雌激性化合物在水環境中之流布－方法開發與應用

Abstract

本研究針對河水、底泥與生物體中之辛基酚（4-tert-octylphenol）、壬基酚4-nonylphenol）、壬基酚單乙氧烯乙酸（nonylphenoxy acetic acid）、壬基酚單乙氧烯醇（nonylphenol monoethoxylate）、壬基酚雙乙氧烯醇（nonylphenol diethoxylate）、雌素酮（estrone）、動情激素（17β-estradiol）、雌素醇（estriol）、乙炔動情激素（17α-ethinyl estradiol）與雙酚A（bisphenol A）等雌激性化合物，發展並驗證一利用極致液相層析�串聯式質譜儀（ultra-high performance liquid chromatography/tandem mass spectrometry, UHPLC/MS/MS）搭配同位素稀釋技術定量之分析方法。河水樣本以圓盤式自動固相萃取設備進行前處理。底泥與生物樣本以基質固相散布法萃取，以C8矽膠為吸附劑，甲醇與丙酮沖提液通過氧化鋁萃取匣進行樣本淨化。本研究亦比較電噴灑游離法（electrospray ionization, ESI）與大氣壓光游離法（atmospheric pressure photoionization, APPI）所產生之質譜訊號強度。層析以極致液相層析C18管柱分離待測物，壬基酚乙氧烯醇之水相為10-mM醋酸銨水溶液，其餘待測物之水相為10-mM甲基嗎啡碄水溶液，有機相為甲醇。利用限制性接觸物質（restricted access material）所進行之二維液相層析之線上樣本淨化效率亦與酸性氧化鋁作一比較。 相較大氣壓光游離法，電噴灑游離法可適用於所有待測物。雖然對兩種壬基酚乙氧烯醇與壬基酚單乙氧烯乙酸而言，大氣壓光游離法未能提供良好訊號，然而其在類固醇類雌激素與酚類產生的訊號為電噴灑游離法之1.0-2.4倍。極致液相層析將層析時間縮短至10分鐘內（包括管柱再平衡時間）。圓盤式自動固相萃取與基質固相散布法大幅改進了樣本前處理效率；地表水的前處理回收率除烷基酚類化合物低於30%，其餘介於60%至91%，底泥、魚與蛤蠣的前處理回收率分別為51-101%、36-109%與30-111%。生物樣本須經氧化鋁淨化，較二維液相層析能有效減低基質效應的影響。地表水中待測物偵測極限為0.81 ng/L至89.9 ng/L，底泥與生物體則為數十pg/g（濕重）至低ng/g（濕重）。此方法具有良好準確度與再現性，三種測試添加濃度之定量偏差與相對標準偏差均小於20%。 此分析方法並應用於淡水河河水、底泥與吳郭魚組織中雌激性化合物調查。每1.5個月採集樣本，連續執行9個月，共採樣6次，取得66個河水、66個底泥及114隻魚體樣本，以評估此類化合物在底泥和魚體內之分布與累積情形。 所有河水樣本中均測得烷基酚類化合物與雙酚A，以雙酚A與壬基酚濃度最高，依序為508 ± 634 ng/L與491 ± 570 ng/L （n = 66）。淡水河中的雙酚A平均濃度（921 ± 635 ng/L，n = 18）為基隆河（392 ± 613 ng/L，n = 48）的兩倍以上。河水中待測物濃度的空間分布情形可能與下游的污染及未經處理的生活污水排放有關。底泥中以壬基酚濃度最高（770 ± 602 ng/g（濕重），n = 66），其中尤以在污水處理廠下游最近的採樣點測得之濃度最高（1,701 ± 1,374 ng/g（濕重），n = 6），顯示壬基酚可長期累積於底泥。 壬基酚是魚體組織中的主要污染物，肌肉中濃度為199 ± 133 ng/g（濕重）（n = 114）；其生物累積係數（bioaccumulation factor）亦為所有待測物中最高者（肌肉生物累積係數967 ± 903，n = 114）。雙酚A雖然在水體中易降解且在某些魚種中清除效率高，在魚體肌肉中測得最高濃度仍達334 ng/g（濕重），且其生物累積係數值得關注（肌肉生物累積係數246 ± 801，n = 114）。肌肉中壬基酚乙氧烯醇及外源性動情激素當量濃度與該採樣點河水中之濃度具顯著正相關性。魚體器官中的雌激性化合物濃度高於肌肉中的濃度，且壬基酚、壬基酚乙氧烯醇與雙酚A的生物累積係數呈現相同趨勢，亦即生殖腺＞肝臟＞肌肉，其中壬基酚在肝臟與生殖腺之生物累積係數大於5,000（依序為9,576 ± 15,048與 27,287 ± 49,587），可能具有生物蓄積性。肝臟形態指標值低下情形（0.16 ± 0.10，n = 114）與河水、肌肉及肝臟中的雌激性化合物濃度顯著相關，顯示雌激性化合物經由河水與攝食進入吳郭魚體內，於標的器官濃縮累積並影響內分泌系統。器官中累積的高濃度此類物質亦可能導致族群性別比偏差（雌雄比為1.92）。這些化合物與其它新興污染物交互作用下對環境生態之進一步危害，則有待未來研究深入調查。

