Martyniuk V. Biochemical mechanisms of the influence of microplastics on the organism of the bivalve mollusk Unio tumidus individually and under combined action

The dissertation is devoted to the investigation of biochemical responses of the freshwater mussels Unio tumidus (Philipson et al., 1788) in the exposures to waterborne microplastics depending on the size of the particles separately and in combination with other adverse factors of chemical and environmental origin. In the first experimental series, we evaluated the accumulative capacity of pearl mussel towards microplastics (MP) with a particle size of 0.1-0.5 mm, 1 mg/l in the exposure during 14 days. Series II - we compared the response of mollusks from two populations - reference (Pr) and anthropogenically loaded (Ct) - to the effects of MP 1 mg/l, ibuprofen (IBU, 8 μg/l) separately and their mixture (Mix). III series - the pearl mussel was exposed to MP 2 μm, 1 mg/l, salinomycin (Sal, 6 μg/l) separately and in a mixture at 18℃ (Mix) and 25℃ (MixT). It was found that the pearl mussel has a high ability to accumulate MP from the environment with a maximum of 8-10 days of incubation. Comparison of control groups from the two populations revealed differences between the populations in the content of metallothionein, zinc, and indicators of antioxidant status and apoptosis. Differences in the range of responses and even opposite reactions to the influence of the same factor in mollusks depending on the population were noted. In the mixture of MP 0.1-0.5 mm with ibuprofen, the influence of the latter was decisive. Under the influence of MP, metallothioneins provided a reserve of zinc in the tissue. In all experimental groups, the ratio of Zn/Cu content in the tissue decreased. Under the exposures, inhibition of antioxidant defense enzymes, an increase in the level and redox status of glutathione, and increased formation of oxidative damage products were observed in the Pr-population mollusks, while the opposite changes were observed in the Ct-population. The NAD + /NADH ratio was twice as high in the Ct- population. In both populations, the exposures caused its decrease. Population peculiarities were also revealed in determining the activity of apoptosis and lysosomal functionality with higher values in the Ct-population. Ibuprofen alone and in combination in Pr-groups reduced caspase-3 activity, while salinomycin activated it. The heating reduced the responses of the to the combined effect of xenobiotics. We observed activation of lysosomal cathepsin D and increased its release from lysosomes, and under the influence of MP 2 μm alone and in combination, a coordinated activation of the cytoplasmic and lysosomal pathways of apoptosis/autophagy was indicated. Thus, we found that the pre-adaptation of mollusks to the effects of unfavorable environmental factors in anthropogenically loaded areas determines the reactions to the effects of MPs separately and in conjunction with pharmaceuticals. Thermal effect neutralizes the body's reactions to xenobiotics. It is proved that the toxicity of MP increases with a decrease in its size. Under the joint influence of MP and pharmaceuticals, a synergistic effect was manifested. An easy-to-implement method for determining the accumulation of MP in the soft tissues of the mollusk and the use of NAD + /NADH and Zn/Cu ratios to assess the response of the mollusk to the damaging effects of the environment are proposed. Key words: accumulation, metallothioneins, zinc, copper, thiol-containing compounds, apoptosis, synergistic effect, cytotoxicity, oxidative stress, protein carbonyls, redox system, hydrobionts, adaptation, pollution.