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Data availability: The databases generated during an/or analysed during the current study are available from the corresponding author on reasonable request. Copyright © The Author(s) 2022. Lentiviral vectors (LV) are attractive for permanent and effective gene therapy. However, integration into the host genome can cause insertional mutagenesis highlighting the importance of understanding of LV integration. Insertion site (IS) tethering is believed to involve cellular proteins such as PSIP1/LEDGF/p75, which binds to the virus pre-integration complexes (PICs) helping to target the virus genome. Transcription factors (TF) that bind both the vector LTR and host genome are also suspected influential to this. To determine the role of TF in the tethering process, we mapped predicted transcription factor binding sites (pTFBS) near to IS chosen by HIV-1 LV using a narrow 20 bp window in infected human induced pluripotent stem cells (iPSCs) and their hepatocyte-like cell (HLC) derivatives. We then aligned the pTFBS with these sequences found in the LTRs of native and self-inactivated LTRs. We found significant enrichment of these sequences for pTFBS essential to HIV-1 life cycle and virus survival. These same sites also appear in HIV-1 patient IS and in mice infected with HIV-1 based LV. This in silco data analysis suggests pTFBS present in the virus LTR and IS sites selected by HIV-1 LV are important to virus survival and propagation. NC3Rs CRACK IT Challenge 21: InMutagene award, sponsored by GSK and Novartis.

Appendix. Supplementary materials: Download all supplementary files included with this article: Download Word document (https://ars.els-cdn.com/content/image/1-s2.0-S0166445X22001552-mmc1.docx 277KB); Download spreadsheet (https://ars.els-cdn.com/content/image/1-s2.0-S0166445X22001552-mmc2.xlsx 43KB). Copyright © 2022 The Authors. Experimental exposures aimed at assessing the risks posed by estrogens in waste-water treatment work (WwTW) effluents to fish populations have rarely considered whether populations differ in their sensitivity to estrogenic compounds. This is despite evidence that selection at genes involved in the estrogen response has occurred in wild populations, and evidence that genotype can influence estrogen-response. In this study we compare the effects of a two-year exposure to a low measured concentration (1.3 ng/L) of ethinylestradiol (EE2) on the sexual development of roach (Rutilus rutilus) whose parental generation was sampled from two river stretches heavily contaminated with WwTW effluent and from two without any known WwTW effluent contamination. Exposure to EE2 significantly reduced the proportion of genetic males and induced a range of feminized phenotypes in males. Significantly, exposure also increased the proportion of genetic females with vitellogenic oocytes from 51 to 96%, raising the possibility that estrogen pollution could impact populations of annually spawning fish species through advancing female reproduction by at least a year. However, there was no evidence that river origin affected sensitivity to estrogens in either sex. Thus, we conclude that chronic exposure to low level EE2 has reproductive health outcomes for both male and female roach, but we find no evidence that the nature or magnitude of the response is affected by the population origin. UK Natural Environmental Research Council (NERC; NE/K004263/1).

Polychlorinated biphenyls (PCBs) are highly toxic and persistent aquatic pollutants that are known to bioaccumulate in a variety of marine mammals. They have been associated with reduced recruitment rates and population declines in multiple species. Evidence to date documents effects of PCB exposures on female reproduction, but few studies have investigated whether PCB exposure impacts male fertility. Using blubber tissue samples of 99 adult and 168 juvenile UK-stranded harbour porpoises (Phocoena phocoena) collected between 1991 and 2017, here we show that PCBs exposures are associated with reduced testes weights in adults with good body condition. In animals with poor body condition, however, the impact of PCBs on testes weights was reduced, conceivably due to testes weights being limited by nutritional stress. This is the first study to investigate the relationship between PCB contaminant burden and testes weights in cetaceans and represents a substantial advance in our understanding of the relationship between PCB exposures and male reproductive biology in cetaceans. As testes weight is a strong indicator of male fertility in seasonally breeding mammals, we suggest the inclusion of such effects in population level impact assessments involving PCB exposures. Given the re-emergent PCB threat our findings are globally significant, with potentially serious implications for long-lived mammals. We show that more effective PCB controls could have a substantial impact on the reproductive health of coastal cetacean species and that management actions may need to be escalated to ensure adequate protection of the most vulnerable cetacean populations. Defra; Natural Environment Research Council (NERC) grant NE/L002485/1; Bertarelli Foundation through the Bertarelli Programme in Marine Science; partially funded by NERC grant NE/S000100/1 supporting the ChemPop project.

