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Removal and degradation characteristics of natural and synthetic estrogens by activated sludge in batch experiments
water research 43 (2009) 573–582
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Removal and degradation characteristics of natural and synthetic estrogens by activated sludge in batch experiments T. Hashimoto*, T. Murakami Research and Development Department, Japan Sewage Works Agency, Shimosasame 5141, Toda, Saitama 335-0037, Japan
article info
abstract
Article history:
The removal and degradation characteristics of natural and synthetic estrogens by acti-
Received 19 July 2008
vated sludge were investigated by a series of batch experiments using the activated sludge
Received in revised form
samples of four actual wastewater treatment plants and synthetic wastewater spiked with
18 October 2008
estrogen. The rapid removal and degradation of 17b-estradiol (E2) and estrone (E1) were
Accepted 28 October 2008
observed by the activated sludge samples of the oxidation ditch process which operated at
Published online 9 November 2008
higher solids retention time (SRT). On the other hand, E1 tended to remain both in the water phase and the sludge phase in the activated sludge samples of the conventional
Keywords:
activated sludge process which operated at lower SRT. The anoxic condition was consid-
Wastewater treatment
ered to be not favorable to the effective removal of estrogens as compared with the aerobic
Activated sludge
condition. The removal and degradation of EE2 showed the lag phase, which neither E2 nor
Estrogen
E1 showed, but EE2 was finally removed and degraded completely after 24 h. The removal
Degradation
of estrogens in the water phase did not follow the first-order-rate reaction because a large
Solids retention time (SRT)
part of the spiked estrogen was immediately removed from the water phase to the sludge phase by adsorption. ª 2008 Elsevier Ltd. All rights reserved.
1.
Introduction
Natural estrogens, i.e., 17b-estradiol (E2) and estrone (E1), and synthetic estrogen, i.e., 17a-ethynylestradiol (EE2), are excreted into wastewater by humans and mammals mainly through their urine. The effluent concentrations of estrogens typically range from a few ng/L to a few tens of ng/L (Johnson and Sumpter, 2001; Khanal et al., 2006), but even these amounts are often high enough to cause endocrine-disrupting effects in some aquatic species such as trouts (Thorpe et al., 2001) and minnows (Panter et al., 2000). Estrogenic activities of estrogens are two or three orders of magnitude higher than those of endocrine-disrupting chemicals such as nonylphenol or bisphenol A (Routledge and Sumpter, 1996; Tanaka et al., 2001). Therefore, estrogens are considered to be the major
source of estrogenic activity in treated wastewater (Desbrow et al., 1998; Snyder et al., 2001), and the effluent of wastewater treatment plants (WWTPs) is responsible for a significant part of the endocrine-disrupting effect in receiving water bodies (Nakada et al., 2004). According to previous studies, estrogens are generally shown to be effectively removed during the secondary treatment of wastewater, particularly in the activated sludge treatment (Johnson and Sumpter, 2001; Khanal et al., 2006). Some batch experiments using activated sludge samples from WWTPs have shown that E2 is readily oxidized to E1, which is further degraded, but EE2 is not degraded significantly (Ternes et al., 1999; Layton et al., 2000; Shi et al., 2004; Li et al., 2005). However, in most of the previous batch experimental studies, initial concentrations of estrogen were 1–2 orders of
* Corresponding author. Tel.: þ81 48 421 2693; fax: þ81 48 421 7542. E-mail address: [email protected] (T. Hashimoto). 0043-1354/$ – see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.10.051
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magnitude higher than the concentrations of estrogens detected in municipal wastewater, and estrogen was spiked into activated sludge as the sole energy and carbon source although many organic substrates coexist with estrogens in wastewater. Therefore, there is a possibility that degradation characteristics of estrogens in actual WWTPs differ from those reported in the previous batch experimental studies. Moreover, field surveys in actual WWTPs have shown that removal efficiencies of estrogens tend to become relatively high and stable with the increase of solids retention time (SRT) in WWTPs (Kreuzinger et al., 2004; Clara et al., 2005; Johnson et al., 2005; Servos et al., 2005; Hashimoto et al., 2007), but no information about the influence of the difference of activated sludge on the degradation characteristic of estrogens has been shown in previous batch experimental studies. The major aim of this study was to investigate the influence of the difference of activated sludge in regard to the removal and degradation characteristics of estrogens, and in addition, to discuss the influence of the initial concentration of estrogens and the coexistence of organic substrates in batch experiments. For this purpose, we conducted a series of batch experiments using activated sludge samples taken from four actual WWTPs, which employed a conventional activated sludge (CAS) process in two plants and an oxidation ditch (OD) process in the other plants, and synthetic wastewater spiked with estrogen that had similar concentrations to the actual level observed in wastewater as possible.
2.
Materials and methods
2.1.
Sample collection
Activated sludge samples used for the batch experiments were taken from four actual WWTPs in the Kanto region of Japan. The characteristics of WWTPs are summarized in Table 1. In two WWTPs CAS was employed, and the other two WWTPs OD was employed. WWTP1, WWTP3 and WWTP4 are located in different adjoining local autonomies in Tochigi Prefecture. WWTP2 is located in Saitama Prefecture and is approximately 50 km away from the other WWTPs. All WWTPs mainly treated domestic wastewater. Activated sludge samples were taken from a return activated sludge channel or a sampling valve of return activated sludge pump on the morning of the experiment. The activated
sludge samples were carried to the laboratory which was situated next to WWTP1 within 1 or 2 h after the sampling, and were used for batch experiments immediately. To evaluate the removal efficiency of estrogens in WWTPs, influent to an aeration tank and effluent from a final settling tank were taken together.
