臺灣博碩士論文加值系統

因為人類科技的急速發展，工業、民生、農業等產業皆會排放許多不同種類的污染物至環境中。某些污染物質被歸類為內分泌干擾物質 (Endocrine disrupting chemicals, EDCs)，其中包括類(抗)雌激素、類(抗)雄激素以及類(抗)鹽皮質激素物質，這些化合物會與生物體中的雌激素受體、雄激素受體、或鹽皮質激素受體結合，並干擾內分泌系統的運作，對生物體造成危害。
本研究使用報導基因酵母菌生物試驗法分析臺灣南部五座污水處理廠各處理單元放流水中的類(抗)雌激素、類(抗)雄激素以及類(抗)鹽皮質激素活性，並且以液相層析串聯式質譜儀分析樣本中選定之EDCs濃度。生物試驗法之結果顯示各污水廠之水相及懸浮固體相樣本中皆無檢測出類雄激素活性及類鹽皮質激素活性，然而在大部分的水相樣本及懸浮固體相樣本中有頻繁檢測出類雌激素活性、抗雄激素活性與抗鹽皮質活性之顯現，而抗雌激素活性則是較容易於懸浮固體相中測得。
液相層析串聯式質譜儀之分析結果指出水相樣本中檢測出雙酚A (8.7~5187.8 ng/L)、單氯雙酚A (ND~71.7 ng/L)、二氯雙酚A (ND~58.2 ng/L)、三氯沙 (ND~1438.6)、壬基酚 (ND~122.3 ng/L)以及天然雌激素物質。比較生物試驗法及液相層析串聯式質譜儀分析之結果，可發現生物試驗法測得之活性較儀器分析所得較高，顯示樣本中可能含有某些非儀器分析目標物質之類雌激素、抗雄激素與抗鹽皮質激素物質，或是因為樣本中物質間的複合效應導致。

SUMMARY
Endocrine disrupting chemicals (EDCs) are synthetic or naturally chemicals which can effect endocrine system of organisms. Different kinds of EDCs would show various kinds of activities, such as estrogen receptor (ER) disrupting activities, androgen receptor (AR) disrupting activities, or mineralocorticoid receptor (MR) disrupting activities, etc. In this work, recombinant yeast bioassays were used to identify ER, AR, and MR disrupting activities in samples collected from wastewater treatment plants (WWTPs) in Taiwan. Also, chemical analysis was carried out using LC-MS/MS to analyze the concentrations of target EDCs. results showed that endocrine disrupting activities could be found in different treatment processes of WWTPs. Estrogenic, anti-androgenic and anti-mineralocorticoid activities were detected frequently in WWTP influent and effluent. LC-MS/MS results revealed that some EDCs, such as bisphenol A, nonylphenol, and triclosan were often detected in samples from WWTPs. More attention should be paid to the potential risk to aquatic organisms and further expansion of other bioassays for endocrine disrupting activity detection is recommended.

Key words: Endocrine disrupting chemicals, Yeast-based reporter gene assays, Liquid chromatography tandem mass spectrometry

INTRODUCTION
Endocrine disrupting chemicals (EDCs) are synthetic or natural chemicals which can affect the endocrine system of organisms, including the disruption of hormone synthesis, secretion, transport, binding, or action in the body. Different EDCs may show various kinds of endocrine disrupting activities, such as estrogen receptor (ER), androgen receptor (AR) or mineralocorticoid receptor (MR) disrupting activities. To detect EDCs in the environment, liquid chromatography tandem mass spectrometry (LC-MS/MS) are often used to quantify target compounds, and yeast bioassays are also reliable tools for assessing potential endocrine disrupting activities in environmental samples. In this work, recombinant yeast bioassays were used to identify ER, AR, and MR disrupting activities in samples collected from wastewater treatment plants (WWTPs) in Taiwan. Also, chemical analysis was carried out using LC-MS/MS to analyze the concentrations of target EDCs.

