利用大型蚤和斑馬魚評(píng)價(jià)腈綸廢水好氧-厭氧處理過(guò)程的急性毒性和遺傳毒性變化

楊京亞，趙璐璐，張洲，劉薇,*，張新，徐曉晨，涂響

1.大連理工大學(xué)環(huán)境學(xué)院工業(yè)生態(tài)與環(huán)境工程教育部重點(diǎn)實(shí)驗(yàn)室，大連116024

2.中國(guó)環(huán)境科學(xué)研究院環(huán)境基準(zhǔn)與風(fēng)險(xiǎn)評(píng)估國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京100012

利用大型蚤和斑馬魚評(píng)價(jià)腈綸廢水好氧-厭氧處理過(guò)程的急性毒性和遺傳毒性變化

楊京亞1，趙璐璐1，張洲1，劉薇1,*，張新1，徐曉晨1，涂響2

1.大連理工大學(xué)環(huán)境學(xué)院工業(yè)生態(tài)與環(huán)境工程教育部重點(diǎn)實(shí)驗(yàn)室，大連116024

2.中國(guó)環(huán)境科學(xué)研究院環(huán)境基準(zhǔn)與風(fēng)險(xiǎn)評(píng)估國(guó)家重點(diǎn)實(shí)驗(yàn)室，北京100012

腈綸廢水毒性較強(qiáng)且難以去除,對(duì)腈綸廢水毒性及污水處理過(guò)程毒性削減能力進(jìn)行評(píng)價(jià),可為開(kāi)發(fā)該類廢水毒性減排技術(shù)提供科學(xué)依據(jù)。利用大型蚤及斑馬魚評(píng)價(jià)某腈綸廢水的急性毒性和遺傳毒性,及厭氧-好氧工藝對(duì)該廢水毒性的削減能力。采用大型蚤活動(dòng)抑制率和斑馬魚致死率表征廢水急性毒性,采用斑馬魚肝細(xì)胞彗星尾矩表征廢水遺傳毒性。腈綸廢水處理前對(duì)大型蚤和斑馬魚的急性毒性單位(TU)分別為1.2和2.9,經(jīng)厭氧-好氧工藝處理后分別降至＜0.4和0.5。遺傳毒性結(jié)果表明腈綸廢水對(duì)斑馬魚肝細(xì)胞造成DNA損傷作用,經(jīng)處理后遺傳毒性仍顯著高于陰性對(duì)照組。理化指標(biāo)與毒性指標(biāo)相關(guān)性分析表明,該廢水氨氮與毒性顯著相關(guān),推測(cè)氨氮可能是該廢水中的重要致毒因子之一。研究結(jié)果表明該腈綸廢水采用現(xiàn)有厭氧-好氧工藝無(wú)法有效削減毒性,對(duì)受納水體水生態(tài)環(huán)境造成潛在危害。

腈綸廢水；大型蚤；斑馬魚；急性毒性；遺傳毒性

腈綸廢水是一種典型有毒廢水,對(duì)微生物有顯著抑制作用,難生物降解[1]。其中干法腈綸在生產(chǎn)過(guò)程中先后加入二甲基甲酰胺、丙烯腈、四乙酸二氨基乙烷和壬基酚聚氧乙烯醚等20余種原料,并且在聚合反應(yīng)中又生成不同分子量的聚丙烯腈,因此腈綸廢水中污染物含量多,且有機(jī)胺和氨氮含量高[2-3]。我國(guó)對(duì)廢水排放的水質(zhì)評(píng)價(jià)與監(jiān)督以理化分析為主,難以反映廢水中多種組分的綜合毒性效應(yīng)[4]。廢水生物毒性監(jiān)測(cè),能彌補(bǔ)理化指標(biāo)監(jiān)測(cè)的不足。有研究者利用蠶豆根尖微核試驗(yàn)、小麥發(fā)芽試驗(yàn)、發(fā)光菌急性毒性試驗(yàn)、斑馬魚急性毒性試驗(yàn)分析腈綸工業(yè)廢水的急性毒性和遺傳毒性,結(jié)果表明腈綸廢水毒性較強(qiáng),排入水體會(huì)對(duì)當(dāng)?shù)厮鷳B(tài)系統(tǒng)乃至人類健康造成一定的危害[5-6]。

