生物炭中持久性自由基對(duì)秀麗隱桿線蟲(chóng)的毒性

張緒超,陳 懿,胡 蝶,趙 力,王 琳,吳 敏*

?

生物炭中持久性自由基對(duì)秀麗隱桿線蟲(chóng)的毒性

張緒超1,2,陳 懿1,2,胡 蝶1,趙 力1,2,王 琳1,2,吳 敏1,2*

(1.昆明理工大學(xué)環(huán)境科學(xué)與工程學(xué)院,云南 昆明 650500；2.云南省土壤固碳與污染控制重點(diǎn)實(shí)驗(yàn)室,云南 昆明 650500)

為了評(píng)價(jià)生物炭的使用對(duì)生態(tài)系統(tǒng),尤其是對(duì)土壤無(wú)脊椎動(dòng)物的毒性影響,使用模式生物秀麗隱桿線蟲(chóng)(,)來(lái)評(píng)估生物炭的環(huán)境風(fēng)險(xiǎn).觀察了生物炭原樣、生物炭顆粒物和生物炭浸提液對(duì)線蟲(chóng)神經(jīng)行為學(xué)評(píng)價(jià)指標(biāo)(身體擺動(dòng)頻率、相對(duì)運(yùn)動(dòng)長(zhǎng)度、排泄間隔時(shí)間、碰觸反應(yīng)率和化學(xué)感知行為指數(shù))的影響;并結(jié)合生物炭的理化性質(zhì)、非金屬元素組成和重金屬元素含量以及環(huán)境持久性自由基(EPFRs)的強(qiáng)度,評(píng)估生物炭對(duì)線蟲(chóng)的生物毒性.結(jié)果顯示,EPFRs信號(hào)強(qiáng)的生物炭和顆粒物對(duì)秀麗隱桿線蟲(chóng)有一定的毒物興奮效應(yīng),EPFRs信號(hào)微弱的浸提液無(wú)顯著性影響.因此,生物炭中的EPFRs對(duì)秀麗隱桿線蟲(chóng)有潛在的神經(jīng)毒性作用.

生物炭；持久性自由基；秀麗隱桿線蟲(chóng)；神經(jīng)毒性；環(huán)境風(fēng)險(xiǎn)

近年來(lái),生物炭因其可以作為土壤修復(fù)改良劑而受到越來(lái)越多的研究關(guān)注.生物炭能通過(guò)提高營(yíng)養(yǎng)元素利用率和土壤保水性而增加農(nóng)作物生產(chǎn)力,因而被大規(guī)模施用到土壤改良中[1-2].生物炭在熱解制備過(guò)程中,不可避免地會(huì)產(chǎn)生大量副產(chǎn)物,如多環(huán)芳烴(PAHs)、重金屬和環(huán)境持久性自由基(EPFRs)等[1,3-4].前人研究報(bào)道了生物炭對(duì)微生物[5-6]和植物[1]的影響,以及其中傳統(tǒng)污染物PAHs和重金屬對(duì)生物的影響[7-8],然而研究中卻很少關(guān)注生物炭中新型風(fēng)險(xiǎn)物質(zhì)EPFRs的影響,尤其是對(duì)土壤重要生物組成部分之一——?jiǎng)游锏挠绊?已有研究發(fā)現(xiàn)生物炭中存在大量的EPFRs,并發(fā)現(xiàn)EPFRs會(huì)對(duì)種子萌發(fā)和幼苗的生長(zhǎng)產(chǎn)生抑制[4].因此,亟需研究生物炭對(duì)土壤動(dòng)物的影響.這將有助于生物炭在被施用到田間土壤前,對(duì)其進(jìn)行有效的風(fēng)險(xiǎn)評(píng)估,從而降低其環(huán)境風(fēng)險(xiǎn),更好地發(fā)揮其土壤修復(fù)作用.

秀麗隱桿線蟲(chóng)(,)作為一種食細(xì)菌類的線蟲(chóng),是土壤生物多樣性的重要組成部分,可以參與并提高土壤肥力和植物生產(chǎn)力[9-10].同時(shí),秀麗隱桿線蟲(chóng)由于其繁殖快、與人類基因同源性、突變體多等眾多的優(yōu)點(diǎn)而成為一種重要的模式生物[11-13].現(xiàn)在,它已經(jīng)被廣泛應(yīng)用于環(huán)境科學(xué)領(lǐng)域中來(lái)評(píng)估污染物的風(fēng)險(xiǎn). Peredney等[14]嘗試?yán)眯沱愲[桿線蟲(chóng)評(píng)估了土壤中重金屬的風(fēng)險(xiǎn),結(jié)果表明秀麗隱桿線蟲(chóng)足夠敏感,并有足夠的生物信息和生態(tài)關(guān)聯(lián)性,可以作為一個(gè)理想的生物選擇來(lái)評(píng)價(jià)土壤中的污染物質(zhì).直到21世紀(jì)初美國(guó)材料與試驗(yàn)協(xié)會(huì)針對(duì)秀麗隱桿線蟲(chóng)制定了標(biāo)準(zhǔn)(E2172?01)[15]并推薦將其作為模式生物來(lái)評(píng)估土壤的風(fēng)險(xiǎn).此后,大量研究將其作為模式生物來(lái)評(píng)價(jià)污染物在多種環(huán)境介質(zhì)中的毒性作用,并發(fā)現(xiàn)其對(duì)神經(jīng)毒性的響應(yīng)比對(duì)其他毒性作用更為敏感[16-21].如,觀察到兩種有機(jī)溴化物混合暴露后對(duì)其有一定的神經(jīng)刺激作用[16-17],量子點(diǎn)可能影響神經(jīng)元系統(tǒng)的正常發(fā)育和功能[18],氧化石墨烯損傷中間神經(jīng)元可能的分子信號(hào)調(diào)節(jié)路徑[19-20],煤燃燒產(chǎn)生的顆粒物(PM2.5)可能因氧化應(yīng)激而誘發(fā)對(duì)秀麗隱桿線蟲(chóng)的毒性作用[21].因此,本研究選取秀麗隱桿線蟲(chóng)為模式生物探究生物炭對(duì)土壤生物的毒性影響.

