Seasonal variability of the reduction in estrogenic activity at a municipal WWTP

In vitro monitored estrogenicity of municipal wastewater influent/effluent samples (collected from September to December from a Northern Canadian biological nutrient removal (BNR) treatment plant serving an urban population of 750,000) were combined with operational, wastewater quality, and climate data to determine which of these latter variables may be related to the levels and reduction in the former parameter. Significant variability was present in operational and wastewater quality parameters throughout the sampling period including a 7 degrees C difference in wastewater temperature Most of the wastewater samples collected during this period show a considerable amount of recombinant yeast assay (RYA) activity with the greatest activity (estradiol (E2)-equivalents of 106-175ng/L) seen in the final effluents collected from mid-September to mid-October. Percent reduction in the levels of RYA measured E2-equivalents varied from -234% to 75%. No correlations were seen in RYA activity reduction with percent reduction in 5-day biochemical oxygen demand (BOD(5)), flow (i.e. inversely related to hydraulic retention time), solids retention time or even rainfall, and the reduction trends for RYA measured activity were explained best by ambient and effluent temperatures in an inverse fashion (% reduction in E2-equivalents=-10.8.(effluent temperature in degrees C)+191, p=0.005). Complementary instrumental analysis of select sample composites revealed that the free/conjugated estrogen ratio was indeed greater in the wastewater sampled during warmer temperatures.     

Survey of contamination of estrogenic chemicals in Japanese and Korean coastal waters using the wild grey mullet (Mugil cephalus)

We monitored the contamination by environmental estrogens (EEs) of coastal areas in Korea and Japan using the wild grey mullet. The grey mullet were collected from Ansan, Jeju, Yeosu, Tongyeong, and Busan in Korea and Nagasaki, Omuta, and Fukuoka in Japan. Contamination by EEs was determined by measuring vitellogenin (VTG) levels in serum and identifying gonadal abnormalities histologically (i.e., testis-ova). In four sites in Korea (Ansan, Yeosu, Tongyeong, and Busan) and two sites in Japan (Nagasaki and Fukuoka), serum VTG in immature and male grey mullet was detected at levels greater than 1.0 μg/ml, which is considered to be an abnormal level. Although, testis-ova were observed in some individuals collected in Ansan, Tongyeong, and Busan in Korea and Omuta in Japan, there was no correlation between individuals with testis-ova and individuals with abnormal levels of VTG. Furthermore, in Japan, serum VTG levels of fish collected from Nagasaki and Fukuoka were also greater than 1.0 μg/ml. Although individuals with testis-ova were found in Omuta, these fish expressed normal levels of serum VTG. Our results suggest that the grey mullets living in these coastal areas are influenced by EEs in the environment. Furthermore, it appears that the production of VTG and the occurrence of testis-ova are caused by different mechanisms.     

A four-hour yeast bioassay for the direct measure of estrogenic activity in wastewater without sample extraction, concentration, or sterilization

The assay described here represents an improved yeast bioassay that provides a rapid yet sensitive screening method for EDCs with very little hands-on time and without the need for sample preparation. Traditional receptor-mediated reporter assays in yeast were performed twelve to twenty four hours after ligand addition, used colorimetric substrates, and, in many cases, required high, non-physiological concentrations of ligand. With the advent of new chemiluminescent substrates a ligand-induced signal can be detected within thirty minutes using high picomolar to low nanomolar concentrations of estrogen. As a result of the sensitivity (EC₅₀ for estradiol is ∼ 0.7 nM) and the very short assay time (2-4 h) environmental water samples can typically be assayed directly without sterilization, extraction, and concentration. Thus, these assays represent rapid and sensitive approaches for determining the presence of contaminants in environmental samples. As proof of principle, we directly assayed wastewater influent and effluent taken from a wastewater treatment plant in the El Paso, TX area for the presence of estrogenic activity. The data obtained in the four-hour yeast bioassay directly correlated with GC-mass spectrometry analysis of these same water samples.     

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.     

