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 Toxicology of Fishes
This up-to-date, comprehensive toxicology handbook is devoted to the effects of environmental pollution on fish. Fish
species represent nearly half of all vertebrates and have become important sentinels for environmental contamination and
model organisms for understanding adverse outcomes from exposures. This new edition is written by recognized experts,
and it highlights the significant research progress in fish toxicology that has resulted from rapid technological develop-
ments in analytical, biochemical, and genomic sciences. The book:

• Discusses fundamental topics such as toxicokinetics in fishes, processes governing biotransformation within
these organisms, and reactive oxygen species and oxidative stress.
• Explains key target organ systems for chemical impacts in fish, such as the nervous and immune systems, and
how fishes can develop resistance to chemical toxicity.
• Covers multi-transgenerational effects on fishes, epigenetics, proteomics and metabolomics, and adverse outcome
pathways.

Replacing the case studies in the first edition, this update delves into the impacts of microplastics, pharmaceuticals, and
oil spills in dedicated final chapters. With nearly 200 illustrations and tables, this comprehensive reference work presents
concepts in a way that is useful for both novices to and experts in the field of fish toxicology.
Toxicology of Fishes

Edited By
Kristine L. Willett
Neelakanteswar Aluru
Cover © shutterstock

Second edition published 2024

by CRC Press
2385 NW Executive Center Drive, Suite 320, Boca Raton FL 33431

and by CRC Press

4 Park Square, Milton Park, Abingdon, Oxon, OX14 4RN

CRC Press is an imprint of Taylor & Francis Group, LLC

© 2024 selection and editorial matter, Kristine L. Willett and Neelakanteswar Aluru; individual chapters, the contributors

Reasonable efforts have been made to publish reliable data and information, but the author and publisher cannot assume responsibility for the
validity of all materials or the consequences of their use. The authors and publishers have attempted to trace the copyright holders of all material
reproduced in this publication and apologize to copyright holders if permission to publish in this form has not been obtained. If any copyright
material has not been acknowledged, please write and let us know so we may rectify in any future reprint.

Except as permitted under U.S. Copyright Law, no part of this book may be reprinted, reproduced, transmitted, or utilized in any form by any electronic,
mechanical, or other means, now known or hereafter invented, including photocopying, microfilming, and recording, or in any information storage or
retrieval system, without written permission from the publishers.

For permission to photocopy or use material electronically from this work, access www.copyright.com or contact the Copyright Clearance
Center, Inc. (CCC), 222 Rosewood Drive, Danvers, MA 01923, 978-750-8400. For works that are not available on CCC, please contact
­[email protected]

Trademark notice: Product or corporate names may be trademarks or registered trademarks and are used only for identification and explanation
without intent to infringe.

ISBN: 978-0-367-74997-2 (hbk)

ISBN: 978-0-367-75000-8 (pbk)
ISBN: 978-1-003-16069-4 (ebk)

DOI: 10.1201/9781003160694

Typeset in Times
by codeMantra
Contents
The Editors............................................................................................................................................................................xii
Contributors........................................................................................................................................................................ xiii

Chapter 1 Introduction.......................................................................................................................................................1
Neelakanteswar Aluru and Kristine L. Willett
References.........................................................................................................................................................2

Chapter 2 Toxicokinetics in Fishes....................................................................................................................................3

John W. Nichols, Jon A. Arnot, and Mace G. Barron
Introduction.......................................................................................................................................................3
Basic Concepts..................................................................................................................................................4
Fish as Wet Vertebrates................................................................................................................................4
The Environment..........................................................................................................................................6
Chemical Hydrophobicity and the Octanol–Water Partition Coefficient (KOW)...........................................8
Absorption, Distribution, and Excretion...........................................................................................................9
Xenobiotic Absorption..................................................................................................................................9
Xenobiotic Distribution.............................................................................................................................. 14
Xenobiotic Excretion.................................................................................................................................. 16
Toxicokinetic Modeling..................................................................................................................................20
Clearance Concepts....................................................................................................................................20
Compartmental Models for Fish................................................................................................................. 22
Physiologically based Toxicokinetic Models..............................................................................................28
Semi-physiological Modeling Approaches................................................................................................. 36
Noncompartmental Analysis...................................................................................................................... 37
Bioaccumulation Assessment and Modeling.............................................................................................. 37
Estimating an Apparent Whole-Body Biotransformation Rate Constant (k B) for Fish.............................. 43
Reverse Toxicokinetics and Quantitative In Vitro-to-In Vivo Extrapolation............................................. 45
Acknowledgments........................................................................................................................................... 47
Disclaimer....................................................................................................................................................... 47
Notes................................................................................................................................................................ 47
References....................................................................................................................................................... 47

Chapter 3 Biotransformation in Fishes............................................................................................................................60

Daniel Schlenk, Jed Goldstone, Margaret O. James, and Peter van den Hurk
Introduction.....................................................................................................................................................60
Phase I Reactions............................................................................................................................................60
Oxidation....................................................................................................................................................60
Cytochrome P450 Family of Drug-Metabolizing Enzymes.......................................................................60
Flavin-Containing Monooxygenases.......................................................................................................... 76
Monoamine Oxidases................................................................................................................................. 78
Alcohol and Aldehyde Dehydrogenases..................................................................................................... 78
Peroxidases................................................................................................................................................. 79
Aldehyde Oxidase...................................................................................................................................... 79
Reductases....................................................................................................................................................... 79
NADPH Oxido-reductase........................................................................................................................... 79
Azo- and Nitroreductases........................................................................................................................... 79
Aldo-Keto Reductases................................................................................................................................ 79
Hydrolysis........................................................................................................................................................80

v
vi Contents

Epoxide Hydrolase......................................................................................................................................80
Carboxylesterases....................................................................................................................................... 82
Phase II Enzymes............................................................................................................................................ 82
UDP-Glucuronosyltransferases.................................................................................................................. 82
Glutathione Transferases............................................................................................................................ 88
Sulfotransferase.......................................................................................................................................... 95
Acetylation................................................................................................................................................ 100
Amino Acid Conjugation.......................................................................................................................... 101
Toxicological Relevance................................................................................................................................ 102
Benzo(a)pyrene......................................................................................................................................... 103
Organophosphate Esters and Carbamates................................................................................................ 104
Conclusions................................................................................................................................................... 106
References..................................................................................................................................................... 106

Chapter 4 Reactive Oxygen Species and Redox Stress.................................................................................................. 121

Alicia R. Timme-Laragy, Richard T. Di Giulio, and Joel N. Meyer
Introduction................................................................................................................................................... 121
Reactive Oxygen Species and Free Radicals................................................................................................ 121
Endogenous Cellular Sources of Reactive Oxygen Species.......................................................................... 123
Antioxidant Defenses.................................................................................................................................... 124
Antioxidant Enzyme Systems.................................................................................................................. 124
Low-Molecular-Weight Antioxidants....................................................................................................... 128
Reactive Oxygen Species and Gene Expression........................................................................................... 131
Overview of ROS-Mediated Modulation of Gene Expression in Eukaryotes......................................... 131
Effects of ROS on Transcription Factors.................................................................................................. 132
Effects of ROS on mRNA Expression..................................................................................................... 132
Antioxidant Response Element (ARE)-Mediated Gene Regulation........................................................ 132
ROS-Mediated Modulation of Gene Expression: Summary.................................................................... 132
Deleterious Cellular Effects of Reactive Oxygen Species............................................................................ 133
Lipid Peroxidation.................................................................................................................................... 133
Protein Oxidations.................................................................................................................................... 134
Oxidative DNA Damage.......................................................................................................................... 134
Redox Status and Energetics.................................................................................................................... 136
Deleterious Organismal Effects of Reactive Oxygen Species...................................................................... 136
Health Relevance...................................................................................................................................... 136
Mechanisms of Chemical-Mediated Oxidative Stress.................................................................................. 137
Enhanced ROS Production....................................................................................................................... 137
Diminished Antioxidant Defense............................................................................................................. 140
Studies with Fishes........................................................................................................................................ 141
Basic Biochemical and Molecular Mechanisms of Redox Stress in Fish................................................ 141
Biomarkers of Oxidative Stress................................................................................................................ 143
Redox Stress and the Health of Wild Fish Populations............................................................................ 144
Future Directions........................................................................................................................................... 145
References..................................................................................................................................................... 145

Chapter 5 Toxic Responses of the Fish Nervous System............................................................................................... 156

Michael J. Carvan III and Steven P. Bradbury
Introduction................................................................................................................................................... 156
Overview of Fish Nervous System Development, Structure, and Function.................................................. 156
Development of Fish Nervous System...................................................................................................... 156
Central Nervous System Anatomy........................................................................................................... 158
Peripheral Nervous System Anatomy and Function: The Autonomic Nervous System.......................... 160
Sensory Organ Systems............................................................................................................................ 160
Basic Mechanisms of Neurotoxicity............................................................................................................. 162
Contents vii

Manifestations of Neurotoxicity in Fish........................................................................................................ 163

Structural Manifestations of Neurotoxicity in Fish.................................................................................. 163
Physiological Manifestations of Neurotoxicity in Fish............................................................................ 163
Examples of Major Classes of Neurotoxicants............................................................................................. 165
Neuroactive Pharmaceuticals................................................................................................................... 165
Cholinesterase Inhibitors.......................................................................................................................... 166
Pyrethroid Insecticides............................................................................................................................. 170
Organochlorine Insecticides..................................................................................................................... 172
Ethanol...................................................................................................................................................... 174
Metals....................................................................................................................................................... 174
Neurotoxins.............................................................................................................................................. 177
Summary....................................................................................................................................................... 178
References..................................................................................................................................................... 178

Chapter 6 The Immune System of Fish: A Target Organ of Toxicity............................................................................ 191

Charles D. Rice, Judy Zelikoff, and Helmut Segner
Immune Cells................................................................................................................................................ 192
Lymphoid Organs and Tissues...................................................................................................................... 193
Thymus..................................................................................................................................................... 193
Anterior Kidney........................................................................................................................................ 194
Spleen....................................................................................................................................................... 194
Mucosal Associated Lymphoid Tissues................................................................................................... 195
Nonspecific Immune Functions.................................................................................................................... 195
Nonspecific Immune Responses: Soluble Components........................................................................... 196
Nonspecific Immune Responses: Cellular Responses.............................................................................. 197
Adaptive Immune Responses........................................................................................................................ 199
Emerging Views of Inflammation in Immunotoxicology............................................................................. 199
Immunomodulation by Chemicals................................................................................................................200
Metals.......................................................................................................................................................202
Pesticides..................................................................................................................................................202
Halogenated Aromatic Hydrocarbons......................................................................................................204
Polycyclic Aromatic Hydrocarbons..........................................................................................................205
Nanoparticles.................................................................................................................................................206
Importance of Nuclear Receptors in Understanding Chemical Biological Interactions...............................206
Nuclear Receptors and Immunotoxicity in Fish............................................................................................207
Future Directions...........................................................................................................................................208
Conclusions................................................................................................................................................... 211
References..................................................................................................................................................... 212

Chapter 7 Toxicity Resistance: Physiological Acclimations and Evolutionary Adaptations to Chemical Stress.......... 226
Diane E. Nacci, Bryan W. Clark, and Andrew Whitehead
Key Points..................................................................................................................................................... 226
Introduction................................................................................................................................................... 226
Defining Tolerance................................................................................................................................... 227
Genetic Adaptation or Evolved Tolerance................................................................................................ 229
Toxicity Defense Mechanisms...................................................................................................................... 230
Detoxification........................................................................................................................................... 230
Altered Biological Targets........................................................................................................................ 235
Toxicity Resistance to Three Major Pollutant Classes.................................................................................. 235
Metal Toxicity Resistance........................................................................................................................ 235
Pesticide Toxicity Resistance................................................................................................................... 239
PAH/PHAH Toxicity Resistance.............................................................................................................. 241
Costs of Toxicity Resistance......................................................................................................................... 253
Tolerance and Individual Fitness Components......................................................................................... 254
viii Contents

Selection and Genetic Diversity............................................................................................................... 255

Evolutionary and Ecological Impacts of Tolerance.................................................................................. 256
Summary and Conclusions............................................................................................................................ 257
Acknowledgments and Disclaimer................................................................................................................ 257
References..................................................................................................................................................... 257

Chapter 8 Endocrine Disruption in Teleosts: Mechanisms of Endocrine Toxicity and Experimental Approaches...... 270
Megan E. Solan and Ramon Lavado
Introduction................................................................................................................................................... 270
Mechanisms of Endocrine Toxicity in Teleosts............................................................................................ 271
Effects of EDCs on Steroidogenesis and Hormone Metabolism.............................................................. 271
Effects of EDCs on the Hypothalamic–Pituitary–Gonadal Axis............................................................ 273
Effects of EDC Exposure on Fish Ovarian Physiology............................................................................ 274
Effects of EDC Exposure on Fish Testis Physiology............................................................................... 275
Evidence of Endocrine Disruption in Wild Fish Populations....................................................................... 275
Experimental Approaches for Characterizing the Effects of Endocrine Disruptors Compounds
(EDCs) in Fish: New Paths Forward............................................................................................................. 276
In Vivo Studies.......................................................................................................................................... 276
In Vitro Studies......................................................................................................................................... 278
Conclusion..................................................................................................................................................... 279
References.....................................................................................................................................................280

Chapter 9 Multi- and Transgenerational Health Effects of Exposure to Toxicants in Fish........................................... 286
Tracie R. Baker and Danielle Meyer
Introduction................................................................................................................................................... 286
Fish as Model Organisms for Toxicant Exposure.................................................................................... 287
The Challenge of Defining Multi- and Transgenerational Effects........................................................... 287
Continuous Exposure Studies................................................................................................................... 288
Multi- and Transgenerational Studies........................................................................................................... 289
Dioxins, PCBs, & PAHs........................................................................................................................... 289
PPCPs....................................................................................................................................................... 291
Heavy Metals: Mercury, Lead, & Arsenic............................................................................................... 292
BPA........................................................................................................................................................... 293
Hormones & Steroids............................................................................................................................... 293
Pesticides & Herbicides............................................................................................................................ 294
PFASs....................................................................................................................................................... 295
Radiation & Mutagens.............................................................................................................................. 295
Other Toxicants........................................................................................................................................ 296
Conclusions................................................................................................................................................... 297
Literature Cited............................................................................................................................................. 297
Figure Citation List.......................................................................................................................................302

Chapter 10 Epigenetics....................................................................................................................................................306
Neelakanteswar Aluru and Kristine L. Willett
Introduction...................................................................................................................................................306
Epigenetic Mechanisms.................................................................................................................................307
DNA Methylation.....................................................................................................................................307
Histone Modifications..............................................................................................................................308
Noncoding RNAs.....................................................................................................................................309
Epigenetics and Energy Metabolism............................................................................................................. 310
Known Effects of Epigenetic Mechanisms on Phenotypic Change.............................................................. 311
Toxicant-Induced Effects on Epigenetic Machinery in Fish......................................................................... 312
Epigenetics, Adverse Outcome Pathways, and Risk Assessment................................................................. 315
Contents ix

Zebrafish Embryonically Exposed to Cadmium...................................................................................... 316

Daphnia Magna and 5-Azacytidine-Mediated DNMT-Inhibition Transgenerational AOP..................... 316
Research Challenges and Future Directions................................................................................................. 316
Conclusions................................................................................................................................................... 317
Acknowledgments......................................................................................................................................... 317
References..................................................................................................................................................... 317

Chapter 11 Proteomics and Metabolomics in Fish.......................................................................................................... 324

Christopher J. Martyniuk and Nancy D. Denslow
Omics in Fish Toxicology: An Overview...................................................................................................... 324
Proteomics..................................................................................................................................................... 326
Proteomics Methods in Fish Toxicology....................................................................................................... 326
Labeling Methods: Two-Dimensional Differential Gel Electrophoresis (2D-DIGE).............................. 326
Labeling Methods: iTRAQ and Tandem Mass Tags................................................................................ 327
Label-Free Methods................................................................................................................................. 330
Phosphoproteomics................................................................................................................................... 332
Proteomics: A Role in Adverse Outcome Pathways..................................................................................... 332
Challenges and Opportunities for Proteomics in Fish Toxicology............................................................... 333
Metabolomics and Lipidomics...................................................................................................................... 334
Metabolomics................................................................................................................................................ 334
NMR......................................................................................................................................................... 335
LC/GC Tandem Mass Spectrometry........................................................................................................ 335
Lipidomics..................................................................................................................................................... 337
Lipidomics Experiments in Fish............................................................................................................... 337
Integration of Proteomics and Metabolomics............................................................................................... 338
Future Opportunities.....................................................................................................................................340
References.....................................................................................................................................................340

Chapter 12 Adverse Outcome Pathways..........................................................................................................................344

Daniel L. Villeneuve and Gerald T. Ankley
Introduction to Adverse Outcome Pathways.................................................................................................344
Principles of AOP Development.................................................................................................................... 345
AOPs Are not Chemical Specific.............................................................................................................346
AOPs Are Modular...................................................................................................................................346
Individual AOPs Are a Pragmatic Unit of Development and Evaluation................................................346
AOP Networks Are the Function Unit of Prediction for Most Real-World Scenarios............................. 347
AOPs Are “Living Documents”............................................................................................................... 347
Assembling Evidence for Causality..............................................................................................................348
Qualitative Support..................................................................................................................................348
Quantitative Understanding...................................................................................................................... 350
The State of Fish-Oriented AOPs (ca. 2021)................................................................................................. 352
Opportunities for Fish AOP Development.................................................................................................... 353
Application of AOPs...................................................................................................................................... 354
Conclusion..................................................................................................................................................... 356
Acknowledgments......................................................................................................................................... 356
References..................................................................................................................................................... 356

Chapter 13 The Potential for Toxicity to Fishes from Micro- and Nanoplastics, and Their Additives........................... 362
Susanne M. Brander, Azora König, Bethanie Carney Almroth, and Leah Thornton Hampton
Introduction................................................................................................................................................... 362
Particle Characteristics..................................................................................................................................364
Particle Behavior in the Environment...........................................................................................................364
Plastic Additives............................................................................................................................................ 365
x Contents

Microplastics as Chemical Vectors............................................................................................................... 366

