Catherine A. Harris

Accurate Prediction of the Response of Freshwater Fish to a Mixture of Estrogenic Chemicals

Existing environmental risk assessment procedures are limited in their ability to evaluate the combined effects of chemical mixtures. We investigated the implications of this by analyzing the combined effects of a multicomponent mixture of five estrogenic chemicals using vitellogenin induction in male fathead minnows as an end point. The mixture consisted of estradiol, ethynylestradiol, nonylphenol, octylphenol, and bisphenol A. We determined concentration–response curves for each of the chemicals individually. The chemicals were then combined at equipotent concentrations and the mixture tested using fixed-ratio design. The effects of the mixture were compared with those predicted by the model of concentration addition using biomathematical methods, which revealed that there was no deviation between the observed and predicted effects of the mixture. These findings demonstrate that estrogenic chemicals have the capacity to act together in an additive manner and that their combined effects can be accurately predicted by concentration addition. We also explored the potential for mixture effects at low concentrations by exposing the fish to each chemical at one-fifth of its median effective concentration (EC50). Individually, the chemicals did not induce a significant response, although their combined effects were consistent with the predictions of concentration addition. This demonstrates the potential for estrogenic chemicals to act additively at environmentally relevant concentrations. These findings highlight the potential for existing environmental risk assessment procedures to underestimate the hazard posed by mixtures of chemicals that act via a similar mode of action, thereby leading to erroneous conclusions of absence of risk.

The Consequences of Feminization in Breeding Groups of Wild Fish

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.

Principles of sound ecotoxicology.

We have become progressively more concerned about the quality of some published ecotoxicology research. Others have also expressed concern. It is not uncommon for basic, but extremely important, factors to apparently be ignored. For example, exposure concentrations in laboratory experiments are sometimes not measured, and hence there is no evidence that the test organisms were actually exposed to the test substance, let alone at the stated concentrations. To try to improve the quality of ecotoxicology research, we suggest 12 basic principles that should be considered, not at the point of publication of the results, but during the experimental design. These principles range from carefully considering essential aspects of experimental design through to accurately defining the exposure, as well as unbiased analysis and reporting of the results. Although not all principles will apply to all studies, we offer these principles in the hope that they will improve the quality of the science that is available to regulators. Science is an evidence-based discipline and it is important that we and the regulators can trust the evidence presented to us. Significant resources often have to be devoted to refuting the results of poor research when those resources could be utilized more effectively.

Benzotriazole is antiestrogenic in vitro but not in vivo

Benzotriazole (BT) is an anticorrosive agent well known for its use in aircraft deicing and antifreeze fluids but also used in dishwasher detergents. It is highly persistent in the environment; therefore, BT is frequently found in runoff emanating from large airports as well as in the surrounding groundwater. In addition, BT has recently been found to be ubiquitous in Swiss wastewater treatment plant effluents and their receiving waters; however, very little chronic toxicity data is available on which to base a sound ecological risk assessment of this chemical. In vitro assays conducted using a recombinant yeast (anti-) estrogen assay indicated that BT possessed clear antiestrogenic properties. This chemical was approximately 100-fold less potent than Tamoxifen, which was used as a positive control. A subsequent in vivo study, however, involving analysis of vitellogenin induction and somatic indices in adult fathead minnows (Pimephales promelas) exposed to BT at concentrations of 10, 100, and 1,000 mug/L for two weeks showed no evidence of antiestrogenic activity by this compound. The possibility exists that higher concentrations of BT may yet induce the type of activity observed in vitro, although the concentrations used here already far exceed those reported in surface-water samples. Furthermore, adverse effects may be observed in fish or other organisms exposed to BT for a longer period than employed here, although such studies are costly and unlikely to be included in standard risk assessment procedures. A rigorous investigation of the chronic toxicity of BT is imperative.

Contamination of Environmental Samples Prepared for PCB Analysis

Archived air-dried soil that had been collected, stored, and sealed in 1914, before the commercial manufacture of PCBs, together with wet freshly sampled ancient peat, which contained little or no PCBs, were exposed to contemporary air in a laboratory. Measurable increases in the concentration of PCBs, particularly the low molecular weight congeners, were detectable after exposure for as little as a few hours. Concentrations after a few days exposure to laboratory air were similar to those measured in contemporary field surface soils (-20-30 pg of CPCB kgl). Laboratory air concentrations ranged between 4.7 and 8.2 ng of CPCB m-3 during the period of exposure, markedly higher than routinely detected in outdoor U.K. urban air. The calculated average net dry deposition flux from air-soil in the laboratory over 25 days was 5 pg of CPCB m-2 day1. Indoor air concentrations might be expected to be higher than those routinely measured outside, exacerbating the potential problems of sample contamination. Extreme caution is needed in the preparation and handling of samples which contain inherently low concentrations of PCBs and before ascribing the presence of these compounds in certain samples to ‘natural production’ mechanisms.