在本研究前期實(shí)驗(yàn)中,發(fā)現(xiàn)生物炭中EPFRs可能會(huì)對(duì)秀麗隱桿線蟲(chóng)有一定的神經(jīng)毒性[22],但未直接排除PAHs和重金屬等潛在有毒物質(zhì)的影響.因此,本研究使用秀麗隱桿線蟲(chóng)來(lái)評(píng)估生物炭原樣、顆粒物以及浸提液的潛在毒性,深入探索生物炭毒性作用的來(lái)源,以期為更好地了解生物炭的環(huán)境風(fēng)險(xiǎn)提供理論基礎(chǔ).

1 材料與方法

1.1 生物炭和顆粒物以及水浸提液的制備

水稻秸稈采自云南省昆明市呈貢區(qū)吳家營(yíng)社區(qū)(24.8°N, 102.8°E),用粉碎機(jī)打碎后,過(guò)60目篩,存放于密封袋置于避光處保存.生物炭制備過(guò)程:(1)將6個(gè)100mL的坩堝置于105℃烘箱干燥2h后,在玻璃干燥器中冷卻到室溫;(2)每個(gè)坩堝進(jìn)行編號(hào),稱重,放入不超過(guò)坩堝體積三分之二的水稻秸稈,記錄質(zhì)量;(3)將坩堝置于馬弗爐內(nèi)腔體中間,通入純度為99.99%的氮?dú)?0min排盡空氣后開(kāi)始升溫,使用轉(zhuǎn)子流量計(jì)控制流速(100mL/min).一直保持氮?dú)猸h(huán)境,升溫到500℃并維持2h后,停止升溫,待溫度降至室溫,稱重.顆粒物的制備和水浸提液提取過(guò)程:將217.4mg的生物炭加入到30mL的無(wú)菌水中(此處由元素分析的結(jié)果換算成碳的濃度即4000mg/L),并設(shè)置一組平行樣;置于搖床中(20℃, 200r/min)避光搖晃24h后,使用0.45μm的濾膜進(jìn)行過(guò)濾,濾膜上的生物炭烘干(50℃,12h)即為顆粒物,過(guò)濾液即為浸提液,未洗的生物炭原樣即為生物炭.此外,實(shí)驗(yàn)中對(duì)生物炭、顆粒物和浸提液3種暴露體系與食物菌液OP50混合之前和混合之后24h的pH值進(jìn)行了檢測(cè),所測(cè)pH值為(5.99±0.04).同時(shí),Khanna等[23]報(bào)道了秀麗隱桿線蟲(chóng)在3.1~11.9的pH值范圍內(nèi)的致死率無(wú)顯著性差異.

1.2 生物炭的元素分析和基本理化性質(zhì)測(cè)定

生物炭的非金屬元素(C、N、H、S、O)使用元素分析儀(vario MICRO cube, Elementar, 德國(guó))檢測(cè),結(jié)果用質(zhì)量比表示.生物炭的金屬元素和類金屬元素使用ICP-MS(PerkinElmer NexION350, 美國(guó))進(jìn)行分析,步驟如下:將0.100g生物炭加入到裝有5mL濃硝酸和5mL氫氟酸的消解管中或1mL浸提液加入到裝有7mL濃硝酸和2mL過(guò)氧化氫的消解管中,把消解管放入智能溫壓雙控微波消解儀(奧普勒MD8h-20H, 成都),在180℃條件下消解45min,消解液冷卻至室溫后進(jìn)入ICP-MS儀器檢測(cè),顆粒物的這些元素含量通過(guò)差量法獲得.PAHs檢測(cè)方法參考中國(guó)生態(tài)環(huán)境部頒發(fā)的標(biāo)準(zhǔn)《土壤和沉積物多環(huán)芳烴的測(cè)定氣相色譜-質(zhì)譜法》(HJ 805-2016),使用20.0g生物炭進(jìn)行實(shí)驗(yàn)[24].

1.3 持久性自由基信號(hào)分析

將2mg左右的生物炭或顆粒物或50μL水浸提液裝進(jìn)石英毛細(xì)管(內(nèi)徑1mm,長(zhǎng)度125mm)中,毛細(xì)管末端使用真空油脂封口,再將毛細(xì)管置于EPR (Bruker X-band A300-6/1,美國(guó))的共振腔中,檢測(cè)EPR信號(hào),調(diào)制頻率100kHz,微波頻率9.2~9.9GHz,調(diào)制幅度1.00G,掃描寬度100G,微波功率31dB (0.131mW).測(cè)到的強(qiáng)度使用質(zhì)量(mg)進(jìn)行標(biāo)準(zhǔn)化.

1.4 秀麗隱桿線蟲(chóng)和菌株的培養(yǎng)

所有實(shí)驗(yàn)使用野生型N2秀麗隱桿線蟲(chóng),同步化后的蟲(chóng)卵在接種有大腸桿菌OP50的96mm的線蟲(chóng)固體生長(zhǎng)培養(yǎng)基(NGM)上培養(yǎng)到L4時(shí)期后,進(jìn)行暴露實(shí)驗(yàn).和OP50來(lái)自明尼蘇達(dá)大學(xué)的線蟲(chóng)遺傳學(xué)中心(CGC).

