Free Synthetic and Natural Estrogen Hormones in Influent and Effluent of Three Municipal Wastewater Treatment Plants

By Chimchirian, Robert F Suri, Rominder P S; Fu, Hongxiang

ABSTRACT: Three municipal wastewater treatment plants (WWTPs) in southeastern Pennsylvania were sampled to determine the presence and concentrations of 12 natural and synthetic estrogen hormones in the wastewater influent and effluent. The target estrogens were 17alpha- estradiol, estrone, estriol, equilin, 17alpha-dihydroequilin, 17beta- estradiol, 17alpha-ethinyl estradiol, gestodene, norgestrel, levonorgestrel, medrogestone, and trimegestone. One WWTP uses a biofilm reactor (packed-bed trickling filter), and the other two use suspended-growth media (continuously stirred activated sludge reactor and sequential batch reactor). Estrone was detected in all the three plants; estriol and estradiol were detected at two WWTPs; and 17 alpha-dihydroequilin and 17 alpha-ethinyl estradiol were detected at one WWTP. The concentration of estrogens in the influent and effluent of the three treatment plants ranged from 1.2 to 259 ng/ L and 0.5 to 49 ng/L, respectively. The percentage removal of estrogens from the aqueous phase ranged from 41 to 99%, except in the case of 17alpha-dihydroequilin; the removal of 17alpha- dihydroequilin was negligible. The suspended-growth media systems showed higher removal efficiencies for estrogens than the biofilm system. The analytical method uses a Varian C-18 solid-phase extraction (Varian Inc., Palo Alto, California), followed by a derivatization with bis(trimethylsilyl)trifluoroacetamide. The detection limits for the estrogen compounds ranged from 0.1 to 10 ng/ L using a sample size of 1 L. The method recoveries ranged from 71 to 120%, and the relative standard deviation ranged from 6 to 14% for all the hormones.

Water Environ. Res., 79, 969 (2007).

KEYWORDS: estrogens, hormone, natural estrogens, synthetic estrogens, wastewater, wastewater treatment plant, gas chromatograph- mass spectrometry.

dol:10.2175/106143007X175843

(ProQuest: … denotes formula omitted.)

Introduction

The increasing release of endocrine-disrupting chemicals, such as estrogen hormones, into surface water, is of growing concern. The major pathway of estrogens entering the environment is by excretion from the body. Humans excrete estrogens through urine in the conjugated (generally inactive) form (Burgess, 2003), with extremely high concentrations coming from pregnant women, who can excrete 7, 2.4, and 4.4 [mu]g/d of estrone, 17 beta-estradiol, and estriol estrogens, respectively (Desbrow et al., 1998). The conjugated estrogens enter the wastewater systems and municipal wastewater treatment plants (WWTPs). Bacteria, such as Escherichia coli, presented in wastewater can produce beta-glucuronidase, an enzyme that can deconjugate estrogens, converting them into the free (active) form (Baronti et al., 2000; Belfroid et al., 1999; D’Ascenzo et al., 2003; Desbrow et al., 1998). Estrogens existing in the firee form can cause detrimental effects to the ecosystem.

Out of a large group of chemicals that produce an unnatural response in the endocrine system, estrogens are the most potent (Lim et al., 2000). Certain estrogens, such as ethinyl estradiol, can affect an endocrine response at levels as low as 0. 1 ng/L, whereas non-estrogen chemicals will induce the same effects in the microgram- per-liter range (D’Ascenzo et al., 2003). Estrogens have been linked to reproduction problems and hormone-dependent diseases in many mammals, including humans (Penalver et al., 2002). Researchers have speculated that estrogens play a role in prostate and testicular cancers, reduced sperm counts, and feminization and lower fertility in humans, birds, and fish (Mol et al., 2000; Snyder et al., 1999; Xiao et al., 2001).

