Endocrine Disrupters: Hazard Testing and Assessment Methods

CHAPTER 1

Ecotoxicity Test Methods for Endocrine-Disrupting Chemicals

AN INTRODUCTION

PETER MATTHIESSEN

Consultant Ecotoxicologist, Old School House, Backbarrow, Cumbria, United Kingdom

1.1 BACKGROUND

1.2 REGULATORY CONCERNS

1.3 INVERTEBRATES

1.4 VERTEBRATES

1.5 TESTING SCHEMES FOR EDCS

REFERENCE

1.1 BACKGROUND

The issue of endocrine disruption has been something of a cause célèbre since it was first identified as an issue about 25 years ago. Few scientists had previously suspected that certain synthetic chemicals might be able to interfere with the workings of the endocrine system at low concentrations. However, in the mid-1990s, Theo Colborn and others [1] brought this subject to the attention of a wide audience when it became clear that many different wildlife species were experiencing effects that were attributable to damaged hormone signaling. Since then, endocrine-disrupting chemicals (EDCs) have come to be treated as a special case rather like carcinogens, so that the mere possession of endocrine-disrupting (ED) properties can be enough to trigger precautionary regulatory action in some jurisdictions, irrespective of the probable environmental risks involved. In other jurisdictions, the risks of EDCs are being evaluated in similar ways to non-EDCs, but these chemicals are the subject of much concern irrespective of the regulatory stance being taken.

It is therefore extremely important that EDCs should be unambiguously identified and their hazards accurately measured. This book represents one of the first attempts to describe and critically evaluate the methods that have been developed for studying the effects of EDCs on mammalian and nonmammalian wildlife in the laboratory.

The chapters in this volume are aimed at scientists and chemical companies that wish to investigate the ecotoxicological properties of EDCs using cutting-edge and (where relevant) internationally agreed techniques and at chemical regulatory authorities that seek to protect the environment from the adverse effects of EDCs through the use of rigorous hazard evaluation programs that employ scientifically sound methods. This is quite an ambitious aim, given that some standardized assays that use wildlife species to assess the toxicity of EDCs are still in development, and there remain whole classes of EDCs for which assays have not yet been standardized or even designed. However, despite these gaps and limitations, it is hoped that the book will provide useful guidance until a more comprehensive array of test methods becomes available.

Chapter 2 by Dick Vethaak and Juliette Legler describes why some EDCs became recognized as an environmental problem in the 1980s, and summarizes the large amount of research that has since discovered many features of this disparate group of chemicals. The chapter brings us up to date about the chemicals that have been found to have ED properties and surveys effects observed in the field and in the laboratory. This review makes it clear that, until recently, chemical risk assessment schemes had failed to prevent some EDCs from entering the environment and causing sometimes severe damage to certain ecosystems. Thus, the chapter sets the scene for the ones that follow.

1.2 REGULATORY CONCERNS

In Chapter 3, Hans-Christian Stolzenberg and coauthors explain why regulatory authorities have become concerned about EDCs and describe in detail how several authorities (especially Japan, the United States, and the European Union) have responded with new programs and regulations designed to identify these chemicals and assess their hazards and associated environmental risks. It became apparent from the early 1990s that existing internationally standardized ecotoxicity assays were largely insensitive to EDCs. As a result of this understanding, member countries of the Organisation for Economic Cooperation and Development (OECD) initiated a program to develop and validate new testing methods. The chapter indicates how these methods are likely to be used in chemical regulatory activities in several jurisdictions, although full details are still being developed and many other jurisdictions have yet to act.

1.3 INVERTEBRATES

The regulatory background is then followed by three chapters that describe testing methods with certain invertebrate groups (insects, crustaceans and molluscs) and five chapters covering methods using vertebrates (fish, amphibians, reptiles, birds, and mammals). At present, regulatory requirements for the testing of suspected EDCs are restricted to vertebrates alone, but this is due mainly to the fact that invertebrate endocrine systems are relatively poorly understood, not because endocrine disruption is not an issue in these phyla.

Chapter 4 by Lennart Weltje concerns testing methods in insects. The endocrine systems of insects, of all the invertebrates, are the best understood due to their overwhelming importance as pests, a fact that has led to the development of pesticides specifically intended to cause endocrine disruption in this group. The chapter not only describes in vivo testing methods covering key endocrine-mediated processes such as growth and reproduction but also a range of in silico and in vitro mechanistic techniques that show promise for the understanding of certain modes of action. This aspect is important given that generally agreed definitions of EDCs require that an apical effect in vivo needs to be plausibly linked to an ED mechanism.

Crustacean test methods are covered by Magnus Breitholtz in Chapter 5. This invertebrate group belonging to the arthropods shares many endocrine similarities with insects and is also economically important, but in this case as a food source. The chapter goes into the endocrinology of various crustacean taxa in considerable detail, and it is clear that a range of mechanistic assays will be developed in the near future. At present, however, available methods include several with in vivo apical endpoints (especially reproductive success) which do not in themselves reveal modes of action. The chapter also includes consideration of some newer techniques including toxicogenomic methods which show promise for the future.

In Chapter 6, Patricia D. McClellan-Green addresses possible endocrine testing methods involving molluscs. In comparison with the invertebrate groups discussed in Chapters 4 and 5, less is known about the endocrine systems in this phylum, although there is good evidence that endocrine disruption can be caused by a variety of substances, some of which (e.g., organotins) are much more potent than in other phyla. For this reason, in vitro techniques are still in their infancy, and we are not yet in a position to standardize mechanistic in vivo molluscan screens, although some biomarkers (e.g., vitellogenin and imposex induction) show promise. Perhaps surprisingly, no mollusc-based toxicity tests of any kind have yet been internationally standardized, but an OECD project led by the United Kingdom, Germany, France, and Denmark is now developing partial and full life cycle apical tests with gastropods that will be useful for the assessment of both EDCs and non-EDCs.

