Abstract: In this study, we examined the toxic effect of sublethal doses of acetochlor (1, 2, 4, 8 mg·kg^-1) on earthworms by exogenous addition. The growth inhibition rate, cytochrome P450 isozymes (CYP1A2, 2C9 and 3A4) activities and the metabolomics were analyzed after seven days of exposure, to infer the toxicity threshold of acetochlor, screen the sensitive biomarkers from the levels of the individual, detoxified enzymes and small molecular metabolites, and elucidate the underlying toxicity mechanism. The results showed that CYP1A2, 2C9 and 3A4 activities were all significantly inhibited, and that the levels of ten metabolites (fructose-6-diphosphate, cytosine monophosphate, uridine monophosphate, adenosine monophosphate, adenosine, xanthine, fumaric acid, dihydroxyglutaric acid, ornithine and 16-hydroxyeicosatetraenoic acid) were significantly decreased by acetochlor exposure. The levels of six metabolites (adenosine succinic acid, succinic acid, arginine, tryptophan, asparagine and phenylalanine) were significantly increased when earthworms being exposed to 2-8 mg·kg^-1 acetochlor. Acetochlor exposure caused oxidative damage to earthworms, weakened the glycolysis, disturbed the tricarboxylic acid cycle, disordered the purine and pyrimidine metabolism, and impaired the amino acids metabolism. Compared with the end point at individual level, the above 16 small molecule metabolites and CYP isozymes activities were more sensitive to acetochlor exposure. It was thus recommended that CYP isozymes (1A2, 2C9, and 3A4) activities and small molecular metabolites could be used as a set of biomarkers to diagnose the acetochlor pollution, given their high sensitivity and accuracy.

Key words: multi-endpoints system, cytochrome P450 isoenzymes, metabolomics, biomarkers, toxicity mechanism