摘要
随着科学技术的进步,工农业生产的发展,人类合成越来越多的化学品,水环境作为工农业废物的最终排放点,污染现象越来越严重。水体有毒有机物污染已成为世界各国科学界和政府关注的新热点。以往所采用的化学分析手段,虽然可以快速而灵敏地分析测定出污染物的种类和浓度及单一因子的直接影响,但因污染物种类繁多、数量巨大,不能反映污染物对生物和生态系统的影响。利用生命有机体对污染物的种种反应,来直接地表征环境质量的好坏及所受污染的程度的生物监测作为化学分析的补充手段是评价水质一个新的方案。采用基于细胞培养的生物方法监测和评估水质相比于藻类、鱼类等的生长繁殖试验具有费用低廉、节省时间、灵敏度高的优点,因此具有推广应用的前景。本论文通过细胞中性红实验、EROD 测试及重组基因酵母增殖实验三种生物毒性测试方法研究水环境中导致急性细胞毒性的物质、芳香烃(Ah)受体效应物质和内分泌干扰物质这几类有毒有机污染物,并选取了北京市北小河污水处理厂的进、出水及不同工艺出水和太湖梅梁湾表层沉积物这两种水环境样品进行测试,从而确定这三种生物测试方法是否适合批量测试水样。
研究结果表明细胞中性红染色法(NR)能快速检测细胞毒性作用,数据处理方便、直观;EROD 生物测试所用的离体大白鼠肝癌细胞对类二恶英物质有着较高的灵敏性,2,3,7,8-四氯二苯并对二恶英(2,3,7,8-TCDD)与EROD 酶活力之间具有良好的剂量-效应关系,适合于对环境样品中的二恶英类化合物进行快速定量筛选;重组受体基因/报道基因的酵母检测技术则是一种筛选和定量分析环境样品中雌激素类污染物的快速、有效方法,本实验中重组基因酵母对雌激素类化合物也有较高的灵敏,17β-雌二醇标准化合物与β-半乳糖苷酶活性诱导的剂量-效应关系良好。在实际水环境样品测试中北京市北小河污水处理厂进、出水以及不同处理工艺出水的生物毒性测试表明城市污水厂出水中含有上述毒性特征的污染物,在再生水不同工艺深度处理中,微滤和反渗透工艺不能有效去除有毒有机污染物,超滤对具有急性毒性特征的物质和类雌激素物质去除效果最好,活性炭处理对Ah 受体效应物质的去除效果最好,这说明本实验所选取的生物毒性测试方法适于探讨再生水不同深度处理工艺技术的适用性,也适于水环境中有毒有机污染物的初步测评,如污水处理厂的不同处理工艺出水;用成组生物毒性测试方法对太湖梅梁湾表层沉积物有毒有机污染物毒性特征的背景值进行测试得知太湖梅梁湾的北部受到Ah 受体效应物质和内分泌干扰物质的严重污染,湖心则相对清洁,但南部且靠近湾中心受急性细胞毒性物质污染较重。这几类有毒有机污染物的分布规
Aqueous environment is continuously loaded with foreign synthetic chemicals released byurban communities、agricultures and industries with the development of science technology.Recently, there has been increased concern regarding the role of aquatic environmental toxinorganic pollutants. The risk assessment of organic pollutants in environments is often basedon the concentrations detector by analytical chemistry and data on the toxicity of singlecompounds. However, all pollutants exert their effects as components of complex mixtures.The great number of compounds with their possible interactions makes it difficult to assessthe overall toxicity of effluents. Bioassays on aquatic concentrates provide a direct functionalresponse that relates to the overall toxic properties of the mixtures of compounds present in asample. Therefore, it is adaptable to evaluate water quality as a feasible scheme. Thecharacteristics of bioassays in vitro are low expenditure, time saving and high sensitivitycompared with the bioassay in vivo such as algae、fish reproducing experiments. In this paper,a battery of bioassays in vitro: neutral red, EROD and recombinant yeast bioassay wasstudied to assess the acute toxicants, Ah-acceptor chemicals and estrogenic substances inaquatic environments. Effluents from different advanced treatment units of the Bei Xiaohesewage treatment plant (STP) in Beijing and surface sediments from Meiliang Bay, TaihuLake were selected as the environmental samples.
