Biochemical responses of the Dreissena polymorpha from municipal pond to caffeine, microplastics, and heating in single and combined exposures

Background. Pharmaceuticals have become the aquatic pollutants of growing concern. Caffeine is one of the most widely distributed in the surface waters among them. However, the environmentally relevant models of its effect, which include combined exposures with probable confounding factors, are unknown. Microplastics are a suspected vector that influences caffeine bioavailability. The temperature dependence of response, considering the increase of temperature in surface waters, can also be anticipated. The aim of this study was to analyze the input of caffeine, microplastics and elevated temperature into their combined effect on the zebra mussel Dreissena polymorpha (Pallas, 1771). Materials and Methods. Molluscs were exposed to caffeine (Caf, 20.0 μg·L−1), microplastics (MP, 1 mg·L−1, 2 μm in size), or elevated temperature (T, 25 °C) in the single and combined (Mix- and MixT-) exposures for 14 days. The concentrations of metallothioneins, metallothioneins-bound zinc, total Zn and Cu concentration in the tissue, total glutathione level, antioxidant (superoxide dismutase) and metabolic (citrate synthase) enzymes activities, acid phosphatase activity as the lysosomal functionality marker were determined. Results and Discussion. The decrease in Zn/Cu concentrations ratio in the soft tissues shared the common response in all exposures, reflecting the metal imbalance as the most sensitive marker. The MP-group was distinguished by the decrease in the levels of total Zn and extra lysosomal acid phosphatase activity, proving injury of Zn transportation and Zn-related enzyme activities. All other exposures (T-, Mix-, MixT-) caused citrate synthase and superoxide dismutase activation. Caf-related groups demonstrated the elevation of the levels of phosphatase lysosomal membrane-linked latency, metallothionein total protein and its apo-form. However, glutathione level was stable in all exposures. Conclusion. These data revealed the adverse effect of MP and shared beneficial effects in the exposures that involved caffeine, which can be explained by the antioxidant activity of this substance. Exposure to elevated temperature partially alleviated the effect of caffeine in the mixture. Thus, the results indicate the importance of multi-stress exposures modeling, which allows the evaluation of environmentally realistic responces of an organism to xenobiotics.