Fish on steroids: Temperature-dependent effects of 17β-trenbolone on predator escape, boldness, and exploratory behaviors

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.