The results showed that neutral red assay is expeditious and the data processing isfacility. In EROD assay the H4IIE rat hepatoma cell used were sensitive to dioxin-likecompounds and there was a good dose-response relationship between 2,3,7,8-TCDD andEROD enzyme activity. This confirmed EROD assay in vitro is a rapid quantitative screeningfor the Ah-receptor-related toxicants in the environmental samples. Meanwhile therecombinant gene yeast assay technique is an efficient and rapid method for determining theactivity of estrogens in pollutants. In this study the recombinant gene yeast had highsensitivity to the estrogenic compounds and the yeast assay also had a good dose-responserelationship between 17β-estradiol and β-galactosidase enzyme activity as well. Additionally,
引文
[1] 肖锦.城市污水处理及回用技术.北京:化学工业出版社,2002,12-30
[2] 唐以剑,章申.我国发展中的水环境问题与对策.地理学报,1989,44(3):302-313
[3] 王晓燕,尚伟.水体有毒有机污染物的危害及优先控制污染物.首都师范大学学报(自然科学版) ,2002,23(3):73-78
[4] 齐文启, 孙宗光编著.痕量有机污染物的监测.北京:化学工业出版社,2001
[5] 苏丽敏,袁星.持久性有机污染物(POPs)及其生态毒性的研究现状与展望.重庆环境科学,2003,25(9):62-65
[6] 岳瑞生.《关于就某些持久性有机污染物采取国际行动的斯德哥尔摩公约》及其谈判背景.世界环境,2001,1:24~28
[7] 李慧蓉.生物监测技术及其研究进展.江苏石油化工学院学报,2002,14(2):57 -60
[8] 修瑞年,许久全,高世荣,郭玖,任致英,印静.几种环境生物测试新技术的研究进展.生态科学,1992,2:18-22
[9] 解庆林,王敦球,李金城.环境生物技术的发展现状.桂林工学院学报,2001,2l(2): 191-194
[10] M.S. Shailaja, ClassyD'Silva.Evaluation of impact of PAH on a tropical fish, Oreochromis mossambicus using multiple biomarkers . Chemosphere, 2003, 53 : 835–841
[11] Chunxia Wang, Yi Wang, F.Kiefer, A.Yediler, Zijian Wang, and A.Kettrup.Ecotoxiocological and chemical charaterization of selected treatment process effluences of municipal sewage treatment plant.Ecotoxicology and Environmental Safety, 2003, 56:211–217
[12] 张凤明,周辉.浅谈生物监测.黑龙江环境通报,2000,24(2):113-114
[13] 王栋枝.综合生物毒性指标在水体污染监测中的应用.环境保护科学,1992 18(1):85-87
[14] Rai, L.C. , Gaur, J.P. , Soeder, C.J. , Eds.Algae and Water Pollution.E. Schweizerbart’sche Ver lagsbuch handlung,Stuttgart,1994
[15] Kenaga, E.E..Test organisms and methods useful for early assessment of acute toxicity of chemicals.Environment Science and Techology,1978,12:1322-1329
[16] Dohety, F.G..Interspecies Correlations of Acute Aquatic Median Lethal Concentrations for four standard testing Species.Environment Science and Techology,1983,17: 661-665
[17] Mayer, F.L. and Ellersieck, M.R..Manual for acute toxicity: interpretation and data base for 410 chemicals and 66 species of freshwater animals.Washington D.C.:U.S. Dep. of the Interior, Fish, and Wildlife Service, Resource Publication,1986:505
[18] U.S.EPA (EPA-821-R-02-013).Short-term Methods for Estimation the Chronic Toxicity of Effluents and Receiving Waters to Freshwater Organisms.Fourth Edition,October 2002
[19] 马梅, 王毅, 王子健.天然水体有毒有机污染物毒性监测的样品前处理方法研究.环 境科学,1999,20(6):80-83.