MATERIALS AND METHODS
Wastewater samples were collected from four municipal WWTPs and one hospital WWTP in Taiwan. Samples were separated into particulate phase (SS) and water phase (W) by filtration. Water samples were extracted by solid phase extraction (SPE) with Oasis HLB Plus cartridges. SS samples were extracted by Soxhlet extraction using hexane:acetone (1:1, v:v) for 24 hrs. Yeast-based reporter gene assays were used to detect ER, AR and MR disrupting activities in these samples. The yeast cells which contained estrogen response element/androgen response element/mineralocorticoid response element and reporter gene Lac-Z would produce β-galactosidase to react with o-nitrophenyl-β-galactoside and chlorophenol red-β-D-galactopyranoside if being coincubated with ER, AR, or MR disruptors. LC-MS/MS was selected to determine target EDCs in WWTP samples. The liquid chromatography system was Agilient 1260 Infinity (Agilent, USA) and the MS/MS system was Thermo TSQ Quantum Ultra (Thermo, USA).

RESULTS AND DISCUSSION
Bioassay results showed that endocrine disrupting activities could be found in different treatment processes of WWTPs. Estrogenic, anti-androgenic and anti-mineralocorticoid activities were detected frequently in WWTP influent and effluent. Higher estrogenic activities were found in water phase samples than particulate phase samples. The highest estrogenic activities were detected in the water phase samples of primary sedimentation tank effluent of HW (Dec 2015) WWTP (132.5 E2-EQ ng/L) and influent of H (Oct 2015) WWTP (119.6 E2-EQ ng/L). The highest anti-androgenic activity was found in the particulate phase sample of H (Oct 2015) influent (3299.6 FLU-EQ μg/L), and effluent of aeration basin (2411.8 FLU-EQ μg/L) was also very high. The results showed a little difference with estrogenic activities, which were seldom detected in particulate phase samples. The results indicated that anti-androgenic compounds might be more likely to exist in particulate phase. Influent of NZ (Mar 2016) showed the highest anti-mineralocorticoid activity (26390.0 SPL-EQ ng/L), and most of the WWTP influent samples also exhibited higher anti-mineralocorticoid activities than other process units. LC-MS/MS results revealed that some EDCs, such as bisphenol A (8.7~5187.8 ng/L), nonylphenol (ND~122.3 ng/L), and triclosan (ND~1438.6) were often detected in samples from WWTPs. Endocrine disrupting activities of chemicals determined by LC-MS/MS were also converted to equivalent factor (EQLC-MS/MS) by using bioassay to determine their endocrine disrupting activities relative to standards and were compared to the endocrine disrupting activities obtained by bioassay analysis (EQbioassay).There is no significant correlation between EQBioassay and EQLC-MS/MS.
CONCLUSION
Our results indicated that WWTPs in Taiwan may need further improvement to remove these harmful compounds, and more attention should be paid to the potential risk to aquatic organisms. Influent of WWTPs showed higher endocrine disrupting activities in most samples. Lower endocrine disrupting activities were detected in particulate phase samples except for anti-androgenic activities. Bioassay and instrumental analyses showed that WWTPs in Taiwan failed to completely remove EDCs in wastewater samples, and these EDCs may adversely impact the environment. Further expansion of other bioassays for endocrine disrupting activity detection is recommended.

