【作者】 朱江；

【导师】 陆贻通；

【作者基本信息】 上海交通大学 ， 环境工程， 2008， 博士

【摘要】 土壤是环境的重要组成部分,承担着环境中大约90%的来自各方面的污染物质,是一个典型的复合污染体系。近年来,国内外已相继开展了重金属-重金属以及有机-有机复合污染方面的研究工作,并取得了富有成效的理论和实践成果。但是,对于有机污染物-重金属复合污染的研究相对较少,对其定量表征及分子生态毒理效应的研究则更少。事实上,土壤中有机污染物-重金属复合污染非常普遍。其中,多环芳烃、重金属是经常共存、广泛分布的典型土壤环境污染物。开展有机污染物-重金属复合污染研究,对于正确评价复合污染条件下污染物迁移转化的行为,帮助人们采取合理的诊治措施,解决土壤的环境污染问题具有非常重要的理论和实践意义。作为土壤动物区系的代表类群——安德爱胜蚓(Eisenia andrei)和白线蚓(Fridericia bulbosa),已日益成为生态毒理学研究的载体。目前,国内外学者对蚯蚓的生态毒理学进行了大量的研究,其中农药、重金属单一及复合污染较多,但关于多环芳烃及其复合污染对蚯蚓的生态毒理的研究相对较少。本文采用室内模拟方法,开展了典型重金属(Cd)-典型多环芳烃(Phe)复合污染对安德爱胜蚓(Eisenia andrei)和白线蚓(Fridericia bulbosa)的生态毒理研究,在此基础上,对Cd-Phe复合污染生态毒理生物标志物的筛选和定量表征进行了重点而深入的研究,以期为有机污染物-重金属复合污染研究与诊治提供科学依据和实践指导,主要研究结果如下:1.从个体水平,分别采用人工土壤法和自然土壤法,研究Cd、Phe单一及复合污染对安德爱胜蚓(Eisenia andrei)和白线蚓(Fridericia bulbosa)的急性毒性。结果表明:安德爱胜蚓(Eisenia andrei)对Cd污染的耐受能力强;Phe对安德爱胜蚓(Eisenia andrei)的急性毒性高。Cd-Phe复合污染对Eisenia andrei的急性毒性效应中Phe为主效应。重金属Cd和Phe之间存在明显的交互作用,Cd-Phe复合污染产生的拮抗作用及其强度与Cd和Phe的不同浓度组合密切相关。这两种污染物对白线蚓(Fridericia bulbosa)都具有潜在的毒性效应,毒性大小顺序为Phe大于Cd;Cd、Phe染毒14d的理论LC50分别为4.548 mg kg-1、4.482mg kg-1。染毒14d后,当Phe的染毒剂量为0.1 mg kg-1和1.6 mg kg-1时,Cd-Phe复合污染对白线蚓的急性毒性效应为协同作用,当Phe的染毒剂量25.6 mg kg-1时,Cd-Phe复合污染对白线蚓的急性毒性效应为拮抗作用,复合污染物的组成及各污染物的不同浓度组合是决定两者之间的交互作用类型和毒性的重要因素。Cd-Phe复合污染对白线蚓(Fridericia bulbosa)的急性毒性效应中Cd为主效应因子。白线蚓(Fridericia bulbosa)是一种可用于表征Cd-Phe复合污染效应的指示生物。2.从组织水平,采用人工土壤法,研究Cd、Phe单一及不同浓度组合复合污染对安德爱胜蚓(Eisenia andrei)抗氧化物酶系:过氧化氢酶(CAT)、超氧化物歧化酶(SOD)和谷胱甘肽过氧化物酶(GSH-PX)活性的影响,结果表明:Cd、Phe单一污染,CAT活力最微弱,各处理组GSH-PX活性与对照组的差异最为显著; CAT活性变化趋势极不稳定,SOD、GSH-PX活性则均具有先升高后降低的趋势;其中复合污染较单一污染对SOD活性表现出较强的抑制作用;而复合污染较单一污染对GSH-PX活性则表现诱导激活作用。GSH-PX可作为Cd、Phe单一及不同浓度组合复合污染的生物标志物,其活性变化可作为表征Cd-Phe复合污染的一项重要的参考指标。3.从细胞和亚细胞水平,优化了蚯蚓体腔细胞提取方法。采用纤维针法与超声波刺激法结合的方法提取的单位体重细胞数较多且存活率较高,为后期的细胞形态学和细胞内DNA损伤的实验打下了基础。采用自然土壤染毒法,研究Cd、Phe单一及复合污染对安德爱胜蚓(Eisenia andrei)体腔细胞溶酶体膜中性红保持时间(NRRT)的影响,并采用细胞流式技术(FCM)研究Cd、Phe单一及复合污染对安德爱胜蚓(Eisenia andrei)体腔细胞质膜的损伤作用,探讨Cd和Phe的跨膜毒理机制。结果表明: NRRT与Cd污染浓度和染毒时间存明显的剂量-效应关系。随染毒时间延长,Cd与Phe复合污染对蚯蚓体腔细胞毒性逐渐增加,蚯蚓染毒7d后和染毒14d后,Cd与Phe复合污染对蚯蚓体腔细胞溶酶体膜损伤的交互作用表现为协同作用;Cd是影响Cd-Phe复合污染对蚯蚓体腔细胞溶酶体膜NRRT影响的主效应因子。NRRT是表征Cd-Phe复合污染毒性的灵敏、准确、重复性好的生物标志物。此外,安德爱胜蚓(Eisenia andrei)体腔细胞在受到Cd、Phe单一及复合污染胁迫下的早期凋亡率可作为污染物毒性评价的敏感指标,可用于对污染物毒性的早期预警。利用细胞流式技术对Cd-Phe复合污染胁迫下蚯蚓体腔细胞的类群的研究,是评价Cd-Phe单一及复合污染及其污染修复程度的一种合理可行的实验方法。4.从分子水平,采用自然土壤法,用单细胞凝胶电泳技术(SCGE)研究Cd、Phe单一及复合污染对安德爱胜蚓(Eisenia andrei)体腔细胞DNA损伤作用。结果显示: Cd、Phe单一污染均表现出致蚯蚓体腔细胞DNA损伤作用,Cd、Phe单一污染与蚯蚓体腔细胞DNA损伤存在明显的剂量-效应关系;Cd-Phe复合污染对蚯蚓细胞DNA的损伤远大与单一的Cd或Phe的污染,单一Cd污染下蚯蚓细胞的OTM值较低,Cd-Phe复合污染下,随着Phe剂量的增加OTM值不断增大,说明Phe对Cd的毒性有显著的增强作用。染毒14d后,Cd-Phe复合污染对蚯蚓体腔细胞DNA损伤表现为拮抗作用。Cd是Cd-Phe复合污染对蚯蚓体腔细胞DNA损伤的主效应因子。更多还原

【Abstract】 With the rapid economic development, more and more pollutants enter the soil environment. It is estimated that more than 90% of the total burden of pollutants resides in the surface soils. Heavy metals (HM) and polycyclic aromatic hydrocarbons (PAHs) are frequently found together as contaminants in soils and have attracted much attention in recent decades. However, they are mostly concentrated on the effect of a single organic pollutants and heavy metals. In fact, contaminated soils usually contain mixtures rather than a pure substance in the real environment. It is very important to investigate the compound pollution between heavy metal and organic pollutant for the management and diagnosis of soil pollution.Earthworms and enchytraeid are the representative organisms in soil animals, which have become a bioindicator of soil contamination. Therefore the acute and chronic