3種有機磷農(nóng)藥對水生生物的乙酰膽堿酯酶抑制效應(yīng)的物種敏感度分析初探

朱巖，王飛飛，張亞輝，曹瑩，曾鴻鵠，劉征濤

1.桂林理工大學(xué)環(huán)境科學(xué)與工程學(xué)院，桂林541004

2.中國環(huán)境科學(xué)研究院環(huán)境基準(zhǔn)與風(fēng)險評估國家重點實驗室國家環(huán)境保護(hù)化學(xué)品生態(tài)效應(yīng)與風(fēng)險評估重點實驗室，北京100012

3種有機磷農(nóng)藥對水生生物的乙酰膽堿酯酶抑制效應(yīng)的物種敏感度分析初探

朱巖1,2，王飛飛1，張亞輝2,*，曹瑩2，曾鴻鵠1，劉征濤2

1.桂林理工大學(xué)環(huán)境科學(xué)與工程學(xué)院，桂林541004

2.中國環(huán)境科學(xué)研究院環(huán)境基準(zhǔn)與風(fēng)險評估國家重點實驗室國家環(huán)境保護(hù)化學(xué)品生態(tài)效應(yīng)與風(fēng)險評估重點實驗室，北京100012

通過篩選敵敵畏、馬拉硫磷和對硫磷3種有機磷農(nóng)藥對水生生物的急性毒性數(shù)據(jù)和乙酰膽堿酯酶抑制效應(yīng)數(shù)據(jù)，構(gòu)建物種敏感度分布曲線進(jìn)行了比較分析。結(jié)果表明，敵敵畏對水生生物的急性毒性和乙酰膽堿酯酶抑制效應(yīng)的大小順序為：酶體內(nèi)抑制效應(yīng)>酶體外抑制效應(yīng)>急性毒性；馬拉硫磷和對硫磷的乙酰膽堿酯酶抑制效應(yīng)數(shù)據(jù)不足但趨勢相似，順序為：酶體內(nèi)抑制效應(yīng)>急性毒性>酶體外抑制效應(yīng)。敵敵畏的急性毒性和酶體外抑制效應(yīng)的5%危害濃度(HC5)分別為2.07 μg·L-1和1.53 μg·L-1，兩者相差1.4倍。在水質(zhì)基準(zhǔn)推導(dǎo)中，乙酰膽堿酯酶抑制效應(yīng)數(shù)據(jù)對有機磷農(nóng)藥的水生生物基準(zhǔn)具有重要的參考價值。

有機磷農(nóng)藥；水生生物；乙酰膽堿酯酶；物種敏感度；水質(zhì)基準(zhǔn)

朱巖,王飛飛,張亞輝,等.3種有機磷農(nóng)藥對水生生物的乙酰膽堿酯酶抑制效應(yīng)的物種敏感度分析初探[J].生態(tài)毒理學(xué)報，2016,11(3):211-218

Zhu Y,Wang F F,Zhang Y H,et al.A preliminary study on species sensitivity analysis of inhibition effect of three organophosphorus pesticides on acetylcholinesterase in aquatic organisms[J].Asian Journal of Ecotoxicology,2016,11(3):211-218(in Chinese)

水生生物基準(zhǔn)作為水質(zhì)基準(zhǔn)(water quality criteria,WQC)的核心組成部分，用于保護(hù)水生態(tài)系統(tǒng)中水生生物的安全。推導(dǎo)污染物的水生生物基準(zhǔn)的方法主要包括生物模型法、評估因子法、統(tǒng)計外推法等[1]。物種敏感度分布(species sensitivity distribution,SSD)[1]作為統(tǒng)計外推法于20世紀(jì)70年代提出，已經(jīng)成為水質(zhì)基準(zhǔn)推導(dǎo)的主要方法之一[2]。SSD方法采用不同函數(shù)，例如對數(shù)-邏輯斯帝函數(shù)[3-4]、對數(shù)正態(tài)分布函數(shù)[5-6]和對數(shù)三角函數(shù)[7]等，將不同生物對同一物質(zhì)的敏感度分布進(jìn)行擬合，計算出保護(hù)一定比例[8]生物的污染物濃度作為水質(zhì)基準(zhǔn)值。目前，該方法主要針對生物個體水平的毒性數(shù)據(jù)進(jìn)行統(tǒng)計分析。

