Removal of Natural Estrogens and Their Conjugates in Municipal

Critical Review pubs.acs.org/est

Removal of Natural Estrogens and Their Conjugates in Municipal Wastewater Treatment Plants: A Critical Review Ze-hua Liu,*,†,‡ Gui-ning Lu,†,‡ Hua Yin,†,‡ Zhi Dang,†,‡ and Bruce Rittmann§ †

College of Environment and Energy, South China University of Technology, Guangzhou 510006, Guangdong China Key Lab Pollution Control & Ecosystem Restoration in Industry Cluster, Ministry of Education, Guangzhou 510006, Guangdong China § Swette Center for Environmental Biotechnology, Biodesign Institute at Arizona State University, Tempe, Arizona 85287-5701, United States ‡

ABSTRACT: This article reviews studies focusing on the removal performance of natural estrogens in municipal wastewater treatment plants (WWTPs). Key factors influencing removal include: sludge retention time (SRT), aeration, temperature, mixed liquor suspended solids (MLSS), and substrate concentration. Batch studies show that natural estrogens should biodegrade well; however, batch observations do not always agree with observations from full-scale municipal WWTPs. To explain this discrepancy, deconjugation kinetics of estrogen conjugates in lab-scale studies were examined and compared. Most estrogen conjugates with slow deconjugation rates are unlikely to be easily removed; others could be cleaved in WWTP settings. Nevertheless, some estrogens cleaved from their conjugates may be found in treated effluent, because deconjugation requires several hours or longer, and there is insufficient rest time for the biodegradation of the cleaved natural estrogens in the WWTP. Therefore, WWTP removals of natural estrogens are likely to be underestimated when estrogen conjugates are present in raw wastewater. This review suggests that biodeconjugation of estrogen conjugates should be enhanced to more effectively remove natural estrogens in WWTPs.

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INTRODUCTION Endocrine disrupting compounds (EDCs) have received attention in the past decades due to their adverse environmental effects.1−5 EDCs include a wide range of chemicals, including natural estrogens/androgens, phytoestrogens, mycoestrogens, synthetic estrogens/androgens, progestins, and industrial chemicals. Among these, natural estrogens, estrone (E1), 17β-estradiol (E2) and estriol (E3) are among the most studied because of their strong estrogenic potencies.1,6−8 The two main sources of these natural estrogens are the human and animal excretions, out of which human excretion is the main source to municipal wastewater.84−88 Therefore, effective natural estrogen removal in municipal WWTPs is an important step to prevent their entry into the natural environment. Many studies have investigated key factors influencing estrogen removal, in both on-site settings and lab-scale batch experiments. Factors including sludge retention time (SRT), hydraulic retention time (HRT), temperature, and configurations are often addressed in on-site full-scale WWTP investigations. Batch studies often focus on natural estrogen biodegradability. Insights from these batch studies may be useful as most operational parameters are controllable and set constant, and results from different sources can be reasonably comparable. It is true natural estrogen biodegradation in batch experiments © 2015 American Chemical Society

may overestimate or underestimate true biodegradation rates in real wastewater, as batch results are obtained under controlled conditions (stable temperature, enhanced aeration, and relatively higher initial target concentrations). However, natural estrogen biodegradability data from lab studies are good indicators of their removal in real wastewater. Many lab-scale studies are available, but there are few summaries and comparisons. Most full-scale WWTP studies have only focused on natural estrogens, with estrogen conjugates rarely addressed. Natural estrogen conjugates differentiate them from their corresponding natural estrogens are that they have sulfate, and/ or glucuronide functional groups (Figure 1), and these conjugates can be deconjugated to their corresponding natural estrogens with arylsulfatase or β-glucuronidase derived from microorganisms or organs of higher organisms.76 With the two different functional groups, these conjugates possess two different properties over their natural estrogens: (1) the polarities of natural estrogen conjugates are stronger than those of natural estrogens, which makes them more favorable for eliminating from human and animal bodies;81,89 (2) the Received: Revised: Accepted: Published: 5288

January 23, 2015 April 2, 2015 April 6, 2015 April 6, 2015 DOI: 10.1021/acs.est.5b00399 Environ. Sci. Technol. 2015, 49, 5288−5300

Critical Review

Environmental Science & Technology

Figure 1. Natural estrogens and their typical conjugates.

