Estrogen Biodegradation Kinetics and Estrogenic Activity Reduction

Jul 30, 2009 - This study investigates two methods to reduce estrogens in wastewater: heterotrophic bacterial degradation in municipal activated sludg...
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Environ. Sci. Technol. 2009, 43, 7111–7116

Estrogen Biodegradation Kinetics and Estrogenic Activity Reduction for Two Biological Wastewater Treatment Methods L I N D A S . G A U L K E , * ,† STUART E. STRAND,† THOMAS F. KALHORN,‡ AND H. DAVID STENSEL† Department of Civil and Environmental Engineering, University of Washington, Box 352700, Seattle, Washington 98195-2700, and Department of Medicinal Chemistry, University of Washington, Box 357610, Seattle, Washington 98195-7610

Received April 21, 2009. Revised manuscript received July 9, 2009. Accepted July 20, 2009.

Estrogens from anthropogenic and livestock sources are a serious concern for aquatic ecosystems at concentrations less than 1 ng/L. Fundamental process parameters to reduce estrogenic activity were investigated for two biotreatment methods: heterotrophic bacterial degradation in municipal activated sludge (AS) and a nitration process that is applicable to high NH4-N wastewaters. Batch tests with estrogen and nitroestrogen compounds were conducted at nanogram per liter concentrations with mixed liquor from an AS wastewater treatment facility (WWTF) operating at a 3 day solids retention time (SRT) and a membrane bioreactor (MBR) WWTF operating at a 30-40 day SRT. The estrogenic activities of estrone (E1), 17βestradiol(E2),and17R-ethinylestradiol(EE2)werereduced80-97% following nitration. First-order biological degradation rate coefficients (kb) of the nitrated estrogens were 10-50% lower than the parent estrogen compounds. The kb values for EE2 in MBR and AS mixed liquors were similar, 1.67 and 1.63 L/gVSSday respectively, indicating that the bacteria responsible for EE2 degradation were present at long and short SRTs. The kb values for E1 and E2 were 2 orders of magnitude greater than for EE2. EE2 degradation was 7.5 times faster in the presence of E1 and E2, and no effect was observed with other estrogen mixtures.

Introduction Exogenous estrogens in aquatic ecosystems pose a serious threat to aquatic species as the ability for estrogens to trigger responses from the endocrine system is not species specific (1). Estrogens can disrupt the endocrine systems of fish at concentrations less than 1 ng/L (2). Major sources of environmental estrogens are anthropogenic: municipal wastewater treatment facilities (WWTFs) and concentrated animal feeding operations (CAFOs). The main source of estrogen in municipal wastewater is urine, which contains 67-80% of estrogens excreted daily (3). Based on observed * Corresponding author phone: 1-206-883-8571; fax: 1-206-6859185; e-mail: [email protected]. † Department of Civil and Environmental Engineering. ‡ Department of Medicinal Chemistry. 10.1021/es901194c CCC: $40.75

