【作者】 周垂帆；

【导师】 俞元春；

【作者基本信息】 南京林业大学 ， 水土保持与荒漠化防治， 2013， 博士

【摘要】 摘要：过去大多数复合污染的研究集中在重金属元素之间，而由于重金属与有机物的性质差异显著，对于重金属-有机污染物之间的复合污染研究相对较少，但重金属-有机污染物的复合污染形式却非常普遍地存在在环境中。除草剂草甘膦，在农业土壤上大量使用，可能对土壤生物造成危害，而环境中草甘膦存在的地方往往重金属含量也很高，如含铜杀菌剂，含镉磷肥，含铜镉的污水灌溉等与除草剂草甘膦在果园、农田等土壤中使用，为两者的共存及复合污染提供了可能，他们的共存可会改变土壤中重金属的环境化学行为和毒性。因为草甘膦中含有羧基、氨基、磷酸基等配位基团对重金属阳离子和有机阳离子有很强的络合能力，能影响重金属的生物有效性、毒性以及重金属在生物体内的积累。本文以除草剂草甘膦（GPS）和重金属铜（Cu）、镉（Cd）为典型的有机物和重金属污染物，对草甘膦与Cu、Cd复合污染生态毒理学生物标记物的筛选和定量表征进行了重点深入的研究，以期为有机物和重金属污染物的研究与诊治提供科学依据，主要研究结果如下:（1）在环境污染物的毒性评价和监测中，发光细菌法是一种具有快速、灵敏和廉价等优点的直接生物测试方法。有毒物质会影响发光菌的呼吸作用、电子传输系统、ATP产生、蛋白质或脂类合成，从而改变发光强度。以明亮发光杆菌T3小种（Photobacteriumphosphorem T3）为例，采用室内模拟实验，研究了Cu、Cd、草甘膦（原药）对发光菌单一，及草甘膦与Cu、Cd的复合的急性毒性效应。实验结果表明，Cu、Cd对发光菌都有很强的毒性，而没有调节pH的草甘膦具有很强的酸性，能够降低溶液pH，对发光菌有很强的毒性。而调节pH后的草甘膦对发光菌无明显毒性，并且能够显著降低重金属Cu、Cd对发光菌的抑制，特别是与Cu复合，随着GPS/Cu摩尔比值的增大而毒性降低，甚至对发光菌无明显抑制。因此本研究认为，在草甘膦和重金属共存的环境中，草甘膦能够降低重金属对发光菌的毒性。（2）采用模拟土壤溶液的方法，研究了Cu、草甘膦（原药）对赤子爱胜蚯蚓（Eisenia fetida）单一，及草甘膦与Cu的复合的48h急性毒性效应。对蚯蚓的死亡、Cu的吸收，和一些生物标记物如超氧化物歧化酶（SOD）活性、谷胱甘肽含量（GSH）、乙酰胆碱酯酶（AchE）活性进行了测定。蚯蚓的死亡率和体内Cu含量随着溶液中Cu浓度的增加而显著增加，Cu产生毒性的原因主要是自由态的Cu²⁺，然而本研究没有发现草甘膦对蚯蚓产生显著毒性，有意思的是由于草甘膦的存在，溶液中自由态的Cu²⁺几乎全部与草甘膦发生络合，显著的降低Cu对蚯蚓的急性毒性，即Cu浓度一定，草甘膦存在的情况下，蚯蚓的死亡率显著降低，体内甚至不再富集Cu。除此之外，蚯蚓的SOD酶活性、GSH含量、AchE酶活性几乎恢复到正常水平。实验结果表明草甘膦能够降低重金属Cu对蚯蚓的急性毒性。（3）采用OECD人工土壤培养方法，研究了Cu和草甘膦以及两者复合对蚯蚓的亚急性毒性效应，将蚯蚓暴露在人工土壤中28d，测定了蚯蚓的体重损失率、产茧量、Cu的吸收动力学、Cu富集量以及一些生物标记物如超氧化物歧化酶（SOD）、过氧化氢酶和丙二醛（MDA）。实验结果表明暴露在Cu处理的土壤中，随着Cu浓度的增加，蚯蚓的体重损失增加以及体内金属含量显著增加，随着时间的增加蚯蚓对Cu的吸收达到平衡，不同的是草甘膦对蚯蚓毒性很低，甚至无明显毒害。有趣的是他们复合之后对蚯蚓的毒性显著降低，即Cu一定时，草甘膦的存在蚯蚓体重损失显著降低、产茧量增加、对Cu的富集减少。除此之外Cu所引起的蚯蚓SOD、CAT、MDA的异常都一定程度上缓解。本实验结果表明暴露28天后Cu对蚯蚓产生了很强的毒性，而草甘膦毒性较弱，两者复合后毒性降低表现为颉颃作用。（4）采用模拟土壤溶液的方法，研究草甘膦存在，Cd对赤子爱胜蚯蚓（Eisenia fetida）的48h急性毒性效应。对蚯蚓的死亡、Cd的吸收和生物标记物如超氧化物歧化酶（SOD）活性、过氧化氢酶（CAT）、谷胱甘肽过氧化物酶（GSH-Px）活性进行了测定。蚯蚓的死亡率和体内Cd含量随着溶液中Cd浓度的增加而显著增加，有意思的是草甘膦的存在能够显著的降低Cd对蚯蚓的急性毒性，即Cd浓度一定，草甘膦存在的情况下，随着GPS/Cd摩尔比值的增加蚯蚓的死亡率降低，体内富集Cd量降低。对Cd的亚细胞测定发现Cd主要分布在E（细胞残渣）、H（热稳定组分）、G（热变性组分），而D（细胞器）和F（微粒体含量）很少。草甘膦的添加在各个组分中都一定程度上有所降低，特别是E（细胞残渣）。暴露在Cd处理中引起了蚯蚓的抗氧化酶应激，但草甘膦的存在蚯蚓的SOD酶活性、CAT酶、GSH-Px酶活性显著降低但与对照相比仍然差异显著。实验结果表明草甘膦能够降低重金属Cd对蚯蚓的急性毒性。（5）采用OECD人工土壤培养方法，研究草甘膦存在，Cd对赤子爱胜蚯蚓（Eisenia fetida）的亚急性毒性效应，将蚯蚓暴露在人工土壤中28d，测定了蚯蚓的体重损失率、产茧量、Cd的吸收动力学、Cd富集量、丙二醛（MDA）含量的变化。实验结果表明Cd处理的土壤中，整个实验过程随着时间的增加蚯蚓对Cd的吸收一直增加未达到平衡，28d后，随着Cd浓度的增加，蚯蚓的体重损失增加、产茧量显著降低、以及体内金属含量显著增加，且引起了蚯蚓的脂质过氧化。当Cd一定，草甘膦的存在蚯蚓体重损失显著降低、产茧量增加、对Cd的富集减少。除此之外Cd所引起的蚯蚓脂质过氧化都一定程度上缓解。本实验结果表明暴露28天后Cd对蚯蚓产生了很强的亚急性毒性，而与草甘膦复合后毒性降低表现为颉颃作用。更多还原

【Abstract】 Herbicide glyphosate (GPS) is widely used in the environments, including farmland andorchard, where heavy metals are very often found in elevated levels. Co-existence of metal andGPS could result in combined pollution of soil environment. Furthermore, GPS affects metalbehaviors in water and soil systems because its functional groups such as amine, carboxylate andphosphonate react with metal ions to form metal complexes.