Single and combined neurotoxic effects of chlorpyrifos and perfluorohexanesulfonic acid (PFHxS) in developing zebrafish (Danio rerio)

Perfluorohexanesulfonic acid (PFHxS), is a per- and polyfluoroalkyl substance (PFAS) and is detected in aquatic ecosystems. Chlorpyrifos (CPF), a widely used organophosphate pesticide, is also frequently detected in surface waters.Although the toxicity of these compounds is well known individually, their combined effects are still poorly understood. In this study, zebrafish embryos were exposed to CPF (0.7–700 µg/L), either alone or combined with PFHxS (10 µg/L), to investigate potential interactive effects. After five days of exposure, endpoints including survival, hatching rate, morphological abnormalities, locomotor behavior, oxidative stress, apoptosis, and transcription of selected genes were analyzed. Survival in the 700 µg/L CPF group was close to 25%, while co-exposure with PFHxS further decreased survival to nearly 5%, suggesting increased toxicity in the combined treatment. A reduction in survival was observed in embryos exposed to 0.7 µg/L CPF alone; however, this effect was not evident in the CPF/PFHxS co-exposure group. Exposure to CPF caused a concentration-related rise in developmental abnormalities, with the highest frequencies detected at 70 and 700 µg/L. Under co-exposure conditions, deformities were detected only at 700 µg/L CPF combined with PFHxS. In general, CPF alone promoted dose-dependent increases in both mortality and malformation rates, whereas the mixture with PFHxS resulted in approximately 30% deformities at 5 dpf. Behavioral analyses demonstrated an overall reduction in locomotor activity depending on both exposure concentration and light/dark phase. No alterations were detected in ROS production or apoptosis levels. However, gene expression assays indicated increased transcription of neurotoxicity-associated genes, including ache, gfap, shha, and syn2a, especially at intermediate CPF concentrations and combined exposure. Genes associated with ROS exhibited distinct expression profiles, with sod1 being upregulated and cat downregulated exclusively in the mixture treatment. These findings highlight the importance of mixture toxicity studies to better understand the ecological risks of co-occurring contaminants.