Degradation of Norgestrel by Bacteria from Activated Sludge

use in contraception, and treatments for abnormal uterine bleeding, symptoms of menopause, and certain types of cancer. .... (29) 16S rDNA gene wa...
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Degradation of Norgestrel by Bacteria from Activated Sludge: Comparison to Progesterone Shan Liu, Guang-Guo Ying,* You-Sheng Liu, Fu-Qiang Peng, and Liang-Ying He State Key Laboratory of Organic Geochemistry, Guangzhou Institute of Geochemistry, Chinese Academy of Sciences, Guangzhou 510640, China S Supporting Information *

ABSTRACT: Natural and synthetic progestagens in the environment have become a concern due to their adverse effects on aquatic organisms. Laboratory studies were performed to investigate aerobic biodegradation of norgestrel by bacteria from activated sludge in comparison with progesterone, and to identify their degradation products and biotransformation pathways. The degradation of norgestrel followed first order reaction kinetics (T1/2 = 12.5 d), while progesterone followed zero order reaction kinetics (T1/2 = 4.3 h). Four and eight degradation products were identified for norgestrel and progesterone, respectively. Six norgestrel-degrading bacterial strains (Enterobacter ludwigii, Aeromonas hydrophila subsp. dhakensis, Pseudomonas monteilii, Comamonas testosteroni, Exiguobacterium acetylicum, and Chryseobacterium indologenes) and one progesterone-degrading bacterial strain (Comamonas testosteroni) were successfully isolated from the enrichment culture inoculated with aerobic activated sludge. To our best knowledge, this is the first report on the biodegradation products and degrading bacteria for norgestrel under aerobic conditions.

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INTRODUCTION Hormone steroids in the aquatic environment have become a public concern because of their potential endocrine disrupting effects on organisms.1,2 However, most environmental studies about steroids focus on estrogens and less on other steroids such as progestagens.3,4 Progestagens (also called progestogens, progestins, or gestagens) are hormones that are involved in the female menstrual cycle, pregnancy, and embryogenesis of humans and other species. Because natural progesterone is inactivated very rapidly in the human body, several synthetic progestagens have been developed for use in contraception, and treatments for abnormal uterine bleeding, symptoms of menopause, and certain types of cancer.5 Norgestrel is a synthetic progestagen used as an oral contraceptive at low doses either alone or in combination with estrogens such as 17β-ethynylestradiol (EE2). It may also be used at high doses as a progestin-only emergency contraceptive.6,7 According to clinical studies,8,9 the progestagenic potency of norgestrel is approximately 1000 times that of progesterone. Besides progestagenic activity, synthetic progestagens may also have other hormonal activities, such as estrogenic, antiandrogenic, and androgenic activities.10,11 Based on a report by Sun et al., norgestrel and progesterone were two of 15 steroidal hormones most used in the Beijing area of China.7 Norgestrel and progesterone are the representatives of the synthetic and natural progestagens, having been detected in municipal wastewaters3,12,13 and livestock wastes,14,15 due to natural excretion by humans and animals and drug application in © XXXX American Chemical Society

various treatments. The reported concentrations of norgestrel and progesterone ranged from 0.99 and the amplification efficiency of 95−110%. Isolation of Norgestrel- and Progesterone-Degrading Bacteria. Enrichment culturing was used to isolate the progestagens-degrading bacteria from the activated sludge with a previously reported method.19,30 The isolation of degrading bacteria was done from an enriched culture obtained through seven transfers into fresh medium. Three replicates (C1, C2, and C3) of the same incubation solutions as mentioned above were used for enrichment culturing of the degrading bacteria for the two target progestagens. After 40 d of enrichment culturing under aerobic and dark conditions at 25 °C, the flask media were used as sources for the isolation of progesterone- and norgestrel-degrading bacterial strains. A 100μL aliquot of the enrichment culture containing approximately 1.5 mg/L progesterone or norgestrel was streaked on nutrient agar plates. The plates were incubated at 25 °C in the dark. After several streakings, morphologically distinct colonies were selected and tested for their ability to degrade progesterone and norgestrel. The isolates showing degradation abilities were further identified by 16S rDNA gene sequencing; detailed procedures provided in the SI. DNA Extraction, Sequencing, and Phylogenetic Tree Construction. Genomic DNA of each isolate was extracted using a TIANamp Bacteria DNA Kit (TIANGEN, China) in accordance with the manufacturer’s instructions. Bacterial universal primers 27F (5′-AGAGTTTGATCCTGGCTCAG3′) and 1492R (5′-GGTTACCTTGTTACGACTT-3′) were used for the extraction and sequencing of 16S rDNA. The PCR assays were carried out in 25 μL of reaction mixture containing: 2 μL of template DNA, 1x PCR buffer (TaKaRa, Japan), 0.2 mM dNTP mix (TaKaRa, Japan), 1.5 mM MgCl2 (TaKaRa, Japan), 2.5 units of Taq polymerase (TaKaRa, Japan), 0.4 μM each primer and distilled water. Amplifications were carried out by denaturing at 94 °C for 5 min, followed by 35 cycles of 94 °C for 30 s, 55 °C for 30 s, 72 °C for 1 min, and a final extension step at 72 °C for 10 min. The PCR product was subjected to sequencing by the Beijing Genomics Institute (BGI, China) and nucleotide sequence comparison was performed by the Basic Local Alignment Search Tool (BLAST) through the National Center for Biotechnology

