To compare aquatic organisms’ responses to the toxicity of copper oxide (CuO) nanoparticles (NPs) with those of CuO microparticles (MPs) and copper (Cu) ions, a global metabolomics approach was employed to investigate the changes of both polar and nonpolar metabolites in microalga Chlorella vulgaris after 5-day exposure to CuO NPs and MPs (1 and 10 mg/L), as well as the corresponding dissolved Cu ions (0.08 and 0.8 mg/L). Unchanged growth, slight reactive oxygen species production, and significant membrane damage (at 10 mg/L CuO particles) in C. vulgaris were demonstrated. A total of 75 differentiated metabolites were identified. Most metabolic pathways perturbed after CuO NPs exposure were shared by those after CuO MPs and Cu ions exposure, including accumulation of chlorophyll intermediates (max. 2.4–5.2 fold), membrane lipids remodeling for membrane protection (decrease of phosphatidylethanolamines (min. 0.6 fold) and phosphatidylcholines (min. 0.2–0.7 fold), as well as increase of phosphatidic acids (max. 1.5–2.9 fold), phosphatidylglycerols (max. 2.2–2.3 fold), monogalactosyldiacylglycerols (max. 1.2–1.4 fold), digalactosylmonoacylglycerols (max. 1.9–3.8 fold), diacylglycerols (max. 1.4 fold), lysophospholipids (max. 1.8–3.0 fold), and fatty acids (max. 3.0–6.2 fold)), perturbation of glutathione metabolism induced by oxidative stress, and accumulation of osmoregulants (max. 1.3–2.6 fold) to counteract osmotic stress. The only difference between metabolic responses to particles and those to ions was the accumulation of fatty acids oxidation products: particles caused higher fold changes (particles/ions ratio 1.9–3.0) at 1 mg/L and lower fold changes (particles/ions ratio 0.4–0.7) at 10 mg/L compared with ions. Compared with microparticles, there was no nanoparticle-specific pathway perturbed. These results confirm the predominant role of dissolved Cu ions on the toxicity of CuO NPs and MPs, and also reveal particle-specific toxicity from a metabolomics perspective.
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