Of the known problems that estrogens can cause in surface water, feminization of fish has been well-documented and is used as a biomarker of estrogen pollution (Baronti et al., 2000; Desbrow et al., 1998; Larsson et al., 1999; Matthiessen and Sumpter, 1998; Routledge et al., 1998; Sumpter and Jobling, 1995). Using WWTPs as a point-source of estrogenic compounds, tests were conducted with caged male rainbow trout downstream of outfall pipes (Jobling et al., 1998; Purdom et al., 1994). Caged fish were held in these areas of the stream for up to 3 weeks and then screened for hormonal changes. Many of these male fish began producing vitellogenin (a female-specific egg yolk protein), which is non-existent or too low to measure in healthy males (Sumpter and Jobling, 1995). Jobling et al. (1998) showed that up to 100% of the populations of male fish downstream from some WWTPs were hermaphrodites. Other studies showed that a concentration of 1 to 10 ng/L 17 beta-estradiol and 0.1 ng/L 17 alpha-ethinyl estradiol can cause vigtellonensis in male rainbow trout (Larsson et al., 1999; Routledge et al., 1998). These concentrations are not absolute, because the estrogen concentration that can cause a hormonal change in male trout is directly related to the length of exposure (Rodgers-Gray et al., 2000). Therefore, a longer exposure time may lower the threshold concentration.

The focus of this research was to examine the presence and concentration of estrogen hormones in municipal wastewater at WWTPs. Twelve natural and synthetic estrogens were examined. Three different WWTPs were sampled. One treatment plant sampled used fixed- growth biofilm, whereas the other two plants used activated sludge and a sequencing batch reactor (SBR). The estrogens of interest were 17alpha-estradiol, estrone, estriol, equilin, 17alphadihydroequilin, 17 beta-estradiol, 17 alpha-ethinyl estradiol, gestodene, norgestrel, levonorgestrel, medrogestone, and trimegestone. In addition, we determined the percentage removal of detected estrogens at treatment plants using different methods.

Materials and Methods

Materials. Hormones were obtained from Sigma Aldrich, Steriloids Inc. (Allentown, Pennsylvania). The hormones (minimum purities) were 17alpha-estradiol (98%); estrone (100%); estriol (100%); equilin (99.9%); 17alpha-dihydroequilin (99.4%); 17 beta-estradiol (97.1%); 17 alpha-ethinyl estradiol (99.1%); gestodene (99.3%); norgestrel (100%); levonorgestrel (100%); 3-O-methyl estrone (internal standard, 98%); medrogestone (99.8%); and trimegestone (99.8%). All solvents (high-performance liquid chromatography-grade) and other chemicals were purchased from Fisher Scientific (Hampton, New Hampshire). Millipore nitrocellulose filters (47-mm white) and amber glass bottles were obtained from Fisher Scientific. Varian Bond Elute 3 mL/500 mg solid-phase extraction (SPE) cartridges were obtained from Varian Inc. (Palo Alto, California). Before use, all glassware was silanized (deactivated with 5% dimethyldichlorosialane in toluene).

Sample Collection and Storage. Wastewater samples were collected from three local municipal WWTPs. All samples were collected in 4-L silanized amber glass bottles with Teflon-lined caps. Two samples were collected at each treatment plant for primary and final effluents. Primary effluent samples were collected after the grit screen, and final effluent samples were collected after chlorination and just before discharge to surface water. All samples collected were grab sample following published methods (Braga et al., 2005; Holbrook et al., 2002). Note that, in this paper, primary effluent is being considered as the influent to the WWTP assuming fliat very little, if any, removal of estrogens occurs in the primary clarifier. Typically, biological activity in the primary clarifier is minimal because of the lack of oxygen. The samples were stored at 4[degrees]C in the laboratory until extraction was performed, within 24 hours. Sampling was restricted to time periods when treatment plants were operating as close to steady-state conditions as possible; therefore, no sampling was done after any sizeable rains. All reported results are an average of triplicate sample analysis.

Filtration of the samples was necessary because of the high suspended solids content and the small particle size (50 to 60 pm) of the SPE adsorbent. All samples were filtered using three different size filter papers in a stepwise method (5, 0.8, and 0.45 pm). This provided consistent flow without clogging the SPE column.