provided by National Institute of Standards and Technology (Gaithersburg, MD, USA) (NIST05). The RRLC-MS/MS instrument used in this study was an Agilent 1200 LC-Agilent 6460 QQQ with an electrospray ionization (ESI) source. The chromatographic separation was performed on an Agilent Zorbax SB-C18 (100 mm × 3 mm, 1.8 μm) column with its corresponding precolumn filter (2.1 mm, 0.2 μm) at 40 °C. Both of the Scan and MRM methods used water containing formic acid (0.01%, v/v) and methanol as the mobile phase with a flow rate of 0.35 mL/min. The gradient program used in the Scan method was as follows: 60% methanol for 50 min, and post time for 5 min. The gradient for the MRM method was as follows: from 60% to 80% (methanol) in 15 min, then from 80% to 60% (methanol) in 0.5 min, and post time for 5 min. Both methods were performed in positive ionization mode (ESI(+)). The results of the degradation products analysis are shown in Figures S2−S6 and Figures S8−S10 (SI). Microbial Abundance Determination. The live microbial abundance in the incubation solutions was reflected by the fluctuations of the copy number of 16S rDNA gene. The solution samples collected from each treatment were immediately stored in a freezer at −18 °C and processed within a short time. Extracted DNA was used as the template in qPCR using the TIANamp Bacteria DNA Kit (TIANGEN, China) in accordance with the manufacturer’s instructions. The 16S rDNA gene was quantified by qPCR with the specific primers listed as follows: forward (P690F) 5′TGTGTAGCGGTGAAATGCG-3′ (690−708 bp); reverse (P829R) 5′-CATCGTTTACGGCGTGGAC-3′ (829−811 bp).29 16S rDNA gene was tested with a 20-μL PCR reaction solution: 2x THUNDERBIRD SYBR qPCR Mix (10 μL), 0.05 mM each primer (0.08 μL), 50x ROX reference dye (0.04 μL), template DNA (2 μL (DNA < 80 ng)), and distilled water (7.8 μL (DNase I treated)). The qPCR assays were run on an Applied Biosystems 7500 Fast Real-Time PCR System (USA). Temperature program for quantification of 16S rDNA genes consisted of initial denaturing at 95 °C for 1 min, followed by 40 cycles for 15 s at 95 °C, 55 °C for 30 s, 72 °C for 30 s, and a final step for melting curve. The external reference method was used to calculate the copy number of 16S rDNA, with the D

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Figure 2. Variation of 16S rDNA during the degradation of nogestrel and progesterone under the aerobic condition.

norgestrel showed an initial increasing trend in the first 2 days and then gradually decreased with time (Figure 2). In the first 2 days, there was a lack of lag phase for norgestrel, demonstrating that the microbial community adapted readily to degrade norgestrel.32 After the first 2 days, the decrease in 16S rDNA abundance could be due to the depletion of available nutrients and carbon source. However, there is a lag phase for progesterone in the first 2 h, suggesting that acclimation is necessary for microorganisms to adapt before the initiation of degradation.33 It should be noted that the incubation times were very different for the two compounds as they had different degradation behaviors (T1/2: 12.5 d for norgestrel and 4.3 h for progesterone). For the synthetic progestagen norgestrel, its aerobic biodegradation followed first order reaction kinetics (Figure 1). But the half-life from the present study was much higher than that found in the only previous study of activated sludge treatment (T1/2 = 1.2 h).13 This could be explained by the different experimental conditions such as different temperatures (e.g., 28 °C in the previous study vs 25 °C in the present study)

Information (NCBI). The phylogenetic tree was constructed using the software program MEGA5. 31 The accession numbers of the sequences in GenBank are from KC189896 to KC189902 for Strains N1−N6 and P1.

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RESULTS AND DISCUSSION Kinetics of Norgestrel and Progesterone Biodegradation. No apparent loss was observed for either norgestrel and progesterone in the sterile controls (Figure 1), indicating that the losses of the two target compounds in the nonsterile treatments under aerobic conditions were due to biological degradation, with little contribution from abiotic degradation. No bacteria were observed in the sterile treatments. The kinetic parameters, including kinetic rate constant (k) and half-life (T1/2), are summarized in Table 1. The copies of 16S rDNA in the nonsterile treatments for norgestrel and progesterone varied from 4.1 × 106 to 9.9 × 108 and from 5.6 × 106 to 5.5 × 107, respectively, indicating the abundance of bacteria in the incubation systems (Figure 2). The abundance of bacteria in the nonsterile treatment for E

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Table 2. Tentative Structures and Relevant Information of the Norgestrel Biodegradation Productsa

a

N/A: not available.