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 (Rutilus rutilus) inhabit river stretches polluted with estrogenic WwTW effluents. In this study, we examine whether such roach populations have evolved adaptations to tolerate estrogenic pollution by comparing frequency differences in single-nucleotide polymorphisms (SNPs) between populations sampled from rivers receiving either high- or low-level WwTW discharges. SNPs within 36 "candidate" genes, selected for their involvement in estrogenic responses, and 120 SNPs in reference genes were genotyped in 465 roaches. There was no evidence for selection in highly estrogen-dependent candidate genes, including those for the estrogen receptors, aromatases, and vitellogenins. The androgen receptor (ar) and cytochrome P450 1A genes were associated with large shifts in allele frequencies between catchments and in individual populations, but there is no clear link to estrogen pollution. Selection at ar in the effluent-dominated River Lee may have resulted from historical contamination with endocrine-disrupting pesticides. Critically, although our results suggest population-specific selection including at genes related to endocrine disruption, there was no strong evidence that the selection resulted from exposure to estrogen pollution.

In vertebrates, the steroidogenesis enzyme 5α-reductase converts testosterone to the more potent androgen 5α-dihydrotestosterone. Homologues of 5α-reductase genes have been identified in molluscs. However, recent findings suggest that vertebrate-type steroid androgens are not utilised in molluscan reproductive development. Genomic searches have revealed that molluscs do not possess many of the steroidogenic enzymes required to make testosterone, nor a nuclear androgen receptor. Consequently, the role of 5α-reductase in molluscs presents a mystery. Here, developmental exposures of Biomphalaria glabrata to selective pharmaceutical 5α-reductase inhibitors elicited a strong, highly reproducible phenotypic response characterised by the development of elongated “banana-shaped” shell morphology. In comparison to untreated snails, the shells are open-coiled and the whorls are unattached. Dutasteride (5α-reductase inhibitor) is approximately 10-times more potent at provoking the banana-shaped shell phenotype than finasteride, paralleling the pharmaceuticals’ efficacy in humans. Other enzyme inhibitors with different modes of action were tested to investigate the specificity of the phenotype. However, only the pharmaceutical 5α-reductase inhibitors provoked the response. Dutasteride elicited the same phenotype in a second gastropod, Physella acuta. In the absence of evidence for de novo androgen steroidogenesis in molluscs, these findings suggest that novel substrates for 5α-reductase exist in gastropods, lending support to the contention that molluscan endocrinology differs from the well-characterised vertebrate endocrine system.

© 2019 The Authors. Endocrine disrupting chemicals (EDCs) are substances that alter the function of the endocrine system and consequently cause adverse effects to humans or wildlife. The release of particular EDCs into the environment has been shown to negatively affect certain wildlife populations and has led to restrictions on the use of some EDCs. Current chemical regulations aim to balance the industrial, agricultural and/or pharmaceutical benefits of using these substances with their demonstrated or potential harm to human health or the environment. A summary is provided of the natural science evidence base informing the regulation of chemicals released into the environment that may have endocrine disrupting effects on wildlife. This summary is in a format (a ‘restatement’) intended to be policy-neutral and accessible to informed, but not expert, policy-makers and stakeholders. This study was funded by the Oxford Martin School, Oxford University.

© 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.

Biomphalaria snails are instrumental in transmission of the human blood fluke Schistosoma mansoni. With the World Health Organization's goal to eliminate schistosomiasis as a global health problem by 2025, there is now renewed emphasis on snail control. Here, we characterize the genome of Biomphalaria glabrata, a lophotrochozoan protostome, and provide timely and important information on snail biology. We describe aspects of phero-perception, stress responses, immune function and regulation of gene expression that support the persistence of B. glabrata in the field and may define this species as a suitable snail host for S. mansoni. We identify several potential targets for developing novel control measures aimed at reducing snail-mediated transmission of schistosomiasis., Biomphalaria glabrata is a fresh water snail that acts as a host for trematode Schistosoma mansoni that causes intestinal infection in human. This work describes the genome and transcriptome analyses from 12 different tissues of B glabrata, and identify genes for snail behavior and evolution.

