Quantitative assessment of estrogenic activity in the water environment of Korea by the E-SCREEN assay

In this study, the E-SCREEN assay was optimized and validated for the sensitive quantitative determination of the total estrogenicity in river samples. River water and sediment samples were collected and analyzed with the E-SCREEN. River water (10 l) was extracted using combined solid-phase extraction in static adsorption mode with Soxhlet extraction. Estrogenic pollutants adsorbed to the XAD-4 resin were recovered with 98.24 +/- 5.90% efficiency by elution with ethyl acetate and dichloromethane (1:9). The detection limit by 17beta-estradiol equivalent concentration (EEQ) of the E-SCREEN assay was 8.03 pg EEQ/l. Among the water samples, the estrogenic activity was observed to be higher downstream of the Kumho river (7.43 ng EEQ/l) and upstream of Kum river (2.05 ng EEQ/l) than in other samples. More than 3 mg of equivalent sediment samples from the Kumho river, Kum river and Miho stream showed partial agonistic effects, and the Mankyung river showed a partial agonistic effect with only 1.5 mg of sediment. The highest value of RPE was 83.34 downstream of the Kumho river, and the lowest value of RPE was 6.52 downstream of the Miho stream. Full estrogen agonistic activities were observed downstream of the Kumho river and upstream of the Kum river. The partial agonistic activity was observed in upstream of the Kumho river, downstream of the Mankyung river, and upstream of the Miho stream, and no agonistic action was observed downstream of the Kum river or Miho stream, or upstream of the Mankyung river. The total estrogenic activity in the river water and sediment samples was between 0.50 pg/L and 7.4 ng/L, 3.39 pg/g and 10.70 pg/g.     

Analysis of environmental endocrine disrupting chemicals using the E-screen method and stir bar sorptive extraction in wastewater treatment plant effluents

Endocrine disrupting chemicals (EDCs) have become a major issue in the field of environmental science due to their ability to interfere with the endocrine system. Recent studies show that surface water is contaminated with EDCs, many released from wastewater treatment plants (WWTP). This pilot study used biological (E-screen assay) and chemical (stir bar sorptive extraction-GC-MS) analyses to quantify estrogenic activity in effluent water samples from a municipal WWTP and in water samples of the recipient river, upstream and downstream of the plant.The E-screen assay was performed on samples after solid phase extraction (SPE) to determine total estrogenic activity; the presence of estrogenic substances can be evaluated by measuring the 17-β-estradiol equivalency quantity (EEQ). Untreated samples were also assayed with an acute toxicity test (Vibrio fischeri) to study the correlation between toxicity and estrogenic disruption activity.Mean EEQs were 4.7 ng/L (± 2.7 ng/L) upstream and 4.4 ng/L (± 3.7 ng/L) downstream of the plant, and 11.1 ng/L (± 11.7 ng/L) in the effluent. In general the WWTP effluent had little impact on estrogenicity nor on the concentration of EDCs in the river water. The samples upstream and downstream of the plant were non-toxic or weakly toxic (0 < TU < 0.9) while the effluent was weakly toxic or toxic (0.4 < TU < 7.6). Toxicity and estrogenic activity were not correlated.At most sites, industrial mimics, such as the alkylphenols and phthalates, were present in higher concentrations than natural hormones. Although the concentrations of the detected xenoestrogens were generally higher than those of the steroids, they accounted for only a small fraction of the EEQ because of their low estrogenic potency. The EEQs resulting from the E-screen assay and those calculated from the results of chemical analyses using estradiol equivalency factors were comparable for all samples and closely correlated.     

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.     

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.     