Evidence of temperature-dependent effects on the estrogenic response of fish: Implications with regard to climate change

Chemical risk assessment is fraught with difficulty due to the problem of accounting for the effects of mixtures. In addition to the uncertainty arising from chemical-to-chemical interactions, it is possible that environmental variables, such as temperature, influence the biological response to chemical challenge, acting as confounding factors in the analysis of mixture effects. Here, we investigate the effects of temperature on the response of fish to a defined mixture of estrogenic chemicals. It was anticipated that the response to the mixture may be exacerbated at higher temperatures, due to an increase in the rate of physiological processing. This is a pertinent issue in view of global climate change. Fathead minnows (Pimephales promelas) were exposed to the mixture in parallel exposure studies, which were carried out at different temperatures (20 and 30 degrees C). The estrogenic response was characterised using an established assay, involving the analysis of the egg yolk protein, vitellogenin (VTG). Patterns of VTG gene expression were also analysed using real-time QPCR. The results revealed that there was no effect of temperature on the magnitude of the VTG response after 2 weeks of chemical exposure. However, the analysis of mixture effects at two additional time points (24 h and 7 days) revealed that the response was induced more rapidly at the higher temperature. This trend was apparent from the analysis of effects both at the molecular and biochemical level. Whilst this indicates that climatic effects on water temperature are not a significant issue with regard to the long-term risk assessment of estrogenic chemicals, the relevance of short-term effects is, as yet, unclear. Furthermore, analysis of the patterns of VTG gene expression versus protein induction gives an insight into the physiological mechanisms responsible for temperature-dependent effects on the reproductive phenology of species such as roach. Hence, the data contribute to our understanding of the implications of global climate change for wild fish populations.

Evidence suggesting that di-n-butyl phthalate has antiandrogenic effects in fish.

Phthalate ester plasticizers are antiandrogenic in mammals. High doses of certain phthalates consistently interfere with the normal development of male offspring exposed in utero, causing disrupted sperm production, abnormal development of the genitalia, and in some cases infertility. In the environment, phthalates are considered ubiquitous and are commonly measured in aquatic ecosystems at low nanograms to micrograms per liter concentrations. Given the similarity between mammalian and teleost endocrine systems, phthalate esters may be able to cause antiandrogenic endocrine disruption in fish in the wild. In the present study, adult male three-spined sticklebacks (Gasterosteus aculetaus; n = 8) were exposed to di-n-butyl phthalate (DBP; 0, 15, and 35 µg DBP/L) for 22 d and analyzed for changes in nesting behavior, plasma androgen concentrations, spiggin concentrations, and steroidogenic gene expression. Plasma testosterone concentrations were significantly higher in males from the 35 µg DBP/L group compared with the solvent control, whereas plasma 11-ketotestosterone concentrations were not significantly affected. Expression of steroid acute regulatory protein and 3β-hydroxysteroid dehydrogenase remained unchanged. Spiggin concentrations were significantly lower in the males exposed to 35 µg DBP/L. Nest building appeared to be slower in some males exposed to DBP, but this was not statistically significant. These results suggest that DBP has antiandrogenic effects in fish. However, further research is required to firmly establish the consequences of chronic DBP exposure in fish.

The Endocrine Disrupting Potential of Phthalates

Phthalate esters are ubiquitous in today’s environment. Both terrestrial and aquatic organisms are subject to a low level but constant exposure to this class of chemicals. Until very recently, it was not thought likely that any phthalates would display endocrine activity, and hence very little, if any, research focused on this possibility. When reproductive effects were observed, they were not interpreted as being due to any intrinsic endocrine activity of phthalates (or their products of metabolism), but rather due simply to a “toxicity” of unknown mechanism. However, recently a small number of phthalates has been found to elicit estrogenic responses in in vitro assays. None of these, however, have been found capable of inducing specifically estrogen-dependent effects in vivo. It is unlikely that phthalates alone are responsible for what may be endocrine-mediated adverse effects observed in wildlife and humans over the past few decades, but it is possible that they are a contributory factor to this phenomenon. Phthalates administered in high doses to adult mammals have caused adverse reproductive development in their offspring. Recent thinking has proposed that these manifestations may be as a result of an anti-androgenic mechanism. This theory should be investigated in greater depth, and at environmentally relevant concentrations of the active phthalates. Before it is possible to assess the risks (if any) of exposure to phthalates, a much wider range of test species, and a wider range of endpoints, particularly endocrine ones, need to be assessed.

