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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.

Copyright @ 2013 The authors. Published by the Royal Society under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0/, which permits unrestricted use, provided the original author and source are credited. The use of human and veterinary pharmaceuticals is increasing. Over the past decade, there has been a proliferation of research into potential environmental impacts of pharmaceuticals in the environment. A Royal Society-supported seminar brought together experts from diverse scientific fields to discuss the risks posed by pharmaceuticals to wildlife. Recent analytical advances have revealed that pharmaceuticals are entering habitats via water, sewage, manure and animal carcases, and dispersing through food chains. Pharmaceuticals are designed to alter physiology at low doses and so can be particularly potent contaminants. The near extinction of Asian vultures following exposure to diclofenac is the key example where exposure to a pharmaceutical caused a population-level impact on non-target wildlife. However, more subtle changes to behaviour and physiology are rarely studied and poorly understood. Grand challenges for the future include developing more realistic exposure assessments for wildlife, assessing the impacts of mixtures of pharmaceuticals in combination with other environmental stressors and estimating the risks from pharmaceutical manufacturing and usage in developing countries. We concluded that an integration of diverse approaches is required to predict 'unexpected' risks; specifically, ecologically relevant, often long-term and non-lethal, consequences of pharmaceuticals in the environment for wildlife and ecosystems.

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.

