青岛李村河—胶州湾水域雌激素水平的研究
摘要
天然雌激素如雌酮、雌二醇、雌三醇或合成雌激素如雌炔醇是一类甾体荷尔蒙。这些雌激素物质对人或野生动物的内分泌系统具有干扰作用。天然雌激素是由人和动物产生，而雌炔醇是合成的避孕药，它们都会随生物排泄物进入环境。随着人口和动物数量的增加，这些物质进入环境的数量也会增加，这将对生态平衡系统造成潜在的威胁。这些物质一旦进入食物链还会影响人类的健康。因此，开展环境中雌激素的研究非常重要。
为了研究青岛近海水环境是否受到雌激素的污染，本文采用气相色谱-质谱法对青岛李村河至胶州湾水域系统中的四种雌激素水平进行测试，通过水、底泥及贝类组织中雌激素的水平对该流域的雌激素污染情况进行研究。本研究同时还对青岛的海伯河河水、石老人海水及栈桥海水的雌激素水平进行了考察。
在这项研究工作中，建立了用改性的吸附剂对水，底泥和贝类样品中的雌激素物质进行了离线固相萃取，用GC-MS测法进行测定的方法。对该实验方法的所有步骤进行了系统的研究。
首先，将三种天然雌激素(E1，E2，E3)和合成雌激素雌炔醇(EE2)分别用三氟乙酸酐(TFA)和N-甲基、N-三甲基硅基三氟乙酰胺(MSTFA)进行衍生化和色谱测定，通过该过程的产率对衍生化条件进行了系统的研究。在优化的条件下，通过GC-FID方法测定的天然雌激素的三氟乙酰衍生物的检出限为6．90～21．90ng／L(采用针尖浓缩进样方式)，四种雌激素硅烷化衍生物的方法检出限为1．34～9．85ng／L，使该方法能够用于样品的加标回收率的准确测定。采用GC-MS方法对实际样品进行分离测定。实际样品中的目标离子的定性分析采取质谱特征诊断离子的方法进行确定，用内标法或内标工作曲线法对实际样品中的雌激素进行定量分析，其方法的检出限为E1=1．98ng／L，E2=1．91ng／L，EE2=6．98ng／L，E3=2．15ng／L．该工作曲线的线性相关系数在0．98～0．99之间。测定值的相对标准偏差为3～15％。
第二，对实际样品中目标物的提取采用了C-18固相萃取法(SPE)或液-固提取法。实验对C-18硅胶颗粒对雌激素的吸附性能，水样体积，pH值和样品的性质等固相萃取操作对回收率的影响以及对萃取物的洗脱和浓缩规程的影响进行了优化。另外，还根据实际水体的污染情况对C-18吸附剂硅胶颗粒进行了修饰，使400mg该C-18吸附剂对目标物的富集回收率达到88～120％。对海泥、河泥中或牡蛎和蛤蛎组织中的雌激素物质的整体分析过程的加标回收率在70～120％(GC-FID法)。
最后，用上述提取方法及气相色谱-质谱法对2005～2007年3～6月的李村河-胶州湾流域的水、底泥和两种贝类(蛤蛎和牡蛎)组织中的雌激素进行了测定。结果显示，该流域的水、底泥样品及贝类样品组织中均含有雌激素，它们主要为E1、E2和E3，也含有少量的EE2。其中在该流域的水样中雌激素的水平为nd(未测出)～306．0ng／L，在底泥样中的浓度为nd～42．0 ng／g干土，在贝类样品组织中为nd～29．7ng／g湿重。在海伯河水样、石老人或栈桥海水样中的雌激素含量分别为31～97．0ng／L、nd～13．3ng／L、nd～10．0ng／L。实验还测定了污水处理厂脱水污泥中的雌激素，其含量为nd～5．9ng／g。水及底泥中的测定结果与国内的某些测定结果相近，三种环境样品中雌激素的测定结果与国外的一些测定值相近或高于他们的报道值。
根据测定结果，该流域雌激素的分布特点为：水中及底泥中雌激素的水平在不同地域水平不同(如海水中的低于河水中)；同一地点的不同时刻水中雌激素的含量不同；雌激素的含量E1大于E3；在大多数采样点，底泥与水中雌激素的含量比值E1为28～444 L／kg，E3为20～140 L／kg。海岸边的牡蛎组织中的雌激素含量明显高于深海中的，海岸底泥中的蛤蛎组织中的雌激素水平高于市售的。在调查期间，连续3个月测得该流域的水、底泥中含有一定量的雌激素物质，蛤蛎组织中的雌激素水平高于栖息的底泥中的。
经调查，李村河水主要来自河道沿途工农业生产、养殖及生活污水的排放。
研究结果表明，雌激素物质已经进入李村河-胶州湾流域的水环境，水中的雌激素水平较高，有可能对敏感野生生物雌性化有影响。至于这种雌激素污染是否造成了该流域水生态系统的危害还有待进一步的研究。
Estrogens such as estril (E3),estradiol (E2) and estrone (El)which are excretedby humans and animals or synthetic compounds such as 17β-ethinyl estradiol (EE2)are a group of hormone steroids.These compounds could affect wildlife and humanhealth by disrupting their normal endocrine systems.With number of populations andanimals increasing,amount of these steroids hormones will be discharged toenvironment and cause potential threat to the ecological balance.Therefore,thestudies on estrogens in environment is very important.