Biofilms......................................................................................................................................................... 367
Effect of Biofilms on Microplastic Fate and Exposure to Fish..................................................................... 368
Effect of Biofilms on the Sorption and Desorption of Chemicals................................................................ 369
The Plastisphere and Importance of Microplastic Microbial Communities................................................. 369
Exposure........................................................................................................................................................ 370
Oral Ingestion........................................................................................................................................... 370
Consumption during Feeding................................................................................................................... 370
Consumption during Drinking or Respiring............................................................................................ 371
Egestion......................................................................................................................................................... 371
Translocation................................................................................................................................................. 371
Molecular-Level Effects on Fishes................................................................................................................ 373
Potential Mechanisms of Toxicity............................................................................................................ 373
Molecular and Cellular Alterations.......................................................................................................... 373
Putative Adverse Outcome Pathways....................................................................................................... 374
Microbiome Effects.................................................................................................................................. 374
Epigenetics, Genetic Variation, and Genotoxicity................................................................................... 375
Organismal-Level Effects on Fishes............................................................................................................. 376
Particle Toxicity........................................................................................................................................ 376
Plastic Additive and Sorbed Pollutant Toxicity........................................................................................ 377
Population-level Effects on Fishes................................................................................................................ 378
Particle Toxicity........................................................................................................................................ 378
Plastic Additive and Sorbed Pollutant Toxicity........................................................................................ 379
Multivariate Approaches for Addressing Microplastic Toxicity................................................................... 380
Conclusion and Future Research Directions................................................................................................. 381
References..................................................................................................................................................... 382

Chapter 14 Effects of Pharmaceuticals............................................................................................................................ 392

Karl Fent, Nadja R. Brun, Kun Zhang, and Yanbin Zhao
Introduction................................................................................................................................................... 392
Analgesics..................................................................................................................................................... 393
Concentrations and Fate in the Aquatic Environment............................................................................. 393
Modes of Action....................................................................................................................................... 394
Effects on Fish.......................................................................................................................................... 394
Risk Assessment....................................................................................................................................... 395
Cardiovascular Drugs.................................................................................................................................... 395
Concentrations and Fate in the Aquatic Environment............................................................................. 396
Modes of Action....................................................................................................................................... 396
Effects on Fish.......................................................................................................................................... 397
Risk Assessment....................................................................................................................................... 397
Lipid-Regulating Agents............................................................................................................................... 398
Concentrations and Fate in the Aquatic Environment............................................................................. 398
Modes of Action....................................................................................................................................... 399
Effects on Fish.......................................................................................................................................... 399
Risk Assessment.......................................................................................................................................400
Neuroactive Compounds...............................................................................................................................400
Concentrations and Fate in the Environment...........................................................................................400
Modes of Action.......................................................................................................................................402
Effects.......................................................................................................................................................403
Risk Assessment.......................................................................................................................................405
Steroid Hormones..........................................................................................................................................406
Concentrations and Fate in the Environment...........................................................................................406
Modes of Action.......................................................................................................................................407
Effects.......................................................................................................................................................408
Mixture Effects......................................................................................................................................... 411
Contents xi

Risk Assessment....................................................................................................................................... 412

Antibiotics..................................................................................................................................................... 412
Concentrations and Fate in the Environment........................................................................................... 412
Modes of Action....................................................................................................................................... 413
Effects....................................................................................................................................................... 413
Risk Assessment....................................................................................................................................... 414
Azole Fungicides: Imidazoles and Triazoles................................................................................................ 415
Concentrations and Fate in the Aquatic Environment............................................................................. 415
Modes of Action....................................................................................................................................... 415
Effects on Fish.......................................................................................................................................... 416
Risk Assessment....................................................................................................................................... 416
Cytostatics..................................................................................................................................................... 417
Concentrations and Fate in the Aquatic Environment............................................................................. 417
Modes of Action....................................................................................................................................... 418
Effects on Fish.......................................................................................................................................... 419
Risk Assessment....................................................................................................................................... 419
Antidiabetics................................................................................................................................................. 419
Concentrations and Fate in the Environment........................................................................................... 420
Modes of Action....................................................................................................................................... 421
Effects....................................................................................................................................................... 421
Risk Assessment....................................................................................................................................... 422
Overall Hazard and Risk Assessment and Conclusions................................................................................ 423
Concepts for Risk Evaluation and Their Limitations............................................................................... 423
Risk Assessment: Comparison of Chronic Effects with Environmental Concentrations........................ 424
Conclusions................................................................................................................................................... 425
References..................................................................................................................................................... 425

Chapter 15 Oil Spills........................................................................................................................................................ 437

Fernando Galvez
Introduction................................................................................................................................................... 437
Global Oil Production and Oil Consumption................................................................................................ 437
Sources of Input of Crude Oil into the Aquatic Environment...................................................................... 438
Composition of Crude Oil............................................................................................................................. 439
Fate of Crude Oil in the Aquatic Environment.............................................................................................440
Standards for the Testing of Crude Oil Toxicity........................................................................................... 442
Description of Toxicity Test Conditions........................................................................................................ 443
Preparation of Oil-in-Water Solutions for Toxicity Testing...................................................................... 443
Exposure Regimes....................................................................................................................................444
Preparation of WAF with Different Head Spaces.................................................................................... 445
Influence of Different Oil-to-Water Ratios............................................................................................... 445
Influence of Oil Droplets on Toxicity.......................................................................................................446
Octanol-Water Partition Coefficient of Crude Oil Constituents....................................................................446
Narcosis and the Models based on the Target Lipid Model.......................................................................... 447
Aryl Hydrocarbon Receptor Pathway........................................................................................................... 447
Current Understanding of Crude Oil Toxicity in Fish Embryos...................................................................448
Cardiovascular Effects.............................................................................................................................448
Other Developmental Effects................................................................................................................... 450
Fitness Level Effects................................................................................................................................ 451
Concluding Remarks..................................................................................................................................... 451
References..................................................................................................................................................... 452

Index���������������������������������������������������������������������������������������������������������������������������������������������������������������������������������� 459
The Editors
Kristine L. Willett, PhD  is Chair and Distinguished Achievement Award and the 2017 Faculty
of the Department of BioMolecular Achievement Award.
Sciences in the School of Pharmacy
at the University of Mississippi (UM). Neelakanteswar Aluru, PhD
A professor of Pharmacology and is an Associate Scientist in the
Environmental Toxicology, she has Biology Department at Woods Hole
taught both graduate and undergradu- Oceanographic Institution (WHOI).
ate courses in toxicology and envi- Dr. Aluru earned his Bachelor of
ronmental toxicology at UM since 2000. Dr. Willett earned Fisheries Science at the Orissa
her BA in Chemistry at the University of North Carolina University of Agriculture and
and a PhD in Toxicology from Texas A&M University. She Technology in Berhampur, India,
was a Dreyfus postdoctoral fellow in environmental chemis- followed by a Master of Fisheries
try at Indiana University followed by an RJR Leon Golberg Science (Aquaculture) from Kerala
postdoctoral fellow in toxicology at Duke University. Agricultural University Cochin, India. He earned a Master
Dr. Willett’s research has been funded over the years by NIDA, of Science from the College of Fishery Science, University
NIEHS, NOAA, USGS, and the Army Corps of Engineers and of Tromsø, Norway. He received his PhD in Biology from
resulted in 80+ manuscripts and book chapters. Throughout the University of Waterloo, Ontario, Canada, where he stud-
her career, she has led research projects which were designed to ied influence of persistent organic pollutants on the endo-
fundamentally understand the molecular mechanisms underly- crine stress axis in rainbow trout. He has held postdoctoral
ing toxicity and/or shed light on the potential adverse outcomes research appointments at the University of Guelph, Ontario,
due to relevant anthropogenic contamination. Her laboratory Canada, and at WHOI. Since 2012, he has been conducting
has studied the developmental, reproductive and multi-genera- research as an independent scientist at WHOI. His research
tional impacts of nanosilver, cannabinoid, and benzo[a]pyrene interests are mainly focused in the field of environmental
exposures using fish models. She also studies consequences epigenetics, particularly the epigenetic processes involved
of environmental stressors on oysters. She serves as a Deputy in determining phenotypic (and/or developmental) plastic-
Editor of Toxicological Sciences. She has been elected to lead- ity in response to environmental cues/stressor exposures.
ership positions in the Society of Environmental Toxicology His laboratory uses a variety of vertebrate and invertebrate
and Chemistry and the Society of Toxicology (SOT). SOT and model systems and employs a number of different molecu-
American Association of Colleges of Pharmacy (AACP) have lar biology methods – gene-specific to high-throughput
also recognized her with undergraduate and graduate educator sequencing to study epigenetic modes of action. He is part
awards, respectively. At UM, she earned the 2022 Research of the Woods Hole Center for Oceans and Human Health.

xii
Contributors
Bethanie Carney Almroth Richard T. Di Giulio
Department of Biology and Environmental Sciences, Environmental Sciences and Policy, Nicholas School of
University of Gothenburg, Göteborg, Sweden the Environment, Duke University, Durham, North
Carolina, USA
Neelakanteswar Aluru
Biology department, Woods Hole Oceanographic Karl Fent
Institution, Woods Hole, Massachusetts, USA ETH Zurich, Institute of Biogeochemistry and Pollutant
Dynamics, 8092 Zürich, Switzerland
Gerald T. Ankley
U.S. Environmental Protection Agency, Center for Fernando Galvez
Computational Toxicology and Exposure, Duluth, Department of Biological Sciences, Louisiana State
Minnesota, USA University, Baton Rouge, Louisiana, USA

Jon A. Arnot Jed Goldstone

ARC Arnot Research and Consulting, Toronto, Ontario, Department of Biology, Woods Hole Oceanographic
Canada Institution, Woods Hole, Massachusetts, USA

Tracie R. Baker Leah Thornton Hampton

Department of Environmental and Global Health, College Toxicology Department, Southern California Coastal
of Public Health and Health Professions, University of Water Research Project, Costa Mesa, California, USA
Florida, Gainesville, Florida, USA
Margaret O. James
Mace G. Barron Department of Medicinal Chemistry, College of
U.S. Environmental Protection Agency (retired), Office Pharmacy, University of Florida, Gainesville,
of Research and Development, Environmental Effects Florida, USA
Research Laboratory, Gulf Breeze,
Florida, USA Azora König
Department of Biology and Environmental Sciences,
Steven P. Bradbury University of Gothenburg, Göteborg, Sweden
Natural Resource Ecology and Management, Iowa State
University, Ames Iowa, USA Ramon Lavado
Department of Environmental Science, Baylor University,
Susanne M. Brander Waco, Texas USA
Department of Fisheries, Wildlife, and Conservation
Sciences, Coastal Oregon Marine Experiment Station, Christopher J. Martyniuk
Oregon State University, Corvallis, Washington USA Department of Physiological Sciences, Center for
Environmental and Human Toxicology, University of
Nadja R. Brun Florida, Gainesville, Florida USA
Department of Biological Sciences, University of Bergen,
5020 Bergen, Norway Joel N. Meyer
Environmental Sciences and Policy, Nicholas School of
Michael J. Carvan III the Environment, Duke University, Durham, North
School of Freshwater Sciences, University of Wisconsin- Carolina, USA
Milwaukee, Milwaukee, Wisconsin, USA
Danielle Meyer
Bryan W. Clark Department of Environmental and Global Health, College
U.S. Environmental Protection Agency, Atlantic Ecology of Public Health and Health Professions, University of
Division, Narragansett, Rhode Island, USA Florida, Gainesville, Florida, USA

Nancy D. Denslow
Center for Environmental and Human Toxicology,
College of Veterinary Medicine, University of Florida,
Gainesville, Florida, USA

xiii
xiv Contributors

Diane E. Nacci Peter van den Hurk

U.S. Environmental Protection Agency (Retired), Office of Department of Biological Sciences, College of Science,
Research and Development, Center for Environmental Clemson University, Clemson, South Carolina, USA
Measurement and Modeling, Atlantic Coastal
Environmental Sciences Division, Narragansett, Rhode Daniel L. Villeneuve
Island, USA U.S. Environmental Protection Agency, Great Lakes
Toxicology and Ecology Division, Duluth, Minnesota,
John W. Nichols USA
U.S. Environmental Protection Agency (retired), Office of
Research and Development, Center for Computational Andrew Whitehead
Toxicology and Exposure, Great Lakes Toxicology and Environmental Toxicology, University of California Davis,
Ecology Division, Duluth, Minnesota, USA Davis California, USA

Charles D. Rice Kristine L. Willett

Department of Biological Sciences, College of Department of BioMolecular Sciences, School of
Science, Clemson University, Clemson, South Pharmacy, University of Mississippi, Mississippi, USA
Carolina, USA
Judy Zelikoff
Department of Medicine, New York University School of
Daniel Schlenk
Medicine, New York, USA
Environmental Sciences Department, University of
California Riverside, Riverside, California, USA
Kun Zhang
State Environmental Protection Key Laboratory of
Helmut Segner Environmental Health Impact Assessment of Emerging
Centre for Fish and Wildlife Health, University of Bern, Contaminants, School of Environmental Science and
Bern, Switzerland Engineering, Shanghai Jiao Tong University, Shanghai
200240, China
Megan E. Solan
Department of Environmental Science, Baylor University, Yanbin Zhao
Waco, Texas USA State Environmental Protection Key Laboratory of
Environmental Health Impact Assessment of Emerging
Alicia R. Timme-Laragy Contaminants, School of Environmental Science and
Department of Environmental Health Science, School Engineering, Shanghai Jiao Tong University, Shanghai
of Public Health and Health Sciences, University of 200240, China
Massachusetts Amherst, Amherst, Massachusetts,
USA
 1 Introduction
Neelakanteswar Aluru
Woods Hole Oceanographic Institution

Kristine L. Willett
University of Mississippi

Toxicology is an applied science focused on understanding estrogenic endocrine disruptors causes expression of the
the adverse outcomes associated with exposure to stressors egg yolk protein vitellogenin in male fish and feminization
that may range from consequences of climatic change, to of testis (Delbes et al., 2022). Induction of liver CYP1A
toxins, metals, pharmaceuticals, and a wide range of envi- activity is used to assess exposure to polycyclic aromatic
ronmental contaminants. The history of toxicology dates hydrocarbons (PAHs) and polychlorinated biphenyls
back to ancient times as humans began to first appreciate (PCBs) (Oris & Roberts, 2007). Adverse consequences
consequences of natural poisons, followed by recogni- of oil spills on wild fish species are becoming well estab-
tion of forensic and occupational aspects of toxicology. As lished and include cardiac toxicity, immune suppression,
pharmacology advanced, toxicology became a fundamen- gill toxicity, and reduced reproductive success (Takeshita
tal aspect of drug development. In the last 50–60 years, et al., 2021). As the diversity of anthropogenic chemicals
the implications of wildlife exposures spawned a subdis- impacting aquatic organisms has grown in the past few
cipline of toxicology coined ecotoxicology. The influence decades, the field of fish toxicology has been in the fore-
of human activities in altering aquatic ecosystems in the front of research in assessing the risk of exposures to these
past few decades has increased in an unprecedented way. chemicals. In 2008, the 1st edition of The Toxicology of
Industrialization, agricultural runoff, and urbanization have Fishes was edited by Drs. Richard Di Giulio and David
resulted in the degradation of the water quality in rivers and Hinton. For years, the first edition has provided a compre-
coastal waters adversely impacting health of aquatic organ- hensive and foundational review of the impacts of various
isms as well as ecosystem health. These changes are also chemicals on different organ systems in fish.
directly influencing human health by threatening water and As the human population grows, fish are also becoming
food security. With the appreciation that effects in wildlife an ever more important source of dietary protein. While
could both be an indicator of potential human effects, and conservation measures have been enacted to preserve some
reflect extreme ecological consequences of toxic exposures, wild fisheries, aquaculture of fish has grown exponentially.
fish became a key species of toxicological interest. The United Nations Food and Agriculture Organization
Fish species account for nearly half of all vertebrate (FAO) 2018 State of the World Fisheries indicates that fin-
species. Of the nearly 30,500 species of ray-finned fish fish aquaculture in 2016 generated more than 54 million tons
(Actinopterygii), teleosts make up 98% of the species live weight primarily driven by ~44 million tons of inland
(Ravi & Venkatesh, 2018). Fish species have evolved with aquaculture in Asia. Marine and coastal aquaculture repre-
distinctive morphologies and physiologies to survive in sented ~6.5 million tons (FAO, 2018). Nile tilapia, carp spe-
diverse aquatic and marine habitats including those (e.g., cies, and salmon are the most commonly cultured. While
Salmonids) that live in both at various life stages. Despite finfish aquaculture may buffer food insecurity, it is not with-
the fact that saltwater covers nearly 70% of the planet, there out criticisms including ecological damage, reduced diver-
are more species of freshwater (~15,150) versus marine fish sity, pharmaceutical overuse, and animal welfare concerns
(~14,740) (Carrete Vega & Wiens, 2012). According to the (Franks et al., 2021). Understanding the adverse effects of
latest assessment report of the Intergovernmental Panel ecological damage due to anthropogenic activities, often
on Climate Change (IPCC, 2022), projected changes in using specific fish species as model organisms, is critical for
the amount and variability of precipitation and increas- both aquatic organismal and human health.
ing temperature could affect the river flows and hence the In addition to the aquatic and ecotoxicology studies using
mobility and dilution of contaminants and surface water fish, fish models are also becoming highly utilized in bio-
quality. Because of the rich genetic and species diver- medical studies because of the many aspects of conserved
sity, fish biodiversity can be used to both track ecosystem physiology. Whole genome sequencing of 60+ teleosts has
stress and conservation efficacy (Manel et al., 2020). In allowed for comparisons across vertebrate genomes (Ravi &
ecotoxicology, fish are often sentinels of contamination Venkatesh, 2018). Zebrafish particularly have become highly
and particular physiological changes have become recog- utilized in laboratories across the world because of their small
nized biomarkers of exposure. For example, exposure to size, high fecundity, and relatively easy and cost-effective

