鹽酸四環(huán)素濃度和食物密度對(duì)萼花臂尾輪蟲(chóng)生活史特征的綜合影響

項(xiàng)賢領(lǐng),朱曄璘,徐秋磊,朱凌云,席貽龍,2

1 安徽師范大學(xué)生命科學(xué)學(xué)院,蕪湖 241000 2 安徽省高校“生物環(huán)境與生態(tài)安全”省級(jí)重點(diǎn)實(shí)驗(yàn)室,蕪湖 241000

鹽酸四環(huán)素濃度和食物密度對(duì)萼花臂尾輪蟲(chóng)生活史特征的綜合影響

項(xiàng)賢領(lǐng)1,2,*,朱曄璘1,徐秋磊1,朱凌云1,席貽龍1,2

1 安徽師范大學(xué)生命科學(xué)學(xué)院,蕪湖 241000 2 安徽省高校“生物環(huán)境與生態(tài)安全”省級(jí)重點(diǎn)實(shí)驗(yàn)室,蕪湖 241000

近年來(lái),抗生素的環(huán)境污染問(wèn)題已引起人們的廣泛關(guān)注,其在環(huán)境中的殘留可對(duì)水生態(tài)系統(tǒng)的結(jié)構(gòu)和功能產(chǎn)生重要影響。迄今,鹽酸四環(huán)素濃度和食物密度對(duì)輪蟲(chóng)生活史特征的影響研究尚未見(jiàn)報(bào)道。以萼花臂尾輪蟲(chóng)(Brachionuscalyciflorus)為受試動(dòng)物,研究了在不同斜生柵藻(Scenedesmusobliquus)密度下,不同濃度的鹽酸四環(huán)素(Tetracycline hydrochloride)對(duì)萼花臂尾輪蟲(chóng)生活史特征的影響。結(jié)果表明,食物密度和鹽酸四環(huán)素濃度對(duì)輪蟲(chóng)的生命期望、凈生殖率、世代時(shí)間、內(nèi)稟增長(zhǎng)率、平均壽命和后代混交率,以及食物密度和鹽酸四環(huán)素濃度間交互作用對(duì)除了內(nèi)稟增長(zhǎng)率外的其他5個(gè)統(tǒng)計(jì)學(xué)參數(shù)均具有顯著影響。在各食物密度下,輪蟲(chóng)的特定年齡繁殖率高峰值隨著鹽酸四環(huán)素濃度的增加呈現(xiàn)先增加后降低的趨勢(shì)；鹽酸四環(huán)素對(duì)輪蟲(chóng)生長(zhǎng)和繁殖參數(shù)的影響呈現(xiàn)“低促高抑”的特點(diǎn)。3個(gè)食物密度下,高濃度的鹽酸四環(huán)素顯著提高了萼花臂尾輪蟲(chóng)的后代混交率,且在1.0×106個(gè)/mL藻密度下毒物濃度與輪蟲(chóng)后代混交率間具有顯著的劑量-效應(yīng)關(guān)系。食物密度的高低顯著影響鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)的毒性效應(yīng)。

鹽酸四環(huán)素；食物密度；萼花臂尾輪蟲(chóng)；生命表；生活史特征

四環(huán)素類抗生素(Tetracyclines, TCs)是由放線菌合成的一類廣譜抗生素,其對(duì)革蘭氏陽(yáng)性菌、革蘭氏陰性菌、立克次體和衣原體等致病微生物均具有較強(qiáng)的抑制和滅殺作用[1]。因其具有價(jià)廉、高效和廣譜等優(yōu)點(diǎn),四環(huán)素作為獸藥和飼料添加劑而被廣泛應(yīng)用于養(yǎng)殖業(yè)和畜牧業(yè)[2- 4]。研究表明,四環(huán)素類抗生素進(jìn)入生物體內(nèi)后,不能被機(jī)體完全吸收,其中有30%—90%會(huì)再以母體化合物或代謝產(chǎn)物的形式隨排泄物釋放到環(huán)境中[5-9],而目前使用的污水處理技術(shù)并不能完全消除抗生素殘留,進(jìn)而對(duì)人體健康和水生態(tài)系統(tǒng)構(gòu)成長(zhǎng)期潛在危害[2]。

目前,我國(guó)長(zhǎng)江三角洲、珠江三角洲等地區(qū)的地表水、地下水和畜禽水產(chǎn)養(yǎng)殖廢水中都可檢出四環(huán)素[10-12],且其在全球水環(huán)境中的濃度有逐漸升高的趨勢(shì)[13-15]。研究表明,殘留在環(huán)境中的四環(huán)素可以誘導(dǎo)環(huán)境中的微生物產(chǎn)生耐藥性,從而使得抗性基因在非靶生物間擴(kuò)散和傳播,且在河流和污水處理廠的出入水與活性污泥中均檢測(cè)到高水平的編碼四環(huán)素抗性基因[16-19],這給公共健康帶來(lái)極大隱患。抗生素的長(zhǎng)期超量使用還會(huì)對(duì)浮游動(dòng)植物和其他水生生物產(chǎn)生影響,最終改變水生態(tài)系統(tǒng)的結(jié)構(gòu)和功能[20-21]?；诎l(fā)光細(xì)菌的微毒(Microtox)急性毒性試驗(yàn)研究表明,鹽酸四環(huán)素(Tetracycline hydrochloride, TCH)在5大類20種常用抗生素中毒性最強(qiáng)[22]。研究表明,四環(huán)素能夠抑制綠藻蛋白質(zhì)的合成,并通過(guò)抑制葉綠體內(nèi)合成酶的活性,對(duì)藻類生長(zhǎng)產(chǎn)生抑制作用[20, 23-24]。此外,四環(huán)素還能夠影響浮游動(dòng)物的生長(zhǎng)與繁殖[25-26],抑制水生動(dòng)物多種酶的活性,如β-半乳糖苷酶,并造成魚(yú)類等較嚴(yán)重的DNA損傷。