長期以來，在推導(dǎo)水生生物基準(zhǔn)中，大多采用水生生物毒性數(shù)據(jù)中存活、生長和繁殖等個體水平的“常規(guī)”毒性終點[9-10]，而“非常規(guī)”的毒性終點，例如雌激素紊亂[11]、酶抑制效應(yīng)、RNA或DNA[12]水平的變化被忽視。在美國的基準(zhǔn)推導(dǎo)文件中，這些“非常規(guī)”的毒性終點數(shù)據(jù)往往歸于“其他數(shù)據(jù)”，但這些毒性終點往往比“常規(guī)”毒性終點更加靈敏[13]，這些指標(biāo)通過個體/亞個體水平表現(xiàn)外來脅迫效應(yīng)，指示環(huán)境中痕量污染物短期或長期暴露的毒性效應(yīng)[14-15]。陳朗等[16]分析了溴氰菊酯的酶類、生物化學(xué)、細(xì)胞學(xué)、遺傳學(xué)、基因及組織學(xué)等生物標(biāo)記物指標(biāo)的SSD曲線，指出這些指標(biāo)可以作為慢性毒性數(shù)據(jù)的一種替代或補充引入到水質(zhì)基準(zhǔn)推導(dǎo)中，彌補“傳統(tǒng)指標(biāo)”的不足。閆振廣等[17]采用基因表達(dá)效應(yīng)分析了重金屬的物種敏感度，結(jié)果發(fā)現(xiàn)銅的基因表達(dá)效應(yīng)較為敏感，具有作為慢性水質(zhì)基準(zhǔn)數(shù)據(jù)的應(yīng)用潛力，而鋅與鎘的基因表達(dá)效應(yīng)與慢性毒性數(shù)據(jù)相比沒有明顯優(yōu)勢。

有機磷農(nóng)藥(organphosphorus pesticides,OPs)具有低成本、藥效高、品種多等特點，是目前生產(chǎn)和使用最為廣泛的一類農(nóng)藥[18]。有機磷農(nóng)藥伴隨降雨和坡地漫流進(jìn)入環(huán)境水體中，對水生生物和人體健康構(gòu)成危害。乙酰膽堿酯酶(acetylcholinestetase, AChE)作為有機磷農(nóng)藥的靶標(biāo)酶，已經(jīng)積累了很多毒性數(shù)據(jù)。本文通過篩選敵敵畏、馬拉硫磷和對硫磷3種有機磷農(nóng)藥對水生生物的急性毒性數(shù)據(jù)和AChE抑制效應(yīng)數(shù)據(jù)，利用物種敏感度分布法比較分析3種有機磷農(nóng)藥的急性毒性數(shù)據(jù)與AChE抑制效應(yīng)的敏感性，計算得到保護(hù)95%水生生物的有機磷農(nóng)藥濃度(HC5)。探討了在水質(zhì)基準(zhǔn)推導(dǎo)中，有機磷農(nóng)藥對AChE抑制效應(yīng)數(shù)據(jù)是否具有補充有機磷農(nóng)藥對水生生物急性毒性數(shù)據(jù)的潛力。

1 材料與方法（Materials and methods）

1.1 數(shù)據(jù)搜集與篩選

本文中3種有機磷農(nóng)藥對水生生物的毒性數(shù)據(jù)主要來自美國環(huán)保署(USEPA)[19]的毒性數(shù)據(jù)庫ECOTOX(http://cfpub.epa.gov/ecotox)，同時利用Web of Science、Sciencedirect、Wiley、Springer和中國知網(wǎng)等文獻(xiàn)數(shù)據(jù)庫搜索公開發(fā)表的中英文文獻(xiàn)，并以所得文獻(xiàn)的引用文獻(xiàn)作為補充。廣泛搜集了敵敵畏(dichlorvos)、對硫磷(parathion)和馬拉硫磷(malathion)3種有機磷農(nóng)藥對水生生物的毒性數(shù)據(jù)。搜索所用關(guān)鍵詞包括“敵敵畏”、“馬拉硫磷”、“對硫磷”、“水生生物”、“乙酰膽堿酯酶”、“有機磷農(nóng)藥”、“水質(zhì)基準(zhǔn)”、“生態(tài)風(fēng)險”、“風(fēng)險評估”、“物種敏感度”等。

