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研究生:盧俊廷
研究生(外文):Chung-Ting Lu
論文名稱: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
論文名稱(外文):The impacts of vinclozolin and its metabolites on soil environment
指導教授:王一雄顏瑞泓
指導教授(外文):Yei-Shung WangJui-Hung Yen
學位類別:碩士
校院名稱:國立臺灣大學
系所名稱:農業化學研究所
學門:農業科學學門
學類:農業化學類
論文種類:學術論文
畢業學年度:97
語文別:中文
論文頁數:103
中文關鍵詞:35-二氯苯胺免克寧降解產物M2免克寧降解產物M1降解產物免克寧變性梯度凝膠電泳
外文關鍵詞:35-dichloroanilineVinclozolin M2Vinclozolin M1VinclozolinPCR-DGGE
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免克寧是已知的內分泌干擾物質,近年研究發現其降解產物2-[[(3,5-dichlorophenyl)-carbamoyl]-oxy]-2methyl-3-butenoic acid (M1)和3’,5’-dichloro-2-hydroxy-2-methylbut-3-enanilide (M2) 與雄性激素受器的親和性較免可寧高。同時,最終降解物3,5-二氯苯胺 (M3) 為二氯苯胺立體異構物中腎毒性最強之化合物。免克寧屬非持久性農藥,隨著降解現象的發生,它的降解物質會存在於土壤或是植物體中。為了得知免克寧及其降解產物 (M1、M2及M3) 在土壤中降解的動態及對土壤微生物族群的影響,本研究利用溶劑萃取法進行土壤中免克寧及降解產物的萃取,再使用高效液相層析法 (HPLC) 及氣相層析儀搭配電子捕獲式偵測器 (GC-ECD) 來進行殘留量的測定。最後藉由變性梯度凝膠電泳 (PCR-DGGE) 來觀測免克寧及降解產物對土壤菌相的影響。免克寧在pH 5之桃園農改場土壤的半生期為12-13天,於pH 7之花蓮農改場土壤的半生期為7-9天。濃度50 mg kg-1免克寧降解試驗結束時 (試驗48天),未滅菌組免克寧的殘量介於3.14-4.01 mg kg-1,免克寧降解產物M1的濃度為14.60-17.77 mg kg-1,免克寧的降解產物M2的含量於試驗結束時介於0.32-0.91 mg kg-1,M3的累積濃度則為3.27-6.27 mg kg-1。滅菌組免克寧的殘留量為3.25-30.90 mg kg-1,M1累積濃度介於1.35-18.48 mg kg-1,M2的累積量達2.16-8.75 mg kg-1,M3的累積量可達0.75 mg kg-1。研究結果顯示,免克寧的降解由化學水解與微生物降解共同作用,未滅菌處理降解至M3的速率較滅菌處理快,各處理的降解情形於試驗期間停留於中間產物階段 (M1) 較長的時間,M3會隨著時間的增加而逐漸累積。PCR-DGGE的實驗結果亦顯示,免克寧或其降解產物對於土壤菌相的影響在施用中期的影響最大,而其土壤微生物菌相有消長的趨勢。
Vinclozolin has found to have the anti-androgen potential, and its metabolites, M1 and M2, are currently found to have more affinity to androgen receptors than their par-ent chemical, vinclozolin. Also, its final metabolite, 3,5-dichloroaniline, is found for more acute nephrotoxicity than other dichloroaniline stereoisomers. To monitor the de-gradation of vinclozolin and its metabolites production, and to investigate the impact of vinclozolin with its metabolites on soil microbial community, we used DNA extracted kits to collect the soil microbe’s DNA, and used solvent phase extraction to extract vin-clozolin and its metabolites. Then we utilized HPLC-UV and GC-ECD to detect vin-clozolin and metabolites in soil sample. The influences of vinclozolin and its metabolites on soil microbial community were visualized by PCR-DGGE. The half-life time of vinclozolin was 12-13 days in Pu soil and 7-9 days in Wl soil, and vinclozolin was re-sidual for more than 40 days. M1 and M2 produced in treated soils within 3 day, and they increased with time. For 48 days of incubation, M1 were amassed from 14.60 mg kg-1 to 17.77 mg kg-1 in non-sterilized soil, and from 1.35 mg kg-1 to 18.48 mg kg-1 in sterilized soil. The concentrations of Vinclozolin M2 were between 0.32 mg kg-1 and 0.91 mg kg-1 in non-sterolized soil, and between 2.16 mg kg-1 and 8.75 mg kg-1 in steri-lized soil. Additional, the degradated product M3, 3,5-dichloroaniline, could accumu-lated to 6.27 mg kg-1. Finally, the soil pH and soil microbial were the two important factors in vinclozolin degradation and metabolites production. Although vinclozolin were rapid degradated, there were other products appear. The application of vilcolzolin to soil will impact the soil microbial community, and it is important to notice the residual in our environment and to re-evaluate whether the regulation of vinclozolin has been updated.
目錄
摘要 I
Abstract II
目錄 III
圖目錄 V
表目錄 IX
一、 前言 1
二、 文獻回顧 3
(一) 免克寧的基本資料 3
(二) 免克寧在環境中的流佈 8
1. 免克寧的殘留 8
2. 免克寧的降解 9
3. 免克寧與其降解產物的宿命 12
(三) 免克寧對環境可能的危害 14
1. 環境荷爾蒙 14
2. 雄性激素和抗雄性激素的作用機制 19
3. 免克寧在表觀遺傳學 (epigenetics) 上的影響 20
4. 免克寧及其代謝物對生物之影響 20
三、 材料與方法 24
(一) 材料 24
1. 土壤樣品及採集地點 24
2. 藥品 24
3. 溶劑 25
4. 氣體 26
5. 儀器設備 26
(二) 方法 27
1. 土壤基本性質測定 28
2. 免克寧的測定條件 31
3. 免克寧的回收率試驗 34
4. 免克寧降解產物的製備 34
5. 免克寧的孵育試驗 38
6. 土壤微生物DNA萃取 38
7. 土壤中免克寧的萃取 39
8. 土壤微生物DNA的聚合酵素鏈鎖反應 39
9. 變性梯度凝膠電泳 42
四、 結果與討論 44
(一) 免克寧的測定 44
(二) 免克寧降解產物的製備 52
(三) 免克寧在土壤中的降解與降解產物的產生 54
(四) 免克寧對土壤細菌菌相的影響 74
五、 結論 97
六、 參考文獻 98

圖目錄

圖2.1美國各地每年之免克寧使用估計量 8
圖2.2免克寧的分解主要途徑 9
圖2.3免克寧的化學水解之轉換 11
圖2.4以蹺蹺板原理表示內分泌系統運作的情形 16
圖2.5 天然荷爾蒙與受器的作用及荷爾蒙拮抗與促進的作用機制 17
圖2.6老鼠睪丸發育的過程與時間 18
圖2.7 抗雄性激素對雄性激素受器的作用機制 19
圖2.8環境荷爾蒙藉由雄性發生株所造成之表觀基因遺傳 22
圖3.1免克寧的檢量標準曲線 31
圖3.2免克寧溶於正己烷的檢量線 33
圖3.3桃園農改場土壤DNA的PCR反應溫度控制條件 41
圖3.4花蓮農改場土壤DNA的PCR反應溫度控制條件 41
圖4.1免克寧標準品使用Hustert與Moza之HPLC測定條件所得到的層析圖 45
圖4.2 3,5-二氯苯胺 (M3) 標準品使用Hustert與Moza之HPLC測定條件所得到的層析圖形 46
圖4.3 合成之M2樣品使用Hustert與Moza之HPLC測定條件所得到的層析圖形 47
圖4.4免克寧標準品使用Vallero與Peirce之HPLC測定條件所得到的層析圖形 48
圖4.5 3,5-二氯苯胺 (M3) 標準品使用Vallero與Peirce之HPLC測定條件所得到的層析圖形 49
圖4.6合成之M1樣品使用Vallero與Peirce之HPLC測定條件所得到的層析圖形 50
圖4.7合成之M2樣品使用Vallero與Peirce之HPLC測定條件所得到的層析圖形 51
圖4.8免克寧降解產物M2的MS圖譜 53
圖4.9濃度5 mg kg-1免克寧於桃園農改場土壤和花蓮農改場土壤的降解情形 55
圖4.10 使用Hustert與Moza之HPLC測定方法測定濃度50 mg kg-1免克寧於滅菌及未滅菌之桃園農改場土壤的降解情形 58
圖4.11 使用Hustert與Moza之HPLC測定方法濃度50 mg kg-1免克寧於滅菌及未滅菌之桃園農改場土壤降解對M2和M3的產生情形 59
圖4.12 使用Hustert與Moza之HPLC測定方法測定濃度50 mg kg-1免克寧於滅菌及未滅菌之花蓮農改場土壤的降解情形 61
圖4.13 使用Hustert與Moza之HPLC測定方法測定濃度50 mg kg-1免克寧於滅菌及未滅菌之花蓮農改場土壤降解對M2和M3的產生情形 62
圖4.14 使用Vallero 與 Peirce 的HPLC測定條件測定濃度50 mg kg-1免克寧於滅菌及未滅菌之桃園農改場土壤的降解與降解產物的生成情形 65
圖4.15 使用Vallero 與 Peirce之HPLC測定方法測定50 mg kg-1免克寧於滅菌及未滅菌之桃園農改場土壤中降解對M1、M2和M3的產生情形 66
圖4.16 使用Vallero 與 Peirce 的HPLC測定條件測定濃度50 mg kg-1免克寧於滅菌及未滅菌之花蓮農改場土壤的降解與降解產物的生成情形 70
圖4.17使用Vallero 與 Peirce之HPLC測定方法測定50 mg kg-1免克寧於滅菌及未滅菌之花蓮農改場土壤中降解對M1、M2和M3的產生情形 71
圖4.18 以PCR-DGGE分析未施用免克寧之桃園農改場土壤微生物的16S rDNA片段變化 76
圖4.19利用UPGMA法進行未施用免克寧之桃園農改場土壤的聚類分析細菌族群結構之結果 77
圖4.20以 PCR-DGGE分析未施用免克寧之花蓮農改場土壤微生物的16S rDNA片段變化 79
圖4.21利用UPGMA法進行未施用免克寧之花蓮農改場土壤的聚類分析細菌族群結構之結果 80
圖4.22 以PCR-DGGE分析施用5 mg kg-1免克寧之桃園農改場土壤微生物16S rDNA片段變化 83
圖4.23利用UPGMA法進行施用5 mg kg-1免克寧之桃園農改場土壤的聚類分析細菌族群結構之結果 84
圖4.24 以PCR-DGGE分析施用5 mg kg-1免克寧之花蓮農改場土壤微生物16S rDNA片段變化 86
圖4.25利用UPGMA法進行施用5 mg kg-1免克寧之花蓮農改場土壤的聚類分析細菌族群結構之結果 87
圖4.26 以PCR-DGGE分析施用50 mg kg-1免克寧之桃園農改場土壤微生物16S rDNA片段變化 89
圖4.27 利用UPGMA法進行50 mg kg-1免克寧處理之桃園農改場土壤的聚類分析細菌族群結構之結果 90
圖4.28 以PCR-DGGE分析施用50 mg kg-1免克寧之花蓮農改場土壤微生物16S rDNA片段變化 93
圖4.29 利用UPGMA法進行施用50 mg kg-1免克寧之花蓮農改場土壤的聚類分析細菌族群結構之結果 94

表目錄

表2.1免克寧的物化性質 4
表2.2免克寧在各種溶劑中的溶解度 5
表2.3免克寧的使用方法及使用範圍 6
表2.4含免克寧之藥劑與防治對象 7
表3.1試驗土樣的基本性質 30
表3.2測定免克寧及其降解產物之梯度動相變化之設定 32
表3.3 GC-ECD分析免克寧的管柱升溫條件 33
表3.4 GC-MS的管柱升溫條件 34
表3.5 0.1 M 磷酸鈉緩衝溶液的製備 37
表3.6本試驗所使用的引子 40
表3.7聚合酶連鎖反應之配方 40
表3.8不同濃度之Acrylamide/bis所能分離之核酸長度 43
表3.9 7%之Acrylamide/bis變性凝膠成分 43
表3.10 10% Ammonium Persulfate之成分 43
表3.11 DCode染劑之成分 43
表4.1免克寧的降解結果代入一級反應方程式所得到的反應常數、半衰期及決定係數 73
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