Evaluation and validation of a novel Arxula adeninivorans estrogen screen (nAES) assay and its application in analysis of wastewater, seawater, brackish water and urine

A novel Arxula adeninivorans yeast estrogen screen (nAES) assay has been developed for detection of estrogenic activity in various liquid samples such as wastewater, seawater, brackish water and swine urine. Two bio-components were engineered to co-express the human estrogen receptor α (hERα) and an inducible reporter gene; either the non-conventional phytase gene (phyK, derived from Klebsiella sp. ASR1) or the non-conventional tannase gene (ATAN1, derived from Arxula). Both reporters were put under the control of an Arxula derived glucoamylase (GAA) promoter, which was modified by the insertion of two estrogen-responsive elements (EREs). The Arxula transformation/expression platform Xplor® 2, which lacks resistance markers and E. coli elements, was used to select stable mitotic transformants. They were then analyzed for robustness and suitability as the bio-component for the nAES assay. Two types of the nAES assay based on the reporter proteins phytase and tannase (nAES-P, nAES-T) were used in this work. The nAES-P type is more suitable for the analysis of seawater, brackish water and urine whereas the nAES-T type exhibited higher robustness to NaCl. Both assay types have similar characteristics for the determination of estrogen in sewage and urine samples e.g. 6-25 h assay period with detection and determination limits and EC₅₀ values for 17β-estradiol of 2.8 ng L^− 1, 5.9 ng L^− 1, 33.2 ng L^− 1 (nAES-P) and 3.1 ng L^− 1, 6.7 ng L^− 1 and 39.4 ng L^− 1 (nAES-T). Substrate specificity and analytical measurement range (AMR) for both assay types are also similar. These characteristics show that the nAES assay based on non-conventional salt tolerant yeast is applicable for a high throughput estrogen analysis in the environmental and regulatory control sectors.     

Occurrence and fate of emerging wastewater contaminants in Western Balkan Region

This paper reports on a comprehensive reconnaissance of over seventy individual wastewater contaminants in the region of Western Balkan (WB; Bosnia and Herzegovina, Croatia and Serbia), including some prominent classes of emerging contaminants such as pharmaceuticals and personal care products, surfactants and their degradation products, plasticizers, pesticides, insect repellents, and flame retardants. All determinations were carried out using a multiresidue analytical approach, based on the application of gas chromatographic and liquid chromatographic techniques coupled to mass spectrometric detection. The results confirmed a widespread occurrence of the emerging contaminants in municipal wastewaters of the region. The most prominent contaminant classes, determined in municipal wastewaters, were those derived from aromatic surfactants, including linear alkylbenzene sulphonates (LAS) and alkylphenol polyethoxylates (APEO), with the concentrations in raw wastewater reaching into the mg/l range. All other contaminants were present in much lower concentrations, rarely exceeding few microg/l. The most abundant individual compounds belonged to several classes of pharmaceuticals (antimicrobials, analgesics and antiinflammatories, beta-blockers and lipid regulators) and personal care products (fragrances). Due to the rather poor wastewater management practices in WB countries, with less than 5% of all wastewaters being biologically treated, most of the contaminants present in wastewaters reach ambient waters and may represent a significant environmental concern.     

Contamination with retinoic acid receptor agonists in two rivers in the Kinki region of Japan

This study was conducted to investigate the agonistic activity against human retinoic acid receptor (RAR) α in the Lake Biwa-Yodo River and the Ina River in the Kinki region of Japan. To accomplish this, a yeast two-hybrid assay was used to elucidate the spatial and temporal variations and potential sources of RARα agonist contamination in the river basins. RARα agonistic activity was commonly detected in the surface water samples collected along two rivers at different periods, with maximum all-trans retinoic acid (atRA) equivalents of 47.6 ng-atRA/L and 23.5 ng-atRA/L being observed in Lake Biwa-Yodo River and Ina River, respectively. The results indicated that RARα agonists are always present and widespread in the rivers. Comparative investigation of RARα and estrogen receptor α agonistic activities at 20 stations along each river revealed that the spatial variation pattern of RARα agonist contamination was entirely different from that of the estrogenic compound contamination. This suggests that the effluent from municipal wastewater treatment plants, a primary source of estrogenic compounds, seemed not to be the cause of RARα agonist contamination in the rivers. Fractionation using high performance liquid chromatography (HPLC) directed by the bioassay found two bioactive fractions from river water samples, suggesting the presence of at least two RARα agonists in the rivers. Although a trial conducted to identify RARα agonists in the major bioactive fraction was not completed as part of this study, comparison of retention times in HPLC analysis and quantification with liquid chromatography-mass spectrometry analysis revealed that the major causative contaminants responsible for the RARα agonistic activity were not RAs (natural RAR ligands) and 4-oxo-RAs, while 4-oxo-RAs were identified as the major RAR agonists in sewage in Beijing, China. These findings suggest that there are unknown RARα agonists with high activity in the rivers.     

