Integrated molecular and metabolomic assessment of copper hydroxide nanopesticide toxicity in zebrafish

Abstract Copper hydroxide (Cu(OH) 2 ) nanopesticides [Cu(OH) 2 NP] are increasingly applied in agriculture, raising concerns about their potential risks to non-target aquatic organisms. In this study, we investigated the toxic effects of Cu(OH) 2 NP exposure in zebrafish ( Danio rerio ) larvae and adults using an integrated gene expression and metabolomic approach. Zebrafish embryos were exposed to sublethal concentrations of Cu(OH) 2 NP until 96 h post-fertilization, while adults were subjected to a 14-day sub-chronic exposure. Cu(OH) 2 NP significantly altered the expression of genes involved in copper homeostasis and redox regulation ( cox16 , atp7a , ccs , and gclm ), accompanied by activation of endoplasmic reticulum stress ( atf6 , perk , and ire1 ) and apoptosis-related pathways ( casp3 , casp9 , bax , and bcl2 ). These responses were dose-dependent and more pronounced in zebrafish larvae and adult liver tissue. Metabolomic analysis revealed substantial metabolic reprogramming, particularly affecting energy metabolism, amino acid pathways, and glutathione metabolism. In adult zebrafish, significant changes in hepatosomatic index (HSI) indicated disrupted energy balance and hepatic stress. Integrated analysis suggests that Cu(OH) 2 NP toxicity is associated with disruption of copper homeostasis and activation of cellular stress-response pathways. Overall, these findings improve our understanding of the molecular and metabolic responses of zebrafish to Cu(OH) 2 NP exposure and highlight potential ecological risks of nano-enabled agrochemicals.