摘要 II
致謝 VI
目錄 VII
表目錄 X
圖目錄 XI
第一章 前言 1
1-1研究動機 1
1-2研究目的 2
第二章 文獻回顧 3
2-1 內分泌干擾物質 3
2-1-1 類(抗)雌激素物質 3
2-1-2 類(抗)雄激素物質 7
2-1-3 類(抗)鹽皮質激素物質 9
2-2 生物試驗法 11
2-2-1 活體內生物試驗 (in-vivo) 11
2-2-2 活體外生物試驗 (in-vitro) 12
2-3儀器分析法 14
2-4 檢測污水處理廠簡介 16
2-4-1臺南H醫院廢水處理廠 16
2-4-2臺南GT水資源回收中心 16
2-4-3臺南HW污水處理廠 16
2-4-4高雄NZ污水處理廠 17
2-4-5高雄FS污水處理廠 17
第三章 實驗方法與步驟 18
3-1 實驗流程 18
3-2 樣本採集 19
3-3 實驗材料與設備 21
3-3-1藥品與試劑 21
3-3-2基因重組酵母菌試驗法 23
3-3-3實驗設備 25
3-4前處理步驟 26
3-4-1水相樣本 26
3-4-2懸浮固體樣本 26
3-4-3樣本稀釋序列 26
3-5基因重組酵母菌生物試驗法 27
3-5-1類(抗)雌激素生物試驗 27
3-5-2類(抗)雄激素生物試驗 30
3-5-3類(抗)鹽皮質激素生物試驗 32
3-5-4生物試驗法當量濃度換算與方法偵測極限 33
3-6液相層析串聯式質譜儀 34
3-6-1型號及操作條件 34
3-6-2回收率及偵測極限 34
第四章 結果與討論 40
4-1 類(抗)雌激素活性 40
4-1-1 H醫院廢水處理廠 40
4-1-2 GT 水資源回收中心 41
4-1-3 HW 污水處理廠 43
4-1-4 NZ 污水處理廠 44
4-1-5 FS 污水處理廠 46
4-2類(抗)雄激素活性 48
4-2-1 H醫院廢水處理廠 48
4-2-2 GT水資源回收中心 49
4-2-3 HW污水處理廠 49
4-2-4 NZ 污水處理廠 50
4-2-5 FS污水處理廠 51
4-2-6 水相樣本與懸浮固體相樣本中抗雄激素活性之分布 52
4-3 類(抗)鹽皮質激素活性 54
4-3-1 H醫院廢水處理廠 54
4-3-2 GT 水資源回收中心 55
4-3-3 HW 污水處理廠 55
4-3-4 NZ 污水處理廠 56
4-3-5 FS 污水處理廠 57
4-3-6水相樣本與懸浮固體相樣本中抗鹽皮質激素活性分布 58
4-4 生物試驗結果討論 60
4-4-1各污水廠水相樣本中類(抗)激素活性變化 60
4-4-2各污水廠懸浮固體相樣本中類(抗)激素活性變化 63
4-4-3各國生物試驗類(抗)激素活性比較與討論 65
4-5 LC-MS/MS儀器分析結果討論 67
4-5-1污水處理廠中BPA濃度變化 67
4-5-2污水處理廠中氯化BPA濃度變化 68
4-5-3污水處理廠中TCS濃度變化 69
4-5-4污水處理廠中NP濃度變化 70
4-5-5污水處理廠中天然雌激素E1、E2、E3濃度變化 71
4-6 生物試驗及儀器分析結果比較 75
4-6-1水相樣本中生物試驗及儀器分析相關性 75
4-6-2檢測物質活性貢獻度 78
第五章 結論與建議 80
參考文獻 82

1.Allner, B., Wegener, G., Knacker, T., ＆ Stahlschmidt-Allner, P. (1999). Electrophoretic determination of estrogen-induced protein in fish exposed to synthetic and naturally occurring chemicals. Science of the Total Environment, 233(1–3), 21-31.
2.Ankley, G. T., Jensen, K. M., Kahl, M. D., Korte, J. J., ＆ Makynen, E. A. (2001). Description and evaluation of a short-term reproduction test with the fathead minnow (Pimephales promelas). Environmental Toxicology and Chemistry, 20(6), 1276-1290.
3.Bettin, C., Oehlmann, J., ＆ Stroben, E. (1996). TBT-induced imposex in marine neogastropods is mediated by an increasing androgen level. Helgoland Marine Research, 50(3), 299-317.
4.Bicchi, C., Schilirò, T., Pignata, C., Fea, E., Cordero, C., Canale, F., ＆ Gilli, G. (2009). Analysis of environmental endocrine disrupting chemicals using the E-screen method and stir bar sorptive extraction in wastewater treatment plant effluents. Science of The Total Environment, 407(6), 1842-1851.
5.Birnbaum, L. S., ＆ Fenton, S. E. (2003). Cancer and developmental exposure to endocrine disruptors. Environmental Health Perspective, 111(4), 389-394.
6.Boyanov, M. A., Boneva, Z., ＆ Christov, V. G. (2003). Testosterone supplementation in men with type 2 diabetes, visceral obesity and partial androgen deficiency. The Aging Male, 6(1), 1-7. doi: 10.1080/tam.6.1.1.7
7.Chang, H., Wan, Y., Wu, S., Fan, Z., ＆ Hu, J. (2011). Occurrence of androgens and progestogens in wastewater treatment plants and receiving river waters: Comparison to estrogens. Water Research, 45(2), 732-740.
