Lead (Pb) and arsenic (As) are commonly occurring heavy metals in the environment and produce detrimental impacts on the central nervous system. Although they have both been indicated to exhibit neurotoxic properties, it is not known if they have joint effects, and their mechanisms of action are likewise unknown. In this study, zebrafish were exposed to different concentrations of Pb (40 μg/L, 4 mg/L), As (32 μg/L, 3.2 mg/L) and their combinations (40 μg/L + 32 μg/L, 4 mg/L + 3.2 mg/L) for 30 days. The histopathological analyses showed significant brain damage characterized by glial scar formation and ventricular enlargement in all exposed groups. In addition, either Pb or As staining inhibited the swimming speed of zebrafish, which was enhanced by their high concentrations in a mixture. To elucidate the underlying mechanisms, we examined changes in acetylcholinesterase (AChE) activity, neurotransmitter (dopamine, 5-hydroxytryptamine) levels, HPI axis-related hormone (cortisol and epinephrine) contents and neurodevelopment-related gene expression in zebrafish brain. The observations suggest that combined exposure to Pb and As can cause abnormalities in swimming behavior and ultimately exacerbate neurotoxicity in zebrafish by interfering with the cholinergic system, dopamine and 5-hydroxytryptamine signaling, HPI axis function as well as neuronal development. This study provides an important theoretical basis for the mixed exposure of heavy metals and their toxicity to aquatic organisms.

The highly hazardous chemical ammonia has been proven to be absorbed by nanoparticles, thereby exerting highly toxic effects on aquatic organisms. As a ubiquitous pollutant in aquatic environments, polystyrene nanomicroplastics (PSNPs) have shown strong adsorption capacity due to their large surface area. Therefore, the potential joint effects of ammonia and PSNPs need to be clarified. In this study, zebrafish embryos were exposed to a water solution with ammonia concentrations (0, 0.1, 1, and 10 mg/L) with or without PSNP (100 μg/L) treatment up to 120 hpf. The results showed that combined exposure increased the accumulation of ammonia and obviously reduced the locomotor speed of zebrafish larvae compared with exposure to ammonia alone. Further studies indicated that PSNPs can aggravate ammonia-induced neurotoxicity by altering the cholinergic system, dopaminergic neurons, and the retinal structure in zebrafish larvae. In addition, our results revealed that ammonia caused significant alterations in the expression of genes related to neurodevelopment and retinal development, and PSNPs exacerbated this adverse effect. In conclusion, PSNPs can aggravate ammonia-induced neurotoxicity in the early stage of zebrafish and their associated health risk to aquatic animals should not be underestimated. The main contribution of this article lies in revealing the synergistic neurotoxicity of ammonia and PSNPs in the early stage of zebrafish. Moreover; it emphasizes that the associated health risks to aquatic animals should not be underestimated.