Microplastics alone or co-exposed with copper induce neurotoxicity and behavioral alterations on zebrafish larvae after a subchronic exposure.

Microplastics (MPs, <5 mm) have been frequently detected in aquatic ecosystems, representing both health and ecological concerns. However data about the combined effects of MPs and other contaminants is still limited. This study aimed to evaluate the impact of MPs and the heavy metal copper (Cu) on zebrafish (Danio rerio) larvae development and behavior. Zebrafish embryos were subchronically exposed to MPs (2 mg/L), two sub-lethal concentrations of Cu (60 and 125 µg/L) and binary mixtures of MPs and Cu using the same concentrations, from 2-h post fertilization until 14 days post fertilization. Lethal and sub-lethal responses (mortality, hatching, body length) were evaluated during the embryogenesis period, and locomotor, avoidance, anxiety and shoaling behaviors, and acetylcholinesterase (AChE) activity were measured at 14 dpf. The results showed that survival of larvae was reduced in groups exposed to MPs, Cu and Cu+MPs. Regarding the behavioral patterns, the higher Cu concentration and mixtures decreased significantly the mean speed, the total distance traveled and the absolute turn angle, demonstrating an adverse effect on swimming competence of zebrafish larvae. Exposure to MPs and Cu, alone or combined, also affected avoidance behavior of zebrafish, with larvae not reacting to the aversive stimulus. There was a significant inhibition of AChE activity in larvae exposed to all experimental groups, compared to the control group. Moreover, a higher inhibition of AChE was noticed in larvae exposed to MPs and both Cu+MPs groups, comparatively to the Cu alone groups. Our findings demonstrate the adverse effects of MPs, alone or co-exposed with Cu, on fish early life stages behavior. This study highlights that MPs and heavy metals may have significant impacts on fish population fitness by disrupting locomotor and avoidance behaviors.