基于定位欄試驗(yàn)的奶牛糞便中雌激素含量特征

李 艷潘 杰梁紫薇胡嘉梧劉姜艷賀德春*李廷真**

基于定位欄試驗(yàn)的奶牛糞便中雌激素含量特征

李 艷1,2,潘 杰1,梁紫薇2,3,胡嘉梧2,劉姜艷1,2,賀德春2*,李廷真1**

(1.重慶三峽學(xué)院,三峽庫區(qū)水環(huán)境演變與污染防治重慶市重點(diǎn)實(shí)驗(yàn)室,重慶 404130；2.生態(tài)環(huán)境部華南環(huán)境科學(xué)研究所,廣東 廣州 510530；3.仲愷農(nóng)業(yè)工程學(xué)院,廣東 廣州 510225)

為探究奶牛糞便中雌激素含量特征,采用定位欄試驗(yàn)收集奶牛糞便樣,采用超高效液相色譜串聯(lián)(UPLC-MS/MS)法對糞便中4種天然雌激素(雌酮;17α-雌二醇,17β-雌二醇,雌三醇)和2種人工合成雌激素(17α-炔雌醇,己烯雌酚)的含量進(jìn)行測定,結(jié)果表明:6種雌激素的檢出率居于8.33%～98.89%之間,4種天然雌激素中,除雌三醇檢出率為8.33%外,其余3種的檢出率均在90%以上;2種人工合成雌激素檢出率分別為52.22%(17α-炔雌醇)和64.44% (己烯雌酚);6種雌激素總濃度范圍為1.51～658.98μg/kg(干重),總雌激素活性當(dāng)量濃度范圍為3.76～478.50μg/kg(干重).不同生長階段奶牛糞便中雌激素含量存在顯著差異,6種雌激素總含量排序?yàn)槊谌榕?犢牛>育成牛,其中位值分別為79.21,47.20,25.72μg/kg.不同季節(jié)采集的糞便樣品中雌激素含量總體表現(xiàn)為冬季與春季相對較高,夏季和秋季相對較低,其中秋季糞便樣品中6種雌激素含量顯著低于其他季節(jié).養(yǎng)殖場糞便堆放場樣品中雌激素總含量在7.63～3921.92μg/kg之間,顯著高于通過定位欄試驗(yàn)采集的鮮糞中的雌激素含量.不同生長階段、不同季節(jié)、不同采樣點(diǎn)采集的牛糞樣品中雌激素含量均存在較大差異,在開展奶牛養(yǎng)殖雌激素排放量調(diào)查及風(fēng)險(xiǎn)評估工作中,應(yīng)充分考慮采樣時間和采樣點(diǎn)位的影響.

奶牛；糞便；雌激素；生長階段；季節(jié)差異

雌激素屬于一類典型的內(nèi)分泌干擾物質(zhì),主要由人類?動物通過尿液和糞便排泄到環(huán)境中[1].環(huán)境中雌激素在痕量濃度下(ng/L)即可干擾生物體內(nèi)分泌系統(tǒng),進(jìn)而影響生物正常的生長、發(fā)育、生殖等過程,導(dǎo)致雌性動物性早熟及雄性動物雌性化等現(xiàn)象,具有極高的生態(tài)風(fēng)險(xiǎn)[2-5].隨著畜禽養(yǎng)殖業(yè)的快速發(fā)展,畜禽養(yǎng)殖對全球雌激素排放的貢獻(xiàn)逐年增加[6].據(jù)測算,2014年我國奶牛、肉牛和豬的雌激素排放量總和約為50.5t[7];其中,通過畜禽糞便排入環(huán)境中的雌激素中約90%源于奶牛和肉牛糞便[8],主要以雌酮(E1)、17α-雌二醇(17α-E2)、17β-雌二醇(17β-E2)和雌三醇(E3 )等天然雌激素為主[9],還包括17α-炔雌醇(17α-EE2)和己烯雌酚(DES)等人工合成雌激素.由于雌激素的分布普遍性、毒性和持久性[10-11],畜禽糞便排放造成的環(huán)境污染問題及其生態(tài)健康風(fēng)險(xiǎn)不容忽視.