Published on Web 07/30/2009

 2009 American Chemical Society

concentrations and estrogenic activities of estrogens in WWTF effluents and CAFOs, the estrogens of greatest environmental concern are the synthetic estrogen, 17R-ethinylestradiol (EE2), and the natural estrogens, estrone (E1) and 17βestradiol (E2). WWTF influent concentrations range from below detection to 70 ng/L for EE2, 670 ng/L for E1, and 150 ng/L for E2, and the range of reported effluent concentrations are from below detection to 5 ng/L for EE2, 72 ng/L for E1, and 30 ng/L for E2 (4, 5). Reported total E1 plus E2 concentrations in CAFO wastewaters include 3030 ng/L in a poultry primary lagoon, 10 700 ng/L in a swine sow primary lagoon (6), and 6000 ng/L in a dairy holding pond (7). Recent research indicates that heterotrophic bacteria are responsible for estrogen degradation in WWTFs, not ammonia oxidizing bacteria (AOB), as previously suggested (8). This research found that AOB play a key role in an abiotic estrogen nitration transformation process by providing NO2-N from NH4-N oxidation. Nitro-E1 and nitro-E2 have a reduced ability to bind the human estrogen receptor (9), suggesting lower estrogenic activity. However, the estrogenic activity of nitro-EE2 has not been reported. Nitration provides a potential method to reduce estrogenic activity. Practical nitration rates require high NO2-N concentrations (>100 mg/L) that are not possible in activated sludge (AS) treatment of municipal wastewater but are applicable to wastewaters with high NH4-N concentrations, where nitrification can be stopped at high NO2-N concentration with appropriate pH, dissolved oxygen (DO), and temperature conditions (e.g., the SHARON process) (10). High NH4-N and high estrogen concentration wastewater applications include treatment of CAFO wastewater and separately collected urine. The nitration reaction rates for E1, E2, and EE2 have been characterized and modeled over pH 6.1-7.0, 10-500 mg/L NO2-N, and 20-32 °C (11), but biodegradation of nitro-estrogens has not yet been studied. Also of interest is the effect of mixtures of nitro-estrogens and estrogens on their respective biodegradation kinetics. Biodegradation is the major estrogen removal mechanism in AS systems in municipal WWTFs (12). Though the degradation pathways are not presently understood, evidence suggests estrogenic activity is fully removed with biotransformation of the parent estrogen compound (13). Significant, but variable, estrogen removal efficiencies have been reported for AS WWTFs (5, 12, 14). Other than Joss et al. (14), information on estrogen biodegradation kinetics relevant to WWTFs at nanogram per liter concentrations is lacking. There is a need to better understand E1, E2, and EE2 biodegradation kinetics so that predictive models can be developed for process designs that reliably reduce effluent estrogen concentrations below 1.0 ng/L concentration. The primary aim of this study was to investigate key fundamental parameters of these two treatment methods for reducing estrogenic activity in the environment. The potential for nitration as a treatment process to reduce estrogenic activity was evaluated by quantifying the estrogenic activity of E1, E2, and EE2 following nitration and monitoring for the return of the parent estrogen compound during nitro-estrogen biodegradation. The biodegradation kinetics of nitro-estrogens and estrogens were compared, and also the effect of estrogen mixtures on biodegradation kinetics was evaluated. Tests were conducted at nanogram per liter concentrations using AS from two different WWTF systems: a membrane bioreactor (MBR) system with nitrification and a long solids retention time (SRT) and a pure oxygen AS system with conventional secondary clarifiers without nitrification and a short SRT. VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1. Operating Conditions for the Activated Sludge (AS) and Membrane Bioreactor (MBR) Wastewater Treatment Facilities operating conditions primary treatment activated sludge process solids retention time, day influent BOD, mg/L configuration MLSS, mg/L nitrification influent

membrane bioreactor (MBR)

activated sludge (AS)