1
DOI: 10.1201/9781003160694-1
2 Toxicology of Fishes

culture needs. A Zebrafish International Resource Center Our hope is that this text will provide a comprehensive
(ZIRC) and the Zebrafish Information Network (zfin.org) sup- resource to both those coming to the field of fish toxicology
port the species as a research organism serving as a strain and as a novice as well as those who have been researching fish
data repository, respectively (Bradford et al., 2022). throughout their careers.
Since the publishing of the first edition of this book
(Di Giulio & Hinton, 2008), there has been significant REFERENCES
research progress in fish toxicology. This stems from the
rapid technological developments in analytical, biochemi- Bradford, Y. M., Van Slyke, C. E., Ruzicka, L., Singer, A., Eagle,
A., Fashena, D., Howe, D. G., Frazer, K., Martin, R.,
cal, and genomic sciences. Mass spectrometry has become Paddock, H., Pich, C., Ramachandran, S., & Westerfield,
a powerful analytical technique in the field of environmen- M. (2022). Zebrafish information network, the knowledge-
tal toxicology and its sensitivity, precision, and dynamic base for Danio rerio research. Genetics, 220(4). https://doi.
range has enabled identification and quantification of org/10.1093/genetics/iyac016
trace levels of chemicals in different matrices. Similarly, Carrete Vega, G., & Wiens, J. J. (2012). Why are there so few fish
advancements in biochemical methods and genomics have in the sea? Proceedings of the Royal Society B: Biological
led to significant process in understanding the effects of Sciences, 279(1737), 2323–2329. https://doi.org/10.1098/
rspb.2012.0075
exposure to toxicants. Over the last 30–40 years, biomark- Delbes, G., Blázquez, M., Fernandino, J. I., Grigorova, P., Hales,
ers have greatly expanded as early and specific endpoints B. F., Metcalfe, C., Navarro-Martín, L., Parent, L., Robaire,
for monitoring cellular responses to exposures and as pre- B., Rwigemera, A., Van Der Kraak, G., Wade, M., & Marlatt,
dictors of health outcomes. Currently, genomic, proteomic, V. (2022). Effects of endocrine disrupting chemicals on
and metabolomic biomarkers represent a continuum of cel- gonad development: Mechanistic insights from fish and
lular responses to chemical exposures and provide linkages mammals. Environmental Research, 204, 112040. https://
to mechanisms of action of chemicals. Until recently, appli- doi.org/10.1016/j.envres.2021.112040
Di Giulio, Richard T., & Hinton, D. E. (Ed.). (2008). The
cation of these approaches was mainly restricted to model
Toxicology of Fishes (1st ed.). CRC Press, Boca Raton, FL.
systems such as zebrafish. In the past 15–20 years, their FAO. (2018). The State of World Fisheries and Aquaculture
application has greatly expanded to any species of interest, 2018- Meeting the Sustainable Development Goals, Rome.
and this has revolutionized aquatic toxicology. License: CC BY-NC-SA 3.0 IGO. https://www.fao.org/
In this revised edition of the book, we have updated the state-of-fisheries-aquaculture/2018/en
scientific discoveries that have taken place in both funda- Franks, B., Ewell, C., & Jacquet, J. (2021). Animal welfare risks
mental and new or emerging subfields within toxicology. of global aquaculture. Science Advances, 7(14), 1–8. https://
doi.org/10.1126/sciadv.abg0677
Here, we have merged the prior chapters on bioavailability
IPCC, 2022: Climate change 2022: Impacts, adaptation and vul-
and toxicokinetics into a single comprehensive chapter. The nerability. Contribution of Working Group II to the Sixth
fish biotransformation chapter includes latest developments Assessment Report of the Intergovernmental Panel on
in P450 diversity. Because receptor-mediated mechanisms Climate Change [H.-O. Pörtner, D. C. Roberts, M. Tignor,
of action permeate much of contaminant toxicology, those E. S. Poloczanska, K. Mintenbeck, A. Alegría, M. Craig,
aspects are integrated across most of the chapters. Please S. Langsdorf, S. Löschke, V. Möller, A. Okem, B. Rama
refer to the first edition for the topics of liver and osmoregu- (eds.)]. Cambridge University Press, Cambridge and New
York, 3056 pp., doi:10.1017/9781009325844.
latory toxicology, cancer, and development. In contrast, in
Manel, S., Guerin, P. E., Mouillot, D., Blanchet, S., Velez, L.,
this edition Chapters 9–15 are all entirely new and highlight Albouy, C., & Pellissier, L. (2020). Global determinants
the recent molecular advances and new emerging contam- of freshwater and marine fish genetic diversity. Nature
inants in the field of fish toxicology. In addition, we have Communications, 11(1), 1–9. https://doi.org/10.1038/
expanded the chapters in oxidative stress, neurological and s41467-020-14409-7
immune effects, toxicity resistance, and oil spills. The new Oris, J. T., & Roberts, A. P. (2007). Statistical analysis of
chapters include epigenetic and multigenerational effects cytochrome P4501A biomarker measurements in fish.
of toxicants, proteomic, and metabolomic responses, endo- Environmental Toxicology and Chemistry, 26(8), 1742.
https://doi.org/10.1897/07-039R.1
crine disruptors and emerging contaminants of concern (e.g., Ravi, V., & Venkatesh, B. (2018). The divergent genomes of
microplastics, pharmaceuticals). This addition includes a teleosts. Annual Review of Animal Biosciences, 6, 47–68.
chapter on Adverse Outcome Pathways, which provide a risk https://doi.org/10.1146/annurev-animal-030117-014821
assessment framework for integrating all the new molecular Takeshita, R., Bursian, S. J., Colegrove, K. M., Collier, T. K., Deak,
initiating events gleaned from the new molecular and ana- K., Dean, K. M., De Guise, S., DiPinto, L. M., Elferink, C.
lytical technologies with population-level adverse impacts. J., Esbaugh, A. J., Griffitt, R. J., Grosell, M., Harr, K. E.,
Our goal with this book was to highlight all of the impor- Incardona, J. P., Kwok, R. K., Lipton, J., Mitchelmore, C. L.,
Morris, J. M., Peters, E. S., ... Hall, A. J. (2021). A review of
tant advances taking place in the area of fish toxicology. We
the toxicology of oil in vertebrates: what we have learned fol-
would like to thank all the authors who participated in this lowing the Deepwater Horizon oil spill. Journal of Toxicology
edition of the book and acknowledge the previous editors and Environmental Health - Part B: Critical Reviews, 24(8),
who provided the framework for us to expand and update. 355–394. https://doi.org/10.1080/10937404.2021.1975182
 2 Toxicokinetics in Fishes
John W. Nichols
U.S. Environmental Protection Agency (retired)

Jon A. Arnot
ARC Arnot Research and Consulting

Mace G. Barron
U.S. Environmental Protection Agency (retired)

Abstract biotransformation, and excretion (ADME1). These processes

The term toxicokinetics refers to the study of absorption,
influence whole-organism dose–response relationships by
distribution, metabolism (biotransformation), and excre- altering the effective chemical concentration over time at
tion (ADME) of toxicants in living organisms in relation to a target site. The degree to which any individual process
time. In this chapter, we employ a physiological perspec- impacts the concentration time course depends on the char-
tive to address the topic of chemical toxicokinetics in fish. acter of the chemical and attributes of the exposed organism.
Introductory sections provide an overview of chemical The role that individual organs play in each process and the
absorption, distribution, and excretion, and highlight how molecular susceptibility of specific tissues and organs provide
adaptations of fish to their aquatic environment shape these the basis for specific toxicities. Most of the literature dealing
processes. An extended section on toxicokinetic modeling
with the ADME of chemicals in living organisms has been
describes how ADME processes may be represented math-
ematically to develop computational models of chemical
obtained in studies with drugs. The term pharmacokinetics is
uptake and disposition in fish. Included are descriptions of dif- used to describe the quantitative study of these ADME pro-
ferent modeling approaches, methods used to estimate model cesses in relation to time. The term toxicokinetics represents
parameter values, and the scientific and regulatory questions an extension of pharmacokinetic principles to chemicals (tox-
to which these models can be applied. Special emphasis is icants) that cause adverse effects in exposed organisms. This
given to the topic of chemical bioaccumulation assessment as distinction aside, the two terms are synonymous.
this historically represents the most important application of For decades, aquatic toxicologists have used mathemati-
toxicokinetic models for fish. Additional sections address the cal models to better understand adverse effects expressed at
need to quantify chemical biotransformation as a means of
the whole-organism level. The goal in such cases is to more
representing this activity in predictive computational models.
Recognizing the desire to reduce the use of live animals in
directly relate an external exposure to the observed effects
chemical testing, a concluding section describes how toxico- by describing the corresponding internal exposure. Models
kinetic models can be used to relate observed in vitro effects developed to describe the toxicokinetic behavior of chemicals
to environmental exposures that could be expected to elicit in fish vary substantially with respect to their complexity and
similar effects in vivo. data requirements. This variability reflects, in turn, the dif-
ferent purposes for which the models are developed, the time
scale of interest, the complexity of ADME processes relevant
INTRODUCTION
to a particular chemical and/or dosing scenario, and the avail-
Fish are intimately linked to their aqueous habitat. They ability of data for model calibration and evaluation.
respire, osmoregulate, achieve acid–base balance, and obtain ADME processes also determine the extent to which xeno-
their thermal character in relation to the surrounding water. biotics accumulate in fish. The term bioconcentration refers to
In addition, water serves as a common conduit for many chemical accumulation that occurs when an aquatic organism
essential life processes. Most fish eat, drink, urinate, defecate, is exposed to a chemical in water only. Bioaccumulation is a
swim, obtain sensory information, reproduce, and spatially more inclusive term that describes chemical accumulation that
orient within a single surrounding and contiguous medium. occurs from all possible route of exposure, including water,
The structural and physiological adaptations that allow fish to diet, and sediments. Biomagnification, which may occur as an
thrive in an aqueous environment strongly impact their inter- outcome of dietary uptake, refers to an increase in chemical
actions with xenobiotic chemicals. The solvent properties of concentrations in organisms that occupy successively higher
water and its contrasting character relative to that of many trophic levels within an aquatic food web.2 Bioconcentration,
environmental contaminants further shape these interactions. bioaccumulation, and biomagnification, particularly of per-
The toxicity of a xenobiotic chemical to fish is substan- sistent chemicals, serve as indicators of past exposure for the
tially determined by the processes of absorption, distribution, animal; as indicators of potential exposure for higher trophic

DOI: 10.1201/9781003160694-2 3
4 Toxicology of Fishes

level consumers; and as markers of potential toxicity. As such, important gases in water also differ. These facts necessi-
bioaccumulation assessment is a critical component of most tate both specialized gas-exchange surfaces and a differ-
chemical risk assessment approaches. ent approach to acid–base balance. The viscosity of water
This chapter builds on a contribution given previously by requires that fish employ specialized approaches to locomo-
Kleinow et al. (2008). As in the original text, our goal was tion and expend considerable energy to support respiration.
to link anatomical, physiological, and environmental deter- Although the physiological and biochemical mechanisms
minants of chemical uptake, accumulation, and elimination available to fish for homeostasis are like those of other verte-
in fish with kinetic modeling approaches that describe this brates, the ways they are used may or may not be the same.
behavior. Introductory sections review physicochemical This is important to keep in mind when comparing the kinet-
concepts relevant to the description of chemical kinetics in ics of xenobiotics among fish and mammals, among fishes of
natural systems, highlight physiological differences between diverse groups, and even among individuals of the same spe-
fish and mammals, and describe mechanisms of xenobiotic cies held under different environmental conditions. Table 2.1
transport across biological membranes. The sections that fol- lists some generalized structural and functional differences
low describe the processes of absorption, distribution, and between fish and mammals (Cunningham, 1997; Evans,
excretion in fish one by one. An extended section on toxico- 1993; Ruckesbusch et al., 1991). Many of these differences
kinetic modeling begins with a discussion of basic clearance influence xenobiotic absorption, distribution, and elimination
concepts. This text is followed by a description of empirical in fishes. As an example, poikilothermy influences mem-
and physiologically based modeling approaches. In compari- brane structure and function, enzymatic isozyme profiles,
son with the chapter provided by Kleinow et al. (2008), the cardiac output, bile flow rate, and gut transit time.
modeling text has been amended to deemphasize the pre- Uptake of xenobiotics by fish and mammals may occur
sentation of mathematical equations while placing relatively by inhalation, ingestion, or dermal exposure (Figure 2.1).
greater emphasis on model evaluation and use. Likewise, the excretion of xenobiotics by fish and mam-
Bioaccumulation modeling is addressed as a special topic mals occurs at respiratory and skin surfaces, as well as by
due to the large amount of focused research in this area and urinary, biliary, and fecal routes. Even with these common-
because prediction of various bioaccumulation metrics repre- alities, there are some obvious and not so obvious differ-
sents the most common application of toxicokinetic models ences between fish and mammals regarding the nature of
for fish. A critical input to these models is the rate of chemical these pathways and their relative importance for different
biotransformation, typically expressed as an apparent whole- environmental contaminants. For example, the mammalian
body elimination rate constant (kB; 1/day). In silico and in lung is exposed primarily to volatile chemicals and chemi-
vitro methods for the prediction of kB are described, and cals associated with or existing as particulates. In contrast,
the domain of applicability of such methods is discussed. A the fish gill is commonly exposed to water-soluble chemi-
concluding section describes the use of reverse toxicokinetic cals, as well as nonpolar chemicals bound to dissolved and
modeling approaches to extrapolate in vitro effects informa- particulate organic matter (DOM and POM, respectively),
tion to a corresponding whole-organism exposure. which are typically quantified as dissolved and particulate
The principal focus of this chapter is on the toxicokinetics organic carbon (DOC and POC, respectively). Branchial
of organic xenobiotic chemicals. In a section on semi-physi- uptake requires diffusion across the gill membranes, which
ological modeling approaches, we discuss models that have possess a hydrophobic core. For nonpolar chemicals bound
been developed to describe the kinetics of several metals and to organic matter in water, uptake requires first that they dis-
metalloids in fish. No attempt is made, however, to review the sociate from the organic material and traverse a polar water
physiological and biochemical determinants of this behavior and mucous interface before diffusing across a compatible
(e.g., the presence/absence and activities of specific metal hydrophobic membrane. The same phase transitions exist
binding proteins) as these determinants tend to be highly sub- for chemical excretion across the gills. In general, the gills
stance specific (Olsson et al., 1998; Roesijadi and Robinson, play a much greater role in the absorption and excretion of
1994). Similarly, this chapter does not address the behavior of xenobiotics by fish than the lung plays in mammals.
engineered nanomaterials or naturally derived toxins. The skin of mammals is dry, dead, and keratinized, and
does not possess a circulatory component. When present,
fur may further limit dermal uptake of xenobiotics in mam-
BASIC CONCEPTS mals. In contrast, the skin of fishes is a living epidermis
that is perfused by an underlying secondary circulation. In
Fish as Wet Vertebrates
very small fish or benthic species that are in contact with
The basic requirements for life are the same for fish as other contaminated sediment, the skin becomes an important
organisms. Life in water, however, presents several chal- exchange surface for xenobiotic chemicals.
lenges to fish that are not equitably shared by most terres- The gastrointestinal tract plays an important role in the
trial animals. Water has a high specific heat capacity, which absorption of xenobiotics in both fish and mammals. The
makes it impossible for all but the largest fish to maintain structure and function of the gastrointestinal tract in both
internal temperatures different from those of the immedi- taxa are similar in many respects. In fish, however, the
ate surroundings. Compared with air, water contains low absence of a lymphatic system and lack of classical mam-
levels of dissolved gases. The solubilities of physiologically malian villi may influence the absorptive process. The feces
Toxicokinetics in Fishes 5

TABLE 2.1
Generalized Structural, Functional, and Environmental Differences between Fish and Mammals
Characteristic Fish Mammals
Media
Surrounding media Water Air
Specific heat High Low
Solvent properties Universal solvent Nonsolvent
Dissolved gases Low levels High levels
Viscosity High Low

Temperature
Modality Poikilotherms Homeotherms
Metabolic rate Slow Fast
Primary energy currency Amino acids and lipid Carbohydrates
Feed/weight conversion Very efficient Less efficient
Membrane composition Homeoviscous adaptation No adaptation necessary
Body temperature regulation Behavioral Internal set point

Respiratory surface
Primary organ Gill Lung
Blood/gas flows Counter current Directional/tidal flow
Gas exchange Yes Yes
Acid–base balance Yes Yes
Nitrogen excretion Yes No
Osmoregulation Yes No
Primary driver of respiration Low oxygen High carbon dioxide

Acid–base balance
Primary organ Gill Lung and kidney
Primary form Ion based Gas (lung) and ion (kidney) based

Nitrogenous waste elimination

Primary organ Gill Kidney
Primary form Ammonia Urea

Circulation
Heart Two chambers Four chambers
Arrangement Heart and gill in series Heart and lung in parallel
Vessels Arteries and veins less distinct Distinct arteries and veins
Lymphatics No Yes
Secondary circulation Yes No
Blood pressure and flow Low High
Red blood cells Nucleated Non-nucleated
Plasma proteins Lower levels Higher levels
Blood pH Higher than mammals Lower than fish

Kidney
Nephron Less complex. SW – no distal tubule; More complex, including glomerulus,
aglomerular. FW, SW – modified loop of Henle distal tubule, and loop of Henle
Blood supply Renal artery and renal portal Renal artery
Hematopoiesis Many fishes NA
Waste excretion Minor site Primary site
Fluid balance Primary site Primary site
Ion balance Secondary site Primary site
Acid–base balance Secondary site Primary site

Liver
Functional unit Tubule, with no triads or zonal relationships Lobule, with triads and zonal arrangement
Gall bladder May or may not be present May or may not be present
Bile flow Slower than mammals Faster than fish
Biotransformation rates Slower than mammals Faster than fish
(Continued )
6 Toxicology of Fishes

TABLE 2.1 (Continued)

Generalized Structural, Functional, and Environmental Differences between Fish and Mammals
Characteristic Fish Mammals
Gastrointestinal tract
Structural divisions Gradual transitions Clear delineation
Gastric stomach Absent in 15% of species Present
Absorptive surface Longitudinal folds Villi
Lipid absorption Primarily into blood Size dependent into lymph
Change in temperature Change in length/morphometry NA

Skin
Epidermis Living, moist Dead, dry, keratinized
Mucous Present Absent

FIGURE 2.1 Pathways for absorption, distribution, and excretion of xenobiotic chemicals in fish. Potential routes of exposure are
underlined. Solid lines represent chemicals movements into and within the animal. Circled portal blood flows represent venous blood
originating from the identified tissues. Dashed lines show pathways by which chemicals are lost from fish to the environment.

