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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. Environ Toxicol Chem 2024;43:589-594. © 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2022 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: John Sumpter reports financial support was provided by Natural Environment Research Council. Damia Barcelo is serving in an editorial capacity for the journal to which we are submitting (STOTEN)., (Copyright © 2022 Elsevier B.V. All rights reserved.)

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

A comprehensive aquatic environmental risk assessment (ERA) of the human pharmaceutical propranolol was conducted, based on all available scientific literature. Over 200 papers provided information on environmental concentrations (77 of which provided river concentrations) and 98 dealt with potential environmental effects. The median concentration of propranolol in rivers was 7.1 ng/L (range of median values of individual studies 0.07 to 89 ng/L), and the highest individual value was 590 ng/L. Sixty-eight EC50 values for 35 species were available. The lowest EC50 value was 0.084 mg/L. A species sensitivity distribution (SSD) provided an HC50 value of 6.64 mg/L and an HC5 value of 0.22 mg/L. Thus, there was a difference of nearly 6 orders of magnitude between the median river concentration and the HC50 value, and over 4 orders of magnitude between the median river concentration and the HC5 value. Even if an assessment factor of 100 was applied to the HC5 value, to provide considerable protection to all species, the safety margin is over 100-fold. However, nearly half of all papers reporting effects of propranolol did not provide an EC50 value. Some reported that very low concentrations of propranolol caused effects. The lowest concentration reported to cause an effect - in fact, a range of biochemical and physiological effects on mussels - was 0.3 ng/L. In none of these 'low concentration' papers was a sigmoidal concentration-response relationship obtained. Although inclusion of data from these papers in the ERA cause a change in the conclusion reached, we are sceptical of the repeatability of these 'low concentration' results. We conclude that concentrations of propranolol present currently in rivers throughout the world do not constitute a risk to aquatic organisms. We discuss the need to improve the quality of ecotoxicology research so that more robust ERAs acceptable to all stakeholders can be completed., Competing Interests: Declaration of competing interest This study was partially funded by the pharmaceutical company AstraZeneca, and one of the authors (SFO) is an employee of that company. However, the literature searches, identification of relevant research, extraction of information, and writing the complete first draft of the paper were all conducted entirely by the university-based authors, with no interference from any employee of AstraZeneca. SFO and other employees of AstraZeneca subsequently improved that initial draft, but none of the data, or their interpretation, were changed during that process., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)

There is concern that psychoactive drugs present in the aquatic environment could affect the behaviour of fish, and other organisms, adversely. There is considerable experimental support for this concern, although the literature is not consistent. To investigate why, fish were exposed to three concentrations of the synthetic opiate tramadol for 23-24 days, and their anxiolytic behaviour in a novel tank diving test was assessed both before and after exposure. The results were difficult to interpret. The positive control drug, the anti-depressant fluoxetine, produced the expected results: exposed fish explored the novel tank more, and swam more slowly while doing so. An initial statistical analysis of the results provided relatively weak support for the conclusion that both the low and high concentrations of tramadol affected fish behaviour, but no evidence that the intermediate concentration did. To gain further insight, UK and Japanese experts in ecotoxicology were asked for their independent opinions on the data for tramadol. These were highly valuable. For example, about half the experts replied that a low concentration of a chemical can cause effects that higher concentrations do not, although a similar number did not believe this was possible. Based both on the inconclusive effects of tramadol on the behaviour of the fish and the very varied opinions of experts on the correct interpretation of those inconclusive data, it is obvious that more research on the behavioural effects of tramadol, and probably all other psychoactive drugs, on aquatic organisms is required before any meaningful risk assessments can be conducted. The relevance of these findings may apply much more widely than just the environmental risk assessment of psychoactive drugs. They suggest that much more rigorous training of research scientists and regulators is probably required if consensus decisions are to be reached that adequately protect the environment from chemicals., (Copyright © 2019 Elsevier B.V. All rights reserved.)

Ill-defined, multi-component mixtures of steroidal pharmaceuticals are present in the aquatic environment. Fish are extremely sensitive to some of these steroids. It is important to know how fish respond to these mixtures, and from that knowledge develop methodology that enables accurate prediction of those responses. To provide some of the data required to reach this objective, pairs of fish were first exposed to five different synthetic steroidal pharmaceuticals (one estrogen, EE2; one androgen, trenbolone; one glucocorticoid, beclomethasone dipropionate; and two progestogens, desogestrel and levonorgestrel) and concentration-response data on egg production obtained. Based on those concentration-response relationships, a five component mixture was designed and tested twice. Very similar effects were observed in the two experiments. The mixture inhibited egg production in an additive manner predicted better by the model of Independent Action than that of Concentration Addition. Our data provide a reference case for independent action in an in vivo model. A significant combined effect was observed when each steroidal pharmaceutical in the mixture was present at a concentration which on its own would produce no statistically significant effect (something from 'nothing'). Further, when each component was present in the mixture at a concentration expected to inhibit egg production by between 18% (Beclomethasone diproprionate) and 40% (trenbolone), this mixture almost completely inhibited egg production: a phenomenon we term 'a lot from a little'. The results from this proof-of-principle study suggest that multiple steroids present in the aquatic environment can be analysed for their potential combined environmental risk., (Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

