Pharmaceuticals in the environment: The effects of clofibric acid on fish

This thesis was submitted for the degree of Doctor of Philosophy and awarded by Brunel University, 10/01/2005.Pharmaceuticals in the aquatic environment is an emerging issue and the risks they pose are mostly unknown. They are used in large amounts throughout the world and can enter the environment, as the active metabolite or unmetabolised, through excretion by people and improper disposal. As these drugs are designed to have specific biological effects in a specific organism (as well as sometimes having other non-specific side effects), their potential to cause effects within the environment is great. Clofibric acid (the major metabolite of the lipid lowering drug, Clofibrate) is non-biodegradable, highly motile, very persistent and frequently detected at μg/I levels in the environment. I studied possible effects of clofibric acid in fish, using different experimental approaches and endpoints. The studies involve two different species, and for one of these species, fish at different stages of development. The chapters within this thesis have presented the first evidence (albeit preliminary) of clofibric acid having effects on both adult and embryo fish. When fathead minnow embryos were exposed to clofibric acid, the effects seen included changes in the eggshell, time to hatch, hatchability, mortality and viability. Adult fathead minnow were similarly exposed and significant effects on specific parameters were also observed. These included effects on lipid metabolism, steroidogenesis and spermatogenesis - thought to be via cholesterol transport - as well as significant effects on the expression of several genes involved in lipid metabolism and detoxification. Exposure of juvenile (sexually undifferentiated) bream also found significant differences in some endpoints. Other results suggested, less pronounced effects of clofibric acid on some other parameters. The results from this research show that there are effects of clofibric acid in pathways which were not only unexpected in fish (for example, steroidogenesis, spermatogenesis and gene expression), but also at concentrations below those previously shown to have any biological effects on fish. These effects indicate that clofibric acid may potentially have an impact on fish fecundity, and even more worryingly, on human health for those people prescribed it

Pharmaceuticals in the aquatic environment: no answers yet to the major questions

The presence of pharmaceuticals in the environment, especially the aquatic environment, has received a lot of attention in the last 20 plus years. Despite that attention, the two most important questions regarding pharmaceuticals in the environment still cannot be answered. It is not possible to put the threat posed by pharmaceuticals into perspective with the many other threats (stressors) facing aquatic organisms, such as low flows due to over-abstraction of water, inhibited passage of migratory species due to dams and weirs, diseases, algal blooms causing low oxygen levels and releasing toxins, eutrophication, climate change, and so on. Nor is it possible to identify which pharmaceuticals are of concern and which are not. Not only can these key questions not be answered presently, they have received extremely little attention, despite being identified 10 years ago as the two most important questions to answer. That situation must change if resources and expertise are to be effectively used to protect the environment

Pharmaceuticals in the environment : the effects of clofibric acid on fish

Pharmaceuticals in the aquatic environment is an emerging issue and the risks they pose are mostly unknown. They are used in large amounts throughout the world and can enter the environment, as the active metabolite or unmetabolised, through excretion by people and improper disposal. As these drugs are designed to have specific biological effects in a specific organism (as well as sometimes having other non-specific side effects), their potential to cause effects within the environment is great. Clofibric acid (the major metabolite of the lipid lowering drug, Clofibrate) is non-biodegradable, highly motile, very persistent and frequently detected at μg/I levels in the environment. I studied possible effects of clofibric acid in fish, using different experimental approaches and endpoints. The studies involve two different species, and for one of these species, fish at different stages of development. The chapters within this thesis have presented the first evidence (albeit preliminary) of clofibric acid having effects on both adult and embryo fish. When fathead minnow embryos were exposed to clofibric acid, the effects seen included changes in the eggshell, time to hatch, hatchability, mortality and viability. Adult fathead minnow were similarly exposed and significant effects on specific parameters were also observed. These included effects on lipid metabolism, steroidogenesis and spermatogenesis - thought to be via cholesterol transport - as well as significant effects on the expression of several genes involved in lipid metabolism and detoxification. Exposure of juvenile (sexually undifferentiated) bream also found significant differences in some endpoints. Other results suggested, less pronounced effects of clofibric acid on some other parameters. The results from this research show that there are effects of clofibric acid in pathways which were not only unexpected in fish (for example, steroidogenesis, spermatogenesis and gene expression), but also at concentrations below those previously shown to have any biological effects on fish. These effects indicate that clofibric acid may potentially have an impact on fish fecundity, and even more worryingly, on human health for those people prescribed it.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

