Environ. Sci. Technol. 2009, 43, 3549–3555
Characterization of Metabolites Formed During the Biotransformation of 17r-Ethinylestradiol by Nitrosomonas europaea in Batch and Continuous Flow Bioreactors J . S K O T N I C K A - P I T A K , †,| W . O . K H U N J A R , ‡ N . G . L O V E , * ,‡,§ A N D D . S . A G A * ,† Department of Chemistry, University at Buffalo, Buffalo, New York 14260, Charles E. Via Jr. Department of Civil and Environmental Engineering, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061, Department of Civil and Environmental Engineering, University of Michigan, Ann Arbor, Michigan 48109, and Department of Environmental Engineering, Cracow University of Technology, 31-155 Krako´w, Poland
Received September 19, 2008. Revised manuscript received April 1, 2009. Accepted April 2, 2009.
The biotransformation of 17R-ethinylestradiol (EE2) by an ammonia oxidizing bacteria, Nitrosomonas europaea, grown in batch (ammonia-rich) and continuous flow (chemostat, ammonia-limited) reactors was investigated. Both C-14 labeled EE2 (10 µg/L) and unlabeled EE2 (1 mg/L) were used to facilitate metabolite identification under environmentally relevant physiological conditions. Whole cell ammonia monooxygenase (AMO) activity was not inhibited at the EE2 concentrations used in this study. Characterization of the primary metabolite formed during batch cultivation by liquid chromatography/iontrap mass spectrometry (LC-ITMS) and nuclear magnetic resonance (NMR) spectroscopy showed modification at the ethinyl group and addition of a carboxyl group. This metabolite (M386) (revealed by m/z 385 in negative mode electrospray LC/ MS) was not formed in the abiotic control. In contrast, biotransformation of EE2 under continuous flow conditions showed formation of a monohydroxylated EE2 (revealed by m/z 311), but not M386. Furthermore, nitrated EE2 derivatives were formed in both batch and continuous flow cultures, as a result of abiotic transformation of EE2 in the presence of high concentrations of nitrite in the bioreactors. Results from this study underscore the importance of physiological state and growth conditions as critical variables that can dictate the metabolic pathway for EE2 biodegradation and the nature of byproducts formed.
* Address correspondence to either author. Phone: (716) 6456800x2226 (D. S. A.); (734) 764-8495 (N. G. L.). Fax: (716) 645-6963 (D. S. A.); (734) 764-4292 (N. G. L.). E-mail: Diana S. Aga:
[email protected] (D. S. A.);
[email protected] (N. G. L.). † University at Buffalo. | Cracow University of Technology. ‡ Virginia Polytechnic Institute and State University. § University of Michigan. 10.1021/es8026659 CCC: $40.75
Published on Web 04/22/2009
2009 American Chemical Society
Introduction Wastewater treatment-based transformation of endocrine disrupting compounds such as the natural and synthetic estrogens (estrone, 17 β-estradiol, estriol, and 17R-ethinylestradiol) has been documented in the past decade (1-5). As these micropollutants possess great potential to pose longterm ecological damage due to endocrine disrupting effects in fish and wildlife (6-8), studies that investigate the precise fate of these micropollutants during activated sludge treatment are needed. Typically, biotransformation of estrogens produces products that are more polar than the parent compound (9, 10). These metabolites may eventually be discharged into the environment together with the parent estrogens. Although prior studies have proposed a relationship between nitrification (the aerobic, microbiological oxidation of ammonia) and transformation of 17R-ethinylestradiol (EE2) (5, 10, 11), very little information is available about the metabolic pathway(s) involved. It is important to know the fate and nature of stable metabolites because they may pose their own ecotoxicological effects on the environment. Consequently, there is a clear need to move beyond mere analytical surveys of wastewater treatment systems to a more comprehensive examination of removal mechanisms in treatment plants. In this study, the biotransformation of the synthetic hormone, EE2, by pure cultures of the ammonia oxidizer Nitrosomonas europaea was investigated to identify the primary metabolites formed during biotransformation in batch and continuous flow cultures. The identities of the metabolites formed were elucidated using a combination of mass spectrometry and nuclear magnetic resonance spectroscopy analyses. It was reasonable to conduct this study with N. europaea because it is a well characterized ammonia oxidizer (12) that has been shown to exist in nitrifying activated sludge cultures (13, 14) and is capable of the cometabolism of multiple organic contaminants ranging from chloroform and trihalomethanes to EE2 (5, 15-17).
