原代培养细胞法测试水中类二噁英物质

采用固相萃取方法对北方某水厂水源水和各处理工艺出水进行富集,对各提取物采用草鱼原代培养肝细胞的7-乙氧基异吩唑酮-脱乙基酶(EROD)体外测试方法进行了类二口恶英物质的检验。结果表明,该水厂水源水和各处理工艺出水中,类二噁英物质浓度相当于0.20～0.50pg/L的2,3,7,8-TCDD(2,3,7,8-四氯代二苯并二噁英)当量,出水TCDD当量远远低于美国环保局饮用水二噁英类化合物含量标准(30pg/L)。研究表明,水样的固相萃取前处理方法结合原代培养细胞EROD分析,可以快速、有效地筛选和定量分析水样中未知的具有EROD效应的物质及其总体效应。     

原代培养细胞法测试水中类二(口恶)英物质

采用固相萃取方法对北方某水厂水源水和各处理工艺出水进行富集,对各提取物采用草鱼原代培养肝细胞的7-乙氧基异吩唑酮-脱乙基酶(EROD)体外测试方法进行了类二噁英物质的检验.结果表明,该水厂水源水和各处理工艺出水中,类二噁英物质浓度相当于0.20～0.50 pg/L的2,3,7,8-TCDD(2,3,7,8-四氯代二苯并二噁英)当量,出水TCDD当量远远低于美国环保局饮用水二噁英类化合物含量标准(30 pg/L).研究表明,水样的固相萃取前处理方法结合原代培养细胞EROD分析,可以快速、有效地筛选和定量分析水样中未知的具有EROD效应的物质及其总体效应.     

液相色谱-电离质谱法测定微囊藻毒素

通过固相萃取(SPE)富集微囊藻毒素(MC),并采用液相色谱-电喷雾电离质谱(HPLC/ESI-MS)测定水源水中的MC-RR、YR和LR。固相萃取回收率在85%以上,固相萃取-HPLC-MS法的线性检测限为0. 5~500ng/L。对北方B市和南方S市的几个饮用水水源和水厂各工艺出水的检测结果表明,两城市源水和水厂出水中的MC含量较低,但随气温升高则B市水库源水中MC含量有升高的趋势。     

“引江济太”工程对太湖水源水雌激素活性的影响

目的调查研究"引江济太"工程对太湖水源水雌激素活性的影响。方法分别于2013年12月、2014年1月和3月采集江苏省W市"引江济太"工程8个相关地点水样,应用固相萃取技术结合重组酵母雌激素筛检试验(recombinant yeast estrogen screen,YES)和液相色谱质谱联用(LC-MS/MS)分析方法,对水样中的有机物雌激素活性强度、酚类雌激素物质含量水平及变化规律进行分析,探讨调水量和相关水源水的雌激素活性及其中酚类雌激素物质的关系。结果各水样均表现出明显的雌激素活性,并且以望虞河入太湖口处的水样雌激素活性效应最强,该水样的酚类环境雌激素物质含量也最高。太湖内各水样雌激素活性强度与靠近望虞河口的距离呈正相关。结论以太湖水为源水的XD水源水和NQ水源水均不同程度受到了长江经望虞河引入太湖水的影响,并且其雌激素活性强度随着引水量增加而升高,呈现出正相关。     

不同工艺出水中有机物的致突变性及类雌激素效应

取南方某市水源水和经5种不同饮用水处理工艺处理后的出水，采用XAD-2大孔树脂分别提取水样中的非挥发性有机物，对其进行Ames试验和重组酵母菌雌激素效应检测，比较了水源水及不同工艺出水中非挥发性有机物（NOCs）的致突变性和类雌激素效应。结果表明，各工艺对水源水中具有致突变性和类雌激素效应的有机物均有一定程度的去除作用，其中经预臭氧氧化-混凝、沉淀-砂滤-GAC过滤-次氯酸钠消毒工艺处理的出水，其NOCs的致突变性和类雌激素效应均较低。     

SPE—GCMS法快速测定水中半挥发性有机物的研究

利用固相萃取-气质联用法对水中半挥发性有机物进行快速定性和定量。采用C18固相萃取小柱对水样进行富集,HP5-MS毛细管柱进行分离分析。该方法操作简单,分析速度快,分离效果好,灵敏度和精密度高。     