For Investigation on whether waters in Qingdao are polluted by estrogens,in thispaper,levels of four hormone steroids in aquatic environment in Licun river -Jiaozhou Bay in Qingdao in China were determined by Gas chromatography Massspectrum (GC-MS) method .According to the levels of estrogens in water,sedimentand shellfish meat tissue from the waters,pollution of estrogens in the aquaticenvironment will be evaluated.Moreover,estrogens in water from Haipo river watersamples and Shilaoren and Zhanqiao seawater sample were studied
In this work,an off-line solid-phase extraction procedure,using a modifiedpolymeric sorbent,in combination with GC-MS detection was proposed for theanalysis of estrogens in waters,sediment and shellfish samples.All steps comprisedin the analytical procedure were systematically studied.
In the first,three natural (E1,E2,E3)and synthetic estrogens (EE2) werederivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA) ortrifluoroacetyl anhydride (TFA) and determined by GC.Derivatization conditions inthe yield of the process was systematically investigated.Under optimal conditions,quantification limits between 6.9 and 21.9ng/L were achieved for trifluoroacetylestrogens by injection way of samples condensed on needle and these of estrogens silylated were 1.3 and 9.8 ng/L by GC-FID mode.The results showed that recoveriesof estrogens spiked in real samples can be accurately detected by the GC-FID mode.Estrogens in real samples were detected by GC-MS mode and the detected limits areE1=1.98ng/L,E2=1.91ng/L,EE2=6.98ng/L,E3=2.15ng/L or ( solid ng/g) byGC-MS mode,respectively.Qualitative analysis of the targets in real samples aretaken by diagnostic ion of mass spectrum,the concentration are calculated usingmethod of internal standard or internal standard calibration curve.Correlationcoefficients of these curves are between 0.98 and 0.99.The relative standard deviationreached 3～15%.
The second,considered estrogens in real samples are extracted by C-18 solidphase extraction (SPE) (or) liquid-solid extraction method.Properties of the C-18silica particles that sorbs estrogens,influence of parameters such as breakthroughvolumes ,PH of water samples,nature of the samples and soon in the recoveries ofthe solid-phase extraction step and in the cleanliness of obtained extracts wereexamined and optimized.Furthermore,according to conditions of real samples,C-18sorbent were modified and recoveries of estrogens spiked in pollution samplesreached 88-120% by using 400mg of the silica particles.The recoveries ofestrogens in the water,sediment and meat samples were 70～120% by extraction andanalysis (GC-FID mode) on the performance of the whole analytical procedure.