[20] 王宏,沈英娃,卢玲,朱红梅,陈宏,许永香,高世荣.几种典型有害化学品对水 生生物的急性毒性.应用与环境生物学报,2003,9(1):49—52
[21] 卢玲,宋福.鱼类急性毒性实验.生物学通报,2002,37(7):52-53
[22] 张娜.鱼类急性毒性实验及其条件选择.水利渔业,1999,19(6):6
[23] 孙皎,高桑美代子,佐藤和子,佐藤温重.中性红染色法定量检测牙科材料细胞毒 性的研究.中国生物医学工程学报,18(1):118-120
[24] 丁竹红,谢标,王晓蓉.生物标志物及其在环境中的应用.农业环境保护,2002, 21(5):465—467
[25] N.C.Bols, K.Schirmer, E.M.Joyce, et al.Ability of polycyclic aromatic hydrocarbons to induce 7-ethoxyresorufin-o-deethylase activity in a trout liver line.Ecotoxicology and Environmental Safety,1999,44(1):118-128
[26] Mikakline Billeret, Philippe Berny, Michelle Mazallon, et al.Bioavailability of polycyclic aromatic hydrocarbons and polychlorinated biphenyls in rats from naturally contaminated soilspreliminary evaluation of the influence of soil parameters.Environment Toxicology, 2000,19(10):2614-2620
[27] Song Lin, Peter L.Bullock, Richard F.Addison, et al.Detection of cytochrome P4501A in several species using antibody against a synthetic peptide derived from rainbow trout cytochrome P4501A1.Environment Toxicology,1998,17(3):439-445
[28] Robin M.Whyatt, Wieslaw Jedrychowski, Kari Hemminki, et al.Biomarkers of polycyclic aromatic hydrocarbon-DNA damage and cigarette smoke exposure in paired
maternal and newborn blood samples as a measure of differential susceptibility.Cancer
Epidemiol., Biomarkers Prev. ,2001,10(6):581-588
[29] M.C.Fossi, S.Casini, L.Marsili.Nondestructive biomarkers of exposure to endocrine disrupting chemicals in endangerer species of wildlife.Chemosphere,1999,39(8): 1273-1285
[30] Payne J.F., Fancey A., Rahimtula D., et al.Review and perspective on thre use of mixd function oxygenase enzymes in biological monitoring.Comp Biochem Physiol,1987, 860:233-245
[31] 许华夏,李培军,刘宛,宋玉芳.生物细胞色素P450的研究进展.农业环境护,2002, 21(2):188-191
[32] 霍传林,王菊英,韩庚辰,关道明.鱼体内EROD活性对多氯联苯类的指示作用.海 洋环境学,2002,2l(1):5-8
[33] Fujita S.P450 in wild animals as a biomarker of environmental impact.Biomarkers,2001, 6(1):19-25
[34] Forlin L., et al . Effects of pulp blench plant effluents on hepatic xenobiotic biotransformation enzymes in fish: laboratory and field studies.Environment Research, 1985,17:109-112
[35] 陈国胜等.鲫鱼肝脏中CYP1A1的诱导作为沉积物中二恶英的毒理学指标的研究.中 国环境科学,1997,17(3):264-267
[36] 朱琳,钱芸,刘广良.细胞色素P450酶系及其在毒理学上的应用.上海环境科学, 2001,202:88-91
[37] Marshall Adams S, Bevelluner Mark S, Mark Stephen Greeley, Levine Daniel A, Trh Swee J.Ecological risk assessment in a large river-reservoil: 6. bioindicators of fish population health.Environmental toxicology and chemistry,1999,18(4):628-640
[38] 王咏,王春霞,王子健,徐镜波.硝基芳烃对鲤鱼肝EROD活性影响的体外研究环境 科学,2001,22(4):120-122
[39] Rowlands J.C., Gustafsson J.A. . Aryl hydrocarbon receptor-mediated signal transduction.Crit Rev Toxicol,1997,27(2):109-134
[40] Swanson H.I., Bradfield C.A. . The Ah-receptor: genetics, structure and function.Pharmacogenetics,1993,3:213-230
[41] Nebert D.W., McKinnon R.A., Puga A..Human drug-metabolizing enzyme polymorphism: effects on risk of toxicology and cancer.DNA and Cell Biology,1996,15(4) :273-280
[42] Schmidt, J.V., Bradfield, C.A..AH receptor signaling pathways.Annual Review of Cell and Developmental Biology,1996,12:55–89.
