废水中典型EDCs的检测新方法及其降解特性研究
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摘要
近年来,内分泌干扰物(EDCs)污染由于人类生产和生活而日益给生态环境带来了巨大的威胁。其被称为第三代环境污染物,即便是低浓度暴露下,也会引起生物体生殖能力降低,并最终导致种群灭绝。EDCs性质稳定,不易降解,通过食物链发生生物富集和生物放大,最终严重危害人类。在我国EDCs亦被发现在很多水域,但相比下国内开展相关的研究还非常不足,其中检测和降解是控制治理EDCs污染的关键环节。开发EDCs的测定技术、掌握EDCs降解特性的研究已经成为摆在我国环境科学工作者面前的重要任务之一。本文在500余种EDCs中,以多环芳烃(PAHs)、阿维菌素(AVMs)、拟除虫菊酯(SPs)和类固醇(Steroids) 4类典型、常见的EDCs物质为目标,分别对其快速、多残留、高精度检测及微生物降解特性展开研究。
经一系列优化实验,最终采用固相萃取方式,真空旋转蒸发作为提纯方式,流动相流速800μL min-1,柱温30℃,在优化的色谱及检测器程序下,得到的16种PAHs线性方程的相关系数均大于0.9950,方法检出限的范围在0.58～11.27 ng.L-1,高中低3浓度下的加标回收实验表明,方法的回收率在80.32%～120.74%,各物质被平行测定5次的相对标准差在2.02%～8.71%,大大满足美国环保局的加标回收率在70～130%之间,RSD小于30%的要求。在此16种PAHs中,分别选取菲、芘作为低环、稠环芳烃的代表,驯化出优势菌株W1、W2,开展在泥水混合相和单一水相中混合菌株及单一优势菌株的降解性质实验。实验结果表明,更接近于自然状况的泥水相、混合菌群下,降解率优于单一水相及单一菌株的降解,但芘降解实验的各组差异不如菲显著。
经实验,选择以含0.1%甲酸的水和甲醇作为流动相,在优化后的色谱及质谱参数条件下,峰形良好,分离度佳,其中丙酸睾酮等7种物质产生了基峰质子化的准分子离子峰[M+H]+,另外6种物质分子脱水产生了准分子离子[M+H-H2O]+,各化合物回归方程线性均高于0.9990,方法的检出限0.15～20 ng.mL-1。13种类固醇加标回收率范围83.75%～111.50%,平行测定6次的相对标准偏差2.02%～14.21%。经实际废水样品测定,该方法能较好的满足其监测需要。驯化、分离得到其优势菌株WS1～3,以炔雌醇为代表物对其降解性能开展实验。72h后,得到的4种降解条件下,各水平的对降解的影响曲线及微生物生长曲线。结果显示,当污染物初始浓度为10mgL-1,35℃,pH=9,NaCl、MgCl2、CaCl2、ZnCl2、FeCl3、CuCl2的浓度分别为20、30、10、10、40、10 mg L-1时,其降解率最高。说明不同条件的不同水平,对降解性能均有不同程度的影响。
对阿维菌素类及拟除虫菊酯类驱虫剂,采用无需衍生化的固相萃取为简单便捷的前处理手段,高效液相色谱-串联四极杆质谱联用技术为高效准确的检测手段,在电喷雾电离正离子模式下,建立了一次进样测定分析两类、7种驱虫剂多残留污染浓度的分析方法,7种化合物定量限范围为0.03～2.47ng mL-1,其校正曲线的相关系数均高于0.9980。从农药底泥中驯化、分离得到1株能同时降解溴氰菊酯、氯氰菊酯和氟氯苯菊酯的高效菌株U1,及能降解阿巴菌素的高效菌株W1,分别以此2株优势降解菌对2类农药化合物进行了降解性能实验。当接种量为OD415nm值0.2,农药浓度为150 mg L-1,25℃,pH=7.0,振荡速率220 rpm时,驯化得到的优势菌株对溴氰菊酯、氯氰菊酯和氟氯苯菊酯3种农药的降解率分别达到最大。在温度为30℃,pH=7.0,污染物初始浓度为100mg L-1,振荡速率300rpm时,其对阿巴菌素的降解率达到峰值。
In recent years, Endocrine Disrupting Chemicals (EDCs) contamination has progressively posed great threat to ecological environment because of the activities of mankind's production and living. It's called the third generation of environmental pollutants, and even under lower exposure concentration, it still can decrease the reproductive capacity of organism and lead to species extinction eventually. The physical and chemical properties of EDCs is stable, it's difficult to degrade,and is absorbed in organism. By food chain, It can also be seriously bioaccumulated and biomagnified, and severely harm human heath finally. In China, EDCs had also been found in many watershed, but the related researchs are very insufficient in contrast, in which determination and degradation are the crucial element for controlling and treating EDCs contamination. For the environmental scientists of our country, the researchs about developing the determination technology of EDCs and mastering degradation characteristics of EDCs have already become one of the most important task. From more than 500 kinds of EDCs, this study selected Polycyclic Aromatic Hydrocarbons (PAHs), Avermectins (AVMs), Synthetic Pyrethroids (SPs) and Steroids 4 types of typical EDCs as research objectives, conducting respectively research about the method of rapid, multi-residue, precision determination and the characteristic of biodegradation.