Finally,the developed method was applied to the determination of estrogens ineffluent sewage water samples,sediment and shellfish tissue such as oysters andcalms from Licun river-Jiaozhou gulf for 3～6 month in 2005～2007 years.The resultsshowed that estrogens such as mainly E1,E2 and E3 were found in every sample butEE2 exited in several samples.Levels of estrogens in water samples were nd( notdetected)～306.0 ng/L,nd～42.0 ng/g dry soil in sediment and nd～29.7ng/g wet weighin shellfish tissue.Moreover,estrogens from water in Haipo river were between 31.0and 97.0 ng/L,nd～13.3ng/L and nd～10.0ng/L in sea water from Shilaoren orZhanqiao,respectively.Estrogens concentration were nd～5.9ng/g dry soil in activesludge from Licun wastewater treatment plant.Compared to some determining valuefrom China,our results are similar with ,but estrogens levels in three kinds of samples are similarly with reported value or higher than these from other countries.
The distribution of estrogens in water and sediment are as follow:First,estrogenslevels in water and sediment were affected by water quality of fiver or sea water,forexample estrogens contents in different location not only exited significant difference(such as the levels in fiver was higher than that in sea) but it did in different time inthe same sampling side.Second,concentrations of E1 in most samples were higherthan these of E3.Third,in most sampling sites,proportions of E1 in sediment andwater ranged from 28～444 L/kg,these of E3 were 20～140L/kg.Forth,estrogenslevels in oysters tissue on rocks in beach were higher than these in blue seawater andmore estrogens in clams in the sediment in estuary were found than these in market.Finally,persistence of these hormone steroids in water or sediment in the zone wasclear for 3 and 5 month and levels of estrogens in shellfish tissue were higher thanthat from sediment in clam habitat。
According to the survey,Licun River mainly came from the discharging ofindustry and agriculture,domestic sewage effluents and disposal of animal waste.
This results show that estrogens have entered the aquatic environment in Licunriver and Jiaozhou Bay,estrogens levels in the water might affect endocrine system ofsensitive wild animals in the zone.However,further investigations are necessary tounderstand whether or not the estrogens pollutions caused harm to ecosystem in thewater.
为了研究青岛近海水环境是否受到雌激素的污染，本文采用气相色谱-质谱法对青岛李村河至胶州湾水域系统中的四种雌激素水平进行测试，通过水、底泥及贝类组织中雌激素的水平对该流域的雌激素污染情况进行研究。本研究同时还对青岛的海伯河河水、石老人海水及栈桥海水的雌激素水平进行了考察。
在这项研究工作中，建立了用改性的吸附剂对水，底泥和贝类样品中的雌激素物质进行了离线固相萃取，用GC-MS测法进行测定的方法。对该实验方法的所有步骤进行了系统的研究。
