电子电器废弃物拆解污染农田土壤微生物生态研究
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摘要
电子电器产品数量的迅速增加导致了大量电子电器废弃物的产生，同时也催生了电子电器废弃物拆解回收利用行业，而不规范的电子电器废弃物拆解作业对周围环境造成了严重的污染。重金属及多氯联苯(polychlorinated biphenyls，PCBs)等持久性有机污染物的污染是其中比较突出的问题，这些污染物的存在对环境安全和民众的健康构成了严重的威胁。对污染物与土壤微生物群落之间相互作用关系的深入了解，有助于了解这些污染物的潜在风险和它们在土壤中的迁移转化，同时也能为受污染土壤的生物修复提供有价值的信息。本项目从两个曾经发生过PCBs泄漏事件的典型电子电器废弃物拆解点(旱地区域D和水稻田区域P)周围被污染的农田采集土壤样品，分析其中重金属、PCBs和有机氯等污染物的含量及分布，并综合运用传统的微生物生态学研究方法和PCR-DGGE、分子克隆等技术，研究因拆解造成的污染及其对农田土壤微生物生态的影响，主要研究结论如下：
(1)D区域和P区域都受到了一定程度的重金属污染。D区域Cd含量超过国家土壤环境质量三级标准一倍，Cu、Pb、Zn浓度超过国家土壤环境质量二级标准。距拆解中心点最近的D1点受到了非常严重的Pb、Zn污染，分别超过国家三级标准2倍和5倍。而P区域Cd含量同样超过国家三级标准1倍，Cu含量超过国家二级标准，另外还存在轻微的Zn、Pb污染。
(2)D拆解点附近土壤PCBs污染物的平均浓度达1．31mg·kg~(-1)，最高浓度达1．67mg·kg~(-1)。超过了加拿大环境委员会规定的具有潜在威胁浓度的6倍，生态风险非常高。随着距拆解中心点距离的增加，土壤中PCBs浓度呈下降趋势。P采样区域PCBs的平均浓度为9．70μg·kg~(-1)，生态风险较低。D旱地采样区域内PCBs主要以三、四、五、六氯代PCBs为主，P水稻田采样区域则以四、五、六氯代PCBs为主。推测D区域可能曾经拆解过国产的含低氯PCBs和进口的含高氯PCBs的电容器，而P区域则主要以拆解进口电容器为主。两个采样区域均有有机氯农药残留，但是其浓度较低，D区域六六六和滴滴涕的平均浓度分别为22．4μg·kg~(-1)和0．5μg·kg~(-1)，P区域中六六六和滴滴涕的平均浓度分别为16．3μg·kg~(-1)和6．6μg·kg~(-1)，残留在土壤中的有机氯农药已经发生了显著的转化。
(3)对污染物含量的相关性分析表明，D区域旱地土壤中PCBs和Cu、Cd含量之间，Zn和Pb含量之间具有显著的相关性。PCBs和Cu、Cd很可能具有相同的来源，Zn和Pb污染物可能来自于另一类拆解物。而在P区水稻田中，主要的重金属污染物Cd与Pb有显著的相关性，Cu与Zn具有显著的相关性，Pb和Cd可能具有相同的来源，Cu和Zn可能来自于另一类拆解物。
(4)土壤呼吸和酶活性分析表明，在旱地采样区域，随着向拆解中心点的靠近，土壤微生物活性有所增强，可能原因是PCBs对土壤微生物代谢具有一定的刺激作用。旱地脲酶活性与Cu、Cd的含量具有极显著的负相关关系，其活性的下降有可能是Cu、Cd重金属的影响导致的。脲酶对重金属污染物较为敏感，可能比较适于评价土壤重金属污染的影响。而在水稻田采样区域，土壤呼吸强度和三种酶的活性呈波动状态，与主要的污染物Cd、Cu、OCPs和PCBs之间没有显著的相关性，这可能是因为各污染物浓度较低，对土壤微生物活性的影响不显著。
(5)平板分离计数表明，随着向拆解中心点的靠近，旱地土壤中细菌和放线菌的数量有逐渐增加的趋势，DGGE图谱Shannon指数分析显示，土壤中总细菌多样性呈上升趋势，而放线菌多样性则呈下降的趋势；DGGE图谱主成份分析显示，随着向拆解中心点的靠近，土壤细菌和放线菌群落结构特征与对照点的差异越大，这可能是由PCBs和Cu等污染物的梯度分布引起的。水稻田区域内各采样点细菌和放线菌数量较均一，真菌数量波动较大，细菌和放线菌Shannon指数较一致，群落结构有一定差异，但无明显变化趋势。
(6)对旱地区域土壤DGGE图谱中特异性条带进行了克隆和测序鉴定，并对其进行了系统发育分析。对克隆所得序列进行分析发现，大部分克隆都属于变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)，并且在属于这两个类群的克隆中发现了大量可能与多氯联苯、多环芳烃、苯类似物等有机污染物降解有密切关系的微生物类群，但未发现与重金属污染物相关的微生物类群。这说明，这两个微生物类群可能在受PCBs污染的土壤微生物生态系统中占有重要地位，并可能对土壤中PCBs污染物的转化和代谢具有重要的作用。同时也说明PCBs污染物对土壤微生物群落结构具有较显著的影响。
Recycling activities of electric and electronic waste (e-waste) are emerging as a global concern as they can contribute to the release of harmful pollutants into environment. Heavy metal and Polychlorinated biphenyls (PCBs) are this kind of harmful pollutants. In this research, we collected soil samples from the farmland around two e-waste recycling sites (dryland region D and paddy soil region P) where the PCBs leaking accident have happened. Concentration and composition of soil pollutants such as heavy metal, PCBs and OCPs were analysed. Combining the use of traditional microbial ecological research methods and PCR-DGGE to explore the changes of microbial ecosystem and community structure under contamination stress. The main conclusions are as follows:
(1) D and P regin were both contaminated by heavy metal. In D region, concentration of Cd was one time higher than the National Environmental Quality Standard level III (NEQS III). Concentration of Cu、Pb、Zn exceeded NEQS II. Concentration of Pb、Zn in D1(the sampling site that was nearest to the recycling site) was 2 and 5 times higher than NEQS III, respectively. In P region, concentration of Cd was one time higher than NEQS III, Concentration of Cu exceeded NEQS II. There were slight contamination of Pb and Zn at the same time.
(2) The average concentration of PCBs around the D region was 1.31 mg·kg~(-1), the highest concentration reached to 1.67 mg·kg~(-1) . The concentratin of PCBs decreased with the increasd of the distance from recycling site. In P region, average concentration of PCBs was just 9.70μg·kg~(-1). in D region PCBs was mainly composed of 3,. But in P region, the main composition were4,5,6-chlorinated PCBs. Both farmland was contaminated by organochlorine pesticide, but the concentration was very low. In D region, the average content of Hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) were 22.4μg·kg~(-1) and 0.5μg·kg~(-1), respectivily. In P region, the content of HCH and DDT were 16.3μg·kg~(-1) and 6.6μg·kg~(-1), respectively. Evident transform had happened on the HCH and DDT in soil.
(3) In D region, there was a significant correlationship between concentration of PCBs and Cu, Cd, and also between Pb, Zn, indicating that they come from the same source, respectivily. In P region, there was a significant correlationship between Cd and Pb. Indicating that they come from the same source.
(4) Analysis of soil respiration and enzymes activity showed that in Dryland region, when getting close to the recycling site, soil microbial activity increased. The PCBs may have a positive effect on the microbial activity. In the paddy soil, soil respiration and enzymes fluctuated from site to site, but have no significant correlationship with the pollutants in soil. In D region, Urase activity showed a significant correlationship with the concentration of Cu and Cd, indicating that the decrease of urease activity may caused by the effect of Cu and Cd in the soil. Urease may be suitable for the assay of the effect of heavy metal contamination.