[43] Gu, Y.Z., Hogenesch, J.B., Bradfield, C.A. . The PAS superfamily: sensors of environmental and developmental signals.Annual Reviews in Pharmacology and Toxicology,2000,40:519-561
[44] 孔昌俊.环境激素对人类健康的影响.辽宁城乡环境科技,2000,20(6):1-4
[45] Zacharewski T.In Vitro bioassay for assessing estrogenic substances.Environment Science Technology。1997,31(3):613-623
[46] 胡军,李杰.环境内分泌干扰物筛检方法研究进展.预防医学文献信息,2002,8(6): 706-708
[47] Arnold SF, Klotz DM, Collins BM, et al.Synergistic antivation of estrogen receptor with combination of environmental chemicals.Science,1996,,272:1489-1492
[48] Zacharewski T.Identification and assessment of endocrine disruptors: limitation of in vivo and in vitro assay.Environ Health Persperct,1998,106 (12):577-582
[49] 张庆华,K.-W.Schramm,徐盈.利用96 孔板和重组基因酵母筛选环境内分泌干扰 物.环境科学学报,2003,23(5):702-704
[50] 楼丽广,胥彬.甾体激素作用的分子机制.见:金正均,王水铭,苏定冯,主编.药 理学进展.北京:人民卫生出版社,1997,114—127
[51] 何世华,梁增浑,战威,贾凌志,晁福寰.环境雌激素重组酵母测评系统的建立.环 境与健康杂志,2002,19(1):57-59
[52] 何世华,梁增浑,战威,贾凌志,晁福寰. 全长人雌激素受体cDNA的克隆及酵母 表达载体的构建. 环境与健康杂志,2001,18(4):198-200
[53] Metzger.D, White.J.H and Chambon.P.The human oestrogen receptor functions in yeast.Nature,1988,334:31-36
[54] 石淑倩,姜志凯,翟海霞.我国城市污水回用现状及前景.环境技术, 2002,(2): 35~38.
[55] 朱喜,张杨文.梅梁湖水污染现状及防治对策.水资源保护,2002,4:28-30
[56] 金相灿,叶春,颜长宙等.太湖重点污染控制区综合治理方案研究.环境科学研究, 1999,12(5):1-5
[57] 李青山.中国水资源保护问题及其对策措施.水资源保护,1999,56(2):28-30
[58] 丁南瑚.增强水的危机感,提高水的自觉性.净化技术,1999,68(2):2-4
[59] 宁振东.水污染·水资源·对策.环境科学进展,1995,3(6):24-27
[60] Keith, L.H.Identification and Analysis of Organic Pollutants in Water.Ann Arbor Science,1976
[61] Zoeteman B.C. J.Sensory Assessment if Water Qunlity.Pergamor Press,1980
[62] Giacco L.L.Water and Water Pollution Handbook.Marcel Dekker,1973
[63] 司徒镇强, 吴军正.细胞培养.世界图书出版,1996
[64] Deml E, Weibel F.J, Oesterle D.Biological activity of 2,4,8-trichlorodibenzo-furan: promotion of rat liver foci and induction of cytochrome P450 dependent monooxygenases.Toxicology,1989,59:229-238
[65] 黎雯.环境样品中二恶英毒性效应的生物标志物研究.武汉:中科院水生生物研究所,1998
[66] Bradford M M.A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle of proteidye binding.Anal.Biochem,1976,72:248-254
[67] 饶凯锋,马梅,王子健, 刘丽君.南方某水厂处理工艺过程中内分泌干扰物的变化规律.环境科学,2004,25(6) :123-126.
