Integrated biomarker response-based investigation of oxidative stress in Hemolymph, gills, and digestive glands of freshwater mussels (Unio delicatus) exposed to Etoxazole

Abstract

Pesticides extensively used in agriculture pose risks to non-target aquatic organisms. This study assessed the acute and sublethal effects of etoxazole (ETX), an isoxazoline-derived acaricide, on the freshwater mussel Unio delicatus. The 96 h LC₅₀ was calculated as 463.97 mg/L (316.29–662.47 mg/L, 95 % CI). Mussels were exposed to sublethal ETX concentrations for 96 h and 7 days. Total hemocyte counts (THCs), total antioxidant status (TAS), and total oxidant status (TOS) were measured in hemolymph. In this study, elevated total hemocyte counts (THCs) indicated activation of cellular immune responses, while reduced TAS together with increased TOS reflected systemic oxidative stress, particularly at higher ETX concentrations. Oxidative stress biomarkers (GSH, GPx, SOD, CAT, MDA) were analyzed in gill and digestive gland tissues. A progressive TAS decline was observed in hemolymph over 7 days. Biomarker responses were evaluated using Min-Max normalization and the Integrated Biomarker Response (IBR) index. IBR profiles revealed tissue-specific stress patterns: in hemolymph, THCs > TOS > TAS suggested pronounced immune activation. In gills, CAT > GSH > GPx > MDA > SOD indicated oxidative defense, while in digestive glands, GSH > GPx > SOD > CAT > MDA reflected detoxification. Although ETX showed low acute toxicity, it significantly altered immune and antioxidant systems. These results demonstrate the sensitivity of U. delicatus to sublethal pesticide exposure and support its use as a sentinel species in ecotoxicological studies.