1.5 毒性暴露條件

參考生物炭田間使用比例和前人毒性研究結(jié)果[4,22],每個(gè)NGM平板上依次加入0,0.5,1,2,4mg的生物炭,相當(dāng)于暴露時(shí)碳的濃度是0,250,500,1000, 2000mg/L,并在培養(yǎng)基表面只加1mL的OP50,待菌液風(fēng)干后開(kāi)始實(shí)驗(yàn).對(duì)于顆粒物加入與生物炭相同的質(zhì)量,對(duì)于浸提液使用含有OP50的K-medium進(jìn)行稀釋得到.神經(jīng)行為學(xué)實(shí)驗(yàn)前,將30條同步化后的L4時(shí)期的線蟲(chóng)放到暴露平板中,置于20℃培養(yǎng)箱中避光培養(yǎng)24h,每個(gè)濃度至少重復(fù)3次實(shí)驗(yàn).

1.6 神經(jīng)行為學(xué)實(shí)驗(yàn)

大量研究表明,神經(jīng)行為學(xué)指標(biāo)可以很好的反映出污染物的神經(jīng)毒性,而且比較快速,易于操作和實(shí)現(xiàn)[16,25].本研究的神經(jīng)行為學(xué)實(shí)驗(yàn)方法參考Lieke等[22]的研究方法.即,身體擺動(dòng)頻率次數(shù)是指60s內(nèi)線蟲(chóng)向前做類似正弦曲線彎曲運(yùn)動(dòng)時(shí)的拐點(diǎn)數(shù);相對(duì)運(yùn)動(dòng)長(zhǎng)度是指在20s內(nèi)線蟲(chóng)運(yùn)動(dòng)的軌跡長(zhǎng)度與其自身長(zhǎng)度的比值;排泄間隔是指線蟲(chóng)每2次排泄行為之間的時(shí)間間隔;機(jī)械感知行為是指線蟲(chóng)應(yīng)對(duì)頭發(fā)絲碰觸的反應(yīng),若向后退縮,記為一次反應(yīng);化學(xué)感知行為指數(shù)()是指線蟲(chóng)對(duì)于氯化鈉(NaCl)的感知能力,并按式(1)進(jìn)行計(jì)算,通常認(rèn)為線蟲(chóng)趨于爬向有NaCl的地方,是因?yàn)镹aCl在其生長(zhǎng)環(huán)境中,當(dāng)線蟲(chóng)感知到NaCl時(shí),會(huì)把NaCl和食物聯(lián)系在一起,如果神經(jīng)受到損傷,會(huì)顯著降低.所有行為學(xué)實(shí)驗(yàn)通過(guò)體式顯微鏡(Nikon SMZ 1500,日本)或顯微鏡(Nikon Eclipse E100,日本)觀察、拍照分析得到.

式中:N和C分別指以NaCl點(diǎn)和以Control點(diǎn)為中心的半徑為1.5cm的圓內(nèi)線蟲(chóng)的個(gè)數(shù);T是放入開(kāi)始點(diǎn)S點(diǎn)的線蟲(chóng)的總個(gè)數(shù).S點(diǎn)位于等腰三角形(腰長(zhǎng)3cm,底邊長(zhǎng)4cm)的頂角所在點(diǎn)的位置,NaCl點(diǎn)和Control點(diǎn)分別位于兩側(cè)底角所在點(diǎn)的位置.本文中在S點(diǎn)放入30條線蟲(chóng)并置于20℃培養(yǎng)箱1h后,開(kāi)始計(jì)算相應(yīng)點(diǎn)的線蟲(chóng)數(shù)目.

1.7 數(shù)據(jù)分析

實(shí)驗(yàn)數(shù)據(jù)以mean ± SEM表示,數(shù)據(jù)使用Microsoft Office Excel 2016進(jìn)行前期處理,圖和單因素方差分析(Holm-Sidak method)由Graphpad Prism 7 完成.*表示<0.05,**表示<0.01.

2 結(jié)果與討論

2.1 生物炭的元素組成和基本理化性質(zhì)

本實(shí)驗(yàn)條件下生物炭的產(chǎn)率為(29.30±2.29)% (表1),通常,生物質(zhì)熱解時(shí)生物炭的產(chǎn)率會(huì)隨著溫度升高而降低,這是由于大量脫水、脫氫反應(yīng)的發(fā)生.這與Wu等[26]報(bào)道的結(jié)果(38.6%)有一定差異,而文獻(xiàn)中玉米秸稈和松木在500℃的產(chǎn)率分別是25.43%和24.12%,導(dǎo)致這一現(xiàn)象差異的可能原因之一是生物質(zhì)來(lái)源不同,如不同木質(zhì)素和纖維素含量的玉米秸稈(CN500)和松木屑(PW500)[27],還有可能是熱解工藝不同,如升溫程序和載氣流速[26,28].

生物炭(R500)的元素組成和原子比見(jiàn)表1,其中生物炭的C含量達(dá)到55.20%,H和O含量較低.H/C和O/C的比值分別是0.47和0.12,與文獻(xiàn)中相同生物質(zhì)和相同溫度的生物炭(表1)差別不大[26].對(duì)比不同溫度的H/C、O/C和(N+O)C發(fā)現(xiàn),這些比值會(huì)隨著溫度升高而降低,這可能與芳香環(huán)結(jié)構(gòu)的增加有關(guān)[26-27].

表1 生物炭的非金屬元素構(gòu)成

注: CN500表示500℃水稻生物炭,PW500表示500℃松木屑生物炭[27]; R300-2:“R”表示水稻秸稈生物炭,“300”表示熱解溫度是300℃,“2”表示熱解時(shí)間是2h,其他數(shù)據(jù)以此類推[26].“—”表示原文沒(méi)有該項(xiàng)數(shù)據(jù).