1.4 VERTEBRATES

Throughout the evolution of the vertebrates, there has been a high degree of conservation of their endocrine systems, with many hormones and receptors being identical or very similar across the vertebrate groups. However, despite these similarities, tests with sensitivity to EDCs are needed for most of the major vertebrate groups because of differences in exposure, metabolic competence, and downstream hormonal interactions. Chapters 7 to 11 address methods involving all vertebrate groups from fish to mammals.

Peter Matthiessen discusses toxicity tests for EDCs using fish in Chapter 7. Some of the earliest widespread effects of EDCs observed in the field involved this group of vertebrates (feminization of male fish exposed to estrogens), and considerable progress has been made in developing and standardizing fish-based test methods with sensitivity not only to (anti)estrogens but also to (anti)androgens and steroidogenesis disrupters. Three different fish-based screening assays have now been published by OECD that are able to provide mechanistic information about the potential of a chemical to interfere with different aspects of the steroid hormone system in vivo, and one of these also provides some apical information about possible effects on reproductive success. A partial life cycle test (the Fish Sexual Development Test) has also been published, providing mechanistic and apical information concerning possible impacts on phenotypic sex ratio. Another partial life cycle test covering the reproductive phase of the life cycle is in development, as are full and multiple life cycle tests. When these are complete, a comprehensive suite of tests for EDCs using fish will be in place.

Chapter 8 by Daniel B. Pickford covers testing of EDCs using amphibians. This group is particularly sensitive to thyroid system disrupters, and the chapter goes into detail about the development and standardization of larval-based screens that are responsive to these chemicals. An amphibian in vivo mechanistic screen (the Amphibian Metamorphosis Assay) has now been published by OECD and shows sensitivity to several different types of thyroid interference. The chapter then goes on to describe possible partial and full life cycle testing in this group, although standardization of such higher-tier tests is still ongoing. Research has already shown that the sexual development of some amphibians can be disrupted by exposure to several different types of EDC including estrogens. However, at present there are no plans to standardize full life cycle tests with amphibians due to the difficulty and expense of culturing the currently used species in the laboratory.

Chapter 9 by Satomi Kohno and Louis J. Guillette Jr. discusses reptiles, for which no internationally standardized tests for EDCs are currently being considered. Reptiles have not traditionally been used in ecotoxicity tests, but several members of this group possess an interesting physiological trait that can be exploited to study endocrine disrupters. In brief, the sex of many young reptiles (e.g., turtles and alligators) is determined by the temperature at which the eggs are incubated, and this process can be subverted by certain EDCs. For example, in crocodilians, lower temperatures produce females alone, intermediate temperatures produce both sexes, and higher temperatures produce males alone; administration of low estrogen doses at male-producing temperatures leads to the induction of females. The chapter describes both the use of estrogen receptor transactivation assays that employ receptors derived from reptiles to measure estrogenic activity in vitro and in vivo assays that exploit interference with sex determination in species such as the American alligator. The drawback of the in vivo methods is that reptile eggs are generally produced only seasonally and are available commercially in relatively small numbers, which may explain why there has been no attempt at standardization to date.

Testing for EDCs using birds is considered in Chapter 10 by Paul D. Jones, Markus Hecker, Steve Wiseman, and John P. Giesy. Life cycle characteristics such as egg laying may make birds particularly sensitive to some EDCs, although the avian endocrine system has many similarities with those of other higher vertebrates. However, although the mechanism of sex determination is not fully understood, it is known that estradiol is the sex-differentiating hormone in birds (testosterone plays this role in mammals), so administration of estrogens to birds during development may cause more profound changes than in mammals. This chapter covers in vitro techniques with avian cell lines and in vivo methods using both embryos and adult birds. Dosing methods comprise egg injection and feeding, and studies can include both partial and full life cycles. An avian partial life cycle reproduction test was published by OECD many years ago, but an avian two-generation test is currently being validated by that organization, and aspects of the test are considered in this chapter.

Chapter 11 by M. Sue Marty covers methods for studying endocrine disruption in mammals. Due to the importance of mammalian tests for predicting chemical effects in humans, they have been more extensively developed than those with lower vertebrates, although some with particular sensitivity to EDCs were standardized and published only recently. This chapter describes an array of five standardized mammalian tests with rodents that can be used to identify ED activity in vivo and indicates how they can be integrated into a screening program for estrogens, androgens, and thyroid-acting compounds. Consideration is then given to more extended rodent-based assays (the two-generation and extended one-generation tests). which could be used at a higher level of testing in order to reveal a fuller range of possible apical effects. The chapter concludes with a discussion of the relevance of these tests for predicting the effects of EDCs in humans and mammalian wildlife.

1.5 TESTING SCHEMES FOR EDCS

Chapter 12 by Thomas H. Hutchinson, Jenny Odum, and Anne Gourmelon describes the five-level OECD Conceptual Framework (CF) that was developed to guide the standardization of screens and tests for EDCs. The CF levels move from Level 1 (data gathering), through Level 2 (in vitro assays), to Levels 3 to 5 covering in vivo assays of increasing complexity. Standardized in vivo assays for both mammals and nonmammals are now available at each of Levels 3 to 5, but it is clear that these levels are not necessarily to be followed in a linear testing scheme. The authors explain why a weight-of-evidence approach is required to assess whether substances have ED properties, and if so, whether those properties are able to cause adverse apical effects. They use two case studies to illustrate how weight-of-evidence assessments might work using the assays in the CF and conclude that the framework provides a logical process for critically evaluating studies that show either positive or negative results. Overall, the assays in the CF are found to provide useful data for identifying EDCs and measuring the type and magnitude of their effects in mammals and other wildlife.