tests of Cadmium (Cd) and Phenanthrene (Phe) to earthworms and enchytraeid were performed in this study in order to know the toxicity and mechanism of toxicosis of these typical heavy metal and typical organic pollutant on environmental organisms. In this paper, quantitative expression and effect of combined pollution between Cd and Phe were studied. The results are showed as follow:1. The acute toxicity of Cd and Phe to earthworms (Eisenia andrei) was studied by the methods of artificial soil test. The results showed that the single toxic sequence of Cd and Phe was Phe > Cd. The values of their LC50 were 788.71 mg kg-1、42.51mg kg-1, respectively. The difference may depend on the biological mechanisms of the earthworm population. Cd at all concentration, it could decrease the toxicity of Phe, and the toxicity of Phe decreased with the decrease of Cd concentration. Phe at low and medium concentration,it could decrease the toxicity of Cd, and the toxicity of Cd decreased with the decrease of Phe concentration, at high Cd concentration, it could increase the toxicity of Phe. Therefore,the component and different concentration of combined pollutant are the significant factors to determine the toxicity of combined pollutant. Phe was the main factor of contribution of acute toxicity on earthworms (Eisenia andrei).. The acute toxicity may be used to express the combined pollution between Cd and Phe. The toxic effects of single and joint stress of Cd (cadmium) and Phe (phenanthrene) on enchytraeid (Fridericia bulbosa) were investigated by natural soil tests. Mortality of single and joint stress of Cd and Phe was significant (p<0.01) except the lowest concentration of single pollutant. There was a positive correlation between mortality and the concentration of pollutant added to soil. Also, the more exposed to single or joint Cd and Phe, the more morality. For joint pollution, Cd was the main contributive factor of toxic effects, the different concentration and interaction of Cd and Phe significantly influenced mortality (p<0.01). The joint effects of Cd and Phe were additive, after a 14-d exposure.When Phe was 0.1 mg kg-1 and 1.6 mg kg-1, the interactive effects between Cd and Phe were synergistic. When Phe was 25.6 mg kg-1, the interactive effects were antagonistic. It suggested that morality may be considered as a valuable and sensitive biomarker to diagnose adverse effects of Cd or Phe in soil environment.2. Enzymatic activities are regarded as fast and prognostic indices of individual reaction to the environmental stress, and should allow prediction of the consequences of pollution at population and ecosystem levels. Our objective was to investigate whether their catalase (CAT),superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX) were influenced by Cd and Phe at different concentrations and exposure times. Three replicates of adult earthworms (Eisenia andrei)were exposed to exposure times ranging from 2-d to 64-d. Results indicated that each change of Cd and Phe in treatments was significantly different compared with the control at the same duration of exposure times. CAT activity was not shown the significant order all the experimentation. In the experiments of combined pollution, the activity of SOD was weaker than single experiment but the activity of GSH-PX was more activate than single experiment. The different concentration of Cd and Phe was significantly influence the activity of SOD