The aquatic environment is polluted with thousands of chemicals. It is currently unclear which of these pose a significant threat to aquatic biota. The typical exposure scenario is now represented by a widespread blanket of contamination composed of myriads of individual pollutants-each typically present at a low concentration. The synthetic steroids, 17α-ethinylestradiol and levonorgestrel, have been widely reported to be present in the aquatic environment in the low ng to sub-ng/l range. They are widely used in contraceptive formulations, both individually and in combination. Our research employed the fathead minnow (Pimephales promelas) 21 day 'pair-breeding' assay to assess reproductive output when pairs of fish were exposed to the single chemicals at low environmentally relevant concentrations, and then to a binary mixture of them. A variety of endpoints were assessed, including egg production, which was inhibited in a concentration-dependent manner by both the individual chemicals and the mixture. Significant, sex specific effects were also seen with both chemicals, at differing levels of biological organisation. Plasma concentrations of EE2 and levonorgestrel were predicted and in the case of levonorgestrel measured, and compared with the human therapeutic plasma concentrations (Read-Across approach) to support the interpretation of the results. A novel quantitative method was developed for the data analysis, which ensured a suitable endpoint for the comparative mixture assessment. This approach compares the reproductive performance from individual pairs of fish during chemical exposure to its pre-treatment performance. The responses from the empirical mixture study were compared to predictions derived from the single substance data. We hypothesised combined responses which were best described by the concept of concentration addition, and found no clear indications against this additivity expectation. However, the effect profiles support the current knowledge that both compounds act in different ways to reduce egg production in fish, and suggest that probably response addition (also called Independent action) is the more appropriate mixture model in this case., (Copyright © 2015 The Authors. Published by Elsevier B.V. All rights reserved.)

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.

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.

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., (Copyright © 2011 SETAC.)

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.

Synthetic progestins, such as Norethindrone (NET), are common ingredients in oral contraceptives and in treatment for post-menopausal problems. Given the widespread use of oral contraceptives and post-menopausal treatments, several reports have targeted and identified progestins in aquatic environments. In fish, progestins play an important role in the stimulation of oocyte final maturation and ovulation in females, stimulation of spermiation and sperm motility in males, and the initiation of meiosis in both sexes. They also have a role as pheromones in some species. Given the pivotal role that progestins play in reproduction, their appreciable daily dose (i.e. microg to mg range in contraceptives and hormone replacement therapies) and continuous use pattern, it is important to understand the potential risk these compounds pose once discharged into the aquatic environment. Since little published data are available on this class of compounds, our research focused on the reproductive effects of NET on the fathead minnow and Japanese medaka. A 28 day static-renewal reproduction study with Japanese medaka indicated that NET produces a significant decrease in fecundity at aqueous concentrations >or=25 ng/L. A 21 day flow-though fathead minnow reproduction study also demonstrated that NET causes a significant decrease in fecundity in the low ng/L range. Fathead minnow morphological changes (i.e. female fin spots) suggest that NET exposure may have a potent androgenic effect on fish; however, plasma 11-Ketotestosterone (11-KT) concentrations were reduced in males at the highest exposure concentration. Collectively, these data indicate that further investigation of reproductive responses associated with synthetic progestins is warranted.

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.

The effects of Clofibric acid (a persistent environmental metabolite of Clofibrate, a human pharmaceutical), on Fathead minnows were studied. Fibrates are used to prevent cardiovascular disease through their antilipidemic activity. In a series of experiments, in which fish were exposed to waterborne Clofibric acid, no convincing, reproducible antilipidemic effects were observed. In contrast, in three separate experiments, Clofibric acid affected the reproductive axis of fish. Spermatogenesis was apparently impaired, leading to a marked reduction in sperm count in two of the three experiments. Various measures of sperm motility were also reduced, although only significantly so at the highest concentration of Clofibric acid tested (1mg/L). There were also indications that plasma androgen concentrations were reduced. These effects of Clofibric acid on the reproductive axis of fish are similar to those that occur in some mammals as a side-effect of the drug. Taken together, a weight-of-evidence argument would suggest that the main discernable effect of Clofibric acid on fish is likely to be a reproductive, not an antilipidemic, one. Although some of these reproductive effects of Clofibric acid occurred only at a high concentration (1mg/L), others occurred at lower concentrations (microg/litre), near or similar to those reported in the aquatic environment (ng to low microg/litre range). Although we recognise that this is not a definitive study of the effects of Clofibric acid on fish reproduction, the results strongly suggest that Clofibric acid could adversely affect sperm parameters and androgen concentrations in adult Fathead minnows. Further studies are warranted. This may be an example of a drug in which an accidentally discovered side-effect found in mammals turns out to be the most important effect in a different vertebrate group, namely fish.