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Polychlorinated biphenyls (PCBs) are toxic, persistent, and lipophilic chemical compounds that accumulate to high levels in harbor porpoises (Phocoena phocoena) and other cetaceans. It is important to monitor PCBs in wildlife, particularly in highly exposed populations to understand if concentrations are declining and how levels relate to toxicological thresholds and indices of health like infectious disease mortality. Here we show, using generalized additive models and tissue samples of 814 U.K.-stranded harbor porpoises collected between 1990 and 2017, that mean blubber PCB concentrations have fallen below the proposed thresholds for toxic effects. However, we found they are still associated with increased rates of infectious disease mortality such that an increase in PCB blubber concentrations of 1 mg kg-1 lipid corresponds with a 5% increase in risk of infectious disease mortality. Moreover, rates of decline and levels varied geographically, and the overall rate of decline is slow in comparison to other pollutants. We believe this is evidence of long-term preservation in the population and continued environmental contamination from diffuse sources. Our findings have serious implications for the management of PCB contamination in the U.K. and reinforce the need to prevent PCBs entering the marine environment to ensure that levels continue to decline.

Exposure of male fish to estrogenic substances from wastewater treatment works (WwTWs) results in feminization and reduced reproductive fitness. Nevertheless, self-sustaining populations of roach (

Polychlorinated biphenyls (PCBs) are toxic, persistent, and lipophilic chemical compounds that accumulate to high levels in harbor porpoises (

© 2015 American Chemical Society. Exposure to environmental estrogens in wastewater treatment works (WwTW) effluents induces feminized responses in male fish, including the development of eggs in male testes. However, the impacts on the offspring of exposed fish are not well understood. In this study, we examined whether roach (Rutilus rutilus) from mothers that had been exposed to an undiluted WwTW effluent from early life to sexual maturity had altered susceptibility to gonadal feminization and an impaired capacity to reproduce. For males from both WwTW effluent exposed mothers and dilution water exposed mothers, effluent exposure for up to 3 years and 9 months induced feminized male gonads, although the intersex condition was relatively mild. There was no difference in the severity of gonadal feminization in roach derived from either WwTW effluent exposed or dilution water exposed mothers. Furthermore, a breeding study revealed that roach with effluent-exposed mothers reproduced with an equal success as roach with mothers exposed to clean water. Roach exposed to the effluent for 3 years in this study were able to reproduce successfully. Our findings provide no evidence for impacts of WwTW effluent exposure on reproduction or gonadal disruption in roach down the female germ line and add to existing evidence that male roach with a mild intersex condition are able to breed competitively. Natural Environmental Research Council (NERC) grant nos. NE/G019355/1, NE/K004263/1; Department for Environment, Food and Rural Affairs, U.K.

© 2015 The Authors. We present a detailed response to the critique of "State of the Science of Endocrine Disrupting Chemicals 2012" (UNEP/WHO, 2013) by financial stakeholders, authored by Lamb et al. (2014). Lamb et al.'s claim that UNEP/WHO (2013) does not provide a balanced perspective on endocrine disruption is based on incomplete and misleading quoting of the report through omission of qualifying statements and inaccurate description of study objectives, results and conclusions. Lamb et al. define extremely narrow standards for synthesizing evidence which are then used to dismiss the UNEP/WHO 2013 report as flawed. We show that Lamb et al. misuse conceptual frameworks for assessing causality, especially the Bradford-Hill criteria, by ignoring the fundamental problems that exist with inferring causality from empirical observations. We conclude that Lamb et al.'s attempt of deconstructing the UNEP/WHO (2013) report is not particularly erudite and that their critique is not intended to be convincing to the scientific community, but to confuse the scientific data. Consequently, it promotes misinterpretation of the UNEP/WHO (2013) report by non-specialists, bureaucrats, politicians and other decision makers not intimately familiar with the topic of endocrine disruption and therefore susceptible to false generalizations of bias and subjectivity.

The reproduction of vertebrates is regulated by endocrine and neuro-endocrine signaling molecules acting along the brain-pituitary-gonad (BPG) axis. The understanding of the neuroendocrine role played in reproductive function has been recently revolutionized since the KiSS1/GPR54 (KiSS1r) system was discovered in 2003 in human and mice. Kisspeptins, neuropeptides that are encoded by the KiSS genes, have been recognized as essential in the regulation of the gonadotropic axis. They have been shown to play key roles in puberty onset and reproduction by regulating the gonadotropin secretion in mammals while physiological roles in vertebrates are still poorly known. In order to provide new knowledge on basic reproductive physiology in fish as well as new tools to assess impacts of endocrine disrupting compounds (EDCs), the neurotransmitter system, i.e., gene/receptor, KISS/GPR54 might constitute an appropriate biomarker. This study provides new understandings on the neuroendocrine regulation of roach reproduction as well as new molecular tools to be used as biomarkers of endocrine disruption. This work completes the set of biomarkers already validated in this species.

Highlights • ER and ERR transcription levels were unaffected in Marisa cornuarietis exposed to 17β-estradiol or 4-tert-Octylphenol. • The mollusc ER protein interacts with the phytoestrogen genistein in transfected HEK-293 cells. • The mollusc ERR protein interacts weakly with bisphenol-A in transfected HEK-293 cells. • The mollusc ER protein binds to the vertebrate consensus estrogen response element (ERE) sequence., Estrogen receptor orthologues in molluscs may be targets for endocrine disruptors, although mechanistic evidence is lacking. Molluscs are reported to be highly susceptible to effects caused by very low concentrations of environmental estrogens which, if substantiated, would have a major impact on the risk assessment of many chemicals. The present paper describes the most thorough evaluation to-date of the susceptibility of Marisa cornuarietis ER and ERR gene transcription to modulation by vertebrate estrogens in vivo and in vitro. We investigated the effects of estradiol-17β and 4-tert-Octylphenol exposure on in vivo estrogen receptor (ER) and estrogen-related receptor (ERR) gene transcription in the reproductive and neural tissues of the gastropod snail M. cornuarietis over a 12-week period. There was no significant effect (p > 0.05) of treatment on gene transcription levels between exposed and non-exposed snails. Absence of a direct interaction of estradiol-17β and 4-tert-Octylphenol with mollusc ER and ERR protein was also supported by in vitro studies in transfected HEK-293 cells. Additional in vitro studies with a selection of other potential ligands (including methyl-testosterone, 17α-ethinylestradiol, 4-hydroxytamoxifen, diethylstilbestrol, cyproterone acetate and ICI182780) showed no interaction when tested using this assay. In repeated in vitro tests, however, genistein (with mcER-like) and bisphenol-A (with mcERR) increased reporter gene expression at high concentrations only (>10−6 M for Gen and >10−5 M for BPA, respectively). Like vertebrate estrogen receptors, the mollusc ER protein bound to the consensus vertebrate estrogen-response element (ERE). Together, these data provide no substantial evidence that mcER-like and mcERR activation and transcript levels in tissues are modulated by the vertebrate estrogen estradiol-17β or 4-tert-Octylphenol in vivo, or that other ligands of vertebrate ERs and ERRs (with the possible exception of genistein and bisphenol A, respectively) would do otherwise.

