Multi-species biomarker assessment of olive mill wastewater toxicity on freshwater bivalves

Olive mill wastewater (OMWW) is a major environmental stressor in Mediterranean regions, producing phytotoxic effluents that may threaten aquatic ecosystems. However, the ecological effects of OMWW discharges are rarely assessed, especially in filter feeders organisms such as freshwater bivalves. Given this gap, the present study evaluated the long-term effects of OMWW exposure (14 days) on four native (Unio delphinus, Unio tumidiformis, Anodonta anatina, and Potomida littoralis) and one non-native (Corbicula fluminea) freshwater bivalve species. Under controlled laboratory conditions specimens from the five species were exposed to three treatments: two OMWW concentrations (C1: 8.77 mg/L and C2: 21.31 mg/L of organic matter) and a control (CTL: 0 mg/L). At the end of the experiment, neurotoxicity (acetylcholinesterase- AChE) and oxidative stress (superoxide dismutase - SOD, Thiobarbituric acid reactive substances - TBARs) biomarkers were assessed in the gills and in the digestive gland. Results demonstrated higher sensitivity of native species-especially U. tumidiformis- showing an overall ineffectiveness of antioxidant mechanisms (SOD) to neutralize reactive oxygen species (ROS), leading to TBARs accumulation and neural inhibition (AChE). In contrast, the non-native species C. fluminea respond to the lower OMWW concentration used reducing basal activities such as SOD and AChE, which may be interpreted as a reduction in their metabolic rate, leading to minimal energy expenditure. Moreover, C. fluminea exhibited a biphasic response characterized by enzymatic downregulation at lower concentrations and enzymatic upregulation at higher levels, suggesting a higher physiological plasticity compared to native species. Overall, this study underscore the ecological importance of bivalves as sentinel species to assess OMWW pollution, supporting the objectives of the EU Water Framework Directive.