Biodegradation of synthetic estrogen using bioelectrochemical system and degradation pathway analysis through Quadrupole-time-of-flight-mass spectrometry.

Synthetic estrogenic compounds such as 17α-ethinylestradiol (EE2) are significant environmental contaminants. This research studied the biodegradation of EE2 utilizing the EE2 adapted cells isolated from a dairy farm waste site in suspension flask vis-a-vis Bioelectrochemical System (BES) and compared the power output in the BES with and without EE2 as a co-substrate. 78% removal of EE2 was observed in the BES as against 60% removal in suspension flasks. The maximum power density in the BES increased about 53% when EE2 is used as a co-substrate. The EE2 biodegradation studied using HPLC and Q-TOF methods, also proposes a hypothetical pathway for EE2 degradation by the newly isolated strain Rhodopseudomonas palustris MDOC01 and reports the significant metabolites like nicotinic acid and oxoproline being detected during bioelectrochemical treatment process of EE2. Study also suggests that Plasma peroxide treatment of anode material enhanced the overall performance in terms of biodegradation efficiency and power output.

Cork sheet as a sorptive phase to extract hormones from water by rotating-disk sorptive extraction (RDSE).

This work reports for the first time the use of laminar cork as a sorptive phase in a microextraction technique, rotating-disk sorptive extraction (RDSE). Typical hormones (estrone, estradiol, estriol and ethinyl estradiol) were selected as analyte models and extracted from wastewater samples on laminar cork with statistically equivalent extraction efficiency to that provided by Oasis HLB. The cork characterization was performed by confocal fluorescence microscopy (CLSM), Fourier-transform infrared spectroscopy (FTIR) and scanning electron microscopy (SEM), allowing the identification of lignin, suberin and polysaccharides (cellulose and hemicellulose) as the main components of the cork. The best conditions for extraction were as follows: rotation velocity of the disk, 2000 rpm; extraction time, 45 min; and sample volume, 20 mL. The analytical features of the developed method show that calibration curves for all analytes have R2 values higher than 0.99. The absolute recoveries were higher than 63%, and the precision, expressed as relative standard deviation, ranged from 2 to 16%. The LOD and LOQ ranges were 3-19 and 10-62 ng L-1, respectively. The proposed method was applied to the analysis of wastewater, and the concentrations of hormones in a wastewater treatment plant in Santiago, Chile, ranged from

UPLC-MS/MS determination of steroid hormones via a novel reaction based on derivatisation by a cyclic-organophosphate.

A cyclic-organophosphate, specifically 2-chloro-5,5-dimethyl-1,3,2-dioxaphosphorinane-2-oxide, was used to derivatise the hydroxyl group at the C3 position of selected steroid hormones to analyse the derivatives using UPLC-MS/MS (ultra-performance liquid chromatography-tandem mass spectrometry). Reactions were performed in an anhydrous pyridine environment in the presence of AlCl3 at 50 °C. The developed reaction is suitable for analytical chemistry applications and was validated by analysis of selected contraceptive drugs. The sensitivity of the method depends on hormone tested and the limit of detection ranges from 130 pg/mL for ß-estradiol to 240 pg/mL for estriol. The estimated efficiency of derivatisation reactions varies in the range from 77.5 to 95.7%, and depends upon the hormone undergoing derivatisation. The method's recovery rate for the lowest concentration tested (800 pg/mL) is 88.1-96.3%. The method exhibits linearity in the 390 pg/mL to 2.5 µg/mL range, with R2 = 0.997. The developed steroid hormone derivatisation reaction was validated experimentally using UHPLC-QTOF-MS (ultra-high performance liquid chromatography quadrupole time of flight mass spectrometry) and NMR (nuclear magnetic resonance) spectroscopy. These studies show that the developed derivatisation reaction provides a precise and repeatable determination of selected steroid hormones in contraceptive drugs. At n = 10, CV (Coefficient of Variation) did not exceed 7%, which is a very good result compared with other analytical methods.

Attenuation, transport, and management of estrogens: A review.