© 2017 The Authors. Understanding the environmental factors driving species-genetic diversity correlations (SGDCs) is critical for designing appropriate conservation and management strategies to protect biodiversity. Yet, few studies have explored the impact of changing land use patterns on SGDCs specifically in aquatic communities. This study examined patterns of genetic diversity in roach (Rutilus rutilus L.) together with fish species composition across 19 locations in a large river catchment, spanning a gradient in land use. Our findings show significant correlations between some, but not all, species and genetic diversity end points. For example, genetic and species differentiation showed a weak but significant linear relationship across the Thames catchment, but additional diversity measures such as allelic richness and fish population abundance did not. Further examination of patterns in species and genetic diversity indicated that land use intensification has a modest effect on fish diversity compared to the combined influence of geographical isolation and land use intensification. These results indicate that environmental changes in riparian habitats have the potential to amplify shifts in the composition of stream fish communities in poorly connected river stretches. Conservation and management strategies for fish populations should, therefore, focus on enhancing connectivity between river stretches and limit conversion of nearby land to arable or urban use to maintain current levels of biodiversity.

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.

Background - The issue of endocrine disrupting chemicals (EDCs) is receiving wide attention from both the scientific and regulatory communities. Recent analyses of the EDC literature have been criticized for failing to use transparent and objective approaches to draw conclusions about the strength of evidence linking EDC exposures to adverse health or environmental outcomes. Systematic review methodologies are ideal for addressing this issue as they provide transparent and consistent approaches to study selection and evaluation. Objective methods are needed for integrating the multiple streams of evidence (epidemiology, wildlife, laboratory animal, in vitro, and in silico data) that are relevant in assessing EDCs. Methods - We have developed a framework for the systematic review and integrated assessment (SYRINA) of EDC studies. The framework was designed for use with the International Program on Chemical Safety (IPCS) and World Health Organization (WHO) definition of an EDC, which requires appraisal of evidence regarding 1) association between exposure and an adverse effect, 2) association between exposure and endocrine disrupting activity, and 3) a plausible link between the adverse effect and the endocrine disrupting activity. Results - Building from existing methodologies for evaluating and synthesizing evidence, the SYRINA framework includes seven steps: 1) Formulate the problem; 2) Develop the review protocol; 3) Identify relevant evidence; 4) Evaluate evidence from individual studies; 5) Summarize and evaluate each stream of evidence; 6) Integrate evidence across all streams; 7) Draw conclusions, make recommendations, and evaluate uncertainties. The proposed method is tailored to the IPCS/WHO definition of an EDC but offers flexibility for use in the context of other definitions of EDCs. Conclusions - When using the SYRINA framework, the overall objective is to provide the evidence base needed to support decision making, including any action to avoid/minimise potential adverse effects of exposures. This framework allows for the evaluation and synthesis of evidence from multiple evidence streams. Finally, a decision regarding regulatory action is not only dependent on the strength of evidence, but also the consequences of action/inaction, e.g. limited or weak evidence may be sufficient to justify action if consequences are serious or irreversible. The workshops that supported the writing of this manuscript were funded by the Swedish Foundation for Strategic Environmental Research “Mistra”. LNV was funded by Award Number K22ES025811 from the National Institute of Environmental Health Sciences of the National Institutes of Health. TJW was funded by The Clarence Heller Foundation (A123547), the Passport Foundation, the Forsythia Foundation, the National Institute of Environmental Health Sciences (grants ES018135 and ESO22841), and U.S. EPA STAR grants (RD83467801 and RD83543301). JT was funded by the Academy of Finland and Sigrid Juselius. UH was funded by the Danish EPA. KAK was funded by the Canada Research Chairs program grant number 950–230607.