represent an important route of elimination in fish, both as a involved in specific target organ toxicity. These differences are
purveyor of bile and as a matrix with affinity for chemicals due to both environmental factors and factors associated with
in the organism. Although bile formation is slow in fishes the fish. The amount of chemical released to the environment,
relative to mammals, many xenobiotics are excreted in bile. its movement among compartments (e.g., air, water, sediment),
In most cases, urinary elimination of xenobiotics is less and susceptibility to biotic and abiotic transformation processes
important in fish than it is for mammals. This is due in part to are primary determinants of chemical exposure to the exchange
the contribution of the gills to chemical elimination and to the surfaces of fish. Conceptually similar processes operate within
modified role of the kidneys in comparison with mammals. In the fish, modulating exposure to the sites of toxicity.
some cases, however, the urine represents the primary route of Water dissolves more substances and dissolves them
elimination. Urinary elimination may be particularly impor- more completely than any other liquid; nevertheless, some
tant for ionized chemicals that are substrates for membrane nonpolar substances are relatively insoluble in water.
transporters within the renal tubular epithelium. In general, polar chemicals that are nonvolatile will remain
unbound in the water column. Alternatively, if the chemical
is nonpolar and nonvolatile, it will bind to DOC and POC
The Environment
in water, reducing the amount that is unbound and available
Large differences may exist between the concentration of a for uptake. These characteristics may influence both chemi-
xenobiotic chemical in the environment, the concentration cal bioavailability to fish and the primary route of exposure.
in the systemic circulation of a fish, and the concentration
Toxicokinetics in Fishes 7

Water in natural systems is a composite of H2O and the Fick’s law is a useful construct insofar as it highlights
materials dissolved and suspended in it. Because these mate- factors that promote or inhibit membrane flux. For exam-
rials influence the complexation, binding, precipitation, and ple, the diffusion coefficient is a function of molecular
form of many chemicals, water cannot be viewed as a uniform size, molecular conformation, and the presence or absence
or generic medium. Water quality as it relates to aquatic toxi- of specific functional groups. In general, diffusion is most
cology includes both biotic and abiotic components. Living rapid for small, moderately hydrophobic molecules that
organisms and organically derived substances contribute to the possess little or no charge character. The chemical con-
biotic character of water. Abiotic factors include pH, hardness, centration gradient refers to the concentrations of diffusing
alkalinity, salinity, dissolved solids, and temperature. Often, chemical species on either side of the membrane. Generally,
the influence of water quality on chemical bioavailability and it is assumed that the principle diffusing species are those
toxicity is a collective action, with each characteristic exerting that are unbound in solution. Factors that reduce this
independent as well as interdependent effects. unbound concentration, such as plasma binding or binding
to DOC in water, can be expected, therefore, to limit flux.
Membrane Transport The membrane–water partition coefficient is a measure of
Xenobiotic absorption, distribution, and elimination are the relative solubility of the chemical in the membrane and
dependent on transport across one or more biological mem- is generally estimated from measured lipid–water partition-
branes. All known cellular membranes are composed of ing or a surrogate measurement such as the octanol–water
lipid bilayers arranged with hydrophilic polar regions fac- partition coefficient (see Chemical Hydrophobicity and the
ing the outer surfaces and hydrophobic regions oriented Octanol–Water Partition Coefficient (KOW)).
toward the interior. On either surface or traversing the entire Empirical evidence suggests that nonionized chemicals
width of the membrane are globular proteins. Hydrophobic diffuse much more readily across membranes than ionized
and electrostatic molecular interactions, along with the forms. These observations have important implications for
cytoskeleton, maintain the association of components, membrane flux of weak organic acids and bases. If the non-
regionalization, and structural integrity of the membrane. ionized moiety has a lipid–water partition coefficient that
Throughout the fish, and even within each cell, membranes favors membrane penetration, it will tend to reach an equilib-
may possess different morphological and biochemical attri- rium concentration on both sides of the membrane, while the
butes. External influences such as diet and temperature may ionized form may be much more limited in its movements.
alter membrane composition and character. As an approximation, therefore, the equilibrium concentra-
tion across the membrane will be based on the concentration
Passive Diffusion of the nonionized form, with Fick’s law of diffusion applying
The primary mechanism of xenobiotic transport across only to this nonionized form. The ratio of the two ionization
lipid membranes is passive diffusion. Moderately nonpo- states is dependent on the dissociation constant (pKa) of the
lar, lipid-soluble chemicals diffuse easily across biologi- chemical and the pH of the surrounding media. This relation-
cal membranes. Polar and very nonpolar chemicals diffuse ship is described by the Henderson–Hasselbalch equation:
across membranes less easily, although for different rea-
For weak organic acids:
sons. Polar chemicals have limited access to the nonpolar
pH = pKa + log (ionized/nonionized) (2.2)
portion of the membrane, due to their low lipid solubility.
In contrast, slow diffusion of nonpolar chemicals across For weak organic bases:
hydrophilic regions may limit flux. pH = pKa + log (nonionized/ionized) (2.3)
Diffusion across a membrane bathed on either side by
an aqueous solution is often described using Fick’s law of For a weak organic acid, a decrease of one pH unit results
diffusion: in a tenfold increase in the concentration of the nonionized
form. Conversely, an increase of one pH unit results in a ten-
Flux = ( D PMW /h )(C1 − C2 ) (2.1) fold increase in the concentration of the ionized form. Weak
organic bases behave in the opposite manner; for example, a
where D is the diffusion coefficient (cm2/s), PMW is the weak organic acid (pKa of 7) in moderately acidic water (pH
membrane–water partition coefficient (unitless), h is the of 5) would exist in a nonionized/ionized ratio of 100/1. For
thickness of the diffusion path or membrane (cm), and C1 a weak organic base, the ratio would be 1/100. The effect of
and C2 are chemical concentrations on each side of the pH on the nonionized/ionized ratio can be extreme; thus, a
membrane (mg/cm3). When aggregated, D, PMW, and h pH change from 5 to 1 would change the nonionized/ionized
define a permeability coefficient with units of cm/sec. For ratio of a pKa 7 organic acid from 100/1 to 1,000,000/1.
a unit area membrane, the rate of chemical transfer across Differences in pH across biological membranes will
the membrane (flux) has units of mg/s/cm2. Multiplication cause the degree of ionization to differ on either side.
by the membrane surface area provides a measure of total The side with the greatest degree of ionization may have
flux with units of mg/sec. This equation indicates that dif- a much higher total xenobiotic concentration because the
fusional flux is first order and linear with respect to the equilibrium distribution of chemical is based on concentra-
chemical concentration gradient and progresses by a frac- tions of the diffusing nonionized form. This phenomenon
tional rate constant. is referred to as ion trapping. The theoretical equilibrium
8 Toxicology of Fishes

concentration ratio (Rxy) of a xenobiotic across a membrane or by coupling transport to the electrochemical potential of a
with two sides, x and y, can be calculated as follows: second solute (secondary active mechanism).
Transporters responsible for transmembrane flux of
For weak organic acids: drugs and toxicologically relevant substrates fall into two
superfamilies: the ATP-binding cassette (ABC) transporter
Rxy = 1 + antilog ( pH x − pK a )  1 + antilog ( pH y − pK a )  superfamily and the solute carrier (SLC) superfamily. ABC
(2.4) transporters act via the primary active mechanism and gen-
erally operate as efflux pumps, moving substrates out of
For weak organic bases: cells or into cell organelles. Included in this family are the
well-known multidrug resistance-related proteins (Deeley
Rxy = 1 + antilog ( pK a − pH x )  / 1 + antilog ( pK a − pH y )  et al., 2006). SLC transporters that act by transporting
(2.5) substrates against their electrochemical gradient do so via
the secondary active mechanism (Nigam et al., 2015; Roth
With respect to these relationships, fish present several et al., 2012). Other SLC transporters operate by facilitating
interesting nuances that are not typically encountered in membrane diffusion. The tissue distribution, regulation, and
mammals. While ion trapping in mammals is generally physiological functions of ABC transporters in fish are rela-
limited to internal membranes (e.g., blood to gastrointes- tively well known (Ferreira et al., 2014; Luckenbach et al.,
tinal tract, blood to milk, blood to urine), ion trapping in 2014). By comparison, the SLC transporters in fish are less
fish may occur across external surfaces such as the gills well described. Nevertheless, functional studies performed
and skin (blood to water). The range of environmental pH using isolated renal proximal tubules (Aslamkhan et al.,
values that will support fish populations is about 5–10. For 2006) and in vitro cellular expression systems (Aslamkhan
a given chemical, therefore, diffusion across the gills and et al., 2006; Popovic et al., 2014) have shown that teleost
skin may occur rapidly or very slowly, depending on the SLC transporters can transport xenobiotic environmental
pH. contaminants against their concentration gradient.
Physiological differences between fish and mammals The different roles that membrane transporters play in sup-
also exist. Most fish species have blood pH values that range porting biliary and urinary elimination of xenobiotics in fish
from 7.7 to 8.0. For most mammals, the blood pH is ~7.4. are described in greater detail below. In mammals, interac-
Finally, the pKa for a particular chemical or functional group tions of xenobiotics with specific nuclear receptors can result
will vary somewhat with temperature. Because fish are poi- in coordinated expression of biotransformation enzymes that
kilotherms, changes in chemical dissociation may occur as metabolize these chemicals as well as membrane transport-
they encounter different temperature environments. ers that promote the elimination of resulting metabolic prod-
Limited diffusion of ions across biological membranes ucts. Similar processes are thought to operate in fish (Bard,
also occurs. In such cases, the driving force for diffusion 2000; Ferreira et al., 2014). Because the total number of mem-
is the electrochemical gradient, which is a function of the brane transporters is finite, there is a potential for the kinet-
chemical concentration gradient across the membrane, the ics of transport to saturate at high substrate concentrations.
charge distribution across the membrane (i.e., the electri- Similarly, there is a potential for one substrate to competi-
cal potential), and the charge on the diffusing species. The tively inhibit the transport of other, similar chemicals. This
transmembrane electrical potential for a typical animal cell type of inhibition is an important cause of drug–drug inter-
is −50 to −70 mV (inside relative to the outside). This elec- actions. Its relevance for chemical mixture interactions in an
trical potential will tend to favor the inward diffusion of ecotoxicological context is largely unknown.
cations while promoting the outward diffusion of anions.
Pinocytosis
Carrier-Mediated Transport Pinocytosis is a process whereby the cell membrane invag-
Biological membranes allow for compartmentalization and inates, capturing external material within the lumen of a
control of biological function by acting as diffusion barri- vesicle. The vesicle may empty its contents within the cell
ers for inorganic ions and polar and/or or charged organic or fuse with another portion of the cell membrane, releasing
molecules, including energy substrates and structural com- its contents to the intercellular space. Uptake of macromo-
ponents (e.g., sugars, fatty acids, and amino acids), signal- lecular proteins by pinocytosis is well documented in the
ing molecules (e.g., hormones and neurotransmitters), and fish intestine (Rombout et al., 1985; Stroband et al., 1979;
substances that regulate osmotic pressure and pH (various Stroband and Kroon, 1981).
organic and inorganic ions). The transport of these ions and
organic molecules across membranes is largely controlled by
membrane-bound channel and transporter proteins. These
Chemical Hydrophobicity and the Octanol–
proteins may facilitate the diffusion of a substance down Water Partition Coefficient (KOW)
its electrochemical gradient or transport it against its elec- While there are several physicochemical properties that
trochemical gradient via an active mechanism. The energy influence chemical toxicokinetics in fish, historically the
required for active transport can be provided by hydrolysis of most relevant is the octanol–water partition coefficient (KOW,
adenosine 5′-triphosphate (ATP; primary active mechanism) sometimes referred to as P or POW). The KOW is defined as
Toxicokinetics in Fishes 9

the ratio of the equilibrium concentrations of a dissolved When a chemical is taken up by a fish, it distributes
substance in a system consisting of n-octanol and water. between water and various organic phases (Bertelsen et al.,
Numerous published relationships exist between KOW and 1998). For many chemicals, the internal distribution at equi-
acute toxicity, bioaccumulation, and other parameters relat- librium can be predicted from KOW and the proximate com-
ing to environmental toxicology and chemistry (Boethling position of blood and individual tissues (e.g., the fractional
and Mackay, 2000; Schwarzenbach et al., 2016). Similar content of water, lipid, and protein). A similar approach can
relationships have been developed and applied in the human be used to predict a partitioning-based equilibrium distribu-
health sciences (Hansch, 1972; Hansch et al., 1995). The tion between the fish and water. In such cases, a proportion-
primary reason that KOW has proven to be a useful property ality constant may be used to relate chemical solubility in
is that n-octanol is a good surrogate for many organic bio- n-octanol to that in different organic phases. A proportion-
logical and environmental phases including neutral storage ality constant of 1.0 generally provides a good prediction
lipids, membrane phospholipids, proteins, POC, DOC, and of chemical partitioning to neutral and membrane lipids
sediment organic carbon. For neutral organic chemicals, sol- (Mackay, 1982; Chiou, 1985; Vaes et al., 1998). A propor-
ubility in n-octanol tends to be relatively constant while solu- tionality constant of 0.05 has been determined for proteins
bility in water can vary enormously (Admire and Yalkowsky, (i.e., the solubility of neutral organic chemicals in protein
2013; Leo, 2000; Mackay et al., 1980; Meylan and Howard, is about 5% of that in octanol; deBruyn and Gobas, 2007).
1995; Pinsuwan et al., 1995; Ran et al., 2001; Sangster, 1989). The KOW is not a suitable parameter for predicting the
For this reason, the term “hydrophobic” (“water-hating”) partitioning behavior of fully ionized substances. For weak
should be used preferentially to “lipophilic” (“lipid loving”) acids and bases, however, it may be useful to characterize
when characterizing a chemical’s relative tendency to parti- partitioning between n-octanol and water using a distribu-
tion out of water and into organic phases such as lipids. tion coefficient (D or log D), which is equal to the ratio of
The KOW may be measured using a variety of standardized the sum of all forms of the chemical (neutral and ionized) in
methods (OECD 1995, 2004, 2006). In a system containing both phases. The ionization of a weak acid or base depends
n-octanol and water, hydrophobic chemicals are thermo- on its dissociation constant and the pH of the local envi-
dynamically driven from water into n-octanol because of ronment. The interpretation of a stated D value requires,
their low aqueous solubility (Admire and Yalkowsky, 2013; therefore, that the pH be identified (typically as a subscript,
Mackay et al., 1980; Meylan and Howard, 1995; Ran et al., e.g., log D 7.4).
2001). At equilibrium, the chemical activity (or fugacity) in Although the KOW has proven to be a useful predictor
each phase is the same but measured chemical concentra- of chemical partitioning to lipids and other organic phases,
tions may differ by many orders of magnitude. Because of more advanced methods for the prediction of chemical
the large range of KOW values, KOW is often expressed as partitioning are being developed and tested. In particular,
log KOW (base 10 logarithm). For chemicals that are very the pioneering work of Abraham and colleagues (Abraham
poorly soluble in water, reliable measurements of KOW may et al., 1999, 2004), as well as Goss and co-workers (Goss
be difficult to obtain. In such cases, it may be necessary to and Schwarzenbach, 2001; Endo and Goss, 2014) has led to
predict KOW using a quantitative structure–activity relation- the development and validation of pp-LFERs for predict-
ship (QSAR; Cappelli et al., 2015) or polyparameter linear ing partition coefficients between water and storage lipids
free energy relationship (pp-LFER; Stenzel et al., 2013a, (Geisler et al., 2012), water and membrane lipids (Endo
2013b). Specific chemical classes may present technical et al., 2011), and water and various proteins (Endo and
challenges that preclude the direct determination of reli- Goss, 2011; Endo et al., 2012). These pp-LFERs are becom-
able KOW values. For example, surfactants may form aggre- ing more accessible to the scientific community (Ulrich
gates in aqueous solution and/or accumulate at the interface et al., 2017) and may in time supplant simple KOW-based
between n-octanol and water (Hodges et al., 2019), while prediction approaches.
some pigments and dyes are essentially insoluble in water
(Anliker and Moser, 1987).
Chemical hydrophobicity is an important determinant of ABSORPTION, DISTRIBUTION,
chemical distribution within the environment, between the AND EXCRETION
environment and biota, and within an organism. Chemicals
Xenobiotic Absorption
bound to POC and DOC are not available for uptake by fish
across the gills (Black and McCarthy, 1988; Freidig et al., 1998; Xenobiotics are absorbed by fish across the gills, skin,
Johnsen et al., 1989; McCarthy and Jimenez, 1985; Schrap and gut. In terms of gross morphology, these structures
and Opperhuizen, 1990). Thus, consideration of bound and differ greatly from one another. All three, however, pos-
unbound fractions in water becomes important when quan- sess two basic features that contribute to their role as
tifying chemical uptake from water or expressing the total chemical exchange surfaces: (1) large surface area; and
concentration in a fish relative to that in water. For neutral (2) separation of the environment, or an extension thereof,
organic chemicals, the issue of bioavailability in the water is from the circulatory system by a membrane consisting of
most relevant chemicals with log KOW > ~5 (Arnot et al., 2010; one, two, or a few cell layers. The path a chemical takes
Gobas and Morrison, 2000; Parkerton et al., 2008). when it is absorbed at one of these surfaces involves: (1)
10 Toxicology of Fishes

presentation to the absorbing epithelium in water or gut water (ventilation) flow. Direct measurements of chemical
contents; (2) transport across the epithelium into blood; uptake across fish gills have been obtained using fish respi-
(3) incorporation into blood, including binding to plasma rometer-metabolism chambers, which separate inspired and
lipid and proteins; (4) transport via the systemic circula- expired water flows (McKim and Goeden, 1982; McKim
tion to various tissues; and (5) transport from blood into and Heath, 1983). Using this model system, McKim and co-
tissues. From mass-balance considerations, the rate of workers (Bradbury et al., 1986; McKim et al. 1985, 1986,
uptake cannot exceed the rate at which chemical is pre- 1987a, 1987b) measured branchial uptake rates in adult
sented to the exchange surface. Rates of diffusion across rainbow trout (Oncorhynchus mykiss) for a heterogeneous
the membrane barrier and removal by the circulatory sys- group of organic chemicals. These measurements suggested
tem also have the potential to limit the overall rate of a consistent relationship between a chemical’s uptake rate
chemical uptake. and its relative hydrophobicity, as indicated by the log of its
octanol–water partition coefficient (log KOW; Figure 2.2A).
The Gills Uptake rates were low for chemicals with log KOW values
Branchial Absorption of Xenobiotics less than 1, increased about four-fold between log KOW 1
The anatomical and physiological features of fish gills that and 3, leveled off between log KOW 3 and 6, and declined
promote efficient exchange of respiratory gases also contrib- when log KOW exceeded 6. Working with a series of phe-
ute to uptake of xenobiotic chemicals from water; namely, nols, anisoles, and carboxylic acids, Saarikoski et al. (1986)
a thin membrane separating blood and water, large surface found a similar relationship between absorption rate and
area, and high rates of counter-current blood (perfusion) and log KOW in the guppy (Poecilia reticulata) (Figure 2.2B).