固相萃取-高效液相色谱法测定水中9种苯环类有机酸

建立了基于双层固相萃取-超高液相色谱(SPE-UPLC)法测定水中苯环类有机酸的检测方法。研究结果表明,80 mg质量比为1∶1的Lichrolut EN/ENVi-18双层填料在1.3 m L/min流量条件下,实现了混合苯环类有机酸的高效富集浓缩,1 m L甲醇洗脱9种苯环类有机酸的萃取率为100%±20%。测定条件如下,以20 mmol/L磷酸盐缓冲溶液(正磷酸调节p H值为2.10)∶纯水∶乙腈=5∶75∶20为流动相,以0.2 m L/min流速进行淋洗,色谱柱为ACQUITY UPLC BEH C18,柱温为25℃,TUV检测器,检测波长为210 nm,进样量为10μL。本方法可以同时测定水中9种苯环类有机酸,在给定浓度范围内各有机酸的峰面积与浓度具有良好的线性关系。平均回收率为79%~102.8%,RSD为0.26%~5.1%(n=6)。将该方法用于东太湖某水厂各工段水样检测,结果令人满意。     

预处理方式对测定渗滤液中有机污染物的影响

垃圾渗滤液中所含的有机污染物成分复杂、浓度高。在采用GC-MS法对其初步定性时预处理方式对检测结果影响较大,考察了液液萃取、固相萃取、大孔树脂吸附三种常见预处理方式对检测结果的影响,固相萃取适于测定垃圾渗滤液中醇酚类为主的物质;而大孔树脂对芳烃类与酯类的富集效果最佳;液液萃取对烯烃类及羧酸类物质萃取效率较高。采用固相萃取测得渗滤液中含有62种物质,其中EPA优先控制污染物有3种。     

污水处理过程中雌激素的测定与去除

雌激素在自然界中普遍存在,其危害较大并且已对水环境造成了威胁.基于近期国内外关于雌激素的研究,介绍了污水厂和自然水环境中雌激素的检测方法以及污水处理过程中雌激素去除的机理.目前应用较为广泛的化学检测方法(气质联用或液质联用)和生物检测方法(重组基因酵母法),在雌激素检测应用上各有优缺点.常规污水处理过程中,雌激素的去除包括污泥吸附和生物降解两个步骤,通过吸附作用雌激素从水相转移至固相活性污泥中,生物降解是雌激素去除的主要途径且遵循一级反应模式.     

HS-SPME-GC-MS/MS测定16种硫醚类嗅味物质

针对可导致饮用水中腥臭味/沼泽味/腐败味的硫醚类物质,基于顶空固相微萃取与气相色谱三重四极杆串联质谱联用,建立了可同时快速分析水中16种硫醚类嗅味物质的方法。对萃取纤维类型、盐浓度、萃取温度、萃取和解吸时间等条件进行了优化,确定的最佳顶空固相微萃取条件为：水样加入20%NaCl,采用DVB/PDMS/Carbon WR萃取纤维于45℃条件下萃取30 min,在250℃条件下解吸180 s。16种硫醚的标准曲线具有较好的线性（R2>0.99）,检出限为0.2～2.9 ng/L,超纯水和原水加标回收率分别为80.4%～105.4%和78.3%～108.2%,相对标准偏差分别为0.7%～13.4%和1.6%～14.1%,可满足饮用水及水源中硫醚类嗅味物质的快速检测。采用该方法对三个水厂的原水进行了分析,有二甲基二硫醚（4.2～45.3 ng/L）、二甲基三硫醚（1.9～6.1 ng/L）和二乙基二硫醚（N.D.～1.5 ng/L）检出,值得关注。     

Determination of estrogens and estrogenic activity in wastewater effluent by chemical analysis and the bioluminescent yeast assay

A scheme of bioassay-directed analysis has been developed which combines a yeast assay screening for estrogenic activity with a liquid chromatographic-mass spectrometric (LC-MS/MS) chemical analysis, chromatographic fractionation, solid phase extraction and freeze-drying. The test scheme was applied on effluent samples collected from a municipal sewage treatment plant. The aim was to determine the substances responsible for main portion of the estrogenic activity in the samples and to compare the efficiency of different procedures for isolation and concentration of estogenicity. LC-MS/MS analyses were used for the quantification of 17beta-estradiol, estrone, estriol and 17alpha-ethinylestradiol, and the measured concentrations compared with the activities found in the yeast assay. Following conversion of the concentrations measured by LC-MS/MS to 17beta-estradiol equivalents it was concluded that freeze-drying, solid phase extraction and the chemical analysis gave comparable activities. Since estrone was the major estrogen in the effluent, this estrogen was also the major contributor to the estrogenic activity in the effluent. The estrogenic activity was equivalent to 4-7 ng/L of 17beta-estradiol. The yeast assay results from the tests of the chromatographic fractions showed that the major activity resides in the fraction where estrone, 17beta-estradiol and 17alpha-ethinylestradiol eluted. The activity of this fraction was substantially higher than the activity of the original wastewater sample. The reason for this could in part be explained by an inhibition of activity occurring in the original water sample.     