estrogenic activities of estrogen conjugates are much weaker when they are compared to natural estrogens.90 The conjugation of natural estrogens in human and animal acts as potential “detoxification” reactions that may protect the cell from estrogen-mediated mitogenicity and mutagenesis.91 Not limited to natural estrogens only, nonpolar organic compounds such as phytoestrogens, mycoestrogens, bisphenol A, nonylphenol, and etc., would end up with their corresponding conjugates once they enter the bodies of humans and animals.6,7,92−95 As early as 1930, it was reported that acidification of human urine for several days before the extraction would give a much greater yield of natural estrogens, suggesting the existence of natural estrogen conjugates.76 The estrogen glucuronide was the first to be isolated and confirmed in 1935, while potassium salt of estrogen sulfate was first isolated from a pregnant mare in 1938.76 Since then, natural estrogens have been well-known as predominantly conjugated in urine. Therefore, these estrogen conjugates likely exist in raw municipal wastewater. This judgment has confirmed by some studies, which have proven that estrogen conjugates not only exist in raw municipal wastewater, but also in some treated effluents.6,9−11 Estrogen conjugates in wastewater are first deconjugated into their free estrogens, and then biodegraded. Thus, the presence of estrogen conjugates in raw wastewater likely affects the normalized natural estrogen removal performance of WWTPs. This is an interesting scientific topic but has not been addressed yet. Similar to natural estrogens, estrogen conjugate biodegradability in lab-scale studies should be good indicator of their fate in real wastewater. Given the similar controlled conditions of batch studies, the overestimation or underestimation of natural estrogens and their conjugates in lab-scale studies should show similar trends; thus, biodegradation differences in batch studies should mirror differences in real wastewater. Given this background, this review’s main objectives are to (1) summarize natural estrogen removal performances by full-scale municipal WWTPs; (2) summarize natural estrogen biodegradation rate constants based on batch studies from different sources, and

discuss key factors influencing WWTP removal; (3) summarize estrogen conjugate biodegradation in batch studies, with a comparison of conjugate biodegradability with natural estrogen biodegradability; and (4) summarize conclusions and suggest important scientific questions for future study.

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NATURAL ESTROGEN REMOVAL BY FULL-SCALE MUNICIPAL WWTPS To provide a complete picture of natural estrogen removal by full-scale municipal WWTPs, data from previously published studies were thoroughly collated. As Table 1 shows, removals vary greatly. The removal efficiencies of E1, E2, and E3 were −477 to 100% (average 37.8%), 0 to 100% (average 75.9%), and −175 to 100% (average 74.8%), respectively. Many factors likely affect WWTP removal performance, including wastewater constitutes, treatment configurations, and operational conditions (SRT, HRT, temperature, and other factors). Based on investigations at 23 WWTPs, biological filter plants (BFP) showed a lower removal of E1 than an activated sludge process (ASP).12 This may result from a shorter HRT than that of ASP. This outcome was confirmed by Gabet-Giraud et al.,13 based on an investigation of 14 WWTPs. However, natural estrogen removal rates showed no observed statistical relationship with HRT for 20 WWTPs in another investigation.14 Investigations by Nakada et al.15 indicated that natural estrogen removal performance was better in the summer (26.4 °C) than in the winter (16.4 °C). This suggested that higher temperatures favor WWTP elimination of natural estrogens. This trend was also observed by Gabet-Giraud et al.,13 who confirmed that operation at 20 °C always resulted in better and stable natural estrogen removal than those operated at 10 °C. Kumar et al.16 and Nie et al.17 found similar results. Johnson et al.,18 however, found no positive correlation between estrogen removal and temperature. A higher SRT should increase natural estrogen removal; Clara et al.19 found that more than 10 days of SRT was needed to remove natural estrogens well. This was confirmed through both full-scale and lab-scale studies. This agrees well with studies by McAdam et al.20 and Petrie et al.21 However, 5289

DOI: 10.1021/acs.est.5b00399 Environ. Sci. Technol. 2015, 49, 5288−5300

Critical Review

Environmental Science & Technology Table 1. Natural Estrogen Removal in WWTPsa target

process

C

ST (°C)

SRT (d)

HRT (h)

LOD (ng/L)

influentp (ng/L)

effluentp (ng/L)

Req (%)

ref

E1

AS AS ASb AS AS AS AS AS AS AS AS AS AS AS AS AS AS ASc ASd SBR TF ASe AS AS ASg SBR AS AS AS/TF/SBR AS/TF AS AS ASj AS AS/ODk AS AS AS AS AS/ODl AS