參考美國水生生物基準(zhǔn)技術(shù)指南[16]的數(shù)據(jù)篩選原則，將搜集到的數(shù)據(jù)進(jìn)行篩選，篩選標(biāo)準(zhǔn)為：數(shù)據(jù)的毒性終點為半數(shù)致死濃度(LC50)或半數(shù)效應(yīng)濃度(EC50)，暴露時間2～4 d，不同暴露時間的數(shù)據(jù)以時間較長的優(yōu)先；同一文獻(xiàn)中對同一種生物的多個毒性數(shù)據(jù)，當(dāng)暴露時間相同時，選擇最敏感的數(shù)據(jù)；同一物種不同生命階段的毒性數(shù)據(jù)以敏感生命階段的數(shù)據(jù)優(yōu)先。

1.2 數(shù)據(jù)處理

將搜集且篩選過的數(shù)據(jù)按照濃度由小到大進(jìn)行排序并編號，同一物種的多個同類毒性數(shù)據(jù)取幾何平均值作為該物種的急性毒性值或AChE抑制效應(yīng)數(shù)據(jù)。計算每個物種的累計概率：

式中，P為累積概率，i是物種排序的秩，N是樣本數(shù)。將排序后的累積概率和濃度的對數(shù)轉(zhuǎn)換值應(yīng)用數(shù)據(jù)分析軟件Origin 9.0，選用某一特定概率模型進(jìn)行參數(shù)擬合構(gòu)建物種敏感度分布曲線。當(dāng)累積概率為0.05時，對應(yīng)的濃度就是水生生物被危害的比例為5%時的污染物濃度(HC5)。采用對數(shù)-正態(tài)分布(公式2)和對數(shù)-邏輯斯蒂函數(shù)(公式3)對SSD曲線進(jìn)行擬合，采用決定校正系數(shù)(Adj.R2)和加權(quán)卡方檢驗系數(shù)(Reduced Chi-Sqr)來判斷模型的擬合優(yōu)度，選擇最佳擬合模型。

式中，y為累計概率，x為有機磷農(nóng)藥濃度對數(shù)轉(zhuǎn)換值(μg·L-1)，α、β、μ和σ均為曲線參數(shù)，erf()為誤差函數(shù)。

2 結(jié)果（Results）

2.1 有機磷農(nóng)藥對AChE的抑制效應(yīng)

敵敵畏、對硫磷和馬拉硫磷3種典型有機磷農(nóng)藥對水生生物的急性數(shù)據(jù)，其中包括敵敵畏的69種生物的急性毒性數(shù)據(jù)，馬拉硫磷的43種生物的急性毒性數(shù)據(jù)，對硫磷的11種生物的急性毒性數(shù)據(jù)(見附表1～3)。

本文搜集的AChE抑制效應(yīng)數(shù)據(jù)明顯少于急性毒性數(shù)據(jù)，共涉及26種水生生物，包括19種生物的敵敵畏毒性數(shù)據(jù)、8種生物的馬拉硫磷毒性數(shù)據(jù)和5種生物的對硫磷毒性數(shù)據(jù)，其中以敵敵畏的AChE抑制效應(yīng)數(shù)據(jù)最為豐富(見表1～2)。26種水生生物中，魚類占19種。

表1 乙酰膽堿酯酶（AChE）體外暴露于3種有機磷農(nóng)藥的半數(shù)效應(yīng)濃度/半數(shù)抑制濃度（EC50/IC50）值Table 1 The EC50/IC50values of AChE fromin vitroexposure to three organophosphorus pesticides(OPs)

