Neilson, Alasdair H. "Frontmatter"Organic Chemicals : An Environmental PerspectiveBoca Raton: CRC Press LLC,2000

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Preface

This book stems from the authors experience with a variety of problems onthe fate, distribution, and toxicity of organic compounds in the aquatic envi-ronment. It became increasingly clear that the procedures for investigatingthese problems crossed the traditional boundaries of organic and analyticalchemistry, microbiology, and biology, and after many years this resulted inthe idea of selecting the relevant aspects of these and writing the presentbook. Environmental problems have become increasingly complex and envi-ronmental impact studies should be based as far as possible on incontrovert-ible scientic facts. In this book the basic issues of chemical analysis,distribution, persistence, and ecotoxicology have therefore been discussedalthough the emphasis has been placed on microbial reactions with which theauthor is most familiar. Throughout the book an attempt has been made toinclude a wide range of structurally diverse compounds as illustration, anda mechanistic approach to degradation and transformation has beenadopted. At the same time, the limitations in this book should be clearlyappreciated: it is not designed for the specialist in any of the traditional dis-ciplines, although it is hoped that the level of detail is acceptable to those whoseek discussions of a range of environmental issues. The book is by no meanscomprehensive but a list of references for those seeking further detail is pro-vided at the end of each chapter.

This volume may be considered a new edition of a previous one

OrganicChemicals in the Aquatic Environment

. It differs from that volume not only inattempting to bring up to date the contents of the original chapters and cor-recting some errors, but in incorporating extensive new material. Thisincludes more extensive discussion in Chapter 2 of recent analytical proce-dures, a more thorough discussion in Chapter 4 of chemical and photochem-ical reactions including those in the troposphere, a presentation in Chapter 7of more recent aspects of ecotoxicological assays, and a new chapter on biore-mediation that makes extensive use of principles introduced in previouschapters. More extensive discussions have been given to the terrestrial andtropospheric environments, and this justies the widening of the title fromthe original one.

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Acknowledgments

When all is said and done, it remains to thank all those who have contributedin many different ways to the making of this book. It is a pleasure to expressmy deep gratitude to the following.

My teachers in the Universities of Glasgow, Cambridge, Oxford and theUniversity of California, Berkeley including many who are no longer withus for illustrating by example the rigors of scientic inquiry.

The late Percy W. Brian FRS for patiently directing my faltering footstepsinto microbiology many years ago.

sten Ekengren, Director, Environmental Technology and Toxicology forallowing me a number of privileges including free access to library and copy-ing facilities.

My collaborators, Ann-Soe Allard, Per-ke Hynning, Marianne Malmberg,and Mikael Remberger not only for their scientic contributions but also fortheir friendship and tolerance over many years.

Ann-Soe Allard for her expertise in producing the numerous gures fromsometimes erratic drafts, and for allowing me to expand and update our ear-lier review that has become Chapter 8.

Mirja S. Salkinoja-Salonen and her colleagues in the Department ofApplied Microbiology and Applied Chemistry, University of Helsinki forfriendship and stimulation during many years.

The Knut and Alice Wallenberg Foundation and its executive director Pro-fessor Gunnar Hoppe for providing instrumentation that opened new hori-zons to our research.

My good and long-standing friends the Sndmr families in versjdalen,Norway for providing the tranquility during which most of the revision ofthe original text was undertaken.

Eva Berg, without whose continued support and encouragement this bookwould never have materialized.

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To A.N. and G.N.M. who no longer tread the Highland hills

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Contents

1. Introduction

1.1 Orientation1.2 Literature Cited1.3 Limitations

1.3.1 Organochlorines1.3.2 Dictates for Inclusion1.3.3 Modeling1.3.4 Enzymes and Relatedness

1.4 Biotechnology1.5 Terrestrial Systems1.6 The Atmosphere1.7 Natural Products and Microbial Metabolites

1.7.1 Organohalogen Compounds1.7.2 Polymeric Compounds1.7.3 Polypyrroles1.7.4 Polycyclic Aromatic Hydrocarbons

1.8 The Effect of Xenobiotics on Microbial-Mediated ProcessesReferences

2. Analysis

2.1 Sampling2.2 Extraction and Cleanup

2.2.1 Solvents and Reagents2.2.2 Cleanup Procedures2.2.3 Toxicity-Directed Fractionation2.2.4 Specic Matrices: Water Samples2.2.5 Specic Matrices: Sediments and Soils2.2.6 Specic Matrices: Biota2.2.7 Volatile Analytes2.2.8 Atmospheric Sampling and Analysis

2.3 Procedures Involving Chemical Reactions: Derivatization2.3.1 Introduction2.3.2 Specic Procedures for Derivatization

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2.4 Identication and Quantication: Basic Denitions2.4.1 Experimental Techniques: Identication

2.4.1.1 X-Ray Diffraction2.4.1.2 Mass Spectrometry2.4.1.3 Nondestructive Procedures: NMR2.4.1.4 Fluorescence Line-Narrowing Spectroscopy

(FLN Spectroscopy)2.4.2 Experimental Techniques: Separation

and Quantication2.4.2.1 GC Systems2.4.2.2 HPLC Systems2.4.2.3 CE Systems

2.4.3 Application of Immunological Assays2.4.4 Stable Isotope Fractionation

2.5 General Comments2.5.1 Introduction2.5.2 Laboratory Practice2.5.3 Flexibility in Operation: An Open Approach2.5.4 The Spectrum of Analytes2.5.5 Multicomponent Commercial Products and Efuents

2.6 ConclusionsReferences

3. Partition: Distribution, Transport, and Mobility

3.1 Partitioning into Biota: Uptake of Xenobioticsfrom the Aqueous Phase3.1.1 Direct Measurements of Bioconcentration Potential

3.1.1.1 Outline of Experimental Procedures3.1.1.2 The Molecular Size of Xenobiotics and the Role

of Lipid Content of Biota3.1.2 The Role of Particulate Matter and Uptake via Food3.1.3 Concentration of Xenobiotics into Algae and Higher

Plants3.1.4 Surrogate Procedures for Evaluating Bioconcentration

Potential3.1.5 Interdependence of Bioconcentration and Metabolism3.1.6 Cautionary Comments

3.2 Partition between the Aquatic and Sediment Phases3.2.1 Outline of Experimental Procedures3.2.2 Reversibility: Sorption and Desorption3.2.3 Aging and Bioavailabilty

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3.2.4 Mechanisms of Interaction between Xenobiotics and Components of Solid Matrices

3.3 Phase Heterogeneity: Dissolved Organic Carbon,Interstitial Water, and Particulate Matter3.3.1 The Inhomogeneity of the Water Column3.3.2 The Role of Interstitial Water3.3.3 The Role of Sediment and Particulate Matter

in the Aquatic Phase3.4 Partitions Involving the Atmospheric Phase

3.4.1 Partitioning between the Aquatic Phaseand the Atmosphere

3.4.2 Partition between Solid Phases and the Atmosphere3.5 Dissemination of Xenobiotics

3.5.1 Transport within Aquatic Systems: The Role of Waterand Sediment

3.5.2 Transport within Aquatic Systems: the Role of Biota3.5.3 The Role of Atmospheric Transport3.5.4 Biomagnication3.5.5 The Role of Models in Evaluating the Distribution

of Xenobiotics3.5.6 Leaching and Recovery from Other Solid Phases

3.6 Monitoring3.6.1 Choice of Samples3.6.2 Temporal Record of Input3.6.3 Choice of Analytes3.6.4 Monitoring and Ecoepidemiology

3.7 ConclusionsReferences

4. Persistence: General Orientation

4.1 Abiotic Reactions4.1.1 Photochemical Reactions in Aqueous and Terrestrial Environments

4.1.2 Reactions in the Troposphere4.1.3 Chemically Mediated Transformation Reactions

4.1.3.1 Hydrolytic Reactions4.1.3.2 Dehalogenation Reactions4.1.3.3 Oxidation Reactions4.1.3.4 Reduction Reactions4.1.3.5 Thermal Reactions during Incineration

4.2 Biotic Reactions4.2.1 Denitions Degradation and Transformation

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4.2.2 Biodegradation of Enantiomers4.2.3 Sequential Microbial and Chemical Reactions

4.3 The Spectrum of Organisms4.3.1 Introduction4.3.2 Aerobic and Facultatively Anaerobic Bacteria4.3.3 Organisms Using Electron Acceptors Other Than

Oxygen4.3.4 Anaerobic Bacteria4.3.5 Phototrophic Organisms4.3.6 Eukaryotic Microrganisms: Fungi and Yeasts4.3.7 Other Organisms