In the final chapter (Chapter 13), Peter Matthiessen continues the discussion of possible testing approaches for EDCs and reiterates the need for weight-of-evidence assessments. Remaining gaps in the testing tool box are identified, but it is clear that a reasonably complete set of assays for so-called EATS modalities (i.e., EDCs with estrogen/androgen/thyroid/steroidogenic action) will be available within the next few years. However, current standardized testing procedures do not account for several new types of endocrine disruption that will need consideration in due course. The chapter then discusses possible integrated procedures for testing EDCs and presents a draft scheme for assessing the hazards posed by chemicals to fish recently discussed at an OECD workshop. This scheme covers all chemicals, not just EDCs, and attempts to integrate the new fish-based tests for EDCs into a wider framework.

To summarize this book, it is clear that the development and standardization of ecotoxicity tests for EDCs remains a work in progress, but great strides have been made during the first decade of the twenty-first century. Enough validated assays are now in place (or will shortly be agreed) to permit the routine operation of hazard assessment schemes for suspected EDCs, providing that the limitations of these assays are borne in mind.

REFERENCE

1. Colborn, T., Dumanoski, D., Myers, J. P. (1996). Our Stolen Future. Penguin Books, New York. 306 pp.

CHAPTER 2

Endocrine Disruption in Wildlife

BACKGROUND, EFFECTS, AND IMPLICATIONS

DICK VETHAAK

Deltares, Marine and Coastal System Unit, Delft, The Netherlands and VU University Amsterdam, Institute for Environmental Studies (IVM), Amsterdam, The Netherlands

JULIETTE LEGLER

VU University Amsterdam, Institute for Environmental Studies (IVM), Amsterdam, The Netherlands

2.1 BACKGROUND TO ENDOCRINE DISRUPTION

2.1.1 Introduction

2.1.2 Brief Account of the History of Endocrine Disruption

2.1.3 What Are Endocrine-Disrupting Chemicals?

2.1.4 Vertebrate Endocrine System

2.1.5 Invertebrate Endocrine System

2.1.6 Mechanisms of Endocrine Disruption

2.1.7 Endocrine Disrupters in the Environment

2.2 EFFECTS OF EDCS ON WILDLIFE

2.2.1 Mammals

2.2.2 Birds

2.2.3 Reptiles and Amphibians

2.2.4 Fish

2.2.5 Invertebrates

2.3 WEIGHT OF EVIDENCE AND ECOLOGICAL SIGNIFICANCE OF ED EFFECTS

2.4 IMPLICATIONS FOR EFFECT ASSESSMENT AND TOXICITY TESTING

2.5 NEED FOR MORE FIELD STUDIES AND AN INTEGRATED APPROACH

2.6 CONCLUDING POINTS

REFERENCES

2.1 BACKGROUND TO ENDOCRINE DISRUPTION

2.1.1 Introduction

It is now well recognized that endocrine-disrupting chemicals (EDCs) pose a potential risk affecting both wildlife and human health on a global scale [1,2]. EDCs are generally defined as substances in our environment, food, and consumer products that can disrupt hormonal balance and result in adverse health effects. An EDC elicits adverse health effects primarily by its interaction with an endocrine mechanism (endocrine disruption), given the right dose and timing of exposure. In the last decades, field and laboratory studies have shown that some EDCs, including natural hormones, pharmaceuticals, some pesticides, and industrial contaminants, can cause developmental, reproductive, neural, immune, and other problems in a range of wildlife taxa [1,3–11]. Nowadays there is clear evidence that a growing number of environmental chemicals may possess endocrine-disrupting (ED) activity and that ED effects can occur at very low concentrations, concentrations that are similar to current exposure levels. ED effects may occur at much lower doses than other types of toxicants that act through different mechanisms. In addition to having adverse effects on wildlife, there are growing indications for associations of certain persistent organic pollutants (POPs) with ED effects in humans at the relatively low doses typically found in the environment—for example, male testicular dysgenesis syndrome (lower sperm count; increases in hypospadias [urogenital abnormalites in male babies], and cryptorchidism [the absence of one or both testes from the scrotum]) and certain cancers [12–14]. Recent research also links human EDC exposure with obesity, metabolic syndrome, and type 2 diabetes (reviewed by [15–17]). The scope and magnitude of EDC harm to wildlife populations and possibly to humans are becoming increasingly apparent, as our knowledge and understanding increase, and demonstrate the need for prompt policy actions and the need for regulation and testing of EDCs.

In this introduction and background chapter, we provide—at the risk of repeating earlier publications—a general description of the issue of endocrine disruption in the environment with particular reference to wildlife. We do not intend to be complete (which is almost impossible given the rapid development in this field), but instead we focus on new developments, the wildlife–human connection, and implications for field monitoring and effect assessment and toxicity testing.

2.1.2 Brief Account of the History of Endocrine Disruption

The effects of EDCs have been evident since the 1940s [18]. Originally reported in peregrines (Falco peregrinus), around 1946, DDE-induced reproduction failure due to eggshell thinning and broken eggs has been described in a large number of raptor species [19]. This is probably also the first example of endocrine disruption in wild populations, even though a plausible mechanism of action has not been elucidated until recently (reviewed by [20]). Since the appearance of Rachel Carson’s Silent Spring in 1962 [21] and the diethylstilbestrol (DES) story [22], the public has been increasingly concerned about those chemicals that could have harmful effects on wildlife and human health. The potential environmental impacts of synthetic hormones, industrial by-products, pesticides, and other chemicals introduced to the environment led in 1979 to the start of a successful and still-continuing series of conferences on estrogens in the environment organized by the U.S. National Institute of Environmental Health Sciences (NIEHS) [23–25]. In 1991, a group of expert scientists at the Wingspread Conference titled Chemically Induced Alterations in Sexual Development: The Wildlife/Human Connection presented a review of the scientific literature from the 1950s through the 1980s, where they found a wide number of pathologies and other abnormalities in wildlife and humans that seemed to be connected to endocrine system dysfunction. A consensus statement released from that group stated:

Many compounds introduced into the environment by human activity are capable of disrupting the endocrine system of animals, including fish, wildlife, and humans. The consequences of such disruption can be profound because of the crucial role hormones play in controlling development. [26, page 1].

