臺灣博碩士論文加值系統

免克寧為一種 dicarboximide 殺菌劑，具抗雄性激素之作用，是一種內分泌干擾物質。免克寧在環境代謝過程中會形成兩個重要之代謝產物 M1 (2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) 與 M2 (3’,5’-dichloro-2-hydroxy-2-methylbut-3-enanilide)。M1 與 M2 會競爭雄性激素受體，造成雄性相關基因表現下降，亦為內分泌干擾物質。因此，本研究欲探討免克寧代謝產物 M1 與 M2 於土壤中降解情形與對土壤微生物菌相的影響，並比較免克寧與代謝產物對本土種圓水蚤 (Daphnia pulex) 急毒性。試驗用 M1 及 M2 試劑自免克寧商品中純化出高純度免克寧 (99.8 %) 後進行水解、收集並純化而得，純度分別為 90.0-96.0%, 91.0-94.0%。試驗土壤採自桃園區改良場之壤質土壤 (Pu) 及花蓮區改良場砂質壤土 (Wl)，降解試驗 M1, M2 添加濃度分別為 20.0 mg kg-1 與 5.0 mg kg-1，利用 HPLC-DAD 分析其殘量。經滅菌與控制組比較發現，M1 在鹼性土壤環境下以生物性降解為主，酸性土壤環境下生物性與化學性降解會同時進行。M2 在酸性及鹼性土壤中皆以生物性的降解為主。且在滅菌土中，代謝產物 M1, M2 的半生期 (10.9-126.6, 87.1-296.5 天) 均較免克寧長 (8.7-35.0 天)。M1 與 M2 對土壤菌相影響，利用萃取土壤中總微生物核酸再以聚合酶連鎖反應串連變性梯度凝膠電泳 (PCR-DGGE) 來觀察。由電泳圖譜可發現土壤中部分優勢微生物，例如：於桃園改良場土壤 Acidobacterium sp. 與 Pseudomonas sp.，或於花蓮改良場土壤之 Ammoniphilus oxalaticus，為原生優勢微生物不易受到代謝產物 M1, M2 影響，造成族群大小與活性改變。某些微生物則會消失或增長，例如：花蓮改良場土壤之 Pseudomonas sp. 與 Bacillus sp. 於添加代謝產物 M1 或 M2 之處理組中較控制組族群更大。顯示免克寧在土壤中降解所產生之 M1, M2 代謝物仍對土壤菌相結構造成影響。水蚤 48 小時急毒性試驗結果，毒性大小依序為 M2 >免克寧> M1。綜觀代謝產物的毒性與持久性之結果，在當評估免克寧對環境的衝擊時，不能忽視代謝產物的影響。

Dicarboximide fungicide vinclozolin was found to have the anti-androgen effects, and its metabolites, M1 (2-[[(3,5-dichlorophenyl)-carbamoyl]oxy]-2-methyl-3-butenoic acid) and M2 (3’,5’-dichloro-2-hydroxy-2-methylbut-3-enanilide), were found to have more affinity to androgen receptors than vinclozoline. In this study, the effect of vinclozolin metabolites in soil on its persistence and soil microbial populations diversity were investigated. The toxicity of vinclozolin on Daphnia pulex, were compared with 2 kinds of its metabolites. First, we extract vinclozolin from commmerical products and then perform chemical hydrolysis to collect the metabolites. The soil sample were collected from Taoyuan District Agricultural Research (Pu) and Extension Center and Hualien District Agricultural Research (Wl). The experimental concentration were 20.0 mg kg-1 for M1 and 5.0 mg kg-1 for M2. We used solvent phase extraction to extract residues from soil, and utilized HPLC-DAD to detect them. In sterilized soil, M1 and M2 have longer half-lives (10.9-126.6 and 87.1-296.5 day) than vinclozolin (8.7-35.0 day). The impact of vinclozolin metabolites on soil microbial community were analysed by PCR-DGGE. In the DGGE patterns, we could discover some original superior populations with the resisence to toxicity of metabolites, such as Acidobacterium sp. and Pseudomonas sp. in Pu soil, and Ammoniphilus oxalaticus in Wl soil had the resistence to M1 or M2. But some populations were disappeared or enlarged by metabolites treatment, such as Pseudomonas sp. and Bacillus sp. did enlarge their population in treated soil. We conducted 48 hr acute toxicity assay on daphnia. The result of toxicity are in the sequence of M2 >vinclozolin>M1. The metabolites from degradation of vinclozolin, still have the hazard on environment. Due to vinclozolin metabolites have characteristics of persistence and toxicity. When we assess the impact of vinclozolin on environment, the effect of metabolites should be taken into account.