遼河流域主要污染物排放量遠(yuǎn)遠(yuǎn)超過(guò)受納水體環(huán)境容量,廢水的排放會(huì)增加區(qū)域環(huán)境負(fù)荷[7],其中化工和石化廢水是遼河流域重要污染來(lái)源之一。遼河流域化工與石化行業(yè)有機(jī)污染特征分析表明,典型企業(yè)排水與受納水體間存在明顯的源-匯響應(yīng)關(guān)系[8]。因此,對(duì)代表性腈綸廢水進(jìn)行毒性評(píng)價(jià),及污水處理過(guò)程毒性削減能力進(jìn)行評(píng)價(jià),對(duì)于遼河流域化工與石化廢水污染控制具有重要意義。

大型蚤和斑馬魚是廢水毒性評(píng)價(jià)國(guó)際通用模式生物,其中斑馬魚70%的基因與人類基因直系同源[9],采用斑馬魚肝細(xì)胞彗星實(shí)驗(yàn)對(duì)廢水遺傳毒性進(jìn)行評(píng)價(jià),有利于預(yù)測(cè)廢水對(duì)高等脊椎動(dòng)物乃至人類健康的潛在危害[10]。本研究采用大型蚤、斑馬魚作為指示生物,對(duì)比分析了撫順市某石化腈綸企業(yè)排水的急性毒性和遺傳毒性特征,以及厭氧-好氧生化處理工藝對(duì)該廢水毒性的削減能力,可為開(kāi)發(fā)腈綸廢水毒性減排技術(shù)提供科學(xué)依據(jù)。

1 材料與方法(Materials and methods)

1.1 試驗(yàn)材料與儀器

1.1.1 試劑

氫氧化鈉(NaOH)、磷酸緩沖鹽溶液(PBS)、乙二胺四乙酸二鈉(Na2EDTA)、三羥甲基氨基甲烷(Tris)、正常融點(diǎn)瓊脂糖(NMA,1.2%)、低熔點(diǎn)瓊脂糖(LMA,1.0%)、聚乙二醇辛基苯基醚(Triton X-100)、二甲基亞砜(DMSO)購(gòu)自北京索萊寶科技有限公司。Gelred染液購(gòu)自美國(guó)Biotium公司。實(shí)驗(yàn)試劑均為分析純。

細(xì)胞裂解液:將2.5 mol·L-1NaCl、10 mmol·L-1Tris、100 mmol·L-1Na2EDTA溶解后,用NaOH調(diào)節(jié)pH=10,置于4℃下備用。用前按體積比加入10%DMSO、1%Triton X-100。

電泳液:1 mmol·L-1Na2EDTA,300 mmol·L-1NaOH等體積混合,pH=13,4℃下保存。

1.1.2 儀器

光學(xué)顯微鏡(YS100,Nikon,日本)；TDZ4-WS低速臺(tái)式自動(dòng)平衡離心機(jī)(湖南湘儀實(shí)驗(yàn)室儀器開(kāi)發(fā)有限公司)；DYY-6C型電泳儀(北京六一儀器廠)；O-lympus IX71倒置式熒光顯微鏡(日本)。

1.1.3供試水樣

取自撫順某石化公司腈綸廠污水處理設(shè)施,該廠采用干法腈綸生產(chǎn)工藝,廢水處理采用厭氧-好氧工藝:經(jīng)調(diào)節(jié)池混合后進(jìn)入?yún)捬跸到y(tǒng),出水進(jìn)入一沉池沉淀,上清液化學(xué)氧化處理后在二沉池內(nèi)混凝沉淀,后經(jīng)流化床、硝化處理進(jìn)入三沉池,出水經(jīng)生物碳塔吸附處理后排放。本研究采集入水、厭氧池、沉淀池出水和最終排放水。水樣采集后,過(guò)濾除去懸浮顆粒物,測(cè)定水質(zhì)常規(guī)理化指標(biāo)(見(jiàn)表1),毒性測(cè)試前置于4℃保存,1周內(nèi)進(jìn)行毒性實(shí)驗(yàn)。毒性測(cè)試過(guò)程中,水中溶解氧＞4mg·L-1,pH為6～8。

