Late lessons from early warnings: science, precaution, innovation

Chapter from collection of essays published by European Environment Agency, 2013. Available online at http://www.eea.europa.eu/publications/late-lessons-2Many decades of research have shown that when released to the environment, a group of hormones known as oestrogens, both synthetic and naturally occurring, can have serious impacts on wildlife. This includes the development of intersex characteristics in male fish, which diminishes fertility and fecundity. Although often sublethal, such impacts may be permanent and irreversible. This chapter describes the scientific evidence and regulatory debates concerning one of these oestrogens, ethinyloestradiol (EE2), an active ingredient in the birth control pill. First developed in 1938, it is released to the aquatic environment via wastewater treatment plants. Although it is now clear that wildlife species are exposed to and impacted by a cocktail of endocrine disrupting chemicals, there is also reasonable scientific certainty that EE2 plays a significant role, and at vanishingly low levels in the environment. In 2004 the Environment Agency of England and Wales accepted this, judging the evidence sufficient to warrant consideration of risk management. In 2012, nearly 75 years after its synthesis, the European Commission proposed to regulate EE2 as a EU-wide 'priority substance' under the Water Framework Directive (the primary legislation for protecting and conserving European water bodies). This proposal was subsequently amended, delaying any decision on a regulatory 'environmental quality standard' until at least 2016. This is in part because control of EE2 will come at a significant price. Complying with proposed regulatory limits in the environment means removing very low (part per trillion) levels of EE2 from wastewater effluents at considerable expense. Is this a price we are willing to pay? Or will the price of precautionary action be simply too high — a pill too bitter to swallow? To what extent is society, which has enjoyed decades of flexible fertility and will also ultimately pay for the control and management of its unintended consequences, involved in this decision? And what could this mean for the many thousands of other pharmaceuticals that ubiquitously infiltrate our environment and which could have sublethal effects on aquatic animals at similarly low levels?European Environment Agenc

Is there a causal association between genotoxicity and the imposex effect?

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

Assessing the Sensitivity of Different Life Stages for Sexual Disruption in Roach (Rutilus rutilus) Exposed to Effluents from Wastewater Treatment Works

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

The Nuclear Receptors of Biomphalaria glabrata and Lottia gigantea : Implications for Developing New Model Organisms

Funding: This work was funded by the National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.Peer reviewedPublisher PD

Is There a Causal Association between Genotoxicity and the Imposex Effect?

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

Health effects in fish of long-term exposure to effluents from wastewater treatment works

The effects of simple mixtures of chemicals, with similar mechanisms of action, can be predicted using the concentration addition model (CA). The ability of this model to predict the estrogenic effects of more complex mixtures such as effluent discharges, however, has yet to be established. Effluents from 43 U.K. wastewater treatment works were analyzed for the presence of the principal estrogenic chemical contaminants, estradiol, estrone, ethinylestradiol, and nonylphenol. The measured concentrations were used to predict the estrogenic activity of each effluent, employing the model of CA, based on the relative potencies of the individual chemicals in an in vitro recombinant yeast estrogen screen (rYES) and a short-term (14-day) in vivo rainbow trout vitellogenin induction assay. Based on the measured concentrations of the four chemicals in the effluents and their relative potencies in each assay, the calculated in vitro and in vivo responses compared well and ranged between 3.5 and 87 ng/L of estradiol equivalents (E2 EQ) for the different effluents. In the rYES, however, the measured E2 EQ concentrations in the effluents ranged between 0.65 and 43 ng E2 EQ/L, and they varied against those predicted by the CA model. Deviations in the estimation of the estrogenic potency of the effluents by the CA model, compared with the measured responses in the rYES, are likely to have resulted from inaccuracies associated with the measurement of the chemicals in the extracts derived from the complex effluents. Such deviations could also result as a consequence of interactions between chemicals present in the extracts that disrupted the activation of the estrogen response elements in the rYES. E2 EQ concentrations derived from the vitellogenic response in fathead minnows exposed to a series of effluent dilutions were highly comparable with the E2 EQ concentrations derived from assessments of the estrogenic potency of these dilutions in the rYES. Together these data support the use of bioassays for determining the estrogenic potency of WwTW effluents, and they highlight the associated problems for modeling approaches that are reliant on measured concentrations of estrogenic chemicals

COMPRENDO: Focus and approach

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)

The nuclear receptors of Biomphalaria glabrata and Lottia gigantea: Implications for developing new model organisms

© 2015 Kaur et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are creditedNuclear 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.The National Centre for the Replacement, Refinement and Reduction of Animals in Research, Grant Ref:G0900802 to CSJ, LRN, SJ & EJR [www.nc3rs.org.uk]

Meta-analysis of the spatial distribution and composition of plastic macro-debris in Indonesia

Plastic pollution is recognised as a global environmental problem with Indonesia identified as a major contributing source. However, a comprehensive understanding of pollution patterns remains hindered by fragmented data and methodological inconsistencies across studies. This study synthesises data from 44 peer-reviewed publications, spanning 68 field locations and 328 individual study sites across 19 provinces, to map the spatial distribution and composition of plastic macro-debris in Indonesia. We identified plastic bags, plastic food packaging, and plastic bottles as the three most prevalent item types, occurring as top three ranked items in 65 %, 63 %, and 40 % of field locations, respectively. Items were recorded across seven different environment types: beach (n = 43 field locations); river (n = 16); mangroves (n = 3); seabed (n = 2); coral reef (n = 2); sea surface (n = 1) and inland areas (n = 1), with research effort concentrated in Java, accounting for 40 % of all field locations. Despite Indonesia\u27s environmental and regional diversity, multivariate analysis revealed no significant differences in litter composition between environment types or provinces, indicating that common plastic items dominate pollution across Indonesia. These findings underscore the need for coordinated national action targeting high-prevalence items and highlight the value of widespread beach litter monitoring as a proxy for broader environmental pollution patterns

Environmental oestrogens and sexual development in fish

This brief paper discusses the assumption that watercourses might be harbouring a chemical(s) affecting the sexual development in fish. Male fish was found with the oestrogen-dependent blood protein, vitellogenin, usually found only in maturing females. The author examines a number of man-made chemicals present in the environment have been found to be oestrogenic. The paper concludes that rivers contain environmental oestrogens that are capable of causing disruptions in the sexual development of fish. Whether or not these environmental oestrogens are causing a widespread disruption in reproduction in wild fish, however, has yet to be determined