海底重燃油對(duì)海膽繁殖及其子代發(fā)育的影響

段美娜,劉泳江,白 雪,高 翔,張欣欣,熊德琪*

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海底重燃油對(duì)海膽繁殖及其子代發(fā)育的影響

段美娜1,劉泳江1,白 雪1,高 翔1,張欣欣2,熊德琪1*

(1.大連海事大學(xué)環(huán)境科學(xué)與工程學(xué)院,遼寧 大連 116026；2.中科海創(chuàng)環(huán)境科技(大連)有限公司,遼寧 大連 116000)

利用室內(nèi)流水式粘油礫石柱模擬實(shí)際環(huán)境中的海底重燃油,研究了重燃油污染的孔隙水對(duì)成年海膽繁殖力、配子質(zhì)量及子代胚胎發(fā)育的影響.結(jié)果表明,暴露結(jié)束后(7d),暴露組海膽的排配子率顯著降低(=0.033),同時(shí)雌海膽繁殖力也顯著降低(=0.036,(1957917±811471)個(gè)卵細(xì)胞);卵細(xì)胞的直徑和精子的受精能力并未受到海底重燃油的影響.子代繼續(xù)暴露48h,發(fā)現(xiàn)親代暴露加劇了子代胚胎畸形程度,表明親代暴露的毒性可傳遞給子代.進(jìn)一步利用整合毒性指數(shù)(ITI)檢測(cè)毒性傳遞的性別差異發(fā)現(xiàn),與母本效應(yīng)相比(24和48h子代的ITI分別為0.54~1.45和1.1~2.57),父本效應(yīng)(24和48h子代的ITI分別為0.82~1.95和1.89~4.04)在毒性傳遞過(guò)程中起著關(guān)鍵作用.

海底重燃油；孔隙水；親本效應(yīng)；海膽；早期發(fā)育

1970~2016年間,世界范圍內(nèi),發(fā)生了超過(guò)1000起船舶溢油事故,其中絕大多數(shù)事故(81%)為小型船舶溢油事故(即溢油量<7t)[1].中小型船舶溢油事故泄漏的油品往往是重燃油(HFO),其導(dǎo)致的HFO溢出量占HFO總溢出量的80%以上[2].HFO是一種原油精煉產(chǎn)品.HFO黏度大(15℃運(yùn)動(dòng)黏度5000~ 30000mPa·s),分散和自然降解十分困難.在風(fēng)浪作用下,HFO可以出現(xiàn)在距溢油事故發(fā)生地點(diǎn)相當(dāng)遠(yuǎn)的地方,使海岸線(xiàn)和敏感海域受到污染[3].此外,HFO密度較高(0.92~1.02g/cm3),在碎波帶與泥沙混合后,更易于沉降.沉降后,在波浪和潮汐的作用下HFO可以在水體和砂礫海灘之間循環(huán),源源不斷地釋放多環(huán)芳烴(PAHs)對(duì)水生生物產(chǎn)生毒害作用,這個(gè)過(guò)程就是孔隙水毒性假說(shuō)[4].

已有學(xué)者在實(shí)驗(yàn)室條件下利用裝填粘油礫石的容器模擬近岸處海底HFO,發(fā)現(xiàn)魚(yú)類(lèi)胚胎的畸形率和死亡率與流經(jīng)粘油礫石的水體中PAHs濃度相關(guān)[5-9].事實(shí)上,海洋底棲生物,由于棲息在海底,不僅會(huì)暴露在有毒孔隙水中,還可能與海底HFO直接接觸,加之其活動(dòng)能力低下難以逃出污染區(qū),勢(shì)必會(huì)受到海底HFO的脅迫,因此海底HFO對(duì)海洋底棲生物的毒性影響亟待研究.

海膽主要棲息于淺海的巖礁、礫石、砂石等海底,對(duì)淺海生物群落的組成、結(jié)構(gòu)和多樣性起著關(guān)鍵的作用[10-13].海膽生活史包括浮游幼體和底棲成體兩個(gè)主要階段.不僅早期浮游幼體對(duì)污染敏感性高[14-21],其成年階段也被視作海洋生態(tài)毒理學(xué)研究和環(huán)境監(jiān)測(cè)的有效生物模型[22-26].成年動(dòng)物的繁殖能力對(duì)整個(gè)物種的延續(xù)起著至關(guān)重要的作用,海底HFO對(duì)成年海膽的毒害作用勢(shì)必會(huì)影響到其體內(nèi)配子的生成、排放、受精過(guò)程,進(jìn)一步影響子代發(fā)育,從而對(duì)整個(gè)種群的生存產(chǎn)生影響[27-34].