Overabundance of endocrine disruptors (EDs), such as steroid estrogens, in the natural environment disrupts hormone synthesis in aquatic organisms. Livestock and wastewater outflows contribute measurable quantities of steroid estrogens into the environment where they are picked up and transported via surface runoff and feedlot effluents into water matrices. E1, E2ß, E2α, E3 and EE2 are the most prevalent estrogens in these environmental systems. Estrogens in soils and water undergo several concurrent attenuation processes including sorption to particles, biotransformation, photo-transformation, and plant uptake. This review summarizes current studies on the attenuation and transport of steroid estrogens with a focus on estrogen attenuation and transport modeling. The authors use this information to synthesize appropriate strategies for reducing estrogenicity in the environment.

Natural and synthetic estrogens in leafy vegetable and their risk associated to human health.

Since the inception of global industrialization, the growth of steroid estrogens becomes a matter of emerging serious concern for the rapid population. Steroidal estrogens are potent endocrine-upsetting chemicals that are excreted naturally by vertebrates (e.g., humans and fish) and can enter natural waters through the discharge of treated and raw sewage. Steroidal estrogens in plants may enter the food web and become a serious threat to human health. We evaluated the uptake and accumulation of ethinylestradiol (EE2) and 17ß-estradiol (17ß-E2) in lettuce plants (Lactuca sativa) grown under controlled environmental condition over 21 days growth period. An effective analytical method based on ultrasonic liquid extraction (ULE) for solid samples and solid phase extraction (SPE) for liquid samples with gas chromatography-mass spectrometry (GC/MS) has been developed to determine the steroid estrogens in lettuce plants. The extent of uptake and accumulation was observed in a dose-dependent manner and roots were major organs for estrogen deposition. Unlike the 17ß-E2, EE2 was less accumulated and translocated from root to leaves. For 17ß-E2, the distribution in lettuce was primarily to roots after the second week (13%), whereas in leaves it was (10%) over the entire study period. The distribution of EE2 at 2000 µg L-1 in roots and leaves was very low (3.07% and 0.54%) during the first week and then was highest (12% in roots and 8% in leaves) in last week. Bioaccumulation factor values of 17ß-E2 and EE2 in roots were 0.33 and 0.29 at 50 µg L-1 concentration as maximum values were found at 50 µg L-1 rather than 500 and 2000 in all observed plant tissues. Similar trend was noticed in roots than leaves for bioconcentration factor as the highest bioconcentration values were observed at 50 µg L-1 concentration instead of 500 and 2000 µg L-1 spiked concentration. These findings mainly indicate the potential for uptake and bioaccumulation of estrogens in lettuce plants. Overall, the estrogen contents in lettuce were compared to the FAO/WHO recommended toxic level and were found to be higher than the toxic level which is of serious concern to the public health. This analytical procedure may aid in future studies on risks associated with uptake of endocrine-disrupting chemicals in lettuce plants.

Multiresidue determination of estrogens in different dairy products by ultra-high-performance liquid chromatography triple quadrupole mass spectrometry.

In this work, a simple and fast methodology has been validated and applied for the analysis of a group of 22 estrogenic compounds including eight phytoestrogens (i.e. daidzein, enterodiol, glycitein, enterolactone, genistein, formononetin, prunetin, biochanin A), six mycotoxins (ß-zearalanol, ß-zearalenol, α-zearalanol, α-zearalenol, zearalanone, zearalenone) as well as four synthetic (i.e. ethynylestradiol, diethylstilbestrol, dienestrol, hexestrol) and four natural estrogens (i.e. estriol, 17ß-estradiol, 17α-estradiol, estrone) in different dairy products. Extraction was carried out using the QuEChERS method while separation, determination and quantification of the target analytes were achieved by ultra-high-performance liquid chromatography coupled to triple quadrupole mass spectrometry with an electrospray ionization interface. The methodology was validated for four dairy product samples with relevant interest for the population including skimmed and whole cheese and goat and cow kefir, using 17ß-estradiol-2,4,16,16,17-d5 as internal standard for natural and synthetic estrogens and ß-zeralanol-10,10,11,12,12-d5 as internal standard for mycotoxins and phytoestrogens. Recovery ranged from 70 to 119% for the four types of matrices with RSD values lower than 14% and the limits of quantification of the method achieved were in the range 0.025-2.50µg/kg for all samples. Finally, the analysis of commercially available products was carried out finding the presence of daidzein, glycitein enterolactone and genistein in some of the studied samples.