輪蟲(chóng)(Rotifer)是廣泛分布于各類水體中的浮游動(dòng)物四大類群之一。因其體型微小、繁殖迅速、世代時(shí)間短和對(duì)污染物變化敏感等特點(diǎn),輪蟲(chóng)已作為模式動(dòng)物在環(huán)境監(jiān)測(cè)和生態(tài)毒理學(xué)研究中發(fā)揮著重要作用[27]。美國(guó)環(huán)保局已于1991年正式把萼花臂尾輪蟲(chóng)(Brachionuscalyciflorus)和褶皺臂尾輪蟲(chóng)(B.plicatilis)分別作為淡水和海水污染的測(cè)試生物列入國(guó)家測(cè)試標(biāo)準(zhǔn)[28]。自20世紀(jì)70年代以來(lái),已有諸多研究以輪蟲(chóng)為受試生物開(kāi)展了重金屬[29-33]、有機(jī)氯農(nóng)藥[34-39]、有機(jī)磷農(nóng)藥[40-41]和擬除蟲(chóng)菊酯類農(nóng)藥[42]等化合物的急、慢性毒性實(shí)驗(yàn)[43]。隨著抗生素在畜牧業(yè)和水產(chǎn)養(yǎng)殖業(yè)中的廣泛應(yīng)用,開(kāi)展輪蟲(chóng)對(duì)抗生素的應(yīng)激響應(yīng)研究顯得尤為重要。已有學(xué)者證實(shí),輪蟲(chóng)的生長(zhǎng)與繁殖對(duì)抗生素濃度的變化非常敏感,大多呈現(xiàn)出“低促高抑”的雙重效應(yīng)[21, 44- 46]。然而,有關(guān)鹽酸四環(huán)素對(duì)輪蟲(chóng)生活史特征的影響研究尚鮮有報(bào)道,僅見(jiàn)Araujo 和 McNair[47]開(kāi)展了鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)和褶皺臂尾輪蟲(chóng)的生活史參數(shù)和種群增長(zhǎng)的影響研究,結(jié)果表明,四環(huán)素對(duì)萼花臂尾輪蟲(chóng)的壽命和繁殖力均具有顯著影響,其中總生殖率對(duì)抗生素濃度的變化最為敏感,其對(duì)鹽酸四環(huán)素的1%抑制濃度(IC1)為3.91 mg/L,上述影響隨著四環(huán)素濃度的加大呈明顯的加劇趨勢(shì)。

作為重要的初級(jí)生產(chǎn)者,藻類在水體中可有效加速污染物降解,減緩毒物對(duì)浮游動(dòng)物的毒害作用,因而,污染物對(duì)浮游動(dòng)物的毒性作用會(huì)常常受到食物密度等因素的制約[34]。近年來(lái),食物密度和污染物濃度對(duì)輪蟲(chóng)種群增長(zhǎng)的交互作用正逐漸引起了人們的關(guān)注[48]。大量研究表明,水體污染物可抑制輪蟲(chóng)的種群增長(zhǎng),而較高的食物密度則能夠降低毒物對(duì)輪蟲(chóng)種群增長(zhǎng)的抑制作用[49- 53]。迄今,不同食物密度下鹽酸四環(huán)素對(duì)輪蟲(chóng)生活史特征的影響研究尚未見(jiàn)報(bào)道。

本文以萼花臂尾輪蟲(chóng)為對(duì)象,采用生命表統(tǒng)計(jì)方法,研究了3個(gè)食物密度下的不同四環(huán)素濃度對(duì)萼花臂尾輪蟲(chóng)生命表參數(shù)的影響,旨在了解亞致死濃度的鹽酸四環(huán)素對(duì)輪蟲(chóng)生長(zhǎng)和繁殖的影響及其與藻類密度間的交互關(guān)系,揭示浮游動(dòng)物種群動(dòng)態(tài)對(duì)抗生素的生態(tài)學(xué)響應(yīng),并為水環(huán)境的監(jiān)測(cè)和化學(xué)污染物對(duì)水生生物的影響提供更為詳實(shí)的參考資料。

1 材料與方法

1.1 輪蟲(chóng)的來(lái)源與培養(yǎng)

實(shí)驗(yàn)用萼花臂尾輪蟲(chóng)采自安徽省蕪湖市鏡湖。采集后的輪蟲(chóng)單克隆培養(yǎng)及后續(xù)實(shí)驗(yàn)均于(25±1)℃、自然光照條件下(光照強(qiáng)度約130 lx)的恒溫培養(yǎng)箱中進(jìn)行。培養(yǎng)液采用EPA配方[54],每天投喂以HB- 4培養(yǎng)基[55]培養(yǎng)的處于指數(shù)增長(zhǎng)期的斜生柵藻(Scenedesmusobliquus),離心濃縮后使用。培養(yǎng)時(shí)間在1個(gè)月以上。實(shí)驗(yàn)前,對(duì)萼花臂尾輪蟲(chóng)進(jìn)行為期兩周的預(yù)培養(yǎng),期間每天更換50%輪蟲(chóng)培養(yǎng)液,并投喂密度分別為1.0×106、2.0×106、4.0×106個(gè)/mL的斜生柵藻為食物,保持輪蟲(chóng)種群處于指數(shù)增長(zhǎng)狀態(tài)。