no

yes

MBR 30-40 370 anoxic/aerobic 15 000 yes casino/ hotel/commercial

pure oxygen 3 110 4-stage aerobic 1400 no domestic

Materials and Methods Batch Biodegradation Tests. A series of batch tests evaluating estrogen transformation were carried out with AS obtained from a MBR and an AS WWTF, with the operating conditions summarized in Table 1. Mixed liquor samples were collected and stored at 4 °C for 1-7 days prior to batch tests. Batch tests were incubated in 250 mL Erlenmeyer flasks in the dark at 20 °C on a shaker. Mixed liquor was diluted to 200-900 mg/L volatile suspended solids (VSS) in pH 7, 10 mM phosphate buffer with inorganic contents and vitamins. Concentrations in 1 L were 220 mg/L MgCl2 · 6H2O, 54.4 mg/L CaCl2 · 2H2O, 15.1 mg/L Na2S2O3 · 5H2O, 1 mg/L FeSO4 · 7H2O, 0.3 mg/L ZnSO4 · 7H2O, 0.3 mg/L MnSO4, 0.06 mg/L CuSO4 · 5H2O, 0.06 mg/L CoCl2 · 6H2O, 0.03 mg/L Na2MoO4 · 2H2O, 0.05 mg/L H3BO3, 0.05 mg/L KI, 0.02 mg/L NiCl2 · 6H2O, 0.14 mg/L Al2(SO4)3 · 18H2O, 6.9 mg/L Na2EDTA, 1.8 mg/L pantothenic acid, 1.8 mg/L niacin, 0.09 mg/L biotin, 0.09 mg/L cyanocobalamin, 0.09 mg/L folic acid, 1.8 mg/L pyridoxine, 1.8 mg/L p-aminobenzoic acid, 1.8 mg/L cocaroxylate, 140 mg/L inositol, 140 mg/L thiamine, 140 mg/L riboflavin, and 140 mg/L choline chloride. At time zero, estrogens were spiked into the flasks from stock solutions of 1 mg/L estrogen or 50 µg/L nitro-estrogen in Milli-q water, and an initial estrogen concentration was 500 ng/L for all batch tests. Batch tests were conducted over either 5 days or 1.67 h, depending on the rate of estrogen degradation (E1 and E2 are degraded much more rapidly than EE2). Estrogen samples were taken once daily during 5 day batch tests or 10 times over the duration of the 1.67 h batch tests. All batch tests were conducted in triplicate and kill controls were autoclaved and cooled to room temperature three times. All glassware was washed with detergent and then rinsed with tap water (all rinses three times), followed by rinses with 10% dilute sulphuric acid, Milli-q water, acetone, and methylene chloride. Total suspended solids (TSS) and VSS were analyzed by Standard Methods (15). The pH was measured with 100 µL samples using a MI-410 pH electrode (Microelectrodes, Inc.). Nitro-estrogens. A 2-nitro-EE2 standard was synthesized as described in Gaulke et al. (8) by the action of excess sodium nitrite on 100 mg of EE2 in 50 mL of 1:1 acetonitrile and acetate buffer (pH 4) at room temperature for 48 h. The acetonitrile was then removed in vacuum, and the steroids were extracted into three 50 mL aliquots of ethyl acetate. Following isolation, the crude product was purified by column chromatography with a 1:4 ratio of ethyl acetate and hexane. The formula was confirmed by high resolution mass spectrometry and 500 MHz proton NMR. Nitro-E1 and nitro-E2 were synthesized by incubation of E1 and E2 with 500 mg/L NO2-N at pH 6.4 and 20 °C, using first-order rate coefficients described in Gaulke et al. (11). Estrogen Analyses. As described in Gaulke et al. (8), estrogens were detected with a Waters Acuity UPLC and 7112

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Micromass Quattro Premier XE MS-MS tandem quadrupole mass spectrometer (LC-MS-MS). Sample aliquots (0.5 mL) were transferred to 13 × 100 mm glass tubes, and 125 pg internal standard (d4EE2) was added. Ascorbic acid (0.1% w/v) was added to samples prior to extraction with methylene chloride, and the resulting solution was mixed for 5 min, after which the organic phase was transferred to a clean tube and evaporated to dryness under nitrogen. Phenolic hydroxyl groups were dansylated as follows: the residue was reconstituted in 100 µL of NaHCO3 buffer (pH 10.5) and vortexed for 1 min, and 100 µL of 1 mg/mL dansyl chloride in acetonitrile was added. The samples were sealed, vortexed for 1 min, and heated at 60 °C for 5 min (16). A Waters Acuity C8 2.2 × 50 mm 1.7 µm dp column was used with a flow rate of 0.4 mL/min acetonitrile and 0.1% formic acid in water. The gradient was initially 30% acetonitrile, adjusted linearly to 87% over 2.5 min, further increased to 100% at 3 min, held at 100% until 4.5 min, and went back to 30% at 4.6 min. The MS-MS was operated in electrospray positive mode with a cone voltage of 40 V and collision energy of 45 eV. During all batch tests, the following products were monitored for E1, E2, EE2, nitro-E1, nitro-E2, nitro-EE2, d4EE2, nonaromatic hydroxylated-estrogens, and A-ring hydroxylated-estrogens; none of the metabolic byproducts that were monitored for during batch tests were detected. Ions monitored for the dansylated compounds were 504 > 171, 506 > 171, 530 > 171, 549 > 171, 551 > 171, 575 > 171, 534 > 171, 520 > 171, 522 > 171, 546 > 171, 615 > 171, 617 > 171, 591 > 171, 753 > 170, 755 > 170, and 779 > 170, respectively. The method level of detection was 1 ng/L, and the method lower level of quantification was 2.5 ng/L. First-Order Kinetic Model of Estrogen Biodegradation. The following pseudo-first-order biodegradation kinetic model was assumed to relate changes in the total estrogen concentration in batch tests to the AS mixed liquor VSS and soluble estrogen concentrations: dST ) -kbXVSSS dt

(1)