Germany Germany Germany Brazil Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Italy Canada Canada Canada Canada Canada UK Spain Spain Australia Australia US US US US US Japan Japan Japan Japan Belgium China China China China

−2 – – 20 9−23 9−23 9−23 9−23 9−23 – – – – – – – – – – 11−12 – – – – – – 19 – – – – – – 16−27 12−29 – – – – – –

– – – – – – – – – – – – – – – – – – – 3c 0.48a 3.7b 0.53−1.21 1.53−1.95 2.74−3.86 0.83−4.27 11.7 19.2 20.7 20.8 7.3

57 58 21 58 21 62 21 63 59 60 64 64 64 61 58 58 57 58 58

aerobic aerobic aerobic aerobic aerobic

100 100 100 1 × 106 1.6 × 106

18 18 18 30 25

1000 1000 1000 2700 192−224

0.34 0.33 0.24 0.011a 0.775b

8 8 8

10 12 30 35 30 12 E2

35 30 27 12 10 7−10 3

12−48

12−48 8 8 4 8

10 10 10 6

E3

a

30 12 35 30 12

12−48

27 10 3

8 8 8

16 16 16 16 16

30 25 5 20 35

(0.80) (1.78) (3.33) (2.32)

21 21 21 59 60

Nitrifying activated sludge with nitrifying medium. bNatural estrogens as the sole carbon sources. cTemperature.

collected from a nitrifying activated sludge process.59 Based on these facts, nitrifying bacteria may achieve lower biodegradation performance than average activated sludge biomass. Prolonged SRT operational conditions may favor the accumulation of slow growing bacteria, such as nitrifying bacteria. If the hypothesis is correct that increased nitrifying bacteria would not enhance natural estrogen biodegradation, there may be some other unknown slow growing bacteria present in the activated biomass, and its existence may enhance natural estrogen biodegradation. More research is needed to explore this possibility. Second, natural estrogen biodegradation in aerobic conditions is far larger than in anaerobic conditions. Anderson et al.57 found that the kbio values of E1 and E2 were 16.9 and 32 L/gSS/h in aerobic conditions; corresponding values in anaerobic conditions were 0.58 and 20.7 L/gSS/h. A study by Joss et al. 58 found similar results. However, E2

and activated sludge at an increased SRT (Figure 2). This conclusion aligns with on-site full-scale investigations indicating that an increased SRT removed natural estrogens better.19,20 Petrie et al.21 studied the biodegradation of three natural estrogens, with nitrifying biomasses at three different SRTs. One data point at an SRT of 27 days, with much lower biodegradation rate constants was not included in Figure 2 as the studied microbial community was far from the activated sludge. The authors also noticed the much lower biodegradation rate constants in their study, and they suggested it might result from the higher starting ratio of substrate to SS. The biodegradation rate constants found by Shi et al.59 were similar to those of Petrie et al.,21 who also used nitrifying activated sludge for the biodegradation experiment. The authors demonstrated that the pure biomass of ammoniaoxidizing bacterium Nitrosomonas europaea showed lower biodegradation rates than those of the nitrifying biomass 5293


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Finally, natural estrogen biodegradation rates at different MLSS and initial target concentrations are mixed. Chen and Hu69 found that biodegradation rates increased with the increase of initial substrate concentration and MLSS. Li et al.64 found similar results; however, Suzuki and Maruyama70 reported no significant difference in degradation between low and high MLSS loading. These different observations may result from different initial concentrations, as the initial concentrations of natural estrogens ranged from 10 to 200 μg/L in the first two studies, but was only approximately 20 ng/L in the latter. Although natural estrogen biodegradation likely increases with higher MLSS loading at relatively high initial natural estrogen concentrations, the biodegradation rate constants (kbio) appear to be mixed. At low temperatures, operations at higher MLSS loading led to a higher kbio, but at higher temperature, the kbio with lower MLSS loading appeared inversely higher.64 Predicted WWTP Natural Estrogen Removal Efficiencies. It is impossible to equally compare natural estrogen biodegradation between lab batch experiments and full-scale WWTPs, because laboratory operating conditions can be well controlled, while full-scale plants cannot be. However, natural estrogen biodegradability using batch studies is a good reference for comparison with full-scale WWTPs, and reflects biodegradability in full-scale WWTPs to a certain extent. This means that if good biodegradability is seen in a batch study, it would likely indicate good removal in a full-scale WWTP. Based on this hypothesis, natural estrogen removal performance by WWTPs can be estimated with biodegradation rate constants from batch experiments and the HRT of the aeration tank. As Figure 4 shows, based on biodegradation rate constants in Table 2 and in eq 2, it takes from several minutes to no more than 11 h for WWTPs to achieve over 99% removal of natural estrogens. The time required to remove 80% of E1, E2, and E3 may even decrease to less than 2 h. Most WWTPs designed for municipal wastewater have a typical HRT of approximately 4−12 h, suggesting that WWTPs will remove natural estrogens well under regular operations. To validate this, the predicted removal of three natural estrogens plus 17α-ethynyl estradiol (EE2) were compared based on biodegradation rate constants and observed removals in one pilot-scale WWTP, for which the original data was collected from Petrie et al.21 The results in Table 3 show that predicted performances aligns with observed removal performance in the WWTP, except for the removal of E1 at a SRT of 3 days (This discrepancy is addressed below). This result confirms that predictions based on kbio can be effective to evaluate WWTP performance, and that natural estrogens can be easily removed. However, some full-scale WWTP investigations indicate good removal, while some did not. Removal efficiencies ranged from 100% to −477%, and the average removal efficiencies were less than 80% (Table 1). Especially the summarized results showed that the removal performance of the activated sludge treatment process was unstable. One possible reason for this difference between predicted and observed values is stated below.

