation and Development; Vol. 10; pp. 565-575. Bharambe, Gokul (2006): Vermifiltration of Wastewater from Food Processing Industries (Brewery and Milk Dairy) in Brisbane; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2006. (Supervisor Dr. Rajiv K. Sinha). Brahambhatt, Ashish (2006): Vermistabilization of Biosolids; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2006. (Supervisor : Rajiv K. Sinha). Bajsa, O., J. Nair, K. Mathew and G.E. Ho (2003) : Vermiculture as a tool for domestic wastewater management; Water Science and Technology; IWA Publishing; Vol. 48: No 11-12; pp. 125-132; (Viewed on 5th May 2006).<www.iwaponline.com/wst/04811/ wst048110125.htm> Bajsa O., Nair J., Mathew K. and G.E.Ho. (2004): Pathogen Die Off in Vermicomposting Process; Paper presented at the International Conference on Small Water and Wastewater Treatment Systems, Perth, 2004. Bajsa O, Nair J, Mathew K and Ho G.E (2005): Processing of sewage sludge through vermicomposting, Water and Environment Management Series; (Eds.) K Mathew and I.Nhapi IWA Publishing London, UK , ISBN: 1-84339-511-8. Binet, F., Fayolle, L., Pussard, M. (1998): Significance of earthworms in stimulating soil microbial activity; Biology and Fertility of Soils; Vol. 27: pp. 79-84. Bhawalkar, V.U. and Bhawalkar, U.S. (1993): Vermiculture: The Bionutrition System;National seminar on Indigenous Technology for Sustainable Agriculture, I.A.R.I, New Delhi, March 23-24 : 1-8. Bhawalkar, U.S and Bhawalkar, V.U. (1994): Vermiculture Eco-technology; Publication of Bhawalkar Earthworm Research Institute (BERI), Pune, India. Bhiday, M.H. (1995): Wealth from Waste : Vermiculturing; Publication of Tata Energy Research Institute (TERI), New Delhi, India; ISBN 81-85419-11-6. Bhatia, Sonu (1998): Earthworm and Sustainable Agriculture : Study of the Role of Earthworm in Production of Wheat Crop; Field Study Report of P.G. Diploma in Human Ecology, University of Rajasthan, Jaipur, India. (Supervisor: Rajiv K. Sinha). Bhatia, Sonu., Rajiv K. Sinha, and Reena Sharma (2000): Seeking Alternatives to Chemical Fertilisers for Sustainable Agriculture: A Study of the Impact of Vermiculture on the Growth and Yield of Potted Wheat Crops (Triticum aestivum Linn); International J. of

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Environmental Education and Information; University of Salford, U.K. ; Vol. 19, No.4 : pp. 295-304. Bogdanov, Peter (2004): The Single Largest Producer of Vermicompost in World; In P. Bogdanov (Ed.), ‗Casting Call‘, Vol. 9 (3), October 2004. (http://www.vermico.com). Buckerfield, J.C., Flavel, T.C., Lee, K.E. and Webster, K.A. (1999): Vermicompost in Solid and Liquid Forms as a Plant – Growth Promoter; Pedobiologia, Vol. 43: pp. 753 – 759. Canellas. L.P., Olivares, F.L., Okorokova, A.L., and Facanha, R.A. (2002): Humic Acids Isolated from Earthworm Compost Enhance Root Elongation, Lateral Root Emergence, and Plasma Membrane H+ - ATPase Activity in Maize Roots; In J. of Plant Physiology, Vol. 130: pp. 1951-1957. Chaoui, H.I., Zibilske, L.M. and Ohno, T. (2003): Effects of earthworms casts and compost on soil microbial activity and plant nutrient availability; Soil Biology and Biochemistry, Vol. 35, No. 2; pp. 295-302. Chaudhari, Uday (2006): Vermifiltration of Municipal Wastewater (Sewage) in Brisbane; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2006. (Supervisor: Rajiv K. Sinha). Chauhan, Krunal (2008): Studies in Vermiculture Biotechnology; 40 CP Honours Project, Griffith University, Brisbane, Australia (Supervisors: Rajiv K. Sinha & Sunil Herat) Collier, J (1978): Use of Earthworms in Sludge Lagoons; In: R. Hartenstein (ed.) ‗Utilization of Soil Organisms in Sludge Management‘; Virginia. USA; pp.133-137. Ceccanti, B. and Masciandaro, G. (1999): Researchers study vermicomposting of municipal and paper mill sludges; Biocycle Magazine, (June), Italy. Cardoso L, Ramirez (2002): Vermicomposting of Sewage Sludge : A New Technology for Mexico; J. of Water Science and Technology; Vol. 46; pp. 153-158. Contreras-Ramos, S.M., Escamilla-Silva, E.M. and Dendooven, L. (2005): Vermicomposting of Biosolids With Cow Manure and Wheat Straw; Biological Fertility of Soils, Vol. 41; pp. 190-198. Contreras-Ramos, Silvia M., Alvarez-Bernal, Dioselina and Dendooven Luc (2006): Eisenia fetida Increased Removal of Polycyclic Aromatic Hydrocarbons (PAHs) from Soil; Environmental Pollution; Vol. 141: pp. 396-401; Elsevier Pub. Davis, B. (1971): Laboratory studies on the uptake of dieldrin and DDT by earthworms; Soil Biology and Biochemistry, .3, pp. 221-223. Dash, M.C (1978): Role of Earthworms in the Decomposer System; In: J.S. Singh and B. Gopal (eds.) Glimpses of Ecology; India International Scientific Publication, New Delhi, pp.399-406. Datar, M.T., Rao, M.N. and Reddy, S. (1997): Vermicomposting : A Technological Option for Solid Waste Management; J. of Solid Waste Technology and Management, Vol. 24 (2); pp. 89-93. Dominguez, J., Edward, C.A. and Webster, M. (2000): Vermicomposting of Sewage sludge: Affect of Bulking Materials on Growth and Reproduction of the Earthworms E.anderi; J. of Pedobiologia, Vol. 44: pp. 24-32. Eijsackers, H., Van Gestel, C.A.M., De Jonge, S., Muijis, B., Slijkerman, D. (2001): PAH Polluted Peat Sediments and Earthworms: A Mutual Inference; J. of Ecotoxicology; Vol. 10: pp. 35-50.

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Eastman, B.R. (1999): Achieving Pathogen Stabilization Using Vermicomposting; Biocycle, pp. 62-64; (Also on Worm World Inc. Available www.gnv.fdt.net/reference/index.html. Seen on 24.7.2001). Eastman, B.R., Kane, P.N., Edwards, C.A., Trytek, L., Gunadi, B., Stermer, A.L. and Mobley, J.R. (2001): The Effectiveness of Vermiculture in Human Pathogen Reduction for USEPA Biosolids Stabilization ; Compost Science and Utilization, Vol. 9 (1); pp. 3841. Edwards, C.A. and Fletcher, K.E. (1988): Interaction Between Earthworms and Microorganisms in Organic Matter Breakdown; Agriculture Ecosystems and Environment; Vol. 24, pp. 235-247. Edward, C.A. (1988): Breakdown of Animal, Vegetable and Industrial Organic Wastes by Earthworms; In C.A. Edward, E.F. Neuhauser (ed). ‗Earthworms in Waste and Environmental Management‘; pp. 21-32; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7. Edward, C.A. (2000): Potential of Vermicomposting for Processing and Upgrading Organic Waste; Ohio State University, Ohio, U.S. Edwards, C.A. and Burrows, I. (1988): The Potential of Earthworms Composts as Plant Growth Media; In C.A. Edward and E.F. Neuhauser (Eds.) ‗Earthworms in Waste and Environmental Management‘; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7; pp. 21-32. Edwards, C.A. and N. Arancon (2004): Vermicompost Supress Plant Pests and Disease Attacks; In REDNOVA NEWS: http://www.rednova.com/display/ ?id =55938. Elvira, C., Sampedro, L., Benitez, E., Nogales, R. (1998): Vermicomposting from Sludges from Paper Mills and Dairy Industries with Elsenia anderi: A Pilot Scale Study; J. of Bioresource Technology, Vol. 63; pp. 205-211. Evans, A.C. and Guild, W.J. Mc. L. (1948) : Studies on the Relationship Between Earthworms and Soil Fertility IV; On the Life Cycles of Some British Lumbricidae; Annals of Applied Biology; 35 (4) : 471-84. Fraser-Quick, G. (2002): Vermiculture – A Sustainable Total Waste Management Solution; What‘s New in Waste Management ? Vol. 4, No.6; pp. 13-16. Frederickson, J. Butt, K.R., Morris, R.M., and Daniel C. (1997) : Combining Vermiculture With Traditional Green Waste Composting Systems; J. of Soil Biology and Biochemistry, Vol. 29: pp. 725-730. Frederickson, Jim (2000): The Worm‘s Turn; Waste Management Magazine; August, UK. Frederickson, Jim (2007): Worms are Killing the Planet; Senior Research Fellow at UK Open University Faculty of Technology ; (Viewed on http://www.content.msn.in on 18.7.2007). Ghabbour, S.I. (1996) : Earthworm in Agriculture : A Modern Evaluation; Indian Review of Ecological and Biological Society; 111(2) : 259-271. Guerero, Angelica (2005): ‗Vermicomposting of Garden Waste (Grass Clippings)‘; Project submitted for the partial fulfillment of the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; June 2005. (Supervisor: Rajiv K. Sinha). Graff, O. (1981): Preliminary experiment of vermicomposting of different waste materials using Eudrilus eugeniae Kingberg; In: M. Appelhof (ed.) Proc. of the workshop on ‗Role

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Kristiana, R., Nair, J., Anda, M., and Mathew, K., (2005): Monitoring of the process of composting of kitchen waste in an institutional scale worm farm; Water Science and Technology; Vol. 51 (10): pp. 171-177. Komarowski, S. (2001): Vermiculture for Sewage and Water Treatment Sludge; WATER, July 2001. Kumar, A. Ganesh and G. Sekaran (2004): The Role of Earthworm, Lampito mauritii in removal of Enteric Bacterial Pathogen in Municipal Sewage sludge; Indian J. of Environmental Protection; Vol. 24, No. 2; pp. 101-105. Kale, R.D., and Bano, K. (1986): Field Trials With Vermicompost, An Organic Fertilizer; In Proc. Of National Seminar on ‗Organic Waste Utilization by Vermicomposting‘; GKVK Agricultural University, Bangalore, India. Kale, R.D., Seenappa S.N. and Rao J. (1993): Sugar factory refuse for the production of vermicompost and worm biomass; V International Symposium on Earthworms; Ohio University, USA. Kale, R.D and Sunitha, N.S. (1995): Efficiency of Earthworms (E.eugeniae) in Converting the Solid Waste from Aromatic Oil Extraction Industry into Vermicompost; Journal of IAEM; Vol. 22 (1); pp. 267-269. Kale, R.D. (1998): Earthworms : Nature‘s Gift for Utilization of Organic Wastes; In C.A. Edward (ed). ‗Earthworm Ecology‘; St. Lucie Press, NY, ISBN 1-884015-74-376. Klein, J., Hughes R.J., Nair, J., Anda, M. and G.E. Ho (2005): Increasing the quality and value of biosolids compost through vermicomposting; Paper presented at ASPIRE Asia Pacific Regional Conference on Water and Wastewater, Singapore, 10-15 July 2005. Kaviraj and S. Sharma (2003): Municipal Solid Waste Management Through Vermicomposting Employing Exotic and Local Species of Earthworms; Journal of Bioresource Technology; Vol. 90 : pp. 169-173. Kanaly, R.A., Harayama (2000): Biodegradation of High Molecular Weight PAHs by Bacteria; J. of Bacteriology, Vol. 182: pp.2059-2067. Lakshmi, B.L. and Vizaylakshmi, G.S. (2000): Vermicomposting of Sugar Factory Filter Pressmud Using African Earthworms Species (Eudrillus eugeniae); Journal of Pollution Research; Vol. 19 (3): pp. 481-483. Lotzof, M. (1999): Very Large Scale Vermiculture on Biosolids Beneficiation; What‘s New in Waste Management ? Dec.-Jan. 1998-1999; pp. 22-26. Lotzof, M. (2000): Advances in Vermiculture a New Technique for Biosolids Management : A Case Study and New Research and Development Results; Paper Presented at Watertech, Sydney, Australia.Also ‗Very Large Scale Vermiculture in Sludge Stabilization‘ (Online)www.eidn.com.au/technicalpapervermiculture.htm (Viewed: 5.8.2002). Lotzof, M. (2000): Vermiculture: An Australian Technology Success Story; Waste Management Magazine; February 2000, Australia. Loehr, R.C., Martin, J.H., and Neuhauser, E.F. (1998): Stabilization of Liquid Municipal Sewage Sludge Using Earthworms; In Edward, C.A and Neuhauser E.F. (ed). ‗Earthworms in Waste and Environmental Management‘; SPB Academic Publishing, The Netherlands; ISBN 90-5103-017-7. Martin, J.P. (1976): Darwin on Earthworms: The Formation of Vegetable Moulds; Bookworm Publishing, ISBN 0-916302-06-7.

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Mitchell, M.J., Horner, S.G., and Abrams, B.L. (1980): Decompostion of Sewerage Sludge in Drying Beds and the Potential Role of the Earthworm Eisenia fetida; Journal of Environmental Quality, Vol. 9; pp. 373-378. McCarthy (2002): Beneficial Reuse of Sewage Sludge Vermicompost in Action; Water, March 2002; pp. 72-76. Masciandaro, G., Ceccanti, B. and Garcia, C. (2000): ‗In-Situ‘ Vermicomposting of Biological Sludges and Impacts on Soil Quality; J. of Soil Biology and Biochemistry; Vol. 32 (7): pp. 1015-1024. Morgan, M., Burrows, I., (1982): Earthworms/Microorganisms interactions; Rothamsted Exp. Stn. Rep. Ma, W.C., Imerzeel and Bodt, J. (1995): Earthworm and Food Interactions on Bioaccumulation and Disappearance of PAHs : Studies on Phenanthrene and Flouranthene; J. of Ecotoxicology and Environmental Safety; Vol. 32: 226-232. Markman, Shai., Irina, A. Guschinna., Sara Barnsleya, Katherine L. Buchanana, David Pascoea and Cartsen T. Mullera (2007): Endocrine Disrupting Chemicals Accumulate in Earthworms Exposed to Sewage Effluents; Cardiff School of Biosciences, Cardiff University, Cardiff, U.K.); J. of Chemosphere; Vol. 70 (1): pp. 119 – 125. Middleditch, Richards (2008): Estimation and Analysis of Greenhouse Gases (GHG) from Composting (Aerobic, Anaerobic and Vermicomposting) of Waste; Honours Project, Griffith University, Brisbane. (Supervisors: Andrew Chan and Rajiv K. Sinha). Malley, Christopher, Jaya Nair and Goen Ho (2006). Impact of heavy metals on enzymatic activity of substrate and on composting worms Eisenia fetida; Journal of Bioresource Technology, Vol 97: pp. 1498-1502. Munroe, Glenn (2007): Manual of On-farm Vermicomposting and Vermiculture; Pub. of Organic Agriculture Centre of Canada; 39 p. Neuhauser, E.F., Loehr, R.C. and Malecki, M.R. (1988): The Potential of Earthworms for Managing Sewage Sludge‘; In Edward, C.A and Neuhauser E.F. (ed). ‗Earthworms in Waste and Environmental Management‘; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7. Nair, Jaya., Vanja Sekiozoic and Martin, Anda (2006): Effect of pre-composting on vermicomposting of kitchen waste, Journal of Bioresource Technology, 97(16):20912095. Nair, Jaya., Kuruvilla Mathew and Goen, Ho (2007): Earthworms and composting wormsBasics towards composting applications; Paper at ‗Water for All Life- A Decentralised Infrastructure for a Sustainable Future‘; March 12-14, 2007, Marriott Waterfront Hotel, Baltimore, USA. Ndegwa, P.M. and Thompson, S.A. (2001): Integrated Composting and Vermicomposting in the Treatment and Bioconversion of Biosolids; J. of Bioresource Technology, Vol. 76 (2); pp. 107-112. Nelson, Beyer W.,Chaney, R.L. and Mulhern, B. (1982): Heavy Metals Concentrations in Earthworms from Soil Amended with Sewage Sludge; J. of Environmental Quality; Vol. 11 (3); pp. 382-385. Neilson, R.L. (1951): Earthworms and Soil Fertility; In Proc. Of 13th Conf. Of Grassland Assoc., New Plymouth, U.S; pp. 158 – 167. Neilson, R.L. (1965). Presence of Plant Growth Substances in Earthworms, Demonstrated by the Paper Chromatography and Went Pea Test; Nature, (Lond.) 208 : 1113-1114.

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NCSU (1997): Large Scale Vermi-composting Operations – Data from Vermi-cycle Organics, Inc.; North Carolina State University, U.S. OECD (2000) : Guidelines for Testing Organic Chemicals, Proposal for New Guidelines : Earthworms Reproduction Tests (E.fetida andrei); Organization for Economic Cooperation and Development. (www.oecd.org). Pajon, Silvio (Undated): ‗The Worms Turn - Argentina‘; Intermediate Technology Development Group; Case Study Series 4; (Quoted in Munroe, 2007). (http://www.tve.org./ho/doc.cfm?aid=1450andlang=English) Parish, Z.D., White J.C., Asleyan, M., Gent, M.P.N., Lannucci-Berger, W., Eitzer, B.D., Kelsey, J.W., and Mattina, M.I. (2005): Accumulation of Weathered PAHs by Plant and Earthworms Species; J. of Chemosphere, Vol. (N.A.): pp. (N.A.). Patil, B.B. (1993): Soil and Organic Farming; In Proc. Of the Training Program on ‗Organic Agriculture‘; Institute of Natural and Organic Agriculture, Pune, India. Patil, Swapnil (2005): Vermicomposting of Fast Food Waste; 20 CP Project submitted for the degree of Master in Environmental Engineering; School of Environmental Engineering, Griffith University, Brisbane; November 2005. (Supervisor : Rajiv K. Sinha). Piearce, T.G. and Piearce, B. (1979): Responses of Lumbricidae to Saline Inundation; Journal of Applied Ecology, Vol. 16 (2): pp. pp. 461 – 473. Pierre, V, Phillip, R. Margnerite, L. and Pierrette, C. (1982): Anti-bacterial activity of the haemolytic system from the earthworms Eisinia foetida andrei; Invertebrate Pathology, 40, pp. 21-27. Parvaresh, A. et, al., (2004): Vermistabilization of Municipal Wastewater Sludge With E.fetida; Iranian J. of Environmental Health, Science and Engineering; Vol. 1(2): pp. 4350. Palanisamy, S. (1996): Earthworm and Plant Interactions; Paper presented in ICAR Training Program; Tamil Nadu Agricultural University, Coimbatore. Ryan, David (2006): Vermiremediation and Other Bioremediation Options for PAHs Contaminated Soil; Project Report submitted for the partial fulfillment of the degree of Bachelor of Environmental Engineering, School of Engineering, Griffith University, Brisbane. (Supervisor : Rajiv K. Sinha). Riggle, D. and Holmes, H. (1994): New Horizons for Commercial Vermiculture; Biocycle, Vol. 35 (10); pp. 58-62. Singleton, D.R., Hendrix, B.F., Coleman, D.C., Whitemann, W.B. (2003): Identification of uncultured bacteria tightly associated with the intestine of the earthworms Lumricus rubellus; Soil Biology and Biochemistry; Vol. 35: pp. 1547-1555. Saxena, M., Chauhan, A., and Asokan, P. (1998): Flyash Vemicompost from Non-friendly Organic Wastes; Pollution Research, Vol.17, No. 1; pp. 5-11. Satchell, J. E. (1983) : Earthworm Ecology- From Darwin to Vermiculture; Chapman and Hall Ltd., London; pp.1-5. Seenappa, S.N. and Kale, R. (1993): Efficiency of earthworm Eudrillus eugeniae in converting the solid wastes from the aromatic oil extraction units into vermicompost; Journal of IAEM; Vol. 22; pp.267-269. Seenappa, S.N., Rao, J. and Kale, R. (1995): Conversion of distillery wastes into organic manure by earthworm Eudrillus euginae; Journal of IAEM; Vol. 22; No.1; pp.244-246. Sinha, Rajiv. K., Sunil Herat, Sunita Agarwal, Ravi Asadi, and Emilio Carretero (2002): Vermiculture Technology for Environmental Management : Study of the action of the

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earthworms Eisinia fetida, Eudrilus euginae and Perionyx excavatus on biodegradation of some community wastes in India and Australia; The Environmentalist, U.K., Vol. 22, No.2. June, 2002; pp. 261 – 268. Sinha, Rajiv K. (2004): Sustainable Agriculture; Surabhee Publication, Jaipur, India; ISBN 81-86599 – 60 – 6; p.370. Sinha, Rajiv K., Sunil Herat, P.D. Bapat, Chandni Desai, Atul Panchi and Swapnil Patil (2005): Domestic Waste - The Problem That Piles Up for the Society: Vermiculture the Solution; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, Canada; pp. 55-62. Sinha, Rajiv K. and Rohit Sinha (2007). Environmental Biotechnology (Role of Plants, Animals and Microbes in Environmental Management and Sustainable Development); Aavishkar Publisher, Jaipur, India; ISBN 978-81-7910-229-9; p. 315. Sinha, Rajiv K. and Gokul Bharambe (2007): Studies on Vermiculture Technologies (Vermicomposting, Vermi-desalinization and Vermi-agroproduction); Center for Environmental Systems Research (CESR) Project, Griffith University, Nathan Campus, Brisbane, Australia. Sharma, Reena (2001) : Vermiculture for Sustainable Agriculture : Study of the Agronomic Impact of Earthworms and their Vermicompost on Growth and Production of Wheat Crops; Ph.D. Thesis, submitted to the University of Rajasthan, Jaipur, India. (Supervision: Dr. Rajiv K. Sinha). Standards Australia (1995 a): Australian StandardsTM Method 6; Thermotolerant Coliforms and Escherichia coli – Estimation of Most Probable Number (MPN), AS 4276.6. Sinha, Rajiv K. (2004): Sustainable Agriculture; Surabhee Publication, Jaipur, India; ISBN 81-86599 – 60 – 6; p.370. Standards Australia (1995 b): Australian StandardsTM Method 8; Water Microbiology – Faecal streptococci - Estimation of Most Probable Number (MPN), AS 4276.8. Standards Australia (1995 c): Australian StandardsTM Method 14; Water Microbiology – Salmonellae- Estimation of Most Probable Number (MPN), AS 4276.14. Safawat, H., Hanna, S., Weaver, R.W. (2002): Earthworms Survival in Oil Contaminated Soil; J. of Plant and Soil; Vol. 240: pp. 127-132. Sims, R.C. and Overcash, M.R. (1983): Fate of Polynuclear Aromatic Hydrocarbons (PNAs) in Soil-Plant Systems; Residue Reviews, Vol. 88: pp. 2-68. Sharma, Reena (2001) : Vermiculture for Sustainable Agriculture : Study of the Agronomic Impact of Earthworms and their Vermicompost on Growth and Production of Wheat Crops; Ph.D. Thesis, submitted to the University of Rajasthan, Jaipur (Supervised by Dr. R.K. Sinha). Sadhale, Nailini (1996) : (Recommendation to Incorporate Earthworms in Soil of Pomogranate to obtain high quality fruits); In Surpala‘s Vrikshayurveda, Verse 131. (The Science of Plant Life by Surpala, 10th Century A.D.); Asian Agri-History Bulletin; No. 1. Secunderabad, India. Subler, Scott., Edwards, Clive., and Metzger, James (1998): Comparing Vermicomposts and Composts; Biocycle, Vol. 39: pp. 63-66. Suhane, R.K. (2007): Vermicompost; Publication of Rajendra Agriculture University, Pusa, Bihar, India. Suhane, R.K, Sinha, Rajiv.K. & Singh, Pancham.K. (2008): Communication of Rajendra Agriculture University, Pusa, Bihar, India.

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Tang, J., Liste, H., Alexander, M. (2002): Chemical Assays of Availability to Earthworms of PAHs in Soil; J. of Chemosphere, Vol.48: pp. 35-42. Tomati, V., Grappelli, A., Galli, E. (1983) : Fertility Factors in Earthworm Humus; In Proc. of International Symposium on ‗Agriculture and Environment : Prospects in Earthworm Farming; Rome, pp. 49-56. Tomati, V.; Grappelli, A. and Galli, E. (1987) : The Presence of Growth Regulators in Earthworm - Worked Wastes; In Proceeding of International Symposium on ‗Earthworms‘; Italy; 31 March- 5 April, 1985; pp. 423-436. Tomati, V. and Galli, E. (1995): Earthworms, Soil Fertility and Plant Productivity; Acta Zoologica Fennica; Vol. 196, pp. 11-14. Taylor et. al (2003): The treatment of domestic wastewater using small-scale vermicompost filter beds; Ecological Engineering; Vol. 21; pp. 197-203. Toms, P. Leskiw, J., and Hettiaratchi, P. (1995): Greenhouse Gas Offsets : An Opportunity for Composting; Presentation at the 88th Annual Meeting and Exhibition, June 8-12, 1995, San Antonio, Texas, USA; pp. 18-23. UNSW, ROU (2002 a): Vermiculture in Organics Management- The Truth Revealed; (Seminar in March 2002) University of New South Wales Recycling Organics Unit; Sydney, NSW, Australia. UNSW, ROU (2002 b): Best Practice Guidelines to Managing On-Site Vermiculture Technologies; University of New South Wales Recycling Organics Unit; Sydney, NSW, Australia; (Viewed on December 2004) www.resource.nsw.gov.au/data/ Vermiculture%20BPG.pdf USEPA (1995): A Guide to the Biosolids Risk Assessment for the EPA; Part 503 Rule EPA/B32-B-93-005; US Environmental Protection Agency Office of Wastewater Management, Washington, D.C. Valani, Dalsukh (2008): Studies in Vermiculture Biotechnology; 40 CP Honours Project, Griffith University, Brisbane, Australia (Supervisors: Rajiv K. Sinha & Sunil Herat) Vermitech (1998): Successful Biosolids Beneficiation With Vermitech‘s Large-Scale Commercial Vermiculture Facility in Redlands; Waste Disposal and Water Management in Australia; Vol. 25 (5); September-October, 1998. White, S. (1997): A Vermi-adventure in India; J. of Worm Digest; Vol. 15 (1): pp. 27-30. Wang, Y.S., Odle, WSI, Eleazer, W.E., and Baralaz, M.A (1997): Methane Potential of Food Waste and Anaerobic Toxicity of Leachate Produced During Food Waste Decomposition; Journal of Waste Management and Research; Vol. 15: pp. 149-167. Wu, XL., Kong, HN, Mizuochi, M., Inamori, Y., Huang, X., and Qian, Y. (1995): Nitrous Oxide Emission from Microorganisms; Japanese Journal of Treatment Biology, Vol. 31 (3): pp. 151-160. Wu, N., and Smith, J.E., (1999): Reducing Pathogen and Vector Attraction for Biosolids; Biocycle; Vol. (November 1999); pp. 59-61. Xing M., Yang, J. and Lu, Z. (2005): Microorganism-earthworm Integrated Biological Treatment Process – A Sewage Treatment Option for Rural Settlements; ICID 21st European Regional Conference, 15-19 May 2005; Frankfurt; Viewed on 18 April 2006. <www.zalf.de/icid/ICID_ERC2005/HTML/ERC2005PDF/Topic_1/Xing.pdf> Yaowu, He., Yuhei, Inamori., Motoyuki, Mizuochi., Hainan, Kong., Norio, Iwami., and Tieheng, Sun (2000): Measurements of N2O and CH4 from Aerated Composting of Food Waste; J.of the Science of The Total Environment, Elsevier, Vol. 254: pp. 65-74.

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Dr. Rajiv Sinha with his vermiculture team at Griffith University, Australia.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 3

HUMAN WASTE - A POTENTIAL RESOURCE: CONVERTING TRASH INTO TREASURE BY EMBRACING THE 5 R‘S PHILOSOPHY FOR SAFE AND SUSTAINABLE WASTE MANAGEMENT Rajiv K. Sinha1, Sunil Herat2, Gokul Bharambe3, Swapnil Patil3, Pryadarshan Bapat3, Krunal Chauhan3 and Dalsukh Valani3 1

School of Engineering (Environment), Griffith University, Nathan Campus, Brisbane, QLD-4111, Australia 2 School of Engineering (Environment), Griffith University 3 School of Engineering (Environment), Griffith University

Keywords: Culture of Consumerism; Culture of Disposables; Packaging Culture; Australians and Americans as Superconsumers and Waste Generators; Waste – A Misplaced Resource; Traditional Societies – Recycling Societies; Modern Society – Throwaway Society; Waste – Source of Greenhouse Gases; Vermiculture Movement for Efficient and Cost-effective Waste Management.

1. INTRODUCTION Waste is being generated by the human societies since ancient times. Ironically waste was not a problem for the environment when men were primitive and uncivilized. Waste is a problem of the modern civilized society. Materials used and waste generated by the traditional societies were little and ‗simple‘ while those by the modern human societies are large and ‗complex‘. With modernization in development drastic changes came in our consumer habits and life-style and in every activity like education, recreation, traveling, feeding, clothing and housing we are generating lots of wastes. The world today generate 

Corresponding Author: [email protected]

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about 2.4 billion tones of solid waste every year in which the Western World alone contributes about 620 million tones / year. Discarded products arising from all human activities (cultural and developmental) and those arising from the plants and animals, that are normally solid or semi-solid at room temperature are termed as solid wastes. Municipal solid waste (MSW) is a term used to represent all the garbage created by households, commercial sites (restaurants, grocery and other stores, offices and public places etc.) and institutions (educational establishments, museums etc.). This also includes wastes from small and medium sized cottage industries. We are facing the escalating economic and environmental cost of dealing with current and future generation of mounting municipal solid wastes (MSW), specially the technological (developmental) wastes which comprise the hazardous industrial wastes, and also the health cost to the people suffering from it. Developmental wastes poses serious risk to human health and environment at every stage – from generation to transportation and use, and during treatment for safe disposal. Another serious cause of concern is the emission of greenhouse gases methane and nitrous oxides resulting from the disposal of MSW either in the landfills or from their management by composting. Dealing with solid household waste in more sustainable ways involves changes not only to everyday personal habits, consumerist attitudes and practices, but also to the systems of waste management by local government and local industry and the retailers. This chapter reviews the causes and consequences of escalating human waste, the increasing complexity of the waste generated, and the policies and strategies of safe waste management. It also provides ‗food for thought‘ for future policy decisions that government of nations may have to take to ‗reduce waste‘ and divert them from ending up in the landfills, drawing experiences from both developed nation (Australia) and a developing nation (India).

2. CITIES AS THE CENTERS OF MOUNTING MUNICIPAL WASTES Cities have become major ‗centers of consumption and waste generation‘ all over the world. In fact a city ‗consumes‘ as well as ‗produce‘. This is called ‗urban metabolism‘. City use some 75 % of world resources and release a similar proportion of wastes. According to UN Population Fund Report (1990), a city with one million population consumes 2000 tones of food and 9,500 tones of fuel, generating 2000 tones of solid wastes (garbage and excreta) and 950 tones of air pollutants; consumes 6,25,00 tones of pure water and secrete 5,00,000 tones of sewage. (UNEP, 1996). United Nation Environment Program (UNEP) worked out the urban metabolism of London city. Greater London with a population of 7 million consumed 2,400,000 tones of food; 1,200,000 tones of timber; 2,200,000 tones of paper; 2,100,000 tones of plastics; 360,000 tones of glass; 1,940,000 tones of cement; 6,000,000 tones of bricks, blocks, sand and tarmac; 1,200,000 tones of metals every year and produced 11,400,000 tones of industrial and demolition wastes; 3,900,000 tones of household, civic and commercial wastes and 7,500,000 tones of wet, digested sewage sludge. Everyday London dispose off some 6,600 tones of household wastes. (UNEP, 1996).

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3. MODERN CULTURE OF CONSUMERISM: THE ROOT OF WASTE PROBLEM The root of waste problem is the ‗culture of consumerism‘ and is directly proportional to the affluency of the human societies. To this has added the ‗culture of disposables‘. Large number of goods in the society are being manufactured for only ‗one time use‘, and to be discarded as waste after use. Modern urban culture of using ‗canned and bottled foods‘, ‗frozen foods‘, ‗take-away foods‘, exchange of ‗greeting cards‘ on all occasions, using ‗disposable‘ home equipments (spoons, cups, plates, tumblers and safety razors), medical instruments (syringes, sharps and needles), office equipments (writing pens and utilities), and plastic bags in all grocery shopping has escalated the solid waste problems. People all over the world consume food and the 5 P‘s (paper, power, petrol, potable water and plastics) in their daily life without realizing the environmental consequences and costs of the waste generated in their production, distribution, and consumption. Every commodity processed from natural resources, and every consumer product, from ‗shampoo to champagne‘ has an environmental cost and generally causes some damage to the environment, ‗before use‘, as a source of pollution during production, and ‗after use‘, as waste (Eklington and Hailes, 1989).

3.1. Waste Generation Is Proportional to Resource Consumption and the Way We Use Resources Consuming resources and generating wastes are ‗two sides of the same coin‘. The way we use resources to maintain our ‗quality‘ of life, assumes as much significance in waste generation as the sheer amount of resources that we use. For instance, one kilogram of steel might be used in a construction that lasts hundreds of years or in the manufacture of several cans thrown away just after a few uses. A few kilograms of PVC plastic materials might be molded into durable home and office furniture, water and sewer pipes, and remain useful for decades or might be used to manufacture plastic bags to be used just once or twice and then thrown away as enduring waste.

Packaging Culture Proliferate Waste Generation Packaging materials have become part of our modern culture and generate huge amounts of waste. Manufacturers and retailers see packaging as a way to attract purchasers. Everything needs fine packaging today, from cornflakes to computers, from gifts to garments, from flowers to foods. Life today cannot be imagined without plastic bags, glass bottles, paper boxes, tins and cans. Plastics are versatile, convenient and light weight — good packaging material — but ultimately end up as a non-biodegradable waste in the landfills to remain intact for centuries. On average each European Union citizen is currently responsible, directly or indirectly, for the generation of some 172 kg of ‗packaging waste‘ every year. Packaging waste generation increased by 10 % in the EU between 1997 and 2002. Per capita consumption of plastics increased by almost 50 % from 64 kg / year in 1990 to 95 kg / year in 2002. Only UK

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managed to actually reduce, and Austria stabilize the generation of packaging waste since 1997. (GEO, 2006). Plastic consumption in Australia has increased from negligible quantities in the early 1940s to enormous quantities today. Plastics made up around one-third of all rubbish collected on Clean Up Australia Day (Clean Up Australia 2004). Even though Australians reduced their use of plastic shopping bags by around one-fifth between 2002 and 2004, each person was still using almost one bag per day (EcoRecyle 2006). Australians use 6 billion plastic bags every year much of that end up in landfills. These bags form litter, infest and block waterways, kill animals.

The ‘Ecological Footprint’ of Global Human Population The Measure of Resource Consumption and Waste Generation. An ecological footprint is an estimate of the average area of productive land and water required to maintain a given population‘s resource consumption and waste generation. (Table 1). In this instance we simply use it to indicate the comparable cost and sheer significance of waste generation. Australia with a small population of just 19.5 million, is part of the North developed world, and Australians are ‗super-consumers‘ and ‗super-waste makers‘. Table 1. Ecological Footprints of Human Population on Earth Reflecting Resource Consumption Vis-à-Vis Waste Generation (2002) Total Population (millions) World High income˚ countries Middle income countries Low income countries Australia United Kingdom China Asia Pacific (regional) Canada USA

6 225.0 925.6 2 989.4 2 279.8 19.5 59.3 1302.3 3448.4 31.3 291.0

Total Ecological Footprint (global ha/person) 2.2 6.4 1.9 0.8 7.0 5.6 1.6 1.3 7.5 9.7

Total Energy Footprint (global ha/person) 1.2 4.1 0.9 0.3 4.0 3.6 0.7 0.6 4.6 6.3

Total Biocapacity* (global ha/person) 1.8 3.4 2.1 0.7 11.3 1.6 0.8 0.7 15.1 4.7

Source: Global Footprint Network (2005). *Biocapacity includes cropland, grazing land, forest and fishing ground. ˚High income countries includes Australia.

In developing Asian countries, consumption and waste generation is growing at an unprecedented pace and populous countries like China and India are becoming consumerist. The ‗consuming class‘ in India is estimated at around 100 million, and in China at 200 million; traditional conservative Indians believing in modesty, simple living and saving, are gradually giving way to rich generations highly influenced by the consumerist North (UNEP, 2006). China manufactures, packages, transports, distributes nationally and for export over 60 billion pairs of disposable wood chopsticks which use up over 32 million trees, harvested

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unsustainably, indeed China risks being without forests within ten years.().

4. WASTE GENERATED IN THE RICH VIS-A-VIS IN THE POOR SOCIETIES OF WORLD The United Nations Environment Program (1992) carried out a study of per capita waste generation in the low, middle and the high-income countries and found it to be 0.5, 1.5 and 3.5 kg. per day per person respectively. The solid wastes generated in the rich affluent societies of the developed nations are exceptionally large in quantity and varied in quality (components). Of these, a considerable part is hazardous waste.

4.1. Waste Generated in the Rich Developed Countries of World The World Watch Institute (1991), Washington, reported that 14 out of 16 members of OECD countries showed increase in generation of MSW per person between 1980 – 85. In the US, each sunset sees a new mountain of nearly 410,000 tones of garbage. (Toth, 1990). The countries of the European Community (EC) throws away an estimated 2 billion tones of solid waste each year. Only Japan and West Germany produced less waste, but after unification MSW in Germany skyrocketed. Americans, Canadians and Australians are great waste makers. They generate roughly twice as much garbage per person as West Europeans or Japanese do. In his /her lifetime an average American wear and discard 250 shirts and 115 pairs of shoes; use and discard 27,50 newspapers, 3900 weekly magazines and 225 pounds of phone directories; consume 12,000 paper grocery bags, use 28,627 aluminum cans weighing 1022 pounds, use 69,250 pounds of steel and 47,000 pounds of cement. The Scandinavian nations generate much less waste than the Americans and Europeans. (WWI, 1991; UNEP, 1996). Table 2. Average Per Capita Municipal Solid Waste (MSW) Generated by Some Developed Nations Country Waste Generation Per Day (in Kg) Country USA Japan France Singapore Germany Italy

Waste Generation Per Day (in Kg) 1.80 – 2.60 1.38 - 2.10 1.10 – 1.90 0.87 – 1.37 0.75 – 1.85 0.69 – 1.75

Source: WWI (1990):‗State of the World‘ (These are 1990 Values which must have increased).

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Urban Waste Generated in Some Developed (Rich) Countries Country Annual Generation (In tones) USA 20,00,00,000 Canada 1,26,00,000 Australia 1,00,00,000 Spain Netherlands 89,28,000 54,00,000 Belgium Sweden 30,82,000 25,00,000 Switzerland 21,46,000 Denmark 20,46,000 Norway New Zealand 17,00,000 15,28,000 Finland 12,00,000 Source: Dorling Kindersley ‗Blueprint for Green Planet‘ ;London (1987).

4.2. Waste Generated in the Poor Developing Countries of World In the low and middle income developing countries of Asia, Asia-Pacific and Africa, waste is a luxury, only produced by the wealthy minority which of course is increasing with the growing economy and the exploding population. What is most concerning is that the waste is not regularly collected by the municipal authorities and often becomes a horrible site of piled and rotting waste on street corners with stray animals (dogs, pigs and cows) feeding on the scraps. Domestic waste heaps often becomes sites of defacation and discharge of human excreta and illegal dumping of hazardous wastes by scrupulous industries with potential health threats. Municipal waste services often swallow between a fifth and a half of city budgets, yet much solid waste is not removed. Even if municipal budgets are adequate for collection, safe disposal of collected wastes often remains a problem. (Holmes, 1984). Another ugly feature in these countries are that waste is often picked up by poor people called ‗rag-pickers‘ for whom waste reuse and recycling is a way of life, and many poor societies survive here by scouring the garbage of the rich for valuable scraps. They collect recyclable wastes (mainly papers, plastics, glasses and metals) from the street corners and even the dumpsites and sell them to the recycling industries to earn for their livelihood. Table 4. Average Per Capita Municipal Solid Waste (MSW) Generated by Some Developing (Poor) Nations Country Waste Generation Per Day (in Kg) Country Pakistan Indonesia India Nigeria

Waste Generation Per Day (in Kg) 0.25 – 0.60 0.33 – 0.55 0.15 – 0.51 0.16 – 0.46

Source:WWI (1990);‘State of the World‘ (These are 1990 Values which must have increased).

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4.3. Typical Waste Components in the MSW Generated in Both Rich Developed and Poor Developing and Underdeveloped Nations Several waste components have been identified in the municipal solid waste (MSW) of both the rich and poor societies of world. It ranged from twelve (12) to fourteen (14) and a considerable portion of this waste is ‗Organic‘ while others are ‗Inorganic.‘ Ironically, there is relatively high amount of food waste in poor developing and underdeveloped nations as compared to the rich developed nations. In contrast, there is high amount of paper and garden wastes in rich nations and low in poor nations. However, very little (or none) food waste finally reach the waste dump-sites in poor nations as they are scoured and scavenged by stray animals – pigs, dogs and cattle and even by the poor street beggars. Paper, cardboard, plastics, leather, wood and metals also do not reach the dump-sites and are picked up by ‗rag-pickers‘. (WHO, 1976). Table 5. Solid Waste Components in MSW of Low, Middle and High Income Countries (In % age) Waste Components Organic Food Waste Paper Cardboard Plastics Textiles Rubber Leather Garden Wastes Wood Inorganic Glass Tin cans Aluminum Other Metals Dirt, Ash etc.

Low-Income

Middle-Income

High-Income

40 – 85 1 – 10 1–5 1–5 1–5 1–5 1 – 10 1–5 1 – 40

20 – 65 8 – 30 2–6 2 – 10 1–4 1 – 10 1 – 10 1–5 1 – 30

6 – 30 20 – 40 5 – 15 2–8 2–6 0–2 0–2 10 – 20 1–4 4 – 12 2–8 0–1 1–4 0 – 10

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995). Low Income = Underdeveloped African, Asian and Pacific Nations; Middle Income = Developing Asian, African, Pacific and South American Nations. High Income = Developed European and North American Nations and Australia.

Table 6. Typical Solid Waste Components in the MSW of an European Society Waste Components Percentage (%) Waste Components 1. Paper and paper products 2. Metals 3. Glass 4. Plastics

Percentage (%) Germany 19.9 8.7 11.6 6.1

Switzerland 26.6 5.6 11.5

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5. Textiles 6. Minerals 7. Wood, leather, bones, rubber 8. Compounded materials 9. Sieving fractions (0-12 mm) 10. Sieving fractions (12-50 mm) 11. Residue 12. Total unidentified fraction

1.5 2.9 2.3 0.8 8.6 15.6 26.8 51.0

2.8 1.1 3.1 0.7 9.2 8.1 27.1 44.4

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Table 7. Typical Solid Waste Components in the MSW in U.S. Society (in % age) Organic 1 Food Waste 2. Paper 3. Cardboard 4. Plastics 5. Textiles 6. Rubber 7. Leather 8. Yard waste

9.0 34.0 6.0 7.0 2.0 0.5 0.5 8.5

Inorganic 10. Glass 11. Tin cans 12. Aluminum 13. Other Metal 14. Dirt, Ash etc.

8.0 6.0 0.5 3.0 3.0

Total = 100.00

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995)

5. CHANGING CHARACTER OF THE MSW IN MODERN SOCIETY: INCREASING AMOUNTS OF TOXIC MATERIALS The character and composition of the municipal solid wastes (MSW) are changing in the modern human society. Significant changes have occurred in the composition of municipal solid waste (MSW) ever since the technological revolution of the 20th century. They are no longer only ‗organic‘ waste, as it used to be in the earlier societies. The technological development which mainly influenced the character of the MSW was the fossil fuel driven ‗industrial revolution‘ and the agro-chemicals driven ‗green revolution‘. Waste components that have an important influence on the composition of the MSW are food waste, paper and plastic wastes, the white goods and the hospital wastes. What is the matter of more serious concern is that all ‗living organisms‘ including the human beings have become exposed to chemicals for which there has been no evolutionary adaptation and experience. The chemicals in the hazardous wastes mixed up with the MSW are completely ‗foreign‘ to living organism. (Sinha and Sinha, 2000).

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5.1. Changing Quality and Quantity of Food Wastes in the MSW The quantity and quality of residential food waste has changed significantly over the years as a result of technical advances in food growing (use of agro-chemicals) and food processing and packaging (use of chemical preservatives), and public attitudes towards food procurement i.e. relying more on processed and packed takeaway foods rather than cooking food at home from raw materials. However, due to education and awareness about the nutritive values of home cooked food, now people are moving back again towards home cooking. Two technological developments that have had a significant effect on generation of food waste are the development of the ‗food processing and packaging industries‘ and the ‗use of kitchen food waste grinders‘ in modern homes. Because of kitchen grinders the grinded food wastes are delivered directly into the sewer systems rather than being disposed as MSW. In these modern homes, the percentage of food wastes, by weight, has decreased from about 14 % in the early 1960s to about 9 % in 1992. In the packed and takeaway food culture, the generation of food wastes in homes have reduced, but it has increased significantly in the food processing industries and the food outlets. In homes, and the food outlets there are more paper and plastic wastes due to over-packaging of processed foods, than food wastes itself.

5.2. Proliferating Plastic Wastes in the MSW Percentage of plastics in MSW has also increased tremendously during the last 50 years. The use of plastic has increased from almost non-measurable quantities in the 1940s to between 7 - 8 %, by weight, in 1992. It is anticipated that the use of plastics will continue to increase, but at a slower rate than during the past 25 years. (UNEP, 1996).

5.3. Escalating Electronics Waste in MSW: Heading for E-waste ‗Tsunami‘? Electronic waste is a growing concern as technology changes and new generations of electronic products and equipments more sophisticated, improved and upgraded versions continue to invade the market and the minds of consumer‘s. The unfortunate part is that the price of the new models and upgraded versions is continuously falling giving more temptation to the consumers for discarding the old ones and replacing with the new. The UNEP working group on Sustainable Product Design described the e-waste essentially as a chemical waste. Electronics industry uses several hazardous chemicals including toxic heavy metals (lead, cadmium, mercury, chromium, barium etc.), acids and plastics, chlorinated and brominated compounds in production process. Developers of electronic products are introducing chemicals on a scale which is totally incompatible with the scant knowledge of their environmental or biological characteristics. (O, Rourke, 2004). More than 2 million tons of e-waste ends up in landfills every year and there is serious threat of leaching lead (Pb) and other heavy metals that may seep into groundwater supplies. Incineration results into emission of dangerous dioxins and furans as e-waste contain considerable amounts of plastics, brominated and chlorinated compounds. (Sinha, et. al., 2006).

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Recent study indicate that e-waste make up approximately 1 % of the MSW waste stream in all developed nations and mercury (Hg) from the e-waste has been cited as the main source of this heavy metal in the general MSW. E-waste in MSW is creating serious health and environmental problems for the MSW landfills and the waste workers, and for the MSW incinerators. In Europe the e-waste is growing at three times the rate of other MSW and mixing with it. ‗USA today is virtually sitting on a mountain of obsolete PCs‘. A report produced by the Silicon Valley Toxics Coalition (a grassroots coalition that performs research and advocacy on health and environmental issues related to electronics industries in the U.S.) in 2001 suggest that if all the consumers decided to throw out their obsolete computer at the same time, the country would face a ‗tsunami‘ of e-waste scraps between 2006 and 2015. The report called ‗Poison PCs and Toxic TVs‘ was released by another grassroots organization California Against Waste (CAW). (Anonymous, 1999).

Developing Countries as the Electronic Junkyards of U.S. and Industrialized Nations : An Untenable Choice Between Poverty and Poison There are reports about Asia, mainly India, China, Taiwan, Vietnam, Singapore and Pakistan, being made as the high-tech dumping ground of U.S. An estimated 20 million computers become obsolete each year in the U.S. and an estimated 200 tons of these computers end up in these countries in the name of ‗reuse‘ and ‗recycling‘. Low labor cost and weak environmental regulations have made these countries dumping grounds of e-waste destined for recycling and final disposal in landfills. A pilot program that collected electronic scrap in San Jose, California estimated that it was10 times cheaper to ship CRT monitors to China than it was to recycle them in the U.S. It is still legal in the U.S., despite international law (The Basel Convention, 1989) to the contrary, to allow export of hazardous waste without controls. (It may be recalled that U.S. has not yet signed the Basel Convention (1989) which prohibits trans-boundary movement of hazardous wastes). Industry insiders indicate that about 80 % of the e-waste goes to Asia and of that 90 % ends up in China. (Puckett et. al, 2002) A report by Basel Action Network and the Silicon Valley Toxics Coalition ‗Exporting Harm: The Techno-Trashing of Asia‘ asserts that 50 to 80 % of e-waste collected for recycling in the U.S. is exported to developing nations. BAN produced a film on the report which shows the Guiyu village in Guangdong province in China as ‗electronics junkyard‘. Some 100,000 men, women and children make US $1.50 a day dismantling e-waste by bare hands to retrieve the valuable metals and materials. Circuit boards are melted over coal grills to release valuable metals giving highly toxic dioxin fumes. Riverbank acid baths are used to extract gold. Lead-containing cathode ray tubes from monitors and televisions are not of much market value and hence are dumped in some wastelands. Toner cartridges are pulled apart manually, sending clouds of toner dust into the air. Soil and drinking water at Guiyu are contaminated by lead much above WHO limits- soil by 200 times and water by 2,400 times. Water has to be trucked from 30 km away. At one point of time both China and India were willing to take the e-waste for almost free. For poor countries of the world it is an untenable choice between ‗poverty and poison‘. (Puckett et. al, 2002) In November, 2002 officials from eight Asian nations met in Tianjin, China, under the auspices of Basel Convention (1989) to prevent their nations from being made the dumping grounds of hazardous e-waste in the name of free trade (export and import) for recycling discarded electronic products. It was represented by India, Malaysia, the Philippines,

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Singapore, Sri Lanka, Thailand, and Vietnam. Resource persons came from Canada, Japan, the U.S. and the Secretariat of the Basel Convention. Financial support was provided by Australia, Japan and Canada. China has now banned and India also follows. (Sinha, et. al., 2006).

5.4. The White Goods and Bulky Items in MSW There are ‗bulky items‘ which include large worn-out or broken household, commercial or industrials goods such as furniture, lamps, bookcase, filing cabinets, etc. There are ‗consumer electronics‘ wastes which include worn-out, broken, and no longer wanted items like stereos, radios, computer and television sets. There are ‗white goods‘ as waste which include worn-out and broken household, commercial and industrial appliances like stoves, refrigerators, dishwashers, cloth washers and driers. The rejected auto-parts like tires, batteries and accessories also constitute important constituents of MSW. About 230 to 240 million rubber tires are disposed off annually in landfills or in tire stockpiles. (UNEP, 1996).

5.5. The Biomedical Wastes in MSW The biomedical waste from hospital and clinics and slaughterhouses contain about 85 % as general refuse, but 10 % is hazardous wastes contaminated with infectious pathological agents, and 5 % is non-infectious but potentially toxic (chemicals) and radioactive and hence hazardous. Dressing and swabs contaminated with blood and body fluids; syringes, needles and sharps; surgically removed placenta, tissues, tumors, organs or limbs are potentially infected wastes from hospitals. There are several ‗disposable‘ items made of PVC and thermocol now being used in medical organizations. Hospital wastes are of special category and require special care for final disposal. WHO has provided strict guidelines for their safe disposal. (WHO, 1976).

6. THE COMPLEX SYNTHETIC WASTES INVADING HUMAN ENVIRONMENT Human ingenuity has created some ‗new and synthetic materials‘ in the wake of technological revolution. They contain both organic and inorganic chemicals and resins and creates more ‗complex‘ type of waste after being discarded. Nature do not possess any organism and mechanism to biodegrade them. The processing of some new materials discovered by technology such as ‗semiconductors‘, ‗optical fibers‘, new class of ‗ceramics‘, and ‗composites‘ requires the use of large amount of toxic chemicals which eventually ends up as hazardous wastes in the MSW. These technological wastes are often toxic and are posing danger not only for the environment, but also for the human health. It has already caused several accidents, deaths and disabilities among the municipal waste workers.

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6.1. The Non-Biodegradable Wastes: Potential to Remain Long in Human Ecosystem The synthetic wastes are ‗non-biodegradable‘ because they cannot be decomposed and can remain in the human ecosystem for years and decades polluting the environment. Common examples are all forms of plastics, x-ray films, celluloid films, cells and batteries, several chemicals and all synthetics. What nature cannot do, human beings are trying to do through the knowledge of environmental biotechnology. Genetically tailored bacteria are being created which would possess the necessary enzymes to degrade the synthetic wastes. Some strains of bacteria and fungi have been identified in nature too, which has the arsenal to degrade some of the complex organic chemicals. Efforts are also being made to create ‗biodegradable synthetics‘ using ‗starch‘ as the raw material. They can be degraded in 4-6 weeks. (Sinha and Sinha, 2007).

6.2. The Hazardous Wastes: Permeating the Human Society Wastes containing toxic chemicals, radioactive substances and infectious materials which poses potential risk to human health and environment are categorized as ‗hazardous wastes‘. Toxicity, radioactivity, flammability, chemical reactivity, corrosivity, nonbiodegradability, carcinogenicity, mutagenicity, infectiousness, oxidizing and leachating are some of the characteristics of hazardous wastes. (WHO, 1983). Many primary and manufacturing industries using toxic chemicals generate hazardous waste (solid or liquid) in the production process. They can be referred as ‗industrial hazardous wastes‘ (IHW). Many of our favorite cultural activities depend on products the manufacture of which creates industrial hazardous waste. Glaring examples are ‗glass and metal‘, ‗paper and plastic‘, ‗leather and textile‘, ‗painting and dyeing‘, ‗printing and publication‘ and ‗photography and dry cleanings‘. Consumer industries today use a variety of chemicals to produce ‗consumer goods‘ a number of which are now ‗disposables‘. When these items are consumed and discarded by society, they eventually end up as hazardous wastes in our homes. This can be referred as ‗household hazardous waste‘ (HHW). Prime examples are torch dry-cells and batteries, pesticides / disinfectant cans and bottles, fluorescent tubes and electric bulbs, detergents and shampoos, lead-acid car batteries, auto tires and waste oils, and the expired medicines. As we enjoy the benefits of consumer goods (furniture and fixtures, white goods, electrical and electronic goods, automobiles, processed and packed food and drinks etc.) produced by consumer industries, we also generate considerable amount of hazardous wastes as by-products when we discard them after use or change the ‗old‘ version with ‗new‘. They can be referred as ‗consumer hazardous waste‘ (CHW). Industries producing products that sustain our modern life-style and living habits generate tremendous amount of hazardous wastes. Glaring examples are the ‗agro-chemical industries‘ (to boost our food production) and the ‗petroleum industries‘ (which drives our automobiles). (Raghupati, 1994; Sinha and Herat, 2004).

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Household Hazardous Wastes (HHW): The Poison in Our Homes Household hazardous wastes are either solids, semi-solids, or liquids. In addition trace chemical compounds can exist as a solute within a liquid solvent, as a gas adsorbed onto a solid, or as a component of the gaseous emissions from MSW. Plastics contain organochlorine compounds, organic solvents in PVC; paints contain heavy metals, pigments, solvents and organic residues; home pesticides contain organochlorine and organophosphates compounds; oil and gasoline contain phenols and organic compounds, heavy metals, salt acids, ammonia and caustics; textiles may have metal dyes and organochlorine compounds; carpets can contain chemical stain resisters, pesticides, solvent-heavy glues, VOCs laden underlayer; curtain treated with stain resisters and backing can give off VOCs; dry-cleaned clothes give off VOCs; chip board and plywood furniture releases formaldehyde; fibre-glass based insulation of ceiling cavity gives off VOCs; window sealants give off toxic fumes; bathroom air fresheners release VOCs; and the carcinogenic benzene can be formed in the garage from the car exhaust. Virtually all of the mercury in MSW is due to the disposal of household dry cell batteries (mercury, alkaline, and carbonzinc types). A smaller amount of mercury may come from the disposal of broken home thermometers. (Sinha, et. al., 2005 a) Several toxic chemicals are commonly used today in modern homes that also results into generation of HHW. They are usually mixed and disposed with the MSW. Some glaring examples of toxic chemicals used in modern homes that contributes in the generation of household hazardous wastes (HHW) are1) Perfumes and cosmetics used by the women contain some 884 ‗neurotoxic‘ chemical compounds. (Report of National Institute of Occupational Safety and Health in the US). 2) Paper whitener is toxic. The chemical used in it has potential to kill. 3) Cadmium is present in food processing equipment, kitchenware enamels, pottery glazes and plastics and relatively high levels in the sea foods. 4) Lead is present in paints and dyes, toys and newspapers, solder and batteries, lead water pipe; 5) The highly toxic polychlorinated biphenyls (PCBs) are added to paints, copying and printing paper inks, adhesive and plastics to improve their flexibility. Fish food contain generally higher levels of PCB‘s. High levels of PCB‘s were reported from the breakfast cereals in Sweden and Mexico as a result of contamination by ‗packaging materials‘. 6) Containers of paints and enamels used in homes and in automobiles. They contain dangerous chemicals like glycol, ether, ammonia, benzene and formaldehyde and continue to give out toxic fumes at least for 7 years. 7) Containers of pesticides, insecticides, herbicides and fungicides are available in modern homes to eradicate pests and insects in garden plants, cockroaches and spiders in kitchens and storerooms. 8) Many relatively innocuous items, such as plastics, glossy magazines, and flashlight batteries used in homes, contain metallic elements. 9) Metals like cadmium (Cd), chromium (Cr), mercury (Hg), and lead (Pb) are present in several household items. After combustion with MSW metals are either emitted as

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Very little is known about the amount of HHW generated in various countries. UNEP (2006) reported that The Netherlands generate 41,000 tonnes of HHW every year. The University of Arizona, US, made a survey and found that about 100 hazardous items (containers) are discarded per household each year. Australian study made in Melbourne in 1990-92 also found 89,576 kg of hazardous wastes from households. (CSIRO, 1996).

Mercury in the MSW from Household Wastes Has the Potential to Kill : A Case Study from U.S. It is interesting to note that if the tons of ‗household batteries‘ generated in California is calculated for whole of U.S., the total amount would be 160,000 tons per year. Given that a typical household battery weighs 50 grams, the corresponding number of batteries is 2,910,000,000. It is estimated that more than 2,700,000,000 battery units were purchased in the US in 1990. If half the household batteries were ‗alkaline‘, and assuming that each battery contained about 1200 mg of mercury (Hg), then, based on the data reported above, 1923 tons of mercury (Hg) would enter the environment each year in California alone. This mercury is enough to kill 8,730,000,000 people based on a lethal dose of 200 mg per person (Tchobanoglous et. al., 1993). Such situation exist in all metropolitan cities of world, in both developingand the developed countries. Clearly, proper disposal of these household batteries in the MSW is an important issues that must be addressed. If not collected separately, all these household batteries get mixed up with MSW and is disposed in the ‗ordinary sanitary landfills‘ instead of the ‗secured landfills‘. The Hazards of Disposable Baby Nappies in the MSW More than 20 billion disposable baby nappies (equivalent to some 2.7 million tons of solid waste) are ending up in the landfills every year the world over. They contain hazardous chemical ‗sodium polyacrylate‘ (a super water absorbent) responsible for several medical conditions in infants, including hampering of genital growth in male child. On an average disposable nappies occupy landfill space of 0.40 m2 per child per year in Australia. They are 2 % by weight in total solid waste but occupy 3.5 % of total landfill space. They are also disturbing the natural microbial biodegradation processes of organic wastes in the closed landfills by absorbing all water internally. (Brahambhatt and Saeed, 2005).

6.3. The Nuclear Waste : Radioactive Substances Invading the Human Environment Nuclear wastes are the result of our urge to generate nuclear energy without emission of greenhouse gases (which is in fact a myth as it requires 18 years of CO2 producing fossil fuel energy (in uranium mining and enrichment, building reactors etc.) to produce one calories of nuclear generated energy. (Report of Friends of Earth, 2000). Nuclear waste are produced regardless of whether nuclear fission is controlled (such as for energy generation in reactors),

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or occurs explosively, as in the atom bomb. The resulting fission products, isotopes of approximately 30 elements, have mass numbers in the range of 72 to 162, are for the most parts solids, and emit beta particles, together with electromagnetic reaction (gamma rays) which are exceedingly penetrating. The chemical separation of fission products and their conversion to nuclear fuel are the most important sources of radioactive wastes. The radioactive wastes can be in all the three forms – solid, liquid and gaseous and two categories of radioactive wastes are mostly encountered- the Low Level Radioactivity Waste (LLRW) and the High Level Radioactive Wastes (HLRW). (IAEA, 1991). Eight tons of liquid radioactive waste result per year from the typical average-size, nuclear reactor. The ‗nuclear reactors‘ mostly generate HLRW in the form of plutonium-239 (Pu239). Other two most significant fission products are strontium-90 (Sr90) and cesium-137 (Cs137) with half-life of 19.9 and 33 years respectively. They are routinely emitted from the reactors and continue to release radiation energy over long periods of time (several generations of the human race). Even dismantling (decommissioning) of retiring nuclear reactors produce enormous amount of radioactive wastes and contaminate vast land area. There are 439 nuclear power reactors in operation around the world mostly in France and Japan. They would all retire in years to come. Uranium mining and processing (enrichment) produces huge amount of solid and liquid radioactive wastes which is highly hazardous. The extraction of uranium from the earth crust leaves vast quantities of wastes as ‗tailings‘ which contain up to 80% of the original radioactivity of the extracted ore. After mining uranium is further enriched to produce ‗nuclear fuel‘. Depleted uranium hexafluoride (DU) is a radioactive waste by-product of enrichment. For every 1000 tones of processed uranium fuel , 100,000 tones of mined wastes as tailings and 3,500,000 liters of liquid waste is produced. They migrate into the environment through air, soil and water. Processing of uranium ores produces considerable volumes of alpha emitters, mainly radium-226. The half-life of Ra226 is 1600 years and gives rise to a toxic gas ‗radon‘. (IAEA, 1991).

7. WASTE : POTENTIAL SOURCE OF GREENHOUSE GASES (METHANE AND NITROUS OXIDE) So far, not much attention was paid towards this aspect of waste generation. Marked increases in the amount of waste generated has also contributed to emission of greenhouse gases carbon dioxide, methane and nitrous oxides. A major issue of concern today is emission of greenhouse gas methane (CH4) resulting from disposal of MSW in landfills and this may be between 45 – 60 %. This is mainly due to anaerobic degradation of the organic waste components in the landfills as oxygen becomes deficient due to compaction. Methane is 2025 times more powerful GHG than carbon dioxide (CO2) in absorbing the infrared solar radiation. Studies have also indicated high emissions of nitrous oxide (N2O) in proportion to the amount of food waste. N2O is mainly formed under moderate oxygen (O2) concentration. (Yaowu et. al., 2000). Molecule to molecule N2O is 296 times more powerful GHG than carbon dioxide (CO2). Yaowu et. al., (2000) has studied the emission of both methane and nitrous oxides from aerated food waste composting.

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In fact, improved recycling of waste can significantly contribute to abatement of GHG emissions. However, convention microbial composting (biological recycling) of organic wastes also emits methane (Wang et, al., 1997) due to ‗anaerobic sites‘ appearing in the inner layers of compost piles. However, it can be reduced significantly by improving ‗aeration‘ in the waste biomass by periodical turning or through mechanical aerating systems. (Toms et. al., 1995). Significantly, vermicomposting of waste by waste eater earthworms decrease the proportion of ‗anaerobic to aerobic decomposition‘, resulting in a significant decrease in methane (CH4). Earthworms can play a good part in the strategy of greenhouse gas reduction and mitigation in the disposal of global organic wastes. Currently we are studying the potential of GHG emissions by various systems of biodegradation of wastes (Aerobic & Anaerobic Composting & Vermicomposting by Earthworms). (Chauhan & Valani, 2008).

8. SAFE MANAGEMENT OF WASTE : A TECHNO-ECONOMIC PROBLEM Both rich developed and the poor developing nations of world have become conscious towards safe waste management in the changing situation where the human waste is no longer ‗simple and organic‘ to be salvaged by nature in course of time but getting more complex and even hazardous, and threatening to remain in the human ecosystem for long time. Safe management of all waste becomes imperative for the safety and security of the society and it require the input of knowledge of diverse disciplines of material science, political science, economics, geography, sociology, demography, urban planning, public and environmental health, communication, conservation, and civil and mechanical engineering. Waste management has been termed as ‗Cradle- to- Grave‘ management i.e. from point of generation to final disposal, involving safe storage, transport and treatment- and in all these steps, the generator owes the main responsibility. The collection and disposal of waste involves huge expenditures in the development of landfills, waste collecting vehicles, precious fuel (petrol and diesel) and labour costs.

8.1. The Nature‘s Technology at Work: Salavaging the Organic Wastes The organic wastes in the MSW are ‗biodegradable‘ and are decomposed in nature by diverse microorganisms – bacteria, fungi, actinomycetes and the protozoa. Among the biodegradable wastes some are ‗rapidly‘ degraded while others are ‗slowly‘ degraded over time. It may take time from few days to several months and years to degrade the organic wastes, but it does happen ultimately. Some organic materials in the waste decomposes rapidly (3 months to 5 years), while others slowly (up to 50 years or more). The relative ease with which an organic waste is biodegraded (decomposed) depends on the ‗genetic makeup‘ of the microorganisms present and the ‗chemical makeup‘ of the organic molecules (mainly carbon structure that the organisms use as a source of energy and biodegrade in the process). Carbons in sugars, lipids and proteins are easily decomposed than the carbon in lignin, while the carbon in plastics are not at all biodegraded.

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The biodegradability depends to a large extent on the lignin content (present in wood fibers) of the waste. Lesser the lignin content, rapid will be the biodegradation rate of that organic material. Nature has those ‗decomposer microorganisms‘ (mainly bacteria and fungi) in soil, air and water which perform the task. This is how the natural ecosystems on earth has been operating since life evolved. Had there been no decomposer organisms, the earth would have been full of animal and human excreta, animal carcasses and vegetable matters (leaves and twigs) and dirt, and life impossible. The biodegradable waste include all plant, animal and human products, the kitchen waste in every home and restaurants, wastes from the agriculture farm, food processing industries, slaughter houses, fish and vegetable markets, and paper and cotton wastes. All these wastes mainly contain organic matters. The process of biodegradation (decomposition) in nature can be enhanced to several times by introducing decomposer organisms such as the earthworms or even the bacterial biomass directly into the waste biomass. This is being done these days to dispose the mounting organic wastes rapidly. The process is called ‗composting‘ and the byproduct is NKP rich biofertilizer. Table 8. Rapidly and Slowly Biodegradable Organic Constituents in MSW Rapidly Biodegradable Food Waste Newspaper Office Paper Cardboard Yard Waste (Leaves and Grass Trimmings)

Slowly Biodegradable Textiles Rubber Leather Yard Waste (Woody portions) Wood Misc. Organics

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Table 9. Biodegradability of Some Organic Components in the MSW Component Food Waste Newspaper Office Paper Cardboard Yard Waste

Lignin Contents (% of volatile solid) 0.4 21.9 0.4 12.9 4.1

Biodegradability (% of vs) 82 22 82 47 72

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

8.2. Sanitary Landfills - The Ultimate Graveyard for Wastes: An Economic and Environmental Burden Sanitary landfills constitute the ultimate graveyard for the safe burial of all human waste in the womb of ‗mother earth‘. There is an enormous range of materials found in landfills. ‗Today‘s landfill is tomorrow‘s time capsule,‘ writes Blatt (2005). Experience have shown that modern landfills although made with great engineering skills are unable to contain the toxic ‗landfill gases‘ and the ‗leachate discharge‘ into the environment. They are proving to

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be a techno-economic burden and a curse in disguise. The cost involved in landfill construction is not only up-front, but also in its monitoring and maintenance, for controlling landfill gases and the leachate collection etc., which is long term, often up to 30 to 50 years. The up-front development costs for new landfills in U.S. varied from US $ 10 million to $ 20 million in 1992, before the first load of waste was placed in the landfill. This must have gone up substantially by now. (Tchobanoglous, 1995).

Health and Environmental Concerns of Waste Landfills : The Uncontrolled Release of Greenhouse and Toxic Gases into the Environment Methane (CH4), carbon dioxide (CO2), carbon monoxide (CO), hydrogen (H2), oxygen (O2) and nitrogen (N2) are the principal landfill gases. Methane and carbon dioxide are known greenhouse gases and molecule to molecule methane (CH4) is 20 – 25 times more powerful greenhouse gas than carbon dioxide in absorbing the infrared solar radiation. Methane can cause explosion when present in air in concentrations between 5 and 15 %. Ammonia (NH3), hydrogen sulfide (H2S) and the Volatile Organic Compounds (VOCs) are in trace amounts. They are products of biochemical reactions occurring in the landfills. Landfill gases migrate horizontally and migration distances greater than 300 m have been observed with 40 % concentration. Gas samples collected from over 66 landfills in UK showed the presence of 116 organic compounds, many of which are classified as VOCs. (UNEP, 1996). VOCs are mostly evolved from the newly placed MSW and specially which also contains hazardous wastes (HW). The increasing quantities of ‗household hazardous wastes‘(HHW) in the modern society and their disposal along with the MSW, is posing a serious problem for the landfill engineers. Table 9. Typical Hazardous Chemical Constituents Found in MSW Landfill Gases Chemical Constituents Methane Carbon dioxide Nitrogen Oxygen Sulfides, disulfides, mercaptans, etc. Ammonia Hydrogen Carbon monoxide Trace constituents (In VOCs)

% (By Dry Volume Basis) 45 – 60 (Greenhouse gas) 40 – 60 (Greenhouse gas) 2–5 0.1 – 1.0 0 – 1.0 0.1 – 1.0 0 – 0.2 0 – 0.2 0.01 – 0.6

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Table 10. Concentrations of Toxic Trace Compounds Found in MSW Landfill Gases Compound Acetone Benzene Chlorobenzene Chloroform

Mean Value in Parts Per Billion (ppb) by Volume 6, 838 2, 057 82 245

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Table 10.- Continued Compound 1,1 Dichloroethane Dichloromethane 1,1 Dichlorethene Diethylene chloride trans- 1,2-Dichloroethane Ethylene dichloride Ethyl benzene Methyl ethyl ketone (MEK) 1,1,1-Trichloroethane Trichloroethylene Toluene 1,1,2,2-Tetrachloroethane Tetrachloroethylene Vinyl chloride Styrenes Vinyl acetate Xylenes

Mean Value in Parts Per Billion (ppb) by Volume 2, 801 25, 694 130 2, 835 36 59 7, 334 3, 092 615 2, 079 34, 907 246 5, 244 3, 508 1,517 5,663 2,651

Source: Tchobanoglous et al, ‗Integrated Solid Waste Management‘; McGraw-Hill (1995).

Some hazardous chemicals like ‗vinyl chloride‘ is going to the landfills by way of plastic bags. Residents are throwing their kitchen wastes mostly packed in grocery plastic bags. Earlier, there was also a practice to dispose some ‗industrial solid wastes‘ (ISW) with MSW in the landfills, which have now been banned. However, to minimize the emission of VOCs, a vacuum is applied and air is drawn through the completed portions of the landfill. Trace gases although present in small amounts, can be toxic and pose grave risk to public health and environment. Trace compounds may carry ‗carcinogenic‘ and ‗teratogenic‘ compounds into the surrounding environment. Half-lives of various trace compounds in the VOCs have been found to vary from fraction of a year to over a thousand years. (Heath, 1983).

The Uncontrolled Release of Leachate and the Threat of Contamination of Groundwater and Surface Water The liquid (waste juice) that collects at the bottom of the landfill is known as ‗leachate‘. It is the result of percolation of precipitation, uncontrolled runoff, and irrigation water into the landfill. Leachate can also include water initially contained in the waste as well as infiltrating groundwater. Leachate seeps downward to the base of the landfill by gravity and poses a potential health risk to public as it can percolate into the groundwater aquifer and contaminate it. Concern is growing worldwide about wastes leaching heavy metals that may seep into groundwater supplies. Leaching into soil and groundwater will occur regardless of whether the landfill is sealed or not. It has become a common knowledge that all landfills leak. Even the best ‗state of the art‘ landfills are not completely tight throughout their lifetimes and a certain amount of chemicals and metal leaching will occur.

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Landfill leachate contains a variety of chemical constituents including heavy metals (Pb, Cu, Ni, Cr, Zn, Cd, Fe, Mn, Hg, Ba, Ag), arsenic, cyanide, fluoride and selenium, and organic acids derived from the solubilization of the materials deposited in the landfill and from the products of chemical and biochemical reactions occurring in the landfills. (Tchobanoglous et, al.,1995). Disposal of consumer electronics mixed with MSW accounts for 40 % of lead (Pb) in the landfills. Mercury (Hg) will leach when circuit breakers are destroyed, PCBs will leach when condensers are destroyed. When plastics with brominated flame-retardants or cadmium containing plastics are landfilled, both PDBE and cadmium (Cd) may leach into the soil and groundwater. (Miller, 2004).

8.3. Thermal Destruction (Incineration) of Waste High temperature incineration of wastes especially the hazardous chemical and biomedical wastes is considered to be the safest remedy to get rid of it. It enables detoxification of all combustible carcinogens, mutagens and teratogens. Destruction is done in stages. In the first stage the waste is thermally decomposed at 800 C in a refractory lined chamber to produce ashes and volatiles. The ashes are removed and the volatiles with gases are led to second stage where they are heated to around 1200C with additional air. It is the only environmentally acceptable means of disposing some complex organic wastes like chlorinated hydrocarbons, polychlorinated biphenyls (PCBs) and dioxins. These chemicals are persistant, non-biodegradable and highly toxic. However, the conventional incinerators emit dangerous ‗dioxins‘, ‗furans‘ and other highly toxic pollutants when inorganic chemical dyes in plastics are incinerated. Dangerous dioxins may also be formed if some organic materials are incinerated at too low temperatures. But, the modern, well-regulated incinerators have dramatically reduced such toxic emissions. It has been observed that injection of lime and activated carbon significantly remove the ‗dioxins‘ and ‗mercury‘ from the gases by 95 %. However, people in most countries are opposing incinerators and landfills in their neighborhood.

Plasma Arc Incineration System High temperature incineration using ‗plasma arc furnaces‘ is a growing technology for the management of hazardous chemical and biomedical wastes. In plasma arc system a thermal plasma field is created by directing an electric current through a low-pressure gas stream. Plasma fields can reach temperature from 5000 C to 15,000 C. This intense high temperature zone can be used to dissociate the wastes into its atomic elements in the combustion chamber. Heat generated from the plasma torch can melt and vitrify solid wastes. Organic components can be vaporized and decomposed by the intense heat and ionised by the air used as the plasma gas. Oxygen may also be added in the primary chamber to enhance combustion. Metal-bearing solids are vitrified into a monolithic non-leachable mass. (Hasselriss, 1995). The system is hermetically sealed and operated below atmospheric pressure to prevent leakage of process gases. Dioxin formation is prevented. The vented gas is held in the tank and recycled into the furnace. The clean gases are released into the atmosphere through an exhaust stack. The destruction and removal efficiency (DREs) of organic compounds are

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greater than 99.99 %. There is less public opposition to such incineration system. Plasma technology‘s inherent ability to eliminate risks of future liabilities from waste disposal through a single step treatment is an added advantage. A mobile plasma arc system would also reduce or eliminate public health risks associated with the hazards of accidents or spills resulting from the transportation of toxic or hazardous wastes by road or rail. Plasma Arc Technology has proved to be an ideal and economically viable method to successfully treat even the carcinogenic asbestos waste, the nuclear power plant wastes and for safe disposal of arms and military waste worldwide. Temperatures in the order of 1,000 C are necessary to irreversibly transform asbestos into a non-hazardous material. Plasma treatment transformed the asbestos waste into a harmless vitrified slag. Japan has been using the plasma arc technology on large scale to vitrify its municipal solid wastes (MSW) to reduce the volume of its MSW going to the landfills. Land starved Japan cannot afford to have large number of landfills on its island. The slag produced by the plasma arc pyrolysis is recycled into glassy bricks used as construction materials in buildings. General cost of waste treatment by plasma arc technology range between US $ 400 and $ 2,000 per ton, depending on the characteristics of the waste. PAT is however, a capital intensive technology due to its initial equipment costs. A new plasma waste processing plant can cost between US $ 3 million and $ 12 million depending on size, the hazardous nature of the waste and the complexity of the treatment process. (Hasselriss, 1995).

Combining Incineration with Energy Generation : Killing two Birds in One Shot Although waste incineration has been discredited worldwide particularly due to emission of ‗dioxins‘ and ‗furans‘, most European nations have waste incinerator plants combined with energy recovery plants, and several categories of wastes (with high calorific value) including the hazardous wastes are used as fuel to achieve the dual objectives of waste disposal and electricity generation. The ‗waste-to-energy‘ movement started with the ‗oil crisis‘ in the Middle East and the increased cost of oil in the 1970s. Denmark and Sweden are leaders. They incinerate 65 % and 55 % of their MSW respectively and also produce thermal electricity from steam generation. Of the 12 MSW incinerator plants in Netherlands, 5 generate thermal electricity. Some large German cities operate combined incinerator plants providing up to 5 – 10 % of the total electricity demand. U.S. has few incinerators and incinerates 16 % of its MSW currently, but recovers energy from almost 80 % of the plants. The U.S. National Energy Strategy (1991), projected 7 fold increase in the electricity generation from MSW incineration plants by 2010. Nearly three quarters of the Japan‘s 1900 MSW incinerators, recover energy. (UNEP, 1996).

9. EMBRACING THE 5 R‘S PHILOSOPHY OF WASTE MANAGEMENT THROUGH WASTE AND CONSUMER EDUCATION As the generation of waste has increased in volume and also become more complex in nature (due to mixing of non-biodegradable plastic wastes and household hazardous wastes) traditional waste management systems have proved inadequate and inappropriate. Landfills are not the lasting solution to the waste problem. Worldwide consensus is emerging to discourage incineration and landfill disposal of wastes. In accordance with the EU Landfill

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Directive of 2001, the UK government is now phasing out the landfills for waste disposal. Several U.S. states have banned landfill disposal of e-waste.

9.1. Waste and Consumer Education for the Wasteful Modern Society Environmental education about consumption and waste is a kind of ‗re-education‘ of the already educated but ignorant modern wasteful society. Waste generation involves the entire population, so broad cooperation is necessary for sustainable and efficient waste management. Partnerships are required between people and governments to deal with waste collection and disposal. Waste and consumer education aims to raise public awareness about the stresses on municipal councils and enable communities to understand their role and share in the financial responsibilities needed for efficient management of solid wastes. Educational programs must involve consciousness-raising on these fundamentals: · · ·

·

Waste is an inevitable by-product of all individual and social activities and is proportionate to daily consumption. Waste is a potential resource and every individual needs to commit to help recover waste by reuse and ‗recycling‘. Waste generation can be minimized — it can never be eliminated — through changing the patterns of production and consumption of goods and materials either used most and/or that are used once then end up as waste. Judicious consumption of food and the 5 P‘s — paper, plastic, power, petrol and potable water — can help minimize waste generation on a large scale.

However, there are series of issues that consumers have no control over and that require government interventions at the level of production and distribution, involving manufacturers and retailers.

The Consumer Power of Acceptance / Rejection Can Positively Influence Consumer Industries Counteracting Vested Commercial Interest : India Showed the Way in 1940s Consumer power can change things provided they are aware. By accepting or rejecting consumer goods for sale, buyers can positively influence the production process, conveying messages to industries and retailers to reduce waste at source both in quantity as well as in quality (hazardous). Consumers can also influence government and industrialists‘ policies to manufacture goods that are durable, and also re-useable and recyclable after one use. Consumer boycotts, which represent the kind of non-violence preached by the great Indian saint, Mahatma Gandhi, back in the 1940s, counteract the vested commercial interest of manufacturing industries promoted by media for commercial gain. Consumer Education: Women Holds the Key Consumerism is a growing human culture all over the world. Shopping is a key activity in market based modern societies and grocery shopping for domestic consumption is done mostly by women all over the world who are keenly aware of the various shelf products. They

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also observe the invasion of diverse consumer products in the market through media and assess their values for family consumption. However, buying less and narrowing choices are seen as infringements on people‘s rights. Individuals and householders who care about the environment can feel powerless. Therefore policy makers, program coordinators, and educators need to address a series of complex issues associated with educating about consumerism and waste generation. A news poll survey conducted in mid-2005 found that five out of ten women compared with three out of ten men were saying ‗No‘ to plastic bags and only two in ten women but more than a third of the men surveyed preferred to use plastic bags rather than reusable ones (Clean Up Australia 2006) ; (Also on ).

International Efforts and Role of UNEP, WWF and WWI The United Nation Environment Program (UNEP) and the World Wide Fund for Nature Conservation (WWF) and the Washington based World Watch Institute (WWI) are playing important roles in educating people at an international level, creating mass awareness about waste problems as well as other environmental issues. UNEP sponsored the first Clean Up the World Campaign, 17-19 September 1993. Several million people, from 79 countries, actively participated in that campaign, which combined dual objectives, environmental sanitation and resource generation. The campaign followed up on the first ‗World Clean Up Movement‘ in Sydney, Australia, initiated by Australian yachtsman, Ian Kiernan, on 8 January 1986. Kiernan gathered around 40,000 Sydney siders to collect more than 5000 tons of rubbish from different parts of the city. The first Clean Up Australia Day, 1990, involved almost 300,000 volunteers. Programs have been developed to influence consumers and to encourage them to rethink about their patterns of consumption which can reduce waste. The Japanese Environmental Agency (JEA) has launched a scheme called ‗Household Eco-Account Books‘ that encourages the citizens to live sustainabily in an environmentally friendly manner and also save money by consuming resources judiciously and by embracing the philosophy of 3 R‘s for waste reduction, reuse and recycling. In Norway NGOs run a program that builds householders awareness on environmental impacts of consumer products, especially on its contribution to piling municipal solid waste (MSW) and also the household hazardous wastes (HHW) as several consumer products in modern homes may contain hazardous chemicals and materials.

9.2. Educating about the Golden Rules of 5 R‘s: Refusal, Reduction, Reuse, Recycling and Responsible Behavior People and policy makers need to embrace the ‗5 R‘s environmental philosophy for sustainable waste management. In the waste management hierarchy ‗waste refusal and reduction‘ is the best option, ‗waste reuse and recycling‘ the better option than the ‗waste disposal‘ in landfills. (Sinha et. al. 2005 c). It is imperative to educate the masses to 1. Refuse to accept articles and materials that generate / create more waste, especially those that generate toxic and hazardous wastes;

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Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al. 2. Reduce waste at source by consuming resources as little as possible and which is enough to enjoy a minimum quality of life; 3. Reuse articles and materials several times and as much as practicable before discarding them as waste and insist on buying durable, reusable and recyclable articles and products and ‗repair‘ household goods before rejection as waste; 4. Recycle / recover / retrieve new materials / energy from discarded waste products and assist the recycling industries by separating the recyclable waste from the nonrecyclables faithfully at source; and to behave with 5. Responsibility (for both consumer societies and producer industries) with regard to judicious use of resources and reduction in waste generation in everyday activities.

If people (society) and producers (industries) embrace the 5 R‘s golden rule majority of the waste will be taken care of and very little will be left for ‗treatment‘ or to ‗contain‘ them and finally to dispose them in landfills. The environmental organization ‗EcoRecycle‘ of Victoria, Australia (2006) proposed a ‗waste management hierarchy‘ which emphasizes that waste ‗reduction / avoidance‘ should be the first option and ‗disposal‘ the last. Waste reuse and recycling comes after reduction. If this management plan is sincerely implemented, there will be very little waste left to be disposed finally. (Figure 1).

Figure 1. Source: EcoRecycle 2006.

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The Power of Refusal : It Can Prevent Misuse of Resources In the 1980s, the Women Environmental Network (WEN) of the UK campaigned against over-packaging of food products and refused to buy those products that were heavily wrapped with plastics and papers. Women groups went into supermarkets, ripping off all the unnecessary extra layers of paper and plastics and handed them back to the managers. WEN has campaigned against several environmentally unfriendly consumer products such as paper handkerchiefs and toilet rolls made from newly produced paper refusing to accept them. (Belamy 1995). Way back in the 1940‘s the great Indian philosopher and educator M.K. Gandhi, used this ‗power of refusal‘ against the mighty British Empire (although in a different context) to generate awareness among the masses. Box 1. Responsibility of Consumers: Check Before Shopping and Refuse to Accept Articles That Can Generate More Waste Upon Use 1. 2.

3.

Refuse to accept non-biodegradable plastic bags for grocery and other shopping — force manufacturers and retailers to offer environmentally friendly alternatives. Refuse to accept any plastic or paper bag for small or just a few articles that you can carry instead in your pockets or personal bags — always carry easily tucked away cloth or alternative bags for shopping. Refuse to accept articles over-packed in plastic and paper, which have no eco-label (are not produced through clean methods of production), and have the potential to generate more waste when used.

The Wisdom of Waste Reduction / Prevention: It Conserves Material Resources This is the best option in the waste management hierarchy. Preventing waste is like preventing a ‗social disease‘ to occur. Waste reduction means conserving resources, that otherwise constitute waste, with further economic and ecological benefits, including: conserving valuable raw geo-chemical and biological resources, water and energy (fossil fuels) used in the manufacture of products; saving money otherwise spent on constructing and maintaining waste landfills; reducing health and environmental impacts of air, water and soil pollution and global warming. Science and technology, the industry and the society all have to play critical role towards waste reduction. If people judiciously use and reduce the consumption of 5 P‘s (paper, plastic, power, petrol and potable water) in daily life, it would dramatically reduce all the three wastes- the solid, liquid and the gaseous from the environment. Society has to begin the process of reducing waste at source – the home, office, or factory- so that fewer materials will become part of the disposable solid wastes of a community. Efforts must be made to reduce the quantity of materials used in both packaging and obsolescent goods. Waste reduction may also occur at the household, commercial, or industrial facility through ‗selective and judicious buying and consuming‘ patterns and the reuse of products and materials. Source reduction is an option that will conserve resources and also has economic viability.

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Technology Has Improved Efficiency of Resource Use and Reduced Waste Generation by Consumers at Source Technological advancement has undergone a process of ‗dematerialization‘ for reducing the consumption of resources (metals, plastics, glasses etc.) in manufacturing products, with consequent reduction in waste generation. Many packaging items (cans and bottles), consumer ‗electronic goods‘ and even the ‗automobiles‘ have become lighter, slikker and smaller. Since 1977, the popular 2-liter PET plastic soft drinks bottles have been reduced from 68 grams each to 51 grams, a 25 % reduction in material used per bottle. One hundred 12 fluid ounce aluminum cans which weighed 4.5 pounds in 1972, only weighed 3.51 pounds in 1992, a 22 % reduction in material use. Steel beverage cans have also been downsized and are now 40 % lighter than they were in 1970. This means that people can still enjoy a good quality of life while consuming smaller amounts of resources from the environment and generating lesser amount of waste. Lesser resource use would also mean ‗lesser energy consumption‘ and ‗lesser waste generation‘, thus benefiting the environment in every way. (WWI, 1991). The Wisdom of Waste Reuse: It Extends the Life of Material Resources There are several articles like bottles and jars made of glasses and tough plastic materials, large tins, metallic cans and cansisters which can remain in our economy and ecosystem for very long time (if not discarded as waste) just by simple cleaning and washing. Box 2. Recipe and Responsibility of Consumers to Reduce and Re-use Waste in Daily Life 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20.

Buy in bulk and concentrates. Buy durable not disposable products. Buy products packed in reusable and refillable containers. Chose products without or as little packaging as possible. Take Your Own Bags, Box or Basket (TYOB). Insist on reusable cotton bags or durable synthetic bags for grocery shopping; keep a few used shopping bags in your car. Select paper bags/wrap only if needed and avoid plastic bags/wrap. Reuse paper/envelopes only printed/photocopied on one side printed. Use rechargeable batteries. Use refillable ink fountain pens and biros. Avoid using greeting/invitation cards. Avoid wrapping, or sparsely wrap, presents etc. Avoid paper napkins — carry cotton handkerchiefs. Save electronic copies of data or print and photocopy on both sides of paper. Prepare meals at home (not take-away/convenience foods). Avoid frequent use of canned, bottled, packed, processed and preserved foods and eat raw, fresh foods (good for human and environmental health). Avoid peeling fruits and vegetables to reduce kitchen wastes (and preserve nutrients for digestion) and compost any food scraps and leftovers. Carry food and edibles to school/workplace/friends‘ place in reusable boxes. Repair clothes and garments, toys, tools and appliances. Go to garage sales rather than throwing away old things or buying new ones. Use safe retreated car tyres.

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Box 3. Responsibility of Producers (Industries) to Reduce Waste 1.

2.

3.

Producers of consumer goods and products MUST embrace the philosophy and principles of ‗cleaner production‘, which emphasize producing ‗more with less‘, thus conserving resources and reducing waste. Producers MUST embrace the ethical principle of producing durable, re-usable and recyclable goods, which can remain in use for longer periods of time even through changes in the purposes of their uses before being discarded. Producers MUST inform consumers about the recycling potential of their products and be committed to ‗take back‘ products after use for recycling, to recover maximum useful materials from them to reduce the amount of final waste.

It do not involve any complex industrial processing, use of chemical and energy for their reuse. They might have been originally made for some other use, but now can be reused for different purpose- especially for storing and packaging. It should rather be termed as ‗resource reuse‘.

The Wisdom of Waste Recycling : Retrieving New Materials / Energy from Waste and Conserving Virgin Raw Materials and Natural Resources Waste recycling is a technological process to reuse waste materials involving physical, chemical and biological processing to recover useful materials from them. It converts waste into a resource, conserves primary and virgin raw materials from environment (geo-chemical and bio-chemical natural resources), saves tremendous amount of water and energy and protect the environment. Hidden environmental protection values of recycled goods, include the energy and water saved, the pollution and deforestation prevented. Government and People’s Support is Paramount for Recycling to Succeed Recycling combines social, economic, and ecological values. The opportunity to recycle provided by local and state governments has seen an almost fourfold increase in the Victorian recycling industry in Australia in the years 1993–2003 (EcoRecycle, 2006). EcoRecycle estimates that re-processors have saved: water equivalent of filling 17,500 Olympic sized swimming pools; greenhouse gas equivalent to that produced by 580,000 cars; and, ‗enough energy to power every household in Victoria (Australia) for 7 months‘ (EcoRecycle, 2006). Consumers can support recycling by buying goods which bear ‗recycled‘ stickers from their manufacturers. People can indirectly support and promote recycling industries by separating the recyclable from non-recyclable wastes and can directly participate by recycling domestic, e.g. kitchen, wastes through composting. Policy makers could encourage or regulate for all recycled goods to bear a tag recording the origin and life history of the goods, i.e. identifying from which waste it was produced and how it has saved damage to the environment. Such initiatives allow consumers to confidently reuse and recycle products. A demand for recycled goods in society can be created by education.

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Rajiv K. Sinha, Sunil Herat, Gokul Bharambe et al. Box 4. Responsibilities of Consumers (Society) in Promoting Waste Recycling 1.

2. 3.

Separate recyclable wastes — glass bottles and jars, aluminum and steel cans, plastic bottles, milk cartons, paper, cardboard, magazines and phone books — from other household wastes faithfully for collection by councils or deliver them to recycling centers / industries. Buy recycled goods. This will encourage promotion of recycling efforts by the government and industries. In order to make recycling an effective strategy, people must understand more about the qualities of materials in everyday items. For example, several vitreous materials that look like, and shatter like, glass are not recyclable. Even a minute amount (such as 5 grams per ton) of these vitreous materials found in some ceramic mugs and plates, cups and crockery, mirror, broken drinking glasses, flower vases, light globes and laboratory glass, can contaminate a load of recyclable glass and render it useless.

Box 5. Responsibilities of Producers (Consumer Industries) Towards Waste Recycling 1.

2.

3.

Producer industries MUST produce / manufacture consumer goods that are ‗potentially recyclable‘. 2. Producers should have a policy to ‗take back‘ their own products to recycle them; Producers should provide necessary information to its prospective buyers on the matter of using, handling, conservation, disposal and recycling potentialities of its products. In designing new products, the industry must assess its potential and even suspected adverse impact on its consumers health and the environment.

10. WASTE RECYCLING : A GROWING GLOBAL BUSINESS WHERE ‗WASTE‘ (TRASH) IS TURNED INTO ‗RESOURCE‘ (TREASURE) Waste is no longer considered as a discarded product to be disposed off from the human ecosystem. Wastes are in fact now considered as a ‗misplaced resource‘ to be brought back into the human ecosystem through the reuse and recycling technologies. Recycling means that otherwise wasted items are returned back to the country‘s economy to make either the same product again, another product or other products. This saves cost on landfill disposal, save landfill space, and prolongs the life of the primary resources used to make the product. (Fairlie, 1992). Recycling can involve mechanical / biological / chemical / thermal processing of waste using some energy, water and chemicals to effectively and efficiently reconvert the waste into ‗secondary raw materials‘ to manufacture new products or recover ‗energy‘ from them in the form of fuel gas or heat. Several of the items of domestic, industrial (non-hazardous) and commercial wastes viz. paper, leather, rubber, cotton rags, metals and glasses, wood and plastics can be recycled in the material recovery and reprocessing industries to get valuable new products. Science and technology has provided a tool in the hands of mankind to renew all those ‗non-renewable resources‘ on earth which otherwise cannot be ‗renewed‘ by nature‘s

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mechanism. This would also save tremendous energy, preserve forest, prevent soil erosion and pollution and above all arrest emissions of greenhouse gases (GHG‘s) and reduce global warming. Some economies in the developed nations of Europe and America, and also in Australia, is retrieving back 80 to 85 % of the materials from the waste as useful products for societal consumption or to be reused in developmental activities and only less than 20 % are going to the landfills for final disposal. (Goldoftas, 1989).

10.1. Global Trade and Exchange for Recycling The old saying ‗one persons trash is another person‘s treasure‘ is becoming true as ‗waste trade and exchange‘ between nations for recycling is entering into new era both economically, ecologically and politically. Japan, Germany, France, Hong Kong, U.K., U.S. and also Australia is recycling their wastes on large scales. Japan recycles 65 % of its MSW into usable products and 40% of its waste paper into high quality paper. A computerized ‗waste exchange register‘ has been prepared to link ‗waste producers‘ with potential ‗waste users‘ and diverse waste items like discarded aluminum cans, paper and card-boards, wooden crates and off-cuts, steel plate off-cuts, plastic products, saw dust, eggshells, caustic soda and chemicals have been listed in this register. The OECD countries have established a central system for moving the non-hazardous ‗recyclable wastes‘ across international borders under the rules of normal trade goods listed in the ‗green list‘ of Basle Convention (1989). In these countries several C and F agents have come up with list of ‗waste available‘ and ‗waste wanted‘. The buyers benefit by the reduced cost of raw materials, while the sellers benefit by the reduced cost of treatment and disposal of wastes. Trade in waste between two countries is economically and politically justified if it is based on ethics. Faced with rising cost of safe waste disposal and recycling at home, many industrialized nations prefer to pass their hazardous wastes along with the ‗recyclable wastes‘ on to the poor Third World countries where there are facilities, cheaper manpower and infrastructure for waste recycling. This has happened greatly in case of hazardous electronics wastes. U.S. has dumped huge pile of e-waste in China and India for recycling.

Trade-in Program for Recycling Electronic Waste Recycling is a good option for the extremely old generation computers such as the PrePentium generation, or the computers (specially the monitors) which are broken. According to the International Association of Electronics Recyclers (IAER) more than 1.5 billion pounds of electronics equipment are recycled annually and is likely to grow by a factor of 4 or 5 by the end of this decade. Eleven countries currently have ‗mandatory‘ electronics recovery laws on the books. These are Denmark, The Netherlands, Norway, Sweden, Switzerland, Japan, Belgium, Taiwan, Portugal and South Korea. Some EU nations have very strong system for ewaste collection, such as the SWICO system in Switzerland and the Netherlands Association for Disposal of Metalectro Products (NVMP). NVMP collect 80 % of e-waste. About 77 % of TVs and 64 % of other small brown goods are recovered for reuse and recycling. (Cui and Forssberg, 2003) Most major computer manufacturers in world e.g. Dell, Hewlett-Packard (HP), Compaq, Gateway have begun to address e-waste problems with their own end-of-life management

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programs which offers a combination of trade-in, take-back and recycling programs. Dell and Gateway lease out their products thereby ensuring they get them back to further upgrade and lease out again. Dell offers both reuse and recycling programs. Dell purchases it computers for a nominal $15 fee. They will recycle both Dell and non-Dell computers, but for corporate customers only. Dell Europe, however, recycles individual consumer‘s computers. Gateway provides $25-50 cash refund for new PC and donate the customer‘s used computers. HP and Compaq‘s trade-in program provides a refund cheque for the value of the used computer if it is traded-in on the purchase of a new HP/Compaq computer. HP has been very active in recycling programs. Since 1987, it has recycled over 500 million pounds of materials from the e-waste and pledge to achieve 1 billion pounds by 2007. HP entered into a joint venture with Micro Metallics in 1996 to recycle materials recovered internally and to recover parts from products returned by customers and reported US $ 72.3 billion as revenue from the recycling program. IBM accepts any type of PC (even other brands) but would charge $ 29.99 to take it back. (Cui and Forssberg, 2003).

10.2. Economics of Recycling: Value Addition When Garbage Becomes Gold Any waste has negative economic and environmental value and is a big techno-economic problem for the local government and involves an economic and environmental cost in safe disposal. But if the same waste is converted into an useful ‗new material‘ its economic and environmental value goes to the positive. (Sinha, 1994).The Brisbane City Council in Queensland, Australia process over 60,000 tones of recyclable materials every year and add $ 20 million to their economy. (BCC, 2002). What was the value of flyash before technology founds its use to reconvert it into building material ? It was an environmental a hazard. What is the value of food scraps and the human excreta ? It is a human waste to be safely disposed off every day and involves cost. Composting technology can convert them into a high value end product i.e. compost (a nutritive organic fertilizer for the farms). Biogas technology can now retrieve ‗methane‘- a clean burning fuel for power generation. (Sinha, 1994; Goldstein, 1995). Recycling also reduces the cost of construction and siting of new ‗landfills‘ and closing it after use. It reduces the economic and environmental cost of incineration of several categories of wastes. Recycling economy is a closed loop in which consumers, manufacturers and waste collectors and haulers, all have a critical sustaining role to play. Many waste articles in the society are ‗potentially recyclable‘, but they may not actually be recycled unless there is a practical way to do so and there is a demand / market for the ‗recycled goods‘. The waste haulers would be encouraged to collect the recyclable wastes if there is a demand by the recycling industries, and the recycling industries will buy these wastes (as secondary raw materials for processing) only if it is less expensive (economically cheaper) than the primary (virgin) raw material. The consumers will buy recycled items only if it is as good as the product made from virgin materials and still less expensive. None of them are bothered about the high ‗environmental cost‘ of the procurement of the primary raw materials from earth or the products made from them. When recycled materials have a high ‗social and economic value‘ despite the cost of collecting and processing, they find a ready market. Much of the gold, silver and other precious metals that were ever mined and extracted from their primary ores centuries ago is

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still circulating (recycling) in our economy (human ecosystem). Materials with low social and economic value relative to the cost of collecting and processing do not find a ready market. The grocery and kitchen wastes, agriculture, dairy and slaughter house wastes and the sewage sludge, with greater organic contents can be biologically recycled to get ‗fuel (biogas) and fertilizer‘ (compost). Even the ‗human hair‘ which are protein materials can be recycled for getting ‗amino acids‘ and making newer and cheaper proteins. It is like getting ‗gold from the garbage‘ and ‗silver from the sewage‘. Recycling of hazardous industrial wastes also minimizes the cost and risk of transporting, storing, treating and disposing of hazardous wastes to distant places. US and Japan recycles a great part of its hazardous wastes. Several recyclable hazardous wastes are ‗exchanged‘ among nations under strict rules of Basel Convention (1989). The recycling potential of wastes from the pharmaceutical industries is 95 %, paints and allied products 40 %, organic chemicals 25 %, petroleum refinery 10 % and small industrial machinery 20 %. Used and discarded products from the automobiles e.g. the lead-acid batteries (LABs), the waste oil and the auto tires are also recycled. In some cases the waste may have to undergo some modifications, such as ‗dewatering‘, in order to become recyclable and salable product. An aluminum die-casting firm developed a market for a by-product of their production process- the ‗fumed amorphous silica‘. After much researches into uses for the product it was found to be a valuable additive to concrete. The firm marketed the waste and now sells all the fumed amorphous silica it generates to cement plants. This is bringing an income of US $ 1 million every year for the company and also saves the enormous cost of disposal. (Noll et. al., 1985). An x-ray film manufacturer in U.S. generates a salable waste product. The company installed equipment that flakes and bales waste polyester –coated film stock which is sold as raw material input to another firm. Over 9 million kg of film stock is exchanged each year. This saves US $ 200,000 annually which would have incurred in collection, transport, and disposal cost. Above that, there is annual profit of US $ 150,000 to the x-ray film manufacturing firm from the sale of the recyclable materials. (Noll et. al., 1985).

10.3. The Environmental Significance of Recycling Nature possess tremendous capacity of recycling of several categories of wastes by ‗biodegradation‘ (biological recycling) aided by the decomposer organisms on earth. The earth provides all the necessary ‗resources‘ for development of mankind and also ‗assimilate‘ all the wastes generated by them in the process of those developmental activities. This is defined as the ‗carrying capacity‘ of Earth. Unfortunately, due to the growing consumerism of resources and the consequent increase in the quantity and concentration of human wastes on earth, this carrying capacity is threatened to be jeopardized with dangerous consequences for the global ecological balance and grave risk of poisoning of the life support systems on earth. The addition of ‗non-biodegradable technological wastes‘ has aggravated the problem. They can remain in the human environment for centuries as nature has not evolved the mechanism to degrade and recycle these new man-made materials.

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Waste Recyled 1. Iron & Steel Scraps 2. Aluminum Cans 3. Papers Wastes 4. Glass Wastes

Energy Savings 60-70 % 90-95 % 60-65 % 30-32 %

Pollution Control

Reduction in Solid Waste

Water Savings

Forest Protection

30 % 95 % 95 % 20 %

95 % 100 % 100 % 60 %

40 % 46 % 58 % 50 %

100 % 100 % 100 % -

Source: WWI (1984) ‗State of the World‘.

By resorting to ‗recycling technologies‘ we can actually ‗renew and sustain‘ the natural ‗carrying capacity‘ of earth ecosystems and not only the natural human wastes can be reconverted into a resource, but also the ‗ man-made synthetic and hazardous wastes‘. Recycling of some municipal and industrial wastes such as papers, metals, glasses can accrue several environmental benefits by way of water and energy saving (consequent reduction of greenhouse gas emission), control of air pollution and forest protection.

Figure 2. Explaining the Economic and Environmental Significance of Recycling.

10.4. Need for Appropriate Recycling Technologies Several municipal as well as industrial solid wastes have potential to be recycled. They only need appropriate technologies for recycling. New recycling techniques are being developed constantly to maximize recovery of useful materials and minimize the amount of waste going to the landfills. Essentially two types of recycling technologies are being developed for waste processing to get valuable end products – mechanical and biological. Mechanical technology involves thermal and chemical processing of waste (both organic and inorganic fractions), while biological technology involves biological processing of waste

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(organic fraction only) by microbes and earthworms. While mechanical technologies incur expenditure of energy, the biological technologies may generate energy.

10.5. Biological Recycling of Wet Organic Wastes into Fertilizer and Fuel: Diverting the Major Part (70- 80 %) of MSW from Landfills Biological recycling involves processing of wet and dry organics such as the food waste from homes and commercial institutions, green garden and farm wastes, cattle and farmyard wastes and the waste organics from the food processing industries). The traditional composting methods are essentially a biological recycling technology which is being revived and improved with new knowledge in microbiology and environmental biotechnology. Other biological recycling methods developed are technology to retrieve ‗biogas‘ and ‗bio-alcohol‘ (cleaner energy sources) from organic wastes. MSW contain 70-80 % by weight of organic materials and the waste biomass is rich in carbon (C) and nitrogen (N) with other valuable minerals like phosphorus (P) and potassium (K) and have potential to be biologically recycled to recover fertilizer and fuel (energy). However, waste with high organic components should preferably be recycled to produce fertilizers (composts) and not fuel. (White, 1996). Table 11. Useful Materials Recovered from Waste by Recycling Technologies Recyclable Materials from MSW

Useful Materials Recovered / Typical Uses

WET RECYCLABLES Organic Fraction of MSW (70-80 %) Food waste, yard & garden waste etc.

Recovery of fertilizer (compost) and fuel (methane and ethanol) ;

DRY RECYCLABLES Paper Old Newspaper Corrugated Cardboard High-grade Paper Plastics Polyethylene Terephthalate (PET) High-density Polyethylene (HDPE) Polyvinyl Chloride (PVC) Low-density Polyethylene (LDPE) Polypropylene (PP)

Polystyrene (PS)

Newsstand and home-delivered newspaper Bulk packaging, fiberboard & roofing material Computer paper, white ledger paper Soft drink bottles, salad dressing and vegetable oil Bottles; photographic film Milk jugs, water containers, detergent and cooking oil bottles. Home landscaping, irrigation pipes, some food packaging, and bottles Thin-film packaging and wraps; dry cleaning film bags; other film material Closures and labels for bottles and containers, battery casings, bread and cheese wraps, cereal box liners Packaging for electronic and electrical component,

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Multilayer and other mixed plastics Glass Metals Ferrous Metals (Iron & Steel) Non-ferrous Metals Textiles Construction & Demolition Wastes

foam cups, fast-food containers, tableware and microwave plates; Packaging, ketchup and mustard bottles Clear, green and brown glass bottles & containers; Tin cans, white goods, reinforcing bars Aluminum cans, copper and lead products Wiping rags Soil, asphalt, concrete, wood, drywall, shingles

HAZARDOUS WASTES Auto Tires Household Batteries Automobile Batteries Incinerator Residues Engine Oil and Lubricants

Road building materials, paving and tire-derived fuel; Recovery of zinc, mercury and silver Recovery of acid, plastic and lead Concrete, road construction material Refined Oil

Fertilizer Production : Retrieving Nutrients from Organic Wastes i) Compost from Waste (Getting Gold from Garbage) There is always greater economic as well as ecological wisdom in converting as much ‗waste into compost‘ (a nutritive fertilizer for farms), so that less and less waste (the noncompostable and non-recyclable residues) finally go to the landfills. The organic fraction of MSW (70-80 %) is rich in nitrogen, potash and phosphorus (NKP) and all macro and micronutrients. This can be easily recycled through microbial biodegradation – process called composting, to retrieve the nutrients from them. If residents all over the world, practice ‗home composting‘ and ‗backyard composting‘ of their ‗kitchen and garden wastes‘, this will significantly reduce burden on the councils and very little MSW will be left for the landfills. Even councils should practice composting of waste on commercial scale instead of sending them to the landfills, earn money by selling them to the farmers, rather than spending money on their landfill disposal. The components that constitute the organic fraction of MSW are food wastes, paper, cardboard, plastics, textiles, rubber, leather, yard wastes, and wood. Yard waste may contain even higher percentage of organic matter. All of these waste materials can be recycled, either separately or as a commingled waste. Certain industrial wastes such as those from the ‗food processing‘, ‗agricultural‘ and ‗paper-pulp industries‘ are mostly organic in composition and can also be composted. Controlling the environmental conditions i.e. the biological, physical and the chemical factors can significantly improve and enhance the composting process without the emission of foul odor and pollution of the environment and without the loss of essential nutrients from the compost. (Epstein, 1997). The conventional composting technology has now been significantly improved with our modern scientific knowledge in ‗microbiology‘ and ‗biotechnology‘ to ‗biodegrade‘ all kinds of organic wastes including the ‗municipal solid wastes (MSW)‘containing sufficient organic components under a completely controlled environmental conditions. We have innovated

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some new, cost-effective and more efficient methods of converting organic waste into compost which besides providing the macro and micronutrients also provide ‗beneficial soil microorganisms‘ to the soil and work as ‗soil conditioner‘ to prevent soil erosion. (Haug, 1993; Epstein, 1997). The relative ease with which an organic material is biodegraded (composted) depends on the ‗genetic makeup‘ of the microorganisms present and the ‗chemical makeup‘ of the organic molecules. Carbons in sugars, lipids and proteins are easily decomposed than the carbon in lignin. A small portion of the carbon is converted to new microbial cells, while a significant portion is converted to carbon dioxide and lost to the atmosphere. The new cells that are produced become part of the active biomass (decomposer microbes), to further enhance and multiply the biodegradation and composting process and on death ultimately become part of the compost. European cities and societies are developing and adopting ambitious technologies for recycling and composting of wastes to divert them from landfills. Some cities have installed sophisticated equipments for composting and resource recovery. Austrian, French and Swiss cities have taken the lead in installing waste recycling and composting systems. Twenty seven (27) composting plants with a combined annual capacity of 60,000 tones of compost from city trashes are currently under construction in German towns and cities (UNEP, 2004).

