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.

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.

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.

A sensitive liquid chromatography/tandem mass spectrometry method for the determination of natural and synthetic steroid estrogens in seawater and marine biota, with a focus on proposed Water Framework Directive Environmental Quality Standards.

RATIONALE: Trace levels of natural and synthetic steroid estrogens estrone (E1), 17ß-estradiol (E2) and 17α-ethynyl estradiol (EE2) have been demonstrated to exert adverse effects in exposed organisms. E2 and EE2 have been proposed for inclusion in the Water Framework Directive (WFD) list of priority pollutants; however, the detection and accurate quantification of these compounds provide significant challenges, due to the low detection limits required. METHODS: A sensitive method combining ultrasonication, solid-phase extraction (SPE) and liquid chromatography/tandem mass spectrometry, with electrospray ionisation in negative mode (LC/ESI-MS/MS), capable of determining E1, E2 and EE2 at concentrations between 0.07 and 60 ng/L for seawater and between 0.4 and 200 ng/g wet weight in Mytilus spp. is reported. Recoveries at the limit of quantification (LOQ) ranged from 95 to 102% and 88 to 100% for water and tissue, respectively. Salinity (12 to 35‰) and typical marine particulate matter loadings (between 10 and 100 mg/L) were not found to affect analyte recoveries. RESULTS: The first detection of E1 by LC/MS/MS in Irish marine waters (Dublin Bay, at 0.76 ng/L) is reported. Steroids were not detected in Galway Bay, or in any mussel samples from Dublin, Galway and Clare. The level of E2 detected in the dissolved water phase was below the proposed WFD Environmental Quality Standard (EQS) in other surface waters. CONCLUSIONS: The proposed method is suitable for the detection of E1, E2 and EE2 at biologically relevant concentrations and, due to the specificity offered, is not subject to potential interferences from endogenous E1 and E2 which often complicate the interpretation of estrogenic biomarker assays.

Occurrence of bisphenol A, estrone, 17ß-estradiol and 17α-ethinylestradiol in Portuguese rivers.

This study focused on the occurrence of several EDCs including bisphenol A, estrone (E1), the 17ß-estradiol (E2) and 17α-ethinylestradiol (EE2) in fourteen rivers of Portugal. Samples analysis revealed a widespread contamination of BPA especially in Ave, Cávado, Douro, Ferro, Sousa and Vizela Rivers. Achieving 98.4 ng/L for the highest concentration. The estrogens achieved above the method quantification limit (MQL) were E1 in Águeda River and E2 in Ave, Lima and Tâmega Rivers. The maximum concentration detected for E1 was 26.9 ng/L. EE2 was detected only below MQL.

A quantitative method evaluating the selective adsorption of molecularly imprinted polymer.

Adsorption isotherms of 4 estrogenic compounds, estrone, 17ß-estradiol, 17α-ethinylestradiol and Bisphenol A, using molecularly imprinted polymer were studied. The isotherms can be simulated by Langmuir model. According to the adsorption isotherms and the template's mass balance, an experimental concept, selective adsorption ratio, SAR, was proposed to assess how many template molecules extracted out of MIP could create selective binding sites. The SAR of the molecularly imprinted polymer was 74.3% for E2. This concept could be used to evaluate quantitatively the selective adsorption.

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.

Occurrence, fate, and biodegradation of estrogens in sewage and manure.

The estrogens estrone (E1), 17alpha-estradiol (E2alpha), 17beta-estradiol (E2beta), and estriol (E3) are natural sex hormones produced by humans and animals. In addition, there are some synthetic estrogens, such as 17alpha-ethinylestradiol (EE2), used for contraception purposes. These compounds are able to produce endocrine disruption in living organisms at nanogram-per-liter levels. In both humans and animals, estrogens are excreted in urine and feces, reaching the natural environment through discharge from sewage treatment plants (STP) and manure disposal units. In STPs, hormone removal depends on the type of treatment process and on different parameters such as the hydraulic and sludge retention times. Thus, hormone elimination rates vary from 0% to 90% in different STPs. Animals are also an important source of estrogens in the environment. Indeed, animals produce high concentrations of hormones which will end up in manure which is typically spread on land. Hence, waste-borne animal hormones may transfer these pollutants to the soil. The purpose of this review is to highlight the significance for both health and the environment of pollution by estrogens and critically review the existing knowledge on their fate and removal in different treatment processes. Relevant information on the microbial degradation of hormones and metabolic pathways is also included.

Removal and degradation characteristics of natural and synthetic estrogens by activated sludge in batch experiments.