Detection of estrogenic activity in sediment-associated compounds using in vitro reporter gene assays

Sediments may be the ultimate sink for persistent (xeno-)estrogenic compounds released into the aquatic environment. Sediment-associated estrogenic potency was measured with an estrogen receptor-mediated luciferase reporter gene (ER-CALUX) assay and compared with a recombinant yeast screen. The ER-CALUX assay was more sensitive to 17beta-estradiol (E2) than the recombinant yeast screen, with an EC50 of 6 pM E2 compared to 100 pM in the yeast screen. Yeast cells were unable to distinguish the anti-estrogens ICI 182,780 and (4-hydroxy)tamoxifen, which were agonistic in the yeast. Acetone-soluble fractions of hexane/acetone extracts of sediments showed higher estrogenic potency than hexane-soluble extracts in the ER-CALUX assay. Sediments obtained from industrialized areas such as the Port of Rotterdam showed the highest estrogenic potency of the 12 marine sediments tested (up to 40 pmol estradiol equivalents per gram sediment). The estrogenic activity of individual chemicals that can be found in sediments including: alkylphenol ethoxylates and carboxylates; phthalates; and pesticides, was tested. Increasing sidechain length of various nonylphenol ethoxylates resulted in decreased estrogenic activity. Of the phthalates tested, butylbenzylphthalate was the most estrogenic, though with a potency approximately 100,000 times less than E2. The organochlorine herbicides atrazine and simazine failed to induce reporter gene activity. As metabolic activation may be required to induce estrogenic activity, a metabolic transformation step was added to the ER-CALUX assay using incubation of compounds with liver microsomes obtained from PCB-treated rats. Results indicate that metabolites of E2, NP and bisphenol A were less active than the parent compounds, while metabolites of methoxychlor were more estrogenic following microsomal incubations.     

Development of a sensitive E-screen assay for quantitative analysis of estrogenic activity in municipal sewage plant effluents

A simplified proliferation test with human estrogen receptor-positive MCF-7 breast cancer cells (E-screen assay) was optimized and validated for the sensitive quantitative determination of total estrogenic activity in effluent samples from municipal sewage plants. After solid phase extraction of 1 l sewage on either 0.2 g polystyrene copolymer (ENV+) or 1 g RP-C18 material and removal of the solvent, analysis of the extracts in the E-screen assay could be performed without any clean-up step. This was even possible with untreated sewage. Parallel extraction of four sewage samples on both different solid phase materials gave comparable quantitative results in the E-screen. A blank sample did not induce cell proliferation. As additive behaviour of the estrogenic response of single compounds was proven for two different mixtures each containing three xenoestrogens, total estrogenic activity in the sewage samples, expressed as 17 beta-estradiol equivalent concentration (EEQ), could be calculated comparing the EC50 values of the samples with those of the positive control 17 beta-estradiol. The detection limit of the E-screen method was 0.05 pmol EEQ/l (0.014 ng EEQ/l), the limit of quantification 0.25-0.5 pmol EEQ/l (0.07-0.14 ng EEQ/l). In total, extracts of nine effluent and one influent sample from five different municipal sewage plants in South Germany were analyzed in the E-screen. All samples strongly induced cell proliferation in a dose-dependent manner which was completely inhibited by coincubation with 5 nM of the estrogen receptor-antagonist ICI 182,780. The proliferative effect relative to the positive control 17 beta-estradiol (RPE) was between 30 and 101%. 17 beta-Estradiol equivalent concentrations were between 2.5 and 25 ng/l indicating a significant input of estrogenic substances via sewage treatment plants into rivers.     

