Neurobehavioral and Molecular Alterations Following Single and Combined Exposure to Chlorpyrifos and PFHxS in Developing Zebrafish (Danio rerio)

Perfluorohexanesulfonic acid (PFHxS) is a per- and polyfluoroalkyl substance (PFAS) frequently detected in aquatic environments, while chlorpyrifos (CPF) is a widely used organophosphate insecticide. Although their individual toxicity is well described, their combined effects remain poorly understood. Here, we evaluated the effects of CPF (0.7 to 700 µg/L), alone or in combination with PFHxS (10 µg/L), in zebrafish embryos. Survival, hatching, malformations, locomotor activity, oxidative stress, apoptosis, and gene expression were assessed after five days of exposure. CPF reduced survival in a concentration-dependent manner, with moderate enhancement under co-exposure, while hatching success was unaffected. Deformities increased with CPF concentration, which remained consistent with PFHxS co-exposure, suggesting toxicity was mediated by CPF. Locomotor activity was largely decreased in a concentration- and phase-dependent manner. No significant changes were observed in ROS levels or apoptosis. Gene expression analysis revealed upregulation of neurotoxicity-related markers (ache, gfap, shha, syn2a), particularly at intermediate CPF concentrations and under co-exposure. Oxidative stress-related genes showed differential responses, with sod1 upregulated and cat downregulated only in the combined treatment. Overall, combined exposure did not substantially enhance toxicity compared with CPF alone, suggesting that CPF was the main contributor to the observed effects, whereas PFHxS had limited influence.