The removal and degradation characteristics of natural and synthetic estrogens by activated sludge were investigated by a series of batch experiments using the activated sludge samples of four actual wastewater treatment plants and synthetic wastewater spiked with estrogen. The rapid removal and degradation of 17beta-estradiol (E2) and estrone (E1) were observed by the activated sludge samples of the oxidation ditch process which operated at higher solids retention time (SRT). On the other hand, E1 tended to remain both in the water phase and the sludge phase in the activated sludge samples of the conventional activated sludge process which operated at lower SRT. The anoxic condition was considered to be not favorable to the effective removal of estrogens as compared with the aerobic condition. The removal and degradation of EE2 showed the lag phase, which neither E2 nor E1 showed, but EE2 was finally removed and degraded completely after 24h. The removal of estrogens in the water phase did not follow the first-order-rate reaction because a large part of the spiked estrogen was immediately removed from the water phase to the sludge phase by adsorption.

Analytical challenges and recent advances in the determination of estrogens in water environments.

Estrogens have been shown to be present in the water compartment, mainly due to the inefficient removal in wastewater treatment plants (WWTP). The concentrations of these compounds, although very low (low ng/L), are sufficient to induce estrogenic responses and alter the normal reproduction and development of wildlife organisms. The compounds have been determined, by a variety of analytical procedures, in the influents and effluents of WWTP, fresh waters, rivers, and even drinking waters. Determination of natural and synthetic estrogens and progestogens in natural water is, however, a difficult analytical task, because of the very low detection limits required and the complexity of the matrix. Thus, in general, complicated, time-consuming extraction and purification processes, usually based on the application of solid-liquid extraction, are performed before final determination by immunoassay, high-performance liquid chromatography, or gas chromatography, very often coupled with mass spectrometry. This paper reviews the analytical methods so far described for the analysis of estrogens, which are currently important environmental pollutants presented in natural and wastewaters. Discuss of the main steps, from sampling up to analysis, and the techniques most commonly used in the determination is presented.

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.

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

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.

Determination of natural and synthetic estrogens and their conjugates in sewage sludge by pressurized liquid extraction and liquid chromatography-tandem mass spectrometry.

In this study we present a pressurized liquid extraction/liquid chromatography-tandem mass spectrometry (PLE/LC-MS-MS) method to determine a group of estrogens and conjugated estrogens in sewage sludge. Parameters that affect the extraction step such as extraction solvent, temperature, pressure, static extraction time, number of cycles, purge time and flush volume have been optimized. In the LC-MS-MS system, electrospray ionization and a triple quadrupole analyzer have been used, and the multiple reaction monitoring mode has enabled low levels of target analytes to be detected. All recoveries were higher than 81% except for estrone 3-glucoronide and estradiol 17-glucoronide which were not extracted and consequently, they were not considered in the present study. The repeatability and reproducibility between days expressed as %RSD (n=3), were lower than 6% and 9%, respectively. The method developed allowed the target analytes to be quantified at low levels of microg/kg. The limits of detection were lower than 26 microg/kg of dry weight (d.w.) of sewage sludge, except for 17 alpha-estradiol, 17beta-estradiol, 17 alpha-ethinylestradiol and estradiol 17-acetate whose values were between 150 and 175 microg/kg (d.w.). The method was applied to determine these compounds in sewage sludge from two domestic sewage treatment plants. Estrone 3-sulfate, estradiol 3-sulfate, diethylstilbestrol, estrone and estriol were determined in some samples and estriol showed the highest value (406 microg/kg d.w.).

Stable isotope-coded quaternization for comparative quantification of estrogen metabolites by high-performance liquid chromatography-electrospray ionization mass spectrometry.

A fast and sensitive LC-ESI-MS method is described for the comparative quantification of 16 estrogen metabolites based on the derivatization of estrogens with a novel derivatizing reagent, N-methyl-nicotinic acid N-hydroxysuccinimide ester (C1-NA-NHS). The process introduces a quaternary amine to the analytes, making the analytes permanently charged regardless of the pH of the high-performance liquid chromatography (HPLC) mobile phase. This quaternization resulted in a highly efficient separation of 16 estrogen metabolites in 7 min at a detection level below 1 ng/mL. By using a deuterated derivatizing reagent (C1-d(3)-NA-NHS), a complete set of deuterated standards was utilized and used as internal standards in a comparative quantification and recovery study, demonstrating acceptable results over a wide concentration range. A pooled breast cancer serum sample was analyzed using the described method, and 15 estrogens were detected in the range of 80-530 pg/mL.

Degradation rate constants of steroids in sewage treatment works and receiving water.