fragment ions at m/z 357.2 (386.3 − CH3CH2) and 319.2 (357.2 − CH−CCH) by GC-EI-MS (SI Figure S4). Product II had a molecular ion (M + 2TMS-2H) at m/z 460.5, and characteristic fragment ion at m/z 431.3 (460.5 − CH3CH2) by GC-EI-MS (SI Figure S4). Product III had a molecular ion (M + TMS-H) at m/z 382.2, and characteristic fragment ion at m/z 353.1 (382.2 − CH3CH2) by GC-EI-MS (SI Figure S4). The same molecular ions (M + H) for the three products could also be found by LC-ESI(+)-MS/MS with scan mode at m/z 315, 317 and 311, respectively (SI Figure S2). In addition, hydrogenation and dehydrogenation processes could easily explain the biodegradation mechanism. Product IV was also found by GC-MS but not identified in the present study due to the low matching degree. Based on the peak areas, the yields for the four products (I, II, III, and IV) were estimated and are shown in SI Table S2. After 58 d, products I and II were still in the medium with high residual levels (15.0% for product I and 21.5% for product II) (SI Table S2). To the best of our knowledge, only two previous studies reported that norgestrel could be transformed by microorganism via hydroxylation reactions at different positions.36,37 This is the first report on the biodegradation products of norgestrel by bacteria from aerobic activated sludge. For progesterone, eight biodegradation products were found and seven of them were identified in the present study (Table 3) by LC-MS/MS scan, LC-MS/MS MRM, and GC-MS methods. Five products (1−4 and 8), including four androgens (androsta-1,4-diene-3,17-dione (ADD), 4-androstene-3,17dione (AED), testosterone (T), 17β-boldenone (17β-BOL)), and 3,20-allopregnanedione were confirmed with the authentic standards (Table 3 and SI Figure S5). These products (1−4) were also quantitatively detected by LC-MS/MS MRM method16 with the authentic standards as shown in SI Figure S6. Products 6, 7, and 8 were tentatively identified based on the mass spectra from GC-MS analyses (SI Figures S3 and S5 and Table 3). The molecular ions for the two products 6 and 7 in

and different media. The previous study simulated activated sludge treatment, while the present study only used activated sludge as inoculum. After 25 days’ incubation, the nutrients were probably depleted, which resulted in low microbial abundances and slow degradation of norgestrel from day 26 to day 49. Natural progestagen progesterone was found biologically degraded within 9 h under aerobic conditions (Figure 1). This is consistent with previous studies on microbial degradation of progesterone.13,25−27 The first order reaction kinetic model was often used to describe the biodegradation of progesterone in activated sludge and animal manure.13,25 In the present study, the fits of both zero order reaction kinetics and first order reaction kinetics for progesterone degradation data were significant (p < 0.0001) and had the r2 values of 0.964 for the zero order model and 0.749 for the first order model (Table 1 and Figure 1). In comparison, it is apparent that the zero order kinetic model explained the degradation data significantly better than the first order kinetic model. Based on the degradation results from the present study, natural progesterone could be easily removed in real WWTP with a hydraulic retention time (HRT) of only 8 h for aerobic tanks, while synthetic norgestrel would be difficult to remove as it behaves like other synthetic steroids such as EE2.32,34,35 It is expected that norgestrel would also be more persistent than progesterone in the receiving environment. Biodegradation Products of Norgestrel and Progesterone. For norgestrel, four biodegradation products (products I−IV) were found, and three of them (products I−III), i.e. 4,5dihydro-norgestrel, 3α,5β-tetrahydro-norgestrel, and 6,7-dehydro-norgestrel,were identified based on the mass spectra from GC-MS analyses of the derivatized extracts, and further confirmed using the AMDIS and NIST05 database searching program (Table 2 and SI Figure S2). After conversion into their trimethylsilyl (TMS, m/z 73.0) derivatives, product I had a molecular ion (M + TMS-H) at m/z 386.3, and characteristic F

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Table 3. Tentative Structures and Relevant Information of the Progesterone Biodegradation Products

the GC-EI-MS were found both at m/z 312. The molecular ions for products 6 and 7 were also found by LC-MS/MS with scan mode (SI Figure S3). Product 5 was found by LC-ESI(+)MS/MS scan mode, but not identified in the present study due to limited data available. Proposed Biotransformation Pathways for Norgestrel and Progesterone. Apparently, most norgestrel was transformed into product I (63.2%) in the first 15 days via hydrogenation at C4 and C5 of Ring-A (SI Table S2 and Figure S4). During the 15th to 43rd day, the yield of product I decreased from 63.2% to 17.1%, while that for product II, III, or IV slightly increased. Product I could be converted into product II (3α,5β-tetrahydro-norgestrel) via hydrogenation at C3 position of A-ring (Figure 3). For product IV, it is difficult to identify its structure and biotransformation pathway due to limited data available. At 43rd day, product III was determined (6.6%), suggesting that the residual norgestrel was transformed

into 6,7-dehydro-norgestrel (product III) via dehydrogenization at C6 and C7 position of Ring-B (Figure 3). Biodegradation experiments by the isolated degrading bacteria (N1−N6) were also performed in the present study. For norgestrel, three degradation products (I−III) were also found in the degradation experiments by the isolated degrading bacterial strains N1, N3, N5, and N6 (SI Figures S8, S10 and Table S8). Based on the estimated yields of the transformation products of norgestrel, the yields of product III (30.4−46.0%) were much higher than those of the other products (3.5−9.1%), suggesting that most norgestrel was transformed into 6,7dehydro-norgestrel (product III) via dehydrogenization at C6 and C7 position of Ring-B. Based on the identified biodegradation products, four biotransformation pathways were tentatively proposed for progesterone (Figure 4). The biotransformation of the first two pathways happened at 17β position at ring-D. Progesterone was transformed via the first pathway into 17α-hydroxyprogesG

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Figure 3. Proposed pathways for the biotransformation of norgestrel (NGT) under aerobic conditions. The biotransformation products of NGT were tentatively identified by GC-MS, and further confirmed by using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) and Version 05 of mass spectral library provided by National Institute of Standards and Technology (Gaithersburg, MD, USA) (NIST05). Product I, 4,5-dihydro-norgestrel; Product II, 3α,5β-tetrahydro-norgestrel; product III, 6,7-dehydro-norgestrel; product IV, unknown.