Environmental estrogens originate from a variety of sources including sewage treatment plant (STP) effluents and adverse physiological effects (endocrine disruption) have been observed in several fish species sampled downstream of STP discharges. In this study we examined common carp (Cyprinus carpio) and roach (Rutilis rutilis) for signs of exposure to environmental estrogens in the iconic Yarra River, Melbourne, Australia. The Yarra River flows through the city of Melbourne and more than 2 million people live within the catchment. Two STPs discharge water into the Yarra River within the middle reaches, and the areas immediately downstream of these discharge locations were the focus of this study. Carp and roach were chosen as test species since both have been utilised extensively for endocrine disruption research throughout Europe, North America and Asia, and data from various international studies was used for comparison with the results of the present study.Neither species showed evidence of exposure to environmental estrogens, with no elevation of plasma vitellogenin levels in males and no incidence of intersex gonads. Most physiological endpoints in both species from this study were within ranges reported in carp and roach from reference sites in other studies, however some degenerative histological changes in both male and female gonads were observed. Surface water samples showed no estrogenic activity (measured by the yeast-estrogen screen, YES), but did display strong anti-estrogenic and weak androgenic activity (measured by the yeast-androgen screen, YAS).Whilst the results show no evidence of impacts from environmental estrogens in the Yarra River, the presence of both anti-estrogenic and androgenic activity in water samples, as well as some gonadal changes in carp is concerning and indicates that our focus needs to broaden, in order to look for biological impacts in resident fauna that might be due to environmental pollutants other than environmental estrogens. Melbourne Water, CSIRO/National Water Commission, The University of Melbourne and The Institute for Environment, Health and Societies, Brunel University.

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.

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 ...

17α-ethinylestradiol (EE2), a synthetic oestrogen in oral contraceptives, is one of many pharmaceuticals found in inland waterways worldwide as a result of human consumption and excretion into wastewater treatment systems. At low parts per trillion (ppt), EE2 induces feminisation of male fish, diminishing reproductive success and causing fish population collapse. Intended water quality standards for EE2 set a much needed global precedent. Ozone and activated carbon provide effective wastewater treatments, but their energy intensities and capital/operating costs are formidable barriers to adoption. Here we describe the technical and environmental performance of a fast- developing contender for mitigation of EE2 contamination of wastewater based upon small- molecule, full-functional peroxidase enzyme replicas called "TAML activators". From neutral to basic pH, TAML activators with H2O2 efficiently degrade EE2 in pure lab water, municipal effluents and EE2-spiked synthetic urine. TAML/H2O2 treatment curtails estrogenicity in vitro and substantially diminishes fish feminization in vivo. Our results provide a starting point for a future process in which tens of thousands of tonnes of wastewater could be treated per kilogram of catalyst. We suggest TAML/H2O2 is a worthy candidate for exploration as an environmentally compatible, versatile, method for removing EE2 and other pharmaceuticals from municipal wastewaters. T.J.C. thanks the Heinz Endowments for support. K.A.-S. thanks the Swiss National Science Foundation for a SNSF Postdoctoral Research Fellowship. M.R.M. thanks the Steinbrenner Institute for a Steinbrenner Doctoral Fellowship. NMR instrumentation at CMU was partially supported by NSF (CHE-0130903 and CHE-1039870).

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.

There is a growing body of evidence that indicates common environmental pollutants are capable of disrupting reproductive and developmental processes by interfering with the actions of endogenous hormones. Many reports of endocrine disruption describe changes in the normal development of organs and tissues that are consistent with genetic damage, and recent studies confirm that many chemicals classified to have hormone-modulating effects also possess carcinogenic and mutagenic potential. To date, however, there have been no conclusive examples linking genetic damage with perturbation of endocrine function and adverse effects in vivo. Here, we provide the first evidence of DNA damage associated with the development of imposex (the masculinization of female gastropods considered to be the result of alterations to endocrine-mediated pathways) in the dog-whelk Nucella lapillus. Animals (n = 257) that displayed various stages of tributyltin (TBT)-induced imposex were collected from sites in southwest England, and their imposex status was determined by physical examination. Linear regression analysis revealed a very strong relationship (correlation coefficient of 0.935, p < 0.0001) between the degree of imposex and the extent of DNA damage (micronucleus formation) in hemocytes. Moreover, histological examination of a larger number of dog-whelks collected from sites throughout Europe confirmed the presence of hyperplastic growths, primarily on the vas deferens and penis in both TBT-exposed male snails and in females that exhibited imposex. A strong association was found between TBT body burden and the prevalence of abnormal growths, thereby providing compelling evidence to support the hypothesis that environmental chemicals that affect reproductive processes do so partly through DNA damage pathways.