FIGURE 2.2 Relationship between branchial uptake of chemicals by rainbow trout and guppies as a function of chemical hydropho-
bicity, expressed as the log of the octanol–water partition coefficient (log KOW). For ionizable chemicals, the log KOW value is that of the
neutral form. The uptake rate for rainbow trout is expressed as a clearance constant; that for guppies is given as the log of the uptake
rate constant from water when pH < chemical pKa. Panel A, Rainbow trout sublethal exposures: A, ethyl formate; B, ethyl acetate; C,
1-butanol; D, nitrobenzene; E, p-cresol; F, chlorobenzene; G, 2,3-dichlorophenol; H, 2,4,5-trichlorophenol; I, 1-decanol; J, 1-dodeca-
nol; K, pentachlorophenol; L, hexachlorobenzene; M, 2,2′,5,5′-tetrachlorobiphenyl; N, dinitrophenol; O, Mirex. Rainbow trout lethal
exposures: 1, benzaldehyde; 2, 2,4-dinitrophenol; 3, MS-222; 4, malathion; 5, 1-octanol (Data from Erickson and McKim, 1990a,
1990b). Panel B, Guppy sublethal exposures: 1, butyric acid; 2, phenol; 3, benzoic acid; 4, 4-phenylbutyric acid; 5, 2,4-dichlorophenol;
6, 2-sec butyl-4,6-dinitrophenol; 7, 3,4-dichlorobenzoic acid; 8, 2,6-dibromo-4-nitrophenol; 9, 2,4,5-trichlorophenol; 10, 2,4,6-trichlo-
rophenol; 11, 2,3,4,6-tetrachlorophenol; 12, tetrachloroverathrol; 13, pentachlorophenol; 14, pentachloroanisole; 15, 2,4,6-trichloro-
5-phenylphenol; 16, DDT; 17, 2,3,6-trichloro-4-nitrophenol (Adapted from Saarikoski et al., 1986).
Toxicokinetics in Fishes 11

Several physiological and chemical factors may account linked; thus, changes in environment and activity increase
for these trends in branchial uptake with chemical hydro- or decrease branchial absorption of xenobiotics by impact-
phobicity. Both McKim et al. (1985) and Saarikoski et al. ing the rate-limiting processes that control this uptake. For
(1986) attributed increases in absorption between log KOW example, Lloyd (1961) observed increases in the toxicity
1 and 3 to greater membrane permeability for the more of several chemicals to rainbow trout as ambient O2 con-
hydrophobic chemicals. Drawing upon the two-phase centrations were reduced from 100% to 30% of saturation.
resistance model given by Flynn and Yalkowsky (1972), Based on these observations, he suggested that the increase
Gobas and co-workers (Gobas et al., 1986; Gobas and in toxicity was caused by an increase in ventilation volume
Mackay, 1987) proposed that branchial uptake was regu- that occurred at reduced O2 concentrations, resulting in a
lated by chemical partitioning into the lipid portion of the larger quantity of chemical being brought in close contact
gill epithelium and diffusion through separate aqueous with the gills.
and organic (gill membrane) layers. Operationally, this McKim and Goeden (1982) measured changes in bran-
description assumes that Fick’s law of diffusion applies chial uptake of endrin in response to a stepwise decrease
to two adjacent membranes possessing different molecu- in afferent O2 concentration. Initial oxygen and endrin
lar properties. Several authors have proposed molecular extraction efficiencies were nearly constant at O2 saturation
size cutoffs (e.g., effective cross-sectional diameter) for values of 100% and 80% but dropped progressively at O2
chemical diffusion across gill membranes (Dimitrov et al., saturation values of 50% and 30%. These observed declines
2003; Opperhuizen et al., 1985) as a means of explaining in extraction efficiency may have been due to an increase
patterns in reported fish bioconcentration factors (BCFs; in ventilatory stroke volume, resulting in greater bypass of
defined as the steady-state ratio of the chemical concen- water around the gill lamellar channels, or an increase in
tration in fish to that in water resulting from a water-only physiological dead space associated with increased water
exposure). Arnot et al. (2010) cautioned against the use of velocities. The primary effect of decreasing O2 content,
strict molecular size criteria to predict chemical bioaccu- however, was to cause an increase in ventilation volume.
mulation potential, arguing that such cutoffs reflect errors The net result of these changes was that the rate of endrin
in the interpretation measured BCFs. Other researchers, uptake at 50% of O2 saturation was approximately double
noting the downward trend in BCFs for chemicals with log that at 100%.
KOW values greater than 6, have proposed that structural Environmental temperature changes can cause dra-
attributes of these chemicals may limit branchial uptake matic changes in the metabolic rates of poikilothermic
(Bruggeman et al., 1984). However, the principal limita- animals, which affects their demand for O2. An increase
tion on branchial uptake of very hydrophobic chemicals is in O2 demand due to increased temperature is especially
thought to be chemical binding to DOC and POC, which problematic for fish because the solubility of O2 in water is
reduces the unbound or bioavailable concentration of the inversely related to temperature. To obtain more O2, a fish
chemical in respired water (Black and McCarthy, 1988; in most cases adjusts its ventilation volume and the num-
Freidig et al., 1998; Johnsen et al., 1989; McCarthy and ber of perfused lamellae in contact with respiratory water
Jimenez, 1985; Schrap and Opperhuizen, 1990). (Randall, 1982). This suggests that, if chemical uptake is
Water and blood flows through the gills maintain xenobi- controlled by water flow across the gills, an increase in
otic diffusion gradients across the gill epithelium. Norstrom temperature will result in an increase in branchial uptake
et al. (1976), Neely (1979), and Bruggeman et al. (1981) sug- rate like that caused by a decrease in O2 content. Changes
gested that branchial uptake should be correlated to the rate in temperature also affect cardiac output and could poten-
of water flow across the gills and thus to respiration rate. tially impact branchial absorption if blood flow to the gills
Barber et al. (1988) presented a hydrodynamic-based model was a limiting factor. Several authors have shown that
for chemical bioconcentration in fish that incorporates chemical uptake correlates with O2 consumption (VO2)
effects of advection and diffusion in water flowing through as temperature changes (Black et al., 1991; Murphy and
the gills. Hayton and Barron (1990) suggested that blood Murphy, 1971; Rodgers and Beamish, 1981). For example,
flow, water flow, and diffusional barriers all have the poten- Black et al. (1991) found that the branchial uptake of three
tial to influence branchial flux, and proposed a model based moderately hydrophobic chemicals – benzo[a]pyrene,
on the concept of serial resistance. Erickson and McKim 2,2′,5,5′-tetrachlorobiphenyl, and naphthalene – changed
(1990a, 1990b) developed counter-current models for bran- in direct proportion to VO2 when rainbow trout were sub-
chial flux of nonionized chemicals that account for both jected to an acute reduction in temperature from 17°C to
flow and diffusion limitations, as well as binding to DOC. 8°C. Finally, it is well known that chemical diffusion rates
in solution vary with temperature. Similar changes are
Physiological Impacts on Branchial Absorption expected to occur in biological membranes and could be
Gill ventilation and blood perfusion rates vary with activ- important if diffusion across the gill epithelium limits the
ity level, temperature, and environmental O2 tension. rate of branchial flux. Fish may also respond to prolonged
Differences in environmental conditions and activity level changes in temperature by altering the molecular structure
also impact the toxicity and bioconcentration of chemi- of the gill epithelium, possibly changing its permeability to
cals in fish. These observations are now understood to be xenobiotic chemicals.
12 Toxicology of Fishes

The Gastrointestinal Tract of chemical consumed by a fish that is absorbed across

Gastrointestinal Absorption of Xenobiotics the gastrointestinal mucosa, while oral bioavailability
(F) refers to the fraction amount of chemical consumed
The gastrointestinal tract (GIT) of fishes functions in diges-
that enters the systemic circulation. The ED for a persis-
tion, nutrient absorption, excretion, and as a barrier to the
tent, poorly metabolized chemical may be estimated from
external environment. For some groups of fishes, the GIT
data collected in a standardized feeding experiment that
also plays a role in osmoregulation, buoyancy, ion regula-
provides information needed to account for the effect of
tion, and placental nutrition. Diverse structural adaptations
chemical elimination (OECD, 2012). For chemicals that
in different species reflect a wide array of digestive strate-
are subject to biotransformation before they enter the
gies. Gross differences among species include the presence
systemic circulation, an “ED” value determined in this
or absence of an acid-secreting stomach, the presence or
manner is more appropriately viewed as an estimate of F
absence of a gizzard, large differences in GIT length, and
(Arnot and Mackay, 2018). An estimate of F may also be
an absence or varying number of blind diverticula called
obtained by dividing the area under the blood concentra-
pyloric ceca that project from the intestine near the pylorus
tion–time curve (AUC) that results from a dietary exposure
of the stomach. In general, nutrient uptake and blood flows
by that which results from an equivalent intravascular dose
are highly regionalized and respond to local stimuli initi-
(Barron et al., 1990). If an ingested chemical undergoes no
ated by the food bolus. As the food bolus moves down the
biotransformation, ED and F will be equal. If a chemical
GIT, the character of the ingesta changes, as does its vol-
undergoes biotransformation in the liver and/or gastrointes-
ume. Nutrients are absorbed and mucus, bacteria, and epi-
tinal mucosa, ED will be greater than F. Biotransformation
thelial cells are added. As a result, different regions of the
in the gastrointestinal lumen would tend to reduce ED and
absorptive surface are presented with ingesta that differs
F in a similar manner.
markedly in composition. Reviews of fish GIT structure and
Oral bioavailability (F) values determined using the
function have been provided by several authors (Bucking
AUC method have been reported for several chemicals in
and Anderson, 2020; Kapoor et al., 1975; Kleinow and
fish (Kleinow et al., 2008), but this limited dataset provides
James, 2001; Smith, 1989).
little information regarding the physicochemical and bio-
Because of its high surface area and role in nutrient
logical factors (e.g., hydrophobicity, metabolic stability)
uptake, the proximal intestine is the most important site for
that control this process. In contrast, ED values have been
xenobiotic absorption. Digestive processes liberate xenobi-
estimated for a substantial number of chemicals (Gobas
otics from the food matrix, transport them to the mucosal
et al., 1988; Arnot and Quinn, 2015; Figure 2.3). Previously,
epithelium, and control their presentation to the absorptive
it was suggested that ED values are relatively constant for
surface. Gut uptake of hydrophobic contaminants has been
chemicals with log KOW values between 4 and 6 (averag-
strongly associated with digestion and absorption of dietary
ing about 50% across studies), then decrease progressively
lipid. Mechanical mixing, pancreatic lipases, and bile salts
with log KOW at log KOW values greater than 6 (Gobas et al.,
disperse dietary lipid and contribute to the formation of
1988; Figure 2.3A). A more recent review of ED data casts
an equilibrium phase consisting of small mixed micelles
some doubt on these generalizations (Arnot and Quinn
(Kleinow and James, 2001). Hydrophobic contaminants
2015; Figure 2.3B). Complicating this assessment is that
reside in the interior of these micelles, while the hydro-
fact that there is considerable scatter in existing ED data,
philic exterior allows the micelles to remain soluble in the
even for the same chemical. The cause of this scatter is not
aqueous milieu of the luminal contents. Upon reaching the
entirely known but likely reflects true biological variability
enterocytes, the micelles are dissociated by the combined
among species, the variable and largely unknown impact
action of pH changes and mucosal lipases, liberating stored
of biotransformation (i.e., whether reported ED values are
xenobiotic. This process creates a highly localized concen-
in fact estimates of F), and differences in experimental
tration gradient for uptake of both xenobiotic and fatty acids
design (e.g., fish size, temperature, diet type, and number
across the absorptive epithelium. Uptake of hydrophobic
and size of individual feedings). It is possible that trends
organic chemicals by fish in an environmental setting may
with respect to the log KOW dependence of ED are obscured
be dominated by the dietary route of exposure (Bruggeman
by variability in existing datasets. Additional research
et al., 1984; Clark et al., 1990; Qiao et al., 2000). This out-
involving a substantial number of chemicals and a highly
come is a product of limitations on branchial uptake that
standardized dietary dosing protocol would be required to
occur because of chemical binding to DOC and POC, as
address this question.
well as chemical accumulation in prey items (e.g., plankton
Empirically based algorithms for the prediction of ED
and benthos) that occupy lower levels of aquatic food webs.
have been developed using data for chemicals with log
An examination of literature of the topic of gastrointes-
KOW values <9 (Gobas et al., 1988; Figure 2.3A; also see
tinal absorption in fish reveals some inconsistencies in the
Bioaccumulation Assessment and Modeling). Presently,
use of terminology. Here, we adopt terminology suggested
there are very few ED measurements for chemicals with log
by Arnot and Mackay (2018), which is consistent with that
KOW values >9. When existing algorithms are applied to a
used in mammalian toxicokinetic studies. Dietary absorp-
chemical with a log KOW >9, dietary uptake is predicted to
tion efficiency (ED) is defined as the fractional amount
be negligible. Accumulating field data indicates, however,
Toxicokinetics in Fishes 13

FIGURE 2.3 Dependence of dietary absorption efficiency (ED) on chemical log KOW. Panel A: ED data presented by Gobas et al.
(1988). The solid line was generated using the fugacity-based model given by the authors. Panel B: ED data summarized by Arnot and
Quinn (2015). The data shown are “high” (open circles) and “medium” (open squares) confidence values from longer term (≥7 day)
exposure tests.

that dietary uptake of such chemicals may contribute to gastric emptying has been shown to increase with acclima-
substantial bioaccumulation (He et al., 2012). It is possible tion temperature (Windell et al., 1976) and decrease with
that diffusive uptake of extremely hydrophobic chemicals, food particle size (Jobling, 1987). Gut transit times tend
while limited, still occurs. Alternatively, chemical uptake to decrease with increased acclimation temperature (Hofer
via pinocytosis in the GIT may provide a “baseline” level of et al., 1982; Shrable et al., 1969).
ED regardless of log KOW. Changes in the character of the ingesta can facilitate or
hinder the absorption of xenobiotics; for example, moder-
Physiological Impacts on Gastrointestinal Absorption ate levels of dietary lipid appear to facilitate the absorption
Little is known regarding the direct effects of GIT struc- of hydrophobic contaminants, but low or very high lipid
ture and function on dietary uptake of xenobiotics by diets are associated with reduced absorption efficiency
fish. Numerous studies indicate, however, that pH gradi- (Van Veld, 1990). High levels of dietary fat may overwhelm
ents, gastrointestinal passage times, blood flow patterns, the ability of the system to digest and remove this mate-
and other aspects of digestive physiology vary with spe- rial. Unprocessed lipid would then provide a partitioning
cies, feeding frequency, meal composition and size, and phase for hydrophobic chemicals within the gut lumen.
environmental influences such as temperature. For fish Qualitative features of dietary lipid such as fatty acid chain
that possess a stomach, gastric evacuation generally pro- length and composition may also determine xenobiotic
ceeds according to an exponential decay model after an solubility in micelles and subsequent systemic availability
initial lag phase (Persson, 1986). The rate constant for (Doi et al., 2000).
14 Toxicology of Fishes

The Skin bass, gill surface area scales to body weight by a fractional
The skin of a fish functions as a physical barrier to separate exponent of about 0.78, consistent with the role of the gill
the internal and external environments. As such, it main- in supporting metabolic activity (Price, 1931). Skin sur-
tains the ionic and osmotic integrity of the internal envi- face area scales instead to a body weight exponent of about
ronment and provides protection from abrasion and disease 0.67 (Schmidt-Nielsen, 1984). Using these values, Lien and
organisms. Physiological functions of fish skin include McKim (1993) predicted that skin surface area approaches
respiration, ion exchange, and acid–base regulation. Based and may even exceed that of the gills in fish weighing less
on morphometric and anatomical information, research- than 5 g.
ers have long suggested that cutaneous O2 flux contributes
significantly to total respiration in larval fishes (McDonald Xenobiotic Distribution
and McMahon, 1977; McElman and Balon, 1980; Oikawa
and Itazawa, 1985; Rombough and Moroz, 1990). Because Primary Determinants
the gill epithelium of both small and large fish consists of A variety of processes act to distribute absorbed xenobiotic
one or a few cell layers, its thickness does not change much chemicals within fish. A portion of the absorbed chemical
with fish size. In contrast, skin thickness tends to decrease may distribute to a site of action where toxic effects are
with decreasing fish size and in small fish may approach the expressed. Other sites serve as repositories for the chemical
thickness of the gill epithelium. In larval Chinook salmon over short or long periods of time, while others are mobile
(Oncorhynchus kisutch), as much as 80% of O2 uptake and provide a means of transport within and out of the
takes place across the skin (Rombough and Moroz, 1990). animal. During its residence in the body, a xenobiotic will
Direct measurements of dermal uptake in rainbow trout redistribute continuously due to changes in concentration
and channel catfish (Ictalurus punctatus) were obtained by gradients that drive chemical movement.
exposing fish confined to respirometer-metabolism cham- Five primary factors control the distribution of xeno-
bers to a mixture of three chloroethanes (McKim et al., biotics from blood to peripheral tissues: (1) the physico-
1996). Although the rates at which both species approached chemical characteristics of the chemical (e.g., pKa, lipid
steady state were close to those observed in inhalation solubility, molecular volume); (2) the concentration gradi-
exposures to the same chemicals, steady-state chemical ent between blood and tissues; (3) the ratio of blood flow
concentrations in blood were much lower. An analysis of to tissue mass; (4) the chemical sorptive capacity of blood
the trout dataset indicated that dermal absorption would and tissue constituents; and (5) the activity of membrane
contribute 2%–4% of initial uptake (dermal plus branchial) transport proteins. For many xenobiotics, transport from
in a hypothetical waterborne exposure. A similar analy- blood to tissues occurs by simple diffusion following a
sis for channel catfish suggested that dermal absorption concentration gradient. When a chemical diffuses rapidly
would contribute 7%–9% of initial uptake. Based on these across biological membranes, an equilibrium may become
findings, it was concluded that dermal uptake is a minor established between a tissue and venous blood exiting the
route of exposure in large fish, except perhaps for species tissue. In other cases, the rate of membrane diffusion limits
that live in intimate contact with contaminated sediments. chemical flux between blood and tissues with the result that
In contrast, several studies have suggested that dermal these phases do not achieve equilibrium at early time points
uptake contributes substantially to total uptake of water- in an exposure. Chemical binding to blood and tissue con-
borne chemicals by small fish and juveniles of larger spe- stituents influences the distribution process by defining the
cies. Tovell et al. (1975) measured anionic detergent uptake concentration gradient that drives diffusion. Distribution
across the skin of small goldfish (Carassius auratus) and processes controlled by blood flow or simple diffusion gen-
found that 20% of total uptake occurred by this route. erally exhibit first-order kinetics. In contrast, distribution
Saarikoski et al. (1986) obtained dermal uptake values for controlled by the activity of a membrane transport protein
guppies exposed to a series of phenols, anisoles, and carbox- could exhibit nonlinear (saturable) kinetics.
ylic acids. To distinguish between gill and skin absorption, At early time points in an exposure, chemical distribu-
fish were positioned into a hole cut in a rubber membrane tion to tissues may be highly influenced by the route of
separating two exposure chambers. Estimates of dermal administration. High concentrations in the skin following
absorption ranged from 25% to 40% of total absorption. exposure to contaminated sediments, the gastrointestinal
Japanese medaka (Oryzias latipes) exposed to 2,2′,5,5′-tet- mucosa following dietary exposure, or muscle following an
rachlorobiphenyl in water accumulated considerably more intramuscular injection are obvious examples. Anatomical
chemical than could be explained by inhalation uptake and physiological peculiarities of fish may also result in
(Lien and McKim, 1993). Similar results were reported for characteristic distribution patterns; for example, xenobiot-
fathead minnows (Pimephales promelas) exposed to three ics administered by intramuscular injection in the trunk
chloroethanes (Lien et al., 1994). Lien and McKim (1993) muscle are initially transported to the kidney because
suggested that dermal uptake should increase in relative venous blood from the trunk muscle collects in the caudal
importance in small fish because of the way that gill and vein, which is part of the renal portal circulation in fish
skin surface areas scale to fish body weight. In small-mouth (Figure 2.1). Similarly, chemicals taken up from the GIT
Toxicokinetics in Fishes 15