Perspectives | Editorial The Impact of Endocrine Disruption: A Consensus Statement on the State of the Science doi:10.1289/ehp.1205448 Ake Bergman, 1 Jerrold J. Heindel, 2,a Tim Kasten, 3,b Karen A. Kidd, 4 Susan Jobling, 5 Maria Neira, 6,c R. Thomas Zoeller, 7 Georg Becher, 8 Poul Bjerregaard, 9 Riana Bornman, 10 Ingvar Brandt, 11 Andreas Kortenkamp, 5 Derek Muir, 12 Marie-Noel Brune Drisse, 6,c Roseline Ochieng, 13 Niels E. Skakkebaek, 14 Agneta Sunden Bylehn, 3,b Taisen Iguchi, 15 Jorma Toppari, 16 and Tracey J. Woodruff 17 1 Department of Materials and Environmental Chemistry, Stockholm University, Stockholm, Sweden; 2 National Institute of Environmental Health Sciences, National Institutes of Health, Department of Health and Human Services, Research Triangle Park, North Carolina, USA; 3 United Nations Environment Programme (UNEP), Geneva, Switzerland; 4 Department of Biology & Canadian Rivers Institute, University of New Brunswick, Saint John, New Brunswick, Canada; 5 Institute for the Environment, Brunel University, Uxbridge, Middlesex, United Kingdom; 6 Department of Public Health and Environment, World Health Organization, Geneva, Switzerland; 7 Biology Department, Morrill Science Center, University of Massachusetts, Amherst, Massachusetts, USA; 8 Division of Environmental Medicine, Norwegian Institute of Public Health, Oslo, Norway; 9 Institute of Biology, University of Southern Denmark, Odense, Denmark; 10 Department of Urology, School of Medicine, Steve Biko Academic Hospital, University of Pretoria, Pretoria, South Africa; 11 Department of Environmental Toxicology, Uppsala University, Uppsala, Sweden; 12 Aquatic Ecosystems Protection Research Division, Water Science & Technology Directorate, Environment Canada, Burlington, Ontario, Canada; 13 Department of Paediatrics and Child Health, Aga Khan University Hospital, Nairobi, Kenya; 14 University Department of Growth and Reproduction, Rigshospitalet, Copenhagen, Denmark; 15 Department of Bioenvironmental Science, Okazaki Institute for Integrative Bioscience, National Institute for Basic Biology, National Institutes of Natural Sciences, Okazaki Aichi, Japan; 16 Departments of Physiology and Paediatrics, University of Turku, Turku, Finland; 17 Department of Obstetrics, Gynecology and Reproductive Sciences, Institute for Health Policy Studies, University of California San Francisco, Oakland, California, USA In 2002, the joint International Programme on Chemical Safety (IPCS) of the World Health Organization (WHO), the United Nations Environment Programme (UNEP), and the International Labour Organisation (ILO) published a report titled Global Assessment of the State-of-the-Science of Endocrine Disruptors (http://www.who.int/ ipcs/publications/new_issues/endocrine_disruptors/en/). Since 2002, intense scientific work has improved our understanding of the impacts of endocrine-disrupting chemicals (EDCs) on human and wildlife health, such that in 2012, the UNEP and WHO, in collaboration with international experts, have produced an updated document on EDCs, State of the Science of Endocrine Disrupting Chemicals - 2012 (http:// www.who.int/ceh/publications/endocrine/en/index.html) that includes scientific information on human and wildlife impacts and lists key concerns for decision makers and others concerned about the future of human and wildlife health. The basis for these key concerns is described in the State of the Science of Endocrine Disrupting Chemicals - 2012 (http://www.who. int/ceh/publications/endocrine/en/index.html) and includes extensive references to the science behind the concerns. A shorter summary, primarily for decision makers, elabo­rates on the key concerns listed below and and also on suggested considerations related to EDCs (State of the Science of Endocrine Disrupting Chemicals - 2012: Summary for Decision-Makers; http://www.who.int/ceh/publications/endocrine/en/ index.html). The key concerns noted in the State of the Science of Endocrine Disrupting Chemicals - 2012 (http://www.who.int/ceh/publications/ endocrine/en/index.html) are as follows: • Human and wildlife health depends on the ability to reproduce and develop normally. This is not possible without a healthy endocrine system. • Three strands of evidence fuel concerns over endocrine disruptors: ǹ ǹ The high incidence and the increasing trends of many endocrine- related disorders in humans; ǹ ǹ Observations of endocrine-related effects in wildlife populations; ǹ ǹ The identification of chemicals with endocrine disrupting proper- ties linked to disease outcomes in laboratory studies. • Many endocrine-related diseases and disorders are on the rise. ǹ ǹ Large proportions (up to 40%) of young men in some countries have low semen quality, which reduces their ability to father children. ǹ ǹ The incidence of genital malformations, such as non-descending testes (cryptorchidisms) and penile malformations (hypospadias), in baby boys has increased over time or levelled off at unfavour- ably high rates. ǹ ǹ The incidence of adverse pregnancy outcomes, such as preterm birth and low birth weight, has increased in many countries. ǹ ǹ Neurobehavioural disorders associated with thyroid disruption affect a high proportion of children in some countries and have increased over past decades. ǹ ǹ Global rates of endocrine-related cancers (breast, endometrial, ovarian, prostate, testicular and thyroid) have been increasing over the past 40–50 years. ǹ ǹ There is a trend towards earlier onset of breast development in young girls in all countries where this has been studied. This is a risk factor for breast cancer. ǹ ǹ The prevalence of obesity and type 2 diabetes has dramatically increased worldwide over the last 40 years. WHO estimates that 1.5 billion adults worldwide are overweight or obese and that the number with type 2 diabetes increased from 153 million to 347 million between 1980 and 2008. • Close to 800 chemicals are known or suspected to be capable of inter- fering with hormone receptors, hormone synthesis or hormone con- version. However, only a small fraction of these chemicals have been investigated in tests capable of identifying overt endocrine effects in intact organisms. ǹ ǹ The vast majority of chemicals in current commercial use have not been tested at all. ǹ ǹ This lack of data introduces significant uncertainties about the true extent of risks from chemicals that potentially could disrupt the endocrine system. • Human and wildlife populations all over the world are exposed to EDCs. ǹ ǹ There is global transport of many known and potential EDCs through natural processes as well as through commerce, leading to worldwide exposure. ǹ ǹ Unlike 10 years ago, we now know that humans and wildlife are exposed to far more EDCs than just those that are POPs [persis- tent organic pollutants]. ǹ ǹ Levels of some newer POPs in humans and wildlife are still increasing, and there is also exposure to less persistent and less bioaccumulative, but ubiquitous, chemicals. Address correspondence to A. Bergman, Department of Materials and Environmental Chemistry, Stockholm University, SE-10691 Stockholm, Sweden. E-mail: ake.bergman@mmk.su.se United Nations Environment Programme (UNEP) address: 13 chemin des Anemones, CH-1219 Chatelaine, Geneva, Switzerland. a The author is an employee of the National Institute of Environmental Health Sciences (NIEHS), National Institutes of Health (NIH); any statements, opinions, or conclusions contained herein do not necessar- ily represent the statements, opinions, or conclusions of the NIEHS, NIH, or the U.S. government. b The authors are staff members of the United Nations Environment Programme (A.S.B. is now retired). The authors alone are responsible for the views expressed in this article, and they do not necessarily represent the decisions or policies of the United Nations Environment Programme. c The authors are staff mem- bers of the World Health Organization (WHO). The authors alone are responsible for the views expressed in this publication, and they do not necessarily represent the views, decisions, or policies of the WHO. This article should not be reproduced for use in association with the promotion of commercial products, services, or any legal entity. The WHO does not endorse any specific organization or products. Any reproduction of this article cannot include the use of the WHO logo. The authors declare they have no actual or potential competing ­financial interests. A 104 Environmental Health Perspectives volume 121 | number 4 | April 2013 •

