Tamsin J. Runnalls

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.

Several Synthetic Progestins with Different Potencies Adversely Affect Reproduction of Fish

Synthetic progestins are widely used as a component in both contraceptives and in hormone replacement therapy (HRT), both on their own and in combination with EE2. Their presence in the environment is now established in wastewater effluent and river water and this has led to concerns regarding their potential effects on aquatic organisms living in these waters. We carried out in vivo experiments to determine the potencies of four different synthetic progestins on the reproductive capabilities of the fathead minnow (Pimephales promelas). We then performed a series of in vitro assays to try and determine the reason for the effects seen in the in vivo experiments. In the first experiment, fathead minnow exposed to a single concentration of 100 ng/L of either Levonorgestrel or Gestodene stopped spawning almost completely. The same nominal concentration of Desogestrel and Drospirenone did not affect reproduction (21 d NOECs of 100 ng/L). The second experiment investigated two progestins of different potency: Gestodene at 1, 10, and 100 ng/L and Desogestrel at 100 ng/L, 1 μg/L, and 10 μg/L. Gestodene concentrations as low as 1 ng/L had significant effects on reproduction over 21 d, whereas concentrations of Desogestrel at or above 1 μg/L were required to significantly reduce egg production. The synthetic progestins also masculinized the female fish in a concentration-dependent manner. Results from yeast-based in vitro assays demonstrated that the progestins are all strongly androgenic, thereby explaining the masculinization effects. The results strongly suggest that synthetic progestins merit serious consideration as environmental pollutants.

Pharmaceuticals in the Aquatic Environment: Steroids and Anti-Steroids as High Priorities for Research

ABSTRACT Many human pharmaceuticals, with many modes of action, are present in the aquatic environment. It is very difficult to know which of these might pose a threat to aquatic organisms, and which are of no environmental concern. Hence, it is difficult to design a research strategy that most effectively maximizes the possibility of identifying those pharmaceuticals that do pose the greatest risk. We consider that steroidal pharmaceuticals, and drugs that inhibit the actions of endogenous steroids, merit more research than they have yet received so far. We analyzed a comprehensive U.K. database covering all prescriptions of pharmaceuticals to determine the annual usage of all steroid and anti-steroid pharmaceuticals. We found that both progestogens and glucocorticoids are used in much greater amounts than estrogens, whereas androgens are used in similar amounts to estrogens. There is also very significant use of anti-estrogens, anti-androgens, and anti-mineralocorticoids. All these pharmaceuticals are likely to be constantly entering the aquatic environment in effluents from sewage treatment works. River concentrations of these pharmaceuticals will be very low; probably in the ng or sub-ng/l range. However, such low concentrations mat still pose a threat to aquatic organisms, especially fish.

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.

Metabolic and Reproductive Effects of Relatively Low Concentrations of Beclomethasone Dipropionate, a Synthetic Glucocorticoid, on Fathead Minnows

Pharmaceuticals present in the aquatic environment could adversely affect aquatic organisms. Synthetic glucocorticoids (GC) are used in large quantities as anti-inflammatory drugs and have been reported to be present in river water. In order to assess the impact of environmental concentrations of GCs, an in vivo experiment was conducted with adult fathead minnows. Fish were exposed to 0.1 μg/L, 1 μg/L, or 10 μg/L beclomethasone dipropionate (BCMD) via a flow-through system over a period of 21 days. Similar duplicate tanks served as control, with no chemical added. There was a concentration-related increase in plasma glucose concentration and a decrease in blood lymphocyte count. Induction of male secondary sexual characters and a decreasing trend in plasma vitellogenin (Vtg) concentrations in female fish were observed with increasing exposure concentration of BCMD. Expression profiles of selected genes (phosphoenolpyruvate carboxykinase - PEPCK, glucocorticoid receptor - GR, and Vtg) in liver also demonstrated concentration-related effects at all three tested concentrations. The results suggest that GCs could cause effects in lower micrograms per liter concentrations that could be environmentally relevant for total GCs present in the environment. Therefore, studies to determine the environmental concentrations of GCs and no effect concentrations are needed to assess if GCs pose a risk to the aquatic environment.

Estrogenic activity of tropical fish food can alter baseline vitellogenin concentrations in male fathead minnow (Pimephales Promelas)

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.

Out-of-season production of 17,20β-dihydroxypregn-4-en-3-one in the roach Rutilus rutilus

In this study, although the highest production of two physiologically significant progestins in teleosts [17,20β-dihydroxypregn-4-en-3-one (17,20β-P) and 17,20β,21-trihydroxypregn-4-en-3-one (17,20β,21-P)] was observed in the period just prior to spawning in both male and female roach Rutilus rutilus, there was also a substantial production (mean levels of 5-10 ng ml(-1) in blood; and a rate of release of 5-20 ng fish(-1)  h(-1) into the water) in males and females in the late summer and early autumn (at least 7 months prior to spawning). During this period, the ovaries were increasing rapidly in size and histological sections were dominated by oocytes in the secondary growth phase [i.e. incorporation of vitellogenin (VTG)]. At the same time, the testes were also increasing rapidly in size and histological sections were dominated by cysts containing mainly spermatogonia type B. Measurements were also made of 11-ketotestosterone (11-KT) in males and 17β-oestradiol and VTG in females. The 3 months with the highest production of 11-KT coincided with the period that spermatozoa were present in the testes. In females, the first sign of a rise in 17β-oestradiol concentrations coincided with the time of the first appearance of yolk globules in the oocytes (in August). The role of the progestins during the late summer and autumn has not been established.

Uptake and Metabolism of Human Pharmaceuticals by Fish: A Case Study with the Opioid Analgesic Tramadol

Recent species-extrapolation approaches to the prediction of the potential effects of pharmaceuticals present in the environment on wild fish are based on the assumption that pharmacokinetics and metabolism in humans and fish are comparable. To test this hypothesis, we exposed fathead minnows to the opiate pro-drug tramadol and examined uptake from the water into the blood and brain and the metabolism of the drug into its main metabolites. We found that plasma concentrations could be predicted reasonably accurately based on the lipophilicity of the drug once the pH of the water was taken into account. The concentrations of the drug and its main metabolites were higher in the brain than in the plasma, and the observed brain and plasma concentration ratios were within the range of values reported in mammalian species. This fish species was able to metabolize the pro-drug tramadol into the highly active metabolite O-desmethyl tramadol and the inactive metabolite N-desmethyl tramadol in a similar manner to that of mammals. However, we found that concentration ratios of O-desmethyl tramadol to tramadol were lower in the fish than values in most humans administered the drug. Our pharmacokinetic data of tramadol in fish help bridge the gap between widely available mammalian pharmacological data and potential effects on aquatic organisms and highlight the importance of understanding drug uptake and metabolism in fish to enable the full implementation of predictive toxicology approaches.