or intraperitoneal cavity are transported first to the liver by 1992). When the primary and secondary circulations are
the hepatic portal vein and then to the gills via the ven- combined and expressed as a percentage of body weight,
tral aorta before distributing into the general circulation. the total circulatory volume places teleost fishes well into
The systemic availability of a chemicals taken up by this the upper range for vertebrates.
route may be influenced, therefore, by elimination path- Although many chemicals are transported in blood as
ways operating in the gut, liver, and gills. For example, the unbound forms, others are transported primarily in asso-
fish anesthetic tricaine methane sulfonate (MS 222) will not ciation with proteins, lipoproteins, lipids, or cellular com-
produce anesthesia when given by intraperitoneal injection ponents. Xenobiotics interact with these components by
anesthesia because of its rapid metabolism in the liver and several mechanisms. Covalent binding usually restricts fur-
the ease with which both metabolites and parent chemi- ther distribution of the chemical. In contrast, specific, non-
cal diffuse out across the gills (Hunn and Allen, 1974). covalent ligand–protein interactions with binding proteins
Anesthetic concentrations of MS 222 in arterial blood can such as albumin may result in reversible binding that fol-
only be achieved by maintaining high MS 222 concentra- lows the law of mass action. When more than one specific
tions in water. binding interaction is possible, the distribution of a chemi-
cal among binding proteins depends on both the binding
Xenobiotic Transport and Binding in Plasma affinity of each protein and their relative concentrations.
Tissues with the highest blood perfusion rates in fish include These binding affinities may change with changes in ionic
the kidney, red muscle, pyloric caeca, intestine, spleen, and strength, pH, temperature, and protein conformation. If this
liver (Barron et al., 1987a). Tissues receiving an intermedi- binding is reversible, however, an equilibrium will tend to
ate level of blood perfusion include the gonads, skin, and be reestablished, providing an efficient means by which
white muscle, while adipose tissue and bone are poorly per- xenobiotics are transported and redistributed. Hydrophobic
fused. The influence of blood flow on chemical distribu- organic chemicals will partition to both lipids and proteins
tion was demonstrated in rainbow trout exposed to linear in blood. This type of binding is reversible, nonspecific and
alkylbenzene sulfonate (LAS) in water (Tolls et al., 2000a). nonsaturable.
Concentrations of LAS in the internal organs (primarily Total plasma protein concentrations in fish are gener-
kidney and GIT) and liver exceeded 80% of their steady- ally lower than those in mammals, possibly resulting in a
state values after 8 hours of exposure, while concentrations reduced number of binding sites. In mammals, plasma albu-
in the muscle and skin continued to increase until 78 hours. min plays an important role in binding weak organic acids,
Blood flow to some tissues may change substantially in while weak organic bases bind to plasma glycoproteins.
response to physiological stimuli. An example of this phe- The structural diversity of plasma albumins in fish exceeds
nomena is provided by the pre- and post-prandial intestine that of albumins from higher vertebrates (Andreeva, 2010).
(Axelsson and Fritsche, 1991; Axelsson et al., 1989, 2000). Some species do not possess plasma albumin at all, includ-
Exercise (Neumann et al., 1983) and hypoxia (Cameron, ing some sharks and rays (Metcalf et al., 1999; Weisiger
1975) were shown to alter blood flow patterns in rainbow et al., 1984), or exhibit only very low concentrations (De
trout and arctic grayling (Thymallus arcticus), respectively. Smet et al., 1998). Others possess relatively high concen-
In rainbow trout, an increase in cardiac output (QC) asso- trations, including some phylogenetically ancient species
ciated with increasing ambient temperature was accom- (Gray and Doolittle, 1992; Metcalf et al., 2007). The plasma
panied by a decrease in relative blood perfusion to most albumin of rainbow trout has been described as para-albu-
tissues (expressed as a percent of QC), resulting in main- min because of significant functional differences from the
tenance of tissue blood flow rates (Barron et al., 1987a). albumin of mammals (Perrier et al., 1977). Thus, the char-
These changes were offset, however, by a redistribution of acteristic binding of xenobiotics to mammalian albumin
QC to white muscle, substantially increasing the perfusion and other plasma proteins may not directly extrapolate to
rate of this tissue. fish. For example, low concentrations of the antibiotic sul-
Another circulatory feature of fish with unknown but fadimethoxine become highly (>90%) bound when added to
potential significance to xenobiotic distribution is the sec- rat plasma. In rainbow trout, sulfadimethoxine binding in
ondary circulation. This network of anastomosing vessels plasma ranges from 13% to 17% over the same concentra-
arises from the walls of the primary arteries and parallels tion range, suggesting a nonsaturable, nonspecific interac-
the primary circulation of arteries, capillaries, and veins tion. This low degree of protein binding in trout facilitates
(Olson, 1996). The secondary circulation shares some char- the elimination of sulfadimethoxine and may result in a
acteristics with the mammalian lymphatic system, such relatively larger apparent volume of distribution when com-
as structural attributes of the vessels and restricted access pared with mammals (Kleinow and Lech, 1988).
for formed elements but is more limited in its distribution. The binding of several neutral and basic drugs was
Tissues perfused by the secondary circulation include the shown to be similar in rainbow trout and human plasma
gill filaments, skin, peritoneal lining, and oral mucosa. (Henneberger et al., 2022). In contrast, acidic drugs exhib-
Limited studies suggest that the secondary circulation has ited up to 70-fold higher binding in human plasma than in
a volume greater than that of the primary circulation and trout plasma. Similar binding was observed for 1-butanol,
a turnover time of several hours (Steffensen and Lomholt, phenol, nitrobenzene, and pentachlorophenol in plasma
16 Toxicology of Fishes

from rainbow trout and rats (Schmieder and Henry, 1988). with rainbow trout demonstrated the redistribution of [14C]
This binding was shown to correlate positively with chemi- 2,2′,5,5′-tetrachlorobiphenyl to eggs and sperm (Guiney
cal log KOW. An empirically based algorithm (Fitzsimmons et al., 1979).
et al., 2001) that predicts binding in trout plasma from The percentage of an accumulated hydrophobic contam-
chemical log KOW provided a good fit to measured values for inant that is redistributed to the developing gonads depends
20 organic chemicals, most of which are neutral at physi- on the lipid content of the fish prior to gonad development,
ological pH (Nichols et al., 2013). the fraction of whole-body lipid content that is mobilized
and incorporated into the gonads, and the size of the gonads
Chemical Partitioning to Neutral Lipid relative to total body weight. Niimi (1983) examined these
Although chemical distribution at the beginning of an features in five fish species: rainbow trout, yellow perch,
exposure is often determined by relative tissue blood flows, smallmouth bass (Micropterus dolomieu), white bass
many chemicals redistribute over time in accordance with (Morone chrysops), and white sucker (Catostomus commer-
their relative affinity for various tissue macromolecules. sonii). Yellow perch were the leanest of these five species
Hydrophobic xenobiotics, such as polychlorinated biphe- (5.1%) and transferred the greatest percentage of accumu-
nyls (PCBs), tend to partition into tissues that have a high lated contaminants (25.5%) to their eggs. In contrast, rain-
neutral (“storage”) lipid content. Distributional differences bow trout had the highest starting lipid content (11.4%) and
among species may therefore occur due to different pat- transferred the lowest percentage of contaminants to their
terns of lipid deposition in organs and tissues. For example, eggs (5.5%). The high percentage of contaminant transfer in
Guiney and Peterson (1980) found that 60% of a dose of spawning yellow perch was due to the large size of its egg
2,2′,5,5′-tetrachlorobiphenyl in rainbow trout was contained mass (22.3% of body weight) and the fact that it transfers a
in skeletal muscle and carcass, but in yellow perch (Perca high percentage of its limited whole-body lipid stores to the
flavescens) 70% was contained in viscera and carcass developing ovaries (27.1%).
(excluding skeletal muscle). These patterns were correlated Reproductive life history may also influence the redistri-
with differences in the lipid content of each tissue, relative bution of hydrophobic contaminants in fish. The Chinook
to that of other tissues. salmon, a semelparous (once-bearing) species, trans-
Tissues that contain a large amount neutral lipid may act fers most of its stored lipid into a single spawn of eggs.
as storage depots for hydrophobic xenobiotics. This storage By comparison, iteroparous (multiple bearing) lake trout
may protect against adverse effects by isolating a chemi- (Salvelinus namaycush) invest a lower proportion of stored
cal away from its sites of toxic action. Conversely, storage lipid into each reproductive effort. In a study of these two
in these sites may prolong the overall residence time of a species in Lake Michigan, Miller (1993) found that female
chemical in the body and promote accumulation during salmon transferred 28%–39% of their accumulated PCB
chronic exposures. By reducing adipose lipid stores, starva- body burden to the eggs during a single spawn. In contrast,
tion may result in a relatively rapid mobilization of accu- female lake trout eliminated 3%–5% of whole-body PCBs
mulated chemical. When the rate of elimination from the during each of what may be several spawns.
whole animal is low, this starvation-induced decrease in
whole-body lipid content may cause chemical concentra- Xenobiotic Excretion
tions in relatively lean tissues to increase substantially, even
as whole-body chemical concentrations remain relatively Branchial Excretion
constant (Gruger et al., 1975; Lieb et al., 1974). In teleosts, the most important route of excretion for
neutral, water-soluble, low-molecular-weight chemicals
Lipid Mobilization and Xenobiotic is across the gills. Working with the Dolly Varden char
Redistribution in Reproducing Fish (Salvelinus malma) in a split chamber system, Thomas
Female fish support egg development by mobilizing lip- and Rice (1981) showed that aromatic hydrocarbons which
ids from body stores. This lipid may be transferred to the exhibit low to moderate hydrophobicity (log KOW 1–4) are
growing egg mass or incorporated into larger energy stor- excreted across the gills at a greater rate than those which
age molecules such as vitellogenin. Additional lipid may are very hydrophobic (log KOW 4–7). A similar finding
be mobilized in males and females to provide energy for was reported for goldfish exposed to a series of substituted
spawning behaviors such as migration and nest defense. In phenols with log KOW values ranging from 1 to 5 (Nagel
either case, xenobiotics may redistribute from fat storage and Urich, 1980). Erickson and McKim (1990b) compiled
depots to the developing gonads. To investigate this phe- data from several studies involving guppies and found that
nomenon, Vodicnik and Peterson (1985) exposed female excretion rates declined linearly with chemical log KOW
yellow perch to [14C] 2,2′,5,5′-tetrachlorobiphenyl in water across a wide range (3–8) of values. This dependence of
for 24 hours and then monitored tissue distribution and branchial excretion on chemical hydrophobicity is due
elimination for 5 months. Two weeks after exposure, 30% largely to nonspecific binding of chemicals in blood. This
of chemical retained by fish was present in the developing binding lowers the diffusion gradient across the gill epithe-
ovaries. This value increased to 50% just prior to spawn- lium by reducing the concentration of chemical in blood
ing, which occurred 4 months into the study. Similar studies that is unbound.
Toxicokinetics in Fishes 17

Biliary Excretion dyes (Plakas et al., 1992a), herbicides (Schlenk and Moore,
The liver plays a central role in the elimination of xenobi- 1993), polycyclic aromatic hydrocarbons (Collier and
otic chemicals, both as the primary site of enzymatic bio- Varanasi, 1991), metals (Grosell et al., 2001), and natural
transformation and via biliary excretion. Here, we consider products (Sahin et al., 1996). Early studies with rainbow
biliary excretion as a process distinct from biotransforma- trout showed that chemical concentrations in bile may be
tion. It should be noted, however, that biotransformation much higher than those in surrounding water (Lech et al.,
may play a pivotal role in biliary excretion of xenobiotics 1973; Statham et al., 1976). Guarino and Lech (1986) sum-
by creating metabolites (e.g., products of phase II conju- marized the results of studies with fish exposed to more than
gation pathways) that are substrates for active membrane 40 drugs and other xenobiotics of varying molecular weight.
transporters within the canalicular membrane. The data were examined to determine bile/plasma concen-
Biliary excretion of xenobiotics and their metabolites tration ratios and the percentage of chemical eliminated in
depends on the formation and secretion of bile, chemical bile or urine. Bile/plasma concentration ratios were greater
uptake by hepatocytes, and the transport of these chemicals than 1 for over 85% of chemicals examined, suggesting that
into bile. A variety of structural arrangements have been fish were capable of concentrating xenobiotics in bile rela-
described for the liver and biliary tree of fish. In general, tive to plasma. The relative importance of biliary secretion
however, fish do not possess the lobular structure and zon- as a route of excretion increased with molecular weight,
ally oriented portal tracts observed in mammalian livers although other factors such as the charge on the molecule
(Eurell and Haensly, 1982; Hampton et al., 1989; Hinton were important. Generally, chemicals that are highly polar
and Couch, 1998; Robertson and Bradley, 1992; Schar et al., and have molecular weights greater than 600 are excreted
1985). Instead, the hepatocytes are commonly arranged largely in bile (Gingerich et al., 1977; Schmidt and Weber,
into tubular elements. In rainbow trout, hepatocyte apices 1973). Xenobiotics of intermediate molecular weight (300–
are directed toward the center of the tubule. At the center 600) and polarity may be excreted in bile as well as by other
of the tubule, plasma membranes of adjacent cells form bile routes (Allen et al., 1979; Hunn and Allen, 1975).
canaliculi. Where a biliary space exists, the biliary epithe- Secretion of bile into the intestinal tract is stimulated by
lial cells form junctional complexes with each other, and nutritional and digestive signals. Due to the episodic nature
the hepatocytes on either side form a wall of the biliary of this process and potential for xenobiotic reabsorption
passageway. These biliary passageways lead in turn to the within the intestine, it is difficult to assess the contribution
cholangioles, small bile ducts, and large bile ducts. The of biliary secretion to the overall process of chemical elimi-
contralateral or basal side of each hepatocyte faces a blood- nation. The gall bladder of a recently fed fish is generally
filled sinusoid surrounding the tubule. Individual tubules void of bile. By necessity, therefore, chemical concentra-
curve, branch, and anastomose to form a complex network tions in bile are generally determined using fasted animals.
of parenchyma. Techniques for continuous collection of bile in unanes-
In salmon, multiple hepatic bile ducts empty into an thetized rainbow trout have been described by several
elongated major duct that runs along the posterior ventral authors (Gingerich et al., 1977; Sanz et al., 1993; Schmidt
margin of the liver. The cystic duct branches from the major and Weber, 1973). Similar methods have been developed for
bile duct and serves to divert bile to the gall bladder. Bile the skate (Raja erinacea) and dogfish shark (Squalus acan-
originating from the major bile duct and gall bladder is col- thias) (Boyer et al., 1976a, 1976b, 1976c). Basal bile flows
lected in the common bile duct and empties into the proxi- measured using these techniques range from 50 to 200
mal intestine just posterior to the pyloric curvature of the μL/h/kg in rainbow trout, 30–70 μL/h/kg in the skate, and
stomach. Some fish species, including members of the cod 80–110 μL/h/kg in the dogfish. Bile flow rates in mammals
family, do not possess a gall bladder. For these species, it is may be 50 times greater than these values (Klaassen and
thought that bile is secreted continuously into the intestine. Plaa, 1967). Differences in the rates of biliary excretion of
Biliary excretion may be facilitated by the activity of bromosulfophthalein between rainbow trout and rats were
ABC transporters and transporters of the SLC family. ABC found to be well correlated with differences in bile flow rate
transporters move substrates out of the hepatocyte and (Gingerich et al., 1977), suggesting that bile formation rate
into bile or blood. Substrates for these transporters include limits the rate of bromosulfophthalein excretion in bile.
ionized xenobiotics and xenobiotics conjugated with sul-
fate, glutathione (GSH) and glucuronic acid. Additional Renal Excretion
substrates include a wide range of endogenous molecules Fish kidneys perform two major functions: (1) ion and
including steroids, lipids, and peptides. SLC transport- water balance; and, (2) excretion of endogenous and exog-
ers residing within the sinusoidal (basolateral) membrane enous solutes. The diversity of kidney structure–function
transport organic anions from blood to the interior of the across species is enormous, but basic features are shared
hepatocyte. by both freshwater and saltwater teleosts (Hickman and
Fish excrete a wide variety of xenobiotics in the bile. Trump, 1969; Nishimura and Imai, 1982; Pritchard, 1981;
A partial listing of these chemicals includes antibiotics Pritchard and Miller, 1993; Pritchard and Renfro, 1984).
(Samuelsen et al., 1995), insecticides (Lech et al., 1973), The functional unit of the freshwater fish kidney consists
18 Toxicology of Fishes

