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.

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