1.2 急性毒性試驗(yàn)

大型蚤(Daphnia magna)購(gòu)自大連海洋大學(xué)生命科學(xué)學(xué)院重點(diǎn)實(shí)驗(yàn)室,采用6～24 h內(nèi)新出生的小蚤,暴露24 h后,記錄不活動(dòng)蚤數(shù)。預(yù)實(shí)驗(yàn)后確定5個(gè)濃度梯度,同時(shí)設(shè)置空白對(duì)照。每個(gè)樣品設(shè)3個(gè)平行,每個(gè)樣品投放5只大型蚤。

斑馬魚(Danio rerio)購(gòu)自大連市花鳥(niǎo)市場(chǎng),曝氣去氯自來(lái)水中馴化1周,期間自然死亡率不超過(guò)5%,試驗(yàn)前1天停止喂食。預(yù)實(shí)驗(yàn)后確定5個(gè)濃度梯度,同時(shí)設(shè)置空白對(duì)照。每個(gè)樣品設(shè)3個(gè)平行,每個(gè)樣品1.2 L水,投放5條魚,低于1克魚/升水。靜態(tài)方式染毒96 h后,記錄存活的斑馬魚數(shù)目。

腈綸廢水對(duì)大型蚤、斑馬魚急性毒性的評(píng)價(jià)采用半數(shù)效應(yīng)濃度(EC50)和毒性單位(TU)。EC50表示腈綸廢水使大型蚤行為抑制率或斑馬魚死亡率達(dá)到50%時(shí)所對(duì)應(yīng)的濃度。TU=1/EC50。若當(dāng)受試生物暴露于未稀釋廢水,急性毒性效應(yīng)未達(dá)到50%,無(wú)法得到EC50,TU通過(guò)以下公式計(jì)算[11]:

TU=死亡率(行為抑制率)×100×0.02

本研究中急性毒性檢出限是20%,因此當(dāng)急性毒性為20%時(shí),TU=0.02×100×20%=0.4。當(dāng)未稀釋水樣的急性毒性低于20%時(shí),TU值用＜0.4表示。

1.3 斑馬魚彗星實(shí)驗(yàn)

采用單細(xì)胞凝膠電泳(single cell gel electrophoresis,SCGE)實(shí)驗(yàn),也稱彗星實(shí)驗(yàn)(comet assay),在單細(xì)胞水平上檢測(cè)DNA損傷[12]。每個(gè)平行測(cè)定樣品采集3條斑馬魚肝臟,混合制備肝細(xì)胞懸液。采用雙層凝膠結(jié)構(gòu)制作電泳膠板,加入細(xì)胞裂解液裂解90 min,加電泳液使DNA解旋、電泳,后用Tris-HCl(pH=7.5)緩沖液浸泡中和。晾干后用Gelred染色,倒置熒光顯微鏡觀察,在自動(dòng)曝光條件下拍照獲取彗星圖像。以尾矩(tail moment,TM,即彗尾長(zhǎng)度和尾部DNA含量的乘積)為指標(biāo)反映暴露于廢水的斑馬魚肝細(xì)胞DNA損傷程度[13]。使用Casp軟件隨機(jī)抓取50個(gè)細(xì)胞圖像測(cè)定尾矩,結(jié)果用(平均值±標(biāo)準(zhǔn)差)表示。

1.4 質(zhì)量控制/質(zhì)量保證

毒性實(shí)驗(yàn)采用空白對(duì)照、陽(yáng)性對(duì)照、平行測(cè)定作為質(zhì)控措施。采用重鉻酸鉀(K2Cr2O7)作為急性毒性試驗(yàn)的陽(yáng)性對(duì)照物,K2Cr2O7對(duì)大型蚤活動(dòng)抑制和斑馬魚致死作用的EC50分別為(0.7±0.1)mg·L-1和(293±16)mg·L-1。遺傳毒性實(shí)驗(yàn)采用4-硝基喹啉-1-氧化物(4-NQO)作為陽(yáng)性對(duì)照物?？瞻讓?duì)照組彗星尾矩為(1.0±0.5),暴露于1μg·L-14-NQO時(shí),彗星尾矩為(7.0±2.2)。