of an encapsulated capillary network called the glomeru- blood. Most of this portal blood bathes the kidney tubules
lus, a tubule lined with epithelial cells, and bladder. The and contributes to the renal secretory system (Pritchard and
kidney tubule can be subdivided further into two proximal Renfro, 1984). Overall, up to 80% of QC flows through the
segments (I and II), a distal tubule and a collecting tubule. fish kidney, as compared with about 20% in mammals.
The functional kidney unit of saltwater fish is like that of Some of the xenobiotics that fish are exposed to exist
freshwater fish but lacks the distal tubule. In some saltwater as organic anions or cations, while others are taken up as
species, the glomerulus may also be absent. neutral species and converted to anions or cations by vari-
Because they are hyperosmotic with respect to their ous biotransformation pathways. In all vertebrates studied
environment, freshwater fish must eliminate large volumes to date, anionic and cationic secretory systems located
of dilute urine and retain salt (principally NaCl). High urine in the proximal tubule transport charged chemicals from
flow rates are supported by a high glomerular filtration rate plasma into the tubule urine (Pritchard and Miller, 1993).
(GFR; L/h), where the GFR defined as the volume of fluid The organic anion secretory system uses metabolic energy
filtered from the glomerular capillaries per unit of time. In to move substrate molecules into tubule epithelial cells
contrast, saltwater teleosts are hypoosmotic with respect against their electrochemical gradient. This mechanism is
to their environment and must overcome dehydration by carrier-mediated, saturable, and inhibited competitively by
drinking seawater and absorbing water and minerals (pri- other substrates (Pritchard, 2001). Organic anion secretion
marily monovalent ions) through the gut. Excess salts are is a three-step process: (1) ATP is hydrolyzed to drive the
eliminated across the gills (both mono- and divalent ions) Na+ pump and create an inward Na+ gradient; (2) the Na+
and kidney (primarily divalent ions), and water loss is mini- gradient into the cell drives the uptake of α-ketoglutarate
mized by producing small quantities of relatively concen- (αKG−2), thereby creating an outward αKG−2 gradient; and
trated urine. The GFR in saltwater fish is much lower than (3) the organic anion moves into the cell in exchange for
that of freshwater fish. Urine osmolarity is maintained by the outward movement of αKG−2. Efflux of the organic
an exchange of divalent and monovalent ions within the anion into the tubule may occur by facilitated diffusion
proximal tubule. Water is then reabsorbed with NaCl in the or through the action of an ABC transporter (Miller and
bladder. Because it comprises up to 40% of total tubular Pritchard, 1997).
surface, the proximal tubule of saltwater fish has been used Organic cation secretion is also a multistep process. In
extensively for both in vitro and in vivo studies of kidney contrast to anion secretion, however, chemical movement
function (Kinter, 1966, 1975; Miller and Pritchard, 1997; into the epithelial cell (by facilitated diffusion) is energeti-
Pritchard and Miller, 1993; Pritchard and Renfro, 1984). cally favored because of the strong negative charge on the
The mechanisms that have evolved for handling ions and cell interior. Cation transport into the lumen of the tubule is
water in the fish kidney also contribute to the excretion of opposed by the membrane potential and must be tied to the
endogenous and exogenous organic solutes. The first step hydrolysis of ATP (Pritchard, 2001). As with anion secre-
in urine formation is ultrafiltration of plasma through the tion, the cationic secretory system is susceptible to sub-
capillary network of the glomerulus. Solutes within this strate competition.
glomerular filtrate may be reabsorbed back into plasma. The mechanisms responsible for renal excretion of chem-
Alternatively, solutes may be added to the filtrate by trans- icals can be distinguished by dividing measured renal clear-
port of chemical from the plasma across the renal tubular ance rates (CL R) by the GFR (McKim et al., 1999; Pritchard
epithelium. The final composition of urine is determined and Renfro, 1984). The CL R is the volume of plasma com-
by characteristics of the epithelium in both the tubule and pletely cleared of chemical by the kidney in a fixed period
bladder (Miller, 1987). and may be calculated by dividing the excretion rate of
Molecules with a radius of less than about 20 Å are chemical in urine by the chemical concentration in plasma
completely filtered at the glomerulus, those with a radius at the midpoint of a sampling period. The GFR is typically
between 20 and 40 Å are partially filtered, and those estimated using an in vivo [14C]-polyethylene glycol method
with a radius greater than 40 Å tend to be retained in (Beyenbach and Kirschner, 1976). Figure 2.4 shows CL R /
plasma (Miller, 1987). In fish as in mammals, chemicals GFR ratios for several chemicals in both freshwater and
with molecular weights greater than about 500 tend to be saltwater fish. In the absence of substantial plasma bind-
excreted in the liver rather than the kidney (Pritchard and ing, a CL R /GFR ratio <1 suggests that a chemical is reab-
Renfro, 1984). Plasma binding is an important determinant sorbed within the kidney tubule, a ratio of 1 indicates no
of urinary excretion because glomerular filtration and tubu- net tubular transport, and a ratio >1 signifies active secre-
lar secretion act primarily on unbound chemicals in plasma. tion into the tubular fluid. Chemical binding in plasma can
Because they bind to plasma proteins, hydrophobic chemi- reduce renal tubular secretion as well as chemical filtration
cals are generally retained by plasma as it flows though the at the glomerulus. In the absence of active section to urine,
glomerulus. Hydrophobic chemicals contained within the a high degree of plasma binding may result in a CL R /GFR
glomerular filtrate may be reabsorbed from the tubule. This ratio close to 0. For this type of chemical, a CL R /GFR ratio
is particularly true of saltwater fish due to the concentrating between 0 and 1 can provide evidence for active secretion
effect of water reabsorption. The caudal vein in fish drains (e.g., PFOA; no. 15 in Figure 2.4). For a given mechanism
into the kidney, providing a substantial supply of portal of renal excretion, CL R /GFR ratios in saltwater fish tend to
Toxicokinetics in Fishes 19

FIGURE 2.4 Influence of tubular transport, biotransformation, and plasma binding on renal excretion in marine teleosts (A–C;
Pritchard and Renfro, 1984) and freshwater teleosts (D, E; McKim et al., 1999; Consoer et al., 2014, 2016). Data for each chemical
are expressed as the ratio of renal clearance (CL R) to glomerular filtration rate (GFR). (A) Reference chemicals: (1) glucose, reab-
sorbed by active transport; (2) 2-deoxyglucose, with no net tubular transport; (3) p-aminohippuric acid, a prototypical substrate for
active anion secretion pathway. (B) Effect of biotransformation: (4) benzo[a]pyrene (BaP), reabsorbed passively; (5) BaP-7-phenol
conjugates (largely glucuronide), limited anion secretion; (6) BaP-7,8-dihydroxydiol conjugates (largely sulfate), a better substrate for
anion secretion; (7) DDT, passive reabsorption; (8) DDA, effective tubular secretion. (C) Effect of plasma binding: (9) DDA, plasma
binding of 97%; (10) 2,4-D, plasma binding of 70%. DDA and 2,4-D are transported similarly in vitro (without binding). (D) Effects of
reabsorption and biotransformation: (11) hydroxyquinone, strong passive reabsorption; (12) phenol, slight passive reabsorption; (13)
phenyl glucuronide, limited anion secretion; (14) phenyl sulfate, better substrate of anion secretion. (E) Renal handling of PFAS: (15)
perfluorooctanoate (PFOA), substantial clearance despite ~100% plasma binding suggests active anion secretion; (16) perfluorooctane
sulfonate (PFOS), binding also close to 100% but no evidence for active anion secretion.

exceed those of freshwater fish. This pattern appears to be Whether or not these metabolites were formed in skin prior
due primarily to higher GFR values in freshwater fish. to their diffusion into mucus was not investigated.

Fecal Excretion Excretion via the Gametes

Xenobiotics incompletely absorbed from the diet will be The redistribution of hydrophobic chemicals from somatic
excreted in feces while the absorbed fraction will be trans- lipid stores to developing gametes was described earlier
ferred to the liver and subjected to biotransformation and in this chapter. Although spawning eliminates xenobiot-
biliary excretion. Parent chemicals and their metabolites ics from the animal, reducing the whole-animal contami-
excreted in bile may be expelled with the feces (Statham nant load, whole-body chemical concentrations may go up
et al., 1976); however, hydrolysis of a biliary conjugate by (Tietge et al., 1998), down (Guiney et al., 1979; Niimi, 1983;
enzymes present in gut microflora may liberate the parent Vodicnik and Peterson, 1985), or stay the same (Niimi,
chemical for reabsorption and enterohepatic recirculation 1983), depending on the chemical concentration in gametes
(Collicutt and Eales, 1974; Fricker et al., 1997; James, 1987; relative to that in the rest of the body. If the contaminant
Layiwola et al., 1983; Schultz et al., 2001). Hydrophobic concentration in the gametes is lower than that of the rest of
xenobiotics that are poorly cleared by hepatic, renal, and the fish, gamete release increases the chemical concentra-
branchial routes may be excreted by passive diffusion from tion expressed on a whole-body basis. The reverse is true if
blood to contents of the intestinal lumen (Ingebrigtsen the contaminant becomes highly concentrated in the gam-
and Solbakken, 1985; Ingebrigtsen et al., 1988; Kleinow etes, relative to the rest of the animal.
et al., 1996). The importance of maternal transfer in viviparous spe-
cies is unknown. Wourms et al. (1988) estimated that,
Dermal Excretion among teleosts, live-bearing strategies have developed in
The extent to which chemicals and their metabolites are only 2%–3% of species; however, two of these species, the
excreted across the skin is poorly known; however, anatom- mosquitofish (Gambusia affinis) and guppy, have been used
ical and physiological factors that promote chemical uptake extensively in aquatic toxicology research. To the extent that
across the skin of some fish species (e.g., high degree of an exchange of blood occurs between female fish and the
vascularization and large ratio of skin surface area to gill developing young (as in Poeciliidae), it can be speculated
surface area) also might be expected to facilitate chemical that maternal transfer of chemicals to offspring occurs in
excretion. Naphthalene was recovered from the mucus of some live-bearing species. In species for which most early
rainbow trout soon after it was administered by intraperito- development takes place after the egg has left the follicle
neal injection or dietary exposure (Varanasi et al., 1978). At (Embiotocidae and live-bearing sharks), the opportunity for
later time points, only naphthalene metabolites were found. maternal transfer may be reduced.
20 Toxicology of Fishes

TOXICOKINETIC MODELING The following text provides an overview of clearance

concepts common to both empirical and PBTK models.
The kinetic behavior of xenobiotic chemicals in fish has These two modeling approaches are then taken up individu-
been described using a variety of modeling approaches. ally. A brief section on noncompartmental modeling is also
Previous reviews of this topic have distinguished between provided. Although seldom employed in aquatic toxicol-
empirical (a.k.a. data-based or compartmental) and ogy, noncompartmental models offer important advantages
physiologically based models (Barron et al., 1990; Gretch when dealing with sparse datasets. Model inputs and cal-
et al., 2016; Kleinow et al., 2008; Landrum et al., 1992). culated terms are accompanied upon first usage by typical
Empirical models are generally developed by fitting mod- units (e.g., L/h for QC). Unless otherwise noted, the listed
eled simulations to concentration time course data from units are for values that have not been normalized to fish
controlled toxicokinetic studies. Common types include body weight.
rate constant-based models that describe chemical move-
ments within a system using first-order rate constants and
clearance-volume models that describe these fluxes using Clearance Concepts
first-order clearance constants. Physiologically based tox- Xenobiotic chemicals taken up by fish may be eliminated by
icokinetic (PBTK) models are based on the premise that several pathways, including biotransformation and excretion
chemical uptake and disposition can be described from in bile, urine, and respiratory water. In some cases, elimina-
anatomical, physiological, and biochemical attributes of the tion rates are extremely slow, and chemicals are retained for
exposed organism and physicochemical characteristics of days, weeks, or even months after fish are transferred to a
the chemical. The compartments in a PBTK model corre- clean environment (Niimi and Oliver, 1983). Alternatively,
spond to actual tissues and organs, and chemical transport elimination pathways may clear >99% of an absorbed dose
within the animal is defined by blood flow relationships. within a few hours. In either case, elimination may be
In principle, it is possible to develop a PBTK model in the characterized by an elimination clearance (CL; L/h). The
absence of measured toxicokinetic data. whole-body clearance (CLwb) is defined as the rate of chemi-
Here, we retain these distinctions, primarily as an aid cal elimination (dX/dt) from the body divided by the chemi-
to describing modeling concepts and equations. In prac- cal concentration in a reference region (here assumed to be
tice, however, these distinctions have become increasingly blood plasma) that is taken to represent the body (CP; mg/L):
blurred. Thus, many recent PBTK models include parame-
ters determined by fitting modeled simulations to measured CLWB = ( dX /dt ) /CP (2.6)
kinetic data. Similarly, some older empirical models have
been amended to include a substantial amount of physio- Normalization to body size (weight or surface area) may
logical information, becoming in the process simple PBTK also be appropriate, particularly when averaging values
models (see Bioaccumulation Assessment and Modeling). from fish of different sizes (Hendriks et al., 2001). The
Finally, there has been a proliferation of models that include CLWB is the sum of the individual clearances for all relevant
compartments corresponding to specific tissues and organs; elimination pathways; for example, the chemical may be
however, most or all intercompartmental rate constants are simultaneously metabolized in the liver and excreted by the
obtained by a process of data fitting. Such models share kidney and at the gills:
attributes of traditional clearance-volume models while
possessing traits more commonly associated with PBTK CLWB = CLH + CLR + CLB (2.7)
models. Models of this type are described in a section on
Semi-physiological Modeling Approaches. where the subscripts refer to hepatic (H), renal (R), and
Most toxicokinetic models consist of one or more com- branchial (B) clearance.
partments, each representing tissues and organs with similar
kinetic properties. Differential equations are then developed Hepatic Clearance
to describe the rate of change of the mass of chemical in each The CLH is limited by the rate of blood flow to the liver (Q L;
compartment (dX/dt; mg/h) as the difference between com- L/h). If blood perfusing the liver is completely cleared of
peting rates of input and output. A listing of commercial soft- chemical, then CLH is equal to Q L. However, CLH may be
ware used to develop and run toxicokinetic models is provided less than Q L if a chemical is poorly metabolized or if it is
on the website for Pharmacokinetics and Pharmacodynamic reversibly bound to plasma proteins, limiting the unbound
Resources (Bourne, 2021). A detailed discussion of software fraction that is available to biotransformation enzymes. A
tools used to perform PBTK modeling was given by Hack physiologically based model of hepatic clearance may be
et al. (2020). It may be noted, however, that while commercial developed by assuming that the liver is a well-mixed com-
modeling software has been provided by numerous vendors, partment (Rowland et al., 1973; Wilkinson and Shand,
there is a trend toward increased use of open source modeling 1975). Chemical concentrations in afferent blood entering
tools. In comparison with proprietary software, open source the liver and venous blood draining the liver are denoted
tools provide greater transparency and may make it easier to as CAFF (mg/L) and CVL (mg/L), respectively. Here the
replicate of other groups’ modeled findings. term −dX/dt is the rate of chemical elimination by the liver
Toxicokinetics in Fishes 21

(the minus sign indicates that chemical is being lost from rate of elimination becomes first order with respect to CL,U
the system). For this well-mixed liver model, the rate of and by extension f U,B CVL:
elimination is:
− dX /dt = (VMAX /K M ) CL,U = (VMAX /K M ) fU,BCVL (2.14)
− dX /dt = QL (CAFF − CVL ) (2.8)
Given values for CLINT,U, f U,B, and Q L, questions can be
If the right-hand side of this equation is multiplied by CAFF/ answered about how CLH, E, and F (oral bioavailabil-
CAFF one obtains: ity) will change if there is a change in hepatic blood flow,
plasma protein binding, or hepatic biotransformation. The
− dX /dt = QL (CAFF − CVL ) CAFF /CAFF (2.9) useful working equations for this model are:

The ratio (CAFF − CVL)/CAFF is termed the hepatic extraction E = fU,BCLINT,U (QL + fU,BCLINT,U ) (2.15)
ratio (E; unitless). Substituting, the rate of elimination can
be written as:
CLH = QL fU,BCLINT,U (QL + fU,BCLINT,U ) (2.16)
− dX /dt = QL ECAFF (2.10)

Because (−dX/dt)/CAFF is equal to CLH, it is apparent that The letter F is commonly used to represent the fraction of
CLH = Q L E. an oral dose that enters the systemic circulation and has
The CLH is controlled by both Q L and the capacity of two contributing parts: ED, which represents the fraction of
hepatic biotransformation enzymes. It is useful, therefore, the dose that is absorbed from the GIT into portal blood
to separate these two influences. This can be accomplished (abbreviated FAB by Kleinow et al., 2008), and FFP, which
by defining the intrinsic clearance (CLINT; L/h). The CLINT represents the fraction of an absorbed dose that escapes
is the rate of chemical elimination divided by the chemical elimination by first-pass clearance in the liver (see also
concentration in liver tissue (CL; mg/L); that is, the concen- The Gastrointestinal Tract, Gastrointestinal Absorption
tration in contact with biotransformation enzymes: of Xenobiotics). The overall bioavailability of an orally
administered chemical is the product of these two fractions:
− dX /dt = CLHCAFF = CLINTCL (2.11)
F = ED FFP (2.17)
and
FFP can also be calculated from CL INT, f U, and Q L using the
CLINT = CLHCAFF /CL = CLH / (1 − E ) (2.12) following relationship:

The rate of chemical elimination by the liver is therefore FFP = 1 − E = QL / (QL + fU,BCLINT,U ) (2.18)
proportional via the hepatic clearance to the chemical con-
centration in blood entering the liver or via the intrinsic The independent variables in Equations 2.15, 2.16, and 2.18
hepatic clearance to the chemical concentration in liver are CLINT,U, f U,B, and Q L. The dependent variables are E,
tissue. CLH, and FFP. The relationships among these independent
A more general model for hepatic clearance that accounts and dependent variables are simplified when the value of E
for the effect of chemical binding in blood may be written is small (<0.25) or large (>0.75); for example, for a large E,
by defining a new reference concentration, the unbound CLINT,U >> Q L, and Equation 2.16 becomes CLH = Q L. For
chemical concentration in the liver (CL,U; mg/L), and a new some toxicants the value of E lies between 0.25 and 0.75,
clearance parameter, the free (unbound) intrinsic clearance and the full equations for E and CLH must be used. In such
(CLINT,U; L/h). From the well-mixed tissue assumption, CL,U cases, there are no direct proportionalities: CL INT,U, f U, and
equals the unbound concentration in venous blood drain- Q L all determine E, CLH, and FFP, and a change in any one
ing the liver (CVL,U), which is equal to the product term f U,B of the former variables produces a less than proportional
CV,L, where f U,B (unitless) is the unbound fraction of chemi- change in the latter.
cal in blood. Accounting for effect of blood binding, the
rate of elimination may therefore be written as: Renal Clearance
The kidney clears blood of xenobiotic chemicals by
− dX /dt = CLINT,UCL,U = CLINT,U fU,BCVL (2.13) filtration and extraction. Filtration involves the passage
of plasma water across the glomerular capillary mem-
The CLINT,U represents the activity of biotransformation brane. Chemicals dissolved in this water are transported
enzymes toward the chemical under first-order conditions across the membrane and end up in the pre-urine. When
and can be understood in terms of the Michaelis–Menten protein binding occurs, protein-bound chemicals remain
relationship: VMAX/(KM + CL,U). When CL,U is low relative to with the protein in plasma. The plasma concentration of
KM (<10%), the relationship simplifies to VMAX/KM, and the unbound chemical remains constant during filtration,
22 Toxicology of Fishes

as both water and unbound chemical are removed by (PMW), which would be expected to correlate positively with
the filtration process. The equilibrium between bound chemical hydrophobicity:
and unbound chemical is therefore not disturbed; conse-
quently, the maximum possible clearance via filtration is kD = DPMW /h (2.22)
the GFR multiplied by the fraction of chemical that is
unbound in plasma. The resulting permeability coefficient is identical to that
The extraction mechanism only applies when a xenobi- given previously in Equation 2.1. Alternatively, if chemi-
otic is a substrate for an active tubular secretory pathway. cal diffusion in the aqueous phase of the membrane lim-
Unbound chemical contained in post-glomerular and renal its flux, Equation 2.21 is appropriate (see Physiologically
portal blood is transported across the proximal tubule into based Toxicokinetic Models, Route of Exposure, Branchial
pre-urine. As in the liver, this may be a high E or low E Uptake). In either case, the value of D applies to the phase
process. The same extraction model described for the liver that constitutes the principal barrier to diffusive flux.
can be used for active tubular secretion. In this case, CL INT Chemical diffusivity in aqueous solution may be calculated
is the activity of the secretory mechanism. from equations given in standard physical chemistry texts
Total clearance by the kidney reflects the net result of and is expected to decline with increasing molecular volume.
filtration, active secretion, and passive reabsorption: If the permeability of the gill epithelium to a chemical
is sufficiently high, branchial uptake may be limited by
CLR = filtration clearance + secretion clearance − reabsorption the rate of blood flow through the gill lamellae. Chemical
(2.19) binding to plasma proteins or to cells in the blood would
increase the capacity of blood to carry chemical away from
Passive reabsorption is expected to be minimal in freshwa- the gills. The uptake clearance when blood flow is the rate-
ter fish because the kidney does not reabsorb much water limiting factor may be expressed as:
from urine and thereby concentrate chemicals contained
therein. In marine fish, the kidney conserves water by reab- CLB = QC PBW (2.23)
sorption and a greater potential exists for chemical reab-
sorption from urine. where QC (L/h) is the total cardiac output, and PBW is an
equilibrium blood–water partition coefficient.
Branchial Clearance Because the value of PBW can be very large, it is pos-
Fish gills are an important site for xenobiotic uptake and sible for water flow across the absorbing surface of the gills
elimination, and both processes can be described using to control the rate of chemical uptake. In this case, CLB is
clearance concepts. Unlike hepatic and renal clearance, equal to the effective respiratory volume (Q W; L/h), which
however, the mathematical description of branchial clear- is the flow rate of inspired water that exchanges with blood:
ance must be bidirectional to reflect the fact that chemical
flux occurs in both directions. Depending on the concentra- CLB = QW (2.24)
tion gradient across the gills, fish may clear chemical from
inspired water or from blood flowing through the gills. The To a first approximation, the potential rate limitations on
principal limitations on chemical uptake at fish gills were branchial flux can be treated as if they were resistances in
identified by Hayton and Barron (1990). If the permeability series in an electrical circuit (Hayton and Barron, 1990):
of the gill epithelium to a chemical is low, diffusion can
limit exchange, and branchial clearance (CLB; L/h) is con- CLB = 1/ ( h /DPMW A + 1/QC PBW + 1/QW ) (2.25)
trolled by the product of a gill permeability coefficient (k D;
m/h) and the surface area for diffusion (A; m2): A more complex model based on the counter-current struc-
ture of fish gills (Erickson and McKim, 1990a, 1990b) is
CLB = kD A (2.20) described below in the section on physiologically based
toxicokinetic models; however, in the case where one resis-
The permeability coefficient is proportional to chemical tance is the principal determinant of chemical flux (by
diffusivity in the gill epithelium (D; m2/h) and inversely virtue of being much greater than the other two), the two
related to the effective thickness of the diffusion barrier models reduce to a common description.
(h; m):
Compartmental Models for Fish
kD = D /h (2.21)
The use of compartmental models to characterize the kinet-
To obtain units of L/h, the CL B calculated by Equation 2.20 ics of absorption, distribution, and elimination of exogenous
must be multiplied by 1,000. and endogenous substances by a variety of species is well
If diffusive flux is limited by chemical diffusion within established (e.g., Derendorf and Schmidt, 2020; Gibaldi
nonaqueous portions of the gill epithelium, it is appropri- and Perrier, 1982; Wagner, 1993). Riggs (1963) defined the
ate to incorporate a membrane–water partition coefficient term compartment as follows:
Toxicokinetics in Fishes 23

If a substance, S, is present in a biological system in sev- Volume of Distribution

eral distinguishable forms or locations, and if S passes The size of a compartment is characterized by an apparent
from one form or location to another form or location at
volume of distribution (V) that has units of volume (e.g., L)
a measurable rate, then each form or location constitutes a
separate compartment for S. or volume normalized to body size (weight or surface area;
L/kg or L/m2). The apparent volume of distribution of the
In the context of toxicokinetics, a xenobiotic and its metab- central compartment (compartment 1 in Figure 2.5B) is
olites would be forms of a substance S, and a location would the amount of chemical in the compartment divided by the
be one or more tissues in which the forms had similar kinetic chemical concentration in a reference region. The reference
behavior. As an example, a tank of exposure water might be region is the fluid or tissue in which the chemical concen-
a compartment from which chemical absorption occurs, the tration is measured, commonly blood or plasma. In the case
blood and highly perfused tissues might constitute a second of plasma, for example, the value of V1 would represent the
compartment, the poorly perfused tissues would be a third volume of plasma that would be required to account for all
compartment, and a metabolite might require specification chemical in the central compartment. In studies with small
of a fourth compartment (Figure 2.5A). fish, the exposure water is commonly used as the refer-
Compartments are assumed to behave like a tank that ence region and the value of V1 represents the volume of
contains a well-mixed fluid; that is, chemical that enters a exposure water that would be required to account for all
compartment is assumed to distribute instantaneously and chemical in the central compartment under equilibrium
linearly, such that the concentration is everywhere propor- conditions, absent clearance from the central compartment.
tional to the amount of chemical in the compartment. Body Under these conditions, the concentration in the exposure
compartments are usually arranged so chemical enters and water equals the concentration of unbound chemical in
exits from only one of the compartments, the so-called plasma water.
central compartment. With multiple-compartment models, The apparent volumes of distribution of the peripheral
the added peripheral compartments are usually attached compartments are based on the same reference region as
to the central compartment so that chemical exchanges that of the central compartment, and the summed volume
between the central and each peripheral compartment; this of all compartments is the apparent steady-state volume
configuration is termed mammillary, as opposed to the of distribution (VSS). The magnitude of the VSS is deter-
catenary configuration, which refers to a chain-like series mined by the chemical sorptive capacity of the reference
of compartments. Typical mammillary configurations are region relative to that of other tissues and fluids. The frac-
shown in Figure 2.5B. Some considerations in the selec- tional water content of most fish tissues is about 65%–80%
tion of the number of compartments include the purpose (Bertelsen et al., 1998). If plasma is used as the reference
of the model, the number of sites from which samples are region, a VSS of about 0.65–0.8 L plasma/kg would be
taken for determination of parent chemical and metabolite expected for a chemical that distributes only into body
concentrations, the number and spacing of individual sam- water and does not bind to plasma proteins. Alternatively,
pling times, and attributes of the resulting dataset including if a chemical is bound extensively to plasma proteins and
apparent mono- or multi-exponential behavior. has relatively little affinity for other tissues, the VSS would

FIGURE 2.5 Examples of compartmental models. (A) A model appropriate for xenobiotic absorbed from the gastrointestinal tract
(GIT) and eliminated by formation of a metabolite. Compartment 1, GIT; compartments 2 and 3, the body lumped into rapidly and
slowly equilibrating tissues; compartment 4, the metabolite. Arrows represent first-order kinetic processes. (B) Mammillary models
that represent the body as one, two, or three well-mixed compartments. The depicted mammillary models indicate that a chemical
taken up by the animal (e.g., by bolus injection) can be eliminated from the body. Such models are termed open models.
24 Toxicology of Fishes

be less than 0.65 L plasma/kg. Conversely, if a chemical has plot gives an estimate of C0 that can be used to calculate a
high affinity for one or more tissues (e.g., high lipid solubil- value for V:
ity resulting in high concentrations in adipose fat), the VSS
could be substantially greater than 1 L plasma/kg. Reported V = dose/C0 (2.30)
VSS values for fish determined using plasma as the refer-
ence region range from 0.08 L plasma/kg for the urinary Ideally, the number of plasma samples should be three times
tract antibiotic nitrofurantoin in channel catfish (Stehly and the number of parameters to be estimated (in this case,
Plakas, 1993) to 6.1 L plasma/kg for the hydrophobic ste- six samples to estimate the two parameters CLWB and V).
roid methyltestosterone in rainbow trout (Vick and Hayton These samples should be uniformly spaced and taken over
2001). Reported VSS values determined using the exposure a time span at least three times the t1/2.
water as the reference region range from 0.55 L water/kg The value of a TK study performed in this manner is dif-
for aminoantipyrine in goldfish (Kaka and Hayton, 1978) ficult to overstate. The resulting kEL estimate is equivalent to
to 19,000 L water/kg for di-2-ethylhexyphthalate in sheeps- that obtained from an aqueous uptake–depuration study pro-
head minnows (Karara and Hayton, 1984). tocol, where measured chemical concentrations are expressed
on a whole-fish basis (e.g., OECD Test Guideline 305; OECD,
One-Compartment Empirical Model 2012). However, because all data are obtained from a single
animal (eliminating the confounding effect of individual vari-
Intravascular Administration
ability), it is possible to obtain estimates of CLWB and V that,
The simplest empirical model assumes that the fish body when evaluated across multiple animals, can be used to char-
behaves like a single, well-mixed compartment. In this acterize the population variance in these parameters.
section, the one-compartment model is developed for a
xenobiotic that is administered by intravascular injection. Infusion Dose
Intravascular administration is usually accomplished by In some cases, it may be advantageous to administer the
injecting a solution of the chemical via an indwelling cath- dose at a constant rate (R0; mg/h). This approach avoids the
eter placed in the dorsal aorta. The dose may be admin- high transient plasma concentration associated with a bolus
istered as a rapid injection (bolus) or as a constant rate dose and provides a larger volume of solvent to dissolve the
infusion using an infusion pump. After the dose is admin- chemical. The plasma concentration will increase expo-
istered, samples of plasma are removed at various times to nentially during the infusion, followed by an exponential
measure the chemical concentration (CP; mg/L). decline after the infusion is stopped. During the infusion,
the predicted plasma concentration is:
Bolus Dose
For a chemical administered as a bolus dose, its rate of (
CP = ( R0 /CLWB ) 1 − e − kEL t ) (2.31)
elimination (dX/dt) is:
After three to four half-lives, the exponential approaches
− dX /dt = CLWBCP (2.26) a value of zero, and a steady-state condition is achieved
in which the rate of infusion equals the rate of chemical
Division of both sides by V, followed by integration, yields elimination.
an equation that predicts the time course of CP: Plasma samples are generally obtained after the infu-
sion has been stopped using the same cannula employed to
CP = C0 e −(CLWB / V )t (2.27) infuse the chemical. The value of k EL is obtained from the
slope of a semi-log plot of the post-infusion plasma data
where t (h) is time and C0 (mg/L) is the plasma concentra- (Equation 2.31). If the infusion is sufficient to achieve a
tion at t = 0 (i.e., the dose/V). This equation predicts that the steady-state condition, CLWB can be calculated from:
plasma concentration will decline exponentially, with the
result that a graph of log CP vs. time gives a straight line CLWB = R0 /CSS (2.32)
(Figure 2.6A). From the slope of this line, the ratio CLWB/V
may be obtained, which is commonly referred to as the where CSS (mg/L) is the steady-state plasma concentration,
elimination rate constant (k EL; 1/h): estimated from the y-axis intercept of the semi-log plot.
The volume of distribution (V) can then be calculated as:
kEL = −2.3 ⋅ slope = CLWB /V (2.28)
V = CLWB /kEL (2.33)
The elimination half-life (t1/2; h) is calculated from k EL as:
If the infusion was stopped prior to steady state, k EL may
t1/ 2 = 0.693/kEL = 0.693V /CLWB (2.29) still be estimated from the slope of the semi-log plot, but
estimation of CLWB and V is less straightforward because
This relationship shows that a change in t1/2 may result from the amount of chemical in the fish at the end of the infu-
a change in CLWB or V. The y-axis intercept of the semi-log sion is unknown. Equation 2.31 may be rearranged to give
Toxicokinetics in Fishes 25

the following expression for estimation of CLWB, which can metabolite and static exposure models were given by
then be used along with k EL to estimate V: Kleinow et al. (2008).

(
CLWB = ( R 0 /CEND ) 1 − e − kEL tINF ) (2.34) Oral Administration
A single oral dose may be administered to a fish that has
where CEND (mg/L) is the concentration in plasma at the end been prepared with an indwelling vascular catheter. If the
of the infusion and t INF (h) is the duration of the infusion. absorption kinetics are first order and monoexponential, the
rate of appearance of chemical in the fish is:
Waterborne Exposure
Kinetic studies with fish may be conducted by exposing dX /dt = k AB X G − CLWBCP (2.38)
small fish or juveniles of larger species to a chemical in
water. Typically, the exposure is initiated with a fixed num- where kAB (1/h) is the absorption rate constant, and XG (mg)
ber of fish, and at each sampling time a subset of animals is the amount of chemical remaining in the GIT. From this
is collected to determine whole-body chemical concentra- equation, it is possible to derive the following relationship:
tions. The exposure may be conducted using a flow-through
system that maintains a constant concentration of chemical ( )
CP = k AB F ⋅ dose e −(CLWB / V )t − e − kABt /V ( k AB − (CLWB /V ))
in the exposure water. Alternatively, fish may be exposed in (2.39)
a static system, which may or may not be renewed during
the progress of a test. The aqueous concentration of chemi- where F (unitless) is the fraction of the dose that reaches
cal in a static exposure system may decline over time due the systemic circulation of the fish (i.e., oral bioavailability).
to absorption by the test organisms. Additional losses may When the dietary exposure is continuous, the rate of
occur due to adsorption to the test container, volatilization, change of the amount of chemical in the fish is:
photodegradation, and microbial biotransformation.
The rate of chemical accumulation in fish dosed in a flow- dX /dt = FRIN − CLWBCP (2.40)
through exposure is proportional to the difference between the
concentration of chemical in the exposure water (CW; mg/L) where RIN (mg/h) is the rate of chemical ingestion. In this
and the concentration unbound in plasma water (CP,U; mg/L): case, the mass of chemical exponentially approaches a con-
stant or steady-state value:
dX /dt = ρ (Cw − CP,U ) (2.35)
(
X SS = ( F RINV / CLWB ) 1 − e − kEL t ) (2.41)
The CW is constant during the exposure, and the CP,U can be
replaced with X/V, where V is the apparent volume of dis-
tribution of the chemical referenced to the exposure water. Two-Compartment Empirical Model
The units of V and ρ are usually normalized to body weight; A one-compartment model assumes that a chemical distrib-
for example, if X had units of mg/kg, and CW and CP,U had utes instantaneously throughout all tissues of the exposed
units of mg/L, then V would have units of L/kg and ρ would animal. In principle, this is impossible because chemi-
have units of L/h/kg. Integration of Equation 2.35 gives an cal distribution to each tissue is linked to blood perfusion
equation for X as a function of time: rate, and tissue-specific perfusion rates vary considerably.
However, if an internal distribution equilibrium is rapidly
(
X = VCW 1 − e − ( ρ / V )t ) (2.36) established, errors associated with application of a one-
compartment modeling assumption are small and can be
This equation is fit to experimentally determined values of ignored. Alternatively, if the time required for establish-
X at various times to determine values for ρ and V. The ment of an internal equilibrium is a significant fraction
elimination half-life can then be calculated as: of total sampling time, this will impact the shape of the
plasma concentration–time profile and a higher order com-
t1/ 2 = ln 2V /ρ = 0.693V /ρ (2.37) partment model may be required. A common example is
when log-transformed plasma concentrations from an intra-
Equation 2.36 predicts that X will exponentially approach a vascular bolus dosing study decrease in a biphasic manner
limiting value equal to V CW. In this case, the value of V is (Figure 2.6B). The early decline in plasma concentration
equivalent to the fish BCF, as defined earlier in the chapter. (α phase) is due primarily to redistribution of the injected
When a metabolite of the test chemical is formed dur- chemical, while the later elimination or β phase reflects
ing a flow-through exposure, the model given above may rate limitations on distribution and elimination. A two-
be extended to permit simultaneous characterization of its compartment model for intravascular administration of a
toxicokinetics. A second modification of the flow-through bolus dose is described in the following section. Space does
model that accounts for chemical accumulation by fish not permit the presentation of other multiple-compartment
can be developed to describe uptake in a static exposure models; however, the principles illustrated by this relatively
scenario. Derivations and equations for the flow-through simple model apply generally to more complex models.
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