2.2.
Three series of batch experiments were performed under the conditions shown in Table 2. The first series (Runs 1–8) was intended to examine the influence of the difference of activated sludge on the removal and degradation characteristics of E2 and E1, and was carried out using each activated sludge samples taken from four WWTPs under aerobic condition. The second series (Runs 9–12) was intended to examine the influence of aeration condition on the removal and degradation characteristics of E2 and E1, and was carried out under aerobic condition and anoxic condition, respectively. The third series (Run 13) was intended to examine the removal and degradation characteristic of EE2 under the condition of a low concentration of estrogen and the coexistence of organic substrates, and was carried out under aerobic condition. In both the second series and the third series, activated sludge samples taken from WWTP1 were used because of the convenience of sampling and the reduction of transport time of sample. Batch experiments were performed using the reactor shown in Fig. 1. The reactor had a volume of 20 L and was maintained at 20 C. An adequate amount of activated sludge sample was added to the reactor and diluted with tap water to a mixed liquor suspended solids (MLSS) concentration of approximately 2000 mg/L. At the beginning of each run, synthetic wastewater (peptone 6 g/L, beef extract 4 g/L, urea 1 g/L, NaCl 0.3 g/L, KH2PO4 1 g/L, KCl 0.14 g/L, CaCl2 0.14 g/L, MgSO4 0.1 g/L; approximately 10,000 mg/L as biochemical oxygen demand (BOD)) was added with the initial BOD of approximately 200 mg/L, and then the stock solution of target estrogen dissolved in methanol was spiked with the initial concentration of approximately 1000 ng/L. In aerobic condition
Table 2 – Experimental conditions of batch experiments. Series Run Activated sludge Spiked estrogen Aeration sample condition 1
Table 1 – Characteristics of wastewater treatment plants from which activated sludge samples were taken. Plant name Treatment Influent flow Population HRT SRT equivalent (h) (day) process (m3/d) (pe) WWTP1 WWTP2 WWTP3 WWTP4
CAS CAS OD OD
13,379 5,799 702 997
58,400 16,200 2,000 2,900
8.5 8.9 42 27
5.5 9.2 28 15
Influent flow and population equivalent are the annual average data in fiscal 2002. HRT and SRT are the data at the sampling day.
Batch experiment protocol
2
3
1 2 3 4 5 6 7 8
WWTP1 (CAS)
9 10 11 12
WWTP1 (CAS)
13
WWTP1 (CAS)
WWTP2 (CAS) WWTP3 (OD) WWTP4 (OD)
E2 E1 E2 E1 E2 E1 E2 E1
Aerobic
E2
Aerobic anoxic Aerobic anoxic
E1
EE2
Aerobic
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Activated sludge
Stainless tank (20L) M
measurement of LC/MS. Waters Alliance 2690 type high performance liquid chromatograph and Micromass Platform LCZ mass spectrometer (Waters Corporation) were used for analysis. Measured ion used in SIM mode detection by LC/MS analysis was m/z 271.4 for E2, m/z 269.4 for E1, m/z 274.4 for EE2 and m/z 269.4 for E2-d3. The minimum limits of detection (LOD) for E2, E1 and EE2 were 0.7 ng/L, respectively.
2.4. Air pump Water-bath
Heater Spherical diffuser
Mixer
Fig. 1 – Schematic diagram of batch experimental apparatus.
experiments, the mixed liquor was stirred gently and aerated to maintain dissolved oxygen (DO) concentration of approximately 1.0–3.0 mg/L. In anoxic condition experiments, the mixed liquor was only stirred. The pH of the activated sludge samples ranged from 7.1 to 7.4, and it showed little change in all experiments. The mixed liquor was taken at just before the spike of estrogen, and at 5 min, 30 min, 1, 2 and 4 h after the spike of estrogen. Additionally, in only the third series of batch experiments, samples were taken at 8 and 24 h after the spike of estrogen. Estrogen concentrations were analyzed separately in the water phase and the solid phase. Total organic carbon (TOC) in the water phase was measured to examine the fate of organic substrates in batch experiments.
2.3.
Analysis of estrogens in water phase
E2, E1 and EE2 were analyzed simultaneously using a liquid chromatograph mass spectrometer (LC/MS). The analytical procedure was based on a method using a liquid chromatograph-tandem mass spectrometer described by Wastewater Examination Methods (JSWA, 2002) with minor modifications. The mixed liquor sample was centrifuged immediately at 1900xg for a few minutes, and the supernatant was filtered with a glass fiber filter of approximately a 1-mm pore size. In cases of influent sample or effluent sample, the sample was filtered as well as the supernatant of the mixed liquor. The filtrate was passed through a Sep-Pack plus C18 cartridge (Waters Corporation) after adding 50 ng of 17b-estradiol16,16,17-d3 (E2-d3) as an internal standard. The cartridge was dried with nitrogen gas, and estrogens were eluted with 20 mL of ethyl acetate/methanol (5:1, v/v). The dry residual was dissolved in 1 mL of hexane/dichloromethane (2:1, v/v), and then cleaned-up with a Sep-Pack plus Florisil cartridge (Waters Corporation). After washing with 5 mL of hexane/ dichloromethane (1:1, v/v), estrogens were eluted from the cartridge with 20 mL of acetone/ dichloromethane (1:2, v/v). The dry residual was dissolved in 1 mL of methanol, and then cleaned-up with a Sep-Pack plus NH2 cartridge (Waters Corporation). Estrogens were eluted from the cartridge with 10 mL of methanol. The dry residual was dissolved in 1 mL of methanol, and the supernatant was used as the sample for
Analysis of estrogens in sludge phase
The extraction procedure of estrogens from sludge samples followed the method described by Wastewater Examination Methods (JSWA, 2002). Fifty mL of mixed liquor sample was centrifuged at 1800xg for 15 min, and the supernatant was decanted. Forty mL of methanol/1 M acetate buffer (pH 5) (9:1; v/v) was added to the precipitate, and it was shaken for 30 min. After centrifugation at 1800xg for 15 min, the supernatant was collected. Moreover, 40 mL of methanol/1 M acetate buffer (pH 5) (9:1; v/v) was added to the precipitate, and it was shaken for 5 min. After centrifugation at 1800xg for 15 min, the supernatant was collected and mixed with the former supernatant. The mixture was then filtered through a glass fiber filter paper (GF/B). The filtrate was concentrated to below 10 mL using a rotary evaporator and ultra-pure water was added until total volume was approximately 200 mL. After homogenized by sonication, solid-phase extraction and LC/MS analysis were carried out in the same procedure as the water phase described above.