Figure 4. Proposed pathways for the biotransformation of progesterone (P) under aerobic condition. The biodegradation products of P were tentatively identified by GC-MS (Products 6−8) further confirmed by using the Automated Mass Spectral Deconvolution and Identification System (AMDIS) and Version 05 of mass spectral library provided by National Institute of Standards and Technology (Gaithersburg, MD, USA) (NIST05), and LC-MS/MS (Products 1−4) based on our previous study.16 17α-OHP, 17α-hydroxyprogesterone; TAC, testosterone acetate; Product 1, ADD, androsta-1,4-diene-3,17-dione; Product 2, 17β-BOL, 17β-boldenone; Product 3, AED, 4-androstene-3,17-dione; Product 4, T, testosterone; Product 6, 6,7-dehydroprogesterone; Product 7, 1,2-dehydroprogesterone; Product 8, 3,20-allopregnanedione. References: a, Jenkins et al.;38 b, Hunter et al.;27 c, Hunter et al.;39 d, Yang et al.;25 e, Egorova et al.;21 f, Al-Awadi et al.;40 g, Haridy et al.;41 h, Stanczyk et al.9.

H

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degrade steroids, not even norgestrel. Thus, this is the first report of the norgestrel-degradation ability of Aeromonas hydrophila. Strain N3 was identified as Pseudomonas monteilii. Pseudomonas sp. could degrade many organic pollutants, such as nitrobenzene,53 chlorpyrifos,54 phenol,55 and bisphenol A.56 Pseudomonas monteilii isolated from a polluted water sample was found very effective in degradation of chlorpyrifos.54 Strains N4 and P1 were identified as Comamonas testosteroni, indicating that both progestagens in the present study could be degraded by Comamonas testosteroni. This is also supported by the fact that steroid degradation by Comamonas testosteroni has been observed during steroid drug synthesis.57 The bacteria degraded the side chains and converted the single/double bonds of some steroids to produce androsta-1,4-diene-3,17dione (ADD) or its derivatives.57 This further supports the detection of ADD in the biodegradation of progesterone in the present study. Studies about the degradation of pollutants by Chryseobacterium indologenes (strain N6) were limited. It was reported that Chryseobacterium sp. isolated from biologically active carbon could degrade geosmin in drinking water,58 and it also could effectively degrade methyl tert-butyl ether (MTBE) and tert-butyl alcohol (TBA).59 Exiguobacterium sp. (including Exiguobacterium acetylicum (strain N5)) has been reported to be involved in the biodegradation of azo dye under high salt conditions.60,61 Environmental Implications. Based on the results from the present study, norgestrel is expected to be more recalcitrant to biological degradation than progesterone in activated sludge treatment plants and receiving environments. In various activated sludge treatment plants, norgestrel was found to have low removal rates.3,12,13 A higher hydraulic retention time may be applied to increase its removal in the aerobic treatment unit of a plant. In fact, due to incomplete removals in WWTPs, norgestrel and progesterone have been detected in sewage effluents and receiving waters at several to hundreds of ng/L levels.12,13,16 The results on their biodegradation kinetics and products from the present study could help to understand their environmental fate and potential ecological effects. The isolated bacterial strains may be used to enhance the biodegradation of the two compounds in a contaminated site. Recent laboratory studies have showed that norgestrel and progesterone could affect fish reproduction and hormone receptor gene expressions at ng/L levels.62−64 Besides progestagenic activity, synthetic norgestrel could also exhibit androgenic activity.10,11 In addition to the parent compounds, those biodegradation products might have different biological effects. For example, the four androgens (Products 1−4) generated from progesterone could cause masculization of fish.65 Further research is required to understand the ecotoxicological impacts due to the presence of the two progestagens and their degradation products in the aquatic environment.