目前國內(nèi)外在奶牛等主要畜禽糞污雌激素含量及周邊環(huán)境雌激素污染特征等方面開展了大量研究[12-14].但關(guān)于糞便中雌激素含量的研究多數(shù)是基于單次采樣或養(yǎng)殖場內(nèi)平均布點(diǎn)混合采樣而開展的,未能考慮畜禽不同生長階段及不同季節(jié)排泄糞便中雌激素含量的差異[15-17].同時,由于這種采樣方式不可避免導(dǎo)致糞便中混入了部分尿液,據(jù)此測定的糞便雌激素含量特征與實(shí)際情況可能存在較大差異,不利于科學(xué)準(zhǔn)確地開展畜禽糞便中雌激素環(huán)境污染的治理與防控.本研究采用定位欄試驗(yàn)對不同生長階段的奶牛開展4個季節(jié)采樣監(jiān)測,系統(tǒng)研究奶牛不同生長階段、不同季節(jié)排放糞便中雌激素的含量與分布特征,以期為科學(xué)準(zhǔn)確測算奶牛糞便雌激素排放量、制定合理的污染與風(fēng)險(xiǎn)防控對策等提供科學(xué)依據(jù).

1 材料與方法

1.1 試驗(yàn)地點(diǎn)與試驗(yàn)動物

本試驗(yàn)在四川省成都市某奶牛養(yǎng)殖場進(jìn)行.試驗(yàn)?zāi)膛７殖扇榕！⒂膳?犢牛3個飼養(yǎng)階段,每個飼養(yǎng)階段奶牛各選取5頭開展定位欄試驗(yàn).整個試驗(yàn)過程的飼料、飲水、環(huán)境與試驗(yàn)點(diǎn)常規(guī)飼養(yǎng)管理一致.

1.2 糞便收集

奶牛采用單頭定位飼養(yǎng),試驗(yàn)動物只能站立、蹲臥,不能轉(zhuǎn)身,保證動物的糞尿不相互混合,地面鋪設(shè)橡膠墊或用水泥地面,保障糞便能全量收集.

1.3 樣品采集與保存

采樣時間與頻率:整個試驗(yàn)周期分4個季節(jié)進(jìn)行,分別在2018年冬季、2019年春季、夏季及秋季開展,每個季節(jié)連續(xù)監(jiān)測3d.

樣品采集:每個定位欄每天分別定時收集試驗(yàn)?zāi)膛５男迈r糞便,稱重后混合均勻,采集1份樣品,樣品量不少于500g;同時分別在奶牛場糞便堆放場中平均布點(diǎn)采集堆積一周左右的糞便樣品,樣品量不少于500g;樣品保存在-20℃冰箱中待測.

1.4 樣品分析

糞便樣品中雌激素的含量測定采用之前報(bào)道的分析方法[18].含水率依據(jù)《復(fù)混肥料中游離水含量的測定真空烘箱法》(GB/T 8576-2010)[19]進(jìn)行測定.

1.5 數(shù)據(jù)處理

1.5.1 雌激素活性 采用雌二醇當(dāng)量EEQ (17β- estradiol equivalents)表征雌激素活性,計(jì)算公式[20]如下:

EEQ=MEC×EEF (1)

式中: MEC為雌激素實(shí)測濃度,μg/kg; EEF為雌二醇當(dāng)量因子,17α-E2、17β-E2、E1、E3、DES、17α-EE2對應(yīng)的EEF[21-22]分別為0.125, 1, 0.59, 0.26, 8.0, 8.71.

