Reductive stress and apoptotic activities in bivalve mollusc Unio tumidus exposed to the combination of microplastics, antibiotic salinomycin and heating

<title>Abstract</title> Multistress effects lead to unpredicted consequences in aquatic ecotoxicology and are extremely concerning. The goal of this study was to trace how specific effects of the antibiotic salynomycin (Sal) and microplastics (MP) on the bivalve molluscs are manifested in the combined environmentally relevant exposures. <italic>Unio tumidus</italic> specimens were treated with Sal (0.6 µg L^{− 1}), MPs (1 mg L^{− 1}, 2 µm size), and both at 18°C (Mix) and 25°C (MixT) for 14 days. The redox stress and apoptotic enzyme responses and the balance of Zn/Cu in the digestive gland were analyzed. The shared signs of stress included a decrease in NAD⁺/NADH and Zn/Cu ratios and lysosomal integrity, and an increase in Zn-metallothioneins and cholinesterase levels. MP caused a decrease in the glutathione (GSH) concentration and redox state, total antioxidant capacity, and Zn levels. MP and Mix induced coordinated apoptotic/autophagy activities, increasing caspase-3 and cathepsin D (CtD) total and extralysosomal levels. Sal activated caspase-3 only and increased by five times Cu level in tissue. Due to the discriminant analysis, the cumulative effect was evident in the combined exposure at 18°C. However, under heating, the levels of NAD⁺, NADH, GSH, GSH/GSSG and metallothionein-related thiols were decreased, and coordination of the cytosolic and lysosomal death stimuli was distorted, confirming that heating and pollution could exert unexpected synergistic effects on aquatic life.