Through a series of optimization experiment, finally taking Solid-Phase Extraction (SPE) as enrichment manner, Rotary Vacuum Evaporation as purification manner, the flow rate of mobile phase was 800μL min-1, the column temperature was 30℃, under the optimized procedures of chromatography and detector, the obtained correlations of linear equations of 16 PAHs were all greater than 0.9950, the limit of detection (LOD) of obtained method were 0.58～11.27 ng·L-1, and the recovery rate experiment at high, middle and low 3 concentration indicated that the recovery rates were 80.32%～120.74%, the relative standard deviations(RSD) were 2.02%～8.71% in 5 duplicates, it far exceeded the request of USEPA that the recovery rate should be 70～130% and RSD should be less than 30%. From the refered 16 PAHs, selecting phenanthrene(Phe) and pyrene(Pyr) to represent respectively low and high molecular weight PAHs, acclimating and obtaining dominant bacterial strains W1, W2, the degradation characteristics experiments were performed in slurry phase and aqueous phase, by individual isolate and mixed consortium. The result indicated, since it's more closer to natural conditions, when in slurry phase and by mixed consortium,the degradation rate was more better than it in other conditions, however, for Pyr,the difference of result under each condition wss not as significant as Phe.
Taking ultrawater and methanol(both of that include 0.1% formic acid) as mobile phases, under optimized parameter conditions of chromatography and mass spectrum, both peak shapes and separation were pretty good. Testosterone propionate etc.7 compounds produced quasi-molecular ion [M+H]+, and other 6 compounds dehydrated to produce quasi-molecular ion [M+H-H2O]+, the corelations of regression equations of each compound were all greater than 0.9990, the LODs were 0.15～20 ng-mL-1, the range of recovery rate of 13 steroid compounds was 83.75%～111.50%, the RSD in 6 duplicates were 2.02%～14.21%. By actual measuring of waste example, the method could better meet the measuring's request. After acclimating, isolating, the dominant bacteria strains of ethynyloestradiol(EE2) WS1-3 were obtained, the degradation experiments of EE2 were carried out. After 72 hours, the curves of degradation effect and microbe growth under each level of 4 conditions were acquired. The result shown that, when the initial concentration of contamination was 10 mgL-1, the experiment temperature was 35℃, the value of pH was 9, the concentrations of NaCl、MgCl2、CaCl2、ZnCl2、FeCl3、CuCl2 were respectively 20、30、10、10、40、10 mg L-1, the degradation rate reached the maximum. It was shown that the different levels of different conditions have effected the degradation performance in varying degree.
For Avermectins(AVMs) and Synthetic Pyrethroids(SPs) compounds, adopting non-derivatization SPE as simple and convenient pre-treatment method, High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MSMS) as efficient and precise measure manner, in electro-spray ion(ESI) soure and positive mode, the multi-residue analysis method of 2 types of 7 kinds of pesticides's concentration in one injection was developed, the range of limit of quantitation(LOQ) of 7 compounds was from 0.03 to 2.47ng mL-1, the corelation of calibration curve were all greater than 0.9980. The U1, a bacteria strain that could efficiently degrade both deltamethrin(DTM), cypermethrin(CPM) and flumethrin(FM) at the same time was acclimated and isolated from pesticide sediment. And another bacteria strain, W1, which could efficiently degrade abamectin(Aba) was obtained as well. Using this 2 dominant strains respectively, the degradation experiments of that 2 types pesticide compounds were conducted. When the inoculum size OD415 nm was 0.2, the initial concentration of contamination was 150 mg L-1, experiment temperature was 25℃, the value of pH was 7.0, shaker rotation was at 220rpm, the degradation rates of DTM, CPM and FM reached the maximum value respectively. When experiment temperature was 30℃, pH=7.0, the initial concentration of contamination was 100 mg L-1, shaker rotation was at 220rpm, the degradation rate of Aba reached the peak value.