1.5.2 雌激素日排泄量 奶牛糞便雌激素日排泄量可以通過糞便中檢測到的雌激素濃度和平均日糞便產(chǎn)量來計(jì)算.每頭奶牛雌激素的日糞便排泄量可通過以下公式[13]計(jì)算:

M=C×g×(1-) (2)

式中:M是選定生長階段通過每頭奶牛每日糞便排泄的目標(biāo)雌激素的估計(jì)質(zhì)量, μg/(d×頭);C是奶牛干糞便中雌激素的測定濃度,μg/kg;g是選定生長階段每頭奶牛的新鮮糞便日產(chǎn)量,kg/d;代表新鮮糞便中含水率,%.

1.5.3 數(shù)據(jù)分析 采用Excel 2016整理數(shù)據(jù),應(yīng)用IBM SPSS Statistics 20.0中非參數(shù)檢驗(yàn)(Kruskal- Wallis檢驗(yàn))對樣本數(shù)據(jù)進(jìn)行顯著性分析,利用Origin pro 2018進(jìn)行作圖.

2 結(jié)果與討論

2.1 奶牛糞便中雌激素的含量特征

奶牛糞便中6種雌激素的檢出率在8.33%～ 98.89%之間(表1),4種天然雌激素中,除雌三醇檢出率較低外(8.33%),其余3種雌激素(E1、17α-E2、17β-E2)的檢出率均在90%以上,2種人工合成雌激素的檢出率分別為52.22%(17α-EE2)和64.44% (DES), 6種雌激素總量檢出范圍為1.51～658.98 μg/kg(干重) .這與文獻(xiàn)報(bào)道中奶牛糞便主要排泄E1、17α-E2、17β-E2相符(表2).人工合成雌激素DES與17α-EE2常被用作激素類藥物和飼料添加劑來提高動物養(yǎng)殖效益[23],本研究中DES與17α-EE2雖然檢出濃度較低,但檢出率均高于50%,表明該養(yǎng)殖場可能存在使用人工合成雌激素的行為.

如圖1所示,泌乳牛、犢牛、育成牛鮮糞中雌激素含量存在顯著差異(<0.05),其6種雌激素總濃度中位值依次為79.21μg/kg(泌乳牛)、47.20μg/kg(犢牛)、25.72μg/kg(育成牛).各生長階段奶牛糞便中的雌激素均以E1、17α-E2、17β-E為主.其中犢牛糞便中未檢出合成雌激素,而17α-E2含量明顯高于育成牛與泌乳牛.泌乳牛糞便中E1含量中位值為60.02μg/kg,占雌激素總含量的76%,約為犢牛與育成牛糞便中E1含量的3倍.Tao等[16]在犢牛、妊娠期奶牛、泌乳牛糞便樣品中自由態(tài)雌激素僅檢出17β-E2,與本研究中各生長階段糞便樣品中17β-E2是同一數(shù)量級. Zheng等[24]報(bào)道的美國一養(yǎng)殖場采集的新鮮奶牛糞便中雌激素素含量要遠(yuǎn)高于本研究.不同地區(qū)對于奶牛養(yǎng)殖場糞污處理方式存在差異,關(guān)于奶牛糞污中雌激素含量部分研究以液態(tài)或半液態(tài)肥料為主[25-27],有限的文章報(bào)道了奶牛糞便中雌激素含量.表2列出了已有研究報(bào)道的奶牛糞便中雌激素含量,不同研究報(bào)道的雌激素含量與雌激素種類差異較大,其主要原因可能與研究對象品種、生長階段、飼養(yǎng)條件、樣品采集方式等不同有關(guān)[28].