(5) Microbial account show that in dryland, when getting close to the recycling site, number of soil bacteria and actinomycete increased the Shannon diversity index of bacteria increased, But that of soil actinomycete decreased at the same time. PCA result show that, when getting close to the cycling site, the difference of the character of soil microbial community between sampling site and background site increased. This may caused by the gradience of PCBs and Cu in the soil. In paddy soil, number of bacteria and actinomycete in different sampling site are similar, number of fungi fluctuated acutely.There was some different between the community structure of different sampling site in paddy soil, but no obvious trend was observed.
(6) Clone and sequence of the specific band on the DGGE gel of dryland showed that most of the specific clone belongs to the Proteobacteria and Bacteroidetes, we also found that many clones were highly similar with the sequences of microbes that are related with organic compound degradation. Bacteria belong to this two phylum may play a very important role in the microbial ecosystem in PCBs contaminated soil, and also in the transformation and degradation of PCBs pollutants.
(1)D区域和P区域都受到了一定程度的重金属污染。D区域Cd含量超过国家土壤环境质量三级标准一倍，Cu、Pb、Zn浓度超过国家土壤环境质量二级标准。距拆解中心点最近的D1点受到了非常严重的Pb、Zn污染，分别超过国家三级标准2倍和5倍。而P区域Cd含量同样超过国家三级标准1倍，Cu含量超过国家二级标准，另外还存在轻微的Zn、Pb污染。
(2)D拆解点附近土壤PCBs污染物的平均浓度达1．31mg·kg~(-1)，最高浓度达1．67mg·kg~(-1)。超过了加拿大环境委员会规定的具有潜在威胁浓度的6倍，生态风险非常高。随着距拆解中心点距离的增加，土壤中PCBs浓度呈下降趋势。P采样区域PCBs的平均浓度为9．70μg·kg~(-1)，生态风险较低。D旱地采样区域内PCBs主要以三、四、五、六氯代PCBs为主，P水稻田采样区域则以四、五、六氯代PCBs为主。推测D区域可能曾经拆解过国产的含低氯PCBs和进口的含高氯PCBs的电容器，而P区域则主要以拆解进口电容器为主。两个采样区域均有有机氯农药残留，但是其浓度较低，D区域六六六和滴滴涕的平均浓度分别为22．4μg·kg~(-1)和0．5μg·kg~(-1)，P区域中六六六和滴滴涕的平均浓度分别为16．3μg·kg~(-1)和6．6μg·kg~(-1)，残留在土壤中的有机氯农药已经发生了显著的转化。
(3)对污染物含量的相关性分析表明，D区域旱地土壤中PCBs和Cu、Cd含量之间，Zn和Pb含量之间具有显著的相关性。PCBs和Cu、Cd很可能具有相同的来源，Zn和Pb污染物可能来自于另一类拆解物。而在P区水稻田中，主要的重金属污染物Cd与Pb有显著的相关性，Cu与Zn具有显著的相关性，Pb和Cd可能具有相同的来源，Cu和Zn可能来自于另一类拆解物。
(4)土壤呼吸和酶活性分析表明，在旱地采样区域，随着向拆解中心点的靠近，土壤微生物活性有所增强，可能原因是PCBs对土壤微生物代谢具有一定的刺激作用。旱地脲酶活性与Cu、Cd的含量具有极显著的负相关关系，其活性的下降有可能是Cu、Cd重金属的影响导致的。脲酶对重金属污染物较为敏感，可能比较适于评价土壤重金属污染的影响。而在水稻田采样区域，土壤呼吸强度和三种酶的活性呈波动状态，与主要的污染物Cd、Cu、OCPs和PCBs之间没有显著的相关性，这可能是因为各污染物浓度较低，对土壤微生物活性的影响不显著。
(5)平板分离计数表明，随着向拆解中心点的靠近，旱地土壤中细菌和放线菌的数量有逐渐增加的趋势，DGGE图谱Shannon指数分析显示，土壤中总细菌多样性呈上升趋势，而放线菌多样性则呈下降的趋势；DGGE图谱主成份分析显示，随着向拆解中心点的靠近，土壤细菌和放线菌群落结构特征与对照点的差异越大，这可能是由PCBs和Cu等污染物的梯度分布引起的。水稻田区域内各采样点细菌和放线菌数量较均一，真菌数量波动较大，细菌和放线菌Shannon指数较一致，群落结构有一定差异，但无明显变化趋势。
(6)对旱地区域土壤DGGE图谱中特异性条带进行了克隆和测序鉴定，并对其进行了系统发育分析。对克隆所得序列进行分析发现，大部分克隆都属于变形菌门(Proteobacteria)和拟杆菌门(Bacteroidetes)，并且在属于这两个类群的克隆中发现了大量可能与多氯联苯、多环芳烃、苯类似物等有机污染物降解有密切关系的微生物类群，但未发现与重金属污染物相关的微生物类群。这说明，这两个微生物类群可能在受PCBs污染的土壤微生物生态系统中占有重要地位，并可能对土壤中PCBs污染物的转化和代谢具有重要的作用。同时也说明PCBs污染物对土壤微生物群落结构具有较显著的影响。