Reconnaissance of selected PPCP compounds in Costa Rican surface waters

Eighty-six water samples were collected in early 2009 from Costa Rican surface water and coastal locations for the analysis of 34 pharmaceutical and personal care product compounds (PPCPs). Sampling sites included areas receiving treated and untreated wastewaters, and urban and rural runoff. PPCPs were analyzed using a combination of solid phase extraction and liquid chromatography tandem mass spectrometry. The five most frequently detected compounds were doxycycline (77%), sulfadimethoxine (43%), salicylic acid (41%), triclosan (34%) and caffeine (29%). Caffeine had the maximum concentration of 1.1 mg L⁻¹, possibly due to coffee bean production facilities upstream. Other compounds found in high concentrations include: doxycycline (74 μg L⁻¹), ibuprofen (37 μg L⁻¹), gemfibrozil (17 μg L⁻¹), acetominophen (13 μg L⁻¹) and ketoprofen (10 μg L⁻¹). The wastewater effluent collected from an oxidation pond had similar detection and concentrations of compounds compared to other studies reported in the literature. Waters receiving runoff from a nearby hospital showed higher concentrations than other areas for many PPCPs. Both caffeine and carbamazepine were found in low frequency compared to other studies, likely due to enhanced degradation and low usage, respectively. Overall concentrations of PPCPs in surface waters of Costa Rica are inline with currently reported occurrence data from around the world, with the exception of doxycycline.     

Effect of chlorination condition and permeability of chlorine species on the chlorination of a polyamide membrane

Most studies on membrane chlorination have been investigated in an unpressurized chlorination mode, even if the polyamide membrane was continuously exposed to chlorine under high operating pressure in real water/wastewater treatment plants. In this study, performance changes due to polyamide membrane chlorination were investigated in both pressurized and unpressurized chlorination modes. Chlorination in an unpressurized mode showed a flux increase at high pH and a flux decline at low pH due to the compaction and swelling of the polyamide chains, respectively. On the other hand, chlorination performed in a pressurized mode decreased the water flux in both acidic and alkaline conditions, showing that compaction is overwhelming compared to swelling. The permeability of HOCl, a dominant species at low pH, through the polyamide membrane was pH independent and almost similar to the system recovery, but the permeability of OCl⁻, which is dominant at high pH, was maxima at a neutral pH. The different performance behaviors of membranes chlorinated at various pH conditions in the presence or absence of applied pressure could be explained by the permeability of chlorine species and compaction/swelling of polymer chains after chlorination. The effect of membrane chlorination on the chemical property changes at the two different modes was confirmed using attenuated total reflection Fourier transform infrared analysis, and a conceptual model of performance change was proposed to explain the performance discrepancy between the two chlorination modes.     

Structural investigations of aquatic humic substances by pyrolysis-field ionization mass spectrometry and pyrolysis-gas chromatography/mass spectrometry

The present state-of-the-art in structural investigations of aquatic humic substances and their fractions such as fulvic and humic acids by pyrolysis-field ionization mass spectrometry is outlined. An integrated approach to interpretation uses complementary data of elemental analysis, functional group determinations, Cu²⁺-complexation, H⁺-capacity and results from u.v.-vis- and FT-i.r.-spectroscopy. Proposed building blocks of the molecular structure are supported by chromatographic investigations using Curie-point Py-GC/MS and the available literature reports. The potential and limitation of the applied methodology are discussed.     

Occurrence of pharmaceutically active compounds in surface waters of the henares-jarama-tajo river system (madrid, spain) and a potential risk characterization

The Henares-Jarama-Tajo river system is the largest drainage basin in the Province of Madrid, Spain. This area is characterized by the presence of intensive urban and industrial activities influenced by a continental Mediterranean climate with rainfalls presenting substantial fluctuations along the different seasons. This research aimed to monitor seasonal variations in concentrations of 22 pharmaceutically active compounds (PhACs) in this river system and to establish the potential risk of sublethal effects on aquatic organisms. A total of 10 sampling sites were selected along the river system with samples collected in each of the four seasons during a year-round schedule. Most of the PhACs detected were present in sampling sites downstream in the vicinity of the most populated cities (i.e. Madrid, Guadalajara and Alcalá de Henares). Only two PhACs, fluoxetine and paraxantine, were detected in all sites regardless of the season, and showed median (± interquartile range) concentrations of 21.4 (± 31.2) ng L^- 1 and 8.5 (± 5.3) ng L^- 1, respectively. Other PhACs were detected with a frequency > 80% and included, caffeine, diphenylhydantoin, hydrochlorotiazide, ibuprofen, ketoprofen, diclofenac, sulfamethoxazole, atenolol, naproxen, carbamazepine and propanolol. Seasonal variations were observed with the highest concentrations in December and the lowest in September. By combining measured environmental concentrations with toxicity data (either publicly available or obtained experimentally in our laboratory), and by calculating an Maximum Risk Index (MaxRI) that each combination of PhACs should have for non exceeding the risk threshold, a high potential for long-term risk (MaxRI < 10) was estimated for most of the sampling sites and sampling dates. This research allowed the characterization of the potential risk for each of the PhACs to exert sublethal effects on aquatic organisms using acute screening methods, justifying the need for chronic data in order to refine the risk of these compounds to aquatic organisms.     

