Reporter cell lines to study the estrogenic effects of xenoestrogens.

In order to characterize the estrogenic activity of chemicals, we established complementary in vitro recombinant receptor-reporter gene assays in stably transfected MCF-7 and HeLa cells. MCF-7 cells which express the endogenous estrogen receptor alpha (ER alpha) were stably transfected with only an estrogen-regulated luciferase gene. These cells enable the detection of compounds which bind to ER alpha or interfere with the induction of ER alpha mediated gene expression. Furthermore, HeLa cells, which do not express endogenous ERs, were transfected with an ER alpha or an ER beta construct together with an estrogen-regulated luciferase gene, or a chimeric GAL4-ER alpha receptor and the corresponding luciferase reporter gene. Finally, we tested these four cellular models as tools to check the estrogenic activities of several potential xenoestrogens and to detect estrogenic activity in wastewater sewage treatment effluents. In all of the models, nonylphenol mixture (NPm), 4n-nonylphenol (4nNP), 2,4'-DDE, 4,4'-DDE and wastewater sewage treatment effluent were active, while PCB mixture (Aroclor 1254), PCB 77, atrazine and lindane (gamma hexachlorocyclohexane) were inactive. Dioxin partially activates the estrogen receptor in MCF-7 cells while in HeLa-derived cell lines, it decreased the estrogenic-induced expression of luciferase.     

Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens

The MCF-7 cell proliferation assay is potentially a simple and highly reproducible tool for the identification of estrogenic compounds. However, its widespread use has been complicated by the lack of a standardised protocol, resulting in considerable inter-laboratory variability. We have explored the sources of variability both in relation to cell lines and test regimens and report on optimised procedures for the identification of estrogenic agents. Two supposedly identical MCF-7 parent cell lines (designated UCL and SOP), and the BUS subline were cultured according to an existing protocol, and responses to 17-estradiol (E2) assessed. Despite yielding almost identical EC50 values, the proliferative response varied widely between cell lines from 0.98-fold over controls (UCL) to 8.9-fold (BUS) indicating major differences between them. The underlying causes may be genetic, and to assess this we used comparative genomic hybridisation (CGH), a technique which allows the detection of DNA sequence copy number changes on a genome-wide scale. Although numerous similarities existed between the different cell lines, the least oestrogen-responsive line (MCF-7/UCL) exhibited the greatest number of cytogenetic changes, many of which were not seen in MCF-7/SOP cells. We suggest that care must be taken, therefore, when choosing a cell line for MCF-7 cell-based experiments. Selecting the MCF-7/SOP line for further work, we carried out a thorough and systematic optimisation of the MCF-7 cell proliferation assay, finding that a 72-h period in oestrogen-free medium before treatment strongly influenced the cells response to E2. With 1 nM E2, proliferation increased from 1.5-fold to 6.5-fold relative to vehicle-treated controls, a response similar to that seen with MCF-7/BUS cells in the E-SCREEN protocol devised by Soto et al. With parent MCF-7 cells, other laboratories have reported only 4.5-fold increases as maximal. Here we present evidence that the choice of cell line and culture conditions are crucial in determining test outcomes, and once chosen and adhered to the assay yields reproducible results.     

Concentration and loading of pesticide residues in Lake Biwa basin (Japan).

The concentrations and loading rates of pesticides used in paddy fields were investigated over a period of 5 years in the Seta River, which is the only natural outlet of Lake Biwa. The lake's water catchment area is 3,174 km2, 20% of which contains paddy fields. Water samples were also collected in six rivers flowing into the lake in order to compare the contamination level and concentration profile. The pesticides analyzed were four herbicides (molinate, simetryn, oxadiazon, and thiobencarb), one fungicide (isoprothiolane), and two insecticides (diazinon and fenitrothion). Molinate, simetryn, oxadiazon and isoprothiolane were found at the higher frequencies with maximum concentrations of 1.1, 0.4, 0.1 and 0.5 microg,/l in the effluent river, one or two order of magnitude higher than that of effluent in influent rivers. These peak concentrations were observed during the application period in influent rivers and two or three weeks after that in effluent river. The frequency of occurrence of thiobencarb, diazinon, and fenitrothion was relatively low and their maximum concentrations in the effluent remained below 0.1 microg/l. The decrease of molinate, simetryn and oxadiazon concentrations in the effluent river were approximated by two straight lines plotted on semilogarithmic scale. Increased loading was induced by intense rainfall, which took place during the application period. Simetryn and isoprothiolane persisted in relatively high concentrations through the year were also influenced on its loading by the heavy rainfall in the following months. The percentages of the total amount of pesticides released through Lake Biwa to the basin in downstream were estimated to be 1.3-2.9% for molinate, 5.4-10.0% for simetryn, 0.6-1.3% for oxadiazon, 0.2-0.9% for thiobencarb, 1.8-6.6% for isoprothiolane, 0.3-2.1% for diazinon. and 0% for fenitrothion.     

