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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 salinomycin (Sal) and microplastics (MP) on the bivalve molluscs are manifested in the combined environmentally relevant exposures. Unio tumidus specimens were treated with Sal (0.6 μg L -1 ), MP (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 the 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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier Ltd.)

The zebra mussel Dreissena polymorpha (Pallas, 1771) is an invasive species and a valuable bioindicator in the inland waters. Nevertheless, the biochemical reasons for the unique competitiveness of zebra mussels are not clear. This study aimed to compare the native and invasive populations of D. polymorpha in their ability to withstand the same multiple environmental challenges (i.e. chemical: microplastics and caffeine; physical: temperatures). The specimens from the invasive population in west Ukraine (Tn) and native population at lower streams of river Dnipro (south Ukraine, Kh) were sampled in the August of 2021 y. Molluscs from both populations were treated simultaneously with microplastics (MP, 1 mg L−1, pore size 2 μm); caffeine (Caf, 20.0 μg L−1) at 18 °C, elevated temperature (25 °C) and MP and Caf combinations at 18 °C and 25 °C for 14 days. Untreated molluscs exposed at 18 °C represented control groups. A set of the 20 markers of oxidative stress, biotransformation, detoxification and apoptosis were assayed in the total soft tissues. From the two controls, Kh-group indicated lower stressful impact. However, both populations increased caspase-3 and GST activities and lysosomal instability in most exposures, and cholinesterase and phenoloxidase activities under the heating and combine exposures, indicating the remarkable properties to respond to new challenges and synergistic effect of mixtures. Inter-population differences were related to the metallothionein, cathepsin D, cytochrome P540 and oxidative stress responses that reflect population-dependent adverse outcome pathways. The discriminant analysis separated two populations with a substantially lesser magnitude of responses in the invasive population as a sign of higher resistance. [Display omitted] • Invasive and native populations of dreissena are compared. • Microplastics, caffeine, heating separately and jointly decreased lysosome integrity. • Responses of native mussels had higher magnitudes. • Interpopulation differences are observed in oxidative stress and CYP450 responses. [ABSTRACT FROM AUTHOR]