When crayfish face painkillers: Tissue-specific cytotoxic and oxidative responses to indomethacin exposure in Procambarus clarkii

Non-steroidal anti-inflammatory drugs (NSAIDs) are increasingly recognized as emerging contaminants in freshwater ecosystems. However, their effects on aquatic invertebrates remain poorly investigated. This study evaluated for the first time both cellular and biochemical responses of the red swamp crayfish (Procambarus clarkii) following a long-term exposure to indomethacin, a widely used NSAID. Specimens were exposed for 28 days to 2.5, 5, and 10 mg/L, and biomarkers indicative of cytotoxicity and oxidative stress were evaluated. Viability of both hemolymph and hepatopancreas cells was assessed using Trypan Blue and Neutral Red assays, while oxidative stress biomarkers, including superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione S-transferase (GST), and malondialdehyde (MDA), were analyzed in hepatopancreas, gill, and muscle tissues. The results showed that indomethacin exposure induced significant, tissue-specific cytotoxic and oxidative responses. Notably, hepatopancreas cell viability decreased significantly at lower concentrations, with partial recovery at the highest dose, suggesting a possible hormetic effect of the contaminant. Oxidative stress biomarkers exhibited concentration-dependent changes: SOD and MDA levels were significantly altered in the hepatopancreas; GST activity was affected in the gills, and GPx activity increased in muscle at higher concentrations. Haemocyte viability remained unaffected, indicating resilience of circulating cells. The Integrative Biomarker Response-Threshold (IBR-T) index highlighted both hormetic and classical dose-response trends, depending on tissue type. From an ecological perspective, these findings suggest that P. clarkii may tolerate relatively high indomethacin concentrations without systemic oxidative collapse, potentially supporting its persistence and competitive advantage in contaminated habitats. However, the hepatopancreas sensitivity indicates possible sublethal metabolic impairment, which could affect long-term fitness and population dynamics. Overall, this study provides novel insights into the ecotoxicological effects of indomethacin in a keystone invasive species and underscore the importance of tissue-specific biomarker analysis for environmental risk assessment.