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.     

Application of short-term bioassay guided chemical analysis for water quality of agricultural land run-off.

The effect of agricultural land run-off on the water quality of Lake Kojima, Japan, was investigated using a short-term bioassay-guided chemical analysis. Water samples were collected for 1 year starting from June 1995 to June 1996. Toxicity of the dissolved and adsorbed extracts in the water samples was evaluated using the Daphnia immobilization test and the concentrations of pesticides and putative toxic substance in the extracts were determined by high performance liquid chromatography. Most of the dissolved extracts caused immobilization of the test Daphnia magna at low concentrations during the period of paddy pesticide application. Some extracts were found to contain pesticides such as dymron, mefenacet and flutolanil, but their concentrations were too low to have a toxic effect on the daphnia. An unknown toxic compound, Peak C, was isolated from some river water samples, but it produced only a relatively weak toxicity to Daphnia. To better understand the impact of agricultural run-off on a receiving water body, the relationship between the observed toxicity and the concentrations of pesticides and Peak C in the water samples was studied both temporally and spatially.     

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.     

Fish model for assessing the in vivo estrogenic potency of the mycotoxin zearalenone and its metabolites.

The in vivo estrogenic potency of zearalenone (ZEA), a mycotoxin produced by different strains of Fusarium fungi, and its metabolites (alpha- and beta-zearalenol), have been studied in fish. Estrogenicity was evaluated using an in vitro competitive receptor binding assay and in vivo induction of vitellogenesis and zonagenesis, two estrogen receptor (ER)-mediated responses that are integral aspects of fish oogenesis. The ER binding affinities of alpha-zearalenol and ZEA in rainbow trout (Oncorhynchus mykiss) were approximately 1/150 and 1/300 to that of estradiol, respectively. Juvenile salmon (Salmo salar) were exposed to a single intraperitoneal injection of ZEA, alpha-zearalenol and beta-zearalenol (each at 1 and 10 mg/kg) and compared to fish injected with estradiol-17 beta (E2; 5 mg/kg) and controls. Using indirect enzyme-linked immunosorbent assay (ELISA) with homologous antibodies, a dose-dependent induction of vitellogenin (Vtg) and eggshell zona radiata proteins (Zr-proteins) were observed 7 days after exposure to ZEA and alpha-zearalenol. beta-Zearalenol did not elevate plasma Vtg levels, but a non-significant elevation of plasma Zr-proteins levels was observed at the highest dose (10 mg/kg). Generally, alpha-zearalenol and ZEA possess estrogenic potencies that are approximately 50% compared to that of E2, and their order of estrogenic potency (in both in vitro receptor competitive binding and in vivo induction of Vtg and Zr-proteins levels) is: alpha-zearalenol > ZEA > beta-zearalenol. Our results show that blood plasma analysis of Vtg and Zr-proteins levels provides a suitable in vivo fish model for assessing the estrogenic potencies of ZEA and its metabolites.     

Understanding the gap between the estrogenicity of an effluent and its real impact into the wild