Steroid estrogens are one of the most important groups of endocrine disrupting chemicals (EDCs) which can cause adverse effects on wildlife species and humans. Natural estrogens, including estrone (E1) and estradiol (E2), and synthetic estrogen 17alpha-ethinylestradiol (EE2) together contribute to most of the estrogenic activity in sewage effluents and receiving water. Degradation, particularly aerobic biodegradation was found to be the dominant removal mechanism in these environments. There are a number of factors such as temperature, pH, SRT, HRT and biomass concentration that can affect the rate of biodegradation. This paper reports the results of investigations in to the relationship between the equivalent biomass concentration and degradation rate constants for compounds E1, E2 and EE2 in various environments. It was found that a higher biomass concentration leads to higher rate constants, and relatively good linear correlations (R2 =0.73, 0.79 and 0.73) between the logarithm of the rate constants and the corresponding logarithm equivalent biomass concentration (EBC) values were obtained.

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.

Combined isolation and purification procedures prior to the high-performance liquid chromatographic-ion-trap tandem mass spectrometric determination of estrogens and their conjugates in river sediments.

A fast and sensitive analytical procedure has been developed for the simultaneous separation and determination of alpha-estradiol, beta-estradiol, estriol, estrone and ethynylestradiol and their sulfate, glucuronide and acetate conjugates in river sediments. The procedure includes a microwave-assisted extraction (MAE) with aqueous methanol (25:75, v/v) at 100 degrees C in 10 min, a clean-up on Oasis WAX cartridge and a high-performance liquid chromatography-ion-trap tandem mass spectrometry (HPLC-IT-MS/MS) with electrospray ionization. The recovery for each compounds ranged from 83 to 107% and the repeatability represented as RSDs ranged from 4.9 to 9.6%. The limits of detection (LODs) were down to 1 ng g(-1). The analytical performance of the method was evaluated using determination of free and conjugated estrogens in river sediments.

Determining estrogenic steroids in Taipei waters and removal in drinking water treatment using high-flow solid-phase extraction and liquid chromatography/tandem mass spectrometry.

River water and wastewater treatment plant (WWTP) effluents from metropolitan Taipei, Taiwan were tested for the presence of the pollutants estrone (E1), estriol (E3), 17beta-estradiol (E2), and 17alpha-ethinylestradiol (EE2) using a new methodology that involves high-flow solid-phase extraction and liquid chromatography/tandem mass spectrometry. The method was also used to investigate the removal of the analytes by conventional drinking water treatment processes. Without adjusting the pH, we extracted 1-L samples with PolarPlus C18 Speedisks under a flow rate exceeding 100 mL/min, in which six samples could be done simultaneously using an extraction station. The adsorbent was washed with 40% methanol/60% water and then eluted by 50% methanol/50% dichloromethane. The eluate was concentrated until almost dry and was reconstituted by 20 microL of methanol. Quantitation was done by LC-MS/MS-negative electrospray ionization in the selected reaction monitoring mode with isotope-dilution techniques. The mobile phase was 10 mM N-methylmorpholine aqueous solution/acetonitrile with gradient elution. Mean recoveries of spiked Milli-Q water were 65-79% and precisions were within 2-20% of the tested concentrations (5.0-200 ng/L). The method was validated with spiked upstream river water; precisions were most within 10% of the tested concentrations (10-100 ng/L) with most RSDs<10%. LODs of the environmental matrixes were 0.78-7.65 ng/L. A pre-filtration step before solid-phase extraction may significantly influence the measurement of E1 and EE2 concentrations; disk overloading by water matrix may also impact analyte recoveries along with ion suppression. In the Taipei water study, the four steroid estrogens were detected in river samples (ca. 15 ng/L for E2 and EE2 and 35-45 ng/L for E1 and E3). Average levels of 19-26 ng/L for E1, E2, and EE2 were detected in most wastewater effluents, while only a single effluent sample contained E3. The higher level in the river was likely caused by the discharge of untreated human and farming waste into the water. In the drinking water treatment simulations, coagulation removed 20-50% of the estrogens. An increased dose of aluminum sulfate did not improve the performance. Despite the reactive phenolic moiety in the analytes, the steroids were decreased only 20-44% of the initial concentrations in pre- or post-chlorination. Rapid filtration, with crushed anthracite playing a major role, took out more than 84% of the estrogens. Except for E3, the whole procedure successfully removed most of the estrogens even if the initial concentration reached levels as high as 500 ng/L.

Biological treatment of estrogenic substances.

The fate and behavior of estrogenic substances in various biological wastewater treatment processes and several advanced sewage treatment processes were examined. The removal of 17beta-estradiol (E2), estrone (E1) and estriol (E3) was investigated by using a pilot-scale activated sludge plant supplied with domestic sewage. Several sewage treatment plants adopting the activated sludge process were evaluated for the removal of estrogenic substances using an in vitro recombinant yeast assay and chemical analysis. The results indicated that E2 significantly contributed to estrogen-like activity particularly in secondary treated effluents. The removal rate of E2 was found to be sufficiently high throughout a one-year study on estrogens in domestic sewage, whereas E1 often remained in the effluent. The optimization of operational conditions based on E1 removal is important for reducing estrogenic activity in treated water.