Where dST/dt is the reaction rate (ng/L-day), ST is the total estrogen concentration (ng/L), kb (L/gVSS-day) is the pseudo first-order rate coefficient for estrogen biodegradation, X is VSS (g/L), and S is the soluble estrogen concentration (ng/ L). The total estrogen concentration is composed of soluble and sorbed estrogen: ST ) S(1 + KPXTSS)

(2)

Where KP is the solids partition coefficient (L/kg TSS), and X is the TSS (kg/L). Equation 3, from combining eqs 1 and 2, shows that an experimental kb value can be determined by total estrogen concentration measurements with time and known values for KP, XVSS, and XTSS:

(

ST dST ) -kbXVSS dt 1 + KPXTSS

)

(3)

Linear regression of the natural log of the fraction of the measured total estrogen concentration remaining versus time was evaluated with SigmaPlot for Windows version 10.0 2006 Systat Software, Inc. The resulting slope of the regression line is slope )

(

kbXVSS 1 + KPXTSS

)

(4)

Rapid solid-liquid equilibrium of estrogens and a partition coefficient of 450 L/kg TSS were assumed on the basis of previous studies (17, 18). The same partition coefficient was

TABLE 2. Estrogenic Activity Reduction of Estrogens Following Nitration As Measured by the Yeast Estrogen Screen (YES)a percent reduction in estrogenic activity with nitration nitro-EE2 nitro-E2 nitro-E1

96-99% 89-104% 67-93%

a

Calculation of estrogenic activity presented in the Supporting Information.

FIGURE 1. Yeast estrogen screen (YES) dose response curves for EE2 and EE2 following 90% transformation to 2-nitro-EE2. Samples were serially diluted in a 96 well plate, and then, 10 µL of each dilution was added to a 96 well plate along with 190 µL of growth medium seeded with YES culture. used for the estrogens and nitro-estrogens, as the nitration of aromatics results in only a slightly more hydrophilic compound (reduction of 0.18 in KOW) (19). kb was then determined by the following relationship: kb )

slope(1 + KPXTSS) XVSS

(5)

Yeast Estrogen Screen Assay. The yeast estrogen screen (YES) has the human estrogen receptor (hER) stably integrated into its chromosome and an expression plasmid with estrogen responsive sequences (ERE) that control expression of a reporter gene allowing for quantification of estrogenic activity. Permission to use the YES was obtained from Dr. Sumpter of Brunel University, and the method used was as described by Routledge and Sumpter (20). Samples were taken following a 4 day incubation of estrogens at pH 6.4 with 10 mM phosphate buffer, using first-order kinetics reported in Gaulke et al. (11), to predict when there would be 90% transformation of parent estrogens to nitro-estrogens. Samples were serially diluted in a 96 well plate, and then, 10 µL of each dilution was added to a 96 well plate along with 190 µL of growth medium seeded with YES culture. Each plate also included serial dilutions of the original parent estrogen and an E2 standard. Plates were sealed and incubated at 30 °C for 3 days, and then, absorbance was read with a Perkin-Elmer Victor (3) V multimode Plate Reader. To determine EC50 ( 95% confidence interval values, regression to a four parameter sigmoidal curve was evaluated with SigmaPlot for Windows version 10.0 2006 Systat Software, Inc.

Results and Discussion Estrogenic Activity of Nitro-estrogens. To evaluate the effect of estrogen nitration on estrogenic activity, the EC50 values of YES dose response curves and estrogen concentrations as quantified by LC-MS-MS were compared for estrogens prior to and following nitration. Representative dose response curves for EE2 at 0 and 90% transformation are shown in Figure 1; the percent reduction in estrogenic activity for E1, E2, and EE2 following nitration are summarized in Table 2, and the calculations for percent removals are presented in the Supporting Information. The estrogenic activity of E2 and EE2 was almost entirely removed by nitration. The estrogenic activity of nitro-E1 was also substantially reduced by 67-93%. Although complete removal of estrogenic activity