Steroid estrogens are thought to be the major cause of feminization (intersex) in wild fish. Widely used wastewater treatment technologies are not effective at removing these contaminants to concentrations thought to be required to protect aquatic wildlife. A number of advanced treatment processes have been proposed to reduce the concentrations of estrogens entering the environment. Before investment is made in such processes, it is imperative that we compare their efficacy in terms of removal of steroid estrogens and their feminizing effects with other treatment options. This study assessed both steroid removal and intersex induction in adult and early life stage fish (roach, Rutilus rutilus). Roach were exposed directly to either secondary (activated sludge process (ASP)), tertiary (sand filtrated (SF)), or advanced (chlorine dioxide (ClO(2)), granular activated charcoal (GAC)) treated effluents for six months. Surprisingly, both the advanced GAC and tertiary SF treatments (but not the ClO(2) treatment) significantly removed the intersex induction associated with the ASP effluent; this was not predicted by the steroid estrogen measurements, which were higher in the tertiary SF than either the GAC or the ClO(2). Therefore our study highlights the importance of using both biological and chemical analysis when assessing new treatment technologies.

Background The feminization of nature by endocrine-disrupting chemicals (EDCs) is a key environmental issue affecting both terrestrial and aquatic wildlife. A crucial and as yet unanswered question is whether EDCs have adverse impacts on the sustainability of wildlife populations. There is widespread concern that intersex fish are reproductively compromised, with potential population-level consequences. However, to date, only in vitro sperm quality data are available in support of this hypothesis. Objective The aim of this study was to examine whether wild endocrine-disrupted fish can compete successfully in a realistic breeding scenario. Methods In two competitive breeding experiments using wild roach (Rutilus rutilus), we used DNA microsatellites to assign parentage and thus determine reproductive success of the adults. Results In both studies, the majority of intersex fish were able to breed, albeit with varying degrees of success. In the first study, where most intersex fish were only mildly feminized, body length was the only factor correlated with reproductive success. In the second study, which included a higher number of more severely intersex fish, reproductive performance was negatively correlated with severity of intersex. The intersex condition reduced reproductive performance by up to 76% for the most feminized individuals in this study, demonstrating a significant adverse effect of intersex on reproductive performance. Conclusion Feminization of male fish is likely to be an important determinant of reproductive performance in rivers where there is a high prevalence of moderately to severely feminized males.

International audience; Unaccounted sources of contamination can be problematic in toxicological studies and can range from the presence of impurities, breakdown products or isoforms of the parent compound to the unexpected compounds leaching from dosing apparatus. As these compounds are not being tested, they may not be measured in the dosed aquaria and hence go undetected, potentially contributing as confounding factors in toxicological assessments. In this paper we report the unexpected detection of butyltin compounds (mono, di and tributyltin) in flow-through aquaria waters of an aquatic toxicological set-up. High and variable leaching rates for dibutyltin of 2.0 and 6.6_g/h were detected during the first week of each of two separate flow-through studies. Following this initial ‘surge' of dibutyltin leachate, a decrease in leachate rate was seen with values of 0.9 and 1.2_g/h by Day 14 (second week of study). The main source of the butyltin leachates was shown, to be the airline tubing used in the assembly of the airsupply into each flow-through tank. A 24 h period of incubation of the airline tubing with clean water led to the leaching of concentrations of 63.8 ng/l TBT-Sn, 1638.8 ng/l DBT-Sn and 4054.6 ng/l MBT-Sn. The concentration of tributyltin detected was within its toxicologically effective range and as such could have potentially confounding effects on the toxicological bioassays being used. These accidental findings could be of enormous relevance to aquatic toxicologists and have an important bearing on the choice of materials used to construct experimental exposure aquaria.

Background: The widespread occurrence of feminized male fish downstream of some wastewater treatment works has led to substantial interest from ecologists and public health professionals. This concern stems from the view that the effects observed have a parallel in humans, and that both phenomena are caused by exposure to mixtures of contaminants that interfere with reproductive development. The evidence for a “wildlife–human connection” is, however, weak: Testicular dysgenesis syndrome, seen in human males, is most easily reproduced in rodent models by exposure to mixtures of antiandrogenic chemicals. In contrast, the accepted explanation for feminization of wild male fish is that it results mainly from exposure to steroidal estrogens originating primarily from human excretion. Objectives: We sought to further explore the hypothesis that endocrine disruption in fish is multicausal, resulting from exposure to mixtures of chemicals with both estrogenic and antiandrogenic properties. Methods: We used hierarchical generalized linear and generalized additive statistical modeling to explore the associations between modeled concentrations and activities of estrogenic and antiandrogenic chemicals in 30 U.K. rivers and feminized responses seen in wild fish living in these rivers. Results: In addition to the estrogenic substances, antiandrogenic activity was prevalent in almost all treated sewage effluents tested. Further, the results of the modeling demonstrated that feminizing effects in wild fish could be best modeled as a function of their predicted exposure to both antiandrogens and estrogens or to antiandrogens alone. Conclusion: The results provide a strong argument for a multicausal etiology of widespread feminization of wild fish in U.K. rivers involving contributions from both steroidal estrogens and xenoestrogens and from other (as yet unknown) contaminants with antiandrogenic properties. These results may add further credence to the hypothesis that endocrine-disrupting effects seen in wild fish and in humans are caused by similar combinations of endocrine-disrupting chemical cocktails.

Experiments were conducted to assess the impacts of exposure to sewage treatment works (STW) effluent upon the growth, reproductive function, and sexual development of the European mollusc, Planorbarius corneus under seasonally varying temperatures and photoperiodic conditions. In river water, a clear seasonal change in the number and weight of egg masses (during both 2003 and 2004), and in the number of eggs produced, was found, providing evidence for profound effects of both changing temperature and photoperiod on reproduction. Exposure to STW effluent caused disturbances in this seasonal reproductive cycle at all concentrations tested. The effects included significant dose-dependent increases in fecundity and in the overall length of the reproductive cycle in adult snails exposed to both 50% and 100% effluent relative to river water for a period of up to 14 weeks. Disturbances in the development of both the male and female gametes of the both the adult snails and their developmentally exposed offspring were also seen. These effects were more evident in the offspring than in the adults.

Feminization of the male roach, Rutilus rutilus, a freshwater, group-spawning fish, is widespread in English rivers; among the causative agents are natural and synthetic steroidal estrogens and chemicals that mimic estrogens. In feminized male roach, concentrations of the egg-yolk protein vitellogenin are elevated, sex steroid hormone dynamics are altered, and gonad development is disrupted (most notably, a female reproductive duct or developing eggs [oocytes] are present in the testis). In some English rivers containing high levels of estrogens, all male roach sampled have been feminized to varying degrees. In the more severely affected males, individuals produce low-quality sperm with a reduced capability for fertilization. Laboratory studies have shown that the environmental estrogens responsible for inducing gonadal feminization in roach can also alter reproductive behavior, disrupting normal breeding dynamics (parentage) in the zebrafish, another group-spawning fish. Together these findings indicate ...