Materials and Methods EE2 Batch Transformation Experiments. Nitrosomonas europaea (ATCC strain 19718) was cultured using batch reactors utilizing autotrophic media listed in the Supporting Information (SI). Batch cultures of N. europaea (2-5 L) were supplemented with either 1 mg/L or 10 µg/L EE2 (final concentration) and incubated for 28 days, during which grab samples (250 mL) were collected at specific time intervals and processed for analysis. Each sample was centrifuged at 13 000g for 30 min at 4 °C to remove suspended solids, and then lyophilized. Negative control experiments (no EE2) and abiotic experiments (with EE2, but no bacteria) were also performed. EE2 Chemostat Transformation Experiments. Feed Stocks for continuous flow cultures of N. europaea (chemostat: 2 L; residence time ) 7 day) were supplemented with EE2 to a final concentration of 1 mg/L unlabeled EE2 and 10 µg/L C-14 labeled EE2 (14C-EE2). Chemostat cultures were maintained for 28 days during which grab samples (100 mL) for ammonia and nitrite analysis were obtained and processed as described for batch samples except that centrifugation was performed at room temperature. A final sample was taken for LC/MS analysis on day 28. Analysis by LC/MS, LC/Radiochromatographic Detection and NMR. Lyophilized samples were reconstituted in Nanopure water (10 mL) and an aliquot (1 mL) from each sampling interval was supplemented with a deuterated VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3549
FIGURE 1. Selected ion chromatograms of EE2 (m/z 295) and M386 (m/z 385) corresponding to various sampling intervals, illustrating relative retention times and signal intensity in LC/MS. internal standard, EE2-d4, to facilitate analysis by liquid chromatography/mass spectrometry (LC/MS). First, samples were analyzed using a single quadrupole Agilent 1100 MSD LC/MS system with electrospray ionization under negative (-ESI) and positive (+ESI) mode, using full scan conditions (m/z 50-1000) to search for the appearance of new peaks unique to the EE2-treated biotic samples. Separation conditions are listed in SI. Then, samples were analyzed in LC with ion-trap mass spectrometer detection (LC-ITMS) under -ESI with selected ion monitoring (SIM). The parent ion of metabolite (m/z of unknown metabolite previously selected using full scan single quad LC/MS) was subjected to MSn fragmentation in LC-ITMS for structural characterization. Analysis of radioactive samples was performed using an LC with online radiochromatographic detector, as described in detail in SI. Transformation products were purified by fractionation using a C-18 semiprep column in HPLC to prepare for analysis by nuclear magnetic resonance spectroscopy (NMR). Separation conditions are listed in SI. Fractions containing M386 were evaporated to dryness, reconstituted in either CD3OD or DMSO-d6 and transferred to NMR tubes. 1H NMR data was performed with Inova-500 MHz NMR Spectrometer (Varian, CA).
Results and Discussion Degradation of EE2 and Characterization of Metabolites under Batch Growth Conditions. The concentration of EE2 decreased by more than 90% during batch incubation in the presence of N. europaea after 9 days incubation (1 mg EE2/ L) and 25 days incubation (10 µg EE2/L) (SI Figure S1). It has been reported in a recent paper by Gaulke et al. (18) that EE2 removal rates in batch tests with N. europaea are proportional to the initial EE2 concentrations, which is similar to what we have observed in this work. To determine if use of the high EE2 concentrations during the experiments (required to allow detection of transformation products) caused AMO inhibition, whole cell AMO activity assays were conducted as described in the SI. Results confirmed that EE2-mediated inhibition was not significant at the concentrations utilized in experiments up to 1 mg/L (see SI Table S1). 3550
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 10, 2009
Analysis of the samples from batch cultures with N. europaea revealed formation of a more polar metabolite with m/z 385 using -ESI LC/MS [corresponding to (M-H)-]. This metabolite was not observed in abiotic control samples, suggesting that this peak is unique to the biotransformation of EE2 in the presence of N. europaea. This metabolite, heretofore called M386, formed a dansyl derivative during reaction with dansyl chloride (see SI Scheme S1) in the same way EE2 forms dansyl derivative (SI Scheme S2), suggesting the presence of the phenolic group in M386. The selected ion chromatograms of underivatized samples from batch N. europaea cultures at 4, 7, 11, and 19 days after EE2 treatment are presented in Figure 1. These chromatograms demonstrate that M386 (with retention