4.4 Mechanisms for the Introduction of Oxygen4.4.1 Microbial Monooxygenase and Hydroxylase Systems

4.4.1.1 Monooxygenases4.4.1.2 Cytochrome P-450 Systems

4.4.2 Bacterial Dioxygenase Systems4.4.3 Eukaryotic Dioxygenases4.4.4 Oxidases, Peroxidases, and Haloperoxidases

4.5 Interactions4.5.1 Single Substrates: Several Organisms4.5.2 Cometabolism and Related Phenomena

4.6 Determinative Parameters4.6.1 Physical Parameters

4.6.1.1 Temperature4.6.1.2 Oxygen Concentration4.6.1.3 Redox Potential4.6.1.4 The Role of Association of Bacteria

with Particulate Material4.6.2 Substrates: Concentration, Transport into Cells,

and Toxicity4.6.3 Bioavailability: Free and Bound Substrates4.6.4 Preexposure: Pristine and Contaminated

Environments4.7 Rates of Metabolic Reactions

4.7.1 Kinetic Aspects4.7.2 Metabolic Aspects: Nutrients

4.8 Regulation and Toxic Metabolites4.8.1 Regulation4.8.2 Toxic or Inhibitory Metabolites

4.9 Catabolic Plasmids4.10 ConclusionsReferences

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5. Persistence: Experimental Aspects

5.1 Abiotic Reactions5.2 Microbial Reactions

5.2.1 Determination of Ready Biodegradability5.2.2 Isolation and Elective Enrichment5.2.3 General Procedures5.2.4 Basal Media5.2.5 Organic Substrates5.2.6 Procedures for Anaerobic Bacteria

5.3 Design of Experiments on Biodegradationand Biotransformation5.3.1 Pure Cultures and Stable Consortia5.3.2 Microcosm Experiments5.3.3 Experiments in Models of Natural Aquatic Systems

5.4 Experimental Problems: Water Solubility, Volatility, Sampling,and Association of the Substrate with Microbial Cells

5.5 Procedures for Elucidating Metabolic Pathways5.5.1 The Principle of Sequential Induction5.5.2 Application of Mutants5.5.3 Application of Metabolic Inhibitors5.5.4 Use of Synthetic Isotopes5.5.5 Application of NMR and Electron

Paramagnetic Resonance5.6 Application of Surrogate Substrates to Establish Enzymatic

Activity5.7 Classication and Identication of Organisms5.8 Procedures for Analysis of Degradative Populations

5.8.1 Specic Metabolic Activity5.8.2 Nondirected Examination of Natural Populations5.8.3 Examination for Established Metabolites or Specic

EnzymesReferences

6. Pathways of Degradation and Biotransformation

6.1 Aerobic Degradation of Nonaromatic Hydrocarbons6.1.1 Alkanes6.1.2 Cycloalkanes6.1.3 Alkenes6.1.4 Alkynes

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6.2 Aerobic Degradation of Aromatic Hydrocarbons and Related Compounds

6.2.1 Bacterial Degradation of Monocyclic AromaticCompounds

6.2.2 Metabolism of Polycyclic Aromatic Hydrocarbonsby Fungi and Yeasts

6.2.3 Metabolism by Bacteria of PAHs and Related Phenolsand Carboxylic Acids

6.3 Aerobic Degradation of Heterocyclic AromaticCompounds6.3.1 Reactions Mediated by Bacteria

6.3.1.1 Hydroxylation Reactions6.3.1.2 Dioxygenation Reactions6.3.1.3 Reductive Reactions6.3.1.4 Hydrolytic Reactions Resulting in Ring

Cleavage6.3.2 Reactions Mediated by Fungi

6.4 Degradation of Halogenated Alkanes and Alkenes6.4.1 Elimination Reactions6.4.2 Hydrolytic Reactions6.4.3 Monooxygenase Systems6.4.4 Reductive Dehalogenation Reactions of Halogenated

Aliphatic and Alicyclic Compounds6.5 Aerobic Degradation of Halogenated Aromatic

Compounds6.5.1 Bacterial Systems

6.5.1.1 Halogenated Aromatic Hydrocarbons6.5.1.2 Halogenated Aromatic Compounds Carrying

Additional Substituents6.5.2 Fungal Systems

6.6 Anaerobic Metabolism of Halogenated AromaticCompounds

6.7 Reactions Carried Out by Anaerobic Bacteria Other Than Dehalogenation

6.7.1 Aliphatic Compounds6.7.2 Biotransformation of Polyalicyclic Compounds

Containing Several Rings6.7.3 Aromatic Compounds

6.7.3.1 Pure Cultures6.7.3.2 Mixed Cultures

6.7.4 Heterocyclic Aromatic Compounds6.7.4.1 Nitrogen-Containing Heterocyclic

Compounds6.7.4.2 Oxygen-Containing Heterocyclic

Compounds

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6.7.4.3 Sulfur-Containing Hetrocyclic Compounds6.8 Aerobic Degradation of Aromatic Compounds Containing

Nitro or Sulfonate Groups6.8.1 Aromatic Sulfonates6.8.2 Aromatic Nitro Compounds6.8.3 Aromatic Azo Compounds

6.9 Aliphatic Compounds Containing Oxygen, Nitrogen, Sulfur,and Phosphorus6.9.1 Ethers6.9.2 Aliphatic Amines6.9.3 Aliphatic Nitro Compounds6.9.4 Suldes, Disuldes, and Related Compounds6.9.5 Phosphonates6.9.6 Organometallic and Related Compounds

6.10 Organouoro Compounds6.10.1 Aliphatic Fluoro Compounds6.10.2 Aromatic Fluoro Compounds6.10.3 Triuoromethyl Compounds

6.11 Biotransformations6.11.1 Hydrolysis of Esters, Amides, and Nitriles6.11.2 Hydroxylations, Oxidations, Dehydrogenations,

and Reductions6.11.2.1 Hydroxylation6.11.2.2 Oxidases6.11.2.3 Halogenation6.11.2.4 Chiral Synthesis

6.11.3 The Formation of Dimeric Products from AromaticAmines

6.11.4 Methylation Reactions6.11.5 Environmental Consequences of Biotransformations

6.12 Summary of Basic Microbial ReactionsReference.

7. Ecotoxicology

7.1 Choice of Test Species in Laboratory Tests7.2 Experimental Determinants

7.2.1 Exposure7.2.2 End Points7.2.3 Test Conditions7.2.4 Evaluation of Variability in the Sensitivity of Test

Organisms7.3 Test Systems: Single Organisms

7.3.1 Aquatic Organisms

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7.3.1.1 The MICROTOX System7.3.1.2 Algae7.3.1.3 Higher Plants7.3.1.4 Crustaceans7.3.1.5 Fish7.3.1.6 Other Aquatic Organisms

7.3.2 Organisms for Evaluating Toxicity of Sediments7.3.3 Assays for Genotoxic Effects7.3.4 Assays for Deformation and Teratogenicity7.3.5 Assays for Estrogenic Activity7.3.6 Terrestrial Organisms

7.4 Test Systems: Several Organisms7.4.1 Introduction7.4.2 Microcosms7.4.3 Mesocosms

7.5 Metabolism of Xenobiotics by Higher Organisms7.5.1 Metabolism by Fish7.5.2 Metabolism by Other Higher Aquatic Organisms7.5.3 Metabolism and Dissemination in Birds7.5.4 Metabolism by Invertebrates7.5.5 Metabolism by Higher Plants

7.6 Biomarkers: Biochemical and Physiological End-Points7.6.1 Biochemical Parameters7.6.2 Physiological Parameters7.6.3 Chromosomal Alterations

7.7 A Wider Perspective7.7.1 Toxic Equivalent Factors (TEF)7.7.2 Effect and Cause: Ecoepidemiology7.7.3 The Structure of the Toxicant and Its Biological Effect

7.8 A Hierarchical System for Evaluating the Biological Effectsof Toxicants

7.9 ConclusionsReference.

8. Microbiological Aspects of Bioremediation

In collaboration with Ann-Soe Allard

8.1 Introduction8.1.1 Orientation8.1.2 Microbial Considerations

8.2 Representative Sites8.2.1 Coal-Distillation Products8.2.2 Renery Waste and Stranded Oil: Petroleum

Hydrocarbons

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8.2.2.1 Terrestrial Habitats8.2.2.2 Marine Habitats8.2.2.3 Conclusion