Occurrence and risk assessment of estrogenic compounds in the East Lake, China.

Forty two surface water samples were collected in May and June of 2013 in five lakelets of the East Lake, China. Four steroid hormones (17ß-estradiol (ßE2), estrone (E1), 17α-estradiol (αE2) and 17α-ethinylestradiol (αEE2)) were analyzed in these samples. Determination of four estrogenic compounds was performed on high performance liquid chromatography/mass spectrometry (HPLC/MS). ßE2 was detected with the highest detection frequency of 62% and its concentration range was from nd to 17.58ng/L. The mean total concentration of four compounds increased with the order: Houhu lake (L5) (8.91ng/L)

Estrogenic compound profiles in an urbanized industry-impacted coastal bay and potential risk assessment by pollution indices and multivariative statistical methods.

The occurrence and distribution of target estrogenic compounds in a highly urbanized industry-impacted coastal bay were investigated, and contamination profiles were evaluated by estimating total estradiol equivalents (∑EEQs) and risk quotients (RQs). Phenolic compounds were the most abundant xenoestrogens, but seldom showed contribution to the ∑EEQs. The diethylstilbestrol (DES) and 17α-ethinylestradiol (EE2) were the major contributors followed by 17ß-estradiol (E2) in comparison with a slight contribution from estrone (E1) and estriol (E3). Both ∑EEQs and RQs indicated likely adverse effects posed on resident organisms. Further, multivariate statistical method comprehensively revealed pollution status by visualized factor scores and identified multiple "hotspots" of estrogenic sources, demonstrating the presence of complex pollution risk gradients inside and particularly outside of bay area. Overall, this study favors the integrative utilization of pollution indices and factor analysis as powerful tool to scientifically diagnose the pollution characterization of human-derived chemicals for better management decisions in aquatic environments.

In situ calibration of three passive samplers for the monitoring of steroid hormones in wastewater.

In situ extraction of steroid hormones from waste water using adsorption-based integrative passive samplers represents a promising approach for their monitoring in water at ultra-trace concentrations. Three passive samplers, namely a POCIS, a Chemcatcher fitted with an Empore SDB-RPS disk, and an Empore SDB-RPS disk-based sampler with enhanced water flow, were calibrated in situ in treated municipal wastewater for the purpose of monitoring five estrogens (17-ß-estradiol, 17-α-estradiol, 17-α-ethinylestradiol, estrone and estriol) at sub ng per litre concentrations. Uptake of steroids to samplers during 14-day exposure in wastewater was compared with steroid concentrations in daily collected composite water samples. Sampling rates were obtained from a numerical solution of first order uptake kinetics equations describing the uptake of compounds into a passive sampler over time. Mass transfer of steroids in the Chemcatcher fitted with naked Empore disks was more than two times faster than in the POCIS sampler. The uptake capacity of the applied Empore disk was not sufficient for integrative uptake of all tested steroids during the entire 14-day exposure. Time-weighted average concentrations of steroids estimated at concentrations in units of ngL-1 using the in situ-calibrated samplers were within a factor of two from values obtained using composite water samples.

Regulation of the synthetic estrogen 17α-ethinylestradiol in water bodies in Europe, the United States, and Brazil.

The synthetic estrogen 17α-ethinylestradiol, the principal component of oral contraceptives, has been identified as one of the main compounds accounting for adverse effects on the endocrine system in various species. This study aimed to analyze the state-of-the-art in legislation and guidelines for the control of this synthetic estrogen in water bodies in Europe and the United States and to draw a parallel with the Brazilian reality. Countries have generally attempted to expand the regulation and monitoring of certain emerging micropollutants not previously covered by legislation. Europe is more advanced in terms of water quality, while in the United States this estrogen is only regulated in water for human consumption. Brazil still lacks legal provisions or standards for this estrogen, which can be explained by the relatively limited maturity of the country's system for controlling water pollutants.