1.2 測(cè)試液的配置

實(shí)驗(yàn)所用的鹽酸四環(huán)素購(gòu)自生工生物工程上海(股份)有限公司,純度為USP Grade(>98.5%)。測(cè)試液按母液稀釋法配置,實(shí)驗(yàn)前用蒸餾水配置1.0 g/L的母液,置于4 ℃冰箱中備用。實(shí)驗(yàn)時(shí)用EPA將其配制成所需濃度的測(cè)試液。

1.3 急性毒性實(shí)驗(yàn)

根據(jù)正式毒性實(shí)驗(yàn)前的預(yù)備實(shí)驗(yàn)結(jié)果將鹽酸四環(huán)素設(shè)置為60.0、80.0、100.0、120.0、140.0、160.0 mg/L共6個(gè)濃度梯度,另設(shè)1個(gè)空白對(duì)照組。實(shí)驗(yàn)在特制的容積為6 mL的玻璃杯(使用前在相應(yīng)濃度溶液中浸泡48 h)中進(jìn)行,每杯放入10只齡長(zhǎng)小于4 h的輪蟲(chóng)幼體和5 mL測(cè)試液,內(nèi)含密度分別為1.0×106、2.0×106、4.0×106個(gè)/mL的斜生柵藻,每個(gè)濃度組設(shè)置3個(gè)重復(fù),共計(jì)63組(7個(gè)測(cè)試液濃度×3個(gè)食物密度×3個(gè)重復(fù))。實(shí)驗(yàn)24 h后分別觀察記錄每個(gè)玻璃杯中輪蟲(chóng)的死亡數(shù)目,采用概率單位法求得鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)的24 hLC50值。

1.4 生命表實(shí)驗(yàn)

根據(jù)急性毒性實(shí)驗(yàn)得出的LC50值,將鹽酸四環(huán)素濃度設(shè)置為0、0.5、1.5、4.5、13.5、40.5 mg/L等6個(gè)濃度梯度,每組均設(shè)3個(gè)重復(fù),共計(jì)54組(6個(gè)鹽酸四環(huán)素濃度×3個(gè)食物密度×3個(gè)重復(fù))。測(cè)試液的配制方法如前所述。實(shí)驗(yàn)時(shí),分別由預(yù)培養(yǎng)的各組輪蟲(chóng)中隨機(jī)挑取若干攜卵非混交雌體的輪蟲(chóng)母體置于與預(yù)培養(yǎng)時(shí)條件相同的燒杯內(nèi)繼續(xù)培養(yǎng)；4 h后,取10只齡長(zhǎng)小于4 h的輪蟲(chóng)幼體置于容積為6 mL的玻璃杯中,每杯添加體積為5 mL、內(nèi)含密度分別為1.0×106、2.0×106、4.0×106個(gè)/mL斜生柵藻的各相應(yīng)濃度鹽酸四環(huán)素溶液(共54個(gè)玻璃杯)。實(shí)驗(yàn)開(kāi)始后,每12 h觀察記錄各玻璃杯中輪蟲(chóng)母體的存活情況及所孵化的幼體數(shù),隨即移出所孵化的幼體置于干凈的玻璃杯中并于相同條件下繼續(xù)培養(yǎng),待其產(chǎn)卵后確定其雌體類型,用于計(jì)算輪蟲(chóng)后代中的混交雌體百分率。實(shí)驗(yàn)期間,每12 h懸浮一次沉積于杯底的藻類食物,每24 h更換一次測(cè)試液。實(shí)驗(yàn)持續(xù)到所有輪蟲(chóng)母體全部死亡時(shí)為止。

1.5 研究參數(shù)定義與計(jì)算

1.6 數(shù)據(jù)處理

采用SPSS 22.0分析軟件和Excel對(duì)數(shù)據(jù)進(jìn)行分析,對(duì)所得數(shù)據(jù)作正態(tài)性分布檢驗(yàn)后,對(duì)符合正態(tài)分布的各組數(shù)據(jù)通過(guò)雙因素方差分析(Two-way ANOVA)檢查食物密度、污染物濃度及兩者間的交互作用對(duì)各參數(shù)影響的顯著性,并對(duì)鹽酸四環(huán)素濃度與各參數(shù)之間的關(guān)系進(jìn)行回歸分析；采用多重比較(LSD檢驗(yàn))分析各參數(shù)的平均數(shù)在各組間(食物密度間和鹽酸四環(huán)素濃度間)的差異顯著性。

2 結(jié)果

2.1 不同藻密度下鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)的急性毒性

急性毒性實(shí)驗(yàn)結(jié)果顯示,在(25±1) ℃、自然光照的條件下,當(dāng)食物密度分別為1.0×106、2.0×106、4.0×106個(gè)/mL時(shí),鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)幼體的24 hLC50值分別是107.95、116.92 mg/L和111.23 mg/L,三者間無(wú)顯著差異(P> 0.05),其95%的置信限分別為100.34—113.69、110.62—123.51 mg/L和104.21—118.34 mg/L。