To study the reliability between in vitro and in vivo data collected downstream 2 sewage treatment plants (STP) as well as from bleached kraft mill industry (BKME), 5 rivers (3 impacted and 2 references) were investigated in the Walloon region (southern of Belgium). For the in vitro part of the work, water samples were collected to measure the estrogenicity of the ‘out’ effluent compared to reference sample point by MCF-7 assay. Results indicated significant estrogenicity of effluents from STP and BKME and a weak estrogenicity in reference sites. However, estradiol equivalents (EEQ) estimated into rivers were probably too low to impact wild population. Chemical analysis of 13 compounds of interest indicated that extraction procedure used in this study gave low recoveries of estrogen-like xenobiotics, leading to probably under-estimated MCF-7 responses. Surprisingly, a full scan mode has revealed an unexpected compound in the sample of BKME which was: 7-isopropyl-1,1,4a-trimethyl-1,2,3,4a,9,10,10a-octahydrophenanthrene, a product of pulp mill manufacture. In parallel to in vitro, in vivo assessment of estrogenic impact of effluent was followed on the gudgeon (Gobio gobio). Samples were achieved during 2 different periods of the reproductive cycle, resting period (RP) and pre-spawning period (pSP). Unspecific physiological parameters to estrogenic exposure (gonadosomatic index and systematic testis cell counting) displayed no significant differences related to endocrine disruption of the reproductive tract, only differences were correlated with the reproductive state of fish (RP versus pSP). Concerning the potent biomarker of estrogen exposure, vitellogenin (vtg), only basal induction was revealed but not related to estrogenic exposure. Nevertheless, vtg over-expression was found for male fish presenting a feminization of the reproductive tract captured downstream the STP station of Wégnez in the Vesdre River. Intersexuality, another indicator of the estrogenicity impact in fish, was observed in every site. Actually, ovotestis was systematically formed by protoplasmic oocyte observed in low percentage in every group analysed (impacted and references). Moreover, in fish captured in Wégnez, oocyte diameter was significantly higher compared to the other groups. In this study, only moderate to none impact in population of gudgeon was noticed. Moreover, in this case no discrepancy between in vitro and vivo was viewed although both approaches revealed gaps in monitoring effluent incidence into the environment. We should remain careful in the interpretation when only partial approaches are used in order to characterize impact in the aquatic milieu.     

Effects of chlorine on the decrease of estrogenic chemicals

The effects of chlorination on the elimination of three estrogenic chemicals such as 17beta-estradiol, nonylphenol and bis-phenol A were investigated using yeast two-hybrid assay (YTA), estrogen receptor (ER) competition assay (ER-CA), and high-performance liquid chromatography/mass spectrometry (LC/MS). The results of YTA, ER-CA and the analysis of LC/MS indicated that the estrogenic activity of the above-mentioned three endocrine disruptors were significantly reduced as a result of chlorination. The decrease in estrogenic activity paralleled a decrease in estrogenic chemicals under the influence of free chlorine. One common characteristic of estrogenic chemicals is the presence of a phenolic ring. Considering that a phenolic ring is likely to undergo some sort of transformation in an aqueous chlorination solution, the above-mentioned results may be applied to the rest of the estrogenic chemicals in natural waters.     

Biological assessments of a mixture of endocrine disruptors at environmentally relevant concentrations in water following UV/H2O2 oxidation

Numerous studies have investigated degradation of individual endocrine disrupting compounds (EDCs) in lab or natural waters. However, natural variations in water matrices and mixtures of EDCs in the environment may confound analysis of the treatment efficiency. Because chemical based analytical methods cannot represent the combined or synergistic activities between water quality parameters and/or the EDC mixtures at environmentally relevant concentrations (microg L(-1)-ng L(-1)), bioanalytical assessments of residual estrogenic activity in treated water were used to evaluate the performance of the UV based advanced oxidation process for estrogenic contaminants in water. Four EDCs including estradiol (E(2)), ethinyl estradiol (EE(2)), bisphenol-A (BPA) and nonylphenol (NP) were spiked individually or as a mixture at mug L(-1)-ng L(-1) in laboratory or natural river water. The removal rates of estrogenic activity were quantitatively evaluated by in vitro yeast estrogen screen (YES) and in vivo Vitellogenin (VTG) assays with Japanese medaka fish (Oryzias latipes). UV in combination with 10 ppm H(2)O(2) as an oxidation process was capable of decreasing in vitro and in vivo estrogenic activity, however, in vivo estrogenic activity of the EDC mixture in natural water was not completely removed at UV fluence up to 2000 mJ cm(-2). The removal rates of in vitro estrogenic activity of the EDC mixtures were lower than those observed for single compounds, and slower in natural waters, likely due to lower steady-state concentrations of hydroxyl radicals (*OH) in the presence of *OH scavengers from the water matrix and EDC mixture.     