Biomagnification of endocrine disrupting chemicals (EDCs) by Pleuronectes yokohamae: Does P. yokohamae accumulate dietary EDCs?

We evaluated the potential for biomagnification of endocrine disrupting chemicals (EDCs) such as nonylphenol (NP), octylphenol (OP), bisphenol A (BP), and natural estrogens such as estrone (E1) and 17ß-estradiol (E2) in a benthic fish, Pleuronectes yokohamae. The assimilation efficiencies (AE) of most EDCs ranged from 88 to 96% suggesting that they were efficiently incorporated and assimilated into P. yokohamae, except for NP (50%). However, the biomagnification factor (BMF) values were <1.0 suggesting that the compounds were not biomagnifying. Additionally, three of the target EDCs were not detected (BP, E1 and E2). Glucuronidation activity towards BP (11.44 ± 2.5 nmol/mg protein/min) and E2 (12.41 ± 3.2 nmol/mg protein/min) was high in the intestine suggesting that EDCs were glucuronidated prior to excretion into bile. Thus, we conclude that biomagnification of dietary EDCs is reduced in P. yokohamae because of effective glucuronidation.

Regulamentação do estrogênio sintético 17-etinilestradiol em matrizes aquáticas na Europa, Estados Unidos e Brasil / Regulación del estrógeno sintético 17-etinilestradiol en matrices acuáticas en Europa, Estados Unidos y Brasil / Regulation of the synthetic estrogen 17-ethinylestradiol in water bodies in Europe, the United States, and Brazil

O estrogênio sintético 17α-etinilestradiol, principal componente utilizado em formulações de contraceptivos orais, tem sido apontado como um dos principais compostos responsáveis por provocar efeitos adversos no sistema endócrino de várias espécies. O objetivo deste estudo foi analisar o estado da arte dos dispositivos legais e normativos referentes ao controle desse estrogênio sintético nas águas da Europa e dos Estados Unidos, e traçar um paralelo com a realidade brasileira. No geral, os países têm buscado ampliar a regulamentação e monitoramento de alguns micropoluentes emergentes que antes não eram objeto de atenção por parte dos dispositivos legais. A Europa está mais avançada no que tange à qualidade dos corpos hídricos, enquanto que nos Estados Unidos esta substância é alvo de regulamentação apenas para a água destinada ao consumo humano. No Brasil, ainda não há nenhum dispositivo legal ou normativo que aborde esse estrogênio, o que pode ser associado a uma baixa maturidade do sistema brasileiro quanto ao controle de poluentes hídricos.

El estrógeno sintético 17α-etinilestradiol, principal componente utilizado en fórmulas de contraceptivos orales, ha sido apuntado como uno de los principales compuestos responsables por provocar efectos adversos en el sistema endócrino de varias especies. El objetivo de este estudio fue analizar el estado de la cuestión de los dispositivos legales y normativos referentes al control de este estrógeno sintético en las aguas de Europa y de los Estados Unidos, y trazar un paralelo con la realidad brasileña. En general, los países han buscado ampliar la regulación y el monitoreo de algunos microcontaminantes emergentes que antes no eran objeto de atención por parte de los dispositivos legales. Europa está más avanzada en lo que se refiere a la calidad de los cuerpos hídricos, mientras que en los Estados Unidos esta substancia es objeto de regulación solamente para el agua destinada al consumo humano. En Brasil todavía no existe ningún dispositivo legal o normativo que aborde este estrógeno, lo que puede ser asociado a una inmadurez del sistema brasileño respecto al control de contaminantes hídricos.

