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.