2.2 不同藻密度下鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)存活率和繁殖率的影響

3個(gè)食物密度和6個(gè)鹽酸四環(huán)素濃度下萼花臂尾輪蟲(chóng)的特定年齡存活率(lx)和特定年齡繁殖率(mx)如圖1所示。在對(duì)照組及低濃度的鹽酸四環(huán)素(0 mg/L和0.5 mg/L)存在下,隨著食物濃度的增加,輪蟲(chóng)的全部死亡時(shí)間逐漸推遲,而其他濃度的鹽酸四環(huán)素(1.5、13.5、40.5 mg/L)下,輪蟲(chóng)的全部死亡時(shí)間則隨著食物密度的增加而逐漸提前。在對(duì)照組及低濃度(0、0.5、1.5 mg/L)的鹽酸四環(huán)素條件下,投喂中等食物密度(2.0×106個(gè)/mL)的輪蟲(chóng)出現(xiàn)死亡的時(shí)間最遲,而在高濃度的鹽酸四環(huán)素(4.5、13.5、40.5 mg/L)條件下,低食物密度(1.0×106個(gè)/mL)下的輪蟲(chóng)開(kāi)始死亡的時(shí)間最遲。

在對(duì)照組及各鹽酸四環(huán)素濃度下,輪蟲(chóng)的特定年齡繁殖率高峰值均隨著食物密度的增加而增大；在各食物密度下,輪蟲(chóng)的特定年齡繁殖率高峰值在低濃度(0.5—4.5 mg/L)的鹽酸四環(huán)素濃度下逐漸增加,而高濃度(13.5 mg/L和40.5 mg/L)的鹽酸四環(huán)素則降低了特定年齡繁殖率高峰值。在鹽酸四環(huán)素脅迫下(除4.5 mg/L外),輪蟲(chóng)的特定年齡繁殖率高峰值出現(xiàn)的時(shí)間在1.0×106個(gè)/mL食物密度組最遲,而在2.0×106個(gè)/mL食物密度組最早。

圖1 不同藻密度和鹽酸四環(huán)素濃度下萼花臂尾輪蟲(chóng)的特定年齡存活率和特定年齡繁殖率Fig.1 The age-specific survivorship (square) and fecundity curves (triangle) of B. calyciflorus at six TCH concentrations and three algal densities

2.3 不同藻密度下鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)生命表參數(shù)的影響

雙因素方差分析表明,食物密度和鹽酸四環(huán)素濃度對(duì)輪蟲(chóng)的生命期望、凈生殖率、世代時(shí)間、內(nèi)稟增長(zhǎng)率、平均壽命和后代混交率,以及食物密度和鹽酸四環(huán)素濃度間交互作用對(duì)除了內(nèi)稟增長(zhǎng)率外的其他5個(gè)統(tǒng)計(jì)學(xué)參數(shù)均具有顯著影響(P< 0.05)(表1)。多重比較分析結(jié)果(表2)表明,與對(duì)照組相比,在各食物密度下,鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)的生命期望、凈生殖率、世代時(shí)間、平均壽命和后代混交率都具有顯著影響。如表2所示,當(dāng)食物密度為1.0×106個(gè)/mL時(shí),1.5 mg/L濃度鹽酸四環(huán)素顯著提高了輪蟲(chóng)的生命期望和平均壽命,1.5、4.5、13.5 mg/L的鹽酸四環(huán)素顯著提高了輪蟲(chóng)的凈生殖率,0.5 mg/L和1.5 mg/L的鹽酸四環(huán)素顯著增加了輪蟲(chóng)的世代時(shí)間,13.5 mg/L和40.5 mg/L的鹽酸四環(huán)素顯著提高了輪蟲(chóng)的混交率。在2.0×106個(gè)/mL食物密度下,0.5 mg/L的鹽酸四環(huán)素顯著提高了輪蟲(chóng)的生命期望、世代時(shí)間和平均壽命,而4.5 mg/L和40.5 mg/L的鹽酸四環(huán)素卻顯著降低了輪蟲(chóng)的生命期望；4.5 mg/L的鹽酸四環(huán)素顯著提升了輪蟲(chóng)的凈生殖率,但40.5 mg/L的鹽酸四環(huán)素顯著降低了輪蟲(chóng)的凈生殖率和平均壽命；40.5 mg/L濃度鹽酸四環(huán)素極顯著提高了輪蟲(chóng)的混交率。當(dāng)食物密度為4.0×106個(gè)/mL環(huán)境下,除0.5 mg/L濃度外,其他濃度鹽酸四環(huán)素顯著縮短了輪蟲(chóng)的生命期望；4.5 mg/L的鹽酸四環(huán)素極顯著提高了輪蟲(chóng)的凈生殖率；1.5和13.5 mg/L的鹽酸四環(huán)素顯著縮短了輪蟲(chóng)的世代時(shí)間；1.5、13.5、40.5 mg/L的鹽酸四環(huán)素顯著縮短了輪蟲(chóng)的平均壽命；40.5 mg/L的鹽酸四環(huán)素極顯著地提高了輪蟲(chóng)的后代混交率。