Vermicomposting: Using Waste Eater Earthworms for Rapid and Odorless Composting of Municipal and Industrial Organic Wastes Vermicomposting is rapid and odorless process triggered by earthworms through enzymatic breakdown of waste organics and also enhancing the microbial degradation by proliferating the microbial population in the waste biomass. Vermicompost is completely free of pathogens but rich in beneficial decomposer microbes including nitrogen fixing bacteria, mycorrhizal fungi and actinomycetes. It is rich in NKP, trace elements, enzymes, growth promoting hormones (gibberlins and auxins) and readily works as ‗soil conditioner‘. Long-term researches into vermiculture have indicated that the Tiger Worm (Elsenia fetida), Red Tiger Worm (E. andrei), the Indian Blue Worm (Perionyx excavatus),the African Night Crawler (Eudrilus euginae),and the Red Worm (Lumbricus rubellus) are best suited for vermi-composting of variety of organic wastes including some of the hazardous wastes like the ‗sewage sludge‘ (biosolids) from the sewage treatment plants and the ‗fly-ash‘ from the coal power plants. Vermiculture was started in the middle of 20th century for management of municipal / industrial organic wastes in Holland in 1970, and subsequently in England, and Canada. Later vermiculture were followed in USA, Italy, Philippines, Thailand, China, Korea, Japan, Brazil, France, Australia and Israel. However, the farmers all over the world have been using worms for composting their farm waste and improving farm soil fertility since long time. In UK, large 1000 mt vermi-composting plants have been erected in Wales. The American Earthworm Technology Company started a 'vermi-composting farm' in 1978-79 with 500 t /month of vermicompost production. Japan imported 3000 mt of earthworms from the USA during the period 1985-87 for cellulose waste degradation. The Aoka Sangyo Co. Ltd., has three 1000 t /month plants processing waste from paper pulp and the food industry. This produces 400 ton of vermicompost and 10 ton of live earthworms per month. The Toyhira Seiden Kogyo Co. of Japan is using rice straw, municipal sludge, sawdust and paper waste for vermicomposting involving 20 plants which in total produces 2-3

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thousands tons of vermicompost per month. In Italy, vermiculture is used to biodegrade municipal and paper mill sludge. Aerobic and anaerobic sludge are mixed and aerated for more than 15 days and in 5000 cum of sludge 5 kg of earthworms are added. In about 8 months the hazardous sludge is converted into nutritive vermicompost. In France, 20 tons of mixed household wastes are being vermi-composted everyday using 1000 to 2000 million red tiger worms (Elsenia andrei) in earthworm tanks. Rideau Regional Hospital in Ontario, Canada, vermi-compost 375 - 400 kg of wet organics mainly food waste everyday. In Wilson, North Carolina, U.S., more than 5 tons of pig manure (excreta) is being vermi-composted every week. (Edward, 1998; Frederickson, 2000; Fraser-Quick, 2002; Sinha et. al., 2005 b).

ii) Fertilizer Pellets from Sludge Cake (Getting Silver from Sewage) A technology has been developed in U.S. by Massachusetts Water Resources Authority to recycle the dewatered ‗sewage sludge‘ into ‗fertilizer pellets‘. The sludge is shipped from sewage treatment plant and pumped directly from barges into the storage tank and then transferred to belt-filter press where water is mechanically squeezed out. The sludge cake is moved through the conveyer belts to rotating heat dryers and heated to convert into small hard pellets. This also destroys the foul smell and harmful bacteria. The pellets are low-grade fertilizers, but if blended with other synthetic nutrients can form a complete fertilizer. Cities all over the US is operating sludge processing plant like MWRA. Fuel Production : Retrieving Cleaner Sources of Energy from Organic Waste The organic municipal solid wastes enriched with biomass and materials with high calorific value (combustible) can be recycled to yield either gaseous fuel ‗methane‘ (biogas) or liquid fuel ‗ethanol‘ by fermentation. The wastes can be directly incinerated and the heat liberated is used for steam generation and electricity production. The new idea is to use the wastes a source of fuel in ‗cement kilns‘ in cement industries to replace the costly fossil fuels. The emission problems accompanying incineration is also minimized to a great extent. But only wastes with high calorific value is useful for energy recovery. (Parker and Roberts, 1985; Porter and Roberts, 1985). i) Biogas There is always greater ecological wisdom in generating ‗biofuel‘ (biogas and bioethanol) from the MSW rather than generating ‗thermal energy‘ from them by combustion technology, with accompanying release of toxic gases especially ‗dioxins‘ and residual ‗ashes‘ which needs landfill disposal. The biogas technology by the use of anaerobic ‗metanobacteria‘ utilizes the organic wastes rich in cellulosic materials with high carbon and nitrogen (C/N) ratio. It produces both fuel and fertilizer. Each ton of organic waste by dry weight yields about 36 cum of biogas and 350 kg of biomanure. Methane is a clean burning substance and on combustion yields 550 BTU of heat per cft of its volume. Even the sewage sludge rich in organic matter and high C/N ratio is efficiently recycled to yield methane. ii) Bio-diesel Bio-diesel is emerging as an alternative cleaner fuel for diesel engines brewed from waste organic feed-stocks, such as animals waste fats (tallows), lard and waste cooking oils. This is

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being produced in Australia and other countries on commercial scales. Bio-diesel is also being produced from offal at a turkey-processing plant in the U.S. Any vegetable oil can form the feedstock to produce bio-diesel. It can produced by recycling the waste oil from fast-food restaurants and the deep friers of French fries which generate huge amount of waste vegetable oils.It can now be produced from household waste and used auto tires. Bio-diesel is completely non-toxic and biodegradable, almost free of sulfur and aromatics, and have lower CO2 emissions than the mineral oil derived diesel. It can be used in existing fuel engines without any modifications. The emissions from bio-diesel are 100 % lower in sulfur, 96 % lower in total hydrocarbons (HC), 80 % lower in polycyclic hydrocarbon (PAH), 45 % lower in carbon monoxide (CO) and 28 % lower in suspended particulate matters (SPM). (UNEP, 2006).

iii) Bio-alcohol (Ethanol) Waste biomass rich in starch and cellulosic materials provides good raw material for ethanol production by enzymatic fermentation carried out by organisms ‗yeast‘ and some bacteria. Bagasse, the waste from sugarcane industry is most appropriate raw material. 6000 kg of bagasse upon fermentation yields 1000 litres of ethanol of 95 % strength. It is a cleaner auto-fuel and Brazil is already using it as an ‗auto-fuel‘ since 1975. New bioconversion technologies could open the gate to the cost-effective use of a wide variety of feed-stocks including agricultural waste products like corn stalks, rice and wheat straws and perennial grasses to produce bio-fuel ethanol iv) Coal Briquetts Technology to recycle the agricultural wastes into a non-polluting fuel has been developed. The dried biomass is crushed and pre-heated to a temperature of 100-120 C and then compacted. v) Fuel Pellets A technology has been developed for converting garbage into non-polluting fuel pellets. The garbage is first shredded and blown dry in rotary kiln. It is then blended with combustible wastes like saw dust and is then pelletized. The pellets have calorific value around 4,000 kcal /kg and there is no harmful emission upon combustion.

10.6. Mechanical Recycling of Dry Recyclables into Original or New Products Mechanical recycling involves processing of dry organic and inorganic recyclables such as papers and cardboards, plastic cans and bottles, metal cans, glass jars and bottles. Mechanical methods include thermal and chemical processing of waste materials and also consumes considerable amounts of water and energy. Now new mechanical technologies is being developed to recycle even hazardous industrial wastes. Metals, glasses, papers and plastics, wood and rubbers are some common domestic and commercial wastes which have high recycling potential to recover goods of mass consumption through mechanical technologies. (WWI, 1984).

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Recycling Potential of Metallic Wastes Iron, aluminum, lead, tin and copper are metals of mass consumption by the society. Their production from their virgin ores by mining activities requires huge amount of energy and water and also cause blatant environmental pollution, solid waste generation (as tailings) and deforestation. Metals like iron and aluminum can be effectively recycled for ever with least impact on environment while conserving huge amount of energy. (AEN, 2000). a) Ferrous Metals (Iron and Steel) The ferrous scraps include autos, household appliances, equipment, bridges, cans and other iron and steel products. The largest amount of recycled steel has traditionally come from large items like road unworthy cars and appliances. It can be reclaimed from the automobile bodies and engines, disused household or industrial equipment and building materials. The shipyards, railyards and the automobile junkyards offer vast amount of waste metallic products to be recycled. They can alone meet more than 50 % of the world‘s metal requirements. Marine ship break is a continuous process throughout the world as the ships lose sea worthiness in 25 to 30 years. Recycling of steel cans is also becoming very popular. They can easily be separated from the mixed recyclables or MSW using large magnets. To protect them from corrosion, all steel cans are coated with a thin layer of ‗tin‘ that must be removed in the recycling process. Immersing the sheets of steel in alkaline bath and transmitting an electric current completes the ‗detinning‘ process. Iron and steel has high recycling potential and can be recycled again and again without reducing the quality of the end products. Nearly half of the iron and steel which has already entered into the human ecosystem is now being used through recycling. World steel production alone consumes as much energy annually as Saudi Arabia produces. Making steel from recycled materials uses only a quarter of the energy needed to make steel from iron ores. (AEN, 2000). Iron scraps costs little more than iron ore but can be converted into steel with much lower economic and environmental cost. Using coke for iron ore reduction produce copious particulate matters including carcinogenic benzopyrene. Recycling of iron reduces this emission by 11 kg/metric tons of steel produced and also cuts iron ore waste and coal mining wastes by 1100 kg/metric tons recycled. Iron and steel scraps are baled into bricks at the material recycling facility (MRF) and melted at 1700 C in the smelters to produce ingots. b) Non-ferrous Metals Non-ferrous scrap metals include aluminum, copper, lead, tin, and precious metals. Recyclable nonferrous metals are recovered from common household items (outdoor furniture, kitchen cookware and appliances, aluminum cans, ladders, tools, hardware); from construction and demolition projects (copper wire, pipe and plumbing supplies, light fixtures, aluminum siding, gutters and downspouts, doors, windows); and from large consumer, commercial and industrial products (appliances, automobiles, boats, trucks, aircraft, machinery) Aluminum: It has high recycling potential. The amount of aluminum which has already entered into the human ecosystem is sufficient to cater the needs of the society through recycling and there is no need to process it from the virgin ores. Aluminum cans are baled into bricks and melted at 700 C in rotary furnaces. The molten aluminum is cast into ingots

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and remade into cans or processed into other aluminum products like saucepans and homewares. Copper: It can be recycled from the boilers of hot water systems, old car radiators and copper pipes. Electric cabling and wiring contains copper and aluminum which can be recycled. Lead: Lead can be recycled from old car batteries and old lead pipes. Lead is recycled in high rate because it is highly toxic and processing from its ore is highly damaging to both man and environment. Silver can also be recovered from silver-plating industries through recycling. A ‗silverplating plant‘ in the US spends about US $ 120,000 a year on waste treatment, of which US $ 60,000 is returned as credit for ‗silver‘ recovered from the waste. Silver and even gold is recovered from electrical industries. Table 12. Metal Consumption Procured through Recycling in USA (1). Lead (4). Aluminum (7). Tungsten

73 % 45 % 29 %

(2). Copper 60 % (5). Zinc 43 % (8). Nickel 26 %

( 3). Iron & Steel (6.) Tin (9). Chromium

56 % 38 % 21 %

Source : WWI, Washington D.C. ‗State of the World‘ (1989).

Environmental and Economic Benefits of Recycling Metallic Wastes World steel production alone consumes as much energy annually as Saudi Arabia produces. Making steel from recycled materials uses only a quarter of the energy needed to make steel from iron ores. Iron scraps costs little more than iron ore but can be converted into steel with much lower economic and environmental cost. Using coke for iron ore reduction produce copious particulate matters including carcinogenic benzopyrene. Recycling of iron reduces this emission by 11 kg/metric tons of steel produced and also cuts iron ore waste and coal mining wastes by 1100 kg/metric tons recycled. Recycling aluminum uses only 5 % of the energy needed to produce new aluminum from its ore ‗bauxite‘. Recycling one aluminum beverage can, saves energy enough to run TV for 3 hours. The energy needed to make 1 ton of virgin aluminum from bauxite could be sufficient to recycle 20 tons of aluminum from its scrap. It takes about 4 tons of bauxite to produce 1 ton of finished aluminum. Recycling aluminum reduces air pollution including the toxic ‗fluoride‘ by 95 %, and cut millions of tones of greenhouse gases (mainly CO2). (AEN, 2000). Recycling Potential of Paper and Cardboard Wastes About 30 % of the paper products which we use today are made from recycled papers and cardboards. Three common grades of paper recycled are corrugated cardboard, high grade office paper and old newsprint. Waste papers and card boards make excellent pulp for making different grades of paper to be used for stationary, magazine and newspapers, game boards, ticket stubbs, cereal and cake mix boxes, grocery bags, tissue papers, paper towels, egg boxes, cards and packaging materials. If cotton rags are mixed with them they make excellent pulp for making other kinds of papers too. Office papers are recycled to manufacture computer papers, writing and printing papers.

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However, infinite recycling of paper is not possible because the fibers become shorter and shorter and the quality of papers declines. Also the tissue papers and the wax coated papers cannot be recycled. The printed papers also need to be de-inked before recycling as it may contaminate the entire fibre stock.

Environmental and Economic Benefits of Recycling Paper Wastes Recycling of waste papers saves green trees, large amount of energy and water and prevent the use of chemicals. Recycling 1 ton of waste paper saves 13 trees, 2.5 barrels of oil, 4100 kWh of electricity and 31,780 litres of water. About 64 % less energy and 58 % less water is needed to make papers from recycled fibers than to make from virgin pulp obtained from the plants. Recycling half of the papers used in the world today would meet almost 75 % of the demand for the new paper and would liberate 8 mha of forest from clear-felling for plantation for paper production. According to one estimate the energy required to produce one ton of paper from the virgin wood pulp is 16, 320 kWh, while only 6000 kWh is needed to obtain the same amount of paper through recycling from paper waste. Reduction in energy use (oil or electricity generated from fossil fuels) proportionately reduce the emissions of greenhouse gas CO2 and other pollutants which would have occurred otherwise. (UNEP, 2004). Recycling of Paper Industry Waste All paper mills recycle and recover their intermediate and untreated effluents called ‗white water‘ (water passing through a wire screen upon which paper is formed) and thus reduces the volume of spray and wash water it uses. Other wastes like ‗sulfite waste-liquor‘ by-product have been found to have multiple uses. Their complete evaporation produces fuel and other salable by-product used in making core binder, road-binder, road bank stabilizer, cattle fodder, fertilizer, insecticides and fungicides, linoleum cement, ceramic hardener, insulating compounds, boiler-water additives, flotation agents, and in the production of alcohol and artificial ‗vanillin‘. The liquor may be ‗fermented‘ to produce ethyl alcohol. About 40 liters of alcohol can be produced per ton of dry solids. Acetone and butyl alcohol can also be produced from the liquor waste. Another product of fermenting the liquor is yeast for cattle feed. The ‗spent cooking liquor‘ produced in the sulfate process (kraft mills) contain recoverable quantities of sodium salts, resins and fatty acids. Caustic soda (Na2CO3) is recovered from them. The resin and fatty acids are further refined and have a variety of applications in industry. In chemical precipitation with lime as coagulant, ‗lignin‘ is precipitated which is used both as a fuel and in the manufacture of plastics, production of tannins, as an anti-scale or antifoam agent in boilers. Wastewater from the paper mills have also been found to be good for ‗irrigation‘ of number of crops. Recycling Potential of Glasses Glasses are 100 % recyclable and can be effectively recycled for ever. The recyclable glasses in MSW are container glass (the clear, green and brown bottles and jars for food and beverages), flat glass (e.g. window panes), and pressed amber or green glass. Glasses to be recycled is often separated by color into categories of clear, green and amber. Broken glasses are known as ‗cullet‘ which are valuable raw material in the production of new glasses.

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Manufacturer adds about 40 tons of cullet to every 100 tons of raw materials silica sand to produce glass. However, several vitreous materials may look and shatter like glasses, but are NOT recyclable. They do not melt like glass. Even a minute amount of these materials can contaminate the whole load of recyclable glasses and render them useless. These are ceramic mugs and plates, cups and crockery such as pyrex and corning ware, mirror, broken drinking glass and flower vase, light globes and laboratory glass. Contamination as little as 5 grams per ton is harmful. One tiny fragment of ceramic material in a load of glass ‗cullet‘ can cause a weakness in the new glass which may crack or even explode when the bottle is filled or opened. Recycled glasses are being used as raw material in cement production. (Wong, 2000). A British firm is building an industry to recycle the used wine bottles into ‗green sand‘ to be used for filtering drinking water and purify sewage. When the recycling plant is fully developed it can save the quarrying of high quality sand and use all the waste wine bottles. The European Union and the UK Department of Environment, Food and Rural Affairs are funding the project. (www.drydenaqua.com).

Environmental and Economic Benefits of Recycling Glass Wastes The cullet (broken glass materials) melt at low temperature than the primary raw materials and hence require 25-30 % less energy on addition and also extends the life of melting furnaces. Every ton of cullet used saves the equivalent of 30 gallons of oil and replaces 1.2 tons of virgin raw materials and proportionately reduce the emissions of greenhouse gas CO2 and other pollutants which would have occurred upon use of oil and other energy sources in the extraction of virgin materials from nature. Recycling Potential of Plastic Wastes Recycling of plastic wastes is not really a good option and is rather a hazard for the human health and the environment. It gives out ‗toxic‘ fumes like ‗dioxins‘ during melting. Technologies are not available for recycling of all categories of plastics. Plastics are recycled by codes. Currently only plastic bottles made of PET (Code 1: soft drink, juice and water bottles and some plastic jars); HDPE (Code 2: milk bottles, cream containers and juice bottles); PVC (Code 3: detergent, shampoo and cordial bottles); PP (Code 5: ice cream containers, takeaway food containers flower pots etc.); PS (Code 6: polystyrene cups, glasses and meat trays), and bottles with ‗R‘ or ‗please recycle‘ symbols are accepted for recycling. Two types of plastics most commonly recycled in world are PET (Polyethylene Terephthalate) and HDPE (High Density Polyethylene). PET is recycled to make soft drink bottles, deli and bakery trays, carpets, fibrefill and geotextile liners for the waste landfills. HDPE is recycled to manufacture plastic milk, disinfectant and detergent bottles, recycling bins, sleeping bags and pillow stuffing, roadside guide posts, irrigation pipes, eskies, water meter boxes, air-conditioning and recycled layer in new PET plastic bottles, agricultural water pipes, bags and motor oil bottles. LDPE (Low Density Polyethylene) is recycled to make new plastic bags and films. PVC (Poly Vinyl Chloride) is recycled to manufacture drainage pipes, non-food bottles and fencing posts. Recycled polypropylene (PP) is used in auto-parts, carpets and geo-textiles. Recycled polystyrene (PS) is used to manufacture wide range of office accessories, video-cassettes and cases.

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Plastic grocery bags made of LDPE or very thin HDPE cannot be recycled as they clog the system. Some supermarkets take back plastic shopping bags, recycling them into bin liners, and hospital waste bags. Other plastics like motor oil containers, plastic takeaway food containers and disposable nappies also cannot be recycled. Also different plastics cannot always be recycled together. The recycled plastic is often hard and brittle. To overcome this problem a ‗compatibilser‘ molecule that sticks together the different plastic molecules have been developed. Such commingled plastic with much gloss and sturdiness as the originals can be used to make ‗car bumpers‘ and fence posts.

Environmental and Economic Benefits of Recycling Plastic Wastes Recycling of plastics at least diverts thousands of tons of plastic materials from ending up in landfills every year and besides saving landfill space also arrest emissions of some very toxic gases and leachate discharge from the landfills. Recycling Potential of Waste Wood It is a waste from the timber industries, forestry and agricultural activities and from building construction and demolitions. Technology found its use in the manufacture of ‗plywood‘ and ‗medium density fiber board‘ (MDF). They have properties like natural wood with additional advantage of not being flammable and absorbing moisture, resistant to pest attack and low cost. They are also shredded and processed as wood chips for fuel or landscaping cover. Recycling Potential of Construction and Demolition (CD) Wastes CD wastes results from construction, renovation, and demolition of buildings; road repaving; bridge repairs; and the cleanup after natural disasters. Typically they are made of about 40-50 % rubbish (concrete, asphalt, bricks, blocks, and dirt), 20-30 % wood and related products (pallets, stumps, branches, forming and framing lumber, treated lumber, and shingles), and 20-30 % miscellaneous wastes (painted or contaminated lumber, metals, tarbased products, plaster, glass, white goods, asbestos and other insulation materials, plumbing, heating and electrical parts). The principal materials that are now recovered from the CD wastes are asphalt, concrete, wood, drywall, asphalt shingles, and ferrous and non-ferrous metals. Reinforcing steel used in foundations, slabs, and pavements are usually recovered and sold as scraps for recycling. Concrete and asphalt are processed for road base, aggregate in asphalt pavement, and as substitute for gravel aggregate in new concrete. Many landfills use the rubbles for road building and as ‗daily cover‘ material for the compacted waste in the landfill. (Hamilton, 2000).

10.7. Recycling Potentials of Some Hazardous Industrial Wastes (IHW) Technologies have been developed to recycle several categories of hazardous industrial wastes. Several hazardous industrial wastes are now finding new use and applications in construction industries through recycling. (Noll et. al., 1985).

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New Use of Phosphogypsum from Phosphatic Fertilizer Industry Radioactively contaminated calcium sulphate slurry called ‗phosphogypsum‘ (CaSO4.2H2O) is a waste byproduct of these industries. For every ton of phosphoric acid produced, 5 tons of phosphogypsum (PG) must be removed. It is now being recovered and reused to manufacture partition panel, ceiling tiles/boards, walling blocks etc. They save cement and steel and minimize the use of timber. Direct reuse of PG, however, presents the potential problem of incorporating radioactivity into building or road products. Several firms in the US and UK is processing the PG into useful cement or plaster. The quality of cement compares favourably with limestone-based cement and is used in all classes of building construction and civil engineering. It possess a ‗compressive strength‘ 3 to 4 times that of Portland cement (1100 kg / cm2 as compared to 300-400 for Portland cement). Moreover the cost of cement production from PG was US $10 / ton as compared to $ 30- $ 40 / ton for Portland cement. The PG is chemically processed into ‗hemihydrate powder‘ to use in cement manufacture. Some South African fertilizer plants disposes about 25 % of its PG as soil conditioner, cement clinker and as cement retarder. (Noll et. al., 1985). Any recovery and reuse of phosphogypsum (PG) would also free up reclaimable land resources for productive purposes by the industry or privately. New Use of Fly-ash from Waste Slurry in Coal-fired Power Plants Fly-ash is a waste byproduct of coal combustion in coal-fired power plants generally consisting of very fine particles. It pollutes water bodies. The wastewater usually contain 6750 gm / liter of fly-ash. A common method of fly-ash removal from the steam power-plant flue gases is by the use of ‗wet scrubbers‘, ‗electrostatic precipitators‘, or ‗cyclone separators‘. Major chemical component of fly-ash are silica (30-50 % by weight as SiO2) and alumina (20-30 % by weight as Al2O). Other materials are sulfur trioxide (SO3), carbon (C) and boron (B). It is now being reused to manufacture clay bonded bricks / blocks, fly-ash ceramics, aerated light weight cellular blocks and slabs, precast blocks for footpath, butiminous concrete for road surfacing and cement concrete etc. Fly-ash bricks have replaced baked earthen bricks and have saved million tones of fertile soil from getting eroded. Technology has found several new uses for fly-ash: 1) 2) 3) 4) 5) 6) 7) 8)

As a pozzolana ingredient in Portland cement; As a pozzolana in soil stabilization; As a soil conditioner in agriculture; As a grout in oil wells; In asphalt roofing and siding materials; As a cement replacement in concrete; As a mineral filler in asphalt pavement; As a coagulant in sewage treatment.

New Use of Red Mud from Aluminum Industry ‗Red Mud‘ is a waste from the aluminum industry being generated in million tones every year. It is a bauxite residue and clay like silt consisting of undissolved minerological components. It is being reused in cement industries both as component of cement raw mix as

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well as additives. Red mud light roofing sheets has been developed as an alternative to the dangerous ‗asbestos‘ roofs. New Use of Metallic Slag from Iron and Steel Industries It is a waste from the Iron and Steel industries and an excellent secondary raw material for cement production.

10.8. Thermal Recycling of Hazardous Wastes to Retrieve Thermal Energy Several hazardous industrial wastes such as paint thinners, degreasing solvents, solvents from ink and printing industries, dry-cleaning fluids, chemical by-products from pharmaceutical industries, waste papers, waste oils and waste auto tires, sewage sludge and municipal solid waste can be used as fuel in industries. Cement Kiln Using Hazardous Industrial Wastes as Fuel and Replacing Fossil Fuels The manufacture of cement from limestone require high kiln temperatures (about 1500 º C) and long residency times. This create an excellent opportunity for hazardous waste to be used as fuel and also get destroyed in the process. As much as 40 % of the fuel requirement of a well operated cement kiln is saved by the use of hazardous wastes.. It is like ‗killing two birds in one shot‘. Further, as the environment inside the kiln is alkaline due to the presence of lime, the acidic gases and the hydrogen chloride generated from the chlorinated wastes (which is poses problem in conventional incinerators) are neutralized. Combustion of wastes in a commercial incinerator also produces ‗ash‘ which needs to be disposed off. Here there is no ash and the only by-product is the ‗dust‘ which is recycled. Any incombustible material such as metals in the waste, becomes vaporized and incorporated in the product. Up to 95-99 % of the chloride and over 99 % of the lead entering the kiln are retained by the process solids. When the kilns are operating properly ‗dioxins‘ and ‗furans‘ and the particulate matters (which are emitted in the conventional incinerators) are significantly cut down and there is no risk to human health and the environment. U.S. uses about 1 million tons of hazardous wastes as a fuel in cement kilns every year. Reduction in use of fossil fuels as the source of energy in cement plants proportionately reduce the emissions of greenhouse gas CO2 and other pollutants which would have occurred otherwise. (Jones and Herat, 1984).

10.9. Recycling Potential of Some Hazardous Consumer Wastes (CHW) Appropriate technologies are now available to recycle several categories of hazardous wastes including the hazardous electronics wastes (e-waste) generated by society.

Recycling of E-Waste – A Hazardous and Costly Process: Reuse – A Better Option Millions of tones of e-waste all over the world are ending up in landfills. According to the Silicon Valley Toxics Coalition in the U.S. cost of recycling obsolete and discarded computers is estimated to be at US $ 10 to $ 60 per unit. And if poorly handled during the clean-up cost of toxic materials from the e-waste, it could go higher. SVTC estimates the minimum costs for recycling and proper disposal of the remaining non-recyclable e-waste in US will reach US $ 10.8 billion by 2015 (Schneiderman, 2004). This could be the approximate cost of recycling of e-waste in all developed nations including the European

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Union, Canada and Australia where labor cost is high. Local governments and councils in these nations have neither the technical ability nor the financial resources to tackle this gigantic techno-economic problem at their own. ‗Reuse‘ is a better option for several categories of e-waste. The term ‗reuse‘ would mean using any old and obsolete electronic product or equipment with or without minor repair and reasonable upgrading, if possible. The best way of reuse is that computers can be sold to the employees of the organizations or the students of institutions at very reasonable price or donated to charitable organizations, schools, orphanage centers, old people homes, women asylums etc. Institutions and organizations in the rich developed nations (where computer models are changing fast) should develop a system based on ethics for donating their old computers to the needy organizations in the developing countries. The term ‗recycling‘ of e-waste would mean to dismantle the equipment or a product and retrieve the valuable components / materials from it for their reuse in other equipment or remanufacture a new equipment / product. The difficulty with electronic waste and many other end of life electronic products is that they are made from a huge range of component materials that are useless for further manufacture until the product is dismantled and the component materials are separated – often a very difficult and expensive process. Recycling may be a good option for the extremely old generation computers such as the Pre-Pentium generation, or the computers (specially the monitors) which are broken. According to the International Association of Electronics Recyclers (IAER) more than 1.5 billion pounds of electronics equipment are recycled annually and is likely to grow by a factor of 4 or 5 by the end of this decade. Eleven countries currently have ‗mandatory‘ electronics recovery laws on the books. These are Denmark, The Netherlands, Norway, Sweden, Switzerland, Japan, Belgium, Taiwan, Portugal and South Korea. Some EU nations have very strong system for e-waste collection, such as the SWICO system in Switzerland and the Netherlands Association for Disposal of Metalectro Products (NVMP). NVMP collect 80 % of e-waste. About 77 % of TVs and 64 % of other small brown goods are recovered for reuse and recycling. (Monchamp, 2000; Cui and Forssberg, 2003; Mathew et. al., 2004)

Recycling Potential of Lead-Acid Car Batteries Billions of lead-acid car batteries are used and replaced every year across the world, 70 to 80 millions in US alone. The average battery contains about 18 pounds of recoverable lead and the worldwide recycling rate is now 90 %. Batteries are crushed and then the lead, plastic, and sulfuric acid are separated. In an innovative recycling process developed in Italy in the 1980s, batteries are crushed in a hammer mill and the components are separated on a vibrating screen. The acid / lead – paste slurry is neutralized, the lead oxide is separated, and the reusable sodium hydroxide and sulfuric acid are recovered from the solution by electrodialysis. Lead oxides are reduced by electrolysis and combined with metallic components, then melted at 400-500 C and cast into ingots. (Vaysgant, et. al., 1995). Recycling Potential of Household Batteries Billions of household dry-cell batteries are used and discarded worldwide, 2.5 billion in the US alone. These batteries are discarded with the general household waste. These batteries contain mercury, cadmium, lead, and other metals, which become toxic contaminants in the landfill leachate or in the air emissions if MSW are incinerated. Study reveals that household

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batteries are the source of more than 50 % of the mercury and cadmium in the MSW. Recycling of these batteries are difficult. Cylindrical 6-volt and 9-volt alkaline and carbonzinc batteries are not recyclable. Only nickel-cadmium cylindrical cells or mercuric oxide and silver oxide button cells can be recycled. Mixed button batteries are difficult to sort out and may present a storage hazard in the MSW waste bin due to mercury vapor emissions.

Recycling Potential of Auto Tires Several millions of auto tires are rejected every year all over the world which turn as hazardous wastes. Earlier they were reused after ‗retreading‘, but with the advent of steelbelted radials and cheaper new tires most are discarded as waste. They cannot be landfilled as they occupy large volume and tend to come to the surface. Auto tires present problem for safe recycling as they are made of ‗hazardous materials‘. The best option is to reuse them as ‗fuel‘ resource in cement kilns. About 70 % of the waste auto tires in the US is being used as source of energy in cement kilns thus also reducing use of coal and emission of greenhouse gases. Power plants, paper and pulp mills and the cement kilns commonly use the shredded tires as fuel. Whole tires are also used to create ‗artificial reefs‘ for erosion control and as highway crash barriers. Split and punched tires are used to make muffler hangers, belts, gaskets, and floor mats. Recycling Potential of Waste Oils and Lubricants from Automobiles Industries Billions of gallons of petroleum-derived waste oil are produced worldwide mostly from the automobiles and some from the industries. Automotive oils include crancase oil, diesel engine oil, transmission, brake and power steering fluids. Waste oil often contains metals like arsenic, cadmium, barium, chromium, and zinc; chlorinated solvents and organic compounds like benzene and naphthalene. Used lubricants can be recycled in two ways- by reprocessing and by re-refining. Reprocessing is done by water and bottom sediment removal of suspended material and ash by gravity settling or chemical treatment to produce partially cleaned fuel oil. Heat is sometimes used to decrease viscosity and improve gravity settling. Distillation is also done to evaporate light fuel fractions. Re-refining produces a clean oil but it is very expensive affair. This is done by solvent treatment / vacuum distillation / hydrotreatment; by acid clay, chemical cleaning / demetalling etc. Recycled lubricating oils are used as transformer oil, equipment oil, motor oil, cutting oil, soluble oil, diesel oil, gasoline, antifreeze, brake fluids and hydraulic oils. Waste oils are mainly recycled to be used as fuel in cement kilns, in commercial, industrial, and marine boilers, and for space heating. Waste oils can also be used as fuel in cement kilns, in commercial, industrial, and marine boilers, and for space heating.

11. SOME POLICY MEASURES ON WASTE MANAGEMENT BY GLOBAL SOCIETY Considerations of environmental sanitation, health and hygiene, contributing to safety and security of society are key factors which has prompted to enact suitable legislation for management of all kinds of waste by the global society. Safe waste management is not only a

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technological pursuit, but also require legal and administrative, economic and ecological planning. Governments can develop policies to encourage and support the manufacture of ‗durable‘ and ‗recyclable‘ goods as well as ‗take-back programs‘ by producers to recycle their products. If required to ‗take-back‘ and recycle their products, manufactures will be compelled to produce durable and recyclable items, thus reducing waste for consumers.

11.1. The European Union‘s Packaging and Packaging Waste (PPW) Directive (1994) On average each EU citizen is currently responsible, directly or indirectly, for the generation of some 172 kg of ‗packaging waste‘ every year. (UNEP, 2006). As early as in 1994 it issued this directive to ‗prevent‘ packaging waste. Yet, the packaging waste (PW) generation increased by 10 % in the EU-15 between 1997 and 2002, in close line with the 12.6 % growth in GDP. Per capita consumption of plastics increased by almost 50 % from 64 kg / year in 1990 to 95 kg / year in 2002. Only UK managed to actually reduce, and Austria stabilize the generation of PW since 1997. The review of the EU-15 PPW in 2005 however, showed that the both the producers (companies) and the consumers (society) made good progress in recycling PW. The EU target of recycling 25 % of packaging waste in 2001 significantly increased to 54 % in 2002 in the 15 member countries then. (UNEP, 2006).

11.2. EU Directives on ‗Extended Producer / Manufacturer Responsibility‘ (EPR) for Reducing Electronic Waste (2001) In Europe, e-waste is projected to reach 12 million metric tones by 2010 (Schneiderman, 2004). In 2001, the European Union adopted a system called ‗Extended Producer / Manufacturer Responsibility‘ that requires the electronics manufacturers to ‗take-back‘ their used products and assume full responsibility for the production of cleaner electronics items, phasing out of hazardous materials in production process, and also dismantling the e-waste products more easily for recycling at the end of their useful life by trading-in the products for recycling. In January 2003, the EU parliament enacted two directives. The first - ‗Waste Electrical and Electronics Equipment‘ (WEE) is based on the concept of Extended Producer Responsibility (EPR) which requires the industries to ‗take-back‘ all their used and obsolete electronic products for safe recycling. The second directive ‗Restriction on Hazardous Substances‘ (RoHS), called for phasing out of heavy metals Hg, Cd, Pb and Cr VI in all electronics items by July 2006 (with a number of exemptions) to reduce hazardous waste when they are discarded. This has now come into effect. (Adam, 2005).

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11.3. The International Legal Instruments for Combating the Problem of Hazardous Wastes at Global Level The United Nations General Assembly initiated to review the international environmental laws in 1981 at Montevideo. The laws were to cover among other things, the transport, handling and disposal of toxic and hazardous wastes. Real negotiations to impose curb on the production, storage and transport of hazardous chemicals and wastes began soon after the Basel Disaster (Switzerland) in 1986 which resulted into the adoption of ‗Basel Convention‘ in 1989.

The Basel Convention on the Control of Transboundary Movement of Hazardous Wastes and Their Safe Disposal (1989) The Convention was adopted in 1989 unanimously by 116 States in the Swiss city of Basel, to reduce the global generation of hazardous wastes and chemicals to a minimum and to prohibit / regularize the illegal traffic in hazardous wastes and to fix the responsibilities of the parties involved. It entered into force on 5 May, 1992 and now have 131 Parties to the Convention except the U.S. It outlines the general obligations of the hazardous waste generating nations for moving their wastes across their boundaries either for disposal or for exchange for recycling. It also outlines the principles of international co-operation to improve and achieve environmentally sound management of all kinds of hazardous wastes. Trade in hazardous waste that does not comply with the terms of the Basel Convention or its control system is illegal, and considered to be criminal. Parties are obliged to enact stringent national laws to prevent and punish the illegal trafficators in hazardous waste. The Convention also cooperates with the Interpol over illegal traffic. The first 10 years of the Convention (1989 – 1999) concentrated on consolidating its control system, legal framework and operation through improving the classification of wastes and refining the work on their hazard characterization. Cooperation to Developing Countries Under the Convention international cooperation is to be extended to developing countries in: 1. Transferring technology and management systems for hazardous waste; 2. Developing and implementing new environmentally sound low-waste technologies, and improving existing ones to eliminate the generation of waste, as far as practicable, and studying the economic, social and environmental effects of adopting the new technologies; 3. Developing and promoting environmentally sound management of hazardous and other wastes; 4. Promoting public awareness about hazardous waste.

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12. SOME INNOVATIVE IDEAS TOWARDS SUSTAINABLE WASTE MANAGEMENT 12.1. The Australian Innovations on Landfill Gas Utilization for Energy Generation While Reducing Emission of Greenhouse Gases An innovative technology to convert the MSW landfills into a bioreactor is being experimented in Australia. In the traditional landfills the ‗anaerobic decomposition‘ of waste is highly retarded and rather inhibited, as the waste has very little moisture. Daily covering and heavy compaction of waste exclude the moisture content. Normal waste typically contains 10-30 % moisture. The rate of waste biodegradation is a function of moisture content. Bioreactor technology significantly reduces the decomposition time and the production of gas begins soon. Bioreactor is a large ‗anaerobic digester‘, in a specially designed void in landfills which receives municipal solid wastes and the also the biosolid (sludge) from the sewage treatment plants. In the landfill bioreactor, the waste is not heavily compacted and sufficient moisture content (45 – 60 %) is maintained through extensive networks of pipes that re-circulate the nutrient rich leachate and inject additional water such as the stormwater and the run-off. Additional microbial inoculum is added to promote rapid anaerobic microbial decomposition and biogas (60 % methane and 40 % carbon dioxide) production is augmented with 98 % collection efficiency and conversion to energy. Each kilogram of waste can generate up to 220 liters of methane. In case there is more paper content in the waste, the methane production is 365 litre / kg. A typical 4000,000 tones per year ‗landfill bioreactor‘ will produce about 7,500 cum of methane per hour, with a generating capacity of 25 MW. This will also save greenhouse gas equivalent to taking 175,000 cars off the road. When biogas generation in the bio-cell declines, the residual partially degraded organic materials can be excavated and used as a ‗soil conditioner‘ or feedstock for composting. Besides, generating biofuel and biofertilizer, the recycling technology will reduce the emission of greenhouse gas (methane) from the landfills. Fugitive emissions of VOCs is decreased to as low as 0.7 %. It would also reduce the environmental risk period of landfills to 5-10 years from 30-50 years.

Power Generation from Landfill Gas The ReOrganic Energy Swanbank in Ipswich, Brisbane, Australia is generating electricity from the landfill biogas. Natural biodegradation processes in the landfills are enhanced in a special configuration to accelerate the production of biogas (methane). This is supplied to the Swanbank Power Plant for electricity generation displacing coal. By end of April 2002, some 20,000 cubic meters per day of biogas was produced and utilized and over 2,300 MWh of electricity generated. It is envisaged that over this period beginning from 2002 to 2016, some 90,000 cubic meters of biogas per day will be utilized, 500,000 MWh of electricity will be generated, while some 250,000 tones of coal will be displaced and approximately 3 million tones of carbon dioxide equivalent greenhouse gas (methane and carbon dioxide) will be arrested from going to the atmosphere. The $ 4 million project took some 20 months to complete.

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After successful utilization of landfill gases for power generation at Ipswich, another $ 5 million landfill gas power generating facility was launched at Rochedale Landfill site in Brisbane in 2004, generating 3 MW of power. This is expected to reduce greenhouse gases by 20,000 tones a year and also supply electricity to 5000 homes. Since the closure of the landfill at Roghan Road, Fitzgibbon in March 2000, BCC has also started generating 2 MW electricity from the methane produced in the old landfill. New waste landfills in Brisbane are now purpose designed to be used as ‗bioreactors‘ for biogas generation and electricity production, killing two birds in one shot (www.thiess-services.com.au).

12.2. Molok Waste Bins: Odor Free, Less Space, Holds More Waste and Emptied Less Often: An Australian Innovation The Molok Pty. Ltd. Of NSW in Australia has invented a new waste collection bin (40 x 120 L size) 60 % of which lies underground and only 40 % is visible. It is installed to a depth of 1.5 meter in ground and takes 80 % less surface area than the conventional waste bins. It comes in 300 L, 1300 L, 3000 L and 5000 L sizes suiting to all locations in residential and commercial areas. The containers are water tight and the waste drop hole opening is approximately 1.1 m above ground level. Within the PVC container is suspended the lifting waste bag made of double layered textile material. While emptying the bag is simply lifted by the hydraulic lifting arm and hoisted into the truck container. Emptying 5000 L Molok bin takes about 3 mins and is one man job. The key advantage of the vertical bin is that gravity forces the old waste to compact as the new waste is added, and the oldest waste materials at the bottom of the container is kept cool because the earth underground is naturally cool due to evaporation. The lowering of temperature at the bottom reduces microbial activity arresting any odor problem. It is also likely that there will be lesser emission of greenhouse gas methane.(www.molok.com.au).

12.3. The Innovative ‗Bio-Bin‘ System of Cleanway for Kerbside Composting of Green Waste Cleanaway‘ is an enterprise of Brambles Industries Limited established in 1970. It operates across Australia and around the world. It provides services in waste recycling, safe hazardous waste disposal, site remediation, and landfill operations. It operates 8 waste recycling plants in four Australian states. It‘s co-mingled bin recycling system services over 770,000 Australian households and has participation rate over 85 %. ‗Cleanaway‘ has introduced a new and innovative concept of ‗Bio-insert‘ system. The ‗Bio-Bins‘ break down ‗green organic waste‘ on the kerbside and reduces the weight of the waste. It promotes oxygen-flow around and through the organic waste, triggering the aerobic decomposition process. Problems like odor, leachate production and greenwaste sticking to the bottom of the bin are eliminated because of oxygenation. The contents of the bio-bins are less compacted, drier, lighter and more uniform than those in the standard bins. It forms good feed-stock material for the ‗compost facility operators‘. A collection frequency of 4 weeks is recommended for green organics, while 2 weeks for the food organics. It is also inexpensive

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to integrate the bio-bin system into the existing waste management services and would save the city councils considerable expenses in collection and landfill costs. Bio-Bins are widely in use in Europe and North America for the past ten years. A trial of bio-bins is being conducted in the City of Mooney Valley, Melbourne, Victoria with very encouraging results.

12.4. The Innovative DiCOM ‗Aerobic-Anaerobic-Aerobic‘ Hybrid System of Bioconversion of MSW into Clean Biofuel and Biofertilizer A new and innovative hybrid biological system for composting of MSW was developed in Australia in 2000 by AnaeCo which process the MSW and produces a finished product in less than half the time required for normal aerobic process of microbial composting without any odor problem. The end products are ‗green energy‘ (biogas methane) and ‗compost‘. This has been termed as DiCOM bioconversion process. The facility was installed at the cost of $ 5 million and is situated in Perth, Western Australia. It receives commingled municipal solid wastes (MSW) from the local councils, removes the non-compostable inert materials (the dry recyclable) such as the metals, glasses and plastics from the waste and then subject the organic materials to a ‗Three (3) Stage Processing‘ that involves an ‗Anaerobic Digestion Phase‘ in between the initial and final ‗Aerobic Composting‘ period. The methane (CH4) gas generated from the anaerobic digestion phase is captured and used as biogas fuel for electricity generation at the facility. The separated recyclable materials are sent to MRF. (www.anaeco.com). The company has patented this technology. The initial bioconversion capacity was 17,000 tons of MSW per annum which increased to 55,000 tons per annum by 2005. The whole plant has small ‗ecological foot print‘ requiring small operational area and with potential for decentralization of waste treatment to multiple sites close to the sources of community waste generation, thus significantly reducing the cost of waste transportation and pollution.

12.5. The Innovative Total Waste Management System‘ (TWMS) in Australia: No Waste to Landfills Berrybank Piggery in Victoria Shows the Way Charles Integrated Farming Enterprises Private Limited company in Victoria developed an innovative concept of ‗Total Waste Management System‘ (TWMS) for its Berrybank Piggery. in Victoria, Australia. The piggery produces on an average 275,000 L of effluents (sewage) a day with ‗solid contents‘ approximately 2 %. The company found that the ‗traditional farming philosophy‘ of ‗wasting nothing‘ and ‗waste from one part of a farm is the input in another‘ makes a good business sense. On regular inventory program the management of piggery found that half of the feed consumed by the animals is actually utilized, and the remaining half goes as waste. The company considered this generation of waste as ‗poor return on investment‘. The TWMS is a seven-stage process which salvages all the waste byproducts in the form of fuel, fertilizer and flush water. Since the introduction of TWMS, the Berrybank Piggery now daily produces –

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Seven (7) tones of waste solids which are utilized as ‗fertilizer‘ for farms; 100,000 L of flush water (recycled water) to be used for flushing toilets; 100,000 L of mineralized water rich in essential micro and macro-nutrients which is utilized as ‗liquid fertilizer‘ for farm irrigation; and 1,700 cum of biogas fuel (methane) which is used for power generation on farm with daily output of 2,900 kW of electricity.

The capital cost of the Berrybank Farm project was approximately AU $ 2 million which was paid back in about 6 years by way of fuel, fertilizer and usable water. From a $ 2 m investment made in the TWMS for the Berrybank Piggery, the Charles IFE brings in AU $ 435,000 return every year. Other environmental benefits was that the odor problem and the risk of groundwater contamination (due to sewage) was completely eliminated and it dramatically reduced the consumption of freshwater (www.environment.gov.au/settlement/industry/corporate/eecp/casestudies/charlesife.html)

12.6. The Indian Innovation on Dumpsite Composting of Commingled MSW: A Cheaper and Safer Alternative to Costly Landfills? An innovative technology to compost the un-segregated municipal solid waste (MSW) biomass on ordinary waste dump-site was developed in India in the 1990‘s and is giving excellent results for managing the solid wastes. No prior segregation of commingled waste is required. Segregation of commingled wastes at source or before composting imposes the biggest obstacle in any composting technology because it is highly labour intensive job to segregate the often sticky and wet biodegradable (compostable) matters from the dry nonbiodegradable ones. It becomes much easier after composting. The technology was developed by Ms. Excel Industries, Mumbai, India and can suit to all countries and is also flexible for 150-700 MT of waste per day. The largest plant with an installed capacity to bioprocess 500 tones /day of MSW is operating in Mumbai. The process can recycle all organic wastes from the households, restaurants and hotels, dairy, agriculture and agro-processing industries, brewing industries and slaughter houses. (Ranjwani, 1996;.Sinha and Herat, 2002 b).

The Bioconversion Process and the Composting Mechanism The dump site is leveled and either cemented or paved with bricks on the bottom to prevent leachate and for easy movement of waste carrying vehicles. Long windrows, about 5 meters wide and 2-3 meters high (deep) are erected and the MSW is then stacked and heaped in the windrows. A ‗microbial consortium‘ slurry containing active decomposer bacteria and enzymes are then added to the windrows to initiate rapid aerobic decomposition of the waste biomass. The slurry is spread on the surface of the garbage and inside the heaps in the windrows with help of probes, so that it reaches deep and in every pocket of the heap. Microbial culture of active decomposer bacteria is prepared from the ‗sewage sludge‘ which contains active decomposer bacteria in millions/gram of the sludge. (Ranjwani, 1996).

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The microbial culture is known as ‗Celrich Substrate DF BC-01. It is prepared after analyzing the composition of the waste and identifying the predominant materials such as celluloses, hemicelluloses, lignins, proteins and fats etc. The microbes produce enzymes such as cellulase, lipase, amylase, protease, pectinase and phospholipase to breakdown the long chain complexes of the substrates. About 1 kg of the consortium in the colloidal emulsion form is mixed with 20 litres of water to be used for spraying on about 3 cubic meter of solid waste and for one ton of waste 200 litres of water is needed. Recycled water can also be used. The waste heaps are turned around once in 7 to 10 days for proper aeration and the inoculant slurry is sprayed in each turning to enhance decomposition and maintain the proper moisture level which is usually 45 - 55 %. The process is ‗exothermic‘ and the windrows reach a temperature of 70-75 C within 24-36 hours, killing the harmful pathogens and repelling all birds, stray animals, flies and mosquitoes from the dump site. (Ranjwani, 1996).

Recovery of Compost The entire process of aerobic decomposition of garbage is completed within 4-6 weeks and as the decomposition is complete the temperature comes down to normal. It recovers over 90 % of the organic matter in the form of compost which may be 25-30 % of the raw waste on dry weight basis. Recovery of compost depends upon the presence of organic matter in the garbage. There will be greater recovery of compost in developed countries as much higher amount of organic wastes reaches the dump-sites (tips) in every city. Retrieving the Dry Recyclables The decomposed waste biomass is passed through rotary and vibratory screens to sieve out the compost. The soft decomposed powdery materials gets easily separated from the plastics, metals, stones and pebbles. About 20-25 % are dry recyclable materials and the rest about 20-25 % are inert materials which are disposed in ordinary land-fills. Same Land for Dumpsite Can be Reused and No Need of Engineered Landfill The same dump-sites can be used again and again after excavating the biodegraded mass (compost) and there is no need of additional land for making more dump-sites. Also the need for ‗engineered land-fills‘ are greatly reduced because very little is left to be disposed off after retrieving the compost and the recyclable materials. Low Emission of Greenhouse Gas Methane The problem of emission of landfill gas methane (CH4) is significantly minimized as the waste is turned constantly and the waste biomass is thoroughly aerated throughout the composting period. Foul Odor at Waste Dumpsite Disappear Soon Giving Relief to Waste Workers Emission of ammonia and hydrogen sulfide which are mainly responsible for foul odor at the waste dump-site (tips), and the leachate discharge is also greatly reduced. The foul odor at the waste dump-site disappear within 2-3 days of sanitization by the microbial culture giving great relief to the waste workers and the local residents.

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13. CONCLUSION AND RECOMMENDATIONS An assessment of waste generation not only involves the production and distribution of commodities and services but also its actual history of use/reuse/recycling. How people act as consumers, re-users and recyclers is as important as how a thing is made and sold to consumers. Any government policy, any waste reducing and recycling technology and strategy cannot succeed unless every member of society is aware and behave responsibly. Sometimes policing becomes essential to change societal behavior. Random check of waste bins by councils and ‗refusal‘ to pick up waste not disposed according to council directives, can force people to behave. The strategy has worked well in some Canadian cities. Economic instruments also seem to affect human behavior. ‗Landfill taxes‘ in Denmark, Austria, Ireland, Italy and the UK, and charging people for plastic bags in supermarkets in Denmark and Ireland (and in France from late 2005 onward) have changed the human behavior in these nations. Manufacturers of consumer goods hold great responsibility in reducing waste in modern consumerist society. Governments of nations need to come out with a policy of ‗durable and recyclable goods‘ by manufacturers, and also ‗take back policy‘ of their products for recycling. It will also be a ‗disincentive‘ for them to change their product versions too frequently to lure people. Society has to play very critical role in all waste management programs. The traditional societies were basically ‗recycling societies‘. They made best use and reuse of all materials several times before discarding them. The modern society is basically a ‗throwaway society‘ which discard most materials after short use. Modern human societies have to revive some of the ‗traditional cultures‘ of resource conservation, resource recycling and their ethical use. Waste recycling is essential for economic stability, ecological sustainability, environmental safety and survival of the global human society. Human society has to begin the process of recycling at the source – the home, office, or factory- so that fewer materials will become part of the disposable solid wastes of a community. A ‗consumer education‘ program is needed to educate the society about the hidden ‗environmental value‘ (the energy and water it has saved, the pollution and deforestation it has prevented) of the recycled goods. All recycled goods should have ‗recycled tag‘ telling about the origin and life history of the goods (from which waste it was produced) and how it has saved the environment. It would develop a sense of ‗civic pride‘ among the consumers that he / she is helping the environment. There is always greater economic and ecological wisdom in reducing waste at source, and diverting more and more of the waste to the ‗mechanical recycling, ‗biological recycling‘ (composting) and ‗thermal recycling‘ (combustion) processes to recover some useful materials and energy from them and reduce their volume, so that less and less waste is left for final disposal in landfills. Government, industries, science and society all have to join hands in fighting the menace of mounting wastes. Industries producing consumer goods have to play more responsible role in waste management program because they have potential to generate waste twice in the lifecycle of the goods produced. First at the ‗production level‘ when they process the virgin raw materials and generate ‗industrial wastes‘. Second at the ‗consumption level‘ when the goods produced by them are used and discarded by the society as ‗municipal solid waste‘ (MSW). Policy makers have carrot and stick options to encourage or enforce industry to contribute to

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reduce waste. Industrialists also owe moral obligation to provide necessary information to its prospective buyers on the matter of using, handling, conservation, disposal and recycling potentialities of its products. In designing new products, the industry must assess its potential and even suspected adverse impact on its consumers health and the environment. We need more and more waste to be converted into resource (by recycling) to sustain our growing population as the several natural resources are either on decline or becoming scarce or are unavailable and beyond our capability to exploit them sustainabily with present technology and within the ecological limits. Government must encourage and promote the recycling industries using waste as raw materials by way of reduced taxation, reduced cost of water and electricity supply etc. Given current technology, not all the municipal or industrial wastes can be readily recycled. Nor do all the waste materials have qualities that currently make them a valuable commodity in the recycling marketplace. Hazardous waste is growing in U.S. industries and very little is being done to reduce or recycle them at source. Most hazardous wastes are being exported to poor developing countries either for dumping or for recycling. (Duke, 1994). Wrong policy decisions of the U.S. government has aggravated the problem. Study made by an environmental organization in the U.S. indicates that subsidies given to the timber, mining, oil, energy, and waste disposal industries undermine and discourage waste recycling industries. These subsidies lower the cost of products made from new and virgin materials, giving them a competitive advantage over those made from recyclable materials. Fifteen government subsidies given to these industries in the U.S. amounted to as much as US $ 13 billion over the next 5 years. It was a great setback for the waste recycling industries in the U.S. They include indirect subsidies such as cheap water and energy supply to these industries.(UNEP, 2006). Life-cycle assessments is also important to determine the recycling potential of a waste product. A number of life-cycle assessments have found that fully recycled paper is not always the most environmentally friendly choice. In some countries paper produced from local agricultural wastes, such as rice straw, may be environmentally more sustainable than that produced from recyclable paper wastes shipped from overseas or transported from distant locations in the same country. One study in Australia found that if the recyclable paper waste is transported more than about 20 km by road, the energy balance (fuel used and pollution generated during transport) is not in favor of recycling. Safe disposal of the radioactive wastes which are accumulating exponentially in the human environment cannot be guaranteed at all even after huge expenditure. A huge pile of radioactive wastes (most in the U.S., France and Japan) remains to be disposed safely. It was just 84,000 tones in 1990 and must have crossed 100,000 tones limit by now. According to the celebrated geologist Konrad – ‗No scientist or engineer can give an absolute guarantee that radioactive waste will not some day leak in dangerous quantities from even the best of repositories‘. The word ‗safe‘ seems to be incompatible with radioactive wastes. However, ‗waste prevention‘ through ‗cleaner production‘ is considered to be even more wiser and sustainable idea of waste management so that any treatment or recycling is not at all needed and tremendous cost can be saved. Waste prevention would also reduce the cost of construction and maintenance of secured landfills because some residual wastes are always left after treatment or recycling which has to be safely dumped in the landfills. Educating the ‗environmentally illiterate‘ society is a big challenge. People need to understand the importance of their activities in the context of global consequences, for instance, the ‗links between waste generation, greenhouse gas emission and climate change‘.

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Waste education needs to take place in a continuous way through schools and universities, through mass and diffuse media and community education programs run by governments at all levels as well as non-government organizations, businesses and industries. Waste education of society should become an integral part of all waste management programs. We have to mend our ways, change our behavior and attitude of life, re-order our priorities, simplify our life-style, and then only the gigantic problem of mounting solid waste, which literally threatens to bury the mankind alive, can be overcome. Only a ‗resource conserving, waste reducing and waste recycling‘ society would be the ‗sustainable human societies‘ of the future and the ‗throwaway wasteful societies‘ would perish.

REFERENCES AND ADDITIONAL READINGS Anonymous (1990): Characterization of the Municipal Solid Wastes in the U.S. : 1990 Update; US EPA, Office of Solid Waste and Emergency Response, Washington, D.C. Anonymous (1999): USA Today, 22nd June, 1999. AEN (2002): Metals Recycling Conserves Energy; Australian Energy News, Issue 24, June 2002; pp. 12-13. Adam Teva V‘Din (2005): Models for E-Waste Management- The European Union Leads the Way; The Israel Union for Environmental Defense; www.softwarelabs.com Viewed February 2005. BCC( 2002): Newsletter ‗Your City Your Say‘; Published by Brisbane City Council, Queensland, Australia; April 2002. Bellamy, Carol (1995): Women and the Environment; Our Planet; Vol. 7 No. 4; Publication of United Nation Environment Program (UNEP), Nairobi, Kenya. Blatt, Harvey (2005): America‘s Environmental Report Card; Are We Making the Grade? MIT Press. Cambridge (Mass.), London. Brahmbhatt, Ashish and Saeed Parshin (2005): Environmental Impact of Disposable Baby Diapers on Landfills; Research Project on Solid Waste Management (7095 EVE); Griffith University, Brisbane, October 2005.(Supervisor Dr. Rajiv Sinha). CSIRO (1996): State of the Environment in Australia; Council of Scientific and Industrial Research Organization, Canberra, ACT. Connor, M.A., Evans, D.G. and Hurse T.J. (1995): Waste Flows in the Australian Economy; Waste Disposal and Water Management in Australia; June 1995, pp. 9-28. Cointreau, S. J. (1982): Environmental Management of Urban Solid Waste in Developing Countries: A Project Guide; Urban Development Technology Papers, No.5, World Bank, Washington D.C. Cave, Shane and Jacquie Ashton (1993): Clean Up the World; Our Planet; Vol. 5, No. 6; UNEP Publication. Cui, J. and Forssberg, E. (2003): Mechanical Recycling of Waste Electric and Electronic Equipment : A Review; Journal of Hazardous Materials; Lulea, Sweden. Chandra, R., Majumdar., A. Roy, S., Joshi, P., Kumar, V., and Marda, V. (2005), E-Waste : The Indian Context; Indian Institute of Management; Kozhikode. Viewed Online April 2005http://www.indiainfoline.com/bisc/ari/ewas.pdf

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Chauhan, Krunal & Valani, Dalsukh (2008): Studies into Aerobic, Anaerobic and Vermicomposting Systems (Part of 40 CP Honours Project – ‗Studies in Vermiculture Biotechnology‘), Griffith University, Brisbane, Australia.(Supervisors: Rajiv K. Sinha & Sunil Herat). Courier Mail (2002): Newspaper Daily; Brisbane, Queensland, 9th May, 2002. Datar, M.T., Rao, M.N. and Reddy, S. (1997): Vermicomposting : A Technological Option for Solid Waste Management; J. of Solid Waste Technology and Management, Vol. 24 (2); pp. 89-93. Dorling, Kindersley (1987): Blueprint for Green Planet; London. Duke, L. Donald (1994): Hazardous Waste Minimization: Is it Taking Roots in U.S. Industry?; Waste Management; Vol.14, No.1; Pergamon Press; pp. 49-59. Edward, C.A. (1988): Breakdown of Animal, Vegetable and Industrial Organic Wastes by Earthworms; In C.A. Edward, E.F. Neuhauser (ed). ‗Earthworms in Waste and Environmental Management‘; pp. 21-32; SPB Academic Publishing, The Hague, The Netherlands; ISBN 90-5103-017-7. Eklington, John and Julia Hailes (1989): The Green Consumer Guide – From Shampoo to Champagne: High Street Shopping for Better Environment; Victor Gollancz Ltd. London. Epstein, E (1997): The Science of Composting;Flintoff, F. (1976): Management of Solid Wastes in Developing Countries; WHO Report, pp. 245. Eawag (2008): Global Waste Challenge : Situation in Developing Countries; Swiss Federal Institute of Aquatic Science & Technology (www.sandec.ch) Fairlie, Simon (1992): Long Distance Short Life, Why Big Business Favors Recycling; The Ecologist; Vol. 22: No. 6; pp. 276-282. Fraser-Quick, G. (2002): Vermiculture – A Sustainable Total Waste Management Solution; What‘s New in Waste Management ? Vol. 4, No.6; pp. 13-16. Frederickson, Jim (2000): The Worm‘s Turn; Waste Management Magazine; August, UK. Gottas, H.B. (1956): Composting ; World Health Organization Monograph, Geneva; p. 25. Global Footprint Network (2005): National Footprint and Biocapacity Accounts. Available from and accessed 15 March 2006 — http://www.footprintnetwork.org. Goldoftas, Barbara (1989): Making Waste Work; SPAN Magazine, United States Information Service (USIS) Publication, New Delhi, India; July 1989. GOI (1989) : The Hazardous Wastes ( Management and Handling Rules) 1989; Department of Environment and Forest, Government of India, New Delhi. GOI (1997): Biomedical Wastes (Management and Handling Rules) 1997; Department of Environment and Forest, Government of India, New Delhi. Goldstein, J. (1995): Recycling Food Scraps into High End Markets; Residuals Biocycle; Vol. 36 (8); pp. 40. Hamilton, John (2000): Construction Waste Recycling at SENT Landfill; Green Valley Landfill Ltd. Proceedings of the Conference on ‗Recycle 2000‘, Hong Kong. Haug, R.T. (1993): The Practical Handbook of Compost Engineering; Lewis Publishers, Boca Raton. Holmes, John, R. (1984): Managing Solid Waste in Developing Countries; John Wiley and Sons; New York. Harding, Ronnie (ed) (1998) Environmental Decision-Making : The Roles of Scientists, Engineers and the Public. Federation Press, Annadale/Leichhardt.

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Heath, C.W., Jr., (1983): Field Epidemiologic Studiesof Population Exposed to Waste Dumps; Environmental Health Perspectives; Vol. 48 : pp. 3-7 Hasselriss. Floyd (1995) : Medical Waste Incineration ; Technical Monitor; US.Menon, Subhadra (1977): Drowning in Trash; India Today; Bi-Monthly Magazine Published in India; May 15, 1977; pp. 78-83. IAEA (1991): Nuclear Power, Nuclear Fuel Cycle and Waste Management: Status and Trends; Report of International Atomic Energy Agency, Vienna, Austria; Part C; pp. 73. Jones, P.H. and Herat, Sunil (1994) : Use of Cement Kilns in Managing Solid and Hazardous Wastes : Implementation in Australia; Journal of the Institution of Water and Environmental Management; Vol. 8; No. 2, April 1994, UK. Komarowski, S. (2001): Vermiculture for Sewage and Water Treatment Sludge; WATER, July 2001. Mathew, J. Realff, Michele Raymonds, and Jane C. Ammons (2004): E-waste : An Opportunity; Materials Today; Georgia Institute of Technology, Atlanta, U.S.; January 2004, pp. 40-45. Miller, C. (2004): E-Waste: Time to Address It? , MAXUS Technology, Inc., Canada. (Viewed online April 2005) http://www.municipalsuppliers.com/MagazineIndex/ 2003/cpwe2003_page18.asp Monchamp, Amanda (2000), The Evolution of Materials Used in Personal Computers; 2nd OECD Workshop on ‗Environmentally Sound Management of Wastes Destined for Recovery Operations‘; Vienna, Austria, September 28-29, 2000. Naidu, Ravi (2004): Report on Hazardous Waste Dump Sites in Australia by Center for Environmental Risk Assessment and Remediation, University of South Australia. In ‗The Australian‘, March 2004. Noll, K.E., C.N. Haas, C. Schmidt and P. Kodukula (ed) (1985): Recovery, Recycle and Reuse of Industrial Waste; Industrial Waste Management Series, Lewis Publishers Inc., Chelsea, Michigan; 196 pp. O‘Rourke, Morgan (2004), Killer Computer: The Growing Problem of E-Waste; Journal of Risk Management; New York; Vol. 51; Issue 10; pp.12-17. Puckett, J., Byster L., Westervelt, S. Gutierrez, R., Davis, S., Hussian, A., and Dutta, M. (2002), Exporting Harm : The High-Tech Trashing of Asia; Basel Action Network and Silicon Valley Toxics Coalition; (Viewed Online April 2005). http://www.ban.org/Ewaste/technotrashfinalcomp.pdf Parker, C, and Roberts, T. (1985): Energy from Waste; Elsevier Applied Science, NY. Porter, R., and J. Roberts (1985): Methods of Recovering Material and Energy from Refuse; In Porter and Roberts (ed.) ‗Energy Savings by Waste Recycling‘; Elsevier Applied Science, NY. Ranjwani, P.U. (1996): City Waste Treatment and Its Bioconversion into Organic Fertilizer; Paper by Ms. Excel Industries Ltd., Mumbai for all Municipal Corporations of India. Rao, Jayshree (1998) Management of Municipal Urban Wastes : Some Innovative Strategies; PhD Thesis, University of Rajasthan, Jaipur, India. (Supervised by Rajiv K. Sinha). Raghupaty, L. (1994): Hazardous Wastes: Management and Minimization; Paper Presented at International Seminar on ‗Solid Waste Management and Training‘; Bangalore, September 19-24, 1994. (Sponsored by Ministry of Environment, New Delhi, India). Schneiderman, Ron (2004), Electronic Waste: Be Part of the Solution; Journal of Electronic Design; Cleveland; Vol. 52; Issue 8, pp. 47-51.

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Sinha, Rajiv K. (1991): Ecological Management of Urban Solid Wastes for Human Survival; Journal of the Institute of Public Administration; University of Lucknow (India), Vol. 6 Nos. 1-4. Sinha, Rajiv K and Ratna Rawat (1991): Waste recycling and reutilisation, essential for environmental safety and sustainable development : Case study of paper and cotton rags recycling industries in Jaipur, India : Journal of Ecobiology : Vol. 11: pp 193-198. Sinha, Rajiv K. (1994): Ecological Economics of Waste Management: Value Addition on Waste, Waste Imports and Waste Exchange for Recycling; Paper Presented at International Seminar on ‗Solid Waste Management and Training‘; Center for Environment Education (South India), Bangalore, September, 19-24, 1994. (Sponsored by IUCN and WWF – Indian Chapter as Resource Person) Sinha, Rajiv. K (1996): Waste Bomb : The Threat to Bury the Humanity Alive; In Self (ed.): Environmental Crisis and Humans at Risk; INA Shree Publication, India; pp. 118-126. Sinha, Rajiv K. and A.K. Sinha (2000): Waste Management: Embarking on the 3R‘s Philosophy of Waste Reduction, Reuse and Recycling; Inashree Publication, India; ISBN 81-86653-32-5. Sinha, Rajiv. K., Sunil Herat, Sunita Agarwal, Ravi Asadi, and Emilio Carretero (2002 a): Vermiculture Technology for Environmental Management : Study of the action of the earthworms Elsinia foetida, Eudrilus euginae and Perionyx excavatus on biodegradation of some community wastes in India and Australia; The Environmentalist, U.K., Vol. 22, No.2. June, 2002; pp. 261 – 268. Sinha, Rajiv K. and Sunil Herat (2002 b): A Cost-effective Microbial Slurry Technology for Rapid Composting of Municipal Solid Wastes on the Waste Dump Sites in India and the Feasibility of its Use in Australia: The Environmentalist, U.K. Vol..22; No.1; pp. 9-12. Sinha, Rajiv K. and Sunil Herat (2004): Industrial and Hazardous Wastes : Health Impacts and Management Plans; Pointer Publishers, Jaipur (India); ISBN 81-7132-365-0; pages 365. Sinha, Rajiv K., Sunil Herat, P.D. Bapat, Chandni Desai, Atul Panchi and Swapnil Patil, (2005 a): Household Hazardous Waste : The Hidden Danger in Every Home : Regulating Their Management; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, CANADA; pp. 45-54. Sinha, Rajiv K., Sunil Herat, P.D. Bapat, Chandni Desai, Atul Panchi and Swapnil Patil, (2005 b): Domestic Waste - The Problem That Piles Up for the Society : Vermiculture the Solution; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, CANADA; pp. 55-62. Sinha, Rajiv K., Jayraman, V.B., and Michael Noronha (2005 c): Waste and Consumer Education for Society about the 3 R‘s and 5 P‘s Holds the Key to Sustainable Waste Management at Source; Proceedings of International Conference on ‗Waste-The Social Context; May 11-14, 2005, Edmonton, Alberta, Canada: 288-293. Sinha, Rajiv K., Sunil Herat, P.D. Bapat and Chandini Desai (2006): Electronics Wastes : Landfill Disposal, Reuse and Recycling; Indian Journal of Environmental Protection; Vol. 26(7): pp. 577-584; ISSN 0253-7141; Regd. No. R.N. 40280/83; Indian Institute of Technology, BHU, Varanasi, India. Sinha, Rajiv K. and Rohit Sinha (2007): Environmental Biotechnology; Pointer Publisher, India. (Under Publication) Tchobanoglous, George; Theisen, Hilary; and Vigil, Samuel (1993):

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Useful Websites Clean Up Australia : Accessed 15 March 2006. EcoRecycle : : Accessed 15 March 2006

Human Waste - A Potential Resource: Converting Trash into Treasure … Womens Environment Network: Accessed 15 March 2006. UNEP Magazine ‗Our Planet‘ : < http://www.ourplanet.com> Global Waste Challenge; www.sandec.ch

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In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 4

EFFECTIVE REMOVAL OF LOW CONCENTRATIONS OF ARSENIC AND LEAD AND THE MONITORING OF MOLECULAR REMOVAL MECHANISM AT SURFACE Yasuo Izumi Department of Chemistry, Graduate School of Science, Chiba University Yayoi 1-33, Inage-ku, Chiba 263-8522, Japan

ABSTRACT New sorbents were investigated for the effective removal of low concentrations of arsenic and lead to adjust to modern worldwide environmental regulation of drinking water (10 ppb). Mesoporous Fe oxyhydroxide synthesized using dodecylsulfate was most effective for initial 200 ppb of As removal, especially for more hazardous arsenite for human's health. Hydrotalcite-like layered double hydroxide consisted of Fe and Mg was most effective for initial 55 ppb of Pb removal. The molecular removal mechanism is critical for environmental problem and protection because valence state change upon removal of e.g. As on sorbent surface from environmental water may detoxify arsenite to less harmful arsenate. It is also because the evaluation of desorption rates is important to judge the efficiency of reuse of sorbents. To monitor the low concentrations of arsenic and lead on sorbent surface, selective X-ray absorption fine structure (XAFS) spectroscopy was applied for arsenic and lead species adsorbed, free from the interference of high concentrations of Fe sites contained in the sorbents and to selectively detect toxic AsIII among the mixture of AsIII and AsV species in sample. Oxidative adsorption mechanism was demonstrated on Fe-montmorillonite and mesoporous Fe oxyhydroxide starting from AsIII species in aqueous solution to AsV by making complex with unsaturated FeOx(OH)y sites at sorbent surface. Coagulation mechanism was demonstrated on double hydroxide consisted of Fe and Mg from the initial 1 ppm of Pb2+ aqueous solution whereas the mechanism was simple ion exchange reaction when the initial Pb2+ concentrations were as low as 100 ppb.

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[email protected]

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Yasuo Izumi

INTRODUCTION Recently, global environment induces even serious debate, e.g. the Novel Prize 2007 for Piece to "An Inconvenient Truth" by Al Gore [1]. The environmental problem is not only the global warming as the major claim in this movie/book by Gore. Contamination of water and soil is one of traditional, major environmental problems and directly affects the human health, e.g. carcinogenic risk via drinking water [2 – 4]. Cadmium contaminated in rice [5], copper contaminated in environmental water from mine, mercury contaminated in fish [6 – 8], and arsenic contaminated in powdered milk are most notorious environmental tragedies occurred in Japan between 1890 and 1960. These accidents are all related to contamination of water derived from human activity (industry) [2]. The health risk of poisonous elements in water has been studied and is becoming clear. Recent environmental regulation sets the minimum level of Mn, Cu, Cd, Pb, Cr, As, and Hg to 400, 125, 5, 10, 50, 10, and 0.5 ppb, respectively. Among these elements, lead is less focused and less intensively studied to adjust to the regulation. Arsenic can be contaminated in ground water not only anthropogenically but from the earth naturally [2 – 4]. Unfortunately, mines containing As mainly distribute in contact with the ground water in developing countries, e.g. Bangladesh, East Bengal, Argentina, Chile, and Vietnam [9]. Especially in Bangladesh, the shallow ground water with arsenic concentrations up to 1 ppm is often used pumped from tube wells without adequate treatment for drinking and thus leading to serious health problem. The arsenic release into ground water is related to microbial metabolism of organic matter [10]. The high capital and maintenance costs of piped water supply are still not acceptable in Bangladesh, especially most affluent villages compared to present individual tube wells [10]. Lead was used as gasoline additive and automobile tail pipes and may be included in drinking water from water supply pipes made of lead, soil contamination, and toys/tools made in developing countries [11, 12]. This chapter reviews recent development of sorbents for low concentrations of Pb and As to adjust to modern environmental regulation for drinking water and monitoring of the molecular removal process by selective spectroscopy in water [13]. The monitoring of surface uptake of Pb [14] and As includes local coordination structure and electronic structure changes in the inner sphere reaction.

METHODS This chapter focuses on the removal of Pb and As by sorption because sorption is economic process to be applicable in developing countries and the reuse is possible by desorption. The concentrations of Pb and As in test aqueous solutions were set between 55 ppb and 32 ppm in this chapter. Various sorbents were evaluated to maintain the concentrations of Pb or As less than 10 ppb or not. Fe-montmorillonite was prepared by mixing 0.43 M ferric nitrate solution with Namontmorillonite (Kunipia F; Na1.5Ca0.096Al5.1Mg1.0Fe0.33Si12O27.6(OH)6.4) [15]. A 0.75 M sodium hydroxide solution was added dropwise to the mixture until the molar ratio Fe3+ added and hydroxide reached 1:2. Iron cations and/or FeOx(OH)y nanoparticles were inserted between negatively-charged montmorillonite clay layers. Recently, some chemical forms of

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FeIII species formed between montomorillonite layers were spectroscopically analyzed [16]. Monomeric and/or dimeric FeIII species was active in oxidative dehydrogenation of propane. In contrast, polymeric FeOx(OH)y nanoparticles were effective for arsenic sorption and unselective propane combustion. FeOx(OH)y porous material was prepared by mixing 0.10 M ferrous chloride with 0.070 M sodium dodecylsulfate followed by the addition of 0.25 M H2O2 [17]. Obtained FeOx(OH)y material was mixed with 0.050 M sodium acetate in ethanol for anion exchange or with pure ethanol for washing. The micro/mesoporous FeOx(OH)y material was characterized by X ray diffraction (XRD), specific surface area measurements and pore volume determination by N2 adsorption/desorption, high-resolution transmission electron microscope (TEM), Fouriertransformed infrared absorption (FT-IR), inductively coupled plasma (ICP) combined with optical emission spectroscopy (OES), electron probe microanalysis (EPMA), thermogravimetric differential thermal analysis (TG-DTA), and Fe K-edge X-ray absorption fine structure (XAFS). Based on these analyses, detailed structural transformation was clarified for the sorbents as depicted in Figure 8 of Ref [17]. Hydrotalcite-like (pyroaurite) layered double hydroxide Mg6Fe2(OH)16(CO3)•3H2O was synthesized via the procedure described in Ref 18. The carbonate anions are sandwiched between positively-charged [Mg3Fe(OH)8]+ layers. To monitor low concentrations of Pb and As, XAFS spectroscopy is most appropriate technique. For several spectroscopic techniques of structural analysis in the application to nanotechnology, the advantage and drawback were summarized in Table 1 [19]. For noncrystalline or hybrid samples, EXAFS (extended X-ray absorption fine structure) gives direct structural information for X-ray absorbing local element sites. XANES (X-ray absorption near-edge structure) is a part of EXAFS spectrum near the X-ray absorption edge region ranging up to 100 eV and gives electronic and (indirectly) structural information [20]. Thus, XAFS spectroscopy (EXAFS, XANES) is essentially single technique for local structure analysis accompanied with valence and coordination symmetry information of nanoparticles and micro/mesoporous materials. The Pb and As adsorbed from low concentrations of aqueous solutions in this chapter are typical examples of nanoparticles and micro/mesoporous material samples. Further, this chapter combines X-ray fluorescence (XRF) spectrometry with the XAFS spectroscopy [21 – 24]. Simply, XRF spectra support valence state information deduced from XAFS. Essentially, high-energy-resolution XRF spectrometry is able to discriminate valence state of Pb and As. In this chapter, the XRF signals originating from PbII, AsIII, and AsV were monitored independently in the XAFS measurements to obtain each coordination structure of PbII, AsIII, and AsV (state-selective XAFS). The experimental setup and measurement conditions for state-selective XAFS were depicted and described in Refs 21, 23, and 24. In brief, XRF spectra and state-selective XAFS spectra measurements were performed at Undulator beamline 10XU of SPring-8 (Sayo, Japan) by utilizing a homemade high-energyresolution Rowland-type fluorescence spectrometer equipped with a Johansson-type Ge(555) crystal (Saint-Gobain) and NaI(Tl) scintillation counter (Oken). The monochromator of beamline used Si(111) double crystal and the X-ray beam intensity in front of sample was monitored using ion chamber (Oken) purged with N2 gas.

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Table 1. Various Analytical Methods for Nano Structure Classified Based on Directness of the Information and the Target to Be Analyzed Method

Directness

Target

TEM (Transmission electron microscope)

Direct

Local

XRD (X-ray diffraction)

Direct

Local

EXAFS (Extended X-ray absorption fine structure)

Direct

Bulk

XANES (X-ray absorption near-edge structure)

Indirect

Bulk

Raman

Indirect

Bulk

Small angle scattering

Indirect

Bulk

NMR (Nuclear magnetic resonance)

Indirect

Local

Mössbauer

Indirect

Local

SPM (Scanning probe microscope)

Indirect

Local (surface)

Reflectivity

Indirect

Local (surface)

RESULTS AND DISCUSSION Arsenic Problem. Adsorption isotherms of arsenite and arsenate at 290 K for 12 h on Femontmorillonite in batch setup are depicted in Figure 1. The Fe-montmorillonite was superior to -FeO(OH) (göthite > 95%) both for arsenite and arsenate sorptions. Fe-montmorillonite consisted of 2-dimensionally distributed Fe3+ ions and FeOx(OH)y nanoparticles between clay layers [16]. The saturated amount of As adsorbed was evaluated to 8.0 and 76 mgAs gsorbent–1 for arsenite and arsenate, respectively, on Fe-montmorillonite. The equilibrium adsorption constant was 1.4×106 ml gAs–1 for arsenite on Fe-montmorillonite. Even better adsorption capacity was found on acetate-exchanged microporous FeOx(OH)y as depicted in Figure 2 for arsenite. The saturated amount and the equilibrium adsorption constant of As adsorbed were evaluated to 21 mgAs gsorbent–1 and 1.0×107 ml gAs–1, respectively. The high specific surface area of microporous FeOx(OH)y (230 m2 g–1) was advantageous compared to Femontmorillonite (100 m2 g–1) with as much as 14 wt% of Fe. Utilizing template synthesis technique for microporous and mesoporous materials, lower coordination FeOx sites were effectively exposed to surface to complex with AsIII(OH)3 [17, 26]. The acetate-exchanged and ethanol-washed FeOx(OH)y consist of 3-dimensionally distributed wormholes exposed with coordinatively unsaturated FeO(OH) sites.

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Figure 1. Adsorption isotherms of arsenite (A) and arsenate (B) at 290 K on Fe-montmorillonite (14.0 wt% Fe) (circles) and -FeO(OH) (triangles). Batch tests for 12h. Observed data were plotted as points and the fits to first-order Langmuir equations were drawn as lines.

The surface uptake mechanism of most toxic arsenite was monitored by XRF and XAFS spectroscopy. Arsenic was adsorbed on Fe-montmorillonite from 200 ppb test aqueous solution of arsenite. The As K1 emission spectrum was depicted in Figure 3. The peak energy position suggested that the adsorbed As state changed to V, not remained at III, compared to the data for KH2AsVO4 and AsIII2O3 [15]. As K-edge XANES spectra for standard inorganic compounds of As0, AsIII, and AsV consist of broad peak feature (Figure 4a – c) and it is complicating to evaluate each valence contribution to a spectrum for sample of mixed valence. In order to demonstrate directly the oxidative adsorption of arsenite suggested above, the author of this chapter observed the uptake of low concentrations of arsenite on Fe-montmorillonite by means of state-selective XAFS. Note that the energy resolution of fluorescence spectrometer (1.3 eV; Figure 3) was smaller than the core-hole lifetime width of As K level (2.14 eV) [27].

Figure 2. Adsorption isotherms of arsenite at 290 K on acetate-exchanged FeOx(OH)y previously heated at 423 K (circles), ethanol-washed FeOx(OH)y previously heated at 423 K (squares), Fe-montmorillonite (14.0 wt% Fe; diamonds), and -FeO(OH) (triangles) [25]. Batch tests for 12h. Observed data were plotted as points and the fits to first-order Langmuir equations were drawn as lines.

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Figure 3. Arsenic K1 emission spectrum for As adsorbed on Fe-montmorillonite (14.0 wt% Fe) from 200 ppb test solution of arsenite (points). A fit to data with pseudo-Voigt function (solid line) and the energy resolution of fluorescence spectrometer (dotted line) were also drawn. The intensity ratio of the Lorentzian and Gaussian components was fixed to 1:1 for the pseudo-Voigt function.

Figure 4. XANES spectra measured at 290 K in transmission mode for As metal (a), AsIII2O3 (b), and KH2AsVO4 (c). Arsenic K1-selecting As K-edge XANES spectra measured at 290 K for As adsorbed on Fe-montmorillonite (14.0 wt% Fe) (d – f) from aqueous test solutions of 16 ppm of KH2AsVO4 (d), 16 ppm of AsIII2O3 (e), and 200 ppb of AsIII2O3 (f). Tune energy of fluorescence spectrometer was 10544.3 eV for spectra d – f.

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Based on the theory discussed in the Appendix section of Ref 24, the As K1-selecting As K-edge XANES spectrum with the energy resolution of 1.3 eV would be shaper and more resolved. The energy values of K absorption edge and first strong peak after the edge were essentially identical for As adsorbed on Fe-montmorillonite from 200 ppb – 16 ppm of AsIII solutions (Figure 4e, f) and from 16 ppm of AsV solution (d). Thus, oxidative adsorption of 200 ppb – 16 ppm of arsenite on Fe-montmorillonite was confirmed based on As K1selecting XANES and As K1 emission spectrum. Proposed molecular surface uptake mechanism was illustrated in Figure 5 over acetate-exchanged microporous FeOx(OH)y. The reaction formula was AsIII(OH)3 + FeO(OH)  (FeO)2AsV(OH)2 + H2O. In summary, oxidative adsorption of low concentrations (200 ppb – 16 ppm) of arsenite was found on coordinatively unsaturated FeOx(OH)y nanoparticles or micro/mesoporous FeOx(OH)y partially covered with acetate anions. The oxidation to arsenate seems to be due to lower coordination of surface FeOx(OH)y species. The lower coordination was also the reason to make the equilibrium sorption constant greater for acetate-exchanged FeOx(OH)y and Femontmorillonite [15, 17]. Lead Problem. Sorption tests for low concentrations (55 ppb) of lead in flow setup were depicted in Figure 6 [18]. The superiority of Mg6Fe2(OH)16(CO3)•3H2O was clearly demonstrated to maintain the Pb2+ concentration less than modern environmental regulation (10 ppb) compared to commercially available activated carbon. Lead L1 emission spectrum for Pb adsorbed on Mg6Fe2(OH)16(CO3)•3H2O from 100 ppb Pb2+ test solution was depicted in Figure 7. The peak energy was identical to that for standard PbII compounds. The energy resolution of fluorescence spectrometer in this measurement condition was 5.0 – 10.1 eV dependent on the measurement conditions of fluorescence spectrometer (Figure 7) [24, 28]. Because the core-hole lifetime widths are 5.81 and 2.48 eV for Pb L3 and M5 levels, respectively [27], the width for L1 is 8.29 eV comparable to the energy resolution of fluorescence spectrometer. Thus, sharper, more resolved spectral feature was expected in Pb L1-selecting Pb L3-edge XANES spectrum if the energy resolution of fluorescence spectrometer was smaller than 5.81 eV, similar to the case of As K1-selecting XANES in previous section.

Figure 5. Proposed adsorption mechanism of low concentrations of arsenite on acetate-exchanged FeOx(OH)y previously heated at 423 K.

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Figure 6. Results of sorption on Mg6Fe2(OH)16(CO3)•3H2O and on activated carbon from 55 ppb of Pb2+ aqueous test solution. The space velocity was 150 min–1.

Figure 7. Lead L1 emission spectrum for 0.12 wt% of Pb adsorbed on Mg6Fe2(OH)16(CO3)•3H2O. The solid line is the experimental data, and (wider) dotted line is a fit with a pseudo-Voigt function. The intensity of the Lorentzian and Gaussian components was fixed to 1:1. The narrower dotted line is the energy resolution of fluorescence spectrometer (10.1 eV).

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Pb L1-selecting XANES spectra are shown in Figure 8a – c. The spectral pattern of b and c resembled each other. The two samples contained 0.30 – 0.12 wt% of lead adsorbed from 100 ppb test aqueous Pb2+ solution. The Pb contents in samples were determined by ICP. Compared to XANES spectra for standard inorganic Pb compounds (Figure 8d – i) and supported Pb species on zeolite [29] or on Fe2O3 [30], the spectra b and c resembled most spectrum d measured for ion-exchanged PbY zeolite (d). Thus, surface uptake mechanism via ion exchange reaction was proposed on Mg6Fe2(OH)16(CO3)•3H2O from relatively low concentration of 100 ppb divalent lead solutions (Figure 9). The reaction formula is [Mg3Fe(OH)8]+ + Pb2+  [Mg3Fe(OH)7(OPb)]2+ + H+. Pb L1-selecting XANES spectrum for Pb adsorbed from 1.0 ppm test Pb2+ solution is depicted in Figure 8a. Compared to XANES spectra for standard inorganic Pb compounds (Figure 8d – i), the spectrum a resembled most spectrum g measured for 2PbCO3•Pb(OH)2. Thus, coagulation uptake mechanism was proposed on Mg6Fe2(OH)16(CO3)•3H2O from relatively high concentration of 1 ppm Pb2+ test solution. Thus-identified reaction formula was Pb2+ + nCO32– + 2(1 – n)OH– ⇄ nPbCO3•(1 – n)Pb(OH)2.

Figure 8. Lead L1-selecting Pb L3-edge XANES spectra measured at 290 K for Pb adsorbed on Mg6Fe2(OH)16(CO3)•3H2O (a – c). Tune energy of fluorescence spectrometer was 10551.5 eV. The Pb content was 1.0 wt% adsorbed from 1.0 ppm Pb2+ aqueous test solution (a) and 0.30 (b) and 0.12 wt% (c) from 100 ppb Pb2+ test solution. XANES spectra measured in transmission mode (d – i) for PbY zeolite (d), PbO (e), Pb(NO3)2 (f), 2PbCO3•Pb(OH)2 (g), Pb6O4(OH)4 (h), and PbCO3 (i).

With closer look of Figure 8a – c, a shoulder feature appeared at 13049 eV. Similar shoulder feature can be found in spectra for PbO, 2PbCO3•Pb(OH)2, and Pb6O4(OH)4 (spectra

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e, g, and h, respectively). However, intense peak at 13060.1 – 13060.5 eV for PbO or Pb6O4(OH)4 did not appear in spectra a – c. Thus, minor coagulation uptake mechanism was suggested from 100 ppb Pb2+ solutions in addition to major ion exchange process (Figure 9).

Figure 9. Pb2+ adsorption mechanism on Mg6Fe2(OH)16(CO3)•3H2O from Pb2+ 1.0 ppm and 100 ppb aqueous solutions.

In summary, Pb2+ uptake mechanism on Mg6Fe2(OH)16(CO3)•3H2O exhibited a switchover from coagulation to (major) ion exchange reactions as the Pb2+ concentration decreased from 1.0 ppm to more environmentally plausible 100 ppb [28].

CONCLUDING REMARKS AND FUTURE PROSPECTS This chapter focused on the removal of low concentrations (55 – 200 ppb) of arsenite and lead by utilizing Fe-montmorillonite, micro/mesoporous FeOx(OH)y effectively porous due to carboxyl-exchange method, and layered double hydroxide consisted of Fe and Mg (pyroaurite). It is still open to discuss the systematic survey of the removal process of other dilute toxic elements, e.g. Cr, Cu, Zn, Cd, or Hg. It is important to formulate the efficiency of surface uptake with respect to critical factors, i.e. pH, chemical species of toxic elements (naked cations, oxyanions, or oxyhydroxyl anions), initial concentrations (10 – 100 ppb) and space velocity of aqueous solutions to be processed, and chemical combination of surface versus chemical species (e.g. FeIIIO(OH) versus As(OH)3 and OH–(clay surface)/CO32– (between layers) versus Pb2+). In the analytical point of view to monitor the destiny of low concentrations of toxic elements, selective XAFS technique, with which the author of this chapter has also

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investigated surface catalytic sites of gold, platinum, tin, and vanadium [31 – 35], needs to be combined with other technique with nanoscale spacial resolution. Spectroscopy with nanoscale spacial resolution is under investigation, but not established to be applicable to nanotechnology (Table 2). At present, spacial resolution of X-ray microscopy is 1 m [36]. Several types of X-ray microscopy/imaging are under investigation, e.g. microbeam XAFS, photoemission electron microscope (PEEM), or phase contrast imaging [37, 38]. The spacial resolution of TEM is already smaller than 1 nm if the sample nanoscopic condition matches to the high-resolution measurement. To monitor the sorption between 2-dimensinal layers (e.g. montmorillonite, hematite), in 2-dimensional mesopores (e.g. FSM-16, MCM-41), and in 3-dimensional micro/mesopores (e.g. ZSM-5, acetate-exchanged FeOx(OH)y [17]), 3-dimensional TEM images would be very helpful by taking series of TEM snapshots from various angles to sample and organizing 3-dimensional image on computer [39]. Scanning probe microscope (SPM), especially scanning tunneling microscopy (STM) and atomic force microscope (AFM), has an advantage of atomic resolution for well-defined surface [40]. To utilize SPM technique to monitor the sorption from low concentrations of toxic elements, the combination with element specific spectroscopy, e.g. XPS, XAFS, is essential to describe the surface chemical mechanism. The author of this chapter is developing this combination (AFM and XPS) based on temporal electron trap phenomena in the metal nano-dots in the front of the AFM tip [41, 42]. Table 2. Spectroscopy Needed to Be Developed to Give Direct Spacial Information of Surface Uptake Mechanism from Low Concentrations (10 – 100 ppb) of Toxic Metal Elements Probe

Method

Factor to be developed for this application

Refs

X-ray Microscope

Smaller X-ray beam (< 10 nm)

[36 – 38]

Electron Microscope

3-dimensional information

[39]

Tip Microscope

Scanning probe microscope to discriminate

[41, 42]

the kind of element X-ray Diffraction

Surface-sensitivity between nano-layers or

[43]

in micro/mesospace X-ray XAFS/XRF

On-site analysis in the field

[44, 45]

Surface-sensitive XRD may be applicable to monitor the application of water purification [43]. The breakthrough is selectivity to internal surface of layered and micro/mesoporous materials used as sorbents. Portable XRF/XAFS apparatus that has been investigated for space science [44, 45] will be applicable to environmental on-site monitoring of the fate of toxic elements in the field.

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ACKNOWLEDGMENTS This article is part 26 in the series of state-sensitive XAFS. The X-ray experiments were performed under the approvals of the SPring-8 Program Review Committee and of the Photon Factory Proposal Review Committee. The works included in this chapter were financially supported by grants from the Grant-in-aid for Encouragement of Young Scientists (B14740401, A12740376) and the Grant-in-aid for Basic Scientific Research (B13555230, C17550073) from the Ministry of Education, Culture, Sports, Science, and Technology, Yamada Science Foundation (2000), Toray Science Foundation (98-3901), and Research Foundation for Opto-Science and Technology (2005 – 2006).

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[23] Izumi, Y.; Kiyotaki, F.; Nagamori, H.; Minato, T. J. Electro. Spectrsc. Relat. Phenom. 2001, 119(2/3), 193 – 199. [24] Izumi, Y.; Nagamori, H.; Kiyotaki, F.; Masih, D.; Minato, T.; Roisin, E.; Candy, J. P.; Tanida, H.; Uruga, T. Anal. Chem. 2005, 77(21), 6969 – 6975. [25] Dixit, S.; Hering, J. G.; Environ. Sci. Technol. 2003, 37, 4182 – 4189. [26] Izumi, Y.; Masih, D.; Aika, K.; Seida, Y. Micropor. Mesopor. Mater. 2007, 99, 355. [27] Zschornack, G. Handbook of X-Ray Data; Springer: Berlin, 2007. [28] Izumi, Y.; Kiyotaki, F.; Minato, T.; Seida, Y. Anal. Chem. 2002, 74(15), 3819 – 3823. [29] Huang, F. T.; Jao, H. J.; Hung, W. H.; Chen, K.; Wang, C. M. J. Phys. Chem. B 2004, 108(52), 20458 – 20464. [30] Pinakidou, F.; Katsikini, M.; Paloura, E. C.; Kalogirou, O.; Erko, A. J. Non-Cryst. Solids 2007, 353(28), 2717 – 2733. [31] Izumi, Y.; Obaid, D. M.; Konishi, K.; Masih, D.; Takagaki, M.; Terada, Y.; Tanida, H.; Uruga, T. Inorg. Chim. Acta 2008, 361(4), 1149 – 1156. [32] Izumi, Y.; Masih, D.; Roisin, E.; Candy, J. P.; Tanida, H.; Uruga, T. Mater. Lett. 2007, 61(18), 3833 – 3836. [33] Izumi, Y.; Masih, D.; Candy, J. P.; Yoshitake, H.; Terada, Y.; Tanida, H.; Uruga, T. "XRay Absorption Fine Structure 13th International Conference", Hedman, B.; Pianetta, P. Eds., AIP Conference Proceedings 2007, Vol. 882, 588 – 590. [34] Izumi, Y.; Konishi, K.; Obaid, D. M.; Miyajima, T.; Yoshitake, H. Anal. Chem. 2007, 79(18), 6933 – 6940. [35] Izumi, Y.; Kiyotaki, F.; Yagi, N.; Vlaicu, A. M.; Nisawa, A.; Fukushima, S.; Yoshitake, H.; Iwasawa, Y. J. Phys. Chem. B 2005, 109(31), 14884 – 14891. [36] Hokura, A.; Kitajima, N.; Terada, Y.; Nakai, I. SPring-8 Research Frontiers 2006, 120 – 121. [37] Tuohimaa, T.; Otendal, M.; Hertz, H. M. Appl. Phys. Lett. 2007, 91(7), 074104. [38] Koshikawa, T.; Guo, F. Z.; Yasue, T. SPring-8 Research Frontiers 2005, 52 – 53. [39] Midgley, P. A.; Thomas, J. M.; Laffont, L.; Weyland, M.; Raja, R.; Johnson, B. F. G.; Khimyak, T. J. Phys. Chem. B 2004, 108(15), 4590 – 4592. [40] Marti, O.; Möller, R. Eds. Photons and Local Probes; Kluwer Academic Publishers: Dordrecht, 1995. [41] Klein, L. J.; Williams, C. C. Appl. Phys. Lett. 2001, 79(12), 1828 – 1830. [42] Klein, L. J.; Williams, C. C.; Kim, J. Appl. Phys. Lett. 2000, 77(22), 3615 – 3617. [43] Takahasi, M. SPring-8 Research Frontiers 2006, 56 – 57. [44] Robinson, A. L. Science 1980, 208, 163 – 164. [45] Frierman, J. D.; Bowman, H. R.; Perlman, I.; York, C. M. Science 1969, 164, 588.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 5

ON THE REDISTRIBUTION OF TISSUE METAL (CADMIUM, NICKEL AND LEAD) LOADS IN MINK ACCOMPANYING PARASITIC INFECTION BY THE GIANT KIDNEY WORM (DIOCTOPHYME RENALE) Glenn H. Parker and Liane Capodagli Department of Biology, Laurentian University, Sudbury, Ontario, Canada, P3E 2C6

ABSTRACT Patterns of metal uptake and accumulation in mink living under conditions of environmental pollution and simultaneously inflicted with the invasive giant kidney worm (Dioctophyme renale) parasite have not been examined, nor is the combined effect of these dual insults on the health and physical condition of the animal known. Using animals collected within the influence of the long-active ore-smelters at Sudbury, Ontario, an examination was made of toxic metal (Cd, Ni and Pb) levels and their tissue distributions within adult male mink bearing different intensities of parasite infection. Higher metal burdens were indicated within infected specimens than those uninfected. Combined renal and hepatic nickel and lead burdens were highest for mink with multiple worm infections, although only lead accumulations reached statistical significance. Cadmium accumulated to the greatest extent in the hypertrophied left kidney and liver, whereas nickel and lead were deposited more readily in the bony spicule of the parasitized right kidney cyst. The relative distribution of cadmium among renal, hepatic and renal cyst tissues (cast, spicule, worms) remained unchanged subsequent to D. renale infection, while the proportions of nickel and lead deposited in hepatic tissue were reduced. Metal burdens in female D. renale were three-fold higher than those of male worms, with the difference being attributable to the substantially greater size of the females. Canonical Correlation Analyses of condition measures and body metal burdens failed to indicate a direct relationship between infection intensity and body fat deposits but did confirm a positive association between metal loads and increased fat levels, along with enhanced gonad weights, neck circumference and reduced spleen weights. Such associations may be productive aspects for future investigation into the combined effects of increased metal loads and parasitic infection on the host system.

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INTRODUCTION Investigating metal contaminant levels in parasites has received considerable attention of recent, with the goal of most studies being to identify useful bioindicators and/or biomonitors of environmental pollutants. The use of parasites as indicators of metal contaminants within fish and aquatic environments has been studied extensively, and has shown that acanthocephalans and to a lesser extent cestodes, can accumulate extremely high concentrations of metal pollutants (Sures et al., 1994 a,b,c; Sures and Taraschewski, 1995; Sures et al., 1997 a,b,c; Siddall and Sures, 1998; Sures et al., 1999; Tenora et al., 1999a; Turcekova and Hanzelova, 1999: Zimmermann et al., 1999; Tenora et al., 2000; Barus et al., 2001b; Turcekova et al., 2002; Palikova and Barus, 2003; Sures, 2003; Williams and Mackenzie, 2003). By comparison, studies focused on bird-parasite systems are relatively few (Barus et al., 2001a; Tenora et al., 2001; Tenora et al., 2002). Virtually no work of this nature has been done on nematode parasites in mammals, with the exceptions of Szefer et al. (1998) who studied the bioaccumulation of trace elements in lung nematodes of harbour porpoises, Greichus and Greichus (1980) and Sures et al. (1998) who reported on roundworms of the pig and Tenora et al. (1999b) who examined metal levels in either gender of Toxocara canis and Protospirura muricola specimens and their respective hosts. The extent to which parasites affect metal uptake and distribution in host tissues is largely unknown and, for the most part, was not investigated in the above studies. It is known from the literature, however, that helminth infestation may affect the hosts‘ sensitivity to toxic metals (Pascoe and Cram, 1977; Poulin, 1992; Sures and Siddall, 1999). Since the giant kidney worm parasite, Dioctophyme renale, produces pathophysiological changes in both the liver through which it migrates and the kidney which it ultimately occupies, and these two organs represent the primary sites of metal accumulation in the vertebrate body, one might expect measurable changes in the tissue distribution of metals within the host animal following infection. The development and life cycle of the giant kidney worm have been summarized by Anderson (2000), and are depicted in Figure 1. Mink (Mustela vison) are the preferred definitive host for D. renale, although other carnivorous species have occasionally been infected (including otter Lontra canadensis, marten Martes americana, short-tailed weasel Mustela erminea, long-tailed weasel M. frenata, wolverine Gulo luscus and Gulo gulo, coyote Canis latrans, wolf C. lupus, dog C. familiaris, red fox Vulpes vulpes, bear Ursus americanus, raccoon Procyon loto, and coati Nasua nasua). Adult kidney worms typically occupy the right kidney (Figs. 2a and 2b) but on occasion have been observed unrestricted within the abdominal cavity or entwined within the liver of their host (Figure 2d). At least one male and one female worm (Figure 2f) must be present in the same kidney for a fertile infection to occur and for the life cycle to continue. Fertilized eggs are released from the female worm, passed down the ureter (which remains open post-infection) to the urinary bladder and voided with the urine. If the eggs are released into water and reach appropriate temperatures (approximately 14 to 30 ºC), they embryonate and are frequently consumed by the aquatic oligochaete Lumbriculus variegatus. The larvae moult twice in this intermediate host, reaching the infective third larval stage. Suitable paratenic hosts (frogs Rana catesbeiana, R. clamitans and R. septentrionalis, pumpkinseed fish Lepomis gibbosus, brown bullheads Ictalurus nebulosus and black bullheads I. melas) eat the parasitized oligochaete, but no futher development of D. renale larvae takes place in these paratenic hosts.

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Definitive Host (final moult and growth to adult worm occupying right kidney)

Incidental direct transfer via infected drinking water

Eggs released with urine into water

Unembryonated Eggs

Paratenic Hosts

Intermediate Host (Lumbriculus variegatus) (eggs hatch and develop to infective 3rd stage larvae)

Figure 1. Development and transmission of the giant kidney worm, Dioctophyme renale.

Although it is possible for mink to become infected through the incidental consumption of parasitized oligochaetes during feeding or drinking, the above-mentioned paratenic hosts are frequent items in the mink‘s diet, and thus typically serve as the vectors through which the parasite is transferred to mink. Once ingested, third-stage larvae penetrate the stomach wall of the definitive host and develop to the adult stage before migrating through the liver. The adult worms make their way to the right kidney, which they penetrate and ultimately excavate by destroying the functional cortical and medullary tissues as they grow to full size. Only a tough thickened capsule or cast (Figure 2c) remains of the right kidney, serving to encase the mature D. renale worms and in most cases a bony spicule (or ‗staghorn‘). The parasitized kidney with its occupants and component structures is commonly referred to as a kidney cyst. The spicule is usually embedded in the lumenal surface of the dorsal wall of the kidney cast; although size, shape and colour have been shown to vary considerably (Dhaliwal and Taylor, 2000), the basic form is a flat plate on the dorsal side, with finger-like projections or spicules radiating ventrally (Figure 2c). Histological examination of the spicule has revealed osteoblasts within lacunae and canaliculi making up the Haversian system of true replacement bone (McNeil, 1948; Mace, 1976). The plate edges and spicule projections have been found to contain osteoclasts and consist of hyaline cartilage (McNeil, 1948). The left kidney of the infected animal hypertrophies (50 to 100% by weight; Figure 2e) to compensate for the loss in function of the right kidney.

190

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Figure 2. Dioctophyme renale infection in mink: a) ventral view of kidneys showing right renal cyst of infected animal b) cyst opened showing several worms present c) cast with bony spicule (‗staghorn‘) embedded, after removing worms d) ‗free-floating‘ abdominal infection showing portions of 3 worms present among visceral organs e) hypertrophied left kidney of infected animal (on right) vs control f) 3 male worms (top) and 3 female specimens (below) removed from renal cyst (bottom). Magnifications: a), b) and d) = 0.75X actual size; c) and e) = 1.25X; f) = 0.38X.

In the Sudbury-area, the prevalence of D. renale infection in mink is approximately 50% (N. Schaffner and G. Parker, unpublished data) and, due to local ore-mining/smelting operations over the past 125 years, wildlife living within the influence of the area-wide smelters are exposed to significantly elevated metal levels in their environment (Wren et al., 1988; Capodagli and Parker, 2007). Patterns of metal uptake and accumulation in mink living under these conditions of environmental pollution and additionally inflicted with the invasive giant kidney worm parasite are currently unknown. As part of an on-going program examining toxic metals and endoparasites in Ontario wildlife species, the opportunity arose to investigate patterns of metal distribution in wild Sudbury-area mink populations infected with the parasite. The objectives of this investigation were as follows:

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1) to determine the effects of D. renale infection (at high and low intensities) on the uptake and renal-hepatic distribution of toxic metals (cadmium, nickel and lead) in wild field-trapped mink. 2) to quantify the extent to which these toxic metals are accumulated within the right kidney cyst and its component structures (namely the parasitic worms, bony spicule and cast tissues) of the infected animal. 3) to compare the extent of metal uptake and accumulation in male versus female specimens of the parasite. 4) to examine the extent to which metals are accumulated in the osseous spicule of the infected animal relative to concentrations occurring in skeletal (femur) bone. 5) to determine the extent to which renal-hepatic metal levels and D. renale infection, either singly or in concert, may influence the health and physical fitness of mink, through an assessment of the effects of these perceived insults on selected morphometric measures and fat reserves of the animal.

MATERIALS AND METHODS Mink carcasses were obtained from local fur trappers in the Sudbury District of Northern Ontario during the October to December trapping seasons of 1997, 1998 and 1999. Metal fallout and environmental contaminant conditions prevailing within the Sudbury Basin, home to intensive mining-smelting operations over the past 125 years, have been described elsewhere (Capodagli and Parker, 2007). A total of 30 adult male specimens were selected based on the intensity of giant kidney worm (Dioctophyme renale) infection present. Three groups were formed; non-infected mink (n=14), those infected with a single worm (n=8) and those infected with 4-6 worms (n=8). Assignment to the adult (>1 yr) male cohort was based on visual inspection of the genitals/gonads and the pattern of temporal muscle coalescence on the dorsal aspect of the skull (E. Addison (pers. comm.). Prior to dissection, the animals were completely thawed and morphometric measurements taken, including total peltless mass (to nearest 0.1 g), body length (from nose to tail base), total body + tail length (nose to tip of tail) and neck circumference (all to nearest 0.5 cm). Subcutaneous fat deposits in the groin and scapular regions were subjectively rated as high (3), medium (2) or low (1). During necropsy, the liver, heart, spleen, kidneys, gonads, omental fat, thoracic postcardinal fat deposit and abdominal mid-ventral fat deposit were removed and weighed (to nearest 0.1 g) immediately upon removal to minimize the effects of dehydration. Livers, femurs and kidneys (or their component parts in the case of infected animals) were retained for determination of metal content. In the case of infected subjects, the right kidney cyst was opened and each individual worm was removed, counted, weighed (to the nearest 0.1 g) and measured (to the nearest mm). Individual D. renale worms were sexed by the presence/absence of the prominent bell-shaped bursa at the caudal end and according to differences in body length and diameter (females ranged from 16-50 cm in length and were approximately 0.5 cm in diameter, while males ranged from 8-17 cm in length and were 0.25 cm in diameter) (Fyvie, 1971). Bony spicules were carefully dissected from the kidney cast of infected mink and kept in small airtight plastic cups at –20 ºC. Kidneys, liver, femur, kidney

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cast and worms were immediately packaged in individual polyethylene (Whirlpak-Nasco) bags and restored at –20 ºC until metal analysis could be performed. All glassware was washed using SparkleenTM dish detergent, rinsed, passed through a 20% HCl bath followed by two deionized distilled water baths and allowed to air dry before use. The right kidney, left kidney, liver, kidney cast, shaft of the right femur, individual whole worms and bony spicule were dehydrated in petri dishes in a drying oven at 60 ºC until constant weight was attained (approximately 3 days). A 2.0 (± 0.005) g sub-sample of dried liver, the shaft of the right femur and the entire sample of each of the remaining tissues were weighed into ceramic crucibles and placed in a muffle furnace. The temperature in the furnace was increased at a rate of 0.7 ºC/minute to 150 ºC and held for 10 hours, and then raised by 0.3 ºC/minute to 500 ºC, where it was held for 15 hours. The samples were allowed to cool to room temperature before digestion began. The ash was then transferred to sterile 15 ml polypropylene centrifuge tubes and a three ml aliquot of 2.5 N reagent grade HNO3 was added to the ash. The centrifuge tubes were capped and vortexed, and the samples then placed in an oven at 60 ºC to digest for 20 hours. The samples were then removed from the drying oven, vortexed again to break up any precipitate and left to stand overnight to ensure complete digestion. The samples were then centrifuged at full speed (approximately 5000 G) for five minutes and the supernatant was extracted with disposable glass Pasteur pipettes and transferred into acid-washed test tubes. Where necessary, the samples were diluted (5 fold) with 20% reagent grade HCl before being analyzed by flame atomic absorption spectrophotometry for Cd, Ni and Pb content using a Perkin Elmer Spectrophotometer (Model 703). Procedural blanks were included in each sample run to control for metals introduced by the digestion process. Metal concentrations were calculated and expressed as µg•g-1 dry weight. Tissue metal burdens (expressed as µg) were derived by multiplying metal concentration by the total tissue dry weight. To determine recovery rates and assess the reliability of the analytical procedures, certified reference materials (oyster tissue from the National Institute of Standards and Technology and citrus leaves from the National Bureau of Standards) were also subjected to the above procedure. As recovery rates (112 to 131%; n=3 per element) were generally deemed acceptable, tissue concentrations were left unadjusted. Where violations of normal distribution and/or homogeneity of variance in the data could not be rectified with standard transformations, the data were statistically evaluated using both parametric and non-parametric procedures. Organ weights and fat deposit measures were standardized for body size using the formula: Standardized measure = organ (or fat) weight x individual body length / mean body length

RESULTS Body and Organ Weights Dried weights of the primary organs of metal accumulation as well as standard body weights for the three infection groups (uninfected mink, mink infected with one kidney worm and mink infected with 4-6 worms) are reported in Table 1.

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Table 1. Body weights and dried organ weights (g) in mink infected with D. renale at different intensities (0, 1 or 4-6 worms). Values are means followed by standard error in parenthesis Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

10.55

(0.43)

a

10.01

(0.35)

a

10.25 (0.72)

a

Left kidney

0.78

(0.02)

a

1.23

(0.08)

b

1.32

(0.05)

b

Right kidney/cyst

0.73

(0.03)

a

0.88

(0.15)

a

2.26

(0.23)

b

a) cast

0.34

(0.03)

a

0.75

(0.06)

b

b) spicule

0.10

(0.04)

a

0.19

(0.06)

a

c) worm(s)

0.44

(0.13)

a

1.32

(0.17)

b

Cyst components:

Body weight

746.68 (24.43) a

766.12 (22.70) a

715.27 (43.39) a

Within organs, mean values bearing the same letter are not significantly different (p > 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test.