time ca. 2 min) is much more polar than EE2 (retention time ca. 10 min) and is more ionizable in LC/MS than EE2 under -ESI mode, as indicated by the high signal intensity of M386 relative to EE2. The peak area for M386 increased over time and corresponded to a decrease in the peak area for EE2 (inset in Figure 1). A second minor metabolite with m/z 311 was also detected at trace levels in samples obtained from the batch reactor studies and was not present in abiotic controls. Based on the LC-ITMS and 1H NMR analyses a structure is proposed for the M386 metabolite formed during the biotransformation of EE2, as shown in scheme 1. The MSn fragmentation pathway for M386 explaining the observed mass spectra in Figure 2 is presented in SI Figure S2. LCITMS characterization suggested that M386 contains a hydroxyl group (loss of 17 in Figure 2A), an aldehyde group (loss of 28 in Figure 2B), and a carboxylic group (loss of 44 in Figure 2C). In addition, the MS transition m/z 385f 368f321 (Figure 2D) supports the proposed conversion of the ethinyl group to a carboxylate group, as shown in SI Scheme 3. In addition, the observed increase in the ionization efficiency of M386 (relative to EE2) in LC/MS under -ESI mode suggests the presence of an easily ionizable group such as a carboxylic moeity. To provide additional information supporting the proposed identitity of M386 metabolite, 1H NMR spectra were obtained for both EE2 standard and isolated M386. The
SCHEME 1. Proposed Metabolite of EE2 upon Biodegradation by N. europaeaa
a
The exact location of carboxylation in the ring could not be established by NMR due to the limited amount of metabolite.
chemical shifts of the aromatic protons and the ethinyl group are key features of the EE2 molecule and hence, were compared with those of the M386 metabolite, as presented in SI Figure S3. The three aromatic protons in ring A of EE2 [H-4 (6.49, s); H-3 (6.56, d, 3J 10 Hz); H-2 (7.11, d, 3J 10 Hz)] (SI Figure S3A) were present in M386 but were slightly shifted (H-4 (5.99, s); H-3 (6.16, d, 3J 15 Hz); H-2 (7.24, d, 3J 15 Hz)) (SI Figure S3B). These features suggest that the substitution pattern is preserved in the aromatic ring of M386, but that a new functional group has been introduced nearby causing this change in the chemical shifts of the aromatic protons. One possible explanation for this change in proton shift is the conversion of the phenol group into a 1,4-diene-3-one structure. However, M386 reacted with dansyl chloride in the same manner as EE2 upon derivatization of the phenol group (see reaction in the SI Schemes S1 and S2), as confirmed by LC/MS (data not shown). Therefore, we infer that M386 still contains a phenol group. In addition, the early retention time in LC and the high ionization efficiency in -ESI MS for M386 (see Figure 1) suggest that a polar functional group has been introduced into the molecule during EE2 biotransformation. As such, we propose the presence of a carboxylic acid group in the nearby ring (see Scheme 1); however, the exact location can not be confirmed because of the limited amount of M386 which did not make it possible to collect 13 C NMR data. It should be noted that the proton shift due to the ethinyl group of EE2 (2.92, s) (SI Figure S3C) was absent in the NMR spectra of M386 (SI Figure S3D) suggesting modification in this functional group. Finally, the chemical shift of the methyl group in EE2 (0.89, s) was also absent in the NMR spectra of M386 (not shown), and a new signal (9.66, s) characteristic of an aldehyde group was observed in M386 (SI Figure S3E). Formation of M386 Metabolite. The AMO enzyme has been noted to catalyze monooxygenation, dehydrogenation, and reductive dehalogenation reactions on a wide range of nonpolar compounds ranging from NH3 to substituted benzene derivatives (19). Unspecific epoxidation of alkenes to corresponding epoxides (20, 21) have also been documented. It is also generally accepted that acetylene oxidation by AMO can yield unsaturated and unstable acetylenic epoxides (22, 23). Along these lines, we propose that under batch growth conditions, N. europaea transforms EE2 to M386 via monooxygenation of the acetylene group at C-17. Such an epoxide reaction has been previously documented to be catalyzed by liver microsomal cytochromes P450 monooxygenase (24) and is not outside the realm of activity for AMO as both monooxygenases have similar substrate ranges (19, 25). Examination of the published genome of N. europaea suggests the presence of genes that encode for an epoxide hydrolase-like enzyme (12) that can hydrolyze epoxides to dihydrodiols, which may be further converted to aldehydes or carboxylic acids (26).