Application of ozone, UV and ozone/UV processes to reduce diethyl phthalate and its estrogenic activity

A research study to monitor the micropollutant levels present in the Han River, a major drinking water source for the Seoul Metropolitan district in Korea, was performed over a five-year period from 2000 to 2004, inclusive. Of the detected micropollutants, phthalates were found to be the major contaminants. In this study, the estrogenic activities of the detected phthalates and raw water samples contaminated with the pollutants were assessed by the E-screen assay using the MCF-7 breast cancer cell line. Of the phthalates, diethyl phthalate (DEP) and di(2-ethylhexyl) phthalate (DEHP) showed relatively high cell proliferation. Using DEP as a phthalate probe, three candidate processes, ozone alone, UV alone, and the ozone/UV combined, were evaluated for their efficiency in removing DEP and reducing its estrogenic activity. The ozone/UV process was shown to have the highest efficiency for the elimination of DEP and its by-products, leading to the complete mineralization of DEP. This study also found that the ozone/UV process is the best candidate to reduce the estrogenicity induced by DEP and its by-products.     

北方某水厂的类雌激素物质变化规律

对北方某水厂春季和夏季源水和各处理单元出水中类雌激素物质的变化规律进行了研究。将水样用固相萃取方法富集后，按不同极性洗脱，得到从非极性到极性的3个组分，分别对总提取物和各分级富集组分进行重组雌激素受体基因的酵母检测。结果表明：源水和各处理单元出水中能够检测到极低水平的类雌激素物质，其中源水中的类雌激素效应仅相当于5．4～11．0pg／L雌二醇当量，主要由非极性与弱极性的类雌激素物质引起；春季和夏季源水中的类雌激素效应相差不大；水厂传统的处理工艺对类雌激素物质的去除效果不明显；重组基因酵母检测技术结合水样的固相萃取、三步纯化分级前处理方法可以快速、有效地筛选和定量分析水样中未知类雌激素物质，并对水处理效果进行评价。     

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.     

Combination of in vitro bioassays encompassing different mechanisms to determine the endocrine-disrupting effects of river water

In this study, the total toxic effects of river water samples were assessed using a series of cell culture bioassays which encompassed different mechanisms, based on specific modes of action. River water samples were collected from three tributaries of the Youngsan River in the western portion of Korea. We confirmed that Youngsan River water was polluted with a complex mixture of estrogenic and dioxin-like compounds. The total toxic effects of the downstream water samples were found to be higher than that of the upstream water samples. In the upstream water samples, total estrogenic activity was measured to be between 0.005 and 0.049 ng-EEQ/l (17beta-estradiol-equivalent concentration) and no CYP1A activity was detected. In the downstream water samples, however, total estrogenic activity was measured to be between 0.021 ng-EEQ/l and 1.918 ng-EEQ/l, and total CYP1A activity was between 0.63 and 3.55 microg-MEQ/l (3-methylcholanthrene-equivalent concentration). When assessed according to a concentration-response curve, downstream water sample extracts exerted dual actions on estrogen receptors, depending on the concentration volume of the samples. The concentration volume range proximal to the original water sample exhibited estrogenic activity, whereas antiestrogenic activity was observed at high concentration volumes (more than 5 times concentration) in the extracts. This study involved a combination of in vitro bioassays, designed to encompass different mechanisms. The bioassays used included the estrogen receptor binding affinity test, E-screen assay, aromatase assay, and EROD assay. These tests provided a great deal of useful information regarding the potency and action modes of estrogenicity and antiestrogenicity inherent in the sampled river water. Although further study is necessary to determine the relationship between toxic responses in in vitro bioassay systems and chronic toxicity in aquatic organisms, our approach is expected to be fairly accurate with regard to the detection of endocrine-disrupting effects in an aquatic environment.     