8.Delyani, J. A. (2000). Mineralocorticoid receptor antagonists: the evolution of utility and pharmacology. Kidney International, 57(4), 1408-1411.
9.Fang, Y.-X., Ying, G.-G., Zhao, J.-L., Chen, F., Liu, S., Zhang, L.-J., ＆ Yang, B. (2012). Assessment of hormonal activities and genotoxicity of industrial effluents using in vitro bioassays combined with chemical analysis. Environmental Toxicology and Chemistry, 31(6), 1273-1282.
10.Fernandez, M. F., Arrebola, J. P., Taoufiki, J., Navalón, A., Ballesteros, O., Pulgar, R., Olea, N. (2007). Bisphenol-A and chlorinated derivatives in adipose tissue of women. Reproductive Toxicology, 24(2), 259-264.
11.Fukazawa, H., Watanabe, M., Shiraishi, F., Shiraishi, H., Shiozawa, T., Matsushita, H., ＆ Terao, Y. (2002). Formation of Chlorinated Derivatives of Bisphenol A in Waste Paper Recycling Plants and Their Estrogenic Activities. Journal of Health Science, 48(3), 242-249.
12.Fuller, P. J., Yao, Y., Yang, J., ＆ Young, M. J. (2012). Mechanisms of ligand specificity of the mineralocorticoid receptor. Journal of Endocrinology, 213(1), 15-24.
13.Fuller, P. J., ＆ Young, M. J. (2005). Mechanisms of mineralocorticoid action. Hypertension, 46(6), 1227-1235.
14.González, S., Petrovic, M., ＆ Barceló, D. (2004). Simultaneous extraction and fate of linear alkylbenzene sulfonates, coconut diethanol amides, nonylphenol ethoxylates and their degradation products in wastewater treatment plants, receiving coastal waters and sediments in the Catalonian area (NE Spain). Journal of Chromatography A, 1052(1–2), 111-120.
15.Gottardis, M. M., ＆ Jordan, V. C. (1988). Development of tamoxifen-stimulated growth of MCF-7 tumors in athymic mice after long-term antiestrogen administration. Cancer Research, 48(18), 5183-5187.
16.Hayes, T. B., Collins, A., Lee, M., Mendoza, M., Noriega, N., Stuart, A. A., ＆ Vonk, A. (2002). Hermaphroditic, demasculinized frogs after exposure to the herbicide atrazine at low ecologically relevant doses. Proceedings of the National Academy of Sciences of the United States of America, 99(8), 5476-5480.
17.Hornbeck, P. V. (2001). Enzyme-Linked Immunosorbent Assays Current Protocols in Immunology: John Wiley ＆ Sons, Inc.
18.Hosnedl, T., Hajšlová, J., Kocourek, V., Tomaniová, M., ＆ Volka, K. (2003). 1-Hydroxypyrene as a Biomarker for Fish Exposure to Polycyclic Aromatic Hydrocarbons. Bulletin of Environmental Contamination and Toxicology, 71(3), 0465-0472.
19.Huang, G.-Y., Ying, G.-G., Liang, Y.-Q., Zhao, J.-L., Yang, B., Liu, S., ＆ Liu, Y.-S. (2013). Hormonal effects of tetrabromobisphenol A using a combination of in vitro and in vivo assays. Comparative Biochemistry and Physiology Part C: Toxicology ＆ Pharmacology, 157(4), 344-351.
20.Hunt, P. A., Lawson, C., Gieske, M., Murdoch, B., Smith, H., Marre, A., . . . VandeVoort, C. A. (2012). Bisphenol A alters early oogenesis and follicle formation in the fetal ovary of the rhesus monkey. Proceedings of the National Academy of Sciences, 109(43), 17525-17530.
21.Ito-Harashima, S., Shiizaki, K., Kawanishi, M., Kakiuchi, K., Onishi, K., Yamaji, R., ＆ Yagi, T. (2015). Construction of sensitive reporter assay yeasts for comprehensive detection of ligand activities of human corticosteroid receptors through inactivation of CWP and PDR genes. Journal of Pharmacological and Toxicological Methods, 74, 41-52.
