Difenoconazole is a type of triazole fungicide that is widely used in the treatment of plant diseases. Triazole fungicides have been shown in several studies to impair the development of the nervous system in zebrafish embryos. There is still little known about difenoconazole-induced neurotoxicity in fish. In this study, zebrafish embryos were exposed to 0.25, 0.5, and 1 mg/L of difenoconazole solution until 120 h post-fertilization (hpf). The difenoconazole-exposed groups showed concentration-dependent inhibitory tendencies in heart rate and body length. Malformation rate and spontaneous movement of zebrafish embryos increased, and the locomotor activity decreased in the highest exposure group. The content of dopamine and acetylcholine was reduced significantly in difenoconazole treatment groups. The activity of acetylcholinesterase (AChE) was also increased after treatment with difenoconazole. Furthermore, the expression of genes involved in neurodevelopment was remarkably altered, which corresponded with the alterations of neurotransmitter content and AChE activity. These results indicated that difenoconazole might affect the development of the nervous system through influencing neurotransmitter levels, enzyme activity, and the expression of neural-related genes, ultimately leading to abnormal locomotor activity in the early stages of zebrafish.

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.