There were no significant differences in mean dry liver weights or overall body weights among the three infection groups. However, substantial weight differences occurred among the kidneys. The left kidneys of mink infected with either 1 worm or 4-6 worms were approximately 58% and 69% heavier, respectively, than the left kidney of uninfected mink. The right kidney cysts that developed in infected mink were likewise heavier than the intact right kidney of non-infected animals. This increase in weight amounted to 21% and 210% in the single worm and 4-6 worm infections, respectively. Despite the 2.6-fold weight difference between single and multiple worm cysts, the contributions of individual structural components were relatively uniform: cast tissue comprised 38.6 versus 33.2%, spicule 11.4 versus 8.4%, and worms 50.0 versus 58.4%, respectively.

Cadmium Levels Cadmium burdens and concentrations in the liver, kidneys, kidney cast, spicule and giant kidney worms of mink with varying intensities of D. renale infection are presented in Table 2. There was an overall increase in cadmium burden in infected mink over uninfected mink. Together renal and hepatic burdens approximated 7 µg in uninfected mink whereas infected mink averaged 13 to 17 µg. The elevated cadmium burdens in parasitized mink were manifested in both the liver and the uninfected left kidney (Figure 3). On average, liver burdens were elevated 2-fold and left kidney burdens by 3.6 fold over values seen in noninfected animals. Cadmium burdens present in the right kidney (non-infected animals) or its component structures (cast, spicule and worms) in infected animals did not differ significantly among the groups (Table 2). Thus the amount of cadmium that was present within the

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confines of the renal cyst appeared to remain unchanged from that present in the right kidney prior to infection, and was distributed among the component tissues/structures (Figure 3). Table 2. Cadmium levels in body tissues of mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis CADMIUM BURDEN (µg) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

4.74

(0.55)

a

10.98

(2.61)

b

7.07

(1.08)

a,b

Left kidney

1.23

(0.23)

a

4.89

(1.41)

b

4.02

(0.88)

b

Right kidney/cyst

1.15

(0.21)

a

1.07

(0.18)

a

1.63

(0.25)

a

a) cast

0.40

(0.10)

a

0.35

(0.05)

a

b) spicule

0.18

(0.04)

a

0.26

(0.05)

a

c) worm(s)

0.48

(0.15)

a

1.02

(0.21)

a

Cyst components:

All above organs/components 7.29 (0.99) a 16.94 (4.06) b 12.75 (2.43) a,b combined Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test. CADMIUM CONCENTRATION (µg•g-1) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

0.47

(0.07)

a

1.14

(0.30)

b

0.70

(0.10)

a,b

Left kidney

1.59

(0.33)

a

4.10

(1.30)

b

3.03

(0.66)

a,b

Right kidney/cyst

1.62

(0.31)

b

1.66

(0.54)

b

0.72

(0.09)

a

a) cast

1.43

(0.57)

b

0.47

(0.07)

a

b) spicule

4.33

(1.36)

a

1.82

(0.23)

a

c) worm(s)

1.73

(0.55)

a

0.80

(0.14)

a

Cyst components:

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test.

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Liver

20

Left kidney Right kidney/cyst Cyst components:

CADMIUM BURDEN (µg)

15

Cast Spicule Worm(s)

D

10

D

C

5

B A, B

A

0

0 worms

1 worm

4-6 worms

INFECTION STATUS

Figure 3. Cadmium burden (µg) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean composite burden. 6

Liver Left kidney

CADMIUM CONCENTRATION (µg • g-1)

5

Right kidney/cyst Cyst components: 4

Cast Spicule Worm(s)

3

B

A

2 A,B A

1

A B

B B

B

0

A A,B

A 0 worms

1 worm

A

A

A

4-6 worms

INFECTION STATUS

Figure 4. Cadmium concentration (µg•g-1 dry wt) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean.

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While cadmium burdens of the left kidney increased 3 to 4-fold as a result of infection (Table 2), organ hypertrophy (58 - 69%; Table 1) reduced mean tissue concentrations to values of 3 - 4 µg•g-1 (Table 2). Nevertheless these values were significantly (1.9 to 2.6-fold) higher than concentrations present in the left kidneys of non-infected subjects. Within the right kidney cyst of infected animals, cadmium was most concentrated in the bony spicule (mean = 4.33 ± 1.36 and 1.82 ± 0.23 µg•g-1; Figure 4). Worm concentrations averaged 1.73 ± 0.55 and 0.80 ± 0.14 µg•g-1 in the single and multiple worm groups, while cast tissues averaged 1.43 ± 0.57 and 0.47 ± 0.07 µg•g-1 in these two respective groups. For all tissues examined, mean cadmium concentration values tended to be higher among animals infected with single versus multiple worms; however, only in the case of the right kidney cyst and cast tissue did these differences reach statistical significance (p = 0.007 and 0.012, respectively).

Nickel Levels Tissue nickel burdens and concentrations for the same three groups of mink are reported in Table 3. Similar to cadmium, nickel burdens were likewise greater in the combined renal and hepatic tissue of infected mink compared to non-infected mink. Unlike cadmium however, the liver was not responsible for any of the increase in nickel burdens of the parasitized animal. The elevated nickel burden was distributed within the renal tissues of the animals, primarily the parasitized right kidney cyst. Approximately twice the nickel burden was present in the left kidney of infected mink, whereas 4 and 8 times more nickel were observed in the right kidney cyst of mink with 1 and 4-6 worm infections respectively, over burdens present in respective uninfected kidneys (Figure 5). Table 3. Nickel levels in body tissues of mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis NICKEL BURDEN (µg) Infection Status Organ

0 worms (n=14)

1 worm (n=7)

4-6 worms (n=8)

Liver

4.47

(0.28)

a

5.14

(0.67)

a

4.63

(0.50)

a

Left kidney

0.52

(0.07)

a

1.03

(0.21)

b

0.98

(0.10)

b

Right kidney/cyst

0.52

(0.08)

a

2.10

(0.50)

a,b

4.08

(0.51)

b

a) cast

1.16

(0.38)

a

1.44

(0.26)

a

b) spicule

0.71

(0.20)

a

1.03

(0.21)

a

c) worm(s)

0.60

(0.19)

a

1.78

(0.78)

b

7.71

(1.13)

a,b

10.19

(0.86)

b

Cyst components:

All above organs/components 5.49 combined

(0.33)

a

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test.

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197

NICKEL CONCENTRATION (µg•g-1) Infection Status Organ

0 worms (n=14)

1 worm (n=7)

4-6 worms (n=8)

Liver

0.43

(0.02)

a

0.51 (0.05)

a

0.45

(0.03)

a

Left kidney

0.65

(0.08)

a

0.83 (0.14)

a

0.75

(0.08)

a

Right kidney/cyst

0.72

(0.10)

a

2.21 (0.36)

b

1.88

(0.19)

b

a) cast

3.73 (1.50)

b

1.95

(0.32)

a

b) spicule

11.47 (2.30)

a

7.78

(1.21)

a

c) worm(s)

0.97 (0.19)

a

1.47

(0.22)

a

Cyst components:

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. 12

Liver Left kidney 10

Right kidney/cyst Cyst components: Cast Spicule Worm(s)

NICKEL BURDEN (µg)

8

6

C C

B 4

A 2

0

A

0 worms

A

1 worm

4-6 worms

INFECTION STATUS

Figure 5. Nickel burden (µg) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Oneway Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean composite burden.

Mean hepatic nickel concentrations ranged between 0.43 ± 0.02 and 0.51 ± 0.05 µg•g-1, and did not differ significantly among the three infection groups (Table 3). Similarly, concentration values in the left kidney were comparable across the three infection groups, indicating that the infection-induced increase in nickel loads within this organ were

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proportional to the degree of hypertrophy undergone. Within the right kidney cyst, the greatest concentration of nickel occurred within the spicules (mean = 11.5 ± 2.3 and 7.8 ± 1.2 µg•g-1 in single worm and multiple worm infections respectively; Figure 6). 14

Liver Left kidney

NICKEL CONCENTRATION (µg • g-1)

12

Right kidney/cyst Cyst components:

10

Cast Spicule Worm(s)

8

A 6

A

4

A A

2

A

A

A

A A

B B

A A

A B

0

0 worms

1 worm

4-6 worms

INFECTION STATUS

Figure 6. Nickel concentration (µg•g-1 dry wt) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean.

Worm concentrations averaged 0.97 ± 0.19 and 1.47 ± 0.22 µg•g-1 while cast concentrations averaged 3.7 ± 1.5 and 2.0 ± 0.3 µg•g-1 respectively in the two infection groups. Relative to non-infected animals, tissue nickel levels (burdens and concentrations) in infected animals were more variable with few statistically significant differences occurring between single-worm and multiple-worm groups.

Lead Levels Lead burden and concentration values in mink from the three infection intensity groups are presented in Table 4. Total lead burdens followed the same trend seen for both cadmium and nickel in that combined kidney and liver lead loads were greater in mink infected with D. renale when compared to uninfected mink. Elevations averaged 21% higher in mink infected with 1 worm and 69% in those with 4-6 worms. As seen for nickel, the infection-induced increase in lead burden was attributable to enhanced deposition in the renal tissues. For the left kidney, lead burdens were elevated 1.6-fold (one worm infections) and 1.9-fold (4-6 worm infections) higher than values seen in the non-parasitized animal. The majority of the increase in total lead burden arose from the right kidney components, which showed

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elevations of more than 3-fold for 1-worm infections and 7-fold for 4-6 worm infections (Figure 7). Although noticeably higher, lead concentrations in the liver and left kidney followed the same trend as nickel in that mean tissue concentrations failed to differ significantly across the three treatment groups (Table 4). Table 4. Lead levels in body tissues of mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis LEAD BURDEN (µg) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

10.28 (0.60) a

10.23

(0.44)

a

11.72

(1.18)

a

Left kidney

0.95

(0.05) a

1.54

(0.07)

b

1.83

(0.17)

b

Right kidney/cyst

0.93

(0.07) a

3.37

(0.73)

b

6.73

(1.03)

c

a) cast

0.54

(0.07)

a

1.11

(0.14)

b

b) spicule

2.19

(0.68)

a

3.44

(0.77)

a

c) worm(s)

0.64

(0.14)

a

2.15

(0.30)

b

Cyst components:

All above organs/components 12.50 (0.61) a 15.13 (0.87) b 21.15 (1.13) c combined Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test. LEAD CONCENTRATION (µg•g-1) Infection Status Organ

0 worms (n=14)

1 worm (n=8)

4-6 worms (n=8)

Liver

0.98

(0.04) a

1.02

(0.02)

a

1.13

(0.07)

a

Left kidney

1.19

(0.05) a

1.26

(0.05)

a

1.40

(0.14)

a

Right kidney/cyst

1.29

(0.10) a

4.07

(0.71)

c

2.93

(0.29)

b

a) cast

1.68

(0.27)

a

1.43

(0.10)

a

b) spicule

40.50

(12.08) a

24.08

(2.84)

a

c) worm(s)

1.78

(0.19)

1.95

(0.10)

a

Cyst components:

a

Within organs, mean values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test.

Glenn H. Parker and Liane Capodagli

200 Liver Left kidney 20

Right kidney/cyst

LEAD BURDEN (µg)

Cyst components: Cast Spicule Worm(s)

15

C B

C

A

A

10

A 5

0

1 worm

0 worms

4-6 worms

INFECTION STATUS

Figure 7. Lead burden (µg) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Oneway Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean composite burden. Liver 50

Left kidney Right kidney/cyst Cyst components:

LEAD CONCENTRATION (µg • g-1)

40

Cast Spicule Worm(s)

30

20

A A 10

C A

A

A

A

A

A

A

B A

A

A

A

0

0 worms

1 worms

4-6 worms

INFECTION STATUS

Figure 8. Lead concentration (µg•g-1 dry wt) in organs of mink infected with D. renale at different intensities. Within organs, values bearing a common letter are not significantly different (p ≥ 0.05) as indicated by a Kruskal-Wallis One-way Analysis of Variance and Duncan‘s Multiple Range Test. Bars indicate ± 1 S.E. of the mean.

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

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Among components of the right kidney cyst, the bony spicule concentrated lead to the greatest extent with mean concentrations reaching 40 ± 12.1 and 24 ± 2.8 µg•g-1 for 1 worm and 4-6 worm infections respectively (Figure 8). Concentrations in cast tissues and in worms averaged less than 2 µg•g-1. Although burden values in the right kidney cyst were substantially higher in the more heavily infected group, concentration values in the structural components of the cyst did not differ significantly between 1 worm and 4-6 worm infection groups.

Proportional Deposition among Tissues To better control for variability in levels of uptake among individuals, the relative distribution of body metal burdens between the liver and renal tissues/structures was calculated and expressed on a percentage basis for both infected and non-infected animals (Table 5). Data available from two mink infected with a single D. renale worm in the abdomen (free-floating infection) and four mink with a single worm in the abdomen and one in the right kidney (double infection) have been included in Table 5 to represent two additional infection groups. The proportions of cadmium, nickel and lead deposited in renal and hepatic tissues of mink with a single abdominal infection were similar to the proportions deposited in non-infected mink. Of the total metal burden in mink with a free-floating infection and in non-infected mink, liver tissue accumulated 67 and 68% of the cadmium, 79 and 82 % of the nickel and 83 and 85% of the lead, respectively. Sixteen percent of the total cadmium, between 8 and 9% of the total nickel and 7-8% of the total lead was deposited in each kidney of non-parasitized mink and mink with an abdominal infection. Although there was a two-fold increase in total cadmium burden in mink infected with 1 worm over uninfected mink (Figure 3), the proportion deposited in the liver remained constant at 65 to 68% (Table 5). Slightly less cadmium (59% and 57%) was deposited in the livers of mink with double and 4-6 worm infections. Approximately 30% of the total body cadmium was found in the functioning renal tissue, regardless of infection status. In noninfected and abdominally infected mink, this was equally distributed between the right and left kidneys, while in mink parasitized in the right kidney, all 30% accumulated in the lone functioning left kidney, regardless of the number of worms present. In single worm and double worm renal infections, only 8 and 9% of the total cadmium was found in the resultant kidney cyst whereas 14% was accumulated in the cyst of animals harbouring 4-6 worms. A higher proportion of cadmium was deposited in worms from multiple worm infections (8%) versus single worms (3%). At all infection intensities, 3% was deposited in the kidney cast and 1-3% was sequestered in the bony spicule. When present, abdominal worms contained only 1-2% of the cadmium burden. In contrast to cadmium, nickel burdens in the liver did not differ with varying degrees of infection intensity (Figure 5). However, the proportion of total nickel burden deposited in the liver did change. In the non-parasitized group, approximately 82% of the total nickel burden accumulated in the liver. Subsequent to infection, the proportion of total nickel found in the liver dropped to 62% in mink infected with 1 renal worm, 44% in mink with a double infection and 46% in mink infected with 4-6 worms.

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202

Table 5. Relative distribution of metals among tissues, expressed as a percentage of total renal-hepatic burden, in mink infected with D. renale at different intensities Metal

Infection Status 0 worms Organ

(n=14)

1 worm Abdomen (n=2)

2 worms

4-6 worms

Right kidney Abdomen + right Right kidney (n=8) kidney (n=4) (n=8)

Cd Liver

68 b

67 a,b

65 a,b

59 a,b

57 a

Left kidney

16 a

16 a

27 b

31 b

29 b

Right kidney/cyst

16 c

15 c

8a

9 a,b

Cyst components a) cast

-

-

3a

3a

3a

b) spicule

-

-

1a

3a

2a

c) worm(s)

-

-

3a

3a

8b

Abdominal worm

-

1

-

2

-

82 c

79 c

62 b

44 a

46 a

14 b,c

Ni Liver Left kidney

9 a,b

9 a,b

12 b

7a

10 a,b

Right kidney/cyst

8a

8a

25 b

47 c

44 c

Cyst components a) cast

-

-

10 a

27 b

14 a

b) spicule

-

-

10 a

7a

11 a

c) worm(s)

-

-

5a

13 a,b

19 b

Abdominal worm

-

4

-

2

-

85 b

83 b

68 a

67 a

58 a

Pb Liver Left kidney

8 a,b

7a

10 c

9 b,c

9 b,c

Right kidney/cyst

8a

7a

21 b

21 b

32 b

Cyst components a) cast

-

-

4a

3a

5a

b) spicule

-

-

13 a

14 a

17 a

c) worm(s)

-

-

4a

3a

11 b

Abdominal worm

-

4

-

3

-

Within organs, values bearing the same letter are not significantly different (p ≥ 0.05) as indicated by a One-way Analysis of Variance and Duncan‘s Multiple Range Test.

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

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The relative proportion of nickel deposited in the left kidney increased only slightly in one worm infections, while that accumulating in the infected right kidney cyst reached as much as 25% and 44% of the body burden in 1 worm and multiple worm animals respectively. The left kidney of animals with an abdominal worm plus a right kidney worm did not differ from that noted for non-infected mink, however 47% of the total nickel burden was deposited in the right kidney cyst. In the case of single worm infections, 10% of the total nickel was deposited in the kidney cast and 5% in the developing worm, whereas in mink with 4-6 worms 14% was observed in the right kidney cast and 19% was taken up by the worms. Mink with both an abdominal and right kidney worm accumulated 27% of the total nickel in the cast and 13% in the right kidney worm. The spicule accounted for 10, 7 and 11 % of body nickel burdens in single, double and 4-6 worm infections respectively. Relative proportions of lead deposited in renal and hepatic tissues paralleled those observed for nickel. Eighty-five percent of the total body burden of lead was deposited in the liver of uninfected mink, while only 68%, 67% and 58% were accumulated in the hepatic tissue of mink with 1, 2 and 4-6 worm infections respectively. In response to infection, proportions in the left kidney increased only slightly whereas total body proportions accumulating in the infected right kidney cyst reached as much as 21% for both single and double infections and 32% for the 4-6 worm infected animals. In each group, more than half of the lead in the right kidney cyst was sequestered within the bony spicule. The worms accounted for 4%, 3% and 11% and the cast tissues for 4%, 3% and 5% of the total lead burden present in single worm, double worm and 4-6 worm infections respectively.

Gender Comparisons A comparison of metal levels in male versus female D. renale taken from mink with varying infection intensities is presented in Table 6. Dry weights for male and for female worms did not vary with infection status as indicated by One-way Analyses of Variance (males p = 0.46 and females p = 0.14). For multiple worm infections, metal levels in male and in female worms occupying the same cyst were averaged and entered in a 2-way Analysis of Co-Variance to assess the effects of infection status and worm gender while covarying for the effects of total worm weight in the infected cyst. Total worm weight in each cyst was not significantly correlated with levels of any of the three metals examined (p > 0.05), thus indicating that competition between male and female parasites within the same cyst had not affected metal uptake by the resident worms. For all three metals, overall mean burdens (i.e. in all worms combined regardless of infection status) tended to be higher in female compared to male worms by a factor of approximately 3 (Table 6). When differences in body mass were factored in by calculating concentration values, significant gender differences were observed only for lead, with males showing approximately 50% higher levels.

Femur versus Spicule Levels The geometric means for metal concentrations in femur sub-samples and in the spicules of parasitized animals were examined for the three infection groups and are presented in Table 7.

Glenn H. Parker and Liane Capodagli

204

Table 6. Metal levels in male and female D. renale taken from mink with varying infection intensities. Values are means followed by standard error in parenthesis Element

Infection Status Variable

1 worm (♂ n=4, ♀ n=4)

4 worms a 5–6 worms a (♂ n=3, ♀ n=4) (♂ n=4, ♀ n=3)

Sex

All groups combined (♂ n=10, ♀ n=11)

Cd Burden (µg) Male

0.29 (0.09) *

Female

0.07 (0.01) **

0.15 (0.04) *

0.18 (0.04) *

0.69 (0.26)

0.34 (0.03)

0.57 (0.14)

0.53 (0.10)

Male

2.27 (0.85)

0.60 (0.13)

0.97 (0.25)

1.34 (0.37)

Female

1.20 (0.70)

0.62 (0.10)

1.14 (0.17)

0.97 (0.25)

0.11 (0.04)

0.16 (0.03)

0.27 (0.03)

0.19 (0.03)

Concentration (µg•g-1)

Ni Burden (µg) Male

** Female

**

0.78 (0.20)

0.55 (0.06)

0.55 (0.16)

0.63 (0.09)

Male

0.82 (0.35)

1.36 (0.24)

1.94 (0.38)

1.43 (0.23)

Female

1.08 (0.25)

0.96 (0.10)

1.10 (0.28)

1.04 (0.11)

Male

0.29 (0.02)

0.29 (0.03)

0.35 (0.07)

0.31 (0.02)

Female

0.99 (0.09)

0.81 (0.07)

0.62 (0.006)

0.82 (0.06)

2.14 (0.17)

2.43 (0.21)

2.20 (0.24)

2.24 (0.12)

Concentration (µg•g-1)

Pb Burden (µg)

**

*

**

Concentration (µg•g-1) Male

* Female a

1.41 (0.20)

1.42 (0.11)

** 1.30 (0.14)

1.38 (0.08)

In multiple-worm infections, values represent the average for all worms of same sex present within the cyst. Gender differences assessed by 2-way Analysis of Variance while covarying for total worm weight within cyst; p < 0.05 *; p ≤ 0.01 **.

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

205

Table 7. Metal concentrations (µg•g-1 dry wt) in femurs and spicules of mink infected with D. renale at different intensities. Values are geometric means (presented as antilogs of the log10 transformed data) followed by standard error in parenthesis Tissue Metal Infection Group Cd

Ni

Pb

(n)

Femur

Spicule

0 worms

(12)

0.87

(+0.035, -0.033)

1 worm

(8 and 6)

0.94

(+0.038, -0.036)

<

2.14 (+0.49, -0.40)

4-6 worms

(8)

0.92

(+0.040, -0.039)

<

1.70 (+0.25, -0.22)

0 worms

(12)

3.39

(+0.12, -0.12)

1 worm

(8 and 6)

3.47

(+0.098, -0.094)

<

9.12 (+1.97, -1.62)

4-6 worms

(8)

3.55

(+0.12, -0.12)

<

7.24 (+1.25, -1.06)

0 worms

(12)

10.71

(+0.30, -0.29)

1 worm

(8 and 6)

11.22

(+0.29, -0.28)

<

23.99 (+2.68, -2.41)

4-6 worms

(8)

11.22

(+0.37, -0.36)

<

22.91 (+2.91, -2.59)

Equalities assessed by Paired-Samples T-Test; p < 0.01.

There were no differences in metal levels across the infection groups in either the femur or the spicule for any of the three metals investigated as indicated by One-way Analyses of Variance, and Analyses of Co-variance (p > 0.05) with spicule dry weight as the co-variant. Thus, infection intensity had not significantly influenced the uptake of metals in either bone tissue. The data were log10 transformed to accommodate for the non-linear relationship between tissue metal concentration and spicule dry weight before a Paired-Samples T-test was performed. Tissue comparisons revealed that the bony spicules of infected mink were consistently higher in concentrations of cadmium, nickel and lead than were the femurs of the same animals. Spicules from single worm infections concentrated 2.6 times more nickel, 2.3 times more cadmium and 2.1 times more lead than the femurs from the same group of mink. Similarly, in multiple worm infections, spicule metal concentrations were approximately twice those found in the femurs.

Effects on Body Condition The Principal Component Analysis of the condition measures in mink for the three infection groups (summarized in Table 8) is presented in Table 9. Four factors, explaining a cumulative 73% of the variance, indicated shared sources of variance within the condition measures. Abdomimal mid-ventral fat weight, thoracic post-cardinal fat weight and neck circumference shared over 56% of their variance with Principal Component 1. These measures were all correlated positively with the first component, indicating a positive relationship with one another.

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Glenn H. Parker and Liane Capodagli

Table 8. Body measures (morphometric measures, organ weights and fat deposits) in adult male mink infected with D. renale at different intensities. Values are means followed by standard error in parenthesis Infection Status 0 worms (n=14)

Measure

1 worm (n=8)

All mink combined (n=30)

4-6 worms (n=8)

Morphometric measures: Peltless body weight (g)

742.81 (22.42)

766.12

(22.70)

715.27 (43.39)

741.68 (16.48)

Total body + tail length (cm)

57.86

(0.69)

57.93

(0.48)

55.88

(0.85)

57.33 (0.44)

Body length (cm)

38.64

(0.55)

39.06

(0.49)

37.75

(0.62)

38.51 (0.34)

Neck circumference (cm)

11.88

(0.21)

12.04

(0.35)

11.75

(0.27)

11.89 (0.15)

Liver

43.19

(2.36)

37.67

(1.23)

40.40

(3.22)

40.97 (1.45)

Right kidney

3.61

(0.14)

4.20

(0.90)

14.20

(1.94)

6.59

(1.01)

Left kidney

3.74

(0.14)

5.69

(0.29)

6.04

(0.43)

4.87

(0.25)

Spleen

3.11

(0.40)

2.73

(0.28)

3.00

(0.38)

2.98

(0.22)

Heart

8.65

(0.33)

9.05

(0.48)

8.47

(0.54)

8.71

(0.24)

Gonads (combined)

0.38

(0.02)

0.66

(0.12)

0.54

(0.06)

0.51

(0.05)

Omental fat

3.54

(0.50)

3.11

(0.20)

4.08

(0.27)

3.55

(0.26)

Thoracic post-cardinal fat

0.20

(0.02)

0.16

(0.03)

0.19

(0.01)

0.19

(0.01)

Abdominal mid-ventral fat

0.64

(0.10)

0.58

(0.07)

0.59

(0.08)

0.61

(0.05)

Organ weights (g):

Fat measures (g):

Fat measures (1=low, 2=moderate, 3=high): Subcutaneous fat

2.14

(0.14)

1.88

(0.23)

2.38

(0.18)

2.13

(0.10)

Groin fat

2.21

(0.15)

1.88

(0.23)

2.50

(0.19)

2.20

(0.11)

Accommodating for less variance but nevertheless indicating shared variance with the first component were heart weight, body length, peltless body weight, scapular fat, groin fat and spleen weight, which were also positively related. The second component presented shared sources of variance for total body + tail length, body length, omental fat weight, peltless body weight, total worm number and spleen weight. In this case, omental fat weight, total worm number and spleen weight were negatively correlated to the second component indicating that as total body + tail length, body length and peltless body weight increased, omental fat weight, total number of D. renale worms and spleen weight decreased. The third component accommodated for shared variance in liver weight, scapular fat, groin fat and peltless body weight, which were all positively associated. The fourth and final component

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

207

indicated that right and left gonad weights, spleen weights and total worm number shared common variance; however, spleen weight was negatively associated, and thus decreased as gonad weight and total number of worms increased. Table 9. Results of the Principal Component Analysis for body condition measures in adult male mink with varying intensities of D. renale infection (n=21) Variables

Correlation Coefficients Component 1

Abdominal mid-ventral fat wt*

0.83

Thoracic post-cardinal fat wt*

0.80

Neck circumference

0.75

Heart wt*

0.48

Component 2

Total body + tail length Body length

0.35

0.83 -0.71

0.46

Total worm number

0.58

0.50

-0.54

Liver wt*

0.49 0.86

Scapular fat

0.40

0.85

Groin fat

0.38

0.80

Right and left gonad wt * Spleen wt*

Component 4

0.87

Omental fat wt* Peltless body wt

Component 3

0.84 0.35

-0.48

-0.55

*Condition measures standardized for body size.

Table 10. Results of the Canonical Correlation Analysis on the Principal Components for body condition measures in adult male mink and their total renal and hepatic burdens (µg) of cadmium, nickel and lead (n=21) Correlation Coefficients for Root 1 Dependent variables: Principal Component 1 (fat deposits, neck circumference)

0.69

Principal Component 4 (gonadal and spleen wts, total worm no.)

0.70

Independent variables: Total Cd burden

0.64

Total Ni burden

0.49

Total Pb burden

0.79

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Glenn H. Parker and Liane Capodagli

These components were used as dependent variables in the subsequent Canonical Correlation Analysis while total body (renal and hepatic) metal burdens of cadmium, nickel and lead were used as independent variables. The results of the Canonical Correlation Analysis are presented in Table 10. One root was significant at p < 0.05 with a Canonical Correlation Coefficient (r) of 0.83. Principal Components 1 and 4 loaded on the dependent (y) component of the root with correlation coefficients of 0.69 and 0.70 respectively. All three total body metal burdens loaded moderately on the independent component (x) of the root. Sixty-two per cent of the variance in total body lead, 41% of the variance in total body cadmium and 24% of the variance in total body nickel burdens were shared with the independent component of the root. In summary, the results of the Canonical Correlation Analysis indicated that as the total body burdens of cadmium, nickel and lead increased, there was an increase in abdominal mid-ventral and thoracic post-cardinal fats, neck circumference, gonadal weight and total number of D. renale worms but the spleen weights decreased.

DISCUSSION Giant kidney worm infection resulted in substantial weight changes among the kidneys, but not the livers or peltless body weights, of mink. Kidneys infected with D. renale were enlarged but the increase only reached significance for multiple worm infections where the development of several worms along with the formation of a bony spicule had taken place within the cyst. Hypertrophy of the left kidney to compensate for the loss in function of the infected right kidney was indicated by the increase in dry organ weight over that seen in noninfected mink. McNeil (1948) and Mace (1976) similarly reported enlargements of both the left kidney and the right kidney cyst in D. renale infected mink. The dry weight of the left kidney in infected animals approximated the combined dry weights of right and left kidneys in non-infected mink; thus the amount of functioning renal (and hepatic) tissue was approximately equal for all infection groups. Consequently, any changes in tissue metal burdens or concentration values noted in the parasitized animal can be deemed to be the result of changes in metal uptake levels and/or the re-distribution of tissue metal loads. Among the most interesting findings of this study was the increased renal plus hepatic metal burden of parasitized (both single and multiple-worm infected) mink over that seen in uninfected animals. Although quantitatively variable, this pattern was apparent for all three metals examined. The nature of this metal-parasite association is of fundamental importance and raises the question of whether the enhanced body metal loads had increased the susceptibility of the animal leading to infection by the kidney worm or conversely whether it was the invasion/presence of the D. renale worm(s) that had resulted in the increased levels of metal uptake within these targeted tissues. The fact that more intense infections (4-6 worms per animal) were generally associated with higher body metal burdens than seen in single worm infections, although interesting, offers little towards answering this question. The latter scenario, inferring a direct enhancing effect of the parasite on metal uptake and retention in the host, appears more likely in this particular host-parasite relationship for a number of reasons: D. renale is known to occur in areas devoid of point source metal emissions and conversely is virtually absent in certain other locales heavily dominated by metal pollution (e.g. Palmerton, Pennsylvania). Furthermore, juvenile and adult mink of the Sudbury area are

On the Redistribution of Tissue Metal (Cadmium, Nickel and Lead) …

209

known to be infected at approximately equal frequencies (N. Schaffner and G.Parker, unpublished data) despite the fact that the 5-6 month old juveniles, when trapped in the fall, have had only brief exposures to prevailing environmental metal conditions and thus would not be expected to have accrued significant metal burdens nor an accompanying increase in susceptibility to the parasite. It is contended, therefore, that parasitic infection by the giant kidney worms, whatever its primary predispositional cause(s) may have been, contributed directly to the increased uptake and/or retention of tissue metals noted in this study. Future laboratory studies investigating experimental infection intensities in captive mink exposed to varying levels of metals within their diet would shed further light on this unresolved contention. Elevated metal burdens in the infected animals were most likely due to an increase in metal uptake subsequent to parasitic infection. Although unconfirmed, it is certainly conceivable that the energy demands imposed on the parasitized animal would be elevated, if for no other reason than to provide the extra requirements needed in the growth and development of the newly-developing cyst structures (namely the cast, worms and spicule), the proliferation of actively hypertrophying tissues within the left kidney and the nutrients necessary to sustain the resident parasites. Inherent in this supposition is the assumption that increased levels of dietary intake would lead to enhanced exposure, uptake and retention of metals by the infected animal. Of the three elements sequestered by the parasitized animal, cadmium was the only one to show significantly increased tissue levels in both the liver and the non-parasitized renal tissue. Cadmium has a high affinity for the metal binding protein metallothionein (MT), which is induced in the liver and to a lesser extent in the kidneys of the exposed animal (Goering and Klaassen, 1984). It has been demonstrated that MT functions as a mechanism for transport, storage and detoxification of metals (Fulkerson and Goeller, 1973). Detoxification of cadmium in the vertebrate body occurs when the divalent form of the metal (the most harmful form of most toxic metals) complexes with the low molecular weight protein. The MT-cadmium complex is then transported to the kidneys for storage where it is kept out of circulation. Once the levels of cadmium in the renal tissue reach a given threshold, which varies among species, irreversible tubular damage results (Friberg et al., 1979). With the loss in function of one kidney, the lone functioning left kidney and liver were responsible for sequestering most of the cadmium taken up subsequent to parasitic infection. The proportion of cadmium deposited in the left kidney of infected mink (27-31%; Table 5) was roughly equivalent to the sum of the proportions deposited in both kidneys of the uninfected animal (32%), and did not differ with increasing infection intensity. Also of interest here is the fact that in infected animals the relative distribution of cadmium between the left (hypertrophied) kidney and the liver remained at a ratio of approximately 3.5:1 – a value comparable to that seen in non-infected mink with both kidneys functional (Table 2). The consistency of the above distribution ratios among infected and non-infected groups suggests that cadmium was distributed between renal and hepatic tissues in a well regulated manner and that the mechanisms and processes involved in the distribution and deposition of cadmium in these two accumulator tissues had not been significantly altered as a result of parasitic infection or increased metal loads. As parasitic burdens increased to 4-6 worms, cadmium was consistently distributed among host tissues, with the exception of a slight decrease in the proportion (57 vs. 65%; Table 5) deposited in the liver. This reduction may have been due to the greater amount of worm tissue available for cadmium deposition and

210

Glenn H. Parker and Liane Capodagli

which accounted for an increased proportion (8 vs. 3%; Table 5) of the total body burden of the more heavily-infected animal, thus leaving less to be sequestered in the liver. Whether the tissue damage induced by the invading D. renale parasite was directly responsible for the augmented hepatic and/or renal cadmium levels noted in parasitized animals is a matter of speculation. Lysis of the internal renal tissues of the right kidney by the new-acquired invading worms might be expected to release bound cadmium from its MT complex, thereby freeing it for incorporation into the growing worms, for reabsorption into systemic circulation leading to increased production of MT followed by redistribution to other host tissues or for elimination via the ureters which are known to remain open in the infected animal (McNeil, 1948; Anderson, 2000). Similarly, migration of the worms as third-stage larvae/subadults through the liver during the initial infective process may have served as a physical or biochemical stimulus for the production of additional MT in the liver. Mace and Anderson (1975) reported D. renale larvae between the diaphragm and the right lobe of the liver in mink as late as 50 days postinfection. In addition, they found numerous lesions throughout the infiltrated liver containing necrotic cells, fibrin, red blood cells, eosinophils, macrophages and lymphocytes. Visual inspection and palpation of the livers of parasitized mink in the present study showed the tissue to be abnormally firm and the ventral surfaces of the lobes to be uneven with evidence of pathological damage. Fibres of connective tissue frequently attached the right kidney cyst to the right lobe of the liver, as well as to other structures including the greater omentum, duodenum and/or ventral surface of the abdominal wall, as has been noted by previous investigators (McNeil, 1948; Mace, 1976). Cadmium, if leached from hepatic storage sites as a result of the physical and/or pathological damage induced by the invading migratory larval worms, could be expected to re-enter circulation, thus re-priming the production of MT within host tissues. Evidence that the cadmium leaking out of necrotic liver tissue results in increased kidney burdens of cadmium has been demonstrated in the mouse (Andersen et al., 1985). The fact that abdominal infections not involving renal-hepatic tissue damage produced little alteration in body cadmium burdens and their distribution confirms the central role of parasite-induced tissue damage in prompting the substantial changes in cadmium accumulation and distribution patterns seen in the more conventionally (i.e. right kidney) infected mink. Unlike the elevations noted in the liver and hypertrophied left kidney, cadmium levels in the right kidney cyst of the parasitized animal remained unchanged from values observed for kidneys prior to infection. On average, individual components within the cyst harboured relatively low levels of cadmium, suggesting the absence of MT-complexing processes in these tissues. The fact that only relatively small amounts of cadmium were taken up by the resident worms may be explained by the absence of a well-developed excretory system, the main site of MT-facilitated accumulation in most species, in these and similar nematodes (Mace and Anderson, 1975). Body burdens of nickel and lead were likewise elevated in response to parasitism, but unlike the pattern noted for cadmium, most of the extra metal accumulated was confined to the renal structures. Both nickel and lead are eliminated from the vertebrate body primarily through the renal system (Friberg et al., 1979). There is no process comparable to the MTcadmium complex known to assist in the detoxification of either nickel or lead. With a reduced ability to excrete these metals through the single remaining kidney, along with an enhanced dietary intake as proposed earlier in this discussion, it would stand to reason that

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some of the excess metals present within the infected animal would be localized within the hypertrophied left kidney as indicated. By comparison, greater amounts of both lead and nickel, however, were accumulated within the dysfunctional right kidney cyst of the parasitized animal. Deposition into the various components of the cyst may have occurred during the growth of these structures but is deemed to have been selective rather than uniform, as concentrations of both lead and nickel within the spicule exceeded those present in the cast tissue and resident worms by a factor of several-fold. The substantial levels deposited within the component structures of the right renal cyst ultimately accounted for as much as 25 to 47% and 21 to 32% of the total renal-hepatic nickel and lead burdens respectively (Table 5) and appear to account for the markedly reduced proportions sequestered within the liver of the parasitized animal. Establishing trends in metal accumulation and distribution in response to infection intensity as attempted here was fraught with several potential confounding factors. Sources of variation that must be acknowledged include individual differences in host reaction to parasitic infection and/or increased metal loads, variability in geographic origin of samples, age-related variations in the host, age of the infection and inherent differences between the sexes of the parasite. The biological response to a stress of any type varies from individual to individual, and when trying to examine the response of individuals to multiple stresses, the variability can be expected to magnify. The Sudbury region was chosen as an appropriate site for this study because of its longstanding history of mining/smelting activity resulting in substantial environmental contamination and because a 50% prevalence of D. renale infections was known to exist within its wild mink populations. However, to collect sufficient numbers, mink had to be taken at varying distances from the point source of metal pollution – a factor which undoubtedly contributed to the variability in metal loads among animals. Only adult male mink were employed in an attempt to reduce variation in metal accumulation as a function of age and/or gender. However, as the adult age class consisting of mink greater than one year probably included some individuals ranging up to 4 or 5 years of age, the period of exposure to metal contaminants cannot be considered uniform. Finally, due to substantial sexual dimorphism in adult D. renale specimens, the gender of the infecting worm in the case of single worm infections and the ratio of male to female worms in multiple worm infections are factors which must be taken into consideration in establishing metal distribution patterns. Female D. renale were consistently longer and heavier than male worms by a factor of 3, which largely accounted for the approximate 3-fold elevation in cadmium, nickel and lead burdens found in female worms versus male worms. However, when metal accumulations were expressed on a per gram dry weight basis, male and female worms indicated nearequivalent concentrations, with the exception of lead which was 1.5 times higher in males. While studies on Ascaris suum, a parasitic nematode of swine, failed to indicate genderdifferences in metal (Cd and Pb) uptake (Greichus and Greichus, 1980; Sures et al., 1998), those involving other nematode species including Conracaecum rudolphii in cormorants (Barus et al., (2001) and Anguillicola crassus in the European eel (Barus et al., 1999; Tenora et al., 1999a) have shown a higher propensity for accumulation in males. In contrast, the archiacanthocephalan Moniliformis moniliformis, experimentally introduced into the gastrointestinal tract of rats and subsequently exposed through dietary supplements, indicated higher Pb accumulations in female specimens – a factor attributed to the substantial levels sequestered within eggs (Sures et al., 2000). No attempt was made to localize the metal accumulations within the giant kidney worms of the present study. Possible sites include the

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cuticle, reproductive structures and/or the gastrointestinal tract, but not excretory tissues as this nematode is not known to possess a structured excretory system (Mace and Anderson, 1975). Analytical methods sensitive enough to evaluate metal contaminants in very small amounts of tissue may provide information identifying the sites of accumulation and explaining differences in uptake between the two sexes. The 2-fold higher concentrations of cadmium, nickel and lead in the developing bony spicule relative to femur levels denote the spicule as an active site for deposition. Previous studies have shown lead to be primarily deposited in bone and secondarily in soft tissue, whereas nickel and cadmium accumulated primarily in soft tissues and secondarily in osseous tissues (Friberg et al., 1979). The present results confirm this pattern in that lead presented the highest concentrations in the spicule while cadmium showed the lowest concentrations. It is tempting to conjecture that the deposition of toxic metals into the spicule structure may be adaptive in allowing the host to store a portion of the increased metal load in a relatively nonexchangeable compartment of the body, thereby reducing the levels of toxic metals in circulation or in storage within more vulnerable tissues. In the case of nickel, the proportion deposited in the spicule was equivalent to the proportion in a single kidney of a nonparasitized mink (Table 5). Likewise, the spicule accounted for an equivalent proportion of lead as seen in the entire renal system of a non-parasitized animal. This conjecture of adaptive sequestration, if valid, could be expected to contribute only within limits as spicules seldom exceeded 0.3 grams dry weight and generally accounted for less than 15% of the total renalhepatic metal burden (Table 5). Few other studies investigating parasite-induced changes in host tissue metal distribution were found in the literature. Palikova and Barus (2003) observed only low levels of mercury accumulation in A. crassus, and concluded that the low efficacy of Hg (and of other elements as reported by variuos authors) sequestering probably had little effect on tissue concentrations in their host species, the eel. Evidence suggesting a significant role by tapeworms in decreasing host tissue metal levels in several fish species has been presented by Turcekov and Hanzelova (1999), Barus et al. (2001b) and Turcekova et al. (2002). Sures and Siddall (1999) examined the effects of lead accumulation by adult intestinal worms (Pomphorhynchus laevis) on the tissue metal levels of their definitive host, the chub (Leuciscus cephalus). The presence of intestinal worms reportedly lowered lead accumulation in the intestinal wall relative to that seen in uninfected fish. The authors suggested that the intestinal parasites had extracted substantial lead from the diet of the host, thereby reducing the amount available for absorption by the host tissues and accounting for the higher concentrations seen in worm versus host tissues (unfortunately, burdens in host and parasite tissues were not reported). The results of the present study in which D. renale infections were consistently associated with elevated cadmium concentrations and unaltered lead and nickel levels within the renal and hepatic tissues of the host are at variances with the above reported trends. The reason(s) for the observed discrepancy is unclear, but may relate to differences in the source and route of metal uptake (dietary versus extraction from the host tissue stores) and/or the level of parasitic damage and host reaction incurred during the process of infection. Assessment of the effects of both a parasitic stress and increased metal loads on mink health revealed a positive relationship for two internal fat deposits, gonadal weight, neck circumference and total number of D. renale worms with combined renal and hepatic burdens of cadmium, lead and nickel. Spleen weight was negatively related to the total metal burdens. These trends substantiate those reported by Capodagli and Parker (2007), in that higher levels

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of fat deposition in both mink and muskrat (non-parasitized) were likewise observed among animals bearing elevated tissue levels of cadmium and lead. If mink infected with the giant kidney worm parasite were consuming more food to meet the increased energy demands of parasitic infection, as suggested above, such dietary enhancement, if excessive, could explain the increase in fat deposits noted for the abdominal mid-ventral and thoracic post-cardinal sites. The extent to which the proposed increase in food consumption may have contributed to the enhanced development of the neck musculature (as denoted by increased circumference), and possibly an improved capacity for successfully capturing larger prey items, is open to speculation. The positive relationship denoted between the intensity of infection (number of worms present) and combined renal-hepatic metal burdens of the animal can best be attributed to the level of tissue damage incurred combined with the nature of the host tissue responses to the invading parasites as outlined earlier in this discussion. The observed increase in gonad weight with increasing renal-hepatic metal levels, although presently unexplained, may be indicative of direct alterations to reproductive processes or indirectly through associated endocrinological functions. Testes have been shown to be sensitive to the effects of cadmium, although low doses of the element have generally been observed to induce necrosis, rather than hypertrophy (Fulkerson and Goeller; 1973). Given the possibility that accrued toxic metal burdens could adversely affect reproductive status and productivity in this fur-bearing species, the metal-prompted increase in testes weight may warrant further investigation including an assessment of histological changes. The negative relationship indicated between spleen weight and body metal burdens is difficult to interpret. The spleen typically functions to breakdown and dispose of aged and discarded red blood cells and has been shown to increase in size (splenomegaly) with increased functional demand in rats experimentally exposed to lead (Ogilvie and Martin, 1981). At the present time, no explanation can be offered for the apparent decrease in fresh spleen weights noted here. It was initially hypothesized that destruction of the right kidney during D. renale infection of the mink, particularly in geographic populations marked by elevated environmental cadmium exposure, might result in critically elevated tissue metal levels leading to functional impairment of the remaining contralateral. The findings of this study support the view that metal levels were substantially elevated (2 to 2.5-fold) in the surviving kidney of the parasitized animal. However, as damage in the human kidney generally does not become apparent until renal cadmium levels reach values of approximately 350 µg•g-1 dry weight (approximately 100 µg•g-1 wet weight) (Friberg et al., 1979) and individual values in this study seldom exceeded 10 µg•g-1 dry weight, the risk to Sudbury-area mink would appear to be only low to moderate. Although the animals had been simultaneously exposed to nickel, lead and possibly other toxic elements within the Sudbury environment, there was no evidence of increased concentrations of these elements within the left kidney tissue. The necrotic effects of lead on renal tissue are well documented (Friberg et al., 1979) and the possibility of synergistic toxicological effects during multi-elemental exposure, even in the absence of tissue accumulations, cannot be ruled out.

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CONCLUSIONS Metal burdens in renal-hepatic tissues were higher in mink infected with the giant kidney worm parasite than in non-infected mink. A trend of increasing metal burdens with greater numbers of worms present was apparent for nickel and lead but not cadmium. Changes in the proportional distribution of nickel and lead in host mink tissues were due to accumulations in new tissue growth induced by parasitic infection. Cadmium consistently accumulated in renal and hepatic tissue with minimal alteration in the proportional distributions subsequent to D. renale infection. The spicule was the tissue of preference for both lead and nickel deposition, whereas cadmium accumulated more readily in the hypertrophying left kidney and the liver. The substantial accumulation of metals (namely lead and nickel) in the bony spicules (but not in the femurs) of infected animals may be an adaptive mechanism whereby the levels found in systemic circulation and soft tissues are reduced and soft tissue damage thus avoided. Female worms accumulated approximately 3-fold higher burdens of cadmium, lead and nickel than did male worms. This gender difference is attributable to the larger mass of females, as concentration values (with the exception of certain lead determinations) did not differ between the sexes. Although toxic metal burdens were enhanced in parasitized mink, concentration values in the hosts‘ soft tissues remained below levels known to elicit damage and disrupt function. Nevertheless, a relationship among certain body measures and increased metal loads of infected mink was noted. Abdominal mid-ventral and thoracic post-cardinal fat deposits, neck circumference and combined gonad weight were positively related to combined renal and hepatic metal burdens while spleen weights were negatively related. These trends substantiate earlier findings reported for non-parasitized mink carrying elevated body metal burdens as a result of residing within the immediate influence of active ore mining/smelting operations (Capodagli and Parker, 2007). These associations may indicate a focal point for future research on the impacts of increased metal loads and parasitic infection on the host system.

ACKNOWLEDGMENTS We gratefully acknowledge the assistance of all members of the Sudbury Fur Trappers Council who contributed carcasses from their traplines. Statistical advice was kindly provided by M.A.Persinger. This work was financially supported through the Laurentian University Research Fund, the Ontario Graduate Scholarship for Scientists and Technologists Program and the Sudbury Game and Fish Protective Association.

REFERENCES Andersen, O., P. Linegaard, M. Unger, and G.F. Nordberg. 1985. Effects of liver damage induced by polychlorinated biphenyls (PCB) on cadmium metabolism in mice. Environmental Research. 38:213-224.

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Anderson, R.C. 2000. Nematode parasites of vertebrates. Their development and transmission. 2nd ed. CABI Publishing, New York, New York. 595-597. Barus, V., F. Tenora, S. Kracmar and M. Prokes. 2001a. Cadmium and lead concentrations in Contracaecum rudolphii (Nematoda) and its host, the Cormorant Phalacrocorax carbo (Aves). Folia Parasitologica 48:77-78. Barus, V., F. Tenora, S. Kracmar, M. Prokes and J. Dvoracek. 1999. Microelement contents in males and females of Anguillicola crassus (Nematoda: Dracunculoidea). Helminthologia 36:283-285. Barus, V., F. Tenora, M. Prokes and M. Penaz. 2001b. Heavy metals in parasite-host systems: tapeworm vs. fish. In: Ochrana zdravi ryb (Health protection of fish) , Vodnany. pp 2028. Capodagli and Parker. 2007. Accumulation and tissue distribution of toxic metals with accompanying effects on body condition measures, in mink (Mustela vison) and muskrat (Ondatra zibethicus) living near mining/smelting operations. In: R. B. Gore (Ed.), Environmental Research at the Leading Edge. pp. 75-112. Hauppauge, NY: Nova Science Publishers, Inc. Dhaliwal, S., and M. Taylor. 2000. Prevalence and characterization of spicule formation in the kidney of mink infected with giant kidney worm. B.Sc. thesis. Laurentian University, Sudbury, Ontario. pp 34. Friberg, L., G.F. Nordberg and V.B. Vouk. (eds.) 1979. Handbook on the Toxicology of Metals. Elsevier North-Holland Inc. New York, New York. pp 709. Fulkerson, W. and H.E. Goeller. (eds.) 1973. Cadmium, the Dissipated Element. ORNL-NSF Environmental Program, Oakridge, Tennessee. pp 426. Fyvie, A. 1971. Dioctophyma renale. Parasitic Diseases of Wild Mammals. J.W. Davis and R.C. Anderson (eds.) pp. 364. Iowa State University Press, Ames, Iowa. Goering, P.L. and C.D. Klaassen. 1984. Tolerance to cadmium-induced toxicity depends on presynthesized metallothionein in liver. Journal of Toxicology and Environmental Health. 14:803-812. Greichus, A., and Y.A. Greichus. 1980. Identification and quantification of some elements in the hog roundworm, Ascaris lumbricoides suum, and certain tissues of its host. International Journal of Parasitology. 10:89-91. Mace, T.F. 1976. Lesions in mink (Mustela vison) infected with giant kidney worm (Dioctophyma renale). Journal of Wildlife Diseases. 12:88-92. Mace, T.F. and R.C. Anderson. 1975. Development of the giant kidney worm, Dioctophyma renale (Goeze, 1782) (Nematoda: Dioctophymatoidea). Canadian Journal of Zoology. 53:1552-1568. McNeil, C.W. 1948. Pathological changes in the kidney of mink due to infection with Dioctophyma renale (Goeze, 1782) the giant kidney worm of mammals. Transactions of the American Microscopical Society. 67:257-261. Ogilvie, D.M. and A.H. Martin. 1981. Splenomegaly and adrenal weight changes in isolated adult mice chronically exposed to lead. Bulletin of Environmental Contamination and Toxicology. 26:647-651. Palikova, M., and V. Barus. 2003. Mercury content in Anguillicola crassus (Nematoda) and its host Anguilla anguilla. Acta Veterinaria Brno. 72:289-294. Pascoe, D., and P. Cram. 1977. The effect of parasitism on the toxicity of cadmium to the three-spined stickleback, Gasterosteus aculeatus L. Journal of Fish Biology. 10:467-486.

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Poulin, R. 1992. Toxic pollution and parasitism in freshwater fish. Parasitology Today. 8:5861. Siddall, R. and B. Sures. 1998. Uptake of lead by Pomphorhynchus laevis cystacanths in Gammarus pulex and immature worms in chub (Leuciscus cephalus). Parasitology Research. 84:573-577. Sures, B. 2003. Accumulation of heavy metals by intestinal helminths in fish: an overview and perspective. Parasitology. 126:S53-S60. Sures, B., G. Jurges and H. Taraschewski. 1998. Relative concentrations of heavy metals in the parasites Ascaris suum (Nematoda) and Fasciola hepatica (Digenea) and their respective porcine and bovine definitive hosts. International Journal for Parasitology. 28:1173-1178. Sures, B., G. Jurges and H. Taraschewski. 2000. Accumulation and distribution of lead in the archiacanthocephalan Moniliformis moniliformis from experimentally infected rats. Parasitology. 121:427-433. Sures, B. and R. Siddall. 1999. Pomphorhynchus laevis: the intestinal Acanthocephalan as a lead sink for its host, chub (Leuciscus cephalus). Experimental Parasitology. 93:1-7. Sures, B., R. Siddal and H. Taraschewski. 1999. Parasites as accumulation indicators of heavy metal pollution. Parasitology Today. 15:16-21. Sures, B. and H. Taraschewski. 1995. Cadmium concentration in two adult acanthocephalans, Pomphorhynchus laevis and Acanthocephalus lucii, as compared with their fish hosts and cadmium and lead levels in larvae of A. lucii as compared with their crustacean host. Parasitology Research. 81:494-497. Sures, B., H. Taraschewski and E. Jackwerth. 1994a. Comparative study of lead accumulation in different organs of perch (Perca fluviatilis) and its intestinal parasite, Acanthocephalus laevis. Bulletin of Environmental Contamination and Toxicology. 52:269-273. Sures, B., H. Taraschewski and E. Jackwerth. 1994b. Lead accumulation in Pomphorhynchus laevis and its host. Journal of Parasitology. 80(3):355-357. Sures, B., H. Taraschewski and E. Jackwerth. 1994c. Lead content of Paratenuisentis ambiguus (Acanthocephala), Anguillicola crassus (Nematodes) and their host Anguilla anguilla. Diseases of Aquatic Organisms. 19:105-107. Sures, B., H. Taraschewski and J. Rokicki. 1997a. Lead and cadmium content of two cestodes, Monobothrium wageneri and Bothriocephalus scorpii, and their fish hosts. Parasitology Research. 83:618-623. Sures, B., H. Taraschewski and M. Rydlo. 1997b. Intestinal fish parasites as heavy metal bioindicators: A comparison between Acanthocephalus lucii (Palaeacanthocephala) and the zebra mussel Dreissena polymorpha. Bulletin of Environmental Contamination and Toxicology. 59:14-21. Sures, B., H. Taraschewski and R. Siddall. 1997c. Heavy metal concentrations in adult acanthocephalans and cestodes compared to their fish hosts and to established free-living bioindicators. Parasitologia. 39:213-218. Szefer, P., J. Rokicki, K. Frelek, K. Skora and M. Malinga. 1998. Bioaccumulation of selected trace elements in lung nematodes, Pseudalius inflexus, of harbor porpoise (Phocoena phocoena) in a Polish zone of the Baltic Sea. The Science of the Total Environment. 220:19-24.

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Tenora, F., V. Barus, S. Kracmar and J. Dvoracek. 2000. Concentrations of some heavy metals in Ligula intestinalis plerocercoids (Cestoda) and Philometra ovata (Nematoda) compared to some of their hosts (Osteichthyes). Helminthologia. 37:15-18. Tenora, F., V. Barus, S. Kracmar, J. Dvoracek and J. Srnkova. 1999a. Parallel analysis of some heavy metals concentrations in the Anguillicola crassus (Nematoda) and the European eel Anguilla anguilla (Osteichthyes). Helminthologia. 36: 79-81. Tenora, F., V. Barus and M. Prokes. 2002. Next remarks to the knowledge of heavy metal concentrations in gravid tapeworm species parasitizing aquatic birds. Helminthologia. 39:143-148. Tenora, F., S. Kracmar, V. Barus and J. Dvoracek. 1999b. Content of microelements of heavy metals in males and females of Toxocara canis and Protospirura muricola (Nematoda). Helminthologia. 36:127. Tenora, F., S. Kracmar, M. Prokes, V. Barus and J. Sitko. 2001. Heavy metal concentrations in tapeworms Diploposthe laevis and Microsomacanthus compressa parasitizing aquatic birds. Helminthologia. 38:63-66. Turcekova, L., and V. Hanzelova. 1999. Concentrations of Cd, As and Pb in non-infected and infected Perca fluviatilis with Proteocephalus percae. Helminthologia 36:31. Turcekova. L., V. Hanzelova and M. Spakulova. 2002. Concentration of heavy metals in perch and its endoparasites in the polluted water reservoir in Eastern Slovakia. Helminthologia. 39:23-28. Williams, H.H., and K. Mackenzie. 2003. Marine parasites as pollution indicators: an update. Parasitology. 126:S27-S41. Wren, C.D., K.L. Fisher and P.M. Stokes. 1988. Levels of lead, cadmium and other elements in mink and otter from Ontario, Canada. Environmental Pollution. 52:193-202. Zimmermann, S., B. Sures and H. Taraschewski. 1999. Experimental studies on lead accumulation in the eel-specific endoparasites Anguillicola crassus (Nematoda) and Paratenuisentis ambiguus (Acanthocephala) as compared with their host, Anguilla anguilla. Archives of Environmental Contamination and Toxicology. 37:190-195.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 6

AEROBICALLY BIODEGRADED FISH-MEAL WASTEWATER AS A FERTILIZER Joong Kyun Kim1 and Geon Lee2 1

Department of Biotechnology and Bioengineering, Pukyong National University, Busan, Korea 2 Research Department, Samrim Corporation, Kimhae, Kyongnam, Korea

ABSTRACT Reutilization of fish-meal wastewater (FMW) as a fertilizer was attempted, and aerobic biodegradation of the FMW were successfully achieved by microbial consortium in a 1-ton bioreactor. During the large-scale biodegradation of FMW, the level of DO was maintained over 1.25 mg∙l-1, and a strong unpleasant smell remarkably disappeared in the end. Although the level of total amino acids and the concentrations of N, P and K in the biodegraded FMW were relatively lower than those in two commercial fertilizers, the concentrations of noxious components in the biodegraded FMW were much lower than the standard concentrations. The phytotoxicity of the biodegraded FMW was almost equal to that of the commercial fertilizers. The fastest growth in hydroponic cultures of red bean and barley was achieved at 100-fold and 500-fold dilution, respectively, the growth of which was comparable to those of 1,000-fold diluted commercial-fertilizers. From all above results, it was concluded that a large-scale biodegradation of FMW was successful and the properties of FMW were acceptable. This is the first study to demonstrate aerobic production of liquid-fertilizer from FMW.

Keywords: fish-meal wastewater, fertilizer, microbial consortium, large-scale biodegradetion, phytotoxicity, hydroponic culture.

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INTRODUCTION The amount of fisheries waste generated in Korea is expected to increase with a steady increase in population to enjoy taste of slices of raw fish. The fisheries waste is reduced and reutilized through the fish meal production. The process, which uses fish wastes such as heads, bones or other residues, is the commonest used in the Korean industries. The first step of the fish-meal manufacturing processes is the compression and crushing of the raw material, which is then cooked with steam, and the liquid effluent is filtered off in a filter press. The liquid stream contains oils and a high content of organic suspended solids. After oil separation, the fish-meal wastewater (FMW) is generated and shipped to wastewater treatment place. FMW has been customarily disposed of by dumping into the sea, since direct discharge of FMW can cause serious environmental problems. Besides, bad smell, which is produced during fish-meal manufacturing processes, causes civil petition. Stricter regulations for this problem also come into force every year in Korea. Therefore, there is an urge to seek for an effective treatment to remove the organic load from the FMW; otherwise the fish meal factories will be forced to shut down. Biological treatment technologies of fish-processing wastewater have been studied to improve effluent quality (Battistoni and Fava, 1995; Park et al., 2001). The common feature of the wastewaters from fish processing is their diluted protein content, which after concentration by a suitable method would enable the recovery and reuse of this valuable raw material, either by direct recycling to the process or subsequent use in animal feed, human food, seasoning, etc. (Afonso and Borquez, 2002). It has been also reported that the organic wastes contain compounds, which are capable of promoting plant growth (Day and Katterman, 1992), and seafood processing wastewaters do not contain known toxic or carcinogenic materials unlike other types of municipal and industrial effluents (Afonso and Borquez, 2002). Although these studies imply that FMW could be a valuable resource for agriculture, potential utilization of this fish wastes has been limited because of its bad smell (Martin, 1999). There is an increasing need to find ecologically acceptable alternatives to overcome this problem. Aerobic biodegradation has been widely used in treatment of wastewaters, and recently references to the use of meso- and thermophilic microorganisms have become increasingly frequent (Cibis et al., 2006). During the biodegradation, the organic matter is biodegraded mainly through exothermic aerobic reactions, producing carbon dioxide, water, mineral salts, and a stable and humified organic material (Ferrer et al., 2001). There have been few reports that presented the reutilization of biodegraded waste products as liquid-fertilizer: a waste product of alcoholic fermentation of sugar beet (Agaur and Kadioglu, 1992), diluted manure streams after biological treatment (Kalyuzhnyi et al., 1999), and biodegraded fish-meal wastewater in our previous studies (Kim et al, 2007; Kim and Lee, 2008). Therefore, aerobic biodegradation is considered to be the most suitable alternative to treat FMW and realize a market for such a waste as a fertilizer. The growth of plants and their quality are mainly a function of the quantity of fertilizer and water. So it is very important to improve the utilization of water resources and fertilizer nutrients. The influence of organic matter on soil biological and physical fertility is well known. Organic matter affects crop growth and yield either directly by supplying nutrients or indirectly by modifying soil physical properties such as stability of aggregates and porosity

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that can improve the root environment and stimulate plant growth (Darwish et al., 1995). Incorporation of organic matter has been shown to improve soil properties such as aggregation, water-holding capacity, hydraulic conductivity, bulk density, the degree of compaction, fertility and resistance to water and wind erosion (Carter and Stewart, 1996; Franzluebbers, 2002; Zebarth et al., 1999). Combined use of organic and inorganic sources of nutrients is essential to maintain soil health and to augment the efficiency of nutrients (Lian, 1994). Three primary nutrients in fertilizers are nitrogen, phosphate, and potassium. According to Perrenoud‘s report (1990), most authors agree that N generally increases crop susceptibility to pests and diseases, and P and K tend to improve plant health. It has been reported that tomato is a heavy feeder of NPK (Hebbar et al., 2004) and total nitrogen content is high in leaves in plants having a high occurrence of bitter fruits (Kano et al., 2001). Phosphorus is one of the most essential macronutrients (N, P, K, Ca, Mg, S) required for the growth of plants, and the deficiency of phosphorus will restrict plant growth in soil (Son et al., 2006). However, the excessive fertilization with chemically synthesized phosphate fertilizers has caused severe accumulation of insoluble phosphate compounds in farming soil (Omar, 1998), which gradually deteriorates the quality as well as the pH of soil. Different fertilization treatments of a long-term field experiment can cause soil macronutrients and their available concentrations to change, which in turn affects soil micronutrient (Cu, Fe, Mn, Zn) levels. Application of appropriate rates of N, P and K fertilizers has been reported to be able to increase soil Cu, Zn and Mn availabilities and the concentrations of Cu, Zn, Fe and Mn in wheat (Li, et al., 2007). It has been also reported that higher rates of fertilizers suppress microbial respiration (Thirukkumaran and Parkinson, 2000) and dehydrogenase activity (Simek et al., 1999). Recently, greater emphasis has been placed on the proper handling and application of agricultural fertilizers in order to increase crop yield, reduce costs and minimize environmental pollution (Allaire and Parent, 2004; Tomaszewska and Jarosiewicz, 2006). Hydroponics is a plant culture technique, which enables plant growth in a nutrient solution with the mechanical support of inert substrata (Nhut et al., 2006). Hydroponic culture systems provide a convenient means of studying plant uptake of nutrients free of confounding and uncontrollable changes in soil nutrient supply to the roots. Thus, it is fit for test of fertilizing ability of liquid fertilizers. The technique was developed from experiments carried out to determine what substances make plants grow and plant composition (Howard, 1993). Water culture was one of the earliest methods of hydroponics used both in laboratory experiments and in commercial crop production. Nowadays, hydroponics is considered as a promising technique not only for plant physiology experiments but also for commercial production (Nhut et al., 2004; Resh, 1993). The technique has been also adapted to many situations, from field and indoor greenhouse culture to highly specialized culture in atomic submarines to grow fresh vegetables for the crew (Nhut et al., 2004). Hydroponics provides numerous advantages: no need for soil sterilization, high yields, good quality, precise and complete control of nutrition and diseases, shorter length of cultivation time, safety for the environment and special utility in non-arable regions. Application of this culture technique can be considered as an alternative approach for large-scale production of some desired and valuable crops. In this study, a large-scale biodegradation was successfully carried out for three days in a 1-ton reactor using the FMW generated from fish-meal manufacturing processes, and the

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properties of the biodegraded FMW, such as phytotoxicity, amino-acid composition, concentrations of major and noxious components, and fertilizing ability on hydroponic culture plants were determined to examine the suitability of the biodegraded FMW as a fertilizer.

MATERIALS AND METHODS Microorganisms and Media The mixed microorganisms used in this study comprised seven microorganisms: Bacillus subtilis, Bacillus licheniformis, Brevibacillus agri, Bacillus coagulans, Bacillus circulans, Bacillus anthracis and Bacillus fusiformis. They were potential aerobically-degrading bacteria, which were isolated from commercial good-quality humus and from compost and leachate collected at three different sites of composting plants (Kim et al., 2007). There was no potential bacterial antagonist among them. Each pure culture was maintained on 1.5% Nutrient agar plate at 4until used, and transferred to a fresh agar plate every month. At the same time, the potential degrading ability of each pure culture was also checked on 1% skim milk agar, 3.215% spirit blue agar, starch hydrolysis agar (5 g∙l-1 of beef extract, 20 g∙l-1 of soluble starch, 10 g∙l-1 of tryptose, 5 g∙l-1 of NaCl, and 15 g∙l-1 of agar, pH 7.4) and cellulose agar (10 g∙l-1 of cellulose powder, 1 g∙l-1 of yeast extract, 0.1 g∙l-1 of NaCl, 2.5 g∙l-1 of (NH4) -1 -1 -1 2SO4, 0.25 g∙l of K2HPO4, 0.125 g∙l of MgSO4∙7H2O, 0.0025 g∙l of FeSO4∙7H2O, 0.025 g∙l-1 of MnSO4∙4H2O, and 15 g∙l-1 of agar, pH 7.2), respectively. All agar plates were incubated at 450C until change of color or a clear zone around each colony appeared.

Liquid-Fertilization of FMW A large-scale biodegradation of the original FMW was carried out in a 1-ton reactor for its liquid-fertilization. The inside of the reactor was sterilized by hot steam for 30 min, and then 600 l of the FMW obtained from a fish-meal factory was added into the reactor by a peristaltic pump under the steaming. The FMW was characterized as: 115,000±13,000 mg∙l-1 of chemical oxygen demand- dichromate (CODCr), 15,400±1,300 mg∙l-1 of total nitrogen (TN), 68,900±7,600 mg∙l-1 of five days biological oxygen demand (BOD5), 2,800±600 mg∙l-1 of NH4+-N, 0 mg∙l-1 of NO3--N and 0 mg∙l-1 of NO2--N. The salt concentration and pH of the FMW were 0.6±0.1% and 6.5±0.2, respectively. After the steaming was quit, the reactor was placed until temperature dropped down to 450C. Then, 30-l liquid broth of seven microorganisms (on the same ratio of cell mass) grown in exponential phase of growth were seeded into the reactor. For faster biodegradation, the inoculated cells were previously acclimated for two days in the original FMW under an aerobic condition. The bioreactor was operated at 42+40C, and air was supplied from two blowers (6.4 m3∙min-1 of capacity) into the reactor through ceramic disk-typed diffusers. The aeration rate was 1,280 l∙min-1. Ten-fold diluted ‗Antifoam 204‘ was used when severe foams occurred during biodegradation. Samples from the reactor were collected periodically. The concentrations of dissolved oxygen (DO), CODCr and TN were measured with oxidation-reduction potential (ORP) and pH.

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Seed Germination Test According to the method of Wong et al.(2001), seed germination tests for 50-, 100-, 250-, 500-, and 1,000-fold diluted final broths, which were taken from the biodegradation of original FMW in a 1-ton reactor, were carried out against control in order to evaluate the phytotoxicity of the biodegraded FMW. Two commercial fertilizers, C-1 (A-company, Incheon, Korea) and C-2 (N-company, Kimhae, Korea), frequently used in Korea were also tested for the comparison of their phytotoxicity. Five milliliters of each sample were pipetted into a sterile petri dish lined with Whatman #1 filter paper. Ten cress (Lepidium sativum) seeds were evenly placed in each dish. The plates were incubated at 25 ºC in the dark at 75% of humidity. Distilled water was used as a control. Seed germination and root length in each plate were measured at 72 h. The percentages of relative seed germination (RSG), relative root growth (RRG) and germination index (GI) after expose to the sample were calculated as the following formula (Zucconi et al., 1981; Hoekstra et al., 2002):

RSG (%) =

Number of seeds germinated in the biodegraded FMW Number of seeds germinated in control

RRG (%) =

Mean root length in the biodegraded FMW Mean root length in control

GI (%) =

× 100

× 100

RS G× RRG 100

Hydroponic Culture To test the fertilizing ability of the biodegraded FMW produced in a 1-ton reactor, a hydroponic culture system was applied to cultivate red bean and barley in a mini-hydroponic culture pot (5×12×8 cm3) against control. Tests were carried out on the biodegraded FMW at various dilutions (50, 100, 250, 500 and 1,000-fold). The tests were also carried out on 1,000fold diluted commercial-fertilizers in order to compare with the fertilizing ability of the biodegraded FMW. The hydroponic culture pot was composed of a glass vessel and a plastic screen inside. In each pot, ten seeds of red bean or twenty-five seeds of barley were put on top of the plastic screen, respectively, and approximately 300-ml solution of the biodegraded FMW at various dilution ratios was filled underneath the plastic screen. After set-up of the hydroponic culture system, each pot was initially covered with aluminum foil to keep seeds in dark before seed germination. After seed germination, the pot placed by the window all day long to provide the necessary sunlight for plant‘s growth. Day and night temperatures of air were maintained at 22±20C and 18±2℃ by natural ventilation and heating. Water temperature was 15±30C, and the relative humidity in the room was 60% on the average. The fertilizer solution was refreshed every four days, and the seeds were soaked all the time. After seed

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germination, roots grew through the pore of the plastic screen. The growth of plants was observed periodically, and height, thickness of stem, number of leaf, and length of leaf of each plant were measured.

Analyses The concentrations of cations (NH4+) and anions (NO2- and NO3-) were estimated by IC (Metrohm 792 Basic IC, Switzerland). The columns used in these analyses were Metrosep C2-150 and Metrosep Supp 5-150 for cation and anion, respectively. The concentrations of CODCr and TN concentrations were analyzed by the Water-quality Analyzer (Humas, Korea). The concentration of BOD5 was analyzed by the OxiDirect BOD-System (Lovibond, Germany). The composition of amino acids, and concentrations of major and noxious components in biodegraded FMW and commercial liquid-fertilizers were analyzed at Science Lab Center Co., Ltd (Daejeon, Korea) by our request.

RESULTS AND DISCUSSION Liquid-Fertilization of FMW To industrialize the biodegraded FMW as liquid-fertilizer, data obtained in laboratory equipment should be transferred to industrial production. Problems of scale-up in a bioreactor are associated with the behavior of liquid in the bioreactor and the metabolic reactions of the organisms. Transport limitation is considered as one of the major factors responsible for phenomena observed at large-scale. For this reason, a large-scale FMW biodegradation were attempted in a 1-ton bioreactor, and the result is shown in Figure 1. As shown in Figure 1, DO level in the reactor started to decrease after 5 h. This implies that active biodegradation of FMW took placed after a lag phase by the mixed microorganisms used in this study, since oxygen consumption has been known to be a general index of microbial metabolism (Tomati et al., 1996). During two days biodegradation, the DO level was maintained over 2 mg∙l-1, but it decreased further thereafter. The final DO level was 1.25 mg∙l-1. In aerobic processes, oxygen is a key substrate and a continuous transfer of oxygen from the gas phase to the liquid phase is decisive for maintaining the oxidative metabolism of the cells because of its low solubility in aqueous solutions. Generally, it has known that DO level in a bioreactor should be maintained over 1 mg∙l-1 for aerobic fermentation (Tohyama et al., 2000). Therefore, it would seem that the supply of oxygen into the 1-ton reactor met the demand of oxygen by the microorganisms during the biodegradation. The pH was 6.99 at the beginning and decreased down to 6.15 after 20 h. Then the pH was recovered slowly and its final value was 6.86. In our previous study (Kim et al., 2008), pH had a tendency to increase during the biodegradation because of insufficient supply of oxygen. These results imply that microorganisms had different metabolism, which was dependent on the availability of dissolved oxygen. The value of ORP started at 18.2 mV. The value of ORP decreased rapidly after 45 h and the final value reached to 0.6 mV. The decrease in the ORP value resulted from the decrease in DO level. During the biodegradation, the values of ORP maintained positive, and a strong unpleasant smell (mainly a fishy smell)

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225

remarkably disappeared in the end. It seemed that a complete aerobic biodegradation might take place to a certain extent with maintenance of ORP in a positive range, since unpleasant odor can be easily produced under incomplete aerobic biodegradation (Zhang et al., 2004). 40 8.5

4

20

2

1

7.5

-20

7.0

-40

ORP (mV)

0

3

pH

DO (mg.l-1)

8.0

-60

6.5

-80 6.0 120000

18000

-1

CODcr (mg.l-1)

14000

TN (mg.l )

16000

100000

80000

12000 10000

60000 8000 6000

40000

4000 0

10

20

30

40

50

60

Time (h) Figure 1. Changes of ORP( ▣ ), DO( ▲ ), pH( ● ), CODcr( ◇ ) and TN( ▼ ) during the biodegradation in a 1-ton reactor.

From all the results, maintenance of DO level during biodegradation was found to be very important and ORP could be a key parameter to operate biodegradation of FMW in a large-scale. The ORP was reported to be used as a controlling parameter for regulation of sulfide oxidation in anaerobic treatment of high-sulfate wastewater (Khanal and Huang, 2003), and on-line monitoring of ORP has been proved to be a practical and useful technique for process control of wastewater treatment systems (Guo et al., 2007; Yu et al., 1997). As a result, process optimization is required in a large-scale operation, especially aeration rate in this case. After 52 h of biodegradation, the concentrations of CODCr and TN in original FMW reduced to 25,100 and 4,790 mg∙l-1, respectively. The removal percentages of CODCr and TN

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were 79.4 and 73.4%, respectively, with slight decrease of CODCr/TN ratio from 6.8 to 5.2. It has been known that the COD/N ratio may influence biomass activity, and therefore on the metabolic pathways of organic matter utilization (Ruiz et al., 2006). Based on this information, the cell activity and metabolic pathways of the mixed microorganisms might be maintained somewhat with their mutualism during the biodegradation.

Properties of Biodegraded FMW as Fertilizer Since the FMW contains various compounds potentially useful for diverse plants, an attractive application is its use as a fertilizer; however, this could have limitations due to some toxic characteristics of the waste. For this reason, it is necessary to prove the non-toxic properties and fertilizing ability of the final biodegraded FMW.

Phytotoxicity of Biodegraded FMW Organic matters hold great promise due to their local availability as a source of multiple nutrients and ability to improve soil characteristics. Sufficient aeration promotes the conversion of the organic matters into nonobjectionable, stable end products such as CO2, SO42-, NO3-, etc. However, an incomplete aeration may result in accumulation of organic acid, thus giving trouble to plant growth if the fertilizer is incorporated into the soil (Jakobsen, 1995). The phytotoxicity of the biodegraded FMW produced in a 1-ton reactor was examined at various dilution ratios, and compared with those of two commercial fertilizers (Figure 2).

Figure 2. Percentages of germination index (GI) at various dilution ratios of the biodegraded FMW produced in a 1-ton reactor. The results are compared to those of 1,000-fold diluted commercial fertilizers, C-1 and C-2.

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227

As shown in Figure 2, the values of GI had a tendency to increase with increase of dilution ratio of the biodegraded FMW. The GI values of the biodegraded FMW were less than 20% at 50- and 100-fold dilution with low root elongation. The reduction in values of GI indicates that some characteristics existed had an adverse effect on root growth. This may be attributed to the release of high concentrations of ammonia and low molecular weight organic acids (Fang and Wong, 1999; Wong, 1985), since cress (Lepidium sativum) used in this study is known to be sensitive to the toxic effect of these compounds (Fuentes et al., 2004). At 250fold dilution, the GI value of the biodegraded FMW was close to 50%. A GI value of 50% has been used as an indication of phytotoxin-free compost (Zucconi et al., 1985). According to this GI criterion, the biodegraded FMW required more than 250-fold dilution to reach stabilization of the organic matter to maintain the long-term fertility in soil. At 1,000-fold dilution, the GI value was found to be close to 90%. This GI value was compared with those of two commercial fertilizers at the same dilution, since 1,000-fold diluted liquid fertilizer was used in horticulture for general purpose. The GI value of the biodegradative FMW was much better than that of C-1 and comparable to that of C-2. This result implies that the biodegradation of FMW in a 1-ton reactor was successfully carried out, and thus the development of a liquid-fertilizer from the FMW was feasible.

Composition of Amino Acids of Biodegraded FMW Amino acids are an essential part of the active fraction of organic matter in a fertilizer. The growth of plants depends ultimately upon the availability of a suitable balance of amino acids, and their composition might also be used as a means of assessing biodegradation. The success of the scale-up process for the production of liquid fertilizer from the FMW can be verified by determining the composition of amino acids. From this point of view, it is necessary to analyze the amino-acid composition of the biodegraded FMW produced in a 1ton bioreactor. The analytical result was tabulated in Table 1 with those of two commercial fertilizers. The level of total amino acids was 5.60 g·100g sample-1, which was a little bit better than that produced in our previous study (Kim and Lee, 2008). The higher content of amino acids is probably due to the higher degree of mineralization of FMW, which indicates release of more nutrients available for plants. However, the level of total amino acids in the biodegraded FMW was lower, compared with those in two commercial fertilizers. This implies that mineralization of FMW in a 1-ton reactor was not as great as that in a lab-scale reactor (Bylund et al., 1998; Xu et al., 1999). Especially, the levels of aspartic acid, alanine and lysine were low, whereas those of proline and glycine were relatively high. The difference may be due to the composition of the original FMW, since it was found to be dependent on the nature of the raw material processed in the factory (Kim et al., 2007). Moreover, the composition of sulfur-containing amino acids, cysteine and methionine, was relatively high in the biodegraded FMW. It has been reported that the sulfur-containing amino acid, methionine is a nutritionally important essential amino acid and is the precursor of several metabolites that regulate plant growth (Amir et al., 2002).

Joong Kyun Kim and Geon Lee

228

Table 1. Comparison of amino-acids composition of the biodegraded FMW with those of commercial fertilizersa Amino acid

Source of liquid fertilizer Biodegraded FMW

a

b

Commercial C-1

Commercial C-2

Aspartic acid

0.49

0.90

0.58

Threonine Serine

0.18 0.21

0.23 0.21

0.21 0.23

Glutamic acid

0.78

2.96

0.89

Proline Glycine Alanine

0.50 1.06 0.60

0.09 0.31 1.00

0.61 1.25 0.98

Valine

0.15

0.39

0.24

Isoleucine

0.14

0.28

0.13

Leucine Tyrosine

0.24 0.07

0.42 0.17

0.26 0.05

Phenylalanine

0.19

0.22

0.18

Histidine

0.20

0.28

0.25

Lysine

0.29

1.54

0.53

Arginine

0.31

0.23

0.35

Cystine

0.04

n.d.c

0.04

Metionine

0.13

n.d.

0.01

Tryptophan

0.02

n.d.

0.02

Total

5.60

9.23

6.81 -1

composition of amino acids was based on dry weight (g•100g sample ). produced in a 1-ton reactor. c n.d. means ‗not detected‘. b

Concentrations of Major and Noxious Components in Biodegraded FMW It is very important to improve the utilization of fertilizer nutrients, since the growth of plants and their quality are mainly a function of the quantity of fertilizer. Organic matter affects crop growth and yield directly by supplying nutrients (Darwish et al., 1995). Combined use of organic and inorganic sources of nutrients is essential to augment the efficiency of nutrients (Lian, 1994). For this reason, concentrations of three primary nutrients (N, P, and K) in the biodegraded FMW were anaylized together with noxious components, and compared with those in commercial fertilizers. The results were tabulated in Table 2. The concentrations of N, P and K in the biodegraded FMW were 1.49, 0.28 and 0.41%, respectively, and were much lower than those in commercial fertilizers.

Aerobically Biodegraded Fish-Meal Wastewater as a Fertilizer

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Table 2. Comparison of concentrations of major and noxious components present in the biodegraded FMW with those present in commercial fertilizers Source of liquid fertilizer

Measurement

N, P, K (%)

Noxious Compounds (mg∙kg-1)

Biodegraded FMWa

Commercial C-1

Commercial C-2

N

1.49

4.83

3.80

P2O5

0.28

2.86

2.83

K2O

0.41

2.10

3.04

Pb

n.d.b

0.63

0.31

As

n.d.

0.88

0.36

Cd

n.d.

0.08

0.03

Hg

0.01

n.d.

n.d.

Cr

0.20

3.52

3.44

Cu

n.d.

3.24

2.24

Ni

n.d.

1.54

2.32

Zn

1.61

4.39

3.51

a

produced in a 1-ton reactor. n.d. means ‗not detected‘.

b

However, the concentrations of noxious components in the biodegraded FMW were much lower than those in commercial fertilizers. The noxious components, Pb, As, Cd, Cu and Ni were not detected, and the other components were much less than the standard concentrations provided by the law.

Hydroponic Culture on Biodegraded FMW The fertilizing ability of the biodegraded FMW at various dilution ratios was tested in a hydroponic culture system, which was applied to cultivate red bean and barley. The tests were also carried out on 1,000-fold diluted commercial-fertilizers simultaneously. Results of hydroponic culture of red bean on diluted biodegraded-FMW and commercial fertilizers were tabulated in Table 3. As seen in Table 3, elongation of root was slow after seed germination in all diluted FMW. However, roots elongated soon after development of root. The fastest growth was achieved at 100-fold dilution, the growth of which was comparable to that of 1,000-fold diluted commercial-fertilizers. This result was somewhat surprising because the GI test on the 100-fold diluted FMW showed low root elongation (Figure 2). The toxic effect of the 100-fold diluted FMW might be not severe on red bean, although it was sensitive on cress.

Joong Kyun Kim and Geon Lee

230

Table 3. Results of hydroponic culture of red bean on diluted biodegraded-FMW and commercial fertilizers Dilution of biodegraded FMW

Control (Water) Measurement

Time (day)

50-fold

100-fold

250-fold

Time (day)

Time (day)

Time (day)

2

5

8

12 14 2

5

8

12 14 2

5

12 14 2

5

8

Height (cm)

-

-

1.5 3.0 6.0 -

-

-

1.0 2.0 -

2.0 4.0 6.0 9.0 -

-

1.0 3.0 4.5

Thickness of stem (cm)

-

-

0.2 0.3 0.5 -

-

-

0.2 0.2 -

0.2 0.3 0.4 0.5 -

-

0.2 0.3 0.3

Number of leaf

-

-

-

-

2

-

-

-

1

-

-

-

2

-

-

-

1

Length of leaf (cm) -

-

-

-

3.0 -

-

-

1.0 2.0 -

-

-

1.5 4.0 -

-

-

1.0 1.5

1

8

Dilution of biodegraded FMW Measurement

Height (cm) Thickness of stem (cm) Number of leaf Length of leaf (cm)

500-fold

Time (day) 12 14 2

8

1,000-fold, C-1

1,000-fold, C-2

Time (day)

Time (day)

2

5

8

-

-

1.0 3.0 4.0 -

1.0 2.0 4.0 5.5 1.0 4.0 4.5 5.0 8.0 -

2.0 3.0 4.0 6.0

-

-

0.2 0.3 0.4 -

0.2 0.2 0.3 0.4 0.2 0.3 0.3 0.4 0.5 -

0.2 0.3 0.4 0.5

-

-

-

-

-

1 2 1.0 1.5 -

5

2

Commercial fertilizer

1,000-fold

Time (day)

2

12 14

-

12 14 2

1 2 1.0 2.0 -

5

-

8

12 14 2

-

2 2 1.5 4.5 -

5

8

-

12 14

2 2 1.0 3.0

Table 4. Results of hydroponic culture of barley on diluted biodegraded-FMW and commercial fertilizers Dilution of biodegraded FMW

Control (Water) Measurement

50-fold

Time (day)

Time (day)

-

1.7 5.8 8.3 8.5 -

1.2 2.6 4.4 5.2 -

2.1 4.1 6.5 7.5 -

2.4 4.3 6.7 7.6

Thickness of stem (cm)

-

0.1 0.2 0.2 0.2 -

0.1 0.2 0.2 0.2 -

0.1 0.2 0.2 0.2 -

0.1 0.2 0.2 0.2

Number of leaf

-

1

1

1

1

2

2

-

8

2

12 14 2

2

2

-

0.7 1.3 3.1 3.5 -

5

8

Time (day)

Height (cm)

1.2 4.6 6.5 6.8 -

5

Time (day)

5

2

12 14 2

250-fold

2

Length of leaf (cm) -

8

100-fold

2

Height (cm) Thickness of stem (cm) Number of leaf Length of leaf (cm)

2

2

-

1.6 3.0 5.0 5.5 -

Dilution of biodegraded FMW Measurement

12 14 2

5

8

2

12 14

2

Commercial fertilizer

500-fold

1,000-fold

1,000-fold, C-1

1,000-fold, C-2

Time (day)

Time (day)

Time (day)

Time (day)

8

12 14 2

5

8

2

1.9 3.0 5.0 5.6

2

5

-

2.9 5.5 7.8 8.1 -

1.5 3.9 5.6 6.3 -

12 14 2

5

2.8 5.6 7.1 7.7 -

8

12 14 2

1.8 4.8 8.6 9.0

-

0.1 0.2 0.2 0.2 -

0.1 0.2 0.2 0.2 -

0.1 0.2 0.2 0.2 -

0.1 0.2 0.2 0.2

-

1 2 2 2 2.4 4.2 5.7 6.1 -

1 2 2 2 0.9 2.7 3.8 4.2 -

1 2

1 2 2 2 1.3 3.8 6.6 7.0

2 2 2 4.3 5.3 5.7 -

5

8

12 14

Aerobically Biodegraded Fish-Meal Wastewater as a Fertilizer

231

In Figure 3, the change of red bean‘s growth from the seed at 100-fold dilution is shown against control. Germination from the seed was observed at day 8 with the 100-fold diluted FMW, whereas the germination was observed at day 12 in control. After 14 days cultivation, much better growth of red bean was observed with the 100-fold diluted FMW, compared with that in control. At the same dilution of 1,000-fold, growth of red bean cultivated on the biodegraded FMW was not as good as that cultivated on commercial fertilizers. A decreasing order of growth in the culture of red bean was C-1 > C-2 > biodegraded FMW. According to Perrenoud‘s report (1990), N, P and K are primary nutrients and considered to improve plant health. The deficiency of phosphorus restricts plant growth in soil (Son et al., 2006), although the excessive fertilization with chemically synthesized phosphate fertilizers has caused severe accumulation of insoluble phosphate compounds (Omar, 1998). Therefore, relatively lower growth of red bean on the biodegraded FMW might be due to the shortage of NPK components (Table 2).

Figure 3. Results of hydroponic culture of red bean in control (A) and 100-fold diluted FMW (B). Each figure shows the growth of red bean from the seed along the cultivation time. (1) day 1; (2) day 2; (3) day 5; (4) day 8; (5) day 12; (6) day 14 and (7) roots on day 14.

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Joong Kyun Kim and Geon Lee

Growth indexes of barley during hydroponic culture were measured on the biodegraded FMW at various dilution ratios, and the measurements were compared with those of commercial fertilizers (Table 4). Even though the growth in root and stem of barley at 50-fold dilution was lower than that of control, it had a tendency to improve with increase of dilution ratio of the biodegraded FMW. The best growth was achieved at 500-fold dilution in which the growth was seen evenly in root and stem of barley (Figure 4).

Figure 4. Results of hydroponic culture of barley in control (A) and 500-fold diluted FMW (B). Each figure shows the growth of barley from the seed along the cultivation time. (1) day 1; (2) day 2; (3) day 5; (4) day 8; (5) day 12; (6) roots on day 12 and (7) day 14.

From the GI test, the biodegraded FMW at 500-fold dilution was found to be phytotoxinfree (Figure 2). With the 500-fold diluted FMW, germination from the seed was observed at day 5, and the same result was observed in control. After 14 days cultivation, however, faster growth of barley was observed with the 500-fold diluted FMW, compared with that in control. This growth of barley with the 500-fold diluted FMW was comparable to that cultivated on 1,000-fold diluted commercial-fertilizers. However, the effect of dilution on barley was not as sensitive as that on red bean.

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233

CONCLUSION Seven thermophilic microorganisms, which had proteolytic, lipolytic and carbohydratedegrading functions, were used in order to reutilize fish-meal wastewater (FMW). With coexistence of the seven microorganisms, a lab-scale aerobic biodegradation of FMW were successfully achieved, and its data were transferred to a 1-ton bioreactor. Although the level of DO was decreased during the large-scale biodegradation of FMW, it was maintained over 1.25 mg∙l-1. The decrease in the level of DO resulted in decrease in ORP value. However, the ORP values were maintained positive, and a strong unpleasant smell remarkably disappeared in the end. After 52 h of biodegradation, the concentrations of CODCr and TN in original FMW reduced to 25,100 and 4,790 mg∙l-1, respectively with slight decrease of CODCr/TN ratio. Properties of the biodegraded FMW were determined to examine the suitability of the biodegraded FMW as a fertilizer. The result of the phytotoxicity test showed that the biodegraded FMW required more than 250-fold dilution to reach stabilization of the organic matter to maintain the long-term fertility in soil. At 1,000-fold dilution, the GI value was found to be close to 90%, which was comparable to those of commercial fertilizers. The level of total amino acids in the biodegraded FMW was 5.60 g·100g sample-1, which was lower than those in two commercial fertilizers. Especially, the levels of aspartic acid, alanine and lysine were low, whereas those of proline and glycine were relatively high. Moreover, the composition of sulfur-containing amino acids, cysteine and methionine, were relatively high in the biodegraded FMW. The concentrations of N, P and K in the biodegraded FMW were 1.49, 0.28 and 0.41%, respectively, which were lower than those in commercial fertilizers. However, the concentrations of noxious components in the biodegraded FMW were much lower than the standard concentrations. The fertilizing ability of the biodegraded FMW at various dilution ratios was tested in a hydroponic culture system. The fastest growth in culture of red bean was achieved at 100-fold dilution, the growth of which was comparable to those of 1,000-fold diluted commercial-fertilizers. In the hydroponic culture of barley, the best growth was achieved at 500-fold dilution in which its growth was seen evenly in root and stem. This growth of barley with the 500-fold diluted FMW was comparable to that cultivated on 1,000-fold diluted commercial-fertilizers. From all above results, it was concluded that a large-scale biodegradation of FMW was successful and the properties of FMW were acceptable as a fertilizer. Consequently, FMW could be a potential industrial resource for liquid-fertilizer production.

ACKNOWLEDGMENTS This research was supported by a grant (B-2004-12) from Marine Bioprocess Research Center of the Marine Bio21 Center funded by the Ministry of land, Transport and Maritime Affairs, Republic of Korea.

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In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 7

EQUITY OF ACCESS TO PUBLIC PARKS IN BIRMINGHAM, ENGLAND Andrew P. Jones, Julii Brainard, Ian J. Bateman and Andrew A. Lovett Centre for Social and Economic Research on the Global Environment, The School of Environmental Sciences, University of East Anglia, Norwich, Norfolk, UK.

ABSTRACT Provision of public parks has long been advocated as an equalising measure between different elements of society. This study assesses equity of park provision for different ethnic and income-status populations in the urban area of Birmingham in central England. Parks in Birmingham were categorized into a two group typology of green areas suited for more solitary and passive activities (amenity parks) or open spaces designed more for informal sports or other physical and group activities (recreational parks). Using a geographical information system, measures of access to these green spaces were computed for populations of different ethnicities and levels of material deprivation, derived from data from the 2001 UK Census and the 2004 Index of Multiple Deprivation. Distance-weighted access scores were calculated and compared for five population groups ranked by relative deprivation, and for five ethnic groups; Bangladeshis, blacks, Indians, Pakistanis and whites. Statistical analysis found that there were strong disparities in access with respect to deprivation whereby the most income-deprived groups were also the most deprived with regard to access to public parks. There was little evidence of unequal access between ethnic groups. The implications of these findings are discussed.

Keywords: Environmental equity, accessibility, deprivation, ethnicity, urban parks.



Author for correspondence: Dr Andrew P Jones, School of Environmental Sciences, University of East Anglia, Norwich, Norfolk, NR4 7TJ. Email: [email protected] Tel: 01603 593127, Fax: 01603 591327.

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INTRODUCTION The establishment of urban public parks has its origins in the social ideal of providing pleasant open space that is free at the point of access, and as such these places have always been intended to be equally accessible for different social groups. Public urban parks were originally established, in part, to provide a place where rich and poor could meet on an equal footing (Young 1996). It is therefore somewhat surprising that relatively few studies concerned with environmental social justice have examined the provision and accessibility of open public space for different social groups. The exact definition of ‗equity of access‘ is difficult to make, and often context specific. Extended discussion of the general concept of equity is given by Harding and Holdren (1993). Crompton and Wicks (1988), Marsh and Schilling (1994) and Wicks and Backman (1994) discuss different approaches to defining or ensuring equity in a planning context. Crompton and Lue (1992) and Nicholls (2001) consider how equity may be defined with relation to urban park usage, and which definitions are likely to be practicable and preferred by the public. We define equity simply as equal opportunity between social groups. Previous research on equity of access to public parks is limited (see Wolch et al. 2005; Estabrooks et al. 2003; Nichols 2001; Talen 1997), and we are not aware of any studies for either a large city or for places outside the USA. Some research from the USA (Payne et al. 2002; Tinsley et al. 2002) reported that ethnic minorities tend to travel further to surveyed parks, but did not question why this might be. In the UK, DTLR (2001) note that ethnic minorities tend to visit urban parks less often than white people. The question arises as to whether this is partly because non-white communities find it more difficult to travel to urban parks. It is also important to consider whether access to parks is reduced for economically or socially deprived populations. The Greater London Authority acknowledge that socially deprived areas in London often have relatively poor access to green spaces (GLA, 2001), and areas with less green space seemed to have more benefit claimants and overcrowded households (GLA, 2003). The relative dearth of work in the UK on the issue of access to urban parks is perhaps surprising when it is considered how much they are a very visible and publicly-owned environmental good. The work reported here focuses on differential access between ethnic minorities and socio-economic groups with respect to urban parks. Our study area is the city of Birmingham, where we had detailed information on parks, demographic statistics and the transport network. Birmingham is located in central England (see Figure 1) and is the second largest city in the UK with just under 1 million residents in the 2001 Census. Approximately 28% of the city population are non-white. In this research we examine equity of access in terms of neighbourhood ethnicity and socio-economic deprivation.

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MATERIALS AND METHODS Data Sources Our analysis focused on urban rather than regional parks provision, and hence the boundaries of the city of Birmingham in central England defined the study area. There were 977,087 residents in the city at the time of the 2001 Census.

Figure 1. Amenity and recreational parks in the study area, with visitor survey parks marked (SF=Summerfield, PHPF=Perry Hall Playing Fields, KHRG=Kinghurst Recreation Grounds).

The project benefited from data derived from a wide variety of sources. These included the 2001 UK Census, and an existing database of Ordnance Survey (OS) digital data encompassing the city of Birmingham and the areas immediately beyond the city boundaries.

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The Ordnance Survey dataset included property boundary features (OS Landline) and a detailed road network (OSCAR). This was augmented with information on the location and type of parks and green spaces that we obtained from a number of sources. Social surveys were also undertaken to collect information on park usage. This information was used to model the relationship between frequency of visits and distance to park entrances. Our analysis made use of the ArcGIS Geographical Information System (GIS) package. The application of these various data sources in the research is discussed below.

Birmingham Road Network We presumed that people would travel to parks via the city road network, either on foot, by bicycle, or in a vehicle. The Ordnance Survey OSCAR digital map database was used to extract road centrelines for the entire city and immediately adjacent areas. The data were produced in early 2000, and hence coincide well with the 2001 Census.

Parks in Birmingham What qualifies as a ―park‖ is a subjective judgement. Examples of different typologies of green and public open spaces are found in Kit Campbell Associates (2001) and DTLR (2002a and 2002b). These typologies tend to include such open spaces as allotments, canal banks or public squares. In our analysis we have consider only spaces that would be used for a particular set of purposes, namely casual recreation. Parks and other green spaces within Birmingham and up to 500m outside of the city boundaries were located with the use of a city atlas (Geographers‘ A-Z Map Co., 2000). Precise boundaries for each park area were located and extracted from the Ordnance Survey digital Landline database. Following the labelling in the A-Z atlas, four categories of park or green space were distinguished: ·

·

· ·

Amenity parks refers to leisure gardens, country parks, wildlife centres, woods, fish ponds, public greens and commons, areas labelled as simply as ‗park‘, and green spaces not allocated for other specific undeveloped purposes (such as canal banks, paved public squares or allotments). Recreational parks are those designated as ‗recreation grounds‘, ‗playgrounds‘, ‗sports grounds‘ and ‗playing fields‘ not attached to any specific school or institution. Specialist sports grounds denotes areas labelled as used for a specific sport, such as tennis courts, hockey pitches, cricket grounds and golf courses. Other facilities included cemeteries, school grounds and college or university grounds.

We consulted both the Birmingham Open Spaces Forum and Birmingham City Council in devising this typology and both organisations agreed with the manner by which we had classified parks. Table 1 provides summary statistics for each type of park. Amenity and recreational parks provide the majority of park provision in the city. Specialist sports grounds

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and ‗other‘ parks were found to not be generally of open access to nearby communities. We therefore focus the rest of our analysis on the former two types of green space. Figure 1 shows amenity and recreational parks in the study area. The large scale of the OS data made it necessary to locate park entrances precisely, and hence the locations of access points were identified for each park included in our analysis, and these were digitised into the GIS. The A-Z map and the Landline database were jointly consulted for this purpose. These sources indicate where parks border roads and where breaks in surrounding buildings exist to apparently allow pedestrian access to the parks, but they do not depict the presence of fencing. Moreover, we had no easy way of detecting informal access routes and points (i.e., cut-throughs over rough ground, or holes in fencing). Table 1. Main types of park included in each typological category General Category Amenity

Including: Public open space/commons/greens Ponds and reservoirs Nature reserves/woods Country parks Park farms Leisure gardens Public playgrounds School rough Other parks not labelled as sporting areas

Number 59 50 31 5 3 2 2 1 89 2821 ha

Recreation grounds Playing fields Sports grounds Paddling pool

105 98 36 1 1308 ha

Golf courses Bowling greens/pavilions Cricket grounds Stadia Tennis courts Football grounds Rugby grounds Hockey pitch Leisure centre

24 20 13 8 7 4 3 1 1 1175 ha

Total area General Recreation

Total area Specialist Recreation

Total area Census area boundaries and 2001 population.

The Office of National Statistics (ONS) supplied data on population totals, populationadjusted geographical centroids (centre points), and geographical boundaries of Output Areas (OAs). These are the smallest geographical units in the 2001 Census. There were 3127 OAs within Birmingham city boundaries, containing an average of 312 persons and 125 households each. Output Area centroids and population totals for them were input to the SurfaceBuilder computer programme (Martin 1996). SurfaceBuilder generates a surface depicting the estimated number of persons resident in regularly spaced square areas, or cells.

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A cell resolution of 20x20m was used. Because the resulting surface is partially a function of original centroid locations, the resultant cell values are an imperfect measure of actual population locations. Furthermore, the fine scale does not necessarily provide an accurate measure of the number of residents in each individual cell. However, the production of the surface enabled the easy estimation of accessibility measures that were weighted by relative population sizes, and it was employed here in the estimation of population accessibility to parks.

Deprivation and Ethnicity Information on material deprivation and ethnicity was collected at the scale of Lower Layer Super Output Areas (LSOAs). These contain small groups (typically 4-6 in number) of contiguous OAs with similar social profiles. There are 641 LSOAs in Birmingham. The LSOA level is appealing because of the availability of the Index of Multiple Deprivation (IMD2004; ODPM 2004) for this geographical scale. The IMD2004 comprises of seven domains describing different types of deprivation including income, access to housing and services, education, employment, health and disability, skills and training, living environment, and crime. For this research only the income domain of the IMD2004 (subsequently abbreviated to Inc-IMD2004) was used. The income domain relates to data collected in 2001 and 2002 on the proportion of the population receiving various types of means-tested income support, including benefits and tax credits. Hence areas scoring more highly on Inc-IMD2004 are more materially deprived. From 2001 Census data, statistics were derived on the percentage population composition of various ethnic groups. The ethnic categorisations that we used (white, Bangladeshi, Pakistani, Indian, and black,) were identical to those reported in the Census, and are based on self-report. For the latter category we combined persons of Caribbean and African heritage who identified themselves as ―black‖ in the 2001 Census with persons of mixed white and black heritage.

Quantification of the Effect of Distance on Accessibility A number of factors will impact upon the decision to visit a park. Purpose of visit is likely to shape decisions regarding which park a person might visit, and both seasonality and time of day can also influence visit patterns (DTLR, 2002a; Scott, 1997). However, the principal consideration for many is likely to be travel distance. Hence, in order to adequately measure accessibility, information on the relationship between distance and park usage was required. We were unable to find previous research that provided information on distances that users in Britain travel to visit urban parks. We therefore conducted our own survey. Visitors to three parks in Birmingham were interviewed during the summer of 2001. Survey parks were chosen by considering deprivation levels in the area around each. Census wards were categorised according to their levels of deprivation into the 33% most deprived, 33% least deprived, and the remaining 33%. One medium-size park was selected for survey in an area

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dominated by each deprivation tercile: Summerfield Park (most deprived), Perry Hall Playing Fields (least deprived) and Kingshurst Recreation Grounds (middle). In total, 117 individuals were interviewed (a minimum of 35 at each park) and asked for their mode of travel, the full postcode of their outset point, estimated one-way travel time to reach the park, the number of persons in their visitor party, and reasons for being in the park that day. Five respondents had to be excluded from subsequent analysis due to them providing incomplete information (such as an invalid postcode). The outset origin postcode supplied by each respondent was related to a 100m resolution reference (on the national grid system) using the Central Postcode Directory held at Manchester University. The road network distance between this origin and the nearest entrance to the park was then calculated within the GIS. Table 2 summarises the characteristics of each park and its visitors. Included are socioeconomic statistics for the origin areas from which visitors came. A trend is apparent where Summerfield visitors tend to come from the most deprived areas, and Perry Hall park users tend to originate from the least deprived areas. Otherwise, the most striking features are the much higher proportion of visitors from predominantly white neighbourhoods to Kinghurst Park, and an inverse association between the proportion of visitors travelling by car and the deprivation levels surrounding each park. Table 2. Summary statistics for the visitor survey locations

Deprivation tercile Origin Inc-IMD2004 score, 10th–90th %ile (median) Park size, ha Median (mean) party size Mode of travel: Number of motorists (%) Number of cyclists (%) Number of pedestrians (%) Travel distance: Median (mean) road distance in metres, all visitors Median (mean) road distance in metres, pedestrians only Maximum stated travel time amongst nonmotorists (mins) Maximum modelled travel distance amongst non motorists (km) Ethnic composition of neighbourhood % White, 10th–90th %ile (median) % Bangladeshi, 10th–90th %ile (median) % Indian, 10th–90th %ile (median) % Pakistani, 10th–90th %ile (median) % Black, 10th–90th %ile (median)

Summerfield

Kinghurst

Perry Hall

Most deprived 0.23-0.45 (0.39)

Middle 0.14-0.40 (0.27)

Least deprived 0.08-0.47 (0.13)

15.7 2 (3.37)

116 1 (2.26)

62.9 2 (2.36)

7 (20%) 0 (0%) 28 (80%)

11 (34%) 2 (5%) 25 (66%)

15 (38%) 3 (8%) 21 (54%)

311 (705)

420 (1232)

1420 (2194)

225 (395)

288 (814)

717 (923)

22

150

53

1.1

9.6

3.1

17.9-51.7 (42.0) 0-2.3 (1.1) 9.9-32.2 (19.2) 2.7-35.0 (13.9) 10.8-28.5 (18.6)

83.2-97.4 (94.1) 0-0 (0) 0-2.2 (0) 0-3.12 (0.5) 1.1-9.4 (4.5)

11.5-95.7 (36.7) 0-10.2 (1.01) 0-39.5 (16.7) 0-42.3 (6.5) 3.2-30.8 (12.1)

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The survey data were used to model the relationship between travel distances and frequency of visits. A model of the decay relationship between proximity and visit frequency was developed for all parks. This was used to generate data indicating the level of park accessibility from any single origin point in Birmingham. There are many possible ways to derive an accessibility index for an environmental amenity and facility such as public parks. Haynes et al. (2003) argued the case for a function to measure relative accessibility taking the form: P = C * exp (Decay_coefficient * Impedance) {1} where P=Potential or accessibility index C=Constant Decay_coefficient = the rate at which accessibility declines per unit of impedance Impedance = difficulty, usually measured in terms of travel time or distance A common way to calculate P would be to predict the number of visitor parties from origin zones as a per capita visitor rate. Unfortunately we did not know what percentage of the total number of visitors we surveyed at each park. In the absence of this information we used the number of visits reported by respondents during the preceding four weeks. This has the advantage of not requiring information about the total number of visitors to the park in any given period, yet visit frequency can be expected to decline with decreasing distance, and to reflect, in large part, ease of access. A distance decay relationship between journey origins and parks in Birmingham was derived by comparing reported frequency with calculated travel distance in the GIS. The relationship was generalised by grouping visitors into seven distance bands: 0-250m, 251500m, 501-750m, 751-1000m, 1001-1500m, 1500-2500m and 2500-3500m. Survey respondents who were only in a park because they were en-route to another location were excluded from this and further analysis. The average number of visits amongst respondents in each distance band is plotted against the average calculated travel distance in Figure 2. A strong and consistent decline is apparent in average visit frequency with increasing average distance. Using the functional form depicted in Equation 1, the average number of visits was regressed against travel distance for Birmingham parks visitors, to produce; P = 32.0085 * exp(-0.0006398 * distance) {2} The high R2 value obtained of 0.922 indicates a strong link between grouped visit frequency and distance between home origin and the nearest park entrance, and this relationship was used to model the way by which accessibility will decline with distance. Based on the results of the above modelling, three accessibility 'potential' surfaces were generated for recreational, amenity, and combined park areas. This process required the identification of both designated outset and destination points. Potential population journey origins were generated at 1420 road junctions throughout Birmingham. Destination points were taken to be park entrances. Visits are known to increase with park size, but the relationship between visit frequency and park size may not be linear. In a study of 516 urban parks in Perth, Australia, Giles-Corti et al. (2005) found that visit frequency was best

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predicted when they weighted their distance decay function by the natural logarithm of park size (in ha) raised to the power of 0.85, or Attractiveness=Distance_Decay_Function * log(size0.85)

{3}

―Attractiveness‖ as Giles-Corti et al. defined it equates to visit frequency in this work. We therefore allocated to each entrance a supply weighting equal to the area (in ha) of that park, raised to a power of 0.85 and then transformed by natural logarithm. For instance, Phoenix Park to the south of the city centre measures 1.58 hectares and is classified as entirely amenity park area. Phoenix has two entrances, each of which was given a supply weighting of 0.388811 (=log(1.580.85)). Very small parks less than 1 ha were given a supply weighting of 0.01 to prevent negative values being calculated. Only the nearest entrance for each origin was considered as an access point, and all of the supply weighting was applied to that.

No. visits in last 4 weeks

40

Average Number Of Visits in preceding four weeks

35 30 25 20 15 10 5 0 0

500

1000

1500

2000

2500

3000

3500

Distance (m)

Figure 2. Average number of visits in preceding four weeks plotted against average distance bands.

Within the GIS the distance in metres between each pair of origin and destination points was calculated. Distances below 110 m were reset to a value of 110, to prevent spuriously high potential being predicted very close to parks. 110m was chosen because of a natural break in the original survey data at that point. Next, an interaction score between each origin and destination was generated using the formula; SCORE = [32.0085 – exp(-0.0006398 * DISTANCE)] * log(HECTARES0.85)

{4}

Subsequently, scores were summed by origin, to yield a single value indicative of park accessibility for that origin. A triangulated irregular network (TIN) (Peuker et al. 1976) was generated to estimate access scores for locations between origin points. The TIN data were sampled on a regular 20 x 20m grid to create potential surfaces. To facilitate interpretation, the potential values were converted to Z-scores with a mean of zero and a standard deviation of one. The greater the

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index value, the better the access to parks for persons travelling from that location. The resulting values are mapped in Figure 3 for amenity and recreational parks, and Figure 4 for both park types combined. Figure 3 shows that the northern-central area and the southwest of the city are well-endowed with amenity type parks. There is a ring of high provision of recreational type parks around the city centre, whilst the city centre itself and the furthest suburbs, especially in the north, are relatively poorly provided with this type of park.

Figure 3. Calculated access scores for amenity (left) and recreational (right) parks in Birmingham.

Figure 4. Calculated access scores for amenity and recreational parks combined.

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Combing the two types of park (Figure 4) the north eastern suburbs and the city centre itself are most disadvantaged. The next step was to overlay these surfaces with LSOA boundaries and the population surface. This enabled us to calculate a population-weighted park access score for each individual LSOA.

RESULTS Equality of access to parks for different communities in Birmingham was examined with respect to both income deprivation and ethnic composition. In order to determine how the estimates of access were distributed across different populations, tests that compared the distributions of access scores were undertaken, as discussed below.