Solar photo-Fenton degradation of nalidixic acid in waters and wastewaters of different composition. Analytical assessment by LC-TOF-MS

This work assessed the solar photo-Fenton degradation of nalidixic acid (NXA), a quinolone antibacterial agent, in several different aqueous solutions. It has been proven that the composition of the water clearly affects the efficiency of the photo-Fenton process. The presence of chlorine ions induces the concurrence of different mechanisms involving Cl and Cl₂⁻ radicals, which slow down the process. Up to 35 transformation products (TPs) were identified and their structures characterized by accurate LC-TOF-MS mass measurements during treatment of the different model waters. Photocatalytic degradation was thus observed to proceed mainly through the attack of the hydroxyl radicals on the double bond C₍₂₎C₍₃₎ which induce further ring opening. All the TPs identified persisted after total degradation of NXA. NXA in real pharmaceutical effluent was treated by photo-Fenton as a first stage before biological treatment. As NXA has been demonstrated to be recalcitrant to biological treatment, photo-Fenton treatment of the effluent was continued until its total degradation. Although NXA was efficiently degraded, LC-MS analyses demonstrated that some of the TPs identified after the photo-Fenton treatment were also recalcitrant to biological treatment, persisting after the combined treatment. These results show that analytical assessment of photocatalytic water treatments is essential to assure they are functioning as intended.     

Effect of residual chlorine on the analysis of geosmin, 2-MIB and MTBE in drinking water using the SPME technique

The effect of chlorine on the analysis of three organic compounds (geosmin, 2-methylisoborneol (2-MIB) and methyl tert-butyl ether (MTBE)) in drinking water is elucidated. Three fibers for solid-phase microextraction (SPME) were employed for the extraction of the organic compounds from drinking water samples with and without free residual chlorine present. A gas chromatograph coupled with a mass spectrometer was used to analyze the compounds trapped by the fibers. The presence of chlorine substantially reduces the observed geosmin, 2-MIB, and MTBE concentrations. Depending on the analyte and chlorine concentrations, an experimental error of 10-70% may be observed due to the presence of free residual chlorine. The impact is larger for lower organic compound concentrations, and under higher residual chlorine conditions. To counteract the effect from residual chlorine, sodium thiosulfate was used to dechlorinate the water. After dechlorination the experimental error was less than 10%, suggesting that dechlorination is necessary when applying SPME for the extraction of organic compounds from chlorinated drinking water.     

Effect of biological and chemical oxidation on the removal of estrogenic compounds (NP and BPA) from wastewater: an integrated assessment procedure

A major source of the wide presence of EDCs (Endocrine Disrupting Compounds) in water bodies is represented by direct/indirect discharge of sewage. Recent scientific literature reports data about their trace concentration in water, sediments and aquatic organisms, as well as removal efficiencies of different wastewater treatment schemes. Despite the availability of a huge amount of data, some doubts still persist due to the difficulty in evaluating synergistic effects of trace pollutants in complex matrices. In this paper, an integrated assessment procedure was used, based on chemical and biological analyses, in order to compare the performance of two full scale biological wastewater treatment plants (either equipped with conventional settling tanks or with an ultrafiltration membrane unit) and tertiary ozonation (pilot scale).Nonylphenol and bisphenol A were chosen as model EDCs, together with the parent compounds mono- and di-ethoxylated nonylphenol (quantified by means of GC-MS). Water estrogenic activity was evaluated by applying the human breast cancer MCF-7 based reporter gene assay. Process parameters (e.g., sludge age, temperature) and conventional pollutants (e.g., COD, suspended solids) were also measured during monitoring campaigns.Conventional activated sludge achieved satisfactory removal of both analytes and estrogenicity. A further reduction of biological activity was exerted by MBR (Membrane Biological Reactor) as well as ozonation; the latter contributed also to decrease EDC concentrations.     