Analytical Methods. Stock solutions of hormones were prepared in methanol at 10 mg/L, using a silanized amber glass volumetric flask and stored at 4[degrees]C. From these stock solutions, calibration standards were prepared by spiking appropriate amounts of stock solution to 1 L of Milli-Q water. The standards ranged from 100 pg/ L to 500 ng/L. A fixed amount of internal standard, 3-O-methyl estrone, was added to each calibration standard and wastewater sample before SPE extraction to account for any analyte loss that may occur during the extraction process. The recovery data for estrogens in the wastewater was obtained by spiking known amount of analytes and then following the extraction and analysis procedures.

The calibration data were regressed, and the standard set was only used if the coefficient of determination (r^sup 2^) value was 0.99 or greater. The calibration data was normalized by taking the ratio of the compound area to the internal standard area (eq 1 ). This process was also applied to each wastewater sample.

… (1) Where

D^sub N^ = normalized data,

A^sub P^ = peak area of analyte, and

IS^sub P^ = peak area of the internal standard.

The detection limit for the analytical method was determined by spiking various concentrations of estrogens in Milli-Q water, and the lowest peak area that was 3 times higher than the noise was cited as the detection limit.

Solid-Phase Extraction. Estrogens in wastewater samples have been mostly reported to be analyzed using SPE and gas chromatograph-mass spectrometry (GC-MS) methods (Braga et al., 2005; Esperanza et al., 2004; Xiao et al., 2001). We used Bond Elute C-18 adsorbent for SPE (Varian Inc., Palo Alto, California). The SPE column was activated with 3 mL of methanol and then rinsed with 3 mL of Milli-Q water. Approximately 1 L of the filtered sample was spiked with internal standard and then passed through an SPE cartridge at a flowrate of 5 mL/min. Because a reverse-phase retention mechanism was used, pH adjustment was not required. After loading, the columns were washed with 3 mL Milli-Q water and twice with 3 mL 60:40 Milli-Q watenmethanol (v/v) solution, to remove any interfering species. The columns were then eluted with 3 mL methanol. The methanol eluant was collected in a clean, silanized test tube and dried in a Genevac centrifugal evaporator (Genevac, United Kingdom) at 45[degrees]C and 12 mbar vacuum. Blank and blank spiked control experiments were performed with Milli-Q water. 1 L Milli-Q water (blank) and estrogen- spiked Milli-Q water (blank spiked) were extracted following the procedures described earlier.

Derivatization. Approximately 15 [mu]L pyridine and 65 [mu]L bis(trimethylsilyl)trifluoroacetamide containing 1% dimethylchlorosilane were added to the dried sample. The sample was allowed to react in a capped test tube for 15 minutes at 26[degrees]C. To the derivatized sample, 0.3 mL toluene was added. Samples were then vortexed and placed in an amber glass gas chromatography vial containing a 0.25-mL insert. The headspace-free vial was placed on the GCMS for analysis.

Gas Chromatograph-Mass Spectrometry Analysis. The GC-MS analysis was performed using an Agilent 6890N gas Chromatograph and a 5973N mass spectrometer (Agilent Technologies, Santa Clara, California). Splitless auto injections were made onto a Pursuit DB-225MS capillary column (30 m x 0.25 mm x 0.25 [mu]m; J&W Scientific, Folsom, California). The initial temperature was 50[degrees]C for 1 minutes, with a flow of 4.5 mL/min; the temperature was then increased to 200[mu]C at 50[degrees]C/min, with a flow of 4.5 mL/ min and held for 95 minutes. Finally, the oven temperature was increased to 220[degrees]C at 10[degrees]C/min and held for 27 minutes. During the post run, the oven temperature was held at 240[degrees]C for 10 minutes, with a flow of 4.8 mL/min. Helium was used as a carrier gas. The inlet and source temperatures were 240[degrees]C, and the relative source voltage was 1447 V. The quad temperature was 150[degrees]C.