The “common sense” intervention by toxicology journal editors regarding proposed European Union endocrine disrupter regulations ignores scientific evidence and well-established principles of chemical risk assessment. In this commentary, endocrine disrupter experts express their concerns about a recently published, and is in our considered opinion inaccurate and factually incorrect, editorial that has appeared in several journals in toxicology. Some of the shortcomings of the editorial are discussed in detail. We call for a better founded scientific debate which may help to overcome a polarisation of views detrimental to reaching a consensus about scientific foundations for endocrine disrupter regulation in the EU., Environmental Health, 12 (1), ISSN:1476-069X

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.

This is an Open Access article: verbatim copying and redistribution of this article are permitted in all media for any purpose, provided this notice is preserved along with the article's original DOI., BACKGROUND In their safety evaluations of bisphenol A (BPA), the U.S. Food and Drug Administration (FDA) and a counterpart in Europe, the European Food Safety Authority (EFSA), have given special prominence to two industry-funded studies that adhered to standards defined by Good Laboratory Practices (GLP). These same agencies have given much less weight in risk assessments to a large number of independently replicated non-GLP studies conducted with government funding by the leading experts in various fields of science from around the world. OBJECTIVES We reviewed differences between industry-funded GLP studies of BPA conducted by commercial laboratories for regulatory purposes and non-GLP studies conducted in academic and government laboratories to identify hazards and molecular mechanisms mediating adverse effects. We examined the methods and results in the GLP studies that were pivotal in the draft decision of the U.S. FDA declaring BPA safe in relation to findings from studies that were competitive for U.S. National Institutes of Health (NIH) funding, peer-reviewed for publication in leading journals, subject to independent replication, but rejected by the U.S. FDA for regulatory purposes. DISCUSSION Although the U.S. FDA and EFSA have deemed two industry-funded GLP studies of BPA to be superior to hundreds of studies funded by the U.S. NIH and NIH counterparts in other countries, the GLP studies on which the agencies based their decisions have serious conceptual and methodologic flaws. In addition, the U.S. FDA and EFSA have mistakenly assumed that GLP yields valid and reliable scientific findings (i.e., “good science”). Their rationale for favoring GLP studies over hundreds of publically funded studies ignores the central factor in determining the reliability and validity of scientific findings, namely, independent replication, and use of the most appropriate and sensitive state-of-the-art assays, neither of which is an expectation of industry-funded GLP research. CONCLUSIONS Public health decisions should be based on studies using appropriate protocols with appropriate controls and the most sensitive assays, not GLP. Relevant NIH-funded research using state-of-the-art techniques should play a prominent role in safety evaluations of 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.

