養豬廢水中四環黴素與磺胺類藥物之測定

Abstract

畜牧業為了能夠快速且大量提供產品以因應市場需求而逐漸發展成為集中型飼育場，其特點為單一種類地飼養牲畜且圈地狹小。狹窄而集中的飼養場地使牲畜較容易患病進而引發大規模的感染，因此使用抗生素作為疾病的治療與預防在畜牧業中相當重要。此外，低於治療劑量的抗生素使用也被發現具有促進牲畜生長的效果，因此抗生素也曾被添加在飼料中投藥給牲畜作為生長促進劑。四環黴素與磺胺類藥物均為曾被廣泛使用的畜牧業用藥，且可能隨著畜牧業廢水排放至環境水體中，造成環境微生物中抗藥性基因的篩選。 本研究為偵測畜牧業廢水中進流水與放流水，以及河水中的四環黴素與磺胺類藥物之方法開發。分析物為三種四環黴素：氯四環黴素(chlortetracycline)、羥四環黴素(oxytetracycline)與四環黴素(tetracycline)以及八種磺胺類藥物：磺胺嘧啶(sulfadiazine)、磺胺塞唑(sulfathiazole)、磺胺甲嘧啶(sulfamerazine)、磺胺二甲嘧啶(sulfamethazine)、磺胺間甲氧嘧啶(sulfamonomethoxine)、磺胺甲噁唑(sulfamethoxazole)、磺胺二甲氧基嘧啶(sulfadimethoxine)與磺胺喹噁啉(sulfaquinoxaline)。 採得之水樣以甲酸調整pH值至pH 3，並保存於4℃中。本方法取10毫升進流水、20毫升放流水與100毫升河水進行樣本前處理。各水樣添加同位素標定的四環黴素與磺胺類藥物作為內對照標準品，並以2 mg/mL比例添加EDTA (ethylenediaminetetraacetic acid)以防止四環黴素與二價金屬離子螯合。水樣於3,000 rpm (1411 xg)離心20分鐘以去除泥沙。水樣上清液通過HLB固相萃取管柱(事先以2毫升甲醇與2毫升Milli-Q純水通過萃取管柱)進行萃取，再以1.6毫升甲醇進行沖提。沖提液以0.22 μm PVDF濾膜過濾後減壓離心濃縮至近乾，再以200微升20%甲醇與水溶液回溶，進樣15微升以極致液相層析/串聯式質譜儀使用電灑游離正離子模式(ESI+)與選擇性反應偵測模式(SRM)分析待測物。 基質效應評估中可見進流水中各待測物訊號僅剩10%－30%，放流水中則為40%－80%，河水中則為85%－100%，顯示進流水中的待測物受到強烈的訊號抑制。本研究將進流水樣本萃取液進一步稀釋2.5倍與5倍，結果發現稀釋確實可降低基質效應的訊號抑制，但待測物的訊號也因稀釋而降低，反而不利於偵測。萃取效率評估中可見進流水中各待測物訊號僅剩40%－70%，放流水中則為100%－120%，河水中則為50%－85%，顯示進流水中待測物可能吸附於水樣中的懸浮微粒使得萃取效率較低。本研究透過進一步實驗，分為同位素內標準品前添加於樣本，與同位素內標準品添加於樣本離心後上清液兩組，發現進流水的待測物訊號在前添加樣本和上清液添加樣本的差異明顯大於放流水與河水，顯示進流水中所含有的較多的懸浮微粒確實會因吸附作用而影響到待測物的萃取效率。進流水中各待測物的回收率為70%－130%，放流水中為65%－140%，河水則為70%－120%。由於三種水樣各待測物的回收率大多介於70%－130%，顯示使用同位素內標的同位素稀釋技術可準確地定量複雜樣本中的待測物。無論日內差與日間差之分析，測定濃度與添加濃度相比的定量誤差在多數待測物中均小於30%，而重複樣本的相對標準偏差(RSD%)在多數待測物中亦小於30%。 四環黴素與磺胺類藥物在進流水中的定量極限分別為2.3 μg/L－2.9 μg/L與1.0 μg/L－2.7 μg/L，在放流水中的定量極限分別為266 ng/L－367 ng/L與93.5 ng/L－351 ng/L，在河水中的定量極限則分別為20.4 ng/L－53.8 ng/L與14.7 ng/L－36.9 ng/L。 本研究分析實際養豬場所採集之進流水與放流水，三種四環黴素在各養豬場進流水中均可被偵測到，濃度範圍大多約為每公升水樣中有數十至數百微克四環黴素。經過廢水處理後，三種四環黴素的濃度降至每公升水樣中有數微克。大多數的磺胺類藥物在進流水與放流水中低於偵測極限，除了sulfamethazine在一座養豬場的進流水和放流水樣本中較高(約600 μg/L)。在鄰近養豬場的河水樣本中，大多數的待測物均低於偵測極限，除了chlortetracycline (160 ng/L－1,070 ng/L)與sulfamethazine (36.8 ng/L)。本研究也分析淡水河流域中各採樣點的河水，發現絕大多數待測物均低於偵測極限，除了sulfamethoxazole在第11號採樣點中測得26.4 ng/L。 本研究開發了在畜牧業廢水與河水中偵測四環黴素與磺胺類藥物的方法，未來可應用於更多實際環境樣本的分析，以確認廢水中待測物的濃度。本研究除了環境監控之外，也可為環境微生物中抗藥性基因篩選的研究提供環境水體中藥物濃度偵測的方法。