terone (17α-OHP), and subsequently into AED and ADD. This could be supported by the previous study of Jenkins et al.,38 who found the same transformation by bacteria. Based on previous studies about steroids biodegradation,21,25,27,38,39 progesterone could be transformed into testosterone acetate (TAC) and four identified products (1−4) T, 17β-BOL, AED, and ADD via the second pathway, where these products could be converted to one another (Figure 4). Compared with the residual progesterone in the media, the concentrations of these four detected steroids (products 1−4) by LC-MS/MS,16 were very low (below 14 μg/L), reaching a peak at 5.7 h, then decreasing gradually (SI Figure S6), indicating that only a relatively low percentage of progesterone was degraded into these androgens, where they were also easily degraded under aerobic conditions. In the third pathway, two dehydro products (1,2-dehydroprogesterone, and 6,7-dehydroprogesterone) were found. Dehydro product has been reported in a study by AlAwadi et al.,40 in which they found that progesterone was transformed into 6,7-dehydroprogesterone by Bacillus stearothermophilus. Progesterone was converted into 3,20-allopregnanedione via hydrogenation in the fourth pathway and the same product could be detected in the previous studies.9,41 In the biodegradation of progesterone by the isolated bacterial strain (P1), only five products (1−4 and 7) were identified (SI Figure S9 and Table S7). It is apparent that the concentration of product 1 was much higher than the other products, indicating that most progesterone was transformed via the first pathway into 17α-hydroxyprogesterone (17αOHP), and subsequently into AED and ADD. Norgestrel- and Progesterone-Degrading Bacteria. Six norgestrel-degrading bacteria (N1−N6) and one progesteronedegrading bacterium (P1) were successfully isolated from the enrichment culture inoculated with aerobic activated sludge. To the best of our knowledge, this is the first report of these six norgestrel-degrading bacteria (N1−N6). Taxonomic analyses based on the 16S rDNA gene sequences revealed that these seven isolates belong to six different genera (Enterobacter, Aeromonas, Pseudomonas, Comamonas, Chryseobacterium, and Exiguobacterium) of two Phyla: Proteobacteria and Firmicutes. Strains P1 and N4 belong to β-Proteobacteria, and N1−N3 and N6 belong to γ-Proteobacteria. Strain N5 belongs to Bacilli. All isolates showed >99% homology in 16S rDNA gene sequences of the selected known bacteria (shown in SI Figure S7). Strain N1 Enterobacter ludwigii has been described as a novel species of clinical relevance42 and also a plant-associated strain with plant beneficial capacities.43 González et al.44 found that the Enterobacter group, including Enterobacter ludwigii, was the dominant bacteria during polycyclic aromatic hydrocarbons (PAHs) degradation in a petroleum polluted soil. Pau-Roblot et al.45 showed that Enterobacter ludwigii was able to produce a high-molecular-mass exopolysaccharide to decrease Cd sequestration in flax seeds. However, little is known about its capability to degrade organic chemicals such as steroids. To our knowledge, only Yousaf et al.46 reported that Enterobacter ludwigii was able to degrade alkanes. Thus, the present study first reported Enterobacter ludwigii with the capability to degrade norgestrel. Strain N2 is Aeromonas hydrophila subsp. dhakensis. Diverse Aeromonas spp. displaying putative virulence traits and distinct antibiotic resistance features were frequently present in wastewaters.47 According to previous studies, Aeromonas hydrophila is able to degrade azo dyes,48,49 diesel oil,50 and quaternary ammonium compounds, 51 and reduce bromate. 52 No previous study has reported that it could

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ASSOCIATED CONTENT

S Supporting Information *

More details on chemicals, sample extraction and analysis, isolation of single degrading bacteria, biodegradation experiments by the isolated bacteria, chromatograms of the two progestagens and their degradation products, phylogenetic tree of the isolated degradation bacteria, and basic information on the selected wastewater treatment plant. This information is available free of charge via the Internet at http://pubs.acs.org/. I