饮用水中土霉味物质测定方法的优化

采用固相微萃取与气相色谱-质谱联用测定饮用水中5种常见的土霉味物质，研究了萃取温度、盐含量、萃取时间和溶液pH值等操作条件对分析方法的影响。得到的优化分析条件如下：萃取温度为65℃、盐含量为25％、萃取时间为1h；该方法的相对标准偏差〈10％，样品的加标回收率≥89％。     

固相萃取—气相色谱/质谱法测定水中7种农药

采用全自动固相萃取法提取水中7种农药,并采用气相色谱/质谱法检测,以保留时间和选择离子定性分析、菲-D10为内标物进行定量分析。结果表明:气相色谱/质谱法检测水中有机磷农药的最低检出限在1.0～2.4μg/L之间,全自动固相萃取测定有机磷农药的相对标准偏差是1.7%～7.2%,回收率在82.6%～92.0%之间。     

顶空固相微萃取法用于测定水中二甲基三硫醚

采用顶空固相微萃取一气相色谱/质谱联用的方法对水中嗅味物质二甲基三硫醚进行了测定.经优化试验,得到了二甲基三硫醚固相微萃取的最佳条件:采用CAR/PDMS(85 μm)纤维头、在水样中加入25%(W/V)的NaCl、65℃水浴下顶空萃取30 min.该方法的检出限为5 ng/L,相对标准偏差为2.2%～7.1%,加标回收率为71.5%～87.3%.利用该方法对不同实际水样的检测发现,不同水源水中均有一定程度的二甲基三硫醚检出(9～40 ng/L),值得关注.     

反相液相色谱/电喷雾质谱法分离水体中6种卤乙酸

建立了能够同时分离水体中6种卤乙酸的反相液相色谱—电喷雾质谱分析方法（RPLC-ESI-MS方法），确定了质谱优化条件（锥孔电压为15V，毛细管电压为4kV，脱溶剂气流量为400L／h，锥孔气流量为30L／h）。根据卤乙酸本身的强极性性质，采用乙腈和水作为流动相，同时在水相中投加25mmoL／L的醋酸铵缓冲盐，并用氨水调节水相pH值为9．7，以JJ50－2D型分析柱为分离柱，淋洗速度采用0．20mL／min，在最优质谱条件下直接进样，可使6种卤乙酸得到很好的分离。该检测方法的平均加标回收率为80％～102％，测定结果较为理想。     

Investigating the chlorination of acidic pharmaceuticals and by-product formation aided by an experimental design methodology

The degradation of seven acidic drugs and two metabolites during chlorination was investigated by liquid chromatography-mass spectrometry (LC-MS). A triple-quadrupole (QqQ) system was used to follow the time course of the pharmaceuticals and by-products, while a quadrupole time-of-flight (Q-TOF) system was also used for the identification of the by-products. Under strong chlorination conditions (10 mg/L Cl₂, 24 h), only four of the target compounds were significantly degraded: salicylic acid, naproxen, diclofenac and indomethacine. The degradation kinetics of these four compounds were investigated at different concentrations of chlorine, bromide and pH by means of a Box-Behnken experimental design. Depending on these factors, measured pseudo-first order half-lives were in the ranges: 23-573 h for salicylic acid, 13-446 min for naproxen, 5-328 min for diclofenac and 0.4-13.4 min for indomethacine. Also, it was observed that chlorine concentration was the overall most significant factor, followed by the bromide concentration (except for indomethacine), resulting in increased degradation kinetics as they are increased. The degradation path of salicylic acid, naproxen and diclofenac consisted of aromatic substitution of one or two hydrogens by chlorine and/or bromide. Moreover, for diclofenac, two other by-products corresponding to a decarboxylation/hydroxylation pathway from the monohalogenated products were also identified. On the other hand, indomethacine degradation did not lead to halogenation products but to oxidation ones. The investigation of these by-products in real samples by LC-MS/MS (QqQ) showed that the halogenated derivates of salicylic acid occurred in all the drinking water and wastewater samples analysed.     

New standards for the determination of geosmin and methylisoborneol in water by gas chromatography/mass spectrometry

Deuterium labelled geosmin and methylisoborneol (MIB) have been synthesized and evaluated as internal standards in the determination of geosmin and MIB in water by closed loop stripping followed by gas chromatography/mass spectrometry (GC/MS). The labelled standards were compared with chloroalkanes added as internal standards either at the time of sampling or immediately before closed loop stripping. When added at sampling time, the new standards enabled accurate determination of the geosmin and MIB present initially, even when the samples were analysed as much as 3 weeks later. The new standards gave better precision and accuracy than the chloroalkanes and overcame the underestimation of initial analyte concentration which usually results from losses of analyte through adsorption, volatilisation, biodegradation etc. during sample storage. Geosmin had a limit of detection of<0.1ng/l and 1 ng/lwas determined with a coefficient of variation (CV) of 1.2% (n = 5). MIB was determined at 1 ng/l with a CV of 3.5% (n = 5).