摘要
人类通过使用农药有效地控制了目标生物,另一方面,施用的农药也通过各种途径对水环境造成污染,它们对水生生态系统和非目标生物的影响日益受到关注。本文报道了除草剂阿特拉津和有机磷杀虫剂毒死蜱对我国重要养殖种类中华绒螯蟹(Eriocheir sinensis)的急性毒性和亚致死效应。
在采用标准毒性试验方法和胚胎离体试验方法评估两种农药对中华绒螯蟹急性毒性基础上,采用生物化学方法、分子生物学方法结合多种免疫学技术(免疫组织化学、酶联免疫吸附试验和免疫印迹)研究了两种农药对胚胎代谢酶和抗氧化酶活力、幼体能量代谢和生长、雌性性腺发育、卵黄发生和蛋白质代谢的影响,并对其机理进行了讨论。研究结果可为丰富水生毒理学内容提供基础资料,同时也为完善水环境标准提供必要的参考数据。
研究结果表明,阿特拉津在试验浓度范围内对幼蟹Ⅰ、幼蟹Ⅱ、成蟹和胚胎均无急性毒性,但10和20mg/L阿特拉津显著缩短胚胎孵化时间。毒死蜱对幼蟹Ⅰ、幼蟹Ⅱ、成蟹和胚胎的96h LC50值分别为22.9、75.9、142.2和54.7μg/L。6.3和12.5μg/L毒死蜱暴露可使胚胎孵化时间显著延长。建立在胚胎试验基础上的阿特拉津安全浓度为100μg/L,毒死蜱安全浓度为0.06μg/L。
首次报道了中华绒螯蟹胚胎发育过程中8个阶段超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、谷胱甘肽转硫酶(GST)以及乙酰胆碱酯酶(AChE)的酶活性变化,SOD和CAT活性随着代谢活动增强活性氧产生增加;GST活性升高表明胚胎解毒能力增强;而AChE活性的升高则反映出神经系统活动增强。抗氧化酶活性变化表明胚胎在阿特拉津和毒死蜱暴露过程中遭受了氧化胁迫,GST参与了胚胎和幼蟹对阿特拉津的代谢,阿特拉津经GST催化直接与GSH结合可能是幼蟹代谢的主要方式。阿特拉津对毒死蜱的毒性不表现出协同效应。
耗氧率和排氨率测定显示阿特拉津通过增加对蛋白质等能量物质的分解来增加能量的产生,而毒死蜱暴露则降低幼蟹代谢水平。两种农药均可减少幼蟹体内能量物质的积累,并对生长产生抑制作用,而幼蟹可以通过延长蜕皮间期的策略来完成生长。幼蟹在蜕皮期间对毒物暴露更为敏感。
首次利用分子生物学手段解决了有关中华绒螯蟹Vg发生场所的争议,并阐明了卵巢发育过程中Vg的发生规律。中华绒螯蟹卵巢和肝胰腺都具有合成Vg
Pesticides are used judiciously to control a wide variety of agricultural pests and most of them pool ultimately into aquatic ecosystems throughout various pathways. Now there is a growing concern worldwide over the toxicity towards non-targeted organisms.However, the toxicity of pesticide to the Chinese mitten-handed crab, E. sinensis, which is economically important crustacean, has not been fully evaluated. Hence, an attempt was made to study the toxic effect of atrazine, a herbicide and chlorpyrifos, an organophosphate insecticide on crab at various developmental stages, including the acute toxic effects on embryo, juvenile and adult; effects of metabolism enzymes and antioxidant enzymes in embryos; effects of energy metabolism and growth in juveniles; effects of gonad mature in female adults. The purpose is to accumulate basal information for aquatic toxicology, and provide reference for criterion of water quality.The acute toxicity tests of herbicides atrazine and insecticides chlorpyrifos to Chinese mitten-handed crab E. sinensis were determined with the semi-static test. The Results of test found no effects on survival of juveniles I, juveniles II, adults crab exposed to atrazine, which indicate that atrazine has slight or no acute toxicity to E.sinensis. The calculated LC50 of 96h for chlorpyrifos to juveniles I, juveniles II, adults and embryos were 22.9, 75.9, 142.2 and 54.7 μg/L, respectively. 96-hr exposure of embryos caused a significant decreased in hatching time compared to controls at 10 and 20mg/L atrazine. However, 6.3 and 12.5μg/L chlorpyrifos treated embryos had a significantly increased hatching time. Based the results of embryos test, the safe concentrations of atrazine and chlorpyrifos are 100 and 0.06μg/L, respectively.The activities of superoxide dismutase (SOD), catalase (CAT), glutathion-S-transferases (GST) and acetylcholinesterase (AChE) were determined in Eriocheir sinensis during embryonic development. The elevated activities of antioxidant enzymes SOD and CAT indicated increased need to detoxify superoxide anion radical and to metabolize hydrogen peroxide. The increased activities of GST
indicated elevated detoxified function. The increased activities of AChE suggested that significant nervous system development is underway.The biochemical stress responses of atrazine and chlorpyrifos were studied in embryos and juvenile of E.sinensis. The antioxidant enzymes activities indicated that the embryos maybe suffered oxidant stress during exposed to atrazine and chlorpyrifos. The results suggested atrazine did not influenced the toxicokinetics of chlorpyrifos (resulting in increased formation of the o-analog), which suggested that direct conjugation to GSH without activation by phase I enzymes may be one main pathway of atrazine detoxication. The GST activities in hepatopancreas might be a promising biomarker of atrazine and chlorpyrifos long-period exposure, and AchE was a specially biomarker of the chlorpyrifos exposure.In the present investigation, laboratory experiment was conduced to study the toxicity of atazine and chlorpyrifos on oxygen-consumption and ammonia-N excretion of E. sinensis juvenile. The results indicated that atrazine exposure caused more catabolism to supply energy for overcome stress while chlorpyrifos exposure decreased the metabolism rate in juvenile crab.In present study, semistatic bioassay was employed to determine the toxicity of atrazine and chlorpyrifos on growth and body composition of E.sinensis juveniles. The results suggested that atrazine and chlorpyrifos decreased the accumulation of energy precursors and inhibited the growth of juvenile through delaying the molting.Using the RT-PCR techniques, the expression of mRNA encoding the Vg were observed in vitellogenic female ovaries and hepatopancreas, while was not detected in any other tissue including muscle, heart, and subepidermal tissue. The positive immunocytological staining with antibody against vitellin have been founded in ovaries and hepatopancreas of vitellogenic female, which is determined by imm
引文
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