This alarming statement of the potential dangers posed by EDCs provided a major impetus for further studies and the discovery of numerous new cases of adverse effects of EDCs in wildlife and humans worldwide. In 1996, Theo Colborn and coworkers Dianne Dumanoski and John Peterson Myers published Our Stolen Future [27] and introduced the endocrine disruptor hypothesis, in which they pointed out the possible negative reproductive effects of EDCs on humans through a study of the mounting evidence of the effects of EDCs on the wildlife populations. They also recommended the precautionary approach to the regulation of EDCs. This book, written for the popular press (with a foreword by Vice President Al Gore), together with TV documentaries, such as the BBC Horizon documentary Assault on the Male, broadcast in 1993, played a crucial role in raising public awareness and concern about endocrine disruption (as evidenced by numerous news media reports at that time). Apart from prompting many more scientific studies, this media attention motivated many environmental groups to call for the banning or strict regulation of all man-made EDCs, suspected or proven, and ultimately influenced government policy and the development of research and regulation initiatives. Since that time, numerous national and international committees and scientific workshops have evaluated the endocrine disruptor hypothesis and generated consensus statements regarding the hazard from endocrine disruptors, mostly in wildlife but also in humans [5,28–34]. In 2002, the World Health Organization (WHO), the United Nations Environment Programme (UNEP), and the International Labor Organization (ILO) International Programme on Chemical Safety (with input from over 60 independent, international scientific experts) provided an objective global assessment of the current state of the science relative to environmental endocrine disruption in humans, experimental studies, and wildlife species. This assessment focused on the global peer-reviewed scientific literature where the associations between environmental exposures and adverse outcomes have been demonstrated or hypothesized to occur via mechanisms of endocrine disruption [1]. The assessment concluded:

Overall the biological plausibility of possible damage to certain human functions (particularly reproductive and developing systems) from exposure to EDCs seems strong when viewed against the background of known influences of endogenous and exogenous hormones on many of these processes. Furthermore, the evidence of adverse outcomes in wildlife and laboratory animals exposed to EDCs substantiates human concerns. The changes in human health trends in some areas for some outcomes are also sufficient to warrant concern and make this area a high research priority, but non-EDC mechanisms also need to be explored. [1, chapter 1, page 3]

In 2009, the Endocrine Society released a scientific statement outlining mechanisms and effects of endocrine disruptors on male and female reproduction, breast development and cancer, prostate cancer, neuroendocrinology, thyroid, metabolism and obesity, and cardiovascular endocrinology and showing how results from experimental and epidemiological studies converge with human clinical observations to implicate EDCs as a significant concern to public health [2, page 1]. However, the statement noted that it is difficult to show that endocrine disruptors cause human diseases. It recommended that the precautionary principle should be followed.

Besides the fact that our knowledge and understanding of the underlying mechanisms and the scope and magnitude of risks posed by EDCs have significantly grown in the past two decades, the ED issue has also triggered a number of scientific controversies. Controversies include the difficulties of adequately assessing the risks linked to EDC exposure (including developmental and nonthresholds effects for EDCs), the ecological relevance of effects at lower levels of biological organization, and the complexity of the EDC mixtures present in the environment, as well as the potential role of EDCs in non–receptor-mediated pathways [10,35]. These critical issues continue to be a challenge for risk assessment and regulation of EDCs.

It is now generally accepted that EDCs are potentially significant environmental risk factors for wildlife health due to their wide distribution throughout the ecosystem [3,36,37]. As such, EDCs have been proposed by some scientists to constitute a serious potential anthropogenic threat to biodiversity and ecosystems [38,39]. The discoveries that have helped build the environmental endocrine hypothesis have caused a paradigm shift in science and policy by influencing the way we think about chemical risks. In fact, some nations are beginning to take precautionary measures based on the weight of evidence that is mounting from diverse sources [18]. Consequently, research on EDCs continues to be a high-priority area and is expected to play possibly an even larger role than ever before in the coming years, in many scientific fields including monitoring and testing guidelines.

2.1.3 What Are Endocrine-Disrupting Chemicals?

There have been several definitions of EDCs from a mode-of-action perspective, ranging in the broadest sense from an exogenous agent that interferes with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processes [36,40] to, in the most limited sense, chemicals that are estrogenic (specifically, estrogen receptor agonists). Other terms used for EDs are: hormonally active compounds, hormone disruptors, hormone-active agents, and endocrine-active substances. Estrogenic compounds are also termed pseudoestrogens or xeno-estrogens.

A general definition used by the U.S. Environmental Protection Agency Endocrine Disruptors Screening and Testing Advisory Committee describes an endocrine disruptor as an exogenous chemical substance or mixture that alters the structure or function(s) of the endocrine system and causes adverse effects at the level of the organism, its progeny, the populations, or subpopulations of organisms, based on scientific principles, data, weight-of-evidence, and the precautionary principle [37]. From this definition, endocrine disruption implies adverse effects and may be suspected on the basis of in vitro tests but can be proven only in vivo [18,41]. However, relationships between exposure to EDCs and long-term effects on wildlife populations are generally difficult to prove. Evaluating the risk to wildlife is complicated by several factors, such as the mixed exposure experienced by wild populations and the difficulties to discriminate population effects by EDCs from those caused by other environmental pressures [42,43]. The use of a weight-of-evidence (WoE) approach can, in those cases, be very useful in the establishment of the likelihood of a causal relationship between the environmental EDCs and adverse health signs. In fact, the WHO published a WoE framework for assessing whether EDCs cause adverse effects in humans and wildlife in 2002 [1].