Results and Discussion

Gas Chromatograph-Mass Spectrometry Analysis. A quantitation ion and two confirming ions were selected in scan mode before the single ion monitor (SIM) analysis. In the SIM mode, the peaks were identified by their specific-mass-to-charge ratio and also by matching retention times with those of previously run standards. Table 1 shows the quantitation ion, confirming ions, and retention time for each analyte. The analytes in Table 1 are listed in the order of elution from the gas Chromatograph column into the mass spectometer. As shown in Table 1 , levonorgestrel and norgestrel are reported together, because they coelute from the gas Chromatograph column and have exactly the same mass-to-charge ratio.

Blank and blank spiked control experiments were performed with Milli-Q water. No analytes were detected in the unspiked blank Milli- Q water. The reproducibility and recovery for the blank spiked samples are presented in Table 1 . It may be observed, from Table 1, that the analytical method showed good reproducibility, with a low relative standard deviation (RSD) of approximately 6. 1 to 14.4%. Most of the estrogen compounds showed high recovery efficiencies with the analysis method; for example, 17 a-estradiol, 17 beta- estradiol, and estrone show recovery higher than 97%. The 17 alpha- ethinyl estradiol, modrogestone, and norgestrel/levonorgestrel showed relatively lower recovery, that is, between 71 and 85%.

The detection limits achieved by this method ranged from 0.1 to 10 ng/L using 1 L milli-Q water. The method detection limit was observed to be 0.1 ng/L for 17 alpha-estradiol, 17 beta-estradiol, 17 alpha-ethinyl estradiol, estriol, and equilin; for 17 alpha- dihydroequilin, it was 1 ng/L. For equilin, medrogestone, norgestrel, levonorgestrel gestodene, and trimegestone, the detection limit was 10 ng/L.

Wastewater Sample Analysis. The effluent of plant 3 was used for the recovery test in wastewater. For the wastewater samples, the analytical method showed good reproducibility, with an RSD between 0.4 and 6.9% for triplicate sample analysis, as shown in Table 1 . The recovery of estrogens from wastewater ranged from 86 to 101%, as shown in Table 1, by processing 1-L samples.

The three WWTPs use biological treatment processes with different types of contactors. Plant 1 uses a trickling filter with attached growth biofilm and was rated at 3400 m^sup 3^/d (0.9 mgd). Plant 2 uses an activated sludge reactor with suspended growth and is rated for 15 000 m^sup 3^/d (4 mgd). Plant 3 has an SBR system and was treating 760 m^sup 3^/d (0.2 mgd) during the sampling period. Selected process details of the WWTPs are listed in Table 2. All treatment plants were designed for municipal wastewater treatment, but had slightly different inputs. Wastewater treatment plants 1 and 3 receive only municipal wastewater, whereas WWTP 2 receives wastewater from municipal sources and from a nearby hospital.

The compounds detected in WWTP 1 were 17 ss-estradiol, 17 aethinyl estradiol, estriol, and estrone, as shown in Table 3. They were detected in both primary and final effluents. The estrogen removal ranged from 41 to 95%. The concentration of the 17 alpha- ethinyl estradiol in both influent and effluent in the plant 1 was very low and close to its detection limit (approximately 5 to 10 times higher than the detection limit).

The three estrogens detected in WWTP 2 were 17 beta-estradiol, estriol, and estrone, and the removal of all three estrogens was 89 to 99%. This treatment plant had a significantly higher influent estrogen concentration than WWTPs 1 and 3. This may be the result of the wastewater contribution coming from a nearby hospital. However, unlike the influent at plant 1, 17 alpha-ethinyl estradiol was not detected at plant 2.

Only two estrogens were detected in WWTP 3-17 alpha- dihydroequilin, which was not detected in plants 1 and 2, and estrone. The removal of 17 a-dihydroequilin during the SBR treatment process was negligible. Estrone was 81% removed; this was similar to removal in plant 2, which also uses suspended-growth media.

