The Combined Effects of Copper Exposure and Hypoxia on Zebrafish Behaviour

Copper (Cu) and hypoxia are two environmentally relevant stressors that are increasing globally due to natural and anthropogenic influences. While the effects of each stressor have been looked at individually in previous work, the present study aimed to understand their combined effect on the behaviour of adult zebrafish (Danio rerio). As a model species, the behavioural ethogram of zebrafish is well-described, allowing for a suite of behaviours to be quantified, utilizing analysis software and by visual observation. In this study, we assessed behaviours associated with movement (velocity and distance metrics), resting, oxygen-seeking, and sociality following a mechanical stimulus. We compared the behaviour of fish after a 96-h exposure to sublethal Cu (45 µg/L) to those not exposed to Cu, with both groups being tested under normoxic (> 90% oxygen saturation [> 7.44 mg/L O₂]) and hypoxic (< 30% oxygen saturation [< 2.48 mg/L O₂]) conditions. Hypoxia influenced behaviours associated with movement (average velocity, darting, total distance travelled, maximum velocity). Exposure to Cu affected bottom-resting and air-seeking behaviours. Cu and hypoxia independently impacted two indices of sociality (shoal area and proximity) in a similar fashion. Many of the effects of hypoxia were dampened by Cu, whereas effects of hypoxia on responses to Cu were variable. This study highlights the value of using both high-throughput automated software and visual observation to reveal subtle interactive effects of Cu and hypoxia on key behaviours.