The proposed carboxylation reaction at ring B is difficult to explain. N. europaea possesses several carboxylases including ribulose 1,5-bisphosphate carboxylate/oxygenase (RuBisCO) (12) but does not possess genes that encode for carboxysomes (12). While it is possible that RuBisCO may be available to interact with cytoplasmic EE2 molecules, transport of EE2 into cells would need to occur via passive transport since few known aromatic compound transporters are present (12). Such action of RuBisCO to carboxylate nonbiosynthetic intermediates in N. europaea has not been demonstrated to our knowledge and is highly unlikely. Degradation Profile of EE2 under Continuous Flow (Chemostat) Conditions. The samples obtained from N. europaea chemostat cultures (500 mL) exposed to 14C-EE2 were concentrated by freeze-drying, and reconstituted with the LC mobile phase (1 mL) for analysis by LC/radiochromatography and LC/MS. Figure 3 shows the radiochromatogram of a concentrated sample from the chemostat culture fed with 14C-EE2, indicating the peaks corresponding to the transformation products and the parent EE2. Interestingly, none of these peaks corresponded with the M386 metabolite formed in batch culture based on LC/MS analysis of the radioactive fractions collected after chromatographic separation. Instead, the more polar monohydroxylated EE2 (retention time ca. 20 min), revealed by the appearance of m/z 311 (corresponding to its molecular ion M-H-), and another currently unidentified polar metabolite (retention time ca. 10 min) were produced. Both of these metabolites were approximately 5% of the total radioactivity in the sample. Futhermore, less polar peaks (eluting after EE2 at ca. 26 and 31 min) were observed. These peaks were identified as 4-nitroEE2 (8% of total radioactivity) and 2-nitro-EE2 (5% of total radioactivity) based on the LC/MS data comparison with the synthesized nitro-EE2 standards (see LC/MS data in SI Figure S4 and crystal structure information of synthesized 2-nitroEE2 in SI Figure S5). The nitrated EE2 are formed abiotically under high concentrations of nitrite (18). These nitrated EE2 were also observed in the batch reactors, including in the abiotic control (sterile media spiked with EE2 and NO2-). As the nitrite concentrations (see SI Figures S1 and S6) in our bioreactors (0-500 mg NO2--N/L in batch reactors and >650 mg NO2--N/L in chemostat cultures, respectively) were within the range of the nitrite concentrations reported to facilitate abiotic nitration of EE2, formation of these nitrated EE2 derivatives is not unexpected. However in domestic treatment plants, such concentrations of nitrite are not typical and thus, nitration of EE2 is not expected to be a significant reaction in activated sludge processes. Further, in the earlier studies investigating nitration of EE2 (18), researchers utilized a sequential batch system which allowed variation of cell physiological state that can affect AMO activity and alter the biotransformation cascade of EE2. VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3551
FIGURE 2. MSn spectra of (A) m/z 385, (B) m/z 385f368, (C) m/z 385f368f340, and (D) m/z 385f368f321.
3552
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 10, 2009
FIGURE 3. Radiochromatogram of EE2 and proposed metabolites generated during chemostat experiments, illustrating relative retention times and signal intensity (cpm) (20-min peak: 6-hydroxy-ethinylestradiol (6-hydroxy-EE2) or 2-hydroxy-ethinylestradiol (2-hydroxy-EE2); 26-min peak: 4-nitro-ethinylestradiol (4-nitro-EE2); 31-min peak: 2-nitro-ethinylestradiol (2-nitro-EE2)). Monohydroxylation of EE2 is hypothesized to occur at either the C-2 or C-6 position resulting in the formation of 2-hydroxy-EE2 or 6-hydroxy-EE2, respectively (27). As NMR was not performed on this compound in the current study due to the limited amount of sample (a consequence of radiolabeled chemostat protocols), the precise location of the hydroxyl group can only be inferred. Both hypothesized metabolites have been observed as byproducts resulting from natural and synthetic estrogen metabolism by P450 monooxygenases in human liver microsomes (24, 27). Whereas 6-hydroxy-EE2 appears to be formed during EE2 degradation by microalgae (28), only 2-hydroxy-EE2 has been previously identified as a byproduct that results from treatment of EE2 by an enriched nitrifying culture (10). However, formation of 4-hydroxy-EE2 is also possible and cannot be discounted. The existence of a monohydroxylated EE2 metabolite detected at trace levels in AOB batch culture and as the primary metabolite in chemostat cultures after 28 days of growth shows that ammonia oxidizing cultures grown under both ammonia-rich conditions (typical of laboratory-scale batch studies 5, 18) or ammonia-limited conditions (which occur in biological wastewater treatment plants that incorporate complete nitrification or nitrogen removal) do biotransform EE2; this observation is in contrast to the conclusion drawn by Gaulke et al. who restricted their studies to nongrowth limited batch growth conditions (18). It should be noted, however, that the EE2 concentrations used in the chemostat and batch cultures are about 3 orders of magnitude higher than the EE2 concentrations typically found in wastewater treatment plants. Estrogenicity of Reactor Effluent and Transformation Products. In addition to monitoring the disappearance of EE2, it is imperative to evaluate the ecotoxicological impact of metabolites that may be generated from wastewater treatment processes. Evaluation of the presence of estrogens such as EE2 in engineered treatment systems (3, 30) and natural waters (29) have been well documented using the YES bioassay. Results from bioassays performed in the current study using protocols outlined in SI indicate that up to 61% of overall estrogenicity was reduced during the course of batch experiments (Figure 4). Analysis of fractionated samples
FIGURE 4. Results from YES assays performed on batch reactor effluent.