8.2.3 Wood Preservation Sites: ChlorophenolicCompounds

8.2.4 Chemical Production Waste8.2.4.1 Chlorinated Alicyclic Hydrocarbons8.2.4.2 Chlorinated and Brominated Aromatic

Hydrocarbons8.2.4.3 Organouoro Compounds8.2.4.4 Chlorinated Anilines8.2.4.5 Conclusion

8.2.5 Military Waste8.2.5.1 Explosives8.2.5.2 Chemical Warfare Agents

8.2.6 Groundwater Contamination8.2.6.1 Benzene/Toluene/Ethylbenzene/Xylenes8.2.6.2 Chloroethenes8.2.6.3 Methyl

t-

Butyl Ether8.2.7 Metabolic Interaction of Metal Cations and Organic Compounds

8.2.8 Municipal Waste8.2.9 Miscellaneous Contaminants

8.3 A Hierarchical Strategy8.4 Concluding CommentsReferences

Neilson, Alasdair H. "Introduction"Organic Chemicals : An Environmental PerspectiveBoca Raton: CRC Press LLC,2000

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1

Introduction

1.1 Orientation

Although the synthesis of 1,1,1-trichloro-bis(4-chlorophenyl)-ethane (DDT)was achieved in 1874 by Othmar Zeidler, the compound elicited little interestuntil its potent toxic effect toward insects was discovered by P.H. Mller in1939. The compound fullled only too well his criteria that a suitable com-pound should be chemically stable and persist for long periods of time.Although its use over the next few years was extensive and effective, DDTwas to become the paradigm for an appreciation of the complex network ofadverse effects brought about by the introduction of synthetic chemicals intothe environment (Dunlap 1982).

It is historically convenient to date the beginning of popular concern overthe environment with the publication of Rachel Carsons

Silent Spring

in 1962.Although this book made no pretense at a scientic exposure of the potentialdangers of pesticides, the date happily coincided with the coming of age ofthe analytical instrumentation needed to provide a rm scientic foundationfor the unease which was expressed.

The succeeding years have seen an increasing degree of sophistication inthe analysis of environmental issues and a clear appreciation of the centralrole of chemical analysis. Indeed, since the birth of quantitative chemistrythere has been an almost continuous search for more sensitive and selectivemethods of analysis. For example, it was the invention of the humble Bunsenburner almost 150 years ago that provided a nonluminous ame, whichmade possible atomic emission spectroscopy, and before the turn of the 19

th

century this had contributed to the discovery of no fewer than 12 new ele-ments. Within the last 40 years or so, the revolution in organic analysis hasbeen no less spectacular: application of gas chromatographic, high-resolutionliquid chromatography, and capillary electrophoresis has become routine,and structural identication using mass spectrometry and nuclear magneticresonance commonplace.

For organic compounds, evaluation of their environmental impact basedon data for acute toxicity to sh and estimation of biochemical oxygendemand has been steadily replaced by the results of experiments on subacuteand chronic toxicity, sophisticated investigations of microbial metabolism,and an appreciation of the true complexity of partitioning processes. It may

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conservatively be stated that during the 1960s, there slowly emerged a newinterdisciplinary activity environmental science.

This book attempts to provide discussions on the analysis, partitioning,metabolism, and toxicity of organic compounds. Most of the principles areapplicable to both the aquatic and terrestrial environments, and the presen-tations are restricted to organic compounds. Although the most-detailedanalysis has been given to aspects of microbiology which has been the maininterest of the author during many years, these discussions that extend overthree chapters have been buttressed by a brief section dealing with chemicalanalysis which is a cornerstone of all aspects of environmental activity, andshort sections devoted to problems of partitioning and ecotoxicology. Yetnone of the chapters stands in isolation; analysis, partitioning, and mecha-nisms of association are intimately bound to metabolism while assessment oftoxicity requires consideration of bioavailability, partition, and metabolism.

There are certain threads that run throughout this series of essays: theimportance of the transformation of xenobiotics, the association betweenxenobiotics and components of environmental matrices, and the dynamicsof ecosystems. By implication, therefore, environmental hazard assess-ments should take into account not only the original xenobiotic but also itspossible transformation products together with the actual state free orassociated in which the compound exists in the environment. It shouldbe clearly appreciated that both the persistence and the toxicity of xenobiot-ics are critically determined by the extent and reversibility of the interac-tions between low-molecular-weight xenobiotics and polymeric material inthe environment. Indeed, the importance of dynamic interactions at boththe physicochemical and the molecular levels cannot be too stronglyemphasized.

The present study is essentially chemical and mechanistic in approach.Attention is drawn to a number of books dealing with the more general issueof hazard assessment even though the emphasis of some is on radiologicalhazard and carcinogenesis in humans (Fischoff et al. 1981; Ottoboni 1984;Lewis 1990; Rodricks 1992), to a provocative sociological book that deals withperceived risk and fear (Furedi 1997), and to a valuable book on randomnessand numerical signicance (Bennett 1998).

1.2 Literature Cited

One aim of this book is to provide a forum for presenting an overview of theissues that are basic to producing an environmental hazard assessment oforganic compounds discharged into the environment. This evaluation requiresthe application of expertise in the analysis of the compounds, knowledge oftheir distribution among the various environmental compartments and their

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dissemination, and appreciation of the factors that determine their persistenceand toxicology. An attempt has been made throughout to illustrate these prin-ciples with concrete examples, even though no effort has been made to providea comprehensive account of any single group of compounds. It was hoped atthe same time that the reader would obtain some appreciation of the complex-ities in the design and interpretation of the relevant experiments, which arebeing carried out in the laboratory. This has therefore necessitated some degreeof compromise both in the depth of the presentation and in the number ofreferences to the literature.

It was decided at the outset that it was clearly impossible to provide cita-tions for every statement; on the other hand, controversial points or possiblyless well-known facts which have not yet reached the textbooks deserve cita-tion of their source. References are almost invariably given to the primary lit-erature, which has been subjected to the scrutiny of peer review. It istherefore assured that even when the present authors interpretations shouldprove faulty and this is inevitable a solid and reproducible basis of factis available to the critical reader. As a result, although the number of refer-ences is considerably greater than had been visualized at the outset, theyrepresent merely an eclectic selection from a vast literature. The choice of ref-erences may, however, seem quixotic on the one hand, unduly historical,and on the other, recent, though incomplete relevant references have nodoubt been omitted, but the writer can assure the authors of these that thereis no malice in the selection. Some older work has been cited when this hasled to lasting concepts, although other early work may be difcult to evalu-ate by the standards of today, and no doubt work at the cutting edge of cur-rent research will rightly require modication and extension in the future.When appropriate, reference has been made to reviews which presentdetailed coverage that lies beyond the scope of this volume. The author hastherefore exercised the privilege of selection. It is too much to hope that anadequate compromise has been reached, and, without doubt, importantstudies have been omitted and errors of interpretation have been made; inthe nal analysis, all that can be done is to offer humble apologies and hopethat the damage done is not too serious. The author hopes he may take ref-uge in the reply given to a lady inquiring why, in his great dictionary, Dr.Johnson had dened pastern as the knee of a horse: Ignorance, madam,pure ignorance.

1.3 Limitations

It is inevitable that the coverage of the areas included for discussion isuneven. The selection of material for inclusion is therefore substantiallydetermined by personal bias and some of these prejudices should be taken

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into account. To quote again Dr. Johnson discussing the state of learning inthe present author s native country: Their learning is like bread in abesieged town: every man gets a little, but no man gets a full meal. This iscertainly true of the discussions presented in this book.

1.3.1 Organochlorines

There is a huge literature on organochlorine compounds since these haveawakened serious and well-merited concern. Although in many cases, inves-tigations concerned with these compounds have yielded principles of gen-eral application, an attempt has been made to redress the balance byincluding illustrative examples using nonchlorinated compounds: the cover-age of, for example, DDT, polychlorinated biphenyls (PCBs), and chlorinateddibenzo[1,4]dioxins does not therefore reect the substantial research effortthat has been directed to these compounds. It is hoped, however, that apart altogether from the specic interest in individual compounds thegeneral principles that have been developed in the course of such investiga-tions have clearly emerged. The other large class of compounds that havebeen extensively studied are the polycyclic aromatic hydrocarbons (PAHs),but it is pointed out in several places that conclusions drawn from studieswith neutral compounds such as PAHs and many of the organochlorinesmay not be directly applicable to compounds with polar substituents such ashydroxyl, amino, or ketonic groups. It is particularly important to under-score the great structural diversity of agrochemicals and pharmaceuticalproducts that have been developed against specic biological targets usingthe increasing sophistication of methods available to the synthetic organicchemist.