3.
Results and discussion
3.1.
Removal efficiencies of estrogens in WWTPs
The concentrations and the removal efficiencies of estrogens in four WWTPs which activated sludge samples used in this study were taken are summarized in Table 3. Removal efficiencies of effluent samples below the LOD were calculated by using the value of LOD (i.e., 0.5 ng/L) for effluent concentrations. EE2 was not detected in all samples. The removal efficiencies of E2 and E1 in WWTP1 employed CAS were clearly inferior to those in the other WWTPs. The effluent concentrations of E2 were below the LOD except for WWTP1. The effluent concentration of E1 in WWTP1 was higher than the influent concentration although the other WWTPs showed a decrease of E1 between the influent and the effluent. Similar
Table 3 – - Estrogen concentrations and removal efficiencies of estrogens in wastewater treatment plants. Plant name Treatment Influent Effluent Removal process (ng/L) (ng/L) efficiency (%)
WWTP1 WWTP2 WWTP3 WWTP4
CAS CAS OD OD
E2
E1
11 12 8.9 5.8
66 68 21 40
E2
E1
E2
E1
1.9 80 <0.5 22 <0.5 9.0 <0.5 0.6
83 98 97 95
21 68 57 99
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increases of E1 during the biological treatment process were reported previously (Baronti et al., 2000; Servos et al., 2005; Hashimoto et al., 2007). Overall, the effluent concentrations of estrogens tended to be high in the CAS plants operated at lower SRT than the OD plants operated at higher SRT. This tendency was consistent with the previous studies reported (Kreuzinger et al., 2004; Clara et al., 2005; Johnson et al., 2005; Servos et al., 2005; Hashimoto et al., 2007).
3.2. Influence of difference of sludge to removal and degradation characteristics of E2 and E1 The concentration profiles of estrogens in the first series of batch experiments (Runs 1–8) conducted using the activated sludge samples taken from four WWTPs spiked with E2 or E1 are shown in Fig. 2. Estrogen concentrations immediately after the spike of estrogen (time at 0 min) were estimated to be the sum of the measured estrogen concentrations just before the spike of target estrogen and the spiked estrogen concentration (1000 ng/L) because the estrogen concentration of synthetic wastewater used for batch experiments was below the LOD. The measurement results of estrogen concentrations were corrected by the volumes of the supernatant and the precipitate, and expressed with the estrogen concentrations in each of the water phase and the sludge phase per 1 L of the mixed liquor. The behaviors of E2 in the batch experiments spiked with E2 (Runs 1, 3, 5 and 7) showed the same tendency regardless of the difference of activated sludge sample. E2 was immediately removed from the water phase after the spike of E2, and approximately 94–98% of spiked E2 was removed from the water phase within 5 min. The concentrations of E1 increased both in the water phase and the sludge phase after 5 min, thereby the conversion from E2 to E1 was observed, as well as in the previous studies (Ternes et al., 1999; Shi et al., 2004). However, the total amount of E2 and E1 both in the water phase and the sludge phase at 5 min after the spike of E2 was lower than the amount of spiked E2. Thus, it was considered that E1 was further degraded immediately after the conversion from E2. More than 99% of spiked E2 was removed from the water phase after 0.5–1.0 h. However, the tendency to remain as mainly E1 in the sludge phase was observed, and this tendency was especially prominent in the activated sludge samples of CAS plants (Runs 1 and 3) in which the effluent concentrations of E1 were significantly high. In contrast, the behaviors of E1 in the batch experiments spiked with E1 (Runs 2, 4, 6 and 8) were different between the activated sludge samples. In Runs 6 and 8, which were conducted using the activated sludge samples taken from OD plants, the behaviors of E1 showed the same tendency of the behavior of E2 in the batch experiments with spiked E2 mentioned above. E1 was immediately removed from the water phase, approximately 84–96% of spiked E1 was removed within 5 min, and more than 99% of spiked E1 was removed after 1 h. On the other hand, in Run 2, which was conducted using an activated sludge sample from WWTP1 which employed CAS and was observed the negative removal of E1, approximately 40% of spiked E1 remained in the water phase and approximately 20% of spiked E1 remained in the sludge phase after 5 min. A clear decrease of E1 both in the water