本文以裝填粘油礫石的容器模擬海底HFO,選擇海洋底棲模式生物海膽為受試生物,研究海底HFO對(duì)親代成年海膽繁殖能力、配子質(zhì)量及其子代胚胎早期發(fā)育的影響.以期為評(píng)估溢油對(duì)海膽種群數(shù)量的影響提供參考數(shù)據(jù),同時(shí)為海洋生態(tài)風(fēng)險(xiǎn)評(píng)估、自然資源損害評(píng)估以及船舶溢油索賠提供重要科學(xué)依據(jù).

1 材料與方法

1.1 材料

1.1.1 實(shí)驗(yàn)油品 HFO380,運(yùn)動(dòng)黏度729800mPa·s (50℃),密度0.9821g/cm3,由大連海洋燃油有限公司提供.

1.1.2 實(shí)驗(yàn)礫石 直徑為10~50mm的表面平整的礫石,取自大連銀沙灘,洗凈烘干后備用.

1.1.3 實(shí)驗(yàn)海水 取自大連市星海灣,鹽度(34±1) PSU,pH=(8.0±0.03).

1.1.4 實(shí)驗(yàn)海膽馴化繁殖期成年海膽購(gòu)自大連海寶漁業(yè)有限公司.每只海膽注射0.5mL的KCl,排黃色配子的為雌海膽,排白色配子的為雄海膽.將成功排配子的海膽在實(shí)驗(yàn)室海水循環(huán)系統(tǒng)(大連匯鋅鈦設(shè)備)中馴化2周,海水溫度為(18±1)°C,光照周期為12h:12h.期間每3d投喂海膽體重5%的新鮮海帶.暴露實(shí)驗(yàn)開(kāi)始前3d停止喂食.馴化期間未出現(xiàn)自發(fā)排配子和死亡個(gè)體.

1.2 方法

1.2.1 粘油礫石制備[5,7,9]將一定量的HFO380與1.8kg潔凈礫石放入混合容器中劇烈晃動(dòng)2min,使油盡可能的均勻的覆蓋在礫石表面.為避免相互干擾,按粘油量由低至高依次制備不同濃度的粘油礫石.然后將粘油礫石置于避光通風(fēng)處24h,用于暴露實(shí)驗(yàn).實(shí)驗(yàn)設(shè)置粘油礫石濃度為0,400,800,1600,3200和6400μg油/g礫石(下文簡(jiǎn)寫(xiě)為μg/g).0μg/g為對(duì)照組.Zhadan和Vaschenko的研究[33]表明暴露于被柴油污染的海水中(總石油烴(TPH)濃度約為300μg/L)50d的雌海膽所產(chǎn)子代的畸形率增加;在此基礎(chǔ)上,通過(guò)預(yù)實(shí)驗(yàn)本文選擇400μg/g濃度組為最低濃度組,該組暴露液中TPH濃度從618.5μg/L降至308.6μg/L.進(jìn)一步預(yù)實(shí)驗(yàn)發(fā)現(xiàn),暴露于16000μg/g濃度組的雌海膽2d后出現(xiàn)自發(fā)排卵現(xiàn)象.暴露于8000和4000μg/g濃度組的雌海膽,在7d暴露期內(nèi)未出現(xiàn)自發(fā)排卵現(xiàn)象,但是8000μg/g濃度組的雌海膽排卵數(shù)目少.不足以用于后續(xù)子代的暴露實(shí)驗(yàn),而4000μg/g組的雌海膽產(chǎn)卵數(shù)量足以用于后續(xù)實(shí)驗(yàn).因此本文最高濃度組的粘油量應(yīng)介于4000~ 8000μg/g之間.因此本文選擇400,800,1600,3200和6400 μg/g,以此保證可以觀察到親代效應(yīng),并保證有足夠的卵細(xì)胞用于后續(xù)暴露實(shí)驗(yàn).

1.2.2 動(dòng)態(tài)暴露裝置 裝置為上下加蓋的聚氯乙烯(PVC)柱(d=10.8cm,H=35cm),距底部3cm處有一進(jìn)水管(d=1.2cm),出水管(d=1.2cm)位于對(duì)面距頂部3cm處,底部配備一個(gè)由PVC支柱(H=3.5cm)支撐的PVC網(wǎng)孔圓板放置礫石(圖1).進(jìn)水流速以針閥控制.粘油礫石制備完成后,轉(zhuǎn)移至該裝置,海水流速20mL/min由下至上流經(jīng)粘油礫石柱,24h后盛接流出液進(jìn)行暴露實(shí)驗(yàn).

