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The purpose of this study was to clarify the effect of fluoxetine, one of the most frequently prescribed antidepressant drugs, on Cyprinus carpio by comparing the variations in its metabolite concentrations after exposure to the drug, using NMR and LC/Orbitrap MS. Three experimental reactors were employed, and the total experimental period was 14 days. Cyprinus carpio were acclimatized for 3 months in the laboratory and then subjected to the study. One reactor was used as a 14-day control, and the others were exposed to fluoxetine at 10 ng/L and 10 μg/L for 7 days and depurate for 7 days. The variations in metabolite concentration after exposure were analyzed by NMR and LC/Orbitrap MS. Before exposure, the concentration trends of fluoxetine and its main metabolite, norfluoxetine were almost the same in blood and liver samples of Cyprinus carpio. However, after the depuration period, the concentrations of norfluoxetine in the liver and blood samples were higher than those of fluoxetine. The concentration of the remaining norfluoxetine in the liver sample was higher than that in the blood sample. This means that the liver mainly metabolized pharmaceuticals. The main change in the concentrations of metabolites of Cyprinus carpio was shown in amino acids such as leucine, isoleucine, glutamate, tyrosine, and valine; organic acids such as acetate and formate; creatine; and glucose. Although after the depuration period, the control, and 10 ng/L and 10 μg/L exposure groups had overlapping results, the exposure and control groups could be classified clearly after 14 days. These results are expected to contribute to identifying the effects of environmental pollutants by tracing metabolite changes and building a chemical metabolite database

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