phase and the sludge phase was not observed within 2 h. After 4 h, approximately 6% of spiked E1 remained in the water phase, and approximately 15% of spiked E1 remained in the sludge phase. In Run 4, which was conducted using an activated sludge sample from WWTP2, more than 99% of spiked E1 was removed from the water phase within in 1 h, like the activated sludge samples taken from the OD plants, but approximately 60% of spiked E1 remained in the water phase and the sludge phase after 5 min, and approximately 30% remained after 30 min. Thus, E1 tended to remain in the activated sludge of the CAS plants as compared with the OD plants because both the removal rate from the water phase and the degradation rate in the sludge phase were slower in the activated sludge of the CAS plants than those of the OD plants. This tendency of the behavior of E1 in the batch experiments agreed with the measurement result of effluent concentrations presented above. It is considered that the removal of estrogens in the activated sludge process is influenced by various factors. Li et al. (2005) suggested that the removal of E2 was strongly dependent on the influence on the influent concentrations of E2, MLVSS and the temperature from the results of aerobic batch experiments using activated sludge samples spiked with E2. The first series of batch experiments in this study was conducted under the same conditions, except for the activated sludge sample. Therefore, the difference in the removal efficiencies of estrogens observed in the first series of batch experiments is considered to be caused by the differences of activated sludge, particularly in between the CAS plants at lower SRT and the OD plants at higher SRT. The SRT indicates the mean retention time of the microorganisms in a reactor. The SRT is related to the growth rate of microorganisms, and only microorganisms which have growth rates more than this time can be detained and enriched in the reactor. According to this definition, high SRT allows the enrichment of slowly growing bacteria and the establishment of a more diverse microbial population as compared with low SRT (Kreuzinger et al., 2004; Clara et al., 2005). Furthermore, the SRT could influence not only the microbial population but also the physical properties of floc particles which would have an important effect on their affinity as sorbents for such compounds as estrogen (Johnson et al., 2000). The results of the first series of batch experiments suggest that the higher SRT is preferable to establish activated sludge adapted to degrade estrogens and tends to lower the effluent concentrations of estrogens.
3.3. Influence of aeration conditions to removal and degradation characteristics of E2 and E1 The concentration profiles of estrogens in the second series of batch experiments (Runs 9–12) conducted under the aerobic condition or the anoxic condition are shown in Fig. 3. Under the aerobic condition, both E2 (Run 9) and E1 (Run 11) were immediately removed from the water phase, and more than 99% of spiked estrogen was removed from the water phase and the estrogen concentrations in the sludge phase decreased to the same level as the initial concentrations before spiked with estrogen within 30 min. On the other hand, under the anoxic conditions, approximately 20% of spiked E2
577
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
water research 43 (2009) 573–582
1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before 0min 5min 30min 1hr spiked
Time
1,400 1,200 1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
2hr
4hr
1,400 1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
Time
1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before spiked 0min 5min 30min 1hr
Time
4hr
1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
(6) Run 6 (WWTP3(OD), spiked E1)
Estrogen concentration [ng/L]
(5) Run 5 (WWTP3(OD), spiked E2)
Estrogen concentration [ng/L]
2hr
Time
1,200
2hr
4hr
1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
Time
2hr
4hr
Time
(7) Run 7 (WWTP4(OD), spiked E2) E2 in water phase
4hr
(4) Run 4 (WWTP2(CAS), spiked E1)
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
(3) Run 3 (WWTP2(CAS), spiked E2)
0
2hr
Time
1,200
0
4hr
(2) Run 2 (WWTP1(CAS), spiked E1)
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
(1) Run 1 (WWTP1(CAS), spiked E2)
0
2hr
Time
E1 in water phase
(8) Run 8 (WWTP4(OD), spiked E1) E2 in sludge phase
E1 in sludge phase
Fig. 2 – Concentration profiles of estrogens in the first series of batch experiments (Runs 1–8) conducted using the activated sludge samples taken from four WWTPs under aerobic condition.
578
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
water research 43 (2009) 573–582
1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before 0min 5min 30min 1hr spiked
Time
1,200 1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before 0min 5min 30min 1hr spiked
Time
2hr
4hr
Time
(3) Run 11 (aerobic condition, spiked E1) E2 in water phase
4hr
(2) Run 10 (anoxic condition, spiked E2)
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
(1) Run 9 (aerobic condition, spiked E2)
0
2hr
Time
(4) Run 12 (anoxic condition, spiked E1)
E1 in water phase
E2 in sludge phase
E1 in sludge phase
Fig. 3 – Concentration profiles of estrogens in the second series of batch experiments (Runs 9–12) conducted under aerobic condition or anoxic condition.
condition, it assumed that the conversion between E2 and E1 was repeated and both E2 and E1 were gradually decreased, and this conversion may be one of the reasons for the difference of removal rates of estrogens between aerobic condition and anoxic condition.