This document is a summary statement of the outcome from the meeting: “Bisphenol A: An Examination of the Relevance of Ecological, In vitro and Laboratory Animal Studies for Assessing Risks to Human Health” sponsored by both the NIEHS and NIDCR at NIH/DHHS, as well as the US-EPA and Commonweal on the estrogenic environmental chemical bisphenol A (BPA, 2,2-bis(4-hydroxyphenyl)propane; CAS# 80-05-7). The meeting was held in Chapel Hill, NC, 28–30 November 2006 due to concerns about the potential for a relationship between BPA and negative trends in human health that have occurred in recent decades. Examples include increases in abnormal penile/urethra development in males, early sexual maturation in females, an increase in neurobehavioral problems such as attention deficit hyperactivity disorder (ADHD) and autism, an increase in childhood and adult obesity and type 2 diabetes, a regional decrease in sperm count, and an increase in hormonally mediated cancers, such as prostate and breast cancers. Concern has been elevated by published studies reporting a relationship between treatment with “low doses” of BPA and many of theses negative health outcomes in experimental studies in laboratory animals as well as in vitro studies identifying plausible molecular mechanisms that could mediate such effects. Importantly, much evidence suggests that these adverse effects are occurring in animals within the range of exposure to BPA of the typical human living in a developed country, where virtually everyone has measurable blood, tissue and urine levels of BPA that exceed the levels produced by doses used in the “low dose” animal experiments.

3 pages.-- PMID: 16818253 [PubMed].-- PMCID: PMC1874174., This article is part of the monograph "The Ecological Relevance of Chemically Induced Endocrine Disruption in Wildlife"., Tens of thousands of man-made chemicals are in regular use and discharged into the environment. Many of them are known to interfere with the hormonal systems in humans and wildlife. Given the complexity of endocrine systems, there are many ways in which endocrine-disrupting chemicals (EDCs) can affect the body’s signaling system, and this makes unraveling the mechanisms of action of these chemicals difficult. A major concern is that some of these EDCs appear to be biologically active at extremely low concentrations. There is growing evidence to indicate that the guiding principle of traditional toxicology that "the dose makes the poison" may not always be the case because some EDCs do not induce the classical dose–response relationships. The European Union project COMPRENDO (Comparative Research on Endocrine Disrupters—Phylogenetic Approach and Common Principles focussing on Androgenic/Antiandrogenic Compounds) therefore aims to develop an understanding of potential health problems posed by androgenic and antiandrogenic compounds (AACs) to wildlife and humans by focusing on the commonalities and differences in responses to AACs across the animal kingdom (from invertebrates to vertebrates)., This project is funded by the European Union (COMPRENDO Project, contract EVK1-CT-2002-00129).

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.

Sexually mature male common carp (Cyprinus carpio) were exposed for a 3-month period to sublethal concentrations of 4-tert-pentylphenol (TPP) and to 17β-estradiol (E2) during spermatogenesis. This was part of a broad research programme investigating the effects of TPP on the life stages of the carp which might prove susceptible to endocrine disruption. Exposure of adult male carp to the pseudo-estrogen TPP, or to E2 during sexual maturation, led to elevated plasma levels of vitellogenin (P

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.

This study assessed whether exposure of male rats to two estrogenic, environmental chemicals, 4-octylphenol (OP) and butyl benzyl phthalate (BBP) during gestation or during the first 21 days of postnatal life, affected testicular size or spermatogenesis in adulthood (90-95 days of age). Chemicals were administered via the drinking water or concentrations of 10-1000 micrograms/l (OP) or 1000 micrograms/l (BBP), diethylstilbestrol (DES; 100 micrograms/l) and an octylphenol polyethoxylate (OPP; 1000 micrograms/l), which is a weak estrogen or nonestrogenic in vitro, were administered as presumptive positive and negative controls, respectively. Controls received the vehicle (ethanol) in tap water. In study 1, rats were treated from days 1-22 after births in studies 2 and 3, the mothers were treated for approximately 8-9 weeks, spanning a 2-week period before mating throughout gestation and 22 days after giving birth. With the exception of DES, treatment generally had no major adverse effect or body weight: in most instances, treated animals were heavier than controls at day 22 and at days 90-95. Exposure to OP, OPP, or BBP at a concentration of 1000 micrograms/1 resulted in a small (5-13%) but significant (p < 0.01 or p < 0.0001) reduction in mean testicular size in studies 2 and 3, an effect that was still evident when testicular weight was expressed relative to body, weight or kidney weight. The effect of OPP is attributed to its metabolism in vivo to OP. DES exposure caused similar reductions in testicular size but also caused reductions in body weight, kidney weight, and litter size. Ventral prostate weight was reduced significantly in DES-treated rats and to minor extent in OP-treated rats. Comparable but more minor effects of treatment with DES or OP on testicular size were observed in study 1. None of the treatments had any adverse effect on testicular morphology or on the cross-sectional area of the lumen or seminiferous epithelium at stages VII-VIII of the spermatogenic cycle, but DES, OP, and BBP caused reductions of 10-21% (p < 0.05 to p < 0.001) in daily sperm production. Humans are exposed to phthalates, such as BBP, and to alkylphenol polyethoxylates, such as OP, but to what extent is unknown. More detailed studies are warranted to assess the possible risk to the development of the human testis from exposure to these and other environmental estrogens. Images Figure 1. Figure 2. Figure 3. A Figure 3. B Figure 3. C Figure 3. D Figure 4.