Ethnicity and Equity of Access to Public Parks The population of Birmingham was 70.35% white, 20.16% Asian/mixed Asian and 7.87% black/mixed black in the 2001 Census. To compare park access between these populations, the percentage of the total Birmingham population in each ethnic group was determined for each LSOA. For example, the LSOA labelled E01009276 had 166 persons recorded as black. This equates to 0.216% of the total 76,930 black persons residing inside the Birmingham city boundaries in 2001. Calculating this percentage of the total for each LSOA allowed us to determine what proportion of each ethnic group had particular parks access scores. Table 3 shows the median access scores for each ethnic group. There are no striking differences between ethnic groups for amenity parks access, although Pakistani populations have the best access. Median scores differ little. However, with respect to recreational or combined parks access, whites seem to have more advantage over other groups, especially Bangladeshis. The detailed distributions of park access scores for different ethnic groups were examined by plotting cumulative frequency distributions against access scores. The resulting plot for access to amenity parks is shown in Figure 5. The plots for access to recreational parks and both types combined were similar and are not reproduced here. Park access scores for LSOAs were divided into fifty categories. Each contains two percent of Birmingham‘s total population. The horizontal axis shows the upper limit of amenity or recreational access scores in each category. Table 3. Median parks access indices for ethnic groups in Birmingham.

Amenity Recreational Combined

Indian

Pakistani

Black

Bangladeshi

White

-0.052 0.069 0.145

0.107 0.109 0.193

-0.071 0.131 0.165

-0.101 -0.027 0.045

0.024 0.218 0.247

Andrew P. Jones, Julii Brainard, Ian J. Bateman et al.

248

Cumulative frequency (%) .

100 90 80 70 60 50 40 30

White Indian Pakistani Bangladeshi

20

Black

10

Amenity park access index 0. 46 0. 63 0. 80 1. 02 1. 24 1. 81

0. 32

0. 15

0. 00

.2 0

.0 8 -0

-0

.3 3 -0

.4 6 -0

.6 5

-0

.1 1

.8 8 -0

-1

-1

.4 6

0

Figure 5. Cumulative probability distributions for specified ethnic groups and access to amenity parks.

The vertical axis plots cumulative percentages. A perfectly straight diagonal line on the plots would indicate that the given range of potential scores occurred equally often across that ethnic group. This is because each category contains an equal proportion of the city population and the horizontal axis is non-linear. Lines that deviate from a perfect diagonal suggest a population displacement towards higher or lower access scores. Percentile lines bulging to the left of a perfect diagonal indicate a population group with relatively lower than average access, whilst lines pushed to the right suggest superior access. The greater the vertical gaps between the cumulative percentile lines, the more likely it is that an inequality is occurring between groups. A robust way to test for differences in the data in cumulative distribution plots is to use the Kolmogorov-Smirnov (KS) test (Connover, 1999). This test assesses the significance of the relative distance between the cumulative frequency lines. A two-sample KS test was used to examine the differences in population distribution between the various subgroups. Table 4 provides the resultant KS values, with the critical values at p=0.1, 0.05 and 0.01 levels. Table 4. Kolmogorov-Smirnov statistics for two-sample tests comparing cumulative probability distributions for ethnic groups and parks access Amenity White Indian Pakistani Black

Indian 0.0776

Pakistani 0.0776 0.0996

Black 0.0704 0.0448 0.1230

Bangladeshi 0.1296 0.0775 0.1572 0.0884

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Table 4 – Continued Recreational Indian 0.0701

White Indian Pakistani Black

Combined Amenity and Recreational Indian White 0.0610 Indian Pakistani Black

Pakistani 0.0823 0.0353

Black 0.0870 0.0535 0.0508

Bangladeshi 0.1375 0.0949 0.1153 0.0986

Pakistani 0.0514 0.0527

Black 0.0739 0.0694 0.0334

Bangladeshi 0.1381 0.0901 0.1010 0.1113

Critical KS values for p=0.1, 0.05 and 0.01 are 0.1725, 0.1923 and 0.2305 respectively.

The KS statistics confirmed the visual impression from the figures that there is no significant inequity between ethnic groups and their relative access to different categories of park type. The greatest differences occur between white and Bangladeshi populations, but these do not reach statistical significance.

Deprivation and Equity of Access to Public Parks Table 5 gives the median park access score for five deprivation groups (Inc-IMD2004 quartile groups and the most deprived decile) and by type of park. Figures 6 to 8 plot the cumulative percentiles of population in each Inc-IMD2004 quartile (plus the top decile) against amenity/recreational/combined parks access scores. Table 6 presents KS statistics for the data in the figures. The table and figures show that, for Amenity parks, there is some indication of disparities in access between the different deprivation groups, with the poorest population cohorts having worse access compared to other deprivation groups (p<0.01). For recreational parks, and both park types combined these disparities are much stronger with the most deprived population groups having significantly poorer access than the others. The middle two deprivation quartiles tended to have better accessibility than the other groups, with the most affluent quartile having comparatively average park access. Table 5. Median access scores for each income deprivation group, by park type

Amenity Recreational Combined

Quartile 1 (least deprived) -0.030 0.027 0.248

Quartile 2

Quartile 3

0.268 0.422 0.423

0 0.3 0.270

Quartile 4 (most deprived) -0.079 -0.220 -0.043

Decile 10 (10% most deprived) -0.148 -0.505 -0.268

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Table 6. Kolmogorov-Smirnov statistics for two-sample tests comparing cumulative probability distributions for Inc-IMD2004 and park access Amenity

Quartile 1 (least deprived) Quartile 2 Quartile 3 Quartile 4 (most deprived)

Quartile 2

Quartile 3

Quartile 4

0.1509

0.1214 0.1878*

0.1778* 0.2349*** 0.1086

Quartile 2

Quartile 3

Quartile 4

0.2573***

0.2261** 0.0968

0.2111** 0.4073*** 0.3239***

Quartile 2

Quartile 3

Quartile 4

0.2416***

0.1960** 0.1470

0.2347*** 0.3478*** 0.2438***

Decile 10 (10% most deprived) 0.2037** 0.2589*** 0.1545 0.0568

Recreational

Quartile 1 (least deprived) Quartile 2 Quartile 3 Quartile 4 (most deprived)

Decile 10 (10% most deprived) 0.4101*** 0.6574*** 0.5961*** 0.2853***

Combined Amenity and Recreational

Quartile 1 (least deprived) Quartile 2 Quartile 3 Quartile 4 (most deprived)

Decile 10 (10% most deprived) 0.4302*** 0.5806*** 0.5048*** 0.2610***

Amenity park access index

Quartile1 Quartile2 Quartile3 Quartile4 Decile10

-1 .4 6 -1 .0 4 -0 .7 1 -0 .4 6 -0 .2 8 -0 .1 2 0. 00 0. 20 0. 43 0. 63 0. 86 1. 17 1. 81

100 90 80 70 60 50 40 30 20 10 0

Cumulative frequency (%)

Critical KS values for p=0.1, 0.05 and 0.01 are respectively 0.1725 (*), 0.1923 (**) and 0.2305 (***).

Figure 6. Cumulative probability distributions for specified deprivation groups and access to amenity parks.

251

Quartile1 Quartile2 Quartile3 Quartile4 Decile10

0. 00 0. 18 0. 32 0. 50 0. 66 0. 90 1. 28 2. 06

.1 7 -0

.3 5 -0

.5 3 -0

.8 2

Recreational park access index

-0

-1

.5 6

100 90 80 70 60 50 40 30 20 10 0

Cumulative frequency (%)

Equity of Access to Public Parks in Birmingham, England

Quartile1 Quartile2 Quartile3 Quartile4 Decile10

0. 05 0. 23 0. 34 0. 48 0. 61 0. 82 1. 10 1. 76

.0 5 -0

.2 3 -0

.4 5 -0

.6 8

Combined park access index

-0

-1

.5 6

100 90 80 70 60 50 40 30 20 10 0

Cumulative frequency (%)

Figure 7. Cumulative probability distributions for specified deprivation groups and access to recreational parks.

Figure 8. Cumulative probability distributions for specified deprivation groups and amenity and recreational parks combined.

DISCUSSION Relatively little previous research has addressed the question of the differential access to green spaces amongst diverse communities residing in urban areas. Using a case study of the

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city of Birmingham England, this study has sought to go some way to rectify this deficiency by assessing the distribution of access to urban parks amongst a diverse urban population. Our analysis found only weak evidence of disparities in access between different ethnic groups in the population. However, we found very statistically significant indications of unequal access to public parks with regard to income deprivation, whereby the most deprived population had the poorest access to parks. Of course this cross sectional observation does not, in itself, provide an insight into the reasons why these disparities may exist. Pearce (2003) contended that environmental equity studies are often flawed because they ignore a fundamental aspect of capitalism, that the rich have the ability to pay for a nicer place to live. He took an economic perspective, arguing that equity could only be properly assessed with respect to how much each community paid for a benefit (or to avoid a disamenity), and whether their benefits or costs were commensurate with those payments. Pearce‘s arguments are supported by many North American studies (see Been and Gupta 1997; Lambert and Boerner 1997; Mitchell et al. 1999; Szasz and Meuser 2000; Yandle and Burton 1996), which concluded that apparently unfair pollution burdens usually result from economic not racist factors, particularly from a move-in of minority and poor social groups, due to lower house prices for instance, after an environmental nuisance was created. Indeed, it is quite possible that the residents of poorer communities in our study may receive other benefits which compensate for their poorer access to urban parks. It is also noteworthy that the most affluent populations did not have the best access, again suggesting that compensatory mechanisms, possibly associated with the availability of private land, were present. Nevertheless, it is important to note that the concept of equal access for all was one of the principles upon which the public parks movement was founded. It could be argued that, due to economic forces, improvements in park provision may only achieve temporary gains for otherwise disadvantaged populations. As the environment in an area improves, wealthier social groups may be attracted to it, making it difficult for the original disadvantaged social group to remain in the now improved area. These exclusionary processes may operate, for example, via rising housing costs or more general issues of social prejudice. This perspective ignores, however, the value of short and medium term gains in environmental quality in the lives of the original area inhabitants. Furthermore, house price rises due to improved environmental conditions may enable existing home-owners in the area to pass on more wealth to their children which is a permanent improvement for the next generation. As with any study, our results will in part be influenced by aspects of our study location and chosen methodology. Our findings are based on a detailed analysis undertaken in a socially and environmentally diverse, yet singular, urban area. We chose to study Birmingham due to the interest generated by this diversity, coupled with availability of very high quality data in the city. From our analysis it is not possible to determine whether similar associations would be apparent elsewhere. However, our research has demonstrated the application of a methodological framework that could be readily applied in other contexts. We also assume that the distribution of households, with respect to deprivation and ethnicity, within each LSOA is uniform. In reality, this will not be the case, although we chose to study LSOAs because of their small size and hence relatively homogeneous nature. In our development of a park typology, we attempted to select locations that would be available and attractive for recreational use amongst the general population. However, any such typological classification will be, at least in part, subjective. It is possible for example that in some areas,

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patches or waste land or other types of green space may compensate for poor availability of parks or enhance current levels of provision by providing some of the same functions. In our study we have measured access in terms of road distances, and our only measure of ‗quality‘ has been the size of the parks. In reality, of course, quality is multifaceted and will be associated with a range of factors surrounding facility provision and standards of maintenance. Research suggests that there is a national trend for deprived areas to contain parks with relatively poorer maintenance, and since the 1960s, support for provision of open spaces in urban areas has declined (ILAM Services and Harding 2000, Urban Parks Forum, 2001). The focus of investment and facility development has shifted instead to suburban and rural country parks (Reeves, 2000). This tendency to suffer from under-investment has led to a loss of perceived amenity value, most noticeably in inner city locations. Given that the poorest populations and members of ethnic minorities in Birmingham (and most big cities) tend to reside in inner city areas, this means that levels of disadvantage may be greater than those apparent from the comparisons we have made if more subtle aspects of quality of provision are considered. The findings of this study reveal reduced access to a public environmental amenity among certain social groups, in particular those living in the most deprived communities. The lack of open-air sports facilities may well have consequences for general health levels in each population, given that public open spaces are increasingly recognised as important places for physical exercise (see Bedimo-Rung et al. 2005; Foster et al. 2005; Giles-Corti et al. 2005; Hillsdon et al. 2006; Jones et al. 2007). Therefore, although our findings relate directly to social inequities, they may have public health implications as well.

ACKNOWLEDGMENTS This research was supported by the Programme in Environmental Decision Making at CSERGE which is funded by the Economic and Social Research Council. We are grateful to to Professor David Martin (University of Southampton) who assisted with the SurfaceBuilder computer program and to Emma Woolf (BOSF) and Valerie Edwards (BCC) for providing feedback on our typology scheme.

REFERENCES Been V, Gupta F, 1997. Coming to the nuisance or going to the barrios? A longitudinal analysis of environmental justice claims. Ecology Law Quarterly 24, 1-56. Bedimo-Rung AL, Mowen AJ, Cohen DA, 2005. The significance of parks to physical activity and public health: A conceptual model. American Journal of Preventive Medicine 28, S2 159-168. Connover WJ, 1999. Practical Nonparametric Statistics 3rd ed. (John Wiley and Sons, New York and Chichester). Crompton JL, Lue CC, 1992. Patterns of equity preferences among Californians for allocationg park and recreation resources. Leisure Studies 14, 227-246.

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Crompton JL, Wicks BE, 1988. Implementing a preferred equity model for the delivery of leisure services in the US context. Leisure Studies 7, 287-304. DTLR (Department for Transport, Local Government and the Regions), 2001, Green Spaces, Better Places: Interim Report (DTLR, London). DTLR (Department for Transport, Local Government and the Regions), 2002a, Improving Urban Parks, Play Areas and Green Spaces (DTLR, London) DTLR (Department for Transport, Local Government and the Regions), 2002b, Green Spaces, Better Places: Final Report (DTLR, London) Estabrooks PA, Lee RE, Gyurcsik NC, 2003. Resources for physical activity participation: Does availability and accessibility differ by neighbourhood socioeconomic status? Annals of Behavioral Medicine 25, 100-104. Foster C, Hillsdon M, Thorogood M, 2005. Environmental perceptions and walking in English Adults. Journal of Epidemiology and Community Health 58, 924-928. Geographer‘s A-Z Map Company Limited, 2000, A-Z Birmingham De Luxe Street Atlas (Geographer‘s A-Z Map Company Limited, Sevenoaks, Kent). Giles-Corti B, Broomhall MH, Knuiman M, Collins D, Douglas K, Ng K, Lange A, Donovan RJ, 2005. Increasing walking: How important is distance to, attractiveness, and size of public open space?. American Journal of Preventive Medicine 28, S2 169-176. Giles-Corti B, Donovan RJ, 2002. The relative influence of individual, social, physical environment determinants of physical activity. Social Science and Medicine 54, 17931812. GLA (Greater London Authority) 2001. Scrutiny of Green Spaces in London (GLA, London). GLA (Greater London Authority), 2003. Valuing Greenness: Green spaces, house prices and London priorities (GLA, London). Gobster PH, 2002. Managing urban parks for a racially and ethnically diverse clientele. Leisure Sciences 24, 143-159. Harding AK, Holdren GR, 1993. Environmental equity and the environmental professional. Environmental Science and Technology 27, 1990-1993. Haynes R, Lovett A, Sünnenberg G, 2003. Potential accessibility, travel time, and consumer choice: geographical variations in general medical practice registrations in Eastern England. Environment and Planning A 35, 1733-1750. Hillson M, Panter J, Foster C, Jones A (2006) The relationship between access and quality of urban green space with population physical activity. Public Health, Vol. 120, No. 12, pp. 1127-1132. ILAM (Institute of Leisure and Amenity Management) Services Ltd. and Harding S, 2000. Local Authority Historic Parks in the UK. Cultural Trends 10, 68-72. Jones, A. P., Bentham, C. G., Foster, C., Hillsdon, M., Panter, J., 2007 Obesogenic Environments: Evidence Review. Foresight Tackling Obesities: Future Choices project Long science review. Office of Science and Innovation, London. Kit Campbell Associates, 2001, Rethinking Open Space (The Scottish Executive, Edinburgh). Lambert T, Boerner C, 1997. Environmental Inequality: Economic Causes, Economic Solutions. Yale Journal on Regulation 14, 195-124. Marsh MT, Schilling DA, 1994. Equity measurement and facility location analysis: A review and framework. European Journal of Operational Research 74, 1-17. Martin D, 1996. An assessment of surface and zonal models of population. International Journal of Geographical Information Systems 10, 973-989.

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Martin D, Nolan A, Tranmer M, 2001. The application of zone-design methodology in the 2001 UK Census. Environment and Planning A 33, 1949-1962. Mitchell J, Thomas DSK, Cutter SL, 1999. Dumping in Dixie revisited: the evolution of environmental injustices in South Carolina. Social Science Quarterly 80, 229-243. Nicholls S, 2001. Measuring the accessibility and equity of public parks: a case study using GIS. Managing Leisure 6, 201-219. ODPM (Office of the Deputy Prime Minister), 2003. £89 million to reclaim parks and public spaces says Cooper‖ News Release 2003/0151: 29 July 2003. http://www.odpm.gov.uk /pns/DisplayPN.cgi?pn_id=2003_0151. ODPM (Office of the Deputy Prime Minister), 2004. ―The English Indices of Deprivation 2004 (revised)‖ http://www.odpm.gov.uk/stellent/groups/ odpm_urbanpolicy/documents /page/odpm_urbpol_029534.pdf Pastor M, Sadd J and Hipp J, 2001. Which came first? Toxic facilities, minority move-in and environmental justice. Journal of Urban Affairs 23, 1-21. Payne LL, Mowen AJ, Orsega-Smith E, 2002. An examination of park preferences and behaviors among urban residents: The role of residential location, race and age. Leisure Sciences 24, 181-198. Pearce D, 2003. The distribution of benefits and costs of environmental policies: conceptual framework and literature survey. Manuscript prepared for the OECD Environment Directorate Workshop on the distribution of benefits and costs of environmental policies, Paris, March 2003, 3rd draft, August 2002. Peuker TK, Fowler RJ, Little JJ, Mark DM, 1976. Digital representation of three-dimensional surfaces by triangulated irregular networks (TIN) Technical Report No. 10 (Revised) Office of Naval Research, Geography Programs. Pulido L, 2000. Rethinking environmental racism: White privilege and urban development in Southern California. Annals of the Association of American Geographers 90, 12-40. Reeves N, 2000. The condition of public urban parks and greenspace in Britain. Water and Environmental Management 114, 157-163. Scott D, 1997. Exploring time patterns in people‘s use of a metropolitan park district. Leisure Sciences 19, 159-174. Szasz A, Meuser M, 2000. Unintended, Inexorable: The production of environmental inequalities in Santa Clara County, California. American Behavioral Scientist 43, 602632. Talen E, 1997. The social equity of urban service distribution: An exploration of park access in Pueblo, Colorado and Macon, Georgia. Urban Geography, 18, 521-541. Tiebout C, 1956, .A pure theory of local expenditures. The Journal of Political Economy 64, 416-424. Tinsley HEA, Tinsley DJ and Croskeys CE, 2002. Park usage, social milieu, and psychosocial benefits of park use reported by older urban park users from four ethnic groups. Leisure Sciences 24, 199-218 Townsend P, Phillimore P, Beattie A, 1988. Health and deprivation: inequality and the North (Croom Helm, London). Urban Parks Forum, 2001. Public Park Assessment: A survey of local authority owned parks focusing on parks of historic interest (Urban Parks Forum, London). Wicks BE, Backman KF, 1994. Measuring equity preferences: A longitudinal analysis. Journal of Leisure Research 26, 386-401.

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Wolch J, Wilson J. P., Fehrenbach, J. 2005. Parks and park funding in Los Angeles: An equity-mapping analysis. Urban Geography 26, 4-35. Yandle T, Burton D, 1996. Reexamining environmental justice: A statistical analysis of historical hazardous waste landfill siting patterns in metropolitan Texas. Social Science Quarterly 77, 477-492. Young T, 1996. Social reform through parks: The American Civic Association's program for a better America. Journal of Historical Geography 22, 460-472.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 8

AN IDEA FOR PHENOMENOLOGICAL THEORY OF LIVING SYSTEMS Svetla E. Teodorova Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, 72 Tzarigradsko chaussee, 1784 Sofia, Bulgaria

ABSTRACT An idea for developing of new science field, biodynamics, as biophysical macroscopic theory is propounded. The functioning of living organism as an organized entirety is the main specificity of the life. Hence it is quite reasonable to describe behaviour of biological systems in terms of own theoretical basis. In this article a new state variable vitality as integral characteristic of biological object and measure unit bion are stated. A quantity biological energy is introduced as energy form related to biological selfregulation. Quantity synergy is suggested as measure of selfregulation quality. Biological principle for maximum synergy in healthy living systems is stated. On the basis of variational principle an equation describing recovery process of a biological object after disturbance is obtained. The quantity optimal vitality, related to homeostasis, decreases in lifespan scale and its evolution is described by ordinary differential equation. The potential lifespan maximum of several species at different life conditions may be calculated at different parameters of the equation. A wide range of environmental influences on the living organism could be promptly and easily assessed in the terms of the biodynamics approach. Such an approach could be used for a simple estimation of a patients‘ health status.

INTRODUCTION XX century brought colossal discoveries in the field of molecular biology. In the same time unified theory of living mater on phenomenological level is not created. Such a theory could help study the behaviour of the native biological object (BO) in its entirety. This may be a new step in exploration of living systems in the context of environmental condition

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changes, diseases etc. The great complexity and different organization levels of BOs embarrass a direct application of physical theories to biological systems. Notwithstanding, serious attempts have been made to describe quantitatively different aspects of the life phenomena on the basis of physical theories – thermodynamics, electromagnetism, hydrodynamics, quantum mechanics etc. Shrödinger (1944) suggested that the essence of the life is that living organisms consume negative entropy (negentropy). Goodwin (1963) tried to create a biological statistical mechanics and thermodynamics on the basis of kinetics of synchronized biochemical oscillators. Rosen (1967) formulated optimal principles in biology. Szent-Györgyi (1968) elucidated some mechanisms of cell regulation in the terms of bioelectronics. Prigogine and Nicolis (1978) developed theory of dissipative structures, as an extended irreversible thermodynamics, to explain self-organization processes in nonequilibrium systems. Davidov (1979) gave a quantum-mechanical interpretation of some biological phenomena. Thermodynamic approach was applied to the structure and functions of macromolecules (Di Cera 2001). Kleidon and Lorenz (2005) presented a coherent study of geosphere-biosphere couplings in the context of maximum entropy production principle. Kurzynski (2005) proposed a thermodynamic approach combined with describing of molecular machines. Many authors emphasized the great importance of information in the functioning of BO. Quastler (1964) represented the problems of the emergence of biological organization on the basis of biological aspect of information. Trincher (1965) tried to extend thermodynamics via introducing of a concept of structural information in BO. Eigen (1971) explained the evolution of macromolecular structures taking into account laws of information exchange. Volkenstein (1994) considered life evolution in its informational aspects. The great significance of all these considerations, however, does not abolish the need of a general phenomenological approach to BOs. Phenomena as thermal conductivity, diffusion, electric current etc. are not describable in the terms of mechanics and thus new fields as thermodynamics and electrodynamics with their concepts and laws were created. On thermodynamic level it is not possible to underline the essence of life. All attempts to construct an extended thermodynamics of irreversible processes, including living matter, remain artificial and not quite adequate. Blümenfeld (1967; 1974) noted that the true way to a general life theory is not a creation of biological thermodynamics. Really, the thermodynamic and electrodynamic laws are valid for different aspects of BO functioning but they do not explain the essence of life – selfregulation and behaviour of BO in its entirety. A new science field – BIODYNAMICS is needed. Indeed, the biological regulation has been regarded in details in cybernetic aspect. Our view is, however, that the biological selfregulation should be an object also of physics. Although the life activity is based on biophysical and biochemical processes the perfect co-ordination of these processes forms already a new quality of matter organization. Thus, it is advisably to put in correspondence of BO behaviour new type quantities as integral characteristics of BO on macroscopic level. Here we introduce the quantity ―vitality‖ as a basic biodynamic quantity. Thus, the following juxtaposition can be stated: Classic dynamics – mass Electrodynamics – charge, field intensity Chromodynamics – colour charge Thermodynamics – temperature, pressure, concentrations etc. Biodynamics – vitality

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Naturally new type of measurements should be expected. The author presumes a creation of a new device and believes that by means of such a device one will be able directly to measure vitality in the future. Such an approach will avoid the great complexity in the quantitative description of living systems via thermodynamics, chemical kinetics etc. The attention in this article is drawn on recovery processes of BO after disturbances running in time intervals much shorter compared to BO lifespan. On the other hand, the evolution of optimal vitality during the lifespan of BO is studied.

A POSSIBLE THEORETICAL BASIS OF BIODYNAMICS We think BO as a macroscopic object, which saves its features and character only in its integrity. The living system demonstrates a new level of material organization non-described previously in physics. We introduce a new quantity, vitality (V) uniquely determining the state of a given BO. Thus V is an integral characteristic of BO functioning in its entirety. In thermodynamics the temperature is a measure of kinetic energy of the molecules. In biodynamics the vitality should be a measure of the health status of BO. Obviously, vitality is related to the informational aspect of BO functioning. We propose the respective measurement unit of the vitality in SI system to be called bion (b). We assume that V could be in principle measured by a new type device – vitalimeter. Such an instrument should measure some electromagnetic wave emitting by BO, a wave (or wave complex) being an integral characteristic of BO functioning. Electromagnetic waves of different frequencies, generated by human, animal and plant organisms in their metabolic activity were measured yet many years ago (Presman 1968). The explorations in this way were continued (Godik and Guljaev 1991; Elizarov 1997). The recent development of advanced technologies have been gone rather far and this fact promises well success in searching of some device, reflecting resultant information about BO. A new constructed apparatus, being able to detect some wave as integral BO characteristic, could be calibrated in a manner that to show V in bions. For instance, one bion may be defined so that the excellent standard of human‘s health corresponds to vitality of 100 bions. Not only the frequency/length of the wave but also the wave amplitude gives information for BO status. Thus, at least two new quantities should be defined. However, here for simplicity we shall take into account only one quantity (understanding, for instance, the wavelength). Here we are not interested in the incidence of the wave in the space. The very value of the vitality at a certain moment is important. We introduce also a quantity optimal vitality (W). That is the optimal vitality value, corresponding to state of excellent health of BO and reflecting homeostasis characteristics evolutionarily established for the respective species. W is genetically determined and in time intervals much shorter compared to lifespan it may be regarded as a constant. During the life, however, W decreases due to aging processes. After different transitory disturbances in BO status V temporarily can deflect from optimal vitality, i. e. V  W. Then a transitional process is running and V → W. That is a recovery process.

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The total energy E of a natural system is sum of the mechanical energy EM, and internal energy EI (Gyarmati 1970): E = EM + E I The internal energy contains heat energy, chemical energy etc. (Prigogine and Defay 1954): dEI = dQ – pdv + Σμidni It is reasonable to establish in biodynamics a respective specific energy. We shall cal it biological energy (B). We could suppose that it is a function of V (t) and V (t) (where V is the rate of change of the vitality in time): B = B (V, V ) The biological energy should be a part of internal energy: dEI = dQ – pdv + Σμidni + dB B is the energy providing biological selfregulation (on the basis of enzyme synthesis, resonance energy transfer between biological macromolecules, electric charge transfer, immune response etc.). All these energies in macroscopic aspect could be denoted as biological energy B. We introduce also a state function synergy G (V), which uniquely determines the state of a given BO:

dG 

dB V

(1)

as a measure of the quality of the BO selfregulation (therefore, of the biological organization). G reflects the degree of coordination and synchronization of the regulatory links in BO. It could be an indicator of BO health and youth. G is genetically determined. G increases in organism growth as well as in training processes. In aging processes, in severe and chronic diseases G decreases. In a mature and healthy BO the synergy G as almost constant G(V) = G(W).

RECOVERY PROCESS AFTER DISTURBANCE When a BO is disturbed (acute disease, trauma, intoxication, environmental influence etc.) and its normal life parameters are violated, i. e. W – V(t)  0, the genetic information potential switches feedback control (repair, immune system reaction, enzyme synthesis etc.) to restore BO to its physiological homeostasis. A recovery process starts. We assume that in case of damages V(t) < W. Here we consider recovery processes, running in time interval much shorter compared to the lifespan of BO. Now we try to define the balance of biological energy in phenomenological aspect.

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We assume the genome energy as composite of two parts: potential genome energy UW and recovery energy UV: U = UW + UV (2) The quantity UW = const is characteristic for a given species. The recovery energy UV, should be proportional to the difference W – V(t), i. e. UV = k (W – V). UV should be a positive function; hence we may present it as a positive determined quadratic form:

UV 

1 K (W  V ) 2 2

(3)

where K ([K] = [kg m2 b–2 s–2]) is the genome inductivity, representing the integral feedback control strength. It is clear that UV has minimum at V(t) = W. Therefore, in a non-disturbed state BO does not spend recovery energy. We assume that the power of immune response P, expressed on phenomenological level, should be proportional to the rate of change of the vitality. To be a positive function P should be constructed as follows:

P  MV 2

(4)

where M ([M] = [kg m2 b–2 s–1]) is coefficient of immune memory. The immune response has a cumulative effect. The state of a BO at a given moment depends not only on the synthesis of immune factors at that moment but on the summary effect of immune response in all prior moments. Therefore the immune reaction energy Z in the recovery process should have the form: t

t

t0

t0

Z (t )   Pd    MV 2 d

(5)

Because BO behavior is considered on phenomenological level, we are interested in the total effect of immune response and here we do not differentiate cell and humoral immunity. We assume that the constant M characterizes the total immune potential of BO. The accomplishment of the recovery process may be embarrassed due to waste products of metabolism (non-fully oxidized substances, macromolecules damaged by free radicals) and toxicants (heavy metals, bacterial and virus toxins etc.) occurring in cell and decreasing the efficiency of metabolic processes and cell selfregulation. Therefore, the total biological energy B could decrease at expense of energy of metabolic resistance R. It is reasonable to suppose that R is proportional to the rate of change of the vitality. To be a positive function R could be defined in the form:

R

1 2 AV 2

where A ([A] = [kg m2 b–2]) is coefficient of metabolic resistance.

(6)

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One can write the following equation for the balance of biological energy B: dB = dU + dZ – dR

(7)

We define

U  

dU  K (W  V ) dV

and

Z 

dP  2 MV dV

(8)

as biological force of feedback control and force of immune reactivity, respectively. Their dimension is: [ΦU] = [ΦZ] = [kg m2 b–1 s–2]. We define also

L

dR  AV dV

(9)

as impulse of metabolic resistance. Its dimension is [L] = [kg m2 b–1 s–1]. We assume that the functioning of the healthy living systems is based on the following biological principle: G(W) = max G(V)

(10)

It means that in its optimal, undisturbed state BO has a synergy maximum. G(W) corresponds to excellent health. For recovery processes it follows from (10): dG > 0

(11)

Taking into account (1), (2), (3), (4), (5), (6), (7), (8), (9) and (11) we can write:

dG 

(dV  LdV ) >0 V

(12)

where  is the total recovery force

1   U   Z 2

(13)

It follows from (12) that the expression

 dV  LdV > 0 is a condition for fully BO recovery.

(14)

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One of the most profound concepts in theoretical physics is that the equations of motion in different fields of physics can be obtained on the basis of integral variational principles. The well known variational principle of Hamilton allows a common treatment of dynamic problems in mechanics, electrodynamics, optics, thermodynamics, quantum mechanics etc. By means of appropriately chosen Lagrangeans the basic equations in physics can be introduced. This approach has a great heuristic concern. The presence of variation principles in all physics fields clearly shows that a basic nature law takes place. This promises that in fields, where no other approaches exist, a variational principle from the Hamilton type could help to obtain adequate equations, describing the characteristic processes. We propose the following integral principle: T

   (U  Z  R)dt  max

(15)

t0

choosing the Lagrangean: L = L (V (t), V (t), t) = U + Z –R, where U, Z and R are determined by the equations (2), (3), (5) and (6). After variation of (15)

( )  0  0

(16)

where ε is an arbitrary parameter, the following biodynamic equation is obtained:

V 

2M K KW V  V A  2 M (T  t ) A  2 M (T  t ) A  2 M (T  t )

(17)

under initial conditions:

V (t0 )  V0

and

V (t0 )  V0

(18)

V0 is the state of disturbed BO from where the recovery process starts and V0 is the start rate of time change of V. T is the time period of the recovery process. The equation (17) has a physical sense and aperiodic solutions when the following conditions are valid: A > 2MT

(19)

M2 > (A – 2MT) K

(20)

Obviously, at a given value of M, in greater contamination of the organism (higher A value) the time T of the recovery process will be longer. The stationary solution of equation (17) is a stable knot.

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Equation (17) is solved numerically. Some solutions at different values of the parameters are displayed in Figure 1. The value of T = 10 days was arbitrary assumed for the recovery process after some disturbance (for instance: influenza, poisoning, burn etc.). We stated that the normal state of BO corresponds to W = 100 bions. We chose an arbitrary value for the start of recovery process: V0 = 60 bions. In principle, on the basis of many empirical data one can determine the real average recovery time periods for different diseases and damages and real W for different species and ages. Using them the characteristic parameters in equation (17) can be calculated.

Figure 1. Time courses of the quantity vitality during the recovery period as numerical solutions of equation (17). The time interval of 10 days for the recovery period after some disturbance was arbitrary chosen (T = 10). A value of 100 bions for optimal vitality was assumed (W = 100). The initial condition was V0 = 60 bions. The displayed time courses were calculated at different values of characteristic constants: A = 9, M = 0.4, K = 0.93 (over-shoot curve); A = 11, M = 0.5, K = 0.17; A = 12, M = 0.5, K = 0.13.

Time courses of several biological parameters, similar to these shown in Figure 1, are very typical for different transitory processes in biological systems.

SINERGY AND ENTROPY CHANGES DURING THE LIFESPAN OF A BIOLOGICAL OBJECT In lifespan scale the following cases could be distinguished regarding the synergy G and entropy S changes: During development and growth dG > 0

dS > 0

dG > dS

Near completion of development and growth

An Idea for Phenomenological Theory of Living Systems G = max

265

dS > 0

3) At mature age dG  0 dS > 0

dG < dS

4) At aging dG < 0

dS > 0

dG  dS

5) Death G=0

S = max

What means the inequality dG < dS at mature age? G can remain almost constant during many years (maximum comfort conditions for BO) or decrease weakly (dG  0). But this decrease is less compared to entropy increase (i. e. notwithstanding that the entropy increase leads to age changes in macromolecules and structures, the integrity of the regulatory links remains or changes slower than structure aging). The synergy is not reciprocal to entropy. The inequality dG < dS shows the stability of BO selfregulation. For instance, the genetic functions demonstrate a relative resistance against to the chronic action of damaging agents, particularly heavy metals (Topashka-Ancheva et al. 2003). The difference in changes of entropy and synergy once more indicates that via thermodynamics (in spite of its adjustment to biological systems) one does not attain adequate description of the life processes.

EVOLUTION OF OPTIMAL VITALITY (W) DURING THE LIFESPAN OF A BIOLOGICAL OBJECT Up to now we considered the optimal vitality W as a constant quantity because our attention was drawn only on processes much shorter compared to the lifespan. The optimal vitality W, however, decreases in lifespan scale due to aging processes (genetic mutations, alterations in the regulatory links of genetic apparatus and hence quantitative and qualitative changes of proteins and attenuation of their self-renewal (Frolkis 1969). The simplest quantitative assumption about W evolution during the life is that the rate of W change is proportional to the time:

dW  qt dt

(21)

where q ([q] = [b s–2]) is a parameter reducing the optimal vitality. We call it aging factor. The parameter q should be a time function. The temp of the aging is most intensive after the age of 25 years (in human) and then it decreases with time (Strehler 1962). Quantitatively this could be expressed by the following differential equation:

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dq  q dt

(22)

under initial condition q0 = q(t0),

t0 = 25 years

(23)

where q0 ([q0] = [b s–2]) and  ([] = [s–1]). The constant  can be called aging correction, because at higher values of  the parameter q decreases faster with time and hence, as follows from (21), the rate of aging decreases. Respectively, when q decreases slowly the aging processes go faster. The analytical solution of (22) is

q  q0 e

 (t  t0 )

(24)

Taking into account (24) the equation (21) can be written as

dW  (t  t0 )  q0 e t dt

(25)

The analytical solution of (25) is

W  W0 

q0 q q (t0  1)  0 te ( t t0 )  02 e ( t t0 ) 2   

(26)

The constant  may be determined from the solution (24) after taking a logarithm: ln q = ln q0 –  (t – t0)

(27)

The equation (27) is an equation of a straight line with angular coefficient . Using the values of q, a plot of ln (q – q0) as a function of t – t0 could be constructed and  determined. The q-values could be calculated in the following way. Human individuals in perfect (accordingly to the respective age) health could be selected in different age groups ranging by five years (25–30, 30–35, 35–40 etc.). Within these groups q may be considered approximately constant. Then the analytical solution of equation (21) has the form:

W  W0 

1 2 qt 2

(28)

and respectively:

q

2(W0  W ) t2

(29)

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W-values for the different age groups could be empirically measured (of course under a reliable statistics) and on the basis of (29) values of q, specific for the respective age periods, could be calculated. The value of q0 may be determined in individuals of 24–26 years old. If the constants q0 and  were known W(t) would be drawn. Thus, we shall have a picture of a normal aging, i. e. aging in individuals, who are not burden with chronic diseases. The same procedure could be applied in people with certain chronic diseases, in smokers, etc. It is seen from (29) that at a greather difference W0 – W the parameter q has a higher walue. For instance, at age 60 years W of a sick man will be less than W of a healthy man (Wsick < Whealthy) and respectively W0 – Wsick > W0 – Whealthy. Therefore, qsick > qhealthy. This indicates a faster aging process in the sick. In illness at young age the value of q0 may be higher than the normal and this could reflect on the further life and aging pace. In most cases the life mode can play a significant role for q modification. The healthful nutrition, going in for sports, natural regimen etc. could increase α-constant in large extent. Consequently the rate of q decrease increases and hence the duration of the individual life could increase.

Figure 2. Evolution of the optimal vitality W during the lifespan. The maximum value of optimal vitality W is assumed of 100 bions. In human context it should correspond to the age of 25 years. A value of 30 bions is assumed as Wcr. Three analytical solutions (26) of W (t) are drawn, calculated at different values of the parameters (q0 = 0.01, α = 0.0061; q0 = 0.01, α = 0.0012; q0 = 0.015, α = 0.0012). The respective curves cross the dotted line Wcr in points corresponding to 150, 125, and 102 years. These points could be considered as possible potential maximums of life duration at different conditions.

In Figure 2 analitical solutions of equation (26) are presented. They are calculated under initial conditions W0 = W(t0) = 100 bions and t0 = 25 years and at different values of the parameters q0 and . The value Wcr corresponds to that value of vitality, under that the selfregulation falls and life recovery processes are impossible. The values of X-axis, corresponding to the points of the dotted line, indicate the lifespan of Homo sapiens in different conditions. The points TWcr correspond to the absolutely life potential in the respective conditions, not to the real end of the individual life. Usually the concrete lifespan is

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(much) shorter than TWcr (often due to different fortuitous factors). In principle TWcr may be considered as genetically determined possible lifespan maximum for a given species but it could vary in a wide range depending on diseases, life conditions, and life mode.

CONCLUSION It is impossible to deduce macro-characteristics of living systems on the basis of the numerous processes on molecular level. Because of that it will be a great benefit to have a measurable (one or a few) macro-characteristic(s) uniquely determining the status of BO as an entire unit. Thus, the behaviour of BO would be explored and predicted. In exemplification of this idea here we considered the simplest case of only one integral variable – vitality V. Such an approach is very important not only for practical purposes. It is also of great theoretical significance. Biological selfregulation provides a new quality of matter. It is quite reasonable to evaluate the state of living matter in terms of specific energy form. No kind of energy known in physics can be put in correspondence to biological selfregulation in order to explain its integrity. The health of BO essentially depends on selfregulation quality and it seems to be very tempting to assess BO via adequate quantities. Thus, the creating of new science field, biodynamics, could be a substantial step to a more profound study of living matter. The general responses of BO to environmental, therapeutic, and other influences as well as to diseases could be effectively studied. Biodynamics approach will be of a great importance in medicine for diagnosis and therapy. A simple and prompt assess of the healthy status of the patients could be made and in patient‘s files abreast of the other data could be added data as vitality V , optimal vitality, and synergy G as important integral biomarkers. Valuable evaluations could be carrid out about the aging rate and life duration via studing of optimal vitality W evolution. Many empirical investigations could provide reliable data for determining the characteristic lifespan TWcr for several species. Essential correlations could be established between environmental conditions specificity and mean human lifespan. Satisfactory values of the parameters introduced here (K, M, A, q0, and α) may be determined via experiments, but it is also of essential interest to attempt to express these constants as functions of some parameters of BO on molecular level. For this purpose many targeted empirical research are needed. Naturally, new device is needed to measure such quantities. The author is optimist regarding the further technical development and the possibility to study the vital status of BO via integral characteristics. The author hopes that the idea shared here may stimulate the scientific thought for further efforts toward development of a phenomenological life theory.

REFERENCES Bljumenfeld, L. A. (1967). Preface to the Russian edition. In: H. Quastler. The emergence of biological organization 5-6. Moskow: Mir Publishing house. Bljumenfeld, L. A. (1974). Problems of Biological Physics. Moskow: Naouka Publishing House.

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Davidiv, A. (1979). Biology and Quantum Mechanics Kiev: Naukova Dumka Publishing House. Di Cera, E. (ed) (2001). Thermodynamics in Biology. Oxford-New York: Oxford University Press. Eigen, M. (1971). Molekulare Selbstorganistion und Evolution (Self organization of matter and the evolution of biological macro molecules). Naturwisswnschaften 58 (10), 465-523. Elizarov, A. A. (1997). Instrumental methods for investigating physical fields of biological objects. Measurement Techniques (Springer) 40 (7), 700-707. Frolkis, V. V. (1969) The Nature of Aging. Moskow: Naouka Publishing House. Godik, E. E. and Guljaev, Yu. V. (1991). The human being through ―Eyes of Radiophysics‖. J. Radio Engineering (Russian) 8, 51-62. Goodwin, B. C. (1963). Temporal Organization in Cells. London: Academic Press Inc. Ltd. Gyarmati, I. (1970). Non-equilibrium thermodynamics: Field theory and variational principles. Berlin-Heidelberg-New York: Springer-Verlag. Kleidon, A and Lorenz, R. D. (2005). Non-Equilibrium Thermodynamics and the Production of Entropy. Life, Eearth, and Beyond. Berlin-Heiderlberg-New York: Springer. Kurzynski, M. (2005). The Thrrmodynamic Machinery of Life. Berlin-Heiderlberg-New York: Springer. Nicolis, G. and Prigogine, I. (1978). Self-organization in Nonequilibrium Systems. From Dissipative Structures to Order through Fluctuations. New York-London-SydneyToronto: John Wiley and Sons. Presman, A. S. (1968) Electromagnetic fields and living nature. Moskow: Naouka Publishing House. Prigogine, I. and Defay, R. (1954). Chemical thermodynamics. London-New York-Toronto: Longmans Green and Co. Quastler, H. (1964). The Emergence of Biological Organization. New Haven and London: Yale University Press. Rosen, R. (1967). Optimality Principles in Biology. London: Butterworths. Shrödinger, E. (1944). What is Life? Cambridge: University Press. Strehler, B. L. (1962). Time, cells, and aging. New York and London: Academic press. Szent-Györgyi, A. (1968). Bioelectronics. A Study in Cellular Regulation, Defense, and Cancer. New York-London: Academic press. Topashka-Ancheva, M., Metcheva, R., Teodorova, S. E. (2003). Bioaccumulation and damaging action of polymetal industrial dust on laboratory mice Mus musculus alba II. Genetic, cell, and metabolic disturbances. Environmental Research 54, 152-160. Trincher, K. S. (1965). Biology and Information. Moskow: Naouka Publishing House. Volkenstein, M. V. 1994). Physical Approaches to Biological Evolution. Berlin-Heidelberg: Springer Verlag.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 9

A NEW TRAIT OF GENTOO PENGUIN: POSSIBLE RELATION TO ANTARCTICA ENVIRONMENTAL STATE? Roumiana Metcheva1, Vladimir Bezrukov2, Svetla E. Teodorova3, and Yordan Yankov4 1

Institute of Zoology, Bulgarian Academy of Sciences, 1, Bd.Tzar Osvoboditel, 1000 Sofia, Bulgaria 2 Department of General and Molecular Genetics, Taras Shevchenko National University of Kyiv, 64 Volodymyrska Str., Kyiv, 01033, Ukraine 3 Institute for Nuclear Research and Nuclear Energy, Bulgarian Academy of Sciences, 72, Tzarigradsko shaussee, 1784 Sofia, Bulgaria 4 Bulgarian Antarctic Institute, 15, Bd.Tzar Osvoboditel, 1000 Sofia, Bulgaria

ABSTRACT Some morphological traits of Antarctic animals could be considered in the context of a trend to more direct relation between environmental conditions and possible adaptive mechanisms of animal‘s organism. Penguins are excellent object for biomonitoring. Here a preliminarily report is presented regarding a new trait, a spot-like coloration (―yellow spot‖) of the bill, observed on the upper mandible of Gentoo penguin (subspecies Pygoscelis papua ellsworthii). The spot varied in size and colour. It was recorded among chicks over two months old and adult birds (normal and molting). The trait had no significant relationship to the animal' s sex. Among all inspected females 31% and among all inspected males 27% exhibited beak spot. Three breeding colonies were investigated at three different geographical locations at the Antarctic Peninsula – Livingston Island, South Shetlands (6238 S), Wiencke Island (64o52 S), and Petermann Island (6510 S). Yellow spot was found with different frequencies at these three locations: 20%, 36%, and 30%, respectively. All spotted penguins from the three colonies were 32% when

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Roumiana Metcheva, Vladimir Bezrukov, Svetla E. Teodorova et al. compared to all non-spotted. The possible reasons for the spot-like coloration are discussed. The trait could be a phenotypic characteristic. It could play some role in the mate choice. A possible connection to carotenoid pigments content does not be eliminated in the context of Gentoo diet. A probable cause for the beak spot appearance seems to be the increasing of ozone depletion above Antarctica. The ultraviolet radiation enhances free radical production. Other studies reported an increase of the flux of transsulfuration pathway as a defense reaction, resulting in elevated level of cysteine. When cysteine concentration is raised, an attaching of cysteine to melanin synthesis pathway occurs and this results in formation of reddish pigments.

INTRODUCTION Antarctica environment is closely related to the global change of our planet and hence it is a topic of increasing importance. Climatic features, atmospheric regime, and temperature and water balance depend on the state of Antarctic continent. The investigation whether Antarctica is influenced by anthropogenic pollution is of great interest. In this context the biomonitoring could provide valuable data. Results reported by Metcheva et al. (2006) suggest that due to annually molting penguin feathers are an excellent monitoring tool of Antarctica environmental state, being precise indicator for detection of metal levels. This allows assess possible contamination trends. During the morphological investigations on Gentoo penguin (Pygoscelis papua ellsworthii) a spot-like coloration (―yellow spot‖) was observed at the base of the upper mandible of some individuals. It varied from clear yellow-orange to reddish. Colour patterns of terrestrial birds have been well studied, but there is little research on seabird coloration (Jones and Hunter 1993; Jouventin et al. 2005). Among the six genera belonging to family Spheniscidae, the genus Pygoscelis is the most widely distributed (Del Hoyo et al. 1992). Three species belong to Pygoscelis: Chinstrap (P. antarctica), Gentoo (P. papua) and Adelie (P. adeliae). Gentoo penguins breed on subantarctic islands and on the Antarctic Peninsula (Stonehouse 1970). There are two subspecies of Gentoo – P. p. papua, J. R. Forster, 1781 and P. p. ellsworthii, Murphy, 1947. The subspecies P. p. papua is distributed in sub Antarctic up to 60 S; P. p. ellsworthii inhabits the Antarctic from 60 S up to 65 S (Del Hoyo et al. 1992). The subspecies P. p. ellsworthii is of smaller size and bill proportion compared to P. p. papua (Martinez 1992). Gentoo penguin populations inhabiting the Antarctic Peninsula have been studied with respect to foraging behavior (Trivelpiece et al. 1986), morphometry (Stonehouse 1970), UV reflectance (Jouventin et al. 2005) etc. However, this trait (―yellow spot‖) observed has not been described in the literature before. Some authors (Stevenson and Anderson 1994; Siefferson and Hill 2005) that study different particular colorations in birds tend to explain the function of these phenomena in terms of health, mate choice behavior or intraspecific signaling. Saks et al. (2003) have established that brighter yellow breast feathers in male greenfinches signals immunocompetence and health status. Thus, females prefer more ornamental males, able to provide parasite resistance genes for the offspring. Some of the colour patches are at the same time also ultraviolet markings. Jouventin et al. (2005) have reported UV beak spots in King penguin (Aptenodytes patagonicus) and Emperor penguin (Aptenodytes forsteri). They have

A New Trait of Gentoo Penguin: Possible Relation to Antartica …

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found UV peaks of reflectance, overlapping with spots of colour on both sides of the lower mandible, which have appeared orange (with variations among individuals from yellow to red). The authors suggested that UV beak spot could be an indicator of sexual maturity having a possible role in pairing. UV reflective patches are of concern in the context of UV vision in birds, which may be used also for foraging (Sűtari et al. 2002) and hunting (Koivula et al. 1997; Koivula et al. 1999). Other interesting problems are whether such kind of markings (colour only or colour and UV reflectivity) might appear as consequence of environmental changes or it depends on the diet, biochemical disturbances and parasite infection. Most probably a complex reason might take place. Similar traits could also be used for comparative population studies. In the present study the distribution of spotted P. p. ellsworthii in three Antarctic locations – Livingston Island, South Shetlands (6238 S), Wiencke Island (64o52 S), and Petermann Island (6510 S) is reported and probable reasons for the beak spot appearance are discussed. Particularly, a possible reason related to the specific Antarctic environment is outlined.

MATERIALS AND METHODS Populations Studied Field measurements were carried out on adult, nonmoulting and molting Gentoo penguins (P. p. ellsworthii), inhabiting Livingston Island (62º38´ S, 60º24´ W, South Shetland Islands), Wiencke Island (64o52 S, 63º30´ W, Palmer Archipelago), and Petermann Island (6510 S, 64º10´ W). The birds were tested during the Antarctic summer seasons, from January to March, – at Livingston Island in years 2002 – 2005; at Petermann Island in 2002-2003, and at Wiencke Island in 2003-2004. At Livingston Island the studied colony varied from 84 to 110 couples. At Petermann and Wiencke Islands the colonies were larger, about 1000 nests per island. Additionally, at Livingston Island twelve marked couples of P. p. ellsworthii as well as the offspring in the crèche were observed in December 2005 - January 2006. The penguins were captured using a hand net. After inspection the morphological traits were measured according to the Commission for the Conservation of Antarctic Marine Living Resources – Ecological Monitoring Programme (CCAMLR EMP) recommendations (CCAMLR 2004). All the penguins were marked with glass-encapsulated TROVAN identification electronic transponders. These transponders, implanted subcutaneously, have demonstrated to be a reliable means to identify individual penguins. The TROVAN system enables long lasting marking, automatic detection of birds and quick identification (Clarke an Kerry 1998). The penguins were checked for the appearance of spot on the beak when they were marked.

Blood Samples for DNA Analysis Blood sampling for sex identification and DNA analyses were performed by two different ways according to the CCAMLR EMP Standard Methods Recommendations. Approximately

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1 ml of blood was drawn from the cubital vein with a syringe and placed into tubes with K3∙EDTA or into heparinzed tubes. DNA was extracted using a standard phenol-chloroform method as described by Sambrook et al. (1989). Sex determination was performed by PCR as described by Itoh et al. (2001).

Discriminant Analysis Sex determination of Gentoo from the Petermann Island was carried out with discriminant analysis, as described EMP Standard methods, Hobart (CCAMLR 2004) with little modification. The equation was Z = 0.922*L + 3.885*H were L and H are the length and high of the bill, respectively. The discriminate value was D = 103.302 instead of the described 112.608, because the localization of the minimum at the male-female distribution of D was at the 103.302 point. Data were verified with the values of body mass differences (only for registered nesting couples) – males were usually heavier than females. The results were confirmed with PCR assay according Savov et al. (2004).

Statistical Treatment The percentages of spotted individuals from Livingston Island, Wiencke Island, and Petermann Island were compared and statistically analyzed for their significance. Differences in the trait distribution between males and females were tested using the 2criterion for table 2 x 2. Trait distribution in the penguins at different locations was tested with exact Fisher‘s test (for two-tailed probability of the II type error) (Sokal and Rohlf 1995; Dubrova 2000).

RESULTS Gentoo penguins from Antarctic Peninsula (Figure 1) were tested for presence/absence of beak spot (Figures 2 and 3) as follows: 157 individuals at Livingston Island (or about 81% of the breeding colony), 114 – at Wiencke Island (11% of the colony), and 201 – at Petermann Island (20% of the colony). The spot is located on the upper mandible of the bird (Figure 2). It begins at the flashy core and extends onto the hardened part of the beak. In different individuals the spot size ranges from very small (1 – 2 mm) to quite large (20 – 25 mm). The largest spots spread over almost all the upper surface of the bill. We named the observed new trait ―yellow spot‖ because of its typical colour. However, the colour of the spot varied from yellowish to red with yellow, orange, and pink intermediate forms in different birds. The scheme in Figure 4 gives a picture regarding size and colour variations of the spot. Other visible peculiarities in body or feather coloration of beak-spotted Gentoo penguins were not recorded. The trait was observed year-round, not only during molt. Over the time of exploration the spots did not change their appearance.

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The ―yellow spot‖ was observed also in adults and chicks inspected. Among adult birds the frequency of this trait was approximately two times higher than among chicks. To prove preliminarily a possible heritable basis and to estimate the age-related process of spot appearance, commencing with the hatching, we observed additionally 3 pairs with spots (both of parents), 2 pairs without spots and 7 mixed pairs (with spot in only one of the parents) at Livingston Island in December 2005 – January 2006. From the beginning of hatching to about 3 weeks of age no beak spot was found in the offspring (Figure 5).

Figure 1. Gentoo penguin distribution map: the white dots indicate the breeding areas of the subspecies Pygoscelis papua papua and the black dots – the breeding areas of the subspecies Pygoscelis papua ellsworthii. 1 – Livingston island; 2 – Wienke island; 3 – Petermann island.

Figure 2. Adult Gentoo penguin (Pygoscelis papua ellsworthii) with spot on the upper mandible.

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Figure 3. Adult Gentoo penguin (Pygoscelis papua ellsworthii) without spot on the upper mandible.

Figure 4. A scheme of the ―Yellow spot‖, observed on the upper mandible of Gentoo penguins (Pygoscelis papua ellsworthii): place of the spot on the mandible and spot size variations.

Figure 5. One-month Gentoo penguin (Pygoscelis papua ellsworthii) without spot on the upper mandible.

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Similar data were obtained at the other locations (Petermann and Wiencke Islands), where recently hatched chicks with beak spot were not found. Spots were observed, however, in two-month chicks (Livingston colony), when they have formed crèche (Figure 6). Among 75 juveniles, 3 two-month chicks (4%) exhibited a small spot in the base of the upper mandible.

Figure 6. Two-month Gentoo penguin (Pygoscelis papua ellsworthii) with an about 2 mm ―Yellow spot‖ on the base of the upper mandible.

The spot distribution among males and females is presented in Table 1. The DNA analysis was carried out on 93 individuals in the Livingston colony and on 185 individuals in Petermann colony. The analyses revealed that at Livingston Island 37 of the samples were females and the rest 56 were males. Respectively, at Petermann Island 94 penguins were females and 91 – males. Among all inspected females 31% exhibited beak spot and among all inspected males colour spot on the beak was observed in 27% of individuals. There was no statistically significant difference in the frequency of spot appearance in both sexes (Table 1). The distribution of the trait in the three Gentoo colonies (at Livingston, Wiencke, and Petermann islands) for the penguins investigated during 2002 – 2005 (without those observed in the summer season 2005/2006) is presented in Table 2. All inspected birds were divided into two categories – ―with spot‖ and ―without spot‖. The average frequency of all spotted individuals is 31.7%. The differences in the percentage of spotted individuals among all the three colonies are statistically significant. The highest frequency of the trait was found in the colony of Wiencke Island followed by the colonies of Petermann and Livingston (Table 2).

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Table 1. Distribution of the ―yellow spot‖ between male and female individuals of Pygoscelis papua ellsworthi

Location

Livingston Island Petermann Island

All groups

n (all inspected)

Sex

With beak spot

Without beak spot

n

Frequency in %

n

2

P

Male

56

15

27

41

Frequency in % 73

Female Total Male

37 93 91

9 24 25

24 26 27

28 69 66

76 74 73

0,069

0.98

Female Total Male Female Total

94 185 147 131 278

31 56 40 40 80

33 30 27 31 29

63 129 107 91 198

67 70 73 69 71

0,66

0.78

0,37

0.93

Table 2. Distribution of the ―yellow spot‖ in the three colonies of Pygoscelis papua ellsworthi, inhabiting different Antarctic geographical areas. Frequencies (%) of spotted individuals and Fisher‘s test data are given

Population

Livingston Island Wiencke Island Petermann Island All

Location

62º38´ S 60º24´ W 64o52 S 63º30´ W 6510 S 64º10´ W

Total

% Spotted

n With beak spot

Without beak spot

157

31

126

19.7

114

58

56

50.9

201

61

140

30.3

472

150

322

31.8

Statistical significance (exact Fisher‘s test) Comparison Twobetween Tailed populations probability Livingston 0.0014 Peterman Peterman 0.0004 Wiencke Livingston 0.00012 Wiencke

DISCUSSION On the basis of the result that the distribution of the revealed beak spot in males and females P. p. ellsworthii does not differ significantly (Table 1) one could safely state that the ―yellow spot‖ is not related to the sex of Gentoo penguin. The ―yellow spot‖ is a characterization both of non-molting and molting penguins. This trait may be a polymorphic characteristic. In such case it will be of interest to study its genetic architecture. The problem of Gentoo population analysis is important today because Gentoo is one of the indicator species of Antarctic ecosystem (Zhu et al. 2005) and a comparison of populations by trait frequencies could be useful for population studies and monitoring programs of Gentoo penguin.

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At the present stage of the study we have not yet collected data supporting an assumption regarding a heritable basis of the trait. The fact that no spot was recorded in Gentoo chicks bellow two months of age suggests a possible correlation between the spot appearance and age, i. e. the spot appears in growing up offspring to play some role related to mature birds. Such an assumption is reasonable taking into account that in the crèche only 4% of the twomonth chicks exhibited a small spot and that in adult birds the frequency of the spot was about five times higher than in juveniles. Much attention has been paid to the significance of colour and UV phenomena in avian intrasexual competition or intrasexual selection. Jouventin et al. (2005) have established that recently paired King Penguins (Aptenodytes patagonicus) had shown higher UV reflectance than courting ones. So, these authors consider the UV ornaments as a factor, which plays a role in pairing of breeding males and females and could serve as an indicator of sexual maturity. Also Mougeot and Arroyo (2006) have noted that UV signals play key roles in social and sexual signaling in birds. European starlings (Sturnus vulgaris) (Benett et al. 1997), Blue tits (Parus caeruleus) (Anderson et al. 1998; Hunt et al. 1998), and Bluethroats (Luscinia s. svecica) (Anderson and Amundsen 1997) use UV-reflecting plumage cues in mate choice. On the other hand, there are studies indicating that UV vision in birds plays an essential role in their foraging and hunting behaviour. So black grouses prefer UV-reflecting berries (Sűtari and al. 2002), kestrels are attracted to the vole urine and faces marks that reflect UV light (Viitala et al 1995; Koivula et al. 1999; Zampiga et al. 2006). The hunting success of nocturnal owls is best during clear nights due to the visibility of the scent markings in UV light (Koivula et al. 1997). Endoparasitism is viewing as a cause for the coloration variety (McGraw and Hill, 2000). It was found (Saks et al. 2003) that coccidian infection reduces the expression of plumage coloration in greenfinches (Carduelis chloris) by creating a deficiency of carotenoids available for deposition in ornamental feathers. Golemanski (2002) first described intestinal coccidiosis in P. papua (Livingston Island). One could suppose some relationship between spot and carotenoid pigments. Red, orange and yellow coloration is usually related to these pigments. Saks et al. (2003) have established that the plumage coloration in male greenfinches, signalizing their immunocompetence and health status, is carotenoid-based. The combination of carotenoid pigments with proteins generates many colors, from the brilliant yellow to red, in crustaceans, fish, and birds. The general distribution and metabolic pathways of carotenoids have been investigated in details (Katayama et al. 1971; Goodwin 1984; Davis 1985; Matsuno and Hirao 1989). We have not analyzed biochemically Gentoo beak but it seems reasonable to suppose at least partial carotenoid contribution in beak coloration because of Gentoo diet. It consists of about 50 – 80% crustaceans, mainly krill, which are a rich source of carotenoids (Berrow at all. 1999). However, there are reports regarding the potential for melanins to produce yellow colors in birds‘ plumage (McGraw et al. 2004). These authors have not detected carotenoid pigments in feathers of five avian species, including King Penguins (Aptenodytes patagonicus) and Macaroni Penguins (Eudyptes chrysolophus). More over McGraw et al. (2004) suggested that the yellow appearance of penguin and domestic chick feathers might be attributed to a new form of plumage pigment, never before described from bird feathers. A probable reason for appearance of the beak spot may be related to some aspects of the changes in Antarctic environment and especially to the increased ozone depletion above Antarctica. As known ozone layer protects the earth surface from the ultraviolet radiation.

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Evidence is provided that UV radiation generates H2O2 (Peus et al. 1998). In presence of H2O2 and under UV rays reactive oxygen species (ROS) are formed. What kind of way might lead from ozone depletion and ROS to ―yellow spot‖? The pigment melanin causes the black colour. It is an essential component in the bill structure. Incorporation of melanin granules into the bill keratin increases the hardness of the bill (Bonser and Witter 1993). Attaching of cysteine to DOPA-quinone after oxidative cyclization and polymerization results in the formation of reddish pigments (Angelov et al. 1995). DOPA-quinone is the third step of the melanin synthesis pathway beginning from tyrosine. Such a reaction is possible to occur in case of an increase of the concentration of cysteine, taking part in desintoxication and antioxidant processes. The low-molecular-weight thiols cysteine and especially glutathione are important antioxidants acting in cells. In the biochemical anabolic pathway the cysteine is a precursor of glutathione. Reduced glutathione (L-gamma-glutamyl-L-cysteinyl-glycine, GSH) is formed in a two-step enzymatic process including, first, the formation of gamma-glutamylcysteine from glutamate and cysteine, and second, the formation of GSH from gamma-glutamylcysteine and glycine (Nikolov 1971; Franco et al. 2007). The production of GSH mainly depends on the amount of available cysteine (van der Crabben et al. 2008). The cysteine synthesis is involved in the transsulfuration pathway. The first step of this pathway, from homocysteine to cystathionine, is catalyzed by cystathionine beta-synthase (CBS). The enzyme transsulfurase (cystathionase) converts cystathionine to cysteine. Persa et al. (2004), exploring some biochemical reactions in eye, showed that a transsulfuration pathway is present in the lens and that oxidative stress of H2O2 could increase the flux of this pathway activating the CBS enzyme. Thus, a lens under oxidative stress accumulates free and protein-bound cysteine. They reported also that oxidative stress transiently up-regulates the gene expression of CBS both in human lens epithelial cells and in pig lens. It seems reasonable to suppose that transsulfuration pathway exists in the penguin‘s bill and likely the accumulation of ROS in the cells as a result of the enhanced UV radiation cooperate to an increase of CBS synthesis and CBS activity. The increase of cysteine production and hence the GSH level may be considered as adequate adaptive reaction to the change of environmental conditions. On the other hand, the elevated cysteine level plays a role for involving of cysteine in melanin synthesis pathway via attaching to DOPA-quinine. The formation of reddish pigments could reflect in beak spot coloration. The frequency of the spot appearance in the three investigated geographical points did not unambiguously show a cline in the trait distribution (Table 2). Still the lowest frequency (19.7%) was found in the colony of the most north point (Livingston Island). The highest percentage of spotted penguins was found at Wiencke Island, situated between Livingston and Petermann Islands. Wiencke Island is by 2o14 southly from Livingston Island. The ozone depletion increases in south direction, to the pole. Longer studies and targeted investigations are required to determine precisely the most probable cause (or cause complex) for the appearance of this trait and to clarify its possible function. \

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ACKNOWLEDGMENTS This work was supported by the grant INTAS-2001–0517 and grant of Ukrainian Antarctic Center 04DF036-01(H/4-2004) and by grant B- 1615/2006 from the Bulgarian National Scientific Fund.

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Jouventin, P; Nolan, PM; Őrnborg, J; Dobson, ES (2005) Ultraviolet beak spots in king and emperor penguins. The Condor 107, 144-150. Katayama, T; Hirat, K; Chichester, CO (1971) The biosynthesis of astaxanthin-IV. The carotenoids in the prawn, Penaeus japonicus Bate (Part I). Bull Japan Soc SciFish 37 (7), 614-620. Koivula, M; Korpimaki, E; Viitala, J (1997) Do Tengmalm‘s owls see vole scent marks visible in ultraviolet light? Animal Behaviour 54, 873–877. Koivula, M; Koskela, E; Viitala, J (1999) Sex and age-specific differences in ultraviolet reflectance of scent marks of bank voles (Clethrionomys glareolus). J. Compar. Physiol. A 185, 561–564. Martinez, I (1992) Family Spheniscidae (Penguins), In: del Hoyo J., Elliott A. R, and Sargatal J., editors Handbook of the birds of the world Vol.1 (pp. 140-158). Barcelona: Lynx Editions ICBP. Matsuno, T; Hirao, S (1989) Marine Carotenoids. In: Ackman R.G., editor Marine Biogenic Lipids, Fats, and Oils Vol. 1 (pp. 251-388). Florida: CRC Press. Mcgraw, KJ and Hill, GE (2000) Differential effects of endoparasitism on the expression of carotenoid- and melanin-based ornamental coloration. Proceed. Roy Soc. London Series B 267, 1525-1531. Mcgraw, KJ; Wakamatsu, K; Ito, S; Nolan, PM; Jouventin, P; Dobson, FS; Austic, RE; Safran, RJ; Siefferman, LM; Hill, GE; Parker, RS (2004). You can‘t judge a pigment by its color: carotenoid and melanin content of yellow and brown feathers in swallows, bluebirds, penguins, and domestic chickens. The Condor 106, 390-395. Mougeot, F; Arroyo, BE; (2006) Ultraviolet reflectance by the cere of raptors. Biology Letters 2, 173–176. Nikolov, T. (1971) General Biochemistry. Naouka and Izkustvo Publ. House, Sofia. (In Bulgarian). Persa, C; Pierce, A; Ma, Z; Kabil, O; Lou, MF (2004) The presence of a transsulfuration pathway in the lens: a new oxidative stress defense system. Exp. Eye Res. 79 (6), 875886. Peus, D; Vasa, RA; Meves, A; Pott, M; Beyerle, A; Squillace, K; Pittelkow, MR (1998) H2O2 is an important mediator of UVB-induced EGF-receptor phosphorylation in cultured keratinocytes. J. Invest. Dermatol. 110 (6), 966-971. Saks, L; Ots, I; Hõrak, P (2003). Carotenoid-based plumage coloration of male greenfinches reflects health and immunocompetence. Oecologia 134, 301-307. Sambrook, J., Fritsch, E. F. and Maniatis, T. 1989. Molecular cloning: a Laboratory Manual, 2nd ed. New York: NY Cold Spring Harbor Laboratory Press, Cold Spring Harbor. Savov, AS; Telegeev, GD; Bichev, SN (2004) Sex identification of Gentoo penguins using PCR with specific primers. Antarctic Peninsula: key region for environment change study. Abstracts of the 2nd Ukrainian Antarctic Meeting. Kyiv, Ukraine, June 22-24. Siefferman, L and Hill, GE (2005) Evidence for sexual selection on structural plumage coloration in female eastern bluebirds (Sialia sialis). Evolution Inter J Org Evol 59 (8), 1819-1828. Sokal, RR and Rohlf, FJ (1995) Biometry: the principles and practice of statistics in biological research, 3rd edition. New York: W. H. Freeman and Co. Stevenson, HM and Anderson, LH (1994) The bird life of Florida. Gainesville: University Press of Florida.

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Stonehouse, B (1970) Geographic variation in Gentoo penguins. IBIS 112, 52-57. Sűtari, H. and Vűtala, J. 2002. Behavioral evidence for ultraviolet vision in a tetraonid species – foraging experiment with black grouse Tetrao tetrix. J. Avian. Biol. 33, 199-202. Trivelpiece, WZ; Bengston, JL; Trivelpiece, SG; Volkman, NJ (1986) Foraging behaviour of Gentoo (Pygoscelis papua) and chinstrap (Pygoscelis antarctica) penguins determined by new radiotelemetry techniques. Auk 103, 777-781. van der Crabben, SN; Wijburg, FA; Ackermans, MT; Sauerwein, HP (2008) Effect of cysteine dosage on erythrocyte glutathione synthesis rate in a patient with cystathionine beta synthase deficiency. J. Inherit. Metab Dis. Jan 24 [Epub ahead of print] Viitala, J; Korplmäki, E; Palokangas, P; Koivula, M (1995) Attraction of kestrels to vole scent marks visible in ultraviolet light. Nature 373, 425–427. Zampiga, E; Gaibani, G; Csermely, D; Frey, H; Hoi, H (2006) Innate and learned aspects of vole urine UV-reflectance use in the hunting behaviour of the common kestrel Falco tinnunculus. J. Avian. Biol. 37, 318–322. Zhu, R; Sun, L; Yin, X; Liu, X (2005) Geochemical evidence for rapid enlargement of a gentoo penguin colony on Barton Peninsula in the maritime Antarctic. Antarctic. Sci. 17 (1), 11-16.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 10

ASSESSING POPULATION VIABILITY OF FOCAL SPECIES TARGETS IN THE WESTERN FOREST COMPLEX, THAILAND Yongyut Trisurat1 and Anak Pattanavibool2 1

Faculty of Forestry, Kasetsart University, Bangkok 10900, Thailand 2 Wildlife Conservation Society Thailand Program, Nonthaburi 11120, Thailand

ABSTRACT The Western Forest Complex (WEFCOM) in Thailand covers approximately 19,000 km2. This protected area complex comprises 11 national parks and 6 wildlife sanctuaries. During 1999-2004, the Danish Government provided financial support to the Royal Forest Department to manage this forest complex through the ecosystem management approach. The WEFCOM Project employed rapid ecological assessment (REA) to determine the current distribution statuses of wildlife species, develop a Geographic Information System (GIS), and define habitat uses of wildlife. This paper is based upon the achievements of the WEFCOM Project. It aims to define suitable habitats of selected key wildlife species in the WEFCOM and to assess the current and desired statuses under a population viability estimate for those species. The focal wildlife species were sambar (Cervus unicolor), gaur (Bos gaurus), banteng (Bos javanicus), Asian elephant (Elephas maximus), and tiger (Panthera tigris. We used logistic multiple regression to determine habitat uses of wildlife and employed minimum dynamic area and landscape matrix surrounding suitable habitats as criteria to assess population viability. The results indicate the current suitable habitat mainly remains in Huai Kha Khaeng and Thung Yai wildlife sanctuaries. In addition, the current viability condition is good for sambar, fair for gaur, elephant and tiger; and poor for banteng. However, landscape matrices outside the suitable habitats for all species range from moderate to high connection of native vegetation. If the project aims to upgrade the viabilities to the next level in the next 10 years, park rangers and multi-stakeholders have to increase the amount of suitable 

Email: [email protected]; Tel: 662-579-0176; Fax: 662-942-8107

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Yongyut Trisurat and Anak Pattanavibool habitats for all species from 12,630 km2 or 67% of the WEFCOM to 16,750 km2 or 89%. By doing this, the number of suitable patches would significantly decrease and the mean patch size would increase substantially, thereby indicating less fragmentation.

Keywords: Western Forest Complex (WEFCOM); population viability; habitat suitability; landscape indices; GIS.

1. INTRODUCTION The World Conservation Union (IUCN) defines a protected area as ―an area of land and/or sea especially dedicated to the protection and maintenance of biological diversity, and of natural and associated cultural resources, and managed through legal or other effective means‖ (IUCN, 1984). In Thailand, there were 282 protected areas, covering about 18% of the country‘s land area in 2004 (Trisurat, 2006). Management of protected areas previously was limited to the boundary legally declared in the National Gazette. Cooperation with park officials beyond the park boundary for effective protection and management was relatively low and multi-stakeholders besides the responsible agency have been rarely involved in management although the New Constitution of Thailand of 1997 clearly defined their roles and responsibility in natural resources management (DARUDEC, 1999). In the past four decades, protected areas have been selected on a site-by-site basis in an ad-hoc way, and often based on the opportunity to preserve remaining forest, as well as to promote tourism. These management practices are unlikely to ensure the long-term protection of biodiversity and the ecological processes upon which biodiversity depends as defined in protected areas‘ management objectives (IUCN, 1984). Therefore, the Royal Forest Department (RFD) and Danish Cooperation for Environment and Development (DANCED) implemented the Western Forest Complex (WEFCOM) Project during 1999-2004. The overall objective for this initiative was to maintain the health of the ecological systems by using the ―Ecosystem Management Approach‖ with community participation. The ecosystem approach is a strategy for management of land, water and living resources that promotes conservation and sustainable use in an equitable way, which was adopted at the Second Conference of the Parties of the Convention on Biological Diversity (Smith and Maltby, 2003). This approach has recognized the need to incorporate ecological processes, disturbances and biological population viability into the planning processes. By conserving viable samples of the whole ecosystem, it is anticipated that all of the species contained within them will, at least, have a fighting chance to survive in the long-term. Viable population is defined as the minimum number in populations (MVP) of species that can persist for long periods of time (Wielgus, 2002). There are many methods for assessing population viability, ranging from the quantitative and data-gathering approaches to more expert-driven and qualitative methods (Beissinger and McCullough, 2002). In practice, there are relatively few species for which population viability analysis has been performed because this technique is available only for a data-rich species. Thus, many scientists use the 50/500 rule of thumb as an effective population size over the short-term (Soule, 1987). For instance, the British Columbia Ministry of Environment established a benchmark population

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of grizzly bears ranging from 100-250 animals and determined the reserve sizes that can accommodate these populations (Wielgus, 2002). More recent analyses suggest that an effective population size of about 1,000 is needed to allow continued evolution and prevent the accumulation of harmful mutation (Allendorf and Ryman, 2002). Based on MVP-related research over the past 30 years, Traill et al. (2007) found that the median MVP for mammals was 3,876 individuals. For practical implementation, there are increasingly fewer populations, especially of large mammals, that would meet the effective population size of 1,000 or more. Thus, a promising approach to maintaining MVP is to increase connectivity among fragmented habitats. The common qualitative technique or expert opinion to assess population viability is based on the criteria of species occurrence, habitat condition and landscape context surrounding the occurrence (Groves, 2003). Recently, the methods for assessing the viability of species that combine static habitat models to accommodate the minimum viable population with population viability analyses has become a promising approach in regional conservation plans, especially when digital data are available (Brito and Figueredo, 2003; Leroux et al., 2007). This approach is named minimum dynamic area (MDA), which describes the smallest area with natural disturbance regime, which maintains internal recolonization sources, and hence minimizes extinction risk (Pickett and Thompson, 1978). This paper is based on the achievements of the WEFCOM project. It aims to define suitable habitats of selected key wildlife species in the WEFCOM and to assess the current and desired status of population viability for these species.

2. STUDY AREA The Western Forest Complex (WEFCOM) is situated along the Thailand-Myanmar border. It comprises 17 contiguous protected areas, including six wildlife sanctuaries, nine national parks and two proposed national parks (Figure 1). The toal land area of WEFCOM is approximately 19,000 km2 or approximately 4% of the country‘s land area. Among these 17 areas, Huai Kha Khaeng Wildlife Sanctuary is the largest protected area, covering 2,780 km2. Salakpra Wildlife Sanctuary is the oldest protected area in this complex having been declared as the first wildlife sanctuary in 1965. There are 162 ranger stations scattered in the WEFCOM landscape mainly situated in the eastern buffer zone of the complex. The WEFCOM is the largest intact forest area in Thailand and also within mainland Southeast Asia. It is situated in six provinces in western Thailand. between latitudes 14 08 16 37 North and longitudes 98 11 - 99 32 East. The WEFCOM is part of the Tenassarim Range extending southward along the Myanmar border. The landscape in the north and the west is rugged highlands. The area slopes gently towards the south and is drained by the Huai Kha Khaeng, which is the main perennial stream in this complex (WEFCOM, 2004). Based on Landsat image interpretations and field assessments in 2000, approximately 90% of the total land area is under forest cover. Mixed deciduous forest is dominant in the complex covering the highest percentage of the WEFCOM area (6,172 km2 or 32%). Approximately 36% of the WEFCOM is comprised of various evergreen forests. Roughly one third can be considered as relatively intact and undisturbed forest. Large tracts of intact and healthy forest still occur in Thung Yai Naresuan and Huai Kha Khaeng Wildlife Sanctuaries but the forest becomes more scattered and fragmented in other areas (WEFCOM, 2003,

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2004). Remaining forest cover along the boundary and surrounding enclave communities is threatened by agricultural encroachment. A socio-economic survey revealed that there were 112 communities situated inside the WEFCOM harboring approximately 5,400 households and 27,700 individuals. Besides this, there were another 103 villages located within a 3-km distance of the WEFCOM, mainly found along the eastern border (WEFCOM, 2002).

Figure 1. Protected area system of the Western Forest Complex in Thailand.

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The WEFCOM has been nationally and internationally recognized as the key area of terrestrial biodiversity in mainland Southeast Asia. It is located at the crossroads of three plant geographies: Indo-Burma, Indo-China and Indo-Malaya; and two zoo-geographies: India Indo-China and Sundaic Sub-region (Nakhasathien and Stewart, 1990; Smitinand, 1987). Based on Wikramanayake et al. (2000), the WEFCOM encompasses two important ecoregions, namely the Kayah-Karen montane rain forests ecoregion and Tenasserim-South Thailand semi-evergreen rain forests. This bio-geographical overlap provides a unique assemblage of Asian species. Therefore, UNESCO designated Huai Kha Khaeng and Thung Yai Naresuan Wildlife Sanctuaries, two of the largest protected area units in the core area of the WEFCOM, as a Natural World Heritage Site in 1991. Some endangered species are tiger (Panthera tigris), Asian elephant (Elephas maximus), gaur (Bos gaurus), banteng (Bos javanicus), tapir (Tapirus indicus), great hornbill (Buceros bicornis), rufous-necked hornbill (Aceros nipalensis), green peafowl (Pavo mulicus), and giant frog (Rana blythii).

3. METHODS The general processes consisted of four steps, namely: 1) identify species conservation targets; 2) estimate habitat suitability for selected species; 3) assess the current and desirable condition of targets‘ viability; and 4) delineate the congregation locations of target species in the WEFCOM landscape. Activities for each step are summarized as follows:

3.1. Identify Species Conservation Targets Conservation targets are those features or elements of biodiversity (ecosystem, species and genetics) that planners seek to conserve within a system of conservation areas (Groves, 2003). In this study, they are those species that make the WEFCOM a globally important conservation site. Wildlife specialists and the planning team comprising protected area staff, scientists and NGO representatives, set four criteria to select the species targets, namely, wide-range distribution, endangered status or ecosystem indicator, specialist availability and public interest.

3.2. Develop Habitat Suitability Maps for Selected Species The WEFCOM Project employed ecological rapid assessment (REA) (Sayre et al., 2000) to gather wildlife occurrences in the study area. Wildlife signs and visual sightings were recorded by park rangers who had been trained in map reading, Global Positioning System (GPS) and identification of wildlife tracks and signs prior to actual survey. In addition, we applied the logistic multiple regression model to generate the suitable habitats for selected species (Trisurat 2003). The present and pseudo-absent data (chosen outside 1 km buffer from present locations) were treated as dependent variables in the habitat suitability model. The independent variables included biophysical and human disturbance factors. The biophysical factors were vegetation type, distance to river and stream, slope, elevation and

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aspect, while the human factors consisted of distance to ranger station and distance to villages. The raster-based modeling in GIS ArcView 3.2 (ESRI, 1992) of 100 m resolution was used to perform all spatial analysis functions. Seventy-five percent of wildlife observations were used to develop the model and the remaining 25% were used to test the model accuracy. According to Petdee (2000) and Prommakul (2003), sambar, banteng and gaur are primary prey for tiger, therefore the suitability maps of these ungulate species were aggregated to derive a prey coverage as an additional layer for tiger habitat. The logistic multiple regression model is written as: Zi

Prob

event

=

e 1 e

Zi

where Z i is the linear combination model of species i The output maps produced from the predicted models were continuous probability values ranking from 0.0 – 1.0. Later these probability values were categorized into 4 classes to determine habitat suitability as follows: Prob. Value 0.0 – 0.2 Prob. Value 0.2 – 0.4 Prob. Value 0.4 – 0.6 Prob. Value 0.6 – 1.0

= = = =

Unlikely Less Likely Likely Most Likely

3.3. Assess the Status of Species Targets‘ Viability We used the Five-S Framework developed by the Nature Conservancy (2000) to evaluate the long-term viability of a focal conservation target‘s occurrence in the WEFCOM landscape. In fact, this framework provides three criteria for assessing the persistence of focal species: size, condition and landscape context. For the size criterion, we used the extent of suitable habitats (likely and most likely) to support MVP or minimum dynamic area (MDA) as a proxy indicator. In addition, we used habitat fragmentation (the percentage of native vegetation surrounding the suitable habitats (2-km buffer) as a landscape context criterion. Condition attributes of wildlife population (e.g. species composition, structure and biotic interactions) were not taken into account due to limited population data. The MVP size of each species was estimated according to the 50/500 rule, carrying capacity and the recommendation of Thai wildlife experts. The MDA is simply derived by multiplying the MVP and the home range of species. For instance, the home ranges of tiger in the Thung Yai are between 50 and 200 km2 and its average coverage is 80 km2 (Prommakul, 2000) and the optimum MVP of tiger in the WEFCOM is 100 individuals. Therefore, the estimated MDA is 8,000 km2. Meanwhile, a simple grading scale (very good, good, fair or poor) was used to assess the current and desired conditions in the next 10 years. Table 1 elaborates the context of each viability class.

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Table 1. Criteria for assessing viability of species conservation targets in the WEFCOM Ranking 1/ Very good: functioning at an ecologically desirable status, and requires little human intervention

Ecological indicators Size 2/ The extent of likely or most likely suitable habitats can accommodate >80% of viable population size.

Good: functioning within its range of acceptable variation; it may require some human intervention Fair: lies outside its range of acceptable variation and requires human intervention

The extent of likely or most likely suitable habitats can accommodate 60-80% of viable population size.

Poor: restoration or preventing extirpation practically impossible

The extent of likely or most likely suitable habitats can accommodate <40% of viable population size.

The extent of likely or most likely suitable habitats can accommodate 40-60% of viable population size.

Landscape context 2/ Highly connected, the suitable habitats (patch > 2 km2) are surrounded by intact natural vegetation ( >60% of intact forest within 2 km). Moderately connected, the suitable habitats (patch > 2 km2) are surrounded by moderately intact natural vegetation (> 4060% of intact forest within 2 km). Moderately fragmented, the suitable habitats (patch > 2 km2) are surrounded by altered vegetation (> 20-40% of intact forest within 2 km). Highly fragmented, the suitable habitats (patch > 2 km2) are entirely or almost entirely surrounded by altered vegetation and human-induced land use (< 20% of intact forest within 2 km).

Remarks: 1/ described by TNC (2000); 2/ defined by the planning team and wildlife experts

3.4. Delineate the Congregation Areas of Target Species The current congregation areas were simply derived from combining the likely and most likely suitable habitats of all target species. The output grid map shows the current location of suitable habitats of focal conservation target species. On the other hand, the extend of suitable habitats for each species to desired level of population viability was obtained from expanding the existing suitable habitat to meet the expected size based on the probability values as defined in the previous step. Later, all desired suitability maps were aggregated as done for the current status.

4. RESULTS AND DISCUSSION 4.1. Selected Target Wildlife Species Five wildlife species were selected by wildlife scientists and a planning team as the conservation targets. These species were tiger, Asian elephant, gaur, banteng, and sambar based on the selection criteria and appropriate observations to run a logistic model (Pattanavibool et al., 2003; Vanichbancha, 2001). The observations for elephant, sambar, gaur, tiger, and banteng were 960, 700, 641, 224, and 131 points, respectively.

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Tiger, elephant and banteng are classified as endangered species by IUCN (2000). According to Lekakul and McNeely (1977), tigers are found in a very wide range of habitat types; they only require sufficient prey species, water and shelter from the sun and occupy a large home range (50-200 km2), the average size of which in Thung Yai is approximately 80 km2 (Prommakul, 2003). They also require good protection from tiger and prey poaching pressures (Karanth et al., 2004). In addition, Simcharoen et al. (2007) used photographic capture-recapture sampling to estimate tiger density in the core area of Huai Kha Khaeng. The sample area yielded a density estimate of 3.98 tigers per 100 km2. Elephant is a top herbivore and found in a variety of forested areas. The home range sizes of elephant herds are between 105 and 320 km2 (Sukumar, 1989). Similarly, banteng lives in loose herds of 2-20 individuals. Prayurasiddhi (1997) reported that the annual home range size was 44 km2 for banteng herds in Huai Kha Khaeng. Gaur is classified as a vulnerable species by IUCN (2000). Gaur live in herds of 3-40 individuals and the home ranges of gaur herds are between 29.9 and 52.1 km2 (Conry, 1989). Sambar still exist in many protected areas in Thailand and make a significant contribution to the long-term integrity and conservation values of the WEFCOM. It is a preferred prey species of several carnivores, including tiger.

4.2. Suitable Habitat Models The results of logistic multiple regressions indicated that three physical factors and two anthropogenic factors were significantly related to the distributions of elephant, sambar, gaur, tiger, and banteng. The environmental factors were elevation, slope, distance to stream, distance to village, and distance to ranger station. The logistic regression models and overall accuracy at cut-off of 0.5 are shown below. Z

sambar

=

1.5191 – 0.0009Alt -0.0002Rst - 0.0136Slp –

0.0003Str+0.0003Vil; overall accuracy 68.06% Z

banteng =

-1.3795 - 0.0018Alt -0.0008Rst - 0.0939Slp –

0.0005Str + 0.0020Vil; overall accuracy 91.97% Z

gaur

=

-3.3434 - 0.0042Alt -0.0001Rst - 0.0946Slp –

0.0002Str + 0.0003Vil; overall accuracy 83.13% Z

=

elephant

1.4603 - 0.0013Alt -0.0001Rst -0.1109Slp –

0.0003Str +0.0002Vil; overall accuracy 83.32% Z

tiger

=

1.1335 + 0.0024Alt -0.0003Rst - 0.1327Slp –

0.0003Str + 0.0003Vil; overall accuracy 84.18% where

Alt = altitude (m);

Rst = distance to ranger station (m);

Assessing Population Viability of Focal Species Targets in the Western Forest …

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Slp = slope (%); Str = distance to stream (m); Vil = distance to village (m). The GIS habitat models indicated that most species prefer to inhabit low altitude, close to ranger stations, low slope, close to streams and far from villages. These areas are safe from illegal poaching and other human disturbances with near year-round water sources. The overall accuracies for all species except sambar, were greater than 83%, being especially high for banteng because its habitat is concentrated in small patches. For sambar, the model indicates low accuracy because it uses various vegetation types. In addition, the pseudoabsent data may contain occurrence locations. Figure 2 shows the likelihood of habitat uses of the five focal species in the WEFCOM, while Table 2 presents the coverage of each suitable class. The distributions of each species are detailed below. Suitable habitats of sambar (likely and most likely) are found in deciduous forest and open areas in the core area and in the southern part of the WEFCOM, covering approximately 8,032 km2 or 43% (Figure 2a). Sambar is unlikely to be present in densely populated areas and the steep terrain mainly in the northern and western parts of WEFCOM. Banteng or wild ox is now restricted to small and fragmented populations congregated along Huai Kha Khaeng stream. Other possible patches are in Khao Laem and Salakpra (Figure 2b). The suitable habitats cover less than 4% of the complex. The main threats to this species are poaching, habitat destruction and human encroachment, and overgrazing by domestic cattle (Prayurasiddhi, 1997). Besides, the habitats of banteng have been degraded in recent years due to the RFD and Department of national Park, Wildlife and Plant Conservation (DNP) have implemented the policy of 100% forest fire prevention nationwide, especially in protected areas. This misunderstood concept influenced vegetation community dynamics and dependent fauna. Parts of open deciduous forests and grassland which are favorable habitats for ungulate species have been invaded by pioneer species such as Cratoxylum spp. and Eupatorium odoratum. Table 2. Predicted suitable habitats for selected wildlife species in the WEFCOM

Species Sambar Banteng Gaur Elephant Tiger 1/

Extent of Suitability Class (%/km2) Unlikely Less Likely 37.9 19.2 7,099 3,596 89.4 7.3 16,731 1,362 41.1 17.1 7,699 3,211 26.6 21.7 4,951 4,058 63.6 16.7 11,922 3,126

= Likely suitability + most likely suitability.

Likely 14.6 2,736 2.6 492 12.8 2,390 18.3 3,425 9.3 1,736

Most Likely 28.3 5,296 0.7 140 29.0 5,427 33.6 6,292 10.4 1,944

Suitable habitat 1/ 42.9 8,032 3.4 632 41.7 7,817 51.9 9,717 19.7 3,680

Figure 2. Current suitable habitats of selected species in the WEFCOM based on probability values.

Figure 3. Comparison of congregation areas of suitable habitats for all species in current condition and desired condition.

Table 3. Assessing population viability in WEFCOM

1

Species target Sambar

Banteng

Good

Very good

Current Rating 2/

Desired Rating 3/

3,0004,500

4,5006,000

6,000-7,500

Very good

Very good

<20%

20-40%

40-60%

> 60%

Very good

NA 5/

<1,700

1,7002,600

2,6003,500

3,500-4,400

Poor

Fair

Category

Key attribute

Indicator

Size

Minimum dynamic area to support viable population (MVP = 3,000; HR4/ = 1.7-4.0 km2 (Sankar 1994); MDA = 7,500 km2) Habitat fragmentation

The extent of likely or most likely suitable habitats in km2

<3,000

% intact forest surrounding 2km buffer of suitable habitats The extent of likely or most likely suitable habitats in km2

Landscape context

2

Viability Assessment 1/ Poor Fair

Size

Minimum dynamic area to support viable population (MVP = 500; HR4/ = 44 km2//herd (Prayurasiddhi, 1997); MDA = 4,400 km2)

Table 3 – Continued

Species target

3

Gaur

Category

Key attribute

Indicator

Poor

Viability Assessment 1/ Fair Good

Landscape context

Habitat fragmentation

<20%

20-40%

40-60%

Size

Minimum dynamic area to support viable population (MVP = 2,000; HR4/ = 29.9 – 52.1 km2/herd (Conry, 1989); MDA = 13,300 km2) Habitat fragmentation

% intact forest surrounding 2km buffer of suitable habitats The extent of likely or most likely suitable habitats in km2

<5,320

5,3207,980

<20%

20-40%

Landscape context

% intact forest surrounding 2km buffer of suitable habitats

Current Rating 2/

Desired Rating 3/

> 60%

Very good

NA

7,98010,640

10,64013,300

Fair

Good

40-60%

> 60%

Good

NA

Very good

Table 3 – Continued

4

Species target Elephant

Viability Assessment 1/ Poor Fair

Category

Key attribute

Indicator

Size

Minimum dynamic area to support viable population (MVP = 500; HR4/ = 105320 km2/herd (Sukumar, 1989); MDA = 20,000 km2) Habitat fragmentation

The extent of likely or most likely suitable habitats in km2

<8,000

% intact forest surrounding 2km buffer of suitable habitats

<20%

Landscape context

Current Rating 2/

Desired Rating 3/

16,00020,000

Fair

Good

> 60%

Very good

NA

Good

Very good

8,00012,000

12,00016,000

20-40%

40-60%

Table 3 – Continued

5

Species target Tiger

Viability Assessment 1/ Poor Fair

Category

Key attribute

Indicator

Size

Minimum dynamic area to support viable population (MVP = 100; HR4/ = 50-200 km2 (Prommakul, 2003); MDA = 8,000 km2) Habitat fragmentation

The extent of likely or most likely suitable habitats in km2

<3,200

% intact forest surrounding 2km buffer of suitable habitats

<20%

Landscape context

Current Rating 2/

Desired Rating 3/

6,4008,000

Fair

Good

> 60%

Good

NA

Good

Very good

3,2004,800

4,8006,400

20-40%

40-60%

1/ Viability assessment is based on measurable size and landscape context criteria; 2/ at present time; 3/ in the next 10 years; 4/ home range; 5/ Not applicable.

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Figure 2c reveals that the distribution of gaur is more limited than that of elephant (Figure 2d) because it is more sensitive to human pressure. The suitable habitats cover approximately 42% of the WEFCOM area and it is most likely found in the core area. On the other hand, the chance to observe gaur in other protected areas is minimal. In fact, elephant can be found in a variety of habitats (Sukumar, 1989) but the GIS habitat suitability map shows that the current suitable habitats are basically situated in the core area of WEFCOM and areas along the Myanmar border (Figure 2d), covering approximately 9,717 km2. Areas in the west, east, north and parts of southern landscapes are not suitable for elephant because there are a lot of human settlements and they are easy accessible. Figure 2e shows that the most suitable habitats for tiger can be found in Huai Kha Khaeng followed by Thung Yai East and Thung Yai West. The total suitable areas cover approximately 20% of the WEFCOM. The results are consistent with the studies of Prommakul (2003) and Simcharoen et al. (2007) which revealed that the home range of tigers in Huai Khai Khaeng is smaller than in Thung Yai due to higher abundance of prey species.

4.3. Current and Desired Conditions of Population Viability The existing suitable habitats of sambar encompass approximately 8,000 km2, thus they have potential to hold more than 3,000 individuals. On the other hand, the suitable habitat of banteng is minimal and ranked as poor condition (Table 3). It can accommodate less than 40% of a viable population size (500 individuals). However, the largest suitable habitat patch is located in the core area of Huai Kha Khaeng and surrounded by dry diptercarp forest and well protected by park rangers. Even though the actual population size of gaur is not known, the existing suitable habitats (7,800 km2) are assumed to accommodate approximately 1,0001,200 individuals (40-60% of viable population). In addition, the adjacent area within a 2 km buffer is highly intact. Thus, the size criterion is classified as fairly viable, while landscape context is in good condition. The existing suitable habitat of elephant covers about 9,700 km2 and could hold approximately 240-250 individuals. The size criterion is ranked as fair. However, the landscape matrix is highly connected and has very high potential to facilitate elephant movement in the WEFCOM landscape. The suitable habitats of tiger encompass approximate 3,700 km2. Based on the average home range size reported by Prommakul (2003), the current suitable habitats are likely to accommodate approximately 40-50 tigers, thus, they are in fair viability status but the landscape context outside occurrence patches is moderately intact with natural vegetation. The project aims to upgrade the viabilities of all species (except sambar) to the next level in the next 10 years. In order to achieve this target, park rangers and multi-stakeholders have to increase the amount of suitable habitats for banteng, gaur, elephant and tiger to an area between 1,700-2,600 km2, 7,980-10,640 km2, 16,000-20,000 km2, and 6,400-8,000 km2, respectively (Table 3).

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4.4. Location of Congregation Areas of Population Viability We overlaid the current suitable habitats of all five species using GIS method to derive current congregation areas, and the desired suitable habitats to derive desired condition to support viable populations. The results showed that the total area of current congregation habitats cover approximately 12,630 km2 or 67% of the WEFCOM (Figure 3a). Concentrations of five focal species are clustered in three places. The largest patch is located in Huai Kha Khaeng and adjoining Thung Yai Naresuan. On the ground, the survey teams observed that there are a number of migratory routes of the landscape species e.g. tiger, elephant, gaur found in these areas. The second dominant area is located in the western part of WEFCOM extending along the Myanmar border. The smallest patch can be found in the north. Meanwhile, the desired congregation habitats cover nearly 16,750 km2 or 89% (Figure 3b). Basically, these areas cover the whole of Huai Kha Khaeng, 80% of Thung Yai East and West and extend to the Myanmar border. In the north, the size of small and fragmented patches increases significantly. The desired areas could hold more wildlife population, in addition to connect fragmented and patchy suitable habitats. However, areas along the western boundary and in the south close to villages are still not feasible to support viable populations.

CONCLUSION The WEFCOM project aims to maintain the health of the ecological systems by using species conservation targets. Five species were selected, namely sambar deer, banteng, gaur, elephant and tiger. Their habitat suitability areas were determined using logistic multiple regression method. The results of GIS habitat models indicated that most species prefer to inhabit low altitude, close to ranger station, low slope, close to stream and far from village locations. The suitable habitats (likely and most likely) of sambar deer, banteng, gaur, elephant and tiger covered approximately, 43%, 3%, 42%, 52% and 20% of the WEFCOM landscape, respectively. The MVP of sambar, banteng, gaur, elephant and tiger in the WEFCOM defined by the planning team and wildlife experts were 3,000, 500, 2,000, 500 and 100 individuals, respectively. We used the MDA or suitable habitats to accommodate MVP population and habitat fragmentation surrounding the suitable to assess the viability of target species. We compared the MDA and predicted suitable habitat derived from the GIS habitat modeling and found that the viability of sambar is in very good condition, while the viability status of gaur, elephant and tiger is in fair condition. More importantly, the current viability status for banteng is extremely poor. However, landscape matrices outside the suitable habitats are in good or very good condition for all species. The planning team of WEFCOM would like to upgrade the level of viabilities of these species to next level in the next 10 years. In order to achieve these targets, park rangers and multi-stakeholders have to increase the congregation habitats from 12,630 km2 or 67% of the WEFCOM to 16,750 km2 or 89%. By doing this, the fragmented suitable patches would become more aggregated.

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The results of GIS modeling may include omission errors (false negative) and commission errors (false positive) derived from pseudo absence data and the grid-based habitat suitability models. In addition, the predicted MDA may overestimate the actual species extent. Nevertheless, the results of this initiative are of value and the approach can become effective tool to implement ecosystem approach when demographic data is limited to do perform traditional population viability analysis.

ACKNOWLEDGMENTS We would like to thank the Danish Government for funding the Western Forest Complex for Ecosystem Management (WEFCOM) Project. In addition, we are grateful to the Superintendents and the staff of the WEFCOM Project for providing information and The Nature Conservancy (TNC) for training on the Five-S Framework. Adrian Hillman is acknowledged for his contribution to edit the manuscript.

REFERENCES Allendorf, F W and Ryman, N. 2002. The role of genetics in population viability analysis. In Beissinger, S R and McCullough, D R (eds). 2002. Population Viability Analysis. The University of Chicago Press, Chicago. Beissinger, S R and McCullough, D R (eds). 2002. Population Viability Analysis. The University of Chicago Press, Chicago. Brito, D and Figueredo, M S L. 2003. Minimum viable population and conservation status of the Atlantic Forest spiny rat Trinomys eliasi. Biological Conservation 112: 153-158. Conry P.J. 1989. Gaur (Bos gaurus) and development in Malaysia. Biological Conservation 49: 47-65. DARUDEC. 1999. Consultancy Services Agreement for WEFCOM Ecosystem Management, Thailand. DANCED. ESRI, 1992. Cell-based Modeling with GRID. Environmental Systems Research Institute Inc., New York. Groves, C R. 2003. Drafting a Conservation Blueprint: A Practitioner‘s Guide to Planning for Biodiversity. The Nature Conservancy, Island Press, Washington. IUCN. 1984. Categories, Objectives and Criteria for Protected Areas. The World Conservation Union (IUCN), Gland, Switzerland. IUCN. 2000. IUCN Red List of Threatened Species. World Conservation Union, Gland, Switzerland. Karanth, U, Chundawat, R S, Nichols, J D and Kumar, N S. 2004. Estimation of tiger densities in the tropical forests of Panna, Central India, using photographic capturerecapture sampling. Animal Conservation 7: 285-290. Lekakul, B and McNeely, J. 1977. Mammals of Thailand. Kurusaphra Press, Laprao, Thailand.

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Leroux, S J, Schmiegelow, F K A, Lessard, R B and Cumming, S G. 2007. Minimum dynamic reserves: a framework for determining reserve size in ecosystems structured by large disturbances. Biological Conservation 138: 464-473. Nakhasathien, S and Stewart-Cox, B. 1990. Nomination of The Thung-Yai-Huai Kha Khaeng Wildlife Sanctuary to be a UNESCO World Heritage Site. Wildlife Conservation Division, Royal Forest Department, Bangkok, Thailand. Pattanavibool, A, Nakhonchai, T, Vinichpornsawan, S and Kieowan, N. 2003. Wildlife Rapid Ecological Assessment Technique Manual. Western Forest Complex, Western Forest Complex. Natural Resources Conservation Office, Royal Forest Department. Petdee, A. 2000. Feeding habits of the tiger (Panthera tigris (Linnaeus)) in Huai Kha Khaeng wildlife sanctuary by fecal analysis. M.S. thesis, Kasetsart University, Bangkok. Pickett, S T A and Thompson, J N. 1978. Patch dynamics and the design of nature reserves. Biological Conservation 13: 27-37. Prayurasiddhi, T. 1997. The Ecological Separation of Gaur (Bos gaurus) and Banteng (Bos javanicus) in Huai Kha Khaeng Wildlife Sanctuary, Thailand. Ph.D. Dissertation, University of Minnesota. Prommakul, P. 2003. Habitat utilization and prey of the Tiger (Panthera tigris (Linnaeus)) in the eastern Thung Yai Naresuan Wildlife Sanctuary. M.S. Thesis, Kasetsart University, Bangkok. Sankar, K. 1994. The ecology of three large sympatric herbivores (chital, sambar and nilgai) with special reference for reserve management in Sariska Tiger Reserve, Rajasthan. Ph.D. Thesis. University of Rajasthan, Jaipur. Sayre. R, Roca, E, Sedaghatkish, G, Young B, Keel, S, Roca, R, and Sheppard, S. 2000. Nature in Focus: Rapid Ecological Assessment. The Nature Conservancy, Island Press, Washington D.C. and California. Simcharoen, S, Pattanavibool, A, Ullas, K, Nichols, J D and Kumar, N S. 2007. How many tigers Panthera tigris are there in Huai Kha Khaeng Wildlife Sanctuary, Thailand? An estimate using photographic capture-recapture sampling. Oryx 41: 447-453. Smith, R D and Maltby, E. 2003. Using the Ecosystem Approach to Implement the Convention on Biological Diversity: Key Issues and Case Studies. IUCN, Gland, Switzerland and Cambridge, U.K. Smitinand, T. 1987. Flora of Thung Yai and Huai Kha Khaeng Wildlife Sanctuaries. Forest Herbarium, Royal Forest Department. Soule, M E. 1987. Viable Populations for Conservation. Cambridge University Press, Cambridge. Sukumar, R. 1989. Ecology of the Asian Elephant in Southern India. I. Movement and Habitat Utilization Patterns. Journal of Tropical Ecology, 5: 1-18. The Nature Conservancy. 2000. The Five-S Framework for Site Conservation: A Practitioner‘s Handbook for Site Conservation Planning and Measuring Success. The Nature Conservancy, Arlington, VA. Traill, L W, Bradshaw, C J A and Brook, B W. 2007. Minimum viable population size: a meta-analysis of 30-years of published estimates. Biological Conservation 139: 159-166. Trisurat, Y. 2003. Ecosystem-based Management Zones of Western Forest Complex in Thailand. In Munro, N W P, Dearden, P, Herman, T B, Beazley, K, and NielsenBondrup, S (Eds). Making Ecosystem-based Management Work. Electronic Proceedings of the Fifth International Conference on Scientific and Management of Protected Areas

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Association, May 6-11, 2003, University of Victoria, Vancouver, Canada. Science and Management of Protected Areas Association, Wolfville, Nova Scotia. 2006. Applying Gap Analysis and a Comparison Index to Assess Protected Areas in Thailand. Environmental Management 39: 235-245. Vanichbancha, K. 2001. Multivariate Analysis Using SPSS for Windows. Chulalongkorn University Printing, Bangkok. WEFCOM. 2002. Rapid Assessment of Socio-economic in Western Forest Complex. Natural Resources Conservation Office, Royal Forest Department. 2003. The Vegetation and the Flora of the Western Forest Complex: Using rapid ecological assessment and vegetation description in the WEFCOM area. Natural Resources Conservation Office, Royal Forest Department. 2004. GIS Database and Its Applications for Ecosystem Management. Natural Resources Conservation Office, Royal Forest Department. Wielgus, R B. 2002. Minimum viable population and reserve sizes for naturally regulated grizzly bears in British Columbia. Biological Conservation 106: 381-388. Wikramanayake, E, Boonratana, R, Rundel, P and Aggimarangsee, N. 2000. Terrestrial ecoregions of the Indo-Pacific: a conservation assessment. Island Press, Washington, D.C.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 11

PROTECTION OF RIPARIAN LANDSCAPES IN ISRAEL Tseira Maruani and Irit Amit-Cohen Department of Geography and Environment, Bar-Ilan University, Ramat-Gan, Isral

ABSTRACT Riparian landscapes are natural habitats of unique ecological and scenic values, which are highly sensitive to human intervention and impact. Yet, due to their qualities, and especially the presence of water, they are also usually attractive for recreation purposes. This is more so in arid and semi-arid zones like Israel. Nevertheless, in the past, the importance of riparian landscapes in Israel did not receive adequate attention in policy and planning. As a result, over the years they were exposed to various negative impacts, including pollution by industrial and agricultural effluents, exploitation of water for agricultural and other purposes, and land use conflicts. Although in recent years with the growing awareness of their ecological and recreational potential, considerable efforts are being invested in the rehabilitation of deteriorated riparian landscapes, their protection is still deficient. This chapter reviews and examines policy tools used for the protection of riparian landscapes in Israel, based mainly on regulations, reports and existing literature. It concludes by offering some lessons for policy-making in general and suggestions for improving the protection of riparian landscapes in Israel in particular.

Keywords: environmental policy, river corridors, conservation, open spaces.

INTRODUCTION Riparian landscapes are natural habitats of unique ecological and scenic values, which are highly sensitive to human intervention and impact (Goren, 2000). Yet, due to their qualities, and especially the presence of water, they are also usually attractive for development as well as for recreation purposes. This is more so in arid and semi-arid zones like Israel (Burmil,

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1999; Patten, 1998). Nevertheless, in the past, the values of riparian landscapes in Israel did not receive adequate attention in policy and physical planning. As a result, they were exposed over the years to various negative impacts, including pollution by industrial and agricultural effluents and household sewage, exploitation of water for agricultural and other purposes, political disputes and land use conflicts (Amir-Shapira and Feldman, 1999; Gabbay, 1998). In recent years, mainly since the year 2000, with the growing awareness of their ecological and recreational potential, considerable efforts are being invested in the rehabilitation of deteriorated riparian landscapes. Nonetheless, according to a recent report most of the rivers in Israel and their surroundings still suffer from pollution and other negative impacts (AmirShapira, 2007). This chapter reviews the protection of riparian landscapes in Israel. It presents the policy tools used for protection of riparian landscapes up to the year 2000, examines their effectivity and reveals embedded deficiencies. The methodology is based on the review and content analysis of various sources, mainly laws, reports and literature. The first part of the chapter introduces the importance of riparian landscapes in general, covering among others ecological, environmental and scenic aspects. It than presents the subject of riparian landscapes in Israel and the factors that affected policy and priorities concerning the use of water in general and protection of riparian landscapes in particular. The second part reviews and examines protection tools, focusing on legislation, institutional structure and physical planning. The third and last part of the chapter discusses the deficiencies and weaknesses that were revealed and proposes improvements to the existing state.

THE IMPORTANCE OF RIPARIAN LANDSCAPES According to Naiman and Décamps (1997) a riparian zone encompasses the stream channel and that adjacent portion of the terrestrial landscape from the high water mark toward the uplands, where vegetation might be influenced by elevated water tables or flooding. The width of a riparian zone and the diversity of its functional attributes are related to the size of the stream, its position within the drainage network, the hydrologic regime and the local geomorphology (Gregory et al., 1991; Naiman and Décamps, 1997). In other words, a riparian zone may be regarded as a linear physical landscape entity composed of aquatic and terrestrial components and the interface between them. Riparian landscapes are unique ecosystems, highly important ecologically and environmentally, and they constitute a visually and functionally outstanding component of the open space system. They form complex habitats, richer than the average with species of plants, animals and microorganisms, thus contributing to overall biodiversity (Gregory et al., 1991; Naiman et al., 1993). These habitats and the richness of species they sustain are especially sensitive to various impacts and interferences, including seasonal or other changes in water physical qualities (like temperature, salinity or electric mobility) or quantities (Allan, 2004; Goren, 2000; Karr, 1994; Naiman and Décamps, 1997; Pollock et al., 1998; Shandas, 2007; Stauffer and Best, 1980). They are considered the most diverse, dynamic and complex habitats (Goren, 2000; Gregory et al., 1991; Naiman and Décamps, 1997). Riparian ecosystems perform a variety of environmental and ecological services (Allan, 2004; Brauman et al., 2007; Hale and Adams, 2007; Naiman and Décamps, 1997; Patten,

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1998). Many of these are considered life-supporting systems (De Groot, 1992; Naveh, 1997), although they are difficult to evaluate in economic terms (Chavas, 2000; Constanza, 2000). Due to their linear nature, riparian landscapes are natural ecological corridors, allowing connectivity between habitats and patches in the landscape (Beier and Noss, 1998; Bentrup and Kellerman, 2004; Jongman, 1995; Machtans et al., 1996; Naiman et al., 1993; Ndubisi et al., 1995; Shkedi and Sadot, 2000; Taylor et al., 1995; Walmsley, 2006; Weber et al., 2006). This is especially important in populated areas, where riparian landscapes may be the last remnants of an open natural space within a built-up area. Another function that is especially important in populated areas is their potential as catchement basins for floods, thus avoiding damage to property and lives (Brody et al., 2007; McHarg, 1969; State Comptroller, 1993). They are also attractive for recreation and leisure activities, and due to their linear form are natural candidates for greenway planning, combining opportunities for recreation with conservation of nature, landscape and heritage values (Ahern, 1995; Baschak and Brown, 1995; Bryant, 2006; Fábos, 2004, 1995; Jim and Chen, 2003; Lewis, 1964; Li et al., 2005; Little, 1990). Open lands along rivers and streams are also often used for agriculture, because of their fertility, due to embedded sediments, and relatively level plains (Bentrup and Kellerman, 2004; Karr, 1994). Riparian landscapes are extremely vulnerable to human impact. Surface water from all over the drainage basin might carry with it soil particles and various pollutants, including pesticides, fertilizers and agricultural wastes into the stream. Their potential for conservation, recreation or agriculture is diminished by conflicts and liabilities from various sources. Pollution by industrial and agricultural effluents and overflow of sewage from treatment plans harms the landscape, disqualifies water for irrigation, prevents recreational water-related activities (e.g. swimming and boating) and repels potential users in general. Exploiting river water for irrigation decreases the availability of water for natural system functions. Though riparian landscapes are attractive for development and recreation, they may be contradictory to certain types of agricultural uses. Moreover, floods may cause damage to agriculture and other uses within the flooded area. Nevertheless, the presence of water is a potentially dominant factor that attracts development. Unfortunately, often the resulting development ends up with the construction of buildings and infrastructure too close to the water line, thus interfering with the ecological functions of the riparian system. Considering their uniqueness and attractiveness on the one hand and their vulnerability to various impacts on the other, riparian landscapes are in need of effective protection from inappropriate uses. In Israel, a hot and dry land, such landscapes are especially important. The next section describes the factors that affect rivers and riparian landscapes in Israel.