(1) A laboratory experiment was therefore conducted to investigate the interactions of GPSand Cu or Cd with respect to acute toxicity to Photobacterium phosphorem T3. Photobacteriumwere exposed to the solution containing different concentration Cu or Cd and GPS. Then theEC50(Median Effective Concentrations) of GPS and Cu or Cd were measured based on linearregression analysis. To study the combined effects, photobacterium were exposed to the solutionscontaining different concentrations of Cu or Cd at the different GPS concentrations, the inhibitionof photobacterium was calculated. Results showed that the toxicity of Cu or Cd tophotobacterium was much higher. The GPS (technical-grade glyphosate acid), unadjusted pH,possessed much higher toxicity. Because GPS is a Lewis acid, the adding of this acid decreasedthe pH of the test media obviously. Nevertheless, toxicity of GPS (pH adjusted) was not observedin this study. Additionally, the presence of GPS could significantly reduce the acute toxicity ofCu or Cd to photobacterium. Particularly, the inhibition to photobacterium decreasedsignificantly wih the increase molar ratio value of GPS/Cu. Based on the observations of thepresent study, it can be concluded that that Cu or Cd exhibited acute toxicities on photobacterium,the toxicity increased when Cu or Cd concentration increased. However, GPS was nearlynon-toxic to them and the presence of GPS could significantly reduce the acute toxicity of heavymetal on photobacterium. Buffered medium or pH-adjustment should be included in the toxicitytests for acidic toxicants to eliminate pH-induced toxicity since natural soil and water has higherbuffering capacity than test media in resisting pH change.(2) A laboratory experiment was conducted to investigate the interactions between GPS andcopper (Cu) expressed with the acute toxicity of soil invertebrate earthworm (Eisenia fetida),which was exposed to aqueous solutions for48h with different mixing concentrations of Cu andGPS (technical-grade Gly acid). The mortality rates, Cu uptake by earthworm, and somebiomarkers such as superoxide dismutase (SOD) activity, glutathione (GSH) content, andacetylcholinesterase (AchE) activity were measured. The mortality rates and whole-worm metalburdens increased significantly with the increasing exposed concentration of Cu. However, therewas no indication that GPS was toxic to earthworms in this study. Furthermore, the presence of GPS could significantly reduce the acute toxicity of Cu to earthworms. The mortality ratesdecreased sharply and the uptake of Cu was nearly halted in the presence of GPS. In addition tothat, the SOD activity, GSH content, and AchE activity almost declined to the levels of thecontrol. These results demonstrate that GPS could control the toxicity as well as thebioavailability of heavy metals in soil solutions where both GPS and heavy metals often coexist.(3) Glyphosate (GPS) is a wildly used pesticide throughout the world. It affects metalbehaviors both in water and soil system because its functional groups such as amine, carboxylateand phosphonate can react with metal ions to form metal complexes. As a result, GPS cantypically decrease the heavy metal bioavailability. A laboratory experiment was conducted toinvestigate the interactions between GPS and copper (Cu) with respect to the subacute toxicity tosoil invertebrate earthworm (Eisenia fetida). Earthworms were exposed to artificial soil for28dwith different concentrations of Cu and GPS (technical-grade GPS acid). The growth inhibitionratio, cocoon production, Cu uptake