The list of proven or suspected EDCs, based on in vitro and in vivo laboratory studies, is now very long and includes diverse natural and synthetic substances. Natural EDCs comprise animal hormones and phyto- and mycoestrogens. Synthetic EDCs include plastics, detergents, pharmaceuticals (e.g., oral contraceptives, androgenic steroids, and lipid-regulating agents), personal care products (such as perfumes, creams), brominated flame retardants (BFRs), herbicides, pesticides, and other industrial chemicals. It also includes chemicals produced as a by-product of industrial processes, such as dioxins, which are suspected of interfering with the endocrine systems of humans and wildlife. The European Union has published a candidate EDC list consisting of 575 chemicals of which 320 substances showed evidence or potential evidence for ED effects. An assessment of the legal status of the substances with evidence or potential evidence of ED effects showed that the majority of them are already subject to a ban or restriction or are addressed under existing European Community legislation, although for reasons not necessarily related to endocrine disruption [44].

2.1.4 Vertebrate Endocrine System

In conjunction with the nervous and immune systems, the endocrine system and its signaling substances (hormones) form the main regulatory mechanism that controls different vital functions in the human or animal body, such as development, reproduction, growth, behaviour and energy balance. The endocrine system is an extremely complex system in which many hormones interact in order to make all the facets of life possible. It consists of a set of glands, such as the hypothalamus, pituitary, thyroid, gonads, and adrenal glands, which produce hormones, such as thyroxine, estrogen, testosterone, and adrenaline. A number of glands that signal each other in sequence are usually referred to as an axis. For example, the neuroendocrine system that regulates reproduction is referred to as the hypothalamic-pituitary-gonad (HPG) axis. While reproduction is largely under the control of reproductive steroids, the production and release of those steroids is under the regulation of a suite of neurotransmitters and hormones that make up the HPG axis [45].

The hormones ensure that molecules, cells, tissues, and organs within an organism function properly, not only with respect to the internal processes but also as regards their interaction with the environment. Hormones influence several essential regulatory, growth, developmental, and homeostatic mechanisms of the organism, such as reproduction, maintenance of normal levels of glucose or ions in the blood, blood pressure, general metabolism, and other muscle or nervous system functions. The balance of the hormones (homeostasis) in the organism is essential in order to prevent functional disorders. For example, sex hormones play a role in all processes relating to reproduction but also perform metabolic functions and are involved in neurological development.

Hormones are transported in the bloodstream either as free molecules or—and this applies to the majority—attached to carrier proteins. Via the bloodstream, hormones reach all living cells, but not all of these cells will react to a particular hormone. Only the so-called target cells have specialized hormone receptors on the cell surface or within the cell (nuclear receptors) that are able to bind specific hormones. This hormone-receptor complex then activates different cell or organ functions. The binding between hormone and receptor is based on steric complementarities, comparable to the key-and-lock principle. The way in which that effect is elicited differs from one type of cell or hormone to another. For example, sex hormones (but also thyroid hormones, glucocorticoids [GCs], and retinoids) bind to nuclear receptors that are located in the cell, forming the hormone receptor complex. Within the nucleus, the hormone receptor complex binds to a specific site on the DNA, the so-called hormone responsive element (HRE), whereupon transcription of one or more genes into messenger RNA takes place. RNA therefore contains the code that is subsequently translated into specific proteins, which, as a result, enables the cell to perform a particular function.

There is much interaction (hormonal cross talk) among systems. Besides sex hormones (estrogens, androgens, and progestogens), various other hormones and growth factors play an important role in reproductive physiology and behavior. In addition, the nervous system exerts a controlling and regulating influence on the reproductive system. Sensory stimuli (including daylight) can stimulate the release of specific neurotransmitters via certain neural networks, and these neurotransmitters subsequently stimulate the release of neurohormones, such as gonadotropin-releasing hormone (GnRH) by the hypothalamus and therefore the pituitary-gonadal axis. Another example is cross talk between the thyroid hormone system (hypothalamic-pituitary-thyroid [HPT] axis) and the sex hormone balance. The thyroid hormone system has a major effect on growth in general and on the formation of specific organs, such as brain and gonads, as well as processes such as metamorphosis in amphibians. In mammals, thyroid hormone level status influences the development of the testes. In frogs, induction of vitellogenin (VTG) by estradiol occurs only if they have first reached a certain level of thyroid hormone. In fish, thyroid hormones most probably play a role in the maturation of the oocytes [32].

Hormonal regulation of biological functions is common to both vertebrates and invertebrates. In general, all vertebrates have similar sex hormone receptors, which have been conserved in evolution. For example, the hormone-binding specificity of estrogen receptors of all vertebrates is virtually identical, and exposure to low concentrations of estradiol or to xeno estrogens leads to estrogen receptor (ER) activation in a wide range of animal species.

2.1.5 Invertebrate Endocrine System

In most invertebrates, hormones (e.g., ecdysteroids and molting hormones) play a similar role as in vertebrates, regulating various biological processes, such as molting of the exoskeleton, growth, reproduction, and development. Although not comprehensively documented, the regulation of these processes by the neuroendocrine system in invertebrates is, therefore, considerably more diverse than that found in vertebrates [46]. The best-characterized invertebrate hormonal system is that of insects, reflecting their economic and ecological significance and especially the need to control insect pests. However, much less is known about other aquatic groups, such as crustaceans and molluscs, and knowledge on the remaining taxa is even more fragmentary. For detailed information the reader is referred to a number of excellent reviews on invertebrate endocrinology [47,48]. Further information on invertebrate endocrine systems can be found in Chapters 4, 5, and 6 of this volume.