TABLE 1. Estrogenic Response of Isolated Metabolites as Measured by YES Assay E2 equivalence (ng/L) a
Fraction
Chemostat
Batch
blank M312 M386 M341 EE2
95 ( 11 2900 ( 371 nac 456 ( 93 264 000 ( 33 400
101 ( 36 nab 4000 ( 873 124 000 ( 22 200
a
Fractionation details are included in the Supporting Information. b insufficient metabolite obtained to perform YES assay. c not present in any chemostat samples.
also suggested a reduction in the estrogenic response of each individual transformation product relative to EE2 (Table 1). Reduced estrogenicity of these metabolites is expected since modifications to the chemical structure of EE2 can induce steric hindrance, which may be manifested by a reduced VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3553
ability of the byproduct to bind to estrogen receptors (11, 30-32). While it appears that the contribution of each transformation product is low compared to EE2, it should be noted that the relative fraction of each byproduct will impact responses observed with mixtures. Indeed, even though up to 90% of EE2 was transformed after 9 days of treatment, estrogenicity was only reduced by approximately 60% (Figure 4). Therefore, transformation of EE2 in nitrifying batch cultures does not completely correlate 1:1 with loss of estrogenicity. Significance of EE2 Biotransformation under Varying Conditions. Our results indicate that both ammonia availability and physiological state can impact the nature and extent of EE2 interaction with N. europaea. Under conditions of nutrient limitation, bacteria typically undergo a metabolic trade-off, upregulating genes associated with stress responses and downregulating genes coding for superfluous functions (33, 34). In this study, ammonia limitation only occurred in the chemostat culture after 20 days of incubation, where effluent ammonia concentrations stabilized below 1 mg/L as N. In contrast, ammonia-rich conditions occurred throughout the exponential growth-phase batch cultures with ammonia concentrations ranging from 720 to 150 mg/L as N. Whole cell AMO activity measurements for batch and chemostat cultures were highest during midlog batch growth when ammonia concentrations were sufficient, and were lower during both late-log growth and in chemostat growth when ammonia was limiting (SI Table S2). The actual role of amoA expression in predicting EE2 biotransformation patterns deserves further evaluation. We hypothesize that the primary reaction under ammonia-rich batch conditions is epoxidation of the acetylene side chain, despite previous studies that report that acetylene constructs are known competitive inhibitors of AMO (23). Previous researchers found that increased concentrations of ammonia enhanced oxidation of methyl/methylene substituents of alkylated benzenes (25) as well as protected against acetylene mediated inhibition of AMO (22). As such, it is possible that under the ammonia-rich, batch growth conditions that were used in this study, AMO was available to oxidize the ethinyl group to an unstable epoxide and hydroxylate the phenolic group of EE2. That chemostat cultured N. europaea did not biotransform EE2 to M386 may be a manifestation of the lower ammonia concentration present at the time of sampling. Physiological adaptation to the ammonia-limited condition may preferentially select for the monohydroxylation reaction as opposed to epoxidation of the acetylene group. Inhibiton of whole cell AMO activity was not oberved to be significant in this study. Such findings are not unexpected since the acetylene associated with concentrations of EE2 used in this study are lower than that used to inactivate AMO in previous studies (22, 23). Finally, we conclude that both byproducts, M386 and M312 were biologically mediated during batch (ammonia-rich) growth of N. europaea, which is in contrast to Gaulke et al. (18) who only found abiotically mediated nitrated EE2 derivatives. Monohydroxylation of EE2 in chemostat grown N. europaea resulted in a different primary byproduct than the primary M386 byproduct detected in batch cultures. While the location of the oxidation reaction varied between both metabolites, the nature of the oxidations that occurred are within the inventory of AMO-facilitated reactions. AMO has also been noted to catalyze oxidation of the polycyclic compound naphthalene resulting in the formation of 1-naphthol and 2-naphthol with the latter comprising 80% of the initial naphthalene input into the system (15). Such action by AMO suggests congruence with the proposed mechanism of formation for the hypothesized hydroxy-EE2 metabolites. The role of physiological states in dictating co-oxidation reactions, as observed in this study, has not been documented 3554