22.Jugan, M. L., Oziol, L., Bimbot, M., Huteau, V., Tamisier-Karolak, S., Blondeau, J. P., ＆ Lévi, Y. (2009). In vitro assessment of thyroid and estrogenic endocrine disruptors in wastewater treatment plants, rivers and drinking water supplies in the greater Paris area (France). Science of The Total Environment, 407(11), 3579-3587.
23.Kanda, R., ＆ Churchley, J. (2008). Removal of encocrine disrupting compounds durring conventional wastewater treatment. Environmental Technology, 29(3), 315-323.
24.Kavlock, R. J., Daston, G. P., DeRosa, C., Fenner-Crisp, P., Gray, L. E., Kaattari, S., Tilson, H. A. (1996). Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop. Environmental Health Perspect, 104 Suppl 4, 715-740.
25.Kelce, W. R., Monosson, E., Gamcsik, M. P., Laws, S. C., ＆ Gray, L. E. (1994). Environmental Hormone Disruptors: Evidence That Vinclozolin Developmental Toxicity Is Mediated by Antiandrogenic Metabolites. Toxicology and Applied Pharmacology, 126(2), 276-285.
26.Laureti, S., Casucci, G., Santeusanio, F., Angeletti, G., Aubourg, P., ＆ Brunetti, P. (1996). X-linked adrenoleukodystrophy is a frequent cause of idiopathic Addison's disease in young adult male patients. The Journal of Clinical Endocrinology ＆ Metabolism, 81(2), 470-474.
27.Lim, Y. C., Li, L., Desta, Z., Zhao, Q., Rae, J. M., Flockhart, D. A., ＆ Skaar, T. C. (2006). Endoxifen, a secondary metabolite of tamoxifen, and 4-OH-tamoxifen induce similar changes in global gene expression patterns in MCF-7 breast cancer cells. Journal of Pharmacology and Experimental Therapeutics, 318(2), 503-512.
28.Liscio, C., Abdul-Sada, A., Al-Salhi, R., Ramsey, M. H., ＆ Hill, E. M. (2014). Methodology for profiling anti-androgen mixtures in river water using multiple passive samplers and bioassay-directed analyses. Water Research, 57, 258-269.
29.Luccio-Camelo, D. C., ＆ Prins, G. S. (2011). Disruption of androgen receptor signaling in males by environmental chemicals. The Journal of Steroid Biochemistry and Molecular Biology, 127(1–2), 74-82.
30.Maletz, S., Floehr, T., Beier, S., Klümper, C., Brouwer, A., Behnisch, P., Hollert, H. (2013). In vitro characterization of the effectiveness of enhanced sewage treatment processes to eliminate endocrine activity of hospital effluents. Water Research, 47(4), 1545-1557.
31.Murk, A. J., Legler, J., Denison, M. S., Giesy, J. P., van de Guchte, C., ＆ Brouwer, A. (1996). Chemical-Activated Luciferase Gene Expression (CALUX): A Novelin VitroBioassay for Ah Receptor Active Compounds in Sediments and Pore Water. Fundamental and Applied Toxicology, 33(1), 149-160.
32.Naylor, C. G., Mieure, J. P., Adams, W. J., Weeks, J. A., Castaldi, F. J., Ogle, L. D., ＆ Romano, R. R. (1992). Alkylphenol ethoxylates in the environment. Journal of the American Oil Chemists Society, 69(7), 695-703.
33.Nyholm, J. R., Norman, A., Norrgren, L., Haglund, P., ＆ Andersson, P. L. (2008). Maternal transfer of brominated flame retardants in zebrafish (Danio rerio). Chemosphere, 73(2), 203-208.
34.Pawlowski, S., Ternes, T. A., Bonerz, M., Rastall, A. C., Erdinger, L., ＆ Braunbeck, T. (2004). Estrogenicity of solid phase-extracted water samples from two municipal sewage treatment plant effluents and river Rhine water using the yeast estrogen screen. Toxicology in Vitro, 18(1), 129-138.
35.Rao, K., Li, N., Ma, M., ＆ Wang, Z. (2014). In vitro agonistic and antagonistic endocrine disrupting effects of organic extracts from waste water of different treatment processes. Frontiers of Environmental Science ＆ Engineering, 8(1), 69-78.