Identification of estrogen-like effects and biologically active compounds in river water using bioassays and chemical analysis

The Nackdong River is the longest river in South Korea and passes through major cities that have several industrial complexes, including chemical, electric, and petrochemical complexes, and municipal characteristics such as apartment complexes. Along the river, the Gumi region has an electric industrial complex and an apartment complex that may be possible point sources of xenoestrogens such as phenolic compounds. To identify the causative chemicals for estrogenic activity in the river water of this region, bioassay-directed chemical analysis was performed. All samples from six sampling sites (an upstream point: S1; hot spot points: S2-1, S2-2, and S2-3; and downstream points: S3, and S4) showed estrogenic activity in the E-screen assay, with bio-EEQs (17β-E₂-equivalent quantities) ranging from 25.35-677.15 pg/L. Samples from S2-2, the sampling point downstream of the junction of stream water, and domestic and industrial wastewater, contained the highest estrogenic activity. Since the bio-EEQ of the organic acid fraction (F2) of the S2-2 sample had the highest activity (823.25 pg-EEQ/L) and F2 may contain phenolic compounds, GC-MS analyses for phenolic xenoestrogens were conducted with the organic acid fractions of the river water samples. Six estrogenic phenolic chemicals, 4-NP, BPA, 4-t-OP, 4-t-BP, 4-n-OP, and 4-n-HTP, were detected, with the highest concentrations (I-EEQ) found in S2-2 (231.80 pg/L). Among these phenolic chemicals, 4-NP was the most potent estrogen (bio-EEF; 8.12 × 10^− 5) and acted as a full agonist. Furthermore, 4-NP was present at levels (2.0 µg/L in S2-2) that can induce VTG induction in fish (>1 µg/L). In addition, we confirmed that river water (S2-2) significantly increased serum VTG levels in crucian carp (Carassius auratus) in a fish exposure experiment under laboratory conditions. Therefore, phenolic xenoestrogens, especially 4-NP, may be the main causative compounds responsible for the estrogenic effect on the Nackdong River.     

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.     

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.     

The use of in vitro bioassays to quantify endocrine disrupting chemicals in municipal wastewater treatment plant effluents

In vitro bioassays are widely used to detect and quantify endocrine disrupting chemicals (EDCs) in the influents and effluents of municipal wastewater treatment plants (WWTP). These assays have sometimes led to false positive or negative results, partly due to the low EDC concentrations in the samples. The objectives of the present study were: (a) to compare the estrogen screen (E-Screen) and the yeast estrogen screen (YES) bioassays using the 17beta-estradiol (E2) or its equivalence and (b) to investigate if a combination of the E-Screen and YES assays can be used to improve the accuracy of EDC detection and quantification. The E-Screen bioassay was conducted with the MCF-7 (BOS) human breast cancer cell line while the YES bioassay employed two different types of recombinant yeast. The influent and effluent samples collected from the five WWTPs operated by the Greater Vancouver Regional District (GVRD) were analyzed by both the E-Screen and the YES bioassays. Since the results of the E-Screen and YES bioassays varied by up to 4-fold on the same split sample of a nominal E2 concentration, the mean value of the E-screen and YES bioassays was used to represent the EDC activity of a given WWTP sample. Results of these studies showed that the E2 equivalent concentration in each WWTP sample was consistently higher than 1 ng/L, a concentration that may potentially cause endocrine disruption in different aquatic species. The composition of selected EDCs in a subset of effluent samples was examined using a gas chromatograph-high resolution mass spectrometer (GC-HRMS). EDC composition in 10 WWTP samples correlated with the mean endocrine disrupting activities of the E-Screen and YES bioassays. Results also indicated that secondary treatment plants are comparable to the primary treatment plants in removing EDCs from the final effluents.     

Rapid loss of estrogenicity of steroid estrogens by UVA photolysis and photocatalysis over an immobilised titanium dioxide catalyst

The presence of low levels of natural and synthetic steroid estrogens in the aquatic environment, and their biological effects on aquatic organisms, are presently issues of concern. In this study, we investigated the temporal removal of estrogenic activity of several potent and environmentally relevant steroid estrogens by photocatalysis over an immobilised titanium dioxide (TiO2) catalyst. We used a recombinant yeast assay to measure estrogenic activity, which provided detection limits within the reactor of 53 ng/l for 17beta-estradiol and 17alpha-ethinylestradiol, and 100 ng/l for estrone. Pseudo-first-order kinetic data showed that photocatalysis over titanium dioxide was equally effective at removing the estrogenic activity of all three steroid substrates in aqueous solutions (initial concentrations of 10 microg/l) with a 50% reduction in estrogenicity within 10 min. In control experiments without TiO2 catalyst, the rate of UVA photolysis of the steroid substrates varied, but was most effective with 17alpha-ethinylestradiol followed by estrone, and was least effective with 17beta-estradiol (0.42, 0.2 and < 0.1 times the rate achieved with photocatalysis, respectively). The application of photocatalysis for the removal of steroid compounds within STW effluent released into the aquatic environment is discussed.     