在對(duì)照組(0 mg/L)中,2.0個(gè)/mL和4.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的生命期望、凈生殖率、內(nèi)稟增長(zhǎng)率和平均壽命；0.5 mg/L的鹽酸四環(huán)素下,2.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的生命期望、凈生殖率、內(nèi)稟增長(zhǎng)率和平均壽命,而世代時(shí)間和后代混交率在各食物密度間無(wú)顯著差異；1.5 mg/L的鹽酸四環(huán)素下,1.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的生命期望、世代時(shí)間和平均壽命,而凈生殖率和內(nèi)稟增長(zhǎng)率在2.0×106個(gè)/mL食物密度下最高；4.5 mg/L的鹽酸四環(huán)素下,2.0×106個(gè)/mL和4.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的凈生殖率和內(nèi)稟增長(zhǎng)率,而其他各參數(shù)在各食物密度間無(wú)顯著差異；在13.5 mg/L的鹽酸四環(huán)素下,2.0×106個(gè)/mL和4.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的凈生殖率和內(nèi)稟增長(zhǎng)率,而1.0×106個(gè)/mL和2.0×106個(gè)/mL的食物密度顯著提高了萼花臂尾輪蟲(chóng)的生命期望和平均壽命,1.0×106個(gè)/mL的食物密度下輪蟲(chóng)的世代時(shí)間和后代混交率最高；40.5 mg/L的鹽酸四環(huán)素下,2.0×106個(gè)/mL和4.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的凈生殖率,而1.0×106個(gè)/mL和2.0×106個(gè)/mL的食物密度顯著提高了萼花臂尾輪蟲(chóng)的生命期望、世代時(shí)間和平均壽命,4.0×106個(gè)/mL的食物密度下輪蟲(chóng)的內(nèi)稟增長(zhǎng)率和后代混交率最高。

表1食物密度和鹽酸四環(huán)素濃度及其交互作用對(duì)萼花臂尾輪蟲(chóng)生命表統(tǒng)計(jì)學(xué)參數(shù)的影響

Table1Effectsoffooddensity,tetracyclinehydrochloride(TCH)concentrationandtheirinteractiononlifetabledemographicparametersofB.calyciflorous

參數(shù)和差異源Parameterandsource平方和SSSumofsquare自由度dfDegreeoffreedom均方MSMeansquareFP生命期望Lifeexpectancyathatching食物密度Fooddensity(A)3181.2921590.6521.42<0.01鹽酸四環(huán)素濃度TCHconcentration(B)3250.115650.028.75<0.01A×B3846.1510384.615.18<0.01Error2672.883674.25凈生殖率Netreproductionrate食物密度Fooddensity(A)932.262466.13113.95<0.01鹽酸四環(huán)素濃度TCHconcentration(B)304.34560.8714.88<0.01A×B210.861021.095.15<0.01Error147.26364.09世代時(shí)間Generationtime食物密度Fooddensity(A)1285.642642.8249.10<0.01鹽酸四環(huán)素濃度TCHconcentration(B)200.04540.013.06<0.05A×B1001.5710100.167.65<0.01Error471.293613.09內(nèi)稟增長(zhǎng)率Intrinsicrateofpopulationincrease食物密度Fooddensity(A)0.0017520.0008861.67<0.01鹽酸四環(huán)素濃度TCHconcentration(B)0.0002550.000053.49<0.05A×B0.00023100.000021.64>0.05Error0.00051360.00001平均壽命Meanlifespan食物密度Fooddensity(A)2959.5721479.7920.05<0.01鹽酸四環(huán)素濃度TCHconcentration(B)3115.655623.138.45<0.01A×B4043.3110404.335.48<0.01Error2656.323673.79后代混交率Mixisrateofoffspring食物密度Fooddensity(A)0.0720.0318.38<0.01鹽酸四環(huán)素濃度TCHconcentration(B)0.1650.0317.79<0.01A×B0.18100.029.72<0.01Error0.07360.00

回歸分析顯示,當(dāng)食物密度為1.0×106個(gè)/mL時(shí),鹽酸四環(huán)素濃度與輪蟲(chóng)的凈生殖率、內(nèi)稟增長(zhǎng)率和后代混交率間均具有顯著的劑量-效應(yīng)關(guān)系；當(dāng)食物密度為4.0×106個(gè)/mL時(shí),鹽酸四環(huán)素濃度與輪蟲(chóng)的生命期望期間具有顯著的劑量-效應(yīng)關(guān)系(表3)。在食物密度為2.0×106個(gè)/mL的條件下,鹽酸四環(huán)素濃度與輪蟲(chóng)的生命表參數(shù)間均不存在顯著的劑量-效應(yīng)關(guān)系。

表2不同食物密度/(×106個(gè)/mL)和鹽酸四環(huán)素濃度/(mg/L)下萼花臂尾輪蟲(chóng)生命表統(tǒng)計(jì)學(xué)參數(shù)(均數(shù)±標(biāo)準(zhǔn)誤)*

Table2LifetabledemographicparametersofB.calyciflorousexposedtodifferentconcentrationsofTCHatthreefooddensities(Mean ± SE)

