Department of Biology, Institute of Natural and Applied Sciences, Adıyaman University, 02040, Adıyaman, Turkey.
Department of Biophysics, Faculty of Medicine, Niğde Ömer Halisdemir University, 51240, Niğde, Turkey.
Arch Environ Contam Toxicol. 2021 Jul;81(1):46-57. doi: 10.1007/s00244-021-00841-3. Epub 2021 Apr 16.
The present study investigates the toxicity of the herbicide tribenuron methyl (TBM) as an anthropogenic agent and ammonia as an abiotic factor on Daphnia magna at environmentally relevant concentrations. These stressors may coexist in surface waters in agricultural regions. To achieve this objective, D. magna were exposed to TBM at a nominal concentration of 0.81 μg/L in association with a low ammonia (LA) concentration of 0.65 mg/L and a high ammonia (HA) concentration of 1.61 mg/L in acute toxicity tests of 96-h duration and chronic toxicity tests of 21-day duration. The D. magna also were exposed to TBM, HA, and LA singly. The D. magna were analysed for various biomarkers of sublethal toxicity. Glutathione peroxidase (GPx), glutathione S-transferase (GST), cholinesterase (ChE) enzyme activities, and levels of thiobarbituric acid reactive substances (TBARS) and total protein were determined spectrophotometrically. Mitochondrial membrane potential (MMP) was analysed by microscopy with fluorescence staining. Cytochrome c and 5' AMP-activated protein kinase (AMPK) were analysed by Western blotting. Morphometric properties were examined microscopically. This is the first study in which AMPK, an indicator of intracellular energy, was measured in D. magna. GST and ChE enzyme activities and TBARS and total protein levels did not change during acute exposures (i.e., 96 h) in all treatments. GPx activity increased in D. magna from the HA + TBM treatment compared with single-exposure groups. The level of cytochrome c protein was elevated in D. magna from the LA and LA + TBM treatments. AMPK protein levels increased in all treatments with daphnids, except in the LA group. MMP was depolarised in D. magna from all treatments, whereas the most notable change was observed in HA + TBM mixture group in chronic exposures. The results show that GST and ChE may not be sensitive biomarkers for evaluating the sublethal toxic effects to D. magna exposed to environmentally relevant concentrations of ammonia and TBM. Acute and chronic exposure to ammonia and TBM probably caused an energetic crisis in D. magna. Therefore, AMPK and MMP are promising biomarkers for these toxicants.
本研究调查了除草剂三甲隆（TBM）作为人为因素和氨作为非生物因素对大型溞（Daphnia magna）的毒性，其浓度与环境相关。这些胁迫因子可能同时存在于农业地区的地表水中。为了实现这一目标，D. magna 在急性毒性试验中以 0.81μg/L 的名义浓度接触 TBM，并在 96 小时的持续时间内接触低氨（LA）浓度 0.65mg/L 和高氨（HA）浓度 1.61mg/L，以及 21 天的慢性毒性试验。D. magna 还单独接触 TBM、HA 和 LA。分析 D. magna 的各种亚致死毒性生物标志物。用分光光度法测定谷胱甘肽过氧化物酶（GPx）、谷胱甘肽 S-转移酶（GST）、胆碱酯酶（ChE）酶活性以及硫代巴比妥酸反应物质（TBARS）和总蛋白的水平。通过荧光染色的显微镜分析线粒体膜电位（MMP）。用 Western blot 分析细胞色素 c 和 5' AMP 激活的蛋白激酶（AMPK）。通过显微镜观察形态学特性。这是首次在大型溞中测量细胞内能量的指示物 AMPK 的研究。在所有处理中，急性暴露（即 96 小时）期间 GST 和 ChE 酶活性以及 TBARS 和总蛋白水平没有变化。与单一暴露组相比，来自 HA+TBM 处理的 D. magna 中的 GPx 活性增加。来自 LA 和 LA+TBM 处理的 D. magna 中的细胞色素 c 蛋白水平升高。除 LA 组外，所有处理组的 AMPK 蛋白水平均升高。除 HA+TBM 混合物组外，在所有处理中 MMP 均去极化，在慢性暴露中观察到最明显的变化。结果表明，GST 和 ChE 可能不是评估大型溞接触环境相关浓度氨和 TBM 的亚致死毒性影响的敏感生物标志物。急性和慢性暴露于氨和 TBM 可能导致 D. magna 发生能量危机。因此，AMPK 和 MMP 是这些毒物的有前途的生物标志物。