1.5 數(shù)據(jù)處理

利用SPSS19.0進(jìn)行統(tǒng)計(jì)分析。采用probit analysis計(jì)算EC50。采用單因素方差分析(One-Way ANOVA)進(jìn)行組間差異檢驗(yàn)。采用Pearson分析考察理化指標(biāo)與毒性指標(biāo)之間的相關(guān)性。

2 結(jié)果與分析(Results and analysis)

2.1 水質(zhì)基本理化指標(biāo)

腈綸廢水的理化參數(shù)測(cè)定結(jié)果可見(jiàn)表1。進(jìn)水、厭氧池及初級(jí)淀池出水COD及氨氮濃度較高,后經(jīng)化學(xué)氧化、二級(jí)沉淀、流化床處理后,出水COD和氨氮顯著降低。三沉池出水COD有所升高,原因可能有污泥指數(shù)高等。最終出水理化指標(biāo)除氨氮外,達(dá)到《城鎮(zhèn)污水處理廠污染物排放標(biāo)準(zhǔn)》(GB18918—2002)的一級(jí)標(biāo)準(zhǔn),厭氧-好氧生化處理工藝對(duì)該腈綸廢水 COD、氨氮的去除率分別為81.6%和57.7%。

表1 污水處理廠各反應(yīng)段出水的水質(zhì)指標(biāo)Table 1 Quality index of wastewater from sewage treatment process

2.2 腈綸廢水對(duì)大型蚤和斑馬魚的急性毒性

腈綸廢水不同處理工藝段出水對(duì)大型蚤的活動(dòng)抑制作用和對(duì)斑馬魚的致死作用結(jié)果見(jiàn)表2。腈綸廢水未經(jīng)處理前對(duì)大型蚤和斑馬魚具有明顯的急性毒性,暴露于原水造成大型蚤活動(dòng)抑制率和斑馬魚死亡率為100%,TU分別為1.2和2.9。經(jīng)過(guò)厭氧-一沉/二沉處理后,急性毒性沒(méi)有明顯變化。處理至流化床,急性毒性顯著降低。最終出水未發(fā)現(xiàn)大型蚤的活動(dòng)抑制作用,對(duì)斑馬魚表現(xiàn)出輕微的致死作用。

圖1 陰性對(duì)照、腈綸廢水進(jìn)水、出水的斑馬魚肝細(xì)胞彗星圖像(×100)注:(A)陰性對(duì)照,(B)進(jìn)水,(C)出水。Fig.1 Comet images of hepatocyte collected fromDanio rerioexposed to negative control,influent and effluent of acrylic fiber wastewaterNote:(A)Negative control,(B)Influent,(C)Effluent.

圖2 腈綸廢水進(jìn)水、出水在不同稀釋度下對(duì)斑馬魚造成的DNA損傷注:(A)進(jìn)水,(B)出水；**代表與對(duì)照組相比P＜0.01。Fig.2 DNA damage in the hepatocytes ofDanio rerioafter exposure to effluents from the typical process stages of acrylic fiber industry at different concentrationsNote:(A)Influent;(B)Effluent.** represent significant differences atP＜0.01,compared with control values.

表2 腈綸廢水進(jìn)水及不同工藝段出水對(duì)大型蚤和斑馬魚的急性毒性Table 2 Acute toxic effects onDaphnia magnaandDanio rerioexposed to acrylic fiber wastewater influent and effluents from different treatment units

2.3 腈綸廢水對(duì)斑馬魚的遺傳毒性

代表性對(duì)照和廢水作用于斑馬魚肝細(xì)胞的彗星圖像見(jiàn)圖1。對(duì)照組細(xì)胞尾矩為(1.0±0.5)(圖2)。隨著暴露稀釋梯度的升高,腈綸廢水不同工藝段出水對(duì)斑馬魚肝細(xì)胞造成的DNA損傷程度增加,暴露在腈綸廢水原水5%稀釋梯度下對(duì)斑馬魚肝細(xì)胞造成的DNA損傷的彗星尾矩為(19.2±3.4),較對(duì)照組呈極顯著差異(P＜0.01)；而出水10%稀釋梯度下對(duì)斑馬魚肝細(xì)胞造成的DNA損傷尾矩為(3.6±1.3),較對(duì)照組呈極顯著性差異(P＜0.01)。