Recycling activities of electric and electronic waste (e-waste) are emerging as a global concern as they can contribute to the release of harmful pollutants into environment. Heavy metal and Polychlorinated biphenyls (PCBs) are this kind of harmful pollutants. In this research, we collected soil samples from the farmland around two e-waste recycling sites (dryland region D and paddy soil region P) where the PCBs leaking accident have happened. Concentration and composition of soil pollutants such as heavy metal, PCBs and OCPs were analysed. Combining the use of traditional microbial ecological research methods and PCR-DGGE to explore the changes of microbial ecosystem and community structure under contamination stress. The main conclusions are as follows:
(1) D and P regin were both contaminated by heavy metal. In D region, concentration of Cd was one time higher than the National Environmental Quality Standard level III (NEQS III). Concentration of Cu、Pb、Zn exceeded NEQS II. Concentration of Pb、Zn in D1(the sampling site that was nearest to the recycling site) was 2 and 5 times higher than NEQS III, respectively. In P region, concentration of Cd was one time higher than NEQS III, Concentration of Cu exceeded NEQS II. There were slight contamination of Pb and Zn at the same time.
(2) The average concentration of PCBs around the D region was 1.31 mg·kg~(-1), the highest concentration reached to 1.67 mg·kg~(-1) . The concentratin of PCBs decreased with the increasd of the distance from recycling site. In P region, average concentration of PCBs was just 9.70μg·kg~(-1). in D region PCBs was mainly composed of 3,. But in P region, the main composition were4,5,6-chlorinated PCBs. Both farmland was contaminated by organochlorine pesticide, but the concentration was very low. In D region, the average content of Hexachlorocyclohexane (HCH) and dichlorodiphenyltrichloroethane (DDT) were 22.4μg·kg~(-1) and 0.5μg·kg~(-1), respectivily. In P region, the content of HCH and DDT were 16.3μg·kg~(-1) and 6.6μg·kg~(-1), respectively. Evident transform had happened on the HCH and DDT in soil.
(3) In D region, there was a significant correlationship between concentration of PCBs and Cu, Cd, and also between Pb, Zn, indicating that they come from the same source, respectivily. In P region, there was a significant correlationship between Cd and Pb. Indicating that they come from the same source.
(4) Analysis of soil respiration and enzymes activity showed that in Dryland region, when getting close to the recycling site, soil microbial activity increased. The PCBs may have a positive effect on the microbial activity. In the paddy soil, soil respiration and enzymes fluctuated from site to site, but have no significant correlationship with the pollutants in soil. In D region, Urase activity showed a significant correlationship with the concentration of Cu and Cd, indicating that the decrease of urease activity may caused by the effect of Cu and Cd in the soil. Urease may be suitable for the assay of the effect of heavy metal contamination.