80
60
TOC [mg/L]
(Run 10) or spiked E1 (Run 12) remained in the water phase and the sludge phase after 4 h. Compared with the aerobic condition, both the removal of estrogens from the water phase and the degradation of estrogens in the sludge phase were slower under the anoxic condition. The aerobic condition is considered to be favorable for the effective removal of estrogens in the wastewater treatment process. The concentration profile of TOC in the second series of batch experiments is shown in Fig. 4. The removal efficiency of TOC in anoxic condition was inferior to that in aerobic condition, and the removal rate of TOC in anoxic condition (5.3–5.7 mg/(L h)) was about one third of that in aerobic condition (14–16 mg/(L h)) The difference of the removal efficiency of organic substrate may be related to the difference of the removal efficiency of estrogens between aerobic condition and anoxic condition because the co-metabolism is considered to be important for the biodegradation of micropollutants (Langford and Lester, 2003). In Run 10 in which E2 was spiked, due to the increase of E1 that was observed both in the water phase and the sludge phase, the conversion from E2 to E1 appeared to occur under the anoxic condition as well as under the aerobic condition. In Run 12 in which E1 was spiked, due to the increase of E2 that was observed both in the water phase and the sludge phase, the reduction from E1 to E2 appeared to occur under the anoxic condition (Joss et al., 2004). Therefore, under the anoxic
40
Run9 (E2,aerobic) 20
Run10 (E2,anoxic) Run11 (E1,aerobic) Run12 (E1,anoxic)
0 0
1
2
3
4
Time [hr] Fig. 4 – Concentration profile of TOC in the second series of batch experiments (Runs 9–12) conducted under aerobic condition or anoxic condition.
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3.4. Removal and degradation characteristic of EE2 under coexistence of organic substances The concentration profile of EE2 in the aerobic batch experiment (Run 13) conducted using the activated sludge sample taken from WWTP1 spiked with EE2 is shown in Fig. 5. The behavior of EE2 was different from the behaviors of E2 and E1. As presented above, both E2 and E1 were removed from the water phase and degraded in the sludge phase immediately after the estrogen was spiked. On the other hand, approximately 50% of spiked EE2 was removed from the water phase to the sludge phase after 5 min, but the total amount of EE2 in the water phase and in the sludge phase was rarely different from the initial spiked amount of EE2. Until 2 h after the spike of EE2, the amount of EE2 in both the water phase and the sludge phase showed little change and the clear degradation of EE2 was not observed. After 8 h, the amount of EE2 in both the water phase and the sludge phase decreased, and approximately 85% of the initial spiked EE2 was degraded, and then the concentrations of EE2 in both the water phase and the sludge phase became below the LOD after 24 h. Thus, the removal and degradation of EE2 showed the lag phase which neither E2 nor E1 showed, but EE2 was finally removed and degraded. The previous batch experiments (Ternes et al., 1999; Vader et al., 2000; Shi et al., 2004) have shown that EE2 is much more resistant as compared with E2 and E1. Ternes et al. (1999) reported that E2 at the initial concentration of 1 mg/L could be degraded using activated sludge taken from a WWTP over a period of 1–3 h, but EE2 at the initial concentration both of 1 mg/L and 1 mg/L was degraded to only 20% within 48 h and 24 h, respectively. Shi et al. (2004) showed that the degradation of estrogens during the batch experiment which used nitrifying activated sludge at the initial concentration of 1 mg/L obeyed first-order reaction kinetics with the degradation rate constants of 1.3 h1 for E2, 0.056 h1 for E1 and 0.035 h1 for EE2. On the other hand, it has been reported that EE2 is removed effectively during wastewater treatment process as a result of previous surveys conducted in actual WWTPs. For example, Baronti et al. (2000) reported that the
EE2 concentration [ng/L]
3.5. Removal rate constants of estrogens in batch experiments It is generally known that the removal of organic substances in the water phase during the activated sludge process can be described by a pseudo-first-order reaction as given in Eq. (1). Previous studies which conducted batch experiments using activated sludge spiked estrogen have been reported that the removal of estrogen in the water phase can be described by a pseudo-first-order reaction as well as organic substances (Layton et al., 2000; Shi et al., 2004; Li et al., 2005). (1)
where C is the concentration of the target substance in the water phase (mg/L), k is the first-order-rate constant (h1) and t is the reaction time (h). Rearranging Eq. (1), the following equation is obtained, C ¼ kt (2) ln C0
1,000 800 600 400 200 0
median concentrations of EE2 in the influents and the effluents of six WWTPs in Rome, Italy, were 3.0 ng/L and 0.4 ng/L respectively, and the average removal rate of EE2 was 85%. In the previous studies mentioned above, the initial spiked concentrations of EE2 ranged from a few 10 mg/L to a few mg/L which were extremely higher than the concentrations of EE2 detected in wastewater, and EE2 was spiked into activated sludge as the sole energy source and/or the sole carbon source although many organic substrates coexist with estrogens in wastewater. Therefore, the previous batch experiments may underestimate the degradation ability of EE2 in actual activated sludge treatment process. It is difficult to validate the recovery efficiencies of estrogens from the sludge phase because of the sorption of estrogens on sludge, so that we could not validate them directly. In Run 13, as shown in Fig. 5, the total amount of EE2 until 2 h after the spike of EE2 was about the same as the initial spiked amount of EE2. It was considered that EE2 detected in Run 13 was derived from the initial spiked EE2, because EE2 was not detected in both the activated sludge sample and the synthetic wastewater. Therefore, we considered that EE2 was almost recovered from both the water phase and the sludge phase, and it seems that the recovery of other estrogens is similar to EE2 because of their relatively similar physicochemical properties. To increase reliability of the results in this study, further experiments will be required.
dC ¼ kC dt
1,200
579
n.d.
n.d.
before spiked 0min 5min 30min 1hr
2hr
4hr
8hr
24hr
Time EE2 in water phase
EE2 in sludge phase
Fig. 5 – Concentration profile of EE2 in the aerobic batch experiment (Run 13). ‘‘n.d.’’ means ‘‘not detectable’’, a.k.a. below the detection limit.
