Toxicity Assessment of Mixture Effects of Insecticides and Perfluorinated Chemicals (PFAS) in Zebrafish ( <scp> <i>Danio rerio</i> </scp> ): A Case Study With Chlorpyrifos and Perfluorohexanoic Acid (PFHxA)

Aquatic ecosystems are increasingly contaminated by complex mixtures of perfluoroalkyl and polyfluoroalkyl substances (PFAS) and pesticides, yet the combined toxicological impacts of these pollutants on aquatic organisms remain poorly understood. This study investigated the effects of chlorpyrifos (CPF) (0.62-620 μg/L) and perfluorohexanoic acid (PFHxA) (10 μg/L) in their isolated forms and as a mixture on developing zebrafish (Danio rerio). We measured several endpoints related to survivability, hatchability, malformations, apoptosis, reactive oxygen species (ROS), locomotor activity, and gene expression following a 7-day exposure. We also employed a computational approach to identify molecular interactions between CPF and PFHxA. Survival decreased with exposure to CPF and the mixture CPF/PFHxA at 620 μg/L. Increased deformity rates were observed at 62 μg/L CPF, but no effects were observed in the hatching rate. In the behavior assay, CPF and the mixture decreased the larvae's locomotor activity. Expression of genes related to neurotoxicity and oxidative stress was altered mainly in the coexposure group. ROS levels were higher in the 62 μg/L CPF than in the solvent control but lower in the coexposure with 0.62 μg/L CPF/PFHxA. Our results demonstrate interactive effects of PFHxA and chlorpyrifos, emphasizing the importance of mixture toxicity in aquatic risk assessment.