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.大連海事大學環(huán)境科學與工程學院,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)

利用室內流水式粘油礫石柱模擬實際環(huán)境中的海底重燃油,研究了重燃油污染的孔隙水對成年海膽繁殖力、配子質量及子代胚胎發(fā)育的影響.結果表明,暴露結束后(7d),暴露組海膽的排配子率顯著降低(=0.033),同時雌海膽繁殖力也顯著降低(=0.036,(1957917±811471)個卵細胞);卵細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.進一步利用整合毒性指數(shù)(ITI)檢測毒性傳遞的性別差異發(fā)現(xiàn),與母本效應相比(24和48h子代的ITI分別為0.54~1.45和1.1~2.57),父本效應(24和48h子代的ITI分別為0.82~1.95和1.89~4.04)在毒性傳遞過程中起著關鍵作用.

海底重燃油；孔隙水；親本效應；海膽；早期發(fā)育

1970~2016年間,世界范圍內,發(fā)生了超過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是一種原油精煉產品.HFO黏度大(15℃運動黏度5000~ 30000mPa·s),分散和自然降解十分困難.在風浪作用下,HFO可以出現(xiàn)在距溢油事故發(fā)生地點相當遠的地方,使海岸線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)對水生生物產生毒害作用,這個過程就是孔隙水毒性假說[4].

已有學者在實驗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)魚類胚胎的畸形率和死亡率與流經粘油礫石的水體中PAHs濃度相關[5-9].事實上,海洋底棲生物,由于棲息在海底,不僅會暴露在有毒孔隙水中,還可能與海底HFO直接接觸,加之其活動能力低下難以逃出污染區(qū),勢必會受到海底HFO的脅迫,因此海底HFO對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,對淺海生物群落的組成、結構和多樣性起著關鍵的作用[10-13].海膽生活史包括浮游幼體和底棲成體兩個主要階段.不僅早期浮游幼體對污染敏感性高[14-21],其成年階段也被視作海洋生態(tài)毒理學研究和環(huán)境監(jiān)測的有效生物模型[22-26].成年動物的繁殖能力對整個物種的延續(xù)起著至關重要的作用,海底HFO對成年海膽的毒害作用勢必會影響到其體內配子的生成、排放、受精過程,進一步影響子代發(fā)育,從而對整個種群的生存產生影響[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對親代成年海膽繁殖能力、配子質量及其子代胚胎早期發(fā)育的影響.以期為評估溢油對海膽種群數(shù)量的影響提供參考數(shù)據,同時為海洋生態(tài)風險評估、自然資源損害評估以及船舶溢油索賠提供重要科學依據.

1 材料與方法

1.1 材料

1.1.1 實驗油品 HFO380,運動黏度729800mPa·s (50℃),密度0.9821g/cm3,由大連海洋燃油有限公司提供.

1.1.2 實驗礫石 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 實驗海水 取自大連市星海灣,鹽度(34±1) PSU,pH=(8.0±0.03).

1.1.4 實驗海膽馴化繁殖期成年海膽購自大連海寶漁業(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驗室海水循環(huán)系統(tǒng)(大連匯鋅鈦設備)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.暴露實驗開始前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處24h,用于暴露實驗.實驗設置粘油礫石濃度為0,400,800,1600,3200和6400μg油/g礫石(下文簡寫為μg/g).0μg/g為對照組.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的雌海膽所產子代的畸形率增加;在此基礎上,通過預實驗本文選擇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.進一步預實驗發(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)子代的暴露實驗,而4000μg/g組的雌海膽產卵數(shù)量足以用于后續(xù)實驗.因此本文最高濃度組的粘油量應介于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,并保證有足夠的卵細胞用于后續(xù)暴露實驗.

1.2.2 動態(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處有一進水管(d=1.2cm),出水管(d=1.2cm)位于對面距頂部3cm處,底部配備一個由PVC支柱(H=3.5cm)支撐的PVC網孔圓板放置礫石(圖1).進水流速以針閥控制.粘油礫石制備完成后,轉移至該裝置,海水流速20mL/min由下至上流經粘油礫石柱,24h后盛接流出液進行暴露實驗.