Investigation of the estrogenic activities of pesticides from Pal-dang reservoir by in vitro assay

Endocrine disruptors, when absorbed into the body, interfere with the normal function by mimicking or blocking the hormone system. To investigate compounds mimicking estrogen in the drinking water source of the residence of Seoul, the Pal-dang reservoir was monitored over a period of 5 years, between 2000 and 2004. Nine kinds of pesticide (carbaryl, DBCP, diazinon, fenitrothion, fenobucarb, flutolanil, iprobenphos, isoprothiolane and parathion) were found to exist in the river water sample. These compounds were detected at low concentrations in the water samples. The total concentration and those of each of these pesticides were below the permissible limits of the National Institute of Environmental Research (NIER), Korea. The estrogenic potencies of the nine pesticides were examined using an E-screen assay with MCF-7 BUS estrogen receptor (ER)-positive human breast cancer cells, with ER-negative MDA MB 231 cell lines also used to compare the results. From this, flutolanil and isoprothiolane were confirmed to have estrogenic activities as shown by the increasing MCF-7 BUS cell growth on their addition. In addition, the estrogen receptor alpha (ERalpha) protein, estrogen receptor-regulated progesterone receptor (PR) and pS2 mRNA levels on the addition of flutolanil and isoprothiolane were measured with MCF-7 BUS cells. It was observed that the levels of ERalpha protein decreased and those of the PR and pS2 genes increased on the addition of either flutolanil or isoprothiolane at concentrations of 10(-4) M, in the same manner as with the addition of 17beta-estradiol, which was used as the positive control. From these results, it was confirmed that flutolanil and isoprothiolane exhibit estrogenic activities, suggesting they might act through estrogen receptors.     

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.     

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.     

An ELISA for brown trout (Salmo trutta) vitellogenin and its use in bioassays for environmental estrogens

An enzyme linked immunosorbent assay (ELISA) was developed for the detection of the egg yolk precursor vitellogenin (Vg) in plasma of brown trout (Salmo trutta). Purified Vg from a 17 beta-estradiol-induced trout was used as the competing antigen in the ELISA which is based on polyclonal antibodies. The ELISA's performance was optimized and characterized. The assay's working range was (25-500 ng ml-1), its sensitivity was (10.5 ng ml-1), and it had an intra-assay coefficient of variation of less than 10% between 30 and 1000 ng ml-1. The ELISA was used in bioassays for the detection of environmental estrogens, including estrogen mimics, in whole and fractionated industrial waste waters. Those bioassays were based on intraperitoneal (i.p.) injection-, static renewal-, and flow through exposure systems. The response threshold of both bioassays is limited to 1-2 micrograms ml-1 Vg by a low level plasma interference that was regularly detected in plasma from non-induced male fish. The responsiveness of the bioassays was characterized using progressive doses of 17 beta-estradiol. The i.p.-based assay, which was responsive to at least 100 micrograms kg-1 of 17 beta-estradiol, was used to screen extracts of pulp mill effluent and black liquor for estrogenic effects. Neither extract induced Vg in our assay. The i.p. assay was also used to test 4-tert-octylphenol (OP) and the PAH derivative, retene, for estrogenic activity. OP induced Vg in the i.p.-exposed fish; no Vg induction was detected in the retene-exposed fish. The static renewal bioassay, which was responsive to at least 0.1 microgram ml-1 of 17 beta-estradiol over a 15-day exposure period, was used to screen whole pulp mill effluents for estrogenic effects. No Vg induction was detected in the effluent-treated fish.     

Uptake of arsenic by New Zealand watercress (Lepidium sativum)

Watercress (Lepidium sativum) is consumed as a vegetable, especially by the indigenous community in New Zealand. An investigation was carried out on the accumulation of arsenic by watercress, following earlier reports of inordinate arsenic concentrations in some aquatic macrophytes collected from the Waikato River, North Island, New Zealand. The Waikato River and some other aquatic systems in Taupo Volcanic Zone, New Zealand have elevated arsenic concentrations due to geothermal activity. Watercress, river water and sediment samples were collected from 27 sites along the Waikato river and analysed for arsenic. Greenhouse trials with watercress grown in beakers containing added arsenic were conducted to confirm the ability of this species to accumulate arsenic. At a number of sites, the concentration of arsenic in both the water and the watercress samples exceeded the World Health Organisation (WHO) limit for drinking water (0.01 mg l(-1)) and foodstuffs (2 mg kg(-1) on a fresh weight basis). The average leaf and stem arsenic concentrations were, respectively, 29.0 and 15.9 mg kg(-1) on a fresh weight basis. Plants grown in solutions of >0.4 mg l(-1) arsenic concentration had fresh weight arsenic concentrations above the WHO limit. Despite these higher concentrations, arsenic levels in plants grown under greenhouse conditions were approximately fivefold lower than in plants growing in the Waikato River, possibly because under natural conditions, the watercress is rooted in sediment containing on average approximately 35 mg kg(-1) arsenic. It is recommended that watercress from the Waikato River, or other areas with elevated water arsenic concentrations, should not be consumed.     

Analysis and occurrence of estrogenic hormones and their glucuronides in surface water and waste water in The Netherlands

An analytical procedure was developed that enables routine analysis of four estrogenic hormones in concentrations below 1 ng/l in surface water and waste water. The recovery was 88-98% with a limit of detection of 0.1-2.4 ng/l depending on the compound and the matrix measured. This method was used to determine the occurrence of 17 beta-estradiol, 17 alpha-estradiol, estrone and 17 alpha-ethinylestradiol in the aquatic environment in The Netherlands. The data show that estrogenic hormones can be detected at low concentrations (up to 6 ng/l) at some locations in surface water. In selected effluents of waste water treatment plants estrone and 17 beta-estradiol were detected in concentrations in the ng/l range. Concentrations of 17 alpha-estradiol and the contraceptive 17 alpha-ethinylestradiol were in most of these samples below the limit of detection. Hormone glucuronides were not detected in most surface water and effluents.     

Responses of Hexaplex (Murex) trunculus to selected pollutants

We report the results of a recent survey of the concentration of natural estrogens (17beta-estradiol, 17alpha-estradiol, estrone, estriol) and the synthetic estrogen, 17alpha-ethynylestradiol in representative animal wastes and sewage treatment plant (STP) effluents in the Waikato region of New Zealand. Dairy farm effluent samples showed high levels of estradiol (19-1360 ng/L) and its breakdown product estrone (41-3123 ng/L) compared with piggery or goat farm effluents. The combined load for these estrogens (excluding beta epimer) varied from 60 to >4000 ng/L. The piggery effluent provided the lowest total estrogen load (46 ng/L), with estrone accounting for nearly 60% of the measured estrogens in this sample. The synthetic analogue, 17alpha-ethynylestradiol was detected only in one wastewater treatment plant sample, albeit at trace level. An estrogen receptor competitive binding assay was used to test the biological activity of the samples and confirmed that most agricultural waste samples contain high levels of estrogenic compounds. The potential of these wastes to cause endocrine disruption in the receiving ecosystem is unknown at present.     

Sample preparation method for the ER-CALUX bioassay screening of (xeno-)estrogenic activity in sediment extracts

The application of bioassays to assess the occurrence of estrogenic compounds in the environment is increasing in both a scientific and statutory context. The availability of appropriate validated methods for sample pre-treatment and analysis is crucial for the successful implementation of bioassays. Here, we present a sample preparation method for the bioassay screening of estrogenic activity in sediment with the in vitro Estrogen Receptor mediated Chemical Activated LUciferase gene eXpression (ER-CALUX) assay. The method makes use of an Accelerated Solvent (ASE) or Soxhlet extraction with a mixture of dichloromethane and acetone (3:1, v/v), followed by clean up of the extract by Gel Permeation Chromatography (GPC). Recoveries of a panel of 17 pollutants differing largely in physical-chemical properties from spiked sediment were determined and appeared to be on average about 86%. Furthermore, the estrogenic potencies of all test compounds were individually assessed by determination of concentration-response relationships in the ER-CALUX assay. Concentration dependent estrogenic potency was found for 14 of the 17 compounds, with potencies of about 10(5) to 10(7) fold lower than the natural estrogenic hormone 17beta-estradiol. Anti-estrogenic potency was assessed by testing combinations of estradiol and individual test compounds, but was found for none of the compounds. The low estrogenic activity of the test compounds in the spiking mixture was well recovered during GPC treatment of the pure mixture, but did not contribute significantly to the background estrogenic activity present in the spiked sediment. Application of the method to field samples showed that estrogenic activity can be found at different types of locations, and demonstrated that levels between locations may vary considerably over relatively short distances.     

Photochemical induced changes of in vitro estrogenic activity of steroid hormones

Steroid estrogens are endocrine disrupting contaminants frequently detected in natural waters. Because these estrogens can elicit significant biological responses in aquatic organisms, it is important to study their rates and pathways of degradation in natural waters and to identify whether the transformation products retain biological activity. Photochemical kinetics experiments were conducted under simulated solar light for the hormones 17β-estradiol (E2), 17α-ethinylestradiol (EE2), estrone (E1), equilin (EQ), and equilenin (EQN) under direct and indirect photolysis conditions. All of these hormones were susceptible to direct photodegradation, with half-lives ranging from 40 min for E1 to about 8 h for E2 and EE2. Indirect photolysis experiments with added Suwannee River fulvic acid (SRFA) lead to faster degradation rates for E2, EE2, and EQ. Added SRFA caused slower photodegradation rates for E1 and EQN, indicating that it acts primarily as an inner filter for these analytes. The well-established yeast estrogen screen (YES) was used to measure the estrogenicity of the analytes and their photoproducts. Results of YES assay experiments show that only the direct photolysis of E1 gave estrogenic products. Lumiestrone, the major E1 direct photolysis product, was isolated and characterized. It formed in 53% yield and exhibited moderate estrogenic activity. When photolysed in the presence of perinaphthenone, a potent synthetic sensitizer, E1 degraded via an indirect photolysis pathway and did not produce lumiestrone or any other active products. These results suggest that under typical natural water conditions photochemical reactions of E2, EE2, EQ, and EQN are expected to produce inactive products while E1 will give the estrogenic product lumiestrone in moderate yield.     

Part I. Identifying anthropogenic markers in surface waters influenced by treated effluents: a tool in potable water reuse

In potable water reuse, treated wastewater becomes part of the drinking water supply. An important question associated with this practice is whether or not the organic quality of the treated wastewater is chemically different from that of non-human impacted water. This question was addressed in a case study of indirect potable water reuse where the organic matrix of the South Platte River was analyzed upstream and downstream of the discharge of treated wastewater effluent using conventional water quality parameters combined with pyrolysis-GC/MS. Effluent-derived organic material (EfOM) was found to be more aliphatic and had higher organic nitrogen and halogen content compared to organic material derived from "natural" (non-anthropogenic) sources (NOM). Seasonal changes that resulted from the change in the contributions of aquatic and terrestrial sources were not observed in EfOM; but they were strongly observed in NOM under the control of natural processes. Using principal component and factor analyses, the pyrolysis fragments of phenol, alkyl-phenols, and acetic acid were identified as the seasonal indicators for the NOM set of samples. In contrast, benzaldehyde, benzonitrile, chlorobutanoic acid, furancarboxaldehyde, and methylfurancarboxaldehyde were identified as the indicators for wastewater inputs for the EfOM set of samples. Overall, the results from conventional water quality parameters and pyrolysis-GC/MS revealed that: (1) EfOM bears a chemical signature distinct from NOM and (2) under the conditions of this study, EfOM discharged to the South Platte River persisted and controlled organic quality at downstream points.