of E1 was not achieved, it should be noted that E1 has only 10-60% the estrogenic activity of E2 or EE2 (21-23). Nitro-estrogen Biodegradation Kinetics. The first-order biodegradation coefficient (kb) values for nitro-E1, nitro-E2, and nitro-EE2 are summarized in Table 3. Nitro-E1 kb values were an order of magnitude slower than nitro-E2 kb values, and 2 orders of magnitude faster than nitro-EE2 kb values. Nitro-E2 was oxidized to nitro-E1, as has been reported with E2 and E1 (24). To evaluate what fraction of nitro-E2 was first oxidized to nitro-E1 and then degraded, versus directly degraded as nitro-E2, the kb value from nitro-E1 batch tests was used to model the test data. As shown in Figure 2, this analysis estimated that about 60% of nitro-E2 was oxidized to nitro-E1 and then degraded, and the remaining nitro-E2 was directly degraded. Although E2 can be oxidized to E1 and then degraded, isolates that are able to degrade E2 directly without oxidation to E1 have also been reported (25, 26). Similar first-order nitro-EE2 kb values were observed in batch tests with the 30-40 day SRT MBR and 3 day SRT AS mixed liquors, indicating that the bacteria that degrade nitroEE2 are present in both short and long SRT mixed liquors. During all batch tests, no accumulation of the parent estrogen compounds was detected, confirming that the nitro-estrogens were not restored to their parent estrogen forms as a result of biodegradation. Effect of Nitration on Estrogen Kinetics. Comparison of kinetic results for estrogen kb values (Table 4) and nitroestrogen kb values (Table 3) indicates that nitration of estrogens reduced their biodegradability, with nitro-EE2 degrading four times slower than EE2, nitro-E2 degrading two times slower than E2, and nitro-E1 degrading 11 times slower than E1. The presence of estrogens and nitro-estrogens together in batch tests did not change their degradation kinetics, indicating that there was no competitive inhibition at this concentration range. Nitro-estrogens have the potential for increased mutagenic and carcinogenic properties (27), and thus, their slower degradation rate may be a concern if biodegradation is not complete prior to discharge to the environment. Estrogen Biodegradation Kinetics. Similar kb values of 1.67 and 1.63 were observed for EE2 in batch tests for the nitrifying MBR and non-nitrifying AS mixed liquors, respectively, as shown in Table 4. This finding indicates that AOB and other slow-growing bacteria present only at long SRTs were not playing a significant role in estrogen degradation. Joss et al. (14) also found little effect of mixed liquor SRT on kb values in batch tests at 16 °C with mixed liquor from parallel MBR and AS systems with the same influent but different operating SRTs. Their reported kb values were 8 L/gTSS-day for a 12 day SRT AS system, and 6 L/gTSS-day for a 30 day SRT MBR system. These results indicate that bacteria that degrade EE2 are present in both short and long SRT mixed liquors, negating the hypothesis that there are critical SRTs for EE2 removal in WWTFs as a result of estrogen degradation by slow growing bacteria. The kb values for E1 and E2 were lower but within the same order of magnitude of values reported by Joss et al. (14) of 350 L/gTSS-day for E1and 950 L/gTSS-day for E2 with their MBR mixed liquor at a similar 30 day SRT. Differences VOL. 43, NO. 18, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 3. First-Order Model Biodegradation Rate Coefficients (kb) with 95% Confidence Interval for Nitro-Estrogen Degradation in Batch Tests with Activated Sludge (AS) and Membrane Bioreactor (MBR) Wastewater Treatment Facilitiesa estrogen rate observed in batch test nitro-EE2

nitro-EE2 nitro-E1 nitro-E2

estrogen combination in batch test

kb L/gVSS-dayd

r2

AS Mixed Liquor nitro-EE2b 0.73 nitro-EE2 and EE2b 0.73

0.567 ( 0.340 a 0.559 ( 0.160 a

0.82 0.95

MBR Mixed Liquor nitro-EE2b nitro-EE2 and EE2b nitro-E1c nitro-E1 and E1c nitro-E2c nitro-E2 and E2c

0.378 ( 0.530 a 0.522 ( 0.150 a 17.5 ( 11.1 b 25.9 ( 14.4 b 176 ( 60 c 239 ( 44 c

0.45 0.96 0.60 0.65 0.82 0.94

VSS/TSS

0.90 0.90 0.91 0.89 0.88 0.92

a Batch tests were conducted at 20°C, and all initial estrogen concentrations were 500 ng/L. Estrogens were quantified with LC-MS-MS. b Batch tests sampled once daily over a period of 5 days. c Batch tests samples 10 times over 1.67 h. d Different letters indicate statistically significant different values (p < 0.05).