THE RIVERS IN ISRAEL The priorities concerning water and riparian landscapes in Israel stem from physical conditions – mainly geomorphology and climate – combined with ideological and geopolitical factors. Israel is situated on the eastern side of the Mediterranean basin, with geomorphology and climate changing both along the north-south axis and the east-west one. The north and center

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of the country lie within the Mediterranean climate zone, characterized by a short rainy winter and a long, hot and dry summer while the south of the country is more desert-like. The mountainous northern areas are the rainiest with an average of 800-1000 millimeters per year, decreasing in the central zone the a yearly average of 500-600 millimeters. Eastward and southward the average decreases to 100 millimeters and less. These quantities may fluctuate from year to year, and, in addition, there is high occurrence of droughts. All these factors add up to a continual condition of water shortage (Menahem, 1999). The area of the State of Israel is intersected longitudinally by a series of mountain ranges that divide it into an eastern basin, where rivers flow towards the Jordan River, the Sea of Galilee and the Dead Sea, along the natural eastern borders, and a western basin, where rivers flow into the Mediterranean Sea. The western basin‘s climate is milder and relatively rainy, while the eastern basin‘s is hot and dry. Due to the limited rain quantities, many river sections are in fact seasonal streams, flowing during winter and spring, and drying out in the summer until the next rainy season. In the dry south, the streams are characterized by short strong flows following rain events, which dry out quickly. Only a few rivers that are fed by yearround springs have water flowing in all seasons. Seasonal streams are exceptionally sensitive because of the fluctuations in water availability for habitat performance in addition to the fact that some of them do not enjoy a strong flow even in the rainy season. These characteristics affect their public conceptual image as landscapes fit for conservation or suitable for recreation purposes. River sections that run through areas declared as nature reserves or national parks are protected, along with the whole area, under the National Parks and Nature Reserves Law of 1963. However, a river or stream crossing an area that is not characterized by special scenic or nature values were usually not grasped as deserving protection. Consequently, they are under pressures for development, especially in areas of level topography, such as the inner and the coastal plains. The most important rivers, considering water discharge amounts and landscape impact are in Israel‘s western basin, crossing the coastal plain where the majority of the population (about 80%) is concentrated. These also have the highest potential for recreation. However, their proximity to densely settled areas has resulted in their deterioration due to various negative impacts, such as: trapping riparian waters for agricultural use and other purposes, pollution by agricultural and industrial effluents, overflow of sewage from treatment facilities, construction or agricultural cultivation close to the water line, neglect and garbage disposal (Amir-Shapira and Feldman, 1999). In addition to this, because of the scarcity of water resources and frequent droughts, the national water policy focused on keeping control over all water sources and prioritizing the use of water for agriculture and other uses. For example, since the mid 1950s, most of the water from the Yarkon springs was captured and transported to agricultural fields in the Negev (the southern region of the land) through the Yarkon-Negev pipeline; as a result, the Yarkon River, the main river in the core, densely-populated area of Israel - once a wide deep stream that the British soldiers had to cross by boats when conquering the land from the Turks in the year 1917 – deteriorated into a narrow and shallow stream, where most of the flow consisted of industrial effluents and initially-treated sewage. The negative impacts on riparian landscapes call for examination of existing protection and management tools and their effectivity.

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PROTECTION OF RIPARIAN LANDSCAPES IN ISRAELI LEGISLATION The protection of landscapes by legislation is a universally accepted model for the conservation of outstanding scenic or natural values (Maruani and Amit-Cohen, 2007). Legislation is a source of authority and constitutes a strongly effective policy tool. Yet this tool has its drawbacks, among them low flexibility, requiring a long, complicated bureaucratic process whenever a change in legislation is desired. This is also a source of conflict between conservationists and developers and landowners. On the other hand, protection based on a statutory declaration is stronger than any other protection tool, and provides the relevant authorities with enforcement measures. In Israel, legislation concerning water resources, including rivers, is complex, involving a multitude of laws and regulations as well as a multitude of authorities and organizations that are meant to enforce them (Kaplan, 2004). This section presents three laws that are the most relevant to the protection of riparian landscapes, as follows: the Water Law, the Drainage Law and the Rivers Authorities Law.

The Water Law The Water Law, enacted in 1959, should be understood in the context of the general scarcity of water resources in Israel, the relative average low rainfall and the frequent droughts, on the one hand, and the centralistic governance style and intensive involvement of the State, on the other (Menahem, 1999). The main objectives of the law were to secure the State‘s sovereignty over all water sources and ensure their utilization for the benefit of Israeli society. The law determines that ―… the water sources in the State are owned by the public, controlled by the State and designated for its population and development needs. The water sources for that matter are the springs, the streams, the rivers…‖ etc. (Water Law, sec.1-2). The law regulates the management of water sources, including allocation for users and preservation of water quality. For these purposes, the law created several institutions, among them a National Water Board, headed by the Water Commissioner. The law gave priority to the agricultural sector, which at the time was regarded not only as a leading sector in the national economy but also as the ideological and political elite of Israeli society (Menahem, 1999; Schiffman, 1999). This was reflected in various attributes of the law, among them its subordination to the Minister of Agriculture, including the power to appoint the Water Commissioner, who was to lead the design and implementation of water policy in Israel. Thus, it is no wonder that the first seven elected commissioners were from the agricultural sector, as were also most of the National Water Board members (Menahem, 1999). Only forty years later, in the 1990s, following ideological, political and social change processes in Israeli society, including the decrease in the economic and ideological importance of agriculture and the acceleration of development (Maruani, 2005; Schiffman, 1999), the Water Law was transferred to the authority of the Minister of National Infrastructures and some of its powers were split among several other institutional structures. Laster (2004) claims that this split in authorities is the reason for the decrease in water quality, in contrast to Western Europe where a centralistic approach was adopted.

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The Water Commissioner, among others, used his powers collect water from springs and rivers, and allocate it to consumers, thus reducing the natural flow, ignoring the ecological consequences, which, in turn, also had a negative impact on the attractiveness and availability of the surrounding area for recreation (Laster, 1995). The only reference in the Water Law to the protection of land near water sources is the available option to define buffer strips for purposes such as ―…protection of water, of water source …‖, that are wide ―… no more than is necessary for the purpose of the buffer strip…‖ (sec.14-15). No references are made to purposes involved in the protection of landscape or other values embedded in the terrestrial area outside the water itself. The Water Law reflects a utilitarian approach towards water resources, and almost completely ignores other values. Some years ago the law was amended, and to the list of uses for water allocation was added the ―…protection and rehabilitation of nature and landscape values, including springs, rivers and wetland habitats‖ (sec.6). However, there is no other reference to such a goal anywhere else in the law, and its implementation is totally dependent on the awareness and the good will on the part of the decision makers. It should also be noted that the interests of the Head of the Water Authority (who replaced the original Water Commissioner, following an amendment in 2006) and the Ministry of National Infrastructures do not coincide with the interests of landscape conservation. This was even truer in the past, when the main interest of the Water Commissioner and the Ministry of Agriculture was the supply of water to the agricultural sector.

The Drainage Law The Drainage Law (or in its full name: the Drainage and Flood Protection Law) was legislated in 1957, after floods in the mid 1950s covered vast areas, mainly agricultural fields, causing severe economic damage. The objective of the law was to prevent the recurrence of floods by forming suitable institutions to manage drainage. This law, too, was subordinated to the Minister of Agriculture – and still is - since agricultural lands were conceived as being most threatened by potential floods. The priority given to agriculture is expressed in the first section of the law, which defines ―drainage‖ as ―…any operation intended to concentrate, to store, to carry or to remove surface or any other water which harm or may harm agriculture, public health…‖ etc. The Drainage Law states that the ―…Minister of Agriculture may… establish a drainage authority…‖ (Drainage Law, sec.11), but the establishment of such an authority is not obligatory. The law also specifies the duties and powers of such an authority, including regulating drainage and initiating drainage projects within its jurisdiction. The roles of a drainage authority as they are specified and defined in the law do not refer to the protection of the relevant riparian landscapes in any other way. In addition to drainage authorities, the law determines the establishment of a National Drainage Board to advise the Minister on matters of drainage, such as the declaration of drainage zones or the approval of drainage projects (sec.2). This is an obligatory body, complementing the relevant institutional structure for regulating drainage and preventing floods. The Drainage Law defines some relevant terms for its purposes including ―channel‖ and ―buffer strip.‖ A Channel is defined as ―…river, stream… and any other water route… where water is flowing or standing always or occasionally.‖ A buffer strip is defined as ―strips of

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land along both sides of a channel (sec.1). The law prohibits agricultural cultivation or construction within a buffer strip. However, according to the law, the overall width of buffer strips on both sides should not exceed half the channel‘s width, and no more than 5 meters each (sec.5-6). It is needless to say that the potential protection of the riparian habitat is very limited given such conditions. The Drainage Law, like the Water Law, expresses the priority given to agriculture. Most drainage projects during the 1960s and 1970s were intended to solve drainage problems in agricultural and open areas, although in populated areas potential damage from floods to property and persons is much higher. Moreover, since the legislation of the Drainage Law in 1957, the scope of development for residential, occupational and infrastructure uses increased greatly, much of it was at the expense of open and agricultural lands where rains could have previously penetrated the soil. As a result, the amounts and intensity of surface flow towards rivers increased considerably, and with them the risk of floods. In spite of that, the Drainage Law was not updated to include instructions regarding measures for the control of drainage in new development plans. This neglect resulted in recurrent flood events in various areas, causing millions dollars worth of damage (Laster, 2004; State Comptroller, 1993, 1999b).

The Rivers Authorities Law The Rivers Authorities Law (RA Law: the full name being ―Rivers and Springs Authorities Law‖) from 1965 complements the Water Law and the Drainage Law by referring not only to the protection of the water but also to the land adjacent to it, thus expressing the values of riparian landscapes for the first time in Israeli legislation. This expression is also reflected in the ministerial subordination; when legislated, the RA Law was subordinated to both the Ministry of Interior and the Ministry of Agriculture; however, it was transferred to the Ministry of Environment after it was created in 1989. The RA Law states that the ―…Minister may… establish an authority for a certain river or part of it… or impose on a drainage authority powers of a river authority…‖ (RA Law, sec.2). That is to say, a river authority – like a drainage authority – is not obligatory. Among the duties of a river authority, the law counts ―protection of the landscape and nature values along the river on both sides…‖ but also regulation of the river‘s water flow and drainage within its jurisdiction (sec.3). This means that there is an overlap between the duties of a Drainage Authority and that of a River Authority, albeit they are related to different ministries. Laster (2004) claims that the law was intended to manage the rivers on a watershed approach basis, but actually it did not enable that, since it demanded subordination to the Water Law and the Water Commissioner. Moreover, the jurisdiction of a River Authority does not necessarily cover a whole watershed. For example, the jurisdiction of the Yarkon River Authority was limited to 20 meters on each side of the river. The RA Law was implemented for the first time in the year 1988 – 23 years after its legislation – with the establishment of the Yarkon River Authority. It took six more years to establish the Kishon River Authority, in 1994, which was the second, and, up–to-now the latest authority based on this law. Laster (2004) comments that the reason it took so long to implement the law was the original subordination to two ministers. Another criticism of the law relates to the absence of a national body, something like the National Drainage Board. In

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other words, the RA Law represents a particularistic approach, referring to each river separately and lacking a broad vision on a national scale.

INSTITUTIONAL STRUCTURE While legislation is a source of regulative powers, there is a need for institutions to use these powers and enforce the regulations. In Israel, there are several institutions that have to do with management and protection of riparian landscapes, the most important of them being the Drainage Authorities, the River Authorities and the Rivers Administration. They differ by their status, composition, main objectives, powers and budgeting.

Drainage Authorities Drainage Authorities (DAs) may be established by the Minister of Agriculture, as already mentioned, subject to the consent of the Minister of Interior and the relevant local municipalities, which are also represented in its composition. The DA is responsible for managing drainage zones within its jurisdiction, including initiating, developing and maintaining drainage projects. The powers of a DA may contribute greatly to the protection of the relevant riparian landscapes, since managing drainage requires, among other things, restrictions on development along the water route. Following the legislation of the Drainage Law, the Minister of Agriculture issued an ordinance in 1960 establishing 26 DAs. The ordinance specified the jurisdiction allocated for each DA, generally consisting of low-elevated plains, mainly agricultural lands (State Comptroller, 1993). Laster (1995, 2004) comments that the large number of DAs was due to political pressures rather than hydrological needs. Most of them actually operated as organs of the relevant Regional Councils - which are the local municipalities in the rural zones – and were dominated by representatives of the agricultural sector, thus reinforcing its control over water sources. The recurrence of severe flood events implies the existence of shortcomings and deficiencies in the Drainage Law‘s implementation in general and in DAs‘ operation in particular. Especially memorable are the floods of winter 1991-2, when river overflows flooded large areas all around Israel, disrupting the course of everyday life: people had to be temporarily evacuated from their homes, roads became impassable, large agricultural areas were under water and there were even some fatal casualties. The overall direct and indirect damage to households, businesses, public property and agriculture amounted to tens of millions of dollars. Following those events, the State Comptroller (1993) issued a report that pointed out various faults in the DAs‘ functioning, stating among others that the drainage infrastructure had been neglected over the years and the sums required for proper planning, regulating and maintenance of many rivers were not allocated and invested as should have been done. In 1996, after studying the State Comptroller‘s report, the Minister of Agriculture issued an ordinance reorganizing the DA system. Their number was decreased from 26 to 11, each covering a jurisdiction overlapping a natural drainage basin, and together covering the whole

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country. Each DA is directed independently of the Regional Council system, intended to apply a whole watershed approach rather than serve local interests (Laster, 2004). Although since this reorganization floods have reccurred, it seems that - especially since 2000 - DAs are taking more active measures to prevent flooding, revealing a more environmentally-oriented approach, including the conservation of landscape and ecological values in the riparian zones, in collaboration with other institutions and organizations, such as the Ministry of Environment, the Nature and Parks Authority and the Society for the Protection of Nature (Carmel Drainage Authority 2004).

Rivers Authorities A River Authority (RA), with powers to limit and control development along the river, is a potentially effective tool in the protection of riparian landscapes. However, this potential cannot be fully realized, partly due to constraints imposed by the RA Law, such as the limited size of the area under the RA‘s jurisdiction. The RA‘s effectivity is also limited by its obligation to comply with the Water Authority (RA Law, sec.4), even in cases when their interests are contradictory. An RA‘s composition is more complex than a DA‘s, including several government officials. Thus, conflicts between government officials and local municipalities‘ representatives may also hamper the RA‘s functioning. Another potential source of conflict is the existence of a DA in the same jurisdiction since the duties and responsibilities according to the Drainage Law and the RA Law partly overlap. The RA Law states that an RA is appointed to manage a particular river and is supposed to operate independently, following its management‘s objectives and policies. However, there is lack of a master organization on a national scale to design and lead a comprehensive policy towards the protection of riparian zones. With the absence of national policy, the influence of local interests increases, thus allowing development and infrastructure on or adjacent to riverbanks that should have been preserved. Nevertheless, an RA is still the only institutional structure that is essentially specified for riparian nature and landscape protection in Israel. This may be exemplified by the Yarkon River Authority (YRA), which since its establishment in 1988 has initiated a master plan for the Yarkon River, seeking, among other goals, to restore the riparian ecosystem and contribute to environmental quality, aesthetic values and climate amelioration (Rachmimov 1996). The YRA develops bicycle trails and areas for recreation along the river and is currently monitoring water quality etc. The YRA was, in fact, a pioneer and a model in river restoration in Israel. Regretfully, however, only one more RA was established till now: the Kishon River Authority (in 1994). Both the Yarkon and the Kishon are large rivers, passing through densely populated areas - the Yarkon in the Tel Aviv metropolis, in the center of the State, and the Kishon in the northern Haifa metropolis - that were in a severely deteriorated state due to pollution from multiple sources, water trapping, etc. However, several other large rivers in a similar condition could have profited from a similar specified institutional structure to manage and restore them.

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The Rivers Administration The Rivers Administration (RAD) was established in 1993 as a mutual initiative of the Ministry of Environment and the Jewish National Fund (JNF), the latter being a historical non-governmental organization that is currently intensively involved in forestry and outdoor recreation. Other institutions and organizations that take part in the RAD are the Nature and Parks Authority, the Ministry of Interior, the Ministry of Agriculture, the Water Authority, the Israel Government Tourist Corporation and the Society for the Protection of Nature which is a non-governmental organization (Amir-Shapira and Feldman, 1999; Gabbay 1998). The declared goals of RAD include: conservation of high-quality open spaces along river channels, controlled development of recreation and tourism foci within riparian zones and rehabilitation of rivers to a state enabling sustainability of unique habitats and landscape values (Amir-Shapira and Feldman, 1999). RAD initiates and promotes plans for the restoration of rivers, issues guidelines for existing and future River Authorities, supports the establishment of local river administrations and is actually involved in many restoration projects that are in progress, especially since 2000. For instance, the RAD takes part in the steering committee of the successful restoration project for the Alexander River – a large coastal river in the Sharon region, and one of the first to enter a restoration program along with the Yarkon River. This is done in collaboration with many other bodies, including the Alexander Drainage Authority, the Emek Hefer Regional Council, the Emek Hefer Streams Society, the Water Authority, the JNF, the Israel Government Tourist Corporation, the Nature Conservation Research Institute, the Ministry of Environment, the Hadera Environmental Association, the Nature and Parks Authority, the Society for the Protection of Nature, the District Planning Commission, the Ministry of Interior and the Israel Lands Administration (Cohen-Shoel 2003). It is clear that when so many organizations are involved, conflicts and clashes of interest are unavoidable and may hamper conservation efforts. For example, the Israel Lands Administration is supposed to manage all public lands, which in Israel amount to 93% of the area of the State. However, since the 1990s, it actually acts as the State‘s main developer, initiating large intensive development plans wherever possible, driven by economic interests (Maruani, 2005). Such a development-oriented organization is bound to confront conservation-oriented bodies like the Nature and Parks Authority and the Society for Protection of Nature. The RAD, which is guided by a statewide vision regarding rivers and riparian landscapes, can bridge such controversies and promote restoration and conservation efforts. Nevertheless, the protection offered by the RAD is limited since this is not a statutory body, and consequently lacks regulative and enforcement powers. Whenever action against polluters or other offenders of the riparian landscape is needed, it is the Ministry of Environment that is supposed to interfere and take steps towards enforcement. Also, due to lack of obligatory organizational procedures, RAD‘s operations are spatially and temporally not consistent in essence, scope and timing. Therefore, in many cases, riparian landscapes in Israel are still subject to the arbitrariness of local authorities, and are not consistently regarded as worthy of protection. In addition, the RAD‘s potential effectiveness is reduced by budget limitations, since budget allocated for riparian management is divided among too many organizations, including DAs and RAs (Laster, 2004).

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The lack of a statutory basis is a source of instability for the RAD, since it may be dismantled by a ministerial decision as quickly as it was established. Nonetheless, since its establishment in 1993, the RAD is a dominant factor in river restoration in Israel.

PHYSICAL PLANNING AS A PROTECTION TOOL Riparian landscapes are integral parts of the land. As such, physical planning of land uses at national, regional and local levels is a potentially strong tool for their protection. In Israel, land uses are determined by a statutory planning system, according to the Planning and Building Law (PBL) of 1965 (which replaced former British mandatory legislation from 1936). The system is three-tiered, with Planning and Building Commissions at national, district and local levels, each having the power to control lower-tier decisions. All planning commissions may initiate outline plans within their jurisdiction.

National Planning The Sharon Plan: The Sharon Plan was the first comprehensive national master plan prepared after the declaration of statehood in 1948. The plan was initiated and prepared within the (then) Planning Department of the Ministry of Labor and Construction, by a team headed by architect Arie Sharon, and published in 1951. This plan was not statutory; nevertheless it had significant long-range effects on spatial planning in Israel. The plan refers to five main facets of planning: agriculture, industry, transportation, parks and new cities. The agricultural plan is based on a national water policy that wishes to divert water out of relatively rich sources – among them rivers in the north and the Yarkon in the center of the State – and carry them to the dry south, thus enabling agricultural settlements that are regarded in the plan as a key factor in development and economic independence (Sharon, 1951). In other words, the Sharon Plan again reflects the economic and ideological values attached to agriculture, and grasps the water of the rivers as input for agriculture and not as a landscape component to be protected. The plan for the parks in the Sharon Plan proposed conservation of certain areas of outstanding nature and landscape values. Four out of those were to be established immediately as national parks, among them two mountainous landscapes: Mount Carmel and Mount Jarmak in the Upper Galilee, and two riparian ones: the Falik Park proposed around the Yarkon and Ayalon rivers in the Tel Aviv metropolis, and a park west of Jerusalem based on the Sorek River (Sharon, 1951). However, although the Sharon Plan as well as the actual implementation of the Carmel and the Jarmak parks significantly affected spatial planning for years to come, the parks proposed around the rivers were ignored as was also the call for using river corridors as buffers between built-up areas. The Sorek corridor area, for example, was reduced over the years due to development (Amit-Cohen et al. 2005). NOP 31: statutory planning in Israel at the national level is delineated in the National Outline Plans (NOPs). Out of almost 40 NOPs prepared up to date, only two represent comprehensive national planning. The first of the two was NOP 31, the Combined National Outline Plan for Construction, Development and Immigration Absorption. NOP 31 was

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prepared and approved in the early 1990s, when Israel was facing a crisis stemming from a sudden unanticipated increase in population due to mass immigration waves from the former USSR (Alterman, 1995). This was the first comprehensive national statutory plan to incorporate environmental considerations on a large scale, stating objectives like: conservation of nature and landscape resources, preservation of surface water quality, nurturing open spaces – among them riparian landscapes - for recreation, and balancing between development and conservation (Feitelson, 1993). The plan enhanced intensification of metropolitan regions in order to preserve open spaces in the rural zone. Despite these good intentions, it has been claimed that they triggered heavy development pressures in the metropolitan core of Israel, even in areas designated for conservation (Maruani, 2005). NOP 35: the Combined National Outline Plan for Construction, Development and Conservation NOP 35 was approved in 2005, substituting NOP 31, which became obsolete in 1998. This plan, too, aspires to balance between development and conservation, taking this a step further by dividing the whole country into zones defined as development-oriented or conservation-oriented in varying degrees. Theplan specifically refers to river strips – including the water route and the banks 100 meters on each side – and requires every statutory plan to issue instructions concerning conservation of the river and its habitats, protection of its drainage functions, bank stabilization and free access to the public. This seems promising indeed, but it will take a number of years before its achievements can be assessed. However, past experience teaches us that NOP‘s instructions are not always kept. One example is the National Outline Plan for the Mediterranean coast, NOP 13, which prohibited construction within 100 meters of the water line. That instruction did not always stand up development pressures (State Comptroller, 1999a). Sectorial NOPs: most national outline plans are sectorial, each dedicated to a specific subject (e.g. roads, power plants, landfills, etc.). None of the plans that have been prepared until now is designated for the protection of rivers and riparian landscapes, although in some cases, there may be a reference to rivers where this is relevant to the main plan‘s design. Such is the case of the National Outline Plan for Nature Reserves and National Parks, NOP 8, which offers protection to sections of riparian landscapes that are found within areas designated and declared as a nature reserve or a national park. Yet, the plan does not ensure protection along the whole water route. The National Outline Plan for Forests and Afforestation proposes plantings along riverbanks (Kaplan, 1993), but only in small limited areas, most of them outside of metropolitan zones where demand for recreation is especially high. The National Outline Plan for Tourism, NOP 12, offers another example. It designates some rivers as recreational spaces, among them the Sorek River (Amit-Cohen et al. 2005). Yet, development attractive for tourism is usually intensive and could harm existing riparian ecological and environmental values.

Regional Planning District Outline Plans (DOPs), like NOPs, are essentially guiding plans, relating to the district under the jurisdiction of a relevant District Commission or part of it. Israel is divided into six administrational districts. Almost all of them had valid DOPs that by the early 1990s were mostly out-of-date, and none of them conceived riparian landscapes as objectives for conservation. An example for the disregard of riparian functions is the Ayalon Highway,

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crossing Tel Aviv through the route that once was the Ayalon River, leaving a rather narrow channel for winter water flow. Planning of this highway started in the early 1970s and it was opened for use at the beginning of the 1990s. In the winter of 1991/2, parts of it flooded, more than 140 cars were trapped in the flood, and transportation along the highway had to be stopped for some time (State Comptroller, 1993). This has since recurred several times, also causing some casualties. The Planning Administration in the Ministry of Interior promoted preparation of new DOPs, some of which have already been approved since 2000. The new plans are much more environmentally oriented than the old ones, including special references and instructions for protection and conservation of rivers and riparian landscapes. For example, the new DOP 3/21 for the Central Area District designates ―river and its surroundings‖ for conservation. Still, there are considerable variations between DOPs in their definitions and conservation instructions; some are guiding while others are obligatory, etc. (Maruani, 2005). In other words, the protection offered by DOPs is not consistent in scope and intensity. Moreover, since most current DOPs are relatively new, it will be some more time before their effectiveness in riparian protection can be evaluated.

Local Planning While national and district outline plans offer general guidelines, Local Outline Plans (LOPs) are more detailed and serve as platform for issuing building permits. The permits are approved and issued by Local Commissions, which are in fact organs of the relevant local municipality, composed of the elected political representatives who constitute the municipal board. Thus, Local Commissions are interested in the promotion of local economic development, even when it is contradictory to conservation needs, and they tend to ignore a broader regional vision. District Commission‘s control is supposed to minimize the influence of local economic and political interests on land use decisions. However, since riparian landscapes are attractive for housing, it is no wonder that initiatives for development within riparian areas recur, assisted by interested Local Commissions which regardless of their negative impact and potential flood risk (see for instance: Shechori, 1999; Shmul, 2000; Vaserman-Amir, 1999). Development decisions are a major causal factor where floods are concerned (Brody et al., 2007; State Comptroller, 1993). The recurrent flood events in Israel, and their negative impacts and costs indicate that local development interests were not restricted by the District Commissions.

DISCUSSION The examination of legislative, organizational and planning tools used for protection of riparian landscapes in Israel revealed various faults and deficiencies that may explain the still distressing state of these landscapes. Despite restoration efforts undertaken, mainly since 2000, almost all rivers are still polluted and ecologically deteriorated with only limited sections available and accessible for recreational uses.

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Table 1 that presents a comparative assessment of protection tools shows that no one tool can be highly rated on all parameters. In addition, no parameter shows a consistently high rating for all tools. One fundamental problem is that no tool – with the possible exception of RAD - is dedicated to the objective of protecting ecological, environmental and scenic values embedded in riparian landscapes. Table 1. Comparison of protection tools Protection tool

Designation

Relevance

Power

Scale

Implementation

Water Law

Low

Medium

High

National

Low

Drainage Law

High

High

High

National

Low to medium

River High Authorities Law

High

High

National

Low to medium

Drainage Authorities

Medium

High

Medium

Regional

Low

River Authorities

High

High

Low to medium

Regional

Low to medium

Rivers Administration

Very high

Very high

Low

National

Medium

Physical

Sharon plan

Low

Low

Low

National

Low

planning

NOP 31

Low

Low

Medium to high

National

Low

NOP 35

Low

Low

Medium to high

National

?

Sectorial NOPs

Low

Low to medium

Medium to high

National

Low

DOPs

Low

Low

Medium to high

Regional

Low

LOPs

Varies with plan

Varies with plan

High

Local

Varies with plan

Legislation

Institutional structure

For example, each of the laws examined makes a partial contribution to this objective although they differ in aims and scope. However, none of them regards the protection of riparian landscapes as its main aim. In addition, none of these laws reflects a watershed approach, theoretically or practically, albeit activities taking place anywhere in the watershed area, especially development and various pollution generators, eventually affect the river and the landscape along it. Moreover, partial overlap between the aims and directives of the Drainage law and those of RA Law are a constant source of ambiguity and fuzziness as to duties and responsibilities, on one hand, and inter-organizational and inter-personal frictions and conflicts, on the other. This situation stands in the way of the conservation interest. The different tools are interconnected. Legislation determines the structure, responsibilities and operational procedures of the organization intended to implement it. Subjects not covered by the law are left to the discretion of organizational decision makers.

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Hence, awareness of riparian values on the part of decision makers is an important factor in effective protection. Statutory physical planning, too, relies on legislative power, and therefore inter-relations between laws may affect organizational actions. For example, the Drainage Law does not refer to statutory physical planning, while on the other hand the Planning and Building Law does not require a preliminary examination of possible impacts on drainage before the approval of a new development plan. The State Comptroller (1993) pointed out that the planning system allowed development too close to water routes and approved plans on large areas without ensuring suitable measures for infiltration of rains within their boundaries. He argues that the severe floods that were the cause for his report could have been prevented, had suitable instructions been embedded in the Planning and Building Law, thus preventing construction within flood retention areas and conditioning plan approval with proper infiltration and drainage solutions. The protection of riparian landscapes as was formulated and implemented in Israel expresses a particularistic approach, where each river is conceived as a discrete entity, with a separate DA or RA. For comparison, nature and landscape values within areas that have been declared as nature reserves or national parks all over the state are protected centrally and managed by the Nature and Parks Authority, which is a national statutory institution, established by the National Parks and Nature Reserves Law. It seems that centralistic approach was efficient even when development pressures in Israel increased considerably towards the end of the 20th century. The establishment of the RAD indicates a conceptual change towards rivers as well but its lack of statutory position and the multitude of other relevant organizations reduce its effectivity. The protection of riparian landscapes also reflects the evolution of environmental awareness in Israel (see also: Fletcher, 2000; Vogel, 1999). For example, the Water Law from 1959 almost completely ignores the environmental functions of water sources, including rivers, while the RA Law from 1965 already regards the protection of riparian environmental values as one of its main aims. The legislation also reflects the prevailing priorities in Israeli society at the time, affected greatly by ideological and national security considerations. In the past, agriculture and development were given priority; agriculture was conceived as a main economic basis and development of spatially dispersed small agricultural settlements also grasped as a tool for dominating national space while environmental needs were overlooked (Hershcovich, 2006; Schiffman 1999). It should be noted that all three laws examined were enacted before the global environmental revolution of the late 1960s. Moreover, as a consequence of the Stockholm Convention in 1972 Israel was one of the first nations to establish an environmentally designated institution in the form of the Environmental Service that was established in 1973 within the Prime Minister‘s office, and later on as part of the Ministry of Interior. However, the assimilation of environmental awareness was slow, especially among decision makers, until the 1990s when development pressures increased considerably, following a sudden increase of population due to large immigration waves, threatening nature and landscape values, especially in the coastal area and in vicinity of water bodies. This also was one of the triggers for initiation of RAD. It should also be noted that split authoritative powers, multiplicities and deficiencies that characterize the protection of riparian landscapes in Israel, tend to be typically characteristic of environmental issues, which are naturally complex, interdisciplinary and bound up with economic and social conflicts. In addition, since its independence in 1948, Israel has been facing enormous challenges more than any other developed state, including an unstable

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geopolitical situation and recurrent wars, absorption of mass immigration waves and serious social conflicts (Alterman, 1995; Fletcher, 2000; Vogel, 1999). Nevertheless, all this still does not explain why former protection frameworks have not been reconstructed in spite of the advances in environmental management and administration in general. For instance, the Drainage Law that was mainly intended to prevent floods is still under the authority of the Ministry of Agriculture although severe damage caused by floods in recent years was mainly in urban areas and not in agricultural fields (State Comptroller, 1993). There is no doubt that the present state of affairs calls for improvement.

CONCLUSION Riparian landscapes constitute extremely vulnerable ecosystems. They need protection to preserve the unique aquatic habitats with their biodiversity richness and ecological processes as well as their value for scenic and recreational purposes. In spite of that, their protection in Israel is defective, lacking comprehensive suitable legislation, a statutory institutional structure on a national scale and a sectorially designated NOP. The existing state of affairs is characterized by a complex array of authoritative powers, some split and others overlap, and by deficiencies in formulation and implementation of policies. Only limited segments of specific riparian landscapes may, in fact, be regarded as functional healthy ecosystems. Several lessons can be drawn from the above discussion. We wish to focus here on those that seem the most important and practical for the State of Israel in the immediate future. First, there is need for a revision of present legislation, integrating together existing laws – mainly Drainage Law and RA Law – rephrasing their aims and directives, and rearranging the institutional structure and its powers according to updated environmental and other needs. This should be done considering a whole watershed approach as has already been suggested by Laster (1995). Second, the prevailing, rather particularistic, approach should be replaced by a comprehensive centralized one, based on a vision and needs on a national scale. This ought to be reflected in all types of protection tools but especially in the institutional structure, as by establishing a national statutory designated organization (similar to the Nature and Parks Authority) or, alternatively, empowering the existing RAD by adequate legislation, including broader definition of its powers and duties. Third, increasing environmental awareness in general, and awareness of riparian landscapes‘ ecological, environmental and scenic aspects in particular, are a key element in promoting protection and conservation of such landscapes. This is true for the general public, as has already been claimed by Lowry (1998), but is even more important where decision makers are concerned. Therefore, educational activities, formal and informal, carry great significance for improving and intensifying riparian protection efforts. Finally, there is hope for riparian landscapes, even when severely deteriorated, providing suitable protection, restoration tools and a plan of action (see for instance: Harnik 2007, Kirk 2005, Tjallingii 2000). This is especially important in a small, densely populated and dry land like Israel.

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SPNI, (2001). The court accepted SPNI‘s appeal and canceled a permit for high-tech building amidst open landscape. Bama, 23 (In Hebrew). State Comptroller, (1999a). Planning land uses on the Mediterranean coast. Annual report 49, 361-371 (In Hebrew). State Comptroller, (1999b). The state drainage infrastructure. Annual report 49, 242-248 (In Hebrew). State Comptroller, (1993). Reports on the state drainage infrastructures and the Ayalon Highways planning, management and maintenance of the Ayalon Channel. Jerusalem (In Hebrew). Stauffer, D. F. and Best, L. B. (1980). Habitat selection by birds of riparian communities: evaluating effects of habitat alterations. The Journal of Wildlife Management, 44 (1), 115. Tal, D. (2003a). Drainage forecast for 2003: rivers clogged; roads, fields and houses inundated. Globes 20.1.03 (In Hebrew). Tal, D. (2003b). Floods in Tel-Aviv and the Dan region: enormous traffic jams, sewers have collapsed. Globes 22.1.03 (In Hebrew). Taylor J., C. Paine and FitzGibbon, J. (1995). From greenbelts to greenways: four Canadian case studies. Landscape and Urban Planning, 33, 47-64. Tjallingii, S.P. (2000). Ecology on the edge: landscape and ecology between town and country. Landscape and Urban Planning, 48 (3-4), 103-119. Vaserman-Amir, N. (1999). Ministry of Interior and SPNI: too vast building rights in the Yarkon River plan. Globes 9.4.99 (In Hebrew). Vogel, D. (1999), Israeli environmental policy in comparative perspective. Israel Affairs, 5 (2), 246-264. Walmsley, A. (2006). Greenways: multiplying and diversifying in the 21st century. Landscape and Urban Planning, 76 (1-4), 252-290. Weber, T., Sloan, A. and Wolf, J. (2006). Maryland‘s green infrastructure assessment: development of a comprehensive approach to land conservation. Landscape and Urban Planning, 77, 94-110.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 12

HYDRAULIC CHARACTERIZATION OF AQUIFER(S) AND PUMP TEST DATA ANALYSIS OF DEEP AQUIFER IN THE ARSENIC AFFECTED MEGHNA RIVER FLOODPLAIN OF BANGLADESH Anwar Zahid1,2, M. Qumrul Hassan1, Jeff L. Imes and David W. Clark3 1

Department of Geology, University of Dhaka, Dhaka, Bangladesh 2 Bangladesh Water Development Board, Dhaka, Bangladesh 3 United States Geological Survey, National Drilling Company-USGS GroundWater Research Program, P.O. Box: 15287, Al Ain, UAE

ABSTRACT To determine the hydraulic characteristics of aquifers and development potential of deep aquifer for sustainable long-term use, study was undertaken by assessing water levels of different aquifer formations and conducting pumping test in deep aquifer under Meghna floodplain area of southeastern Bangladesh. Because of arsenic contamination in shallow groundwater, characterization of deeper aquifers and assess their hydraulic connectivity is now an important issue in Bangladesh. Study shows that groundwater pumping for irrigation and other uses cause large seasonal water level fluctuations that is between 2 and 4.5m, 6.5 and 11m and 6.5m in the shallow, main and deep aquifers, respectively. The trend of groundwater level fluctuations supports the hydraulic connectivity of these aquifers. Aquitards separating aquifers are not continuous regionally. This implies that uncontrolled development of deep aquifers may cause leakage of arsenic from contaminated shallow depths to aquifers below. Water levels dropping below sea level for over withdrawal may also cause saline water intrusion as well. However, during the constant-discharge pumping test for deep aquifer, water levels in observation wells open to the shallow and main aquifers showed no noticeable effect from pumping i.e. under conditions of moderate groundwater use for public supply, arsenic-rich groundwater in the shallow aquifer are not likely to be drawn into the deep 

E-mail: [email protected], Tel-+880-2-7287176; +880-1819 105 871

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aquifer. The transmissivity values of the aquifer is generally favorable for groundwater development and ranged from about 1,070 m2/day to 2,948 m2/day at a distance of 44 m from the pumped well. Transmissivity ranged between 1,570 m2/day and 2,956 m2/day at a distance of 120m. Transmissivity was calculated as 2,385 m2/day using recovery data. Estimated storage coefficient values ranged between 0.0000375 and 0.00268, indicates that the aquifer is confined to leaky-confined or semi-confined in nature.

Keywords: Arsenic contamination, groundwater level, irrigation abstraction, fluctuation, aquifer test, transmissivity, storage coefficient.

NOTATION c L Q r s S S' sm

D'/K': hydraulic resistance of the semi-pervious layer (day) √Tc: leakage factor (meter) constant discharge rate at the well (meter3/day) radial distance from the pumping well (meter) drawdown at the well (meter) aquifer storage coefficient is the residual drawdown (meter) maximum drawdown (meter) in a piezometer at distance r (meter) from the pumped well Δs the drawdown per log cycle of time (meter) T transmissivity of the aquifer (meter2/day) t time from the start of pumping (minutes) t' time from the cessation of pumping (minutes) t0 time, where the straight line intersects the zero-drawdown axis (minutes)

INTRODUCTION Groundwater is the main source for drinking and irrigation water in the lower floodplain areas of south-eastern Bangladesh and mainly withdrawn from shallow aquifer. The upper aquifer system of the area can yield large quantities of water, however, is not completely suitable for sustainable development because of quality problems. The arsenic contamination of shallow (generally up to 50m depth) groundwater has changed the potentiality of it‘s use. Besides, high concentration of iron, manganese and salinity at different depth levels of main aquifer makes it unsuitable for drinking use. Considering the increasing demand for municipal and rural water supplies, agricultural, industrial and other uses and quality problems in shallow and main aquifers, development of deep aquifer has already been started in some areas. However, before large scale withdrawal of groundwater from deep aquifers, understanding the natural distribution of groundwater for long term sustainability is very important. Sustainable yield from aquifers, effective use of the water stored in aquifers, preservation of water quality, and movement of water between different aquifer formations into a comprehensive management system needs to be studied carefully. Rapid and

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uncontrolled development of the deep aquifer could severely limit the usefulness and the productive duration of the aquifer. Groundwater levels of different aquifers were monitored throughout Kachua upazilla (sub-district) area under Chandpur district and an aquifer test was conducted for the deep aquifer at Sreerampur village of Kachua to determine the aquifer hydraulic characteristics, potability of the aquifer system, and the response of the deep aquifer pumping to upper aquifers and to development stresses. In particular, it is essential to understand whether pumping stresses can induce arsenic contaminated water from the shallow aquifer or high salinity water from depth or the shallow aquifer to migrate into the aquifer. The electric conductivity (EC) of water pumped from the production well was also monitored during the test to determine if higher salinity water was being captured by the well during the test. The long-term constant-discharge pumping test was performed as the aquifer properties determined from the analysis of constant-discharge pumping test represent the regional hydraulic properties (Nury et al, 1998) in alluvial aquifers like Bengal Delta.

STUDY AREA Most of Bangladesh including the study area lies within Bengal basin which began forming during the Late Mesozoic as the continental landmass of Gondwana fragmented and continued to form during the Tertiary when the Indian plate collided with the Eurasian plate resulted in the formation of the Himalayan ranges. The Bengal basin contains a 15-km to 22km thick sequence of Cretaceous to Recent sediments and occupies some 100,000 km2 of lowland floodplain and delta (DPHE-BGS 2001). Alluvial deposit carried by the GangesBramaputra-Meghna (GBM) river systems have gradually built up the delta and Meghna estuary (Brammer 1996). Physiographically, the study area lies within Meghna estuarine floodplain under Tippera surface (Morgan et al. 1959) that is bounded by the Meghna river in the west, Lalmai hills in the east and Old Meghna estuary at its south. The total discharge of the lower Meghna river was contributed from the eastern territory and the huge discharge hit the western bank and caused erosion in the western bank and subsequent deposition in the eastern bank towards the study area (Hussain and Huq 1998). The surface of the sequence, composed of silt, silty clay, silty loam and grayish clay, has been assigned to the Chandina Formation (Bakr 1977), while the status of the underlying medium sand aquifer is not well known. The Geological Survey of Bangladesh (GSB) has dated the surface of the Chandina deltaic plain as between 3,000 and 6,000 years. The underlying sands locally contain brackish water and may belong to the Dupi Tila Formation. Based on elevation and morphological features the area is divided into three units (BWDB-USGS 2005). Southeastern part of the upazilla has been defined as Chandina Alluvium High that always remains above normal flood level. The sediments mainly consist of clayey silt, silty clay and very fine sand. Below this oxidized zone grey colored fresh sandy silt and fine sand are present. The northwestern and southwestern part have been defined as Chandina Alluvium Medium. This part normally remains under water for about 3 to 4 months. Maximum and minimum elevation of this unit is 6.0 and 4.6 m above mean sea level (AMSL) respectively. The northeastern and central-western part of the area is defined as Chandina Alluvium Low. The surface remains under water for longer periods than the other

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units. In part of the southeast Bangladesh, multi-layered aquifer conditions exist. On a regional basis BWDB-UNDP (1982) described three aquifers between Holocene and PlioPleistocene formations. In Kachua area aquifers may be classified as; (Figure 1), (a) the shallow (1st) aquifer, extends down to 40 to 80m, below the 3 to 6 m thick upper clay and silt unit.

Figure 1. Aquifer system under Kachua Upazila area (Zahid et al 2007).

The aquifer sediments are composed of sand with lenses of clay. Water of this unit is severely contaminated by arsenic, (b) the main (2nd) aquifer, extends down to 250 to 350m and is generally underlain and overlain by silty clay bed, and composed mainly of fine to medium sand, grey to light brown in color, occasionally inter-bedded with clay lenses. It is either semi-confined/leaky or consists of stratified interconnected, unconfined water-bearing zones. Irrigation water in drawn predominantly from these strata, and (3) the deep (3rd) aquifer, encountered to depths of 400m below a 10 to 15m thick silty clay bed. This aquifer is composed mainly of grey to dark grey fine to medium sand that in places alternates with thin sandy shale/clay lenses. This deeper water bearing unit is separated from the overlying main aquifer by one or more clay layers of varied thickness.

METHODOLOGY OF THE STUDY Observation of Water Levels in Different Aquifers 1998 to 2003 groundwater level data of 4 Bangladesh Water Development Board (BWDB) monitoring wells (20-30m deep) installed in the shallow aquifer (Table 1), 2004-05 to 2005-06 data of 13 selected Department of Public Health Engineering (DPHE) hand tubewells (180-225m deep) installed in the main aquifer (Table 2) and 2003 to 2004 data of 2 BWDB deep monitoring wells (336-352m deep) installed in the deep aquifer (Table 3) were used to asses the response of groundwater level above or below mean sea-level (MSL) considering recharge and withdrawal for different uses. Locations of monitoring wells are shown in Figure 2.

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Figure 2. Location of groundwater level monitoring wells.

Table 1. Information on BWDB observation wells screened in shallow aquifer Location Jagatpur Machimpur Sacher Muradpur

Latitude (North) 231555.0 231897.7 232649.6 232000.0

Longitude (East) 910000.0 905532.1 905051.2 905000.0

Depth (m) 19.20 27.44 27.44 28.66

Measuring point (m) Above MSL 12.70 6.48 5.36 6.53

Table 2. Tube wells selected for monitoring groundwater movement in main aquifer Location Nalua, Karaia Sahedapur, Karaia North Dumuria, Karaia Hossainpur, Kachua South Ghagra, Kachua South Tetaia, Kachua North Ujani, Kachua North Hasimpur, Gohat North Gobindapur, Gohat South Singua, Sahadevpur West Khilmeheb, Sahadevpur West Kadla, Kadla Chaumuhari, Kadla

Latitude (North) 231827.3 231833.6 231906.1 232123.7 232134.5 232249.4 232219.0 232006.9 231724.1 232303.3 232342.0 231852.7 231946.9

Longitude (East) 905440.7 905456.4 905240.9 905515.9 905222.8 905331.4 905522.0 905651.1 905638.8 904953.9 904942.8 905128.9 904946.6

Depth (m) 215 215 220 220 218 213 211 191 218 223 220 210 225

measuring point Above MSL (m) 6.288 6.376 5.848 7.016 6.335 6.317 6.28 6.342 6.649 5.992 6.236 5.987 7.188

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Setting of Pumping Well and Observation Wells For a well performance test, yield and drawdown are recorded to calculate the specific capacity of the well. The aquifer test at Sreerampur village was conducted mainly to provide data from which the principal factors of aquifer performance [transmissivity (T) and storage coefficient (s)] can be calculated. An aquifer test with the setup of a 365-m deep pumping well with a constant discharge of 708 gpm (gallons per minute), and 5 (five) observation wells installed at different depth levels from 25 to 352 m (Figure 3, Table 3), were performed to obtain a picture of the general hydraulic properties of the aquifer and also to predict the effect of withdrawals on the aquifer system, the drawdown in the tested well with time, and different discharges and the radius of the zone of influence for individual or multiple wells. In a uniform and homogeneous aquifer the piezometer should be installed at about the same depth as the middle of the well screen in the pumped well. Amongst observation wells, two have been installed in the pumped aquifer and others are above the confining layer that separates the two aquifer systems. The test continued for 98.5 hours and water levels were measured in both the pumped well and five piezometers. The constant-discharge pumping test was followed by recovery test. Important water quality parameters were also monitored and samples were collected at different time intervals during the test.

Figure 3. (a) Location and (b) position of pumping well and observation well screens.

Table 3. Major features of pumping and observation wells Well ID PW P-1 P-2 P-3 P-4 P-5

Aquifer Deep Main Deep Deep Shallow Main

Depth (m) 365 281 336 352 25 183

Screen length (m) From To 326 362 269 278 324 333 333 349 20 23 171 180

Diameter of well (m) 0.35 0.076 0.076 0.076 0.076 0.076

Distance from PW (m) 0 14.5 44.1 105 7.5 9.25

measuring point (m) Above MSL 6.84 6.3 6.94 6.28 6.72 6.54

The flow rate of 3,860 m3/day was determined using standard tables for the discharge pipe diameter considering a water height of 8.33 m in a manometer attached to the side of the pumping-well discharge tube. The rate at which the discharge water stream dropped from the

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horizontal was also measured. A flow rate of 3880 m3/day was determined using standard tables that relate the rate of fall to discharge. The two methods were in good agreement and an average value of 3870 m3/day flow was used to analyze the aquifer-test data. The constant-rate test for the time duration of 98.5 hours was conducted at Sreerampur. The pumping rate was estimated and monitored using two different methods. The circular orifice weir was used to measure and monitor the discharge rate of the pump. The height of the water column, the static water level just before the test started, time since the pump started, pumping rate, dynamic groundwater levels at various intervals during the pumping period, and time the pump stopped were recorded. Measurement of water levels after the pump stopped (recovery data) were also measured as these are extremely valuable to verify the aquifer storage coefficient calculated during the pumping phase of the test. The test was started on November 13, 2003 at 12:00 noon. Before the test began, water level transducers were placed in the pumping and observation wells. Drawdown was also recorded manually by measuring tapes. To obtain better and more reliable results pumping continued till the depression cone had reached a stabilized position. The depression cone continues to expand until the recharge of the aquifer equals the pumping rate.

Best Fit Analytical Methods for Pump Test Data Amongst different analytical methods, it is important to select a numerical solution which is more appropriate to actual field conditions. Two analytical solutions were studied in detail to determine the most appropriate solution to the deep observation well aquifer-test data. The Papadopolous-Cooper (1967) solution for a confined aquifer with well-bore storage and the Hantush-Jacob (1955) solution for a leaky confined aquifer were determined to be the best choices. Besides, Jacob (1950) straight line plot was drawn manually and Jacob (1950), Chow (1952) and Theis recovery methods were applied using computer programs developed by Abdin (2004) for manually measured drawdown data. The following assumptions and conditions were considered for analyzing aquifer-test data using different methods valid for confined or leaky-confined aquifer. ·

·

·

All geologic formations are horizontal and the aquifer has a seemingly infinite areal extent. In reality, hydrogeologic settings rarely have aquifers that can be considered of infinite areal extent, rather they change laterally in grain size, shape or lithology that affect the shape of a time-drawdown curve. Although such aquifers do not exist in lower delta, many aquifers are of such wide extent that all practical purposes they can be considered infinite. The aquifer is homogeneous, isotropic and of uniform thickness over the area influenced by the pumping test. Homogenous aquifers seldom occur in lower delta of Bengal basin and most aquifers are stratified to some degree. As a result of this stratification the drawdown observed at a certain distance from the pumped well may be different at various depths within the aquifer, because of differences in hydraulic conductivity in vertical and horizontal direction. These differences in drawdown diminish with increased pumping itme. Groundwater has a constant density and viscosity.

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Prior to pumping, the piezometric surface were nearly horizontal over the area influenced by the pumping test. The aquifer was pumped at a constant discharge rate of about 3870 m3/day, All changes in the position of the potentiometric surface were due to the effect of the pumping well alone, Pumping well was 100% efficient

Another important assumption is that the pumped well fully penetrations the aquifer and thus receives water from the entire thickness of the aquifer by horizontal flow. If the well only partially penetrates the aquifer, like Sreerampur study, the flow paths have a vertical component to them. The flow paths are, therefore, longer and converge on a shorter well screen, resulting in an increase in head loss (Driscill 1986). However, observation wells for pumping tests were placed far enough away from the pumping well to avoid partial penetration effects. If the observation well is partially penetrating and more than 1.5b(K h / K y )0.5 away from the pumping well, the effects are negligible (Hantush 1964) (b is the saturated thickness, Kh and Kv are the horizontal and vertical hydraulic conductivities). This condition is valid for Sreerampur aquifer. If this condition is not satisfied, there will be an upward inflection in the response, similar to that obtained in the leaky method or for some sort of recharge boundary (Domenico and Schwartz 1997).

Theis Method for Predicting Drawdown in a Confined Aquifer A major advance was made by Theis (1935), who was the first to develop a nonsteadystate formula which introduces the time factor and the storage coefficient. Theis developed the analytical solution for flow to a well in a confined aquifer. This is generally the base of other methods applied to similar conditions. Theis noted that when a well penetrating an extensive confined aquifer, is pumped at a constant rate, the influence of the discharge extends outward with time. The rate of decline of head, multiplied by the storage coefficient and summed over the area of influence, equals the discharge. Because the water must come from a reduction of storage within the aquifer, the head will continue to decline as long as the aquifer is effectively infinite. Since all sources of recharge are moving through a semiconfining unit, the flatness of the recharge effect on a Theis curve will continue in a horizontal manner (Lohman 1979). However, when the semi-confining layer is saturated and has a head higher than the head in the aquifer being pumped, this head differential may cause the aquitard to release water from storage in an attempt to reach equilibrium (Weight and Sonderegger 2000). As in the semi-confining scenario, the initial drawdown tends to follow the confined Theis curve. As the aquifer becomes stresses and the head lowers in the pumping well, the head change stimulates leakage from above and below to act as recharge to the system. The result is a flattening of the curve rather than following the Theis curve. Besides the assumptions mentioned above the following assumptions are also considered for Theis solution. ·

Aquifer is fully confined and discharge is derived exclusively from storage in the aquifer.

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The flow to the well is in unsteady state, i.e. the drawdown differences with time are not negligible nor is the hydraulic gradient constant with time. The water removed from storage is discharged instantaneously with decline of head. The storage in the well can be neglected.

The nonsteady-state or Theis equation which was derived from the analogy between the flow of groundwater and the conduction of heat for predicting drawdown (s) at the well is as follows: 

s

Q e  y dy Q Q  W (u ) and, consequently T  W (u )  4T u y 4T 4s

The well function W (u ) is the infinite series part of the analytical solution to the nonsteady, radial groundwater flow equation that is approximated by:

W (u )  0.577216  ln u  u 

where, u 

u2 u3 u4    ... 2  2! 3  3! 4  4!

r 2S 4Ttu and, consequently S  2 4Tt r

u determines the radius of a cone of depression (Theis 1940). The radius not only increases with increasing time but, for a given time, is larger for decreasing values of storativity and increasing values of transmissivity. Theis‘ type curve u versus W(u) is unique only because it pertains to a particular set of conditions at the pumped well and in the aquifer (Stallman 1976).

RESULTS AND DISCUSSION Water Level Fluctuations in Different Aquifers Long-term groundwater hydrograph of four of BWDB wells, installed in the shallow aquifer, show maximum depth to water table in dry season and in the monsoon it regains (Figure 4). No permanent declining is observed. The average maximum and minimum water level was observed as 6 and 0.5m above MSL respectively (Table 4). Seasonal groundwater table fluctuation ranges in between 2 and 4.5m in the Upazila area. Generally, groundwater withdrawal from shallow aquifer for domestic purposes and during dry period by shallow irrigation wells is balanced with the vertical percolation of rain water and inflow from surrounding aquifers in monsoon.

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Table 4. Average groundwater levels above or below mean sea-level in different aquifers Aquifer Formation

Nos. of Wells 04 13 02

Shallow (1st) Main (2nd) Deep (3rd)

Depth of Wells (m) 20-30 180-225 336-352

Groundwater Level (m) Maximum Minimum 6 0.5 4 -8 4.5 -2

CM 014

CM020

7 Groundwater Elevation (m)

Groundwater Elevation (m)

6 5 4 3 2 1 0

6 5 4 3 2

Jan-00

2003

1998

Aug-01

1/ 0/ 00

10/ 26/ 00

2003

1998

Year

Year CM032

CM043

5

6 Groundwater Elevation (m)

Groundwater Elevation (m)

Fluctuation (m) Maximum Minimum 4.5 2 11 6.5 6.5 6.5

4

3

2

1 1/ 0/ 00

10/ 26/ 00 2003

1998

Year

5 4 3 2 1 1/ 0/ 00

9/ 6/ 00

1998

2003

Year

Figure 4. Groundwater level hydrograph of four BWDB wells screened in shallow aquifer at Kachua sub-district.

There is a clear difference between the amount of water that can potentially recharge the aquifer system and the actual quantity. Actual recharge is the quantity of water when the groundwater table has risen to the ground surface and no more water can enter the aquifer system. Its quantity depends on the degree of depletion of this reservoir during the dry season.

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Potential recharge is the maximum amount of recharge that can occur if enough storage reservoir in the aquifer system is available. Any surplus rainfall is then rejected and contributes to surface flooding. The ultimate limit on groundwater development is controlled by the long-term average amount of potential recharge. Assessment done by WARPO (2000) indicates that when the clay is relatively thin, but exhibits low vertical permeability, the piezometric level may drop below the base of the clay. In this case the total gross quantity of groundwater abstracted from the aquifer is balanced by infiltration through the upper clay. When the upper clay varies in thickness, the piezometric level in the aquifer may locally drop below the base of the clay, creating local unconfined conditions within the aquifer and groundwater abstraction is partially balanced by leakage through the clay and partially by unconfined storage change in the aquifer. The recharge to the aquifer, which is the leakage during the period of no abstraction, may be less than the potential recharge, particularly when the vertical permeability is low. During the peak irrigation season in March and April, the hydrograph is fairly smooth from year to year and steepest rise in the hydrograph is observed immediately after the irrigation pumps are switched off (mid-April to mid-May) rather than at the start of the monsoon (late June) as might be expected. During the monsoon, the hydrographs rise steadily until the levels are within about 1-2m of the surface which indicates that a dynamic equilibrium is established between the water table, deep rooted vegetation and surface water bodies (Ravenscroft 2003). The aquifer full condition i.e. no more storage capacity is attributed before the end of June. A shallow aquitard is common in this region at depth about 75m. Below this aquitard all deep irrigation wells are installed in the upper part while DPHE domestic and community hand tubewells are screened in the deeper part of main aquifer. Groundwater pumping for irrigation by deep irrigation wells causes large seasonal water level fluctuations (Figure 5), ranges from 6.5 to maximum of 11m (Table 4). The average maximum and minimum water level was observed as 4 and -8m above and below MSL respectively. Withdrawal by shallow irrigation wells may also influence on huge fluctuation of water level in the main aquifer. However, these levels in the deeper part get recovered rapidly when seasonal pumping stops. This rapid recovery, parallel to water levels in shallow aquifer, reveals that shallow and main aquifers are hydraulically connected and rainfall also contributes recharge to main aquifer through shallow aquifer. However, with intensive abstraction, groundwater levels may fall towards a permanent new equilibrium state. At Sreerampur village a deep confining layer is encountered at 302 to 320m depth, which is also common in surrounding areas of Meghna floodplain. Only two monitoring wells are installed at entire Kachua area in this deep aquifer. The average maximum and minimum water level of one hydrologic year was observed as 4.5 and -2m above and below MSL respectively (Figure 6a). Hence the seasonal fluctuation is 6.5m that is less compare to average water level fluctuation of main aquifer (Figure 6b), but still high though the development stress in deep aquifer is almost nil. This huge fluctuation indicates the influence of irrigation withdrawal in upper aquifers.

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Hydrographs for wells (2nd Aquifer) in Northern Kachua

Water levels (elevation) in meter

6 4 2 0 -2 KH-28 KH-40 KH-44 KH-45 KH-46 KH-63

-4 -6 -8

Mar-06 Feb-06 Jan-06 Dec-05 Nov-05 Oct-05 Sep-05 Aug-05 Jul-05 Jun-05 May-05 Apr-05 Mar-05 Feb-05 Jan-05 Dec-04 Nov-04 Oct-04 Sep-04 Aug-04 Jul-04 Jun-04 May-04 Apr-04 Mar-04 Feb-04

a Hydrographs for wells (2nd Aquifer) in southern Kachua

Water level (elevation) in meter

6 4 2 0 -2

KH-22 KH-25 KH-34 KH-36 KH-55 KH-56 KH-60

-4 -6 -8

Mar-06 Feb-06 Jan-06 Dec-05 Nov-05 Oct-05 Sep-05 Aug-05 Jul-05 Jun-05 May-05 Apr-05 Mar-05 Feb-05 Jan-05 Dec-04 Nov-04 Oct-04 Sep-04 Aug-04 Jul-04 Jun-04 May-04 Apr-04 Mar-04 Feb-04

b Figure 5. Groundwater level hydrographs of hand tubewells screened in the deeper part of main aquifer.

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Water level (elevation) in meter

Hydrographs for wells (3rd Aquifer) at Srirampur 6 5 4 3 2 1 0 -1 Well (3rd Aquifer) 335 m

-2

Well (3rd Aquifer) 350 m

-3

Nov-04

Oct-04

Sep-04

Aug-04

Jul-04

Jun-04

May-04

Apr-04

Mar-04

Feb-04

Jan-04

Dec-03

Nov-03

Oct-03

Sep-03

Aug-03

Jul-03

Jun-03

May-03 Water level (elevation) in meter

a Hydrographs for wells (3 Aquifers) at Srirampur 6 4 2 0 -2 Well (2nd Aquifer) 280 m Well (3rd Aquifer) 335 m Well (3rd Aquifer) 350 m Well (1st Aquifer) 25 m Well (2nd Aquifer) 180 m

-4 -6 -8

Nov-04

Oct-04

Sep-04

Aug-04

Jul-04

Jun-04

May-04

Apr-04

Mar-04

Feb-04

Jan-04

Dec-03

Nov-03

Oct-03

Sep-03

Aug-03

Jul-03

Jun-03

May-03

b Figure 6. (a) Groundwater level hydrograph of observation wells screened in the deep aquifer; (b) Response of irrigation pumping on water level of different aquifers

In Figure 6b, water level hydrographs of five wells installed in all three aquifers within 500m2 area are drawn for comparison study. It is clear that maximum water level i.e. static water level is almost same (about 4.5m above MSL) for three aquifers and during irrigation period, all five wells response similarly with different declining intensity. Because of continuous percolation of rain water through unsaturated zone, lowering of water level in

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shallow aquifer is very low (about 1.0m) that is about 10.5m for main aquifer and about 6.5 for deep aquifer. As most of the irrigation wells are installed in the main aquifer, lowering of water level is highest in this aquifer. However, the trend of water level fluctuations in different aquifers support the hydraulic connectivity of these aquifer i.e. the aquitards separating aquifers are not continuous regionally rather locally extended. This implies that uncontrolled development of deep aquifers may cause both qualitative and quantitative degradation of groundwater. Water levels dropping below sea level for over withdrawal in dry season may eventually cause saline water intrusion as well as leakage of arsenic from shallow aquifer to upper part of main aquifer.

Response of Groundwater Levels to Pumping The water level in the pumping (deep) aquifer declined abruptly during the first 3 minutes of pumping, then stabilized at about 12.5 to 13.0m drawdown for the duration of the test (Figure 7a). The pumping phase of the aquifer test was terminated after 5,911 minutes (98.5 hours), and data was collected during the recovery phase of the test until 7,168 minutes (119.5 hours) after the test had begun. During the pumping test, water levels in observation wells open to the shallow and main aquifers showed no noticeable effect from pumping in the deep aquifer (Figure 7b). The overall fluctuation of water levels (as caused by factors other than long-term declines or barometric pressure changes) measured in P-1, P-4, and P-5 was less than about 0.04m during the test. This indicates that the 12 to15m confining unit that separates the main and deep aquifer retards the movement of ground water between the aquifers. Therefore, under conditions of moderate groundwater use for public supply, arsenic-rich, iron-rich, and saline ground water in the shallow aquifer are not likely to be drawn into the deep aquifer. Groundwater levels in the deep aquifer observation wells responded to the withdrawal of water from the pumping well. Water levels in P-2 (44 m from the pumping well) declined about 1.1 m in response to pumping (Figure 7a). Water levels in P-3 (120m from the pumping well) declined about 0.68m in response to pumping. Small fluctuations in the measured water levels may be partly caused by variations in the pumping rate during the test as voltage in the power supply fluctuated. Water levels in both observation wells rose rapidly at the end of the aquifer test, and had returned to within 0.05m of the pre-test water levels when data collection was stopped. Comparison of water level altitudes in the shallow and deep observation wells shows that water levels in the deeper aquifer were lower than water levels in the shallower aquifer in an area about 100m radius centered on the pumping well after about 3,600 (60 hours) minutes into the test.

Analysis of Drawdown Data Gunther Theim (1962) published the first formula on the response equation for steady radial flow (Ferris et al. 1962), based on the work of Darcy and Dupuit, and computed the hydraulic characteristics of a water-bearing formation by pumping a well and observing the effect of this pumping in a number of other wells. Now, most of the more or less complicated

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flow problems can be solved by applying proper mathematical methods. Aquifer test analyses may provide unrealistic estimates of hydraulic properties (Halford and Kuniansky. 2002). However, it makes no difference in performing a test whether response curves are obtained as analytical solutions or by other methods, e.g. flow nets, digital computers, etc (Stallman 1976).

P-3

P-2

a

P-1, P-4 and P-5

b Figure 7. Monitoring of water level (drawdown) during aquifer test (a) Observation wells in deep (3rd) pumping aquifer below aquitard; (b) Observation wells in upper aquifers.

The investigated deep aquifer under Sreerampur village may be considered as a confined aquifer as it is overlain by a 10 m to 12 m thick nearly-impervious silty clay layer, 295m

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below the surface. The lower boundary of the aquifer was not encountered above the investigated depth of 362 m; however, from the depositional history of the region it may be assumed that another impermeable formation can exist beneath the tested depth. A confined aquifer is a completely saturated aquifer whose upper and lower boundaries are impervious layers. However, completely impervious layers (aquatard) rarely exist in nature. A semiconfined or leaky aquifer, is a completely saturated aquifer that is bounded above by a semipervious layer and below by a layer that is either impervious or semi-pervious. Physically, there is induced recharge during an aquifer test through the semi-pervious layer. The rate of leakance is determined by the hydraulic conductivity of the aquitard and head difference across the aquitard. In a semi-confining scenario, confining units may pinch out laterally and part of the aquifer may become unconfined. This may true for the studied aquifer, too.

Hantush-Jacob Leaky Aquifer Model For all practical purposes, the time-drawdown behavior before the inflection represents a withdrawal of water from storage from the aquifer, no part of which was contributed from other sources. This part of the curve, along with its straight line extension, may be analyzed with methods discussed for time-drawdown behavior. There are several ways in which this condition may be compromised in field conditions: direct recharge from streams, recharge across bounding low permeable materials etc. The problem of leakage has been extensively investigated by Hantush and Jacob (1955) and Hantush (1956, 1960, 1964). In a leaky aquifer, the drawdown curve initially follows the nonleaky curve. However after a finite time interval, the lowered hydraulic head in the aquifer induces leakage from the confining layer. If there is storage in the confining layer, the rate of drawdown is slower than that in the case where there is no storage in the confining layer (Hantush 1960). In many aquifer tests, water is contributed from the less permeable confining units, in addition to the aquifer that is pumped (Halford and Kuniansky 2002). Hantush and Jacob (1955) presented a solution for drawdown in a pumped aquifer that has an impermeable base and a leaky confining unit above. Conceptually, this would be a four layer system, from top to bottom, a water table aquifer, a leaky confining unit, a confined aquifer, and an extremely low permeability bed rock. During the early time of pumpage, water is coming out of storage from the pumped aquifer and the leaky confining unit. Eventually, the discharge comes into equilibrium with the leakage through the confining unit from the unstressed water-table aquifer and the system is at steady-state. The additional assumptions for the analysis are: ·

Aquifer is leaky, horizontal flow stressed aquifer, vertical flow through confining unit. · Drawdown in the water table or unstressed aquifer is negligible. · Well storage can be neglected. · Water instantaneously comes out of storage in the aquifer. · Confining unit storage is negligible. The equation is based on the drawdown of a well pumped at a constant discharge rate in a leaky aquifer (Hantush and Jacob 1955).

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s

341

Q W (u, r ) B 4T

where, u 

r 2S is dimensionless time, 4Tt

K Z b' T

1  B

KZ/b' is the leakance (1/T), where KZ is vertical hydraulic conductivity of the confining unit (L/T) and b' is thickness of the confining unit (L). Early drawdown data from the field curve match the non-leaky part of the curve, but they soon deviate and follow one of the leaky r/B curves. The match point method yields values of w(u, r/B), 1/u, t and s. In addition, the r/B curve followed by the field data is noted. T and S are readily determined from

T

Q r 4uTt W (u, ) and S  2 4s B r

Unaware of the work done many years earlier by De Glee (1930, 1951), Hantush and Jacob also derived the same equation, which expresses the steady-state distribution of drawdown in the vicinity of a pumped well in a semi-confined aquifer in which leakage takes place in proportion to the drawdown. Hantush (1956, 1964) noted that if r/L is small (r/L≤0.05), the equation may, for practical purposes, be approximated by

sm 

2.30Q L (log 1.12 ) 2T r

Thus a plot of sm against r on semi-logarithmic paper, with r on the logarithmic scale, shows a straight-line relationship in the range where r/l is small. The slope of the straight portion of the curve, i.e. the drawdown difference Δsm per log cycle of r, is expressed by

s m 

2.30Q 2.30Q , consequently T  2s m 2T

The Hantush-Jacob leaky aquifer solution was chosen to analyze the aquifer test data because of the possibility that there was some induced leakage of ground water across the confining unit during the test. With the advent of computer programs to analyze aquifer test data, manually plot graphs and calculate aquifer parameters by hand is generally no longer in use. This makes it much easier to analyze the data, but makes much less aware of the assumptions behind the analytical solutions. Drawdown data for the individual deep

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observation wells can be matched well to about 500 minutes using the Hantush-Jacob solution. Drawdown from about 200 to 500 minutes becomes nearly constant at about 1 m for P-2 and nearly constant at about 0.6 m for P-3. This response may be caused by equilibrium between leakage across the confining unit and drawdown from well pumpage. After about 500 minutes, drawdown in both observation wells begins to increase in an approximately linear manner, at a rate of about 0.00001 m/minute in P-2 and about 0.00002 m/minute in P-3. This may indicate decreased leakage through the confining unit, draining of stored water in a permeable sand lens, or the presence of a lateral barrier. Of the three possibilities, a lateral barrier seems unlikely considering the geological setting. The best fit curve match (Figure 8a) to P-2 yielded an aquifer storage of 0.0013, aquifer transmissivity of 2,300 m2/day, vertical to lateral aquifer hydraulic conductivity ratio of 0.0044 (Kz/Kr), and a confining unit vertical hydraulic conductivity of 0.33 m/day (r/B = 0.1533). The best fit curve match (Figure 8b) to P-3 yielded an aquifer storage of 0.0022, aquifer transmissivity of 2,956 m2/day, vertical to lateral aquifer hydraulic conductivity ratio of 0.0044, and a confining unit vertical hydraulic conductivity of 0.43 m/day (r/B = 0.365). Because the aquifer values are similar for the individual observation well data curve matches, both sets of data can be approximately fit to a single set of values (Figure 8c). The best fit curve match to both observation wells taken together yielded an aquifer storage of 0.0017, aquifer transmissivity of 2,386 m2/day, vertical to lateral aquifer hydraulic conductivity ratio of 0.0044, and a confining unit vertical hydraulic conductivity of 0.35 m/day (1/B = 0.0035). The parameter 1/B in the Hantush-Jacob solution is related to the confining unit vertical hydraulic conductivity by the relation Kv' = (T*b')/2B, where T is the deep aquifer transmissivity and b' is the confining unit thickness. The lateral hydraulic conductivity of the deep aquifer is estimated as 23 m/day using Kh=T/b, where b is the aquifer thickness. The vertical hydraulic conductivity of the deep aquifer as determined from the solution derived ratio of the aquifer vertical hydraulic conductivity to aquifer lateral hydraulic conductivity (Kz/Kr = 0.0044) is about 0.10 m/day, about 200 times smaller than the lateral hydraulic conductivity of the deep aquifer. This may indicate that the cumulative hydraulic effect of disseminated clay and silt in the aquifer is similar to the clay-rich confining unit. During the long-term monitoring period from May 2003 to November 2003 water levels in P-2 (deep aquifer) were from 0.15 m to 0.60 m higher than water levels in P-1. Therefore, under natural flow conditions experienced during the summer months, the groundwater gradient is upward from the deep aquifer to the shallow aquifer, and the specific discharge through the confining unit is estimated to be from about 0.004 m/day to about 0.017 m/day. Assuming a porosity of 0.30, the average velocity of water through the 12.2 m thick confining unit is 0.014 m/day to 0.057 m/day. The time for groundwater to move through the confining unit under these gradients at the estimated average velocities range from about 200 days to about 850 days.

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b

c Figure 8. Best fit Hantush-Jacob solution to drawdow.n data in semilog scale collected from (a) P-2; (b) P-3; (c) Average drawdown data for P-2 and P-3.

Papadopulos-Cooper Solution to Drawdown Data A solution for drawdown in large diameter wells that takes into consideration the storage within the well, which is assumed to be negligible in the Theis method, has been presented by

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Papadopulous and Cooper (1967). Besides the general assumptions mentioned earlier, the added conditions are · Storage in the well cannot be neglected as well diameter is relatively large. · The aquifer is confined. · The well losses are negligible. The general flow equation inside a large diameter well is (Kruseman and Ridder, 1983),

sw 

Q F (u w,  ) 4T

where F (uw,  ) is a function for which numerical values are given. 2

2 r S rw S and   w 2 uw  4Tt rc

The index w stands for ‗at the pumped well‘ and rc is the radius of the unscreened part of the well. The best visual fit to the complete data sets, using each observation well individually, is the Papadopolous-Cooper solution (Figures 9a and 9b). However, the best match for each set of observation well data required aquifer properties and well bore diameters that were not reasonable.

A Figure 9. (Continued)

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b

c Figure 9. Best fit Papadopulos-Cooper solution to drawdown data in semilog scale collected from (a) P2; (b) P-3; (c) Average drawdown data collected from both P-2 and P-3.

Aquifer storage values were extremely small (on the order of 10-9). The size of the well bore needed to match the delayed drawdown response was much larger than the actual bore of the pumping well for both data sets. Both observation well P-2 and P-3 have an actual casing

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radius of 0.175 m. The predicted casing radius for P-2 was 1.735 m, and for P-3 that was 3.372 m. Also, the transmissivity value predicted from the data for P-2 (6921 m2/day) differed considerably from the value predicted from the data for P-3 (8401 m2/day). Because the transmissivity and well-bore radius values predicted from the individual observation well curve matches differed considerably, the drawdown data cannot be matched simultaneously for both observation well P-2 and P-3 using average transmissivity values (Figure 9c).

Jacob‘s Straight-Line Plot The Cooper-Jacob (1946) method of time-drawdown and Jacob (1950) method of distance-drawdown are a modification of the Theis equation and assume that u is small. However, the conditions for its application are more restricted than for the Theis and Chow method. Cooper-Jacob and Jacob methods are valid where the time (t) is sufficiently large or radial distance (r) is sufficiently small. The Cooper-Jacob time-drawdown approach is modified to plot drawdowns at various radial distances from the pumping well. By graphing drawdown on a linear scale versus distance on a logarithmic scale, a straight line fit results (Jacob 1950). Estimates of the hydraulic properties can also be made, if the assumption of u being sufficiently small (0.02) is valid. The intercept where the straight-line fit crosses the zero drawdown represents the range of influence of the cone of depression (Weight and Sonderegger 2000). The same assumptions apply to the Cooper-Jacob analytical solution as the Theis solution, but the well function W(u) is calculated for u<0.01 in order to neglect all but the first two terms of the infinite series of the well function. A straight-line approximation of W(u) is adequate for most applications even where u is as great as 0.1 (Halford and Kuniansky 2002). In the Theis formula, the exponential integral can be expanded in a convergent series, so that the drawdown (s) may be written as (Kruseman and Ridder 1983)

Q u2 u2 s (0.5772  ln u   ) 4T 2.2! 3.3! From u 

r 2S it will be seen that u decreases as the time of pumping t increases. 4Tt

Accordingly, for large values of t and/or small values of r the terms beyond ln u in the series of the equation become negligible. So for small values of u (<0.01) the drawdown can be expressed as

s

Q r 2S (0.5772  ln ) 4T 4Tt

After rewriting and changing into decimal logarithms this equation reduces to

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s

347

2.30Q 2.25Tt log 2 4T r S

Therefore, a plot of drawdown versus the logarithm of t forms a straight line. If this line is extended till it intercepts time-axis where s=0, the interception point will have the coordinates s=0 and t=t0. Substitution of these values into the drawdown equation gives

0

2.25Tt 0 2.30Q , log 4T r 2S

and because

2.25Tt 0 2.25Tt 0 2.30Q  1 or S   0 , it follows that 2 4T r S r2

If t/t0=10 and hence log t/t0 =1, s can be replaced by Δs, i.e. by the drawdown difference per log cycle of time, and it follows that

T

2.30Q . 4s

Jacob straight-line plot was applied to interpret the time-drawdown data for Sreerampur test as time was sufficiently large and radial distance is small. Drawdown recorded manually at selected intervals was used to create the hand-drawn data graph in Figure 10.

Figure 10. (Continued)

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Figure 10. Jacob straight-line time-drawdown method for a confined aquifer plotted on semilog papers for (a) P-2 and (b) P-3.

In this method, the field data are plotted on semi-logarithmic paper and a straight line is drawn through the field data points and extended backward to the zero drawdown axis. This is the distance at which the well is not affecting the water level. It may intercept the axis at some positive value of time (t0). The value of drawdown per log cycle (ΔS) is obtained from the slope of the graph. Drawdown of the observation wells installed in the deep aquifer were used to calculate transmissivity and storativity values of the deeper formation. The rate of drawdown after 1000 minutes is negligible because the water table was nearly in the steady condition. For P-2 two straight lines were drawn and transmissivity and storativity values were calculated as 1,070.784 m2/day and 0.00095 (first cycle) and 2,078.496 m2/day and 0.00268 (third cycle) respectively. The average values are 1,574.64 m2/day and 0.001815 respectively for transmissivity and storativity. These values are more appropriate as this well (P-2) is within a reasonable distance from the pumped well. Two straight lines were also drawn for P-3 and transmissivity and storativity values were calculated as 1,570.464 m2/day and 0.00068 (second cycle) and 2,944.656 m2/day and 0.00010 (third cycle) respectively. The average values are 2,257.56 m2/day and 0.00039 respectively for transmissivity and storativity. Using computer program, transmissivity and storativity were estimated as 2,468 m2/day and 0.000546 respectively (Figure 11).

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Figure 11. Jacob straight-line time-drawdown method for a fully confined aquifer using computer program for P-2.

Chow‘s Method Chow (1952) developed a method which has the advantage of avoiding the curve fitting of the Theis method and not being restricted to small values of r and large values of t as is the Jacob method (Kruseman and Ridder 1983). The same assumptions and conditions are generally satisfied as for the Theis method because this method is directly based on the Theis equation.

s

Q W (u ) 4T

To find the values of W(u) and u corresponding with the drawdown s measured at a certain moment t, Chow introduced the function

W (u )e u F (u )  2.30 F(u) can be calculated from the drawdown (s) versus the corresponding time (t) on single logarithmic paper (t on logarithmic scale). Using a computer program, transmissivity and storativity were estimated as 2,948 m2/day and 0.0000357 respectively (Figure 12).

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Figure 12. Analysis of data with the Chow method for P-2.

Theis Recovery Data Analysis for Confined Aquifer The analysis of recovery data involves the measurement of the rise in water levels, also referred to as residual drawdowns, following the cessation of a period of pumping at a constant rate. The field procedure requires a drawdown measurement at the end of the pumping period (t) and recovery measurements during the recovery period (t'). The residual drawdown is plotted on a linear axis and the value of t/t' on the logarithmic axis. The analytical method is based on the Theis theory and applies to confined aquifers with fully penetrating wells. The method relies on the theory of superposition in that the water level rise after the test is assumed to be the combined response to an imaginary well recharging the aquifer and continued pumping. Imaginary recharge occurs at an identical rate to the constant discharge during the pumping test. The equation for residual drawdown after a pumping test with constant discharge is:

s' 





Q W (u )  W (u ' ) 4T

where, u 

r 2S r 2S ' and u  4Tt 4Tt '

If u and u' are small, less than 0.01, then the above equation can be simplified to:

s' 

2.3Q t log 10  '  4T t 

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A semilog plot of s' versus t/t' will yield a straight line. The slope of which is:

s ' 

2.3Q 4T

where, Δs' is the change in residual drawdown in one log cycle of t/t'. The same assumptions as for the Cooper-Jacob, straight-line method must be met, and the flow to the well is in an unsteady state when t'>(25 r2S)T and u<0.01. Using computer program, transmissivity was estimated as 2,385 m2/day (Figure 13).

Figure 13. Analysis of recovery data with the Theis recovery method for P-2.

Comparison of Results for Different Methods The results of the pumping test and recovery data analysis using different methods are presented in table 5. The estimated transmissivity of the aquifer using drawdown data collected at P-2 varied between 1,070 m2/day for first cycle analysis using Jacob‘s straight line method and 2,948 m2/day applying Chow‘s method from constant-discharge test, to 6921 m2/day for Papadopulous-Cooper method. For P-3, the estimated transmissivity ranged between 1,570 m2/day using Jacob‘s straight line method from second cycle analysis and 2,956 m2/day using Hantush-Jacob solution, to 8,401 m2/day using Papadopulous-Cooper method. Because of solution match difficulties, the Papadopolous-Cooper solution was not considered as the appropriate solution to the aquifer-test data. Transmissivity was calculated as 2,385 m2/day using recovery data analyzed by the Theis recovery method. As these are graphical methods of solution, there is often slight variation in the results, depending upon the accuracy of the graph construction and subjective judgments in matching field data to type curves (Fetter 1994).

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Table 5. Aquifer properties determined from constant-discharge pumping test data using different methods Method Papadopulous-Cooper Hantush-Jacob Jacob (Hand Drawn) 1st Cycle 2nd Cycle 3rd Cycle Jacob Chow Theis Recovery

Transmissivity (m2/day) P-2 P-3 6,921 8,401 2,300 2,956

Storage Coefficient P-2 P-3 0.0013 0.0022

1,071 2,078 2,468 2,948 2,385

0.00095 0.00268 0.000546 0.0000357 -

1,570 2,945 -

0.00068 0.00010 -

The accuracy of the numerical values of the hydraulic characteristics of water-bearing layers and less permeable strata determined during the graphical analyses and the accuracy of the assumed boundary conditions play an important role in the reliability of the results obtained by these methods.The transmissivity values estimated from the Theis curve method and the Cooper-Jacob method are generally are comparable (Weight and Sonderegger 2000), and the resulting answers are almost the same. Well losses and partial penetration have a minimal effect on transmissivity values that are estimated using the Cooper-Jacob method. Additional drawdown at later times is due to declining heads in the aquifer and the rate of decline is controlled mostly by the transmissivity of the aquifer. Analyzing the change in drawdown at later times negates the effect of a fixed offset due to well losses and partialpenetration on the determination of transmissiviy (Halford and Kuniansky 2002). Estimated storage coefficient values ranged from 0.0000375 to 0.00268 for P-2 and from 0.00010 to 0.0022 for P-3. The estimated storage coefficients indicate that the aquifer is confined to leaky-confined or semi-confined in nature. Bouwer (1978) and Fetter (1994) suggest that storage coefficients for confined aquifer can vary from 0.00001 to 0.001, and Weight and Sonderegger (2000) suggest that storage coefficients for leaky-confined or semiconfined aquifers can vary from 0.001 to 0.03. The sorting of unconsolidated sediments largely controls the expected range of hydraulic conductivity. Well-sorted sediment has a much larger hydraulic conductivity than poorlysorted sediment, because finer material fills the voids between coarser grains in poorly-sorted sediment. The hydraulic conductivity of unconsolidated sediment can be estimated empirically from the grain-size distribution (Vukovic and Soro 1992). Hydraulic conductivity estimates from grain-size distributions typically have a greater uncertainty than estimates from aquifer tests (Halford and Kuniansky 2002). Using the Hantush-Jacob solution, the vertical to lateral aquifer hydraulic conductivity ratio was estimated at 0.0044, and the confining unit vertical hydraulic conductivity was estimated at 0.35 m/day. Drawdown in both observation wells becomes constant in the interval from about 200 to 500 minutes. During the aquifer test, water levels in P-1 were about 0.4 m higher than water levels in P-2. Therefore, under pumping stresses similar to those induced during the aquifer test, the ground-water gradient is downward from the main aquifer to the deep aquifer, and the groundwater flux through the confining unit is estimated at about 0.011 m/day. Assuming a

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porosity of 0.30, the average velocity of water through the confining unit is 0.037 m/day. The time for ground water to move through the confining unit under this gradient at the estimated average velocity is about 330 days.

CONCLUSION Three distinct aquifer units have been classified in the studied Meghna floodplain areas to an investigated depth of about 400m. However, there is a complex aerial variability of the hydrogeologic characteristics as a consequence of the floodplain depositional history of the formations. Groundwater level in the shallow and main aquifers varies in places due to localscale lithologic variations, leakage, the impact of irrigation abstraction and partially by unconfined storage change in the aquifer. During the dry irrigation period pumping water mainly from the lower part of the shallow and upper part of the main aquifers, water levels in all three aquifers response to the pumping stress. The trend of water level fluctuations in different aquifer units supports the hydraulic connectivity of aquifer formations. The aquitards separating aquifers are not continuous regionally but locally extended. During the pumping test, water levels in observation wells open to the shallow and main aquifers showed no noticeable effect from pumping in the deep aquifer that indicates at least local hydraulic separation of aquifers and under conditions of moderate groundwater use for domestic and municipal supply, arsenic and/or chloride-rich groundwater in the upper aquifers are not likely to be drawn into the deep aquifer. Aquifer test results as well as aquifer properties and characteristics of the deep aquifer show that the deep aquifer can yield significant amounts of potable water. As different hydrogeologic factors control the aquifer parameters and the graphical methods of solution depend upon the accuracy of the graph construction and subjective judgments in matching field data to type curves, solution results have varied for different method of analysis. However, all results indicate that the aquifer is confined to leaky-confined or semi-confined in nature. Slight deviations are not prohibitive to the application of different methods. When greater deviations from the above assumptions occur, special flow problems may raise. So, it is useful to evaluate data using as many methods as possible. Each may help to provide a different perspective and aid in a better interpretation. The Hantush-Jacob solution for a leaky confined aquifer was chosen as the most representative of the physical situation and this gives better results considering field condition in the deltaic floodplain aquifers.

ACKNOWLEDGMENT The authors are grateful to the respective officials of Ground Water Hydrology (GWH) of Bangladesh Water Development Board (BWDB) and Bangladesh Arsenic Mitigation Water Supply Project (BAMWSP) for undertaking such an extensive aquifer pumping test component at Sreerampur. Gratitude goes to Mr. Alamgir Hossain, MA Karim, Satish C Das GWH, BWDB for supporting authors to participate during the pump test, Mr. M Zainal Abdin to support data analysis using computer programs developed by him and to all field personnel of BWDB participated in performing pump test.

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REFERENCES Bouwer, Herman (1978) Groundwater Hydrology: McGraw-Hill, New York, 480 p. Brammer H (1996) The Geography of the soils of Bangladesh. University Press Limited. BWDB-UNDP (1982) Groundwater Survey: the Hydrogeological conditions of Bangladesh. UNDP Technical Report DP/UN/BGD-74-009/1, 113p. BWDB-USGS (2005) Report on Deep Aquifer Characterization and Mapping Project, Phase-I, Ground Water Hydrology Division-I, BWDB. Chow VT, (1952) On the determination of transmissivity and storage coefficients from pumping test data. American Geophysical Union Transactions, v.33, 397-404. Cooper HH and Jacob CE (1946) A generalized graphical method for evaluating formation constants and summarizing well field history, American Geophysical Union Transactions, v.27,526-534. De Glee GJ (1930) Over grondwaterstromingen bij wateronttrekking door middle van putten. Thesis, J. Waltman, Delft (The Netherlands): 175p. De Glee GJ (1951) Berekeningsmethoden voor de winning van groundwater. In: Drinkwatervoorziening, 3e Vacantie cursus: 38-80. Moorman‘s periodieke pers. The Hague (The Netherlands). Domenico PA, Schwartz FW (1997) Physical and Chemical Hydrogeology, 2nd edition, John Wiley and Sons Inc., New York, 506p. DPHE-BGS (2001) Arsenic contamination of groundwater in Bangladesh, British Geological Survey and Department of Public Health Engineering, Govt. of Bangladesh; rapid investigation phase, Final Report. Driscoll FG (1995) Groundwater and Wells, US Filter/Johnson Screens, St. Paul, MN 55112, 1089p. Ferris JG, Knowles DB, Brown RH, Stallman RW (1962) Theory of aquifer tests: U.S. Geological Survey Water Supply Paper 1536-E, P. 69-174. Fetter CW (1994) Applied Hydrogeology, Third Edition: Macmillan, New York, 691 p. Halford KJ and Kuniansky EL (2002) Documentation of spreadsheets for the analysis of aquifer test and slug test data: U.S. Geological Survey, open-file report 02-197, 51 p. Hantush MS (1956) Analysis of data from pumping tests in leaky aquifers: Transactions of the American Geophysical Union, vl.37, 702-714. Hantush MS (1964) Drawdown around wells of variable discharge. Journal of Geophysical Resources, vol, 69:4221-4235. Hantush MS (1960) Modification of the theory of leaky aquifer. Journal of Geophysical Resources, vol, 65:3713-25. Hantush MS and Jacob CE (1955) Non-steady flow in an infinite leaky aquifer: Transactions of the American Geophysical Union, vl.36, 95-100. Hussain SI and Huq NE (1998) Late Quaternary morphostratigraphy and paleogeography of Chandpur-Shariatpur area, south-central Bangladesh: Bangladesh Journal of Geology, Vol. 17, P 1-9. Jacob CE (1947) Drawdown test to determine the effective radius of artesian well: Transaction of the American Society of Civil Engineers, Paper 2321, v.112, 1047-1064 p. Jacob CE (1950) Flow of ground-water. In Engineering Hydraulics. H. Rouse (ed.), John Wiley, New York, pp 321-386.

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Kruseman GP and Ridder NAD (1983) Analysis and evaluation of pumping test data: International Institute for Land Reclamation and Improvement/ILRI, The Netherlands, 200p. Lohman SW (1979) Ground-water Hydraulics, U.S. Geological Survey professional paper 708, 70pp. Morgan JP and McIntire WG (1959) Quaternary geology of the Bengal Basin, East Pakistan and India, Geological Society of America Bulletin, Vol. 70, pp. 319-342. Nury SN, Bashar K, Chowdhury KR (1998) Determination of transmissivity and storage coefficient from step-drawdown pumping test data of Rajshahi and Dhaka using Birsoy and Summers‘s method: Bangladesh Journal of Geology, Vol. 17, P 43-54. Papadopulos IS and Cooper HH (1967) Drawdown in a well of large diameter. Water Resources Research, v. 3, p. 241-244. Ravenscroft P (2003) Overview of the Hydrogeology of Bangladesh. In. Groundwater Resources Development in Bangladesh: Background to the Arsenic Crisis, Agricultural Potential and the Environment. Editors: A Atiq Rahman and Peter Revenscroft. The University Press Limited, Bangladesh. P 43-86. Stallman RW (1976) Aquifer test design, observation and data analysis, Techniques of waterresources investigations of the U.S. Geological Survey, Book 3, Chapter B1, 26p. Theim G (1906) Hydrologic methods: Leipzig, J.K. Gebhardt, 56p. Theis CV (1935) The relation between the lowering of the piezometric surface and the rate and duration of discharge of a well using ground water storage: Transaction of American Geophysical Union, v. 16, 519-524 p. Theis CV (1940) The source of water derived from wells-essential factors controlling the response of an aquifer to development. Civil Eng., American Society of Civil Engineers, p. 277-280. Vukovic M and Soro (1992) Determination of hydraulic conductivity of porous media from grain-size composition: Water Resources Publications, Littleton, Colorado, 83 p. WARPO (2000) Draft Development Strategy (DDS), Estimation of groundwater resources, Annex-C, Appendix 6, National Water Management Plan, WARPO, Dhaka, Bangladesh, 2000. Weight WD and Sonderegger (2000) Manual of Applied Field Hydrogeology, McGraw-Hill, New York, 608p. Zahid A, Hassan MQ, Balke K-D, Flegr M, Clark DW (2007) Groundwater Chemistry and Occurrence of Arsenic in the Meghna Floodplain Aquifer, Southeastern Bangladesh. Journal of Environmental Geology, DOI 10.1007/s00254-007-0907-3.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 13

APPLICATION OF DNA MICROARRAYS TO MICROBIAL ECOLOGY RESEARCH: HISTORY, CHALLENGES, AND RECENT DEVELOPMENTS John J. Kelly Department of Biology, Loyola University Chicago 6525 N. Sheridan Road, Chicago, IL 60626

ABSTRACT During the last three decades, molecular methods have dramatically expanded our view of microbial diversity in natural and engineered systems. A variety of molecular approaches, including both PCR-based and hybridization-based techniques, have been applied extensively to the analysis of complex microbial communities and have yielded new insights. However, the majority of molecular methods that are widely used in microbial ecology are limited in their ability to encompass the incredible diversity of microbial communities. DNA microarrays, which were first introduced in the early 1990s, are one of the fastest growing technologies in biology, and they offer tremendous potential for microbial ecologists. DNA microarrays consist of nucleic acids spotted within a very small area on some solid support, and they enable the immobilization and simultaneous hybridization of hundreds of thousands of nucleic acids. This represents a dramatically higher degree of multiplexing than is possible with other widely used technologies. In addition, microarrays offer the advantages of increased speed of detection, low cost, and the potential for automation. Microarray technology has been used extensively for measuring gene expression in a wide variety of organisms, including human cells, plants, yeast, and bacteria, but its application to microbial ecology has been more limited. There are significant challenges to the use of microarrays in microbial ecology studies, including optimization of specificity and sensitivity and quantification of targets. However, in recent years several research groups have made significant progress in overcoming these challenges, and microarrays are beginning to be applied more frequently to microbial ecology studies in a variety of systems including terrestrial soils,  Phone: 773.508.7097 ; Fax: 773.508.3646 ; E-mail: [email protected]

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John J. Kelly wetland sediments, and freshwater and marine ecosystems. This article will provide a brief history of the use of molecular methods in microbial ecology, and will then review the development of microarray technology, the challenges that exist for application of microarrays to microbial ecology, the available strategies for overcoming these challenges, and some recent applications of microarrays to studies in microbial ecology.

PURE CULTURE TECHNIQUES One of the key innovations that spurred the development of the science of microbiology was the pure culture technique, which was introduced by Robert Koch in the late 1800s (Beck, 2000). The pure culture technique made it possible for the first time to isolate individual microbial species in the lab and to study their physiological and biochemical properties, and it enabled Koch in 1876 to prove the germ theory of disease by demonstrating conclusively that Bacillus anthracis was the causative agent of anthrax (Koch, 1876). Subsequently Koch used the pure culture technique to identify other disease-causing microorganisms, including Mycobacterium tuberculosis and Vibrio cholerae (Beck, 2000), thus revolutionizing our understanding of human and animal disease. The pure culture technique also allowed microbiologists to explore the incredible diversity of microorganisms in the natural world. In 1887 Sergei Winogradsky discovered the process of chemolithotrophy by isolating the marine bacterium Beggiatoa and observing that it could obtain the energy it needed for growth from the oxidation of an inorganic substrate (hydrogen sulfide), and in 1890 he elucidated another chemolithotrophic process when he isolated the first nitrifying bacteria from soil: Nitrosomonas and Nitrosococcus (Beck, 2000). In 1888 Martinus Beijerinck isolated the first nitrogen fixing bacterium (Bacillus radicicola) from the root nodules of the pea plant, and subsequently he isolated the first organism capable of anaerobic respiration: the sulfate reducing Spirillum desulfuricans, which is now known as Desulfovibrio desulfuricans (Beck, 2000). More recently, Konneke et al. (2005) were the first to isolate an autotrophic ammoniaoxidizing Archaeon in pure culture. Autotrophic ammonia-oxidation was previously thought to be limited to the Bacterial domain, but the possibility of Archaeal ammonia oxidation was suggested by several recent studies: in a metagenomic analysis of the Sargasso Sea, Venter et al. (2004) found a unique ammonia monooxygenase gene (ammonia monooxygenase catalyzes the first step of ammonia oxidation) on an Archaeal-associated scaffold. In addition, Treusch et al. (2005) found genes encoding a potential ammonia monooxygenase on a metagenomic soil clone alongside an Archaeal ribosomal RNA operon. These studies suggested that an Archaeon might be able to oxidize ammonia, but it was not until Konneke et al. (2005) isolated the first pure culture of an Archaeal ammonia oxidizer, named Nitrosopumilus maritimus, that the ability of an Archaean to catalyze this process was conclusively proven. Culture-based studies have thus made tremendous contributions to our understanding of microbial diversity and microbial physiology, and they continue to be a vital component of microbiology. However, culture-based studies, which require growth of microorganisms in the laboratory, are severely limited by the fact that most microorganisms are difficult or impossible to culture. Direct microscopic counts of bacteria in most habitats routinely exceed culture-based counts (plate counts or most probable number counts) by several orders of

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magnitude (Amann et al., 1995). For example, Jones (1977) found that the culturable fraction represented 0.25% of the total population in both freshwater and freshwater sediment, and Torsvik et al. (1990) reported that culturable organisms represented only 0.3% of the total community in soil. Staley and Konopka (1985) termed this phenomenon ―the great plate count anomaly‖, and this anomaly represents a significant challenge to microbial ecologists, as it indicates that culture-based studies provide an extremely restricted view of the diversity of the natural microflora. Fortunately, molecular techniques, which do not rely on laboratory culture, have led to significant advances in the study of microbial communities (Kelly, 2003).

SSU RRNA TECHNIQUES The most widely used molecular methods for the analysis of microbial communities are based on the small subunit ribosomal RNA (SSU rRNA) gene. The use of SSU rRNA as a phylogenetic marker was first proposed by Carl Woese in 1977 (Fox et al., 1977). The SSU rRNA molecule is a useful phylogenetic marker because it is present in all cells and because its sequence is fairly well conserved across phylogenetic groups. Since the pioneering work of Carl Woese, SSU rRNA gene sequences have been used to develop a comprehensive phylogenetic framework for the analysis of microbial communities (Amann et al., 1995). The most current version of the Ribosomal Database Project (release 9.56) contains 451,545 aligned and annotated SSU rRNA sequences (Cole et al., 2007). Molecular techniques that analyze prokaryotic SSU rRNA (i.e. 16S rRNA) genes have been widely applied to the study of microbial ecology (Macrae, 2000), and there are a wide variety of methods that have been used. For example, a phylogenetic inventory of the prokaryotic component of a microbial community can be assembled using the Polymerase Chain Reaction (PCR) to amplify the 16S rRNA genes contained within the community, followed by cloning and sequencing this collection of amplified genes. This approach, known as clone library sequencing, was first applied by Giovannoni et al. (1990) for the analysis of marine microorganisms from the Sargasso Sea. Phylogenetic inventories of this type can be produced for the entire bacterial component of a microbial community by using ―universal primers‖ designed to amplify 16S rRNA genes from all of the bacteria within a sample (e.g. Borneman et al., 1996; Kuske et al., 1997), or they can be produced for a specific phylogenetic group by using primers designed to amplify only the 16S rRNA genes of the group of interest. For example, Bruns et al. (1999) used primers specific for the 16S rRNA genes of autotrophic ammonia oxidizers to create an inventory of ammonia oxidizing bacteria from soil. Clone library-based inventories have significantly increased our understanding of microbial diversity by revealing much greater diversity than was detected by classical culturebased studies (Becker et al., 2000). However, clone library preparation and sequencing is time consuming, labor intensive, and expensive. Although the cost and time required for large scale sequencing projects are decreasing quickly, the incredible diversity of microbial communities makes it impractical at this point to attempt to sequence every amplicon produced from PCR amplification of an environmental sample. For example, soil from a beech forest showed total bacterial counts of 1.5 x 1010 g soil-1 and approximately 4,000 unique bacterial genomes based on DNA reassociation analysis (Torsvik et al., 1990), and a

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more recent study used reassociation kinetics to determine that a pristine soil contained more than one million distinct genomes (Gans et al., 2005). Attempting to capture all of this biodiversity by sequencing a clone library is beyond our current capabilities. Therefore, although phylogenetic inventories of microbial communities based on cloning and sequencing 16S rRNA genes can provide a great deal of useful information, these inventories will generally be incomplete, so populations comprising very small fractions of the overall community may not be detected. In addition, clone library sequencing is too slow to be useful for routine profiling and comparison of large numbers of environmental samples. As an alternative to cloning and sequencing, techniques such as denaturing gradient gel electrophoresis (DGGE; Muyzer et al., 1993) and terminal restriction fragment length polymorphism analysis (T-RFLP; Liu et al, 1997) can be used to analyze the amplicons produced by PCR amplification of DNA extracted from a microbial community. DGGE and T-RFLP do not identify individual bacterial species. They instead generate profiles for microbial communities based on differences in the gene sequences of their constituents, and these profiles can be used to compare microbial communities and assess their degree of similarity or difference. DGGE and T-RFLP have been applied to 16S rRNA amplicons produced with universal PCR primers (e.g. Crump et al., 2003; Janus et al., 2005) and to amplicons produced with group-specific PCR primers (e.g. Cebron et al., 2004; Rosch and Bothe, 2005), and these approaches have been used to compare microbial communities from different habitats (e.g. Ludemann et al., 2000), and to assess changes in microbial communities over time (e.g. Duineveld et al., 1998), seasonal changes (e.g. Crump et al., 2003), and changes resulting from different experimental treatments (e.g. Klamer et al., 2002).

LIMITATIONS OF PCR-BASED METHODS Clone libraries, DGGE, and T-RFLP are all useful techniques, but they are all reliant on PCR amplification, and there are significant limitations to all techniques that are based on PCR amplification of DNA extracted from environmental samples. First, PCR-based assays cannot provide reliable quantitative information on microbial community composition due to the potential for bias in PCR amplification. PCR bias can be caused by differences in DNA extraction efficiency (Miller et al., 1999), differences in gene copy number (Farrelly et al., 1995), and differences in the efficiency of the PCR reaction itself (Suzuki and Giovannoni, 1996). PCR bias can result in misrepresentation of phylogenetic diversity (Lueders and Friedrich, 2003), and thus it can be problematic in microbial ecology studies that rely on PCR. A second limitation of PCR-based assays is that DNA can persist in the environment outside of a cell for significant periods of time. For example, free DNA can bind to soil particles (Blum et al., 1997) and this binding can protect DNA from degradation (Demaneche et al., 2001). Thus, when working with environmental samples, it is possible for PCR to amplify DNA from cells that are no longer living, which would be a confounding factor in studies of microbial community structure. Both of these limitations, PCR bias and DNA persistence, should be considered when interpreting results from PCR-based community analyses such as clone library sequencing, T-RFLP, and DGGE (Kelly, 2003).

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PROBE-BASED METHODS As an alternative to PCR-based techniques, probe-based methods can be used to detect specific target bacteria in environmental samples. DNA probes, which are composed of short segments of DNA, typically 15 to 25 nucleotides in length, can be synthesized in the lab and can be designed to target regions of the 16S rRNA gene that are unique to a particular phylogenetic group (Stahl, 1995). Ribosomal RNA is a good target for probing because active cells contain thousands to tens of thousands of copies of ribosomal RNA per cell, making it a naturally amplified target which can often be detected without PCR amplification (Amann and Ludwig, 2000). DNA probes have generally been applied using either in-situ or membrane-based hybridization techniques. The in-situ technique, also known as fluorescence in-situ hybridization (FISH), hybridizes a fluorescently labeled probe to fixed whole cells. The fixation process renders the cells permeable to the probe, so cells containing RNA matched by the probe will be fluorescently labeled and can be observed and counted with an epifluorescence microscope. FISH, which was first developed by DeLong et al. (1989), can provide information on cell morphology as well as the abundance and spatial distribution of targeted organisms (Amann et al., 1995). FISH can be especially useful for examining spatial relationships between different phylogenetic groups of bacteria, as several unique probes can be applied to a sample simultaneously if the probes are labeled with different fluorescent markers (Mobarry et al., 1996). DNA probes can also be utilized in a membrane hybridization format known as dot-blot hybridization, which is similar to a conventional Southern hybridization. In dot-blot hybridization, RNA isolated from a microbial community is immobilized on a nylon or nitrocellulose membrane filter and exposed to a radiolabeled probe. Hybridization of a probe to an immobilized target can be detected based on the radioactive tag and can provide information on the presence or absence as well as relative abundance of the group targeted by that probe. This approach was first applied to monitoring population changes in the rumen of cattle (Stahl et al., 1988), and since then it has been used extensively for the analysis of microbial communities from a variety of habitats (e.g. Raskin et al., 1996; Rooney-Varga et al., 1997; Weber, 2001). However, membrane hybridization has several significant limitations. Since the probes are labeled with radioactive tags, only one probe can be applied per membrane, so a separate membrane must be prepared for each probe, which can become quite cumbersome. In addition, membrane hybridization can provide information on relative rRNA abundance, but relative rRNA abundance can not be directly translated into cell numbers since cellular rRNA contents can vary significantly with growth rate (Amann et al., 1995). In-situ and membrane hybridization techniques are extremely useful tools for microbial ecologists, and both methods have been widely applied. However, the scope of such studies is often constrained by the fact that these hybridization techniques severely limit the number of probes that can be applied simultaneously, thus limiting the amount of information that can be acquired (Kelly, 2003). Microarrays offer the opportunity to overcome some of the limitations of in-situ and membrane hybridization techniques.

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DNA MICROARRAY TECHNOLOGY DNA microarrays (also known as DNA microchips or DNA chips) generally consist of a set of nucleic acids spotted within a very small area on some solid support (Small et al., 2001). The concept of microarray hybridization of nucleic acids was first proposed 20 years ago by Bains and Smith (1988) as a method for sequence determination. In 1994 Pease et al. built one of the first DNA microarrays using photolithography to synthesize a miniaturized array of 256 densely packed oligonucleotide probes on a glass surface (Pease et al., 1994). Pease et al. demonstrated that these surface immobilized oligonucleotides could be hybridized in parallel to fluorescently labeled oligonucleotide targets, and hybridization could be detected by epifluorescence microscopy. Pease et al. used their microarrays for sequencing by hybridization, as had been proposed by Bains and Smith (1988). In 1995 Schena et al. took a different approach and built a microarray by immobilizing 45 Arabidosis thaliana cDNAs on poly-L-lysine-coated microscope slides using a custom-built arraying machine with a single printing tip. All of the microarray-immobilized cDNAs were hybridized simultaneously with a mixture of fluorescently labeled cDNAs produced from total Arabidopsis mRNA by a single round of reverse transcription. Hybridization to the microarray-immobilized cDNAs was quantified by measuring fluorescence with a laser scanner. This microarray was able to quantify the relative expression of 45 Arabidopsis genes simultaneously (Schena et al., 1995). Since the pioneering work of Pease et al. (1994) and Schena et al. (1995), microarrays have been widely used: according to a search of Web of Science (Thompson Scientific, Philadelphia, PA) on February 1, 2008, Pease et al., (1994) had received 674 citations and Schena et al. (1995) had been cited 3,462 times. In addition, rapid developments in both robotics and miniaturization technology have made it possible to immobilize higher numbers of nucleic acids, from thousands to hundreds of thousands, in even smaller areas, thus providing extremely high hybridization capacity (Gentry et al., 2006). The main application of microarrays to date has been analysis of gene expression (Gentry et al., 2006). In these studies, mRNAs are isolated from different samples or different experimental treatments and each mRNA pool is then either directly labeled with different fluorescent dyes (typically Cy3 and Cy5) (e.g. Taniguchi et al., 2001) or used to produce differentially labeled cDNAs via reverse transcription (e.g. Lashkari et al., 1997). Differentially labeled nucleic acid samples can then be hybridized simultaneously to a microarray containing gene specific probes, and comparison of the intensities of the two dyes provides relative expression levels of the genes contained on the microarray. This technique allows for the analysis of the differential expression of thousands of genes in a single experiment, and it has been used to analyze gene expression in a wide variety of organisms, including human cell lines (DeRisi et al., 1996), plants (Schena et al., 1995), yeast (Lashkari et al., 1997), and bacteria (de Saizieu et al., 1998). Microarrays have also been used for the detection of single nucleotide polymorphisms (Hacia, 1999; Straub, 2002), the detection of mutations (Cronin et al., 1996; Gerry et al., 1999), and the comparison of microbial genomes (Cho and Tiedje, 2001; Murray et al., 2001).