參數(shù)Parameters食物密度Fooddensities鹽酸四環(huán)素濃度TCHconcentrations00.51.54.513.540.5e0(h)1105.20±12.94Bb116.00±8.51Bb145.00±2.40Aa107.60±3.67Ab114.00±8.40Ab113.20±2.50Ab2130.40±2.77Ab142.40±9.77Aa127.20±9.8ABbc116.00±1.83Acd125.60±6.61Abcd114.80±3.67Ad4120.40±12.26ABa124.00±3.46Ba104.40±7.3Bbc106.80±7.3Ab94.80±1.2Bbc93.20±4.54BcR0(個(gè))19.23±1.08Bde11.12±1.21Ccd14.93±2.19Ba12.52±0.71Bbc13.85±1.17Bab8.34±0.99Be219.93±0.53Ab22.72±0.72Aab22.45±1.96Aab24.39±3.04Aa20.81±2.97Aab14.78±1.97Ac420.87±3.48Ab19.48±1.92Bb17.50±1.29Bb27.79±3.6Aa17.26±1.3ABb17.43±2.16AbT(h)177.04±4.64Ac84.68±6.62Aab98.15±4.98Aa75.89±0.37Ac83.30±1.14Ab82.57±3.81Abc278.12±1.84Ab84.53±5.81Aa78.07±3.76Bb80.67±3.98Aab80.26±1.37Bab80.90±1.46Aab475.00±4.05Aa73.90±3.67Aab68.74±0.87Cbc75.92±3.33Aa68.10±1.14Cc70.56±3.8Babcrm(d-1)10.0371±0.005Bab0.0370±0.003Bab0.0378±0.002Ca0.0419±0.001Ba0.0418±0.002Ba0.0320±0.003Cb20.0502±0.0003Aab0.0545±0.013Aa0.0526±0.002Aab0.0513±0.002Aab0.0485±0.002Aab0.0433±0.004Bb40.0504±0.002Aab0.0493±0.002ABb0.0488±0.002Bb0.0533±0.004Aa0.0482±0.008Ab0.0498±0.009AabML(h)193.20±12.94Bb104.00±8.51Bb133.20±1.03Aa95.60±3.67Ab102.00±8.40Ab101.20±2.50Ab2118.40±2.77Ab130.40±9.77Aa115.20±9.8ABb104.00±1.83Abc113.60±6.61Abc102.80±3.67Ac4108.40±12.6ABab112.00±3.46Ba92.40±7.3Bcd98.80±7.62Abc82.80±1.2Bd81.20±20.45BdM(%)10.19±0.01Bc0.21±0.06Abc0.20±0.01Ac0.28±0.02Abc0.45±0.02Aa0.31±0.02Bb20.19±0.01Bb0.18±0.02Ab0.15±0.05Ab0.16±0.03Ab0.16±0.02Bb0.29±0.04Ba40.27±0.04Ab0.17±0.01Ac0.16±0.02Ac0.19±0.02Ac0.22±0.04Bc0.42±0.02Aa

*多重比較方法(最小顯著差數(shù)法)：不同字母分別表示同列(A,B和C)或同行(a,b,c,d和e)存在顯著性差異(5%),字母從前到后表示平均數(shù)逐漸減?。槐碇蟹?hào)的含義如下：e0：生命期望,Life expectancy at hatching；R0：凈生殖率,Net reproductive rate;T：世代時(shí)間,Generation time；rm：內(nèi)稟增長(zhǎng)率,Intrinsic rate of population growth；ML：平均壽命,Mean lifespan；M：后代混交率,Mixis rate of offspring

表3 各食物密度下萼花臂尾輪蟲(chóng)的生命表參數(shù)(Y)與鹽酸四環(huán)素濃度(X, mg/L)間的關(guān)系

3 討論

抗生素類藥物被廣泛應(yīng)用于人類的疾病治療與預(yù)防,同時(shí)也作為牲畜用藥和生長(zhǎng)促進(jìn)劑在農(nóng)業(yè)、水產(chǎn)和畜牧業(yè)中普遍使用。急性毒性研究表明,鹽酸四環(huán)素對(duì)秀麗線蟲(chóng)(Caenorhabditiselegans)的24h半致死濃度LC50為82.9 mg/L[56],對(duì)浮游動(dòng)物大型溞(Daphniamagna)的最大無(wú)觀察效應(yīng)濃度(NOEC)為340 mg/L[25]。本研究中,當(dāng)食物密度分別為1.0×106、2.0×106、4.0×106個(gè)/mL時(shí),鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)的24hLC50值分別是107.95、116.92、111.23 mg/L。可見(jiàn),萼花臂尾輪蟲(chóng)對(duì)鹽酸四環(huán)素的敏感性較秀麗線蟲(chóng)弱,但比浮游動(dòng)物大型溞敏感。因而,在水環(huán)境生物監(jiān)測(cè)尤其是對(duì)鹽酸四環(huán)素的生物監(jiān)測(cè)中,萼花臂尾輪蟲(chóng)是較為理想的受試生物。