Inhibition of progesterone receptor activity in recombinant yeast by soot from fossil fuel combustion emissions and air particulate materials

Numerous environmental pollutants have been detected for estrogenic activity by interacting with the estrogen receptor, but little information is available about their interactions with the progesterone receptor. In this study, emission samples generated by fossil fuel combustion (FFC) and air particulate material (APM) collected from an urban location near a traffic line in a big city of China were evaluated to interact with the human progesterone receptor (hPR) signaling pathway by examining their ability to interact with the activity of hPR expressed in yeast. The results showed that the soot of a petroleum-fired vehicle possessed the most potent anti-progesteronic activity, that of coal-fired stove and diesel fired agrimotor emissions took the second place, and soot samples of coal-fired heating work and electric power station had lesser progesterone inhibition activity. The anti-progesteronic activity of APM was between that of soot from petroleum-fired vehicle and soot from coal-fired establishments and diesel fired agrimotor. Since there was no other large pollution source near the APM sampling sites, the endocrine disrupters were most likely from vehicle emissions, tire attrition and house heating sources. The correlation analysis showed that a strong relationship existed between estrogenic activity and anti-progesteronic activity in emissions of fossil fuel combustion. The discoveries that some environmental pollutants with estrogenic activity can also inhibit hPR activity indicate that further studies are required to investigate potential mechanisms for the reported estrogenic activities of these pollutants.     

Urinary excretion rates of natural estrogens and androgens from humans, and their occurrence and fate in the environment: A review

Endocrine disrupting compounds (EDCs) are pollutants with estrogenic or androgenic activities at very low concentrations and are emerging as a major concern for water quality. For sewage of municipal wastewater treatment plants in cities, one of the most important sources of EDCs are natural estrogens and natural androgens (NEAs) excreted from humans. Therefore, estrogenic/androgenic potencies or relative binding affinity of the NEAs were first outlined from different sources, and data of urinary excretion rates of NEAs were summarized. To evaluate their estrogenic activities, their excretion rates of estrogen equivalent (EEQ) or testosterone (T) equivalent (TEQ) were also calculated. Based on our summary, the total excretion rates of EEQ by estrone (E1), 17β-estradiol (E2), and estriol (E3) only accounted for 66-82% of the total excretion rate of EEQ among four different groups, and the other corresponding natural estrogens contributed 18-34%, which meant that some of the other natural estrogens may also exist in wastewater with high estrogenic activities. Based on the contribution ratio of individual androgens to the total excretion rate of TEQ, five out of 12 natural androgens, T, dihydrotestosterone (DHT), androsterone (AD), 5β-androstanediol (β-ADL), and androstenediol (ANL) were evaluated as the priority natural androgens, which may exist in wastewater with high androgenic activities. Published data on occurrence and fate of the NEAs including natural estrogen conjugates in the environment were also summarized here.     

Degradation and estrogenic activity removal of 17beta-estradiol and 17alpha-ethinylestradiol by ozonation and O3/H2O2

This work investigated the degradation of a natural (17beta-estradiol) and a synthetic (17alpha-ethinylestradiol) estrogens (pure or in the mixture) and the removal of estrogenic activity by the ozonation and O3/H2O2 process in three different pHs (3, 7 and 11). The effect of oxidation via OH radical was evaluated adding a radical scavenger (t-butanol) in the medium. Estrogenic activity was performed using the YES assay. 17beta-estradiol and 17alpha-ethinylestradiol presented similar estrogenic potential and the association of these estrogens resulted in an addictive effect for estrogenic activity. Ozonation and O3/H2O2 processes were effective in removing the estrogens in aqueous solution. In the mixture at pH 11, removals were higher than 98% and 96% for 17beta-estradiol and 17alpha-ethinylestradiol, respectively. In pH 3, 17beta-estradiol and 17alpha-ethinylestradiol removals were 100% and 99.7%, respectively. When estrogens were treated separately, the removals in pH 11 were superior to 99.7 and 98.8%, while in pH 3 were 100% and 99.5% for 17beta-estradiol and 17alpha-ethinylestradiol, respectively. 17alpha-ethinylestradiol has been always removed at lower rates (pure or in the mixture) for all applied conditions. Estrogenic activity was completely removed in pH 3 for ozonation or O3/H2O2. The samples oxidized in pH 11 presented higher estrogenic activity than those in pH 7. Estrogens removal was lower at pHs 7 and 11, when the scavenger was added to the media. The higher estrogen residual concentrations found in ozonation in presence of tert-butanol are contributing for higher estrogenic activity observed in pHs 7 and 11. By-products with estrogenic activity were formed by oxidation via OH radical. Only a few compounds could be identified in pHs 7 and 11 and they have a phenolic ring, which, probably is contributing to the estrogenic activity observed.     

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.