1.2.3 親代海膽暴露方法每個(gè)濃度組設(shè)置3個(gè)重復(fù),每個(gè)重復(fù)包括6只雌海膽和6只雄海膽,雌雄海膽分開(kāi)暴露(圖1).暴露容器上部開(kāi)孔,保證暴露溶液體積為6L.暴露時(shí)間為7d,暴露期間不充氣不喂食,并虹吸出容器底部排泄物.

1.2.4 子代胚胎獲取及暴露方法 親代海膽暴露結(jié)束后,用0.45μm濾膜過(guò)濾海水(FSW)清洗體表,然后經(jīng)圍口膜向海膽體腔內(nèi)注射1mL現(xiàn)配的KCl溶液(0.5M).雌海膽口面向上置于盛滿(mǎn)FSW錐形瓶口處,雄海膽口面向上,擦干體表后置于干燥的培養(yǎng)皿上.30min后將雌海膽取下,將每重復(fù)組6只雌海膽所產(chǎn)卵細(xì)胞混合,置于18℃的FSW中暫放待受精.每重復(fù)組的6只雄海膽所排精子混合后置于4℃待受精.取10μL干精子稀釋于5mL的FSW中,加入到500mL密度為100個(gè)/mL的卵細(xì)胞溶液中,輕輕晃動(dòng)進(jìn)行受精[35].胚胎親本組合有4種(如圖2所示):對(duì)照組(對(duì)照組卵細(xì)胞和對(duì)照組精子受精),母本暴露組(暴露組卵細(xì)胞和對(duì)照組精子受精),父本暴露組(對(duì)照組卵細(xì)胞和暴露組精子受精)和雙本暴露組(暴露組卵細(xì)胞和同組精子受精).受精15min后,虹吸法洗卵3次以去除多余的精子.

來(lái)自于暴露親本的胚胎暴露濃度與其親代暴露濃度相同.對(duì)照組胚胎均分為6份,分別于FSW和粘油礫石柱流出液中培養(yǎng).因此對(duì)于子代暴露實(shí)驗(yàn),共有5個(gè)暴露濃度,每個(gè)濃度組包括4種親本組合的胚胎.胚胎于黑暗處(18±1)℃的水浴中培養(yǎng),期間無(wú)需換水和喂食.

圖1 粘油礫石柱和成年海膽動(dòng)態(tài)暴露方法示意

該示意圖為6個(gè)粘油礫石濃度組(0,400,800,1600,3200和6400μg/g)之一的模式圖;I、II和III指親代暴露實(shí)驗(yàn)的3個(gè)重復(fù)組;水流動(dòng)方向在圖中以黑色箭頭標(biāo)注

1.2.5 親代海膽相關(guān)參數(shù)測(cè)定 排配子率指排配子的雌或雄海膽個(gè)數(shù)與對(duì)應(yīng)性別的海膽總數(shù)的比值.繁殖力指雌海膽開(kāi)始排卵的30min內(nèi)排出的卵的個(gè)數(shù)[36],其中將未排卵的雌海膽繁殖力記為0.卵細(xì)胞計(jì)數(shù)使用0.1mL的浮游生物計(jì)數(shù)框進(jìn)行,每只海膽重復(fù)計(jì)數(shù)4次并取平均值作為一只海膽的繁殖力.

1.2.6 配子質(zhì)量 將每重復(fù)組6只雌海膽的卵細(xì)胞混合,取3個(gè)1mL重復(fù)樣品,滴入幾滴40%福爾馬林,置于4℃待測(cè).顯微鏡(OLYMPUS IX73)觀測(cè)并拍照,使用Cell Standard軟件測(cè)量卵細(xì)胞直徑,每個(gè)樣品至少測(cè)量100個(gè)卵細(xì)胞.于受精15min后取3個(gè)1mL重復(fù)樣品,加入幾滴40%福爾馬林置于4℃待觀察受精率.以受精膜鼓起為受精成功標(biāo)志,每個(gè)樣品至少觀察100個(gè)受精卵.

1.2.7 子代海膽胚胎早期發(fā)育觀察 分別于受精后24h(原腸胚期)和48h(長(zhǎng)腕幼蟲(chóng)期)取樣,每重復(fù)組各取4個(gè)2mL重復(fù)樣品并滴入40%福爾馬林放于4 ℃,在24h內(nèi)完成觀察,并記錄畸形率.正常發(fā)育的胚胎形態(tài)應(yīng)滿(mǎn)足4個(gè)條件[37-38]:(1)胚胎在受精后24h進(jìn)入原腸胚時(shí)期,受精后48h進(jìn)入長(zhǎng)腕幼蟲(chóng)期;(2)胚胎呈現(xiàn)左、右和背、腹側(cè)對(duì)稱(chēng);(3)原腸期具備發(fā)育良好的原腸,長(zhǎng)腕幼蟲(chóng)期具備結(jié)構(gòu)完整的消化道(口、胃、腸);(4)長(zhǎng)腕幼蟲(chóng)期具備發(fā)育良好的骨針和腕.