Figure 2. Relationship between degradation rate constants and different SRT. Data of kbio was adopted from Table 2 study summary.

biodegradation was affected less than E1 when operational conditions changed from aerobic to anaerobic. This may be due to the easy biotransformation of E2 into E1 under different conditions by different microorganisms.65−67 E1 biodegradation rates are greater in aerobic conditions than in anoxic conditions, which were greater than in anaerobic conditions. The E2 biodegradation rate is greater in aerobic conditions than in anaerobic conditions, which was greater than in anoxic conditions. Recent results show that under aerobic conditions, natural estrogen biodegradation is effectively done by biomass, regardless of whether it is derived from activated sludge or digestion sludge. There were no evident differences in biodegradation between different types of derived sludge.60 This indicates the importance of oxygen and some facultative bacteria in biodegradation. Third, increased temperature enhances natural estrogen biodegradation. Temperatures ranging from 5 to 35 °C were used in different studies (summarized in Table 2); it is very difficult to isolate the influence of temperature, as other operational parameters differed across their studies. Li et al.64 compared the influence of temperature on E2 biodegradation; the result showed that biodegradation rate constants linearly increased with increasing temperature (Figure 3). This result aligns well with findings from Layton et al.68

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ESTROGEN CONJUGATE BEHAVIOR IN WASTEWATER Deconjugation Rate Constants. Natural estrogens in urine are mainly in their conjugated forms. Some have proposed these conjugates could be cleaved before WWTP entry,71 however, sophisticated analytical methods have yielded increasing evidence that estrogen conjugates exist in both raw

Figure 3. Biodegradation rate constants of E2 at different temperature range. The original data was collected from Li et al.64 5294


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Figure 4. Predicted time necessary for WWTPs to achieve certain removal based on biodegradation rate constants kbio in Table 2 under aerobic conditions and SS concentration of 3000 mg/L (a: E1; b: E2; c: E3).

Table 3. Comparison between Observed and Estimated Removal Based on the Batch Experiment. The Original Data of Observed Removal and Biodegradation Rate Constant Were Collected from Petrie et al.21 steroidal estrogen

SRT(d)

HRT (h)

biodegradation rate constant (L/ gSS*h)

influent (ng/ L)

effluent (ng/ L)

observed removal (%)

estimated removal (%)

E1

3 10 27

8

0.204 0.346 0.329

92 101 94

75 7.1 15

19 93 84

80 94 93

E2

3 10 27

8

0.142 0.271 0.271

30 29 30

9.7 2.9 2.4

68 90 92

68 89 89

E3

3 10 27

8

0.242 0.325 0.342

30 220 214

17 6.8 6.2

92 97 97

86 93 93

EE2

3 10 27

8

0.067 0.058 0.067

33 20 15

1.3 0.7 0.9

30 29 41

41 37 41

municipal wastewater and some treated effluents.11,72−81 There are two reasons for paying attention to estrogen conjugates with weak estrogenic potencies. First, estrogen conjugates may adversely affect the environment, as they can be biotransformed into their corresponding free estrogens once entering the natural environment. Second, estrogen conjugates in raw

wastewater may indicate an underestimation of WWTP removal on natural estrogens. This aspect is important but is always neglected. Estrogen conjugate biodegradation in WWTPs mainly involves two pathways: (i) deconjugation to corresponding free natural estrogens, followed by (ii) biotransformation of 5295


