Reductive stress and cytotoxicity in the swollen river mussel (Unio tumidus) exposed to microplastics and salinomycin.

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.

Biochemical basis of resistance to multiple contaminations in the native and invasive populations of Dreissena polymorpha.

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.

Combined effect of microplastic, salinomycin and heating on Unio tumidus.

Microplastic (MP) and heating (T) suspected to modulate biological effects of aquatic contaminants. Salinomycin (Sal) is veterinary antibiotic and anticancer agent. The goal of this study was to examine the multistress effect of MP, Sal and T on the bioindicator bivalve mollusc. The Unio tumidus were treated with MP (1 mg L-1), Sal (0.6 µg L-1), their combination under 18° C (Mix) and 25° C (MixT) for 14 days. The digestive glands were analyzed. MP and Sal did not cause changes of Mn- and Cu,Zn-SOD, lipid peroxidation and Cyp-450-depended EROD levels, whereas catalase, GST and protein carbonyls (Sal-group) increased compared to control. In the Mix-group, enzymes, particularly EROD and GST (by 34% and 115% respectively) were up-regulated. However, in the MixT-group, they were corresponding to control or lesser (EROD, catalase). Our findings emphasize the need to take into account multistress interactions in the MP environmental risk assessment.

Stress responses of bivalve mollusc Unio tumidus from two areas to ibuprofen, microplastic and their mixture.

Even though bivalve molluscs are recognized as bioindicators of freshwater quality, their responses to multiple stressors are unpredictable. This study aims to elucidate the inter-population peculiarities of the effect in the sub-chronic environmentally relevant exposure to novel contaminants. The specimens of Unio tumidus from reference (Pr) and contaminated (Ct) areas were treated with ibuprofen (IBU, 0.8 µg L-1), microplastic (MP, 1.0 mg L-1, size 0.1-0.5 mm), or their combination (Mix) for 14 days. Untreated mussels (PrC- and CtC-groups) served as controls. The PrC-group had higher levels of antioxidants Mn-SOD, Cu,Zn-SOD, catalase, and cholinesterase (AChE) as well as lesser levels of oxidative lesions (TBARS and protein carbonyls) in digestive glands, indicating lower environmental impact than in the CtC-group. However, lysosomal stability was similar in both control groups. Among antioxidants, Mn-SOD activity was affected most prominently, increasing in all exposed Ct-groups. TBARS level was increased only in PrMP-group compared to responsive control. IBU and Mix enhanced protein carbonyl concentration in the Pr-groups, and decreased it in the Ct-groups. AChE was induced in the CtIBU- and PrMix-groups, and lysosomal integrity increased in the CtIBU and CtMix-groups. Discriminant analyses indicated lesser differences between Pr-groups, demonstrating lower cumulative stress compared to Ct-groups. Generally, the most remarkable response was revealed in the CtIBU-group, and distortion of individual effects was established in combined exposures. The qualification of stress-neutral and stress-positive populations was proposed for Pr- and Ct-populations correspondingly. Inter-site peculiarities must be taken into consideration when the environmental impact of MP and pharmaceuticals is evaluated.

Indication of the impact of environmental stress on the responses of the bivalve mollusk Unio tumidus to ibuprofen and microplastics based on biomarkers of reductive stress and apoptosis.

The vulnerability of bivalve mollusks to micropollutants is estimated mainly in single model exposures. However, chronic environmental stress and complex exposures can modulate their responses. To evaluate the impact of population-dependent adaptations on the ability to react to common micropollutants, we compared freshwater bivalves Unio tumidus from two distinct populations, pure (Pr) and contaminated (Ct), in their exposures to microplastics (MP, 1 mg L-1, size 0.1-0.5 mm), pharmaceutical ibuprofen (IBU, 0.8 µg L-1), or their combination (Mix) for 14 days. Control groups from both sites showed remarkable differences, with lower levels of total antioxidant capacity (TAC), metallothionein protein (MTSH), NADH and NAD+, cytochrome P450-related EROD, glutathione-S transferase (GST), and citrate synthase (CS) but higher levels of GSH, GSSG, caspase-3 and cathepsin D (CTD) in the Ct-control group. These data indicate a chronic stress impact in the Ct population. Under exposures, we found an almost common strategy in both populations for NAD+/NADH and MTSH suppression and CTD induction. Additionally, Mix exposure caused an increase in CS, and IBU did not change GSH in both populations. However, the expected response to IBU - the suppression of caspase-3 - was indicated only in PrIBU- and PrMix-mollusks. CTD efflux increased dramatically only in PrMP- and PrMix- groups, and suppression of EROD and GST was detected in the PrMix-group. According to discriminant analysis, exposed Pr-groups were highly differentiated from control, whereas Ct-control and exposed groups had common localization demonstrating high resistance to environmental stress. Thus, the same exposures resulted in different adverse outcome pathways depending on the population.