有關(guān)環(huán)境中殘留的抗生素對(duì)浮游生物的影響,目前主要表現(xiàn)為低濃度暴露可以促進(jìn)種群的生長(zhǎng)與繁殖,此后隨著暴露濃度的增大轉(zhuǎn)為抑制作用[44, 46]。例如,低濃度(0.0125 mg/mL)的氯霉素和低劑量組(0.25 mg/mL和0.50 mg/mL)的煙酸氟哌酸可促進(jìn)褶皺臂尾輪蟲(chóng)的種群增長(zhǎng),隨著染毒濃度的升高(0.0250 mg/mL和0.0500 mg/mL的氯霉素及0.75 mg/mL和1.00 mg/mL的煙酸氟哌酸),褶皺臂尾輪蟲(chóng)的種群增長(zhǎng)則受到不同程度的抑制作用[44]。當(dāng)紅霉素和氯霉素濃度為6.25 μg/mL時(shí)可促進(jìn)萼花臂尾輪蟲(chóng)的種群增長(zhǎng),當(dāng)濃度增加到12.5—50 μg/mL時(shí),則有不同程度的抑制作用,且抑制作用隨著濃度的增大而增大,當(dāng)濃度達(dá)到100 μg/mL時(shí),抑制作用最為明顯。本研究中,在各食物密度下,輪蟲(chóng)的特定年齡繁殖率(mx)高峰值均隨著鹽酸四環(huán)素濃度的增加呈現(xiàn)先增加后降低的趨勢(shì)；在食物密度1.0×106個(gè)/mL的情況下,1.5、4.5、13.5 mg/L 3個(gè)濃度的鹽酸四環(huán)素均顯著提高了萼花臂尾輪蟲(chóng)的凈生殖率(R0),而40.5 mg/L濃度則降低了萼花臂尾輪蟲(chóng)的凈生殖率；食物密度為2.0×106、4.0×106個(gè)/mL時(shí),4.5 mg/L的鹽酸四環(huán)素顯著提高了輪蟲(chóng)的凈生殖率(R0),但隨著染毒濃度的升高,輪蟲(chóng)的凈生殖率有不同程度的降低。同樣,在1.0×106個(gè)/mL和2.0×106個(gè)/mL的食物密度下,低濃度(0.5 mg/L或1.5 mg/L)的鹽酸四環(huán)素延長(zhǎng)了萼花臂尾輪蟲(chóng)的生命期望、世代時(shí)間和平均壽命,這進(jìn)一步驗(yàn)證了在低濃度污染物下藻密度的上升有利于輪蟲(chóng)對(duì)毒物毒性的降解,而暴露于高濃度污染物下的輪蟲(chóng)對(duì)毒物的實(shí)際攝入量增加,導(dǎo)致毒性影響顯著提高。這一結(jié)果與Chopra等[1]、Araujo等[47]和Huang等[57]的研究結(jié)果基本一致,即毒物對(duì)輪蟲(chóng)的生長(zhǎng)和繁殖具有“低促高抑”的特點(diǎn)。這種“毒性興奮效應(yīng)”的雙相劑量—反應(yīng)現(xiàn)象已在各類生物、各類毒物及各類生命現(xiàn)象中都有發(fā)現(xiàn),其范圍幾乎涵蓋了包括重金屬化合物、氰化物、多環(huán)芳烴、多氯聯(lián)苯、有機(jī)砷化物以及農(nóng)藥和一些抗生素在內(nèi)的所有有毒物質(zhì)[58]。

萼花臂尾輪蟲(chóng)為營(yíng)周期性孤雌生殖的單巢目輪蟲(chóng),在環(huán)境適宜時(shí)其非混交雌體以孤雌生殖的方式進(jìn)行繁殖,此時(shí)的種群后代混交率極低或沒(méi)有混交雌體產(chǎn)生；當(dāng)受到混交刺激時(shí),非混交雌體以減數(shù)分裂的方式產(chǎn)生混交卵,輪蟲(chóng)進(jìn)入有性生殖繁殖方式,后代混交率提高。有性生殖可使輪蟲(chóng)種群以休眠卵的形式渡過(guò)不利的環(huán)境條件,并通過(guò)基因重組增加了種群遺傳多樣性,從而提高輪蟲(chóng)種群對(duì)環(huán)境變化的適應(yīng)能力。引起輪蟲(chóng)由無(wú)性生殖轉(zhuǎn)向有性生殖產(chǎn)生的因素包括諸多外源性因素(如光照周期、低溫刺激、培養(yǎng)液pH值、食物密度、種群密度、生育酚、保幼激素、增塑劑、重金屬和農(nóng)藥等)及內(nèi)源性因素(如遺傳因素、母體的年齡和孤雌生殖的累積世代數(shù))[59-60]。有關(guān)抗生素的研究表明,2.0—10.0 μg/mL的利福平各濃度均顯著提高了萼花臂尾輪蟲(chóng)的后代混交率；在各藻密度下,后代混交率與利福平濃度間均具有劑量—效應(yīng)關(guān)系,表明萼花臂尾輪蟲(chóng)的有性生殖顯著受到利福平毒性的脅迫影響。本研究中,在3個(gè)食物密度下鹽酸四環(huán)素濃度為40.5 mg/L時(shí),以及1.0×106個(gè)/mL的食物密度下13.5 mg/L的鹽酸四環(huán)素均顯著提高了萼花臂尾輪蟲(chóng)的后代混交率,且鹽酸四環(huán)素濃度與輪蟲(chóng)后代混交率間具有顯著的劑量-效應(yīng)關(guān)系。可見(jiàn),鹽酸四環(huán)素像利福平一樣可有效促進(jìn)萼花臂尾輪蟲(chóng)有性生殖的發(fā)生。