首先，将三种天然雌激素(E1，E2，E3)和合成雌激素雌炔醇(EE2)分别用三氟乙酸酐(TFA)和N-甲基、N-三甲基硅基三氟乙酰胺(MSTFA)进行衍生化和色谱测定，通过该过程的产率对衍生化条件进行了系统的研究。在优化的条件下，通过GC-FID方法测定的天然雌激素的三氟乙酰衍生物的检出限为6．90～21．90ng／L(采用针尖浓缩进样方式)，四种雌激素硅烷化衍生物的方法检出限为1．34～9．85ng／L，使该方法能够用于样品的加标回收率的准确测定。采用GC-MS方法对实际样品进行分离测定。实际样品中的目标离子的定性分析采取质谱特征诊断离子的方法进行确定，用内标法或内标工作曲线法对实际样品中的雌激素进行定量分析，其方法的检出限为E1=1．98ng／L，E2=1．91ng／L，EE2=6．98ng／L，E3=2．15ng／L．该工作曲线的线性相关系数在0．98～0．99之间。测定值的相对标准偏差为3～15％。
第二，对实际样品中目标物的提取采用了C-18固相萃取法(SPE)或液-固提取法。实验对C-18硅胶颗粒对雌激素的吸附性能，水样体积，pH值和样品的性质等固相萃取操作对回收率的影响以及对萃取物的洗脱和浓缩规程的影响进行了优化。另外，还根据实际水体的污染情况对C-18吸附剂硅胶颗粒进行了修饰，使400mg该C-18吸附剂对目标物的富集回收率达到88～120％。对海泥、河泥中或牡蛎和蛤蛎组织中的雌激素物质的整体分析过程的加标回收率在70～120％(GC-FID法)。
最后，用上述提取方法及气相色谱-质谱法对2005～2007年3～6月的李村河-胶州湾流域的水、底泥和两种贝类(蛤蛎和牡蛎)组织中的雌激素进行了测定。结果显示，该流域的水、底泥样品及贝类样品组织中均含有雌激素，它们主要为E1、E2和E3，也含有少量的EE2。其中在该流域的水样中雌激素的水平为nd(未测出)～306．0ng／L，在底泥样中的浓度为nd～42．0 ng／g干土，在贝类样品组织中为nd～29．7ng／g湿重。在海伯河水样、石老人或栈桥海水样中的雌激素含量分别为31～97．0ng／L、nd～13．3ng／L、nd～10．0ng／L。实验还测定了污水处理厂脱水污泥中的雌激素，其含量为nd～5．9ng／g。水及底泥中的测定结果与国内的某些测定结果相近，三种环境样品中雌激素的测定结果与国外的一些测定值相近或高于他们的报道值。
根据测定结果，该流域雌激素的分布特点为：水中及底泥中雌激素的水平在不同地域水平不同(如海水中的低于河水中)；同一地点的不同时刻水中雌激素的含量不同；雌激素的含量E1大于E3；在大多数采样点，底泥与水中雌激素的含量比值E1为28～444 L／kg，E3为20～140 L／kg。海岸边的牡蛎组织中的雌激素含量明显高于深海中的，海岸底泥中的蛤蛎组织中的雌激素水平高于市售的。在调查期间，连续3个月测得该流域的水、底泥中含有一定量的雌激素物质，蛤蛎组织中的雌激素水平高于栖息的底泥中的。
经调查，李村河水主要来自河道沿途工农业生产、养殖及生活污水的排放。
研究结果表明，雌激素物质已经进入李村河-胶州湾流域的水环境，水中的雌激素水平较高，有可能对敏感野生生物雌性化有影响。至于这种雌激素污染是否造成了该流域水生态系统的危害还有待进一步的研究。
Estrogens such as estril (E3),estradiol (E2) and estrone (El)which are excretedby humans and animals or synthetic compounds such as 17β-ethinyl estradiol (EE2)are a group of hormone steroids.These compounds could affect wildlife and humanhealth by disrupting their normal endocrine systems.With number of populations andanimals increasing,amount of these steroids hormones will be discharged toenvironment and cause potential threat to the ecological balance.Therefore,thestudies on estrogens in environment is very important.
For Investigation on whether waters in Qingdao are polluted by estrogens,in thispaper,levels of four hormone steroids in aquatic environment in Licun river -Jiaozhou Bay in Qingdao in China were determined by Gas chromatography Massspectrum (GC-MS) method .According to the levels of estrogens in water,sedimentand shellfish meat tissue from the waters,pollution of estrogens in the aquaticenvironment will be evaluated.Moreover,estrogens in water from Haipo river watersamples and Shilaoren and Zhanqiao seawater sample were studied
In this work,an off-line solid-phase extraction procedure,using a modifiedpolymeric sorbent,in combination with GC-MS detection was proposed for theanalysis of estrogens in waters,sediment and shellfish samples.All steps comprisedin the analytical procedure were systematically studied.