This review assesses the effects of environmental concentrations of bisphenol-A (BPA) on wildlife. Water concentrations of BPA vary tremendously due to proximity to point and non-point sources, but reported concentrations in stream/river water samples are less than 21 microg/L, and concentrations in landfill leacheate are less than 17.2mg/L. Extensive evidence indicates that BPA induces feminization during gonadal ontogeny of fishes, reptiles, and birds, but in all cases the amount of BPA necessary to cause such ontogenetic disruption exceeds concentrations in the environment. Extensive evidence also exists that adult exposure to environmental concentrations of BPA is detrimental to spermatogenetic endpoints and stimulates vitellogenin synthesis in model species of fish. Most of the reported effects of BPA on vertebrate wildlife species can be attributed to BPA acting as an estrogen receptor agonist, but mechanisms of disruption in invertebrates are less certain. A comparison of measured BPA environmental concentrations with chronic values suggests that no significant margin of safety exists for the protection of aquatic communities against the toxicity of BPA. Further studies should examine the most vulnerable vertebrate and invertebrate species.

It has been hypothesized that a wide variety of reproductive and developmental problems observed in some wildlife populations and in humans are caused by exposure to environmental contaminants that can interfere with endocrine signaling pathways (reviewed by Vos et al. 2000). This hypothesis is supported by a growing body of evidence from laboratory studies that show that pollutants present in the environment can disrupt the endocrine functioning of wildlife species, thus causing permanent alterations in the structure and function of the endocrine system [reviewed by Tyler et al. (1998)]. There are, however, significant gaps in our knowledge of endocrine disruption in wildlife and humans, particularly in the causes and mechanisms of these phenomena. Although the interaction of pollutants with endogenous hormone receptors (e.g., the estrogen receptor) continues to dominate the literature, there are many associations between exposure to endocrine-disrupting chemicals (EDCs) and a variety of biological outcomes for which the mechanisms of action are poorly understood. This makes it difficult to distinguish between direct (hormone-receptor–mediated) and indirect effects and between primary and secondary outcomes. Moreover, wildlife species are rarely exposed to single chemicals but instead are exposed to complex, fluctuating mixtures of contaminants that may act in various ways (Silva et al. 2002; Sumpter 2003; Thorpe et al. 2001, 2003) and that may induce combination effects (Rajapakse et al. 2002) via the same or different mechanisms. Wastewater treatment works (WwTW) effluent, for example, contains a mixture of natural and synthetic xenobiotics, household and agricultural chemicals, pharmaceuticals, hormones, and other compounds, many of which remain unidentified (Stevens et al. 2003). Studies have correlated exposure to WwTW effluent with alterations in sex steroid hormone levels in adult and juvenile fish (Folmar et al. 1996, 2001a; Hecker et al. 2002), impaired gonadal development in adults and juveniles (Hemming et al. 2001; Jobling et al. 2002; Sheahan et al. 2002), altered sexual differentiation in early life stages (Rodgers-Gray et al. 2001), and induction of the egg-yolk precursor protein vitellogenin (VTG) in adult male and juvenile fish of both sexes (Harries et al. 1999; Purdom et al. 1994; Rodgers-Gray et al. 2001). These effects have been associated with the presence of chemical contaminants in the effluents that act as estrogen receptor agonists, including natural and synthetic steroids (Desbrow et al. 1998; Routledge et al. 1998), alkylphenol polyethoxylates (Gimeno et al. 1997, 1998; Gronen et al. 1999; Jobling et al. 1996; Seki et al. 2003), and phthalates and pesticides (Ankley et al. 2001; Christiansen et al. 2000; Jobling et al. 1995; Sohoni and Sumpter 1998). These disorders, however, may not necessarily be a consequence of estrogenic effects alone. Many EDCs are reactive chemicals that exhibit multiple mechanisms of toxicity by acting at different sites within the body. Consequently, alterations to the endocrine immune and nervous systems and damage to genetic material may each contribute toward the overall toxic impact of a particular chemical (Choi et al. 2004; Galloway and Handy 2003; Handy 2003; Roy and Liehr 1999). It is pertinent to note that a recent comprehensive literature survey of 48 EDCs revealed that 79% of these were also carcinogenic or mutagenic, 52% were also immunotoxic, and 50% were also neurotoxic (Choi et al. 2004). Essentially half of all the chemicals tested (including both natural and synthetic) cause tissue injury at high enough doses (and thus could result in disruptions in immune function and in cancers, etc.). Both 4-tert-nonylphenol (4-NP) and bisphenol A (BPA), for example, are contaminants found at appreciable concentrations in the aquatic environment that can cause endocrine disruption by interacting with both the estrogen receptor (as agonists; Gaido et al. 1997) and the androgen receptor (Sohoni and Sumpter 1998). In addition, 4-NP can disrupt steroidogenesis in the liver and can interfere with the dynamic control of follicle-stimulating hormone release from the pituitary (Harris et al. 2001). Moreover, recent studies have also shown that concentrations of 4-NP and BPA that inhibit gonadal development and reproductive function in fish can also cause damage to the kidneys (as a consequence of VTG induction) and decreased body weight and induce stressed behavior (Magliulo et al. 2002) as well as result in damage to DNA [in barnacles, Atienzar et al. (2002)] and to the immune system [in rats, Karrow et al. (2004)]. Similarly, steroid estrogens that are known to be present in WwTW effluents (Desbrow et al. 1998) and to cause feminizing effects in fish have been reported to be genotoxic in mammals both in cell lines and in vivo (Banerjee et al. 1994; Floyd et al. 1990; Han and Liehr 1994; Nutter et al. 1991, 1994). With these issues in mind, we need a better understanding of the relative importance of the impact of endocrine disruption on the physiology of wild organisms compared with the other forms of toxicity. Furthermore, studies are also needed to help unravel how some of the health effects seen in wildlife and ascribed to endocrine disruption are actually mediated. To date, virtually nothing is known of the potential importance of interacting modes of toxicity in complex cocktails of pollutants. Several studies have documented either the immunological (Kakuta and Murachi 1997) or genotoxic (White et al. 1996) activity/effects of WwTW effluents separately, but there are no studies in which the impacts of WwTW effluents on these axes have been examined simultaneously or that have compared the relative importance and sensitivity for these disruptive/toxic effects. Our goal in this present study was to fill these knowledge gaps by examining the integrated health effects (reproductive, endocrinological, immunological, genotoxic, and nephrotoxic) of long-term (300-day) exposure to a range of concentrations of a treated WwTW effluent on the roach (Rutilus rutilus), a common European fish species in which a widespread incidence of endocrine disruption has been described and reported (Jobling et al. 1998; Nolan et al. 2001). The exposure regime adopted covered the period of embryogenesis and development during early life.