This study developed and validated a method for measuring ten feminizing chemicals 4-tert-octylphenol, 4-nonylphenol (NP), nonylphenoxy acetic acid (NP1EC), nonylphenol monoethoxylate (NP1EO), nonylphenol diethoxylate (NP2EO), estrone, 17β-estradiol, estriol, 17α-ethinyl estradiol and bisphenol A (BPA) in river water, sediment, and tissues using ultra-high performance liquid chromatography/tandem mass spectrometry (UHPLC/MS/MS) and isotope-dilution techniques. Analytes in water samples were extracted using disk-type automated solid-phase extraction (SPE). Solid samples of sediment, fish, and clams were treated with matrix solid-phase dispersion (MSPD) using C8 adsorbent; eluents of methanol and acetone were directly passed through following alumina cartridges for cleanup. The signal intensities of analytes on electrospray ionization (ESI) were compared with that of atmospheric pressure photoionization (APPI). The analytes were separated on a UHPLC C18 column with aqueous 10-mM ammonium acetate for NPEOs and aqueous 10-mM N-methylmorpholine for the other compounds. The organic mobile phase was methanol. Two-dimensional liquid chromatography (2-D LC) with a C4 restricted access material column was evaluated for on-line cleanup. ESI provided satisfactory response for all of the analytes. Although APPI provided better signal intensities for the steroid estrogens (1.0-2.4 times higher) and the phenols (3.2-4.4 times higher) than ESI, it did not offer good responses on NP1EO, NP2EO and NP1EC. UHPLC shortened chromatographic time to less than 10 min (including re-equilibration). Disk-type automated SPE and MSPD dramatically increased the throughput of sample preparation. The extraction efficiency of most analytes on surface water ranged from 60% to 91%, but that of alkylphenolic compounds were lower than 30%. The extraction efficiency of MSPD on sediment, fish, and clams was 51-101%, 36-109%, and 30-111%, respectively. Acidic alumina cleanup was essential for the analysis of the tissue samples, and reduced matrix effects more significantly than 2-D LC on-line cleanup. The limits of detection (LODs) in water ranged from 0.81 ng/L to 89.9 ng/L. The LODs in sediment and tissues ranged from tens of pg/g wet weight (w.w.) to only a few ng/g w.w. This method proved to be accurate and reproducible, as both quantitative biases and relative deviations remained smaller than 20% at three spiked levels. The method was applied to investigate the distribution of feminizing chemicals in river water, sediment and fish (Oreochromis niloticus) in the Dan-Shui River, Taipei, Taiwan. Sampling was conducted every one and a half months for nine months, in the total of six times. Sixty six water samples, 66 sediment ones and 114 fish were collected. The concentrations in fish tissues were measured to estimate the distribution and the bioaccumulation of these chemicals within fish. Alkylphenolic compounds and BPA were detected in all of the water samples. BPA and NP were the most abundant analytes in river water (508 ± 634 ng/L and 491 ± 570 ng/L, respectively, n = 66). The average concentrations of BPA in the main stream of the Dan-Shui River (921 ± 635 ng/L, n = 18) was more than twice higher than that in the tributary (353 ± 567 ng/L, n = 48). The spatial variation of concentrations in water may be associated with the downstream pollution and the emission of untreated municipal wastewater. NP was the most abundant compound in sediment (770 ± 602 ng/g w.w., n = 66) and the highest concentration was detected at the closest downstream site from a wastewater treatment plant (1,701 ± 1,374 ng/g w.w., n = 6), indicating long-term accumulation of NP in sediment. NP was also the predominant compound in fish tissues (199 ± 133 ng/g w.w. in muscle, n = 114) and had the highest bioaccumulation factor (BAF, 967 ± 903 in muscle, n = 114) among the analytes. Although BPA is easy to degrade in water and is eliminated rapidly in some fish species, up to 334 ng/g w.w. was detected in fish muscle and considerable BAFs (246 ± 801 in muscle, n = 114) were found. Concentrations of feminizing chemicals in fish organs were found to be higher than that in muscle and the BAFs of NP, nonylphenol ethoxylates and BPA showed similar trends in organs: gonad > liver > muscle. The high BAFs of NP in liver and in gonad (9,576 ± 15,048 and 27,287 ± 49,587, respectively, n = 114) indicated possible bioaccumulation. Low hepatosomatic indices (0.16 ± 0.10, n = 114) were significantly correlated with the concentrations of feminizing chemicals in river water, in fish muscle and in liver, indicating that feminizing compounds entered tilapia via river water and ingestion, concentrated and accumulated in the target organ, and affected the endocrine system. The accumulation of these compounds in the organs may attribute to the skewed sex ratio of fish (female:male = 1.92). Future studies are desired to investigate the impacts of the interaction between these analytes and other emerging contaminants on biologial environments.