Synergistic Activation of Immune Pathways by Pesticide Chlorpyrifos and Its Metabolite 3,5,6-Trichloro-2-Pyridinol Mixtures in Fish

Various pesticides and their metabolites commonly co-occur in aquatic environments, yet their combined effects are often overlooked. In this study, alterations in blood physiology (e.g., routine blood parameters, erythrocyte damage, respiratory burst, serum metabolomics, and expression of immune- and inflammation-related genes) in crucian carp (Carassius auratus) following single and combined exposure to chlorpyrifos (CPF) and its metabolite 3,5,6-trichloro-2-pyridinol (TCP) were investigated. CPF mainly affected oxidative metabolism and immune activation, while TCP induced erythrocyte damage and hematological disorders. Notably, CPF + TCP coexposure markedly upregulated immune- and inflammation-related genes, activated toll-like receptors, and induced distinct hematological toxicity compared to single exposure. Coexposure triggered proinflammatory cytokines (e.g., TNFα, IL-1β, IL-6), and altered inflammation-related metabolites (e.g., prostaglandins, leukotrienes), forming a synergistic inflammatory cascade. These findings indicate that pesticide–metabolite mixtures synergistically activate innate immune pathways in aquatic organisms, offering insight into combined pesticide-metabolite toxicity in nontargeted aquatic species.