9
ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 43, NO. 10, 2009
in the literature. At the same time though, it is known that bacterial cells can utilize alternative anabolic pathways under varying physiological conditions while utilizing/yielding the same reactants/products (35). While such observations do not necessarily explain the mechanisms proposed in this current study, they may explain results obtained from various heterotrophic cultures in the literature. The results from this study show that biotransformation of EE2 by N. europaea does occur with both batch (ammoniarich) and chemostat (ammonia-limited) cultures. Detection of a biotransformation intermediate under ammonia-limited chemostat conditions is most applicable to practice because that growth condition most closely reflects what is found in biological WWTPs. The WWTP condition incorporating nitrogen removal processes has been observed to have enhanced levels of pharmaceutical biotransformation. (11, 36, 37). In contrast to prior conclusions (18), this result indicates that ammonia oxidizing bacteria may be important contributors to EE2 biotransformation in biological wastewater treatment systems; however, the role that coexisting heterotrophic bacteria play in competing for EE2 or in further biotransforming the metabolic product generated by nitrifiers is unknown. It should be noted that the abiotic formation of nitrated EE2 is not expected to occur in full-scale wastewater treatment plants because the typical nitrite levels are below the concentrations at which nitration takes place. It is important to recognize that the different EE2 metabolites observed in batch and chemostat cultures underscore the importance of monitoring growth condition as a critical parameter that can dictate the metabolic pathway for EE2 biodegradation and the nature of byproduct formed. Hence, the differences in the reported literature on the nature and rate of EE2 biodegradation under laboratory conditions using cultures of N. europaea, mixed cultures from nitrifying activated sludge, membrane bioreactors, as well as those studies based on full-scale WWTPs could significantly be due to the varying physiological states of the microorganisms involved in EE2 metabolism. Finally, it is important to recognize that EE2 biotransformation did not result in mineralization by N. europaea in either the chemostat or batch culture studies performed here. Therefore, understanding the fate of AOB-transformed metabolites is important to understanding the fate of estrogens.
Acknowledgments This material is based upon work supported by the National Science Foundation (Grant No. 0504477 and 0504359). Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. We thank Erwin Garcia (UB), for his help in acquiring NMR data, Julie Petruska (VT) and Doug Smiley (VT) for assistance during development of radioisotope protocols, and Lindsay Tayloe (undergraduate assistant VT) for assisting with N. europaea reactor maintenance. We also acknowledge Dawn Celiz, Susan Mackintosh, Tony Baik, and Jerry Tso (all from the Aga Research group at UB) for all their help in LC/MS analysis and compound isolation. We thank Professor Philip Coppens (UB) for use of his X-ray diffractometer and Dr. Mateusz Pitak for crystal structure determination. We are also deeply grateful to Dr. Sumpter of Brunel University for supplying the original yeast culture used in these experiments. J.S.-P. and W.O.K. contributed equally to this work.
Supporting Information Available Additional details including experimental protocols, three schemes, six figures, two tables, and additional citations. This material is available free of charge via the Internet at http://pubs.acs.org.
