Endocrine Disruption Is Reduced but Still Widespread in Wild Roach (Rutilus rutilus) Living in English Rivers.

Endocrine disruption of wild fish, primarily resulting in the feminization of males, has been reported in English river sites for several decades. Estrogenic activity emanating from wastewater treatment works (WwTW) has been conclusively demonstrated to be the main driver of these feminized phenotypes. Here, we revisit 10 English river sites previously surveyed in the late 1990s and early 2000s to assess how the frequency and severity of feminization now compare with the historical surveys. In the contemporary assessment, 60% of the sites revisited still showed endocrine disruption at the tissue organization level (oocytes present in otherwise male gonads; intersex) and 90% of sites had average male plasma vitellogenin concentrations (female-specific yolk protein; a sensitive biomarker of estrogen exposure) above natural baseline levels. In contrast to the historic surveys, none of the males sampled in the contemporary survey had ovarian cavities. At one of the larger WwTW, improvements to treatment technology may have driven a significant reduction in intersex induction, whereas at several of the smaller WwTW sites, the frequencies of feminization did not differ from those observed in the late 1990s. In conclusion, we show that although the severity of feminization is now reduced at many of the revisited sites, endocrine-disrupting chemicals are still impacting wild fish living downstream of WwTW in England.

Spatiotemporal Trends Spanning Three Decades Show Toxic Levels of Chemical Contaminants in Marine Mammals.

Despite their ban and restriction under the 2001 Stockholm Convention, persistent organic pollutants (POPs) are still widespread and pervasive in the environment. Releases of these toxic and bioaccumulative chemicals are ongoing, and their contribution to population declines of marine mammals is of global concern. To safeguard their survival, it is of paramount importance to understand the effectiveness of mitigation measures. Using one of the world's largest marine mammals strandings data sets, we combine published and unpublished data to examine pollutant concentrations in 11 species that stranded along the coast of Great Britain to quantify spatiotemporal trends over three decades and identify species and regions where pollutants pose the greatest threat. We find that although levels of pollutants have decreased overall, there is significant spatial and taxonomic heterogeneity such that pollutants remain a threat to biodiversity in several species and regions. Of individuals sampled within the most recent five years (2014-2018), 48% of individuals exhibited a concentration known to exceed toxic thresholds. Notably, pollutant concentrations are highest in long-lived, apex odontocetes (e.g., killer whales (Orcinus orca), bottlenose dolphins (Tursiops truncatus), and white-beaked dolphins (Lagenorhynchus albirostris)) and were significantly higher in animals that stranded on more industrialized coastlines. At the present concentrations, POPs are likely to be significantly impacting marine mammal health. We conclude that more effective international elimination and mitigation strategies are urgently needed to address this critical issue for the global ocean health.

HIV- 1 lentivirus tethering to the genome is associated with transcription factor binding sites found in genes that favour virus survival.

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.

Levels of Polychlorinated Biphenyls Are Still Associated with Toxic Effects in Harbor Porpoises (Phocoena phocoena) Despite Having Fallen below Proposed Toxicity Thresholds.

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

Investigation into Adaptation in Genes Associated with Response to Estrogenic Pollution in Populations of Roach (Rutilus rutilus) Living in English Rivers.

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.

A restatement of the natural science evidence base on the effects of endocrine disrupting chemicals on wildlife.

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.

Scientific principles for the identification of endocrine-disrupting chemicals: a consensus statement.

Endocrine disruption is a specific form of toxicity, where natural and/or anthropogenic chemicals, known as "endocrine disruptors" (EDs), trigger adverse health effects by disrupting the endogenous hormone system. There is need to harmonize guidance on the regulation of EDs, but this has been hampered by what appeared as a lack of consensus among scientists. This publication provides summary information about a consensus reached by a group of world-leading scientists that can serve as the basis for the development of ED criteria in relevant EU legislation. Twenty-three international scientists from different disciplines discussed principles and open questions on ED identification as outlined in a draft consensus paper at an expert meeting hosted by the German Federal Institute for Risk Assessment (BfR) in Berlin, Germany on 11-12 April 2016. Participants reached a consensus regarding scientific principles for the identification of EDs. The paper discusses the consensus reached on background, definition of an ED and related concepts, sources of uncertainty, scientific principles important for ED identification, and research needs. It highlights the difficulty in retrospectively reconstructing ED exposure, insufficient range of validated test systems for EDs, and some issues impacting on the evaluation of the risk from EDs, such as non-monotonic dose-response and thresholds, modes of action, and exposure assessment. This report provides the consensus statement on EDs agreed among all participating scientists. The meeting facilitated a productive debate and reduced a number of differences in views. It is expected that the consensus reached will serve as an important basis for the development of regulatory ED criteria.

A proposed framework for the systematic review and integrated assessment (SYRINA) of endocrine disrupting chemicals.

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.

No evidence of exposure to environmental estrogens in two feral fish species sampled from the Yarra River, Australia: A comparison with Northern Hemisphere studies.

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.

Effects of Exposure to WwTW Effluents over Two Generations on Sexual Development and Breeding in Roach Rutilus rutilus.

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.

Manufacturing doubt about endocrine disrupter science--A rebuttal of industry-sponsored critical comments on the UNEP/WHO report "State of the Science of Endocrine Disrupting Chemicals 2012".

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.

Populations of a cyprinid fish are self-sustaining despite widespread feminization of males.

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 (N(e)) of wild roach (Rutilus rutilus L.) living in English rivers contaminated with estrogenic effluents. N(e) was estimated from DNA microsatellite genotypes using approximate Bayesian computation and sibling assignment methods. We found no significant negative correlation between N(e) 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 N(e) of up to 65% is still possible for the most contaminated sites because of the wide confidence intervals associated with the statistical model.

A path forward in the debate over health impacts of endocrine disrupting chemicals.

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.

Assessing the exposure risk and impacts of pharmaceuticals in the environment on individuals and ecosystems.

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.

Modeling of steroid estrogen contamination in UK and South Australian rivers predicts modest increases in concentrations in the future.

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.

Science and policy on endocrine disrupters must not be mixed: a reply to a "common sense" intervention by toxicology journal editors.

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.

Additional treatment of wastewater reduces endocrine disruption in wild fish--a comparative study of tertiary and advanced treatments.

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.

Feminizing effects of ethinylestradiol in roach (Rutilus rutilus) populations with different estrogenic pollution exposure histories.

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.

Polychlorinated biphenyls are associated with reduced testes weights in harbour porpoises (Phocoena phocoena).

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.