Distribution of estrogens, 17beta-estradiol and estrone, in Canadian municipal wastewater treatment plants

The distribution of female hormones, 17beta-estradiol and estrone, was determined in effluents of 18 selected municipal treatment plants across Canada. Replicate 24-h composite samples were collected from the influent and final effluent of each treatment plant, and the removal efficiency compared to the operational characteristics of the plants. In conventional activated sludge and lagoon treatment systems, the mean concentrations of 17beta-estradiol and estrone in influent were 15.6 ng/l (range 2.4-26 ng/l) and 49 ng/l (19-78 ng/l). In final effluents, the mean concentrations of both 17beta-estradiol and estrone were reduced to 1.8 ng/l (0.2-14.7 ng/l) and 17 ng/l (1-96 ng/l), respectively. 17beta-estradiol was removed effectively, >75% and as high as 98%, in most of the conventional mechanical treatment systems with secondary treatment. The removal of estrone was much more complex with removal varying from 98% to situations where the concentrations in the effluent were elevated above that detected in the influent. The estrogenicity, measured using a transfected estrogen receptor in yeast (YES) assay, was also variable, ranging from high removal to elevations of estrogenicity in final effluent. Although the apparent removals were not statistically correlated with either hydraulic (HRT) or solid (SRT) retention times, plants or lagoons with high SRT were very effective at reducing the levels of hormones. Well-operated plants that achieved nitrification also tended to have higher removal of hormones than those that did not nitrify. Laboratory aerobic reactor experiments confirmed the rapid removal of 17beta-estradiol, estrone, and estrogenicity when exposed to sewage slurries.     

Behavior and occurrence of estrogens in municipal sewage treatment plants--I. Investigations in Germany, Canada and Brazil

The developed method enables the quantification of estrogens in sewage samples down to 1 ng/l and in river water down to 0.5 ng/l. Mean recoveries of the analytes in ground water after SPE extraction, clean-up and derivatization generally exceeded 75%. The determined R.S.D. varied from 0 to 14% at a spiking level of 0.05 microgram/l. Even in the raw influent and the final effluent from municipal STPs the mean recoveries of estrogens were mostly above 70%. Using this method the behavior and occurrence of natural estrogens and synthetic contraceptives in municipal sewage treatment plants (STP) were investigated in German and Canadian facilities. In the sewage of a German municipal STP close to Frankfurt/Main 17 beta-estradiol and estrone were determined, with mean concentrations of 0.015 microgram/l and 0.027 microgram/l, respectively. In two investigated municipal STPs, 17 beta-estradiol and 16 alpha-hydroxyestrone were eliminated with a higher efficiency than 17 alpha-ethinylestradiol and estrone. In Canadian and German STP discharges estrone, 17 beta-estradiol, 17 alpha-ethinylestradiol and 16 alpha-hydroxyestrone were frequently detected within the lower ng/l-range. A maximum concentration was found for estrone with 70 ng/l. In 15 investigated German rivers and streams only estrone was present with a maximum concentration of 1.6 ng/l.     

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.     

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.     

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.     

Sources of pharmaceutical pollution in the New York City Watershed

An investigation was carried out in the New York City Watershed for the presence of selected pharmaceuticals. In four seasonal sampling events between August 2003 and May 2004, surface water was collected from eight reservoir keypoints and effluent was collected from four wastewater treatment plants. We evaluated the following twelve compounds: amoxicillin, atenolol, caffeine, carbamazepine, cephalexin, estrone, 17alpha-ethinylestradiol, 17beta-estradiol, ibuprofen, sulfamethoxazole, trimethoprim, and valproic acid. In the treated effluents, carbamazepine was detected most frequently (100%; concentration range: 22-551 ng/L), followed by atenolol (94%; ND - 14,200 ng/L), trimethoprim (83%; ND - 37,000 ng/L), ibuprofen (61%; ND - 14,600 ng/L), and caffeine (49%; ND - 37,200 ng/L), while estrone was detected once (56 ng/L). In the reservoir keypoint samples, only ibuprofen (2.5%; ND - 932 ng/L) and caffeine (2.9%; ND - 177 ng/L) were detected. The other analytes were not detected in any sample. It is expected that investigation of other wastewater treatment plants in the New York City Watershed would show that their effluents are also a potential source of pharmaceuticals, but that these pharmaceuticals are unlikely to be detected in the Watershed's surface waters.     

Behavior of pharmaceuticals, cosmetics and hormones in a sewage treatment plant

Two cosmetic ingredients (galaxolide, tonalide), eight pharmaceuticals (carbamazepine, diazepam, diclofenac, ibuprofen, naproxen, roxithromycin, sulfamethoxazole and iopromide) and three hormones (estrone, 17beta-estradiol and 17alpha-ethinylestradiol) have been surveyed along the different units of a municipal Sewage Treatment Plant (STP) in Galicia, NW Spain. Among all the substances considered, significant concentrations in the influent were only found for the two musks (galaxolide and tonalide), two anti-inflammatories (ibuprofen and naproxen), two natural estrogens (estrone, 17beta-estradiol), one antibiotic (sulfamethoxazole) and the X-ray contrast medium (iopromide), where the other compounds studied were below the limit of quantification. In the primary treatment, only the fragrances (30-50%) and 17beta-estradiol (20%) were partially removed. On the other hand, the aerobic treatment (activated sludges) caused an important reduction in all compounds detected, between 35% and 75%, with the exception of iopromide, which remained in the aqueous phase. The overall removal efficiencies within the STP ranged between 70-90% for the fragrances, 40-65% for the anti-inflammatories, around 65% for 17beta-estradiol and 60% for sulfamethoxazole. However, the concentration of estrone increased along the treatment due to the partial oxidation of 17beta-estradiol in the aeration tank.     

Comparison of natural estrogen removal efficiency in the conventional activated sludge process and the oxidation ditch process

The presence of natural estrogens, 17beta-estradiol (E2), estrone (E1) and estriol (E3), as well as estrogenic activity in wastewater influents and secondary effluents were investigated in 20 full-scale wastewater treatment plants in Japan. In all of the influent samples, natural estrogens were detected at concentrations above the minimum limits of detection (0.5ng/L). The concentrations of natural estrogens detected in the effluent of oxidation ditch plants were generally lower than previously reported values. On the other hand, in the conventional activated sludge plants, increments of E1 during biological treatment were frequently observed although E2 and E3 were removed effectively in the process. The removal rates of natural estrogens or estrogenic activity show no observed statistical relationship with the solids retention time (SRT) and the hydraulic retention time (HRT). However, the plants with high SRT or HRT generally showed high and stable removal of both natural estrogens and estrogenic activity.     

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.     

Pharmaceuticals and personal care products (PPCPs) in surface and treated waters of Louisiana,USA and Ontario,Canada

A newly developed analytical method was used to measure concentrations of nine pharmaceuticals and personal care products (PPCPs) in samples from two surface water bodies, a sewage treatment plant effluent and various stages of a drinking water treatment plant in Louisiana, USA, and from one surface water body, a drinking water treatment plant and a pilot plant in Ontario, Canada. The analytical method provides for simultaneous extraction and quantification of the following broad range of PPCPs and endocrine-disrupting chemicals: naproxen; ibuprofen; estrone; 17beta-estradiol; bisphenol A; clorophene; triclosan; fluoxetine; and clofibric acid. Naproxen was detected in Louisiana sewage treatment plant effluent at 81-106 ng/l and Louisiana and Ontario surface waters at 22-107 ng/l. Triclosan was detected in Louisiana sewage treatment plant effluent at 10-21 ng/l. Of the three surface waters sampled, clofibric acid was detected in Detroit River water at 103 ng/l, but not in Mississippi River or Lake Pontchartrain waters. None of the other target analytes were detected above their method detection limits. Based on results at various stages of treatment, conventional drinking-water treatment processes (coagulation, flocculation and sedimentation) plus continuous addition of powdered activated carbon at a dosage of 2 mg/l did not remove naproxen from Mississippi River waters. However, chlorination, ozonation and dual media filtration processes reduced the concentration of naproxen below detection in Mississippi River and Detroit River waters and reduced clofibric acid in Detroit River waters. Results of this study demonstrate that existing water treatment technologies can effectively remove certain PPCPs. In addition, our study demonstrates the importance of obtaining data on removal mechanisms and byproducts associated with PPCPs and other endocrine-disrupting chemicals in drinking water and sewage treatment processes.     

An assessment of estrogenic organic contaminants in Canadian wastewaters

A suite of 30 primarily estrogenic organic wastewater contaminants was measured in several influent/effluent wastewater samples from four municipal wastewater treatment plants and effluents from one bleached kraft pulp mill (BKME) using an ultra-trace analytical method based on gas chromatography-high resolution mass spectroscopy (GC-HRMS). In vitro recombinant yeast assay detection of the estrogenic equivalent (EEq) on whole and solid phase extracted (SPE) and fractionated wastewater was also performed. 19-norethindrone was the most frequently detected and abundant (26-224 ng/L) of all the synthetic estrogens/progesterones in the influent samples. 17alpha-ethinylestradiol was the more frequently detected synthetic estrogen/progesterone in the effluents occurring at or below 5 ng/L with some sporadic occurrences of up to 178 ng/L. The greatest levels of steroidal estrogens in municipal effluents were E1>E2>E3 which were all <20 ng/L. Nonylphenol and di(2-ethylhexyl) phthalate were found to be the highest non-steroidal synthetic compounds surveyed in both municipal influent and effluent samples, both occurring at 6-7 microg/L in municipal effluents. BKME contained relatively large amounts of the plant sterol stigmasterol (4 microg/L) but low amounts of fecal sterols, and steroidal estrogens (E2 only at 6 ng/L) when compared to the municipal effluents. In vitro EEq in the wastewater surveyed ranged from 9-106 ng E2/L and ranked from municipal influent>municipal effluent approximately BKME, with most of the estrogenicity fractionating in the 100% methanol SPE fraction followed by a secondary amount in the diethyl ether (for municipal) or methyl-tert butyl ether (for BKME) SPE fractions. Most correlations between chemical and in vitro estrogenic equivalency were weak (p>0.05 in most cases). Unexpected inverse correlations between in vitro estrogenic activity and concentrations of the estrogenic contaminant bisphenol A were found which likely contributed to the weakness of these correlations. A modified toxicity identification and evaluation procedure was continued with the SPE extracts from the more potent 100% methanol SPE fractions of municipal effluent. High performance liquid chromatography band elution retention times, based on in vitro estrogen detection, indicated that steroidal estrogens such as E2 were responsible for most of the estrogenicity of the samples. Subsequent collection and GC-MS analysis of active bands did not confirm the presence of steroidal estrogens, but expanded the possibility of phthalate esters (i.e. dibutyl phthalate) and natural sterols (i.e. beta-sitosterol) contributing to the overall estrogenic load.     

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.     

Occurrence and removal of pharmaceuticals and endocrine disruptors in South Korean surface, drinking, and waste waters

Liquid chromatography/tandem mass spectrometry (LC-MS/MS) with electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI) was used to measure the concentrations of 14 pharmaceuticals, 6 hormones, 2 antibiotics, 3 personal care products (PCPs), and 1 flame retardant in surface waters and wastewater treatment plant effluents in South Korea. Tris (2-chloroethyl) phosphate (TCEP), iopromide, naproxen, carbamazepine, and caffeine were quite frequently observed (>80%) in both surface waters and effluents. The analytes of greatest concentration were iopromide, TCEP, sulfamethoxazole, and carbamazepine. However, the primary estrogen hormones, 17alpha-ethynylestradiol and 17beta-estradiol, were rarely detected, while estrone was detected in both surface water and wastewater effluent. The elimination of these chemicals during drinking water and wastewater treatment processes at full- and pilot-scale also was investigated. Conventional drinking water treatment methods were relatively inefficient for contaminant removal, while efficient removal (approximately equal to 99%) was achieved by granular activated carbon (GAC). In wastewater treatment processes, membrane bioreactors (MBR) showed limited target compound removal, but were effective at eliminating hormones and some pharmaceuticals (e.g., acetaminophen, ibuprofen, and caffeine). Membrane filtration processes using reverse osmosis (RO) and nanofiltration (NF) showed excellent removal (>95%) for all target analytes.     

Electrophoretic determination of estrogen-induced protein in fish exposed to synthetic and naturally occurring chemicals.

In a first step we investigated whether Leuciscus idus, a fish routinely used to assess the quality of surface water in Germany, is suitable to indicate the presence of endocrine-disrupting chemicals. An electrophoretic method was used to determine estrogen-induced protein in fish serum (presumed vitellogenin). This method was tested by exposing juvenile L. idus to 17 alpha-ethinylestradiol, 17 beta-estradiol and estrone. Nominal concentrations of the steroid were compared with concentrations determined analytically by using GC/MS techniques. Under flow-through conditions, 7 days of exposure to 6 ng/l EE2 significantly induced serum vitellogenin in juvenile L. idus. In juvenile rainbow trout (Oncorhynchus mykiss) 4 ng/l EE2 induced significantly elevated levels of serum vitellogenin. In Danio rerio oral application of 40 micrograms 4-nonylphenol/fish per day during a 3-week exposure period caused a major increase of serum vitellogenin. Physiological protein induction could also be measured in Abramis brama, Cyprinus carpio, Oncorhynchus mykiss, Perca fluviatilis, Rutilus rutilus obtained from natural habitats and in Rhodeus sericeus. The minimum amount of blood required for electrophoretic analysis was 1 microliter which should allow monitoring of small sized fish species as well as juvenile fish.     

Behaviour and occurrence of estrogens in municipal sewage treatment plants--II. Aerobic batch experiments with activated sludge

Aerobic batch experiments containing a diluted slurry of activated sludge from a real sewage treatment plant (STP) near Frankfurt/Main were undertaken, in order to investigate the persistence of natural estrogens and contraceptives under aerobic conditions. The batch experiments showed that while in contact with activated sludge the natural estrogen 17 beta-estradiol was oxidized to estrone, which was further eliminated in the batch experiments in an approximate linear time dependence. Further degradation products of estrone were not observed. 16 alpha-hydroxyestrone was rapidly eliminated, again without detection of further degradation products. The contraceptive 17 alpha-ethinylestradiol was principally persistent under the selected aerobic conditions, whereas mestranol was rapidly eliminated and small portions of 17 alpha-ethinylestradiol were formed by demethylation. Additionally, two glucuronides of 17 beta-estradiol (17 beta-estradiol-17-glucuronide and 17 beta-estradiol-3-glucuronide) were cleaved in contact with the diluted activated sludge solution and thus 17 beta-estradiol was released. The glucuronidase activity of the activated sludge was further confirmed by the cleavage of 4-methylumbelliferyl-beta-D-glucuronide (MUF-beta-glucuronide) in a solution of a activated sludge slurry and Milli-Q-water (1:100, v/v). The turnover rate obtained was approximately steady state, with a turnover rate of 0.1 mumol/l for the released MUF. Hence, it is very likely that the glucuronic acid moiety of 17 beta-estradiol glucuronides and other estrogen glucuronides become cleaved in a real municipal STP, so that the concentrations of the free estrogens increase.     

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.     

Pharmaceuticals and personal care products (PPCPs) and endocrine disrupting chemicals (EDCs) in stormwater canals and Bayou St. John in New Orleans, Louisiana, USA

Samples were collected from two stormwater canals and a recreational urban waterway known as Bayou St. John in New Orleans, Louisiana, USA and analyzed for a range of pharmaceuticals and personal care products (PPCPs) and endocrine disrupting chemicals (EDCs). Concentrations of 7 PPCPs and EDCs were measured by a method that provides for simultaneous extraction and quantification of the following compounds: clofibric acid, naproxen, ibuprofen, fluoxetine, clorophene, triclosan, bisphenol A. The method also was used as an indicator of the occurrence of estrogenic compounds by targeting estrone and 17beta-estradiol. The two canals (Orleans and London) are used to drain a portion of the city's stormwater directly into the Mississippi River or Lake Pontchartrain. Bayou St. John is located between the two canals and supplied with water from Lake Pontchartrain. Results from the 6-month sampling period indicated the following concentration ranges for the two stormwater canals: naproxen (ND - 145 ng/l), ibuprofen (ND - 674 ng/l), triclosan (ND - 29 ng/l) and bisphenol A (1.9-158 ng/l). Concentrations of these target analytes increased with cumulative rainfall. For bayou waters, only naproxen (2.1-4.8 ng/l) and bisphenol A (0.9-44 ng/l) were detected. Estrone was detected but determined non-quantifiable for multiple sampling events at the 3 sites. None of the other target analytes (clofibric acid, fluoxetine, clorophene, and 17beta-estradiol) were detected above their method detection levels. Results of this study demonstrate the occurrence of PPCPs and EDCs in New Orleans stormwater canals and Bayou St. John. Results also demonstrate the use of this analytical technique as an indicator of non-point source sewage contamination in New Orleans stormwater canals.     

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.     

Contribution of known endocrine disrupting substances to the estrogenic activity in Tama River water samples from Japan using instrumental analysis and in vitro reporter gene assay

To quantitatively characterize the substances contributing to estrogenic activity in river water, in vitro bioassay using MVLN cells and instrumental analysis using liquid chromatograph–mass spectrometer (LC/MS) or liquid chromatograph–tandem mass spectrometer (LC/MS/MS) were applied to river water extracts taken from various locations in the Tama River, Japan. Tama River water samples were extracted using solid phase extraction and the crude extracts were fractionated by high-performance liquid chromatography (HPLC) into 10 fractions. The sixth fraction contained nonylphenol (NP) and octylphenol (OP) at concentrations in the range of 51.6–147 and 6.9–81.9 ng/L, respectively (concentrations corresponding to the original sample volumes). No estrogenic activity, expressed as 17β-estradiol equivalents (E2-EQ_B), however, was observed in this fraction (<0.6 ng-E2eq/L). Instrumentally determined estrogenic activity (E2-EQ_C), which is the concentrations of NP and OP multiplied by their corresponding relative potency, was below the detection limit of the MVLN cell bioassay. Estrogenic activities were detected only in HPLC fraction nos. 7, 8 and 9. Estrone (E1), estradiol (E2) and bisphenol A (BPA) were detected in these fractions. Estriol (E3) and ethynylestradiol (EE2) were not detected (<0.2 ng/L) in these fractions. The calculated E2-EQ_C for BPA was below the detection limit of bioassay. The E2-EQ_C for E1 and E2 were on the same order as the estrogenic activity determined by the bioassay (E2-EQ_B). The ratios of E2-EQ_C and E2-EQ_B for E1 and E2 in the three factions collectively (nos. 7–9) were 0.49–0.97 and 0.29–1.12, respectively. Above results indicated that the major causal substances to the estrogenic activity in the Tama River were E1 and E2.