中文摘要 .......................................I
Abstract .....................................III
目錄 ......................................IV
圖目錄 ......................................VI
表目錄 ....................................VIII
一、前言 .................................................1
（一）土壤環境與生態......................................2
（二）免克寧的簡介........................................3
（三）免克寧代謝產物......................................9
（四）研究相關分子生物技術...............................18
（五）水蚤急毒性試驗.....................................28
（六）研究目的...........................................34
二、材料與方法...........................................36
（一）免克寧代謝產物於土壤中之降解試驗...................36
（二）代謝產物對土壤微生物菌相影響之研究.................54
（三）水蚤急毒性試驗.....................................62
三、結果與討論...........................................66
（一）免克寧代謝產物的製備結果...........................66
（二）代謝產物測定的情形.................................71
（三）免克寧代謝產物在土壤中的降解.......................74
（四）免克寧代謝產物對土壤細菌族群的影響.................88
（五）免克寧代謝產物對水蚤 (Daphnia pulex) 的急毒性試驗.109
四、結論 ...............................................114
五、參考文獻............................................115
附錄....................................................122

王一雄。1997年。土壤環境污染與農藥。國立編譯館。 P 9-12, 81-97.
林豊祺。2009年。硫醯尿素類除草劑Chlorsulfuron及依速隆於土壤中的消散及對土壤細菌族群結構的影響。臺灣大學農業化學系碩士論文。
潘泊原。2010年。奈米和微米級TiO2, SiO2和Al2O3對本土水蚤、大腸桿菌、小白菜和空心菜的毒性及土壤菌相的影響。臺灣大學農業化學系碩士論文。
盧俊廷。2009年。殺菌劑免克寧在土壤中之降解與降解產物的生成對土壤菌相之影響。臺灣大學農業化學系碩士論文。
李宏萍、李仁厚、廖合堂、翁愫慎。2004年。鋅錳乃浦農民施藥暴露量安全評估。植物保護學會會刊 46：333-343。
張仲民。1987年。普通土壤學。國立編譯館。 P 1-10.
行政院環境保護署環境檢驗所。1994年。土壤中陽離子交換容量－醋酸銨法 (NIEA S201.60T)。土壤檢測方法彙編。(http://www.niea.gov.tw/analysis/method/methodfile.asp?mt_niea=S201.60T)
行政院環境保護署環境檢驗所。2002年。土壤水分含量測定方法－重量法 (NIEA S280.61C)。土壤檢測方法彙編。(http://www.niea.gov.tw/analysis/method/methodfile.asp?mt_niea=S280.61C)
行政院環境保護署環境檢驗所。2005年。水樣急毒性檢測方法－水蚤靜水式法 (NIEA B901.12B)。環境生物檢測方法彙編。 (http://www.niea.gov.tw/analysis/method/methodfile.asp?mt_niea=B901.12B)
行政院環境保護署環境檢驗所。2008年。土壤酸鹼值 (pH 值) 測定方法－電極法 (NIEA S410.62C)。土壤檢測方法彙編。(http://www.niea.gov.tw/analysis/method/methodfile.asp?mt_niea=S410.62C)
Amann, R.I., W. Ludwig, and K.H.Schleifer. 1995. Phylogenetic identification and in situ detection of individual microbial cells without cultivation. Microbiol. Rev. 59:143-169.
Andrade J., A. Karmali, M. A. Carrondo, and C. Frazão. 2007. Structure of Amidase from Pseudomonas aeruginosa Showing a Trapped Acyl Transfer Reaction Intermediate State. J. Biol. Chem. 282: 19598–19605.
Baker, V.A. 2001. Endocrine disrupters -testing strategies to assess human hazard. Toxicol. In Vitro. 15:143-149.
Barthold, J.S., K. Kumasi-Rivers, J. Upadhyay, B. Shekarriz, and J. Imperato-McGinley. (2000). Testicular position in the androgen insensitivity syndrome: Implications for the role of androgens in testicular descent. J. Urol. 164:497-501.
Benke, G.M., and S.D. Murphy. 1975. The influence of age on the toxicity and metabolism of methyl parathion and parathion in male and female rats. Toxicol. Appl. Pharmacol. 31:254-269.
Campbell, A.K., K.T. Wann, and S.B.Matthews. 2004. Lactose causes heart arrhythmia. Comp. Biochem. Physiol., B. 139: 225-234.