Chemical compounds and toxicological assessments of drinking water stored in polyethylene terephthalate (PET) bottles: A source of controversy reviewed

A declaration of conformity according to European regulation No. 10/2011 is required to ensure the safety of plastic materials in contact with foodstuffs. This regulation established a positive list of substances that are authorized for use in plastic materials. Some compounds are subject to restrictions and/or specifications according to their toxicological data. Despite this, the analysis of PET reveals some non-intentionally added substances (NIAS) produced by authorized initial reactants and additives.Genotoxic and estrogenic activities in PET-bottled water have been reported. Chemical mixtures in bottled water have been suggested as the source of these toxicological effects. Furthermore, sample preparation techniques, such as solid-phase extraction (SPE), to extract estrogen-like compounds in bottled water are controversial. It has been suggested that inappropriate extraction methods and sample treatment may result in false-negative or positive responses when testing water extracts in bioassays. There is therefore a need to combine chemical analysis with bioassays to carry out hazard assessments.Formaldehyde, acetaldehyde and antimony are clearly related to migration from PET into water. However, several studies have shown other theoretically unexpected substances in bottled water. The origin of these compounds has not been clearly established (PET container, cap-sealing resins, background contamination, water processing steps, NIAS, recycled PET, etc.).Here, we surveyed toxicological studies on PET-bottled water and chemical compounds that may be present therein. Our literature review shows that contradictory results for PET-bottled water have been reported, and differences can be explained by the wide variety of analytical methods, bioassays and exposure conditions employed.     

Changes in estrogen/anti-estrogen activities in ponded secondary effluent

Total estrogenic activity, measured using the yeast estrogen screen reporter gene bioassay, decreased from 60 pM (equivalent 17alpha-ethinylestradiol concentration) to an estimated 1.4 pM during a 24-hour period in which secondary effluent was held in a shallow infiltration basin. Over the same period, anti-estrogenic activity, measured as an equivalent concentration of tamoxifen, increased from 35 to 260 nM, suggesting that antagonists produced during secondary effluent storage played a role in the apparent loss of estrogenic activity. Androgenic activity, measured over the same 24-hour period using the yeast androgen screen, was near or below the method detection limit (0.7 pM as testosterone). However, the same pond samples were clearly anti-androgenic. When whole-sample extracts were separated via adsorption and stepwise elution in alcohol/water solutions consisting of 20, 40 and 100% ethanol, the sum of estrogenic activities in derived fractions was always lower than the measured estrogenic activity in the whole-sample extracts. Summed anti-estrogenic activities in the same fractions, however, always exceeded values for corresponding whole-sample extracts. Results reinforce the importance of sample preparation steps (concentration of organics followed by estrogen/anti-estrogen separation) when measuring endocrine-related activities in chemically complex samples such as wastewater effluent. The potential complexity of relationships among estrogens, anti-estrogens and matrix organics suggests that additive models are of questionable validity for estimating whole-sample estrogenic activity from measurements involving sample fractions.     

Contribution of known endocrine disrupting substances to the estrogenic activity in Tama River water samples from Japan using instrumental analysis and in vitro reporter gene assay

To quantitatively characterize the substances contributing to estrogenic activity in river water, in vitro bioassay using MVLN cells and instrumental analysis using liquid chromatograph–mass spectrometer (LC/MS) or liquid chromatograph–tandem mass spectrometer (LC/MS/MS) were applied to river water extracts taken from various locations in the Tama River, Japan. Tama River water samples were extracted using solid phase extraction and the crude extracts were fractionated by high-performance liquid chromatography (HPLC) into 10 fractions. The sixth fraction contained nonylphenol (NP) and octylphenol (OP) at concentrations in the range of 51.6–147 and 6.9–81.9 ng/L, respectively (concentrations corresponding to the original sample volumes). No estrogenic activity, expressed as 17β-estradiol equivalents (E2-EQ_B), however, was observed in this fraction (<0.6 ng-E2eq/L). Instrumentally determined estrogenic activity (E2-EQ_C), which is the concentrations of NP and OP multiplied by their corresponding relative potency, was below the detection limit of the MVLN cell bioassay. Estrogenic activities were detected only in HPLC fraction nos. 7, 8 and 9. Estrone (E1), estradiol (E2) and bisphenol A (BPA) were detected in these fractions. Estriol (E3) and ethynylestradiol (EE2) were not detected (<0.2 ng/L) in these fractions. The calculated E2-EQ_C for BPA was below the detection limit of bioassay. The E2-EQ_C for E1 and E2 were on the same order as the estrogenic activity determined by the bioassay (E2-EQ_B). The ratios of E2-EQ_C and E2-EQ_B for E1 and E2 in the three factions collectively (nos. 7–9) were 0.49–0.97 and 0.29–1.12, respectively. Above results indicated that the major causal substances to the estrogenic activity in the Tama River were E1 and E2.     