1.2.8 綜合毒性指數(shù)(ITI)[35]根據(jù)發(fā)育是否延遲和形態(tài)是否畸形,每個(gè)胚胎賦予從0~10不同分值.24h正常晚期原腸胚為0分,原腸胚、囊胚和桑椹胚各得1,3和4分;當(dāng)胚胎出現(xiàn)畸形形態(tài)時(shí),原腸胚、囊胚和桑椹胚各得5,7和10分.48h正常長(zhǎng)腕幼蟲(chóng)為0分,早期長(zhǎng)腕幼蟲(chóng)、棱柱幼蟲(chóng)、原腸胚、囊胚和桑椹胚各得2,3,4,5和5.5分;當(dāng)胚胎出現(xiàn)畸形形態(tài)時(shí),長(zhǎng)腕幼蟲(chóng)、早期長(zhǎng)腕幼蟲(chóng)、棱柱幼蟲(chóng)、原腸胚、囊胚和桑椹胚各得6,7,7.5,8,9和10分.ITI根據(jù)以下公式計(jì)算:

式中:S為每類(lèi)畸形胚胎得分;F為這類(lèi)胚胎出現(xiàn)的頻率(=10).

圖2 不同親本組合的子代海膽暴露方法示意

Fig.2 Schematic exposure regime of offspring derived from different parental crosses of sea urchins

I,II和III指子代暴露實(shí)驗(yàn)的3個(gè)重復(fù)組.步驟(1):對(duì)照組海膽的配子平均分成11份;步驟(2):各暴露組海膽的配子均分2份;步驟(3):受精過(guò)程.另有一組來(lái)自于對(duì)照雙親的子代胚胎在FSW中培養(yǎng)

1.2.9 暴露溶液組分分析方法暴露期間每隔24h取水樣分析暴露液TPH濃度和PAHs濃度. TPH采用紫外法測(cè)定(BIOTEK EPOCH2)[39].各濃度組各取3個(gè)混合流出液重復(fù)樣品,正己烷萃取,并于225nm下正己烷調(diào)零測(cè)定吸光度.根據(jù)以下標(biāo)準(zhǔn)曲線(xiàn)公式計(jì)算TPH濃度:

y

= 0.051

x

-0.0033 (2)

式中:為樣品萃取液吸光度與正己烷吸光度差值;為萃取液TPH濃度,mg/L;2=0.9996.

PAHs濃度采用氣相色譜/質(zhì)譜聯(lián)用法(GC/MS)測(cè)定.樣品前處理步驟參照GB/T 21247-2007《海面溢油鑒別系統(tǒng)規(guī)范》[40].儀器型號(hào)GC(HP 6890GC)- MS(5975),選擇SIM模式,內(nèi)標(biāo)法進(jìn)行定量分析.色譜柱為DB-5MSUI,長(zhǎng)30m,內(nèi)徑0.32mm,膜厚度0.25μm.載氣為高純氮?dú)?流量1mL/min.升溫程序:50℃保持2min,以8℃/min的速度升溫至150℃,保持3min;再以3℃/min速度升溫至300℃,保持15min.分析16種PAHs:萘,苊烯,苊,芴,菲,蒽,熒蒽,芘,苯并[a]蒽,苯并[b]熒蒽,苯并[k]熒蒽,苯并[a]芘,茚并[1,2,3-cd]芘,二苯并[a,h]蒽,苯并[ghi]芘.

1.2.10 數(shù)據(jù)統(tǒng)計(jì)分析 所有生物測(cè)定結(jié)果均以3個(gè)重復(fù)組測(cè)定值的(平均值±標(biāo)準(zhǔn)差)表示.采用SPSS 19.0軟件進(jìn)行數(shù)據(jù)的正態(tài)性檢驗(yàn)(Shapiro–Wilk法)和方差同質(zhì)性檢驗(yàn)(Levene法).若數(shù)據(jù)滿(mǎn)足以上條件則進(jìn)行方差分析,事后檢驗(yàn)方法采用Tukey HSD,若方差不齊則事后檢驗(yàn)采用Dunnett法.若數(shù)據(jù)不滿(mǎn)足以上2個(gè)條件,則采用非參數(shù)檢驗(yàn)的Kruskal Wallis法和Mann Whitney法進(jìn)行顯著性差異分析.不同發(fā)育時(shí)期之間的差異采用配對(duì)T檢驗(yàn)法.<0.05表示差異顯著.TPH濃度為3個(gè)重復(fù)樣品的(平均值±標(biāo)準(zhǔn)差),使用OriginPro軟件對(duì)TPH濃度和PAHs濃度進(jìn)行擬合.

