Ozone is often used to disinfect process water in recirculating aquaculture systems. Here, ozone rapidly leads to the formation of secondary oxidants, called ozone produced oxidants (OPO). We studied the impact of OPO on juvenile tilapia Oreochromis niloticus in brackish water at 0, 0.05, 0.1 and 0.15 mg/L chlorine equivalents after 2 d, 9 d and 21 d of exposure, assessing gill histopathology and lesions of the mitochondrial DNA. Damage of the gills was already observed after 2 d, comprising an increased occurrence of lamellar lifting, fusion of the lamellae and edema at 0.15 mg/L. After 21 d, occurrence of lamellar fusion and edema was elevated at 0.15 mg/L. Increased incidence of hyperplasia was already observed at 0.05 mg/L after 9 d. DNA fragmentation increased to 3.92 lesions per 10 kb at 0.15 mg/L compared to 2.62 lesions per 10 kb in the control. In conclusion, first adverse effects were observed at 0.05 mg/L and severe impact is subsequently recorded at 0.1 mg/L chlorine equivalents. Therefore, OPO concentrations need to be kept below 0.05 mg/L chlorine equivalents to avoid adverse effects on animal welfare.