生物炭中的重金屬元素含量會(huì)因其原生質(zhì)的不同而有所差異.本實(shí)驗(yàn)使用的生物炭中Fe和Mn含量分別為204.5,218.2μg/g(表2),比一般生物炭中含量偏高,這可能與實(shí)驗(yàn)所采用的水稻秸稈生產(chǎn)于云南地區(qū)有關(guān),云南地區(qū)主要以紅壤為主,紅壤中含有較多的鐵錳化合物.參考中國(guó)生態(tài)環(huán)境部頒布的《土壤環(huán)境質(zhì)量標(biāo)準(zhǔn)農(nóng)田地土壤污染風(fēng)險(xiǎn)管控標(biāo)準(zhǔn)(試行)》(GB 15618-2018)[28]中農(nóng)用地土壤污染風(fēng)險(xiǎn)篩選值的基本項(xiàng)目,即As(20mg/kg)、Cd (0.3mg/kg)、Cu(50mg/kg)、Pb(70mg/kg)、Hg(0.5mg/ kg)、Ni(60mg/kg)、Zn(200mg/kg)(標(biāo)注的是較為嚴(yán)格的風(fēng)險(xiǎn)篩選值),對(duì)比發(fā)現(xiàn):生物炭和浸提液中的金屬元素和類金屬元素含量均低于基本項(xiàng)目中的風(fēng)險(xiǎn)篩選值.值得注意的是,參考《土壤和沉積物多環(huán)芳烴的測(cè)定氣相色譜-質(zhì)譜法》(HJ 805-2016)[24],在生物炭中未檢測(cè)到PAHs,可能其濃度低于儀器的檢測(cè)限.

生物炭性質(zhì)的差異可以導(dǎo)致不同的反應(yīng)活性和吸附性能,如非金屬元素構(gòu)成、金屬元素含量以及比表面積等.這些差異對(duì)于生物毒性的影響是未知的.

表2 生物炭和浸提液中的金屬和類金屬元素含量(μg/g)

注: “—”表示沒(méi)有檢測(cè)到該項(xiàng)值.

2.2 不同濃度生物炭對(duì)線蟲(chóng)的毒性影響

自主行為是神經(jīng)行為學(xué)中的常見(jiàn)評(píng)價(jià)指標(biāo),可以反映出有毒物質(zhì)對(duì)于線蟲(chóng)神經(jīng)行為的基本影響以及線蟲(chóng)的行為能力和損傷情況[29].感知行為是線蟲(chóng)對(duì)于來(lái)自生存環(huán)境中物理或化學(xué)或其他刺激作用的反應(yīng),可以體現(xiàn)出線蟲(chóng)特定感知神經(jīng)元的變化[29].

在自然界中,運(yùn)動(dòng)行為(Locomotion, 即身體擺動(dòng)頻率和相對(duì)運(yùn)動(dòng)長(zhǎng)度)能力是線蟲(chóng)生存的必備能力,可以幫助線蟲(chóng)獲取食物和躲避捕食者以及逃離不利環(huán)境[25].圖1顯示,在生物炭低濃度(250mg/L)暴露時(shí),線蟲(chóng)身體擺動(dòng)頻率從49次/min提高到53次/ min(圖1(a)),相對(duì)運(yùn)動(dòng)長(zhǎng)度從4.51/20s增加到4.89/ 20s(圖1(b)),而在高濃度時(shí)(2000mg/L)2個(gè)指標(biāo)依次減小到42次/min(=0.0003)和3.7/20s(=0.0031).暴露于生物炭中的線蟲(chóng),運(yùn)動(dòng)行為表現(xiàn)為低濃度時(shí)運(yùn)動(dòng)活力增加,高濃度時(shí)被抑制.

排泄間隔時(shí)間反映的是線蟲(chóng)腸道內(nèi)食物的消化時(shí)間長(zhǎng)短,通常,時(shí)間變長(zhǎng)時(shí),線蟲(chóng)對(duì)于自身能量的需求也就減少,也有可能是神經(jīng)的損傷.排泄間隔時(shí)間在生物炭低于500mg/L濃度時(shí)有一定降低的趨勢(shì)但是無(wú)顯著影響,在高濃度時(shí)(1000,2000mg/L)被延長(zhǎng)到77s左右(圖1(c),=0.0020和=0.0004).在低濃度時(shí),排泄間隔時(shí)間縮短可能是因?yàn)榫€蟲(chóng)接觸一些能被它感知的有毒物質(zhì)時(shí)出于對(duì)自身保護(hù)的反應(yīng)[25,30],高濃度時(shí),神經(jīng)被損傷導(dǎo)致時(shí)間延長(zhǎng).Lieke等[25]在研究和比較天然水體產(chǎn)物中的二溴乙酸(DBAA)和人工合成外源性化合物的四溴雙酚-A(TBBP-A)對(duì)線蟲(chóng)排泄間隔時(shí)間的影響時(shí),也觀察到了類似的結(jié)果,同時(shí)指出GABAergic神經(jīng)元AVL和DVB的調(diào)節(jié)起到了重要作用.

機(jī)械感知行為能力是線蟲(chóng)對(duì)于其外部環(huán)境和捕食者以及食物等的反應(yīng),通常,碰觸反應(yīng)率降低時(shí),意味著線蟲(chóng)的生存能力會(huì)因缺少足夠的食物來(lái)源或者適宜的環(huán)境而降低[25].圖1(d)中線蟲(chóng)的碰觸反應(yīng)率總體上沒(méi)有太大的變化,有趣的是即使未添加任何生物炭或顆粒物或浸提液,碰觸反應(yīng)率也只有97%,但在500mg/L時(shí)提高了約3%,在高濃度的時(shí)候(2000mg/L)降低了約3%.化學(xué)感知行為能力可以反映出線蟲(chóng)對(duì)于食物和不利環(huán)境的感知能力,通常,化學(xué)感知行為指數(shù)降低時(shí),線蟲(chóng)可能面臨食物短缺的威脅,也會(huì)遭受到有毒物質(zhì)的傷害[17,25].圖1(e)中線蟲(chóng)的化學(xué)感知行為指數(shù)在低于濃度1000mg/L時(shí)有一定升高,高濃度時(shí)(2000mg/L)被抑制,并且誤差較大.機(jī)械感知行為和化學(xué)感知行為通過(guò)感覺(jué)神經(jīng)元ASH接受外界的刺激[31],然后信號(hào)由谷氨酸能神經(jīng)元ASE、AWC和ASH進(jìn)行傳遞,這3個(gè)神經(jīng)元調(diào)節(jié)線蟲(chóng)感知鈉和氯化物的能力[25,32].當(dāng)有毒化學(xué)物質(zhì)引起轉(zhuǎn)錄減少的時(shí)候,化學(xué)感知行為指數(shù)有可能也會(huì)因此降低[33].此外,排泄間隔時(shí)間的減少和碰觸反應(yīng)率的降低都有可能會(huì)引起線蟲(chóng)壽命的縮短[25].