where C0 is the initial concentrations of the target substance in the water phase (mg/L). This equation suggests that a plot of ln(C/C0) versus t yields a straight line and the first-order rate constant k is obtained from a slope of the straight line. The concentration profiles of TOC and estrogen in the first series of batch experiments (Runs 1–8) plotted in the manner of ln(C/C0) versus t are shown in Fig. 6. As shown in Fig. 6 (1) and (3), in the removal of TOC, the linear relationships between ln(C/C0) and t were observed at all experiments, although the first-order rate constants k were different between activated sludge samples. Therefore, the removal of organic substance
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0
0 Run 1 (WWTP1) Run 3 (WWTP2) Run 5 (WWTP3) Run 7 (WWTP4)
Ln(C/C0) [-]
Ln(C/C0) [-]
-2 -1
Run 1 (WWTP1) Run 3 (WWTP2) Run 5 (WWTP3) Run 7 (WWTP4)
-2
-4
-6
-3
-8
0
1
2
3
4
0
1
2
t [hr]
(1) TOC (Runs spiked E2)
(2) E2 concentration Runs spiked E2
0
4
0 Run 2 (WWTP1) Run 4 (WWTP2) Run 6 (WWTP3) Run 8 (WWTP4)
Ln(C/C0) [-]
-2
Ln(C/C0) [-]
3
t [hr]
-1
Run 2 (WWTP1) Run 4 (WWTP2) Run 6 (WWTP3) Run 8 (WWTP4)
-2
-4
-6
-8
-3
0
1
2
3
4
0
1
2
3
4
t [hr]
t [hr]
(3) TOC (Runs spiked E1)
(4) E1 concentration (Runs spiked E1)
Fig. 6 – Description of the concentration profiles of TOC and estrogen in the first series of batch experiment (Runs 1–8) by the first-order rate expression.
was considered to follow the first-order-rate reaction given in Eq. (1). On the other hand, in the removal of estrogens as shown in Fig. 6 (2) and (4), the slopes between ln(C/C0) and t within 5 min immediately after the spike of estrogen were significantly steep when compared with those 5 min later. Thus, the concentration profiles of estrogens plotted in the manner of ln(C/C0) and t did not exhibit linear features, and the removal of estrogens was not considered to follow the first-order-rate reaction unlike the previous studies. In actual wastewater, estrogens exist at extremely low concentrations of ng/L level and many other substances coexist at much higher concentrations of mg/L level. On the other hand, estrogen was spiked into activated sludge with synthetic wastewater in this study, but estrogen was spiked into activated sludge as the sole energy and carbon source in the previous studies (Layton et al., 2000; Shi et al., 2004; Li et al., 2005). Furthermore, the initial concentrations of estrogens in these previous studies ranged from a few 10 mg/L to a few mg/L, and they were very high compared with the concentrations of estrogens observed in wastewater. Therefore, it is considered that the batch experiments in the previous studies mentioned above were conducted on the condition which distinctly differed from the actual condition in wastewater treatment processes, and these results would not accurately
reflect the actual reaction in activated sludge. From the results in this study, we assume that estrogens are immediately removed from the water phase to the sludge phase by adsorption at the first step of the reaction, and then the further removal of estrogens remaining in the water phase progress gradually with the degradation of estrogens in the sludge phase. However, the initial concentrations of estrogens in this study, i.e., 1000 ng/L, were about two orders of magnitude higher than the concentrations of estrogens observed in wastewater, although those were relatively lower than the initial concentrations of estrogens in the previous works. Further study is required about the mechanism of the removal and degradation of estrogens by activated sludge under the really similar conditions in actual wastewater treatment processes. The results of our study will give significant suggestions to future studies.
4.
Conclusions
The removal and degradation characteristics of estrogens by activated sludge were investigated by a series of the batch experiments using activated sludge samples taken from four
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actual WWTPs and synthetic wastewater spiked with estrogen. The conclusions are summarized as follows: 1. The removal and degradation characteristics in the aerobic batch experiments agreed with the actual removal efficiencies in the WWTPs from which the activated sludge samples were taken. E2 and E1 were immediately removed from the water phase and then further degraded in the sludge phase by the activated sludge samples of the OD plants which operated at higher SRT and showed the effective removal of estrogens. On the other hand, the removal and degradation of E1 were slower and E1 tended to remain in the activate sludge samples of the CAS plants which operated at lower SRT and showed higher effluent concentrations of E1. The higher SRT was considered to be preferable to establish the activated sludge adapted to degrade estrogens. 2. Under anoxic conditions, both E2 and E1 remained both in the water phase and the sludge phase after 4 h. The anoxic condition was considered to be not favorable for the effective removal of estrogens in wastewater treatment. 3. The removal and degradation of EE2 showed the lag phase during 2 h after the spike of EE2 which neither E2 nor E1 showed, but EE2 was finally removed and degraded completely after 24 h. 4. The large part of the spiked estrogen was immediately removed from the water phase to the sludge phase by adsorption within 5 min after the spike, and then estrogen was further removed gradually 5 min later. The removal of estrogens in the water phase did not follow the first-orderrate reaction.