The synthetic estrogen 17α-ethinylestradiol, the principal component of oral contraceptives, has been identified as one of the main compounds accounting for adverse effects on the endocrine system in various species. This study aimed to analyze the state-of-the-art in legislation and guidelines for the control of this synthetic estrogen in water bodies in Europe and the United States and to draw a parallel with the Brazilian reality. Countries have generally attempted to expand the regulation and monitoring of certain emerging micropollutants not previously covered by legislation. Europe is more advanced in terms of water quality, while in the United States this estrogen is only regulated in water for human consumption. Brazil still lacks legal provisions or standards for this estrogen, which can be explained by the relatively limited maturity of the country's system for controlling water pollutants.

An Ultrasensitive (Parts-Per-Quadrillion) and SPE-Free Method for the Quantitative Analysis of Estrogens in Surface Water.

An analytical method is presented here that is sensitive to the parts-per-quadrillion (pg/L) for estrogens in surface water. The estrogens included for study were estrone, 17ß-estradiol, estriol, 17α-ethinylestradiol, and equilin. The method consisted of the small-scale liquid-liquid extraction of surface water followed by derivation with dansyl chloride. Analyte separation and detection were performed by high-pressure liquid-chromatography and tandem mass-spectrometry. A large volume (100 µL) of the sample was injected on-column to increase the analyte mass sent to the detector. The detection limits of the method were 0.045 ng/L for estrone, 0.086 ng/L for 17ß-estradiol, 0.030 ng/L for estriol, 0.049 ng/L for 17α-ethinylestradiol, and 0.13 ng/L for equilin. The whole-method accuracy ranged from 93 ± 5.8% to 105 ± 4.5% for all the analytes at two different spike levels. Similarly, the precision of the method was less than 8.0% relative standard deviation. The final method was used to analyze a series of samples from the Mississippi River spanning 51 river miles. Estrone was detected in all of the samples and 17ß-estradiol was detected in one. Concentrations of estrone ranged from between the detection and quantification limits up to 0.63 ng/L. Increases in the concentration of estrone could be observed downstream from potential sources including a drinking water treatment plant. 17ß-estradiol was detected below its quantitation limit in a sample taken downstream from a wastewater treatment plant.

Determination of hormones, a plasticizer, preservatives, perfluoroalkylated compounds, and a flame retardant in water samples by ultrasound-assisted dispersive liquid-liquid microextraction based on the solidification of a floating organic drop.

Dispersive liquid-liquid microextraction based on the solidification of a floating organic drop (DLLME-SFO) is a novel extraction technique commonly applied for the extraction on a specific group of compounds. In this paper, the applicability of ultrasound-assisted DLLME-SFO for multiresidue extraction has been evaluated. A method for the simultaneous extraction of four hormones (17α-ethinylestradiol, 17ß-estradiol, estriol and estrone), a plasticizer (bisphenol A), three preservatives (methyl-, ethyl- and propylparaben), six perfluoroalkylated compounds (perfluorooctane sulfonic acid and five perfluoroalkyl carboxylic acids, from C4 to C8), and a brominated flame retardant (hexabromocyclododecane) has been developed and validated for their extraction from surface water and tap water. Determination was carried out by high-performance liquid chromatography-tandem mass spectrometry in negative ionization mode. Recoveries of the target compounds were highly dependent on their log K(ow) values. Linear relationship between recoveries and log K(ow) values was observed for compounds from the same group (hormones, preservatives and perfluoroalkylated carboxylic acids). The lowest recoveries were obtained for the less hydrophobic compounds (estriol (43%), methylparaben (32%), ethylparaben (45%) and the perfluorinated compounds of shorter alkyl chain (C4: 17%, C5: 41% and C6: 57%)). Recoveries of the other pollutants were higher than 80%. Precision, expressed as relative standard deviation, was in the range from 1% to 16%. Method detection limits were in the range 0.001-1.126 µg L(-1), for surface water, and 0.001-1.446 µg L(-1) for tap water. No important matrix effect was observed.

Recovery of a wild fish population from whole-lake additions of a synthetic estrogen.

