Common and particular biochemical responses of Unio tumidus to herbicide, pharmaceuticals and their combined exposure with heating

The priority list of freshwater pollutants is increasingly amended by pharmaceuticals. Their impact on the aquatic biota can be modulated by the presence of typical pollutants, like pesticides, and/or abnormal heating. The aim of this study was to elucidate potentially hazardous impact of combined environmental factors on the freshwater mussels by analyzing various sets of biochemical markers. We treated the bivalve molluscs of Unio tumidus with non-steroidal anti-inflammatory drug diclofenac (Dc, 2 nM), calcium antagonist and antihypertensive drug nifedipine (Nf, 2 nM) or organophosphonate glyphosate-based herbicide Roundup MAX (Rn, 79 nM of glyphosate) at 18 °C as well as with the mixture of these substances at 18 °C (Mix) or 25 °C (MixT) during 14 days. The concentrations used were correspondent to the environmentally relevant levels. The biomarkers of stress and toxicity were evaluated in digestive gland, except the lysosomal membrane stability measured in hemocytes. Exposures caused an oxidative stress due to the decreased SOD and GST activities and GSH/GSSG ratio, increased levels of thiobarbituric acid-reactive substances and protein carbonyls (with some exceptions). Dc increased cathepsin D activity in lysosomes. Nf increased lysosomal membrane stability and caspase-3 activity. Rn caused a dramatic distortion of metallo-thiolome due to increased levels of GSH and metallothionein-related thiols (MTSH) as well as depletion of Zn, Cu and Cd in the composition of metallothioneins, and decreased Zn/Cu molar ratio in the tissue. The particular toxicity of Rn was also attested by decreased lysosomal membrane stability and cholinesterase activity. Canonical discriminant analysis separated Rn-, Mix- and MixT-groups from the joint set of C-, Dc- and Nf-groups. Generally, compound-specific effects were expressed in U. tumidus responses to the mixtures, but in MixT-group some effects were particular or extremely strong. Multi-marker approach and integrative analysis proved to be a useful tool for understanding possible future risks to freshwater mussels under a combination of xenobiotics and warming climate

Environmental concentrations of Roundup in combination with chlorpromazine or heating causes biochemical disturbances in the bivalve mollusc Unio tumidus

Bivalve molluscs represent the most recognized bioindicators of freshwater pollution. However, their ability to indicate specific xenobiotics in complex exposures is unclear. In this study, we aimed to track the particular effects of the pesticide Roundup (Rnd) and the antipsychotic drug chlorpromazine (Cpz) on the mussel Unio tumidus at the simpler environmentally relevant models. We treated the mussels by Rnd (17 μg L−1), Cpz (18 μg L−1), the mixture of Rnd and Cpz at 18 °C (RndCpz), and Rnd at 25 °C (RndT) and examined their digestive glands after 14 days of exposure. We analyzed total antioxidant capacity, glutathione (GSH&GSSG) and protein carbonyls levels, total and Zn-related concentrations of metallothioneins (MT and Zn-MT, respectively), the activities of CYP450-related EROD, glutathione S-transferase, cholinesterase, caspase-3, citrate synthase (CS), lysosomal membrane integrity (NRR), and Zn level in the tissue. Shared responses were indicated as the increase of the antioxidant, Zn-MT, and EROD levels, whereas the changes of Zn concentration, NRR, and caspase-3 activity were most diverse compared to control. According to discriminant analysis, complex exposures abolished the individual response traits and intensified the harmful effects that caused a decrease in the Zn level in the RndCpz- and RndT-groups and the loss of lysosomal integrity in the RndT-group. We concluded that multi-marker expertise with the application of integrated indices had benefits when evaluating the effects of complex exposures. Graphical abstract: [Figure not available: see fulltext.

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