Acknowledgement The authors thank the local authorities for kindly providing activated sludge samples and wastewater samples.
references
Baronti, C., Curini, R., D’Ascenzo, G., Di Corcia, A., Gentili, A., Samperi, R., 2000. Monitoring natural and synthetic estrogens at activated sludge sewage treatment plants and its receiving river water. Environ. Sci. Technol. 34 (24), 5059–5066. Clara, M., Kreuzinger, N., Strenn, B., Gans, O., Kroiss, H., 2005. The solids retention time – a suitable design parameter to evaluate the capacity of wastewater treatment plants to remove micropollutants. Water Res. 39 (1), 97–106. Desbrow, C., Routledg, E.J., Brighty, G.C., Sumpter, J.P., Waldock, M., 1998. Identification of estrogenic chemicals in STW effluent. 1. Chemical fractionation and in vitro biological screening. Environ. Sci. Technol. 32 (11), 1549–1558. Hashimoto, T., Onda, K., Nakamura, Y., Tada, K., Miya, A., Murakami, T., 2007. Comparison of natural estrogen removal efficiency in the conventional activated sludge process and the oxidation ditch process. Water Res. 41 (10), 2117–2126.
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Japan Sewage Works Association (JSWA), 2002. Wastewater Examination Methods, the Additional Temporary Edition. Japan Sewage Works Association, Tokyo, Japan (in Japanese). Johnson, A.C., Belfroid, A., Di Corcia, A., 2000. Estimating steroid oestrogen inputs into activated sludge treatment works and observations on their removal from the effluent. Sci. Total Environ. 256 (2–3), 163–173. Johnson, A.C., Sumpter, J.P., 2001. Removal of endocrinedisrupting chemicals in activated sludge treatment works. Environ. Sci. Technol. 35 (24), 4697–4703. Johnson, A.C., Aerni, H.-R., Gerritsen, A., Gibert, M., Giger, W., Hylland, K., Ju¨rgens, M., Nakari, T., Pickering, A., Suter, M.J.-F., Svenson, A., Wettstein, F.E., 2005. Comparing steroid estrogen and nonylphenol content across a range of European sewage plants with different treatment and management practices. Water Res. 39 (1), 47–58. Joss, A., Anderson, H., Ternes, T., Richle, P.R., Siegrist, H., 2004. Removal of estrogens in municipal wastewater treatment under aerobic and anaerobic conditions: consequences for plant optimization. Environ. Sci. Technol. 38 (11), 3047–3055. Khanal, S.M., Xie, B., Thompson, M.L., Sung, S., Ong, S.-K., Van Leeuwen, J., 2006. Fate, transport, and biodegradation of natural estrogens in the environment and engineered systems. Environ. Sci. Technol. 40 (21), 6537–6546. Kreuzinger, N., Clara, M., Strenn, B., Kroiss, H., 2004. Relevance of the sludge retention time (SRT) as design criteria for wastewater treatment plants for the removal of endocrine disruptors and pharmaceuticals from wastewater. Water Sci. Technol. 50 (5), 149–156. Langford, K., Lester, J.N., 2003. Fate and behavior of endocrine disrupters in wastewater treatment processes. In: Birkett, J.W., Lester, J.N. (Eds.), Endocrine Disrupters in Wastewater and Sludge Treatment Processes. Lewis Publishers, Boca Raton, USA, pp. 103–143. Layton, A.C., Greory, B.W., Seward, J.R., Schultz, T.W., Sayler, G.S., 2000. Mineralization of steroidal hormones by biosolids in wastewater treatment systems in Tennessee U.S.A. Environ. Sci Technol. 34 (18), 3925–3931. Li, F., Yuasa, A., Obara, A., Mathews, P.A., 2005. Aerobic batch degradation of 17-b estradiol (E2) by activated sludge: effects of spiking E2 concentration, MLVSS and temperature. Water Res. 39 (10), 2065–2075. Nakada, N., Nyunoya, H., Nakamura, M., Hara, A., Iguchi, T., Takada, H., 2004. Identification of estrogenic compounds in wastewater effluent. Environ. Toxicol. Chem. 23 (12), 2807–2815. Panter, G.H., Thompson, R.S., Sumpter, J.P., 2000. Intermittent exposure of fish to estradiol. Environ. Sci. Technol. 34 (13), 2756–2760. Routledge, E.J., Sumpter, J.P., 1996. Estrogenic activity of surfactants and some of their degradation products assessed using a recombinant yeast screen. Environ. Toxicol. Chem. 15 (3), 241–248. Servos, M.R., Bennie, D.T., Burnison, B.K., Jurkovic, A., McInnis, R., Neheli, T., Schnell, A., Seto, P., Smyth, S.A., Ternes, T.A., 2005. Distribution of estrogens, 17 b-estradiol and estrone, in Canadian municipal wastewater treatment plants. Sci. Total Environ. 336 (1–3), 155–160. Shi, J.H., Fujiwara, S., Nakai, S., Hosomi, M., 2004. Biodegradation of natural and synthetic estrogens by nitrifying activated sludge and ammonia-oxidizing bacterium Nitrosomonas Europaea. Water Res. 38 (9), 2323–2330. Snyder, S.A., Villeneuve, D.L., Snyder, E.M., Giesy, J.P., 2001. Identification and quantification of estrogen receptor agonists in wastewater effluents. Environ. Sci. Technol. 35 (18), 3620–3625.
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Tanaka, H., Yakou, Y., Takahashi, A., Higashitani, T., Komori, K., 2001. Comparison between estrogenicities estimated from DNA recombinant yeast assay and from chemical analyses of endocrine disruptors during sewage treatment. Water Sci. Technol. 43 (2), 125–132. Ternes, T.A., Kreckel, P., Mueller, J., 1999. Behaviour and occurrence of estrogens in municipal sewage treatment plants-II. Aerobic batch experiment with activated sludge. Sci. Total. Environ. 255 (1–2), 91–99.