暴露于顆粒物后的線蟲(chóng),與暴露于生物炭后的線蟲(chóng)的結(jié)果相似,運(yùn)動(dòng)行為表現(xiàn)為低濃度時(shí)運(yùn)動(dòng)活力增強(qiáng),高濃度時(shí)被抑制.只是化學(xué)感知行為指數(shù)呈現(xiàn)出隨著濃度升高而降低的趨勢(shì),說(shuō)明顆粒物對(duì)于化學(xué)感知行為有潛在的損傷能力.暴露于浸提液后的線蟲(chóng),與生物炭和顆粒物表現(xiàn)出明顯的區(qū)別.總體上來(lái)看,線蟲(chóng)的頭部擺動(dòng)頻率、相對(duì)運(yùn)動(dòng)長(zhǎng)度、碰觸反應(yīng)率隨濃度均變化不大,對(duì)于排泄間隔時(shí)間高濃度(2000mg/L)只增加了3s,化學(xué)感知行為指數(shù)的波動(dòng)也不大.說(shuō)明浸提液中的潛在有毒物質(zhì)對(duì)線蟲(chóng)的神經(jīng)行為能力影響不是很大.

生物炭與顆粒物對(duì)線蟲(chóng)運(yùn)動(dòng)行為的這種低濃度促進(jìn)高濃度抑制(即呈現(xiàn)出神經(jīng)毒性)的現(xiàn)象,稱之為毒物興奮效應(yīng).毒物興奮效應(yīng)通常是指某一物質(zhì)在低濃度時(shí)對(duì)生命體沒(méi)有什么損害甚至有一定刺激和有益的作用,而在高濃度時(shí)有害的現(xiàn)象[22,34-35].目前還沒(méi)有一個(gè)統(tǒng)一的機(jī)理來(lái)解釋毒物興奮效應(yīng),但是它被發(fā)現(xiàn)廣泛存在于生態(tài)系統(tǒng)中[34]. Steinberg等[36]通過(guò)研究自然界中外源性化合物對(duì)秀麗隱桿線蟲(chóng)的影響后,發(fā)現(xiàn)轉(zhuǎn)錄反應(yīng)可能是誘發(fā)毒物興奮效應(yīng)的原因之一.

a.頭部擺動(dòng)頻率;b.相對(duì)運(yùn)動(dòng)長(zhǎng)度;c.排泄間隔時(shí)間;d.碰觸反應(yīng)率;e.化學(xué)感知行為

2.3 持久性自由基及其對(duì)線蟲(chóng)的神經(jīng)毒性影響

生物炭中的EPR信號(hào)強(qiáng)度達(dá)到了5.6×105mg-1,洗過(guò)后的生物炭也就是顆粒物其強(qiáng)度與生物炭仍然維持在一個(gè)數(shù)量級(jí)上,保持在3.7×105mg-1,而浸提液只有1.2×103mg-1,與空白管1.1×103mg-1基本一樣,見(jiàn)圖2.g-因子(g-Factor)是EPR儀器中用來(lái)分析EPFRs類型的值,通常g-Factor的數(shù)值小于2.0030時(shí),EPFRs屬于以碳為中心的自由基,如木材燃燒過(guò)程產(chǎn)生的的自由基[37];g-Factor大于2.0040時(shí),則屬于典型的以氧為中心的自由基,如煤炭中的有機(jī)自由基[38-39];g- Factor介于2.0030~2.0040之間時(shí),則可能是以碳和氧為中心的混合自由基,如公路上收集的顆粒物中的半醌類自由基[40].本實(shí)驗(yàn)中生物炭和顆粒物的g-Factor分別是2.00318和2.00320,介于2.0030~ 2.0040之間屬于以碳和以氧為中心的混合自由基,這與Liao等[4]和Lieke等[22]的研究結(jié)果基本一致.

研究發(fā)現(xiàn),當(dāng)水稻秸稈熱解溫度從200℃升到500℃時(shí),生物炭中的EPFRs強(qiáng)度隨著溫度升高而快速增強(qiáng),而當(dāng)溫度從500℃升到1000℃時(shí),EPFRs強(qiáng)度則隨溫度升高而迅速降低[4].因此,本實(shí)驗(yàn)選用EPFRs強(qiáng)度最強(qiáng)時(shí)的溫度500℃生物炭作為實(shí)驗(yàn)材料.從圖1中可以看出,生物炭和顆粒物對(duì)線蟲(chóng)的神經(jīng)行為能力影響趨勢(shì)基本一致,而浸提液對(duì)線蟲(chóng)則基本沒(méi)有影響.對(duì)比生物炭、顆粒物和浸提液三者之間的EPFRs信號(hào)發(fā)現(xiàn)(圖2),生物炭和顆粒物的EPFRs強(qiáng)度保持在一個(gè)數(shù)量級(jí)上,而浸提液則基本上與空白對(duì)照的強(qiáng)度一樣,因此,本文推測(cè)生物炭和顆粒物對(duì)線蟲(chóng)的神經(jīng)毒性來(lái)源于EPFRs的影響.