2.1.6 Mechanisms of Endocrine Disruption

It is because of the highly complex nature of hormonal systems that there are a large number of points at which disruption can occur. In general, EDCs can affect hormonal systems in several ways: (1) agonistic/antagonistic effect at the receptor level (hormone mimics); (2) disruption of production, transport, metabolism or secretion of natural hormones, including all associated proteins and enzymes; and (3) disruption of production and/or function of hormone receptors [49]. Moreover, EDCs can influence the endocrine system at various points simultaneously, apparently depending on the dose given to the organism. For example, in studies with Atlantic salmon (Salmo salar), 4-nonylphenol can act as an estrogen mimic, as a steroid metabolism disruptor, and by modulating ER levels [50]. Table 2.1 summarizes representative examples of the endocrine actions of various environmental contaminants on wildlife or laboratory species. It can be seen that the range and diversity of ED mechanisms is diverse and ubiquitously represented across vertebrate and invertebrate taxa.

Table 2.1 Representative examples of receptor-mediated and non–receptor- mediated endocrine actions of various environmental contaminants on wildlife or laboratory species

The various EDCs differ greatly in their potencies relative to natural hormones and in their affinity for target receptors. Classification of EDCs has been performed according to their known or suspected activity in relation to hormone receptors and pathways. While most attention has focused on EDCs that are mediated through the ERs (estrogen agonists) and affect development and reproductive functions in wildlife and humans, numerous laboratory and field studies show that many EDCs can also target the androgen receptors (ARs), the thyroid hormone receptors (THRs), glucocorticoid receptors (GCRs), progesterone receptors (PRs), aryl hydrocarbon receptor (AhR) and retinoid X receptor (RXR) and other signaling pathways [1].

In addition to nonylphenol (NP), a wide variety of chemical compounds are known to act as ER agonists, including:

The pesticides methoxychlor, aldrin, dieldrin, certain polychlorinated biphenyls (PCBs), bisphenol A (BPA; a high-production-volume chemical used to make polycarbonate plastic)

Pharmaceutical estrogens, such as diethylstilbestrol (DES) and ethinyl estradiol (EE2; a major active component in birth control pill)

Natural steroid hormones excreted by humans and livestock (estradiol [E2], estrone [E], etc.)

Phyto-estrogens (which occur naturally in many plants, most notably in soybeans in the form of genistein and related substances)

A number of chemical mixtures (reviewed by [51])

There are a few known ER antagonists, or antiestrogens, including certain OH-PCBs [52]. AR antagonists comprise chemical compounds such as vinclozolin, procymidone, linuron, fenitrothion and chlorinated pesticides such as p,p′-DDE and lindane as well as some of the phthalate plasticizers (a group of chemicals used to soften polyvinyl chloride plastics), phytosterols (present in pulp mill effluents), and certain PCBs (reviewed by [53,54]). Polycyclic aromatic hydrocarbons (PAHs) are suspected of having a range of weak ED effects (depending on structure) via mediation through ER, AR, and Ah receptors (reviewed by [18]). Chemicals such as PCBs, perchlorates, and BFRs are AhR agonists and characteristic disruptors of thyroid hormone homeostasis [55]. The BFRs polybrominated diphenyl ethers (PBDEs) are known also to disrupt thyroid hormone transport and metabolism [56].

Thus, in addition to the reproductive system, many other different receptors and tissues can be affected by EDCs, including endocrine glands such as pituitary, thyroid, thymus, and adrenal and a number of other endocrine-mediated physiological systems, including the immune and neurological systems, although underlying mechanisms are poorly understood. Increasing evidence from laboratory and field studies demonstrates that the neuroendocrine stress response is a sensitive target for disruption by a range of environmental contaminants, at a number of discrete loci. For example, it has been established that interrenal dysfunction, involving an impairment of the secretion of corticosteroid hormones such as cortisol, can be caused in wild fish and other vertebrates by chronic exposure to a range of organic and inorganic pollutants, including heavy metals, PAHs, and PCBs (see [57,58]). Until now, however, relatively few studies have investigated links between endocrine disruptors and stress hypothalamo-pituitary-interrenal/adrenal (HPI/HPA) axis. Corticosteroid hormones in vertebrates are critical for metabolism, growth, reproduction, immunity, and ion homeostasis, and are an important part of the coping mechanisms involved in the stress responses [59]. Furthermore, chemical activation of the HPA axis by PCBs or through interactions with the GCRs [60] can have adverse effects on a number of different systems, thereby expanding the number of potential targets for EDCs [10]. The underlying mechanisms of the neurocrine stress response and how precisely this affects the fitness of the individual (via reduced growth, immunosuppresion and reproductive failure, etc.) and potentially the population level is not well understood (reviewed by [57]).

Although there is considerable information on the early molecular events involved in hormone response, there is very little knowledge concerning the relationship between those molecular events and adverse health effects such as reproductive toxicity, behavior, and cancer. Immune function, long known to be sensitive to steroids, has also been identified as an EDC target [61]. Examples of chemicals interfering with immune function via endocrine interactions have been described for numerous compounds, including androgens [62], estrogens, organotins, and dioxins [10,61]. EDC exposure may also reduce the production of immune-related proteins in fish, which makes them more susceptible to disease. A recent study demonstrated that a PCB mixture (A1248) modulates both immune function and endocrine physiology in brown bullhead [63]. The results suggest that EDCs may make fish more susceptible to disease by blocking production of hepcidin and other immune-related proteins that help protect fish against disease-causing bacteria, viruses, and parasites [64].

