Elucidating Toxicodynamic Differences at the Molecular Scale between ZnO Nanoparticles and ZnCl₂ in Enchytraeus crypticus via Nontargeted Metabolomics

Much effort has been devoted to clarifying the comparative toxicity of ZnO nanoparticles (NPs) and Zn ions; however, little is known about their toxicodynamic processes at the metabolic level. Here, we investigated the acute (2d) and chronic (7d) effects to a soil species, Enchytraeus crypticus, of two sublethal doses of ZnO-NPs and ZnCl₂ (10 and 30 mg/L Zn) using ultrahigh performance liquid chromatography-quadrupole-time-of-flight/mass spectrometry-based metabolomics. The metabolomics analysis identified 99, 128, 121, and 183 significantly changed metabolites (SCMs) in E. crypticus exposed to ZnO-NPs for 2d, ZnCl₂ for 2d, ZnO-NPs for 7d, and ZnCl₂ for 7d, respectively, suggesting that ZnCl₂ induced stronger metabolic reprogramming than ZnO-NPs, and a longer exposure time caused greater metabolite changes. Among the SCMs, 67 were shared by ZnO-NPs and ZnCl₂ after 2d and 84 after 7d. These metabolites were mainly related to oxidative stress and antioxidant defense, membrane disturbance, and energy expenditure. The targeted analysis on physiological and biochemical responses further proved the metabolic observations. Nevertheless, 32 (33%) and 37 (31%) SCMs were found only in ZnO-NP treatments after 2 and 7d, respectively, suggesting that the toxicity of ZnO-NPs cannot be solely attributed to the released Zn ions. Metabolic pathway analysis revealed significant perturbations of galactose metabolism, amino sugar and nucleotide sugar metabolism, and glycerophospholipid metabolism in all test groups. Based on involvement frequency, glucose-1-phosphate, glycerol 3-phosphate, and phosphorylcholine could serve as universal biomarkers for exposure to different Zn forms. Four pathways perturbed by ZnO-NPs were nanospecific upon acute exposure and three upon chronic exposure. Our findings demonstrated that metabolomics is an effective tool for understanding the molecular toxicity mechanism and highlighted that time-series measurements are essential for discovering and comparing modes of action of metal ions and NPs.