Cowan, D.A. 2000. Microbial genomes – the untapped resource. Trends Biotechnol.18:14-16.
Current Protocols in Microbiology, Appendeix 2 Commonly used reagents and equipment.
Damstra, T., S. Barlow, A. Bergman, R. Kavlock, and G. Van Der Kraak (Eds.), International Programme on Chemical Safety, Global Assessment of the State-of-the-Science of Endocrine Disruptors, World Health Organization, 2002.
Dhananjeyan, M.R., P.W. Erhardt, and C. Corbitt. 2006. Simultaneous determination of vinclozolin and detection of its degradation products in mouse plasma, serum and urine, and from rabbit bile, by high-performance liquid chromatography. J. Chromatogr. A.115:8-18.
Ebert , D.. 2005. Ecology, Epidemiology and Evolution of Parasitism in Daphnia.
Available from (http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=Books)
Fry, D.M. ,and Toone, C. K. 1981. DDT-induced feminization of gull embryos. Science 213:922-924.
Gray, L.E., J.S. Ostby, and W.R. Kelce. 1994. Developmental effects of an environmental antiandrogen: The fungicide vinclozolin alters sex differentiation of the male rat. Toxicol. Appl. Pharmacol.129:46-52.
Gray, L.E., J.S. Ostby, E. Monosson, and W.R. Kelce. 1999. Environmental antiandrogens: Low doses of the fungicide vinclozolin alter sexual differentiation of the male rat. Toxicol. Ind. Health 15:48-64.
Goleveva, L. A., Z. I. Finkelstein, A. V. Polyakova, B. P. Baskunov, and M. Y. Hefedova. 1991, Microbial conversion of fungicide vinclozilin. J. Env. Sci. Health B26, 293–307.
Kelce, W.R., E. Monosson, M.P. Gamcsik, S.C. Laws, and L.E. Gray 1994. Environmental hormone disruptor: Evidence that vinclozolin developmental toxicity is medidated by antiandrogenic metabolites. Toxicol. Appl. Pharmacol. 126:276-285.
Kelce, W.R., and E.M. Wilson. 1997. Environmental antiandrogens: developmental effects, molecular mechanisms, and clinical implications. J. Mol. Med. 75:198–207.
Mansour, S.A.,and M.F. Gad. 2010. Risk assessment of pesticides and heavy metal contaminants in vegetables: A novel bioassay method using Daphnia magna Straus. Food Chem. Toxicol. 48:377-389.
Molina-Molina, J.M., A. Hillenweck, I. Jouanin, D. Zalko, J.P. Cravedi, M.F Fernández, A. Pillon, J.-C. Nicolas, N. Olea, and P. Balaguer. 2006. Steroid receptor profiling of vinclozolin and its primary metabolites. Toxicol. Appl. Pharmacol. 216:44-54.
Muyzer, G., E.C. WAAL, and A.G. Uitterinden. 1993. Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl. Environ. Microbiol. 59:695-700.
Muyzer G., and K. Smalla. 1998. Application of denaturing gradient gel electrophoresis (DGGE) and temperature gradient gel electrophoresis (TGGE) in microbial ecology. Antonie van Leeuwenhoek. 73:127-141.
Myers, R.M., S.G. Fischer, T. Maniatis, and L.S. Lerman. 1985. Modification of the melting properties of duplex DNA by attachment of a GC-rich DNA sequence as determined by denaturing gradient gel electrophoresis. Nucleic Acids Res. 13:3111-3129.
Monosson, E., W.R. Kelce, C. Lambright, J. Ostby, and L.E. Gray Jr.. 1999. Peripubertal exposure to the antiandrogenic fungicide, vinclozolin, delays puberty, inhibits the development of androgen-dependent tissues, and alters androgen receptor function in the male rat. Toxicol. Ind. Health 15:65-79.
Nitche, E.M., and O. Hiort. 2000. The molecular basis of androgenin sensitivity. Horm. Res. 54:327-333.
Saiki, R. K., D. H. Gelfand, S. Stoffel, S. J. Scharf, R. Higughi, G. T. Horn, K. B. Mullis, and H. A. Erlich. 1988. Primer-directed enzymatic amplification of DNA with a thermostable DNA polymerase. Science. 239: 487-491.
Saitou, N., and M. Nei. 1987. The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4:406-425.
Schloter, M., M. Lebuhn, T. Heulin, and A. Hartmann. 2000. Ecology and evolution of bacterial microdiversity. FEMS Microbiol. Rev. 24: 647-660.