Sex hormones play a critical role in both developmental and adult expression of behavior through actions on the brain. These compounds interact with brain neurochemistry to mediate many social behaviors in vertebrates. Even small deficits in brain function could render the animal less able to escape predation, catch fast-moving prey, attract a mate, and rear offspring. A rapidly increasing body of scientific research is revealing that a large number of EDCs (e.g., dichlorodiphenyltrichloroethane (DDT), PCBs, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), tributyltin [TBT], BPA, methylmercury, many pesticides) can have profound effects on neuroendocrine-regulated behavior in wildlife (and humans), and suggests that such altered behavior may detrimentally affect survival (reviewed by [65]). Recent examples include altered reproductive behavior of the male three-spined stickleback (Gasterosteus aculeatus) caused by the organophosphorous pesticide fenitrothion [66] and impaired courtship and aggressive behavior of the male sand goby (Pomatoschistus minutus) after exposure to EE2 [67]; for more examples, see Section 2.3.

It is important to note that, for some EDCs, the parent compound may have no ED activity whereas the metabolites of the same chemical may be biologically active. For example, methylsulfonyl(MeSO2)-PCB metabolites (which are persistent and bioaccumulative contaminants) are potentally antiestrogenic [68]. Another example is 17β-trenbolone (a synthetic androgen), which is a metabolite of a steroidal growth promoter administered to cattle that can cause reproductive effects in fish [69]. And there are other types of EDCs that affect particular endocrine targets (see also Table 2.1). The highly complex nature of hormonal systems and the many points at which disruption can occur is well demonstrated by a recent in vitro toxicity testing of 27 individual BFRs [70]. The results revealed a scala of ED potencies, some of which had not or had only marginally been described before (AR antagonism, PR antagonism, estradiol sulfotransferase [E2SULT] inhibition, and potentiation of T3-mediated effects). For some BFRs, the potency to induce AR antagonism, E2SULT inhibition, and transthyretin receptor (TTR) competition was higher than for natural ligands or clinical drugs used as positive controls. A number of these BFRs have shown ED effects in vivo, including altered thyroid hormone homeostasis and effects on neurodevelopment [71].

Low-dose effects are commonly associated with the endocrine system. These low-dose effects refer to biological changes that may occur at much lower doses than would normally be expected to have an effect or at doses insensitive to traditional testing methods. A wide range of adverse effects has been reported in experimental animals exposed to low doses of BPA exposed both during development and in adulthood. These findings have been related to the potential involvement of EDCs in a range of human disease processes, such as the increase in prostate and breast cancer, hypospadias a decline in semen quality in men, and various metabolic and neurological disorders [72–77]. The available evidence in the case of BPA also illustrates the issue of a very long latency for effects that may not become apparent until long after EDC exposure during development has occurred. These developmental effects may be irreversible and can occur due to low-dose exposure during brief sensitive periods in development, even though no BPA may be detected when the damage or disease is expressed [75].

2.1.7 Endocrine Disrupters in the Environment

Both natural and synthetic environmental EDCs enter into the different environmental compartments (atmosphere, freshwaters, seawater, soils, and marine sediments) through active application, industrial and domestic waste water discharges, incineration, and/or livestock runoff. The aquatic environment may act as a sink for many contaminants that originate from wastewater, air deposits, runoff, and other sources and could therefore pose a high risk to aquatic organisms and fish-eating top predators. Common EDCs in domestic and some industrial effluents and their receiving surface waters are estrogenic hormones (E2 and E), the synthetic EE2, and other pharmaceuticals including glucocorticosteriods (GCs) and personal care products [51,78,79]. The majority of EDCs are POPs and other bioaccumulating chemicals. They are distributed around the globe through atmospheric transport and can contaminate areas far removed from the original site of contamination. Persistent synthetic EDCs have been detected in all environmental media, although concentrations of some legacy compounds, such as PCBs, DDT, and TBT, have declined markedly in some regions, because they are no longer produced or used in those countries. Many of these chemicals exist within complex mixtures (wastewater effluents) and are mobile in water.

The relative importance of direct uptake from the water and uptake from food will depend on the characteristics of the chemical. If the chemical is persistent, and particularly if it is also lipophilic, food chain effects can be expected to predominate as they can move through food chains and represent a threat to top predators in the aquatic and terrestrial ecosystem, such as birds and mammals. For most aquatic organisms, hydrophilic chemicals are readily taken up via the gills, digestive tract, and skin. Furthermore, the eggs of most aquatic animals are deposited into water, and thus the developing embryos may be directly exposed to EDCs and other toxicants at susceptible stages in their development. Benthic invertebrates may be exposed to EDCs through direct contact and ingestion of sediment/soil particles and pore water or by eating contaminated food. For other terrestrial (land-living) wildlife, the major route of exposure is via the diet [80].

Indeed, significant concentrations of legacy and newly emerging POPs with known or suspected ED proporties and other EDCs are increasingly reported in especially (aquatic) top predators at locations remote from human activity (such as the Pacific Ocean and the Arctic) and might perhaps pose the most serious threat of EDCs to wildlife populations and biodiversity (reviewed by [81,82]). Recent reports that document these threats from putative or known EDCs to marine top predators, such as polar bears, sperm whale, dolphins, tuna, albatross and other bird species, include organochlorines [82,83], TBT [84,85], toxaphenes [86], PBDEs [86–89], perfluorooctane sulfonate (PFOS) and perfluorooctanoic acid (PFOA) [90–93], and phthalate esters [94].