The influence of a surfactant, linear alkylbenzene sulfonate, on the estrogenic response to a mixture of (xeno)estrogens in vitro and in vivo

The effect of the presence of a surfactant on the activity of a mixture of environmental estrogens was assessed. In their natural habitat, fish are subject not only to exposure to mixtures of estrogenic compounds, as has been addressed in previous publications, but also to other confounding factors (chemical, physical and biological), which may, in theory, affect their responses to such compounds. To assess the potential for such interference, the commonly occurring surfactant, linear alkylbenzene sulfonate (LAS), was applied to the yeast estrogen screen at various concentrations, independently and together with a mixture of estrogens at constant concentrations. LAS enhanced the estrogenic activity of the mixture, an effect which became less pronounced over the course of time. This information was used to design an in vivo study to assess induction of vitellogenin in fathead minnows exposed to the same mixture of estrogens plus LAS. A similar trend was observed, that is, the response was enhanced, but the effect became less pronounced as the study progressed. However, the enhanced response in vivo occurred only at the highest concentration of LAS tested (362microg/L), and was transient because it was no longer apparent by the end of the study. Although LAS is a significant contaminant in terms of both concentration and frequency of detection in the aquatic environment, these data do not suggest that it will have a significant impact on the response of fish to environmental estrogens.

Could the Quality of Published Ecotoxicological Research Be Better

A increasing number of scientific publications (including some describing ecotoxicological research) are found to contain poor quality experiments and unreproducible results. This is widely recognized as being a problem not only from an ethical and even obligatory point of view (scientists have a responsibility both to the funders and to the wider society to undertake accurate and objective research), but also from a more practical standpoint, that is, that of the use of peerreviewed data to underpin the management of the environment. The use of erroneous data in the latter field can lead to costly mistakes, both with respect to wildlife and economics. Evidently this issue of poor-quality science is not new. For over a decade, Ioannidis and colleagues have been pointing out that a very significant percentage of all claims in biomedical research are false (i.e., not reproducible), and have been providing the reasons why this is so. There is now wide acceptance that many claims in the biomedical literature cannot be substantiated. Direct attempts to replicate more than 50 of the key findings of recent years reported in the biomedical literature have shown that a high proportion cannot be replicated, albeit to different degrees. The situation in other fields of research is less clear. There has, for example, been no systematic analysis of the ecotoxicology literature conducted in the way it has been in the biomedical field. It is nonetheless clear that many ecotoxicologists have become concerned about the quality of published research in their field, and some have started to address the issue objectively. For example, Agerstrand and colleagues have built on the start provided by Klimisch to develop and use criteria capable of evaluating ecotoxicology data. In 2014 we published a paper in this journal presenting 12 “Principles of Sound Ecotoxicology” that, in our opinion, should be adhered to in order to produce a reliable set of data in this area of research. The paper also aimed to provide guidance for reviewers/regulators assessing publications, as well as for young researchers beginning their careers. We have recently been using some of these principles to assess the current state of affairs with respect to the quality of published ecotoxicological research. We embarked on this as a first step toward gauging the scale of the problem at hand, as well as to start to pinpoint which areas of scientific (specifically, ecotoxicological) experiments are most often neglected, in order that we can start to consider practical measures to improve the quality of this research. We were not aiming to identify all of the causes of poor science in this instance, but rather to identify some of the most regularly occurring “mistakes” that can lead to irreproducible data. We selected two journals specializing in toxicological publications (Environmental Toxicology and Chemistry (ET&C), and Aquatic Toxicology); and one (ES&T) which has a high number of papers published in this field each month. From these, we analyzed ecotoxicology papers published during the first 6 months of 2013. All (66) of the relevant publications from this time period were analyzed from ET&C; a random selection (up to 10 from each issue; 58 in total) was analyzed from Aquatic Toxicology, and all (49) of the ecotoxicology articles from ES&T were analyzed. Our primary aim was to get a preliminary feel for the overall situation and not to draw comparisons between the individual journals. Any apparent differences between the journals may partly be a reflection of the somewhat different remit of the three journals. Rather than using all 12 of the ‘Principles of Sound Ecotoxicology’, which would have included several somewhat subjective end points, we instead chose the three most objective Principles, in order to try to avoid the potential for bias. These were: