免克寧代謝產物降解及對土壤菌相與生物毒性影響

Abstract

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.