Potential adverse outcome pathway (AOP) of emamectin benzoate mediated cardiovascular toxicity in zebrafish larvae (Danio rerio)

Emamectin benzoate (EMB) is an efficient insecticide which widely used as an anthelmintic drug additive in aquaculture fish. However, its extensive use has resulted in widespread pollution in the aquatic environment. Previous studies have identified the potential developmental and neurotoxic effects of EMB, however, systematic studies pertaining to the cardiovascular toxic effects of EMB on fish are scarce. In this study, zebrafish embryos were exposed to EMB at concentrations of 0, 0.1, 0.25, 0.5, 1, 2, 4, and 8 mg/L for 3 days, aiming to investigate the cardiovascular toxic effects of EMB via examining morphology, cardiac function, and vascular development phenotypes. It revealed that EMB exposure led to marked deteriorated effects, including adverse effects on mortality, hatching rate, and general morphological traits, such as malformation, heart rate, body length, and eye area, in zebrafish embryos/larvae. Furthermore, EMB exposure resulted in abnormal cardiac function and vascular development, triggering neutrophil migration and aggregation toward the pericardial and dorsal vascular regions, and finalized apoptosis in the zebrafish heart region, these phenomena were further deciperred by the transcriptome analysis that the Toll-like receptor pathway, P53 pathway, and apoptotic pathway were significantly affected by EMB exposure. Moreover, the molecular docking and aspirin anti-inflammatory rescue assays indicated that TLR2 and TLR4 might be the potential targets of EMB. Taken together, our study provides preliminary evidence that EMB may induce apoptosis by affecting inflammatory signaling pathways and eventually lead to abnormal cardiovascular development in zebrafish. This study provides a simple toxicological AOP framework for safe pesticide use and management strategies.