Fish gills are in direct contact with the surrounding pollutants, and thus, potentially more vulnerable to microplastics (MPs) and heavy metals. The present study aimed to evaluate the long-term exposure effects of MPs and copper (Cu) in the gills of adult zebrafish (Danio rerio). To this end, zebrafish were exposed to MPs (2 mg/L), Cu (Cu25, 25 μg/L) and their mixture (Cu25 + MPs) for 30 days, and then oxidative stress, detoxification, antioxidant, metabolic and neurotoxicity enzymes/genes, as well serotonergic system and apoptosis genes, were evaluated in gills. In the mixture group, ROS levels were increased, while CAT and GPx activities were inhibited, indicating the induction of oxidative stress in zebrafish gills. This was followed by an increase of LPO levels and potential oxidative damage in zebrafish gills. The tryptophan hydroxylase 1a (tph1a) and caspase-3 (casp3) genes were significantly upregulated in Cu25 + MPs group, indicating a potential dysregulation of serotonin synthesis and apoptosis pathways, respectively. Overall, the present study contributes to improving the knowledge about the response of aquatic organisms to MPs and the potential ecological risk that these particles represent to the ecosystems.

The knowledge regarding the neurological and behavioral toxic effects associated with microplastics (MPs) and heavy metals exposure is still scarce. The present study aimed to evaluate the potential chronic (30 days) toxic effects of MPs (2 mg/L) and copper (Cu, 25 µg/L), alone or combined, in the zebrafish (Danio rerio) brain antioxidant system, cell proliferation/death, cholinergic-, serotonergic- and dopaminergic pathways and, consequently, in locomotor, anxiety, and social behaviors. Our findings showed that MPs and Cu exposure modulated the antioxidant system of zebrafish brain, with superoxide dismutase (SOD) and glutathione reductase (GR) having higher activity in the Cu25 +MPs group, but glutathione peroxidase (GPx) being inhibited in MPs, Cu25 and Cu25 +MPs. Moreover, an increase in acetylcholinesterase (AChE) activity was observed in all exposed groups. When considering neurogenesis genes, a downregulation of proliferating cell nuclear antigen (pcna) was noticed in zebrafish exposed to the mixture treatment, while for dopaminergic system-related genes (th and slc6a3) an upregulation was observed in MPs, Cu25 and Cu25 +MPs groups. An increase in apoptosis-related genes expression (casp8, casp9 and casp3) was observed in the MPs exposed group. Changes in zebrafish behavior, particularly in mean speed, total distance moved, inactivity in the aquaria, and social/shoaling behavior was also observed in the MPs and Cu exposed groups. Overall, our results highlight the multiplicity of toxic effects of MPs, alone or combined with Cu, in zebrafish brain, namely apoptosis and alterations in adult neurogenesis, neurocircuits and, consequently, behavior.