1.2.3 親代海膽暴露方法每個濃度組設置3個重復,每個重復包括6只雌海膽和6只雄海膽,雌雄海膽分開暴露(圖1).暴露容器上部開孔,保證暴露溶液體積為6L.暴露時間為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 親代海膽暴露結束后,用0.45μm濾膜過濾海水(FSW)清洗體表,然后經圍口膜向海膽體腔內注射1mL現(xiàn)配的KCl溶液(0.5M).雌海膽口面向上置于盛滿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,將每重復組6只雌海膽所產卵細胞混合,置于18℃的FSW中暫放待受精.每重復組的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/mL的卵細胞溶液中,輕輕晃動進行受精[35].胚胎親本組合有4種(如圖2所示):對照組(對照組卵細胞和對照組精子受精),母本暴露組(暴露組卵細胞和對照組精子受精),父本暴露組(對照組卵細胞和暴露組精子受精)和雙本暴露組(暴露組卵細胞和同組精子受精).受精15min后,虹吸法洗卵3次以去除多余的精子.

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

圖1 粘油礫石柱和成年海膽動態(tài)暴露方法示意

該示意圖為6個粘油礫石濃度組(0,400,800,1600,3200和6400μg/g)之一的模式圖;I、II和III指親代暴露實驗的3個重復組;水流動方向在圖中以黑色箭頭標注

1.2.5 親代海膽相關參數(shù)測定 排配子率指排配子的雌或雄海膽個數(shù)與對應性別的海膽總數(shù)的比值.繁殖力指雌海膽開始排卵的30min內排出的卵的個數(shù)[36],其中將未排卵的雌海膽繁殖力記為0.卵細胞計數(shù)使用0.1mL的浮游生物計數(shù)框進行,每只海膽重復計數(shù)4次并取平均值作為一只海膽的繁殖力.

1.2.6 配子質量 將每重復組6只雌海膽的卵細胞混合,取3個1mL重復樣品,滴入幾滴40%福爾馬林,置于4℃待測.顯微鏡(OLYMPUS IX73)觀測并拍照,使用Cell Standard軟件測量卵細胞直徑,每個樣品至少測量100個卵細胞.于受精15min后取3個1mL重復樣品,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志,每個樣品至少觀察100個受精卵.

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(長腕幼蟲期)取樣,每重復組各取4個2mL重復樣品并滴入40%福爾馬林放于4 ℃,在24h內完成觀察,并記錄畸形率.正常發(fā)育的胚胎形態(tài)應滿足4個條件[37-38]:(1)胚胎在受精后24h進入原腸胚時期,受精后48h進入長腕幼蟲期;(2)胚胎呈現(xiàn)左、右和背、腹側對稱;(3)原腸期具備發(fā)育良好的原腸,長腕幼蟲期具備結構完整的消化道(口、胃、腸);(4)長腕幼蟲期具備發(fā)育良好的骨針和腕.

1.2.8 綜合毒性指數(shù)(ITI)[35]根據發(fā)育是否延遲和形態(tài)是否畸形,每個胚胎賦予從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;當胚胎出現(xiàn)畸形形態(tài)時,原腸胚、囊胚和桑椹胚各得5,7和10分.48h正常長腕幼蟲為0分,早期長腕幼蟲、棱柱幼蟲、原腸胚、囊胚和桑椹胚各得2,3,4,5和5.5分;當胚胎出現(xiàn)畸形形態(tài)時,長腕幼蟲、早期長腕幼蟲、棱柱幼蟲、原腸胚、囊胚和桑椹胚各得6,7,7.5,8,9和10分.ITI根據以下公式計算:

式中:S為每類畸形胚胎得分;F為這類胚胎出現(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指子代暴露實驗的3個重復組.步驟(1):對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):受精過程.另有一組來自于對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采用紫外法測定(BIOTEK EPOCH2)[39].各濃度組各取3個混合流出液重復樣品,正己烷萃取,并于225nm下正己烷調零測定吸光度.根據以下標準曲線公式計算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/質譜聯(lián)用法(GC/MS)測定.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GC(HP 6890GC)- MS(5975),選擇SIM模式,內標法進行定量分析.色譜柱為DB-5MSUI,長30m,內徑0.32mm,膜厚度0.25μm.載氣為高純氮氣,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)據統(tǒng)計分析 所有生物測定結果均以3個重復組測定值的(平均值±標準差)表示.采用SPSS 19.0軟件進行數(shù)據的正態(tài)性檢驗(Shapiro–Wilk法)和方差同質性檢驗(Levene法).若數(shù)據滿足以上條件則進行方差分析,事后檢驗方法采用Tukey HSD,若方差不齊則事后檢驗采用Dunnett法.若數(shù)據不滿足以上2個條件,則采用非參數(shù)檢驗的Kruskal Wallis法和Mann Whitney法進行顯著性差異分析.不同發(fā)育時期之間的差異采用配對T檢驗法.<0.05表示差異顯著.TPH濃度為3個重復樣品的(平均值±標準差),使用OriginPro軟件對TPH濃度和PAHs濃度進行擬合.

2 結果與討論

2.1 暴露溶液組分分析

2.1.1 TPH濃度隨時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態(tài)系統(tǒng)[41].結果表明隨著海水流經粘油礫石柱,所有濃度組的流出液中的TPH濃度呈指數(shù)形式降低,并具有良好相關性(2>0.95)(見表1).這與溢油事故發(fā)生后實際測得的近岸處海水TPH濃度變化趨勢一致[42].此外,根據擬合公式可對毒性進行定量表征,克服了動態(tài)暴露系統(tǒng)中暴露溶液濃度難以預測的缺點.

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

表1 流出液中TPH濃度隨時間變化擬合參數(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隨時間的變化及其與TPH的關系 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降低到結束時的3.530μg/L.開始暴露時,暴露溶液中最主要的PAHs為萘(84.4%),其次為菲(7.3%).萘的分子量相對低且易揮發(fā),暴露結束時萘的含量僅占了38.2%,菲的相對含量增加到了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,各組分的相對含量也發(fā)生改變,萘相對含量由28.4%降低到15.8%,而菲的相對含量由23.1%升高到28.2%.

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

2.2 對親代海膽生長繁殖的影響

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).粘油礫石對海膽排配子率的影響無性別差異性(Mann Whitney,>0.05).另外,與對照組相比((5551667±714587)個卵細胞),6400μg/g組雌海膽排卵數(shù)量減少((1957917± 811471)個卵細胞),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)境下海膽性腺內性細胞再吸收作用相關,性細胞的再吸收可以使海膽將更多的能量分配到細胞解毒和保護過程中.

2.3 對配子質量的影響

暴露組卵細胞的大小與對照組相比無顯著性差異(1-way ANOVA,>0.05).結果表明,粘油礫石暴露對卵細胞的尺寸無顯著效應,說明卵細胞的大小不適合作為監(jiān)測短期粘油礫石暴露對成年海膽繁殖狀態(tài)影響的指標.已有研究發(fā)現(xiàn)海膽種群密度大的區(qū)域雌海膽所排卵細胞較小,而種群密度小的區(qū)域雌海膽所排卵細胞較大,說明卵細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,抑制海膽性腺內未成熟卵細胞的生長.據此推斷本研究中卵細胞尺寸未發(fā)生變化可能是因為暴露時間較短,雌海膽未能及時調整卵細胞大小.

親代海膽暴露在粘油礫石柱流出液中7d對受精率并無顯著影響(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,與其他學者的研究結果一致[28,46].

2.4 對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長腕幼蟲期(圖4)觀測子代畸形率,并計算綜合毒性指數(shù)(ITI,表3).生長在FSW中的對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.對照指雙親為對照組海膽的子代,母本、父本和雙本指來自暴露親本的子代.虛線指來自對照組雙親的子代在FSW中培養(yǎng)的畸形率.下同

礫石粘油量(μg/g)

2.4.1 親代暴露史對子代的影響 由圖3和圖4可知,受精后24和48h,對于來自于對照組雙親的胚胎,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對子代影響中的母本效應和父本效應,暴露組的卵細胞和精子分別與對照組的精子和卵細胞受精,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應組對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,對子代胚胎發(fā)育產生毒性.Zhadan等[33]將海膽暴露于0.04~0.3mg/L的柴油水溶液中50d,并測定了卵細胞內鈣濃度和子代發(fā)育情況,發(fā)現(xiàn)長期暴露于低濃度石油烴中雌海膽的卵細胞內具有顯著升高的鈣離子濃度,并且由這種卵細胞發(fā)育而來的子代胚胎畸形率也相應升高,這與本研究結果一致.