经一系列优化实验,最终采用固相萃取方式,真空旋转蒸发作为提纯方式,流动相流速800μL min-1,柱温30℃,在优化的色谱及检测器程序下,得到的16种PAHs线性方程的相关系数均大于0.9950,方法检出限的范围在0.58～11.27 ng.L-1,高中低3浓度下的加标回收实验表明,方法的回收率在80.32%～120.74%,各物质被平行测定5次的相对标准差在2.02%～8.71%,大大满足美国环保局的加标回收率在70～130%之间,RSD小于30%的要求。在此16种PAHs中,分别选取菲、芘作为低环、稠环芳烃的代表,驯化出优势菌株W1、W2,开展在泥水混合相和单一水相中混合菌株及单一优势菌株的降解性质实验。实验结果表明,更接近于自然状况的泥水相、混合菌群下,降解率优于单一水相及单一菌株的降解,但芘降解实验的各组差异不如菲显著。
经实验,选择以含0.1%甲酸的水和甲醇作为流动相,在优化后的色谱及质谱参数条件下,峰形良好,分离度佳,其中丙酸睾酮等7种物质产生了基峰质子化的准分子离子峰[M+H]+,另外6种物质分子脱水产生了准分子离子[M+H-H2O]+,各化合物回归方程线性均高于0.9990,方法的检出限0.15～20 ng.mL-1。13种类固醇加标回收率范围83.75%～111.50%,平行测定6次的相对标准偏差2.02%～14.21%。经实际废水样品测定,该方法能较好的满足其监测需要。驯化、分离得到其优势菌株WS1～3,以炔雌醇为代表物对其降解性能开展实验。72h后,得到的4种降解条件下,各水平的对降解的影响曲线及微生物生长曲线。结果显示,当污染物初始浓度为10mgL-1,35℃,pH=9,NaCl、MgCl2、CaCl2、ZnCl2、FeCl3、CuCl2的浓度分别为20、30、10、10、40、10 mg L-1时,其降解率最高。说明不同条件的不同水平,对降解性能均有不同程度的影响。
对阿维菌素类及拟除虫菊酯类驱虫剂,采用无需衍生化的固相萃取为简单便捷的前处理手段,高效液相色谱-串联四极杆质谱联用技术为高效准确的检测手段,在电喷雾电离正离子模式下,建立了一次进样测定分析两类、7种驱虫剂多残留污染浓度的分析方法,7种化合物定量限范围为0.03～2.47ng mL-1,其校正曲线的相关系数均高于0.9980。从农药底泥中驯化、分离得到1株能同时降解溴氰菊酯、氯氰菊酯和氟氯苯菊酯的高效菌株U1,及能降解阿巴菌素的高效菌株W1,分别以此2株优势降解菌对2类农药化合物进行了降解性能实验。当接种量为OD415nm值0.2,农药浓度为150 mg L-1,25℃,pH=7.0,振荡速率220 rpm时,驯化得到的优势菌株对溴氰菊酯、氯氰菊酯和氟氯苯菊酯3种农药的降解率分别达到最大。在温度为30℃,pH=7.0,污染物初始浓度为100mg L-1,振荡速率300rpm时,其对阿巴菌素的降解率达到峰值。
In recent years, Endocrine Disrupting Chemicals (EDCs) contamination has progressively posed great threat to ecological environment because of the activities of mankind's production and living. It's called the third generation of environmental pollutants, and even under lower exposure concentration, it still can decrease the reproductive capacity of organism and lead to species extinction eventually. The physical and chemical properties of EDCs is stable, it's difficult to degrade,and is absorbed in organism. By food chain, It can also be seriously bioaccumulated and biomagnified, and severely harm human heath finally. In China, EDCs had also been found in many watershed, but the related researchs are very insufficient in contrast, in which determination and degradation are the crucial element for controlling and treating EDCs contamination. For the environmental scientists of our country, the researchs about developing the determination technology of EDCs and mastering degradation characteristics of EDCs have already become one of the most important task. From more than 500 kinds of EDCs, this study selected Polycyclic Aromatic Hydrocarbons (PAHs), Avermectins (AVMs), Synthetic Pyrethroids (SPs) and Steroids 4 types of typical EDCs as research objectives, conducting respectively research about the method of rapid, multi-residue, precision determination and the characteristic of biodegradation.