Whereas agrochemicals have attracted attention as a result of their potentialadverse effects on nontarget organisms and concern with their persistence,the same intensity of effort has not been directed so far to pharmaceuticalcompounds for human use. Although this presumably reects the muchsmaller quantities that are involved, the widespread distribution of some ofthem including, for example, antibiotics and steroids merits attention.Indeed, recent interest in the possible estrogenic effects of xenobiotics hasrevealed low levels of the pharmaceutical diclofenac in lake water in Switzer-land (Buser et al. 1998) and the contraceptive 17

-ethynlyestrone in treateddomestic sewage efuent (Desbrow et al. 1998). On the other hand, largequantities of veterinary chemotherapeutic agents are used in large-scale hus-bandry and may be eliminated into the terrestrial environment: an illustrativeexample is provided by the uoroquinolone carboxylic acids. Enrooxacin,which is eliminated largely unchanged and as the

N

-deethylated metabolite,is strongly sorbed to clay minerals (Nowara et al. 1997), so that its degrada-tion is photochemically mediated (Burhenne et al. 1997).

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1.3.2 Dictates for Inclusion

Areas that have attracted intense research effort may not have done so exclu-sively because of their scientic interest, but as a result of economic oreven social pressure. Two illustrative examples are the 2,3,7,8-tetrachloro-dibenzo[1,4]dioxins and the phthalates. The long-standing uncertaintysurrounding the hazard associated with exposure to 2,3,7,8-tetrachloro-dibenzo[1,4]dioxin (U.S. EPA 1993) in spite of massive international effortillustrates both the inuence of public opinion and the intrinsic difculty ofthe hazard assessment process. Enormous attention has been directed tophthalate esters, which is not reected in this book, although the biodegrada-tion of these is addressed in Sections 6.2.1 and 6.7.3. It should be noted that

o

-phthalate is an intermediate in several degradation pathways: for example,the bacterial degradation of phenanthrene (Section 6.2.3) and quinoline(Section 6.3.1), and the fungal degradation of anthracene (Section 6.2.2).

1.3.3 Modeling

There is at least one major area of activity pertaining directly to the environ-ment for which the reader will seek in vain. The complexity of environmentalproblems and the availability of personal computers have led to extensivestudies on models of varying sophistication. A discussion and evaluation ofthese lie well beyond the competence of an old-fashioned experimentalist;this gap is left for others to ll but attention is drawn to a review that coversrecent developments in the application of models to the risk assessment ofxenobiotics (Barnthouse 1992), a book (Mackay 1991) that is devoted to theproblem of partition in terms of fugacity a useful term taken over fromclassical thermodynamics and a chapter in the book by Schwarzenbach etal. (1993). Some supercial comments are, however, presented in Section 3.5.5in an attempt to provide an overview of the dissemination of xenobiotics innatural ecosystems. It should also be noted that pharmacokinetic modelshave a valuable place in assessing the dynamics of uptake and elimination ofxenobiotics in biota, and a single example (Clark et al. 1987) is noted paren-thetically in another context in Section 3.1.1. In similar vein, statistical proce-dures for assessing community effects are only supercially noted inSection 7.4. Examples of the application of cluster analysis to analyze bacte-rial populations of interest in the bioremediation of contaminated sites aregiven in Section 8.2.6.2.

1.3.4 Enzymes and Relatedness

The discussion of enzymology is both limited and uneven. An area of increas-ing interest is the relation between enzymes based on sequences of amino

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acids or nucleotides. This has been noted in a few examples, but a satisfactorydiscussions lies beyond both the competence of the author and the limits ofthis volume.

1.4 Biotechnology

In Chapters 4 and 6, considerable emphasis is placed on the environmentalsignicance of biotransformation as opposed to biodegradation. It thereforeseems appropriate to draw brief attention also to their use in biotechnology.Microorganisms play important roles in widely diverse technical processesranging from the leaching of ores and the preservation of food, to the produc-tion of antibiotics and food additives. It is worth drawing attention to somefeatures of these applications.

1. Some of these processes are carried out by specic groups of organ-isms: for example, by acid-tolerant thiobacilli in the leaching ofores, by lactobacilli in food preservation, or by fungi and actino-myces in the production of antibiotics.

2. The production of food additives such as fructose, glutamate,lysine, citric acid, ascorbic acid, and vanillin implies that thesecompounds are not used as substrates for growth. This is generallyaccomplished by use of mutant strains that, as a bonus, producehigh yields of the desired products.

Increasing attention has been directed to the use of microbial transforma-tions in the production of valuable compounds or chemical intermediates. Asurvey has been given of biochemical transformations in organic chemistry(Faber 1997) and covers many reactions (including the formation of CCbonds) that have made use of microorganisms to accomplish an importantstep in organic syntheses. Some of the salient reactions are discussed inSection 6.11.

Biotechnology is often interpreted to include biological waste treatment, sothat one important application of studies in biodegradation is in the designand optimization of biological systems for the treatment of liquid industrialwaste. Attention is therefore directed to a review that draws attention to thepossible application of organisms not traditionally considered in this context(Kobayashi and Rittman 1982). Biological waste treatment has not been dis-cussed at all here since most of the fundamental physicochemical and engi-neering principles are not included in this book. On the other hand, manycritical and in some cases unresolved issues that are directly relevanthave been taken up in the appropriate chapters of this book: partition into thesolid phase, into biota, and into the atmosphere together with the reversibility

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of these processes (Chapter 3), persistence to biotic and abiotic attack and theformation of metabolites (Chapters 4 and 6), and toxic effects (Chapter 7).Microbiological issues relevant to the bioremediation or biorestoration of con-taminated areas are discussed in Chapter 8, and the contamination of ground-water by leachate from landlls clearly interfaces with that of the aquaticenvironment. One important issue which may be of dominant signicance insome of these articial terrestrial environments and which is clearly not soin the aquatic environment is the water concentration, which may be a fac-tor that limits the growth and metabolism of the relevant organisms.

Some other aspects of biological treatment systems both aqueous andterrestrial are clearly germane to the principles discussed in this book andattention is drawn to the following issues.

1. In most situations a number of structurally diverse substrates aresimultaneously present, and because of toxicity or metabolicincompatibility, this may give rise to problems in assessing theirtoxicity and degradability.

2. Care must be exercised not to confuse the aims of the investigation:for example, anaerobic reactors that are developed to treat waste-water with the object of producing methane may not degrade recal-citrant xenobiotics for which inoculation with specicmicroorganisms may be necessary (Ahring et al. 1992).

3. There exist unresolved issues in the application of genetically engi-neered organisms for biological treatment (a) from the potentialrisk of dissemination of the organisms and (b) from the problemof maintaining these organisms in a mixed bacterial population incompetition with other organisms.

4. Addition of nutrients such as nitrogen and phosphorus must becarefully adjusted so that excess is not discharged into receivingwaters, while for efuents with high concentrations of readilydegraded substrates, advantage might be taken of nitrogen-xingbacteria to diminish or even eliminate additions of combined nitro-gen (Neilson and Allard 1986).

1.5 Terrestrial Systems

There is considerable interest and concern over the fate of agrochemicals inthe terrestrial environment and over their possible effect on nontarget organ-isms. These problems are considered only parenthetically here, although thetransport of agrochemicals is governed by the principles of distribution dis-cussed in Chapter 3, their fate by those given in Chapters 4 and 6, and theirtoxicology by those in Chapter 7. Extensive results on the persistence of

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agrochemicals have also provided gratuitous support for the principles ofelective enrichment that are discussed in Chapter 5.

Considerable interest has arisen in the environmental problems associatedwith the disposal of solid waste; again this interfaces closely with the aquaticenvironment through leaching of organic compounds from landlls bothas solutes and as particulate material into watercourses, rivers, and lakes.There has been considerable interest in the bioremediation of sites that havebeen contaminated with both municipal and industrial solid waste. A chapteron bioremediation is therefore provided for two additional reasons: (1) inimportant respects, bioremediation involves an extension of the principlesoutlined in Chapters 3, 4, 5 and 6, and (2) it illustrates that many of the prin-ciples developed within the aquatic environment that were the subject of pre-vious chapters can be applied with suitable and relatively minor modicationto the terrestrial environment.

1.6 The Atmosphere

It is apparent that separate consideration of the atmospheric, terrestrial, andaquatic phases is articial since there are strong interactions among them.Two examples, which support this view, will be used as illustration.

