We describe the androgen receptor (AR) agonistic/antagonistic effects of 140 veterinary drugs regulated in Republic of Korea, by setting maximum residue limits. It was conducted using two in vitro test guidelines of the Organization for Economic Cooperation and Development (OECD)—the AR-EcoScreen AR transactivation (TA) assay and the 22Rv1/MMTV_GR-KO AR TA assay. These were performed alongside the AR binding affinity assay to confirm whether their AR agonistic/antagonistic effects are based on the binding affinity to AR. Prior to conducting the AR TA assay, the proficiency test was passed the proficiency performance criterion for the AR agonist and AR antagonist assays. Among the veterinary drugs tested, four veterinary drugs (dexamethasone, trenbolone, altrenogest, and nandrolone) and six veterinary drugs (cymiazole, dexamethasone, zeranol, phenothiazine, bromopropylate, and isoeugenol) were determined as AR agonist and AR antagonist, respectively in both in vitro AR TA assays. Zeranol exhibited weak AR agonistic effects with a PC₁₀ value only in the 22Rv1/MMTV_GR-KO AR TA assay. Regarding changing the AR agonistic/antagonistic effects through metabolism, the AR antagonistic activities of zeranol, phenothiazine, and isoeugenol decreased significantly in the presence of phase I + II enzymes.These data indicate that various veterinary drugs could have the potential to disrupt AR-mediated human endocrine system. Furthermore, this is the first report providing information on AR agonistic/antagonistic effects of veterinary drugs using in vitro OECD AR TA assays.

Globally, amphibian species are experiencing dramatic population declines, and many face the risk of imminent extinction. Endocrine-disrupting chemicals (EDCs) have been recognised as an underappreciated factor contributing to global amphibian declines. In this regard, the use of hormonal growth promotants in the livestock industry provides a direct pathway for EDCs to enter the environment—including the potent anabolic steroid 17β-trenbolone. Emerging evidence suggests that 17β-trenbolone can impact traits related to metabolism, somatic growth, and behaviour in non-target species. However, far less is known about possible effects of 17β-trenbolone on anuran species, particularly during early life stages. Accordingly, in the present study we investigated the effects of 28-day exposure to 17β-trenbolone (mean measured concentrations: 10 and 66 ng/L) on body size, body condition, metabolic rate, and anxiety-related behaviour of tadpoles (Limnodynastes tasmaniensis). Specifically, we measured rates of O₂ consumption of individual tadpoles as a proxy for metabolic rate and quantified their swimming activity and their time spent in the upper half of the water column as indicators of anxiety-related behaviour. Counter to our predictions based on effects observed in other taxa, we detected no effect of 17β-trenbolone on body size, metabolic rate, or behaviour of tadpoles; although, we did detect a subtle, but statistically significant decrease in body condition at the highest 17β-trenbolone concentration. We hypothesise that 17β-trenbolone may induce taxa-specific effects on metabolic function, growth, and anxiety-related behaviour, with anurans being less sensitive to disruption than fish, and encourage further cross-taxa investigation to test this hypothesis.

The environmental impact of endocrine-disrupting chemicals (EDCs)—compounds that interfere with endocrine system function at minute concentrations—is now well established. In recent years, concern has been mounting over a group of endocrine disruptors known as hormonal growth promotants (HGPs), which are natural and synthetic chemicals used to promote growth in livestock by targeting the endocrine system. One of the most potent compounds to enter the environment as a result of HGP use is 17β-trenbolone, which has repeatedly been detected in aquatic habitats. Although recent research has revealed that 17β-trenbolone can interfere with mechanisms of sexual selection, its potential to impact sequential female mate choice remains unknown, as is true for all EDCs. To address this, we exposed female guppies (Poecilia reticulata) to 17β-trenbolone at an environmentally relevant level (average measured concentration: 2 ng/L) for 21 days using a flow-through system. We then compared the response of unexposed and exposed females to sequentially presented stimulus (i.e., unexposed) males that varied in their relative body area of orange pigmentation, as female guppies have a known preference for orange colouration in males. We found that, regardless of male orange pigmentation, both unexposed and exposed females associated with males indiscriminately during their first male encounter. However, during the second male presentation, unexposed females significantly reduced the amount of time they spent associating with low-orange males if they had previously encountered a high-orange male. Conversely, 17β-trenbolone-exposed females associated with males indiscriminately (i.e., regardless of orange colouration) during both their first and second male encounter, and, overall, associated with males significantly less than did unexposed females during both presentations. This is the first study to demonstrate altered sequential female mate choice resulting from exposure to an endocrine disruptor, highlighting the need for a greater understanding of how EDCs may impact complex mechanisms of sexual selection.