There were several estrogens that were not detected in this study, but may be present in the wastewater at concentrations below the detection limits or sorbed onto biosolids. The estrogens that were not detected at all three WWTPs were 17 a-estradiol, equilin, medrogesterone, levonorgestrel, norgestrel, gestodene, and trimegestone. Estrone was detected in all three plants; estriol and 17 beta-cstradiol were detected at two WWTPs; and 17 a- dihydroequilin and 17 a-ethinyl estradiol were detected at only one WWTP. The concentrations measured in this study correlate well with the work of other researchers (Belfroid et al., 1999; Desbrow et al., 1998; Johnson et al., 2000; Kuch and Ballschmiter, 2001; Larsson et al., 1999; Rodgers-Gray et al., 2000; Temes et al., 1999). Lopez de Alda and Barcelo (2001) reported concentrations of 2 to 500 ng/L of 8 different estrogens, including estriol, estradiol, ethinyl estradiol, estrone, norgestrel, and levonorgestrel from a sample size of 0.5 L of wastewater effluent. Komori et al. (2004) reported concentrations of 0.1 to 1.3 ng/L of 12 estrogens, including 17 ss-estradiol, 17 aethinyl estradiol, estriol, and estrone in effluent wastewater. Kuch and Ballschmiter (2001) reported estrone, 17 a-estradiol, 17 beta-estradiol, and ethinyl estradiol at concentrations ranging from 0. 1 to 5.1 ng/L in wastewater treatment effluent. Desbrow et al. (1998) detected estrone, 17 ss-estradiol, and ethinyl estradiol effluent concentrations ranging from 0.2 to 48 ng/L.

The daily mass load of the detected estrogen hormones in effluent wastewater is shown in Table 3. It can be observed, from Table 3, that the daily load of estrone in plant 1 is the highest. The daily load of estrogens in effluent was between 2 to 167 mg/d at plant 1 and 22 to 95 mg/d at plant 2. The daily loads of 17 alpha- dihydroequilin and estrone in the effluent of plant 3 were 18 and 9 mg/d, respectively.

Estrogens have low solubility in water, a high partitioning coefficient (log Kovl), and are nonvolatile (low Henry’s constant), as shown in Table 4. Mass-transfer analysis indicates that there will be high sorption onto sludge. It must be mentioned that the concentrations of estrogens reported in this paper are for the aqueous phase. The wastewater samples were filtered to remove suspended solids particles. The suspended solid particles may have a significant amount of sorbed estrogens. The high removal of estrogens at WWTPs, as shown in Table 3, is likely the result of biological degradation and sorption onto sludge and suspended particles. This is likely to produce estrogen-contaminated biosolids. If the biosolids are land-applied as a soil supplement, the estrogens could leach and contaminate the surface water and groundwater. Many researchers have reported estrogenic activity in the sludges of various WWTPs (Holbrook et al., 2002; Temes et al., 2002).

The removal of estrogen hormones at WWTPs has been examined by several researchers (Esperanza et al., 2004; Holbrook et al., 2002; Joss et al., 2004; Komori et al., 2004; Komer et al., 2000). Esperanza et al. (2004) examined the removal-of-estrogens process at pilot scale. They reported removal of estradiol, estrone, and ethinyl estradiol as 58 to 78% and 50 to 94% in anaerobic and aerobic processes, respectively. Joss et al. (2004) reported a >90% removal of estrone, estradiol, and ethinyl estradiol in an activated sludge process. All three plants have biochemical oxygen demand (BOD) removal of 96 to 99%. Wastewater treatment plants 2 and 3 show much higher estrogen removal than plant 1. Both the activated sludge reactor in plant 2 and the SBR in plant 3 are based on suspended- growth media, while the trickling filter in plant 1 is fixed biofilm. Comparison of the three WWTPs shows that the suspended- growth media provides a higher percentage removal than the biofilm system. This could be a result of the type of biomass and age of the sludge. The age of the bacteria in suspended-growth media processes (activated sludge system in plant 2 and SBR in plant 3) is generally younger, and more sludge is produced when operating at steady-state conditions. Moreover, with a larger quantity of sludge produced, there is more solid material for estrogens to sorb onto, which will result in a better percentage removal from the aqueous phase. The age of sludge in the biofilm is longer than in the suspended-growth systems, and the biomass type is also different. The older sludge of the biofilm system (trickling filter) may get saturated with estrogens and result in a lower percentage removal.