采用雌二醇當(dāng)量(EEQ)表征雌激素活性.犢牛、育成牛、泌乳牛糞便雌激素雌二醇當(dāng)量濃度中位值分別為27.52,42.16,71.93μg/kg.如圖1所示,育成牛與泌乳牛糞便中雌激素活性主要由E1、17α-EE2、DES貢獻(xiàn),占比為16.71%～49.22%.其原因?yàn)镋1是糞便樣品中檢出濃度最高的雌激素;雖然17α-EE2、DES檢出含量相對較低,但其雌激素活性較高,排放到環(huán)境中對水生生物具有較強(qiáng)的危害性.根據(jù)Partridge等[29]研究,即使在短期的低劑量(1ng/L)暴露情況下,17α-EE2也會通過影響雄性海龍對雌性海龍的吸引力來影響交配行為.與育成牛和泌乳牛相比,犢牛糞便中雌二醇當(dāng)量濃度相對較低;因?yàn)闋倥＜S便中未檢出合成雌激素,檢出的雌激素以E1和17α-E2為主,所以犢牛糞便樣品的雌激素活性相對較低.雖然17α-E2雌激素活性較低,但E2異構(gòu)體在不同的生物體內(nèi)可能具有不同的雌激素活性.Huang等[3]發(fā)現(xiàn),17α-E2在魚類中的雌激素作用大于哺乳動物;而在青鳉魚中,17α-E2的雌激素活性大約是17β-E2的30倍.因此,由奶牛糞便中17α-E2造成的環(huán)境污染風(fēng)險(xiǎn)不容被忽視[30-31].

表1 奶牛糞便中雌激素含量(μg/kg,干重)分布 (n=180)

注:n.d.表示未檢出或低于檢出限,按1/2檢出限參與統(tǒng)計(jì)處理;

表2 文獻(xiàn)報(bào)道中奶牛糞便中雌激素含量(μg/kg)

續(xù)表2

注:采用中位值描述其平均水平;-表示無相關(guān)數(shù)據(jù).

圖1 各生長階段奶牛糞便雌激素含量特征

2.2 奶牛糞便中雌激素含量特征的季節(jié)差異

不同生長階段奶牛糞便中雌激素含量具有顯著的季節(jié)性差異(圖2).泌乳牛糞便樣品中雌激素總含量為春季(193.22μg/kg)>冬季(136.00μg/kg)>夏季(69.67μg/kg)>秋季(13.33μg/kg),秋季泌乳牛糞便樣品中雌激素含量顯著低于其他季節(jié)(<0.05).泌乳牛糞便樣品中E1貢獻(xiàn)較高,占每個季度雌激素總量的31.96～85.69%.春季與夏季的泌乳牛糞便樣品中17α-E2?DES顯著高于其他季節(jié)(<0.05),育成牛糞便樣品中雌激素總含量為依次為冬季(39.65μg/kg)>夏季(36.65μg/kg)>春季(29.46μg/kg)>秋季(7.31μg/kg),冬季與秋季育成牛糞便樣品中DES與17α-E2顯著低于春季與夏季(<0.01).17α-EE2在春季沒有檢出,顯著低于其他季節(jié)(<0.05).犢牛糞便樣品中雌激素總含量依次為春季(56.26μg/kg)>夏季(35.70μg/kg)>秋季(32.96μg/kg)>冬季(56.14μg/kg),春季與冬季雌激素含量相對高于夏季與春季;犢牛糞便中的E3、17α-E2、17α-EE2、17β-E2檢出濃度較低,不同季節(jié)間無顯著差異(>0.05);冬季犢牛糞便中E1濃度顯著高于秋季(<0.05),約為秋季含量的4倍.春季、夏季、秋季、冬季糞便樣品中雌激素總含量中位數(shù)分別為60.76, 55.12, 12.78, 69.24μg/kg,總體表現(xiàn)為冬季與春季含量相對高于夏季、秋季,該結(jié)果與Hill等[46]研究結(jié)果相似.奶牛生理周期與季節(jié)間不同的環(huán)境條件(溫度、濕度、光照、氣壓、環(huán)境污染程度等)變化可能是造成奶牛糞便中雌激素含量季節(jié)性差異的主要原因.研究表明,奶牛畜舍溫度濕度表現(xiàn)出顯著的季節(jié)差異性[47].奶牛舒適的環(huán)境溫度為5～25℃,當(dāng)夏季環(huán)境溫度過高時,奶牛遭受熱應(yīng)激可能引起采食量的波動,營養(yǎng)消耗增加和牛體不適等,直接影響卵泡發(fā)育、生殖器官蛋白和激素的合成,進(jìn)而影響雌激素的分泌及排泄,并且這種影響會持續(xù)到秋季[48-49].