2 結(jié)果與討論

2.1 暴露溶液組分分析

2.1.1 TPH濃度隨時(shí)間和礫石粘油量的變化 粘油礫石柱是一種模擬近岸處沉降溢油的動(dòng)態(tài)系統(tǒng)[41].結(jié)果表明隨著海水流經(jīng)粘油礫石柱,所有濃度組的流出液中的TPH濃度呈指數(shù)形式降低,并具有良好相關(guān)性(2>0.95)(見(jiàn)表1).這與溢油事故發(fā)生后實(shí)際測(cè)得的近岸處海水TPH濃度變化趨勢(shì)一致[42].此外,根據(jù)擬合公式可對(duì)毒性進(jìn)行定量表征,克服了動(dòng)態(tài)暴露系統(tǒng)中暴露溶液濃度難以預(yù)測(cè)的缺點(diǎn).

分析不同粘油量礫石柱在經(jīng)海水沖洗相同時(shí)間后流出液中TPH濃度變化,發(fā)現(xiàn)TPH濃度與礫石粘油量呈正相關(guān)(2>0.84)(見(jiàn)表2).

表1 流出液中TPH濃度隨時(shí)間變化擬合參數(shù)

注:擬合公式:=1exp(/1) +0,式中為流出液中TPH濃度,μg/L;為暴露時(shí)間,d.

表2 流出液中TPH濃度隨礫石粘油量變化擬合參數(shù)

注:擬合公式:=x,式中為流出液中TPH濃度,μg/L;為礫石粘油量,μg/g.

2.1.2 PAHs隨時(shí)間的變化及其與TPH的關(guān)系 親代海膽暴露期間溶液中PAHs濃度逐漸減小,并且各種組分之間的相對(duì)含量也有所變化.例如,6400μg/g組暴露溶液中的PAHs含量在親代暴露期間由最初的13.140μg/L降低到結(jié)束時(shí)的3.530μg/L.開(kāi)始暴露時(shí),暴露溶液中最主要的PAHs為萘(84.4%),其次為菲(7.3%).萘的分子量相對(duì)低且易揮發(fā),暴露結(jié)束時(shí)萘的含量?jī)H占了38.2%,菲的相對(duì)含量增加到了27.5%[7].6400μg/g組粘油礫石上的PAHs濃度由9.806μg/g降低到了6.408μg/g,各組分的相對(duì)含量也發(fā)生改變,萘相對(duì)含量由28.4%降低到15.8%,而菲的相對(duì)含量由23.1%升高到28.2%.

通過(guò)分析6400μg/g組暴露溶液中PAHs濃度及其對(duì)應(yīng)的TPH濃度在7d內(nèi)的變化趨勢(shì),發(fā)現(xiàn)盡管PAHs的各種組分相對(duì)含量有所不同,但是PAHs濃度和TPH濃度在暴露期間呈現(xiàn)良好相關(guān)性(2= 0.866).考慮到TPH濃度與礫石粘油量呈良好相關(guān)性,因此本文中毒性均以礫石粘油量進(jìn)行對(duì)比分析.

2.2 對(duì)親代海膽生長(zhǎng)繁殖的影響

暴露在粘油礫石柱流出液中7d后,海膽排配子率顯著降低(Kruskal-Wallis,=0.033).粘油礫石對(duì)海膽排配子率的影響無(wú)性別差異性(Mann Whitney,>0.05).另外,與對(duì)照組相比((5551667±714587)個(gè)卵細(xì)胞),6400μg/g組雌海膽排卵數(shù)量減少((1957917± 811471)個(gè)卵細(xì)胞),繁殖力顯著降低(1-way ANOVA,=0.036),表明粘油礫石流出液7d暴露影響了海膽的繁殖狀態(tài).這可能與壓力環(huán)境下海膽性腺內(nèi)性細(xì)胞再吸收作用相關(guān),性細(xì)胞的再吸收可以使海膽將更多的能量分配到細(xì)胞解毒和保護(hù)過(guò)程中.