Sierra-Santoyo, A., H.A. Barton, and M.F. Hughes. 2004. Liquid chromatography determination of the anti-androgen vinclozolin and its metabolites in rat serum. J. Chromatogr. B Analyt. Technol.Biomed. 809 :105-110.
Sheffield, V. C., D. R. Cox, L. S. Lerman, and R. M. Myers. 1989. Attachment of a 40-base-pair G＋C-rich sequence (GC-clamp) to genomic DNA fragments by the polymerase chain reaction results in improved detection of single-base changes. Proc. Natl. Acad. Sci. USA. 86:232-236.
Spencer, E. Y. Guide to the chemicals used in crop protection, 7th ed.; Publication 1043; Agriculture Canda Research Branch:Ottawa, 1982; p585.
Sumpter, J.P. 1999. Xenoendocrine disrupters - environmental impacts. Toxicol. Lett.28:337-342.
Szeto, S. Y., N. E.Burlinson, J. E Rahe, and P. C Oloffs. 1989a. Kinetics of hydrolysis of the dicarboximide fungicide vinclozolin.J. Agric. Food Chem. 37:523-529.
Szeto, S. Y., N. E.Burlinson, J. E Rahe, and P. C Oloffs. 1989b. Persistence of the fungicide vinclozolin on pea leaves under laboratory conditions. J. Agric. Food Chem. 37:529-534.
Szeto, S. Y., N. E.Burlinson, S.J. Rettig, and J. Trotter. 1989c. Identification of hydrolysis products of the fungicide vinclozolin by spectroscopic and x-ray crystallographic methods. J. Agric. Food. Chem. 37:1103-1108.
Tomoya, M., N. Yasunori, M. Kazuo, I Makoto, A. Susumu, and K. Makoto. 2007. Bacterial community in plant residues in a Japanese paddy field estimated by RFLP and DGGE analyses. Soil Biol. Biochem. 39:463-472.
Trucco, R.G., et al., F.R. Engelhardr, B. Stacey. 1983. Toxicity, accumulation and clearance of aromatic hydrocarbons in Daphnia pulex. Environ. Pollut.A. 31: 191-202.
US Environmental Protection Agency. (2000). Reregistration eligibility decision (RED). Vinclozolin. Case No. 2740. Office of Prevention, Pesticides, and Toxic Substances. EPA 738-R-00-023 (www.epa.gov/REDs/2740red.pdf).
US Environmental Protection Agency. (2002). Methods for measuring the acute toxicity of effluents and receiving waters to freshwater and marine organisms. (http://www.epa.gov/waterscience/methods/wet/disk2/atx.pdf)
United nations environment programme. Stockholm convention on persistent organic pollutants. (http://www.pops.int/documents/convtext/convtext_en.pdf)
Vallero, D. A. 2000. Dicarboximide fungicide flux to the lower troposphere from an aquic hapludult soil. Doctoral Dissertation, Duke Univ., Durham, N.C.
Villadieu, J. C., de Savignac, A., and J. P. Calmon. 1995. Kinetics and mechanisms of hydrolysis of dicarboximide fungicides in micellar media. J. Agric. Food Chem., 43:1948-1953.
Walker, A. 1987. Further observations on the enhanced degradation of iprodione and vinclozolin in soil. Pestic. Sci., 21:219-231.
Wittke, K., K. Wittke, H. Hajimiragha, L. Dunemann, and J. Begerow. 2001. Determination of dichloroanilines in human urine by GC-MS, GCMS-MS and GC-ECD. J. Chromatogr. B Biomed. Sci. Appl. 755:215-228.
Wolf, C.J., G.A. LeBlanc, J.S. Ostby ,and L.E. Gray, Jr.. 2000. Characterization of the Period of Sensitivity of Fetal Male Sexual Development to Vinclozolin. Toxicol. Sci. 55:152-161.
Wong, C., W.R. Kelce, M. Sar, and E.M. Wilson. 1995. Androgen receptor antagonist versus agonist activities of the fungicide vinclozolin relative to hydroxyflutamide. J. Biol. Chem. 270: 19998-20003.
Zaitsev, G.M., I. V. Tsitko , F.A.. Rainey, Y.A. Trotsenko, J.S. Uotila, Erko Stackebrandt, and M.S. Salkinoja-Salonen. 1998. New aerobic ammonium-dependent obligately oxalotrophic bacteria: description of Ammoniphilus oxalaticus gen. nov., sp. nov. and Ammoniphilus oxalivorans gen. nov., sp. nov.. Int. J. Syst. Bacteriol 48: 151-163.