On basis of the information of the many facets of endocrine disruption in wildlife (and humans) just presented, EDCs in the environment are a matter of great concern because:

Hormonal systems can be disrupted by numerous different anthropogenic chemicals through various mechanisms.

EDCs may be effective at very low concentrations, and their effects depend on timing of exposure as well as level of exposure.

Disruption by EDCs may have widespread implications for many organisms, as hormonal regulation of biological functions is common to both vertebrates and invertebrates.

The ubiquity of exposure: both naturally occurring and man-made hydrophilic and bioaccumulative substances can be EDs and are widely distributed over the globe.

Mixtures can produce additive responses at individually negligible concentrations.

The persistence of effects: the effects of exposure to EDCs can be observed long after the actual exposure has ceased. For this reason, the effects may be delayed and subtle and often difficult to discern before they are discovered in the field.

Beside reproductive effects, there is scope for a wide range of potential effects on the immune and neurological systems as well as epigenetic effects.

Our knowledge about the precise endocrine mechanisms and how EDCs interact with and influence endocrine, immune, neural, and other systems is still fragmentary.

2.2 EFFECTS OF EDCS ON WILDLIFE

Here we present an overview of the documented reproductive and nonreproductive effects of EDCs in different vertebrate and invertebrate taxa. We focus on recent literature and new findings, and describe commonalities in observed effects among various wildlife taxa.

2.2.1 Mammals

Numerous field, and some (semi-)experimental studies have shown that aquatic mammals, particularly those high in the food chain, such as pinnipeds, odontocete cetaceans, otters, and polar bears, may accumulate high levels of some EDCs in their fatty tissue, in particular organochlorines and other POPs, and are sensitive to the toxicological effects of these EDCs [7,95].

Although it is generally accepted that persistent contaminants (PCBs, DDT) have played a major role in the population declines of seals populations in northwest Europe in the second half of the twentieth century, the evidence is not conclusive. Field studies on Baltic gray (Halichoerus grypus) and ringed seals (Phoca hispida baltica) and semi-field studies on harbor seals (Phoca vitulina) by Dutch research groups have attributed impairment of both reproduction and immune function to organic contaminant (OC) contamination, notably PCBs, in the food chain. Reproductive effects resulted in population declines, whereas suppression of immune function likely contributed to the mass mortalities due to morbillivirus infections. These historic cases have been described and evaluated at length elsewhere (reviewed by [1,7]). In brief, Baltic seals exhibited sterility and a suite of reproductive and nonreproductive disorders including skull lesions, uterine stenosis, occlusions, uterine smooth muscle tumors (leiomyomas), and adrenocortical hyperplasia (Baltic seal disease syndrome). This syndrome has been associated with high levels of PCB and DDT and their metabolites, notably PCB- and 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (DDE)-methyl solfones and 1,1-dichloro-2,2-bis(p-chlorophenyl)ethane (DDD), which are known to affect the function of the HPG axis and adrenal axes [1,7]. Several of these EDCs are capable of disrupting both glucocorticosteroid hormone synthesis and receptor-mediated action (reviewed by [7]). The available evidence supports an etiological role of PCB for parts of the disease syndrome, particularly for lesions connected to the reproductive failure among the seals but also involvement of an ED component of physiological stress (reviewed by [7]). In a two-year feeding experiment, female harbor seals fed fish from the polluted Wadden Sea displayed a lower reproductive success than seals fed less contaminated fish from the Atlantic Ocean [96]. Reduced levels of E2, retinol, and thyroid hormones in plasma were found in the group with the highest PCB uptake [96–98]. In the same study, implantation failure was found to be associated with lower levels of E2 [99]. Plausible explanations for the observed effects include: PCB-induced reduction in E2 levels due to alterations in enzyme metabolism and interference by PCB or DDE and their metabolites with receptors in target tissues [7]. However despite the strong correlation of the various reproductive effects in seals with OC exposure, there is still an incomplete understanding of the specific compounds responsible for the reproductive and pathological effects and their mechanism of action(s) [1]. In a second long-term feeding study, it was shown that ambient levels of environmental contaminants, notably PCBs, are immunotoxic to harbor seals [100]. Various immune function parameters were suppressed in the contaminant-fed group including natural killer (NK) cell activity and proliferative lymphocyte responses after stimulation, suggesting an impaired T cell function. These functions are known to be important in the clearance of virus infections [7]. The results obtained in captive seals fed contaminated fish are consistent with the effects observed in laboratory animals exposed to Ah-receptor binding PCBs, polychlorinated dibenzodioxin (PCDDs), and polychlorinated dibenzofuran (PCDFs) [7]. The results of these studies make it likely that chemical-induced immunosuppression could have contributed to the virus-associated mass mortalities among seals inhabiting northwestern Europe in 1988–1989 [7] and possibly also in 2002. Another group of authors found indications for a link between thyroid hormone levels and exposure to PBDEs in gray seals from the United Kingdom during their first year of life [101]. The molecular mechanisms involved in contaminant immunosuppression and susceptibility related to those determining viral susceptibility are, however, still unresolved [102].

There are a several cases in which recent declines in endangered mammalian populations are unexplained and where exposure to environmental EDCs may be involved. Examples are California sea otter declines in the United States and Steller sea lion (Eumetopias jubatus) declines in Alaska [103]. Kannan and coworkers [104] found PCBs, DDTs, and butyltins to be major contaminants in sea otters and their prey collected from California (United States). They noted that California sea otters suffer from various fatal infections and showed that the 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) equivalents of non- and mono-ortho PCBs in sea otters and certain prey species were at or above the theoretical threshold for toxic effects, indicating a possible role of EDCs in the disease events. Also, elevated tissue levels of butyltins, mercury, PCBs, DDTs, chlordanes, and hexachlorobenzene have been reported in Alaskan Steller sea lions. However, the impacts