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MICROARRAYS FOR MICROBIAL COMMUNITY ANALYSIS David Stahl‘s group first proposed the use of microarrays for the detection of microbes within complex microbial communities (Guschin et al., 1997). The Guschin et al. approach was essentially the reverse of the dot-blot membrane hybridization technique in that Guschin et al. immobilized a set of oligonucleotide probes on a microarray, hybridized a fluorescently labeled target to all of these immobilized probes simultaneously, and detected hybridization via epifluorescence microscopy. Guschin et al. successfully demonstrated this approach by fabricating a microarray that included a set of eight oligonucleotide probes targeting the 16S rRNA genes of several groups of nitrifying bacteria. When this microarray was hybridized with fluorescently labeled RNA from several reference strains, the immobilized probes were shown to selectively capture RNA from the appropriate target organisms, thus providing specific identification of the target organisms. Guschin et al. suggested that this approach could be used for the detection of different microbial populations within complex communities, and that the extremely high probe capacity of microarrays should allow for the detection of hundreds to thousands of target organisms simultaneously. This would represent much higher throughput than is possible with other probe hybridization formats (i.e. membrane hybridization and FISH). After the pioneering work of Guschin et al. (1997), several groups demonstrated the effectiveness of microarrays for the detection of bacteria in complex communities based on hybridization of microarrays with RNA isolated directly from environmental samples: Small et al. (2001) detected Geobacter in soil samples; Koizumi et al. (2002) detected bacteria in oil-contaminated marine sediments; El Fantroussi et al. (2003) detected Acidobacteria as well as Alpha, Beta, and Gamma Proteobacteria in estuarine sediments; Kelly et al. (2005) detected nitrifying bacteria in wastewater treatment plant samples; and Smoot et al. (2005) detected bacteria in human saliva. One significant advantage of microarrays for the analysis of complex microbial communities via 16S rRNA sequences is that their extremely high probe capacity enables researchers to create highly redundant and hierarchically nested probe sets. Redundant probes are multiple distinct probes that target the same organism or group; for example, in Figure 1 probes 1 and 2 are redundant probes for organism A and probes 3 and 4 are redundant probes for organism B. Hierarchically nested probes target organisms at multiple phylogenetic levels; for example, in Figure 1 probes 1, 6, and 7 are hierarchically nested. Due to the fact that the 16S rRNAs include both highly conserved and highly variable regions, it is possible to design hierarchically nested probes that target organisms at various phylogenetic levels (e.g. species, genus, phylum and domain), with highly conserved sequences giving broad taxonomic resolution and hypervariable sequences giving genus- and species-level resolution. For example, SSU rRNA probes have been designed for the highest taxonomic level, the domains Archaea, Bacteria, and Eukarya; for intermediate levels such as the alpha, beta, and gamma subclasses of Proteobacteria; and for many genera, species, and subspecies (Amann et al., 1995).

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Figure 1. Model of a redundant and nested probe set. The model shows the phylogenetic realtionships of three hypothetical bacterial species (A, B, and C) as well as the coverage of hypothetical probes 1-7.

The use of redundant and hierarchically nested probes is not unique to microarray technology. These approaches are often incorporated to some degree in FISH and membrane hybridization experiments. However, the high probe capacity of microarrays enables redundant and hierarchically nested probes to be used on an unprecedented scale. Microarrays offer the possibility of including redundant and hierarchically nested probes for virtually every target, which would enable the detection of target organisms to be confirmed by multiple probes, thus greatly reducing false-positive errors. In addition, hierarchical nesting can provide a more complete picture of the overall phylogenetic composition of a microbial community than is possible if only genus- or species-specific probes are utilized, since broadly targeted probes will include organisms not targeted by more specific probes. A number of microarray studies have included both redundant and hierarchically nested probes (Liu et al., 2001; Loy et al., 2002; Kelly et al., 2002; Loy et al., 2005; Sanguin, Herrera et al., 2006).

MICROARRAY FORMATS One interesting aspect of microarray technology is the fact that multiple formats are available. The most widely-used format is the planar or 2-D array (Starke et al., 2006) in which nucleic acids are immobilized on a glass slide in a single layer (e.g. Schena et al., 1995). Planar arrays are widely available because the equipment required for fabricating these arrays has been commercialized and is rapidly dropping in price due to competition between a number of manufacturers, making this format accessible to many laboratories (Li and Liu, 2003). An alternative to planar arrays are gel-based microarrays (also known as 3-D arrays) in which individual acrylamide gel pads or drops are arrayed on a glass slide, and nucleic acids are covalently cross-linked to the acrylamide within these three-dimensional gel pads (Yershov et al., 1996). Gel-pad microarrays have not been used as widely as planar arrays because these arrays are not available commercially and currently they are only fabricated by a limited number of research laboratories (Li and Liu, 2003). However, gel-pad microarrays do offer some advantages over planar arrays. For example, the three dimensional nature of the gel pads makes it possible to immobilize a much higher concentration of oligonucleotide probes in the same microarray surface area: planar arrays can immobilize a maximum of approximately 10 pmol/cm2, whereas gel-pad microarrays can immobilize several nmols/cm2 (Li and Liu, 2003). Higher probe concentration is significant as it can improve detection of low abundance targets (Starke et al., 2006).

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One limitation of gel-pad microarrays is that they require targets to diffuse into the gel pad in order to hybridize with probes located within the gel interior, so the pore size of the gel will limit target access. Targets ranging in size from 20-150 nucleotides have been shown to penetrate the gel effectively and hybridize to probes throughout the entire three-dimensional gel pad (Starke et al., 2006). Therefore, when native RNAs or other large nucleic acids are used as the target for gel-pad microarrays, fragmentation of the nucleic acids is necessary prior to hybridization to allow access to the gel interior. Interestingly, fragmentation has also been shown to result in stronger hybridization signals for planar arrays (Liu et al., 2007). Fragmentation is beneficial in both gel-based and planar microarrays because it eliminates the three dimensional structure of the target nucleic acids which can interfere with hybridization, and it reduces steric hindrance during hybridization which can occur with large target molecules (Liu et al., 2007). Fragmentation of nucleic acids can be accomplished by treatment with divalent ions (Chee et al., 1996), alkali (Proudnikov and Mirzabekov, 1996), or nucleases (Gunderson, 1998). In addition, Kelly et al. (2002) developed an effective fragmentation method based on radical-generating coordination complexes. This method, which included simultaneous fluorescent labeling of the targets, resulted in random fragmentation of nucleic acids and effective hybridization of targets to DNA microarrays (Kelly et al., 2002). This method has been used in several microarray studies of environmental microbial communities (El Fantroussi et al., 2003; Smoot et al., 2005; Kelly et al., 2005; Siripong et al., 2006). The gel used in manufacturing gel-pad microarrays can also be modified to increase its porosity. Several new gel polymers with significantly larger pore sizes are under development, which should help reduce the impact of the gel on target diffusion and should allow for the hybridization of larger targets (Starke et al, 2006).

FUNCTIONAL GENE MICROARRAYS Bacteria can be placed into taxonomic groups based on phylogenetic affiliations (species, genera, etc.), but bacteria can also be grouped into functional guilds which can be defined by some metabolic capability (e.g. ability to denitrify) (Kelly, 2003). In some cases bacteria within a phylogenetically defined group share some metabolic functions. For example, among the bacteria autotrophic ammonia oxidation is restricted to two monophyletic groups: the first group belongs to the gamma subdivision of the Proteobacteria and includes one genus, Nitrosococcus; the second group belongs to the beta subdivision of the Proteobacteria and includes two genera: Nitrosomonas and Nitrosospira (Avrahami et al., 2002). In contrast, some metabolic functions are more widely distributed across numerous phylogenetic groups. An example is denitrification, which is found in about 50 bacterial genera (Rich et al., 2003). Molecular approaches based on 16S rRNA genes can target microorganisms based on their phylogenetic affiliation, which can provide functional information in some cases (e.g. ammonia oxidizing bacteria). However, 16S rRNA approaches are not useful for the detection of specific functional guilds when the function is widely distributed over the phylogenetic tree (e.g. denitrification) (Braker et al., 2001). Functional guilds can be targeted using so called ―functional genes‖ that code for enzymes critical to some metabolic process. Functional genes can be useful for detecting and

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monitoring functional guilds, and sequence variations within these functional genes can in some cases be used to differentiate members of a functional guild (e.g. Purkhold et al., 2000). Several prokaryotic functional genes involved in biogeochemical cycling processes have been identified and widely studied. For example, amoA codes for the membrane-associated activesite polypeptide of ammonia monooxygenase, which catalyzes the first step in nitrification (Rotthauwe et al. 1997); nirS and nirK code for structural variants of nitrite reductase, which catalyzes a key step in denitrification (Braker et al. 1998); nifH codes for one of the subunits of nitrogenase, the enzyme that catalyzes nitrogen fixation (Zehr et al., 2003) ; and dsrA and dsrB code for the alpha and beta subunits of dissimilatory sulfite reductase, which catalyzes a key step in sulfate reduction (Dahl et al., 1993). These functional genes have been used to analyze microbial communities via many of the same molecular approaches discussed above, including clone library sequencing (Zehr et al., 1998; Braker et al. 2000), T-RFLP (Ohkuma et al ., 1999; Avrahami et al., 2002; Angeloni et al., 2006), and DGGE (Ibekwe et al., 2003). A number of research groups have also applied microarray technology to the detection of functional genes. For example, Wu et al. (2001) built a functional gene microarray targeting different variants of several genes involved in the nitrogen cycle (amoA, nirK, and nirS). In this array, large PCR products (0.76 kb) from each of the functional gene variants were spotted on the array and used as hybridization probes. Taroncher-Oldenberg et al (2003) designed a microarray that included smaller oligonucleotide probes (70-mer) targeting different variants of amoA, nifH, nirK, and nirS. Zhou (2003) built a microarray that included 50-mer probes targeting genes involved in both nitrogen cycling (nirS, nirK, nifH, and amoA) and sulfur reduction (dsrA and dsrB), and Zhang et al. (2007) built an oligonucleotide microarray (15-25 mers) targeting variants of nifH.

CHALLENGES FOR MICROARRAY TECHNOLOGY: SPECIFICITY The application of microarrays to the assessment of microbes in the environment poses a number of technical challenges. One of the main challenges is the specific detection of target nucleic acids against a complex background of non-target sequences. In any hybridization format, the potential exists for non-specific hybridization, i.e. hybridization between a target and probe that are not perfectly complementary. To achieve highly specific detection the discrimination of perfect and imperfect duplexes is important, and for detection of specific targets with short oligonucleotide probes this often requires the discrimination of single-base mismatches. In membrane-based hybridizations, single base mismatch discrimination can be achieved by optimizing the hybridization and wash conditions (buffer composition, salt concentration, and temperature) for each oligonucleotide probe (Stahl et al., 1988). This approach is effective for membrane hybridization since each membrane is hybridized with a single probe. However, the challenge for microarrays is that a large number of probes, which can vary in duplex stability due to differences in length and base composition, are hybridized and washed simultaneously under the same conditions, making it impossible to optimize the conditions for all of the probes on the microarray. One approach to addressing this challenge is to design sets of probes such that all probes on an array have nearly identical melting temperatures. In this way, the microarray can be hybridized and washed under conditions (buffer composition, salt concentration, and

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temperature) that will minimize non-specific hybridizations for all of the probes. This approach can be effective (e.g. Bodrossy et al., 2003; Zhang et al., 2007), but it places significant constraints on probe design, which could make it difficult to build microarrays with very large numbers of probes. Another approach to optimizing specificity is the inclusion of tetramethylammonium chloride (Maskos and Southern, 1993) or betaine (Rees et al. 1993) in the hybridization buffer. These compounds equalize the melting points of oligonucleotides with different base compositions by stabilizing AT base pairs (Peplies et al., 2003) so that a single wash temperature can be used for all probes on a microarray (for an example of this approach see Loy et al., 2002). This approach is reasonably effective and it is simple, but it does require that all probes on the microarray be identical in length, which again places a constraint on probe design. Specificity on microarrays can also be optimized via non-equilibrium dissociation kinetics, first demonstrated by Liu et al. (2001). In this approach, the microarray is hybridized with the targets, a thermal platform is used to control the temperature of the microarray, and the dissociation of targets from each of the probes is monitored simultaneously by measuring the fluorescent signal for each probe as the temperature of the array is incrementally increased. The resulting melting profiles (signal versus temperature) can be used to discriminate target hybridizations from non-target hybridizations, since duplexes containing one or more mismatches are generally less stable and dissociate at a lower temperature than perfect-match duplexes (e.g. see Figure 2). Liu et al (2001) first demonstrated the effectiveness of the non-equilibrium dissociation approach in gel-based microarrays, and Li et al. (2004) and Wick et al. (2006) subsequently confirmed its effectiveness in planar microarrays. The non-equilibrium dissociation approach has been applied to microarray analysis of microbial communities (Kelly et al., 2005; Siripong et al., 2006), so it has been shown to be effective, but this approach is time consuming and technically challenging. For example, effective discrimination of single base mismatches depends on mismatch position (Urakawa et al., 2003; Wick et al., 2006), which places constraints on probe design. In addition, the characterization and analysis of large numbers of melting profiles is difficult. However, several recent publications have developed software tools to address this challenge (Urakawa et al., 2002; Pozhitkov et al., 2005). Another approach to optimizing specificity is the use of mismatch probes that are designed to differ in sequence relative to a perfect-match probe. Bavykin et al. (2008) used this strategy on a microarray that contained probes targeting 16S rRNA and 23S rRNA gene sequences in Bacillus anthracis and closely related microorganisms. This microarray was designed to include perfect match / mismatch probe pairs for each target, and Bavykin et al. found that analysis of perfect / mismatch signal ratios enabled specific identification of targets both singly and in mixtures (Bavykin et al., 2008). Wilson et al. (2002) used a similar approach with a SSU rRNA-based microarray and demonstrated specific detection of bacteria in pure cultures and simple mixtures. Specificity can also be addressed by designing microarrays in which single base mismatch discrimination is not a necessity. For example, Zhou (2003) built a microarray which included 50-mer probes targeting specific variants of genes involved in nitrogen cycling (nirS, nirK, nifH, amoA and pmoA) and sulfur reduction (dsrA and dsrB). Zhou found that genes with less than 86–90% sequence identity were clearly differentiated using hybridization conditions of 50ºC and 50% formamide. Although this does not reflect a single

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base mismatch level of specificity, Zhou suggested that this level of specificity should provide species level resolution, since the average similarity of these functional genes at the species level ranged from 74 to 84% (Zhou 2003).

Figure 2.Example of non-equilibrium dissociation curves (signal versus temperature) obtained with a DNA microarray containing oligonucleotide probes. S0 represents a perfectly matched probe-target duplex, S30 represents a probe-target duplex with a single mismatch, and S59 represents a probe-target duplex with two mismatches. Data reprinted from Urakawa et al. (2003).

CHALLENGES FOR MICROARRAY TECHNOLOGY: SENSITIVITY Sensitivity is another critical parameter for microarray applications. Planar microarrays have been shown to be 105-fold less sensitive than membrane hybridization (Cho and Tiedje, 2002), due to significant differences in the amount of nucleic acid immobilized: a membrane immobilizes > 1ug per dot, whereas a planar microarray immobilizes 10 to 20 pg per spot (Cho and Tiedje, 2002). As mentioned above, gel-pad microarrays have a higher probe binding capacity than planar arrays due to their three-dimensional nature. Gel-pad arrays have been used to immobilize 3 pmol of oligonucleotide probe per gel pad (Urakawa et al., 2002), which corresponds to approximately 18 ng for a typical 20 base pair probe. Therefore, gel-pad microarrays should result in an increase in sensitivity over planar arrays, but no direct comparisons of the sensitivity of these two formats are available in the literature. Several design and methodological approaches can also be used to increase sensitivity, such as increasing probe length and reducing the stringency of the hybridization and/or wash conditions. Several studies have demonstrated that increasing probe length results in increased sensitivity of microarray hybridization (Zhou , 2003). In addition, a number of studies have demonstrated higher hybridization signals at lower stringency: Guschin et al.

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(1997) showed higher hybridization signals at lower hybridization temperatures, and several studies have reported higher hybridization signals at lower denaturant (formamide) concentrations (Urakawa et al., 2002; Zhang et al., 2007). However, increasing probe length and reducing stringency also have the drawback of decreasing hybridization specificity, so it is necessary in any microarray application to find an appropriate balance between specificity and sensitivity. Increasing probe concentration is another approach that has been shown to increase sensitivity (Guschin et al., 1997), and this approach has the advantage of not decreasing specificity. Several groups have demonstrated that microarrays can be designed with adequate sensitivity to detect nucleic acids directly from environmental samples. As mentioned above, ribosomal RNA is a good target for hybridization because active cells contain thousands to tens of thousands of copies of ribosomal RNA per cell (Amann and Ludwig, 2000). Several groups have demonstrated successful microarray-based detection of ribosomal RNAs from a variety of environmental samples: Small et al. (2001) detected Geobacter chapellei 16S rRNA directly from a total-RNA soil extract; El Fantroussi et al. (2003) successfully detected Acidobacteria as well as Alpha, Beta, and Gamma Proteobacteria by hybridizing RNA extracted directly from estuarine sediments to a microarray containing 16S rRNA targeted oligonucleotide probes; and Kelly et al. (2005) detected nitrifying bacteria by hybridizing RNA extracted directly from a wastewater treatment plant aeration tank to a microarray containing 16S rRNA-targeted oligonucleotide probes. Detection of bacterial DNA in environmental samples via direct hybridization is more challenging since most bacterial cells contain only a single chromosome which generally includes one or perhaps a few copies of each gene. Nevertheless, Wu et al. (2001) successfully hybridized DNA isolated from soil and sediment samples to a microarray using large PCR products (0.76 kb) as hybridization probes. These very large probes enabled high sensitivity (1 ng of target DNA), but low specificity (genes had to be at least 15% to 20% different in sequence in order to be discriminated). Zhou (2003) used much smaller oligonucleotide probes (50 mer) and successfully hybridized DNA isolated from soil. These shorter probes resulted in improved specificity (genes that were at least 10% to 14% different in sequence were discriminated), but lower sensitivity (8ng of target DNA). However, Zhou (2003) suggested that this level of sensitivity should be sufficient for many studies in microbial ecology since DNA yields from soil and sediment samples typically range between 10 and 400 g of DNA per gram of soil dry weight.

AMPLIFICATION OF TARGETS PRIOR TO MICROARRAY HYBRIDIZATION Although the studies cited above have shown some success in detecting bacteria in environmental samples by direct hybridization of either RNA or DNA, the sensitivity of these approaches may not be adequate to detect nucleic acids in low biomass systems or to detect nucleic acids that represent a small fraction of the total nucleic acid pool in a sample. For example, microarrays may not be sensitive enough to directly detect organisms whose rRNA makes up a small fraction of the total community rRNA pool either because the organisms are present in low numbers or because their cellular rRNA content is low due to a low level of

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metabolic activity. In addition, functional genes present in low concentrations may not be detectable by direct hybridization. For this reason, several groups have used PCR amplification prior to microarray hybridization to increase target concentration. Because highly conserved universal primers for amplifying rRNA genes are available, PCR amplification can be run with universal 16S primers to simply increase the overall 16S rRNA gene concentration prior to hybridization with a microarray containing specific 16S rRNA probes. This approach was used by Loy et al. (2002) to detect sulfate-reducing prokaryotes in a hypersaline cyanobacterial mat, by Wang et al (2002) to detect a variety of human intestinal bacteria in fecal samples, and by Loy et al. (2005) to detect members of the betaproteobacterial order ―Rhodocyclales‖ in activated sludge from an industrial wastewater treatment plant. This approach has the advantage of increasing sensitivity, and general amplification of all 16S rRNA genes requires only a single PCR reaction. PCR can also be run with group-specific primers to selectively amplify the 16S rRNA genes from specific phylogenetic groups. For example, Siripong et al. (2006) improved their detection of ammonia oxidizing bacteria (AOB) in wastewater treatment plant samples by selectively amplifying the 16S rRNA genes of beta-proteobacterial AOB with specific PCR primers prior to microarray hybridization, and Loy et al. (2005) found that the selective amplification of ―Rhodocyclales‖ 16S rRNA genes prior to microarray hybridization allowed the detection of rare ―Rhodocyclales‖ groups in activated sludge. Group-specific PCR amplification has the advantage of improving detection of the targeted group, but it does require separate PCR reactions for each group, which can limit the number of groups detected in a particular experiment. Several groups have also used PCR amplification prior to microarray hybridization to detect functional genes in environmental samples. For example, Taroncher-Oldenberg et al. (2003) amplified nirS genes from sediment samples using general nirS primers and detected specific nirS variants by hybridization of amplicons to a microarray containing a set of 64 nirS-specific probes. Zhang et al. (2007) used a similar approach to detect nifH variants in roots of wild rice. One limitation of PCR amplification of functional genes prior to microarray hybridization is that each functional gene targeted by the array must be amplified via a separate PCR reaction. This was not a problem for the Zhang et al. (2007) study, as their array targeted only one functional gene, nifH, so universal nifH primers were used to amplify all nifH sequences in the samples followed by hybridization of amplicons to a set of 56 specific oligonucleotide probes on the microarray. However, the need for separate PCR reactions for each targeted gene could be problematic for microarrays targeting a large number of different functional genes. For example, the microarray used by TaroncherOldenberg et al (2003) included probes targeting four distinct functional genes (amoA, nifH, nirK, and nirS), each of which would have required a separate PCR reaction prior to microarray hybridization. Running multiple PCR reactions prior to microarray hybridization is not desirable for a number of reasons: 1) it will significantly increase the time and labor involved in microarray analysis, 2) it could introduce experimental error due to sample splitting, and 3) it limits the main advantage of DNA microarrays, namely the potential for highly parallel analysis of a high number of targets. One way to avoid multiple PCR reactions is via Multiplex PCR, in which multiple primer sets are included in a single PCR reaction. Several groups have utilized multiplex PCR amplification prior to microarray hybridization. Panicker et al. (2004) demonstrated detection of Vibrio parahaemolyticus in shellfish using multiplex PCR amplification of four gene

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targets followed by microarray hybridization. Gonzalez et al. (2004) detected five marine fish pathogens by multiplexing nine primer sets and subsequently hybridizing PCR products to a microarray that included nine specific oligonucleotide probes. However, multiplex PCR is restricted in the number of targets that can be amplified simultaneously due to primer-primer interactions (Edwards and Gibbs, 1994). For example, Panicker et al. (2004) were able to multiplex four primer pairs targeting Vibrio parahaemolyticus, but were unable to multiplex ten primer pairs. Pemov et al. (2005) developed a unique approach to multiplex PCR called multiplex microarray-enhanced PCR (MME-PCR) that avoids some of the issues associated with standard multiplex PCR. MME-PCR enables PCR amplification of multiple targets via specific primers immobilized within a gel-pad based microarray. MME-PCR prevents primerprimer interactions because each primer pair is immobilized within a separate gel-pad, so each primer pair is physically separated, and the specific amplification occurs within the gel pad. Amplification within each gel pad is detected by subsequent hybridization of fluorescently labeled, gene-specific probes. Pemov et al. (2005) demonstrated successful, specific on-chip amplification via MME-PCR of six genes from Bacillus subtilis using six different, gene-specific primer pairs. This approach could also be useful for many microbial ecology applications. For example, our lab is currently designing a MME-PCR chip for the detection of specific variants of nitrogen cycling functional genes. Another way to avoid multiple PCR reactions prior to microarray hybridization is through a process known as multiple displacement amplification (MDA). The MDA reaction uses DNA polymerase and random primers to amplify entire genomes (Raghunathan, 2005). MDA has been used to amplify whole genomes from pure cell cultures (Raghunathan, 2005), and Wu et al. (2006) recently developed an MDA method for the amplification of entire genomes from mixed microbial communities, which they termed whole-community genome amplification (WCGA). Wu et al. (2006) used WCGA to amplify all of the genes in a microbial community prior to hybridization to an oligonucleotide (50 mer) functional gene array in order to improve sensitivity. They compared direct hybridization of genomic DNA isolated from groundwater to WCGA amplification of dilutions of this DNA prior to hybridization, and they found that WCGA-assisted microarray hybridization with as little as 1 ng of community DNA produced results that were highly similar to direct DNA hybridization. These results suggest that WCGA may be a useful method for improving the sensitivity of microarray-based assays for low biomass samples. As demonstrated above, the addition of an amplification step to a microarray detection protocol can increase sensitivity and enable the detection of low abundance targets, but the incorporation of an amplification step does create some challenges as well. For example, double stranded nucleic acids such as PCR amplicons do not hybridize as effectively as single stranded nucleic acids (i.e. ssDNA or ssRNA) due to the potential for double stranded DNAs to reanneal instead of hybridizing to the microarray immobilized probes (Zhang et al., 2007). Several studies have avoided this problem by removing one of the DNA strands prior to hybridization via amplification with a 5‘-biotin-labeled reverse primer and subsequent removal of this strand via streptavidin-coated paramagnetic beads (Peplies et al., 2003; Zhang et al., 2007). Another potential challenge related to amplification of targets prior to microarray hybridization is the fact that amplification may introduce biases, as was discussed above for PCR-based assays. Since an amplification step may not amplify all targets in an

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unbiased manner, amplification prior to microarray hybridization may interfere with the ability to extract quantitative information from microarray data.

CHALLENGES FOR MICROARRAY TECHNOLOGY: QUANTIFICATION The value of microarrays as a tool for microbial ecologists would be greatly enhanced if microarrays could not only detect target nucleic acids but also provide quantitative information on their abundance. In theory, it should be possible to extract quantitative information from microarray hybridization if the amount of hybridization to a probe (i.e. the fluorescent intensity) correlates to the amount of target present in the sample. Several studies have confirmed linear relationships between target concentrations and signal intensities for specific probes: Wu et al (2001) found a log-linear relationship between the concentration of target genomic DNA and the hybridization signal for a 760 bp probe, and Rhee et al. (2004) found strong log-linear relationships between signal intensity and DNA concentrations for a large set of 50-mer oligonucleotide probes. Subsequently, Cho and Tiedje (2002) developed a method that involved co-immobilization of functional gene probes (500-900 bp) with a control probe (500 bp) and simultaneous hybridization of differentially labeled target genes and control DNA, and they achieved good log-linearity between signal ratio and DNA concentration ratio for three gene targets. These data confirmed that a correlation exists between the hybridization intensity for a microarray immobilized probe and the concentration of that probe‘s target. However, the challenge for microbial ecologists is that quantification of distinct bacterial populations in a complex community would require the comparison of hybridization signals from multiple probes with a microarray. This is a challenge because different probes can vary significantly in hybridization potential due to differences in probe length (Wu et al., 2001) and G+C content (Siripong et al., 2006) among other factors. These differences in hybridization potential have significant implications. Several studies have shown that redundant probes targeting the same organism often do not produce identical signal intensities when hybridized with RNA from the target organism (e.g. Peplies et al., 2003; Guschin et al., 2007). For example, Loy et al. (2005) demonstrated that probes targeting different regions of the 16S rRNA gene of the same organism can vary by a factor of 240 in signal intensity when hybridized with DNA from that organism. In addition, different probes can yield dramatically different hybridization signals even when hybridized to equal amounts of their respective targets (Ward et al., 2007). These situations make it extremely challenging to extract quantitative information on relative target abundance from multiple probes within a microarray. Since linear relationships have been shown to exist between target concentration and hybridization signal (Wu et al., 2001; Rhee et al., 2004), a standard curve could be developed for each probe on an array (Cho and Tiedje, 2002), as is routinely done for dot-blot membrane hybridizations. However, having to develop a standard curve for every probe on a microarray would be extremely labor intensive, and would limit the main advantage of microarrays, their high probe capacity. Therefore, attempting to determine the relative abundance of distinct bacterial populations in a complex community based on microarray hybridization is a very difficult task, and further work is needed to determine if microarrays can provide this type of information.

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MICROARRAY-BASED MONITORING OF GENE EXPRESSION IN MIXED MICROBIAL COMMUNITIES Functional gene arrays in which the presence of specific functional genes is detected via either direct hybridization of DNA extracted from an environmental sample (e.g. Zhou 2003) or via hybridization of functional genes amplified by PCR (e.g. Taroncher-Oldenberg et al., 2003; Zhang et al., 2007) can provide valuable information on the distribution of specific functional guilds in the environment as well as insight into the functional potential of microbial communities. However, the presence of a functional gene in an environmental sample does not necessarily mean that the gene is being expressed. One way to determine expression is by detection of gene transcripts (mRNAs). As discussed above, microarrays have been used extensively to investigate gene expression patterns in eukaryotic cells, such as human cell lines (DeRisi et al., 1996) and yeast (Lashkari et al., 1997), via analysis of mRNAs. Microarray analysis of mRNAs has been less extensively applied to prokaryotic cells, due to difficulties in priming cDNA synthesis from bacterial mRNA (Dennis et al., 2003). In addition, most microarray studies examining prokaryotic mRNA have been conducted under controlled laboratory conditions on single cell lines or pure bacterial cultures (e.g. de Saizieu et al., 1998; Methé et al., 2005) due to the difficulties associated with obtaining sufficient quantities of high quality mRNA from environmental samples (Parro et al., 2007). One of the first studies to demonstrate the detection of bacterial mRNAs from mixed communities used a microarray containing near-full length amplicons from 25 catabolic genes involved in the degradation of chlorinated aromatic compounds (Dennis et al., 2003). The steps used in this study were extraction of total RNA, synthesis of cDNAs via reverse transcription, labeling of cDNAs, and hybridization of labeled cDNAs to the microarray. This approach demonstrated the induction of catabolic genes in response to substrate additions in pure cultures, in an artificial six member microbial community, and in sludge-fed pulp mill effluent (Dennis et al., 2003). Zhang et al. (2007) used a similar approach to detect expression of nifH gene variants in roots of wild rice samples with a microarray containing short (15-25 mer) oligonucleotide probes. Recently Parro et al. (2007) took a slightly different approach and built a DNA microarray containing a gene library from a single target organism, Leptospirillum ferrooxidans. They used this microarray to assess the relative expression of L. ferrooxidans genes in two habitats with low bacterial diversity that differed in salt and oxygen contents. The results demonstrated increased expression of genes required for adaptation to each of the two habitats: for example, genes related to halotolerance were preferentially expressed in the high salinity environment (Parro et al., 2007). One of the challenges associated with microarray detection of bacterial mRNAs in environmental samples such as soil and sediments is the isolation of sufficient quantities of high quality mRNA for analysis (Parro et al., 2007). Several recent studies have addressed this challenge by developing strategies for amplification of low quantity bacterial mRNAs prior to microarray hybridization. Moreno-Paz and Parro (2006) used random-primed reverse transcription coupled to in vitro transcription as a method for total bacterial RNA amplification, and they demonstrated their ability to amplify as little as 250 ng of total bacterial RNA from a pure culture and successfully hybridize the product to a microarray. Gao et al. (2007) utilized a similar approach, which they termed whole-community RNA

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amplification (WCRA), to amplify RNA from mixed bacterial communities. The WCRA approach used fusion primers (six to nine random nucleotides with an attached T7 promoter) for the first-strand synthesis, followed by second strand synthesis and in vitro transcription. This approach resulted in representative microarray detection from as little as 50 to 100 ng total RNA, and Gao et al. (2007) demonstrated that bacterial mRNAs could be detected in groundwater samples via WCRA amplification followed by hybridization to an oligonucleotide (50-mer) functional gene array. The publications described above illustrate the potential for microarrays to monitor bacterial gene expression in environmental samples, which would provide extremely valuable insights into bacterial community function. The success of these pioneering studies should lead to further applications of this approach.

PARALLEL ANALYSES OF MICROBIAL COMMUNITY COMPOSITION AND FUNCTION WITH MICROARRAYS One of the key goals in microbial ecology research is the elucidation of the relationship between the composition of a microbial community (i.e. the species present) and the function of that community, but assessing this relationship for complex microbial communities in environmental samples has always been extremely challenging. As described above, numerous studies have demonstrated the ability of microarray technology to assess microbial community composition, via hybridization of either 16S rRNAs, 16S rRNA genes, or functional genes, and a few studies have demonstrated the ability of microarrays to assess microbial community function through the detection of mRNAs (Dennis et al., 2003; Gao et al., 2007; Parro et al., 2007). Several recent studies have expanded upon this work and developed highly innovative, microarray-based approaches to examine both microbial community composition and function in parallel. Adamczyk et al. (2003) developed an isotope array approach. In this approach, a microbial community is incubated with a 14C-labelled substrate, and after an incubation period, total RNA is extracted from this community, fluorescently labeled, and hybridized with a microarray containing 16S targeted oligonucleotide probes. The array can then be scanned for fluorescence as well as for radioactivity. For each probe on the array, fluorescence indicates the presence of the target organism in the community, and radioactivity indicates that the target organism has incorporated 14C into RNA, indicating active growth and utilization of the labeled substrate. Adamczyk et al. (2003) demonstrated this approach using a microarray containing oligonucleotide probes targeting 16S rRNA genes of ammoniaoxidizing bacteria. In this study, activated-sludge samples were incubated with 14C-labelled bicarbonate, and detection of radioactivity for specific probes on the array was taken as an indication of CO2 fixation by the targeted organisms. This study demonstrated the potential use of an isotope array for the simultaneous assessment of community composition and function. Another novel approach to examining community composition and function was developed by Zhang et al. (2007). They assessed both the presence and expression of different variants of nifH in roots of wild rice using a microarray containing 56 oligonucleotide probes (15-25 mers) targeting nifH variants. In this study, DNA and RNA were coextracted from

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plant roots, and separate aliquots were treated with either DNase I or RNase A to yield pure RNA and DNA samples, respectively. DNA was amplified prior to microarray hybridization via PCR with universal nifH primers. RT-PCR with a universal nifH primer was used to convert mRNA to cDNA, and PCR with universal nifH primers was then used to amplify the RT-PCR products prior to microarray hybridization. In this creative approach, hybridization of amplified DNA indicated the presence of specific nifH variants, and hybridization of amplified mRNA indicated the expression of specific nifH variants. The results of this study demonstrated that only a small subset of the nifH-containing organisms found in the roots were expressing the nifH gene (Zhang et al., 2007). This approach has tremendous potential for simultaneous exploration of microbial community composition and function, which could provide fascinating insights into the functioning of microbial communities in the environment.

APPLICATIONS OF DNA MICROARRAYS TO MICROBIAL ECOLOGY Since the pioneering work of Guschin et al. (1997) the use of microarray technology in microbial ecology research has increased rapidly (Figure 3). The vast majority of microarray studies published to date in the field of microbial ecology have focused on demonstrating the capabilities of microarrays and developing the technology, but recently a growing number of studies have been applying microarrays as tools in studies asking ecological questions.

Figure 3.Number of publications per year that include application of microarray technology to microbial ecology. Numbers are based on a search of the Web of Science database on February 1, 2008 using the following search string: (TS=microarray* OR TS=microchip*) AND (TS=microbial ecology OR TS=microbial community OR TS=microbial detection OR TS=microbial identification). This approach was based on Wagner et al.(2007).

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Some examples include the following: Taroncher-Oldenberg et al. (2003) used a microarray to reveal differences in the distribution of nirS variants in sediment samples from two stations in the Choptank River that differed in salinity, inorganic nitrogen, and dissolved organic carbon. Loy et al. (2002) used a 16S rRNA gene-based oligonucleotide microarray to detect sulfate-reducing prokaryotes in an unusual habitat, a low-sulfate acidic fen, and they found differences in the distribution of these sulfate-reducers in fen samples from different locations. Rhee et al. (2004) demonstrated that the community composition of naphthalenedegraders in soil microcosms differed depending on incubation conditions based on analysis with a functional-gene microarray containing oligonucleotide probes. Sanguin, Remenant, et al. (2006) used a 16S rRNA-based microarray to reveal a significant maize rhizosphere effect on soil bacterial community composition. Ward et al. (2007) demonstrated variations in the composition of ammonia oxidizing communities across a freshwater/marine transect extending from the Choptank River through the Chesapeake Bay and out into the Sargasso Sea using an oligonucleotide (70 mer) functional gene microarray targeting amoA genes. This study also revealed correlations between amoA guilds and environmental parameters, suggesting that different amoA-containing organisms occupy different ecological niches within the estuarine/marine environment (Ward et al., 2007). All of the studies cited above are excellent illustrations of the effective use of microarrays in microbial ecology, and based on these and other successes it is likely that the uses of microarrays in this field will continue to expand rapidly.

CONCLUSIONS Microarrays have the potential to revolutionize the field of microbial ecology via high throughput analysis of microbial community structure, function, and population dynamics. There are significant challenges to the use of microarrays in microbial ecology studies, including optimization of specificity and sensitivity and quantification of targets. However, as reviewed above, the last decade has seen tremendous progress in addressing these challenges, and this progress suggests that microbial ecologists are now on the verge of finally being able to realize the tremendous potential of microarray technology.

ACKNOWLEDGMENTS JJK‘s efforts in preparing this review were supported by a Research Support Grant from Loyola University Chicago and by USDA Grant 2005-35107-16098.

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Small, J, DR Call, FJ Brockman, TM Straub, DP Chandler (2001) Direct detection of 16S rRNA in soil extracts by using oligonucleotide microarrays. Appl. Environ. Microbiol. 67: 4708-4716. Smoot, LM, JC Smoot, H Smidt, PA Noble, M Könneke, ZA McMurry, DA Stahl (2005) DNA microarrays as salivary diagnostic tools for characterizing the oral cavity's microbial community. Adv. Dent. Res. 18: 6-11. Stahl, DA, B Flesher, HR Mansfield, L Montgomery (1988) Use of phylogenetically based hybridization probes for studies of ruminal microbial ecology. Appl. Environ. Microbiol. 54: 1079–1084. Stahl, DA (1995) Application of phylogenetically based hybridization probes to microbial ecology. Mol. Ecol. 4: 535-542. Staley JT, A Konopka (1985) Measurement of in situ activities of nonphotosynthetic microorganisms in aquatic and terrestrial environments. Ann. Rev. Microbiol. 39: 321346. Starke, EML, JC Smoot, LM Smoot, WT Liu, DP Chandler, HH Lee, DA Stahl (2006) Technology development to explore the relationship between oral health and the oral microbial community. BMC Oral Health 6: S10. Straub TM, DS Daly, S Wunshel, PA Rochelle, R DeLeon, DP Chandler (2002) Genotyping Cryptosporidium parvum with an hsp70 single-nucleotide polymorphism microarray. Appl. Environ. Microbiol. 68: 1817-1826. Suzuki, MT, SJ Giovannoni (1996) Bias caused by template annealing in the amplification of mixtures of 16S rRNA genes by PCR. Appl. Environ. Microbiol. 62: 625-630. Taniguchi, M, K Miura, H Iwao, S Yamanaka (2001) Quantitative assessment of DNA microarrays—comparison with northern blot analyses. Genomics 71: 34-39. Taroncher-Oldenburg, G , EM Griner, CA Francis, BB Ward (2003) Oligonucleotide microarray for the study of functional gene diversity in the nitrogen cycle in the environment. Appl. Env. Microbiol. 69: 1159-1171. Torsvik, V, J Goksoyr, FL Daae (1990) High diversity of DNA of soil bacteria. Appl. Environ. Microbiol. 56: 782-787. Treusch, AH, S Leininger, A Kletzin, SC Schuster, H-P Klenk, C Schleper (2005) Novel genes for nitrite reductase and Amo-related proteins indicate a role of uncultivated mesophilic crenarchaeota in nitrogen cycling. Environ. Microbiol. 7: 1985-1995. Urakawa, H, PA Noble, S El Fantroussi, JJ Kelly, DA Stahl (2002) Single-base-pair discrimination of terminal mismatches by using oligonucleotide microarrays and neural network analyses. Appl. Environ. Microbiol. 68: 235-244. Urakawa, H, S El Fantroussi, H Smidt, JC Smoot, EH Tribou, JJ Kelly, PA Noble, DA Stahl (2003) Optimization of single-base-pair mismatch discrimination in oligonucleotide microarrays. Appl. Environ. Microbiol. 69: 2848-2856. Venter, JC et al. (2004) Environmental genome shotgun sequencing of the Sargasso Sea. Science 304: 66-74. Wagner, M, H Smidt, A Loy, J Zhou (2007) Unravelling microbial communities with DNAmicroarrays: challenges and future directions. Microb. Ecol. 53: 498–506. Wang, R-F, ML Beggs, LH Robertson, CE Cerniglia (2002) Design and evaluation of oligonucleotide-microarray method for the detection of human intestinal bacteria in fecal samples. FEMS Microbiol. Lett. 213: 175–182.

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Ward, BB, D Eveillard, JD Kirshtein, JD Nelson, MA Voytek, GA Jackson (2007) Ammoniaoxidizing bacterial community composition in estuarine and oceanic environments assessed using a functional gene microarray. Environ. Microbiol. 9: 2522–2538. Weber, S, S Stubner, R Conrad (2001) Bacterial populations colonizing and degrading rice straw in anoxic paddy soil. Appl. Environ. Microbiol. 67: 1318-1327. Wick, LM, JM Rouillard, TS Whittam, E Gulari, JM Tiedje, SA Hashsham (2006) On-chip non-equilibrium dissociation curves and dissociation rate constants as methods to assess specificity of oligonucleotide probes. Nucleic Acids Res. 34: e26. Wilson KH, WJ Wilson, JL Radosevich, TZ DeSantis, VS Viswanathan, TA Kuczmarski, GL Andersen (2002) High-density microarray of small subunit ribosomal DNA probes. Appl. Environ. Microbiol. 68: 2535–2541. Wu, L, X Liu, CW Schadt, J Zhou (2006) Microarray-based analysis of subnanogram quantities of microbial community DNAs using whole-community genome amplification. Appl. Environ. Microbiol. 72: 4931–4941. Wu L, D Thompson D, G Li, RA Hurt, JM Tiedje, J Zhou (2001) Development and evaluation of functional gene arrays for detection of selected genes in the environment. Appl. Environ. Microbiol. 67: 5780-5790. Yershov G, V Barsky, A Belgovskiy, E Kirillov, E Kreindlin, I Ivanov, S Parinov, D Guschin, A Drobishev, S Dubiley, A Mirzabekov (1996) DNA analysis and diagnostics on oligonucleotide microchips. Proc. Nat. Acad. Sci. 93: 4913-4918. Zehr, M, MT Mellon, S Zani (1998) New nitrogen-fixing microorganisms detected in oligotrophic oceans by amplification of nitrogenase (nifH) genes. Appl. Environ. Microbiol. 64: 3444-3450. Zehr, JP, BD Jenkins, SM Short, GF Steward (2003) Nitrogenase gene diversity and microbial community structure: a cross-system comparison. Environ. Microbiol. 5: 539– 554. Zhang, L, T Hurek, B Reinhold-Hurek (2007) A nifH-based oligonucleotide microarray for functional diagnostics of nitrogen-fixing microorganisms. Microb. Ecol. 53: 456-470. Zhou, JZ (2003) Microarrays for bacterial detection and microbial community analysis. Curr. Opin. Microbiol. 6: 288-294.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 14

FOOD SAFETY IN INDIA: CHALLENGES AND OPPORTUNITIES Wasim Aktar* Pesticide Residue Laboratory, Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India

1. INTRODUCTION Rising incomes and urbanization, an expanding domestic consumer base concerned about food quality and safety, and rapidly growing agricultural exports have been important drivers for the increased attention to food safety in India. But the development of effective food safety systems is hampered by a number of factors, including: restrictive government marketing regulations, weak policy and regulatory framework for food safety, inadequate enforcement of existing standards, a multiplicity of government agencies involved, weak market infrastructure and agricultural support services. The small farm structure further limits farmer capacity to meet increasing domestic and export food safety and SPS requirements. Addressing food safety concerns in India will require adoption of appropriate legislation, strengthening capacity to enforce rules, promoting adoption of good agricultural, manufacturing and hygiene practices, greater collective action, and some targeted investments. Implementing these actions will require joint efforts by the government and the private sector. Developing countries are paying increased attention to food safety, because of growing recognition of its potential impact on public health, food security, and trade competitiveness. Increasing scientific understanding of the public health consequences of unsafe food, amplified by the rapid global transmission of information regarding the public health threats associated with food-borne and zoonotic diseases (e.g. E. coli and salmonella, bovinespongiform encephalopathy (BSE), severe acute respiratory syndrome (SARs) and H5N1 avian flu) through various forms of media and the internet has heightened consumer awareness about food safety risks to new levels globally (Lindsay 1997, Unnevehr 2003, *

Correspondence to: [email protected]

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Buzby and Unnevehr 2003, Kafersteing 2003, Ewen et al. 2006, Bramhmbatt 2005). Increased understanding of the impact of mycotoxins, which can contaminate dietary staples such wheat, maize, barley and peanuts, has further raised food security and public health concerns in many developing countries (Dohlman 2003, Bhat and Vasanthi 2003, Unnevehr 2003). As developing countries seek to expand agricultural exports especially to OECD countries, many are receiving a wake-up call on the challenges of meeting both government and private sanitary and phyto-sanitary (SPS) standards in export markets (Otsuki et al. 2001, Henson 2003, Unnevehr 2003, World Bank 2005a). Private standards or supplier protocols have grown in prominence over the past decade as a means to further ensure compliance with official regulations, to fill perceived gaps in such regulations, and/or to facilitate the differentiation of company or industry products from those of competitors. Trends in private standards increasingly tend to blend food safety and quality management concerns (i.e. the recent creation of ISO 22000), or to have protocols which combine food safety, environmental, and social (child labor, labor conditions, animal welfare) parameters (Willems et al. 2005, World Bank 2005). At the same time, increasing globalization of trade introduces greater risks of cross-border transfer of food-borne illnesses. Recent cases of disease episodes in the United States resulting from imported food produce, such as cyclospora from raspberries, hepatitis A from strawberries and salmonella from cantaloupe (Calvin 2003), illustrate to developing countries the potential food safety challenges that can arise in a more globalized market. Weaknesses in food safety systems can have a high cost to society and the global economy. The World Health Organization (WHO) estimates that 2.2 million people worldwide die from diarrheal diseases caused by a host of bacterial, viral and parasitic organisms, which are spread by contaminated water (WHO 2006a). In India, it is estimated that 20% of deaths among children under five are caused by diarrheal disease (WHO 2006b). The SARs outbreak in 2003 in East Asia is estimated to have caused an immediate economic loss of about 2% of the Region‘s GDP in the second quarter of that year, even though only 800 people died from the disease (Brahmbatt 2005).1 The Lowy Institute for International Policy (2006) estimates that a mild global outbreak of the avian flu can cost the world 1.4 million lives and close to 0.8% of GDP (US$330 billion) in lost economic output. At the same time, country reactions to protect its citizens from food safety risks can also have large consequences for exporting countries. Otsuki et al (2001) examined the projected impact of the EU‘s new harmonized aflatoxin standard on the value of trade flows to 15 European countries from 9 African countries and found that it could decrease African exports by 64% (US$670 million). Food safety concerns are getting widespread attention in India. The country‘s rural development strategy, for which a key element is the promotion of increased agricultural exports as a means to foster rural growth and poverty reduction, is coming up against tightening food safety and SPS standards in prospective markets (World Bank 2006a, 2006b). From a domestic perspective, the large national market of 1.2 billion people is undergoing rapid change. Increasing incomes, a growing middle class, increased urbanization and 1

The large economic impact resulted primarily from uncoordinated efforts of individuals to avoid becoming infected, contributing to a contraction in services sectors (tourism, mass transportation, retail, hotel and restaurant sales) and workplace absentiism (Brahmbatt 2005).

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literacy, and a population highly tuned to international trends fueled by the information technology boom are creating a large consumer base giving increasing value to food quality and safety. Improving food safety systems, to meet domestic and export requirements, however, face a number of policy, regulatory, infrastructural and institutional obstacles.

2. OBJECTIVES (i) To review the main drivers for the increased priority to addressing food safety risks in India in both the export and domestic markets, (ii)To examine the nature and effectiveness of government and private responses to the food safety challenges, with special focus on high value agriculture; (iii)To identify the constraints to more effective responses; (iv) To examine the implications for policy; v) To review food safety with special relation to Pesticides; and vi) To discuss briefly about the food safety from consumer point of view.

3. TYPES OF FOOD SAFETY RISKS

Figure 1. Food Supply Chain: Potential Sources of Food Safety Hazards

Food safety risks, as they relate to human health, arise from of a number of factors. These include: (i) microbial pathogens (bacteria, viruses, parasites, fungi and their toxins); (ii) pesticide residues, food additives, livestock drugs and growth hormones; (iii) environmental toxins such as heavy metals (e.g. lead and mercury); (iv) persistent organic pollutants (e.g.

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dioxins); and (v) zoonotic diseases (e.g.Avian flu, Japanese encephalitis, tuberculosis) (Buzby and Unnevehr 2003, Ewen et al. 2004).2 The health risks associated with these agents impact the whole food supply chain, starting from input supply to the farm to the consumer table (Figure 1). Common use of pesticides in modern farming inevitably leaves some residues on food crops.

Potential food safety hazards at HOME can be divided into three categories:

2

There remains considerable debate regarding the food safety risks associated with genetically modified organisms (GMOs). The paper will not be covering issues relating to GMOs.

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1. Biological

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2. Chemical

3. Physical

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While all the above type of hazards are important from viewpoint of prevention, the focus here will be on the microbiological hazards and in that on foodborne bacteria, which can lead to illness if the food is mishandled, particularly for those more at risk -- the very young, the elderly and the immuno-compromised. Certain processes or handling practices by consumers in the home have been identified as being essential or critical in preventing foodborne illness. These practices, which prevent or control the "meals" microbial contamination associated with foodborne illness, are under the direct control of the consumer, from food acquisition through disposal. They are purchasing, storing, pre-preparation, cooking, serving, and handling leftovers. Failure to take appropriate action at these critical points could result in foodborne illness.

4. PESTICIDES AND FOOD SAFETY Fruits, vegetables and cereal crops treated with pesticides are perceived by some as a health risk, and this belief along with affordability, and time pressures may all play a role in limiting consumption of plant foods, such as cereal grains, fruit and vegetable consumption of consumers in Asia. The World Health Organisation (WHO), the World Cancer Research Fund (WCRF) and many other national and inter-governmental agencies recommend that adults consume at least 400g of fruit and vegetables per day and 25-30 grammes of dietary fibre per day, but analysis of current dietary patterns around the world indicate that many consumer are not achieving these dietary goals, particularly those who are less affluent. AFIC‘s Short Briefing on Pesticides, Food Safety and Health is intended to provide a science-based factual overview of the issue, to enable consumers to make better informed choice about their diet, in particular fruit, vegetables and grains consumption, and allay unwarranted anxieties and concerns.

Definition of Pesticide The Food and Agriculture Organisation (FAO) defines a pesticide as ‗any substance or mixture of substances intended for preventing, destroying, attracting, repelling, or controlling any pest including unwanted species of plants or animals during the production, storage, transport, distribution, and processing of food, agricultural commodities, or animal feeds or which may be administered to animals for the control of ectoparasites‘

Natural Toxins Substances that are capable of causing cancer are virtually everywhere, even in natural compounds. The FDA estimates that the intake of carcinogens from man-made pesticide residues is extremely small compared to carcinogenic residues that plants produce naturally. According to Bruce Ames, a professor of molecular biology and biochemistry at the University of California, more than 99.99 percent of the pesticides Americans ingest are "nature's pesticides" or "natural toxins" (Hotchkiss, 1992; Moore, 1989).

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Natural toxins are present in all plants and such food products as beans, lettuce, apple juice, wine, black pepper, spinach, peanut butter and many others. Of the known natural toxins, which concentrate in parts per thousand versus parts per billion in synthetic pesticides, none has been shown to cause cancer (Hotchkiss, 1992; Moore, 1989).

Reasons of Pesticide Residues in Food Pesticide residues may be present in food because of the following reasons: 1) Direct use of pesticides on food crops; 2) Animal feeding on pesticide treated feed; 3) Environmental contamination

Pesticide Use on the Farm Many of today's food producers are taking an Integrated Pest Management (IPM) approach to preventing, reducing or eliminating pest problems. Growers and processors must make complicated decisions prior to planting, during the growing season, and during postharvest handling. Scientific IPM strategies give the grower economic incentives for sustaining long-term crop protection with minimal disruption to the environment. The agricultural community typically will use pesticides judiciously as part of the IPM strategy whenever proven alternatives are not available for pest control. Growers are hiring professional crop consultants with increasing frequency for advice on maintaining or increasing production through the utilization of IPM programs structured toward their specific agronomic situations.

Integrated Pest Management It is an ecological approach to pest management in which all available control techniques are consolidated into a unified program so that pest populations can be managed in such a manner that economic damage is avoided and adverse side effects are minimized. Practices used as a part of this management philosophy include the following: 1) destruction of crop debris, 2) having pests feed and concentrate on trap crops, 3) crop rotation, 4) selectivity of planting and harvest dates, 5) soil test analysis for crop nutrient needs, 6) planting crop species adapted for local conditions, 7) using genetically improved crop varieties with resistance to specific pests, 8) using biological control, 9) predicting pest outbreaks with computers, 10) pheromones for trapping pests, 11) scouting and monitoring for pests, 12) economic thresholds as guides to pest control, 13) better timing and application of pesticides, 14) use of biological insecticides, 15) improved pesticide application efficiency, 16) adapting promising technology, including the use of infrared scanners, satellite photos, gene-splicing biotechnology, and new pesticide delivery systems that incorporate farm-specific information on tractor mounted computers.

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Pesticide Limits and Regulation Approval for use of any pesticide in a country is subject to its safety evaluation. Safety levels for any pesticide are calculated over a number of formal assessments. The Codex Alimentarius Commission is an international body which sets international guidelines on many elements of food safety, including pesticides residues on food. These guidelines are not mandatory, but many countries in Asia use these guidelines, sometimes with additional scientific data determined by their national regulatory agencies to establish limits on use and also acceptable residue levels at point of sale.

Acceptable Daily Intake One of the most important tools in the safety evaluation of pesticide use on food crops is the calculation of what is an Acceptable Daily Intake (ADI). The ADI for any given pesticide is a measure of the quantity of a particular chemical in food that can be consumed daily over a lifetime without any known risk to health. It is expressed in relation to bodyweight. ADI is derived by first conducting diet trials on laboratory animals and observing the maximum level of pesticide that can be consumed by the animal with no observable adverse effect on health. This level expressed as percentage of body weight is known as the No Observable Adverse Effect Level (NOAEL or NOEL), The investigations include checks for birth defects, cancer, reproductive changes, damage to the nervous system, harm to organs such as the kidney or liver, and many other measurable health indicators. A safe level for human consumption is estimated by dividing the NOAEL on humans by an uncertainty factor (usually 100) to allow for the possibility that humans may more sensitive than the animals used for testing and also to account for possible variation in sensitivity to the pesticide between human individuals, for example adults and children. These results in an ADI for humans which is 100 times lower than the NOAEL consumption rate established from trials on laboratory animals.

Acute Reference Dose Safety evaluation of all pesticides also requires an estimate of the acute refrence dose (ARfD). The ARfD is an estimate of the amount of a substance in food or drinking water expressed as percentage of body weight, that can be consumed over a short period of time, usually one meal or one day, without any known effect on health. This figure is also expressed as a percentage of body weight.

Maximum Residue Levels A maximum reside levels (MRL) is the maximum permissible quantity of pesticide that may still be present on the crop at point of sale. It is derived from an assessment of the residues found when the crop is treated according to good agricultural practices. The MRL is

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the maximum concentration of a pesticide residue that is legally permitted in, or on, a food commodity, and is set by national governments if the approval is given for the use of the pesticide on specified crops. MRLs are set to determine legal trading limit, and are not an indicator of risk to health. MRLs are set at levels which would result in consumption of any residue at a level substantially lower than the ADI or the ARfD for the pesticide, and any pesticide whose MRL could result in dietary intake which might exceed the ADI or ARfD would not receive approval.

Pesticide Residue Monitoring Under FFDCA, the Food and Drug Administration (FDA) and USDA share responsibility for monitoring levels of pesticide residues on foods. FDA enforces pesticide tolerances for all domestically produced food shipped in interstate commerce and in imported foods, except for meat, poultry and some egg products, which are monitored by USDA. Many agriculturallyintensive states such as California and Florida also conduct extensive pesticide residue monitoring programs. FDA uses three approaches for pesticide residue monitoring: 1) incidence/level monitoring, 2) regulatory monitoring, and 3) Total Diet Study (FDA, 1994).

Total Diet Studies To assess potential health problems from contaminants, both natural and man-made in the food supply, the WHO recommends total diet studies (TDS) as the one of the most costeffective means for assuring that people are not exposed to unsafe levels of toxic chemicals through food. TDS provides an additional tool to assess whether or not any pesticides may be present in the diet at levels which might pose a risk to health. A TDS is conducted by purchasing through standard retail outlets a typical selection of foods commonly consumed in the country or region. The ‗basket‘ of foods is processed and prepared as if for normal consumption and then analysed in the laboratory to measure total levels of the substances of interest, for example pesticides. Drinking water and water used in cooking are also included in the assessments. The TDS provides a measure of the average amount of the pesticide consumed by different age/sex groups living in a country. See box for an example of an actual TDS and results for estimate of pesticide consumption.

Risk Calculation Risk = exposure x toxicity. Risk of harm from a chemical depends on both the level of exposure to the chemical and on the toxicity of the chemical (Chaisson et al., 1991). Therefore, to quantify potential risks from consuming minute quantities of a particular chemical residue in food, scientists consider the toxicity of the chemical, the residue content of foods and the amounts of these foods eaten by population subgroups. Population subgroups such as infants, children, women, women of child-bearing age and ethnic subgroups may be considered in risk assessments in addition to the total population. The groups considered

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depend on the toxicologic characteristics of a particular chemical. Risk assessments that consider regional and seasonal variations also are performed.

Exposure = residue concentration in food x amount of food consumed. Potential exposure to a chemical in a specific food is assessed by multiplying the residue concentrations in food times the amount of food consumed by each person in the population. This exposure is expressed as milligrams of residue per kilogram of body weight per day (mg/kg BW/day). Potential dietary exposure to a chemical is assessed by adding together residue intakes from all foods. Different assumptions regarding residue concentrations in food may be used to assess exposure. A worst-case exposure scenario may be calculated using tolerance levels for pesticides in food. This exposure assessment is the theoretical maximum residue contribution. Exposure may also be calculated using anticipated residue levels (Chaisson et al., 1991; California Agriculture,1994).

5. FOOD SAFETY AND THE INDIAN DOMESTIC MARKET Increasing incomes, urbanization, and literacy, improved infrastructure and closer ties to global trends, especially during the last decade, are driving changes in consumer demand and preferences in India. Sustained economic growth (6.0% per year in real terms from 1990/91 to 2003/04) resulted in GDP per capita increasing by about 70%, from about US$315 in 1990 to US$538 in 2004 (constant 2000 dollars). National poverty rates (headcount) declined from 38.9% (Central Statistical Organization 2002) in 1987/88 to 28.5% in 1999/00 (Deaton and Dreze 2002).3 The middle class, which now accounts for about 15% of the 1.2 billion people in India, is the fastest growing income group and is a major force shaping the diet revolution that is occurring (Landes and Gulati 2003). 3

There continues to be a debate on the headcount poverty rate in 1999/00, arising from the adjustment in the design of the 1999/00 National Sample Survey. Depending on the methodology used, the poverty estimates range from 26.1% (Planning Commission) to 28.9% (Sundaram and Tendulkar) (Virmani 2006).

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Figure 2. Diversification on Food Consumption Expenditures

These structural changes are reshaping consumer demand. The Indian food consumption basket is diversifying away from cereals towards higher value and more perishable products, such as fruits and vegetables, dairy, meat and fish (Figure 2). Increasing female participation in the work force and higher disposable incomes to spend on non-home cooked foods are driving growth in demand for prepared and semi-prepared foods, and thus the growth of the processed food industries (Pingali and Khwaja 2004). These trends bring increased attention to safety concerns in the handling, processing and marketing of foods. In addition, growing consumer preference for shopping convenience, increased exposure to the media (TV, cable and the internet) and ownership of durables such as refrigerators and cars are fostering the growth of modern retailing (i.e. supermarkets and hypermarkets), which in turn demand greater efficiency and food quality and safety standards in the supply chain Mukherjee and Patel 2005, Chenggapa, et al 2005). Increased vigilance by NGOs, consumer groups, and local research institutes is also raising awareness and spurring action among consumers and policy makers to address food safety risks. Findings of high levels of pesticides in bottled water and soft drinks in 2003 by the Centre for Science and Environment (CSE), an NGO, shook the country and forced the Government of India (GOI) to take swift action (Mathur et al 2003, CSE 2004). The CSE tested 30 bottled water brands from the major cities of Delhi and Mumbai in Maharashtra and found that all except one contained pesticide residues. The Delhi brands on average contained pesticide residues 36.4 times the maximum pesticide residues stipulated by the European Union standards for bottled water (CSE 2004). Shortly thereafter, Mathur et al. (2003) tested 12 brands of soft drinks sold in Delhi for 16 organochlorine and 12 organophosphorus pesticides and 4 synthetic pyrethroids commonly used in agricultural fields and homes in India. Their analysis found that all brands exceeded the EU maximum pesticide residue limit of 0.0005 ppm (Figure 3).

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Figure 3. Pesticide Residues in Soft Drinks in India, 2003

To deal with the back-to-back crises, the GOI established a special Joint Parliamentary Committee on ―Pesticide Residues in and Safety Standards for Soft Drinks, Fruit Juice and Other Beverages‖ in August 2003 to investigate the allegations. Two GOI Laboratories were instructed to conduct tests on the 12 brands (but using different samples) and their findings showed that 9 of the 12 samples exceeded the EU limits (Hindu Business Line 2003). Weak regulations and inadequate standards were major causes of these high profile food safety crises. In the case of bottled water, while the existing norm set out by the Bureau of Indian Standard (BIS) required that ―no pesticides should be detectable,‖ the prescribed methodology could only detect pesticides at extremely high levels. Consequently, GOI issued a notification revising the standards for pesticide residues on bottled water, adopting the EU single residue limit of 0.0001 ppm and multiple residue limit of 0.0005 ppm (CSE 2004). In the case of soft drinks, the BIS only had voluntary standards, not mandatory standards for pesticide residues. To address the problem, BIS constituted a 39 member committee, consisting of representatives from the soft drinks industry, government scientists, NGOs and consumer groups to formulate the new BIS standards. The outcome was the Indian Ready to Serve Non-Alcoholic Beverages Specifications, which established the limits for 16 pesticides in the finished product (0.0001 mg/l for individual pesticides and total pesticide residue limit of 0.0005 mg/l) (CSE 2004). Even the government-sponsored Mid-day Meals program encountered serious food safety incidents. The National Program for Nutritional Support to Primary Education (NPNSPE), more popularly known as the Mid-Day Meals Scheme, aims to improve child enrollment in primary school and encourage regular attendance by providing supplementary feeding, while improving their nutritional status. It covers children enrolled in classes I to IV in government

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and government-aided schools in the whole country (Jha and Umali-Deininger 2003). In June 2006, 85 students from a Chennai primary school were admitted to the hospital because of food poisoning after consuming food prepared under mid-day meal scheme.4 In February 2004, 281 children attending municipal schools in Delhi fell ill and were admitted to the hospital after consuming their mid-day meal.5 There have been many other cases, despite quality norms being established for the mid-day meal program. While issues related to pesticides in bottle water and carbonated drinks, and out-breaks of food-borne illnesses received wide media attention, there are other serious domestic food safety concerns that have been identified including heavy metal contamination in foods. Marshall, et al. (2003), tested fresh cauliflower, okra, and spinach — common vegetables in the Indian diet — in 5 production sites around the Delhi region and in Delhi‘s Azadpur wholesale market from May 2001 to June 2003. They found that 72% of the 222 spinach samples exceeded the Indian MRLs for lead of 2.5 mg/kg, and 100% exceeded the Codex MRL of 0.3 mg/kg. They attributed the high lead content to a number of possible causes, including contamination of the irrigation water by sewage and industrial effluent and industrial pollution.6 Contamination was exacerbated by their locations—the production sites and market were in peri-urban and urban areas. When tested for zinc, 21% of samples exceeded both the Indian and international standards. Currently, however, no regular testing for heavy metals in vegetables is undertaken by government agencies in India. Tests undertaken by the Indian Council for Agricultural Research found pesticide residues above the MRL in 5.3% of 666 samples of vegetables in 2003 and 15% of 468 samples of milk tested in 2001 (Directorate of Plant Protection and Quarantine 2006). The long term use of pesticides in agriculture and for disease control (e.g. DDT for malaria control) is manifesting itself in the blood, human milk and fatty tissue in the population in many states. Table 1 presents the results of micro-research studies in selected states in India from 1980 to 2005. Table 1. Level of DDT and HCH Content in Human Blood Samples in Selected States in India. Location

Year

Number of Samples

Total DDT (ppm)

Total HCH (ppm)

Lucknow, Uttar Pradesh Delhi Lucknow, Uttar Pradesh Delhi Ahmedabad, Gujarat (rural) Ahmedabad, Gujarat (urban) Punjab (rural)

1980 1982 1983 1985 1992 1997 2005

25 340 48 50 31 14 20

0.020 0.710 0.028 0.301 0.048 0.032 0.0652

00.022 0.049 0.075 0.148 0.039 0.057

Note: HCH - Hexachlorocyclohexane Source: ICMR 2001, Mathur et al. 2005. 4

http://www.newkerala.com/news3.php?action=fullnews&id=11595 http://www.hindu.com/2004/02/27/stories/2004022713760300.htm 6 Potential sources of industrial pollution include emissions from vehicles, industrial plants, coal power generation plants, and diesel generator sets and re-suspended road dust. Marshall et al. found that washing the spinach twice reduced the lead contamination by 50% indicating that a large proportion of the lead was air-borne. 5

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6. FOOD SAFETY CONCERNS IN INDIAN EXPORTS Increased globalization and liberalization of markets, facilitated by the World Trade Organization (WTO), are opening new export markets for Indian agricultural products, both fresh and processed. Indian agricultural exports grew at an average annual rate of 7.2% from 1990/91 to 2003/04. In response to these new opportunities, India‘s agriculture exports diversified from traditional exports of tea, spices, and coffee to include horticultural, fish and livestock products. Between the triennium ending (TE) 1991/92 and TE 2003/04, the value of fresh and processed fruit and vegetable exports rose from US$84 million to US$394 million in real terms (1993/94 dollars) while marine product exports rose from US$516 million to US$1.5 billion during the same period (Figure 4). As Indian agricultural exports diversified, and the value of exports to high income countries increased, India has had to confront new food safety challenges. Concerns over numerous rejections of Indian agro-food exports on food safety grounds have spilled over domestically, generating greater domestic attention to pervasive food safety problems in the supply chain including high levels of pesticide residues, presence of heavy metals in food, and micro-biological contamination. The following section describes recent food safety challenges in Indian horticultural, spice and fisheries exports.

Figure 4. Trend in Agricultural Exports, Triennium Ending (TE) 1990/91 to TE 2003/04

Horticultural Exports In 2004, India exported US$575 million of fresh and processed fruits, vegetables and flowers. Traditionally India‘s fresh fruit and vegetables exports were targeted to markets in neighboring South Asian countries, to the Middle East and to East Asia. Since the early 1990s India achieved some success in exporting fresh horticultural produce to Western Europe. India has been quite proud of its penetration into the U.K, Netherlands and German fresh

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grape markets. Grapes are a highly seasonal crop and Indian exporters have been targeting a crucial March to April window in the European market, which falls at the end of the main southern hemisphere production season (in South Africa and Chile) and before Egypt and Turkey enter the market. Virtually all of India‘s grape exports are of the Thompson Seedless variety. The Indian grape export crisis in May 2003 was a pivotal wake-up call to Indian exporters concerning the costs of failing to meet food safety standards. In the midst of a commercial dispute with an Indian grape exporter, a Dutch importer had samples of the Indian grapes tested by a private laboratory. On finding that the grapes contained residues of the insecticide methomyl in excess of the EU maximum residue limit (0.05 microgram/kg.), the importer placed an advertisement in the local paper warning that grapes from this Indian supplier contained ―poison‖ (World Bank, 2006b). Dutch authorities, who were alerted about the finding, tested samples from the 28 containers of Indian grapes then in Rotterdam port and found that about 75% of the samples exceed the MRLs for methomyl and/or acephate.7 The problem was reported on the EU Rapid Alert system, causing not only significant short term economic losses, but also considerable longer term reputation damage. The price of Indian grapes dropped sharply, and the Indian grape shippers incurred losses, either in Dutch sales or by diverting the shipments to other markets.

Spice Exports India is the world‘s largest consumer and producer of spices and is also a significant exporter of spices (Jaffee, 2005). In 2004/05, India‘s spice exports totaled US$399 million. India, however, has encountered a number of food safety problems in its spice exports including high pesticide residues, aflatoxin contamination and the use of prohibited food colorants. In the mid-nineties, Indian dry chili exports faced several rejections including rejections in Spain due to pesticide residue in excess of permissible MRLs, and in the United States because residues of quinalphos, a pesticide not registered in the United States (Jaffee, 2005). Between 1998 and 2000, Indian dry chili exports also faced rejection in Germany, Italy, Spain and the U.K. due to the presence of aflatoxin.8 More recently, exports of chili and curry powder faced problems due to the use of the prohibited red dye Sudan 1 (Jaffee, 2005). In February 2005, a massive recall of some 600 food products took place in the UK because of the detection of Sudan 1 in Worcester sauce. This was the largest ever food recall in the U.K. and it affected all major retailers as well as large numbers of food manufacturers and food service companies, as the Worcester Sauces had been used in the preparation of a large number of different products. It is estimated that this recall, and associated expenses, cost the U.K. and other European food manufacturers some 200 million Euros (Jaffee, 2005). The source of the Sudan 1 dye in the Worcester sauce was traced to chili powder imported from India in 2002.

7

Of the twenty Indian samples with violative levels of methomyl, six exceeded the MRL by ten times, but most of the others were also far in excess of the MRL (Schee 2004). 8 Aflatoxin may emerge in dried chilies as a result of improper dying (Jaffee, 2005).

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Fish and Fish Product Exports Fish and fish products are one of India‘s largest agricultural export earners, totaling US$1.3 billion in 2004/05. Over the years, India has encountered several food safety problems with its fish and fish product exports. Most prominent, in 1997, the European Commission found the industry to be non-compliant in maintaining hygiene standards in fish processing plants. In May 1997 the European Commission banned Indian exports of fresh crustaceans and cephalopods and imposed border testing for Salmonella and Vibrio spp. for frozen products (Henson, Saqib and Rajasena, 2005). Because of continued detection of salmonella, all exports of fish and fishery products to the EU from India were banned in 1997. While India has for the most part been able to address the hygiene-related problems plaguing its export of fishery products in the late nineties, Indian exports are now under scrutiny because of problems related to antibiotic residues and bacterial inhibitors (antibiotics, preservatives and chlorine) (Henson, Saqib and Rajasena, 2005). It is widely acknowledged that in the future, heavy metals and other contaminants could be an emerging issue particularly because of the increased attention to heavy metals in the EU. Surveillance of fisheries products for heavy metals has already begun in the U.K. Although India has been able to broadly comply with food safety requirements for each of the export commodities mentioned above, it continues to face problems across a range of agro-food exports. Evidence of continuing trouble is clearly apparent from Import Refusal Reports issued each month by the USFDA for food and drug imports into the United States. Most recently, in both April and May 2006, India had one of the highest rejections among all countries exporting to the USA; India faced 176 rejections in May, 2006 and 211 rejections in April, 2006.9 While a significant number of the 176 rejections were issued for drugs and cosmetics, the grounds for rejection among the various food items included salmonella and/or filth in raw peeled shrimp, prepared Indian breads (paratha, roti), basmati rice, sesame seeds, pepper, coriander and chili powder; pesticide residues in lentils; failure to declare the color additive FD & C Yellow No. 5 in banana chips; and unsafe coloring in cream biscuits. The number of rejections and the range of problems reveal extensive safety problems in Indian food products. It is also reasonable to assume that the extent of the problems faced by domestic consumers is far more serious as there many more micro, small and medium enterprises that cater to domestic consumers and generally pay less attention to food safety issues. By contrast, exporters are likely to be more well-established and larger firms with better technology and relatively more cognizant about food safety concerns.

7. CHALLENGES TO IMPROVE FOOD SAFETY IN INDIA Improving food safety in India, whether for the domestic market or for export trade, is hampered by a number of structural, policy, institutional, technical and cultural barriers.

9

India had the most rejections of any country in May and the second highest number of rejections, behind Mexico, in April, 2006. http://www.fda.gov/ora/oasis/5/ora_oasis_cntry_lst.html.

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Policy and Regulatory Environment A number of policies and regulations governing agricultural marketing and food processing complicate the implementation of food safety measures by the government and by the private sector. Two critical marketing regulations are the State level Agricultural Produce Marketing (Development and Regulation) Acts and the Small Scale Industry Reservation Policy. Almost all states in India have an Agricultural Produce Marketing (APM) Act, which gives state governments the sole authority to establish and manage wholesale markets.10 The Act, adopted by most states in the 1960s and 1970s, prescribes the setting up of a network of state controlled ―regulated markets‖ or mandis and the establishment of Market Committees to operate each. All ―notified‖ agricultural commodities grown in areas surrounding the market are required by law to be sold only through these markets, with the number of notified commodities varying by state and market. Implementation of the Act and its enforcement vary considerably by state. In 2005, there were nearly 8,000 regulated markets in the whole country.11 The requirement that all agricultural commodities be channeled through the regulated markets not only increases transactions costs, but is also a major obstacle to preserving produce quality and traceability. In 2003, the GOI formulated a model Agricultural Produce Market Act for state governments to adopt, which removes the restrictions on farmer direct sales and permits entities outside of government to establish and operate wholesale markets. To date only 10 of the 28 states and Union Territories have adopted the model Act.12 The Small Scale Industry (SSI) Reservation restricts the processing of certain commodities to the small scale sector. Although the list of commodities subject to this restriction has been reduced significantly during the last decade, several processed agricultural products are still subject to SSI reservation, such as rapeseed, mustard and ground nut oil,13 bread, pastry, pickles and chutneys, and hard boiled sugar candy (Department of Small Scale Industries 2006). The SSI reservation imposes constraints on enterprises‘ ability to undertake the necessary investments (e.g. HACCP) and certifications required to meet the domestic and international food safety and SPS requirements.14 There is a complex web of laws governing the processed food sector which complicate implementation of food safety measures. These laws are enforced by 8 different ministries. Some of the most critical are: Prevention of Food Adulteration Act 1954 implemented by the Ministry of Health and Family Welfare; Milk and Milk Products Order 1992 and Agricultural Produce Grading and Marking Act 1937 implemented by the Ministry of Agriculture; the Essential Commodities Act 1955, Standards of Weights and Measures Act 1976, Consumer Protection Act 1986, and Bureau of Indian Standards Act 1986 implemented by the Ministry of Food, Consumer Affairs and Public Distribution; the Fruit Products Order 1955 10

The states of Kerala, Jammu and Kashmir, Manipur, Andaman and Nicobar Islands, Dadra and Nagar Haveli, and Lakshadweep do not have the regulation. 11 In 2003, there were 7,383 wholesale markets in the country of which 7,360 were regulated markets. In addition, there were 27,294 rural periodic markets (Ministry of Agriculture as cited in www.indiastat.com). 12 The states that adopted the model Act include : Punjab, Madhya Pradesh, Andhra Pradesh, Orissa, Maharashtra, Rajasthan, Chhattisgarh, Himachal Pradesh, Sikkim and Nagaland. 13 Exceptions are rapeseed, mustard, and ground oil through solvent extraction and those processed by growers cooperatives and state agro-cooperatives (Ministry of Small Scale Industries 2005) 14 This issue is more serious for domestic consumers since food processing units exporting more than 50% of production are not subject to the SSI reservation.

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implemented by the Ministry of Food Processing Industries; import and export regulations implemented by the Ministry of Commerce; Trade in Endangered Species Act implemented by the Ministry of Forest and Environment; Atomic Energy Act 1962/Control of Irradiation of Food Rule 1991 implemented by the Ministry of Science and Technology; and Infant Milk Substitutes, Feed Bottles and Infant Foods (Regulation of Production, Supply and Distribution) Act 1992 implemented by the Ministry of Human Resource Development (Patnaik 2005). These laws also authorize several agencies to lay down standards for food products: (i) Bureau of Indian Standards (BIS) of the Ministry of Food, Consumer Affairs and Public distribution under the BIS Act, (ii) Ministry of Food Processing Industry under the Fruit Products Order, (iii) Ministry of Agriculture under ―Ag Mark‖ and the FPO, (iv) Ministry of Health and Family Welfare (MOHFW) under the PFA Act; (v) Export Inspection Council under the Export-Import Policy, and (vi) the Defense Ministry for their own purchases. These laws and associated regulations in some cases prescribe contradictory or differing standards. For example, while the Fruit Products Order (FPO) allows the use of artificial sweeteners in fruit products, the Prevention of Food Adulteration (PFA) Act bans it. Mandatory declaration labels required by the PFA differ from those of the Packaged Commodity Regulation Rules (1977) under the Standard Weights and Measures Act. The emulsifier and stabilizers permitted for use in jams and chutneys under the PFA differ from those allowed under the FPO. In 1998, the GOI began the process of rationalizing the legal and regulatory framework for food and food processing. The Prime Minister‘s Council on Trade and Industry established a Task Force on Food and Agro-Industries Management Policy to recommend options for rationalizing the various policies and regulations. The outcome was a new Food Safety and Standards Bill, which was submitted to Parliament in August 2005 and is awaiting approval. The Bill aims to consolidate the laws relating to food. The key provisions of Bill include: (i) the repeal of a number of Acts and Orders;15 (ii) the establishment of a Food Safety and Standards Authority of India; (iii) definition of the standards for food additives, contaminants, genetically modified and organic foods, packaging and labeling, and food imports; (iii) accreditation of laboratories, research institutions and food safety auditors; (iv) licensing and registration of food business and setting penalties for offenses; and (v) establishment of a Food Safety Adjudication Tribunal (Ministry of Food Processing Industries 2005). Approval of the Bill will be an important milestone in strengthening food safety systems in India. There are a large number of government agencies involved in agricultural marketing activities, more broadly or with respect to specific commodities, which complicates effective implementation of a coherent food safety strategy for the country. As in the case of the soft drink contamination, the multiple laws and agencies added to the confusion. The BIS was charged with setting the standards for pesticides in soft drinks, while the MOHFW is charged with setting the pesticide standards for bottled water.

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The laws and orders repealed are the: Prevention of Food Adulteration Act 1954 (37 of 1954), Fruit Products Order 1955, Meat Food Products Order 1973, Vegetable Oil Products (Control) Order 1947, The Edible Oils Packaging (Regulation Order) 1998, Solvent Extracted Oil, De-oiled Meal and Edible Flour (Control) Order 1967, Milk and Milk Products Order 1992, and other orders under the Essential Commodities Act 1955 (10 pf 1955) relating to food.

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Smallholder Agriculture The current structure of the farm sector in India constrains farmer capacity to meet domestic and international food safety standards. Farming in India is dominated by small farmers — the average farm size in 1990/00 was 1.8 ha (NABARD 2002). Most farmers face credit constraints (World Bank 2004), and literacy rates are low.16 These constraints impose limits on the number of farmers capable to adopt more sophisticated farm practices and undertake the necessary investments (e.g. land improvements, obtaining necessary certifications, cold storage) to meet more stringent food quality and safety requirements. They increase the cost of transacting business and monitoring compliance with food safety standards. Stringent land policies, e.g. land ceilings and restrictions on land rental, limit possibilities for greater land amalgamation (World Bank 2006c). International experience indicates, however, that farm size constraints may be overcome through innovative interventions such as organizing farmers into producer groups, establishing collection centers (by supermarkets and exporters), using contract farming arrangements, and by creating public-private partnerships to assist farmers in a variety of ways, including help in obtaining the capital required to make on-farm improvements and other investments (e.g. grading or cooling facilities), developing and improving farming skills through joint extension provision, and assistance in acquiring the required national and international certifications (Berdegué et al. 2003, Boselie et al. 2003, Dries et al 2004, Reardon and Swinnen 2004, Reardon and Timmer 2005a, 2005b). In order to address various food safety concerns in both the spices and fresh and processed fruit and vegetable sectors, some exporters initiated contract farming operations or ―vendor screening‖ programs. One industry that has been especially successful in establishing contract farming arrangements and meeting stringent food safety and quality standards is the pickled gherkin industry. The industry, consisting of some 42 companies and nearly 50,000 smallholder outgrowers, is concentrated in Karnataka, Andhra Pradesh, and Tamil Nadu. The leading gherkin exporting companies each have several thousand farmers under contract. The companies provide intensive oversight and maintain extensive records of farmer practices, especially related to pesticide use. At least one company began the process of getting outgrowers certified under EurepGAP (World Bank 2006b). Contract farming has worked relatively well in the case of gherkins as almost the entire production from India is exported and there is no local market. Hence contract enforcement has not been a major challenge as in the case of other commodities where the export intensity is much lower and the majority of production is consumed domestically. Until recently, contract farming was illegal in India as per the provisions of the APM Act. The only way entrepreneurs can legally enter into contract farming with farmers is to obtain a special waiver from the APM Act from the State Government. The new model APM Act provides the legal framework and guidelines for contract farming. The provisions in the model Act allow contract buyers to directly purchase commodities from farmers under individual contracts or from farmers‘ markets. It also allows the direct sale of farm produce at the farmers‘ fields without having them routed through regulated markets. Adoption of the

16

The rural literacy rate in 1999/2000 was 50% http://www.indiastat.com/india/ShowData.asp?secid=16611 &ptid=367635&level=5

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model Act by state governments will therefore facilitate not only more efficient marketing, but also improved food safety and the adoption of improved agricultural practices.

Weak Extension Systems The public agricultural extension systems at the state level are very weak and have not effectively caught up to the changing needs of farmers and the market (World Bank 2005b). In view of the GOI‘s earlier concentration on food self-sufficiency, the state-level Department of Agriculture (DoA) extension systems generally focused on cereals, particularly rice and wheat, with an emphasis on the transfer of improved varieties and management practices. The weak coordination between the state DoAs and the other line departments (e.g. Departments of Irrigation, Horticulture, Livestock, Marketing, etc) and the limited staff capacity beyond the Department of Agriculture also often translated to limited extension activities beyond cereals, limiting its impact on agricultural and market diversification trends. The weak coordination with research at the central level further increased the difficulty of ensuring effective research-extension-farmer linkages at the state level. In many states, tight fiscal constraints contributed to the breakdown of the state extension machinery (Hanumantha Rao 2003). Private extension provision (fee for service) is emerging. There are an increasing number of input suppliers, traders, contract buyers, supermarkets, and exporters which provide extension services to farmers as an integral part of their trading arrangements (World Bank 2005b). However in the national context, private extension remains limited. Table 2. Farmer Sources of Information.

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The findings of a World Bank agricultural marketing survey, covering 1,579 farmers producing high value crops (tomatoes, potatoes, mangoes, maize and tumeric) in four states in India (Orissa, Tamil Nadu, Uttar Pradesh, and Maharashtra) conducted during February to May 2005, confirm the limited effectiveness of the national extension system. Farmers primarily depended on personal observation or on other farmers for information about crop prices, post harvest practices, irrigation, fertilizer and pesticide use (Table 2). Although food safety concerns have not been a major focus in the extension program, it is partly addressed through the increased Ministry of Agriculture (MoA) priority to integrated pest management (IPM). MoA established the National Center for Integrated Pest Management in1988 to develop and promote IPM technologies. Notably there has been a decline in total pesticide consumption in India from 75,000 mt in 1990/91 to 48,400 mt in 2003/03 (Directorate of Plant Protection and Quarantine 2006).

Poor Infrastructure and Services in the Marketing System Reducing food safety risks from the farm to domestic and export markets is constrained by inadequate infrastructure and facilities, particularly at the wholesale markets. The World Bank Agricultural Marketing Survey also collected information on the operations of 78 wholesale markets in the four states. The survey found that the infrastructure and facilities in these markets are limited and rudimentary. Overall, Maharashtra and UP had slightly better infrastructure than the other two states. About 83% of markets had covered shops, but only 18% had paved roads within the market and 51% had public toilets. Access to warehouses is limited, except in Maharashtra (85%). Less than 40% of markets had a drying area and no markets in Orissa or Uttar Pradesh had cold storage facilities (compared to 5% in Tamil Nadu and 20% in Maharashtra). Waste management and pest control in the markets are very weak. Officials working in the wholesale markets were asked how the spoiled produce and waste products were disposed off. Fifty-four percent responded that market employees or contracted firms handled garbage disposal and waste management; 29% reported that they were just left to rot in the market, while 13% reported that they were left for the animals to eat. Market officials were also asked about the pest control measures they undertake. Fifty-nine percent indicated that no particular control measure for rats and insects are implemented in their market, 32% indicated it was up to the individual shop owners to take care of their rat problems. Only 8% reported the market management or association or a subcontracted firm took care of rat problems. Reducing food safety risks will require significant public and private investments to upgrade the market infrastructure and services. For regulated markets, this will also require improving the operational and fiduciary management to ensure that more resources are re-invested back into the markets.

Cultural Issues Religious beliefs further constrain the kinds of food safety measures that could be adopted in India. The sacred value attached to cattle imposes limits on disease control

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measures to address food safety and public health (BSE, foot and mouth disease), such as culling to limit disease spread or to create disease free zones.

Inadequate Grades and Standards for the Domestic Market and Poor Enforcement The Directorate of Marketing & Inspection under the Department of Agriculture and Cooperation is responsible for enforcing and implementing the Agricultural Produce (Grading and Marking) Act. Its mandate includes promoting standardization and grading of agricultural products. Grades and standards have been prescribed for 164 commodities under the APM Act for domestic trade, for export trade and for grading at the producer‘s level. The AGMARK grades are primarily voluntary grades covering aspects such as size, variety, weight, color, and moisture levels. For certain items they also cover parameters such acceptable levels of organic and inorganic foreign matter (in pulses, for example) and other chemical properties such as specific gravity for essential oils. Different grades and standards are laid out under AGMARK for domestic consumption versus exports. The Directorate provides third party certification under the AGMARK quality certification scheme. The ‗AGMARK‘ seal is supposed to ensure quality and safety. Any consumer, trader or manufacturer can have products tested at one of the 23 regional AGMARK laboratories for designated commodities. Typically, testing is only carried out for adulteration prone commodities such as oils, ghee, whole and ground spices, honey, and whole and milled food grains. Blended edible vegetable oils and fat spreads are compulsorily required to be certified under AGMARK. The Prevention of Food Adulteration Act also sets standards for food products including aspects such as permissible food colorings, preservatives, pesticide residues, packaging and labeling. As illustrated by the bottled water and soft drink pesticide residue incidents, inadequate standards and weak enforcement remain a problem. The grades specified under AGMARK and standards as laid out in the PFA are designed to facilitate trade as well as ensure food safety. The food safety standards under the PFA in general need to be aligned with international standards. However there are many commodities that are not grown or consumed outside of India. For these commodities it may not be possible to align domestic standards with international standards because there are no established international standards. In these instances it is important for research to be conducted in India to set appropriate standards for the domestic market.

Lack of Pro-Activity in Addressing Food-Safety Issues Domestic food safety scares and the more notable food-safety problems faced by Indian agro-exports, reveal the overall absence of any pro-activity in addressing food safety concerns in India. Several factors contribute to this. In the case of exports, many if not most of the emerging SPS and international standards are widely viewed as not scientifically based and as representing unfair ―barriers to trade‖ (World Bank, 2006b). These measures are viewed as efforts to protect foreign farmers or processors from competition, or are being fueled by

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unreasonable consumer fears in high income countries and improved technologies for detecting hazards. Consequently, the approach of the government and private sector has been to try to negotiate away the problems with trading partners and, failing that, addressing the various measures in international standard-setting or dispute flora. As a consequence, insufficient attention is devoted to monitoring the requirements of official and private standards, interpreting their implications for Indian agriculture and using current and anticipated requirements as catalysts to upgrade existing operations and strengthen supply chain management (World Bank 2006b). This absence of pro-activity has meant that India has either had to adopt a ―defensive‖ strategy avoiding markets with more stringent food safety and agricultural health standards or launch into a fire-fighting mode when it faces potential disruption or loss of trade due to noncompliance with standards.17 The absence of pro-activity is well illustrated through examples of problems faced with exports of fishery products in the late nineties and the more recent troubles with grape exports to Europe. In both cases, although there were signs of potential problems for a considerable period of time, the food safety problems were not given serious attention until India was faced with a crisis. In the case of exports of fish and fishery products, necessary monitoring and enforcement measures for ensuring that exports complied with food safety concerns were not put in place until the loss of EU markets in 1997 (Henson, Saqib and Rajasena, 2005). This was despite the fact that India had continually faced rejections because of failure to meet hygiene standards and other food safety requirements since the 80s, and in spite of regulatory reforms to provide safety assurance for fish and fishery products undertaken in 1995 (Henson, Saqib and Rajasena, 2005). Similarly, in the case of grape exports to the EU, pesticide residue problems had surfaced since the late nineties. During this period, some limited testing was done for pesticide residues in export-oriented grapes. Testing was made mandatory in 2000, but most of the available testing equipment was not up to date, could not test to the same level of detection as was common in Europe and was unable to detect certain heat-sensitive chemicals such as acephate and methomyl (World Bank, 2006b).18 Only after EU Rapid Alerts were issued in 2003 did the Government and industry step into action to address the problem. In general India has not viewed complying with food safety and agricultural health standards as a means to both improve its competitive position and to enhance the effectiveness of its negotiations on particular technical and commercial matters, which is in stark contrast to the approach of leading agro-food exporting countries (World Bank, 2006b). A consequence of the lack of pro-activity and the crisis management mode of operation has been the adoption of very rigorous and strict controls for commodities threatened with the loss or disruption of trade. This has led to extremely high costs of compliance in some cases (e.g. grapes) (World Bank, 2006b) or rather onerous requirements (e.g. requirements for processing facilities exporting fishery products) (Henson, Saqib and Rajasena, 2005). In the case of grapes, the Government of India (GOI) Agricultural and Processed Food Products Export Development Authority (APEDA), formulated an integrated system of intensive grape supply chain oversight that included: 17

An example of a defensive strategy is the existing trend where many of India‘s mango pulp exporters are forced to sell to less remunerative markets because they are not HACCP compliant. 18 As reported in Buurma et al 2001.

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A requirement that all farms growing grapes for export to Europe have to register with the Department of Agriculture. About 6200 growers registered for the 03/04 season; Three field inspections (for registered exporters) during the crop cycle by a newly constituted cadre of horticultural field inspectors. Some 244 such officers were initially appointed and trained. There are now 291 such officers; The inspection and registration of all grape export packinghouses by APEDA. Mandatory pesticide residue testing from each registered field of export grapes. Testing would be done prior to harvest and only if the tests were passed would authorization be given for harvesting for export. Grapes from fields with failed results would need to be sold in other markets or re-tested. Every consignment would be checked by AGMARK to ensure conformity with EU quality specifications for grapes. AGMARK would issue certificates. Obtaining a phytosanitary certificate issued by Plant Protection, Quarantine and Storage for every consignment; and Later, in 2005, another procedure was added whereby National Research Center for Grapes would take a 5% sample of ex-packhouse grape consignments to re-test for pesticide residues.

The extensive system of checks and controls primarily focused on end-of-the-pipeline solutions. In addition to the protocols that potential exporters to the EU have to follow, the government also invested heavily in upgrading laboratory testing equipment, training field inspectors, subsidizing packhouse upgrades, and strengthening the National Research Centre for Grapes. Overall, it is estimated that the cost of this control system for pesticide residues (to government and the private sector) is about US$1.2 million, equivalent to 7.9% of the FOB value of India‘s grape trade to Europe in 2005 (Table 3). If all other costs associated with the oversight of the grape supply chain are added to the costs of pesticide residue testing, SPS compliance costs are estimated to account for 13% of this FOB value. Table 3. Estimated Annual Cost of Meeting EU SPS Standards—2005 US $

While it is arguable that there are many spillovers and important lessons that have been learned from the handling of the pesticide residue problem with grape exports, and that these measures have been ―successful‖ in that they have not resulted in further alerts or rejections, the heavy handed approach with which the problems were addressed, and the costs involved,

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clearly suggest that it is not a strategy that should be replicated. Although India has not faced further rejections of exports to the EU, routine laboratory testing still reveals violative residues, indicative of the continuing need to focus on improving overall agricultural practices to assure food safety.

Lack of Good Agricultural, Manufacturing and Hygiene Practices In addition to constraints that arise due to small farm sizes, the lack of good agricultural, manufacturing and hygiene practices remain a major challenge for improving food safety both for the domestic and export market. It is only recently that efforts are being made to promote good practices. For example, Marine Products Export Development Authority (MPEDA) promoted codes of good practice, particularly with regards to addressing antibiotic use. To this extent the organization was involved in monitoring antibiotic usage levels, providing training and disseminating information (Henson, Saqib and Rajasena, 2005). In the spices sector, the Spices Board (SB) undertook measures to address problems with regards to pesticide residues and aflatoxin. The SB, in conjunction with State Departments of Agriculture and various NGOs, supported measures to promote integrated pest management (IPM) and the production of organic spices (Jaffee, 2005). They helped address the aflatoxin concern by promoting better drying practices. The Ministry of Food Processing Industries and APEDA have both been promoting adoption of HACCP and ISO certification among processed food manufacturers through a range of training initiatives and private sector investment grant for upgrading processing plants to obtain HACCP/ISO certification. However, the adoption of good practices remains limited. Much remains to be done in improving practices with regards to the manufacture and use of pesticides and improving post harvest techniques. Although there have been some limited spillovers from the export sector into the domestic market, in terms of improving production practices, for most commodities, including spices and fresh fruit and vegetables, farmers do not necessarily see any advantages or necessity for altering their production practices since the vast majority of production is consumed in the domestic market. Until domestic consumer awareness and willingness to pay for improved food safety becomes more widespread, it is unlikely that addressing food safety concerns will become standard practice nationally. Similarly, significant measures are needed to improve the safety of processed foods. In the food processing sector there are a growing number of firms with modern factories and good quality assurance systems, but this segment co-exists with large numbers of small and older firms that would need to make significant upgrades to implement HACCP and other quality assurance systems.19 In the short term, developments in the food retail sector in India are likely to bring about improvements in food safety. International experience shows that modernization of the food retail sector is an important driver for change not only in the structure of production and wholesale marketing of produce, but also in fostering adoption of improved grades and food safety standards (Berdegué et al 2003, Reardon and Timmer 2005a, 2005b). Despite the ban on foreign direct investments in food retailing, the supermarket industry is growing rapidly,

19

For instance, in recent work on the mango pulp sector in India one company reported costs of $35,000 to put it in a position to implement a proper HACCP system (World Bank, 2006b).

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driven by investments from the Indian corporate sector.20 Many of the modern retail outlets are beginning to undertake direct procurement from individual farmers or farmers‘ associations. In some cases farmers or associations supplying these outlets are required to follow a code of practice to meet quality and safety requirements of their buyers. The retail outlets are also involved in disseminating new agricultural techniques and information to their suppliers as well as providing training on quality control of produce handling, grading and packaging. There are also efforts by the public sector to promote good agricultural practices among producer groups and to help establish linkages with the organized food retail sector.21 The Government of India and State governments are working closely with the supermarket industry (with support from USAID) to develop an India Good Agricultural Practice standard for agricultural produce (INDIA-GAP), which will in turn also provide the framework for government extension support to farmers.

Need for More Collective Action International experience highlights the importance of collective action within the private sector to promote awareness of SPS matters, find solutions to emerging challenges, promote good agricultural and manufacturing practices, and otherwise provide a degree of selfregulation, which in turn reduces the need for government agencies to play enforcement roles. While there are some examples of successful collective action in both the spice and fishery export industries in India, it has been lacking in many other sectors, notably in horticulture (World Bank, 2006b). For example, the Seafood Exporters Association of India (SEAI) has developed a model to provide a number of pre-processing units with common water, ice and effluent infrastructure. SEAI in collaboration with MPEDA has also been involved in developing a system to ensure traceability for shrimp from aquaculture in order to address quality problems (Henson, Saqib and Rajasenan, 2005). In the Spices sector, the All India Spice Exporters Forum has been an important player in trying to influence standards for pesticides in spices grown under tropical conditions and in finding solutions to address food safety concerns in its export markets (Jaffee, 2005).

20

Corporate manufacturers such as Hindustan Lever Ltd, International Tobacco Company, Godrej, Bharti,Reliance, DCM Sriram Conolidated, RPG Group, Pantaloon Group are setting up or have set hypermarkets, supermarkets and retail outlets in rural areas, recognizing the huge untapped potential (World Bank 2006a). Gasstation stores are also another growing retail outlet. Petroleum companies like Hindustan Petroleum Corporation Limited, Indian Oil, and Bharat Petroleum have introduced branded outlets like Speedmart (around 60-65 in number), ConveniO‘s (around 150), and In&Out Stores (around 100) which sell food items (Singh 2004). 21 The Marashtra Agricultural Agricultural Marketing Board in collaboration with USAID is trying to promote good agricultural practices among mango farmers in the state and link these farmers with various supermarkets and other retail outlets that are interested in procuring better quality and safer fruit.

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8. CONSUMER CONTROL POINTS Minimizing Possible Consumption of Pesticide Residues For those consumers who wish to take additional measures to reduce any possible pesticide residues on their foods, here are some tips from AFIC. 1) Raw foods should be washed thoroughly before cooking and/or consumption. Washing in dilute vinegar solution, or solution of sodium bicarbonate, then rinsing with clean water will help to remove any chemical residues and also any soil or other foreign matter on the produce. 2) Many chemicals applied to crops to protect from insects and disease are sprayed onto external surfaces, so peeling outer layer or skin when preparing fresh fruit and vegetables will remove any surface residues. 3) Look out for the many of the quality assurance schemes, which guarantee chemical treatment of produce has strictly followed manufacturers recommendations and residues levels at point of harvest are either zero or very low. There are also an increasing number of retailers and growers offering ‗organic‘ produce, however, be aware that ‗organic‘ farming often uses some pest control substances, approved by the various associations established to promote this form of cultivation. 4) Do not consumer berries, leaves or other edible plant material picked from roadsides or other public areas, as these plants as it is not possible to know if these plants have been sprayed intentionally or unintentionally contaminated with pesticides or other substances and will not be subject to safety restrictions of designated food crops. Certain processes or handling practices by consumers in the home have been identified as being essential or critical in preventing foodborne illness. These practices, which prevent or control the "meals" microbial contamination associated with foodborne illness, are under the direct control of the consumer, from food acquisition through disposal. They are purchasing, storing, pre-preparation, cooking, serving, and handling leftovers. Failure to take appropriate action at these critical points could result in foodborne illness.

Critical Point 1: Purchasing  

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Purchase as far as possible perishables on a daily basis. Procure processed food items only after checking properly the `Best Before date ‗ validity and prefer processed branded food items with product certification marks viz. ISI, FPO & AGMARK. Procure milk and milk products, fish, seafood, poultry, meat and meat products, eggs and other perishables last when out on purchasing and keep packages of raw meat and meat products separate from other foods, particularly foods that will be eaten without further cooking. Consider using plastic bags to enclose individual packages of raw meat and meat products.

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Make sure milk and milk products, fish, seafood, poultry, meat and meat products, eggs and other perishables are refrigerated as soon as possible after purchase. Plan to drive directly home after purchases from the grocery store. Canned food items, if purchased, should be free of leaks, dents, cracks or bulging lids.

Critical Point 2: Home Storage 

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Verify the temperature of your refrigerator and freezer with an appliance thermometer - refrigerators should run at 5°C or below; freezers at -18°C. Most foodborne bacteria grow slowly at 5°C, a safe refrigerator temperature. However, freezer temperatures of -18°C stops bacterial growth. Keep High risk products such as fish, seafood, poultry, meat and meat products in the freezer cabinet of refrigerator immediately on reaching home. Keep the milk and milk products in the designated racks in the refrigerator or on the first topmost rack just under the freezer cabinet. To prevent raw juices drippings & pieces of meat and meat products from falling on cooked to other foods kept in the refrigerator. Keep them in shelves below the stand cooked food. Use plastic bags or place meat and poultry on a plate. Wash hands with soap and water for 20 seconds before and after handling any raw fish, seafood, poultry, eggs, meat and meat products. Store canned foods in a cool, clean dry place and as per the labeling directions of the manufacturer. Avoid extreme heat or cold which can be harmful to canned food items. Never store any foods directly under a sink and always keep foods off the floor, on a rack or pallet and completely separate from cleaning supplies.

Critical Point 3: Pre-Preparation 

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The importance of hand washing cannot be overemphasized. This simple practice is the most economical, yet often forgotten way to prevent contamination or crosscontamination. Wash hands with soap and water thoroughly (for 20 seconds): before beginning preparation; after handling raw meat, poultry, seafood or eggs; after touching animals; after using the toilet/bathroom; after changing diapers; or after blowing the nose. Don't let juices from raw meat, poultry or seafood come in contact with cooked foods or foods that will be eaten raw, such as fruits or salads. Wash hands, kitchen slabs / tables, equipment, utensils, and cutting boards with soap and water immediately after use. Kitchen slabs / tables, equipment, utensils and cutting boards can be sanitized with a chlorine solution of 1 teaspoon liquid household bleach per quart of water. Let the solution stand on the board after washing, or follow the instructions on sanitizing products. Before processing frozen fish, seafood, poultry, meat and meat products, thaw the same properly in the refrigerator but NEVER ON THE KITCHEN SLAB / TABLE.

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It is also safe to thaw in cold water in an airtight plastic wrapper or bag, changing the water every 30 minutes till thawed. Thawing may also be carried out in the microwave and followed immediately by cooking. Marinate foods in the refrigerator and NEVER ON THE KITCHEN SLAB / TABLE.

Critical Point 4: Cooking 

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Make sure to cook food thoroughly, particularly the meat products, Depending upon the type of dish made, thoroughly cooking would be signified by colour, texture and taste. For example boiled/steamed rice shall have grains double the size of original rice grains, with each grain separate; Fried item viz cutlets, pokoras etc should be brought to golden brown coloir at medium flame, which ensures thorough cooking upto the core of the fried item. If harmful bacteria are present, only thorough cooking will destroy them (core temperature of product to be higher than 75 °C) ; remember freezing or rinsing the foods in cold water is not sufficient to destroy bacteria. Avoid interrupted cooking. Never refrigerate partially cooked products to later finish cooking on the grill or in the oven. Fish, seafood, poultry, meat and meat products must be cooked thoroughly the first time and then they may be refrigerated and safely reheated later. When microwaving foods, carefully follow manufacturers instructions. Use microwave-safe containers, cover, rotate, and allow for the standing time, which contributes to thorough cooking.

Critical Point 5: Serving:  

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Wash hands with soap and water before serving or eating food. Serve cooked products on clean plates with clean utensils and clean hands. Never put cooked foods on a dish that has held raw products unless the dish is washed with soap and hot water. Hold hot foods above 60°C and cold foods below 5°C. Never leave foods, raw or cooked, at room temperature longer than 2 hours.

Critical Point 6: Handling Leftovers      

Wash hands before and after handling leftovers. Use clean utensils and surfaces. Divide leftovers into small units and store in shallow containers for quick cooling. Refrigerate within 2 hours of cooking. Discard anything left out too long. Never taste a food to determine if it is safe. When reheating leftovers, reheat thoroughly (temperature of 75 °C) until the dish is hot and steamy. Bring soups, sauces and gravies to a rolling boil. If in doubt, throw it out.

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Ten Steps to a Safe Kitchen: Step 1 Keep your refrigerator at 4° C or less.A temperature of 4° C or less is important because it slows the growth of most bacteria. The fewer bacteria there are, the less likely you are to get sick from them. Step 2 Refrigerate cooked, perishable food as soon as possible but within two hours after cooking. A temperature of 4° C or less is important because it slows the growth of most bacteria. The fewer bacteria there are, the less likely you are to get sick from them. Date leftovers so that they can be used within one day. If in doubt, throw it out! Step 3 Sanitize your kitchen dishcloths and sponges regularly. Wash with a solution of one teaspoon (5 ml) chlorine bleach to one litre of water, or use a commercial sanitizing agent, following product directions. Step 4 Wash your cutting board with soap and hot water after each use to prevent any subsequent contamination in food during preparation. Never allow raw meat, poultry, and fish to come in contact with each other as they have generally high bacteria count including pathogens which cause food poisoining .Washing with only a damp cloth will not remove bacteria. Periodically washing in a bleach solution is the best way to prevent bacteria from remaining on your cutting board. Step 5 Cook meats , seafood and poultry products thoroughly so as to ensure that cooked food is free from harmful bacteria. Cooking meat products on a law/medium flame such that the core temperature reaches at least 75° C usually protects against foodborne illness. Welldone meats reach that temperature. Step 6 Don't consume raw or lightly cooked eggs as they may contain the harmful Salmonella bacteria. Always cook the eggs thoroughly before eating them. Step 7 Clean kitchen counters and other surfaces that come in contact with food with hot water and detergent or a solution of bleach and water. Bleach and commercial cleaning agents are best for getting rid of pathogens. Hot water and detergent are good, but may not kill all strains of bacteria. Keep sponges and dishcloths clean because, when wet, these materials harbor bacteria and may encourage their growth. Step 8 When washing dishes by hand, it‘s best to wash them with warm water and detergents all within two hours--before bacteria can begin to form. Allow dishes and utensils to air-dry in order to eliminate re-contamination from hands or towels. Step 9 Wash hands with soap and warm water immediately after handling raw meat, poultry, or seafood. Wash for at least 20 seconds before and after handling food, especially raw meat. If you have an infection or cut on your hands, wear rubber or plastic gloves. Step 10 Defrost frozen meat, poultry and fish products in the refrigerator, microwave oven, or cold water that is changed every 30 minutes. Cook microwave-defrosted food immediately after thawing. Changing water every 30 minutes when thawing foods in cold water ensures that the food is kept cold, an important factor for slowing bacterial growth on the outside while inner areas are still thawing.

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CONCLUSION The Indian experience illustrates the many challenges faced by developing countries in addressing food safety concerns in domestic and export markets. Despite a large number of food safety incidents in the past, it is only in the past five years or so that food safety issues have begun to receive greater attention. As elaborated in this paper, this has partly been due to greater consumer awareness arising from campaigns led by NGOs, increased coverage of food-safety incidents in the media, wider access to media and the internet, and the problems encountered with agro-food exports in high income markets. Despite this, considerable efforts are still needed to give the issue of food safety the attention it warrants. Because of low consumer awareness, the private sector engaged in agriculture, food processing and the food retail industry in India, for the most part, has not taken the necessary steps to improve the quality and safety of food products. In most cases, the responsibility of ensuring food safety fell into the hands of government through enacting and enforcing legislation and setting standards. While government has taken actions in instances where there have been immediate public health scares or disease outbreaks, less attention has been given to food safety concerns whose impact is only apparent in the medium to long-term. One of the positive results of globalization and the emergence of modern food retailing in India is the increased attention to quality and safety issues. As incomes are increasing, consumers are also more willing and able to pay for better quality and safer food. Addressing food safety issues in India will require the adoption of more appropriate legislation and their enforcement (Table 4). Parliamentary approval of the Food Safety and Standards Bill will be critical to removing the uncertainty arising from, and the associated additional cost of dealing with, overlapping and conflicting food safety regulations. Broad based adoption of the model APM Act and the removal of the remaining agricultural commodities from the SSI reservation will foster both increased market efficiency and facilitate adoption by firms of appropriate food safety measures. Joint efforts by the government and the private sector will be needed in a number of areas. These include better risk management, the promotion and adoption of good agricultural, manufacturing and hygiene practices, greater collective action and some targeted public investments. Responsibilities for these functions need to be shared between the private and public sectors. While there are many critical regulatory, research and management functions that are normally carried out by governments, the private sector also has an important role in the actual compliance with food safety requirements. Quality grades should be voluntary for fruits, vegetables and for most other fresh produce, since they are set primarily to facilitate trade and are not a regulatory instrument. Yet, for matters of food safety, standards should be mandatory rather than voluntary. These standards would apply for pesticide residues, heavy metal and other forms of environmental contamination, and especially for microbiological contaminations for which there could be acute health risks. A coordinated program of food safety product surveillance can be used to highlight the nature and scope of pertinent problems and also be used as a basis for developing consumer and supply chain awareness and good practice promotion. Overall there is a large role for extension service providers to promote good practices in order to ensure that farmers follow recommended dosages for agro-chemicals and observe appropriate pre-harvest

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intervals. Soil and water testing should also be routinely conducted through the extension apparatus (World Bank, 2006a). Table 4. Role of Public and Private Sector in Enhancing Food Safety Capacity. Role of Public Sector Policy and Regulatory Environment • Adopt domestic food safety legislation and standards suited to local risk conditions and preferences and consistent with India‘s WTO and other treaty obligations. Risk Assessment and Management: • Strengthen national or state-level systems of pest and animal disease surveillance and market surveillance programs to gauge the incidence of various food safety hazards in the domestic agrofood system. • Find solutions to animal health constraints that limit domestic (for imports) and foreign (for exports) market access. This might entail, product inspection, agreed development of disease-free areas. etc Awareness building and promoting good practices: • Consumer awareness campaigns about food safety risk and improve hygiene in the home • Raise stakeholder awareness about and promote good agricultural, hygiene, and manufacturing practices and quality management. Incorporate these areas into curricula of public agricultural/ technical institutes and universities. • Incorporate food-safety focused practices in extension program, including through public-private partnerships • Accredit private laboratories and conduct reference/consistency testing. • Facilitate technical, administrative and institutional change and innovation within the private sector for example through publicprivate partnerships Public Expenditures • Investments in water supply and sanitation, marketing facilities, to reduce food safety hazards • Support research to address food safety and agricultural health concerns International Trade Diplomacy: • Undertake continuous dialogue and periodic negotiations to address emerging constraints or opportunities.

Source: Adapted from World Bank 2006a.