Literature Cited (1) de Mes, T.; Zeeman, G.; Lettinga, G. Occurrence and fate of estrone, 17β-estradiol and 17R-ethynylestradiol in STPs for domestic wastewater. Rev. Environ. Sci. Bio/Technol. 2005, 4, 275–311. (2) Gomes, R. L.; Scrimshaw, M. D.; Lester, J. N. Determination of endocrine disrupters in sewage treatment and receiving waters. Trends Anal. Chem. 2003, 22, 697–707. (3) Holbook, R. D.; Novak, J. T.; Grizzard, T. J.; Love, N. G. Estrogen receptor agonist fate during wastewater and biosolids treatment processes: A mass balance analysis. Environ. Sci. Technol. 2002, 36, 4533–4539. (4) Hugget, D. B.; Foran, C. M.; Brooks, B. W.; Weston, J.; Peterson, B.; Marsh, K. E.; La Point, T. W.; Schlenk, D. Comparison of in vitro and in vivo bioassays for estrogenicity in effluent from North American municipal wastewater facilities. Toxicol. Sci. 2003, 72, 77–83. (5) Shi, J.; Fujisawa, S.; Nakai, S.; Hosomi, M. Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia oxidizing bacterium Nitrosomonas europaea. Water Res. 2004, 38, 2323–2330. (6) Jobling, S.; Nolan, M.; Tyler, C. R.; Brighty, G.; Sumpter, J. P. Widespread sexual disruption in wild fish. Environ. Sci. Technol. 1998, 32, 2498–2506. (7) Mortensen, A. S.; Arukwe, A. Effects of 17R-ethynylestradiol on hormonal responses and xenobiotic biotransformation system of Atlantic salmon (Salmo salar). Aquat. Toxicol. 2007, 85 (2), 113–123. (8) Lavado, R.; Thibaut, R.; Raldua, D.; Martın, R.; Porte, C. First evidence of endocrine disruption in feral carp from the Ebro River. Toxicol. Appl. Pharmacol. 2004, 196, 247–257. (9) Cirja, M.; Zuehlke, S.; Ivashechkin, P.; Hollender, J.; Schaeffer, A.; Corvini, P. F. X. Behavior of two differently radiolabelled 17R-ethinylestradiols continuously applied to a laboratory-scale membrane bioreactor with adapted industrial activated sludge. Water Res. 2007, 41, 4403–4412. (10) Yi, T.; Harper, W. F., Jr. The Link between nitrification and biotransformation of 17R-Ethinylestradiol. Environ. Sci. Technol. 2007, 41, 4311–4316. (11) Vader, J. S.; Ginkel, C. G.; van Stokman, F. M.; Sperling, G. M.; Jong, J.; de Boer, W.; de Graaf, J. S.; de Most, M.; van der Stokman, P. G. W. Degradation of ethinyl estradiol by nitrifying activated sludge. Chemosphere 2000, 41, 1239–1243. (12) Chain, P.; Lamerdin, J.; Larimer, F.; Regala, W.; Lao, V.; Land, M.; Hauser, L.; Hooper, A. B.; Klotz, M. G.; Norton, J. M.; Sayavedra-Soto, L. A.; Arciero, D. M.; Hommes, N. G.; Whittaker, M. R.; Arp, D. J. Complete genome sequence of the ammoniaoxidizing bacterium and obligate chemolithoautotroph Nitrosomonas europaea. J. Bacteriol. 2003, 185, 2759–2773. (13) Kelly, J. J.; Siripong, S.; McCormack, J.; Janus, L. R.; Urakawa, H.; El Fantroussi, S.; Noble, P. A.; Sappelsa, L.; Rittman, B. E.; Stahl, D. A. DNA microarray detection of nitrifying bacterial 16S rRNA in wastewater treatment plant samples. Water Res. 2005, 39, 3229–3238. (14) Mobarry, B. K.; Wagner, M.; Urbain, V.; Rittman, B. E.; Stahl, D. A. Phylogenetic probes for analyzing abundance and spatial organization of nitrifying bacteria. Appl. Environ. Microbiol. 1996, 62, 2156–2162. (15) Chang, S. W.; Hyman, M. R.; Williamson, K. J. Cooxidation of naphthalene and other polycyclic aromatic hydrocarbons by the nitrifying bacterium Nitrosomonas europaea. Biodegradation 2002, 13, 373–381. (16) Hyman, M. R.; Wood, P. M. Methane oxidation by Nitrosomonas europaea. Biochem. J. 1983, 212, 31–37. (17) Wahman, D. G.; Katz, L. E.; Speitel, G. E., Jr. Cometabolism of trihalomethanes by Nitrosomonas europaea. Appl. Environ. Microbiol. 2005, 71, 7980–7986. (18) Gaulke, L. S.; Strand, S. E.; Kalhorn, T. F.; Stensel, H. D. 17Rethinylestradiol transformation via abiotic nitration in the presence of ammonia oxidizing bacteria. Environ. Sci. Technol. 2008, 42, 7622–7627.