by earthworm, and some biomarkers such as superoxidedismutase (SOD) activity, catalase (CAT) activities, and malondialdehyde (MDA) content weremeasured. The growth inhibition ratio and whole-worm metal burdens increased significantlywith the increasing exposed concentration of Cu. However, the toxicity of GPS was much lowerthan copper in this study. Furthermore, The joint toxicity data showed that the presence of GPScould reduce the toxicity of Cu to earthworm, The growth inhibition were alleviated obviously,the cocoon production were increased, and Cu absorption by earthworms were decreased. Inaddition, the SOD, CAT and MDA were alleviated by Cu when GPS was present to a certaindegree. Therefore, it is shown that the herbicide glyphosate can affect the toxicity andbioavailability of heavy metals in the soil ecosystems acts.This may be due to GPS is similar tothe well-known complexing agents for Cu like EDTA and NTA.(4) A laboratory experiment was therefore conducted to investigate the interactions of GPSand Cd with respect to acute toxicity to earthworm (Eisenia fetida). To study the combinedeffects, earthworm were exposed to the solutions containing different concentrations of Cd at thedifferent GPS concentrations. Results showed that the toxicity of Cd to earthworm was muchhigher. The presence of GPS could significantly reduce the acute toxicity of Cd to earthworm.Particularly, the motility, accumulation of Cd decreased significantly wih the increase molar ratiovalue of GPS/Cd. Analyses of metal subcellular distribution in E. fetida showed that the theinternal metals Cd were major in fraction E (intact cells), H (heat-stable proteins fraction),followed by the G (denatured proteins fraction), F (microsomal fraction), D (microsomalfraction). In the presence of GPS, each fraction, especially the fraction E, were decreased to acertain extent, respectively. In addition to that, the SOD activity, CAT activity, and GSH-Pxactivity also declined. Based on the observations of the present study, it can be concluded that,GPS was nearly non-toxic to them and the presence of GPS could significantly reduce the acutetoxicity of Cd on earthworm.(5) A laboratory experiment was conducted to investigate the interactions between GPSand Cd with respect to the subacute toxicity to soil invertebrate earthworm (Eisenia fetida). Earthworms were exposed to artificial soil for28d with different concentrations of Cd and GPS(technical-grade GPS acid). The weight loss ratio, cocoon production, Cd uptake by earthworm,and malondialdehyde (MDA) content were measured. The weight loss ratio, cocoon production,whole-worm metal burdens, MDA content were all changed significantly with the increasingexposed concentration of Cd. Furthermore, the joint toxicity data showed that the presence ofGPS could reduce the toxicity of Cd to earthworm. The growth inhibition were alleviatedobviously, the cocoon production were increased, and Cd absorption by earthworms weredecreased. In addition, the MDA were alleviated by Cd when GPS was present to a certain degree.Therefore, it is shown that the herbicide glyphosate can affect the toxicity and bioavailability ofCd in the soil ecosystems acts.更多还原