1. It has been repeatedly demonstrated that organic compounds even those of apparently low volatility may be transported viathe atmosphere over great distances and may therefore be encoun-tered in samples of precipitation and particulate deposition col-lected from areas remote from the point of initial discharge. Thisis discussed further in Section 3.5.3.

2. The atmosphere is not an inert medium, and compounds dis-charged into it may be transformed by photochemical reactions orby interaction with atmospheric constituents such as oxygen, orthe oxides of sulfur and nitrogen; this is discussed briey inSection 4.1. These transformation products may subsequently enterthe aquatic and terrestrial environments in the form of both par-ticulate matter and precipitation. This important issue has beenaddressed in Section 4.1.3.

For these reasons, a thorough discussion of the role of atmospheric pro-cesses ought to be presented if the aquatic environment is to be adequatelydiscussed. This is, however, a monumental task far beyond the competenceof the author; all that can be offered instead are brief comments at the appro-priate places in the various chapters. Reference should be made to the

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comprehensive discussion of principles given by Finlayson-Pitts and Pitts(1986) that is referred to in Sections 3.5.3 and 4.1.2, and to the range of com-pounds produced by higher plants including hydrocarbons (Cao and Hewitt1995) and oxygenated derivatives (Knig et al. 1995) which may enter theatmospheric environment.

1.7 Natural Products and Microbial Metabolites

This account is directed almost exclusively to low-molecular-mass xenobiot-ics so that a discussion of biosynthetic reactions mediated by microorganismsand of biopolymers lies beyond its assigned limits. A few brief commentsseem justied, however, because of an upsurge of interest in various groupsof naturally occurring compounds, the structure of these biopolymers, andtheir role in carbon cycling in the environment. Attention is therefore drawnto a few of these, some of which may be persistent.

1.7.1 Organohalogen Compounds

There has been considerable interest in these due to widespread concern overpossibly adverse effects of some anthropogenic organohalogens, but since thequantitative signicance of almost none of these microbial metabolites hasbeen evaluated and the toxicology of only very few has been investigated invariably in other contexts it seems justiable to accord a low priority tosuch problems in the light of more urgent issues.

Organohalogen metabolites are produced by bacteria, fungi, and algae by

haloperoxidases

that contain protoporphyrin IX, vanadium (V), or are withoutmetals, and catalyze their synthesis in the presence of H

2

O

2

, formally by pro-duction of Hal

+

(Neidleman and Geigert 1986).

1. Biosynthetic reactions carried out by microorganisms produce astructurally diverse range of chlorinated and brominated com-pounds (Strunz 1984; Gribble 1996; van Pe 1996; de Jong and Field1997), while some higher plants produce uorinated compounds.Brominated compounds are widely distributed in marine environ-ments, and bromoform is the major haloform produced by marinemacroalgae (Nightingale et al. 1995). An unusual 1,1

-dimethyl-2,2

-bipyrrole containing four bromine and two chlorine atoms has beenfound in the eggs of seabirds from both the Atlantic and PacicOceans but not from the Great Lakes (Tittlemeier et al. 1999).Comparable highly halogenated compounds of marine origin maytherefore have considerable bioconcentration potential. A plethora

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of organobromo compounds is produced by higher organisms inparticular, sponges and their possible therapeutic application hasresulted in intense activity (Munro et al. 1987). Attention has alsobeen drawn to the possibility of exploiting the synthetic capabilityof marine bacteria for producing compounds of pharmaceuticalinterest (Austin 1989), and to the use of chlorinated phenylpyrrolesproduced by

Pseudomonas cepacia

for controlling fruit spoilage fungi(Roitman et al. 1990).

2. Halogenated compounds are produced both by

de novo

synthesisinvolving direct incorporation of halide ion and by transmethyla-tion reactions (Section 6.11.4). The former include reactions medi-ated by haloperoxidases in the presence of hydrogen peroxide andhalide ion, and these enzyme systems have wide biosynthetic capa-bility (Neidleman and Geigert 1986; van Pe 1996) including oxi-dative dimerization resulting, for example, in the formation of2,3,7,8-tetrachlorodibenzo[1,4]dioxin from 2,4,5-trichlorophenol(Svenson et al. 1989).

3. A few naturally occurring compounds such as methyl chlorideand halogenated phenols are important industrial products,although the relative quantitative contribution of the natural prod-ucts on a global basis cannot be estimated reliably. In addition, itseems unlikely that the more highly chlorinated phenols andin particular, for example, 2,4,5-trichlorophenol are producedin signicant concentrations by such reactions (Wannstedt et al.1990).

4. It has been hypothesized on the basis of the formation of trichlo-roacetate from aliphatic compounds, especially acetate, by theaction of chloroperoxidase in the presence of hydrogen peroxideand chloride that this might be a naturally occurring metabolite(Haiber et al. 1996). Plausible mechanisms for the formation oftrichloroacetic acid by atmospheric reactions involving trichloro-ethane and tetrachloroethene are discussed in Section 4.1.2.

5. Although the distribution, persistence, and toxicity of these halo-genated compounds such as the chlorinated grisans, chloram-phenicol, 7-chlorotetracycline, and clindamycin are antibiotics are subject to the same principles as those outlined in this book,this aspect has seldom been examined. One exception that mayserve as an illustration is the debromination of naturally occurringbromophenols by bacteria under anaerobic conditions (King 1988).

6. 3-Chloro- and 3,5-dichloroanisyl alcohols produced by the white-rot fungus

Bjerkandera

sp. may play a physiological role by pro-ducing H

2

O

2

during oxidation to the aldehydes by aryl

alcohol

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oxidase; the chlorinated metabolites are poor substrates for ligninperoxidases so that the H

2

O

2

may therefore be used for oxidationof lignin while the anisyl aldehydes are readily recycled by reduc-tion to the alcohols (de Jong et al., 1994). The metabolite 2-chloro-1,4-dimethoxybenzene is a cofactor in the oxidation of anisyl alco-hol by lignin peroxidase, and is superior to veratryl alcoholalthough it does not protect the enzyme against inactivation byH

2

O

2

(Teunissen and Field 1998).7. Chlorinated orcinols have been identied from the bulbs of the

edible lily

(Lilium

maximowiczii

) infected with the fungus

Fusariumoxysporum

f.sp.

lilii

. They were not, however, fungal metabolites;these could also be induced by ultraviolet radiation of bulb scales,and the more highly chlorinated metabolites inhibited conidialgermination of

Bipolaris leersiae

(Monde et al. 1998).

1.7.2 Polymeric Compounds

There are many important groups of macromolecules. A discussion of theselies beyond the expertise of the author, but it seems appropriate to note brieya few of these groups due to their environmental relevance and since all ofthem make a contribution to organic carbon cycling in the environment.

1. Lignin is an important constituent of many higher plants althoughthe amounts vary with the taxon, the part of the plant, and thetime of year. A great deal of attention has been given to the deg-radation of these compounds (Jeffries 1994), and to attempts torecover useful monomeric products including methane. A fewaspects of the enzymes involved are given in Section 4.4.4, whilethe role of the lignin-degrading white-rot fungi including

Phanero-chaete chrysosporium

in degrading xenobiotics is noted in Section4.3.5 and in Chapter 6 (Sections 6.2.2, 6.5.2, and 6.8.2). The greatermetabolic potential of other white-rot fungi is, however, emerging.

2. The empirically dened terms humic acids and fulvic acidshave been used to denote important macromolecular constituentsof aquatic systems. Even though their structures are incompletelyresolved (Section 3.2.3 and 4.5.3), their association with low-molecular-mass organic compounds and with metal cations is important in determining the bioavailability and hence biode-gradability and toxicity of organic compounds. Their role in thegeneration of OH radicals under anaerobic conditions is noted inSection 4.1.1, and as intermediates in electron-transfer reactionsunder anaerobic conditions in Section 5.5.5.

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3. Considerable attention has been directed to particulate organicmatter (POM) in the marine environment that is plausibly derivedfrom organisms in the euphotic zone. This is supported by MScomparison of pyrolysis products from samples collected in sedi-ment traps in the

Mediterranean Sea with that from the diatom

Biddulphia sinensis

. Although the structures are unknown, a widevariety of compounds have been identied in the pyrolysatesincluding aliphatic hydrocarbons and nitriles, pyrroles, indoles,and aromatic hydrocarbons (Peulv et al. 1996).