(19) Hooper, A. B.; Vannelli, T.; Bergmann, D. J.; Arciero, D. M. Enzymology of the oxidation of ammonia to nitrite by bacteria. Antonie van Leeuwenhoek 1997, 71, 59–67. (20) Arp, D. J.; Yeager, C. M.; Hyman, M. R. Molecular and cellular fundamentals of aerobic cometabolism of trichloroethylene. Biodegradation 2001, 12, 81–103. (21) Hyman, M. R.; Murton, I. B.; Arp, D. J. Interaction of ammonia monooxygenase from Nitrosomas europaea with alkanes, alkenes, and alkynes. Appl. Environ. Microbiol. 1988, 54, 3187– 3190. (22) Hyman, M. R.; Wood, P. M. Suicidal inactivation and labelling of ammonia mono-oxygenase by acetylene. Biochem. J. 1985, 227, 729–735. (23) Hyman, M. R.; Arp, D. J. 14C2H2 and l4C02 Labeling studies of the de novo synthesis of polypeptides by Nitrosomonas europaea during recovery from acetylene and light inactivation of ammonia monooxygenase. Biol. Chem. 1992, 267, 1534–1545. (24) Kent, U. M.; Mills, D. E.; Rajnarayanan, R. V.; Alworth, W. L.; Hollenberg, P. F. Effect of 17R-ethynylestradiol on activities of cytochrome P450 2B (P450 2B) enzymes: Characterization of inactivation of P450s 2B1 and 2B6 and identification of metabolites. J. Pharmacol. Exp. Ther. 2002, 300, 549–558. (25) Keener, W. K.; Arp, D. J. Transformations of aromatic compounds by Nitrosomonas europaea. Appl. Environ. Microbiol. 1994, 60, 1914–1920. (26) Ensign, S. A.; Allen, J. R. Aliphatic epoxide carboxylation. Annu. Rev. Biochem. 2003, 72, 55–76. (27) Lee, A. J.; Kosh, J. W.; Conney, A. H.; Zhu, B. T. Characterization of the NADPH-dependent metabolism of 17β-estradiol to multiple metabolites by human liver microsomes and selectively expressed human cytochrome P450 3A4 and 3A5. J. Pharmacol. Exp. Ther. 2001, 298, 420–432. (28) Della Greca, M.; Pinto, G.; Pistillo, P.; Pollio, A.; Previtera, L.; Temussi, F. Biotransformation of ethinylestradiol by microalgae. Chemosphere 2008, 70, 2047–2053. (29) Beck, I. C.; Bruhna, R.; Gandrassa, J. Bioassay-directed fractionation for analyzing estrogens in surface waters of the German Baltic Sea. Acta Hydrochim. Hydrobiol. 2006, 34, 560–567. (30) Fang, H.; Tong, W.; Perkins, R.; Soto, A. M.; Prechtl, N. V.; Sheehan, D. M. Quantitative comparisons on in vitro assays for estrogenic activities. Environ. Health Perspect. 2000, 108, 723– 729. (31) Huber, M. M.; Ternes, T. A.; Von Gunten, U. Removal of estrogenic activity and formation of oxidation products during ozonation of 17R-ethinylestradiol. Environ. Sci. Technol. 2004, 38, 5177–5186. (32) Miller, D.; Wheals, B. B.; Beresford, N.; Sumpter, J. P. Estrogenic activity of phenolic additives determined by an in vitro yeast bioassay. Environ. Health Perspect. 2001, 109, 133–138. (33) Guina, T.; Wu, M.; Miller, S. I.; Purvine, S. O.; Yi, E. C.; Eng, J.; Goodlett, D. R.; Aebersold, R.; Ernst, R. K.; Lee, K. A. Proteomic analysis of Pseudomonas aeruginosa grown under magnesium limitation. J. Am. Soc. Mass Spectrom. 2003, 14, 742–751. (34) Tao, H.; Bausch, C.; Richmond, C.; Blattner, F.; Conway, T. Functional genomics: expression analysis of Escherichia coli growing on minimal and rich media. J. Bacteriol. 1999, 181, 6425–6440. (35) Fiske, M. J.; Whitaker, R. J.; Jensen, R. A. Hidden overflow pathway to L-phenylalanine in Pseudomonas aeruginosa. J. Bacteriol. 1983, 154, 623–631. (36) Batt, A. L.; Kim, S.; Aga, D. S. Enhanced biodegradation of iopromide and trimethoprim in nitrifying activated sludge. Environ. Sci. Technol. 2006, 40, 7367–7373. (37) Joss, A.; Zabczynski, Z.; Gobel, A.; Hoffmann, B.; Loffler, D.; McArdell, C. S.; Ternes, T. A.; Thomsen, A.; Siegrist, H. Biological degradation of pharmaceuticals in municipal wastewater treatment: Proposing a classification scheme. Water Res. 2006, 40, 1686–1696.
ES8026659
VOL. 43, NO. 10, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
9
3555