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ionization tandem mass spectrometry. J. Chromatogr. A 2011, 1218, 1367−1378. (17) Ternes, T. A.; Kreckel, P.; Mueller, J. Behaviour and occurrence of estrogens in municipal sewage treatment plants - II. Aerobic batch experiments with activated sludge. Sci. Total Environ. 1999, 225 (1−2), 91−99. (18) Barr, W. J.; Yi, T.; Aga, D.; Acevedo, O.; Harper, W. F. Using electronic theory to identify metabolites present in 17α-ethinylestradiol biotransformation pathways. Environ. Sci. Technol. 2012, 46, 760− 768. (19) Li, Z.; Nandakumar, R.; Madayiputhiya, N.; Li, X. Proteomic analysis of 17β-estradiol degradation by Stenotrophomonas maltophilia. Environ. Sci. Technol. 2012, 46, 5947−5955. (20) Zheng, W.; Li, X.; Yates, S. R.; Bradford, S. A. Anaerobic transformation kinetics and mechanism of steroid estrogenic hormones in dairy lagoon water. Environ. Sci. Technol. 2012, 46, 5471−5478. (21) Egorova, O. V.; Nikolayeva, V. M.; Sukhodolskaya, G. V.; Donova, M. V. Transformation of C-19-steroids and testosterone production by sterol-transforming strains of Mycobacterium spp. J. Mol. Catal. B-Enzym. 2009, 57, 198−203. (22) Bradley, P. M.; Barber, L. B.; Chapelle, F. H.; Gray, J. L.; Kolpin, D. W.; McMahon, P. B. Biodegradation of 17 beta-estradiol, estrone and testosterone in stream sediments. Environ. Sci. Technol. 2009, 43, 1902−1910. (23) Khan, B.; Lee, L. S.; Sassman, S. A. Degradation of synthetic androgens 17 alpha- and 17 beta-trenbolone and trendione in agricultural soils. Environ. Sci. Technol. 2008, 42, 3570−3574. (24) Chang, H.; Wu, S. M.; Hu, J. Y.; Asami, M.; Kunikane, S. Trace analysis of androgens and progestogens in environmental waters by ultra-performance liquid chromatography-electrospray tandem mass spectrometry. J. Chromatogr. A 2008, 1195 (1−2), 44−51. (25) Yang, Y. Y.; Borch, T.; Young, R. B.; Goodridge, L. D.; Davis, J. G. Degradation kinetics of testosterone by manure-borne bacteria: Influence of temperature, pH, glucose amendments, and dissolved oxygen. J. Environ. Qual. 2010, 39, 1153−1160. (26) Safiarian, M.; Faramarzi, M.; Amini, M.; Soltani, N.; TabatabaeiSameni, M.; Hasan-Beikdashti, M. Microalgal transformation of progesterone by the terrestrial-isolated cyanobacterium Microchaete tenera. J. Appl. Phycol. 2012, 24, 777−781. (27) Hunter, A. C.; Rymer, S. J.; Dedi, C.; Dodd, H. T.; Nwozor, Q. C.; Moghimi, S. M. Transformation of structurally diverse steroidal analogues by the fungus Corynespora cassiicola CBS 161.60 results in generation of 8β-monohydroxylated metabolites with evidence in favour of 8β-hydroxylation through inverted binding in the 9αhydroxylase. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2011, 1811, 1054−1061. (28) Healy, J. B.; Young, L. Y. Anaerobic biodegradation of 11 aromatic-compounds to methane. Appl. Environ. Microbiol. 1979, 38, 84−89. (29) Duan, Q. J.; Shang, S. Q.; Wu, Y. D. Rapid diagnosis of bacterial meningitis in children with fluorescence quantitative polymerase chain reaction amplification in the bacterial 16S rRNA gene. Eur. J Pediat. 2009, 168, 211−216. (30) Kimura, H. S.; Sawada, H.; Omura, H.; Watanabe, H. M.; Takashi Makino, K.; Takeshi Yoshimoto, F. N.; Junji Fujimoto, K. Degradation of estrogens by Rhodococcus zopf ii and Rhodococcus equi isolates from activated sludge in wastewater treatment plants. Appl. Environ. Microbiol. 2004, 70, 5283−5289. (31) Tamura, K.; Peterson, D.; Peterson, N.; Stecher, G.; Nei, M.; Kumar, S. MEGA5: Molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 2011, 28, 2731−2739. (32) Robinson, B. J.; Hellou, J. Biodegradation of endocrine disrupting compounds in harbour seawater and sediments. Sci. Total Environ. 2009, 407, 5713−5718. (33) Hashimoto, T.; Murakami, T. Removal and degradation characteristics of natural and synthetic estrogens by activated sludge in batch experiments. Water Res. 2009, 43, 573−582.

AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86 20 85290200; e-mail: guangguo.ying@gmail. com or [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the support from the National Natural Science Foundation of China (NSFC 41273119, U1133005, and 41121063) and Chinese Academy of Sciences Key Project (KZCX2-EW-108, KZCX2-YW-JC105, and KZZD-EW-09). Thanks to Hao-Chang Su for his assistance during the experiment. This is a Contribution No. 1730 from GIG CAS.

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REFERENCES

(1) Coe, T. S.; Soffker, M. K.; Filby, A. L.; Hodgson, D.; Tyler, C. R. Impacts of early life exposure to estrogen on subsequent breeding behavior and reproductive success in zebrafish. Environ. Sci. Technol. 2010, 44, 6481−6487. (2) Mills, L. J.; Chichester, C. Review of evidence: Are endocrinedisrupting chemicals in the aquatic environment impacting fish populations? Sci. Total Environ. 2005, 343, 1−34. (3) Fan, Z.; Wu, S.; Chang, H.; Hu, J. Behaviors of glucocorticoids, androgens and progestogens in a municipal sewage treatment plant: Comparison to estrogens. Environ. Sci. Technol. 2011, 45, 2725−2733. (4) Ying, G. G.; Kookana, R. S.; Ru, Y. J. Occurrence and fate of hormone steroids in the environment. Environ. Int. 2002, 28, 545−551. (5) Chang, H.; Wan, Y.; Hu, J. Y. Determination and source apportionment of five classes of steroid hormones in urban rivers. Environ. Sci. Technol. 2009, 43, 7691−7698. (6) Qiao, Y. W.; Yang, H.; Wang, B.; Song, J.; Deng, A. P. Preparation and characterization of an immunoaffinity chromatography column for the selective extraction of trace contraceptive drug levonorgestrel from water samples. Talanta 2009, 80, 98−103. (7) Sun, L.; Yong, W.; Chu, X. G.; Lin, J. M. Simultaneous determination of 15 steroidal oral contraceptives in water using solidphase disk extraction followed by high performance liquid chromatography-tandem mass spectrometry. J. Chromatogr. A 2009, 1216, 5416−5423. (8) King RJB, W. M. Assessment of the potency of orally administered progestins in women. Fertil. Steril. 1986, 46, 1062−1066. (9) Stanczyk, F. Z. All progestins are not created equal. Steroids 2003, 68, 879−890. (10) Schindler, A. E.; Campagnoli, C.; Druckmann, R.; Huber, J.; Pasqualini, J. R.; Schweppe, K. W.; Thijssen, J. H. H. Classification and pharmacology of progestins. Maturitas 2003, 46 (Supplement 1), 7− 16. (11) Rozenbaum, H. Les Progestatifs, 2nd ed.; ESKA: Paris, 2001. (12) Liu, S.; Ying, G. G.; Zhao, J. L.; Zhou, L. J.; Yang, B.; Chen, Z. F.; Lai, H. J. Occurrence and fate of androgens, estrogens, glucocorticoids and progestagens in two different types of municipal wastewater treatment plants. J. Environ. Monit. 2012, 14, 482−491. (13) Chang, H.; Wan, Y.; Wu, S.; Fan, Z.; Hu, J. Occurrence of androgens and progestogens in wastewater treatment plants and receiving river waters: Comparison to estrogens. Water Res. 2011, 45, 732−740. (14) Liu, S.; Ying, G. G.; Zhou, L. J.; Zhang, R. Q.; Chen, Z. F.; Lai, H. J. Steroids in a typical swine farm and their release into the environment. Water Res. 2012, 46, 3754−3768. (15) Liu, S.; Ying, G. G.; Zhang, R. Q.; Zhou, L. J.; Lai, H. J.; Chen, Z. F. Fate and occurrence of steroids in swine and dairy cattle farms with different farming scales and wastes disposal systems. Environ. Pollut. 2012, 170, 190−201. (16) Liu, S.; Ying, G. G.; Zhao, J. L.; Chen, F.; Yang, B.; Zhou, L. J.; Lai, H. J. Trace analysis of 28 steroids in surface water, wastewater and sludge samples by rapid resolution liquid chromatography-electrospray J