26種水生生物的離體實驗中，根據(jù)試驗方法的不同分為體內(nèi)染毒和體外染毒。有10篇文獻(xiàn)中采用體外染毒的方法：提取生物體內(nèi)的AChE與不同種類、濃度的有機磷農(nóng)藥混合，在一定條件下培養(yǎng)后，測定有機磷農(nóng)藥對AChE的抑制效應(yīng)[20-22]；有8篇文獻(xiàn)中采用體內(nèi)染毒的方法：將待測生物暴露在不同種類、濃度的有機磷農(nóng)藥中，經(jīng)過一定時間后，提取并測定酶活，得到有機磷農(nóng)藥在不同暴露時間內(nèi)對AChE的抑制效應(yīng)[23-28]?，F(xiàn)有的數(shù)據(jù)表明，在AChE的體外抑制中，敵敵畏的16種水生生物中，最敏感的是歐洲魚銜，最不敏感的是鯽魚，兩者相差約350倍；馬拉硫磷的4種水生生物中，最敏感的是黑頭軟口鰷，最不敏感的是鰱魚，兩者相差約10倍；對硫磷的2種淡水水生生物中，最敏感的是大型溞，最不敏感的是羅非魚，兩者相差5倍。在AChE的體內(nèi)抑制中，敵敵畏毒性相關(guān)的3種淡水水生生物中，最敏感的是大型溞，最不敏感的是搖蚊幼蟲，兩者相差約35倍；馬拉硫磷毒性相關(guān)的4種淡水水生生物中，最敏感的是側(cè)邊底鳉，最不敏感的是藍(lán)鮎，兩者相差1萬倍；對硫磷毒性相關(guān)的4種淡水水生生物中，最敏感的是大型溞，最不敏感的是金頭鯛，兩者相差約110倍。另外，體外染毒方法中暴露時間也相差很大，最短為3 min(鰱魚)，最長為1 h(太平洋牡蠣)。

2.2 SSD最佳擬合函數(shù)

表2 乙酰膽堿酯酶（AChE）體內(nèi)暴露于3種有機磷農(nóng)藥的半數(shù)效應(yīng)濃度/半數(shù)抑制濃度（EC50/IC50）值Table 2 The EC50/IC50values of AChE fromin vivoexposure to three organophosphorus pesticides(OPs)

表3 2個數(shù)學(xué)函數(shù)對SSD曲線的統(tǒng)計參數(shù)Table 3 The statistics of two mathematical functions for SSD curves

通過對數(shù)-正態(tài)分布函數(shù)和對數(shù)-邏輯斯蒂函數(shù)擬合構(gòu)建SSD曲線，2個函數(shù)擬合的統(tǒng)計參數(shù)(見表3)。整體而言，2個函數(shù)擬合的參數(shù)值比較接近，其校正決定系數(shù)均大于0.937，加權(quán)卡方檢驗系數(shù)均較小。對數(shù)-邏輯斯帝函數(shù)對敵敵畏和馬拉硫磷毒性數(shù)據(jù)擬合的校正決定系數(shù)大于對數(shù)-正態(tài)分布函數(shù)擬合值，并且對數(shù)-邏輯斯帝函數(shù)擬合的加權(quán)卡方檢驗系數(shù)小于對數(shù)正態(tài)分布函數(shù)擬合值；對數(shù)-邏輯斯帝函數(shù)對對硫磷毒性數(shù)據(jù)擬合的矯正檢驗系數(shù)和加權(quán)卡方檢驗系數(shù)均小于對數(shù)-正態(tài)分布函數(shù)的擬合值。綜上所述，本文中SSD曲線的擬合采用對數(shù)-邏輯斯蒂函數(shù)。