Despite widespread recognition that municipal wastewaters contain natural and synthetic estrogens, which interfere with development and reproduction of fishes in freshwaters worldwide, there are limited data on the extent to which natural populations of fish can recover from exposure to these compounds. We conducted whole-lake additions of an active component of the birth control pill (17α-ethynylestradiol; EE2) that resulted in the collapse of the fathead minnow (Pimephales promelas) population. Here we quantify physiological, population, and genetic characteristics of this population over the 7 years after EE2 additions stopped to determine if complete recovery was possible. By 3 years post-treatment, whole-body vitellogenin concentrations in male fathead minnow had returned to baseline, and testicular abnormalities were absent. In the spring of the fourth year, adult size-frequency distribution and abundance had returned to pretreatment levels. Microsatellite analyses clearly showed that postrecovery fish were descendants of the original EE2-treated population. Results from this whole-lake experiment demonstrate that fish can recover from EE2 exposure at the biochemical through population levels, although the timelines to do so are long for multigenerational exposures. These results suggest that wastewater treatment facilities that reduce discharges of estrogens and their mimics can improve the health of resident fish populations in their receiving environments.

Impact of climate change and population growth on a risk assessment for endocrine disruption in fish due to steroid estrogens in England and Wales.

In England and Wales, steroid estrogens: estrone, estradiol and ethinylestradiol have previously been identified as the main chemicals causing endocrine disruption in male fish. A national risk assessment is already available for intersex in fish arising from estrogens under current flow conditions. This study presents, to our knowledge, the first set of national catchment-based risk assessments for steroid estrogen under future scenarios. The river flows and temperatures were perturbed using three climate change scenarios (ranging from relatively dry to wet). The effects of demographic changes on estrogen consumption and human population served by sewage treatment works were also included. Compared to the current situation, the results indicated increased future risk:the percentage of high risk category sites, where endocrine disruption is more likely to occur, increased. These increases were mainly caused by changes in human population. This study provides regulators with valuable information to prepare for this potential increased risk.

Parameterization of pharmaceutical emissions and removal rates for use in UK predictive exposure models: steroid estrogens as a case study.

Newly available prescription data has been used along with census data to develop a localised method for predicting pharmaceutical concentrations in sewage influent and effluent for England, and applied to a case study: the steroid estrogens estrone, 17ß-estradiol, and 17α-ethinylestradiol in a selected catchment. The prescription data allows calculation of the mass consumed of synthetic estrogens, while use of highly localised census data improves predictions of naturally excreted estrogens by accounting for regional variations in population demographics. This serves two key purposes; to increase the accuracy of predictions in general, and to call attention to the need for more accurate predictions at a localised and/or catchment level, especially in light of newly proposed regulatory measures which may in the future require removal of steroid estrogens by sewage treatment facilities. In addition, the general lack of measured sewage works data necessitated the development of a novel approach which allowed comparison of localised predictions to average national measurements of influent and effluent. Overall in the case study catchment, estrogen predictions obtained using the model described herein were within 95% confidence intervals of measured values drawn from across the UK, with large improvements to predictions of EE2 being made compared with previous predictive methods.

Occurrence, removal, and fate of progestogens, androgens, estrogens, and phenols in six sewage treatment plants around Dianchi Lake in China.