4. Although polysaccharide, lipid, and polypeptide components ofmicroorganisms may be presumed to be readily degraded in avariety of ways, it has been shown that algae synthesize a numberof more resistant compounds. The algaenans represent an impor-tant group of aliphatic macromolecules; these have been examinedin a number of marine algae and consist of linear C

28

C

34

methylenegroups linked by oxygen atoms (Gelin et al. 1996), and have plau-sibly been suggested as an important sink for organic carbon inthe marine environment.

5. Biphytanes derived from membrane ether lipids of archaea havebeen found in water column particulates, and sedimentary organicmatter (Hoefs et al. 1997).

In addition, a number of anthropogenic oligomers are produced in sub-stantial quantities.

1. Important groups of detergents consist of alcohol ethoxylates, alky-lphenyl ethoxylates, and alkylethoxy sulfates, and their biodegra-dation has been reviewed (White et al. 1996). Only parentheticalcomments on this group of compounds are given in Chapter 6.

2. Silicones have found diverse and increasing use and are generallyconsidered highly stable. Although the levels in efuents frommunicipal treatment plants were generally below the limits ofquantication, they have been found in substantial amounts in thesludges, in sediments in the vicinity of the outfalls, and in agricul-tural soil that was amended with sewage sludge (Fendinger et al.1997). Polydimethylsiloxanes are, however, at least partiallydegraded in soil to dimethylsilanediol (Carpenter et al. 1995;Fendinger et al. 1997), and the mineralization in soil of themonomer (dimethylsilanediol) has been shown (Sabourin et al.1996). The interesting observations were made that in liquid cul-tures degradation could be obtained with

F. oxysporium

growingconcurrently with propan-2-ol or acetone, and by a strain of

Arthro-bacter

sp. growing concurrently with dimethylsulfone.

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1.7.3 Polypyrroles

Chlorophylls are produced by all photosynthetic organisms and even bysome nonphotosynthetic bacteria and details of their structures depend ontheir source. Collectively they represent a considerable reserve of organic car-bon and nitrogen, although little seems to have been established on their per-sistence. A wide range of transformation products of chlorophylls has beenrecovered from the sediments of a freshwater eutrophic lake, and theseincluded the unusual sterol esters of pyrophaeophorbides (Eckardt et al.1995). It is also presumable that such chlorophyll transformation productsproduce the pyrroles and indoles that have been described in sedimentpyrolysates noted above.

1.7.4 Polycyclic Aromatic Hydrocarbons

These have been encountered in a wide variety of environmental samplesranging from sedimentary rocks to lake sediments and street dust. Althoughthermal processes during incineration of fossil fuels represent a major inputinto the environment, it seems plausible that many of these compounds aretransformation products of naturally occurring steroids and terpenoids, andit is possible that these reactions are microbially mediated. A summary ofmany of the structures involved has been given (Simoneit 1998), and of plau-sible reactions for their formation (Neilson and Hynning 1998). The occur-rence of these in environmental samples could seriously compromisenonspecic analysis for aromatic compounds; this is noted again in Section2.4.2. Detailed reviews of many aspects of PAHs including their sources, dis-semination, mechanisms of their carcinogenic activity, their uptake and tox-icity in aquatic biota, and their microbial metabolism have been given in amultiauthored book (Neilson 1998).

1.8 The Effect of Xenobiotics on Microbial-Mediated Processes

The present discussion is directed to the degradation and transformation ofxenobiotics by microorganisms. The converse issue the inhibitory effect ofxenobiotics toward microbial processes that are not directly involved indegradation, but may play an important role in natural geochemical cycles is not explored. This is of enormous importance, particularly in the terrestrialenvironment where nontarget organisms may be subjected to exposure toagrochemicals. Some of these organisms have an autotrophic way of life andutilize CO

2

as principal, or exclusive, source of carbon rather than aheterotrophic metabolism that is characteristic of those organisms that are

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primarily responsible for degradation of xenobiotics. The autotrophic organ-isms include algae, ammonia-oxidizing bacteria, and many thiobacilli. In thecontext of degradation, however, the relevant microorganisms are generallysufciently resistant to the xenobiotics to preclude serious inhibition. Someuseful examples of the reactions that may be involved are found in a review(Smit et al. 1992) that discusses the hazard of genetically manipulated organ-isms released into the terrestrial environment.

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Neilson, Alasdair H. "Analysis"Organic Chemicals : An Environmental PerspectiveBoca Raton: CRC Press LLC,2000

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2

Analysis

SYNOPSIS

Chemical analysis is an integral part of all environmentalinvestigations. Brief attention is directed to aspects of sampling in order toavoid interference from possible artifacts, to various procedures for extrac-tion and concentration of analytes, and to the importance of cleanup proce-dures before identication and quantication. Procedures for the analysis ofwater, soil, and sediment samples and biota are outlined, and some impor-tant reactions for derivatizing functional groups are summarized. Methodsfor the identication of components of environmental samples are brieysummarized, and attention is directed to developments in ionization proce-dures for mass spectrometry (MS) analysis, high-performance liquid chroma-tography (HPLC) MS interfaces, and the application of nondestructiveprocedures such as nuclear magnetic resonance (NMR) spectroscopy. Theimportance of access to authentic reference compounds is emphasized. Gaschromatographic (GC) procedures for quantication are briey discussed,and attention is directed to recent developments including the use of chiralsupport phases and a range of detector systems. The use of supercritical u-ids for the extraction of samples and as mobile phases for chromatography isbriey noted, and developments in liquid chromatography (LC) and capil-lary electrophoresis (CE) are discussed. The potential for application ofimmunologically based assays is noted, and attention is drawn to the poten-tial for application of stable isotope fractionation. The range of analytes including transformation products which may be encountered in environ-mental samples is discussed including the problems in the analysis of com-mercial mixtures and complex efuents. There is an inherent indeterminacyin assessing the recoverability of analytes from naturally aged sediment sam-ples as a result of dynamic associations between analytes and macromolecu-lar components in environmental matrices.

Introduction

It is appropriate to begin this book with a discussion of chemical analysiswhich lies at the heart of almost all environmental investigations whether

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they are devoted to monitoring the distribution of a xenobiotic, evaluating itspersistence and toxicity, or determining its partition among environmentalmatrices. Analytical support will be incorporated into all of these programsand its central role should be clearly appreciated at the planning stage; oth-erwise, the conclusions that are drawn from the results of the investigationmay be equivocal. These comments should not, however, be interpreted asnegating the fundamental contributions that analytical chemistry itself hasmade, not the least of which is in illuminating the role of metabolites andtransformation products, and in revealing the global distribution of hithertounsuspected compounds.

There have been revolutionary instrumental developments during the last35 years or so, and these have completely altered the scope and possibilities ofenvironmental research. The following examples may be given as illustration.

1. After early concern over the accumulation of DDT in biota, analysisof DDT and its metabolites was carried out by nitration and col-orometric measurement after treatment with alkali. This was super-seded by GC some 15 years later, and this development facilitatedmore rapid analysis, greater precision, and simultaneous unambig-uous analysis of DDT metabolites such as DDE and DDD.

2. Although polychlorinated biphenyls (PCBs) were rst synthesizedin 1881 and introduced into industrial use as electrical insulatorsin the 1920s, they were detected in environmental samples onlysome 35 years later in 1966 by Jensen (Jensen 1972; Jensen et al.1972). This discovery was facilitated by the development of theelectron-capture (EC) detector in GC, and it also illustrates an earlyexample of the application of MS to the tentative identication ofenvironmental contaminants. Since that date, PCBs have becomerecognized as virtually universal contaminants of environmentalsamples from all parts of the world.

3. The interest of the synthetic organic chemist in stereospecic syn-thesis has resulted in the need for methods for the analysis ofchirons. This has led to the development of both chiral reagentsand of chiral supports for GC analysis, and the use of both HPLCand CE. The application of these methods to environmental sam-ples is beginning to draw attention to the environmental input ofenantiomers without established biological activity as well as totheir possible persistence. The inherent complexity when the asym-metric atom carries an electron-attracting group such as carboxylis illustrated by the example of phenoxypropionates (Buser andMller 1997) is discussed in Section 4.2.2.

4. The identication of xenobiotics at the concentrations existing inenvironmental contaminants and of metabolites formed duringlaboratory experiments in biodegradation and transformation hasbeen completely revolutionized by the availability of modern

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instrumentation for structure determination. For example, infra-red (IR) and NMR spectroscopy and MS have been widely used,and their sensitivity has been facilitated by Fourier transform (FT)techniques for signal processing.