It is now well established that there is a diverse array of chemicals discharged into the environment that can mimic or antagonise the action of hormones. These endocrine-disrupting chemicals (EDCs) can thus interact with physiological systems and cause alterations in development, growth and reproduction in wildlife that are exposed to them. As yet, however, there is little information on the relative sensitivities of different wildlife groups to these chemicals and/or mixtures of them (e.g. estrogenic effluents) and hence, there are fundamental shortfalls in our knowledge of the ecological importance of endocrine disruption in wildlife. In this study, the effects of exposure to individual estrogenic chemicals (17alpha-ethinylestradiol; EE2, bisphenol-A, and 4-tert octylphenol) and a mixture containing these chemicals (treated sewage effluent) on embryo production in the prosobranch mollusc, Potamopyrgus antipodarum, were studied and compared with the effects of EE2 and the same estrogenic effluent on vitellogenin induction and/or egg production in various species of freshwater fish (fathead minnow; Pimaphales promelas, rainbow trout (Oncorhynchus mykiss); Cyprinus carpio, carp; Cyprinus carpio). The lab-based studies demonstrated that all of the tested chemicals (known to be estrogenic and to cause reproductive effects in fish) also affected embryo production in P. antipodarum. Furthermore, exposure to EE2 induced similar reproductive responses in the snails as in the fathead minnow (Pimephales promelas), stimulating egg/embryo production at low doses (up to 1 ng/l in the minnow and 25 ng/l in the snail) and causing inhibitory effects at higher doses. A similar pattern of embryo production occurred in P. antipodarum when it was exposed to a graded concentration of treated sewage effluent containing mixtures of estrogenic EDCs and hence, the total number of new embryos produced by the snails increased steadily over the 9 weeks exposure period in treated snails. Plasma vitellogenin concentrations in two species of male fish (the rainbow trout and the carp) also increased over the same time period. These data indicate that both the nature of the response and the relative sensitivities to environmental estrogens in P. antipodarum and three different fish species fish are comparable. P. antipodarum is thus, potentially a sensitive test organism for assessing estrogenicity of chemicals with a relevance to their activity in vertebrates.

Disruption of the endocrine system has been shown to occur in wild freshwater fish populations across the globe. Effects range from subtle changes in the physiology and sexual behaviour of fish to permanently altered sexual differentiation, impairment of gonad development and/or altered fertility. A wide variety of adverse environmental conditions may induce endocrine disruption, including sub-optimal temperatures, restricted food supply, low pH, environmental pollutants, and/or parasites. Furthermore, it is conceivable that any/all of these factors could act simultaneously to cause a range of disparate or inter-related effects. Some of the strongest evidence for a link between an adverse health effect, as a consequence of endocrine disruption, and a causative agent(s) is between the condition of intersex in wild roach (Rutlius rutilus) in UK rivers and exposure to effluents from sewage treatment works. The evidence to indicate that intersex in roach (and other cyprinid fish living in these rivers) is caused by chemicals that mimic and/or disrupt hormone function/balance in treated sewage effluent is substantial. There are a few parasites that affect the endocrine system directly in fish, including the tape worm Ligula intestinalis and a few parasites from the micropsora phylum. L. intestinalis acts at the level of the hypothalamus restricting GnRH secretion (resulting in poorly developed gonads) and is one of the very few examples where an endocrine disrupting event has been shown to result in a population-level effect (reducing it). It is well established that many parasites affect the immune system and thus the most common effect of parasites on the endocrine system in fish is likely to be an indirect one.

Endocrine disruption has been reported in freshwater fish populations around the world. This phenomenon ranges from subtle changes in the physiology and sexual behavior of fish to permanently altered sexual differentiation and impairment of fertility. Despite widespread reports of endocrine disruption in fish (and this is well characterized at the individual level), few studies have demonstrated population-level consequences as a result of exposure to endocrine-disrupting chemicals (EDCs). An exception to this is in Lake Ontario Lake trout where precipitous declines in the population have been linked with periods of high exposure to organochlorine chemicals (known EDCs). Recently, it has been established that roach (Rutilus rutilus) exposed to treated sewage effluent (that contains complex mixtures of EDCs) in UK rivers, have a reduced reproductive capacity. This, in turn, may have population-level consequences. Evidence for a link between exposure to effluents from kraft mill (BKME) and sewage treatment works (STWs) and altered reproductive function in freshwater fish is compelling. In most cases, however, a causal link between a specific chemical and a physiological effect has not been established. Indeed, identifying specific chemical(s) responsible for adverse effects observed in the wild is difficult, given that tens of thousands of man-made chemicals enter the aquatic environment and that mixtures of chemicals can have combination (e.g., additive) effects. Some EDCs are known to act at a number of different body targets to affect a variety of physiological processes, further complicating the identification of the causative agent(s). Endocrine disruption appears to be particularly widespread in freshwater fish populations. There is little evidence, however, to suggest fish are more susceptible to EDCs relative to other wildlife. Notwithstanding this, there are some features of the endocrine physiology of fish that may be particularly susceptible to the effects of EDCs, including the processes of sex-determination and smoltification (in salmonids). Furthermore, their aquatic existence means that fish can be bathed constantly in a solution containing pollutants. In addition, uptake of chemicals readily occurs via the gills and skin, as well as via the diet (the major exposure route for most EDCs in terrestrial animals). The exposure of fish early life stages to the cocktail of EDCs present in some aquatic environments may be of particular concern, given that this is an especially vulnerable period in their development. The challenge, from the point of view of ecological risk assessment, is to determine effects of EDCs on freshwater fish populations and freshwater ecosystems. In order to meet this challenge, high-quality data are required on the population biology of freshwater fish, on the effects of EDCs on their various life history characteristics, and comprehensive and appropriate population models. Basic information on the population biology of most species of wild freshwater fish is, however, extremely limited, and needs significant improvement for use in deriving a sound understanding of how EDCs affect fish population sustainability. Notwithstanding this, we need to start to undertake possible/probable predictions of population level effects of EDCs using data derived from the effects found in individual fish. Furthermore, information on the geographical extent of endocrine disruption in freshwater fish is vital for understanding the impact of EDCs in fish populations. This can be derived using published statistical associations between endocrine disruption in individual fish and pollutant concentration in receiving waters. Simplistic population models, based on the effects of EDCs on the reproductive success of individual fish can also used to model the likely population responses to EDCs. Wherever there is sufficient evidence for endocrine disruption in freshwater fish and the need for remediation has been established, then there is a need to focus on how these problems can be alleviated. Where industrial chemicals are identified as causative agents, a practical program of tighter regulation for their discharge and/or a switch to alternative chemicals (which do not act as EDCs) is needed. There are recent examples where such strategies have been adopted, and these have been successful in reducing the impacts of EDCs from point source discharges on freshwater fish. Where EDCs are of natural origin (e.g., sex steroid hormones from human and animal waste), however, remediation is a more difficult task. Regulation of the release of these chemicals can probably be achieved only by improvements in treatment processes and/or the implementation of systems that specifically remove and degrade them before their discharge into the aquatic environment.

Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = -0.603; P < 0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.

As a result of the introduction of tighter discharge limits and effluent treatment processes at source, the concentration of alkylphenol ethoxylates and nonylphenol present in the final effluent discharge from a sewage treatment works that treats trade effluent from the textiles industry was reduced. The estrogenic effects of the final effluent discharge to the Aire River were compared over a four-year period during which various treatment measures were introduced. Male rainbow trout exposed to the effluent on four occasions in consecutive years (1994–1997) showed a reduction in the level of induced vitellogenesis between 1994 and 1997. A marked decrease in gonadosomatic index (GSI) and increase in heptaosomatic index (HSI) was measured in fish exposed to the effluent in 1994. In successive years, these differences diminished, and in the case of the GSI no measurable difference was observed between fish exposed to the final effluent or those in the control group in 1997. However, an increase in HSI was still measurable in 1997 in fish exposed to the final effluent and at sites farther downstream. The reduction in the effects of the effluent paralleled the reduction in the concentration of nonylphenol as well as its mono- and diethoxylates, which have been demonstrated to produce estrogenic effects in trout exposed to these compounds in the laboratory. This study demonstrates that the setting of more restricted discharge limits for known estrogenic chemicals of industrial origin can lead to significant reductions in the estrogenic activity of the watercourses into which the effluents are discharged.

