Abstract
Background: Zebrafish have been widely used to study anxiety-related phenotypes using the novel tank test (NTT). Although the NTT is well-characterized and commonly used by researchers, there is still a lack of information regarding how different experimental variables such as water quality can influence NTT performance. Zebrafish use different chemical cues and olfactory stimuli to communicate in water, so we predicted that water change frequency would affect cortisol, locomotion and anxiety-related parameters in the NTT. New methods: After extensive literature research, we found that only about 18% of papers using NTT report partial or complete water changes between subjects. Here, we tested multiple zebrafish in the NTT using the same water up to 9 consecutive times (with no water change) and analyzed cortisol levels, as a stressrelated marker. Results: We found that when using the same water for more than 4 trials, data variability is increased and a higher number of extreme values is observed for the time spent in the top zone and immobility. Moreover, after 4 trials with no water change, increased cortisol levels are observed, indicating that animals show increased stressrelated responses with the lack of water changes. Conclusions: This study shows that lack of water change can significantly influence zebrafish stress-responses in the NTT. Altogether, behavioral experiments should avoid using the same water when testing multiple fish in the task, especially when looking at anxiety in the NTT.