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.

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.

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 3ß-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 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.

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.

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.

Evidence of estrogenic mixture effects on the reproductive performance of fish.

Recent research into the effects of mixtures of estrogenic chemicals has revealed the capacity for similarly acting chemicals to act in combination, according to the principles of concentration addition. This means that, collectively, they may pose a significant environmental risk, even when each component is present at a low and individually ineffective concentration. The aim of this study was to investigate the ecological significance of mixture effects at low-effect concentrations by assessing the combined effect of estrogenic chemicals on the reproductive performance of fish. Pairs of fathead minnows were exposed to five estrogenic chemicals. Endpoints analyzed included fecundity, the expression of male secondary sexual characteristics, somatic indices, and vitellogenin induction. In the first phase of the study, a concentration-response analysis was performed to investigate the relative sensitivity of these endpoints. In the second phase, mixture effects at low-effect concentrations were explored by exposing fish to each of the mixture components, individually and in combination. Data from these experiments provide evidence of mixture effects on fitness and fecundity, demonstrating the capacity for chemicals to act together to affect reproductive performance, even when each component is present belowthe threshold of detectable effects. This has important implications for hazard assessment and contributes to our understanding of mixture effects at increasing levels of biological complexity.

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.