Disruption in gonadal development of wild roach living in U.K. rivers receiving large volumes of treated sewage effluent is manifest in a variety of ways, ranging from malformation of the germ cells and/or reproductive ducts to altered gamete production. Intersex fish were also found to have an altered endocrine status and an elevated concentration of plasma vitellogenin. Gonadal growth was inhibited only in severely intersex fish, whereas progression of spermatogenesis was delayed in a large proportion of all intersex and exposed male fish. In contrast to the effects observed in the intersex and exposed male fish, the maturation of ovaries in female fish inhabiting effluent-contaminated rivers appeared to be less obviously affected, although a higher incidence of oocyte atresia was found in the effluent-exposed fish compared with the reference fish. A positive correlation was found between the proportion of female tissue in the gonads of intersex fish and their plasma vitellogenin concentration, suggesting that vitellogenin can be an indicator for the level of gonadal disruption in intersex roach. The estradiol-17β concentration in intersex fish was intermediate between the concentration found in males and females, and the plasma testosterone was between 2- and 3-fold higher in intersex fish compared with male fish. These data suggest a link between altered endocrine status in intersex and female fish and gonadal disruption. Spermiation was also affected in roach living in effluent-impacted rivers: a lower proportion of fish were found releasing sperm, and in those intersex fish that were spermiating, a reduced milt volume and a reduced sperm density were found. All intersex fish had malformations of the reproductive duct(s), and in severely affected fish, the ducts were occluded, thus preventing release of gametes. In view of the widespread occurrence of intersexuality in wild fish populations in rivers throughout the United Kingdom, assessment of the reproductive capabilities of these intersex roach is clearly needed to understand the impact of this phenomenon on roach fertility.

Nuclear receptors (NRs) are transcription regulators involved in an array of diverse physiological functions including key roles in endocrine and metabolic function. The aim of this study was to identify nuclear receptors in the fully sequenced genome of the gastropod snail, Biomphalaria glabrata, intermediate host for Schistosoma mansoni and compare these to known vertebrate NRs, with a view to assessing the snail's potential as a invertebrate model organism for endocrine function, both as a prospective new test organism and to elucidate the fundamental genetic and mechanistic causes of disease. For comparative purposes, the genome of a second gastropod, the owl limpet, Lottia gigantea was also investigated for nuclear receptors. Thirty-nine and thirty-three putative NRs were identified from the B. glabrata and L. gigantea genomes respectively, based on the presence of a conserved DNA-binding domain and/or ligand-binding domain. Nuclear receptor transcript expression was confirmed and sequences were subjected to a comparative phylogenetic analysis, which demonstrated that these molluscs have representatives of all the major NR subfamilies (1-6). Many of the identified NRs are conserved between vertebrates and invertebrates, however differences exist, most notably, the absence of receptors of Group 3C, which includes some of the vertebrate endocrine hormone targets. The mollusc genomes also contain NR homologues that are present in insects and nematodes but not in vertebrates, such as Group 1J (HR48/DAF12/HR96). The identification of many shared receptors between humans and molluscs indicates the potential for molluscs as model organisms; however the absence of several steroid hormone receptors indicates snail endocrine systems are fundamentally different.

To establish whether the intersex condition seen in the roach (Rutilus rutilus) in United Kingdom (UK) rivers was species specific or a more general phenomenon in fish, evidence for sexual disruption was sought in a second cyprinid species, the gudgeon (Gobio gobio). Gudgeon were collected from the Rivers Aire and Lea (at locations that receive high-volume discharges of sewage treatment works [STW] effluent and that contain intersex roach) and from two still waters, and their gonads were examined histologically for evidence of intersexuality (the simultaneous presence of oocytes and testicular tissue). Intersex gonads were found at all sites, with the highest incidences occurring at one of the still waters (Lakeside Fisheries: 15%) and at sites on the River Aire (Thwaite Weir, Silsden Bridge, and Knostrop: 14, 13, and 12%, respectively). In the River Lea and Longton Park Lake, the incidence of intersexuality in gudgeon was 6%. In most cases, intersex gonads were characterized by a few primary oocytes/gonad section in an otherwise normal testis. However, at some sites on the River Aire (Thwaite Weir and Knostrop), the intersex condition was more severe. At Thwaite Weir, for example, more than half of the gonad in 40% of the intersex fish was comprised of ovarian tissue. Elevated concentrations of plasma vitellogenin both in male and in intersex fish indicated that fish had been exposed to estrogen(s). Some of the gudgeon were found at sites several kilometers downstream of any point discharge of STW effluent; therefore, the results likely are representative of this species in wild populations found in typical UK river ecosystems. Together with the findings in the roach, these data on the gudgeon confirm that sexual disruption in fish in UK rivers is not species specific.

This paper is an illustrative guide to intersex in the roach Rutilus rutilus, based on 150 intersex individuals. Most intersex roach had female germ cells, or oocytes, within a predominantly male gonad (testis), and/or malformed/intersex reproductive ducts. The number, pattern and developmental stage of oocytes within testicular tissue in intersex fish varied greatly. In most intersex fish, a few primary oocytes, or numerous primary and secondary oocytes, were scattered randomly throughout the testicular tissue (multifocal intersex). In other, more severely feminized individuals, large areas of ovarian tissue were separated clearly from testicular tissue (focal intersex). Almost all intersex individuals had a female-like reproductive duct (ovarian cavity). In mild cases of intersex (in which the majority of the germ cells were male) the ovarian cavity was present together with the male sperm duct/vas deferens, whilst in certain severe cases, the sperm duct was absent or vestigial.

Sewage treatment works (STW) effluents throughout the U.K. have been shown to be estrogenic, inducing vitellogenin (VTG) synthesis in caged and wild fish for considerable distances (up to several kilometers) downstream of the effluent discharge. Thresholds for vitellogenin induction in caged fish in those studies have been derived from short-term exposures, and may not necessarily be representative of thresholds for estrogenic responses in wild fish living in rivers that contain STW effluent. In addition, very little is known about the long-term fluctuations in the concentrations of the estrogenic components of STW effluents. In this paper, it was established that the concentrations of natural steroid estrogens and xenoestrogens (alkylphenolic chemicals) in a treated sewage effluent fluctuated temporally (over 8 months), from between 36 and 308 ng/L and between

Rainbow trout were exposed for 3 weeks (in a flow-through system) to various dilutions of treated effluent (25, 50, 75, and 100%) from Harpenden and Chelmsford sewage treatment works (STW) during November 1994 and August 1996, respectively, and the induction of plasma vitellogenin was measured. Significant (p < 0.05) increases in plasma vitellogenin concentrations occurred in fish held in effluent from Harpenden STW at concentrations of 50% and above. At Chelmsford STW, the effluent appeared to be more potent, causing induction of vitellogenin in fish at concentrations as low as 25% (compared with 50% for the Harpenden effluent). Exposure to lower concentrations of effluent emanating from Chelmsford in April of the following year indicated that estrogenic activity was diluted to no-effect levels at this STW at concentrations between 12 and 25%.

A number of chemicals present in the environment have been shown to mimic or antagonize the actions of steroid hormones, an issue often described as “endocrine disruption/modulation”. There is very little evidence, however, to support the hypothesis that exposure to endocrine-disrupting chemicals is a global environmental health problem. In this paper, we demonstrate a high incidence of intersexuality in wild populations of riverine fish (roach; Rutilus rutilus) throughout the United Kingdom. These reproductive disturbances are consistent with exposure to hormonally active substances and are associated with discharges from sewage treatment works that are known to contain estrogenic chemicals. This is the first documented example of a widespread sexual disruption in wild populations of any vertebrate and indicates that reproductive and developmental effects do result from exposure to ambient levels of chemicals present in typical British rivers.

In recent years, a number of man-made chemicals have been shown to be able to mimic endogenous hormones, and it has been hypothesized that alterations in the normal pattern of reproductive development seen in some populations of wildlife are linked with exposure to these chemicals. Of particular importance are those compounds that mimic estrogens and androgens (and their antagonists), because of their central role in reproductive function. In fact, the evidence showing that such chemicals actually do mimic (or antagonize) the action of hormones in the intact animal is limited. In only a few cases have laboratory studies shown that chemicals that mimic hormones at the molecular level (in vitro) also cause reproductive dysfunction in vivo at environmentally relevant concentrations. In addition, the reported studies on wild populations of animals are limited to a very few animal species and they have often centered on localized 'hot-spots' of chemical discharges. Nevertheless, many of these xenobiotics are persistent and accumulate in the environment, and therefore a more widespread phenomenon of endocrine disruption in wildlife is possible. This article reviews the evidence, from both laboratory and field studies, that exposure to steroid hormone mimics may impair reproductive function and critically assesses the weight of evidence for endocrine disruption in wildlife.

It was recently demonstrated that most, if not all, effluents of sewage-treatment works (STWs) in the United Kingdom are estrogenic to fish. As many STWs discharge into rivers, it is possible that some stretches of rivers downstream of where the effluent enters might also be estrogenic. To assess this possibility, the induction of vitellogenin synthesis in caged male trout placed at various distances downstream of the effluent entry point was used as a biomarker of estrogen exposure. Individual discharges into five rivers in England were studied. In four cases, fish placed in the neat effluent, or close to where it entered a river, showed very marked and rapid increases in their plasma vitellogenin concentrations, demonstrating that the effluent was estrogenic. In two of these four cases, none of the downstream sites were estrogenic, whereas in one of the four, fish placed at a site 1.5 km downstream did respond by synthesizing appreciable amounts of vitellogenin, although sites further downstream were not estrogenic. The situation in the fourth river was quite different; not only was the effluent extremely estrogenic (a maximum vitellogenin response in the mg/ml range was attained), but so were all the other study sites on the river, the last of which was 5 km downstream of where the effluent entered. This particular river receives trade effluent from wool-scouring mills, which contains much higher concentrations of alkylphenolic chemicals than any of the other discharges studied. It is suggested that these chemicals probably account for the estrogenic activity of this river. The final (fifth) river showed no estrogenic activity, not even in the neat effluent. This discharge comes from a very small STW, which receives no trade waste, and one or both of these factors may account for why the effluent (and hence the river) was not estrogenic.

The prediction of risks posed by pharmaceuticals and personal care products in the aquatic environment now and in the future is one of the top 20 research questions regarding these contaminants following growing concern for their biological effects on fish and other animals. To this end it is important that areas experiencing the greatest risk are identified, particularly in countries experiencing water stress, where dilution of pollutants entering river networks is more limited. This study is the first to use hydrological models to estimate concentrations of pharmaceutical and natural steroid estrogens in a water stressed catchment in South Australia alongside a UK catchment and to forecast their concentrations in 2050 based on demographic and climate change predictions. The results show that despite their differing climates and demographics, modeled concentrations of steroid estrogens in effluents from Australian sewage treatment works and a receiving river were predicted (simulated) to be similar to those observed in the UK and Europe, exceeding the combined estradiol equivalent’s predicted no effect concentration for feminization in wild fish. Furthermore, by 2050 a moderate increase in estrogenic contamination and the potential risk to wildlife was predicted with up to a 2-fold rise in concentrations.

Vitellogenin, a yolk-precursor protein normally found only in the blood plasma of sexually mature female teleosts and other egg-laying vertebrates, was used as an indicator of exposure of male rainbow trout (Oncorhynchus mykiss) to exogenous estrogens. Vitellogenin concentrations were measured using a specific radioimmunoassay for trout. Cages containing adult male trout were placed at the points of discharge and at varying distances downstream of treated sewage effluent outfalls into the River Lea (UK) during the summer and winter months of 1992. After 3 weeks exposure at the majority of sites, fish held up to 15 km downstream of inputs showed an increase in plasma vitellogenin concentration, with statistically significant elevations up to 4.5 km downstream. A repeat survey in November 1992 below Harpenden sewage treatment works showed that the only two stations to give a significant response were 3 m and 1.6 km downstream of the outfall. This reduced effect compared to the first survey is thought to be due to dilution of both the influent sewage to the treatment works and of the river water itself by increased rainfall, the overall increase in dilution being approx. 36%. Trout were also placed in 15 raw water storage reservoirs in southeast England during the summer of 1993 for an exposure period of 6 weeks. No biologically significant increases in plasma vitellogenin concentration were observed in any of the reservoirs.

It is becoming evident that an increasing number of widely used industrial and agricultural chemicals are estrogenic. The biodegradation products of a major group of nonionic surfactants, the alkylphenol polyethoxylates, are one such group. Some of these chemicals are widespread aquatic pollutants, and bioconcentrate in aquatic biota. Exposure of male rainbow trout (Oncorhynchus mykiss) to four different alkylphenolic chemicals caused synthesis of vitellogenin, a process normally dependent on endogenous estrogens, and a concomitant inhibition of testicular growth. The magnitude of these estrogenic effects was dependent on the estrogenic potency of the chemical, the stage of reproductive development of the fish, and the concentration of the chemical in the water. These results support the contention that exposure of wildlife to environmentally persistent estrogenic chemicals can result in deleterious reproductive consequences.

A rapidly increasing number of chemicals, or their degradation products, are being recognized as possessing estrogenic activity, albeit usually weak. We have found that effluent from sewage treatment works contains a chemical, or mixture of chemicals, that induces vitellogenin synthesis in male fish maintained in the effluent, thus indicating that the effluent is estrogenic. The effect was extremely pronounced and occurred at all sewage treatment works tested. The nature of the chemical or chemicals causing the effect is presently not known. However, we have tested a number of chemicals known to be estrogenic to mammals and have shown that they are also estrogenic to fish; that is, no species specificity was apparent. Many of these weakly estrogenic chemicals are known to be present in effluents. Further, a mixture of different estrogenic chemicals was considerably more potent than each of the chemicals when tested individually, suggesting that enhanced effects could occur when fish are exposed simultaneously to various estrogenic chemicals (as is likely to occur in rivers receiving effluent). Subsequent work should determine whether exposure to these chemicals at the concentrations present in the environment leads to any deleterious physiological effects.

Alkylphenol-polyethoxylates (APnEO, n = 1–40) are a major group of surfactants and are normally present in raw sewage. Many of the products of the biodegradation of these compounds are both persistent and present in substantial quantities in effluent and in river water. We report here on the use of an in vitro bioassay to determine the oestrogenic potencies of these compounds to fish. The bioassay is based on the fact that the synthesis of vitellogenin by hepatocytes is oestrogen dependent. Of the compounds tested, 4-nonylphenol, 4-tert-octylphenol, 4-tert-butylphenol, 4-nonylphenol-diethoxylate, Tergitol-NP9, and 4-nonylphenoxycarboxylic acid were all weakly oestrogenic, with potencies between about 1 × 10−4 to 1 × 10−6 the activity of 17β-oestradiol The oestrogenic activity observed appeared to be confined to para or 4 substituted compounds, because 2-tert-butylphenol and 3-tert-butylphenol were inactive. The polyethoxylate compounds became less oestrogenic with increasing lengh of the ethoxy chain. Thus cells exposed to Tergitol NP40EO (with a chain length of 40) did not secrete vitellogenin. Simultaneous exposure of the hepatocytes to Tamoxifen (an oestrogen antagonist) and effective doses of representative compounds caused an inhibition of the oestrogenic effect in all cases, suggesting that the action of these compounds is mediated by the oestradiol receptor.

Whilst the effects of oestrogenic contaminants in the aquatic environment are well documented in fish, effects in invertebrate species has been subject to debate, possibly due to differences in experimental conditions (temperature, timing and duration of exposure) between studies. It has been suggested that molluscs are only susceptible to oestrogens in periods either following the main spawning or leading up to the maturation of gametes. To investigate this possibility, two temperate, seasonally reproducing gastropods (Planorbarius corneus and Viviparus viviparus) were exposed to two concentrations of 17β-oestradiol (E2; 10 ng/l and 100 ng/l nominal) in an outdoor mesocosm (subject to natural seasons). In addition, P. corneus was also exposed to E2 (1, 10 and 100 ng/l) in the laboratory at temperatures and photoperiods to simulate summer and autumn. In the mesocosm, both snail species produced similar numbers of eggs/embryos as reference groups in the summer, but the groups exposed to 10 ng E2/l (nominal) had significantly higher productivities after the onset of autumn, when entering their quiescent phase, whilst the snails exposed to a higher concentration (100 ng/l, nominal) had an increased rate of mortality, and did not experience increased reproduction. In the laboratory, the rate of egg laying in P. corneus was unaffected in simulated summer (20 °C, 16 h photoperiod), but snails exposed to 10 and 100 ng/l (nominal) in simulated autumn (15 °C, 12 h photoperiod) showed a concentration-dependent inhibition of the natural decline in egg laying observed in the control snails. Overall, rather than an increase in reproductive rate, the response of this species was a perpetuation of summer reproductive rates into autumn. We conclude that exposure to E2 can affect reproduction in the freshwater gastropods studied, but in P. corneus at least, this is dependent on the seasonal conditions (temperature and photoperiod) at which exposures are made.

The official published version can be obtained at the link below - copyright @ 2011 John Wiley and Sons Vitellogenin (VTG) is a precursor of egg-yolk protein and is therefore present at high concentrations in the plasma of female fish. In male fish, VTG concentrations are usually undetectable or low but can be induced upon exposure to estrogenic substances either via the water or the diet. This work was performed to determine the reason for the apparently elevated VTG concentrations in unexposed stock male fathead minnow maintained in our laboratory. The results showed clearly that some Of the food given to the fish was estrogenic and that replacement of this with nonestrogenic food led to a significant reduction in the basal VTG levels measured in male fish after a six-month period. This reduction in male VTG concentrations drastically increased the sensitivity of the VTG test in further studies carried out with these fish. Moreover, a review of published concentrations of VTG in unexposed male fathead minnow suggests that this problem may exist in other laboratories. The fathead minnow is a standard ecotoxicological fish test species, so these findings will be of interest to any laboratory carrying out fish tests on endocrine-disrupting chemicals.

Wild male roach (Rutilus rutilus) living in U.K. rivers contaminated with estrogenic effluents from wastewater treatment works show feminized responses and have a reduced reproductive capability, but the chemical causation of sexual disruption in the roach has not been established. Feminized responses were induced in male roach exposed to environmentally relevant concentrations of the pharmaceutical estrogen 17alpha-ethinylestradiol, EE2 (up to 4 ng/ L), during early life (from fertilization to 84 days posthatch, dph), and these effects were signaled by altered patterns of expression of two cloned roach estrogen receptor (ER) subtypes, ERalpha. and ERbeta, in the brain and gonad/ liver. Transactivation assays were developed for both roach ER subtypes and the estrogenic potencies of steroidal estrogens differed markedly at the different ER subtypes. EE2 was by far the most potent chemical, and estrone (E1, the most prevalent environmental steroid in wastewater discharges) was equipotent with estradiol (E2) in activating the ERs. Comparison of the EC50 values for the compounds tested showed that ERbeta was 3-21-fold more sensitive to natural steroidal estrogens and 54-fold more sensitive to EE2 as compared to ERalpha. These findings add substantial support to the hypothesis that steroidal estrogens play a significant role in the induction of intersex in roach populations in U.K. rivers and that the molecular approach described could be usefully applied to understand interspecies sensitivity to xenoestrogens.

We have isolated novel molluskan steroid receptor transcripts orthologous to vertebrate estrogen receptors (ERs) and estrogen receptor-related receptors (ERRs) from the freshwater snail Marisa cornuarietis. Radiolabeled ligand binding analyses showed that neither recombinant receptor protein specifically bound 17beta-estradiol over the range applied (0.3-9.6 nM). These novel receptor transcripts have thus been designated mcER-like and mcERR respectively. Quantitative PCR revealed mcER-like to be expressed ubiquitously throughout a range of male and female structures studied, including neural and reproductive tissues. Highest absolute levels were seen in the male penis-sheath complex. The mcERR mRNA was also expressed ubiquitously throughout all male and female tissues analyzed here, with very low absolute transcript numbers in female accessory sex structures compared to other tissues.

Surveys of U.K. rivers have shown a high incidence of sexual disruption in populations of wild roach (Rutilus rutilus) living downstream from wastewater treatment works (WwTW), and the degree of intersex (gonads containing both male and female structural characteristics) has been correlated with the concentration of effluent in those rivers. In this study, we investigated feminized responses to two estrogenic WwTWs in roach exposed for periods during life stages of germ cell division (early life and the postspawning period). Roach were exposed as embryos from fertilization up to 300 days posthatch (dph; to include the period of gonadal sex differentiation) or as postspawning adult males, and including fish that had received previous estrogen exposure, for either 60 or 120 days when the annual event of germ cell proliferation occurs. Both effluents induced vitellogenin synthesis in both life stages studied, and the magnitude of the vitellogenic responses paralleled the effluent content of steroid estrogens. Feminization of the reproductive ducts occurred in male fish in a concentration-dependent manner when the exposure occurred during early life, but we found no effects on the reproductive ducts in adult males. Depuration studies (maintenance of fish in clean water after exposure to WwTW effluent) confirmed that the feminization of the reproductive duct was permanent. We found no evidence of ovotestis development in fish that had no previous estrogen exposure for any of the treatments. In wild adult roach that had previously received exposure to estrogen and were intersex, the degree of intersex increased during the study period, but this was not related to the immediate effluent exposure, suggesting a previously determined programming of ovotestis formation.