3 討論(Discussion)

本研究選擇大型蚤及斑馬魚為受試生物,對(duì)腈綸廢水的毒性進(jìn)行評(píng)價(jià)。結(jié)果表明腈綸廢水對(duì)大型蚤和斑馬魚產(chǎn)生明顯的急性毒性作用,并對(duì)斑馬魚肝細(xì)胞產(chǎn)生DNA損傷作用。大型蚤和斑馬魚作為浮游動(dòng)物和脊椎動(dòng)物的代表,其毒性測(cè)試結(jié)果對(duì)于廢水的潛在生態(tài)危害具有重要指示作用。經(jīng)過(guò)厭氧-好氧生化工藝處理后,腈綸廢水的COD、氨氮及毒性降低,且單位COD的急性毒性降低,表明處理過(guò)程中強(qiáng)毒性物質(zhì)被降解為低毒物質(zhì),該工藝對(duì)急性毒性和遺傳毒性具有一定的削減能力。但最終出水仍具有顯著的遺傳毒性,表明該廢水經(jīng)處理后雖然毒性顯著降低,但對(duì)于水生態(tài)系統(tǒng)仍存在一定風(fēng)險(xiǎn)。張凱等[5]發(fā)現(xiàn)丙烯腈模擬廢水經(jīng)Fenton、活性炭吸附、加成法處理后,對(duì)小麥的毒性降低,但對(duì)發(fā)光菌的毒性升高,提示該類廢水毒性削減難度較大。

單細(xì)胞凝膠電泳(彗星實(shí)驗(yàn))應(yīng)用于檢測(cè)污染物對(duì)DNA造成的損傷程度[12],DNA損傷是指由化學(xué)或物理因素引起的DNA鏈斷裂、分子機(jī)體修飾和DNA交聯(lián)等,若這種損傷沒(méi)有得到及時(shí)的修復(fù),就會(huì)引起突變,乃至癌變[13]。該腈綸廢水氨氮、COD均與遺傳毒性顯著相關(guān)(P＜0.05),表明遺傳毒性是該腈綸廢水的重要毒性特征,廢水中的主要成分能引起DNA損傷。Chang等[14]研究表明丙烯腈可引起原代培養(yǎng)的成人支氣管上皮細(xì)胞DNA單鏈斷裂。丙烯腈及其代謝產(chǎn)物可損傷生物膜,產(chǎn)生活性氧攻擊DNA大分子,造成DNA損傷,使小鼠精細(xì)胞DNA產(chǎn)生交聯(lián)作用[15]。有研究表明,二甲基酰胺在有氧的條件下會(huì)降解產(chǎn)生H2O2,且會(huì)導(dǎo)致DNA的損傷[16]。

此外,該腈綸廢水氨氮與急性毒性也顯著相關(guān)(P＜0.05),推測(cè)氨氮是造成腈綸廢水對(duì)水生生物產(chǎn)生毒性作用的關(guān)鍵致毒因子之一。當(dāng)水中氨氮濃度超過(guò)25mg·L-1時(shí),會(huì)對(duì)水體生態(tài)環(huán)境產(chǎn)生嚴(yán)重風(fēng)險(xiǎn)[17]。已有研究表明,氨氮對(duì)大型蚤和斑馬魚急性毒性的 LC50分別為 165.97mg·L-1和 101mg·L-1[19-20]。與本研究中腈綸廢水經(jīng)在氨氮為30.7mg·L-1下對(duì)斑馬魚產(chǎn)生輕微致死效應(yīng)的結(jié)果相近。氨氮對(duì)水生動(dòng)物的急性毒性主要是非離子氨的作用,非離子氨通過(guò)細(xì)胞膜進(jìn)入血液后轉(zhuǎn)化為NH+4,使神經(jīng)元去極化,最終導(dǎo)致細(xì)胞死亡,即使是在劑量較低的情況下,也能對(duì)魚類神經(jīng)系統(tǒng)產(chǎn)生影響[18]。腈綸廢水毒性鑒別仍需進(jìn)一步研究,以確定廢水中的有毒有害污染物,有助于實(shí)現(xiàn)污染物源頭控制。