表3 親代海膽暴露于粘油礫石柱產生的子代胚胎繼續(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)與畸形率變化趨勢不同的組別.

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

2.4.2 親代暴露對子代影響的性別差異 隨著胚胎發(fā)育的進行,48h時雙本和父本暴露的子代畸形率顯著高于對應濃度組的母本暴露子代和對照組子代的畸形率(圖3,1-way ANOVA,<0.05).母本暴露子代的畸形率與對照組子代的畸形率無顯著差異(圖3,1-way ANOVA,>0.05).當暴露親代為雌海膽時,對子代發(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.Sch?fer等[44]報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]研究菲對雌海膽性腺損傷時發(fā)現(xiàn)主要受到影響的是未成熟的卵細胞,而已成熟的卵細胞并未遭受氧化損傷.Lister等[29]在研究膳食PAHs暴露對海膽繁殖損傷和子代的影響時,發(fā)現(xiàn)暴露組海膽產生的卵細胞的氧化損傷程度與對照組無顯著性差異.據此作者推斷,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已成熟卵細胞具有保護作用,因此與精子相比,卵細胞受到的損傷更小,對子代的影響也較小.

海膽屬體外受精動物,在受精之前精子和卵細胞被排放到海水中,精子和卵細胞對子代的發(fā)育具有同等的重要性[49].然而大多數(shù)野外和室內的毒理研究側重于雌海膽暴露對子代胚胎發(fā)育的影響[29,31-34].本研究通過對來自父本暴露和對照組雙親的海膽子代胚胎畸形率比較,發(fā)現(xiàn)暴露在HFO中的父本對子代具有不可忽視的效應.這種父本效應可能來源于3種機制.其一,父本的生活環(huán)境對精子的形成具有選擇性作用[50],屬于環(huán)境誘導的父本效應.其二,父本受到的環(huán)境壓力會使精子的基因表達情況發(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[51].其三,在環(huán)境壓力的脅迫下父本的DNA受到損傷[46],也會引起子代畸形率的增加.事實上,在海洋無脊椎動物種群動態(tài)研究中發(fā)現(xiàn)精子是主要的限制因素[52-53].綜合上述結果,本研究表明粘油礫石柱所模擬的海底HFO對雄海膽的精子損傷將會嚴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)育時期畸形率的差異 對比同一組胚胎在不同發(fā)育時期畸形率的差異,發(fā)現(xiàn)48h畸形率顯著高于24h畸形率(圖3和4,配對T檢驗,<0.05),表明隨著暴露時間延長,致畸效應更加明顯.隨著胚胎發(fā)育的進行,48h母本暴露對子代的致畸作用逐漸降低,而父本暴露對子代的致畸作用越加明顯.這可能與海膽早期胚胎發(fā)育過程中基因表達模式有關.有研究證明,囊胚期之前的胚胎發(fā)育過程由卵細胞中的母源mRNA控制,隨著發(fā)育的進行受精卵的基因開始表達并逐步占據主導地位[54-55].因此父本效應隨著胚胎發(fā)育而逐步顯現(xiàn)出來.

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

3 結論

3.1 本文采用粘油礫石柱模擬海底HFO,在7d內間隙水中TPH濃度呈現(xiàn)指數(shù)式衰減,與溢油事故發(fā)生后實時監(jiān)測的近岸TPH濃度變化趨勢一致,因此該暴露裝置能有效的模擬實際環(huán)境中HFO污染情況.

3.2 試驗濃度下(400~6400μg/g)海底HFO對近岸底棲生物海膽產生毒性效應,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脅迫會顯著增加子代胚胎發(fā)育的畸形率,HFO對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程中,父本效應起著關鍵作用.

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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,黑龍江七臺河人,大連海事大學博士研究生,主要從事溢油毒理相關研究.發(fā)表論文7篇.

2018-05-10

國家自然科學基金資助項目(41276105)

* 責任作者, 教授, xiongdq@dlmu.edu.cn