[68] Wan X., Ma T., Wu W., Wang Z..EROD activities in a primary cell culture of grass carp (Ctenopharyngodon idellus) hepatocytes exposed to polychlorinated aromatic hydrocarbonas.Ecotoxicol Environ Saf,2004,58:84-89
[69] 万小琼,吴文忠,贺纪正.利用草鱼原代肝细胞培养评价二恶英毒性效应.中国环境科学,2002,22(2):114-117
[70] 张庆华,K.-W.Schramm,徐盈.利用96 孔板和重组基因酵母筛选环境内分泌干扰物.环境科学学报,2003,23(5):702-704
[71] Routledge E J,Sumpter J P.Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen . Environment Toxicology and Chemidtry,1996,15(3):241-248
[72] Gaido K W, Leonard L S, Lovell S,et al.Evaluation if chemicals with endocrine miodulating activity in a yeast-based steroid hormone receptor gene transcription
assay.Toxicol Appl Pharmacol,1997,143:205-212
[73] Jingxian Wang,Wenzhong Wu,Berhard Henkelmann,Li You,Antonius Kettrup and Karl-Werner Schramm.Presence of estrogenic activity from emission of fossil fuel combustion as detected by a recombinant yeast bioassay.Atmospheric Environment, 2003,37:3225-3235
[74] 马梅,王毅,王子健.城市污水生物处理过程中有毒有机污染物浓度及毒性变化的 规律.工业水处理,1999,19 (6):9-12
[75] 徐盈,吴文忠,张甬元.利用EROD 生物测试法快速筛选二恶英化合物.中国环境 科学,1996,16(4):279-284
[76] 黎雯,徐盈,吴文忠,K.-W.Schramm,A.Kettrup.利用EIA 生物测试法快速定量 筛选环境样品中的二恶英污染物.环境科学,2000,21(4):69-72
[77] 黎雯,徐盈,吴文忠.鱼肝EROD 酶活力诱导作为二恶英的水生态毒理学指标.水 生生物学报,2000,24(3):201-207
[78] Yutaka Tashiro , Akihiro Takemura, Haruhiko Fujii, Kaneshi Takahira,Yasuhiro Nakanishi.Livestock wastes as a source of estrogens and their effects on wildlife of Manko tidal flat, Okinawa.Marine Pollution Bulletin,2003,47:143–147
[79] Kurunthachalam Kannan, Daniel.L. Villeneuve, Nobuyoshi Yamashita, Takashi Imagawa, Shinya Hashimoto, Akira Miyazaki, and John P. Giesy.Vertical Profiles of Dioxin-like and Estrogenic Activities Associated with a Sediment Core from Tokyo Bay, Japan.Environ. Sci. Technol,2000,34:3568-3573
[80] Seung-Min Oh, Se-Young Choung, Yhun-Yhong Sheen, Kyu-Hyuck Chung.Quantitative assessment of estrogenic activity in the water environment of Korea by the E-SCREEN assay.The Science of the Total Environment,2000,263:161-169
[81] 蒋学之.环境雌激素对人类健康的潜在影响.中国公共卫生,1997,13(4):251-252
[82] 申继忠.内分泌干扰的农药.农药市场信息,2002,24:35
[83] Safe,S.H. . Polychlorinated biphenyls(PCBs), dibenzo-p-dioxin(PCDD), dibenzofurans(PCDF) and related compounds: environmental and mechanistic considerations which support the development of toxin equivalency factors(TEFs).Toxicol,1990,2:51-88