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Variation in Acute Toxicity and Genotoxicity of Acrylic Fiber Wastewater on Daphnia magna and Danio rerio during Anaerobic－aerobic Treatment Process

Yang Jingya1,Zhao Lulu1,Zhang Zhou1,Liu Wei1,*,Zhang Xin1,Xu Xiaochen1,Tu Xiang2
1.Key Laboratory of Industrial Ecology and Environmental Engineering(MOE),School of Environmental Science and Technology, Dalian University of Technology,Dalian 116024,China
2.State Key Laboratory of Environmental Criteria and Risk Assessment,Chinese Research Academy of Environmental Sciences,Beijing 100012,China

24 March 2015 accepted 5 April 2015

The acrylic fiber wastewater exhibits relatively high toxicity,which is difficult to be removed.The evaluation for the toxicity of the acrylic fiber wastewater and its reduction during the treatment process would facilitate the improvement of toxicity removal when optimizing the wastewater treatment technique.Daphnia magnaandDanio reriowere applied to evaluate the acute toxicity and genotoxicity of acrylic fiber wastewater,and the toxicity removal by anaerobic－aerobic(A/O)treatment process.The acute toxicity of the acrylic fiber wastewater was tested byDaphnia magnaimmobilization andDanio reriomortality.The genotoxicity was evaluated by comet tailmoment ofDanio reriohepatocyte.The acrylic fiber wastewater samples were collected from a representative chemical industry located in Fushun,Liaoning Province.The removal of COD and ammonia by the A/O process were 81.6%and 57.7%,respectively.The COD and the ammonia of the effluent from the treatment process were 36.2 and 30.7mg·L-1,respectively,which fell within the“Discharge Standard of Pollutants for Municipal Wastewater Treatment Plant”in China.The wastewaters induced acute toxicity toDaphnia magnaandDanio rerio, with the acute toxicity units at 1.2 and 2.9 before treatment,＜0.4 and 0.5 after A/O treatment,respectively.Acrylic fiber wastewater caused DNA damage toDanio reriohepatocyte,and the genotoxicity of the effluent was still significantly higher than that of the negative control group.Correlation analysis between physiochemical indexes and toxicity indicator showed that ammonia concentrations were significantly correlated with the toxicity,suggesting that ammonia may be one important toxicity factor in the acrylic fiber wastewater.The results suggested that the current A/O treatment process was limited in remove the toxicity of the acrylic fiber wastewater,and the discharged wastewater would pose potential hazard to the aquatic ecosystems of the receiving water.

acrylic fiber wastewater;Daphnia magna;Danio rerio;acute toxicity;genotoxicity

2015-03-24 錄用日期:2015-04-05

1673-5897(2016)1-225-06

X171.5

A

10.7524/AJE.1673-5897.20150324003

楊京亞,趙璐璐,張洲,等.利用大型蚤和斑馬魚評(píng)價(jià)腈綸廢水好氧-厭氧處理過(guò)程的急性毒性和遺傳毒性變化[J].生態(tài)毒理學(xué)報(bào),2016,11(1):225-230

Yang J Y,Zhao L L,Zhang Z,et al.Variation in acute toxicity and genotoxicity of acrylic fiber wastewater onDaphnia magnaandDanio rerioduring anaerobic－aerobic treatment process[J].Asian Journal of Ecotoxicology,2016,11(1):225-230(in Chinese)

國(guó)家水體污染控制與治理科技重大專項(xiàng)(2012ZX07202-002)

楊京亞(1990-),女,碩士,研究方向?yàn)樗鷳B(tài)毒理學(xué),E-mail:nearlyyang@163.com；

),E-mail:liu_wei@dlut.edu.cn

簡(jiǎn)介:劉薇(1980-)，博士，副教授，主要研究方向?yàn)榄h(huán)境污染物暴露評(píng)估、毒性效應(yīng)和機(jī)理,發(fā)表學(xué)術(shù)論文30余篇。