36.Routledge, E. J., ＆ Sumpter, J. P. (1996). Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environmental Toxicology and Chemistry, 15(3), 241-248.
37.Sebire, M., Allen, Y., Bersuder, P., ＆ Katsiadaki, I. (2008). The model anti-androgen flutamide suppresses the expression of typical male stickleback reproductive behaviour. Aquatic Toxicology, 90(1), 37-47.
38.Shappell, N. W., Billey, L. O., Forbes, D., Matheny, T. A., Poach, M. E., Reddy, G. B., ＆ Hunt, P. G. (2007). Estrogenic Activity and Steroid Hormones in Swine Wastewater through a Lagoon Constructed-Wetland System. Environmental Science ＆ Technology, 41(2), 444-450.
39.Shi, W., Wang, X., Hu, W., Sun, H., Shen, O., Liu, H., Yu, H. (2009). Endocrine-disrupting equivalents in industrial effluents discharged into Yangtze River. Ecotoxicology, 18(6), 685-692.
40.Singer, H., Müller, S., Tixier, C., ＆ Pillonel, L. (2002). Triclosan:  Occurrence and Fate of a Widely Used Biocide in the Aquatic Environment:  Field Measurements in Wastewater Treatment Plants, Surface Waters, and Lake Sediments. Environmental Science ＆ Technology, 36(23), 4998-5004.
41.Solomon, K. R., Baker, D. B., Richards, R. P., Dixon, K. R., Klaine, S. J., La Point, T. W., Williams, W. M. (1996). Ecological risk assessment of atrazine in North American surface waters. Environmental Toxicology and Chemistry, 15(1), 31-76.
42.Soto, A. M., Sonnenschein, C., Chung, K. L., Fernandez, M. F., Olea, N., ＆ Serrano, F. O. (1995). The E-SCREEN assay as a tool to identify estrogens: an update on estrogenic environmental pollutants. Environmental Health Perspect, 103(Suppl 7), 113-122.
43.Su, S.-J., Yeh, T.-M., Chuang, W.-J., Ho, C.-L., Chang, K.-L., Cheng, H.-L., Chow, N.-H. (2005). The novel targets for anti-angiogenesis of genistein on human cancer cells. Biochemical Pharmacology, 69(2), 307-318.
44.Ternes, T. A., Kreckel, P., ＆ Mueller, J. (1999). Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge. Science of the Total Environment, 225(1-2), 91-99.
45.Thomas, P. M., ＆ Foster, G. D. (2005). Tracking acidic pharmaceuticals, caffeine, and triclosan through the wastewater treatment process. Environmental Toxicology and Chemistry, 24(1), 25-30.
46.Tollefsen, K.-E., Harman, C., Smith, A., ＆ Thomas, K. V. (2007). Estrogen receptor (ER) agonists and androgen receptor (AR) antagonists in effluents from Norwegian North Sea oil production platforms. Marine Pollution Bulletin, 54(3), 277-283.
47.Vethaak, A. D., Lahr, J., Schrap, S. M., Belfroid, A. C., Rijs, G. B. J., Gerritsen, A., de Voogt, P. (2005). An integrated assessment of estrogenic contamination and biological effects in the aquatic environment of The Netherlands. Chemosphere, 59(4), 511-524.
48.Ying, G.-G., Kookana, R. S., ＆ Ru, Y.-J. (2002). Occurrence and fate of hormone steroids in the environment. Environment International, 28(6), 545-551.
49.李宗益. (2015). 利用生物試驗及液相層析串聯式質譜儀檢測臺灣河川中之類(抗)糖皮質激素及類(抗)鹽皮質激素.
50.陳廣育. (2014). 臺灣河川中內分泌干擾物質之流布及其複合效應之評估.
51.臺灣質譜協會. (2015). 質譜分析技術:原理與應用. 新北市: 全華圖書.
52. 於望聖＆黃文彥.(2011). 下水道誌. 新北市:營建署.
53. 葉奕伯. (2014) 以生物試驗法及液相層析串聯式質譜儀分析污水處理廠中內分泌干擾物質及其衍生物之變化