2.3 對(duì)配子質(zhì)量的影響

暴露組卵細(xì)胞的大小與對(duì)照組相比無(wú)顯著性差異(1-way ANOVA,>0.05).結(jié)果表明,粘油礫石暴露對(duì)卵細(xì)胞的尺寸無(wú)顯著效應(yīng),說(shuō)明卵細(xì)胞的大小不適合作為監(jiān)測(cè)短期粘油礫石暴露對(duì)成年海膽繁殖狀態(tài)影響的指標(biāo).已有研究發(fā)現(xiàn)海膽種群密度大的區(qū)域雌海膽所排卵細(xì)胞較小,而種群密度小的區(qū)域雌海膽所排卵細(xì)胞較大,說(shuō)明卵細(xì)胞的大小具有可塑性[43].Sch?fer等[44]的研究發(fā)現(xiàn)菲的濃度為500μg/L暴露20d,抑制海膽性腺內(nèi)未成熟卵細(xì)胞的生長(zhǎng).據(jù)此推斷本研究中卵細(xì)胞尺寸未發(fā)生變化可能是因?yàn)楸┞稌r(shí)間較短,雌海膽未能及時(shí)調(diào)整卵細(xì)胞大小.

親代海膽暴露在粘油礫石柱流出液中7d對(duì)受精率并無(wú)顯著影響(2-way ANOVA,>0.05),最大受精率為800μg/g組的雙本暴露受精卵((99.23± 0.69)%).結(jié)果表明,雄海膽短期暴露于粘油礫石流出液中并未影響精子的受精能力,與其他學(xué)者的研究結(jié)果一致[28,46].

2.4 對(duì)子代海膽胚胎早期發(fā)育的影響

將各組子代繼續(xù)暴露在相同濃度的流出液中培養(yǎng)48h,并在胚胎受精后24h原腸期(圖3)和48h長(zhǎng)腕幼蟲(chóng)期(圖4)觀測(cè)子代畸形率,并計(jì)算綜合毒性指數(shù)(ITI,表3).生長(zhǎng)在FSW中的對(duì)照組雙親子代畸形率始終低于10%.

礫石粘油量(μg/g)

不同字母表示具有顯著性差異,大寫(xiě)字母表示同一親本組合不同濃度組之間存在顯著性差異,<0.05;小寫(xiě)字母表示同一濃度不同親本組合之間存在顯著性差異,<0.05.對(duì)照指雙親為對(duì)照組海膽的子代,母本、父本和雙本指來(lái)自暴露親本的子代.虛線(xiàn)指來(lái)自對(duì)照組雙親的子代在FSW中培養(yǎng)的畸形率.下同

礫石粘油量(μg/g)

2.4.1 親代暴露史對(duì)子代的影響 由圖3和圖4可知,受精后24和48h,對(duì)于來(lái)自于對(duì)照組雙親的胚胎,隨著子代暴露濃度增加,畸形率逐漸升高(ITI分別為0.58~0.92和1.38~2.76)(1-way ANOVA,<0.05).為了研究親代海膽暴露于海底HFO對(duì)子代影響中的母本效應(yīng)和父本效應(yīng),暴露組的卵細(xì)胞和精子分別與對(duì)照組的精子和卵細(xì)胞受精,胚胎繼續(xù)暴露于其親本的暴露濃度.隨著親本暴露濃度和子代暴露濃度的增加,母本暴露(ITI分別為0.54~1.45和1.1~ 2.57)、父本暴露(ITI分別為0.82~1.95和1.89~4.04)和雙本暴露(ITI分別為0.88~2.08和1.91~4.14)的子代的畸形率顯著增加(1-way ANOVA,<0.05).受精后24h,高濃度組(1600,3200和6400μg/g)的母本暴露、父本暴露和雙本暴露的子代畸形率均顯著高于對(duì)應(yīng)組對(duì)照雙親的子代畸形率(1-way ANOVA,<0.05).表明親代海膽暴露在粘油礫石流出液7d后,對(duì)子代胚胎發(fā)育產(chǎn)生毒性.Zhadan等[33]將海膽暴露于0.04~0.3mg/L的柴油水溶液中50d,并測(cè)定了卵細(xì)胞內(nèi)鈣濃度和子代發(fā)育情況,發(fā)現(xiàn)長(zhǎng)期暴露于低濃度石油烴中雌海膽的卵細(xì)胞內(nèi)具有顯著升高的鈣離子濃度,并且由這種卵細(xì)胞發(fā)育而來(lái)的子代胚胎畸形率也相應(yīng)升高,這與本研究結(jié)果一致.

表3 親代海膽暴露于粘油礫石柱產(chǎn)生的子代胚胎繼續(xù)暴露24和48h的綜合毒性指數(shù)

注:加粗字體為綜合毒性指數(shù)與畸形率變化趨勢(shì)不同的組別.