FIGURE 2. Nitro-E2 and nitro-E1 transformation in batch tests with sludge from the membrane bioreactor (MBR) wastewater treatment facility. Modeled values were obtained using kb values in Table 3. Batch tests were conducted at 20 °C, and all initial estrogen concentrations were 500 ng/L. between the kb values in this study and Joss et al. (14) may be related to differences in the composition of the VSS of the respective AS systems, as the VSS is composed of varying ratios of bacteria biomass and inert VSS. As with nitro-E2, the conversion of E2 to E1 was not complete, as shown in Figure 3. Pauwels et al. also observed a 50% conversion of E2 to E1 prior to degradation in a study with bacterial isolates (28). Effect of Estrogen Mixtures on E1, E2, and EE2 Biodegradation Kinetics. There was not a significant difference in EE2 kb values with the addition of nitro-EE2. However, the presence of E1 and E2 increased the EE2 degradation rate, with kb values that were 7.5 times greater (Table 4). Pauwels et al. (28) also observed greater EE2 degradation rates with E2 in batch tests with estrogen-degrading bacterial isolates exposed to 5 mg/L E2 and EE2. At an initial E2/EE2 ratio of 5, the EE2 degradation rate was 2.5 times higher than that for an initial E2/EE2 ratio of 1. Despite these increases, the EE2 degradation rate was 20-50 times slower than the E1 and E2 degradation rates. Application to Biotreatment Methods for Reduction of Estrogenic Activity. These results indicate that estrogen removal is not limited to slow growing bacteria. The kb values for E1 and E2 were characteristic of kb values derived for domestic wastewater biodegradable chemical oxygen demand (COD) of 375 L/gVSS-day based on typical kinetic values (29). The estrogen degradation rates are most likely 7114

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too high to be due to the extremely low concentration of bacteria that would exist if they were growing solely on estrogen compounds in the wastewater influent. Thus, it appears that heterotrophic, estrogen-degrading bacteria grow on other carbon sources in WWTFs. The importance of other substrates has also been noted for EE2 degradation rates in surface waters; EE2 degradation rates increased with increased biodegradable dissolved organic carbon concentrations (30). A more detailed understanding of the influence of mixed substrates will be critical in developing accurate predictive biological treatment models that can be used in design and modification of WWTFs. It is important to note that the observed estrogen degradation rates were based on the VSS concentration and not the actual estrogen-degrading biomass. Further research is needed to determine if the firstorder rates based on VSS are applicable to other facilities with different wastewater characteristics, reactor configurations, and operating SRTs. Pure culture studies of estrogen metabolism and nonculture based studies of VSS populations are likely to contribute to that understanding. CAFO wastewater is frequently treated in anaerobic lagoons, however, lagoons frequently produce off-site odors, ammonia gas, and water pollution from runoff, discharge, and seepage (31). More environmentally friendly treatment methods that incorporate liquid discharge to the environment employ nitrification and then denitrification prior to discharge (31). One example is a system for CAFO swine wastewater treatment that includes oxidation of 765 mg/L NH4-N to NO3-N (31). If nitrification were stopped at NO2-N in these systems, it could result in reduced estrogenic activity through the nitration of estrogens and an energy savings for aeration. Similar systems could be employed for CAFO poultry and dairy wastewater treatment, which have NH4-N concentrations 0.5-2.5 times that of CAFO swine wastewater (32). Additional research is required to determine the proportion of estrogens that would be directly biodegraded versus nitrated and then further degraded in such systems and determine whether the kinetics determined with mixed liquor in this study could be applied to CAFO systems for estrogen and nitro-estrogen degradation. CAFO wastewater typically has a biological oxygen demand (BOD) to estrogen ratio that is an order of magnitude lower than the WWTFs from which mixed liquor was obtained in this study. Further research is necessary to determine whether estrogen removal efficiencies are related to influent BOD to estrogen ratios. If this is the case, it suggests that the nitration reaction in CAFO treatment could be more important than degradation by heterotrophic bacteria.