Role of Private Sector ‗Good‘ Management Practices: • Implement appropriate management practices to minimize food safety risks. Examples include ‗good‘ agricultural, hygiene, and manufacturing practices and HACCP principles. • Where commercially valuable, gain formal certification for such adopted systems. • Develop incentives, advisory services and oversight systems to induce the similar adoption of the above ‗good practices‘ by supply chain partners. Traceability: • Develop systems and procedures to enable the traceability of raw materials and intermediate and final products in order to identify sources of hazards, manage product recalls or other emergencies, etc. Develop Training, Advisory, and Conformity Assessment Services: • On a commercial basis provide support services to agriculture, industry, and government related to quality and food safety management. Invest in the needed human capital, physical infrastructure and management systems to competitively supply such services. Collective Action and Self-Regulation: • Work through industry, farmer, and other organizations to share the costs of awarenessraising and systems improvement, alert government to emerging issues, advocate for effective government services, and provide a measure of self-regulation through the adoption and oversight of industry ‗codes of practice‘

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The use of agricultural chemicals in relation to food safety will continue to be a complex subject. Some studies have shown that pesticides may affect ground-water, wildlife and occupational workers if the chemicals are not used in accordance with the law. But the future looks promising as food scientists, research-ers, government officials and manufacturers search for methods to improve agricultural techniques while reducing pesticide-related risks. Today's consumers can feel confident that they can choose from an abundant and safe food supply for themselves and their families. There is also a need for regular inspection of health and sanitary conditions at certain types of food premises that may be associated with more severe consumer health risks, (abattoirs, for example). Inspection should not be random, but should be targeted based upon risk assessments that government may do on different types of food establishments to help pinpoint areas requiring particular attention, not only in the form of inspection, but also including awareness-raising, training, periodic licensing, etc. The challenges for ensuring food safety in the domestic market and in its food exports remain large. India has made some progress in the last decade to strengthen food safety measures at home and in meeting food safety and SPS standards abroad. The challenge for the future will be to adopt a more strategic, rather than crisis management approach. This will be essential to ensuring the sustainability and cost effectiveness of these efforts.

REFERENCES Bhat, Ramesh V. and Siriguri Vasanthi, 2003, ―Mycotoxins Food Safety Risk in Developing Countries‖, in L. Unnevehr (Ed), Food Safety in Food Security and Trade, 2020 Vision for Food Agriculture and the Environment, Focus 20, Brief 3, International Food Policy Research Institute, Washington, D.C. Boselie, David, Spencer Henson, and Dave Weatherspoon, 2003, ―Supermarket Procurement Practices in Developing Countries: Redefining the Roles of the Public and Private Sectors‖, American Journal of Agricultural Economic 85 (number 5): 1155-1161. Brahmhatt, Milan, 2005, ―Avian Influenza: Economic and Social Impacts,‖ http://web.worldbank.org/WBSITE/EXTERNAL/NEWS/0,,contentMDK:20663668~pagePK:3 4370~piP K:42770~theSitePK:4607,00.html Buurma, J., M. Mengelers, A. Smelt, and E. Muller. 2001. Developing Countries and Products Affected by New Maximum Residue Limits of Pesticides in the EU. Agricultural Economics Research Institute,The Hague. Buzby, Jean C. and Laurian Unnevehr, 2003, ―Introduction and Overview,‖ in in Jean C. Buzby (Ed), International Trade and Food Safety, Economic Theory and Case Studies, Agricultural Economic Report No. 828, USDA, Chapter 1, pp.1-9. Calvin, Linda, 2003, ―Produce, Food Safety and International Trade, Response to US Foodborne Illness Outbreaks Associated with Imported Produce‖, in Jean C. Buzby (Ed), International Trade and Food Safety, Economic Theory and Case Studies, Agricultural Economic Report No. 828, USDA, Chapter 5, pp.74-96. California Agriculture, 48(1):6-35, January/February 1994. Center for Science and Environment, 2004, ―Poison vs Nutrition, A Briefing Paper on Pesticide

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Contamination and Food Safety,‖ Center for Science and Environment, New Delhi, mimeo. Central Statistical Organization, 2002, ―Socio Economic Statistics 2002,‖ New Delhi: Central Statistical Organization. Chaisson, C.F., Petersen, B., and Douglass, J.S. Pesticides in Food, A Guide for Professionals. American Dietetic Association, Chicago, IL, 1991. Chengappa, P.G., Lalith Achoth, Prasanna Rashmi K.K., Vijayalakshmi Dega, B. M. Ramachandra Reddy, and P. K. Joshi, March 23-25, 2005, Emergence of Organised Retail Chains in India During Post Liberalization Era, Paper presented at the South Asia Regional Conference of International Association of Agricultural Economists Globalisation of Agriculture in South Asia which was held at Hyderabad, The World Bank., Washington, D.C. Deaton, A., and J. Dreze. 2002. ―Poverty and Inequality in India: A Re-Examination.‖ Economic and Political Weekly (September): 3729–48. Department of Small Scale Industries, 2006, http://www.laghu-udyog.com/publications/reserveditems/dereserve03.htm Directorate of Plant Protection and Quarantine, 2006, http://www.ncipm.org.in/asps/ DisplayPesticides.asp Dohlman, Eric, 2003, ―Mycotoxin Hazards and Regulations, Impacts on Food and Animal Feed Crop Trade,‖ in Jean C. Buzby (Ed), International Trade and Food Safety, Economic Theory and Case Studies, Agricultural Economic Report No. 828, USDA, Chapter 6, pp.97-108. Dries, L, T Reardon and J Swinnen. 2004. ―The Rapid Rise of Supermarkets in Central and Eastern Europe: Implications for the Agrifood Sector and Rural Development.‖ Development Policy Review 22(5): 525-56 Ewen, C., D. Todd and C. Narrod, 2003, ―Understanding the Links between Agriculture and Health, Agriculture, Food Safety, and Food Borne Diseases,‖ 2020 Vision for Food, Agriculture and the Environment, Focus 10, Brief 5, International Food Policy Research Institute, Washington, D.C. Food and Drug Administration Pesticide Program. Residue Monitoring—1993. Washington, D.C., 1994. Hanumantha Rao, C.H, 2003. ―Reform Agenda for Agriculture.‖ Economic and Political Weekly (February 15). Henson, S, M Saqib, D Rajasena. 2005. Impact of Sanitary Measures on Exports of Fishery Products from India: The Case of Kerela, Agriculture and Rural Development Discusion Paper, World Bank, Washington, D.C. Henson, Spencer, 2003, ―Food Safety Issues in International Trade‖ in L. Unnevehr (Ed), Food Safety in Food Security and Trade, 2020 Vision for Food Agriculture and the Environment, Focus 20, Brief 10, International Food Policy Research Institute, Washington, D.C. Hindu Business Line, 2003, ―Pesticides in soft drinks — Industry may present views to JPC today‖, http://www.thehindubusinessline.com/2003/10/21/stories/2003102101880500.htm Hotchkiss, J.H. Pesticide residue controls to ensure food safety. Critical Reviews in Food Science and Nutrition, 31(3):191-203, 1992. Indian Council of Medical Research, 2001, ―Pesticide Pollution: Trends and Perspectives,‖ ICMR Bulletin, Vol 31, No. 9. Indian Council of Medical Research, New Delhi.

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Jaffee, Steven. 2005. ―Delivering and Taking the Heat: Indian Spices and Evolving Product and Process Standards,‖ Agriculture and Rural Development Discussion Paper #19, World Bank, Washington, D.C. Jha, S., and D. Umali-Deininger. 2003. ―Public Expenditures on Food and Nutrition Security Programs in India: Are They Meeting the Challenge?‖ Working Paper. South Asia Rural Development Unit, World Bank. Kafferstein, Fritz, K., 2003, ―Food Safety as a Public Health Issue for Developing Countries‖, in L. Unnevehr (Ed), Food Safety in Food Security and Trade, 2020 Vision for Food Agriculture and the Environment, Focus 20, Brief 2, International Food Policy Research Institute, Washington, D.C. Landes, M. and A. Gulati. 2003. ―Policy Reform and Farm Sector Adjustment in India‖ Paper presented at the Wye Imperial College sponsored workshop on Policy Reform and Adjustment, London, October 23-25. Lindsay, J.A., 1997, ―Chronic Sequelae of Foodborne Disease,‖ Emerging Infectious Diseases, Vol 3 No 4 (Oct-Dec 1997), www.cdc.gov/Ncidod/eid//vol3/no4/lindsay.htm Lowy Institute for International Policy, 2006, ―Global Macroeconomic Consequences of Pandemic Influenza‖, Analysis, Australian National University (February 2006). Marshall, Fiona Ravi Agarwal, Dolf te Lintelo, D.S. Bhupal, Rana P.B. Singh, Neela Mukherjee, Chandra Sen, Nigel Poole, Madhoolika Agrawal, and S.D. Singh, 2003. ―Heavy Metal Contamination of Vegetables in Delhi.‖ Report prepared for the UK Department for International Development. Mimeo. Mathur, H.B., H.C. Agarwal, Sapna Johnson, and Nirmali Saikia, 2005, ―Analysis of Pesticide Residues in Blood Samples From Village of Punjab,‖ CSE/PML/PR-21/2005, Center for Science and Environment, New Delhi, mimeo. Mathur, H.B. Sapna Johnson, and Avinash Kumar, 2003, ―Analysis of Pesticide Residues in Soft Drinks, ― Center for Science and Environment, New Delhi. Mimeo. Ministry of Commerce, 2005, Annual Report 2004/05, New Delhi: Ministry of Commerce Ministry of Food Processing Industries, 2005, Annual Report 2004/05, New Delhi: Ministry of Food Processing Industries. Moore, M. Health Risks and the Press—Perspectives on Media Coverage of Risk Assessment and Health. American Medical Association, The Media Institute, Washington, D.C., 1989 Mukherjee, A. and N. Patel, 2005, FDI in Retail Sector India, New Delhi: Academic Foundation. National Center for Integrated Pest Management, 2006, http://www.ncipm.org.in/aboutus/htm Otsuki, Tsunehiro, John S. Wilson, and Mirvat Sewadeh, 2001, ―Saving Two in a Billion: Quantifying the Trade Effect of European Food Safety Standards on African Exports‖, Food Policy, Vol 26: 495-514. Pingali, P. and Y. Khwaja. 2004. ―Globalization of India diets and the transformation of food supply systems.‖ Keynote address XV11 annual Conference of The Indian Society of Agricultural Marketing. 5th February , 2004. Acharya N.G. Ranga Agricultural University and Indian Society of Agricultural Marketing. Reardon, T and J F M Swinnen. 2004. ―Agrifood Sector Liberalization and the Rise of Supermarkets in Former State-Controlled Economies: A Comparative Overview.‖ Development Policy Review 22(5):515- 23 Reardon, T and C P Timmer. 2005a. ―Transformation of Markets for Agricultural Output in Developing Countries Since 1950: How Has Thinking Changed? In R E Evenson, P

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Pingali and T P Schultz (ed) Handbook of Agricultural Economics (Vol.3): Agricultural Development: Farmers, Farm Production and Farm Markets. Reardon, T and C P Timmer., 2005b, ―The Supermarket Revolution with Asian Characteristics‖, Paper presented during the SEARCA International Conference ―Agricultural and Rural Development in Asia: Ideas, Paradigms, and Policies Three Decades After,‖on November 10-11, 2005, Mandarin Oriental, Makati City, Philippines. Singh, S.K., 2004, India Retail Food Sector Report 2004, United States Department of Agriculture Foreign Agricultural Service Global Agriculture Information Network Report No. IN4126, New Delhi. Unnevehr, Laurian J., 2003, ―Overview‖ in L. Unnevehr (Ed), Food Safety in Food Security and Trade, 2020 Vision for Food Agriculture and the Environment, Focus 20, Brief 1, International Food Policy Research Institute, Washington, D.C. Unnevehr, Laurian and Nancy Hirschhorn, 2002, Food Safety Issues in the Developing World, World Bank Technical Paper No. 469. Washington, D.C. Virmani, Arvind, 2006, ―Poverty and Hunger in India: What is Needed to Eliminate Them,‖ Planning Commission Working Paper No. 1/2006-PC, Planning Commision, New Delhi. Willems, Sabine, Eva Roth, and Jan van Roekel, 2005, Changing Public and Private Food Safety and Quality Requirements in Europe—Challenges for Fresh Produce and Fish Exporters in Developing Countries, Agriculture and Rural Development Discussion Paper 15, World Bank, Washington, D.C. World Health Organization, 2006a, ―Water-related diseases‖ in Water Sanitation and Health, www. Who.int/water_sanitation_health/diseases/diarrhea/en/ World Health Organization, 2006b, Core Health Indicators, www3/who/int/whois/core/ core_select_process.cfm World Health Organization , 2004c, India: Scaling-up Access to Finance for India‘s Rural Poor, Report No. 30740-IN,Finance and Private Sector Development Unit, South Asia Region, Washington, D.C. World Bank, 2005a, Food Safety and Agricultural Health Standards: Challenges and Opportunities for Developing Country Exports, Report No. 31207, Poverty Reduction and Economic Management Sector Unit, Washington, D.C. World Bank, 2005b, India Re-energizing the Agricultural Sector to Sustain Growth and Reduce Poverty, New Delhi: Oxford University Press. World Bank, 2006a, India Taking Agriculture to the Market, South Asia Agriculture and Rural Development Department, World Bank, Washington, D.C. forthcoming World Bank, 2006b, India‘s Emergent Horticultural Exports: Addressing Sanitary and Phyto-Sanitary Standards and Other Challenges, South Asia Agriculture and Rural Development Department, World Bank, Washington, D.C. forthcoming World Bank, 2006c, ―Land Policies for Growth,‖ South Asia Agriculture and Rural Development Department, World Bank, Washington, D.C. forthcoming

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 15

IMPACT OF PESTICIDE USE IN INDIAN AGRICULTURE THEIR BENEFITS AND HAZARDS Wasim Aktar* Pesticide Residue Laboratory, Department of Agricultural Chemicals, Bidhan Chandra Krishi Viswavidyalaya, Mohanpur-741252, Nadia, West Bengal, India

1. INTRODUCTION The term pesticide covers a wide range of compounds including insecticides, fungicides, herbicides, rodenticides, molluscicides, nematicides, plant growth regulators and others. Among these, organochlorine (OC) insecticides, used successfully in controlling a number of diseases, such as malaria and typhus, were banned or restricted after the 1960s in most of the technologically advanced countries. The introduction of other synthetic insecticides – organophosphate (OP) insecticides in the 1960s, carbamates in 1970s and pyrethroids in 1980s and the introduction of herbicides and fungicides in 1970s - 1980s contributed greatly in pest control and agricultural output. Ideally a pesticide must be lethal to the targetted pests, but not to non-target species, including man. Unfortunately, this is not, so the controversy of use and abuse of pesticides has surfaced. The rampant use of these chemicals, under the adage, ―if little is good, a lot more will be better‖ has played havoc with human and other life forms.

1.1. Production and Usage of Pesticide in India The production of pesticides started in India in 1952 with the establishment of a plant for the production of BHC near Calcutta, and India is now the second largest manufacturer of pesticides in Asia after China and ranks twelfth globally[9]. There has been a steady growth in the production of technical grade pesticides in India, from 5,000 metric tonnes in 1958 to 102,240 metric tonnes in 1998. In 1996-97 the demand for pesticides in terms of value was *

Correspondence to: [email protected]

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estimated to be around Rs. 22 billion (USD 0.5 billion), which is about 2% of the total world market. The pattern of pesticide usage in India is different from that for the world in general. As can be seen from Figure 1, in India 76% of the pesticide used is insecticide, as against 44% globally.[9] The use of herbicides and fungicides is correspondingly less heavy. The main use of pesticides in India is for cotton crops (45%), followed by paddy and wheat.

Figure 1. Consumption pattern of pesticides

2. BENEFITS OF PESTICIDES 2.1. Improving Productivity Tremendous benefits have been derived from the use of pesticides in forestry, public health and the domestic sphere - and, of course, in agriculture, a sector upon which the Indian economy is largely dependent. Food grain production, which stood at a mere 50 million tonnes in 1948-49, had increased almost fourfold to 198 million tonnes by the end of 1996-97 from an estimated 169 million hectares of permanently cropped land. This result has been

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achieved by the use of high-yield varieties of seeds, advanced irrigation technologies and agricultural chemicals.[1] Similarly outputs and productivity have increased dramatically in most countries, for example, wheat yields in the United Kingdom, corn yields in the USA. Increases in productivity have been due to several factors including use of fertiliser, better varieties and use of machinery. Pesticides have been an integral part of the process by reducing losses from the weeds, diseases and insect pests that can markedly reduce the amount of harvestable produce. Warren (1998) also drew attention to the spectacular increases in crop yields in the United States in the twentieth century. Webster et al. (1999) stated that "considerable economic losses" would be suffered without pesticide use and quantified the significant increases in yield and economic margin that result from pesticide use. Besides this, most of the pesticides, in environment, undergo photochemical transformation to produce metabolites which are relatively non-toxic to the human beings as well as environment.[47]

2.2. Protect Crop Losses/Yield Reduction In medium land rice even under puddle conditions during the critical period warranted an effective and economic weed control practice to prevent a reduction in rice yield due to weeds that ranged from 28 to 48% based on comparisons that included control (weedy) plots[43].Weeds reduce yield of dry land crops[43] by 37-79%. Severe infestation of weeds particularly in early stage of crop establishment ultimately accounts for a yield reduction of 40%. Herbicides provided an economic and labour benefit too.

2.3. Vector Disease Control Vector-borne diseases are most effectively tackled by killing the vectors. Insecticides are often the only practical way to control the insects that spread deadly diseases such as malaria that results in an estimated 5000 deaths each day (Ross, 2005). In 2004, Bhatia wrote that malaria is one of the leading causes of morbidity and mortality in the developing world and a major public health problem in India.

2.4. Quality of Food In the countries of first world, it is now observed that a diet containing fresh fruit and vegetables far outweigh potential risks from eating very low residues of pesticides in crops [27]. Increasing evidence (Dietary Guidelines, 2005) shows that eating fruit and vegetables regularly reduces the risk of many cancers, high blood pressure, heart disease, diabetes, stroke, and other chronic diseases. Lewis et al (2005) discussed the nutritional properties of apples and blueberries in the US diet and concluded that their high concentrations of antioxidants act as protectants against cancer, heart disease. Lewis attributed doubling in wild blueberry production and subsequent increases in consumption chiefly to herbicide use that improved weed control.

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2.5. Other Area-Transport, Sport Complex, Building The transport sector makes extensive use of pesticides, particularly herbicides. Herbicides and insecticides are used to maintain the turf on sports pitches, cricket grounds and golf courses. Insecticides protect buildings and other wooden structures from damage by termites and wood boring insects.

3. HAZARDS OF PESTICIDES 3.1. Direct Impact on Human Being If the credits of pesticides include enhanced economic potential in terms of increased production of food and fibre, and amelioration of vector-borne diseases, then their debits have resulted in serious health implications to man and his environment. There is now overwhelming evidence that some of these chemicals do pose potential risk to humans and other life forms and unwanted side effects to the environment [17-19]. No segment of the population is completely protected against exposure to pesticides and the potentially serious health effects, though a disproportionate burden is shouldered by the people of developing countries and by high risk groups in each country.[20] The world-wide deaths and chronic illnesses due to pesticide poisoning number about 1 million per year[21]. The high risk groups exposed to pesticides include the production workers, formulators, sprayers, mixers, loaders and agricultural farm workers. During manufacture and formulation, the possibility of hazards may be more because the processes involved are not risk free. In industrial settings, the workers are at increased risk since they handle various toxic chemicals including pesticides, raw materials, toxic solvents and inert carriers. In India, the first report of poisoning due to pesticides was from Kerala in 1958, where over 100 people died after consuming wheat flour contaminated with parathion.[2] This prompted the Special Committee on Harmful Effects of Pesticides constituted by the ICAR to focus attention on the problem[3]. Further, Carlson in 1962 warned that OC compounds could pollute the tissues of virtually every life form on the earth, the air, the lakes and the oceans, the fishes that live in them and the birds that feed on the fishes.[4] Later, the US National Academy of Sciences stated that the DDT metabolite, DDE causes eggshell thinning and that the bald eagle population in the United States declined primarily because of exposure to DDT and its metabolites[5]. Certain environmental chemicals including pesticides termed as endocrine disruptors are known to elicit their adverse effects by mimicking or antagonising natural hormones in the body and it has been postulated that their long-term, low-dose exposure are increasingly linked to human health effects such as immunosuppression, hormone disruption, diminished intelligence, reproductive abnormalities and cancer[6-8]. A study on workers (N=356) in four units manufacturing HCH revealed neurological symptoms (21%) which were related to the intensity of exposure[22]. The magnitude of the toxicity risk involved in the spraying of methomyl, a carbamate insecticide, in field conditions was assessed by the National Institute of Occupational Health (NIOH) [24]. Significant changes were noticed in the ECG and the levels of serum LDH and ChE activities in the spraymen indicating the cardiotoxic effects of methomyl.

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Observations confined to health surveillance in male formulators engaged in production of dust and liquid formulations of various pesticides (malathion, methyl parathion, DDT and lindane) in industrial settings of the unorganised sector revealed a high occurrence of generalized symptoms (headache, nausea, vomiting, fatigue, irritation of skin and eyes) besides psychological, neurological, cardiorespiratory and gastrointestinal symptoms coupled with low plasma cholinesterase (ChE) activity [23]. Data on reproductive toxicity were collected from 1,106 couples when the males were associated with the spraying of pesticides (OC, OP and carbamates) in cotton fields.[25] A study in malaria spraymen was initiated to evaluate the effects of a short term (16 week) exposure in workers (N=216) spraying HCH in field conditions.[26]

3.2. Impact through Food Commodities The UK Pesticide Residue Committee annual report (2002) showed that over 70% of the food in the UK contained no pesticide residues at all and only 1.09% contained residues above the statutory maximum residue levels (MRLs). It concluded that ―none of these residues caused concern for people's health‖. Yet these very small quantities of chemicals in our food, detected at ever lower levels due to increasingly sensitive laboratory equipment, are now easy targets for the media. In India, a study revealed that 50% of the vegetable samples taken from farm gate were found contaminated with various pesticides (0.01-2.23 ppm) of which 16% were above MRL.[48]

3.3. Impact on Environment Pesticides can contaminate soil, water, turf, and other vegetation. In addition to killing insects or weeds, pesticides can be toxic to a host of other organisms including birds, fish, beneficial insects, and non-target plants. Insecticides are generally the most acutely toxic class of pesticides, but herbicides can also pose risks to non-target organisms.

3.3.1. Surface Water Contamination Pesticides can reach surface water through runoff from treated plants and soil. Contamination of water by pesticides is widespread. The results of a comprehensive set of studies done by the U.S. Geological Survey (USGS) on major river basins across the country in the early to mid- 90s yielded startling results. More than 90 percent of water and fish samples from all streams contained one, or more often, several pesticides.45 Pesticides were found in all samples from major rivers with mixed agricultural and urban land use influences, and 99 percent of samples of urban streams.[28] The USGS also found that concentrations of insecticides in urban streams commonly exceeded guidelines for protection of aquatic life 41. Twenty-three pesticides were detected in waterways in the Puget Sound Basin, including 17 herbicides. According to USGS, more pesticides were detected in urban streams than in agricultural streams. [29]

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3.3.2. Ground Water Contamination Pesticides, including herbicides, can and do leach to contaminate ground water. According to the USGS, at least 143 different pesticides and 21 transformation products have been found in the ground water, including pesticides from every major chemical class. Over the past two decades, detections have been found in the ground water of more than 43 states[30]. Contamination of ground water is of concern because ground water supplies 50 percent of the U.S. population with Drinkingwater.[31] During one survey in India it has been found that 58% of drinking water samples drawn from various hand pumps and wells around Bhopal are contaminated with Organ Chlorine pesticides above the EPA standards.[46] Once ground water is polluted with toxic chemicals, it may take many years for the contamination to dissipate or be cleaned up. Cleanup may also be very costly and complex, if not impossible [32-34]. 3.3.3. Soil Contamination Pesticides have various characteristics that determine how they act once in soil. Mobility refers to how much a pesticide will move around in the soil. The half life of a pesticide refers to the length of time it takes for half of the pesticide to degrade. Persistence refers to the length of time until all measurable residues of a pesticide are gone. 3.3.4, Effect on Soil Fertility (Beneficial Soil Microorganisms) One spoonful of healthy soil has millions of tiny organisms including fungi, bacteria, and a host of others. These microorganisms play a key role in helping plants utilize soil nutrients needed to grow and thrive. Microorganisms also help soil store water and nutrients, regulate water flow, and filter pollutants.[38] The heavy treatment of soil with pesticides can cause populations of beneficial soil microorganisms to decline. Sometimes pesticides have a negative impact on the available NPK from soil.[49] According to soil scientist Dr. Elaine Ingham, ―If we lose both bacteria and fungi, then the soil degrades. Overuse of chemical fertilizers and pesticides have effects on the soil organisms that are similar to human overuse of antibiotics. Indiscriminate use of chemicals might work for a few years, but after awhile, there aren‘t enough beneficial soil organisms to hold onto the nutrients.‖ [40]. 3.3.5. Contamination of Air, Soil, and Non-Target Vegetation Pesticide sprays can directly hit non-target vegetation, or can drift or volatilize from the treated area and contaminate air, soil, and non-target plants. Some pesticide drift occurs during every application, even from ground equipment.[35] Drift can account for a loss of 2 to 25% of the chemical being applied, which can spread over a distance of a few yards to several hundred miles. There are thousands of reported complaints of off target spray drift each year in the U.S. [36]. Many pesticides can volatilize (that is, they can evaporate from soil and foliage, move away from the application, and contaminate the environment.)[38]. As much as 80-90 percent of an applied pesticide can be volatilized within a few days of application[39]. Despite the fact that only limited research has been done on the topic, studies consistently find pesticide residues in air. According to the USGS, pesticides have been detected in the atmosphere in all areas of the USA sampled.[40] Nearly every pesticide investigated has been detected in rain, air, fog, or snow across the nation at different times of

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the year.[41] Many pesticides have been detected in air at more than half the sites sampled nationwide.

3.3.6, Non-Target Organisms: Pesticides are found as common contaminants in soil, air, water and on non-target organisms in our urban landscapes. Once there, they can harm plants and animals ranging from beneficial soil microorganisms and insects, non-target plants, fish, birds, and other wildlife.[37]

CONCLUSION Pesticides are often considered a quick, easy, and inexpensive solution for controlling weeds and insect pests in urban landscapes. However, pesticide use comes at a significant cost. Pesticides have contaminated almost every part of our environment. Pesticide residues are found in soil and air, and in surface and ground water across the nation, and urban pesticide uses contribute to the problem. Pesticide contamination poses significant risks to the environment and non-target organisms ranging from beneficial soil microorganisms, to insects, plants, fish, and birds. Contrary to common misconceptions, even herbicides can cause harm to the environment. In fact, weed killers can be especially problematic because they are used in relatively large volumes. The best way to reduce pesticide contamination (and the harm it causes) in our environment is for all of us to do our part to use safer, nonchemical pest control (including weed control) methods.

REFERENCES [1] [2] [3]

[4] [5] [6]

[7]

Employment Information: Indian Labour Statistics 1994. Chandigarh: Labour Bureau, Ministry of Labour, 1996. Karunakaran, (1958), C.O. The Kerala food poisoning. J Indian Med Assoc, 31: 204. Eds. A.M. Wadhwani and I.J. Lall. (1972) Harmful Effects of Pesticides. Report of the Special Committee of ICAR, Indian Council of Agricultural Research, New Delhi, p. 44. Carlson, R. (1962) Silent Spring. Houghton-Mifflin Co, Boston. Liroff, R.A. (2000) Balancing risks of DDT and malaria in the global POPs treaty. Pestic Safety News 4: 3. Crisp, T.M., Clegg, E.D., Cooper, R.L., Wood, W.P., Anderson, D.G., Baeteke, K.P., Hoffmann, J.L., Morrow, M.S., Rodier, D.J., Schaeffer, J.E., Touart, L.W., Zeeman, M.G. and Patel, Y.M. (1998) Environmental endocrine disruption: An effects assessment and analysis. Environ Health Perspect, 106: 11. Hurley, P.M., Hill, R.N. and Whiting, R.J. (1998) Mode of carcinogenic action of pesticides inducing thyroid follicular cell tumours in rodents. Environ Health Perspect 106: 437.

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Wasim Aktar Brouwer, A., Longnecker, M.P., Birnbaum, L.S., Cogliano, J., Kostyniak, P., Moore, J., Schantz, S. and Winneke, G. (1999) Characterization of potential endocrine related health effects at lowdose levels of exposure to PCBs. Environ Health Perspect 107:639. Mathur, S.C. (1999) Future of Indian pesticides industry in next millennium. Pesticide Information;XXIV(4):9-23. Warren, G.F. (1998) Spectacular Increases in Crop Yields in the United States in the Twentieth Century, Weed Technology, Vol. 12, P.752. Webster, J.P.G., R.G. Bowles, and N.T. Williams (1999) Estimating the Economic Benefits of Alternative Pesticide Usage Scenarios: Wheat Production in the United Kingdom, Crop Production, Vol. 18, P.83. MAF (Ministry of Agriculture and Forestry) New Zealand. Motivation for Growing Organic Products (available at http://www.maf.govt.nz/mafnet/rural-nz/sustainableresourceuse/organic-production/organic-farming-in-nz/org30004.htm). Oerke, E.C. and Dehne, H.W. (2004) Safeguarding Production - Losses in Major Crops and the Role of Crop Protection, Crop Protection, Vol. 23, P.275. Ross, G., (2005) Risks and benefits of DDT, The Lancet, Vol. 366, No.9499, P.1771November. Lewis, Nancy M., Jamie Ruud, (2005) Blueberries in the American Diet, Nutrition Today, Vol. 40, No.2, P.92March-April. Dietary guidelines for Americans (2005). U.S. Department of Health and Human Services U.S. Department of Agriculture. Forget, G. (1993) Balancing the need for pesticides with the risk to human health. In: Impact of Pesticide Use on Health in Developing Countries. Eds. G. Forget, T. Goodman and A. de Villiers, IDRC, Ottawa, p. 2. Igbedioh, S.O. (1991) Effects of agricultural pesticides on humans, animals and higher plants in developing countries. Arch Environ Health 46: 218. Jeyaratnam, J. (1985) Health problems of pesticide usage in the third world. BMJ 42: 505. WHO. Public Health Impact of Pesticides Used in Agriculture. World Health Organization, Geneva, p. 88, (1990). Environews Forum. Killer environment. Environ Health Perspect 107: A62, (1999). Nigam, S.K., Karnik, A.B., Chattopadhyay, P., Lakkad, B.C., Venkaiah, K. and Kashyap, S.K. (1993) Clinical and biochemical investigations to evolve early diagnosis in workers involved in the manufacture of hexachlorocyclohexane. Int Arch Occup Environ Health 65: S193. Gupta, S.K., Jani, J.P., Saiyed, H.N. and Kashyap, S.K. (1984) Health hazards in pesticide formulators exposed to a combination of pesticides. Indian J Med Res, 79: 666. Saiyed, H.N., Sadhu, H.G., Bhatnagar, V.K., Dewan, A, Venkaiah, K. and Kashyap, S.K. (1992) Cardiac toxicity following short term exposure to methomyl in spraymen and rabbits. Hum Exp Toxicol, 11: 93. Rupa, D.S., Reddy, P.P. and Reddy, O.S. (1991) Reproductive performance in population exposed to pesticides in cotton fields in India. Environ Res 55: 123. Gupta, S.K., Parikh. J.R., Shah, M.P., Chatterjee, S.K. and Kashyap, S.K. (1982) Changes in serum exachlorocyclohexane (HCH) residues in malaria spraymen after short term occupational exposure. Arch Environ Health 37: 41.

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[27] Brown, Ian UK Pesticides Residue Committee Report (2004) (available online http://www.pesticides.gov.uk/uploadedfiles/Web_Assets/PRC/PRCannualreport2004 .pdf also available on request). [28] Bortleson, G. and D. Davis. (1987-1995). U.S. Geological Survey & Washington State Department of Ecology. Pesticides in selected small streams in the Puget Sound Basin. pg. 1-4. [29] US Department of the Interior. (1995). Pesticides in ground water: current understanding of distribution and major influences. U.S. Geological Survey. National Water Quality Assessment. Factsheet number FS-244-95. [30] Waskom, R. (1994). Best management practices for private well protection. Colorado State Univ. Cooperative Extension (August). http://hermes.ecn.purdue.edu:8001/cgi/. [31] O‘Neil, W. and Raucher, R. (1998). Groundwater public policy leaflet series #4: The costs of groundwater contamination. Wayzata, MN:Groundwater Policy Education Project. http:// www.dnr.state.wi.us/org/water/dwg/gw/costofgw.htm (Aug). [32] US EPA. (2001). Managing small-scale application of pesticides to prevent contamination of drinking water. Water protection practices bulletin, Washington, DC: Office of Water (July). EPA 816-F-01-031. [33] Johnson, J. and Ware, W.G. (1991). Pesticide litigation manual 1992 edition. Clark Boardman Callaghan Environmental Law Series, New York, NY. 65. US EPA. 1999. Spray drift of pesticides. Washington, DC: Office of Pesticide Programs (December). http:// www.epa.gov/pesticides/citizens/spraydrift.htm#1. [34] US EPA. (1999). Spray drift of pesticides. Washington, DC:Office of Pesticide Programs (December). http://www.epa.gov/pesticides/citizens/spraydrift.htm#1. [35] Glotfelty and Schomburg. (1989). Volatilization of pesticides from soil in Reactions and Movements of organic chemicals in soil. Eds. BL Sawhney and K. Brown. Madison, WI: Soil Science Society of America Special Pub. [36] Que, S. et al. (1975). Factors effecting the volatility of DDT, dieldrin, and dimethylamine salt of (2,4-dichlorophenoxy) acetic acid (2,4-D) from leaf and glass surfaces. Bull. Environ. Contam. Toxicol. 13(3):284-290. [37] USGS. (1995). Pesticides in the atmosphere: current understanding of distribution and major influences. Fact Sheet FS- 152-95. http://water.wr.usgs.gov/pnsp/atmos/ [38] Marx, J et al. (1999). The relationship between soil and water, how soil amendments and compost can aid in salmon recovery. Soils for Salmon 1-18. [39] Majewski, M. and P. Capel. (1995). Pesticides in the atmosphere: distribution, trends, and governing factors. Volume one, Pesticides in the Hydrologic System. Ann Arbor Press Inc. pg. 118. [40] Savonen, C. (1997). Soil microorganisms object of new OSU service. Good Fruit Grower. http://www.goodfruit.com/archive/ 1995/6other.html. [41] U.S. Geological Survey. (1999). The quality of our nation‘s waters – nutrients and pesticides. Circular 1225. Reston VA: USGS. http://water.usgs.gov/pubs/circ/circ1225/ [42] Bhatia, Mrigesh R., Fox-Rushby, J and Mills, M. (2004) Cost-effectiveness of malaria control interventions when malaria mortality is low: insecticide-treated nets versus inhouse residual spraying in India. Soil Science and Medicine, Vol. 59, p-525. [43] Behera, Basudev, Gauri Shankar Singh. (1999) Studies on Weed Management in Monsoon Season Crop of Tomato. Indian Journal of Weed Science, Vol. 31, No.1+2, p67.

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[44] Porwal, M.K. (2002) Relative Economics of Weed Management Systems in Winter Sweet Potato (Ipomoea batatus L.) in Command Area of Southern Rajasthan. Indian Journal of Weed Science, Vol. 34, No.1+2, P.88. [45] Kole R.K., Banerjee H. and Bhattacharyya A. (2001) Monitoring of market fish samples for Endosulfan and Hexachlorocyclohexane residues in and around Calcutta. Bull. Envir. Contam. Toxicol 67: 554-559. [46] Kole R.K. and Bagchi M.M. (1995) Pesticide residues in the aquatic environment and their possible ecological hazards. J. Inland Fish. Soc. India. 27(2): 79-89. [47] Kole R.K., Banerjee H., Bhattacharyya A., Chowdhury A. and AdityaChaudhury N. (1999) Photo transformation of some pesticides. J. Indian Chem. Soc. 76:595-600. [48] Kole R.K., Banerjee H. and Bhattacharyya A. (2002) Monitoring of pesticide residues in farm gate vegetable samples in west Bengal. Pest. Res. J. 14(1): 77-82. [49] Sardar D. and Kole R.K. (2005) Metabolism of Chlorpyriphos in relation to its effect on the availability of some plant nutrients in soil. Chemosphere 61: 1273-1280.

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 16

OZONE DECOMPOSITION BY CATALYSTS AND ITS APPLICATION IN WATER TREATMENT: AN OVERVIEW J. Rivera-Utrilla, M. Sánchez-Polo, J. D. Méndez-Díaz Inorganic Chemistry Department, Faculty of Science, University of Granada, Granada, Spain

ABSTRACT Ozone has recently received much attention in water treatment technology for its high oxidation and disinfection potential. The use of ozone brings several benefits but has a few disadvantages that limit its application in water treatment, including: i) low solubility and stability in water, ii) low reactivity with some organic compounds and iii) failure to produce a complete transformation of organic compounds into CO2, generating degradation by-products that sometimes have higher toxicity than the raw micropollutant. To improve the effectiveness of ozonation process efficiency, advanced oxidation processes (AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on ozone decomposition into hydroxyl radicals (HO·), which are high powerful oxidants. This chapter offers an overview of AOPs, focusing on the role of solid catalysts in enhancing ozone transformation into HO· radicals. Catalytic ozonation is a new way to remove organic micropollutants from drinking water and wastewater. The application of several homo- and heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation has largely been limited to laboratory applications, the good results obtained have led to investigations now under way by researchers worldwide. It is therefore timely to provide a summary of achievements to date in the use of solid materials to enhance ozone transformation into HO· radicals.

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1. INTRODUCTION Demographic development and an exponential increase in industrial activity have largely been responsible for the large rise in the demand for water for domestic, public, and industrial use, and for the high volume of effluents discharged into waters. Legislation has been introduced to reduce permissible contamination levels. However, conventional treatment systems cannot completely remove a large amount of organic and inorganic contaminants present in waters, because most cannot be metabolized by microorganisms as carbon source and can even inhibit the activity of these microorganisms, leading to their bioaccumulation in the food chain. Hence, there is an increasing demand for more effective treatments to reduce the potential environmental impact of effluents and to comply with increasingly strict legislations. Successful water treatment requires the use of more sophisticated methods, including: 





  



Biological systems for nitrogen removal: Ammonium can be transformed into nitrate by using nitrifier microorganisms in aerobic medium. Nitrate can be removed in a subsequent stage under anaerobic conditions, when it is transformed by denitrifying bacteria into molecular nitrogen. Advanced oxidation processes for removal of toxic organic compounds, chromophore compounds, and other non-biodegradable organic compounds. These are based on the use of highly oxidizing agents, e.g., ozone or hydrogen peroxide. A greater effectiveness is observed when these oxidizing agents are used in the presence of UV radiation. Ionic exchange for ion removal. This is highly effective for removing cations and anions from the aqueous phase but transfers the problem to the solid phase by concentrating the pollutant in the adsorbent medium. Adsorption on activated carbon for removal of metals, organic compounds, etc. This has the same drawback as described above for ionic exchange. Chemical precipitation for phosphorus removal: Chemical agents (Al2(SO4)3, Fe2(SO4)3 or FeCl3) are used to precipitate phosphorus. Distillation for removal of volatile organic compounds. This is only appropriate when there are high concentrations of the contaminant and its recovery brings economic benefits. Liquid-liquid extraction. This is also only useful under the above conditions.

Chemical oxidation processes currently play a very important role in water treatment. Table 1 lists the most common chemical oxidants used in water treatments and their corresponding reduction potentials [1]. Oxidants can be used to remove both inorganic pollutants and toxic organic compounds (pesticides, hydrocarbons, toxins, etc.) [2,3]addition, oxidants are widely used to degrade compounds responsible for odor, color or taste [4,5,6] Ozone, due to its high oxidation and disinfection potential, has recently received much attention in water treatment technology. Despite several advantages of using ozone, it has a few disadvantages that limit its application in water treatment, including: i) low solubility and stability in water, ii) low reactivity with some organic compounds, and iii) failure to produce a complete transformation of organic compounds into CO2, generating degradation by-

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products that sometimes have a higher toxicity than the raw micropollutant. To improve the effectiveness of ozonation, advanced oxidation processes (AOPs) have recently been developed (O3/H2O2, O3/UV, O3/catalysts). AOPs are based on the decomposition of ozone into hydroxyl radicals (HO·), a very powerful oxidant. This chapter offers an overview of the role of different solid catalysts in enhancing ozone transformation into HO· radicals. Catalytic ozonation is a new way to remove organic micropollutants from drinking water and wastewater. The application of several homo- and heterogeneous ozonation catalysts is reviewed, describing their activity and identifying the parameters that influence the effectiveness of catalytic systems. Although catalytic ozonation has largely been limited to laboratory applications, the good results obtained have led to investigations now under way by researchers worldwide. It is therefore timely to provide a summary of achievements to date in the use of solid materials to enhance ozone transformation into HO· radicals. Table 1. Reduction potentials of different oxidants used in water treatments. Oxidant Ozone

Reduction semireaction O3 (aq) + 2H+ + 2e-  O2 (aq) + H2O

E0 (V) 2.08

Permanganate

2MnO4- + 8H+ + 6e-  2MnO2 (s) + 4H2O

1.68

-

-

Chlorine dioxide

ClO2 + e  ClO2

Hypochlorous acid

HOCl + H+ + 2e-  Cl- + H2O -

+

-

0.95 1.48

-

Hypochlorite ion Dichloramine

ClO + 2H + 2e  Cl + H2O NHCl2 + 3 H+ + 4e-  2Cl- + NH4+

1.64 1.34

Oxygen Hydroxyl radical

O2 (aq) + 4H+ + 4e-  2H2O HO· + H+ + e-  H2O

1.23 2.85

Hydrogen peroxide

H2O2 + 2H+ + 2e-  2H2O

1.78

2. OZONE Ozone, O3, discovered by Schönbein in 1840 [7], is an allotrope of oxygen consisting of three atoms. It is a diamagnetic compound that is an unstable gas at room temperature with a characteristic sharp odor. Experimental results show a bond angle of 116.8 ± 0.5º and an interatomic distance of 127.8 pm between central oxygen and each terminal [8]. Fig 1 depicts the two resonant forms of the ozone molecule according to the valence bond theory:

+ ·· O

:O :

··

·· O+ :O :

Figure 1. Chemical structure of ozone.

:O :

O : :

··

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Due to its configuration, ozone is a highly oxidant compound (Eº = +2.08 V), and its natural tendency is to transfer an oxygen atom and release O2. Applications of ozone applications in water treatment can be grouped into three categories: i) as disinfectant or biocide, ii) as oxidant for the removal of organic pollutants and, iii) as pre- or post-treatment in another procedure (coagulation, flocculation, sedimentation, biological oxidation, adsorption on activated carbon, etc.). Ozone began to be used as bactericide agent to treat drinking waters in Nice (France) at the beginning of the 20th century. As a result of the large amount of resources invested in the study of ozone, other advantages of its use in treatment plants [9] are now widely known, including: i) removal of compounds that produce odor, taste or color in water, ii) oxidation of inorganic chemical compounds, e.g., iron and manganese, iii) removal of algae and other aquatic microorganisms, iv) oxidation of organic micropollutants, v) absence of the increase in the presence of organochlorinated compounds found when chlorine is used for the treatment, and vi) enhanced performance of adsorption and coagulation processes. Nevertheless, its large-scale application to treat industrial liquid waste did not become widespread due to the high economic costs involved and the chemical complexity of industrial effluents. However, ozone has now become an attractive option for the treatment of these effluents because conventional systems are inadequate to reduce the toxic organic compounds they contain to levels required by new environmental legislation. O3 + OH-

O2-· + HO2·

Start 2HO2·

O3 + H2O HO2·

H+ + O2-·

O2-· + O3

O2 + O3-·

(2) (3) (4)

HO· + O2

(5)

O3-·

O-· + O2

(6)

HO·

O-· + H+

(7)

O3-· + H+

Propagation

HO· + O3

End

(1)

HO2· + O2

(8) (9)

HO2· + HO2·

H2O2 + O2

HO2· + O2-·

HO2- + O2

(10)

2HO- + 2O2

(11)

O3-· + O2-· + H2O

Figure 2. Mechanism of ozone decomposition in aqueous medium.

Over the past two decades, there has been a notable increase in research into the reaction between ozone and numerous organic and inorganic compounds, especially aromatic compounds [10,11,12,13,14]. Because it is highly reactive, ozone can interact with a large number of organic and inorganic substances by direct oxidation/reduction reaction, cycloaddition-substitution, or nucleophilic addition [15,16,17]. The direct reaction between

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ozone and a given compound is not the only pathway by which ozone can act to degrade pollutants, since ozone is very unstable in aqueous solution and spontaneously decomposes by a complex chain mechanism (Fig 2) in which different radical species participate [18]. The radicals generated are of great interest for water treatment because some of them, e.g., the hydroxyl radical HO·, are even more reactive than ozone and play an essential role in removing pollutants in solution [19]. These systems will be described in greater depth in next section, which is devoted to AOPs. Despite its high efficacy in some systems, ozone has inadequate capacity to degrade surfactants and tensioactive substances, among other pollutants. Narkis et al. [20] observed that ozone treatment favored the biodegradation of non-ionic surfactants but was unable to remove them. Other authors also reported the lack of reactivity of saturated cationic surfactants [21]. Regarding anionic surfactants, although some researchers achieved a high degradation of alkylbenzene-sulphonates with ozone [22], the reaction rate constant values were subsequently determined to be low [23], suggesting that an indirect mechanism was largely responsible for the degradation.

3. ADVANCED OXIDATION PROCESSES BASED ON OZONE Polluted waters can generally be effectively treated by biological, adsorbent or conventional chemical approaches (chlorination, ozonation or oxidation with permanganate). However, as stated earlier, these methods are sometimes inadequate to degrade pollutants to levels required by law or necessary for subsequent utilization of the effluent. AOPs have proven highly effective in the oxidation of numerous organic and inorganic compounds and are based on the generation of free radicals, notably hydroxyl radical HO·. These free radicals are highly reactive species that can successfully attack most organic and inorganic compounds with very high reaction rate constants (106-109 M-1s-1). The numerous systems that can be produced by these radicals (Table 2) account for the high versatility of AOPs. Advanced oxidation processes based on the use of ozone are briefly described below, highlighting catalytic ozonation (also see section 4). Table 2. Water treatment technologies based on advanced oxidation processes.



Non-photochemical processes Oxidation in sub/supercritical water





Fenton‘s reagent (Fe2+/H2O2)



Photochemical processes Photolysis of water with vacuum UV (VUV) UV/hydrogen peroxide



Electrochemical oxidation



UV/ozone

    

Radiolysis Non-thermal plasma Ultrasound Ozonation in alkaline medium (O3/OH-) Ozonation in the presence of hydrogen peroxide (O3/H2O2) Catalytic ozonation

 

Photo-Fenton Heterogeneous photocatalysis



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3.1. Ozonation in Alkaline Medium Based on studies by Hoigné et al.[10,11,12], Aieta et al. [24] published an illustrative diagram in 1988 describing ozone in aqueous solution. Figure 3 summarizes the two reaction pathways of molecular ozone: i) direct reaction of the substrate with molecular ozone, which is selective but slow or null with some species, and ii) decomposition and generation of HO· hydroxyl radicals, which attack rapidly but not selectively. Direct oxidation of the substrate

O3

Products

Slow / Selective

OHRadical formation

HO·

Radical oxidation Fast / Non-selective

Products

Figure 3. Reaction pathways of ozone.

Moreover, hydroxyl radicals react rapidly with molecular ozone, contributing towards autocatalytic decomposition of the ozone. Several researchers have studied the mechanism and reaction kinetics involved in ozone decomposition in aqueous phase [18,25,26,27,28,29,30,31,32,33]. Ozone stability largely depends on the aqueous matrix, especially the pH, type of organic matter present, and alkalinity [34]. The water pH is especially important, because hydroxyl ions considerably increase the ozone decomposition rate [18]. Thus, according to equations 12 and 13, ozone decomposition spontaneously accelerates with an increase in the solution pH, leading to an AOP. However, it must be taken into account that a high pH increase can have a negative effect on the degradation, depending on the composition of the water, because of the inhibiting action of HO· radical scavengers, e.g., bicarbonate or carbonate ions [35]. O3 + OH-  HO2- + O2 k = 70 M-1s-1 (13) O3 + HO2-  HO· + O2·- + O2 k = 2.8 · 106 M-1 s-1

(12)

3.2. Ozonation in the Presence of Hydrogen Peroxide The addition of hydrogen peroxide favors the ozonation of organic compounds in the medium. The combination of ozone and hydrogen peroxide is largely used to oxidize pollutants that require high ozone consumption. Hydrogen peroxide is a weak acid (pKa = 11.6), a powerful oxidant (See Table 1), and an unstable compound that easily dismutes (equations 14-16). H2O2  HO2- + H+ (14) H2O2 + 2e- +2H+  2 H2O

(15)

Ozone Decomposition by Catalysts and its Application in Water Treatment: … H2O2 + HO2-  H2O +O2 +HO-

439

(16)

The mechanism by which H2O2 favors free radical generation was described by Forni et al. [25], who demonstrated that the conjugated base of hydrogen peroxide initiates ozone decomposition in aqueous phase via an electronic transference reaction. HO2- + O3  HO2 + O317) Taking advantage of the capacity of H2O2 to initiate ozone decomposition in aqueous phase (Figure 4), numerous researchers have used this process for a faster and more effective oxidation of organic matter [36,37,38,39,40,41,42,43. The presence of H2O2 in the system favors oxidation, although the possibility that added H2O2 is consumed in reactions with other contaminants has hampered application of this method to treat industrial effluents. Fernández et al. [44] compared the efficacy of the O3/H2O2 system and photolysis to remove linear alkylbenzene-sulphonates (LAS), observing the complete degradation of the mixture of surfactants after 20 min of ozonation. HO2- + H+

H2O2

O3 O3O2- *

HO2*

HR+ HO3 RHO2* Degradación HO* HR* O2

HRH

Figure 4. Mechanism of oxidation of an organic compound (HRH) by means of O3/H2O2.

3.3 Ozonation in the Presence of UV Radiation Ozonation coupled with UV radiation is one of the most effective chemical oxidation techniques to treat polluted waters. This process is capable of oxidizing organic substances at room temperature and generates products that are innocuous to the environment. As in the O3/H2O2 system, the UV radiation of O3 generates hydroxyl radicals in solution [45]. The reactions involved in this process are: O3 + h (<310 nm) O2 + O (18) O + H2O 2HO· (19)

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Prengle et al. [46] of ―Houston Research Inc.‖ were the first researchers to describe the application of an O3/UV-based system for water treatment. Glaze et al. [47,48] subsequently analyzed the effectiveness of the O3/UV system to remove microcontaminants and determined the reactions involved in the mechanisms of this process [49]. They demonstrated that compounds not degraded by UV light behave very similarly in response to O3/UV and O3/H2O2 systems [50]. The O3/UV process has been mainly applied in the oxidation of aromatic derivatives [51,52] but it has also shown high efficacy to remove surfactants [53,54,55], pesticides [56], and other organic compounds [57]. Taking advantage of the high photocatalytic capacity of TiO2, a new treatment process has been developed based on the simultaneous use of O3/UV/TiO2. Table 3 displays information on the type of pollutant, experimental conditions and the main conclusions of publications on the application of the O3/UV system to remove organic pollutants from waters; in the majority of these studies, TiO2 was used as catalyst. Table 3. Application of systems based on O3, UV radiation, and catalysts for removing pollutants from waters. System

Catalyst

O3, H2O2, UV, 3/UV, O3/H2O2, UV/H2O2, O3/UV/H2O2

Pollutants

Experimental conditions Observations

Semi-continuous; pH 8; 17ºC; flow 1-1.5 L/min; Phenolic compounds low-pressure lamps (254nm)

Ref.

Treatments without ozone are less effective in pollutant 58 removal

UV, V/TiO2, V/Fenton

Different TiO2 (Degussa P25) wastewaters

Better performance of Semi-continuous; 25ºC; catalyzed experiments; Fenton medium-pressure Hg lamp 59 process more cost-effective 400 W; 2 g/L of catalyst; than UV

O2/UV/cat

Ag/ZnO

Semi-continuous; pH 3-7; Total removal of toxicity of medium-pressure Hg lamp certain effluents, but little 125 W; flow 10 mL/min reduction in TOC

O2/UV/cat

TiO2 (Degussa P25), Colorants ZnO

Semi-continuous; pH 5; 20ºC; medium-pressure Hg lamp 125 W; gas flow10 mL/min; 0.8 g/L catalyst

UV/TiO2

TiO2

Formic acid

Discontinuous with Better performance in ecirculation; pH 3.8; 20ºC; combined use; determination 62 Lamp 6W of reaction constants

UV/H2O2,

2-methyl benzimidazole carbamate

Discontinuous; lowpressure Hg lamp

O3/UV

Oxalic acid

Semi-continuous; pH 7; 1.5 Kinetic model proposed; study L/min; UV lamps 3-9·10-6 of effect of operational 64 E/L-s variables

O2/UV/TiO2

O3, H2O2, UV, O3/UV, O3/H2O2, UV/H2O2,

Textile effluent

TiO2 (Degussa P25) Tetracyclines

Diazinon

Semi-continuous; flow 0.2L/min; 3 lamps of different power; 0.5-1 g/L catalyst

60

High mineralization; better catalytic activity of ZnO; 61 synergic effect between TiO2 and ZnO

Determination of radical reaction rate constants (hydroxyl and carbonate); identification of degradation products

63

Degradation is not observed in the absence of TiO2; 65 significant mineralization with use of catalyst

Kinetic study and comparison Discontinuous; pH 2-9; 20- between systems; 40ºC; low-pressure Hg determination of quantum 66 lamp 15W performances and reaction constants

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441

Table 3. (Continued) System

UV, O3/UV, O3/TiO2, O3/UV/TiO2

Catalyst

Pollutants

Experimental conditions Observations

TiO2

Acetic acid, monochloroacetic acid, phenol, dimethyl-2,2,2trichlorine-1hydroxyethylphosphate

Semi-continuous; lowpressure Hg lamp 6 W

O3, UV/TiO2, TiO2 O3/UV/TiO2

Ref.

Proposal of the reaction mechanism; detection of formic, acetic, glyoxylic, and 67 glycolic acids as reaction intermediates

Study of kinetics and Semi-continuous; pH 3-11; operational variables; O3/UV 2,4-dichlorine 18-22ºC; flow 1-220 mg improves treatment efficiency; 68 phenoxyacetic acid O3/L; lamp 20W identification of reaction intermediates

O3/UV/TiO2

TiO2

Semi-continuous; pH 3; 25ºC; flow 5·10-4 mol O3/min; medium-pressure Hg lamp 125 W; 2 g/L of catalyst

Proposal of reaction mechanism; simultaneous use of O3/UV/TiO2 is more 69 effective versus O3 and UV/TiO2 in parallel

O3, O3/UV, UV/TiO2, O3/UV/TiO2

Alkylamines, Semi-continuous; pH 6.5; alkanolamines, 20ºC; flow 35 L/h; Xe TiO2 (Degussa P25) nitrogenated Lamp 450 W; 0-3 g/L of heterocyclic and catalyst aromatic compounds

Proposal of reaction mechanism; identification of intermediate reaction 70 products; study of effect of structure and concentrations on reaction rate

UV/TiO2, O3/UV, O3/UV/TiO2

Pyridine; TiO2 (Degussa P25) monochloroacetic acid

Semi-continuous; pH 3; 20ºC; flow 20 L/h; UV Lamp; 0.5g/L catalyst

Proposal of reaction mechanism; much higher 71 performance of system based on O3/UV/TiO2

3

, UV/TiO2, O3/UV/TiO2

TiO2 (Degussa P25) Textile effluent

Semi-continuous; pH 11; flow 6 L/h; high-pressure Hg lamp 125 W; 0.1 g/L catalyst

Determination of toxicity and TOC and color of effluent as a 72 function of treatment time

O3/UV/TiO2

TiO2

Aniline

Semi-continuous; pH 6; Higher efficiency of the 4flow 1 L/min; low-pressure combined system; detection of 73 chlorobenzaldehyde Hg lamp 10 W; 1 mg/mL degradation by-products of catalyst

UV/TiO2, 2-dimethyl-pyrazine, Semi-continuous; highO3/UV/TiO2, TiO2(Degussa P25) monochloroacetic pressure Hg lamp 125W UV/Fenton acid

O3/UV/TiO2

TiO2 (Degussa P25 Cyanides y BDH)

Proposal of disjunctive mechanism: UV promotes electrons in TiO2 or radical reaction generation

74

Kinetic study and mechanism; Semi-continuous; pH 11.3; formation of carbonate, 20ºC; medium-pressure Hg 75 cyanate, and nitrate; strong lamp 400 W O3/TiO2 interaction

O3, O3/H2O2, O3/H2O2/UV O3/UV/TiO2, TiO2 O3/H2O2/UV/T iO2

Wastewater

O3/H2O2/UV/TiO2 is the only Semi-continuous; pH 4.4; one able to appreciably reduce flow 500 L/h; Hg Lamp TOC; after treatment, 76 (200-280 nm); 2 g/L of anaerobic digestion improves catalyst CH4 production

UV/TiO2, O3, TiO2 O3/UV/TiO2

Formic acid

Semi-continuous; pH 2-12; Diffusion effects worsen 10-50ºC; gas flow 5·10-3 performance 3 m /min; 6W Lamp

O3/UV/TiO2

Cyanides

Mechanisms is proposed and Continuous; pH 10; gas products identified; effluent is flow 4 L/min; low-pressure 78 reutilized to obtain deionized lamp 40 W water

TiO2

77

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Table 3. (Continued) System O3/UV/TiO2

Catalyst

TiO2 (Degussa P25 , Phenol SG)

O2, O3, O3/UV, UV/TiO2, Carbon-TiO2 O3/UV/TiO

O3/UV/TiO2

Pollutants

Catechol

TiO2 (Degussa P25) Humic acid

O3, O3/UV, UV/TiO2, Acetic acid, formic TiO2 (Degussa P25) O2/UV/TiO2, acid, propionic acid O3/UV/TiO2 O3/Foto fenton, O3/UV/TiO2

Experimental conditions Observations

Ref.

Semi-ontinuous; 25ºC; Detectionof intermediates; flow 2.1 mg O3/L; Hg improvement of Lamp (220-380nm); 15 g/L mineralization with use of of catalyst combined system

79

Semi-continuous; lowpressure lamp 15 W

Determination of rate constant and reaction order; higher 80 efficacy of O3/UV/TiO2 system to remove TOC

Kinetic study; UV Semi-continuous; flow 4.8 compensates for low capacity 81,82, mg O3/min; 125 W Lamp; of O3 to remove organic 83 0.25-1 g/L of catalyst matter; adsorption study of pollutant on catalyst Semi-continuous; pH 2; 25ºC; flow 0.9 L/min; medium-pressure Hg lamp 6 W; 1 g/L catalyst

Identifies intermediates; better performance of combined 84 process versus ozone and photocatalysis separately

Semi-continuous; pH 3, 7; Alachlor, atrazine, Photo-fenton system (order 1) 25ºC; flow 1.6 g O3/h; TiO2 (Degussa P25) diuron, isoproturon, shows better results than 85 UVA Lamp 6W; 0.25 g/L pentachlorophenol O3/UV/TiO2 (order 0) of catalyst

O3/UV, O3/TiO2, O3/V- TiO2, V-O/TiO2 O/TiO2

Higher removal efficacy of Semi-continuous; pH 4, 7, O /V-O/TiO2 system; Sulphosalicylic acid 9; 20ºC; Lamp (254 nm) 14 3 86 maintains activity even in the W; 1.6 g/L of catalyst presence of carbonates

O3, O3/TiO2, Phenol, pO3/UV/TiO2, TiO2 (Degussa P25) chlorophenol, pO3/UV, UV, nitrophenol UV/TiO2 O3, UV/TiO2, TiO2 O3/UV/TiO2

Semi-continuous; pH 5-6; flow 51 L/h; high-pressure Hg lamp 700 W; 1.5 g/L catalyst

Semi-continuous; UV Water with fungicide Lamp 6W

Kinetic study and identification of intermediates; 87 determination of reaction constants Synergic effect; O3/UV/TiO2 system removes organic 88 compounds and inhibits germination of fungi

4. CATALYTIC OZONATION Research into novel alternatives to conventional advanced oxidation processes has significantly increased over the past few decades. These new treatments are based on the addition of reagents to the system, generally heavy metals that increase the effectiveness of ozone as oxidizing agent. These substances, called catalysts, participate in the reaction mechanism but are not consumed in the process. Depending on the state of the species that acts as catalyst, two types can be distinguished: homogeneous catalysis (catalyst dissolved in aqueous phase) and heterogeneous catalysis (solid or supported catalyst). Each of these catalytic processes is studied in depth below.

Ozone Decomposition by Catalysts and its Application in Water Treatment: …

443

4.1. Homogeneous Catalytic Ozonation One of the first investigations in this field was by Hewes and Davison [89], who observed that addition of certain salts during ozonation of phenolic compounds increased the mineralization of organic matter. Subsequently, other researchers [90,91] found that these catalysts could improve the decoloration of effluents from textile industries and accelerate the oxidation of compounds at acid pH values. Unlike for other AOPs, a general reaction mechanism cannot be established for homogeneous catalytic ozonation because the system can behave very differently according to the compounds involved. Whereas some researchers indicated that the presence of transition metals does not promote the generation of free radicals [92], explaining that the higher oxidation of the substrate is due to the formation of a complex that is highly reactive to ozone, others explained the enhancement of substrate degradation by the generation of hydroxyl radicals [93]. A review of the literature shows that metals (either in cation or anion form) that are susceptible to oxidization in the ozonation process can act as initiators/promoters of the transformation of ozone into HO· radicals. Despite the improvement in performance, the need to add metal species to the system increases the difficulties in applying homogeneous catalytic ozonation in liquid waste treatments, because it introduces highly contaminant species into the system. Table 4 summarizes data from the main published studies on the use of homogeneous catalytic ozonation to remove organic microcontaminants from waters. Table 4. Most relevant data from publications on homogeneous catalyzed ozonation. System

Catalyst

ContaminantsExperimental Conditions

Observations

Ref.

Ozone/H2O2

Fe2+, Co2+, Ni2+, Cu2+ Sulfates

*

Discontinuous; acid pH

Study of the catalytic effect of several cations; reaction mechanism is proposed

94

Ozone

Co2+

*

Discontinuous; acid pH; 0ºC

Study of mechanism and kinetics; 95 acid pH inhibits the process

Ozone

Co2+

Acetic acid

Discontinuous; acid pH; 0ºC

Study of mechanism and kinetics 96

Ozone

Co2+, Ti2+, Mn2+

Secondary effluent

Discontinuous and Improvement of DQO removal recirculation; pH 5-10, 30-60ºC

*

Discontinuous; acid pH; [O3] 3·10-4M

2+

Determination of reaction stoichiometry; mechanism proposal; influence of several radicals

89

Ozone

Fe

97

Ozone

Cu2+, Zn2+, Cr2O3

Azoic colorants

Semi-continuous; flow 100 L/h Improvement in discoloration rate 90

Ozone

Fe2+, Mn2+

*

Discontinuous, pH 5-8, 25ºC, 1mg/L O3

Determination of reaction stoichiometry and rate constant

98

Ozone

NOM

Mn2+

Discontinuous, 0.5-2 mg/L

Significant removal of Mn2+; influence of O3 dose

99

Ozone

Mn2+

Oxalic Acid

Discontinuous and semiKinetic study and determination of continuous (flow 36 L/h); acid rate constant; proposal of reaction 91 pH; 20ºC mechanism

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Table 4. (Continued) System

Catalyst

ContaminantsExperimental Conditions

Observations

Ozone

Fe2+

*

Kinetic study and determination of Reactor with flow stop; pH 0-2; rate constant at the beginning of 100 25ºC; 1·10-4-6·10-5 M O3 the process; mechanism is proposed

Ozone

NOM

Mn2+, DQO

Continuous; diffusion plate

Efficacy is tested in different waters

Ref.

101

-5

Ozone

Mn2+

*

Discontinuous; pH 2-8; 4-5·10 Reaction mechanism is proposed 102 M O3

Ozone

Mn2+

Pyrazine, oxalic acid

Semi-continuous; pH 4; flow 36 Oxalic- Mn complex as catalyst L/h

+

2+

103

3+

Ozone

Ag , Fe , Fe , Co2+, Cu2+, Mn2+, TOC Zn2+, Cd2+

Discontinuous; pH 7; flow 1.5g Intermediates are identified; TOC 104 O3/h reduction improves with catalyst

Ozone

Mn2+

Atrazine

Semi-continuous and recirculation; pH; 20ºC

Considerably increases atrazine removal with Mn2+

105

Ozone

Mn2+

TOC

Semi-continuous; pH 8; flow 1.6g/ O3/h

TOC removal increases with the catalyst; intermediates are identified

106

Ozone

Mn2+

Pyruvic acid

Semi-continuous; pH 2-4; 25ºC; Only removes pyruvic acid if flow 36 L/h catalyzed; negative effect of pH

107

Ozone

Mn2+

Atrazine, DQO

Semi-continuous and recirculation; pH 7; 20ºC; 1 mg/L Mn2+

108

Ozone

Fe2+, Fe3+, Mn2+

Chlorobenzene Semi-continuous; pH 7 s

Ozone

Mn2+

Glyoxylic acid

Semi-continuous; pH 2-5.4; flow 36 L/h

Ozone

Mn2+

Pyruvic acid

Semi-continuous; pH 1-3; flow Kinetic study; similar behavior of 111 36 L/h Mn2+ and Mn4+

Ozone

Mn2+

p-Nitrotoluene Determination of the kinetic Semi-continuous; acid pH; flow in acetic constant and reaction -2 10 L/s anhydride intermediates

112

Ozone/H2O2

Fe2+

Acids and colorants

113

+

2+

Humic acids improve atrazine removal; reaction mechanism is proposed

Presence of the catalyst improves 109 contaminant removal Reaction intermediates are 110 identified; mechanism is proposed

Semi-continuous; 4-10.5; flow Kinetic study 29mg O3/min

2+

Ozone

Pb , Cu , Zn , Fe2+, Mn2+ Nitrates

OrthoSemi-continuous; pH 3; flow chlorophenol mg O3/min

Ozone

Co2+

Oxalic acid

Semi-continuous; acid pH; 20ºC; flow 24 L/h

Ozone

Co2+

*

Discontinuous; pH 1.6-8.4; 10- Influence of pH, O3 dose and 5 -2·10-4 M catalyst; mechanism is proposed

116

Ozone

Fe3+

Oxalic acid

Improvement in oxalic acid Semi-continuous; pH 2.5; 20ºC, removal; reaction mechanism is flow 24 L/h proposed

117

Ozone

Mn2+, Cu2+

Phenol

Semi-continuous; pH 3 and 10

*Publications on ozone decomposition in the absence of pollutants

Mechanism is proposed; order of metal catalyst activity is 114 established Kinetic study and reaction stoichiometry determination

Phenol and TOC removal improved with catalyst

115

118

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4.2. Heterogeneous Catalytic Ozonation Heterogeneous catalysis takes place in systems with two or more phases, most frequently with catalyst in solid phase and reagents in gas or liquid phase. Most publications in this field have centered on the study of reactions in gas phase, although increasing numbers of researchers have studied reactions in liquid phase in recent years. Since Chen et al. [119] published results on heterogeneous catalytic ozonation of some compounds using Fe2O3 as catalyst, numerous catalysts have been tested, including metal oxides [120,121], supported metal oxides [122,123], supported metals [124], mesoporous materials [125], and activated carbons [126]. In general, the catalytic activity of these catalysts is mainly based on the generation of radical species such as hydroxyl; therefore, the efficacy of heterogeneous catalysis in ozone decomposition will be largely influenced by solution characteristics (pH, temperature, ionic strength, etc.) and by chemical and textural properties of the catalyst. Among the numerous materials that have been used as heterogeneous catalysts, there are four major types: [1] [2] [3] [4]

Supported metals. Metal oxides. Supported metal oxides. Activated carbons.

4.2.1. Supported Metals After the first results published by Heinig et al. [127], the use of catalysts based on metals deposited on different supports began to be studied in the late 1990s. At present there is considerable uncertainty about the mechanisms involved and the influence of different operational variables on this process. Results obtained by Karpel Vel Leitner et al. [128] indicated that adsorption of dissolved humic matter on the surface of the catalyst reduces the catalytic activity of metal catalysts supported on alumina, whereas catalysts supported on TiO2, where there is little such adsorption, show a better performance. Although the value of solution pH and the adsorption process are important, they do not always determine the activity of the catalyst in the ozonation of organic contaminants. Thus, the catalyst synthesis method and consequently its structure are critical for its capacity to transform ozone into HO· radicals[129,130]. Lin et al.[131] recently studied the catalytic activity of more than 20 catalysts (alumina, silica, activated carbons impregnated with metals, etc.) in the ozonation of formic acid, concluding that the transformation of ozone into HO· radicals is mainly influenced by its diffusion rate on the catalyst surface. Table 5 lists the catalysts tested in some of the most important research studies in this field, including data on the pollutants studied, the experimental conditions, and some observations.

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Table 5. Ozonation catalyzed by supported metals. System

Catalyst

Contaminants

Experimental Conditions

Observations

O3, O2

Ag/Al2O3

Microorganisms

Fixed bed; 500 g of catalyst

Bacteria and virus removal; strong catalytic interaction with dissolved 127 O2

O3

Cu supported on Organic compounds alumina, anatase, and (TOC), succinic acid attapulgite, Ru/CeO2

Discontinuous; and semi-continuous (flow 1.25 g O3/h); 0.2-1.2 g/L catalyst

Demonstrates the catalytic activity of supported metals; proposes 132 mechanism by adsorption and/or hydroxyl radical attack

O3

Continuous (6.5 mg TOC removal increases with certain Metals supported on Salicylic, peptide acid, O3/L) and semicontaminants; low adsorption on 128 Al2O3, TiO2 and clay humic substances continuous; acid pH catalyst surface

O3

Ru/CeO2

Succinic acid

Semi-continuous; pH High TOC mineralization in 3.4; flow 2.75·10-4 catalysts prepared by impregnation 129 mol O3/h; 0.8 g/L of and reduction catalyst

O3

Ru/CeO2

Succinic acid

Semi-continuous; pH Effect of the catalyst structure on its 3.4; flow 1.25 g O3/h; efficacy; organic molecules produce 130 0.8-3.2 g/L of catalyst metal sintering

O3

Ru/CeO2-TiO2

Chloroacetic acid, chlorosuccinic acid

Semi-continuous; pH 2.6-3.6; flow 15.6 High contaminant removal rates L/h; 0.8 g/L of catalyst

O3

Cu/Al2O3

Alachlor

Semi-continuous; Use of the catalyst increases TOC, 134 flow 0.5 mg O3/min chloride and nitrate formation

O3

Mg and Al on Fe, Co, Phenol, oxalic acid Ni and Cu oxides

Ref.

133

Semi-continuous; pH Identifies oxalic acid as product of 2; 20ºC; flow 200 phenol ozonation; oxalic acid 135 mL/min; 1 g/L of appears to cause losses of Cu and Ni catalyst

4.2.2. Metal Oxides Besides its chemical stability, the most important parameter determining the ability to use a metal oxide as catalyst of ozone decomposition is its acidity or basicity. Thus, active centers of the surface of a catalyst are located in groups that act as Brönsted or Lewis acids, which is largely conditioned by the medium pH. Adsorption of organic compounds on the catalyst surface plays an essential role in the catalytic activity of the oxide. This is because: i) it is the first step in the catalytic mechanism of ozone decomposition, and ii) the affinity of the metal oxide for certain inorganic species in solution (carbonates, phosphates, etc) can block active sites and reduce the efficacy of the catalyst. Researchers have used numerous metal oxides to study ozone decomposition and/or the ozonation of organic contaminants in solution. Thus, different groups successfully degraded oxalic acid by using catalysts such as TiO2, MnO2 and Al2O3[120,136,137] Several authors have studied the optimization of experimental conditions and have investigated reaction mechanisms (Table 6), proposing catalysts of different origin to achieve or improve the degradation of organic contaminants and mineralize dissolved organic matter in waters. Table 6 shows the most recent investigations into the simultaneous use of ozone and metal oxides to remove pollutants from waters, including the main study conclusions.

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Table 6. Most recent research on catalyzed ozonation with metal oxides. System

Catalyst

Contaminants

Experimental conditions

Observations

Ozone

Fe2O3

Phenol, TOC

Continuous; pH 4.3-6.3

High efficacy to remove phenol and 119 DQO; mechanisms is proposed

Ozone

CuO, Fe2O3, NiO, Aniline, DQO Cr2O3, Co2O3

Semi-continuous; flow Good catalytic 12 L/h; 55 g/L of leaching catalyst

Ozone

Mo, Zn, Sn, V, Ni, Co, Cu, Pb, Mn, * Fe, Ce, Ag and Bi oxides

Continuous; 25ºC; flowDeactivation of certain oxides due to 0.15 L/min; 0.5 g ofpossible adsorption of H2O on active139 catalyst sites

Ozone

α-FeOOH

Chlorobenzene, bicarbonates

Semi-continuous; flow Increased chlorobenzene removal in 0.2 L/min; 0.05-0.2 g/L 140 the presence of catalyst of catalyst

Ozone

MnO2

Oxalic acid

Semi-continuous; pH 3Improved performance with lower pH; 7, flow 36 L/h; 0.25 g/L 120 reaction on surface of catalyst

Ozone

γ-Al2O3

2-chlorophenol

Semi-continuous; pH 3Kinetic study; toxicity and TOC 9; flow 18 mg O3/h; 2 141 behaviors g/L of catalyst

Ozone

TiO2

Oxalic acid

Semi-continuous; pH Total removal of TOC; zero-order 2.5, 5; 30ºC; 5 g/L 136 reaction mechanism is proposed catalyst

Ozone

Tierra, Fe(O), α* FeOOH

Continuous; catalyst

Ozone

FeOOH

Semi-continuous, pH 7, Proposal of radical flow 0.5 L/min; 10 g/L improved TOC removal catalyst

Ozone

Mn2+, β-MnO2, α-Sulfosalicylic andSemi-continuous, pH 1-Study of effects of pH and state of Mn 144 MnO2, MnSO4 propionic acid 8.5 on catalytic activity

Ozone

Mixed metal oxides, metal oxides

p-chlorobenzoic acid

Discontinuous (pH 7 and deionized water); semi- No catalysis in deionized water; Co continuous (natural and Cu-Zn-Al oxides catalyze in145 water); 43 mg/L of natural water catalyst

Ozone

CuO-Al2O3

Phenol, chlorophenol, nitrophenol

Reactor tubular; pH 3-Intermediates are identified; the 11; 21ºC; flow 20 L/h;catalyst reduces ozone consumption146 0.25-2 g/L catalyst and increases rate

Ozone

Perfluorinated alumina

Methyl tert-butyl Semi-continuous with ether, isopropyl Alumina does not catalyze; kinetic and without recirculaether, ethyl tertconstant is determined and reaction by-147 tion; pH 5; 10-60 g/L of butyl ether, tertproducts are identified catalyst amyl methyl ether

Ozone

Al2O3

Discontinuous and semiOxalic, acetic, continuous (flow 100Removal increase depends on type of salicylic, and suc137 L/h); pH 5.5; 20-50 g/Lcontaminant cinic acids of catalyst

Ozone

Al2O3

NOM (DQO)

Semi-continuous; pH 7-Increased NOM removal and 8; flow 0.4 mg O3/L; 75decreased formation of reaction by-148 g/L of catalyst products

Ozone

TOCCATA®

Wastewaters (DQO)

Semi-continuous; pH 7-Improvement in DQO removal by 149 8; flow 40 mg O3/L oxidation and precipitation

Ozone

Sludge with metals * and metal oxides

DQO,

NOM (TOC)

5-50

Ref.

activity;

catalyst

138

Study of operational variables; effect g/L of metal oxides and NOM on the142 process mechanism;

143

Semi-continuous; pH 3;Feasibility study on the use of sludge 150 25ºC as catalyst of ozone decomposition

*Publications on ozone decomposition in absence of contaminants

J. Rivera-Utrilla, M. Sánchez-Polo and J. D. Méndez-Díaz

448

4.2.3. Supported Metal Oxides Attempts have been made to enhance the catalytic activity of metal oxides in the transformation of ozone into HO· radicals by increasing their surface area, supporting them on alumina, clays, silica, or zeolites. However; results obtained were not completely satisfactory because of leaching. According to Copper et al. [151] the presence of a heterogeneous surface facilitates the transference of ozone in solution and its decomposition into radical species. For this reason, many organic compounds were more effectively degraded when ozonation was carried out in the presence of these supported catalysts. Table 7 summarizes the main conclusions of different research groups, and describes the experimental conditions for the simultaneous use of ozone and supported metal oxides to remove organic contaminants from waters. Table 7. Removal of organic contaminants by systems based on the simultaneous use of ozone and supported metal oxides. System

Catalyst

Ozone

CuO, MnO2, Pd, supported on Phenol Al2O3

Contaminants

Experimental conditions Observations

Ref.

Semi-continuous; pH 6-9; Without catalyst activity 20ºC; flow 1.43 L/min

152

Catalyst activity removes more Semi-continuous; acid pH TOC; it does not oxidize hydroquinone and phenol

153,154

Continuous; 25ºC; flow 0.15 L/min; 0.5 g of catalyst

Ozone

Fe2O3/Al2O3

Phenol, hydroquinone, carboxylic acids, aldehydes

Ozone

Ag y Ni supported on zeolite

*

Ozone

High removal of TOC and TiO2, TiO2/Al2O3, Fulvic acid, proteins, Semi-continuous; pH 8; 30 pollutants in the presence of the 155 TiO2/arcilla disaccharides g of catalyst catalyst

High efficacy of Ag to decompose O3; it does not appear 139 to affect adsorbed H2O

Ozone

MnO2/Al2O3

*

Semi-continuous

Decomposition of O3, in the presence of the catalyst is zero order with respect to water; mechanism proposed with superoxide and peroxide intermediates

Ozone

TiO2/Al2O3

Fulvic acid

Discontinuous; pH 7.5; 20ºC; 10 g/L de catalyst

Use of the catalyst increases mineralization

Ozone

Al2O3, Fe2O3/Al2O3, TiO2/Al2O3

Use of the catalyst increases Chlorophenol, oxalic Semi-continuous; flow 24 solubility and decomposition of 151 acid, chloroethanol mg O3/(L·h) O3,improving pollutant removal

Ozone

TiO2/Al2O3

Humic acid, natural river water (TOC)

Semi-continuous; pH 7.2; By-products are identified; TOC 123, flow 0.4 g O3/h; 2.5-10 g/L removal increases at low humic 158 catalyst acid concentration

Ozone

V-O/SiO2, VO/TiO2, MnO2

Sulfosalicylic acid

Semi-continuous, pH 3, flow 0.67 L/min

Ozone

Metal oxides on Al2O3, SiO2, TiO2 * y SiO2·Al2O3

Fixed-bed; flow 0.175 Proposes a mechanism of ozone L/min of aqueous solution decomposition; catalytic efficacy 160 with O3; 1.5 g of catalyst of noble metals; catalyst effect

Ozone

MnOx/Al2O3, MnOx/SiO2, MnOx/TiO2, MnOx/ZrO

Fixed-bed; gas flow of Catalyst deactivation and 0.25-1L/min; 20-75 mg/L subsequent regeneration at high 161 of catalyst temperatures

Benzene

Formation of oxalic acid; improved TOC removal and reaction rate with catalysts; MnO2 does not catalyze

156

157

159

Ozone Decomposition by Catalysts and its Application in Water Treatment: …

449

Ozone

TiO2/Al2O3

Oxalic acid

The catalyst notably improves Semi-continuous; pH 2.5; pollutant removal; Eley-Rideal 10-40ºC; flow 12-36 L/h; mechanism; reaction order and 1.25-3.75 g/L of catalyst activation energy determined

Ozone

MnOx/activated carbon

Nitrobenzene

Higher catalytic activity at acid Continuous (flow 0.075 pH; pollutant oxidation and L/h) and semi-continuous; 163 adsorption is detected on catalyst pH 2-9 surface

Ozone

MnOx/Al2O3

Cyclohexane

Indicates formation of Semi-continuous; flow 0.5 intermediate compounds on L/min; 0.1 g/L of catalyst catalyst surface

Ozone

Co oxides supported on alumina and hydrotalcite

Naphthol blue black

Co leaching; dispersion is studied Semi-continuous; 25ºC; 30 in catalysts and reutilization in 3 165 L/h; 0.3 g/L of catalyst cycles

162

164

*Publications on ozone decomposition in the absence of contaminants

4.2.4. Activated Carbons The porous texture and chemical nature of activated carbons makes them ideal materials for use as adsorbents and also as catalysts or catalyst supports [166]. Although there has been far less study on the use of activated carbons as catalyst or metal catalyst support for reactions in aqueous solution than on their role as adsorbent, research interest has been increasing over the past 10 years. In the 1990s, it was observed that the presence of activated carbon catalyses ligand substitution between complex compounds [167] and can produce oxidation of sulfide into sulfate [168]. These studies found a relationship between the catalytic activity of activated carbon and its porous structure, although they did not consider the influence of the surface chemistry of the carbon, which was subsequently addressed by other authors[169,170] Table 8. Relevant publications on catalyzed ozonation with activated carbons. System Catalyst

Contaminants

Experimental conditions Observations

Ref.

Ozone

Granular activated carbon

Phenols

Semi-continuous; pH 2; 15-35ºC

Lower consumption of O3 with AC; AC improves selectivity but not the 179 reaction constant

Ozone

Activated carbon

Chlorinated hydrocarbons, COD

Fixed bed ―ECCOCLEAR‖

Combines catalyzed ozonation with biological treatment and nano 187 filtration; low influence of carbonates as scavengers

Ozone

Activated carbon

Subterranean waters, domestic and industrial wastewaters

Fixed bed ―ECCOCLEAR‖

Results of industrial plants; catalyzed ozonation requires less ozone and is effective over a wide pH range

Ozone

Activated carbon, carbon black

Radical pathway process; Inhibitor p-Chlorobenzoate, Discontinuous; 20 mg/L and promoter effects of ozone 178 acetate, methanol of catalyst decomposition

Ozone

Activated carbon

Textile wastewaters

Ozone

Granular activated carbon

Fixed bed; pH 2, 8; 25ºC; Solid-gas catalytic reaction; 1,2 flow 5 L/min; 4 g/L of increases % of oxidized groups after 190 dihydroxybenzene catalyst activated carbon ozonation

Ozone

Granular activated carbon

*

Semi-continuous; flow Kinetic and stability study and 60-360 L/h; 200 g of catalyst activity; removes color activated carbon column

188

189

Proposes mechanism; heterogeneous Discontinuous; pH 2-9; 5- and homogeneous processes 191 30ºC; 2 g/L de CA simultaneously; influence of variables and kinetic study

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450

Table 8. (Continued) System Catalyst

Contaminants

Experimental conditions Observations

Ref.

Higher catalytic activity of basic activated carbon with/without metals in mineral matter

180

181

Ozone

Granular activated carbon

1,3,6 naphthaleneDiscontinuous; pH 2.3 trisulfonic acid

Ozone

Granular activated carbon

(mono,di,tri) naphthalenetrisulfonic acids

Semi-continuous; pH 2Study of adsorption capacity and 12; flow 76 mg O3 /min; surface chemical modification of 0.5-2 g/L of activated ozonated activated carbon carbon

Ozone

Granular activated carbon

(mono,di,tri) naphthalenetrisulfonic acids

Semi-continuous; pH 2; flow 76 mg O3/min

Ozone

Granular activated carbon

Identification of intermediates; 1,3,6 naphthalene- Discontinuous; pH 2.3; 2 reduction in catalytic activity of trisulfonic acid g/L of catalyst activated carbon with ozonation

Ozone

Activated carbon

Pyruvic acid

Catalytic and non-catalytic reaction Semi-continuous; pH 7.5; rate constants determined; transfer 194 20ºC; 2.5 g/L CA of O3 mass is limiting step at high carbon doses

Ozone

Granular activated carbon

*

Discontinuous; 22ºC; 1 g/L of catalyst

Ozone

Granular activated carbon, pellets

Succinic acid

Use of activated carbon permits Semi-continuous; pH 7; 5, almost complete mineralization; 15, 25ºC; flow 20-60 L/h; 195 reaction via 2 pathways: water and 10-20 g/L CA activated carbon surface

Ozone

Activated carbon (0.1* 0.3 mm)

Because of activated carbon Semi-continuous; pH 3-9; oxidation, the surface chemistry is 25ºC; 4-15 mg/L O3; 0.2 only important in the first cycles, 196 g/L CA after which the texture is more influential

Ozone

Activated carbon

Columns in line; 100 g CA; 3 mg/L O3

Rainwater (DQO)

Reduces genotoxicity of ozonation products when catalyzed with 192 activated carbon 193

Higher catalytic activity of basic activated carbon and larger surface 183 area; greater ozone decomposition with higher concentration

Optimization of operational variables; synergic effect in the O3/AC /CAB system

197

Effect of operational variables Ozone, Discontinuous; pH 7; Granular and powdered Sodium dodecyl(activated carbon size and dose, O3 ozone/H2 25ºC; 2·10-5M; 0.1 g/L of 198 activated carbon benzenesulfonate dose, etc.); activated carbon reduces O2 activated carbon effect of radical scavengers Ozone, Granular activated ozone/H2 carbon O2

Ozone

Carbon aerogels with Mn2+, Co2+y Ti4+

Ozone

Granular activated carbon

Removal of TOC and Organic and Discontinuous; pH 7, 9; microcontaminants; similar inorganic -5 25ºC; 2·10 M O3; 0.5 g/L performance of O3/GAC and compounds in CA O3/H2O2 in generation of HO· natural lake water radicals

199

Metals susceptible to oxidation with Discontinuous; pH 7; Para-chlorobenzoic O improve the generation of HO· -5 25ºC; 2·10 M O3; 2.5-10 3 200 acid radicals; activated (more basic) mg/L aerogel samples are more effective Benzothiazole

Proposes mechanism and Semi-continuous; pH 2,7, determines kinetic constants; high 11; 15ºC; flow 54 L/h; 1 201 radical contribution to contaminant g/L CA removal Notable catalytic activity in phenol removal; in situ regeneration of activated carbon fiber during 202 ozonation, although its chemical and textural properties are modified

Ozone

Activated carbon fiber Phenol

Semi-continuous; 25ºC; flow 1.5 L/min; 4 g/L activated carbon fiber

Ozone

Granular activated

Semi-continuous; pH 2.5, Identification of reaction by-

Polyphenols

203

Ozone Decomposition by Catalysts and its Application in Water Treatment: … carbon

Ozone

Activated carbon (0.1Oxalic acid, 0.3 mm), oxidized with oxamic acid HNO3

451

5; 25ºC; flow 30 L/h; 10 products; improves TOC removal; g/L CA generation of H2O2 Study of the effect of pH, presence Semi-continuous; pH 3, 7; of radical scavengers and surface 25ºC; flow 0.15 L/min; chemistry; proposes mechanism; 204 0.5 g/L CA better result with basic activated carbons

The catalytic activity of activated carbon in aqueous solution has been studied in the synthesis[171,172]and degradation of ammonic nitrate [173], phenolic compounds[174,175]or chlorinated compounds [176,177] among others. The main interest in this behavior of carbon is related to the oxidation of organic matter. Thus, it has been observed that activated carbon can decompose ozone in aqueous solution into radicals with a high oxidizing power, increasing the extent of ozonation [178]. Therefore, the combined use of ozone and activated carbon in a single process was proposed as an attractive option to destroy toxic organic compounds. Until recently, the major advantage of adding activated carbon to the treatment system was considered its high adsorption capacity [179]. However, Jans and Hoigné [178] showed that both carbon black and activated carbon catalyze the decomposition of ozone in aqueous phase. These authors indicated that both types of carbon initiate the radical-type chain reaction of ozone, which continues in aqueous phase and accelerates the transformation of ozone into free hydroxyl radicals. Zaror et al.[179] reported that the presence of activated carbon drastically reduced the stability of ozone in aqueous solution, which they attributed to a combination of the decomposition catalyzed by the surface and the participation of activated carbon surface groups. Subsequently, in-depth research into ozone/activated carbon systems by Rivera-Utrilla et al.[180,181,182,183] demonstrated that the surface chemistry and textural characteristics of activated carbon play a major role in its behavior as catalyst of ozone decomposition into hydroxyl radicals. These authors ascribed this effect to basic carbon groups, e.g., pyrone and chromene [180]. Zaror et al. found that addition of activated carbon to the system favored the oxidation rate of 1,2-dihydroxybenzene with ozone[179]. Hu et al.[184] reported a more effective removal of organic matter from textile industry effluents by using reduced Cu supported on activated carbon. Table 8 summarizes the results of some of the most relevant studies on the simultaneous use of ozone and activated carbon in water treatments. It is necessary to study the mechanisms underlying this catalytic process in order to achieve its economic and effective large-scale optimization and implementation. Various researchers have investigated the mechanisms involved in heterogeneous catalytic ozonation[185,186], but these have not yet been fully elucidated because of the complexity of these simultaneous pollutant oxidation and adsorption processes.

CONCLUSIONS There is now a considerable body of knowledge on advanced oxidation processes (O3/H2O2, O3/UV) in the literature, including data on the mechanisms and reactions involved and on the influence of operational variables in the transformation of ozone into HO· radicals.

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This information has allowed the large-scale application of these processes to remove a wide variety of organic microcontaminants. The use of dissolved or supported metals to transform ozone into HO· radicals is not very effective. Studies show that i) the simultaneous use of ozone and dissolved metals must be approached in a more systematic manner; and ii) there is considerably controversy about the reactions involved in the process and the metal characteristics that promote the transformation of ozone into HO· radicals. Research is required into the mechanisms that underlie this catalytic process in order to achieve its economical and effective optimization and implementation on a large scale. Because of the complexity of simultaneous pollutant oxidation and adsorption processes, the mechanisms involved in heterogeneous catalytic ozonation have not yet been fully elucidated. Finally, activated carbon appears to be the most appropriate solid catalyst to use for enhancing micropollutant ozonation. Besides considerably accelerating the process of ozone transformation into HO· radicals, thereby increasing the rate of organic contaminant removal, activated carbon also has a high adsorbent capacity, further contributing towards the removal of microcontaminants from waters.

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[190] Zaror C., Soto G., Valdes H., Mansilla H., Ozonation of 1,2-dihydroxybenzene in the presence of activated carbon, Water Sci. & Tech., 44, 125-130 (2001). [191] Beltran F.J., Rivas J., Alvarez P., Montero-de-Espinosa R., Kinetics of heterogeneous catalytic ozone decomposition in water on an activated carbon, Ozone: Sci. & Eng., 24, 227-237 (2002). [192] Rivera-Utrilla J., Sanchez-Polo M., Mondaca M. A., Zaror C. A., Effect of ozone and ozone/activated carbon treatments on genotoxic activity of naphthalenesulfonic acids, J. of Chem. Tech. & Biotech., 77, 883-890 (2002). [193] Sanchez-Polo M., Rivera-Utrilla J., Effect of the ozone-carbon reaction on the catalytic activity of activated carbon during the degradation of 1,3,6-naphthalenetrisulphonic acid with ozone, Carbon, 41, 303-307 (2003). [194] Beltran F.J., Acedo B., Rivas F.J., Gimeno O., Pyruvic acid removal from water by the simultaneous action of ozone and activated carbon, Ozone: Sci. & Eng., 27, 159-169 (2005). [195] Beltran F.J., Garcia-Araya J.F., Giraldez I., Masa F.J., Kinetics of activated carbon promoted ozonation of succinic acid in water, Ind. & Eng. Chem. Res., 45, 3015-3021 (2006). [196] Faria P.C.C., Orfao J.J.M., Pereira M.F.R., Ozone decomposition in water catalyzed by activated carbon : Influence of chemical and textural properties, Ind. & Eng. Chem. Res., 45, 2715-2721 (2006). [197] Li L., Zhu W., Zhang P., Zhang Q., Zhang Z., AC/O3-BAC processes for removing refractory and hazardous pollutants in raw water, J. of Hazard. Materials, 135, 129-133 (2006). [198] Rivera-Utrilla J., Mendez-Diaz J., Sanchez-Polo M., Ferro-Garcia M.A., BautistaToledo I., Removal of the surfactant sodium dodecylbenzenesulphonate from water by simultaneous use of ozone and powdered activated carbon : Comparison with systems based on O3 and O3/H2O2, Wat. Res., 40, 1717-1725 (2006). [199] Sanchez-Polo M., Salhi E., Rivera-Utrilla J., von Gunten U., Combination of ozone with activated carbon as an alternative to conventional advanced oxidation processes, Ozone: Sci. & Eng., 28, 237-245 (2006). [200] Sanchez-Polo M., Rivera-Utrilla J., von Gunten U., Metal-doped carbon aerogels as catalysts during ozonation processes in aqueous solutions, Wat. Res., 40, 3375-3384 (2006). [201] Valdes H., Zaror C.A., Heterogeneous and homogeneous catalytic ozonation of benzothiazole promoted by activated carbon : Kinetic approach, Chemosphere, 65, 1131-1136 (2006). [202] Qu X., Zheng J., Zhang Y., Catalytic ozonation of phenolic wastewater with activated carbon fiber in a fluid bed reactor, J. of Colloid & Interf. Sci., 309, 429-434 (2007). [203] Giraldez I., Garcia-Araya J.F., Beltran F.J., Activated Carbon Promoted Ozonation of Polyphenol Mixtures in Water : Comparison with Single Ozonation, Ind. & Eng. Chem. Res., 46, 8241-8247 (2007). [204] Faria P.C.C., Orfao J.J.M., Pereira M.F.R., Activated carbon catalytic ozonation of oxamic and oxalic acids, Appl. Catal. B: Environ., 79, 237-243 (2008).

In: Advances in Environmental Research, Volume 13 Editor: Justin A. Daniels

ISSN: 2158-5717 © 2011 Nova Science Publishers, Inc.

Chapter 17

USE OF MICROARRAYS TO STUDY ENVIRONMENTALLY RELEVANT ORGANISMS: A UK PERSPECTIVE Michael J. Allena, Andrew R. Cossinsb, Neil Hallb, Mark Blaxterc, Terry Burked and Dawn Fielde a

Plymouth Marine Laboratory, Prospect Place, Plymouth, UK. NERC Molecular Genetics Facility, School of Biological Sciences, University of Liverpool, Liverpool, UK c NERC Molecular Genetics Facility, Institute of Evolutionary Biology, University of Edinburgh, Edinburgh, UK d NERC Molecular Genetics Facility, Dept of Animal & Plant Sciences, University of Sheffield, Sheffield, UK e NERC Environmental Bioinformatics Centre, NERC Centre for Ecology and Hydrology, Mansfield Road, Oxford, UK; b

ABSTRACT Historically, the majority of microarray work has been restricted to well-defined model organisms. This was primarily due to the limited availability of genomic or transcriptomic sequence data and the then high cost involved in developing microarrays. However, recent technological developments have greatly enhanced the speed of generating the underpinning sequence data for non-model species, and have opened up more cost-effective approaches for microarray production to make them far more affordable for researchers at the lower end of the budget range. These developments have been seized upon by the environmental genomics community within the UK. The creation of a network of closely integrated facilities for sequencing, microarray printing and bioinformatics has opened the gateway for the study of environmentally relevant organisms. Here, we describe the infrastructure for microarray development within the UK, and the diverse applications for which they are currently being used.

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INTRODUCTION The advent of the genomic era has heralded a new dawn in the study of biological systems. The intensive study of model species such as E. coli, yeast, Drosophila and the mouse has provided a wealth of information on the molecular workings inside the cell. These advances were largely dependent on large-scale sequencing projects which determined the entire genome sequence of several well studied model strains [1-4]. The development of microarray technology for massively parallel detection of sequence targets defined from large-scale genome and EST sequencing projects, has allowed transcriptional profiles from cells, tissues and organisms at various life stages to be analysed and mapped on a global scale with increasing precision [5]. It cannot be denied that the intensive study of model organisms is of great benefit. However, the study of ecologically important, non-model species in their natural environment is arguably of greater relevance when analysing the response of species to real-world environmental conditions. Model organisms are often chosen for reasons of experimental power and convenience, rather than for ecological interest or evolutionary significance [6]. In an era of increasing awareness of the environmental changes occurring through global warming, habitat destruction, deforestation, globalisation and ocean acidification there has never been a more important time to study the impact on the Earth‘s ecosystem, and this means adapting advanced genomic tools for this purpose. Historically, tools such as microarrays have been restricted to model species due to limitations in the genomic sequence available and the high cost involved in developing microarrays. However, technical advances have led to a significant decrease in the costs involved, opening the field to researchers working on non-model organisms. The uptake of microarray technology by environmental researchers, in particular the marine sciences, has been relatively slow partly due to a lack of both focused funding and technical expertise in the community. In the UK this has been addressed by the predominantly NERC-funded environmental genomics community which has created a dedicated network of facilities and resources to aid the use and development of microarrays.

FUNDING The United Kingdom Natural Environment Research Council (NERC) has invested over £26 million in two directed science programmes; (i) Environmental Genomics (EG) and (ii) Post-Genomics & Proteomics (PGP). To complement these initiatives, NERC has also funded the creation of dedicated technical support centres covering DNA sequencing, genotyping, microarray design and fabrication (through NERC‘s Services and Facilities branch), and bioinformatics (through the thematic programmes themselves). Each centre offers a strong technical capability built on the existing expertise in established laboratories, and also consultation, training, and support not only to undertake the lab work but also in the subsequent data analysis and management. Access is open to all UK-based scientists, but priority is given to NERC grant holders based on an appraisal of science quality. Applicants for NERC-funded research are advised to take advantage of these facilities whenever possible.

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In addition, to promote the use of these advanced technologies in environmental science, an annual small projects competition has allowed researchers to conduct pilot studies in support of full-scale future project applications. National and international collaboration between environmental scientists often working in vastly different areas is promoted through this system. The diverse range of subjects for which new microarrays have been developed include: marine phytoplankton viruses (see Case Study 1); adaptive osmoregulatory physiology of euryhaline teleosts; ecotoxicological responses of terrestrial annelids; genetic control of avian plumage traits; sexual differentiation and endocrine disruption in fish; and responses to cold and hypoxia in carp (see Case Study 2).

INFRASTRUCTURE Every microarray experiment is dependent on a number of steps, all of which are essential to produce results that are scientifically competent and of publishable quality. These steps are listed in Table 1, and can be grouped into 3 main categories: the acquisition of genomic sequence data, the design and construction of the array and the experimental use of the array. Of course, this is only the beginning of a researcher‘s challenges: correct experimental design with the appropriate number and type of replicates, in combination with the large volume of data produced, demands the use of complicated statistical analysis to provide meaningful results. An environmental scientist working alone on a microarray project will need to be expert in a range of disciplines, such as molecular biology, bioinformatics and statistics. In addition, ready access to sequencing, array printing and hybridisation facilities is essential. It is important that the researcher has an understanding of the processes involved at each stage, and also that the correct expertise is available at each stage in line with current opinion in a rapidly developing field. Thus the use of microarrays is a daunting prospect to any researcher who lacks significant experience or expertise in the technologies involved. In particular this includes environmental researchers whose field has been historically dominated by non-molecular scientists (i.e. large numbers of field biologists). It is perhaps for these reasons that environmental scientists studying non-model organisms have been relatively slow to embrace microarray technology. Fortunately for the UK-based environmental science community, NERC foresaw this problem and put in place the infrastructure required to facilitate the technical development of microarray platforms and their application to environmental science. The new provision was based on the pattern established by an existing NERC genotyping centre based in Sheffield (MGF-Sheffield) and consists of three additional Molecular Genetics Facilities (MGF) nodes: two facilities are involved with sequencing (MGF-Edinburgh and MGF-Liverpool through its Advanced Genomics Facility, AGF-Liverpool), and a third facility with microarray construction (MGF-Liverpool through its Liverpool Microarray Facility, LMF-Liverpool). A fourth but separate facility, The NERC Environmental Bioinformatics Centre (NEBC) provides the necessary bioinformatic expertise required at all levels of the process. Combined these facilities provide a comprehensive suite of technical platforms and expertise, and they interact seamlessly with one another to satisfy the needs of the researcher.

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GENOMIC SEQUENCING There are three currently applicable methods for generating sequence data for environmentally relevant organisms: capillary Sanger sequencing, massively parallel pyrosequencing, and massively parallel sequencing-by-synthesis. Each method has its benefits and drawbacks, and any particular environmental genomics project will choose the route(s) most suited to its needs. Both MGF-Edinburgh and AGF-Liverpool are dedicated sequencing facilities and thus generally not directly involved in physical sample acquisition: it is assumed that a researcher working on a particular environmental organism has the necessary expertise for culture and/or sample extraction to provide suitable genomic material (quantity and quality) for sequence analysis. Capillary Sanger sequencing, the workhorse of DNA sequencing for over 30 years, is offered through the MGF-Edinburgh. This generates 500–700 bases per read, and is suited to both survey sequencing (such as expressed sequence tag sequencing, or ESTs) and also whole-genome sequencing. The MGF-Edinburgh offers medium-throughput (~1000 samples per day) Sanger sequencing using ABI3730 instruments. This suits projects from ~100 to 50,000 sequencing reads from targets such as PCR products, cloned inserts in plasmids and other vectors provided by the user. It also carries out the complete sequencing of large DNA fragments (>30 kb; for example from fosmid and BAC clones) using shotgun strategies. Bioinformatic support is offered by MGF- Edinburgh, including preliminary sequence trace processing, assembly and annotation. For EST projects, a suite of in-house developed sequence annotation tools can also be applied to sequence datasets using programs such as PartiGene (databasing tool for EST datasets) [7], trace2dbEST (for processing EST sequencing traces) [7], prot4EST (for prediction of peptides from neglected genome sequence datasets) [8], and annot8r (annotation of peptide or nucleotide sequences with GO, KEGG and EC tags based on sequence similarity). The Advanced Genomic Facility (AGF) based at the University of Liverpool offers access to the ultra-high throughput pyrosequencing Genome Sequencer FLX system (GS FLX, Roche), which at present can generate in excess of 100 Mb of genome sequence in a single run with a mean length of 250 bases. The FLX pyrosequencing platform incorporates single-molecule amplification and parallel sequencing of up to ~400,000 individual molecules for 250 bases, making large-scale scientific projects feasible and more affordable. By mid2008 this instrument is expected to yield up to 1 Gb with read lengths approaching 500 bp. FLX pyrosequencing is suited to deep surveying of transcriptomes, complete sequencing of large-insert clones and smaller genomes, resequencing of larger genomes, and metagenomic analyses. AGF-Liverpool also has the latest computational tools and trained staff to deliver bioinformatic services for data handling and analysis, in particular for assembling sequence reads (Newbler assembly) and sequence alignment (FlowMapper). Sequencing by synthesis is also massively parallel, but generates ~30 million reads of ~35 bases per run, generating 1 Gb of sequence. This technology is ideal for resequencing of complex genomes, metagenomic surveys of mesocosms, digital transcriptomics and de novo sequencing of smaller genomes. MGF-Edinburgh also offers a sequencing-by-synthesis service using an Illumina Genome Analyser instrument. The production of raw sequence data is only the start of the process: the genomic or transcriptomic sequence must be correctly assembled and annotated in order to provide

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meaningful information that can be used at the next stage for microarray design. As shown above, both facilities have their own particular expertise in certain areas. However, in addition to the bioinformatic facilities offered by MGF-Edinburgh and AGF-Liverpool, additional bioinformatic support for genomic annotation is offered by the NERC Environmental Bioinformatics Centre (NEBC). The bioinformatic support provided by MGFSheffield is focused on population genetic data analyses, including genomic mapping.

MICROARRAY DESIGN AND FABRICATION The key requirement for generating a DNA array is access to suitable genomic resources. For arrays printed in-house using amplified DNA, this comprises cloned cDNAs or largeinsert genomic fragments isolated from libraries [9]. Alternatively, arrays can be printed from oligonucleotides synthesised in bulk with defined sequences as probes against known DNA targets of interest. Both techniques are expensive in time (for cloning and amplification) and costs (for oligonucleotide purchase). However, for situations where sequence data already exist it is now preferable to design probes in silico against all known targets for a particular species using one of several design algorithms, followed by on-chip synthesis by one of several manufacturers, notably Agilent. Advancing fabrication techniques now makes this a relatively inexpensive yet highly effective approach to array provision. The design of oligonucleotide probes from a known target DNA sequence can be achieved using any one of several open source or commercial packages [10]. The MGFLiverpool uses a commercial package (ArrayDesigner, Premier Biosoft) as an alternative to the widely used open source package OligoArray2 [11]. Design algorithms continue to advance, and one algorithm might suggest different probe designs to another. Commercial providers of oligonucleotides or arrays offer a design service (e.g. Agilent‘s eArray system), but the precise algorithms employed remain confidential to the company and so one cannot be totally confident of design quality or the criteria used in its execution. It is also worth noting that the outputs of any design algorithm do not necessarily provide for optimal function as a hybridisation probe, since they are based on thermodynamic criteria that are surrogates of the hybridisation process. This is why the MGF-Liverpool uses an optimisation approach, comparing the properties of several competing probe designs generated from a single algorithm against stringent performance criteria [12] in order to define the best performing design. All of these options are available at the MGF-Liverpool which has access to both contact and non-contact (piezo) printing robots for array fabrication, has service centre status for both Affymetrix and Agilent platforms, and has access to the Illumina BeadStation, this being the third major commercial platform. As a central array facility, it has negotiated special rates for purchasing large numbers of synthesised oligos and all of the other necessary consumables, including catalogue arrays. It is thus well able to provide support to users with all aspects of microarray design, construction and commissioning. In the past few years MGF-Liverpool has constructed cDNA arrays for a wide range of species (e.g., common carp, flounder, rainbow trout, ground squirrel and eel) all of which were preceded by large-scale cDNA sequencing projects. It has also been involved in a series of microbial metagenomics projects printing commercially synthesised oligoarrays directed against rRNA targets defined by the

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user. MGF-Liverpool has also some experience in constructing oligoarrays for environmental model species directly from public EST data, taking advantage of the rapidly increasing capacity of new platforms. Thus, an oligoarray directed against 22,000 BLAST-identified genes in the rainbow trout was fabricated by Oxford Gene Technology to a MGF-Liverpool design, and this has been expanded to 44,000 probes [12]. This design was optimised in the sense that multiple predicted probes were quantitatively compared using several different tests and the best performing probe identified. The resulting arrays displayed a dynamic range and sensitivity that far exceeded that of conventional cDNA arrays [12] .

ANALYSING THE RESULTS OF MICROARRAY EXPERIMENTS Once a microarray has been designed and fabricated the actual experimental biology can begin. The first important issue is the statistical design of the array experiment. since the shift from the early reference-based designs to more complex ANOVA-based loop designs [13], and the need to account for multiple sampling corrections in such large-scale statistical comparisons, makes this the realm of specialist statistical advice. Only rarely do biologists who develop skills in laboratory technique have the numerical and statistical skills for highlevel data analysis and pattern searching. The staff of the MGF-Liverpool and NERC Environmental Bioinformatics Centre (NEBC) provide specialist advice to define the most appropriate and cost-effective experimental plan to success. The researcher also needs to understand the very fine definition offered by array experiments to ensure that the experiment is adequately controlled and that sufficient independent biological replicates are generated for detailed statistical analysis. Dedicated microarray bioinformaticians at both the MGFLiverpool and at NEBC keep abreast of the latest developments and issues with microarray analysis and are best positioned to advise on correct experimental design. The MGFLiverpool also supports all of the stages of array usage, including sample preparation, hybridisation protocols, imaging/data acquisition and interpretation of the resulting data. This includes the training of research students and staff from NERC-funded laboratories. The fundamental responsibility for sample acquisition and for extraction of DNA or RNA lies with the client. Indeed, sample processing, hybridisation and data acquisition can be performed at the researchers own laboratory if facilities are available. Alternatively the entire programme can be performed as a service by MGF-Liverpool staff or the latter can provide the necessary training for clients to undertake the work. Emphasis is placed on the researcher remaining in control of the experiment, but with the full benefit of state-of-the-art protocols and equipment. MGF-Liverpool offers several scanning platforms for image acquisition including Agilent, Perkin-Elmer and Axon. It also advises on the critical issue of interpreting the image file in order to quantify the distribution of fluorescence intensities across the population of probes. Once microarray images have been processed and quantified, it is strongly recommended that that the researcher interacts further with the MGF-Liverpool or the NEBC if they require help progressing the statistical analysis. NEBC offers access to the commercial GeneSpring (Agilent) and the open-source maxD analysis platforms, and both the NEBC and the MGF-Liverpool routinely use algorithms from within the Bioconductor package written in the R statistical language. This combined provision has three main advantages. Firstly, the

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researcher is directed to well established statistical and data visualisation algorithms. Second, he/she can be trained in analytical technique through interactions with the facilities. Third, he/she can be exposed to objective approaches for drawing inferences both in terms of which genes are responding to experimental treatment, but also how this relates to the biological pathways and processes, and to the questions being addressed. This avoids biasing the analysis to favoured genes or to genes that fit with particular hypotheses. Bioinformaticians at any NERC facility can explain the data output, its strengths and its limitations. However, it is entirely up to the researcher to interpret and publish the data. This involves the use of gene ontology profiling tools such as GOMatrix, GoMiner, Reactome etc, as well as the newer gene set analysis techniques originated in the Broad Institute, MIT. At the final stage of publication, it is necessary for all microarray data to be submitted to a public repository such as GEO or ArrayExpress [14, 15]. This is aided by the development of the Biolinux based maxdload2 data submission program, developed by NEBC partners at the University of Manchester, which ensures it is MIAME-compliant [16]. Indeed, in response to the issues associated with environmental microarrays, the NEBC has developed its own guidelines for MIAME for environmental projects, designated MIAME/Env [17]. Researchers funded by NERC are also required to submit their data to the NERC-supported database EnvBase (http://nebc.nox.ac.uk/).