dx.doi.org/10.1021/es304688g | Environ. Sci. Technol. XXXX, XXX, XXX−XXX

Environmental Science & Technology

Article

(34) Zhou, X. K.; Oleszkiewicz, J. A. Biodegradation of oestrogens in nitrifying activated sludge. Environ. Technol. 2010, 31, 1263−1269. (35) Ying, G. G.; Toze, S.; Hanna, J.; Yu, X. Y.; Dillon, P.; Kookan, R. S. Decay of endocrine-disrupting chemicals in aerobic and anoxic groundwater. Water Res. 2008, 42, 1133−1141. (36) Hu, S. H.; Tian, X. F.; Han, G. D. Novel microbial hydroxylation of 13-ethyl-17β-hydroxy-18,19-dinor-17α-pregn-4-en-20-yn-3-one. Steroids 1998, 63, 88−92. (37) Hu, S. H.; Tian, X. F.; Sun, Y. H.; Han, G. D. Microbial hydroxylation of 13-ethyl-17β-hydroxy-18,19-dinor-17α-pregn-4-en20-yn-3-one. Steroids 1996, 61, 407−410. (38) Jenkins, R. L.; Wilson, E. M.; Angus, R. A.; Howell, W. M.; Kirk, M.; Moore, R.; Nance, M.; Brown, A. Production of androgens by microbial transformation of progesterone in vitro: A model for androgen production in rivers receiving paper mill effluent. Environ. Health Perspect. 2004, 112, 1508−1511. (39) Hunter, A. C.; Elsorn, J.; Ross, L.; Barrett, R. Ring-B functionalized androst-4-en-3-ones and ring-C substituted pregn-4en-3-ones undergo differential transfonnation in Aspergillus tamarii KITA: Ring-A transformation with all C-6 substituted steroids and ring-D transfonnation with C-11 substituents. Biochim. Biophys. Acta Mol. Cell Biol. Lipids 2006, 1761, 360−366. (40) Al-Awadi, S.; Afzal, M.; Oommen, S. Studies on Bacillus stearothermophilus: Part II. Transformation of progesterone. J. Steroid Biochem. Mol. Biol. 2002, 82, 251−256. (41) Haridy, M. S. A.; Hegazy, M. E. F.; Mohamed, A. H.; Pare, P. W.; Hirata, T. Biotransformation of progesterone by cultured cells of Marchantia polymorpha. Z. Naturforsch., C: J. Biosci. 2010, 65, 599− 602. (42) Hoffmann, H.; Stindl, S.; Stumpf, A.; Mehlen, A.; Monget, D.; Heesemann, J.; Schleifer, K. H.; Roggenkamp, A. Description of Enterobacter ludwigii sp. nov., a novel Enterobacter species of clinical relevance. Syst. Appl. Microbiol. 2005, 28, 206−212. (43) Khan, S.; Afzal, M.; Iqbal, S.; Khan, Q. M. Plant-bacteria partnerships for the remediation of hydrocarbon contaminated soils. Chemosphere 2013, 90, 1317−1332. (44) González, N.; Simarro, R.; Molina, M. C.; Bautista, L. F.; Delgado, L.; Villa, J. A. Effect of surfactants on PAH biodegradation by a bacterial consortium and on the dynamics of the bacterial community during the process. Bioresour. Technol. 2011, 102, 9438− 9446. (45) Pau-Roblot, C.; Lequart-Pillon, M.; Apanga, L.; Pilard, S.; Courtois, J.; Pawlicki-Jullian, N. Structural features and bioremediation activity of an exopolysaccharide produced by a strain of Enterobacter ludwigii isolated in the Chernobyl exclusion zone. Carbohydr. Polym. 2013, 93, 154−162. (46) Yousaf, S.; Afzal, M.; Reichenauer, T. G.; Brady, C. L.; Sessitsch, A. Hydrocarbon degradation, plant colonization and gene expression of alkane degradation genes by endophytic Enterobacter ludwigii strains. Environ. Pollut. 2011, 159, 2675−2683. (47) Carvalho, M. J.; Martínez-Murcia, A.; Esteves, A. C.; Correia, A.; Saavedra, M. J. Phylogenetic diversity, antibiotic resistance and virulence traits of Aeromonas spp. from untreated waters for human consumption. Int. J. Food Microbiol. 2012, 159, 230−239. (48) Hsueh, C. C.; Chen, B. Y.; Yen, C. Y. Understanding effects of chemical structure on azo dye decolorization characteristics by Aeromonas hydrophila. J. Hazard. Mater. 2009, 167, 995−1001. (49) Chen, B. Y.; Lin, K. W.; Wang, Y. M.; Yen, C. Y. Revealing interactive toxicity of aromatic amines to azo dye decolorizer Aeromonas hydrophila. J. Hazard. Mater. 2009, 166, 187−194. (50) Kaczorek, E.; Urbanowicz, M.; Olszanowski, A. The influence of surfactants on cell surface properties of Aeromonas hydrophila during diesel oil biodegradation. Colloids Surf., B 2010, 81, 363−368. (51) Patrauchan, M. A.; Oriel, P. J. Degradation of benzyldimethylalkylammonium chloride by Aeromonas hydrophila sp K. J. Appl. Microbiol. 2003, 94, 266−272. (52) Davidson, A. N.; Chee-Sanford, J.; Lai, H. Y.; Ho, C. H.; Klenzendorf, J. B.; Kirisits, M. J. Characterization of bromate-reducing