圖2 不同季節(jié)奶牛糞便中雌激素含量特征

2.3 牛糞堆場樣品中雌激素含量特征

奶牛糞便利用之前在堆場內(nèi)暫時自然堆放,是中國傳統(tǒng)奶牛養(yǎng)殖場的普遍管理方式.由表3可知,牛糞堆場樣品中檢出的雌激素以E1、17β-E2、17α-E2為主,E3、17α-EE2?DES檢出率與檢出濃度均較低,這與奶牛糞便主要排泄的雌激素種類一致.其中E1、17β-E2在糞便堆場樣品中檢出率為100%,E1檢出濃度最高,其含量中位值為124.43μg/ kg.糞便堆積場樣品中6種雌激素總含量范圍為7.63～3921.92μg/kg.

與定位欄鮮糞樣品比,牛糞堆場樣品中雌激素含量明顯較高,這可能是由于糞便堆場樣品中混入了部分尿液[28],且堆存過程中結(jié)合態(tài)雌激素轉(zhuǎn)化為自由態(tài)雌激素[50];M?stl等[51]研究發(fā)現(xiàn),奶牛糞便堆體表面樣品中總雌激素含量高達(dá)1000μg/ kg,而堆體內(nèi)部樣品只有約27μg/kg.另有研究表明,E1是E2兩種異構(gòu)體的降解產(chǎn)物[52].E1通常是堆放糞便樣品中最常檢測到的雌激,本研究中牛糞堆場樣品中E1的含量明顯高于其他雌激素.這表明糞便在堆存的過程中,糞便中的雌激素存在轉(zhuǎn)化、降解等過程,堆放的時間也對堆積糞便中雌激素含量具有一定的影響.因此,在評估奶牛養(yǎng)殖通過糞便排放的雌激素負(fù)荷時,如采用從糞便堆場樣品的濃度進(jìn)行估算,可能會導(dǎo)致估算結(jié)果存在偏差.

表3 牛糞堆場樣品中雌激素含量(μg/kg)分布(n=24)

2.4 不同生長階段奶牛糞便雌激素日排放量

研究采集4個季度的犢牛、育成牛、泌乳牛糞便樣品各60份,經(jīng)計(jì)算得到犢牛、育成牛、泌乳牛每日產(chǎn)糞量均值分別為0.91, 20.43, 23.32kg,含水率均值分別為70.38%、78.22%、80.64%.犢牛日產(chǎn)糞量明顯低于育成牛、泌乳牛.張振偉等[53]的研究表明,產(chǎn)糞量與奶牛的體重有關(guān),隨著體重的增加,產(chǎn)糞量也會相應(yīng)增加.

結(jié)合各生長階段奶牛糞便中雌激素含量,參照1.5.2計(jì)算各生長階段奶牛每天通過糞便排放的雌激素量,結(jié)果見表4.泌乳牛、育成牛、犢牛每天通過糞便排泄的雌激素含量分別為364.28, 115.55, 12.70μg/(d×頭),明顯低于Hui等[7]研究中估算的每頭奶牛雌激素日排泄量1159.86～1533.12μg/(d×頭),其主要原因可能是估算方法及基礎(chǔ)數(shù)據(jù)來源不同導(dǎo)致的.每頭泌乳牛糞便中雌激素日排泄量(364.27μg/ (d×頭))與Tao等[16](192～671μg/(d×頭))和Johnson等[54](199μg/(d×頭))的研究結(jié)果非常接近.犢牛、育成牛、泌乳牛糞便雌激素日排泄量差異較大,主要是由于泌乳牛日產(chǎn)糞量與糞便中雌激素含量較高于育成牛、犢牛、泌乳牛通過糞便排泄的雌激素對環(huán)境中雌激素污染貢獻(xiàn)較大.