杜青平等[47]的研究結(jié)果表明,成年斑馬魚(yú)暴露在三氯苯中引起子代發(fā)育畸形.而Carls等[48]的研究結(jié)果表明,母本暴露在油中并沒(méi)有對(duì)魚(yú)的子代胚胎早期發(fā)育產(chǎn)生影響.這種差異可能與物種對(duì)污染物的敏感性不同有關(guān)[19].另外,本研究結(jié)果表明雄海膽的暴露對(duì)其子代發(fā)育也會(huì)產(chǎn)生影響.Lewis等[46]同樣發(fā)現(xiàn)暴露在苯并芘中72h的雄貽貝所產(chǎn)子代的畸形率增加.

2.4.2 親代暴露對(duì)子代影響的性別差異 隨著胚胎發(fā)育的進(jìn)行,48h時(shí)雙本和父本暴露的子代畸形率顯著高于對(duì)應(yīng)濃度組的母本暴露子代和對(duì)照組子代的畸形率(圖3,1-way ANOVA,<0.05).母本暴露子代的畸形率與對(duì)照組子代的畸形率無(wú)顯著差異(圖3,1-way ANOVA,>0.05).當(dāng)暴露親代為雌海膽時(shí),對(duì)子代發(fā)育的影響最小,這可能與雌海膽具有較高的抗氧化能力有關(guān).Sch?fer等[44]報(bào)道雌海膽性腺與雄海膽相比具有更高的抗壞血酸濃度和較低的氧化損傷程度.Sch?fer等[45]研究菲對(duì)雌海膽性腺損傷時(shí)發(fā)現(xiàn)主要受到影響的是未成熟的卵細(xì)胞,而已成熟的卵細(xì)胞并未遭受氧化損傷.Lister等[29]在研究膳食PAHs暴露對(duì)海膽繁殖損傷和子代的影響時(shí),發(fā)現(xiàn)暴露組海膽產(chǎn)生的卵細(xì)胞的氧化損傷程度與對(duì)照組無(wú)顯著性差異.據(jù)此作者推斷,親代海膽暴露在粘油礫石柱流出液中7d,由于雌海膽具有較高的抗氧化能力和對(duì)已成熟卵細(xì)胞具有保護(hù)作用,因此與精子相比,卵細(xì)胞受到的損傷更小,對(duì)子代的影響也較小.

海膽屬體外受精動(dòng)物,在受精之前精子和卵細(xì)胞被排放到海水中,精子和卵細(xì)胞對(duì)子代的發(fā)育具有同等的重要性[49].然而大多數(shù)野外和室內(nèi)的毒理研究側(cè)重于雌海膽暴露對(duì)子代胚胎發(fā)育的影響[29,31-34].本研究通過(guò)對(duì)來(lái)自父本暴露和對(duì)照組雙親的海膽子代胚胎畸形率比較,發(fā)現(xiàn)暴露在HFO中的父本對(duì)子代具有不可忽視的效應(yīng).這種父本效應(yīng)可能來(lái)源于3種機(jī)制.其一,父本的生活環(huán)境對(duì)精子的形成具有選擇性作用[50],屬于環(huán)境誘導(dǎo)的父本效應(yīng).其二,父本受到的環(huán)境壓力會(huì)使精子的基因表達(dá)情況發(fā)生改變,表觀遺傳突變不僅可以穩(wěn)定的遺傳給子代并且可以在子代中表達(dá)[51].其三,在環(huán)境壓力的脅迫下父本的DNA受到損傷[46],也會(huì)引起子代畸形率的增加.事實(shí)上,在海洋無(wú)脊椎動(dòng)物種群動(dòng)態(tài)研究中發(fā)現(xiàn)精子是主要的限制因素[52-53].綜合上述結(jié)果,本研究表明粘油礫石柱所模擬的海底HFO對(duì)雄海膽的精子損傷將會(huì)嚴(yán)重威脅到其子代胚胎的發(fā)育和種群的生存.

2.4.3 子代不同發(fā)育時(shí)期畸形率的差異 對(duì)比同一組胚胎在不同發(fā)育時(shí)期畸形率的差異,發(fā)現(xiàn)48h畸形率顯著高于24h畸形率(圖3和4,配對(duì)T檢驗(yàn),<0.05),表明隨著暴露時(shí)間延長(zhǎng),致畸效應(yīng)更加明顯.隨著胚胎發(fā)育的進(jìn)行,48h母本暴露對(duì)子代的致畸作用逐漸降低,而父本暴露對(duì)子代的致畸作用越加明顯.這可能與海膽早期胚胎發(fā)育過(guò)程中基因表達(dá)模式有關(guān).有研究證明,囊胚期之前的胚胎發(fā)育過(guò)程由卵細(xì)胞中的母源mRNA控制,隨著發(fā)育的進(jìn)行受精卵的基因開(kāi)始表達(dá)并逐步占據(jù)主導(dǎo)地位[54-55].因此父本效應(yīng)隨著胚胎發(fā)育而逐步顯現(xiàn)出來(lái).