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Environmental Science & Technology Table 4. Deconjugation Rate Constants of Estrogen Conjugatesa derived on-site sludge target E1−3S E1−3G E3−16G E2−3S E3−3G

SRT(d) 5 5 5 ---

HRT(h) b

------

operation

Ci (ng/L)

Td (°C)

MLSS (g/L)

kbio (L/gSS/h)

ref

aerobic aerobic aerobic aerobic aerobic

2348 2517 2323 5E4 5E4

17 17 17 ---

4 4 4 4−12.5 4−12.5

0.00319 0.0875 0.06 0.0728−0.13 (0.0976)c 0.26−0.42(0.34)c

82 82 82 69 69

a Simultaneous biotransformation of one estrogen conjugate to another kind of estrogen conjugate may occur. b--, Not available. cEstimated from the work, and number in the parentheses mean the mean value. dTemperature.

these free estrogens. The second pathway is the same as with free estrogens, so this discussion focuses on the first step. Like natural estrogens, the deconjugation rates of these conjugates are described in eq 3: dA = −r = −kdec × MLSS × A dt

(3)

In this expression, r, t, A, MLSS, and kdec denote deconjugation rate (ng/L*h), reaction time (hours), concentrations of target conjugates (ng/L), mixed liquor suspended solid concentrations (gSS/L), and deconjugation rate constant (L/gSS·h), respectively. Based on eq 3, we further derive: ⎛A⎞ ln⎜ ⎟ = −kdec × MLSS × t ⎝ A0 ⎠

(4) Figure 5. Predicted estrogen conjugate biodegradation in activated sludge at a MLSS concentration of 3000 mg/L.

Here, A and A0 are the concentrations of estrogen conjugates at time t and zero. Unlike natural estrogens, only limited batch studies are available related to estrogen conjugate biodegradation.9,11,69,82,83 All these batch studies indicated that estrogen glucuronide biodegradation was far faster than estrogen sulfate biodegradation. Table 4 presents the limited available deconjugation rate constants. Biodegradation of Estrogen Conjugates. Estrogen conjugates in wastewater can be cleaved by enzymes, such as arylsulfatase and β-glucuronidase derived from wastewater bacteria; deconjugation is favored at high temperature. Zheng et al.83 showed that the aerobic degradation rate of 17αestradiol-3-sulfate increased approximately 92 times when the operational temperature increased from 15 to 35 °C; there was a corresponding 11 times enhancement in anaerobic operation. This implies that the frequency and concentrations of estrogen conjugates in municipal wastewater would be expected to be higher in colder seasons than in warmer ones. In terms of matrix type, Kumar et al.11 showed that estrogen conjugate degradation in activated sludge was the fastest, followed by in raw sewage, followed by river water. This indicates the important role of activated sludge in deconjugation. However, in Kumar et al’s study,11 the E1−3S and E2−3S degradations were almost complete within 100 min, surprisingly far higher than other studies.9,69,82 The paper by Kumar et al.11 did not include comparisons with other studies. However, the presence of these estrogen sulfate conjugates in both influent and effluent in three fullscale WWTPs, based on on-site investigations, appeared to contradict their batch studies. With the deconjugation rate constants in Table 4 and eq 4, the time required for deconjugation in activated sludge can be predicted. As Figure 5 shows, the deconjugation of the five studied estrogen

conjugates can be categorized into three groups: low, moderate, and high deconjugation rate conjugates. The E1−3S deconjugation rate is very slow, and the deconjugation ratio is only 25% for up to 30 h. The E3−16G deconjugation rate is very fast, and nearly 90% of this conjugate can be cleaved within 2 h. The other three estrogen conjugates had moderate deconjugation rates. For 60% deconjugation, 3−5 h is needed. To achieve 90% deconjugation, 8−13 h are needed. These levels are conversely larger than the time needed to remove 90% of their corresponding natural estrogens. In most cases, WWTP natural estrogen removal efficiency (R) is calculated using eq 5: R=

E in − Eout × 100% E in

(5)

In this expression, Ein and Eout represent natural estrogen concentrations in influent and effluent. However, if estrogen conjugates also exist, R should be calculated using eq 6: R=

E in − Eout + C in − Cout × 100% E in + C in − Cout

(6)