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 (EC(50)). 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

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

This document is the unedited Author’s version of a Submitted Work that was subsequently accepted for publication in Environmental science & technology, copyright © American Chemical Society after peer review. To access the final edited and published work see https://doi.org/10.1021/acs.est.7b03441Recent species-extrapolation approaches to predict 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 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/plasma concentration ratios were within the range of values reported in mammalian species. This fish species was able to metabolise the pro-drug tramadol into the highly active metabolite O-desmethyl tramadol and the inactive metabolite N-desmethyl tramadol in a similar manner to 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.We would like to thank members of the Ecotoxicology Research Group, Brunel University London, particularly J. Walker, N. Brodigan, and A. Ferreira for fish husbandry, and T. Thrupp, E. Lawton, and A. Baynes for fish sampling. The research at Brunel University London was internally funded by the university. This study was also supported by the Ministry of Education, Culture, Sports, Science and Technology, Japan (MEXT) to a project on Joint Usage/Research Center– Leading Academia in Marine and Environment Pollution Research (LaMer), and Research Fellowships from the Japan Society for the Promotion of Science (JSPS) for Young Scientists in Japan (PD) provided to R. Tanoue (26·2800), Grants in Bid (KAKENHI) for Scientific Research (A) (25257403), Scientific Research (A) (16H01784). This study was also funded by the Sasakawa Scientific Research Grant from The Japan Science Society

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

The official published version can be obtained from the link below - Copyright @ 2008 Elsevier BV.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.This work was funded by a grant from the Natural Environment Research Council NE/D00389X/1). Additional support was provided by a small research grant from the Fisheries Society of the British Isles

Renewing and improving the environmental risk assessment of chemicals

The processes underpinning the environmental risk assessment (ERA) of chemicals have not changed appreciably in the last 30 years. It is unclear how successful these processes are in protecting the environment from any adverse effects of chemicals. To ascertain if the current methodology can be improved, and if so, how, we invited experts to suggest how the current ERA process could be improved. They were not asked to select from a list of suggestions. The 36 experts made 109 suggestions for improvement, which could be grouped into 33 categories. The category that received the most support, from 12 experts, was to utilise a broader range of scientific information, including all up-to-date information, in ERAs. The second most popular category, supported by 10 experts, was the suggestion to regulate mixtures of chemicals; the current regulatory process involves chemical-by-chemical assessment. Two quite radical proposals were suggested. One was to replace the regulator with artificial intelligence. The other was to establish a new competent authority that would appoint groups of experts, each including representatives of the range of stakeholders, to decide which studies were required, commission those studies, then conduct the ERA based on the results of those studies. These two radical proposals, which the authors support strongly, are not necessarily mutually exclusive. We conclude that the present ERA process could be improved to better protect the environment from the myriad of chemicals in use

A 'Limitations' section should be mandatory in all scientific papers

It is unusual, and can be difficult, for scientists to reflect in their publications on any limitations their research had. This is a consequence of the extreme pressure that scientists are under to 'publish or perish'. The inevitable consequence is that much published research is not as good as it could, and should, be, leading to the current 'reproducibility crisis'. Approaches to address this crisis are required. Our suggestion is to include a 'Limitations' section in all scientific papers. Evidence is provided showing that such a section must be mandatory. Adding a 'Limitations' section to scientific papers would greatly increase honesty, openness and transparency, to the considerable benefit of both the scientific community and society in general. This suggestion is applicable to all scientific disciplines. Finally, we apologise if our suggestion has already been made by others.The UK authors are grateful to NERC for grant NE/S000100/1 supporting the ChemPop project on the impacts of chemicals on wildlife. We also thank the three reviewers for their excellent reviewing, which allowed us to significantly improve our paper.Peer reviewe

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