5. The detection and quantication of extremely low concentrationsof chlorinated dibenzo[1,4]dioxins in environmental sampleswould not have been possible without the development of high-resolution mass spectrometers. As a dividend, the instrumentationthereby developed has been adapted to other analytes and hassignicantly lowered both their limits of detection and the level ofquantication.

6. The increased sensitivity of NMR instrumentation has made pos-sible the application of

13

C NMR to study the pathways and kineticsof microbial reactions in cell suspensions. These procedures haveprovided direct evidence for the structure of intermediate metab-olites without the need for their isolation. Determination of thestructure of environmental analytes has also become possible usingnatural levels of

13

C. Techniques have also been developed to over-come the negative magnetogyric ratio of nuclei such as

15

N and

29

Si(Morris and Freeman 1979), and these procedures have beenapplied to a number of environmental problems that were previ-ously inaccessible.

Today, few modern laboratories engaged in environmental studies lackfacilities for carrying out analysis using GC, HPLC, CE, or MS, and manyhave access to high-eld NMR instrumentation. Most of these instrumentsare coupled to systems for automatic injection of samples and to data systemsfor processing the output signals and reducing background interference. Thismakes possible the analysis of extremely small amounts of material, and thissingle advantage can hardly be overestimated since it is seldom possible toisolate environmental contaminants in sufcient quantity for identicationby conventional chemical procedures.

At the outset, brief mention should possibly be made of sum parameterssince these have traditionally dominated environmental analysis. Some ofthese, such as total organic carbon (TOC), dissolved organic carbon (DOC),particulate organic carbon (POC), and their nitrogen and phosphorus ana-logues have been included in conventional water quality criteria, and are stilluseful in wide-ranging monitoring, as in oceanography for example. With theupsurge of interest in halogenated xenobiotics, extensive effort has thereforebeen directed to devising comparable methods for organohalogen com-pounds. Analysis for total PCBs which depends on estimation of decachloro-biphenyl after chlorination (Lin and Hee 1985) or by dechlorination withLiAlH

4

(de Kok et al. 1981), or estimation of halogenated compounds in gen-eral by reduction with dispersed sodium followed by potentiometric analysis(Ware et al. 1988) have been used, but are now primarily of historic interest

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although they might nd application to other chemically stable analytes.Methods have also been developed for analyzing total organic chlorine, bro-mine, or iodine by neutron activation analysis (Gether et al. 1979) or for non-specic halogen by coulometry or potentiometric titration after combustion(Sjstrm et al. 1985). None of these procedures merits inclusion in contem-porary environmental hazard assessment programs since it has been consis-tently demonstrated that differences in the persistence, the toxicity, and thephase partitioning among, PCB congeners for example, and polycyclic aro-matic hydrocarbons (PAHs) make the use of sum parameters quite inappro-priate; such parameters should be replaced by analysis for the concentrationsof specic compounds. Although sum parameters have been used quiteextensively in some monitoring programs (Martinsen et al. 1988), they arereally justiable only when it can be established that a restricted range ofstructurally similar compounds is present.

The discussion presented here makes no attempt at being comprehensive,nor does it provide experimental details of standardized methodologies. Thisaccount is therefore not directed to the professional analyst, nor is it intendedto serve as a handbook although some general comments on laboratory prac-tice are made in Section 2.5. The needs of the professional are fullled by sev-eral complementary books (Keith 1988; 1992; Heftmann 1992) and byreferences to reviews cited in the text. A few examples may be given to illus-trate the substantial limitations in the present account:

1. There is no systematic presentation of analytical procedures devel-oped by the U.S. EPA, nor of those suggested by the OECDalthough attention is directed to a valuable critique of EPA proce-dures (Hites and Budde 1991).

2. No attempt is made to evaluate, for example, the range of columnswhich may advantageously be used in gasliquid or high-resolu-tion liquid chromatography, those used for solid-phase concentra-tion of analytes from water samples, or commercially availablecolumns for cleaning up samples.

3. There is no discussion of technical aspects of MS or NMR, nordetails of the various procedures that are nding increasing appli-cation. Attention is directed to a review (Barcel 1992) that containsreferences to a number of books dealing with these specializedtopics.

Experimental details may be found in reviews dealing with specic groupsof compounds including polycyclic aromatic compounds (Bartle et al. 1981;Colmsj 1998; Poster et al. 1998), chlorinated hydrocarbons (Hale andGreaves 1992), PCBs (Duinker et al. 1991; Lang 1992; Schmidt and Hesselberg1992; Creaser et al. 1992), and pesticides in general (Barcel 1991). Increasedinterest in aromatic nitro compounds has led to development of proceduresfor their analysis (Lopez-Avila et al. 1991), and some of the material presented

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here is covered in a review dealing specically with marine organic pollut-ants (Hhnerfuss and Kallenborn 1992). Attention is also directed to specialissues of the

Journal of Chromatography

(1993: 642 and 643) that are devoted toa broad spectrum of procedures that have been applied to the analysis of awide range of analytes in environmental samples.

In broad outline, the following steps will be incorporated into virtually allenvironmental investigations: (1) sampling from a predetermined site,(2) extraction and cleanup of the sample, (3) identication of the components,and (4) their quantication. The objective of the investigation will naturallydetermine the degree of sophistication of the procedures that will be applied.It should also be appreciated that unforseeable difculties may arise duringthe investigation, and it is important to resolve these at as early a stage as pos-sible. Attempts will be made in this chapter to discuss all of the steps outlinedabove, although no effort has been made to provide a comprehensive accountof any single aspect. Instead, this chapter will attempt to erect signpostsalong the wayside, and to provide an overview of analytical problems and aperspective on their application to specic problems in partitioning, estimat-ing persistence, and evaluating toxicity. It is hoped, however, that some of thepitfalls awaiting the unwary have been revealed, and that at least some of themajor unresolved issues have emerged. The available literature is both spe-cialized and extensive, and no attempt has been made to provide either acomplete or a systematic coverage.

2.1 Sampling

Sampling from laboratory experiments, for example, on partitioning, toxicity,or on degradation and transformation generally presents few problems: theconcentrations of analytes are relatively high, only small volumes of samplesare required, and these systems are homogeneous. Samples may be frozenafter collection if they are not analyzed immediately. On the other hand, themagnitude of the problem with eld material is substantially greater sincethe systems are less homogeneous and the concentrations of the analytes maybe low. Although no attempt at a comprehensive treatment will be madehere, it cannot be emphasized too strongly that the quality of an investigationis critically determined by the care devoted to the selection of samples, theircollection, and their preservation before analysis. The relative costs of thesampling and the subsequent analyses should be kept in proportion. The fol-lowing issues merit brief notice.

1. Attention should be paid not only to the cleanliness of the samplecontainers but also to their composition; use of plastic containersis attractive due to their physical inertness and robustness, but their

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use may inevitably contaminate the sample with plasticizers suchas phthalate esters. Glass containers are therefore generally pre-ferred for samples of water and sediment. Problems arising fromsorption of analytes to glass surfaces may exist, and may be sig-nicant for particular groups of compounds; in the case of sterolanalysis, this has been circumvented by silylation of glassware towhich the analyte is exposed (Fenimore et al. 1976). Samples ofbiota should be wrapped in aluminum foil rather than any kind ofplastic material.

2. Water samples present fewer problems than other matrices,although if low concentrations of xenobiotics are to be analyzed, itmay be necessary to use high-volume samplers and process thesamples on board the ship. One issue which should be recognized even if it cannot be resolved is that presented by sampleshaving particulate material, and this is discussed in greater detailin Sections 2.2.4 and 2.2.5. Freezing of such samples may bring aboutalterations in their chemical composition and hence in their toxicity(Schuytema et al. 1989).

3. Sediment sampling may present a number of problems includingthe patchiness of the area being investigated (Downing and Rath1988; Brandl et al. 1993), and the difculty of obtaining a trulyrepresentative subsample from a possibly heterogeneous bulk sam-ple. These problems may be particularly severe where monitoringis directed to providing a historical record of deposition: ambigu-ities may arise (Sanders et al. 1992) where the sediment surface isnonuniform or where diffusion and bioturbation are signicant.There is probably no single optimal procedure for preserving sed-iment samples after collection; for chemical analysis, althoughfreezing may eliminate or at least minimize the possibility of chem-ical reactions occurring after sampling, analytes may be releasedfrom sedimentxenobiotic associations during subsequent thaw-ing. Preservation at 4

C will generally minimize microbial alter-ations of the analyte, but the addition of inhibitors of microbialactivity such as mercuric chloride or sodium azide may introduceproblems during subsequent analysis (Section 2.2.4) and thusshould be avoided.

4. Particular care should be exercised in the collection of samples ofsh and attempts should be made to minimize the inevitable stressof capture; this is particularly critical for analysis of compoundssuch as steroid hormones or for assays of enzymatic activity (Mun-kittrick et al. 1991; Hontela et al. 1992).

5. There has been increased interest in samples from remote areassuch as the Arctic and Antarctic. There areas are, however, increas-ingly visited both by tourists and by scientic expeditions so that

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particular care should be exercised in the conclusions drawn fromthe results of such sampling. Atmospheric input may not be theonly source of xenobiotics, and truly undisturbed localities arebecoming increasingly rare throughout the world.

6. Attention is drawn briey to a few examples of procedures forpassive sampling:a. Xenobiotics may be concentrated from soil by passive diffusion

into solid adsorbents or into organic solvents in dialysis mem-branes (Zabik et al. 1992). Desorption of the solid supports suchas C-18 bonded silica or XAD resins is then carried out foranalysis of the xenobiotics.

b. Xenobiotics have been concentrated from aquatic systems intodialysis membranes containing solvents such as hexane or tri-olein, and this procedure has been suggested as simulating up-take by biota (Sdergren 1987; Huckins et al. 1990a; Meadowset al. 1998). Although these procedures are valuable for demon-strating the presence of compounds, they are of restricted appli-cability to hazard assessment for which the concentrations ofthe analyte are clearly needed.

c. Xenobiotics may be concentrated from the air using a passivesampler that uses a thin lm of polythene containing triolein,and this has been evaluated for monitoring of PCBs and gavegood agreement with conventional high-volume samplingmethods (Ockenden et al. 1998).

d. Application of laser-desorption from membrane introductionMS is noted in Section 2.4.1.2 for the analysis of PAHs in watersamples.

2.2 Extraction and Cleanup

After collection and transfer to the laboratory, the samples have to be ana-lyzed. Two factors combine to magnify the problem: (1) the low concentrationof the analyte which generally necessitates concentration and (2) interferencefrom other compounds that may occur in much greater concentration andwhich necessitates purication or cleanup procedures to reduce their concen-tration. These factors have resulted in increasing demands upon the skill ofthe analyst, leading to the need for the application of sophisticated proce-dures for the pretreatment of samples before identication and quantica-tion. The analysis will therefore generally encompass at least the followingsteps: extraction, concentration, and cleanup with or without derivatization,identication, and quantication. A range of matrices including samples of

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water, sediment, and biota ranging from microbial cultures to higher organ-isms, and samples of atmospheric deposition may be submitted for analysis.Each of these presents its own special problem, so that procedures for theirextraction and cleanup will vary considerably. An attempt will be made topresent only an outline of the methods that have been widely used and tosummarize some of the problems that may be encountered.

2.2.1 Solvents and Reagents

Organic solvents are used at virtually all stages of the analytical procedures for extraction, during cleanup, and in identication and quantication. Itis therefore appropriate to preface this section with general comments onsome of the important issues that should be addressed. The purity of the sol-vents is of cardinal importance: the initial extracts from the samples whichmay have volumes of tens of milliliters are generally concentrated to volumesof the order of hundreds of microliters, and volumes in the microliter rangeare then used for the nal analysis. Concentration procedures should bedesigned to minimize the loss of compounds with low boiling points andappreciable volatility; for this reason, solvents such as dichloromethane, pen-tane, or diethyl ether or

t

-butyl methyl ether are frequently used, while a lessvolatile solvent such as isooctane may be added as a keeper to retain a liq-uid phase. A gas atmosphere of N

2

is generally maintained during concentra-tion to minimize oxidation of sensitive components in the sample. There is,unfortunately, no single extraction solvent which is optimal for all analytes,and the choice depends both upon the nature of the matrix and upon thestructure of the analyte. Solvents with limited capacity for dissolving waterand ranging in polarity from hexane and dichloromethane through tolueneor benzene to diethyl and

t

-butyl methyl ethers and ethyl acetate may be use-fully exploited, although no single one of these is invariably optimal. Forsome purposes, such as extraction of wet sediment samples, water-misciblesolvents such as methanol, propan-2-ol, tetrahydrofuran, acetonitrile, ordimethylformamide may be applicable; the analyte is then subsequently par-titioned into a water-immiscible phase. The major drawback to the use of sol-vents such as ethyl acetate, and to a lesser extent the dialkyl ethers forextracting aqueous solutions, is the high solubility of water in these solvents:such extracts should therefore be dried, for example, with anhydrous sodiumsulfate or preferably by azeiotropic distillation, before attempting derivatiza-tion with water-sensitive reagents such as acid anhydrides, acid chlorides, orisocyanates.

It is pointed out later (Sections 3.2.3 and 4.6.3) that substantial amounts ofxenobiotics become associated with components of the soil and sedimentwith time, and therefore only a fraction of the total is recoverable by solventextraction.

Most common solvents are now available commercially in a high state ofpurity; these have extremely low residue levels and they can be purchased as

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glass-distilled quality to obviate this step in the laboratory. Care should beexercised in their storage after purchase to prevent contamination with vola-tile laboratory chemicals, and especially with standards used for analysis.Particular caution should be exercised in the use of solvents which containstabilizers: for example, 4-

t

-butyl-2-methylphenol in diethyl ether, or cyclo-hexene in dichloromethane. Dichloromethane is recommended for manyextraction procedures because of its volatility and the simplication in extrac-tion resulting from its greater density than water; reactions of the analyte withthe cyclohexene inhibitor may, however, result in the production of artifactssuch as halogenated cyclohexanes and cyclohexanols (Campbell et al. 1987;Fayad 1988), and the presence of iodocyclohexanol has been associated withlow recoveries of phenolic compounds using EPA standard procedures (Chenet al. 1991). Attention is also drawn to the reaction of methanol with carbonylcompounds that may result in the formation of acetals or ketals (Hatano et al.1988) or of esters from chlorinated acetic acids (Xie et al. 1993). Care shouldtherefore be exercised in the analysis of such compounds particularly byHPLC for which methanol is a widely used mobile phase. For the extractionand analysis of compounds containing reactive carbonyl groups especiallyaldehydes their chemical reactivity clearly precludes the use of solventswith active methylene groups such as acetone, acetonitrile, dimethyl-sulfoxide, or nitromethane.

As a result of the increase in the number of laboratories carrying out envi-ronmental analysis and the corresponding increase in the volumes that areused primarily for extraction and have to be disposed of, there has beenincreasing use of solid-phase extraction (SPE) methods including solid-phasemicroextraction (SPME) procedures using ber-coated poly(dimethylsilox-ane), methyl silicone, or polyacrylate. These procedures are discussed in Sec-tions 2.2.4 and 2.2.7.

Although most widely used reagents are available in an acceptable degreeof purity, for some analytical applications interfering impurities must beremoved. For solids, this can usually be accomplished by successive recrys-tallization from a suitable solvent. Solid phases for open chromatographymay have been activated by ignition so that organic impurities have beenremoved. It is worth pointing out, however, that analytically pure Na

2

SO

4

that is widely used as a drying agent may contain interfering organic resi-dues that should also be removed by ignition.

2.2.2 Cleanup Procedures

Extracts from environmental samples are seldom sufciently free of contam-inants that they can be analyzed directly, since the high-resolution columnsused for GC or HPLC analysis must not be overloaded with compounds otherthan the analyte. Pretreatment of the sample before GC, GC-MS, or HPLCanalysis is therefore often a critical step in the analysis of extracts from com-plex matrices, and this is particularly important when the concentrations of

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the interfering compounds greatly exceed that of the analyte. Thesecompounds may then seriously compromise the analytical procedure. Thecleanup procedures that will be used subsequent to the initial extractiondepend both on the nature of the matrix and on that of the analyte in par-ticular the sensitivity of the analyte to chemical reagents such as strong acids(Bernal et al. 1992) or bases (Vassilaros et al. 1982) or oxidizing agents includ-ing molecular oxygen (Wasilchuk et al. 1992). All of these may bring aboutsignicant chemical changes in the analytes and should therefore be avoidedunless the stability of the analyte toward them has been unequivocally estab-lished. For example, although the quantication of chlorinateddibenzo[1,4]dioxins and some of the PCB congeners is facilitated by takingadvantage of the great stability of these compounds to chemical reagents suchas sulfuric acid for removing interfering compounds, comparable cleanupprocedures are clearly not applicable to more sensitive compounds. Lessdrastic cleanup procedures should ther