Sublethal effects of diclofenac and ibuprofen, individually and in mixture, in a Neotropical fish: A multi-biomarker assessment under environmentally realistic concentrations.

Pharmaceutical contamination of aquatic ecosystems is a growing global concern, particularly in tropical regions where non-steroidal anti-inflammatory drugs (NSAIDs) such as diclofenac (DCF) and ibuprofen (IBU) are frequently detected. This study investigated the sublethal effects of environmentally relevant concentrations of DCF, IBU, as well as their mixture (IBU+DCF) on the freshwater Neotropical fish Astyanax lacustris, using a multi-biomarker approach. Fish were exposed for 7 days, and biomarkers related to oxidative stress, detoxification, osmoregulation, genotoxicity, and endocrine function were evaluated. All treatments induced significant, tissue-specific biochemical alterations. The liver and testis were particularly affected, showing impaired antioxidant defences and increased lipid peroxidation (LPO), especially under mixture exposure. The gills exhibited the same response pattern in all treatments, characterized by increased glutathione S-transferase (GST) activity. The responses of antioxidant enzymes in the brain differed according to treatment. There was a decrease in GST activity in animals exposed to IBU and an increase in SOD activity in the IBU, DCF and IBU+DCF groups. The IBU+DCF mixture also stimulated gill Na⁺/K⁺-ATPase activity, suggesting disruption of prostaglandin-mediated osmoregulatory pathways. Erythrocyte DNA damage was significant across all treatments, with the mixture causing the most pronounced genotoxic effects. The Integrated Biomarker Response (IBR) index indicated higher overall biological disturbance in fish exposed to the mixture than to individual compounds. These findings highlight additive or synergistic toxic effects of DCF and IBU and underscore the ecological relevance of pharmaceutical mixtures. This study contributes important insights into the sublethal impacts of NSAIDs on native Neotropical fish and reinforces the need for more realistic environmental risk assessments that consider the complexity of contaminant mixtures in freshwater ecosystems.