(5) Microbial account show that in dryland, when getting close to the recycling site, number of soil bacteria and actinomycete increased the Shannon diversity index of bacteria increased, But that of soil actinomycete decreased at the same time. PCA result show that, when getting close to the cycling site, the difference of the character of soil microbial community between sampling site and background site increased. This may caused by the gradience of PCBs and Cu in the soil. In paddy soil, number of bacteria and actinomycete in different sampling site are similar, number of fungi fluctuated acutely.There was some different between the community structure of different sampling site in paddy soil, but no obvious trend was observed.
(6) Clone and sequence of the specific band on the DGGE gel of dryland showed that most of the specific clone belongs to the Proteobacteria and Bacteroidetes, we also found that many clones were highly similar with the sequences of microbes that are related with organic compound degradation. Bacteria belong to this two phylum may play a very important role in the microbial ecosystem in PCBs contaminated soil, and also in the transformation and degradation of PCBs pollutants.
引文
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[38] Kim S,Picardal Fw. Microbe growth on dichloro biphenyls chlorinated on both rings as asole carbon and energy sourse. Appl. Environ. Microbiol. 2001, 67: 1953 —1955.
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[43] Loomis D, Browning SR, Schenck AP, et al. Cancer mortality among electric utility workers exposed to polychlorinated biphenyls . Occup Environ Med. 1997, 54:720-728.
[44] Mackay D, Paterson S. Evaluation the Multimedia Fate of Orgamic Chemicals: A Level Fugacity III Model. Environ. Sci. Technol. 1991, 25:427-432
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[48] MF Yueh, M Kawahara, J Raucy. Cell-based high-throughput bioassays to assess induction and inhibition of CYP1A enzymes. Toxicol In Vitro. 2005. 19: 275-287
[49] MH Wong, SC Wu, WJ Deng, et al. Export of toxic chemicals e A review of the case of uncontrolled electronic-waste recycling. Environmental Pollution, 2007, 1-10
[50] Mi Y, Zhang C. Toxic and hormonal effects of polychlorinated biphenyls on cultured testicular germ cells of embryonic chickens. Toxicol Lett. 2005, 155(2): 297-305.
[51]Moza P, Scheunert L, Klein W et al. Studies with 2,4',5-Trichlorobiphenyls' 14C and 2,2',4,4',6-Penchloribiphenyl-14C in Carrots, Sugar Beets and Soil", J. Agric. Food Chem. 1997,27:1120-1124.
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[58] Schulz Bull DE, Petrick G, Duinker JC. Complete characterization of polychlorinated biphenyl congeners in commercial aroclor and clophen mixtures by multi-dimentional gas chromatography-electron capture detection. Environ Sci Technol. 1989, 23:852-859.
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[65] Suneja S, Dogra RC. Effect of aldrin and lindine seed treatment on symbiosis of chickpea rhizobium with Bengal gram. Trop Agric , 1984, 62 :110-114.
[66] Svensson BG, Hallberg T, Nilsson A, et al. Parameters of immunological competence in subjects with high consumption of fish contaminated with persistent organochlorine compounds. Int Arch Occup Environ Health. 1994, 65:351-358.
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??Soil Science Socity of America,Wisconsin WI,1994. 775-833.
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[69] Tryphonas H. Immunotoxicity of PCBs(Aroclors)in relation to GreatLakes. EnvironHealth Perspect. 1995,103:35-46.
[70] Tyson Gene W., Chapman Jarrod, Philip Hugenholtz,et al., Community structure andmetabolism through reconstruction of microbial genomes from the environment. Nature,2004, 428(4):37-43
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[74] Wu QZ, Bedard D L, Wiegel J. Effect of Incubation temperature on the Route ofMicrobial Reductive Dechlorination of 2, 3, 4, 6-Tetrachlorobiphenyl in Poly chlorinatedBiphenyl (PCB)-Contaminated and PCB-Free Freshwater Sediments", Appl. Environ.Microbiol. 1997, 63:2836-2843
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