Towards improved behavioural testing in aquatic toxicology: Acclimation and observation times are important factors when designing behavioural tests with fish

- Behavioural toxicology is currently under scrutiny for irreproducibility.
- Baseline behaviours and influencing factors must be understood.
- We identified acclimation and observation time as key factors influencing behaviour in fish.
- Both have the capacity to influence behavioural outcomes and interpretations.
- Review of the literature revealed that both vary substantially between studies.

The quality and reproducibility of science has recently come under scrutiny, with criticisms spanning disciplines. In aquatic toxicology, behavioural tests are currently an area of controversy since inconsistent findings have been highlighted and attributed to poor quality science. The problem likely relates to limitations to our understanding of basic behavioural patterns, which can influence our ability to design statistically robust experiments yielding ecologically relevant data. The present study takes a first step towards understanding baseline behaviours in fish, including how basic choices in experimental design might influence behavioural outcomes and interpretations in aquatic toxicology. Specifically, we explored how fish acclimate to behavioural arenas and how different lengths of observation time impact estimates of basic swimming parameters (i.e., average, maximum and angular velocity). We performed a semi-quantitative literature review to place our findings in the context of the published literature describing behavioural tests with fish. Our results demonstrate that fish fundamentally change their swimming behaviour over time, and that acclimation and observational timeframes may therefore have implications for influencing both the ecological relevance and statistical robustness of behavioural toxicity tests. Our review identified 165 studies describing behavioural responses in fish exposed to various stressors, and revealed that the majority of publications documenting fish behavioural responses report extremely brief acclimation times and observational durations, which helps explain inconsistencies identified across studies. We recommend that researchers applying behavioural tests with fish, and other species, apply a similar framework to better understand baseline behaviours and the implications of design choices for influencing study outcomes.