圖2 生物炭、顆粒物和浸提液中的持久性自由基信號(hào)圖

生物炭中含有一些潛在有毒物質(zhì)(金屬、類金屬元素和PAHs),若這些元素超過(guò)一定量或長(zhǎng)期暴露后,同樣會(huì)對(duì)線蟲(chóng)有一定的損傷,但是本實(shí)驗(yàn)中金屬與類金屬元素含量(表2)遠(yuǎn)遠(yuǎn)低于閾值[14,41];此外,生物炭在高溫?zé)峤膺^(guò)程中可能會(huì)產(chǎn)生PAHs等致癌物質(zhì),但是本實(shí)驗(yàn)并未檢測(cè)到生物炭中PAHs的存在,這可能與水稻秸稈生物炭中的PAHs含量較少以及生物炭的強(qiáng)吸附性能有關(guān).Hale等[7]研究了50種不同來(lái)源和溫度的生物炭后,發(fā)現(xiàn)慢速熱解的生物炭中16種PAHs的質(zhì)量濃度總和(SPAHs)最高的也只有3.27μg/g,這個(gè)濃度是遠(yuǎn)低于土壤環(huán)境質(zhì)量標(biāo)準(zhǔn)中SPAHs濃度的.本實(shí)驗(yàn)使用浸提液進(jìn)行毒性實(shí)驗(yàn)的結(jié)果也說(shuō)明潛在有毒物質(zhì)對(duì)線蟲(chóng)影響不大.所以,本研究生物炭中的金屬、類金屬元素和PAHs對(duì)線蟲(chóng)造成影響的可能性也非常小.因此,線蟲(chóng)的神經(jīng)毒性極有可能來(lái)源于生物炭中EPFRs的暴露.

生物炭中的EPFRs不僅穩(wěn)定時(shí)間長(zhǎng)久,能長(zhǎng)達(dá)1a以上[4,22],而且實(shí)驗(yàn)表明水稻秸稈等生物炭可以在液相環(huán)境中刺激活性氧(ROS)的產(chǎn)生[4,42],從而損傷種子萌發(fā),降低種子發(fā)芽率,抑制幼苗根長(zhǎng)和莖長(zhǎng)[4]. EPFRs不僅可以在體外誘導(dǎo)產(chǎn)生ROS[43],而且可以隨著環(huán)境介質(zhì)進(jìn)入到體內(nèi),刺激體內(nèi)ROS的形成[44-45]. ROS的性質(zhì)非?；顫?能夠攻擊和損傷包含脂質(zhì)、蛋白質(zhì)和多聚核苷酸在內(nèi)的幾乎所有生物大分子,造成氧化應(yīng)激損傷[46],進(jìn)而破環(huán)機(jī)體正常身體功能,引發(fā)神經(jīng)退行性病等疾病.氧化應(yīng)激可以損傷神經(jīng)元,因而氧化應(yīng)激在阿茲海默癥和帕金森病等病中被看作是一個(gè)重要原因[46].因此,生物炭和顆粒物中大分子斷裂形成的EPFRs可能是引發(fā)線蟲(chóng)神經(jīng)毒性的主要原因.未來(lái)的工作可以嘗試使用不同溫度和不同生物質(zhì)來(lái)源的生物炭(含有不同強(qiáng)度的EPFRs)來(lái)進(jìn)行毒性實(shí)驗(yàn),以進(jìn)一步證實(shí)EPFRs的神經(jīng)毒性作用.

3 結(jié)論

3.1 觀察了生物炭、顆粒物和浸提液對(duì)秀麗隱桿線蟲(chóng)的5個(gè)神經(jīng)行為學(xué)指標(biāo)后發(fā)現(xiàn),生物炭和顆粒物對(duì)秀麗隱桿線蟲(chóng)有毒物興奮效應(yīng),而浸提液的影響基本可以忽略.

3.2 生物炭的EPFRs強(qiáng)度為5.6×105,顆粒物的強(qiáng)度為3.7×105;浸提液的強(qiáng)度遠(yuǎn)低于生物炭和顆粒物;且生物炭和顆粒物的g-Factor介于2.0030~2.0040之間,屬于以碳和氧為中心的混合自由基.

3.3 生物炭中潛在有毒物質(zhì)(金屬元素和PAHs)的含量遠(yuǎn)低于國(guó)家標(biāo)準(zhǔn),因此EPFRs可能是引起毒性的主要原因.

[1] Hussain M, Farooq M, Nawaz A, et al. Biochar for crop production: Potential benefits and risks [J]. Journal of Soils and Sediments, 2017, 17(3):685-716.

[2] Van Zwieten L, Kimber S, Morris S, et al. Effects of biochar from slow pyrolysis of papermill waste on agronomic performance and soil fertility [J]. Plant and Soil, 2009,327(1/2):235-246.

[3] Lian F, Xing B. Black carbon (biochar) in water/soil environments: molecular structure, sorption, stability, and potential risk [J]. Environmental Science & Technology, 2017,51(23):13517-13532.

[4] Liao S, Pan B, Li H, et al. Detecting free radicals in biochars and determining their ability to inhibit the germination and growth of corn, wheat and rice seedlings [J]. Environmental Science & Technology, 2014,48(15):8581-8587.

[5] Quilliam R S, Glanville H C, Wade S C, et al. Life in the ‘charosphere’ – does biochar in agricultural soil provide a significant habitat for microorganisms? [J]. Soil Biology & Biochemistry, 2013,65(6):287- 293.

[6] 張尚毅,劉國(guó)濤,謝夢(mèng)佩.有機(jī)垃圾熱解炭對(duì)紫色土細(xì)菌群落結(jié)構(gòu)的影響 [J]. 中國(guó)環(huán)境科學(xué), 2017,37(2):669-676.Zhang S, Liu G, Xie M. Influence of organic fraction of municipal solid waste-based biochar on microbial community structure in a purple soil [J]. China Environmenatl Science, 2017,37(2):669-676.

[7] Hale S E, Lehmann J, Rutherford D, et al. Quantifying the total and bioavailable polycyclic aromatic hydrocarbons and dioxins in biochars [J]. Environmental Science & Technology, 2012,46(5):2830-2838.

[8] Cang L, Zhu X, Wang Y, et al. Pollutant contents in biochar and their potential environmental risks for field application [J]. Transactions of the Chinese Society of Agricultural Engineering, 2012,28(15):163- 167.

[9] Trap J, Bonkowski M, Plassard C, et al. Ecological importance of soil bacterivores for ecosystem functions [J]. Plant & Soil, 2016,398(1/2): 1-24.

[10] Gebremikael M T, Steel H, Buchan D, et al. Nematodes enhance plant growth and nutrient uptake under C and N-rich conditions [J]. Scientific Reports, 2016,6:32862.

[11] Tejeda-Benitez L, Olivero-Verbel J. Caenorhabditis elegans, a biological model for research in toxicology [M]. Switzerland: Springer International Publishing, 2016:1-35.

[12] Leung M C K, Williams P L, Benedetto A, et al. Caenorhabditis elegans: An emerging model in biomedical and environmental toxicology [J]. Toxicological Sciences, 2008,106(1):5-28.

[13] Shaye D D, Greenwald I. Ortholist: A compendium of c. Elegans genes with human orthologs [J]. PloS One, 2013,6(5):1573-1574.

[14] Peredney C, Williams P. Utility of caenorhabditis elegans for assessing heavy metal contamination in artificial soil [J]. Archives of Environmental Contamination and Toxicology, 2000,39(1):113-118.

[15] E2172-01Standard guide for conducting laboratory soil toxicity tests with the nematode Caenorhabditis elegans [S].

[16] Saul N, Stürzenbaum S R, Chakrabarti S, et al. Adsorbable organic bromine compounds (AOBr) in aquatic samples: A nematode-based toxicogenomic assessment of the exposure hazard [J]. Environmental Science and Pollution Research, 2015,22(19):14862-14873.

[17] Ju J, Lieke T, Saul N, et al. Neurotoxic evaluation of two organobromine model compounds and natural aobr-containing surface water samples by a Caenorhabditis elegans test [J]. Ecotoxicology and Environmental Safety, 2014,104:194-201.

[18] Zhao Y, Wang X, Wu Q, et al. Quantum dots exposure alters both development and function of d-type gabaergic motor neurons in nematode Caenorhabditis elegans [J]. Toxicology Research, 2015,4(2): 399-408.

[19] Zhi L, Qu M, Ren M, et al. Graphene oxide induces canonical wnt/β- catenin signaling-dependent toxicity in caenorhabditis elegans [J]. Carbon, 2017,113:122-131.

[20] Chen H, Li H, Wang D. Graphene oxide dysregulates neuroligin/nlg- 1-mediated molecular signaling in interneurons in Caenorhabditis elegans [J]. Scientific Reports, 2017,7:41655.

[21] Wu Q, Han X, Wang D, et al. Coal combustion related fine particulate matter (PM2.5) induces toxicity in Caenorhabditis elegans by dysregulating microrna expression [J]. Toxicology Research, 2017, 6(4):432-441.

[22] Lieke T, Zhang X, Steinberg C E W, et al. Overlooked risks of biochars: Persistent free radicals trigger neurotoxicity in Caenorhabditis elegans [J]. Environmental Science & Technology, 2018,52(14):7981-7987.

[23] Khanna N, Cressman C P, Tatara C P, et al. Tolerance of the nematode Caenorhabditis elegans to ph, salinity, and hardness in aquatic media [J]. Archives of Environment Contamination and Toxicology, 1997, 32(1):110-114.

[24] HJ 805-2016 土壤和沉積物多環(huán)芳烴的測(cè)定氣相色譜-質(zhì)譜法 [S].HJ 805-2016 Soil and Sediment-Determination of polycyclic aromatic hydrocarbon by Gas Chromatography-Mass Spectrometry Method [S].

[25] Lieke T, Steinberg C E, Ju J, et al. Natural marine and synthetic xenobiotics get on nematode’s nerves: Neuro-stimulating and neurotoxic findings in Caenorhabditis elegans [J]. Marine Drugs, 2015, 13(5):2785-2812.

[26] Wu W X, Yang M, Feng Q B, et al. Chemical characterization of rice straw-derived biochar for soil amendment [J]. Biomass & Bioenergy, 2012,47(4):268-276.

[27] 田路萍,常兆峰,王 朋,等.利用苯多酸生物標(biāo)記物表征生物炭的含量及特性 [J]. 環(huán)境化學(xué), 2017,36(4):738-744. Tian L, Chang Z, Wang P, et al. Characterization of biochars properities with benzene polycarboxylic acid biomarker [J]. Environmental Chemistry, 2017,36(4):738-744.

[28] GB 15618-2018 土壤環(huán)境質(zhì)量標(biāo)準(zhǔn)農(nóng)田地土壤污染風(fēng)險(xiǎn)管控標(biāo)準(zhǔn)(試行) [S]. GB 15618-2018 Soil environmental quality-Risk control standard for soil contamination of agricultural land [S].

[29] 居靜娟.應(yīng)用秀麗隱桿線蟲(chóng)模型研究藍(lán)藻毒素的神經(jīng)毒性及其作用機(jī)制 [D]. 南京:東南大學(xué), 2014. Ju J. Using Caenorhabditis elegans to study the neurotoxicity and mechanism of microcystins [D]. Nanjing:Southeast University, 2014.