An assessment of estrogenic organic contaminants in Canadian wastewaters

A suite of 30 primarily estrogenic organic wastewater contaminants was measured in several influent/effluent wastewater samples from four municipal wastewater treatment plants and effluents from one bleached kraft pulp mill (BKME) using an ultra-trace analytical method based on gas chromatography-high resolution mass spectroscopy (GC-HRMS). In vitro recombinant yeast assay detection of the estrogenic equivalent (EEq) on whole and solid phase extracted (SPE) and fractionated wastewater was also performed. 19-norethindrone was the most frequently detected and abundant (26-224 ng/L) of all the synthetic estrogens/progesterones in the influent samples. 17alpha-ethinylestradiol was the more frequently detected synthetic estrogen/progesterone in the effluents occurring at or below 5 ng/L with some sporadic occurrences of up to 178 ng/L. The greatest levels of steroidal estrogens in municipal effluents were E1>E2>E3 which were all <20 ng/L. Nonylphenol and di(2-ethylhexyl) phthalate were found to be the highest non-steroidal synthetic compounds surveyed in both municipal influent and effluent samples, both occurring at 6-7 microg/L in municipal effluents. BKME contained relatively large amounts of the plant sterol stigmasterol (4 microg/L) but low amounts of fecal sterols, and steroidal estrogens (E2 only at 6 ng/L) when compared to the municipal effluents. In vitro EEq in the wastewater surveyed ranged from 9-106 ng E2/L and ranked from municipal influent>municipal effluent approximately BKME, with most of the estrogenicity fractionating in the 100% methanol SPE fraction followed by a secondary amount in the diethyl ether (for municipal) or methyl-tert butyl ether (for BKME) SPE fractions. Most correlations between chemical and in vitro estrogenic equivalency were weak (p>0.05 in most cases). Unexpected inverse correlations between in vitro estrogenic activity and concentrations of the estrogenic contaminant bisphenol A were found which likely contributed to the weakness of these correlations. A modified toxicity identification and evaluation procedure was continued with the SPE extracts from the more potent 100% methanol SPE fractions of municipal effluent. High performance liquid chromatography band elution retention times, based on in vitro estrogen detection, indicated that steroidal estrogens such as E2 were responsible for most of the estrogenicity of the samples. Subsequent collection and GC-MS analysis of active bands did not confirm the presence of steroidal estrogens, but expanded the possibility of phthalate esters (i.e. dibutyl phthalate) and natural sterols (i.e. beta-sitosterol) contributing to the overall estrogenic load.     

重庆五座水厂水的有机物致突变活性比较

采用固相萃取技术（GDX-120大孔树脂）于春、夏、冬季对以嘉陵江为水源的A厂和B厂，以长江为源的C厂和D厂以及以两江汇水为水源的E厂提取进厂水和出厂水中的有机物，以Ames试验评估其致突变活性。结果表明：①现行水处理工艺在低温季节及源水污染较为严重的情况下可降低出厂水的致突变活性，而在高温季节及源水污染不太严重的情况下可导致出厂水的致突变活性升高；②以嘉陵江为水源的水厂出水致突变危害均高于以长江为水源的水厂出水；③水中有机物的致突变活性以移码型为主，TA98是测试的敏感菌株。     

Identification of estrogen receptor agonists in sediments from Wenyu River, Beijing, China

Assignment of ecological impacts of contamination to specific classes of contaminants is a prerequisite for risk assessment and remediation. In this study, the combination of polarity-based fractionation, two-hybrid yeast bioassay, and chemical analysis were used to evaluate and identify estrogen receptor agonists (ER-agonists) in sediments from Wenyu River, Beijing, China. By bioassay, organic raw sediment extracts could induce significant estrogenicity and the bioassay-derived 17β-estradiol equivalents (EEQs) of raw extracts (EEQ_raws) ranged from 0.8 to 19.8 ng/g dry weight. By polarity-based fractionation, the raw extracts were separated into three fractions, i.e. non-polar, moderately polar, and polar fractions, which were subjected to bioassay and chemical analysis. The highest estrogenicity was observed in the polar fraction, which accounted for more than 78% of the total. The medium polar fraction contains PAHs and OCPs, and the estrogenic activities in this fraction contributed 3%-12% of the total in raw extract. An estrogenic activity of non-polarity fraction was negligible in compare to other two fractions. By chemical analysis and toxic equivalent calculation, major part of the estrogenicity in polar fraction could be attributed to six natural/synthetic estrogens (16%-63%), i.e. 17β-estradiol, estrone, estriol, 17α-ethynylestradiol, diethylstilbestrol, and β-estradiol-17-valerate, and to nonylphenols (26%-55%). The proposed approach has been successfully used for characterization of ER-agonists in this case study.