and GSH-PX, and their interaction was significantly influence the activity of CAT and GSH-PX. It suggested that GSH-PX may be considered as a valuable and sensitive biomarker to diagnose adverse effects of Cd or Phe in soil environment.3. Four different coelomcytes extraction methods for earthworms (Eisenia andrei) were evaluated. They are fiber needle extraction, ultrasound protocol, electrical extraction and combined method with fiber needle extraction and ultrasound protocol. The effect of them was compared based on the total of coelomcytes and viability of coelomcytes. The results showed that the combined method with fiber needle extraction and ultrasound protocol is most effective. It has higher total and viability of coelomcytes. It could be found a good base for the tests about the neutral red retention time (NRRT) and single cell gel electrophoresis (SCGE). Lysosome is used as the biomarker. NRRT of single and joint stress of Cd and Phe on earthworms (Eisenia andrei) were investigated by natural soil tests. The results showed there was an opposite relation between NRRT and the concentration of pollutant added to soil. Also, the more exposed to single Cd, the lower NRRT. For joint pollution, the joint effects of Cd and Phe were synergistic. After the 7-d or 14-d exposure,Cd was the main contributive factor of toxic effects. It suggested that NRRT may be considered as a valuable and sensitive biomarker to diagnose adverse effects of combined Cd and Phe in soil environment. The integrality of plasmolemma of single and joint stress of Cd (cadmium) and Phe (phenanthrene) on coelomcytes of Earthworms (Eisenia andrei) were investigated by natural soil tests. The results showed there was an opposite relation between integrality of plasmolemma and the concentration of pollutant added to soil. It founds a good theoretical base for the mechanism of stride membrane of Cd and phenanthrene. Moreover, the rate of early apoptotic coelomcytes could be considered as a valuable and sensitive biomarker to diagnose adverse effects of combined Cd and Phe in soil environment. Using FCM to detect the subpopulations of coelomcytes is feasible to indicate the combined pollution of heavy metal and PAHs.4. The DNA-damaging effects of the combined pollution of heavy metal and organic contamination on earthworms were evaluated by SCGE assay. Earthworms (Eisenia andrei) were exposed to single or combined test compounds in different doses of cadmium (Cd) 5, 10, 50 mg kg-1 and phenanthrene (Phe) 0.5, 2.5, 12.5 mg kg-1 with a treatment of 14 days. In SCGE assay, isolated coelomcytes and electrophoresis were employed to determine DNA damage degree after a 14-d treatment by test compounds. The results showed that there was significant statistical difference between earthworms treated with Cd combined Phe with them treated alone with Cd or Phe. The Olive tail moment (OTM) of SCGE assay using earthworm coelomcytes appears to be a sensitive biomarker for evaluating exposure to genotoxic compounds. These tests also revealed that the interaction between Cd and Phe to DNA damaging effects was negative, and was strongly dependent on the concentration of pollution. This study corresponds to exploratory phase in order to reveal interaction effects of heavy metal and organic contamination on earthworms and then to set up more in-depth analysis to increase progressively the understanding of the genotoxicity mechanisms involved.更多还原