The animal husbandry industry demands a large amount of veterinary antibiotics in concentrated animal feeding operations (CAFOs) on treating and preventing from diseases as well as on promoting growth. To evaluate the potential impact of antibiotics releasing into the environment, a good analytical method is essential for measuring veterinary antibiotics in environmental matrixes. Tetracyclines and sulphonamides are broad-spectrum antibacterial agents, and were once heavily used in animal husbandry and as feed additives. This study developed a method to determine three tetracyclines, including chlortetracycline, oxytetracycline, as well as tetracycline, and eight sulphonamides, including sulfadiazine, sulfathiazole, sulfamerazine, sulfamethazine, sulfamonomethoxine, sulfamethoxazole, sulfadimethoxine in swine wastewater and river water using deuterium-labeled tetracycline (tetracycline-deuterium 6) and sulphonamides (sulfamerazine-deuterium 4, sulfamethoxazole-deuterium 4, sulfadimethoxine-deuterium 4) as internal standards. All water samples were adjusted to pH 3 by formic acid, and then were stored at 4℃. Ten millimeters of influent, 20 mL of effluent, and 100-mL river water were used and isotope-labeled standards were added at the beginning of sample preparation. EDTA was also added (2 mg/mL) to prevent the tetracyclines from chelating with divalent metal ions. The samples were centrifuged under 3,000 rpm (1,411 xg) for 20 minutes, then the supernatants underwent solid-phase extraction (SPE) with hydrophilic-hydrophobic balance (HLB) cartridges, which were conditioned with 2 mL methanol and 2 mL Milli-Q water before extractions. Analytes were eluted with 1.6 mL of methanol, filtered with 0.22-μm PVDF filters, and were concentrated to barely dry by a SpeedVac concentrator. The residues were reconstituted with 200 μL of 20% methanol/80% water, and 15 μL were injected onto the ultra-high performance liquid chromatography/tandem mass spectrometry at positive electrospray ionization and with selected reaction monitoring. Matrix effect factors of analytes in influent, effluent and river water were 10%－30%, 40%－80%, and 85%－100%, respectively, indicating serious ion suppression in influent. This study diluted post-spiked influent samples with 2.5 and 5 times, respectively, and found that the dilutions decreased not only matrix suppression effect but also the signal intensities of analytes; thus, the eluent was not further diluted before the instrumental analysis. The extraction efficiencies of analytes in influent, effluent and river water were 40%－70%, 100%－120%, and 50%－85%, respectively. The signal intensity of internal standards in pre-spiked samples of influent, effluent, and river water were 45%－65%, 83%－90%, and 88%－120%, respectively, comparing with those in spiked supernatant samples, these indicated that analytes may adsorb to the particles in influent and may be removed at the step of centrifugation. The recoveries of analytes in influent, effluent and river water were 70%－130%, 65%－140%, and 70%－120%, respectively; recoveries of most analytes were 70%－130%, demonstrating that isotope-labeled internal standards could compensate the influence on quantitation from the matrix effect and analytes adsorption to the particles. Most inter-day and intra-day quantitative biases (accuracy) were below 30%, and inter-day and intra-day RSD% precision were also below 30% on most analytes. The limits of quantitation of tetracyclines and sulphonamides were 2.3 μg/L－2.9 μg/L and 1.0 μg/L－2.7 μg/L in influent, 266 ng/L－367 ng/L and 93.5 ng/L－351 ng/L in effluent, and 20.4 ng/L－53.8 ng/L and 14.7 ng/L－36.9 ng/L in river water, respectively. The concentrations of tetracyclines in influent and effluent of piggery farms were detected at tens to hundreds of micrograms per liter in influent and were below 10 μg/L in effluent. Most sulphonamides in influent and effluent of piggery farms were below detection limits, except for sulfamethazine from one site (around 600 μg/L in the influent and effluent). The concentrations of most tetracyclines and sulphonamides in river water nearby a piggery farm were below detection limits, except for chlortetracycline (160 ng/L－1,070 ng/L) and sulfamethazine (36.8 ng/L) in one sample. Most analytes were below detection limits in Dan-Shui River, except for sulfamethoxazole (26.4 ng/L) in one sample. This study provides an accurate and precise method for determining tetracyclines and sulphonamides in swine wastewater and river water. The method is useful for researches in environment fate and monitoring of tetracyclines and sulphonamides. More analyses on field samples are desired for clarifying the background concentrations of tetracyclines and sulphonamides in swine wastewater and river, and their potential impact on the environment.