Hormonal growth promoters (HGPs), widely used in beef cattle production globally, make their way into the environment as agricultural effluent—with potential impacts on aquatic ecosystems. One HPG of particular concern is 17β-trenbolone, which is persistent in freshwater habitats and can affect the development, morphology and reproductive behaviors of aquatic organisms. Despite this, few studies have investigated impacts of 17β-trenbolone on non-reproductive behaviors linked to growth and survival, like boldness and predator avoidance. None consider the interaction between 17β-trenbolone and other environmental stressors, such as temperature, although environmental challenges confronting animals in the wild seldom, if ever, occur in isolation. Accordingly, this study aimed to test the interactive effects of trenbolone and temperature on organismal behavior. To do this, eastern mosquitofish (Gambusia holbrooki) were subjected to an environmentally-relevant concentration of 17β-trenbolone (average measured concentration 3.0 ± 0.2 ng/L) or freshwater (i.e. control) for 21 days under one of two temperatures (20 and 30 °C), after which the predator escape, boldness and exploration behavior of fish were tested. Predator escape behavior was assayed by subjecting fish to a simulated predator strike, while boldness and exploration were assessed in a separate maze experiment. We found that trenbolone exposure increased boldness behavior. Interestingly, some behavioral effects of trenbolone depended on temperature, sex, or both. Specifically, significant effects of trenbolone on male predator escape behavior were only noted at 30 °C, with males becoming less reactive to the simulated threat. Further, in the maze experiment, trenbolone-exposed fish explored the maze faster than control fish, but only at 20 °C. We conclude that field detected concentrations of 17β-trenbolone can impact ecologically important behaviors of fish, and such effects can be temperature dependent. Such findings underscore the importance of considering the potentially interactive effects of other environmental stressors when investigating behavioral effects of environmental contaminants.

The capacity of pharmaceutical pollution to alter behaviour in wildlife is of increasing environmental concern. A major pathway of these pollutants into the environment is the treatment of livestock with hormonal growth promotants (HGPs), which are highly potent veterinary pharmaceuticals that enter aquatic ecosystems via effluent runoff. Hormonal growth promotants are designed to exert biological effects at low doses, can act on physiological pathways that are evolutionarily conserved across taxa, and have been detected in ecosystems worldwide. However, despite being shown to alter key fitness-related processes (e.g., development, reproduction) in various non-target species, relatively little is known about the potential for HGPs to alter ecologically important behaviours, especially across multiple contexts. Here, we investigated the effects of exposure to a field-realistic level of the androgenic HGP metabolite 17β-trenbolone—an endocrine-disrupting chemical that has repeatedly been detected in freshwater systems—on a suite of ecologically important behaviours in wild-caught female eastern mosquitofish (Gambusia holbrooki). First, we found that 17β-trenbolone-exposed fish were more active and exploratory in a novel environment (i.e., maze arena), while boldness (i.e., refuge use) was not significantly affected. Second, when tested for sociability, exposed fish spent less time in close proximity to a shoal of stimulus (i.e., unexposed) conspecific females and were, again, found to be more active. Third, when assayed for foraging behaviour, exposed fish were faster to reach a foraging zone containing prey items (chironomid larvae), quicker to commence feeding, spent more time foraging, and consumed a greater number of prey items, although the effect of exposure on certain foraging behaviours was dependent on fish size. Taken together, these findings highlight the potential for exposure to sub-lethal levels of veterinary pharmaceuticals to alter sensitive behavioural processes in wildlife across multiple contexts, with potential ecological and evolutionary implications for exposed populations.

The interaction of xenobiotics is common in aquatic ecosystems; therefore, we wanted to evaluate if trenbolone (TB) modulates the effects of 17α-ethinylestradiol (EE2). Male eelpout (Zoarces viviparus) were exposed to 5 ng L−1 EE2 continuously for 19 d (EE2-C) or discontinuously (11 d, EE2-D) alone or in combination with low (50 ng L−1, TBL) or high (500 ng L−1, TBH) concentrations of TB (19 d). Exposure to EE2 caused reduced gonadosomatic index, increased plasma vitellogenin concentrations, up-regulated vtg and era mRNA expression and severe alterations in gonadal histology. TBL and TBH did not affect plasma vitellogenin, era or vtg mRNA expression. TBL and TBH did not counteract the EE2-induced increase in plasma vitellogenin and reduction in 11-ketotestosterone whereas TBH counteracted the EE2 induced increase in vtg and era mRNA expression. Exposure to TBH and EE2-C + TBH lead to severe gonadal histology alterations. TBL and EE2-D + TBH exposed fish showed less histopathological alterations.

The synthetic growth-promoting hormones trenbolone and melengestrol acetate have been detected in the environment near beef cattle feedlots and are reportedly transported via wind-borne particulate matter. Therefore, movement of synthetic hormones from beef cattle feedlots to water bodies via particulate matter is possible. Our objective was to evaluate potential effects of 17α-trenbolone (17α-TB), melengestrol acetate (MGA), and combinations of both on growth, development, and survival of Xenopus laevis larvae. On post-hatch day 2 (stage 33/34), X. laevis larvae were exposed to three nominal concentrations of 17α-TB (10, 100, and 500 ng/L), MGA (1, 10, and 100 ng/L), a combination of both (1/10, 10/100, and 100/500 ng/L MGA/17α-TB), frog embryo teratogenesis assay-Xenopus medium, or a solvent control. Significant increases in all X. laevis growth metrics were observed among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB and 10 ng/L MGA + 100 ng/L 17α-TB treatments. Stage of development was increased among larvae in the 1 ng/L MGA + 10 ng/L 17α-TB treatment group and significantly decreased among those in the 500 ng/L 17α-TB treatment. Total body mass and snout–vent length of X. laevis larvae were significantly reduced in the 100 ng/L MGA and 100 ng/L MGA + 500 ng/L 17α-TB treatment groups. Larvae exposed to 500 ng/L 17α-TB had decreased total body mass, snout–vent length, and total length. In general, growth measurements decreased with increasing concentration of MGA, 17α-TB, or a combination of both. Survival among all treatments was not significantly different from controls. Amphibians exposed to MGA and 17α-TB in the environment may experience alterations in growth and development.