[30] Sanger G J, Yoshida M, Yahyah M, et al. Increased defecation during stress or after 5-hydroxytryptophan: selective inhibition by the 5-HT4receptor antagonist, SB-207266 [J]. British Journal of Pharmacology, 2000,130(3):706-712.

[31] Kaplan J M, Horvitz H R. A dual mechanosensory and chemosensory neuron in Caenorhabditis elegans [J]. Proceedings of the National Academy of Sciences, 1993,90(6):2227-2231.

[32] Bargmann CI. Chemosensation in C. elegans. [EB/OL]. https://www. ncbi.nlm.nih.gov/books/NBK19746/2006-10-25.

[33] Takeshi I, Yuichi I, Akiko M, et al. Hen-1, a secretory protein with an ldl receptor motif, regulates sensory integration and learning in Caenorhabditis elegans [J]. Cell, 2002,109(5):639-649.

[34] Calabrese E J. Hormesis: Why it is important to toxicology and toxicologists [J]. Environmental Toxicology and Chemistry, 2008, 27(7):1451-1474.

[35] Calabrese E J, Baldwin L A. The dose determines the stimulation (and poison): Development of a chemical hormesis database [J]. International Journal of Toxicology, 1997,16(6):545-559.

[36] Steinberg C E, Pietsch K, Saul N, et al. Transcript expression patterns illuminate the mechanistic background of hormesis in Caenorhabditis elegans maupas [J]. Dose-Response, 2013,11(4):573.

[37] Tian L, Koshland C P, Yano J, et al. Carbon-centered free radicals in particulate matter emissions from wood and coal combustion [J]. Energy & Fuels, 2009,23(5):2523.

[38] Di Valentin C, Neyman K M, Risse T, et al. Density-functional model cluster studies of EPR g tensors of centers on the surface of MgO [J]. Journal of Chemical Physics, 2006,124(4):044708.

[39] Wang P, Pan B, Li H, et al. The overlooked occurrence of environmentally persistent free radicals in an area with low-rank coal burning, Xuanwei, China [J]. Environmental Science & Technology, 2018,52(3):1054-1061.

[40] Dellinger B, Pryor W A, Cueto R, et al. Role of free radicals in the toxicity of airborne fine particulate matter [J]. Chemical Research in Toxicology, 2001,14(10):1371-1377.

[41] Wang D, Xing X. Assessment of locomotion behavioral defects induced by acute toxicity from heavy metal exposure in nematode Caenorhabditis elegans [J]. Journal of Environmental Sciences, 2008, 20(9):1132-1137.

[42] Fang G, Liu C, Wang Y, et al. Photogeneration of reactive oxygen species from biochar suspension for diethyl phthalate degradation [J]. Applied Catalysis B: Environmental, 2017,214:34-45.

[43] Dalal N S, Suryan M M, Vallyathan V, et al. Detection of reactive free radicals in fresh coal mine dust and their implication for pulmonary injury [J]. The Annals of occupational hygiene, 1989,33(1):79-84.

[44] Thevenot P T, Saravia J, Jin N, et al. Radical-containing ultrafine particulate matter initiates epithelial-to-mesenchymal transitions in airway epithelial cells [J]. American Journal of Respiratory Cell and Molecular Biology, 2013,48(2):188-197.

[45] Saravia J, Lee G I, Lomnicki S, et al. Particulate matter containing environmentally persistent free radicals and adverse infant respiratory health effects: A review [J]. Journal of Biochemical and Molecular Toxicology, 2013,27(1):56-68.

[46] Pham-Huy L A, He H, Pham-Huy C. Free radicals, antioxidants in disease and health [J]. International journal of biomedical science: IJBS, 2008,4(2):89-96.

Neurotoxic effect of environmental persistent free radicals in rice biochar to

ZHANG Xu-chao1,2, CHEN Yi1,2, HU Die1, ZHAO Li1,2, WANG Lin1,2, WU Min1,2*

(1.Faculty of Environmental Science and Engineering, Kunming University of Science and Technology, Kunming 650500, China；2.Yunnan Provincial Key Lab of Carbon Sequestration and Pollution Control in Soils, Kunming 650500, China)., 2019,39(6)：2644~2651

As an effective soil amendment, biochars have attracted increasing research attention, while their environmental risks have not been fully explored. Studies on the effects of biochars on ecosystem, especially on soil invertebrates, are still scarce. The model organism,(, nematode), was used to assess the potential neurotoxicity of biochar. The neurobehaviors of, including their body bends, relative move length, defecation, touch response and chemical index, were investigated after 24h-exposed in biochars, the washed biochars and biochar supernatants. The contents of metallic and non-metallic elements, biochar physicochemical properties and the environmentally persistent free radicals (EPFRs) were characterized. The results showed a hormesis behavior ofwhen exposed in biochar particles with high EPFRs intensity. No significant effect towas observed when exposed in supernatants. Compared to the other endogenous pollutants in biochars, EPFRs have a potential neurotoxic effects to nematodes. This study provides a new angle to assess the potential environmental risks of biochar application.

biochar；persistent free radicals；；neurotoxicity；environmental risk

X503.22,R994.6

A

1000-6923(2019)06-2644-08

張緒超(1994-),男,安徽六安人,昆明理工大學(xué)碩士研究生,主要研究方向?yàn)槌志眯宰杂苫沫h(huán)境風(fēng)險(xiǎn).發(fā)表論文3篇.

2018-11-22

國(guó)家自然科學(xué)基金資助項(xiàng)目(41663013);國(guó)家自然科學(xué)基金資助重點(diǎn)項(xiàng)目(U1602231);云南省重點(diǎn)研發(fā)計(jì)劃資助項(xiàng)目(2018BC004)

*責(zé)任作者, 教授, minwup@hotmail.com