Endocrine-disrupting chemicals, known to be present in the environment, have great potential for interfering with reproductive health in wildlife and humans. There is, however, little direct evidence that endocrine disruption has adversely affected fertility in any organism. In freshwater and estuarine fish species, for example, although a widespread incidence of intersex has been reported, it is not yet known if intersexuality influences reproductive success. The purpose of this study was, therefore, to determine gamete quality in wild intersex roach (Rutilus rutilus) by assessing sperm characteristics, fertilization success, and ability to produce viable offspring. The results clearly demonstrate that gamete production is reduced in intersex roach. A significantly lower proportion of moderately or severely feminized fish (17.4% and 33.3%, respectively) were able to release milt compared with normal male fish from contaminated rivers (in which 97.6% of the males were able to release milt), reference male fish (97.7%), or less severely feminized intersex fish (experiment 1: 85.8%, experiment 2: 97%). Intersex fish that did produce milt produced up to 50% less (in terms of volume per gram of testis weight) than did histologically normal male fish. Moreover, sperm motility (percentage of motile sperm and curvilinear velocity) and the ability of sperm to successfully fertilize eggs and produce viable offspring were all reduced in intersex fish compared with normal male fish. Male gamete quality (assessed using sperm motility, sperm density, and fertilization success) was negatively correlated with the degree of feminization in intersex fish (r = -0.603; P0.001) and was markedly reduced in severely feminized intersex fish by as much as 50% in terms of motility and 75% in terms of fertilization success when compared with either less severely feminized intersex fish or unaffected male fish. This is the first evidence documenting a relationship between the morphological effects (e.g., intersex) of endocrine disruption and the reproductive capabilities of any wild vertebrate. The results suggest that mixtures of endocrine-disrupting substances discharged into the aquatic environment could pose a threat to male reproductive health.

As a result of the introduction of tighter discharge limits and effluent treatment processes at source, the concentration of alkylphenol ethoxylates and nonylphenol present in the final effluent discharge from a sewage treatment works that treats trade effluent from the textiles industry was reduced. The estrogenic effects of the final effluent discharge to the Aire River were compared over a four-year period during which various treatment measures were introduced. Male rainbow trout exposed to the effluent on four occasions in consecutive years (1994-1997) showed a reduction in the level of induced vitellogenesis between 1994 and 1997. A marked decrease in gonadosomatic index (GSI) and increase in heptaosomatic index (HSI) was measured in fish exposed to the effluent in 1994. In successive years, these differences diminished, and in the case of the GSI no measurable difference was observed between fish exposed to the final effluent or those in the control group in 1997. However, an increase in HSI was still measurable in 1997 in fish exposed to the final effluent and at sites farther downstream. The reduction in the effects of the effluent paralleled the reduction in the concentration of nonylphenol as well as its mono- and diethoxylates, which have been demonstrated to produce estrogenic effects in trout exposed to these compounds in the laboratory. This study demonstrates that the setting of more restricted discharge limits for known estrogenic chemicals of industrial origin can lead to significant reductions in the estrogenic activity of the watercourses into which the effluents are discharged.

Wild roach (Rutilus rutilus) have been found with intersex gonads in rivers throughout the United Kingdom. The incidence of intersexuality is strongly correlated with discharges of estrogenic treated sewage effluent into those rivers, and this has led to the hypothesis that estrogenic chemicals in effluents are feminizing wild male fish. In this study, early-life stage roach (50 days post hatch, dph) were exposed for 150 days to a graded concentration (0%, 12.5%, 25%, 50%, and 100%) of treated sewage (primarily domestic) effluent to examine the effects of exposure on sexual differentiation and development. Measurement of steroid estrogens and alkylphenolic chemicals in the effluent and a resulting dose-dependent induction of vitellogenin (VTG; a female-specific, estrogen-dependent plasma protein) confirmed that the fish had been exposed and responded to "estrogen" in the effluent. Exposure to treated sewage effluent induced feminization of the reproductive ducts in "male" roach in a dose-dependent manner (in full-strength effluent, 100% of the fish had feminized ducts), indicating that the disruption of the gonad ducts seen in wild roach is the result of exposure to treated sewage effluents during early-life stages. There were no effects of treated sewage effluent exposure on germ cell development; therefore, no oocytes occurred in the testes of the feminized male roach. Subsequent, depuration of the effluent exposed fish in "clean" water for 150 days resulted in a reduction in plasma VTG but no alteration of the feminized ducts, indicating that the effect of the treated sewage effluent on reproductive duct development was permanent. The causality of oocytes in the testes of wild male roach therefore remains to be elucidated.

Over the last two decades, there has been increasing scientific concern and public debate regarding the adverse effects of chemical pollutants in the environment that can interfere with the normal functioning of the endocrine system in wildlife and in humans (the so-called endocrine-disrupting chemicals, EDCs). These concerns have been fueled primarily by reports of disrupted reproductive function and development in certain wildlife—mammals, birds, fish, amphibians, and mollusks—and by the increased incidence of certain diseases of the endocrine system in humans. Investigators hypothesize that EDCs are the cause. Some of the adverse effects observed in wildlife species are strongly associated with exposure to chemicals that mimic or interfere with hormone function, particularly estrogen function, but in many cases, the causal link between exposure to EDCs and endocrine disruption is unclear. Because of the diverse effects of EDCs on the thyroid, retinoid, androgen, estrogen, and corticosteroid systems of a wide range of animals, it is imperative that research continues to address the extent of the risk posed by EDCs to wildlife. The ecological relevance of endocrine disruption in wildlife is, however, difficult to quantify, as there is limited understanding of how physiological changes affect the individual animal and how individual responses affect population and community. Furthermore, a major challenge faced by environmental biologists is the need to place endocrine disruption into context with other environmental pressures faced by our wildlife populations, for example, global warming.

Sewage, a complex mixture of organic and inorganic chemicals, is considered to be a major source of environmental pollution. A random screen of 20 organic man-made chemicals present in liquid effluents revealed that half appeared able to interact with the estradiol receptor. This was demonstrated by their ability to inhibit binding of 17 beta-estradiol to the fish estrogen receptor. Further studies, using mammalian estrogen screens in vitro, revealed that the two phthalate esters butylbenzyl phthalate (BBP) and di-n-butylphthalate (DBP) and a food antioxidant, butylated hydroxyanisole (BHA) were estrogenic; however, they were all less estrogenic than the environmental estrogen octylphenol. Phthalate esters, used in the production of various plastics (including PVC), are among the most common industrial chemicals. Their ubiquity in the environment and tendency to bioconcentrate in animal fat are well known. Neither BBP nor DBP were able to act as antagonists, indicating that, in the presence of endogenous estrogens, their overall effect would be cumulative. Recently, it has been suggested that environmental estrogens may be etiological agents in several human diseases, including disorders of the male reproductive tract and breast and testicular cancers. The current finding that some phthalate compounds and some food additives are weakly estrogenic in vitro, needs to be supported by further studies on their effects in vivo before any conclusions can be made regarding their possible role in the development of these conditions. Images Figure 1. Figure 2. Figure 3. Figure 4.