In the first,three natural (E1,E2,E3)and synthetic estrogens (EE2) werederivatized with N-methyl-N-(trimethylsilyl)trifluoroacetamide(MSTFA) ortrifluoroacetyl anhydride (TFA) and determined by GC.Derivatization conditions inthe yield of the process was systematically investigated.Under optimal conditions,quantification limits between 6.9 and 21.9ng/L were achieved for trifluoroacetylestrogens by injection way of samples condensed on needle and these of estrogens silylated were 1.3 and 9.8 ng/L by GC-FID mode.The results showed that recoveriesof estrogens spiked in real samples can be accurately detected by the GC-FID mode.Estrogens in real samples were detected by GC-MS mode and the detected limits areE1=1.98ng/L,E2=1.91ng/L,EE2=6.98ng/L,E3=2.15ng/L or ( solid ng/g) byGC-MS mode,respectively.Qualitative analysis of the targets in real samples aretaken by diagnostic ion of mass spectrum,the concentration are calculated usingmethod of internal standard or internal standard calibration curve.Correlationcoefficients of these curves are between 0.98 and 0.99.The relative standard deviationreached 3～15%.
The second,considered estrogens in real samples are extracted by C-18 solidphase extraction (SPE) (or) liquid-solid extraction method.Properties of the C-18silica particles that sorbs estrogens,influence of parameters such as breakthroughvolumes ,PH of water samples,nature of the samples and soon in the recoveries ofthe solid-phase extraction step and in the cleanliness of obtained extracts wereexamined and optimized.Furthermore,according to conditions of real samples,C-18sorbent were modified and recoveries of estrogens spiked in pollution samplesreached 88-120% by using 400mg of the silica particles.The recoveries ofestrogens in the water,sediment and meat samples were 70～120% by extraction andanalysis (GC-FID mode) on the performance of the whole analytical procedure.
Finally,the developed method was applied to the determination of estrogens ineffluent sewage water samples,sediment and shellfish tissue such as oysters andcalms from Licun river-Jiaozhou gulf for 3～6 month in 2005～2007 years.The resultsshowed that estrogens such as mainly E1,E2 and E3 were found in every sample butEE2 exited in several samples.Levels of estrogens in water samples were nd( notdetected)～306.0 ng/L,nd～42.0 ng/g dry soil in sediment and nd～29.7ng/g wet weighin shellfish tissue.Moreover,estrogens from water in Haipo river were between 31.0and 97.0 ng/L,nd～13.3ng/L and nd～10.0ng/L in sea water from Shilaoren orZhanqiao,respectively.Estrogens concentration were nd～5.9ng/g dry soil in activesludge from Licun wastewater treatment plant.Compared to some determining valuefrom China,our results are similar with ,but estrogens levels in three kinds of samples are similarly with reported value or higher than these from other countries.
The distribution of estrogens in water and sediment are as follow:First,estrogenslevels in water and sediment were affected by water quality of fiver or sea water,forexample estrogens contents in different location not only exited significant difference(such as the levels in fiver was higher than that in sea) but it did in different time inthe same sampling side.Second,concentrations of E1 in most samples were higherthan these of E3.Third,in most sampling sites,proportions of E1 in sediment andwater ranged from 28～444 L/kg,these of E3 were 20～140L/kg.Forth,estrogenslevels in oysters tissue on rocks in beach were higher than these in blue seawater andmore estrogens in clams in the sediment in estuary were found than these in market.Finally,persistence of these hormone steroids in water or sediment in the zone wasclear for 3 and 5 month and levels of estrogens in shellfish tissue were higher thanthat from sediment in clam habitat。
According to the survey,Licun River mainly came from the discharging ofindustry and agriculture,domestic sewage effluents and disposal of animal waste.
This results show that estrogens have entered the aquatic environment in Licunriver and Jiaozhou Bay,estrogens levels in the water might affect endocrine system ofsensitive wild animals in the zone.However,further investigations are necessary tounderstand whether or not the estrogens pollutions caused harm to ecosystem in thewater.
引文
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