圖1 馬拉硫磷、敵敵畏和對硫磷對水生生物的急性和酶抑制效應(yīng)數(shù)據(jù)的SSD曲線

2.3 急性毒性數(shù)據(jù)與AChE抑制效應(yīng)數(shù)據(jù)的對比

為比較急性毒性數(shù)據(jù)和AChE抑制效應(yīng)數(shù)據(jù)之間的敏感度差異，利用對數(shù)-邏輯斯蒂函數(shù)分別對有機磷農(nóng)藥敵敵畏、對硫磷和馬拉硫磷的急性毒性和AChE抑制效應(yīng)數(shù)據(jù)進(jìn)行分布擬合，根據(jù)所獲得的參數(shù)得到SSD曲線，它可以直觀地表示不同水生生物對有機磷農(nóng)藥的敏感度分布情況(見圖1)。其中馬拉硫磷和對硫磷的AChE抑制效應(yīng)數(shù)據(jù)太少(n< 10)僅在圖中顯示位置，沒有構(gòu)建SSD曲線[1]。由圖1可以看出，在橫坐標(biāo)相同的情況下，對硫磷和馬拉硫磷對水生生物急性毒性的SSD曲線斜率相對較小，而敵敵畏的SSD曲線斜率則較大，表明大部分水生生物對敵敵畏的敏感度相對接近，而不同種水生生物對于馬拉硫磷和對硫磷的敏感度則差異較大。對于急性毒性和AChE抑制效應(yīng)這2類數(shù)據(jù)的敏感度來說，不同的有機磷農(nóng)藥顯示出差異性。敵敵畏的數(shù)據(jù)敏感度排序為“酶體內(nèi)抑制效應(yīng)>酶體外抑制效應(yīng)>急性毒性效應(yīng)”；在低濃度區(qū)間，對硫磷對AChE的體內(nèi)抑制效應(yīng)數(shù)據(jù)與急性毒性數(shù)據(jù)接近，馬拉硫磷與敵敵畏類似；在高濃度區(qū)間，馬拉硫磷對AChE的抑制效應(yīng)數(shù)據(jù)與急性毒性數(shù)據(jù)接近；從趨勢上看，3種有機磷農(nóng)藥的共同之處都是AChE的體內(nèi)抑制效應(yīng)比AChE的體外抑制效應(yīng)更靈敏。

為便于對3種有機磷農(nóng)藥的急性毒性和AChE抑制效應(yīng)數(shù)據(jù)進(jìn)行量化對比，分別對能保護(hù)95%的水生生物的污染物濃度(HC5)進(jìn)行計算，HC5是計算保護(hù)水生生物的水質(zhì)基準(zhǔn)的重要依據(jù)。由于欠缺馬拉硫磷和對硫磷的AChE抑制效應(yīng)數(shù)據(jù)，只有敵敵畏的數(shù)據(jù)比較豐富，可以進(jìn)行擬合計算HC5。敵敵畏的急性毒性和AChE體外抑制效應(yīng)的HC5分別為2.07 μg·L-1和1.53 μg·L-1，兩者相差1.4倍。

3 討論（Discussion）

總體而言，3種有機磷農(nóng)藥對AChE的體內(nèi)抑制效應(yīng)比體外抑制效應(yīng)更靈敏。這可能是由于在體外染毒方法中沒有考慮到有機磷農(nóng)藥在生物體內(nèi)代謝作用的影響，使得測定農(nóng)藥對AChE的毒性數(shù)據(jù)與實際環(huán)境中同濃度農(nóng)藥對AChE的毒性數(shù)據(jù)有一定偏差。例如，對氧磷對AChE的毒性大于對硫磷，但由于對硫磷在生物體內(nèi)可以轉(zhuǎn)化為對氧磷，使得體內(nèi)抑制所得到的IC50遠(yuǎn)遠(yuǎn)小于體外抑制所得到的IC50[45]。那么，幾乎可以肯定對硫磷對水生生物的AChE抑制效應(yīng)還存在更小的IC50值，即AChE抑制效應(yīng)的敏感度還會提升。

