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Juveniles Rhamdia quelen fish species were exposed to diclofenac for 96 h at concentrations of 0.2, 2, and 20 μg/L. Biochemical, genetic, and hematological biomarkers were assessed in the liver, kidney, and blood in order to evaluate the toxic effects. No oxidative stress was observed in liver. In kidney the superoxide dismutase activity increased in all concentrations, suggesting an alteration in the hydrogen peroxide production, but DNA damage and lipid peroxidation were not detected. Diclofenac exposure increased the red blood cells number at concentrations of 0.2 and 2 μg/L, and monocytes and neutrophils at 2 and 20 μg/L, respectively. These results suggest that acute exposure to diclofenac, even at low concentrations, caused hematologic and renal enzymatic alterations in R. quelen.