The estrone concentration was high in the influent of all three plants and was the highest in the effluent of WWTPs 1 and 2. It was also high in the effluent in plant 3. The influent concentration ranged from approximately 57 to 83 ng/L, and the effluent concentration ranged from approximately 6 to 49 ng/L. The removal of estrone in the three plants ranged from 41 to 89%. For both treatment plants 1 and 2, estrone removal was lower than estriol and estradiol. This may be the result of the breakdown of other estrogen hormones, such as estradiol, into estrone in biological oxidation processes. Degradation of other estrogens into estrone has been reported (Christensen, 2002; Hanselman et al., 2003; Holbrook et al., 2002; Xiao et al., 2001). In addition, estriol and estrone have much higher solubility than the other estrogen hormones (Table 3), leading to lower sorption on suspended solids. This may be the cause of high influent concentrations of these two chemicals. In all the compounds detected in the wastewater, 17 alpha-dihydroequilin showed very low removal efficiency. It was not detected in two treatment plants.

Of the estrogens detected, 17 beta-estradiol, 17 alpha- dihydroequilin, estriol, and estrone are naturally produced in the body, and ethinyl estradiol is synthetically produced for use in oral contraceptives. The concern with 17 alpha-ethinyl estradiol is its high estrogenicity-higher than the other estrogens detected in this study (Kuch and Ballschmiter, 2001; Larsson et al, 1999). Higher estrogenicity implies that lower concentration levels can cause more adverse effects. In addition, this chemical is more stable and can last in the environment for long periods of time. This also implies that it can travel long distances in surface water and affect a larger area. The reason for its resistance to breakdown is the structure of the 17 alpha-ethinyl group, which differs from natural estrogens, as reported by Routledge et al. (1998).

Conclusions

The estrogens detected in the treatment plant wastewaters were 17 beta-estradiol, 17 alpha-dihydroequilin, ethinyl estradiol, estriol, and estrone. Estrone was detected at all the three plants; estriol and estradiol were detected at two WWTPs; and 17 alpha- dihydroequilin and 17 alpha-ethinyl estradiol were detected at one WWTP. The influent concentrations ranged from 1.2 to 259 ng/L, and the effluent concentrations ranged from 0.5 to 49 ng/L. Estrone and estriol concentrations were the highest compared with estradiol, 17 alpha-dihydroequilin, and 1 7 a-ethinyl estradiol. The estrogens that were not detected during this study were 17 alpha-estradiol, estriol, medrogestone, levonorgestrel, norgestrel, gestodene, and trimegestone. The WWTPs reduced the concentration of estrogens; however, the effluent concentrations were not safe for the environment. The percentage removal of estrogen hormones at treatment plants ranged from 41 to 99%, except the negligible removal of 17 alpha-dihydroequilin in plant 3, although the BOD removal was 95 to 99% at all WWTPs. It should be noted that these data represent the removal from the aqueous phase and do not reflect the solid-phase concentration. Of the three types of treatment reactors examined, the suspended-growth processes (activated sludge reactor and SBR) provide a better removal of estrogens from the aqueous phase than the biofilm (trickling-filter reactor) process. The existing WWTP processes need to be improved and/or new treatment methods developed to prevent these chemicals from entering the environment.

Credits

Assistance in the laboratory by John Stofey and Sandhya Meduri is acknowledged. The authors thank the WWTPs for providing the samples for this study. Opinions, findings, and conclusions expressed in this paper are those of the authors.

Submitted for publication November 17, 2005; revised manuscript submitted November 21, 2006; accepted for publication December 18, 2006.

The deadline to submit Discussions of this paper is December 15, 2007.

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Robert F. Chimchirian, Rominder P.S. Suri*, Hongxiang Fu

Villanova Center for the Environment, Villanova University, Villanova, Pennsylvania.

* Villanova University, Civil and Environmental Engineering, 139 Tolentine Hall, 800 Lancaster Ave., Villanova, PA 19085; e-mail: rominder.suri@villanova.edu.

Copyright Water Environment Federation Sep 2007

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