bacterial isolates and their potential for drinking water treatment. Water Res. 2011, 45, 6051−6062. (53) Lu, W.; Yan, X.; Zhang, J.; Li, S. Isolation, identification and characteristics of nitrobenzene-degrading bacterium XY-1. Chin. J. Appl. Environ. Biol. 2011, 17, 243−246. (54) Li, X. H.; Jiang, J. D.; Gu, L. F.; Ali, S. W.; He, J.; Li, S. P. Diversity of chlorpyrifos-degrading bacteria isolated from chlorpyrifoscontaminated samples. Int. Biodeterior. Biodegrad. 2008, 62, 331−335. (55) Cao, J. W.; Dong, C. M.; Cao, H. B.; Shao, Z. Z. Isolation of phenol-degrading bacteria from coking wastewater and their degradation gene. Chin. J. Environ. Sci. 2011, 32, 560−6. (56) Masuda, M.; Yamasaki, Y.; Ueno, S.; Inoue, A. Isolation of bisphenol A-tolerant/degrading Pseudomonas monteilii strain N-502. Extremophiles 2007, 11, 355−362. (57) Horinouchi, M.; Hayashi, T.; Kudo, T. Steroid degradation in Comamonas testosteroni. J. Steroid Biochem. Mol. Biol. 2012, 129, 4−14. (58) Zhou, B.; Yuan, R.; Shi, C.; Yu, L.; Gu, J.; Zhang, C. Biodegradation of geosmin in drinking water by novel bacteria isolated from biologically active carbon. J. Environ. Sci. 2011, 23, 816−823. (59) Zhang, R. L.; Huang, G. Q.; Lian, J. Y.; Li, X. G. Degradation of MTBE and TEA by a new isolate from MTBE-contaminated soil. J. Environ. Sci. 2007, 19, 1120−1124. (60) Tan, L.; Qu, Y. Y.; Zhou, J. T.; Li, A.; Gou, M. Identification and characteristics of a novel salt-tolerant Exiguobacterium sp. for azo dyes decolorization. Appl. Biochem. Biotechnol. 2009, 159, 728−738. (61) Chen, B. Y.; Hsueh, C. C.; Chen, W. M.; Li, W. D. Exploring decolorization and halotolerance characteristics by indigenous acclimatized bacteria: Chemical structure of azo dyes and dose− response assessment. J. Taiwan Inst. Chem. Eng. 2011, 42, 816−825. (62) Zeilinger, J.; Steger-Hartmann, T.; Maser, E.; Goller, S.; Vonk, R.; Länge, R. Effects of synthetic gestagens on fish reproduction. Environ. Toxicol. Chem. 2009, 28, 2663−2670. (63) Zucchi, S.; Castiglioni, S.; Fent, K. Progestins and antiprogestins affect gene expression in early development in zebrafish (Danio rerio) at environmental concentrations. Environ. Sci. Technol. 2012, 46, 5183−5192. (64) DeQuattro, Z. A.; Peissig, E. J.; Antkiewicz, D. S.; Lundgren, E. J.; Hedman, C. J.; Hemming, J. D. C.; Barry, T. P. Effects of progesterone on reproduction and embryonic development in the fathead minnow (Pimephales promelas). Environ. Toxicol. Chem. 2012, 31, 851−856. (65) Jenkins, R.; Angus, R. A.; McNatt, H.; Howell, W. M.; Kemppainen, J. A.; Kirk, M.; Wilson, E. M. Identification of androstenedione in a river containing paper mill effluent. Environ. Toxicol. Chem. 2001, 20, 1325−1331.

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NOTE ADDED AFTER ASAP PUBLICATION The structures for norgestrel and progesterone in Table 1 were transposed in the version of this paper published on August 27, 2013. The correct version published August 29, 2013.

K

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