本文選擇了對確定水質(zhì)基準(zhǔn)具有重要參考價值的HC5對敏感度進(jìn)行定量分析，對硫磷和馬拉硫磷對水生生物體內(nèi)的AChE抑制效應(yīng)數(shù)據(jù)不足無法計算HC5。由于數(shù)據(jù)量的限制，僅將敵敵畏對水生生物體內(nèi)AChE的抑制效應(yīng)數(shù)據(jù)進(jìn)行推算來比較數(shù)據(jù)靈敏度。敵敵畏的急性毒性和AChE體外抑制效應(yīng)的HC5分別為2.07 μg·L-1和1.53 μg·L-1，兩者相差1.4倍，比較接近。陳朗等[16]對于溴氰菊酯，采用酶類、生物化學(xué)、細(xì)胞、遺傳、基因及組織學(xué)等生物標(biāo)記物指標(biāo)的最低可觀測效應(yīng)濃度(LOEC)的SSD曲線，計算得到HC5值為1.3 ng·L-1，與水生生物的基準(zhǔn)連續(xù)濃度(CCC)值0.9 ng·L-1相近，與基準(zhǔn)最大濃度(CMC)值7.5 ng·L-1，相差較大，因此作者指出這些生物標(biāo)記物指標(biāo)可以作為水生生物基準(zhǔn)中慢性數(shù)據(jù)的替代或補充。因此，鑒于試驗動物福利和AChE體外抑制試驗數(shù)據(jù)的快速可獲得性上來說，在推導(dǎo)水質(zhì)基準(zhǔn)中，AChE抑制效應(yīng)數(shù)據(jù)可以作為有機磷農(nóng)藥的急性毒性數(shù)據(jù)的補充，具有重要的參考價值。

一般認(rèn)為，在分子水平上的AChE對污染物脅迫發(fā)生應(yīng)激響應(yīng)要早于個體、種群或群落[46]。由于AChE的檢測手段能更好地評價有機磷農(nóng)藥對水生生物的危害程度，但現(xiàn)階段的數(shù)據(jù)量難以支持其在實際水環(huán)境中的應(yīng)用。只有通過積累更多有機磷農(nóng)藥對水生生物的AChE抑制數(shù)據(jù)，才能正確評價AChE抑制效應(yīng)指標(biāo)在水質(zhì)基準(zhǔn)研究中的作用。因此有機磷農(nóng)藥對水生生物的AChE抑制效應(yīng)在水質(zhì)基準(zhǔn)上具有廣泛的研究前景。

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A Preliminary Study on Species Sensitivity Analysis of Inhibition Effect of Three Organophosphorus Pesticides on Acetylcholinesterase in Aquatic Organisms

Zhu Yan1,2,Wang Feifei1,Zhang Yahui2,*,Cao Ying2,Zeng Honghu1,Liu Zhengtao2

1.College of Environmental Science and Engineering,Guilin University of Technology,Guilin 541004,China
2.State Key Laboratory for Environmental Criteria and Risk Assessment,State Environmental Protection Key Laboratory of Ecological Effects and Risk Assessment of Chemicals,Chinese Research Academy of Environmental Sciences,Beijing 100012,China

30 November 2015 accepted 21 January 2016

The data of the acute toxicity and acetylcholinesterase(AChE)inhibition effect of aquatic organisms of three organophosphorus pesticides(OPs),including dichlorvos,parathion and malathion,were selected and analyzed by species sensitivity distribution.The results showed that the order of acute toxicity and AChE inhibition effect for dichlorvos wasin vivoAChE inhibition>in vitroAChE inhibition>acute toxicity.The trends of speciessensitivity distribution of malathion and parathion were similar based on their insufficient data.The orders of acute toxicity and AChE inhibition effect of malathion and parathion werein vivoAChE inhibition>actue toxicity>in vitroAChE inhibition.The 5%hazard concentration(HC5)of acute toxicity and inhibition effect of dichlorvos were 2.07 μg·L-1and 1.53 μg·L-1,respectively,which differs by 1.4 times from each other.The data of AChE inhibition effect of OPs could be of great reference value when deriving the water qulity criteria.

organophosphate pesticide;aquatic organism;acetylcholinesterase;species sensitivity;water quality criteria

2015-11-30 錄用日期：2016-1-21

1673-5897（2016）3-211-08

X171.5

A

10.7524/AJE.1673-5897.20151130006

簡介：張亞輝(1979—)，女，副研究員，主要從事污染物聯(lián)合毒性及毒性評估與預(yù)測研究，發(fā)表學(xué)術(shù)論文20余篇。

國家水體污染控制與治理科技重大專項(2012ZX07501-003)；科技基礎(chǔ)性工作專項(2014FY120600)；國家自然科學(xué)基金(51268008，21407139)

朱巖(1990-)，男，碩士研究生，研究方向為水處理理論與技術(shù)，E-mail:zhuyan_craes@foxmail.com；

*通訊作者（Corresponding author），E-mail:zhangyahui@craes.org.cn