DATA ANALYSIS AND LONG-TERM MANAGEMENT While each of the sequencing facilities have specialist informatics services focused on specific sequencing platforms, the NEBC is focused on delivering multi-omic approaches to analysis and data management. NEBC was formed in 2002 to specifically provide support for the NERC Environmental Genomics Programme, and subsequently for the sister PostGenomics and Proteomics thematic programme. NEBC‘s primary remit is to archive all NERC-funded data generated under these programs subscribing to the NERC data policy of spending up to 15% of the programme budget on data management and archiving activities. This was one of the earliest data policies operated by a major UK funding agency and its wider implementation is mediated by a set of seven designated data centres that receive core funding from NERC for long-term data management. To guide the data management of the programme, and the stewardship of ‗omic data in NERC as a whole, NEBC has also produced an extension of the NERC data policy to cover ‗omic data. This policy includes very specific mechanisms for submission of data. All data are catalogued in the EnvBase portal and must be submitted to an appropriate public repository where one exists. EnvBase contains discovery-level information about all listed projects, and for each data-holding includes a list of relevant hyperlinked accession numbers leading to databases such as ArrayExpress [14, 15]. EnvBase also provides a mechanism to house all data for which a recognized database does not exist. NEBC was designed to build informatic capacity within the newly forming NERC environmental genomic community. NEBC is therefore staffed by bioinformaticians and computer scientists, in addition to data managers, who provide direct consultation to the wider community. NEBC was established in part to realize the vision of providing the entire Environmental Genomics community with a powerful and uniform computing environment

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based on Linux. This system, called Bio-Linux, is one of a growing suite of highly customized Linux distributions [18]. It contains over 60 software packages and is distributed free of charge to all labs supported by the program. The Bio-Linux system has been used to build teaching labs and computer clusters in several locations across the UK and is used by the core facilities and researchers to coordinate research. The primary focus of NEBC efforts under these programs has been support for research that is generating microarray and EST data. Microarrays were a focus because of their widespread use among funded researchers, the expense and effort of tooling up to produce and interpret them for new users, and the added complexity that several NERC-funded groups aimed to build custom arrays for novel species and sets of genes. In order uphold its remit to provide stewardship over the collective data holdings of these programmes; the centre has become progressively more involved in standardization activities. While at first, this simply meant monitoring emerging standards and compliance activities, over time this led to active participation and leadership in standardization activities that expressly benefit the environmental ‗omics community and that have made submission of data to public repositories possible. The NEBC spearheaded a standardization project in response to the difficulties NERC researchers were facing in complying to the MIAME standard, a requirement for submission to ArrayExpress [19]. The MIAME/Env project emerged out of work aimed at simplifying this process for researchers across the board. This initiative extended the core MIAME specification to capture additional information about the environmental context of samples (for example geographical location, biological interactions, and environmental conditions) [17]. Work on MIAME/Env has since been extended into the metabolomics and genomics domains [20]. Because of participation in such projects, and its focus on multi-omic data sets, NEBC is also a founding member of the ―Minimum Information about a Biological and Biomedical Investigation‖ (MIBBI) community [21].

CONCLUSION The UK has developed a powerful network of specialist facilities to promote the use of high-density microarrays that help researchers overcome the myriad challenges of working with non-model but environmentally-focussed organisms. Depending on the resources available to them and their level of expertise, environmental researchers can selectively choose the facilities while having as much or as little control in the development, construction and processing of the microarrays as they are comfortable with. The nature of the organisms involved can vary dramatically, as can the type of experiments undertaken, but help is available at every stage of the process. This has had a strong beneficial effect on the UKbased environmental community by promoting the use of microarrays and driving environmental sciences into the 21st century. To illustrate this, the following are two case studies, typical of the type of work that has been undertaken, by researchers in the environmental genomics community.

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NON-MODEL ORGANISM CASE STUDY 1: THE COCCOLITHOVIRUS MICROARRAY The Science Emiliania huxleyi is a marine coccolithophorid with worldwide distribution capable of forming vast blooms that can cover up to 10 000 km2 [22, 23]. Its production of calcium carbonate coccoliths and its role in CO2 cycling and dimethyl sulphide (DMS) production makes E. huxleyi an important species with respect to past, present and future marine primary productivity. Viruses have been shown to be a major cause of bloom termination [24-26]. Emiliania huxleyi virus 86, EhV-86, is a giant virus that was originally isolated from a bloom in the English Channel off the south coast of England in 1999 [27]. EhV-86 is the type species of the genus Coccolithovirus within the family Phycodnaviridae and is currently its largest fully sequenced member [28]. The 407,339 bp genome is the second largest virus genome sequenced and is predicted to encode around 472 protein coding sequences (CDSs) [29]. The majority of EhV-86 CDSs exhibit no similarity with proteins in the public databases; a mere 21% of the CDSs contain protein–protein basic local alignment search tool (BLAST) results that matched an E value lower than 0.01 [29]. The large size (and availability) of the EhV-86 genome and its highly unknown nature easily justified the construction of a microarray to aid in the molecular characterisation of this unique virus family.

The Array The EhV-86-based coccolithovirus microarray has been through 2 stages of development. Version 1 was dominated by probes for the 472 predicted CDSs of EhV-86 and contained only a handful of E. huxleyi probes, and consisted of around 1,600 features. As the work has progressed and developed, and the nature of the questions asked of the array changed, so the latest version 2 of the array (printed at MGF-Liverpool) has around 25,000 features and contains probes for multiple coccolithovirus strains and for ESTs from the host species E. huxleyi (developed with the aid of MGF-Edinburgh).

The Research The majority of the EhV-86 genome is composed of genes of which the function is totally unknown [29]. Indeed, unusually among sequenced genomes, a number of predicted genes fail to find any matches whatsoever in the public databases [30]. This novelty of the genome makes the task of annotation difficult: without functional information it is difficult to assess whether what is predicted to be a gene is actually a gene. Thus, the first use to which the coccolithovirus array was put was simply to detect the presence of viral transcripts. The likelihood of an open reading frame being a functional coding sequence and gene is vastly increased by detecting the presence of a mRNA transcript. This approach was used effectively

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during the annotation of the EhV-86 genome, where message for 65% of the 472 predicted genes of EhV-86 was confirmed from a lytic phase total RNA extraction [29]. Following the lytic phase transcriptional work the microarray was used to outline the transcriptional profile generated by EhV-86 during the early stages of infection. Using a highly sensitive amplification method, the precise expression profile of the EhV-86 infection of E. huxleyi was determined over the first 4 h of infection [31]. This strategy allowed the designation of EhV-86 CDSs into three groups on the basis of when their transcripts are first detected (1, 2 or 4 h post infection). The results obtained suggested two distinct phases to the infection process: a primary phase dominated by a group of CDSs localized to a sub-region of the genome, which have no database homologues and are associated with a unique promoter element; and a secondary phase during which CDSs are transcribed from the remainder of the genome [30-32]. Since the majority of EhV-86 CDSs are unknown, the designation of CDSs into transcriptional categories is a significant step forward in determining their function. The distinctive transcriptional profile has answered and raised many interesting questions about the infection strategy and life style of the virus, in particular the role and function of the virally encoded RNA polymerase. It is important to note that a microarray based on sequence data for a particular strain is not restricted for sole use with that strain. The Plymouth Virus Collection (PVC) contains 12 virus strains all capable of infecting E. huxleyi [33]. These strains were collected over the 4year period between 1999 and 2003 from sampling sites in the English Channel and in a Norwegian Fjord [25, 27, 34]. The construction of the coccolithovirus microarray allowed the genetic diversity of the viruses in the PVC to be assessed. A major disadvantage of using a specific strain/species microarray for this purpose is that the microarray can only tell you what is highly variable or not present in a particular genome. Genomic additions go undetected since there is no probe on the array to detect it. However, the use of the array for screening for genomic content can provide a wealth of information on genetic diversity within a family of closely related species. At least 70 genes found in EhV-86 are absent or highly variable from one or more of the genomes of the coccolithoviruses found in the PVC [33]. The distinctive pattern of genetic content displayed by each of the strains (essentially a genomic fingerprint) suggested a complicated series of gene loss/gain events. In particular, a high frequency of deleted or variable genes was found in a 100 kb region of unknown function suggesting this section of the genome may be under a high selection pressure [30, 33]. Thus the microarray method is a relatively cheap, efficient and quick way to glean an insight into the genetic content of a family of closely related species. Genes of interest that are conserved among all strains or are lacking and/or variable in some strains can be identified rapidly for further downstream analysis. In summary, over a relatively short period of time, a predominantly uncharacterized virus has had its genome annotated, the kinetic profile of its transcripts determined and the diversity within its family assessed through the use of microarrays [29, 31, 33, 35]. The results have indicated a plethora of new and novel genes, a distinctive biphasic transcriptional pattern during the infection process and an unexpectedly large degree of genomic content variability among the coccolithoviruses. The construction of the microarray rapidly increased the understanding of the virus and its infection strategy and serves an excellent model for other researchers to follow.

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CASE STUDY 2: GENE DISCOVERY AND SYSTEM-WIDE OVERVIEWS OF ENVIRONMENTAL STRESS RESPONSES IN THE COMMON CARP The Science Abiotic environmental stress, such as heat and cold, hypoxia, UV and pollution pose major problems for all forms of life, and organisms respond to stress by both protecting themselves from the damaging effects of extreme stress, but also by adjuting themselves to allow life processes continue unaffected by the change in environmental conditions. Animals from temperate climatic zones and which inhabit small freshwater ponds are routinely exposed to seasonal fluctuations of temperature and daily fluctuations of environmental oxygen concentrations, and survival depends upon the existence of powerful stress-coping mechanisms [36-38]. Understanding the underpinning mechanisms of stress responses and adaptation is important not only academically but practically in agriculture and in human society [39]. Cold presents special problems both in the form of reduced biochemical activity but also from the damaging effects of freezing [36]. Our understanding of response patterns to chronic cold in complex animals has been transformed by microarray analyses, from one composed of a few key or ‗candidate‘ genes to one where the system-wide effects on largescale networks of genes and proteins can be appreciated [40, 41]. Since 2000 NERC has funded a large-scale analysis of response patterns at the transcript level in the tissues of the common carp, Cyprinus carpio L..

The Array Given that there were no genomic resources for the carp, it was first necessary to generate a collection of cDNA clones, representing genes, all of which was accomplished by the MGFLiverpool. Fish were exposed to the full range of environmental treatments under investigation, including hypoxia, and RNA isolated, normalised to rebalance representation the representation of genes, and then cloned directionally into a suitable vector. The inserts were sequenced from the 5‘ end, clustered clones into groups containing overlapping sequences and then their identity established by homology searching. The inserts of 13,500 clones were amplified by PCR and the amplicons were used to print a microarray on glass slides using a contact-printing robot. More recently, another 20,000 clones have been added to the collection which were ‗cherry-picked‘ to reduce redundancy. The new, more representative collection consists of 26,000 clones, which have been printed using a noncontact, piezo-deposition robot. The evolution of the carp array has been detailed in Williams et al (2008) [42]

The Research Our first experiment [9] followed changes in the expression of genes in 7 different tissues of the carp during long-term transition from warm (30°C) to cold conditions (10°C). The fish

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were cooled over a 3-day period and then held at 3 cooled temperatures (23, 17 and 10°C) for up to 21 days. On each sampling interval 6-8 animals were killed and the tissues dissected into a plastic (‗sushi) tray for long-term freezing at -80°C. Samples of these tissues were used to generate target cDNA, which were hybridised to over 400 high-density cDNA arrays. The data was subjected to refined statistical analysis to reveal a very complex picture of gene responses [9]. Over 3000 probes were found to be significantly different between warm and cooled specimens in at least one tissue. The first focus was on genes that were differentially expressed (DE) in all 7 tissues, which generated a list of 260 genes of which 180 were identified. This list was composed of genes that were largely involved in cell homeostasis, with the greatest and most consistent responses being for the ∆9-desaturase and CIRBP. The former gene is widely invoked in cold responses as being the key gene in transforming lipids from saturated to more unsaturated, thereby fluidising the cold-ordered lipids in cell membranes [43, 44]. The latter gene is an RNA-binding protein of unknown function, but its prominent responses indicate that it is important to all tissues during cold adaptation. A large number of genes involved in protein turnover were found, indicating strong up-regulation in all tissues. The second focus was on the remaining ~1,700 identified genes that were regulated in one or more tissues. Clustering of this data using unsupervised techniques generated 23 groups of genes with characteristic tissue-specific responses. Each cluster was profiled by statistical analysis of the Gene Ontology annotations [45] and this pointed directly at the nature of gene responses in relation to tissue function. For example, cluster 5 was composed of genes that were up-regulated exclusively in the intestinal mucosa. This cluster included an unusually large number of transport-associated genes, which is entirely consistent with the role of the intestinal epithelium in nutrient absorption and ion/pH regulation. This gene profiling technique allows the broader gene expression properties across treatment space to be interpreted within the context of tissues function. Comparing between clusters allowed the identification of major effects in the pathways of intermediary metabolism [9] [46]. Thus, it was found that genes of glycolysis were generally up-regulated in heart and brain, but down-regulated in the other tissues, indicating a shift between tissues in the patterns of energy provision and storage. Consistent responses were identified in all of the major tissues, despite them being mediated by different genes or isoforms. Thus brain, liver, heart and liver all showed a very high representation of genes involved in energy pathways and carbohydrate metabolism in the responding gene clusters, which is consistent with a cold-induced modification of the substrates and sources of energy metabolism, and of the pathways involved. A second major experiment involved the exposure of carp to chronic hypoxia [47]. Carp is unusually tolerant to hypoxia which allows it to prosper in natural ponds. This experiment involved over 700 microarrays, comprising responses to hypoxia in 7 tissues but also at two environmental temperatures, 17°C and 30°C. Again this revealed strong patterns within and between tissues which were surprisingly different between the two exposure temperatures. Curiously, a transcript corresponding to myoglobin was substantially up-regulated by hypoxia in carp liver. Myoglobin is an oxygen-binding protein which was famous for its role in protecting oxidative muscle cells including cardiac myocytes. Expression of this gene in nonmuscle tissues is linked to the presence of myoglobin protein in the liver of hypoxia-treated fish [48] and that the amount of protein increases in carp exposed to chronic hypoxia. It was also found that carp possess a second isoform of this important gene, whose expression was

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limited to the brain, this being the only known vertebrate species possessing two myoglobin isoforms. This fact is likely to be linked to a whole genome duplication event some 15MYA in a clade of fish that includes the goldfish and crucian carp [49]. This discovery of non-muscle expression of this gene was an entirely serendipitous and unexpected finding, arising directly from an open microarray screen that was directed at the discovery of both genes and their environmental responses. It is notable because it flies in the face of conventional respiratory physiology of vertebrates animals. Similar myoglobin expression has been found in zebrafish and the mouse system is now being explored. This phenomenon is likely to have major implications for understanding hypoxia responses in vertebrate animals generally but particularly in humans where hypoxia injury caused by ischaemia and infarct are major clinical conditions in the developed world. Table 1. Integration of different UK research facilities in the microarray research What? DNA sequencing Sample acquisition Sequencing Annotation Microarray Construction Oligo design Oligo synthesis cDNA clones and EST sequencing Microarray printing or submission of in silico oligoprobes to 3rd part fabrication Microarray experiment Experimental design Sample acquisition Array hybridisation Image acquisition and quantification Data analysis Interpretation Publication

Researcher   

Who? MGF- MGF-Liverpool MGF-Liverpool NEBC Edinburgh (AGF) (LMF)  

 

 

   









   

  

  

  





  

The combination of data from both cold and hypoxia treated carp constitutes a very large data set which can be used to explore the correlation of gene expression properties between pairs of genes. This allows the regulatory relationships between genes to be explored, as whether the correlated profiles between genes are conserved between species. More than 400 gene pairs have been identified whose close expression relationships are conserved between carp and human (Li, W and AR Cossins, to be published), and thus by extension across all vertebrate animals. Finally, the expression data collected on such a large scale and across several different tissues and stress modalities, constitutes another resource with which to differentiate the identities of closely-related members of gene families, which completes the

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more usual sequence-based approaches. This is a particular problem in recently duplicated genomes, such as the common carp [49] and the salmonid fish. Indeed, genes given a common identity by homology alignment proved to have distinguishable expression properties. On further investigation these expression forms have been found to constitute separate genes.

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[6] [7] [8] [9]

[10] [11]

[12]

[13] [14] [15]

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[16] Hancock, D., et al., maxdLoad2 and maxdBrowse: standards-compliant tools for microarray experimental annotation, data management and dissemination. BMC Bioinformatics, 2005. 6(1): p. 264. [17] Morrison, N., et al., Annotation of environmental OMICS data: application to the transcriptomics domain. Omics, 2006. 10(2): p. 172-8. [18] Field, D., et al., Open software for biologists: from famine to feast. Nat Biotechnol, 2006. 24(7): p. 801-3. [19] Brazma, A., et al., Minimum information about a microarray experiment (MIAME)toward standards for microarray data. Nat Genet, 2001. 29(4): p. 365-71. [20] Sansone, S.A., et al., The metabolomics standards initiative. Nat Biotechnol, 2007. 25(8): p. 846-8. [21] Taylor, C.F., Standards for reporting bioscience data: a forward look. Drug Discov Today, 2007. 12(13-14): p. 527-33. [22] Westbroek, P., et al., A Model System Approach to Biological Climate Forcing - the Example of Emiliania huxleyi. Global and Planetary Change, 1993. 8(1-2): p. 27-46. [23] Westbroek, P., et al., Emiliania huxleyi as a key to biosphere-geoshere interactions., in The Haptophyte Algae., J.C. Green and B.S.C. Leadbeater, Editors. 1994, Clarendon Press: Oxford. p. 321-334. [24] Jacquet, S., et al., Effects of inorganic and organic nutrient addition on a coastal microbial community (Isefjord, Denmark). Marine Ecology-Progress Series, 2002. 228: p. 3-14. [25] Schroeder, D.C., et al., Coccolithovirus (Phycodnaviridae): Characterisation of a new large dsDNA algal virus that infects Emiliania huxleyi. Archives of Virology, 2002. 147(9): p. 1685-1698. [26] Wilson, W.H., G. Tarran, and M.V. Zubkov, Virus dynamics in a coccolithophoredominated bloom in the North Sea. Deep-Sea Research Part Ii-Topical Studies in Oceanography, 2002. 49(15): p. 2951-2963. [27] Wilson, W.H., et al., Isolation of viruses responsible for the demise of an Emiliania huxleyi bloom in the English Channel. Journal of the Marine Biological Association of the UK., 2002. 82: p. 369-377. [28] Wilson, W.H., et al., Family: Phycodnaviridae., in Virus Taxonomy, VIIIth ICTV Report, C.M. Fauquet, et al., Editors. 2005, Elsevier/Academic Press: London. p. 163175. [29] Wilson, W.H., et al., Complete Genome Sequence and Lytic Phase Transcription Profile of a Coccolithovirus. Science, 2005. 309(5737): p. 1090-2. [30] Allen, M.J., et al., Evolutionary History of the Coccolithoviridae. Mol Biol Evol, 2006. 23(1): p. 86-92. [31] Allen, M.J., et al., Locus-specific gene expression pattern suggests a unique propagation strategy for a giant algal virus. J Virol, 2006. 80(15): p. 7699-705. [32] Allen, M.J., D.C. Schroeder, and W.H. Wilson, Preliminary characterisation of repeat families in the genome of EhV-86, a giant algal virus that infects the marine microalga Emiliania huxleyi. Arch Virol, 2006. 151: p. 525–535. [33] Allen, M.J., et al., Use of microarrays to assess viral diversity: from genotype to phenotype. Environ Microbiol, 2007. 9(4): p. 971-982. [34] Schroeder, D.C., et al., Virus Succession Observed during an Emiliania huxleyi Bloom. Applied and Environmental Microbiology, 2003. 69(5): p. 2484-2490.

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[35] Allen, M.J. and W.H. Wilson, The coccolithovirus microarray: an array of uses. Brief Funct Genomic Proteomic, 2006. 5(4): p. 273-9. [36] Cossins, A.R. and K. Bowler, The Temperature Biology of Animals. 1987, London: Chapman & Hall. 339. [37] Wendelaar Bonga, S., The stress response in fish. Physiological Reviews, 1997. 77: p. 591-625. [38] Bickler, P.E. and L.T. Buck, Hypoxia tolerance in reptiles, amphibians, and fishes: Life with variable oxygen availability. Annual Review of Physiology, 2007. 69: p. 145-170. [39] Feder, M., Key issues in achieving an integrative perspective on stress. J. Biosciences, 2007. 32: p. 433-440. [40] Gracey, A.Y. and A.R. Cossins, Application of microarray technology in environmental and comparative physiology. Annual Reviews of Physiology, 2003. 65: p. 231-259. [41] Cossins, A.R. and D.L. Crawford, Opinion - Fish as models for environmental genomics. Nature Reviews in Genetics, 2005. 6(4): p. 324-333. [42] Williams, D., et al., Genomic resources and microarrays for the common carp (Cyprinus carpio L). J. Fish Biology 2008: p. In press. [43] Tiku, P.E., et al., Cold-induced expression of ∆9-desaturase in carp by transcriptional and posttranslational mechanisms. Science, 1996. 271: p. 815-818. [44] Polley, S.D., et al., Differential expression of cold- and diet-specific genes encoding two carp liver delta 9-acyl-CoA desaturase isoforms. American Journal of Physiology; Regulatory, Integrative and Comparative Physiology, 2003. 284(1): p. R41-50. [45] Ashburner, M., et al., Gene Ontology: tool for the unification of biology. Nature Genetics, 2000. 25: p. 25-29. [46] Cossins, A., et al., Post-genomic approaches to understanding the mechanisms of environmentally induced phenotypic plasticity. Journal of Experimental Biology, 2006. 209: p. 2328-2336. [47] Nikinmaa, M. and B.B. Rees, Oxygen-dependent gene expression in fishes. Am J Physiol Regul Integr Comp Physiol, 2005. 288(5): p. R1079-90. [48] Fraser, J., et al., Hypoxia-inducible myoglobin expression in nonmuscle tissues. Proceedings of the National Academy of Sciences USA, 2006. 103: p. 2977-2981. [49] David, L., et al., Recent duplication of the Common Carp (Cyprinus carpio L.) genome as revealed by analyses of microsatellite loci. Molecular Biology and Evolution, 2003. 20: p. 1425-1434.

Reviewed by William H. Wilson of Bigelow Laboratory for Ocean Science Research, Maine, USA.

INDEX A abatement, 126, 458 absorption, x, 43, 64, 77, 173, 175, 176, 179, 192, 212, 320, 322, 476 abstraction, 326, 335, 353 abuse, xv, 423 accessibility, 237, 238, 242, 244, 245, 249, 254, 255 acclimatization, 235 accounting, 21, 212 accreditation, 403 accuracy, 27, 290, 292, 293, 351, 352, 353 acetic acid, 431 acid, 17, 19, 27, 33, 120, 122, 141, 144, 153, 155, 156, 192, 222, 226, 227, 228, 233, 235, 236, 362, 368, 369, 377, 379, 431, 435, 438, 440, 441, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 455, 456, 457, 458, 459, 460, 461, 462, 463 acidity, 446 activated carbon, 130, 179, 180, 434, 436, 445, 449, 450, 451, 452, 461, 462, 463 activation energy, 449, 457 active centers, 446 active site, 212, 446, 447 adaptability, 96 adaptation, 118, 373, 475, 476 additives, 31, 150, 154, 387, 403 adjudication, 403 adjustment, 97, 265, 395 adsorption, x, 18, 65, 68, 70, 97, 173, 175, 176, 177, 179, 182, 436, 442, 445, 446, 447, 449, 450, 451, 452, 459, 461, 462 advantages, xiii, 25, 31, 61, 62, 64, 81, 221, 357, 364, 378, 410, 434, 436, 470 advocacy, 120 aerobic bacteria, 48

aerogels, 450, 463 aflatoxin, xv, 386, 400, 410 AFM, 183 Africa, 5, 17, 20, 35, 116, 400 agar, 25, 26, 34, 222 agencies, xiv, 385, 391, 393, 398, 403, 411 aggregation, 221 aging process, 259, 260, 265, 266, 267 agricultural exports, xiv, xv, 385, 386, 399 agricultural market, 402, 403, 406 agricultural sector, 309, 310, 312 agriculture, 54, 75, 84, 100, 127, 141, 153, 162, 220, 307, 308, 309, 310, 311, 312, 315, 319, 387, 398, 399, 408, 416, 417, 424, 475 AIDS, 24, 25 air emissions, 155 air pollutants, 112 alanine, 227, 233 aldehydes, 448 algae, 24, 36, 436 algorithm, 469 allele, 27 alters, 379 aluminum oxide, 460 amino acid, 227, 235 amino acids, x, 63, 85, 141, 219, 224, 227, 228, 233 ammonia, 55, 82, 123, 163, 227, 358, 359, 365, 366, 370, 374, 376, 377, 378, 380, 382 ammonium, 63, 82, 377, 462 amphibians, 480 amplitude, 259 amylase, 82, 163 anaerobic bacteria, 60 anaerobic sludge, 46, 146 anatase, 446 anger, 468

482

Index

animal disease, 358, 417 animal welfare, xiv, 386 anionic surfactants, 437, 455 annealing, 383 annotation, 468, 469, 473, 479 annual rate, 399 ANOVA, 470 anoxia, 478 antagonism, 85 anthrax, 358, 380 antibiotic, 401, 410 antibody, 28 antioxidant, 280 anus, 45 apples, 425 aquaculture, 411 aquatic habitats, 320 aquatic life, 427 aqueous solutions, 174, 175, 182, 224, 453, 454, 457, 458, 459, 460, 461, 463 aquifers, xii, 16, 325, 326, 327, 328, 331, 333, 334, 335, 337, 338, 339, 350, 352, 353, 354 architecture, 278 Argentina, 100, 107, 174 Aristotle, 43 aromatic compounds, 373, 436, 441, 454 aromatic hydrocarbons, 43, 61, 76, 454 aromatics, 147 arrest, 45, 139, 152 arrests, 75 ARs, xiv, 385 arsenic, ix, x, xiii, 130, 156, 173, 174, 175, 325, 326, 327, 328, 338, 353 arthritis, 20, 38 arthropods, 84 asbestos, 131, 152, 154 aseptic, 16 Asia, xv, 17, 20, 76, 105, 114, 116, 120, 168, 287, 289, 386, 391, 393, 399, 419, 420, 421, 423 asian countries, 114, 399 assessment, xii, 5, 6, 31, 164, 191, 213, 254, 285, 289, 299, 304, 318, 324, 366, 374, 382, 383, 393, 395, 429 assimilation, 319 atmosphere, vii, 3, 130, 428 atmospheric pressure, 130 atomic force, 183 atomic force microscope, 183 atoms, 435 atypical pneumonia, 24

Austria, 37, 114, 157, 164, 168 authorities, 100, 116, 309, 310, 314, 400 automation, xiii, 29, 357 automobiles, 122, 123, 136, 141, 148, 156 avoidance, 134 awareness, xii, xiv, 31, 119, 132, 133, 135, 158, 305, 306, 310, 319, 320, 385, 396, 410, 411, 416, 417, 466

B BAC, 463, 468 Bacillus subtilis, 222, 371 bacteria, viii, xiii, 12, 13, 19, 20, 23, 24, 25, 29, 32, 36, 39, 43, 48, 55, 60, 64, 75, 78, 82, 83, 85, 97, 99, 107, 122, 126, 127, 145, 146, 147, 162, 222, 357, 358, 359, 361, 362, 363, 365, 367, 369, 370, 374, 377, 379, 381, 382, 383, 387, 391, 413, 414, 415, 428, 434 bacterial infection, 24 bacterium, 19, 25, 26, 98, 358, 382 Bangladesh, vi, xiii, 174, 325, 326, 327, 328, 353, 354, 355 banks, 240, 316, 323 barium, 119, 156 barometric pressure, 338 barriers, 30, 156, 401, 407 base pair, 367, 368, 382 basicity, 446 baths, 120, 192 batteries, 121, 122, 123, 124, 136, 141, 149, 155 bauxite, 149, 153 beef, 18, 222 behaviors, 255, 447 Beijing, 323 Belgium, 116, 139, 155 beneficial effect, 472 benefits, xv, 122, 142, 162, 242, 252, 255, 424, 430, 433, 468 benzene, 123, 129, 156, 461, 462 benzo(a)pyrene, 98 beta particles, 125 beverages, 150 bias, 29, 360, 377, 381 bicarbonate, 374, 412, 438 bile, 25 bioaccumulation, 77, 80, 188, 434 biochemistry, 391 bioconversion, 147, 161 biodegradability, 122, 127

Index biodegradable wastes, 47, 126 biodegradation, viii, x, 42, 43, 49, 51, 58, 59, 64, 65, 78, 82, 97, 98, 99, 108, 124, 126, 127, 141, 144, 145, 159, 169, 219, 220, 221, 222, 223, 224, 225, 227, 233, 234, 235, 382, 437 biodiversity, 286, 289, 306, 320, 322, 323, 360 biofuel, 146, 159 biogas, 146, 159, 160, 161, 162 bioindicators, 188, 216 bioinformatics, xvi, 465, 466, 467 biological control, 392 biological stability, 13 biological systems, xi, 257, 258, 264, 265, 466 biomarkers, 268 biomass, vii, 3, 7, 43, 45, 47, 48, 58, 59, 60, 62, 63, 65, 67, 76, 97, 105, 126, 127, 143, 145, 146, 147, 162, 163, 226, 235, 236, 369, 371 biomonitoring, xi, 271, 272 bioremediation, 81 biosensors, 29, 36 biosphere, 100, 258, 479 biosynthesis, 282 biotechnology, viii, 42, 82, 122, 143, 144, 392 biotic, 63, 290 biotin, 371 birds, xi, 154, 160, 163, 217, 271, 272, 273, 274, 275, 277, 279, 281, 282, 323, 324, 426, 427, 429 bisphenol, 75 blood pressure, 425 body fat, x, 187 body fluid, 121 body size, 192, 207 body weight, 43, 192, 193, 206, 208, 393, 395 boilers, 149, 150, 156 bonds, 453 bone, 189, 191, 205, 212 bones, 44, 52, 118, 220 boundary conditions, 352 brain, 16, 476, 477 Brazil, 45, 145, 147 breakdown, 99, 145, 163, 213, 405 breeding, xi, 271, 274, 275, 279 brevis, 30 bridges, 148 Britain, 242, 255 Brno, 215 buffalo, 49 buildings, 131, 152, 241, 307, 426 Bulgaria, 257, 271 Burma, 289

483

burn, 264 bursa, 191 butyl ether, 447 buyers, 132, 138, 139, 165, 404 by-products, xv, 70, 122, 154, 433, 435, 441, 447, 450, 460

C cabbage, 84 cabinets, 121 cadmium, x, 54, 57, 64, 76, 77, 119, 123, 130, 155, 156, 187, 191, 193, 196, 198, 201, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217 calcium, 56, 59, 77, 85, 153, 473 calcium carbonate, 59, 473 campaigns, 416, 417 cancer, 17, 378, 391, 392, 393, 425, 426 candidates, 307 capillary, 468 capital intensive, 131 capitalism, 252 capsule, 127, 189 carbohydrate, 233, 476 carbohydrate metabolism, 476 carbohydrates, 44, 49 carbon, vii, 3, 4, 7, 11, 33, 35, 61, 62, 63, 64, 123, 125, 126, 128, 130, 143, 145, 146, 147, 153, 156, 159, 179, 180, 220, 234, 376, 434, 436, 449, 450, 451, 452, 453, 454, 457, 459, 461, 462, 463 carbon dioxide, 61, 62, 125, 128, 145, 159, 220 carbon materials, 461 carbon monoxide, 64, 128, 147 carboxylic acids, 448, 459 carcinogenicity, 122 Caribbean, 242 carotenoids, 279, 282 carp, 467, 469, 475, 476, 477, 480 cartilage, 189 case study, 251, 255, 323, 377 cash, 140 casting, 141 catalysis, 442, 445, 447, 458, 461 catalyst, 440, 441, 442, 444, 445, 446, 447, 448, 449, 450, 451, 452, 456, 458, 459, 460, 461, 462 catalytic activity, 440, 445, 446, 447, 448, 449, 450, 451, 463 catalytic effect, 443 catalytic reaction, 449, 450 catalytic system, xvi, 433, 435

484

Index

category a, 121 cation, 224, 443 cattle, 21, 45, 48, 50, 56, 59, 61, 63, 85, 86, 87, 89, 90, 117, 143, 150, 235, 293, 361, 406 CBS, 280 cDNA, 373, 375, 378, 469, 475, 476, 477 cell culture, 27, 371 cell line, 25, 362, 373 cell membranes, 476 cellulose, 46, 48, 49, 145, 222 cement, 21, 112, 115, 141, 146, 150, 151, 153, 154, 156 cement plants, 141, 154 census, xi, 237, 238, 239, 240, 241, 242, 247, 255 ceramic, 138, 150, 151, 192, 222 certificate, 409 certification, 407, 410, 412, 417 cesium, 125 cestodes, 188, 216 charitable organizations, 155 cheese, 143 chemical, viii, 17, 19, 21, 42, 43, 44, 46, 48, 54, 55, 56, 58, 62, 67, 70, 76, 77, 78, 81, 82, 85, 87, 89, 90, 91, 92, 95, 96, 99, 100, 119, 122, 123, 124, 125, 126, 130, 135, 137, 138, 142, 144, 145, 147, 150, 153, 154, 156, 174, 182, 183, 222, 235, 236, 259, 260, 378, 393, 394, 395, 407, 412, 428, 429, 434, 436, 437, 439, 445, 446, 449, 450, 463 chemical kinetics, 259 chemical properties, 236, 378, 407 chemical reactions, 58 chemical reactivity, 122 chemical stability, 446 chemicals, viii, ix, 42, 43, 44, 54, 55, 61, 66, 75, 76, 77, 78, 82, 84, 90, 98, 118, 119, 121, 122, 123, 129, 130, 133, 138, 139, 141, 158, 394, 408, 412, 416, 418, 426, 427, 428, 431 chemiluminescence, 25 chemoreceptors, 44 Chicago, 302, 376 chicken, 53, 281 child labor, xiv, 386 childhood, 34 Chile, 174, 400 China, 45, 67, 114, 120, 139, 145, 289, 322, 323, 423 chitin, 21, 84 chlorinated hydrocarbons, 130 chlorination, viii, 12, 13, 19, 30, 36, 437 chlorine, viii, 12, 21, 30, 401, 413, 415, 436, 452 chlorobenzene, 447, 460

chloroform, 274 cholera, 20, 23, 35 cholinesterase, 427 chromatography, 454 chromium, 77, 119, 123, 156 chromosome, 369 chronic diseases, 260, 267, 425 chronic hypoxia, 476 chronic illness, 426 chronology, 377 circulation, vii, 3, 209, 210, 212, 214 cities, 12, 124, 131, 145, 396 citizens, xv, 133, 386 city, 11, 55, 112, 140, 161, 166, 168, 240, 421 civilization, viii, 42, 97 class, 114, 121, 211, 290, 293, 427, 428 classes, 153, 290, 397 classification, 5, 6, 158, 252 cleaning, 46, 68, 76, 136, 143, 154, 156, 413, 415, 456 cleanup, 152 clients, 470 climate, vii, 3, 4, 5, 6, 7, 50, 165, 307, 308, 313 climate change, 5, 7, 50, 165 clitellum, 44 clone, 358, 359, 360, 366 cloning, 282, 359, 360, 378, 469 closure, 160 clusters, 472, 476 coagulation process, 436 coal, 78, 120, 145, 148, 149, 153, 156, 159, 398 coastal management, 322 cobalt, 457, 458 cocoon, 45 coding, 381, 473 coffee, 399 coke, 148, 149 colonization, 24, 31, 39, 68 color, 56, 150, 222, 282, 328, 401, 407, 434, 436, 441, 449 colour patches, 272 combined effect, x, 187 combustion, vii, 3, 123, 130, 146, 147, 153, 164, 175 commercial crop, 221 commodity, 113, 165, 394 communication, 26 community, xvi, 13, 24, 29, 32, 38, 39, 40, 61, 108, 135, 161, 164, 166, 169, 252, 286, 293, 335, 359, 360, 361, 364, 369, 371, 372, 373, 374, 375, 376,

Index 377, 378, 379, 380, 381, 382, 383, 384, 392, 452, 465, 466, 467, 471, 472, 479 competition, 203, 279, 364, 407, 467 competitive advantage, 165 competitiveness, xiv, 385 competitors, xiv, 386 complaints, 428 complement, 466 complementary DNA, 382 complexity, ix, 112, 131, 258, 259, 436, 451, 452, 472 compliance, xiv, 386, 404, 408, 409, 416, 472 complications, 19 composites, 121 composition, 46, 54, 118, 144, 163, 221, 222, 224, 227, 228, 233, 242, 243, 247, 290, 312, 313, 355, 360, 364, 366, 374, 376, 377, 379, 382, 384, 438 compost, 43, 46, 47, 54, 55, 59, 60, 61, 62, 63, 78, 79, 80, 82, 85, 86, 87, 89, 90, 93, 94, 95, 97, 99, 102, 105, 126, 136, 140, 141, 143, 144, 145, 146, 160, 161, 162, 163, 222, 227, 235, 236, 431 composting, viii, ix, 42, 43, 44, 45, 47, 49, 50, 55, 57, 58, 59, 60, 61, 62, 63, 87, 96, 97, 98, 99, 105, 106, 107, 108, 112, 125, 126, 127, 137, 143, 144, 145, 159, 161, 162, 163, 164, 222, 234, 236 compounds, xv, 17, 62, 63, 76, 78, 79, 80, 82, 98, 119, 123, 128, 129, 130, 150, 156, 177, 179, 181, 220, 221, 226, 227, 231, 367, 373, 391, 423, 426, 433, 434, 436, 437, 438, 440, 441, 442, 443, 445, 446, 448, 449, 450, 451, 452, 453, 454, 455, 456, 460, 462 compression, 220 computing, 471 conceptual model, 253 conditioning, 151, 319 conductance, 25, 34 conduction, 333 conductivity, 65, 221, 258, 327, 331, 340, 341, 342, 352, 355 conference, 104 configuration, 159, 436 conflict, 309, 313 conformity, 409 connective tissue, 210 connectivity, xiii, 287, 307, 321, 325, 338, 353 consciousness, 132 consensus, 131 consent, 312 conservation, 126, 138, 164, 165, 286, 287, 289, 290, 291, 292, 301, 302, 304, 305, 307, 308, 309,

485

310, 313, 314, 315, 316, 317, 318, 320, 321, 322, 323, 324 constant rate, 332, 350 constitution, 286 construction, 30, 113, 128, 131, 140, 144, 145, 148, 152, 153, 165, 307, 308, 311, 316, 319, 351, 353, 467, 469, 472, 473, 474 consumer choice, 254 consumer demand, 395, 396 consumer education, 132, 164 consumer electronics, 121, 130 consumer goods, 122, 132, 137, 138, 164 consumers, 13, 30, 114, 119, 120, 132, 133, 137, 138, 140, 157, 164, 165, 310, 391, 396, 401, 402, 412, 416, 418 consumption, vii, 11, 16, 17, 20, 22, 33, 38, 64, 112, 113, 114, 132, 133, 135, 136, 139, 147, 148, 157, 162, 164, 189, 213, 224, 391, 393, 394, 396, 406, 407, 412, 425, 438, 447, 449 contact time, 17 contaminant, 31, 99, 188, 191, 434, 443, 444, 446, 447, 450, 452 contaminated sites, 76 contaminated soil, 78, 79, 80, 98 contaminated soils, ix, 42, 43, 76, 78, 99 contaminated water, xiv, 13, 24, 30, 327, 386 contamination, viii, xiii, 12, 13, 16, 17, 25, 31, 35, 123, 162, 174, 211, 263, 272, 325, 326, 354, 391, 392, 398, 399, 400, 403, 412, 413, 415, 416, 428, 429, 431, 434 content analysis, 306 contract enforcement, 404 control measures, 406, 407 controversies, 314 convention, 126 cooking, 48, 119, 143, 146, 150, 391, 394, 412, 414, 415 cooling, 48, 64, 404, 414 coordination, 174, 175, 176, 179, 260, 365, 379, 405 copper, 54, 77, 85, 144, 148, 149, 174 copulation, 44 corporate sector, 411 correlation, 208, 279, 372, 477 correlation coefficient, 208 correlations, 268, 376 corrosion, 148 corrosivity, 122 cosmetics, 123, 401 cost, viii, ix, xiii, xiv, xvi, 12, 13, 22, 25, 29, 30, 32, 40, 54, 76, 96, 97, 98, 99, 112, 113, 114, 120, 128,

Index

486

131, 138, 139, 140, 141, 145, 147, 148, 149, 152, 153, 154, 161, 162, 165, 357, 359, 386, 394, 400, 404, 409, 416, 418, 429, 440, 465, 466, 470 cost effectiveness, 418 costs of compliance, 408 cotton, 127, 136, 138, 149, 169, 424, 427, 430 covering, 47, 159, 286, 287, 293, 300, 306, 312, 388, 406, 407, 466 coxsackievirus, 17 cracks, 413 crew, 221 crisis management, 408, 418 critical period, 425 critical value, 248 criticism, 311 crop production, 221 crops, 18, 39, 84, 85, 87, 89, 90, 95, 96, 100, 150, 221, 235, 388, 391, 392, 393, 394, 406, 412, 424, 425 cross-fertilization, 44 crude oil, 43 crust, 125 cryptosporidium, 37 crystalline, 175 cues, 279 cultivation, 221, 231, 232, 308, 311, 377, 412 cultural barriers, 401 culture, 25, 26, 27, 28, 36, 37, 65, 113, 119, 132, 162, 163, 219, 221, 222, 223, 229, 230, 231, 232, 233, 234, 235, 236, 358, 359, 373, 468 culture media, 25, 37 cumulative frequency, 247, 248 cumulative percentage, 248 current limit, 380 curricula, 417 customers, 140 cuticle, 44, 212 cyanide, 130, 456 cycles, 7, 19, 21, 38, 449, 450 cycling, 7, 366, 367, 371, 383, 473 cyst, x, 24, 39, 187, 189, 190, 191, 193, 194, 196, 197, 198, 199, 201, 202, 203, 204, 208, 209, 210, 211 cystathionine, 280, 283 cysteine, xii, 227, 233, 272, 280, 283

D damages, 260, 264 danger, 30, 121

Darwin, Charles, 43, 96, 100 data analysis, 29, 351, 353, 355, 466, 470 data collection, 32, 338 data set, 344, 345, 472, 477 database, 5, 239, 240, 241, 375, 378, 471, 474, 478 Dead Sea, 308 deaths, xv, 18, 121, 386, 425, 426 decay, 75, 244, 245 decentralization, 161 decision makers, 310, 318, 319, 320 decomposition, vii, xv, 3, 5, 6, 7, 43, 48, 50, 59, 61, 63, 64, 96, 126, 127, 159, 160, 162, 163, 235, 236, 433, 435, 436, 438, 439, 444, 445, 446, 447, 448, 449, 450, 451, 453, 454, 456, 457, 458, 460, 461, 463 defects, 393 deficiencies, 306, 312, 317, 319, 320 deficiency, 221, 231, 252, 279, 283 deforestation, 137, 148, 164, 466 degradation, viii, xv, 17, 42, 46, 49, 50, 52, 55, 58, 59, 65, 86, 98, 99, 125, 145, 338, 360, 373, 377, 378, 433, 434, 437, 438, 439, 440, 441, 443, 446, 451, 455, 456, 457, 459, 460, 461, 463 degradation process, 46 Degussa, 440, 441, 442 dehydration, 22, 54, 191 dematerialization, 136 demographic data, 302 demography, 126 denitrification, 365, 366 denitrifying, 236, 377, 382, 434 Denmark, 20, 116, 131, 139, 155, 164, 479 Department of Agriculture, 405, 407, 409, 421, 430 Department of Energy, 83 Department of Health and Human Services, 33, 430 Department of the Interior (DOI), 355, 431 dependent variable, 208, 289 deposition, 6, 68, 198, 209, 212, 213, 214, 279, 323, 327, 475 deposits, x, 40, 187, 191, 206, 207, 212, 214 depression, 331, 333, 346 deprivation, xi, 237, 238, 242, 243, 247, 249, 250, 251, 252, 255 derivatives, 440 desorption, x, 173, 174, 175 destination, 244, 245 destiny, 182 destruction, viii, 42, 59, 63, 130, 213, 293, 392, 458, 459, 460, 466

Index detection, xiii, 17, 25, 26, 27, 28, 29, 31, 32, 33, 34, 35, 36, 37, 39, 40, 272, 273, 357, 362, 363, 364, 365, 366, 367, 369, 370, 371, 373, 374, 375, 377, 378, 379, 380, 381, 382, 383, 384, 400, 401, 408, 441, 466 detection system, 28 detergents, 122, 415 detoxification, 130, 209, 210 developed countries, vii, 11, 12, 17, 21, 22, 30, 31, 32, 124, 163 developed nations, 115, 117, 120, 139, 154, 155 developing countries, vii, xiv, 11, 17, 19, 20, 21, 116, 155, 158, 165, 174, 386, 416, 426, 430 developing nations, 76, 120, 126 deviation, 245 diabetes, 425 diagnosis, 26, 268, 430 diamonds, 177 diaphragm, 210 diarrhea, 18, 38, 421 diesel engines, 146 diet, xii, 189, 209, 212, 272, 273, 279, 281, 391, 393, 394, 395, 398, 425, 480 dietary intake, 209, 210, 394 diffraction, 175, 176 diffusion, 258, 365, 444, 445 digestion, 23, 136, 161, 192, 441, 456 dimorphism, 211, 281 dioxin, 43, 120 dioxins, 119, 130, 131, 146, 151, 154, 388 direct investment, 410 directives, 157, 164, 318, 320 dirt, 127, 152 disability, 242 disadvantages, xv, 433, 434 discharges, 330 discriminant analysis, 274 discrimination, 366, 367, 383 disinfection, viii, xv, 12, 13, 21, 24, 30, 34, 36, 37, 42, 433, 434, 458 dispersion, 82, 449 displacement, 29, 248, 371 disposable income, 396 dissipative structure, 258 dissociation, 367, 368, 379, 380, 384 dissolved oxygen, 222, 224 distillation, 156 distilled water, 192 disturbances, 259, 273, 286, 293, 303 diversification, 405

487

diversity, xiii, 13, 252, 286, 306, 323, 357, 358, 359, 360, 373, 377, 379, 380, 381, 382, 383, 384, 474, 479 DNA, vi, xiii, 28, 29, 32, 37, 273, 277, 357, 359, 360, 361, 362, 365, 368, 369, 370, 371, 372, 373, 374, 375, 377, 378, 379, 380, 381, 382, 383, 384, 466, 468, 469, 470, 477, 478 DNA extraction, 360, 381 DNA polymerase, 371 DNA sequencing, 466, 468, 477 DNase, 375 DOC, 454 dogs, 21, 116, 117 domestic markets, 387 DOP, 317 dosage, 283 double bonds, 453 draft, 255 drainage, 62, 82, 151, 306, 307, 310, 311, 312, 316, 319, 324 drawing, ix, 112, 471 drinking water, viii, ix, xv, 12, 13, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 31, 32, 33, 34, 35, 36, 37, 38, 40, 120, 151, 173, 174, 393, 428, 431, 433, 435, 436, 452, 453, 454, 460 drosophila, 466, 478 drug resistance, 20 drugs, 387, 401 drying, 192, 308, 406, 410 dumping, 99, 116, 120, 165, 220 duodenum, 210 dyeing, 122 dyes, 27, 123, 130, 362, 455, 457, 458

E E.coli, 18, 60, 61 early warning, 38 earthworms, viii, ix, 41, 42, 43, 44, 45, 46, 47, 48, 50, 51, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 104, 107, 108, 126, 127, 143, 145, 146, 169 East Asia, xv, 386, 399 Eastern Europe, 419 ecology, xiii, 13, 31, 32, 303, 322, 323, 324, 357, 359, 360, 369, 371, 374, 375, 376, 377, 379, 380, 381, 383 economic consequences, 13

488

Index

economic development, 317 economic growth, 395 economic incentives, 392 economic independence, 315 economic losses, 22, 400, 425 economic problem, 140, 155 economy, 97, 116, 136, 138, 140, 141, 309, 424 ecosystem, xii, 122, 126, 136, 138, 141, 148, 278, 285, 286, 289, 302, 313, 321, 322, 466 editors, 38, 282 education, 31, 119, 132, 166 educators, 133 efficiency, 50, 72, 105, 107, 136, 462 effluent, 18, 67, 220, 373, 379, 398, 411, 437, 440, 441, 443, 455, 456 effluents, xii, 36, 46, 67, 150, 161, 220, 305, 306, 307, 308, 378, 434, 436, 439, 440, 443, 451, 455, 460, 462 egg, 21, 52, 139, 149, 394 Egypt, 400 electric charge, 260 electric conductivity, 327 electric current, 130, 148, 258 electricity, 131, 146, 150, 159, 160, 161, 162, 165 electrolysis, 155 electromagnetic, 125, 259 electromagnetism, 258 electron, 175, 176, 183 electronic structure, 174 electrons, 441 electrophoresis, 26, 27, 29, 360, 378, 380, 381 elongation, 83, 227, 229 elucidation, 374 emission, ix, 48, 61, 64, 96, 112, 119, 124, 125, 129, 131, 142, 144, 146, 147, 148, 149, 156, 159, 160, 163, 165, 175, 177, 178, 179, 180 emitters, 125 employment, 242 encephalitis, 16, 388 encephalopathy, xiv, 385 encoding, 358, 380, 480 endangered species, 289, 292 endocrine, 75, 98, 426, 429, 430, 467 endonuclease, 26 energy, viii, xi, 42, 62, 74, 75, 124, 125, 126, 131, 134, 135, 136, 137, 142, 143, 146, 148, 149, 150, 151, 154, 156, 164, 165, 177, 178, 179, 180, 181, 209, 213, 235, 257, 259, 260, 261, 262, 268, 358, 449, 457, 476 energy consumption, 136

energy recovery, 131, 146 energy supply, 165 energy transfer, 260 enforcement, xiv, 309, 314, 385, 402, 404, 407, 408, 411, 416 engineering, 126, 127, 153 England, vi, xi, 37, 39, 45, 145, 237, 238, 239, 252, 254, 473 enlargement, 283 enrollment, 397 enterovirus, 16, 33 entrepreneurs, 404 entropy, 258, 264, 265 environmental awareness, 319, 320 environmental change, 273, 466 environmental conditions, xi, 21, 144, 252, 268, 271, 280, 466, 472, 475 environmental contamination, 31, 211, 416 environmental degradation, 17 environmental effects, 158 environmental factors, 292, 379 environmental impact, 133, 135, 434 environmental influences, xi, 257 environmental issues, 120, 133, 319 environmental management, viii, 41, 320 environmental policy, 305, 324 environmental protection, 137 Environmental Protection Agency (EPA), 13, 22, 109, 166, 428, 431 environmental quality, 252, 313 environmental regulations, 120 environmental temperatures, 34, 476 enzymatic activity, 106 enzymes, 25, 48, 49, 59, 81, 82, 84, 99, 122, 145, 162, 163, 365, 382 eosinophils, 210 epidemic, 17 epidemiology, 22, 26, 32, 40 epithelial cells, 280 epithelium, 476 EPR, 157 equilibrium, 176, 179, 269, 332, 335, 340, 342, 367, 368, 380, 384 equilibrium sorption, 179 equipment, 29, 123, 131, 139, 141, 148, 155, 156, 224, 364, 408, 409, 413, 427, 428, 470 equity, xi, 237, 238, 252, 253, 254, 255, 256 erosion, 6, 139, 145, 156, 221, 327 esophagus, 55, 56 EST, 466, 468, 470, 472, 477

Index ethanol, 143, 146, 147, 175, 176, 177 ethics, 139, 155 ethnic groups, xi, 237, 242, 247, 248, 249, 252, 255 ethnicity, 237, 238, 242, 252 ethyl alcohol, 150 etiology, 22, 23, 37, 380 eukaryotic cell, 373 European Commission, 401 European Community, 115 European Union (EU), xv, 113, 131, 139, 151, 155, 157, 166, 386, 396, 397, 400, 401, 408, 409, 410, 418 evaporation, 150, 160 EXAFS, 175, 176, 184 excretion, 77, 99 execution, 469 exercise, 253 expenditures, 126, 255 experiences, ix, 112 experimental condition, 440, 445, 446, 448 experimental design, 467, 470 expertise, 466, 467, 468, 469, 472 exploitation, xii, 305, 306 exploration, 255, 257, 274, 375 exporter, 400 exports, xiv, xv, 385, 386, 399, 400, 401, 407, 408, 409, 416, 417, 418 exposure, 12, 17, 209, 211, 213, 394, 395, 396, 426, 427, 430, 476 expressed sequence tag, 468, 478 external environment, 21 extinction, 287 extraction, 47, 107, 125, 151, 212, 360, 373, 381, 402, 434, 468, 470, 474

F fabrication, 466, 469, 477 factor analysis, 22 factories, 220, 410 false negative, 302 false positive, 302 famine, 479 farm size, 404, 410 farmers, 43, 45, 82, 85, 96, 100, 144, 145, 404, 405, 406, 407, 410, 411, 416 farmland, 76, 82, 100 farms, 43, 54, 97, 140, 144, 162, 235, 241, 409 fat, x, 187, 191, 192, 205, 206, 207, 212, 214, 407 fatty acids, 150

489

faults, 312, 317 fauna, 43, 293 FDA, 391, 394 fears, 408 feces, 18, 20, 47, 48 feedback, 253, 260, 261, 262 femur, 191, 192, 203, 205, 212 fencing, 151, 241 fermentation, 146, 147, 220, 224, 236 ferrous ion, 457, 458 fertility, viii, 42, 45, 82, 83, 96, 100, 145, 220, 227, 233, 307 fertilization, 44, 221, 222, 231, 235 fertilizers, x, 81, 85, 86, 87, 89, 90, 91, 92, 95, 96, 99, 100, 143, 146, 219, 221, 223, 224, 226, 227, 228, 229, 230, 231, 232, 233, 307, 428 fever, 16, 18 fiber, 152, 450, 463 fibers, 121, 127, 150 fibrin, 210 fibrosis, 378 field trials, 84 films, 122, 151, 457 filters, 40 filtration, viii, 18, 25, 30, 42, 65, 66, 68, 74, 75, 76, 97, 98, 449 financial resources, 155 financial support, xii, 285 Finland, 18, 40, 116 fish, x, 53, 63, 127, 174, 188, 212, 215, 216, 219, 220, 221, 222, 233, 234, 235, 240, 279, 371, 379, 396, 399, 401, 408, 412, 413, 415, 427, 429, 432, 467, 475, 476, 478, 480 fisheries, 220, 235, 399, 401 fishing, 114 fission, 124, 125 fitness, 191 fixation, 99, 361, 366, 374, 381 flame, 130, 192, 414, 415 flammability, 122 flatness, 332 flexibility, 123, 309 flocculation, 30, 436 flooding, 306, 313, 335 flora, 48, 80, 408 flotation, 150 flour, 426 fluctuations, xiii, 308, 325, 335, 338, 353, 475 flue gas, 153 fluid, 20, 47, 64, 75, 136, 463

Index

490

fluorescence, 25, 28, 175, 177, 178, 179, 180, 181, 361, 362, 374, 470 fluorophores, 29 foams, 222 food additives, 387, 403 food industry, 46, 145 food poisoning, 398, 429 food production, 46, 122 food products, 20, 54, 97, 135, 392, 400, 401, 403, 407, 416 food safety, xiv, xv, 385, 386, 387, 388, 393, 396, 397, 398, 399, 400, 401, 402, 403, 404, 405, 406, 407, 408, 410, 411, 416, 417, 418, 419 food security, xiv, 386 foodborne illness, 18, 391, 412, 415 foreign direct investment (FDI), 410, 420 formaldehyde, 123 formamide, 367, 369 formula, 179, 181, 192, 223, 245, 332, 338, 346 fouling, viii, 12, 13 foundations, 152 fragments, 378, 468, 469 framing, 152 France, 45, 46, 115, 125, 139, 145, 146, 164, 165, 436 free radicals, 261, 437, 443, 454 free trade, 120 freezing, 414, 475, 476 frequencies, xi, 209, 259, 271, 278 frequency distribution, 247 freshwater, xiii, 38, 162, 216, 358, 359, 376, 377, 475 fruits, 84, 85, 96, 108, 136, 221, 396, 399, 413, 416 funding, 31, 151, 256, 302, 466, 471 fungi, 24, 43, 60, 78, 82, 83, 84, 97, 122, 126, 127, 145, 235, 387, 428, 442 fungus, 52, 55, 60, 75, 81, 84 fusion, 24, 25, 374

G gamma rays, 125 garbage, ix, 61, 96, 112, 115, 116, 141, 147, 162, 163, 308, 406 gastroenteritis, 16, 18, 20, 22, 32, 37 gastrointestinal tract, 20, 24, 211 GDP, xv, 157, 386, 395 GDP per capita, 395 gel, 26, 27, 360, 364, 365, 367, 368, 371, 378, 380, 381, 382

gender differences, 203 gene arrays, 28, 373, 384 gene expression, xiii, 280, 357, 362, 373, 374, 378, 380, 382, 476, 477, 478, 479, 480 genes, 24, 27, 28, 272, 358, 359, 360, 362, 363, 365, 366, 367, 369, 370, 371, 372, 373, 374, 376, 377, 378, 379, 380, 381, 382, 383, 384, 470, 471, 472, 473, 474, 475, 476, 477, 478, 480 genetic diversity, 474 genetic information, 260, 378 genetic mutations, 265 genetics, 289, 302 genome, 27, 28, 40, 261, 371, 378, 379, 380, 383, 384, 466, 468, 473, 474, 477, 478, 479, 480 genomics, xvi, 465, 466, 468, 472, 480 genotype, 479 geography, 126 geology, 355 Georgia, 168, 255 Germany, 62, 115, 117, 139, 224, 400 germination, 83, 87, 95, 223, 226, 229, 231, 232, 235, 442 gizzard, 49, 56, 81, 97, 99 glasses, 116, 136, 138, 142, 147, 150, 151, 161 global climate change, 5 global consequences, 165 global economy, xiv, 386 globalization, xiv, 386, 399, 416 glucose, 33 glutamate, 280 glutathione, 280, 281, 283 glycine, 227, 233, 280 glycol, 123 glycolysis, 476 gonads, 191 Gore, Al, 174 governance, 309 government intervention, 132 government policy, 164 governments, 31, 132, 137, 155, 166, 394, 402, 405, 411 GPS, 289 grades, 149, 407, 410, 416 gradient formation, 234 grading, 290, 404, 407, 411 granules, 280 graph, 347, 348, 351, 353 grass, 48, 59 grasses, 47, 48, 147 grassroots, 120

Index gravity, 21, 69, 75, 129, 156, 160, 407 grazing, 114 Greece, 38 green revolution, 82, 118 greenhouse gases, vii, ix, 3, 61, 112, 124, 125, 128, 139, 149, 156, 160 greening, 323 groundwater, xiii, 22, 29, 32, 40, 119, 129, 130, 162, 325, 326, 328, 329, 331, 333, 334, 335, 338, 342, 352, 353, 354, 355, 371, 374, 431, 453, 454 group activities, xi, 237 grouping, 244 growth factor, 234 growth hormone, 48, 85, 99, 387 growth rate, 361 Guangdong, 120 guidelines, 4, 121, 314, 317, 393, 404, 427, 430, 471

H habitats, xii, 24, 32, 285, 287, 289, 290, 291, 293, 294, 295, 296, 297, 298, 299, 300, 301, 305, 306, 307, 310, 314, 316, 320, 358, 360, 361, 373 hair, 141 half-life, 125 hammer, 155 hardener, 150 hardness, 280, 281 harvesting, 35, 95, 96, 409 hazardous materials, 156, 157, 454 hazardous waste, 120, 122, 154, 157, 158, 160, 256 hazardous wastes, 99, 116, 118, 120, 121, 122, 123, 124, 128, 131, 133, 139, 141, 142, 145, 154, 156, 158, 165 hazards, 30, 35, 131, 388, 391, 408, 417, 426, 430, 432 HDPE, 56, 78, 143, 151, 152 headache, 427 Health and Human Services, 430 health care, 13, 24 health effects, 23, 38, 426, 430 health problems, 394 health status, xi, 257, 259, 272, 279 heart disease, 425 heavy metals, 43, 47, 54, 55, 56, 57, 58, 59, 61, 64, 74, 75, 76, 77, 78, 96, 98, 106, 119, 123, 129, 130, 157, 216, 217, 234, 261, 265, 387, 398, 399, 401, 442 height, 86, 224, 330, 331 hematite, 183

491

hemisphere, 5, 6, 400 hepatitis, xiv, 17, 386 herbicide, 425 heterogeneous catalysis, 442, 445 high blood pressure, 425 highlands, 287 HIV, 20 homeostasis, xi, 257, 259, 260, 476 homocysteine, 280 homogeneity, 192 Hong Kong, 139, 167, 170, 236 host, x, xiv, 20, 24, 28, 31, 33, 37, 39, 187, 188, 189, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 386, 427, 428, 473 hotel, 386 hotels, 162 house, 281 housing, ix, 111, 242, 252, 317, 323 hub, 216 human activity, 174 human behavior, 164 human capital, 417 human genome, 28 human health, ix, 112, 121, 122, 151, 154, 174, 387, 426, 430 human immunodeficiency virus, 33 human milk, 398 humoral immunity, 261 humus, 49, 81, 82, 100, 222 Hunter, 19, 35, 272, 281 hunting, 273, 279, 283 hyaline, 189 hybrid, 161, 175 hybridization, xiii, 28, 29, 357, 361, 362, 363, 364, 365, 366, 367, 368, 369, 370, 371, 372, 373, 374, 375, 378, 380, 381, 382, 383 hydrocarbons, 76, 78, 80, 98, 99, 130, 147, 434, 449, 454 hydrogen, 56, 59, 64, 70, 128, 154, 163, 358, 434, 437, 438, 439, 453, 454, 455, 457, 460, 461, 462 hydrogen chloride, 154 hydrogen peroxide, 434, 437, 438, 439, 453, 454, 455, 457, 460, 461, 462 hydrolysis, 222, 462 hydroponics, 221, 235 hydroquinone, 448 hydroxide, ix, x, 155, 173, 174, 175, 182, 453 hydroxyl, xv, 433, 435, 437, 438, 439, 440, 443, 445, 446, 451, 453 hygiene, xiv, 156, 385, 401, 408, 410, 416, 417

Index

492 hypertrophy, 196, 198, 213 hypoxia, 467, 475, 476, 477

I ice, 151, 411 ideal, 65, 68, 76, 97, 131, 238, 449, 468 identity, 367, 475, 478 ideology, 322 image, 183, 287, 308, 470 image interpretation, 287 images, 27, 183, 470 immigration, 316, 319, 320, 321, 322 immobilization, xiii, 77, 78, 357, 372 immune memory, 261 immune reaction, 261 immune response, 260, 261 immune system, 260 immunity, 261 immunocompromised, 13 immunodeficiency, 33 immunosuppression, 426 impacts, xii, 61, 87, 89, 90, 93, 95, 214, 305, 306, 307, 308, 317, 319, 323 imports, 401, 403, 417 impregnation, 446 impurities, 68 incidence, viii, 12, 17, 20, 36, 37, 84, 90, 259, 394, 417 income support, 242 incomplete combustion, vii, 3 incubation period, 374 independence, 315, 319 independent variable, 208, 289 India, vi, ix, xiv, xv, 41, 44, 46, 49, 50, 83, 85, 87, 89, 100, 101, 102, 104, 105, 107, 108, 109, 112, 114, 116, 120, 132, 139, 162, 167, 168, 169, 289, 302, 303, 355, 385, 386, 387, 395, 396, 397, 398, 399, 400, 401, 402, 403, 404, 406, 407, 408, 409, 410, 411, 416, 417, 418, 419, 420, 421, 423, 424, 425, 426, 427, 428, 430, 431, 432 Indians, xi, 114, 237 individual differences, 211 Indonesia, 116 induction, 373 industrial processing, 137 industrial revolution, 118 industrial wastes, ix, 112, 141, 142, 144, 147, 152, 154, 164, 165 inequality, 248, 255, 265

inequity, 249 infants, 124, 394 inferences, 471 inflammation, 17 information exchange, 258 information technology, xv, 387 infrastructure, xiv, xvi, 139, 307, 311, 312, 313, 324, 385, 395, 406, 417, 465, 467 ingest, 49, 54, 55, 63, 70, 75, 77, 81, 98, 391 ingestion, 12, 13, 17, 43, 64 initiation, 319, 453 inoculum, 159 insecticide, 400, 424, 426, 431 insects, 84, 123, 406, 412, 425, 426, 427, 429 inspections, 409 inspectors, 409 institutional change, 417 institutions, ix, 112, 155, 310, 312, 313, 314 insulation, 123, 152 integration, 30 intelligence, 426 intercepts, 347 interface, 38, 306 interference, x, 173 International Atomic Energy Agency, 168 international law, 120 international standards, 398, 407 intervention, xii, 31, 32, 291, 305 intervention strategies, 31 intestinal malabsorption, 21 intestine, 43, 48, 49, 56, 60, 81, 107 intoxication, 260 invertebrates, 22 investments, 31, 402, 404, 406, 411 ion-exchange, 181 ionic strength, 445 ions, 70, 176, 365, 438, 453, 456, 458 Iowa, 215 Ireland, 11, 40, 44, 61, 76, 77, 104, 164 iron, 77, 85, 148, 149, 326, 338, 436, 457, 460 irradiation, 30, 455 isolation, 25, 28, 373 isotherms, 176, 177 isotope, 7, 374, 376 Israel, vi, xii, 45, 145, 166, 305, 306, 307, 308, 309, 312, 313, 314, 315, 316, 317, 319, 320, 321, 322, 323, 324 Italy, 43, 45, 46, 102, 109, 115, 145, 146, 155, 164, 400

Index

J Jammu and Kashmir, 402 Japan, 45, 46, 115, 121, 125, 131, 139, 141, 145, 155, 165, 173, 174, 175, 282, 456 Jordan, 308 Jordan River, 308 jurisdiction, 310, 311, 312, 313, 315, 316 juveniles, 209, 277, 279

K Kenya, 166, 170 keratin, 21, 280, 281 keratinocytes, 282 kidney, x, 187, 188, 189, 190, 191, 192, 193, 194, 196, 197, 198, 199, 201, 202, 203, 206, 208, 209, 210, 211, 212, 213, 214, 215, 393 kidneys, 18, 190, 191, 193, 196, 201, 208, 209, 210 kill, 21, 45, 84, 114, 124, 415 kinetic constants, 450 kinetic model, 454, 455 kinetics, 258, 259, 360, 367, 438, 441, 443, 452, 455, 457, 460 Korea, 45, 139, 145, 155, 219, 220, 223, 224, 233

L labeling, 365, 373, 376, 380, 382, 403, 407, 413 lack of confidence, 13 lakes, viii, 12, 22, 38, 379, 426 landfills, ix, 46, 48, 54, 55, 62, 76, 96, 99, 112, 113, 114, 119, 120, 121, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 139, 140, 142, 144, 145, 151, 152, 154, 159, 160, 164, 165, 316 landscape, xii, 6, 285, 286, 287, 289, 290, 299, 300, 301, 306, 307, 308, 310, 311, 313, 314, 315, 316, 318, 319, 321, 322, 323, 324 landscapes, xii, 300, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 317, 318, 319, 320, 321, 429 Latin America, 17 leaching, 119, 129, 447, 448, 449 lead, ix, x, 18, 44, 57, 63, 76, 77, 85, 119, 120, 123, 130, 144, 145, 149, 154, 155, 173, 174, 179, 181, 182, 187, 198, 201, 203, 205, 207, 208, 209, 210, 211, 212, 213, 214, 215, 216, 217, 280, 309, 313, 374, 387, 391, 398 lead content, 398

493

leadership, 472 leakage, xiii, 130, 325, 326, 332, 335, 338, 340, 341, 353 leaks, 413 legislation, xiv, 156, 306, 309, 311, 312, 315, 319, 320, 385, 416, 417, 436 leisure, vii, 11, 12, 240, 254, 307 lens, 280, 282, 342 lesions, 210 Lewis acids, 446 liberalization, 399 liberation, 17 life cycle, 188 lifetime, 115, 177, 179, 393 ligand, 449, 461 lignin, 69, 126, 127, 145, 150, 455 limestone, 59, 153, 154 limitations, 360 lipases, 49 lipids, 21, 126, 145, 476 liquid chromatography, 454 liquid phase, 224, 445 liquids, 123 listeria monocytogenes, 23 literacy, xv, 387, 395, 404 literacy rates, 404 litigation, 431 liver, x, 17, 187, 188, 189, 191, 192, 193, 196, 198, 201, 203, 206, 209, 210, 211, 214, 215, 393, 476, 480 liver cancer, 17 liver damage, 214 livestock, 12, 18, 19, 20, 22, 31, 34, 46, 47, 67, 387, 399 living environment, 242 local authorities, 314 local government, ix, 48, 112, 140 localization, 274 logging, 47 low temperatures, vii, 11, 130 lubricants, 156 lubricating oil, 156 lupus, 188 lymphocytes, 210 lysine, 44, 63, 227, 233, 362 lysis, 23 lysosome, 24

Index

494

M machinery, 141, 148, 405, 425 macromolecules, 258, 260, 261, 265 macronutrients, 221 macrophages, 25, 210 magazines, 115, 123, 138 magnesium, 62, 82, 85 magnetic resonance, 176 magnets, 148 magnitude, 359, 426 Maine, 480 majority, xiii, xvi, 5, 12, 21, 30, 134, 198, 240, 308, 357, 375, 404, 410, 440, 465, 473, 474 malabsorption, 21 malaria, xv, 398, 423, 425, 427, 429, 430, 431 Malaysia, 120, 235, 302 management, viii, ix, xii, xiv, 30, 35, 41, 43, 45, 46, 61, 67, 96, 101, 112, 126, 130, 131, 132, 133, 134, 135, 139, 145, 156, 158, 161, 164, 165, 166, 285, 286, 303, 308, 309, 312, 313, 314, 320, 321, 322, 324, 326, 386, 392, 405, 406, 408, 410, 416, 417, 418, 431, 466, 471, 479 mandarin, 421 mandible, xi, 271, 272, 273, 274, 275, 276, 277 manganese, 77, 326, 436, 457, 458, 459, 461 manpower, 139 manufacture, 113, 122, 132, 135, 138, 149, 150, 151, 152, 153, 154, 155, 157, 410, 426, 430 manufacturing, xiv, 47, 122, 132, 136, 141, 220, 221, 365, 385, 410, 411, 416, 417, 426 manure, 18, 19, 46, 55, 56, 59, 61, 107, 146, 220, 234, 235 mapping, 256, 469, 478 marine environment, 376 markers, 361, 377 market access, 417 marketing, xiv, 385, 396, 402, 403, 405, 406, 410, 417 marketplace, 165 marsh, 382 Marx, 431 Maryland, 324 mastitis, 20 mathematical methods, 339 matrix, xii, 28, 285, 300, 438 meat, 20, 52, 151, 394, 396, 412, 413, 414, 415 mechanical engineering, 126 media, xiv, 23, 25, 34, 37, 69, 132, 133, 166, 355, 385, 396, 398, 416, 427, 454

median, 243, 247, 249, 287 medicines, 122 Mediterranean, 307, 308, 316, 324 Mediterranean climate, 308 MEK, 129 melanin, xii, 272, 280, 282 melt, 130, 151 melting, 151, 190, 211, 214, 215, 366, 367, 382 melting temperature, 366 membrane separation processes, 234 membranes, 24, 476 memory, 261 meningitis, 16 mercury, 54, 76, 77, 119, 120, 123, 124, 130, 144, 155, 174, 212, 387 mesoporous materials, 175, 176, 183, 445 messages, 132 meta-analysis, 303 metabolic disturbances, 269 metabolic pathways, 226, 279 metabolism, 59, 61, 112, 174, 214, 224, 236, 261, 281, 476 metabolites, 227, 425, 426 metal oxides, 445, 446, 447, 448, 459 metals, 43, 44, 47, 54, 55, 56, 57, 58, 59, 61, 64, 74, 75, 76, 77, 96, 98, 106, 112, 116, 117, 119, 120, 123, 129, 130, 136, 138, 140, 142, 148, 152, 154, 155, 156, 157, 161, 163, 188, 190, 191, 192, 202, 203, 205, 208, 209, 210, 212, 214, 215, 216, 217, 234, 261, 265, 387, 398, 399, 401, 434, 442, 443, 445, 446, 447, 448, 450, 452, 457 meter, 65, 68, 75, 97, 98, 151, 160, 163, 326 methanol, 449 methodology, 252, 255, 306, 395, 397 metropolitan areas, 12 Mexico, 102, 123, 401 mice, 25, 214, 215, 269 microarray detection, 371, 373, 379, 382 microarray technology, xiii, 37, 358, 364, 366, 374, 375, 376, 380, 382, 466, 467, 478, 480 microbial cells, 145, 377 microbial communities, xiii, 24, 28, 29, 30, 34, 357, 359, 360, 361, 363, 365, 366, 367, 371, 373, 374, 375, 377, 378, 379, 382, 383 microbial community, 13, 29, 40, 61, 359, 360, 361, 364, 371, 373, 374, 375, 376, 377, 379, 381, 383, 384, 479 microbial metagenomics, 469 microcosms, 376 micrometer, 28

Index micronutrients, 81, 82, 90, 95, 144, 145 microorganism, 17 microscope, 175, 176, 183, 361, 362 microscopy, 183, 362, 363 microspheres, 29 middle class, xv, 386, 395 Middle East, 131, 399 migration, 128, 210 military, 131 milligrams, 395 mineral water, 34 mineralogy, 6, 67 miniaturization, 362 mining, 76, 124, 125, 148, 149, 165, 190, 191, 211, 214, 215 Ministry of Education, 184 minorities, 238, 253 misconceptions, 429 missions, vii, 3, 5, 139, 147, 152 mixing, 46, 64, 79, 80, 86, 120, 131, 174, 175 modeling, 290, 301, 302, 458 modelling, 244, 455 models, vii, 3, 6, 24, 119, 155, 254, 287, 290, 292, 293, 301, 480 modern society, 128, 164 modernization, ix, 111, 410 modification, 25, 34, 267, 274, 346, 450, 476 moisture, 43, 45, 55, 57, 58, 76, 78, 85, 95, 97, 152, 159, 163, 407 moisture content, 55, 78, 85, 159 molecular biology, 26, 257, 391, 467 molecular weight, 209, 227, 455 molecules, 126, 145, 152, 259, 269, 365, 446, 454, 468 monitoring, viii, 12, 28, 29, 30, 31, 35, 37, 128, 174, 183, 225, 272, 278, 313, 328, 329, 335, 342, 361, 366, 380, 382, 392, 394, 404, 408, 410, 472 monoclonal antibody, 28 morbidity, vii, 11, 20, 22, 425 morphology, 361 morphometric, 191, 206 mortality rate, 20 mosquitoes, 163 mRNA, 362, 373, 375, 473 mRNAs, 362, 373, 374 mucoid, 19 mucosa, 476 mucous membrane, 24 mucus, 49, 81 multiple regression, xii, 285, 289, 290, 292, 301

495

multiplication, 23, 48, 78, 83 muskrat, 213, 215 mutation, 287, 378 mutations, 265, 362, 379 Myanmar, 287, 300, 301 mycobacteria, 13, 19, 23, 24, 38 mycorrhiza, 235 mycotoxins, xiv, 386 myocarditis, 16 myoglobin, 476, 477, 480

N NaCl, 83, 222 NAD, 355 nanoparticles, 174, 175, 176, 179 nanotechnology, 31, 175, 183 naphthalene, 156, 376, 450 National Bureau of Standards, 192 national parks, xii, 285, 287, 308, 315, 319 national policy, 313 National Research Council, 11 national security, 319 natural disasters, 152 natural habitats, xii, 305 natural resources, 113, 137, 165, 286 nausea, 427 necrosis, 213 needy, 155 negative relation, 213 neglect, 308, 311, 346 nematode, 188, 211 nervous system, 393 Netherlands, 103, 105, 106, 116, 124, 131, 139, 155, 167, 354, 355, 399 neural network, 382, 383 New England, 37, 39 New South Wales, 109 New Zealand, 46, 48, 76, 116, 430 next generation, 252 NGOs, 133, 396, 397, 410, 416 nickel, x, 156, 187, 191, 196, 197, 198, 201, 203, 205, 207, 208, 210, 211, 212, 213, 214 Nigeria, 116 nitrate, 174, 434, 441, 446, 451, 462 nitrates, 49, 63, 81, 82 nitrification, 366 nitrifying bacteria, 358, 363, 369, 379, 381, 382 nitrobenzene, 461

Index

496

nitrogen, 44, 48, 54, 59, 62, 63, 64, 67, 78, 81, 82, 83, 85, 87, 90, 95, 99, 128, 143, 144, 145, 146, 221, 222, 234, 235, 236, 358, 366, 367, 371, 376, 377, 381, 383, 384, 434, 453, 456 nitrogen fixation, 99, 366, 381 nitrogenase, 384 nitrogen-fixing bacteria, 82, 99 nitrous oxide, ix, 61, 62, 112, 125 NMR, 176 noble metals, 448 nodes, 467 nodules, 78, 358 nonequilibrium, 258 nonequilibrium systems, 258 non-renewable resources, 138 normal aging, 267 normal distribution, 192 North America, 21, 117, 161, 252, 323 North Sea, 479 Northern Ireland, 11, 40 Norway, 116, 133, 139, 155 nuclear energy, 124 nuclear power, 125, 131, 455 nucleic acid, xiii, 17, 27, 28, 33, 357, 362, 364, 365, 366, 368, 369, 371, 372, 377, 380 nucleotides, 361, 365, 374 nuisance, 252, 253 null, 438 nutrients, 20, 23, 48, 54, 55, 62, 63, 64, 65, 67, 78, 82, 83, 85, 86, 99, 100, 136, 144, 146, 162, 209, 220, 221, 226, 227, 228, 231, 428, 431, 432 nutrition, 83, 221, 267

O obstacles, xv, 387 oceans, 384, 426 offenders, 314 oil, 43, 47, 76, 85, 107, 123, 131, 141, 143, 147, 150, 151, 152, 153, 156, 165, 220, 363, 379, 402 oligonucleotide arrays, 378, 379, 381 omentum, 210 omission, 302 open spaces, xi, 237, 240, 253, 305, 314, 316 operating costs, 75 operations, 29, 190, 191, 214, 314, 404, 406, 408 operon, 358 opportunities, 32, 307, 399, 417 optical fiber, 121 optimization, xiii, 225, 357, 376, 381, 446, 451, 452

oral cavity, 383 oral health, 383 ores, 125, 140, 148, 149 organ, 30, 91, 192, 193, 196, 197, 206, 208 organic chemicals, 122, 141, 431 organic compounds, xv, 63, 123, 128, 130, 156, 433, 434, 436, 438, 440, 442, 446, 448, 451, 452, 454, 455, 456, 462 organic food, 403 organic materials, 43, 46, 75, 126, 130, 143, 159, 161 organic matter, vii, 3, 6, 43, 48, 49, 55, 59, 63, 64, 66, 69, 70, 77, 81, 82, 85, 96, 99, 100, 127, 144, 146, 163, 174, 220, 226, 227, 233, 234, 236, 378, 438, 439, 442, 443, 446, 451, 460, 461 organic solvents, 123 organism, xi, 26, 27, 29, 30, 118, 121, 257, 260, 263, 271, 358, 363, 372, 373, 374, 468 organizing, 183, 404 organochlorine compounds, 123 osmosis, 98 overgrazing, 293 overlap, 289, 311, 313, 318, 320 overlay, 247 oversight, 404, 408, 409, 417 ownership, 396 oxidation, xv, 179, 222, 225, 358, 365, 433, 434, 436, 437, 439, 440, 442, 443, 447, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 oxidation rate, 451 oxidative stress, 280, 282 oxygen, 58, 59, 61, 64, 67, 69, 70, 97, 125, 128, 160, 222, 224, 235, 280, 373, 381, 435, 436, 459, 475, 476, 480 oxygen consumption, 64, 224 oyster, 192 oysters, 40 ozonation, xv, 433, 435, 437, 438, 439, 443, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 ozone, xii, xv, 21, 30, 272, 279, 280, 433, 434, 435, 436, 437, 438, 439, 440, 442, 443, 444, 445, 446, 447, 448, 449, 450, 451, 452, 453, 454, 455, 456, 457, 458, 459, 460, 461, 462, 463 ozone reaction, 462 ozonization, 458, 460 ozonolysis, 456

Index

P Pacific, 16, 40, 76, 105, 114, 116, 117, 304, 377 pain, 400 paints, 123, 141 pairing, 273, 279 Pakistan, 116, 120, 355 palladium, 461 palpation, 210 parallel, 29, 335, 362, 370, 374, 380, 441, 455, 466, 468 paralysis, 16 parasite, viii, x, 12, 30, 34, 187, 188, 189, 190, 191, 208, 210, 211, 212, 213, 214, 215, 216, 272, 273 parasites, 21, 188, 203, 212, 213, 215, 216, 217, 281 parasitic infection, x, 187, 209, 211, 213, 214 parliament, 403 partition, 153 pathogens, viii, 11, 12, 13, 16, 18, 19, 20, 21, 22, 23, 24, 26, 28, 29, 30, 31, 32, 34, 36, 37, 38, 40, 45, 47, 48, 54, 55, 56, 57, 59, 60, 61, 75, 84, 96, 97, 98, 145, 163, 371, 379, 387, 415 pathology, 32 pathophysiology, 281 pathways, 226, 279, 438, 450, 471, 476 peat, 459 penalties, 403 peptides, 17, 459, 468 percentile, 248 percolation, 66, 129, 333, 337 performance, 35, 87, 308, 330, 430, 436, 440, 441, 442, 443, 445, 447, 450, 469 peri-urban, 398 permeability, 335, 340 permit, 324 peroxide, 434, 435, 437, 438, 439, 448, 453, 454, 455, 457, 460, 461, 462 person-to-person contact, 19 Perth, 101, 161, 244 pest populations, 392 pesticide, xv, 61, 76, 387, 391, 392, 393, 394, 396, 397, 398, 399, 400, 401, 403, 404, 406, 407, 408, 409, 410, 412, 416, 418, 423, 424, 425, 426, 427, 428, 429, 430, 432 pesticides, xv, 43, 76, 85, 100, 122, 123, 307, 388, 391, 392, 393, 394, 395, 396, 397, 398, 403, 410, 411, 412, 418, 423, 424, 425, 426, 427, 428, 429, 430, 431, 432, 434, 440, 457 pests, xv, 84, 85, 90, 123, 221, 392, 423, 425, 429 PET, 136, 143, 151

497

petroleum, 122, 141 phagocytosis, 24 PHB, 236 phenol, 274, 441, 446, 447, 448, 450, 456, 459, 460, 461, 462 phenotype, 479 Philippines, 45, 120, 145, 421 phosphates, 49, 81, 83, 236, 446 phosphorus, 48, 54, 63, 64, 67, 78, 82, 83, 90, 95, 143, 144, 221, 231, 236, 434 phosphorylation, 282 photocatalysis, 437, 442, 456, 457 photodegradation, 454, 455 photoemission, 183 photolithography, 362 photolysis, 78, 439, 454 phylogenetic tree, 365 phylum, 363 physical activity, 253, 254 physical environment, 254 physical exercise, 253 physical fields, 269 physical fitness, 191 physical properties, 220, 234 physics, 258, 259, 263, 268 physiology, 76, 221, 358, 467, 477, 478, 480 phytoplankton, 467 pigs, 116, 117 pilot study, 67 pioneer species, 293 pitch, 241 placenta, 121 plants, ix, xiii, 21, 30, 45, 54, 55, 63, 83, 84, 85, 90, 91, 92, 94, 95, 96, 97, 99, 112, 123, 131, 141, 145, 150, 153, 154, 156, 159, 160, 220, 221, 222, 224, 226, 227, 228, 234, 235, 306, 316, 357, 362, 391, 392, 398, 401, 410, 412, 427, 428, 429, 430, 436, 449, 455 plasmid, 378 plastic products, 139 plasticity, 480 plastics, 112, 113, 116, 117, 119, 122, 123, 126, 130, 135, 136, 138, 144, 147, 150, 151, 152, 157, 161, 163 platform, 68, 317, 367, 468, 469 platinum, 183, 462 PLS, 5 plutonium, 125 pneumonia, 24 point mutation, 379

498

Index

poison, 120, 400 policy makers, 31, 133, 396 polio, 16, 33 pollutants, 76, 130, 150, 151, 154, 188, 387, 434, 436, 437, 438, 440, 445, 446, 448, 454, 455, 458, 463 polluters, 314 pollution, x, xii, 25, 46, 113, 135, 137, 139, 142, 144, 148, 149, 161, 164, 165, 184, 187, 190, 208, 211, 216, 217, 221, 234, 252, 272, 305, 306, 308, 313, 318, 398, 475 polychlorinated biphenyls (PCBs), 48, 76, 123, 130, 430 polycyclic aromatic hydrocarbon, 43, 61, 76 polymerase, 27, 36, 40, 371, 381, 474 polymerase chain reaction (PCR), xiii, 26, 27, 28, 29, 33, 35, 36, 39, 274, 281, 282, 357, 359, 360, 361, 366, 369, 370, 371, 373, 375, 377, 378, 379, 380, 381, 382, 383, 468, 475 polymerization, 280 polymers, 365 polymorphism, 26, 38, 360, 377, 380, 381, 382, 383 polymorphisms, 362, 379, 380 polypeptide, 366 polypropylene, 68, 151, 192 polystyrene, 151 pools, vii, 3, 21, 22, 137 population density, 65, 67 population group, xi, 237, 248, 249 population size, 242, 286, 291, 300, 303 porosity, 62, 66, 82, 220, 342, 353, 365 porous media, 355 portability, 29 Portland cement, 153 Portugal, 139, 155 positive relationship, 205, 212 potassium, 54, 62, 63, 82, 85, 90, 95, 143, 221 potato, 39, 47, 52, 53, 234, 235 poultry, 18, 31, 34, 394, 412, 413, 414, 415 poverty, xv, 46, 120, 386, 395 poverty reduction, xv, 386 power plants, 145, 153, 316, 455 pozzolana, 153 precipitation, 129, 150, 434, 447 predation, 17 predators, 24 prejudice, 252 preparation, 29, 69, 317, 359, 400 prevention, 22, 27, 165, 235, 293, 391 primary school, 397

priming, 210, 373 prioritizing, 308 private investment, 406 private sector investment, 410 probability, 63, 248, 250, 251, 274, 278, 290, 291, 294 probability distribution, 248, 250, 251 probe, 175, 176, 183, 361, 363, 364, 366, 367, 368, 372, 374, 378, 469, 470, 474 process control, 34, 225 process gas, 130 procurement, 119, 140, 411 producers, 134, 139, 157, 392 productivity, viii, 5, 6, 22, 42, 82, 83, 96, 99, 213, 425, 473 profit, 141 project, xii, 151, 159, 162, 239, 254, 285, 287, 300, 301, 314, 378, 467, 468, 472 prokaryotes, 370, 376, 380 prokaryotic cell, 373 proliferation, 33, 60, 99, 209 promoter, viii, 42, 87, 95, 99, 374, 449, 474 propagation, 479 propane, 175 prophylaxis, 25 proteases, 49 protected areas, 286, 287, 292, 293, 300 proteins, 21, 49, 63, 82, 126, 141, 145, 163, 234, 265, 279, 383, 448, 473, 475 prototype, 382 Pseudomonas aeruginosa, 23 public awareness, 132, 158 public health, xiv, 12, 27, 34, 38, 40, 129, 131, 253, 310, 385, 407, 416, 424, 425 public interest, 289 public investment, 416 public parks, xi, 237, 238, 244, 252, 255 public policy, 323, 431 public sector, 411, 416 public-private partnerships, 404, 417 pulp, 46, 47, 63, 144, 145, 149, 150, 156, 373, 408, 410, 460 pumps, 335, 428 pure water, 112 purification, viii, 16, 29, 42, 65, 67, 183, 381, 458 PVC, 68, 69, 113, 121, 123, 143, 151, 160, 474 pyrolysis, 131

Index

Q quality assurance, 410, 412 quality control, 378, 411 quality of life, 134, 136 quality standards, 404 quantum mechanics, 258, 263 quartile, 249 Queensland, 55 quinone, 280

R race, 125, 255 racism, 255 radial distance, 326, 346, 347 radiation, vii, xii, 3, 125, 128, 272, 279, 280, 434, 439, 440, 454, 455 radical mechanism, 447 radical reactions, 452, 453 radicals, xv, 261, 433, 435, 437, 438, 439, 443, 445, 448, 450, 451, 452, 453, 454, 462 radioactive waste, 125, 165 radium, 125 radius, 330, 333, 338, 344, 346, 354 radon, 125 rain forest, 289 rainfall, 6, 309, 335 rash, 16 raw materials, 119, 137, 138, 139, 140, 151, 164, 165, 417, 426 RDP, 378 REA, xii, 26, 285, 289 reaction mechanism, 441, 442, 443, 444, 446, 447, 460 reaction order, 442, 449 reaction rate, 437, 440, 441, 448, 450 reaction rate constants, 437, 440, 450 reactions, xv, 27, 29, 58, 59, 128, 130, 182, 220, 224, 280, 370, 371, 386, 439, 440, 445, 449, 451, 452, 453, 457, 461, 462 reactive arthritis, 20, 38 reactive oxygen, 280 reactivity, vii, xv, 3, 122, 262, 433, 434, 437 reading, 289, 473 reagents, 29, 442, 445 real terms, 395, 399 reality, 252, 253, 331 recall, 400

499

recognition, xiv, 17, 26, 385 recommendations, 273, 412 recovery plan, 131 recovery processes, 259, 260, 262, 267 recreation, ix, xii, 111, 240, 253, 305, 307, 308, 310, 313, 314, 316 recurrence, 310, 312 recycling, 116, 120, 126, 132, 133, 134, 137, 138, 139, 140, 141, 142, 143, 145, 147, 148, 149, 150, 151, 152, 154, 155, 156, 157, 158, 159, 160, 164, 165, 166, 169, 220 red blood cells, 18, 210, 213 Red List, 302 redistribution, 210 redundancy, 475 reflectivity, 273 reforms, 408 regeneration, 448, 450 regression, xii, 285, 289, 290, 292, 301 regression method, 301 regression model, 289, 290, 292 regulatory framework, xiv, 385, 403 rehabilitation, xii, 305, 306, 310, 314 rehydration, 20 rejection, 134, 400, 401 relevance, 466 reliability, 192, 281, 352 relief, 6, 163 remediation, viii, 42, 81, 83, 98, 99, 160 repair, 134, 155, 260 reparation, 460 replacement, 153, 189 replication, 25, 39 reporters, 28 reprocessing, 138, 156 reproduction, 45, 58 reproductive organs, 92, 94, 95 reputation, 400 requirements, 34, 78, 209, 401, 408, 411 research facilities, 477 research institutions, 403 researchers, xvi, 95, 363, 433, 435, 437, 438, 439, 440, 443, 445, 451, 465, 466, 467, 470, 472, 474 reserves, 191, 241, 303, 308, 319 residuals, 13 residues, 59, 61, 76, 96, 123, 144, 220, 236, 387, 388, 391, 392, 393, 394, 396, 397, 398, 399, 400, 401, 407, 408, 409, 410, 412, 416, 425, 427, 428, 429, 430, 432 resins, 121, 150

Index

500

resistance, viii, 12, 13, 20, 25, 84, 99, 221, 261, 262, 265, 272, 326, 392 resolution, 175, 177, 178, 179, 180, 183, 242, 243, 290, 363, 368 resources, 84, 96, 112, 113, 133, 134, 135, 136, 137, 138, 141, 153, 155, 165, 220, 253, 286, 308, 309, 310, 316, 355, 406, 436, 466, 469, 472, 475, 480 respiration, 63, 221, 236, 358 restaurants, ix, 51, 112, 127, 147, 162 restriction enzyme, 382 restriction fragment length polymorphis, 26, 360, 377, 380, 381, 382 retail, 386, 394, 410, 411, 416 retardation, 22 revenue, 55, 140 ribosomal RNA, 358, 359, 361, 369, 378, 379 rights, 133, 324 rings, xv, 53, 433 risk assessment, 394, 418 risk factors, 39 risk management, 416 risks, xiv, xv, 32, 115, 131, 385, 386, 387, 388, 394, 406, 425, 427, 429 river basins, 427 river systems, 327 RNA, 28, 358, 359, 361, 363, 369, 372, 373, 374, 377, 378, 379, 380, 381, 382, 470, 474, 475, 476 robotics, 362 rodents, 429 room temperature, ix, 112, 192, 414, 435, 439 root hair, 98 roundworms, 188 rowing, 409 rubber, 118, 121, 138, 144, 415 rubbers, 147 rules, 139, 141 runoff, 129, 427 rural areas, 97, 411 rural development, xv, 386 rural women, 46 Russia, 100

S salinity, viii, 42, 83, 306, 326, 327, 373, 376 saliva, 363 salmon, 431 salmonella, xiv, 385, 386, 401 salts, 150, 220, 443 sanctuaries, xii, 285, 287

Saudi Arabia, 148, 149 sawdust, 46, 60, 145 scarcity, 308, 309 scattering, 176 scavengers, 438, 449, 450, 451 scientific knowledge, 144 scientific understanding, xiv, 385 screening, 47, 404, 474 sea level, xiii, 325, 327, 338 seafood, 220, 234, 412, 413, 414, 415 sea-level, 328, 334 seasonal changes, 360, 378 seasonality, 30, 242 secrete, 49, 82, 112 secretion, 24, 99 sediment, 7, 66, 156, 352, 359, 369, 370, 376, 377, 381, 382 sedimentation, 30, 436 sediments, viii, xiii, 5, 6, 7, 12, 64, 307, 327, 328, 352, 358, 363, 369, 373, 377 seed, 83, 86, 87, 95, 223, 229, 231, 232 seedlings, 83, 95 segregation, 162 selectivity, 183, 392, 449 selenium, 130 self-organization, 258 self-regulation, 411, 417 semiconductors, 121 sensitivity, xiii, 7, 28, 29, 30, 183, 188, 357, 368, 369, 370, 371, 376, 393, 470 sequencing, xvi, 28, 39, 359, 360, 362, 366, 378, 383, 465, 466, 467, 468, 469, 471, 477, 478 serum, 426, 430 service provider, 416 settlements, 300, 315, 319 severe acute respiratory syndrome, xiv, 385 sewage, 18, 36, 43, 45, 46, 54, 55, 56, 60, 61, 62, 63, 64, 65, 67, 68, 70, 71, 72, 73, 74, 75, 76, 96, 97, 98, 101, 112, 141, 145, 146, 151, 153, 154, 159, 161, 162, 234, 306, 307, 308, 398 sex, xi, 204, 271, 273, 278, 281, 394 sexual dimorphism, 211, 281 shade, 78 shape, 189, 242, 331 shear, 13, 36 sheep, 21 shellfish, 22, 370, 381 shelter, 13, 292 shingles, 144, 152 ships, 148

Index shoot, 87, 264 shortage, 231, 308 shrimp, 401, 411 side effects, 392, 426 signals, 175, 272, 279, 365, 368, 372 signs, 16, 289, 408 silica, 141, 151, 153, 445, 448 Silicon Valley, 120, 154, 168 silver, 96, 140, 141, 144, 149, 156, 460 Singapore, 105, 115, 120, 121 single-nucleotide polymorphism, 379, 383 sintering, 446 skin, 24, 58, 412, 427 slag, 131 Slovakia, 217 sludge, 43, 45, 46, 47, 54, 55, 56, 57, 59, 60, 62, 63, 64, 68, 74, 75, 77, 97, 98, 101, 102, 105, 112, 141, 145, 146, 154, 159, 162, 234, 236, 370, 373, 374, 447, 460 social activities, 132 social change, 309 social conflicts, 319 social group, 238, 252, 253 social justice, 238 social problems, viii, 42 socioeconomic status, 254 sociology, 126 sodium, 35, 124, 150, 155, 174, 175, 412, 455, 463 sodium hydroxide, 155, 174 software, 367, 472, 479 soil erosion, 6, 139, 145 soil particles, 81, 307, 360 soil pollution, 135 solid phase, 434, 445 solid surfaces, 29 solid waste, viii, ix, 18, 42, 43, 48, 96, 98, 107, 112, 113, 115, 116, 117, 118, 124, 129, 130, 131, 132, 133, 135, 142, 144, 146, 148, 154, 159, 161, 162, 163, 164, 166 solubility, xv, 224, 433, 434, 448 solvents, 123, 154, 156, 426 sorption, 174, 175, 179, 180, 183 South Africa, 153, 400 South Asia, 399, 419, 420, 421 South Korea, 139, 155 Southeast Asia, 287, 289 sovereignty, 309 SPA, 378, 381 Spain, 116, 400, 433 specialists, 289

501

speciation, 234 species richness, 323 specific gravity, 21, 407 specific surface, 65, 175, 176 specifications, 27, 409 spectrophotometry, 192 spectroscopic techniques, 175 spectroscopy, x, 173, 174, 175, 177, 183 speculation, 210, 213 spicule, x, 187, 189, 190, 191, 192, 193, 194, 196, 197, 199, 201, 202, 203, 205, 208, 209, 211, 212, 214, 215 spiders, 123 spillovers, 409, 410 spleen, x, 187, 191, 206, 207, 208, 213, 214 splenomegaly, 213 spreadsheets, 354 Spring, 282, 429 Sri Lanka, 121 SSI, 402, 416 stabilization, viii, 42, 54, 55, 63, 65, 153, 227, 233, 236, 316 stabilizers, 403 stakeholders, xii, 285, 286, 300, 301 standard deviation, 245 standard error, 194, 196, 199, 204, 205, 206 standardization, 407, 472 starch, 122, 147, 222 state control, 402 State Department, 410, 431 statehood, 315 states, 132, 310, 311, 313, 394, 398, 402, 406 statistics, 13, 238, 240, 242, 243, 248, 249, 250, 267, 282, 467 steel, 113, 115, 138, 139, 148, 149, 152, 153, 156 sterile, 47, 60, 192, 223 stimulus, 210 STM, 183 stoichiometry, 443, 444, 457 stomach, 189 storage, xiii, 29, 54, 61, 68, 76, 126, 146, 156, 158, 209, 210, 212, 234, 326, 330, 331, 332, 333, 335, 340, 342, 343, 345, 352, 353, 354, 355, 391, 404, 406, 476 stormwater, 159 stoves, 121 stratification, 234, 331 streams, viii, 12, 47, 220, 235, 293, 307, 308, 309, 340, 427, 431 streptococci, 108

502

Index

stroke, 425 strontium, 125 structural changes, 396 structural gene, 382 style, ix, 111, 122, 309, 474 subgroups, 248, 394 subjective judgments, 351, 353 submarines, 221 substitution, 436, 449, 461 substitution reaction, 461 substrates, 25, 163, 476 Sudan, 400 sugar beet, 220 sugarcane, 47, 83, 147 sulfate, 150, 225, 235, 358, 366, 376, 449 sulfur, 62, 82, 147, 153, 227, 233, 366, 367 sulfuric acid, 155 Sun, 109, 170, 283, 379, 456 supermarkets, 135, 152, 164, 396, 404, 405, 411 supplier, xiv, 386, 400 suppliers, 411 supply chain, 388, 396, 399, 408, 409, 416, 417 support services, xiv, 385, 417 suppression, 84 surface area, 65, 160, 175, 176, 364, 448, 450 surface chemistry, 449, 450, 451 surfactant, 453, 463 surplus, 65, 335 surrogates, 32, 469 surveillance, 16, 30, 416, 417, 427 survey, 124, 133, 182, 239, 242, 243, 244, 245, 255, 288, 289, 301, 406, 428, 468 survival, vii, 11, 13, 24, 35, 58, 164, 475 susceptibility, 208, 221 suspensions, 456 sustainability, 164, 314, 326, 418 sustainable development, viii, 41, 169, 326 Sweden, 36, 37, 116, 123, 131, 139, 155, 166 Switzerland, 116, 117, 139, 155, 158, 224, 235, 302, 303 symmetry, 175 symptoms, 16, 17, 19, 426, 427 synchronization, 260 syndrome, xiv, 18, 385 synergistic effect, 456 synthesis, xii, 176, 260, 261, 272, 280, 283, 373, 374, 445, 451, 468, 469, 477

T tags, 361, 468, 478 Taiwan, 120, 139, 155, 235 tanks, 46, 146 tannins, 150 tapeworm, 215, 217 tar, 152 target, xv, 27, 28, 29, 157, 289, 290, 291, 296, 297, 298, 299, 300, 301, 361, 363, 364, 365, 366, 367, 368, 369, 370, 372, 373, 374, 380, 423, 427, 428, 429, 469, 476 taxation, 165 taxonomy, 40 technical support, 466 technological developments, xvi, 119, 465 technological revolution, 118, 121 technologies, viii, xiii, 29, 42, 59, 74, 97, 142, 145, 147, 154, 220, 259, 357, 408, 425, 437, 467 technology, xiii, xv, 37, 64, 76, 96, 97, 98, 99, 119, 121, 130, 131, 138, 140, 142, 143, 144, 146, 147, 158, 159, 162, 164, 165, 357, 362, 364, 366, 374, 375, 380, 382, 387, 401, 433, 459, 462, 466, 468, 478, 480 TEM, 175, 176, 183 temperature, vii, ix, 3, 4, 16, 43, 45, 47, 48, 50, 58, 59, 60, 67, 76, 96, 97, 112, 124, 130, 147, 151, 160, 163, 170, 192, 222, 223, 258, 259, 272, 306, 366, 367, 368, 413, 414, 415, 435, 439, 445, 462, 475 territory, 327 test data, 278, 331, 341, 351, 352, 354, 355 testing, 26, 393, 398, 401, 407, 408, 409, 410, 417 tetrachlorodibenzo-p-dioxin, 43 textiles, 123, 144, 151 textural character, 451 texture, 5, 47, 56, 67, 414, 449, 450 Thailand, vi, xii, 33, 45, 121, 145, 285, 286, 287, 288, 289, 292, 302, 303, 304 therapy, 268 thermal analysis, 175 thermal energy, 146 thermodynamics, 258, 259, 263, 265, 269 thermometer, 413 thin films, 457 thinning, 426 Third World, 139 threats, xiv, 116, 293, 385 threonine, 234 thyroid, 429

Index time periods, 264 time pressure, 391 time use, 113 tin, 148, 183 tissue, x, 149, 150, 187, 188, 192, 193, 196, 198, 199, 201, 203, 205, 208, 209, 210, 211, 212, 213, 214, 215, 398, 476 titanium, 456, 459 TLR, 238 toluene, 380 tones, ix, 83, 112, 115, 116, 125, 140, 145, 149, 153, 154, 157, 159, 160, 162, 163, 165 total energy, 260 tourism, 286, 314, 316, 386 toxic effect, 227, 229 toxic gases, 48, 146, 152 toxic metals, 188, 190, 191, 209, 212, 215 toxic waste, 76, 97 toxicity, xv, 215, 379, 394, 426, 427, 430, 433, 435, 440, 441, 447, 456 toxin, 18, 20 toys, 123, 136, 174 trace elements, 54, 99, 145, 188, 216 tracks, 289 trading partner, 408 training, 37, 242, 260, 302, 409, 410, 411, 418, 466, 470 traits, xi, 271, 273, 467 transactions, 402 transcription, 27, 362, 373 transcriptomics, 468, 479 transcripts, 373, 473, 474 transference, 439, 448 transformation, xv, 20, 55, 175, 420, 425, 428, 432, 433, 434, 435, 443, 445, 448, 451, 452, 454, 462 transformation processes, 55 transformation product, 428 transformations, 192 transition metal, 443, 457 translocation, 20 transmission, xiv, 16, 17, 19, 21, 22, 28, 30, 31, 35, 36, 38, 156, 175, 178, 181, 189, 215, 385 transport, 6, 55, 126, 141, 158, 165, 209, 238, 391, 426, 476 transportation, ix, 31, 112, 131, 161, 315, 317, 386 trauma, 260 treatment methods, 98 trial, 161 trickle down, 67 triggers, 319

503

tropical forests, 302 trucks, 148 tsunami, 120 tuberculosis, 20, 38, 358, 388 tumors, 121 tumours, 429 tunneling, 183 Turkey, 400 Turks, 308 turnover, vii, 3, 4, 476 typhoid, 18 typhoid fever, 18 typhus, xv, 423 typology, xi, 237, 240, 252, 253 tyrosine, 228, 280

U U.S. Geological Survey, 354, 355, 427, 431 Ukraine, 271, 282 UNESCO, 289, 303 uniform, 63, 68, 85, 160, 193, 211, 252, 330, 331, 471 united, xiv, 11, 14, 16, 18, 24, 33, 38, 112, 114, 115, 133, 158, 166, 167, 170, 322, 380, 386, 400, 401, 421, 425, 426, 430, 466 United Kingdom (UK), vi, xi, xvi, 11, 21, 45, 49, 50, 101, 103, 113, 114, 128, 132, 135, 145, 151, 153, 157, 164, 167, 168, 237, 238, 239, 254, 255, 400, 420, 425, 427, 430, 431, 465, 466, 467, 471, 472, 477, 479 United Nations (UN), 112, 115, 158, 354 universities, 46, 166, 417 uranium, 124, 125 urban area, xi, 237, 251, 252, 253, 320, 398 urban population, 252 urbanization, xiv, xv, 385, 386, 395 urea, 82, 85, 87 ureter, 188 ureters, 210 urinary bladder, 188 urine, 20, 64, 188, 279, 283 USDA, 376, 394, 418, 419 USSR, 316 UV irradiation, 30, 455 UV light, 279, 440 UV radiation, 280, 434, 439, 440, 455

Index

504

V vacuum, 129, 156, 437 valence, ix, 173, 175, 177, 435 validation, 377, 382, 478 valuation, 321 vanadium, 183 vapor, 156 variables, 35, 208, 289, 445, 449, 450, 451 variations, 211, 254, 273, 274, 276, 317, 338, 353, 366, 376, 395 varieties, 392, 405, 425 vector, 426, 475 vegetable oil, 47, 143, 147, 407 vegetables, 18, 20, 136, 221, 391, 396, 398, 399, 410, 412, 416, 425 vegetation, xii, 285, 289, 290, 291, 293, 300, 304, 306, 335, 427, 428 vehicles, 17, 126, 162, 398 vein, 274 velocity, 180, 182, 342, 353 ventilation, 223 versatility, 437 vertebrates, 22, 215, 477 vertical transmission, 31 video, 151 Vietnam, 120, 121, 174 vinasse, 456 vinyl chloride, 129 violence, 132 viral meningitis, 16 virus infection, 33, 37 viruses, 13, 16, 17, 25, 27, 32, 60, 387, 467, 474, 479 viscosity, 156, 331 vision, 273, 279, 281, 283, 312, 314, 317, 320, 471 visual impression, 249 vitamins, 85 VOCs, 123, 128, 129, 159 volatility, 431 volatilization, 78 vomiting, 427 vulnerability, 307

W waiver, 404 Wales, 45, 109, 145 walking, 254

Washington, 109, 115, 133, 149, 166, 170, 302, 303, 304, 323, 377, 418, 419, 420, 421, 431, 455 waste disposal, 131, 132, 133, 139, 160, 165 waste incineration, 131 waste incinerator, 131 waste management, ix, 61, 96, 112, 126, 131, 132, 133, 134, 135, 156, 161, 164, 165, 166, 406 waste treatment, 131, 149, 161, 443, 452 wastewater, viii, x, xv, 13, 28, 34, 36, 42, 43, 44, 47, 54, 64, 65, 66, 67, 68, 69, 70, 74, 75, 76, 96, 97, 98, 101, 104, 109, 153, 219, 220, 225, 233, 234, 235, 236, 363, 369, 370, 378, 379, 382, 433, 435, 453, 455, 456, 460, 462, 463 water policy, 308, 309, 315 water quality, viii, 12, 13, 17, 25, 28, 33, 38, 40, 309, 313, 316, 326, 330 water resources, 220, 308, 309, 310 water supplies, 12, 17, 18, 22, 24, 29, 326, 428, 452 watershed, 30, 311, 313, 318, 320 waterways, 114, 427 weakness, 151 wealth, 46, 252, 466, 474 wear, 115, 415 web, 402, 418 websites, 170 weight changes, 208, 215 weight loss, 22 welfare, xiv, 386 wells, xiii, 29, 153, 174, 325, 328, 329, 330, 331, 332, 333, 334, 335, 337, 338, 339, 342, 343, 348, 350, 352, 353, 354, 355, 428 Western Europe, 309, 399 wetlands, 323 wholesale, 398, 402, 406, 410 wild animals, viii, 12, 13, 17 wildlife, xii, 12, 18, 20, 31, 38, 190, 240, 285, 287, 289, 290, 291, 293, 301, 303, 418, 429 windows, 148 withdrawal, xiii, 325, 326, 328, 333, 335, 338, 340 wood, 114, 117, 127, 138, 144, 147, 150, 152, 426 workers, 120, 121, 163, 418, 426, 427, 430 working hours, 32 workplace, 136, 386 World Bank, xiv, xv, 166, 386, 400, 404, 405, 406, 407, 408, 410, 411, 417, 419, 420, 421 World Health Organisation, 40, 391 World Trade Organization (WTO), 399, 417 worldwide, ix, xiv, xvi, 6, 12, 13, 16, 21, 36, 129, 131, 155, 156, 173, 386, 433, 435, 473

Index worms, x, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 54, 56, 58, 59, 60, 61, 62, 64, 65, 66, 67, 75, 76, 78, 79, 81, 82, 83, 84, 85, 87, 90, 91, 92, 94, 95, 97, 98, 99, 100, 106, 145, 146, 187, 188, 189, 190, 191, 192, 193, 194, 196, 197, 198, 199, 201, 202, 203, 204, 205, 206, 208, 209, 210, 211, 212, 214, 216

X x-axis, 267 XPS, 183 x-ray, x, 173, 175, 176, 183, 184 x-ray diffraction (XRD), 175, 176, 183

505

Y Yale University, 269 yeast, xiii, 147, 150, 222, 357, 362, 373, 466, 478 yield, 83, 87, 89, 90, 146, 220, 228, 234, 245, 326, 330, 351, 353, 372, 375, 425, 468

Z zeolites, 448 Zimbabwe, 104 zinc, 54, 76, 77, 85, 123, 144, 156, 398 ZnO, 440, 455