表4 不同生長階段奶牛糞便中雌激素日排泄量[μg/(d×頭)]

3 結(jié)論

3.1 新鮮奶牛糞便中4種天然雌激素和2種人工合成雌激素均有檢出,但以3種天然雌激素E1、17β-E2、17α-E2為主.不同生長階段奶牛糞便中雌激素含量存在顯著差異,6種雌激素的總含量排序?yàn)槊谌榕?犢牛>育成牛.

3.2 奶牛糞便中雌激素含量存在顯著的季節(jié)性差異,冬季與春季的含量相對高于夏季與秋季.在評估奶牛養(yǎng)殖通過糞便排放的雌激素負(fù)荷時,應(yīng)考慮采樣時間對結(jié)果的影響.

3.3 奶牛場牛糞堆場樣品中雌激素含量明顯高于定位欄的鮮糞樣品.在評估奶牛養(yǎng)殖通過糞便排放的雌激素負(fù)荷時,如采用牛糞堆場樣品的雌激素含量進(jìn)行估算,可能會導(dǎo)致結(jié)果存在偏差.

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Characterizing the estrogen content in cow feces based on gestation crate experiment.

LI Yan1,2, PAN Jie1, LIANG Zi-wei2,3, HU Jia-wu2, LIU Jiang-yan1,2, HE De-chun2*, LI Ting-zhen1**

(1.Chongqing Key Laboratory of Water Environment Evolution and Pollution Control in Three Gorges Reservoir, Chongqing Three Gorges University, Chongqing 404130, China；2.South China Institute of Environmental Science, Ministry of Ecology and Environment, Guangzhou 510530, China；3.Zhongkai University of Agriculture and Engineering, Guangzhou 510225, China)., 2022,42(1)：119～126

To explore characteristics of estrogen content in cow feces, fecal samples were collected from the gestation crates to quantify the contents of 4 natural estrogens (including estrone, 17α-estradiol, 17β-estradiol, estriol) and 2 synthetic estrogens (17α-ethinylestradiol, diethylstilbestrol) with ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The results showed that the detection frequency (DF) of 6 estrogens ranged from 8.33% to 98.89%. The DF of detected natural estrogens was higher than 90%, except for estriol (8.33%); while the DF of synthetic estrogens were 52.22% and 64.44% for 17α-estradiol and diethylstilbestrol, respectively. The total content of 6 estrogens was in the range of 1.15～658.91μg/kg (dry weight, dw). The total estrogenic activity was equivalent to the content of 3.76～478.50μg/kg (dw). A significant difference in the total content (median) of 6 estrogens among different growth stages was observed in the order of: lactating cows (79.21μg/kg) > calves (47.20μg/kg) > heifers (25.72μg/kg). The content of estrogens in fecal samples demonstrate large seasonal variations: relatively higher in winter and spring than in summer and autumn andthe lowest concentration of 6 estrogens was observed in autumn. The total concentration of 6 estrogens in the feces collected from fecal stock dump ranged from 7.63 to 3921.92μg/kg which were significantly higher than that in fresh feces from gestation crate. Clearly, cow growth stage, sampling time (season) and sampling sites influenced the content of estrogens in cow feces, which should be fully considered in the investigation and risk assessment of estrogen emissions for cow breeding.

dairy cows；feces；estrogen；growth stage；seasonal variation

X713,171.5

A

1000-6923(2022)01-0119-08

李 艷(1997-),女,湖南郴州人,重慶三峽學(xué)院碩士研究生,研究方向?yàn)樾滦臀廴疚锟刂萍夹g(shù)研究.

2021-05-31

中央級公益性科研院所基本科研業(yè)務(wù)專項(xiàng)項(xiàng)目(PM-zx097- 202104-121);三峽庫區(qū)水環(huán)境演變與污染防治重慶高校市級重點(diǎn)實(shí)驗(yàn)室開放基金(WEPKL2019ZD-01,WEPKL2016XX-01);國家重點(diǎn)研發(fā)計(jì)劃項(xiàng)目(2017YFD0800701)

* 責(zé)任作者, 賀德春, 研究員, hedechun@scies.org; 李廷真, 教授, litingzhen@163.com