Metabolomics approach to evaluate diclazuril-induced developmental toxicity in zebrafish embryo

Anticoccidials, commonly used in veterinary medicine to treat coccidiosis in food-producing animals, particularly in poultry farming, are associated with potential environmental risks due to their excretion in manure and subsequent land-spreading. Diclazuril, a widely used anticoccidial, has been detected in groundwater, raising concerns about its impact on non-target species. This study investigates the developmental toxicity of diclazuril in zebrafish embryos over a 96-hour exposure period, utilizing biomarkers such as oxidative stress indicators and metabolomic profiles. The acute toxicity assessment determined an LC₅₀ of 255 µg/L for diclazuril. Observed sublethal effects included pericardial edema, curved spine, and yolk sac edema, which worsened with increasing concentrations from 106 µg/L to 515 µg/L. Based on the Lowest Observed Adverse Effect Level (LOAEL), further experiments were conducted at concentrations of 50 µg/L, 100 µg/L, and 200 µg/L. Significant increases in reactive oxygen species (ROS) were noted at 100 µg/L and 200 µg/L, alongside notable reduction in superoxide dismutase (SOD) and glutathione S-transferase (GST) activities at concentrations ≥100 µg/L, while no significant changes observed in catalase (CAT) activity. Metabolomic analysis using GC-MS/MS revealed significant disturbances in pathways such as pyruvate metabolism, the citric acid cycle, and amino acid metabolism, indicating potential mitochondrial dysfunction in groups exposed to concentrations ≥100 µg/L. Furthermore, alterations in histological lesions in brain region and altered neurotransmitter activity suggests possible neurobehavioral disorders. Increased oxidative stress, along with decreased ATP and NADH levels, points to mitochondrial dysfunction, which is further supported by ultrastructural analysis and locomotor behavior confirming mitochondrial disruption. The disruption of cellular energetics is likely a key factor contributing to the neurotoxic effects observed in zebrafish embryos exposed to ≥100 µg/L of diclazuril.