2.4.4 子代綜合毒性指數(shù)ITI 暴露海膽親本產(chǎn)生的子代ITI見(jiàn)表3.結(jié)果表明除最高濃度組(6400μg/g),母本暴露組24h子代與來(lái)自于對(duì)照組雙親24h子代的ITI無(wú)顯著性差異(表3,1-way ANOVA,>0.05).而畸形率在這兩類(lèi)親本組合的24h子代之間存在顯著性差異(圖3,1-way ANOVA,<0.05).導(dǎo)致畸形率與ITI變化趨勢(shì)不同的原因是母本子代胚胎的畸形類(lèi)型中延遲發(fā)育的胚胎較多,致使ITI降低.母本暴露組48h子代的ITI與來(lái)自對(duì)照組親本的48h子代無(wú)顯著性差異(表3,1-way ANOVA,>0.05),這與48h畸形率變化趨勢(shì)相同,除了3200μg/g組.在3200μg/g組,母本暴露的子代延遲發(fā)育較多,使其ITI顯著低于對(duì)照組子代ITI.上述結(jié)果表明,與父本暴露相比,母本暴露對(duì)子代的影響較小,主要表現(xiàn)為胚胎畸形程度較輕.

3 結(jié)論

3.1 本文采用粘油礫石柱模擬海底HFO,在7d內(nèi)間隙水中TPH濃度呈現(xiàn)指數(shù)式衰減,與溢油事故發(fā)生后實(shí)時(shí)監(jiān)測(cè)的近岸TPH濃度變化趨勢(shì)一致,因此該暴露裝置能有效的模擬實(shí)際環(huán)境中HFO污染情況.

3.2 試驗(yàn)濃度下(400~6400μg/g)海底HFO對(duì)近岸底棲生物海膽產(chǎn)生毒性效應(yīng),雌雄海膽的排配子率和雌海膽繁殖力在最高濃度組(6400μg/g)受到顯著抑制.

3.3 暴露于被海底HFO污染的孔隙水中7d,海膽卵細(xì)胞的大小和精子的受精能力并未受到顯著影響.

3.4 繁殖期海膽受到HFO脅迫會(huì)顯著增加子代胚胎發(fā)育的畸形率,HFO對(duì)親代海膽的毒性傳遞給了子代.

3.5 與母本暴露相比,父本暴露對(duì)子代的影響較大,主要表現(xiàn)為胚胎畸形程度較重,并且ITI較高,即在HFO毒性由親代傳遞給子代的過(guò)程中,父本效應(yīng)起著關(guān)鍵作用.

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Exposure of adult sea urchins to sunken heavy fuel oil affects the reproductive status and the development of their offspring.

DUAN Mei-na1, LIU Yong-jiang1, BAI Xue1, GAO Xiang1, ZHANG Xin-xin2, XIONG De-qi1*

(1.Department of Environmental Science and Engineering, Dalian Maritime University, Dalian 116026, China；2.Technology of Oily Sludge Eco-Treatment, Dalian 116000, China)., 2018,38(12)：4720~4729

The present study investigated effects of exposure to sunken heavy fuel oil (HFO) on the fecundity, gamete quality and development of the offspring in the sea urchin. Adult sea urchins were exposed to effluents from HFO-oiled gravel columns for 7days to simulate an oil contaminated gravel shore. The spawning ability of adults and the fecundity ((1957917±811471) eggs) of females significantly decreased (=0.033 and=0.036, respectively). No effect was observed on the egg size and fertilization success. However, a significant increase in the percentage of abnormality of the offspring were observed, demonstrating that parental exposure (especially paternal exposure) had adverse effects on the offspring. The offspring from exposed fathers showed higher ITI values (ITI values of 24 and 48h offspring were 0.82~1.95 and 1.89~4.04, respectively) with a higher number of malformed embryos compared to maternal exposure (ITI values of 24 and 48h offspring were 0.54~1.45 and 1.1~2.57, respectively), indicating that detrimental effects of sunken HFO on the early development of sea urchin embryos may be largely attributed to paternal effects.

sunken heavy fuel oil；interstitial water；parental effects；sea urchins；early development

X55

A

1000-6923(2018)12-4720-10

段美娜(1991-),女,黑龍江七臺(tái)河人,大連海事大學(xué)博士研究生,主要從事溢油毒理相關(guān)研究.發(fā)表論文7篇.

2018-05-10

國(guó)家自然科學(xué)基金資助項(xiàng)目(41276105)

* 責(zé)任作者, 教授, xiongdq@dlmu.edu.cn