In these equations, Cin and Cout refer to natural estrogen concentrations derived from estrogen conjugates, if the conjugate is 100% deconjugated in raw and treated wastewater. It is clear that R in eq 6 is no less than to R in eq 5. When estrogen conjugates in wastewater do not exist or are negligible, the R in eq 6 is equal to R in eq 5. Two important conclusions can be drawn based on these facts. First, most estrogen conjugates with low deconjugation rates, such as E1−3S in raw wastewater, would more likely 5296


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Environmental Science & Technology remain in the treated effluent, as they resist deconjugation. Second, most estrogen conjugates with a moderate or fast deconjugation rate would be largely cleaved in the WWTP. Some of the cleaved natural estrogens from the parent conjugates likely exist in treated effluent; this deconjugation process would result in an underestimation of the natural estrogen removed by the WWTP. The observed removal of E1 at an SRT of 3 days (Table 3) is lower than the predicted value based on biodegradation rate constant. This may result from the release of E1 from E1 conjugates; E2 conjugates may also contribute to some as E2 derived from E2 conjugates can be further biotransformed into E1.11 One question remains it is possible that the direct biotransformation of E2 into E1 rather than the deconjugation of E1 or E2 conjugates plays a core role. As can be seen from Table 3, the amount from E2 only accounts for 22% reduced removal of E1 even the decreased E2 at an SRT of 3 days were all biotransformed into E1. Based on the above fact, it is not difficult to estimate that E1 and/or E2 conjugates at least accounts for 39% reduced removal of E1, which is far larger than that of the direct biotransformation of E2 into E1. Therefore, it is evident that estrogen conjugates plays an important role on the reduced removal of E1. As has been stated that the removal performance of the activated sludge treatment process on natural estrogens is unstable based on our summarization as shown in Table 1. It seems very difficult to draw solid conclusion that why the system is unstable. However, if we realize the important role of estrogen conjugates, a reasonable explanation below can be proposed. Deconjugation of estrogen conjugates in wastewater by arylsulfatase or β-glucuronidase is temperature-dependent. When the temperature is high, the deconjugation efficiencies of estrogen conjugates would be high, which results in low estrogen conjugate concentrations in raw wastewater. That is to say the influence of estrogen conjugates on the removal performance of natural estrogens is small. Conversely, relative high concentrations of estrogen conjugates likely exist in raw wastewater during cold season, and their following deconjugation during wastewater treatment gives an important effect on the observed removal performances of WWTPs on natural estrogens. Therefore, it is reasonable to conclude that estrogen conjugates likely be a crucial reason for the unstable removal performance of WWTPs on natural estrogens. To improve WWTP performance in reducing the adverse effects of natural estrogens, both natural estrogens and their conjugates should be considered. Enhancing the deconjugation rates of these conjugates may more effective, as this step is time-limited. More work is needed to elucidate this. Final Remarks. Six important conclusions can be drawn from this summary of natural estrogen removal in full-scale municipal WWTPs, and this overview of natural estrogen and estrogen conjugate biodegradation rate constants. (1) Natural estrogen removal capabilities of full-scale municipal WWTPs vary greatly, ranging from complete removal to −477%. The average removals of E2 and E3 seem similar, with approximately 75% or more removed. Average removals for E1 were less, at an average removal of only 38%. (2) Prolonged SRT can improve a WWTP’s natural estrogen removal performance; slow-growing nitrifying bacteria seems to not play a decisive role in this improvement. (3) Natural estrogen removal in aerobic conditions is faster than that in anaerobic conditions; E2 is the least affected

estrogen when the operation transitions from aerobic to anaerobic. (4) If only natural estrogens exist in raw wastewater, WWTP removal should be excellent under regular operations. (5) Estrogen deconjugation rates vary greatly between different groups. Normally, estrogen glucuronides deconjugate faster than estrogen sulfates. Most conjugates with low deconjugation rates, such as E1−3S, likely remain in the treated effluent. Estrogen glucuronides should be well cleaved in the WWTP. Yet, the cleaved natural estrogens from the conjugates may result in the underestimation of WWTP removal of these natural estrogens. (6) Strategies to enhance the deconjugation rates of estrogen conjugates may facilitate a decrease in natural estrogen concentrations in treated effluent.

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AUTHOR INFORMATION

Corresponding Author

*Phone/fax: +86-20-39380507; e-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was financially supported by The Key Program of National Natural Science of China (No.41330639), the Program for National Science Foundation of China (No. 21107025) as well as the Fundamental Research Funds for the Central Universities (2014ZM0073).

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