Through a series of optimization experiment, finally taking Solid-Phase Extraction (SPE) as enrichment manner, Rotary Vacuum Evaporation as purification manner, the flow rate of mobile phase was 800μL min-1, the column temperature was 30℃, under the optimized procedures of chromatography and detector, the obtained correlations of linear equations of 16 PAHs were all greater than 0.9950, the limit of detection (LOD) of obtained method were 0.58～11.27 ng·L-1, and the recovery rate experiment at high, middle and low 3 concentration indicated that the recovery rates were 80.32%～120.74%, the relative standard deviations(RSD) were 2.02%～8.71% in 5 duplicates, it far exceeded the request of USEPA that the recovery rate should be 70～130% and RSD should be less than 30%. From the refered 16 PAHs, selecting phenanthrene(Phe) and pyrene(Pyr) to represent respectively low and high molecular weight PAHs, acclimating and obtaining dominant bacterial strains W1, W2, the degradation characteristics experiments were performed in slurry phase and aqueous phase, by individual isolate and mixed consortium. The result indicated, since it's more closer to natural conditions, when in slurry phase and by mixed consortium,the degradation rate was more better than it in other conditions, however, for Pyr,the difference of result under each condition wss not as significant as Phe.
Taking ultrawater and methanol(both of that include 0.1% formic acid) as mobile phases, under optimized parameter conditions of chromatography and mass spectrum, both peak shapes and separation were pretty good. Testosterone propionate etc.7 compounds produced quasi-molecular ion [M+H]+, and other 6 compounds dehydrated to produce quasi-molecular ion [M+H-H2O]+, the corelations of regression equations of each compound were all greater than 0.9990, the LODs were 0.15～20 ng-mL-1, the range of recovery rate of 13 steroid compounds was 83.75%～111.50%, the RSD in 6 duplicates were 2.02%～14.21%. By actual measuring of waste example, the method could better meet the measuring's request. After acclimating, isolating, the dominant bacteria strains of ethynyloestradiol(EE2) WS1-3 were obtained, the degradation experiments of EE2 were carried out. After 72 hours, the curves of degradation effect and microbe growth under each level of 4 conditions were acquired. The result shown that, when the initial concentration of contamination was 10 mgL-1, the experiment temperature was 35℃, the value of pH was 9, the concentrations of NaCl、MgCl2、CaCl2、ZnCl2、FeCl3、CuCl2 were respectively 20、30、10、10、40、10 mg L-1, the degradation rate reached the maximum. It was shown that the different levels of different conditions have effected the degradation performance in varying degree.
For Avermectins(AVMs) and Synthetic Pyrethroids(SPs) compounds, adopting non-derivatization SPE as simple and convenient pre-treatment method, High Performance Liquid Chromatography-Tandem Mass Spectrometry (HPLC-MSMS) as efficient and precise measure manner, in electro-spray ion(ESI) soure and positive mode, the multi-residue analysis method of 2 types of 7 kinds of pesticides's concentration in one injection was developed, the range of limit of quantitation(LOQ) of 7 compounds was from 0.03 to 2.47ng mL-1, the corelation of calibration curve were all greater than 0.9980. The U1, a bacteria strain that could efficiently degrade both deltamethrin(DTM), cypermethrin(CPM) and flumethrin(FM) at the same time was acclimated and isolated from pesticide sediment. And another bacteria strain, W1, which could efficiently degrade abamectin(Aba) was obtained as well. Using this 2 dominant strains respectively, the degradation experiments of that 2 types pesticide compounds were conducted. When the inoculum size OD415 nm was 0.2, the initial concentration of contamination was 150 mg L-1, experiment temperature was 25℃, the value of pH was 7.0, shaker rotation was at 220rpm, the degradation rates of DTM, CPM and FM reached the maximum value respectively. When experiment temperature was 30℃, pH=7.0, the initial concentration of contamination was 100 mg L-1, shaker rotation was at 220rpm, the degradation rate of Aba reached the peak value.
引文
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