The occurrence and behavior of endocrine disrupting chemicals (EDCs) in sewage treatment plants (STPs), especially estrogens and phenols, have been closely concerned in previous studies. However, the systematical researches about progestogens and androgens were scarce in STPs adopting different treatment technologies. This work investigated the occurrence, removal, and fate of one progestogen, three androgens, four estrogens, and six phenols in six STPs around Dianchi Lake in China, where the influents, effluents of primary treatment, secondary treatment, and advanced treatment, as well as excess sludge samples, were analyzed. All of the above EDCs were detected out in influents of the six STPs. Bisphenol A, nonylphenol-mono-ethoxylate, and nonylphenol-diethoxylate were the dominant EDCs detected in those influent samples with the concentrations that varied from 637.6 to 1,684.0 ng/L, 633.8 to 1,540.0 ng/L, and 648.7 to 2,246.0 ng/L, respectively; E1 and dihydrotestosterone were the major steroids with the mean concentration of 126.8 and 277.4 ng/L. For effluents and sludges, phenols showed higher concentration (366.8-1,233.0 ng/L and 1,478.1-6,948.9 ng/g dry weight (dw)) and detection rate (100 %). The total removal rates were more than 80 % for most compounds in wastewater treatment processes, and high removal efficiency (86-100 %) was found for androgens and progestogens compared with estrogens (75-92 %) and phenols (62-85 %). The secondary treatment processes play significant roles on degrading EDCs, whereas the primary sedimentation has little effects. The treatment capacity of anoxic-anaerobic-anoxic membrane bioreactor and anaerobic/anoxic/oxic technologies was superior to the conventional oxidation ditch in the degradation of EDCs. The advanced treatment process, two units of filter (D-type or V-type), and ultraviolet disinfection were adopted and presented effective to remove these compounds. According to fate analysis, it was obvious that biological degradation was the main pathway on the removal of EDCs in STPs compared with adsorption. Risk quotients were calculated to assess ecological risks of those EDCs. Risk quotients of 54 and 61 % were more than 1 in effluents and sludges, respectively, showing potential hazard of effluents and sludges to the environment.

Diagnostic investigation of steroid estrogen removal by activated sludge at varying solids retention time.

The impact of solids retention time (SRT) on estrone (E1), 17ß-estradiol (E2), estriol (E3) and 17α-ethinylestradiol (EE2) removal in an activated sludge plant (ASP) was examined using a pilot plant to closely control operation. Exsitu analytical methods were simultaneously used to enable discrimination of the dominant mechanisms governing estrogen removal following transitions in SRT from short (3d) to medium (10d) and long (27d) SRTs which broadly represent those encountered at full-scale. Total estrogen (∑EST, i.e., sum of E1, E2, E3 and EE2) removals which account for aqueous and particulate concentrations were 70±8, 95±1 and 93±2% at 3, 10 and 27d SRTs respectively. The improved removal observed following an SRT increase from 3 to 10d was attributable to the augmented biodegradation of the natural estrogens E1 and E2. Interestingly, estrogen biodegradation per bacterial cell increased with SRT. These were 499, 1361 and 1750ng 10(12) viable cells(-1)d(-1). This indicated an improved efficiency of the same group or the development of a more responsive group of bacteria. In this study no improvement in absolute ∑EST removal was observed in the ASP when SRT increased from 10 to 27d. However, batch studies identified an augmented biomass sorption capacity for the more hydrophobic estrogens E2 and EE2 at 27d, equivalent to an order of magnitude. The lack of influence on estrogen removal during pilot plant operation can be ascribed to their distribution within activated sludge being under equilibrium. Consequently, lower wastage of excess sludge inherent of long SRT operation counteracts any improvement in sorption.

Co-occurrence of estrogenic and antiestrogenic activities in wastewater: quantitative evaluation of balance by in vitro ERα reporter gene assay and chemical analysis.

Endocrine-disrupting chemicals are exogenous substances that alter the function of the endocrine system, with adverse health effects on organisms or their progeny. In vitro estrogen receptor (ER) reporter gene assays have long been used to measure estrogenic activity in wastewater. Nevertheless, there is still uncertainty about their usefulness in environmental monitoring on account of a discrepancy between the estrogenic response of the in vitro assay and concentrations of estrogenic compounds determined by chemical analysis. Here, we measured estrogenic and antiestrogenic activities in wastewater by ERα reporter gene assay. All samples were simultaneously analyzed for estrone, 17ß-estradiol, estriol, and 17α-ethynylestradiol, and the concentrations were used to predict estrogenic activity. All samples in which measured estrogenic activity was significantly lower than predicted showed strong antiestrogenic activity. In addition, we confirmed that the fraction that did not have antiestrogenic activity showed stronger estrogenic activity than the unfractionated wastewater extract. These results indicate that antiestrogenic compounds in wastewater suppress the activity of natural estrogens, and the reporter gene assay represents the net activity.