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.

Urgent public debate is needed over a European proposal to regulate environmental levels of the active ingredient in birth-control pills, say Richard Owen and Susan Jobling.

This article has been made available through the Brunel Open Access Publishing Fund. Sexual disruption in wild fish has been linked to the contamination of river systems with steroid oestrogens, including the pharmaceutical 17α-ethinylestradiol, originating from domestic wastewaters. As analytical chemistry has advanced, more compounds derived from the human usage of pharmaceuticals have been identified in the environment and questions have arisen as to whether these additional pharmaceuticals may also impact sexual disruption in fish. Indeed, pharmaceutical anti-androgens have been shown to induce such effects under laboratory conditions. These are of particular interest since anti-androgenic biological activity has been identified in the aquatic environment and is potentially implicated in sexual disruption alone and in combination with steroid oestrogens. Consequently, predictive modelling was employed to determine the concentrations of two anti-androgenic human pharmaceuticals, bicalutamide and cyproterone acetate, in UK sewage effluents and river catchments and their combined impacts on sexual disruption were then assessed in two fish models. Crucially, fish were also exposed to the anti-androgens in combination with steroid oestrogens to determine whether they had any additional impact on oestrogen induced feminisation. Modelling predicted that the anti-androgenic pharmaceuticals were likely to be widespread in UK river catchments. However, their concentrations were not sufficient to induce significant responses in plasma vitellogenin concentrations, secondary sexual characteristics or gross indices in male fathead minnow or intersex in Japanese medaka alone or in combination with steroid oestrogens. However, environmentally relevant mixtures of oestrone, 17β-oestradiol and 17α-ethinylestradiol did induce vitellogenin and intersex, supporting their role in sexual disruption in wild fish populations. Unexpectedly, a male dominated sex ratio (100% in controls) was induced in medaka and the potential cause and implications are briefly discussed, highlighting the potential of non-chemical modes of action on this endpoint.

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Background: Treated effluents from wastewater treatment works can comprise a large proportion of the flow of rivers in the developed world. Exposure to these effluents, or the steroidal estrogens they contain, feminizes wild male fish and can reduce their reproductive fitness. Long-term experimental exposures have resulted in skewed sex ratios, reproductive failures in breeding colonies, and population collapse. This suggests that environmental estrogens could threaten the sustainability of wild fish populations.Results: Here we tested this hypothesis by examining population genetic structures and effective population sizes (Ne) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. Ne was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between Ne and the predicted estrogen exposure at 28 sample sites. Furthermore, examination of the population genetic structure of roach in the region showed that some populations have been confined to stretches of river with a high proportion of estrogenic effluent for multiple generations and have survived, apparently without reliance on immigration of fish from less polluted sites.Conclusions: These results demonstrate that roach populations living in some effluent-contaminated river stretches, where feminization is widespread, are self-sustaining. Although we found no evidence to suggest that exposure to estrogenic effluents is a significant driving factor in determining the size of roach breeding populations, a reduction in Ne of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model. © 2014 Hamilton et al.; licensee BioMed Central Ltd. Natural Environmental Research Council (NERC) grant numbers NE/G019355/1 and NE/K004263/1 and the Environment Agency (England and Wales).