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 analysing 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 18oC as well as with the mixture of these substances at 18oC (Mix) or 25oC (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. All exposures caused an oxidative stress due to the decreased SOD and GST activities and decreased GSH/GSSG ratio, increased levels of thiobarbituric acid-reactive substances and protein carbonyls (with exceptions of Dc and MixT treatments). Diclofenac increased lysosomal cathepsin D activity. Nifedipine increased lysosomal membrane stability and caspase-3 activity. Roundup caused a dramatic distortion of metallo-thiolome due to highly 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, and by the highest value of integrated biomarker index. Generally, compound-specific effects were expressed in U. tumidus responses to the mixture of pharmaceuticals and herbicide at both temperatures. 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.

淡水污染物优先级名录正日益因药物类污染物而更新完善。药物对水生生物群落的影响，可受到典型污染物（如农药）或异常升温的调控。本研究旨在通过分析多组生物化学标志物，阐明复合环境因素对淡水贻贝的潜在危害效应。本研究以肿珠蚌（Unio tumidus）的双壳类软体动物为实验材料，分别在18℃条件下采用非甾体抗炎药双氯芬酸（diclofenac, Dc, 2 nM）、钙拮抗剂类降压药硝苯地平（nifedipine, Nf, 2 nM）或有机膦酸盐类草甘膦基除草剂农达MAX（Roundup MAX, Rn, 以草甘膦计为79 nM）进行单独暴露处理，同时设置18℃下的混合处理组（Mix）与25℃下的混合处理组（MixT），处理时长均为14天。所采用的药物浓度均符合环境相关实际暴露水平。本研究对消化腺中的应激与毒性生物标志物进行了检测，仅溶酶体膜稳定性指标采自血淋巴细胞。所有暴露处理均引发了氧化应激，具体表现为超氧化物歧化酶（SOD）与谷胱甘肽S-转移酶（GST）活性下降、还原型谷胱甘肽/氧化型谷胱甘肽（GSH/GSSG）比值降低，同时硫代巴比妥酸反应物质与蛋白质羰基水平升高（双氯芬酸与MixT处理组除外）。双氯芬酸可上调溶酶体组织蛋白酶D的活性。硝苯地平可提升溶酶体膜稳定性与半胱氨酸天冬氨酸蛋白酶3（caspase-3）活性。农达处理可引发金属巯基组的显著紊乱，具体表现为还原型谷胱甘肽与金属硫蛋白相关巯基（MTSH）水平大幅升高，同时金属硫蛋白结合的锌（Zn）、铜（Cu）与镉（Cd）出现耗竭，且组织内锌/铜摩尔比降低。农达的特殊毒性还可通过溶酶体膜稳定性与胆碱酯酶活性下降，以及生物标志物综合指数达到峰值得到证实。总体而言，在两个温度条件下，肿珠蚌对药物与除草剂混合暴露的响应均表现出化合物特异性效应。多标志物分析法与整合分析被证实是解析外源性化合物复合暴露与气候变暖背景下淡水贻贝潜在未来风险的有效工具。