Steroidal estrogens, originating principally from human excretion, are likely to play a major role in causing widespread endocrine disruption in wild populations of the roach (Rutilus rutilus), a common cyprinid fish, in rivers contaminated by treated sewage effluents. Given the extent of this problem, risk assessment models are needed to predict the location and severity of endocrine disruption in river catchments and to identify areas where regulation of sewage discharges to remove these contaminants is necessary. In this study we attempted to correlate the extent of endocrine disruption in roach in British rivers, with their predicted exposure to steroid estrogens derived from the human population. The predictions of steroid estrogen exposure at each river site were determined by combining the modeled concentrations of the individual steroid estrogens [17beta-estradiol (E2), estrone (E1), and 17alpha-ethinylestradiol (EE2)] in each sewage effluent with their predicted dilution in the immediate receiving water. This model was applied to 45 sites on 39 rivers throughout the United Kingdom. Each site studied was then categorized as either high, medium, or low "risk" on the basis of the assumed additive potency of the three steroid estrogens calculated from data derived from published studies in various cyprinid fish species. We sampled 1,438 wild roach from the predicted high-, medium-, and low-risk river sites and examined them for evidence and severity of endocrine disruption. Both the incidence and the severity of intersex in wild roach were significantly correlated with the predicted concentrations of the natural estrogens (E1 and E2) and the synthetic contraceptive pill estrogen (EE2) present. Predicted steroid estrogen exposure was, however, less well correlated with the plasma vitellogenin concentration measured in the same fish. Moreover, we found no correlation between any of the end points measured in the roach and the proportion of industrial effluents entering the rivers we studied. Overall, our results provide further and substantive evidence to support the hypothesis that steroidal estrogens play a major role in causing intersex in wild freshwater fish in rivers in the United Kingdom and clearly show that the location and severity of these endocrine-disrupting effects can be predicted.

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.

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.

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.

Nature Communications 8: Article number: 15451 (2017); Published 16 May 2017; Updated 23 August 2017 The original version of this Article contained an error in the spelling of the author Leon Di Stefano, which was incorrectly given as Leon di Stephano. This has now been corrected in both the PDF andHTML versions of the Article.

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.

Several recent publications reflect debate on the issue of “endocrine disrupting chemicals” (EDCs), indicating that two seemingly mutually exclusive perspectives are being articulated separately and independently. Considering this, a group of scientists with expertise in basic science, medicine and risk assessment reviewed the various aspects of the debate to identify the most significant areas of dispute and to propose a path forward. We identified four areas of debate. The first is about the definitions for terms such as “endocrine disrupting chemical”, “adverse effects”, and “endocrine system”. The second is focused on elements of hormone action including “potency”, “endpoints”, “timing”, “dose” and “thresholds”. The third addresses the information needed to establish sufficient evidence of harm. Finally, the fourth focuses on the need to develop and the characteristics of transparent, systematic methods to review the EDC literature. Herein we identify areas of general consensus and propose resolutions for these four areas that would allow the field to move beyond the current and, in our opinion, ineffective debate. The authors gratefully acknowledge the following agencies: Swedish governmental academic research through Research Council, Stockholm University, Karolinska Institute, Stockholm County Council, Knut and Alic Wallenberg Foundation (alb); The Research Council Formas, and the Swedish Foundation for Strategic Environmental Research (Mistra) through the MistraPharma research program (ib), The Danish Innovation fund (nek); European Commission DG Environment, the European Food Safety Authority, DG Research (FP7 Quality of Life), the Danish EPA, the French ANSES, the OAK Foundation Geneva (ak); National Research Fund, the Medical Research Council and the University of Pretoria (rb); Academy of Finland and Sigrid Juselius Foundation, EU Fp7 Quality of Life, and Turku University Hospital (jt); National Institutes of Environmental Health Science (rtz).

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

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.

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.