TABLE 4. First-Order Model Biodegradation Rate Coefficients (kb) with 95% Confidence Interval for Estrogen Degradation in Batch Tests with Activated Sludge (AS) and Membrane Bioreactor (MBR) Wastewater Treatment Facilitiesa estrogen rate observed in batch test EE2

EE2

E1 E2

estrogen combination in batch test

VSS/TSS

kb L/gVSS-dayd

r2

AS Mixed Liquor EE2b EE2 and nitro-EE2b

0.73 0.73

1.62 ( 0.30 d 1.48 ( 0.29 d

0.98 0.98

MBR Mixed Liquor EE2b EE2 and nitro-EE2b EE2 and E1c EE2 and E2c EE2, E1, and E2c E1c E1and EE2c E1 and nitro-E1c E2c E2 and EE2c E2, E1, and EE2c E2 and nitro-E2c

0.90 0.90 0.88 0.88 0.89 0.89 0.88 0.89 0.89 0.88 0.89 0.92

1.66 ( 0.07 d 1.58 ( 0.41 d 8.24 ( 0.01 e 13.1 ( 0.004 e 15.6 ( 0.002 e 229 ( 64 f 270 ( 0.03 f 230 ( 0.02 f 432 ( 20 g 527 ( 0.02 g 441 ( 80 g 524 ( 0.02 g

0.99 0.96 0.33 0.71 0.85 0.89 0.94 0.96 0.99 0.99 0.99 0.99

a Batch tests were conducted at 20°C, and all initial estrogen concentrations were 500 ng/L. Estrogens were quantified with LC-MS-MS. b Tests sampled once daily over a period of 5 days. c Batch tests sampled 10 times over 1.67 h. d Different letters indicate statistically significant different values (p < 0.05).

bacteria. Estrogens in urine are initially in conjugated forms (34). Further research is needed to determine whether the estrogens would be deconjugated during the proposed urine treatment process.

Acknowledgments

FIGURE 3. E2 and E1 transformation in batch tests with mixed liquor from the membrane bioreactor (MBR) wastewater treatment facility with an initial estrogen concentration of 500 ng/L E2. Modeled values were obtained using average values for kb values in Table 4. Batch tests were conducted at 20 °C, and all initial estrogen concentrations were 500 ng/L. The separate collection and treatment of urine is increasingly being considered as a alternative to centralized wastewater treatment, with successful implementations of separate urine collection, treatment, and reuse in Austria, Germany, The Netherlands, and Sweden (3). Collection of urine provides a way to isolate the majority of anthropogenic estrogens and greatly reduce estrogens entering WWTFs. Estrogen removal prior to the beneficial use of nutrients in urine could prevent estrogens from entering the environment while stabilizing NH4-N, preventing volatilization during transport. Depending on the collection method, reported urine NH4-N concentrations range from 1691 to 8100 mg/L (3). A 1:1 ratio of NO2-N to NH4-N can be achieved with reactors employing partial nitrification of urine and has been reported for a continuous flow stirred tank reactor (CSTR) and a sequencing batch reactor (SBR) without the addition of alkalinity (33). In a system with a 56 h HRT and 2000 mg/L NO2-N at 20 °C and pH 6.4, 90% nitration of E1, E2, and EE2 could be achieved (11). Urine is also high in COD (1600-10000 mg/L); however, during storage in nonsterile conditions, urea is rapidly hydrolyzed to NH4-N and carbonate (3), which is not likely to support heterotrophic estrogen-degrading

The authors are grateful to Saburo Matsui’s laboratory at Kyoto University for assistance with the YES method. This research was developed under STAR Research Assistance Agreement FP 916858 awarded by the U.S. Environmental Protection Agency, fellowships awarded by the King County Department of Natural Resources and Parks Wastewater Treatment Division, and the National Science Foundation IGERT program, and by NSF grant CBET-0652109. This paper has not been formally reviewed by the EPA, King County, or NSF. The views expressed in this document are solely those of the authors. The EPA, King County, and the NSF do not endorse any products or commercial services mentioned in this publication.

Supporting Information Available The Supporting Information contains the calculation for reduction of estrogenic activity following nitration of estrogens, an evaluation of the assumption of rapid liquid-solids sorption equilibrium in the first-order kinetic model of estrogen biodegradation, and results of a regression analysis for EE2 and nitro-EE2 transformation in batch tests. This material is available free of charge via the Internet at http:// pubs.acs.org.

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