萼花臂尾輪蟲(chóng)是濾食性浮游動(dòng)物。當(dāng)輪蟲(chóng)暴露于低濃度污染物時(shí),藻密度的上升加大了其對(duì)毒物毒性的降解,進(jìn)而降低了毒物對(duì)輪蟲(chóng)的影響；此外,藻密度的上升也提高了輪蟲(chóng)的攝食率,從而增加了輪蟲(chóng)對(duì)毒物的抵抗力,促進(jìn)了輪蟲(chóng)的種群增長(zhǎng)[48]。然而,當(dāng)輪蟲(chóng)暴露于高濃度污染物時(shí),由于輪蟲(chóng)濾水率的增加進(jìn)而導(dǎo)致實(shí)際攝入毒物劑量的增加,這使得毒物對(duì)輪蟲(chóng)的毒性影響顯著提高[21]。本研究中,在對(duì)照組及低濃度的鹽酸四環(huán)素(0 mg/L和0.5 mg/L)存在下,隨著食物濃度的增加,輪蟲(chóng)的全部死亡時(shí)間逐漸推遲,而其他濃度的鹽酸四環(huán)素(1.5、13.5、40.5 mg/L)下,輪蟲(chóng)的全部死亡時(shí)間則隨著食物密度的增加而逐漸提前。在對(duì)照組及低濃度(0、0.5、1.5 mg/L)的鹽酸四環(huán)素條件下,投喂中等食物密度(2.0×106個(gè)/mL)的輪蟲(chóng)出現(xiàn)死亡的時(shí)間最遲,而在高濃度的鹽酸四環(huán)素(4.5、13.5、40.5 mg/L)條件下,低食物密度(1.0×106個(gè)/mL)下的輪蟲(chóng)開(kāi)始死亡的時(shí)間最遲。雙因素方差分析表明,除了4.5 mg/L的鹽酸四環(huán)素對(duì)萼花臂尾輪蟲(chóng)各生命表參數(shù)在食物密度間基本未見(jiàn)顯著性差異外,在對(duì)照組及低濃度的鹽酸四環(huán)素(0 mg/L和0.5 mg/L)中,2.0×106個(gè)/mL和4.0×106個(gè)/mL的食物密度顯著增加了萼花臂尾輪蟲(chóng)的生命期望、凈生殖率、內(nèi)稟增長(zhǎng)率和平均壽命；而在1.5、13.5、40.5 mg/L的鹽酸四環(huán)素下,4.0×106個(gè)/mL的食物密度顯著降低了萼花臂尾輪蟲(chóng)的生命期望、世代時(shí)間和平均壽命。因此,食物密度可顯著影響污染物濃度對(duì)浮游動(dòng)物的毒性效應(yīng),且低污染物濃度下高食物密度表現(xiàn)為有利影響,而高污染物濃度下的高食物密度則加劇了毒性效應(yīng)。藻密度對(duì)鹽酸四環(huán)素毒性的干擾作用還有待進(jìn)一步研究。

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ComprehensiveeffectsoftetracyclinehydrochlorideconcentrationonlifehistorytraitsofBrachionuscalyciflorusunderdifferentfooddensities

XIANG Xianling1, 2,*, ZHU Yelin1, XU Qiulei1, ZHU Lingyun1, XI Yilong1, 2

1CollegeofLifeSciences,AnhuiNormalUniversity,Wuhu241000,China2KeyLaboratoryofBioticEnvironmentandEcologicalSafetyinAnhuiProvince,Wuhu241000,China

Increasing attention has been paid to the challenging issue of environmental pollution by antibiotics in recent years, and the structure and functioning of aquatic ecosystems could be disturbed by residues remaining in the environment. To date, however, few attempts have been made to investigate the effects of tetracycline hydrochloride on life history traits of rotifers under different food densities. In this study, the effects of tetracycline hydrochloride concentration on life table demography ofBrachionuscalyciflorusunder differentScenedesmusobliquusdensities were studied. The results showed that tetracycline hydrochloride concentration and food density had significant effects on life expectancy at hatching, net reproductive rate, generation time, intrinsic rate of population increase, average lifespan, and proportion of sexual offspring ofB.calyciflorus. In addition, the interaction of tetracycline hydrochloride concentration and food density had a marked influence on five demographic parameters, except for the intrinsic rate of population increase, in rotifers. The peak of age-specific fecundity inB.calyciflorusincreased to a maximum and then decreased with increasing concentration of tetracycline hydrochloride under each food density. The effects of tetracycline hydrochloride on the growth and reproduction ofB.calycifloruspresented a pattern of hormesis. Mixis rate of offspring in rotifers increased at the high concentration of tetracycline hydrochloride under the three food densities, and there were significant dose-effect relationships between tetracycline hydrochloride concentration and the proportion of sexual offspring at the 1.0×106cells/mL food density. Levels of food density played an important role in the poisoning effects of tetracycline hydrochloride onB.calyciflorus.

Tetracycline hydrochloride; food density;Brachionuscalyciflorus; life table; life history trait

國(guó)家自然科學(xué)基金(31470015, 31200324)；安徽省自然科學(xué)基金(1208085QC47, 1708085MC79)；“重要生物資源保護(hù)和利用研究”安徽省重點(diǎn)實(shí)驗(yàn)室專項(xiàng)基金

2016- 10- 19;

2017- 02- 17

*通訊作者Corresponding author.E-mail: xiangxianling@163.com

10.5846/stxb201610192135

項(xiàng)賢領(lǐng),朱曄璘,徐秋磊,朱凌云,席貽龍.鹽酸四環(huán)素濃度和食物密度對(duì)萼花臂尾輪蟲(chóng)生活史特征的綜合影響.生態(tài)學(xué)報(bào),2017,37(22):7718- 7728.

Xiang X L, Zhu Y L, Xu Q L, Zhu L Y, Xi Y L.Comprehensive effects of tetracycline hydrochloride concentration on life history traits ofBrachionuscalyciflorusunder different food densities.Acta Ecologica Sinica,2017,37(22):7718- 7728.