Thorpe, K.L., Hutchinson, T.H., Hetheridge, M.J., Scholze, M., Sumpter, J.P., Tyler, C.R., 2001. Assessing the biological potency of environmental estrogens using vitellogenin induction in juvenile rainbow trout (Oncorhynchus mykiss). Environ. Sci. Technol. 35 (12), 2476–2481. Vader, J.S., van Ginkel, C.G., Sperling, F.M.G.M., de Jong, J., de boer, W., de Graaf, J.S., van der Most, M., Stokman, P.G.W., 2000. Degradation of ethinyl estradiol by nitrifying activated sludge. Chemosphere 41 (8), 1239–1243.
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Removal and degradation characteristics of natural and synthetic estrogens by activated sludge in batch experiments T. Hashimoto*, T. Murakami Research and Development Department, Japan Sewage Works Agency, Shimosasame 5141, Toda, Saitama 335-0037, Japan
article info
abstract
Article history:
The removal and degradation characteristics of natural and synthetic estrogens by acti-
Received 19 July 2008
vated sludge were investigated by a series of batch experiments using the activated sludge
Received in revised form
samples of four actual wastewater treatment plants and synthetic wastewater spiked with
18 October 2008
estrogen. The rapid removal and degradation of 17b-estradiol (E2) and estrone (E1) were
Accepted 28 October 2008
observed by the activated sludge samples of the oxidation ditch process which operated at
Published online 9 November 2008
higher solids retention time (SRT). On the other hand, E1 tended to remain both in the water phase and the sludge phase in the activated sludge samples of the conventional
Keywords:
activated sludge process which operated at lower SRT. The anoxic condition was consid-
Wastewater treatment
ered to be not favorable to the effective removal of estrogens as compared with the aerobic
Activated sludge
condition. The removal and degradation of EE2 showed the lag phase, which neither E2 nor
Estrogen
E1 showed, but EE2 was finally removed and degraded completely after 24 h. The removal
Degradation
of estrogens in the water phase did not follow the first-order-rate reaction because a large
Solids retention time (SRT)
part of the spiked estrogen was immediately removed from the water phase to the sludge phase by adsorption. ª 2008 Elsevier Ltd. All rights reserved.
1.
Introduction
Natural estrogens, i.e., 17b-estradiol (E2) and estrone (E1), and synthetic estrogen, i.e., 17a-ethynylestradiol (EE2), are excreted into wastewater by humans and mammals mainly through their urine. The effluent concentrations of estrogens typically range from a few ng/L to a few tens of ng/L (Johnson and Sumpter, 2001; Khanal et al., 2006), but even these amounts are often high enough to cause endocrine-disrupting effects in some aquatic species such as trouts (Thorpe et al., 2001) and minnows (Panter et al., 2000). Estrogenic activities of estrogens are two or three orders of magnitude higher than those of endocrine-disrupting chemicals such as nonylphenol or bisphenol A (Routledge and Sumpter, 1996; Tanaka et al., 2001). Therefore, estrogens are considered to be the major
source of estrogenic activity in treated wastewater (Desbrow et al., 1998; Snyder et al., 2001), and the effluent of wastewater treatment plants (WWTPs) is responsible for a significant part of the endocrine-disrupting effect in receiving water bodies (Nakada et al., 2004). According to previous studies, estrogens are generally shown to be effectively removed during the secondary treatment of wastewater, particularly in the activated sludge treatment (Johnson and Sumpter, 2001; Khanal et al., 2006). Some batch experiments using activated sludge samples from WWTPs have shown that E2 is readily oxidized to E1, which is further degraded, but EE2 is not degraded significantly (Ternes et al., 1999; Layton et al., 2000; Shi et al., 2004; Li et al., 2005). However, in most of the previous batch experimental studies, initial concentrations of estrogen were 1–2 orders of
* Corresponding author. Tel.: þ81 48 421 2693; fax: þ81 48 421 7542. E-mail address: [email protected] (T. Hashimoto). 0043-1354/$ – see front matter ª 2008 Elsevier Ltd. All rights reserved. doi:10.1016/j.watres.2008.10.051
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magnitude higher than the concentrations of estrogens detected in municipal wastewater, and estrogen was spiked into activated sludge as the sole energy and carbon source although many organic substrates coexist with estrogens in wastewater. Therefore, there is a possibility that degradation characteristics of estrogens in actual WWTPs differ from those reported in the previous batch experimental studies. Moreover, field surveys in actual WWTPs have shown that removal efficiencies of estrogens tend to become relatively high and stable with the increase of solids retention time (SRT) in WWTPs (Kreuzinger et al., 2004; Clara et al., 2005; Johnson et al., 2005; Servos et al., 2005; Hashimoto et al., 2007), but no information about the influence of the difference of activated sludge on the degradation characteristic of estrogens has been shown in previous batch experimental studies. The major aim of this study was to investigate the influence of the difference of activated sludge in regard to the removal and degradation characteristics of estrogens, and in addition, to discuss the influence of the initial concentration of estrogens and the coexistence of organic substrates in batch experiments. For this purpose, we conducted a series of batch experiments using activated sludge samples taken from four actual WWTPs, which employed a conventional activated sludge (CAS) process in two plants and an oxidation ditch (OD) process in the other plants, and synthetic wastewater spiked with estrogen that had similar concentrations to the actual level observed in wastewater as possible.
2.
Materials and methods
2.1.
Sample collection
Activated sludge samples used for the batch experiments were taken from four actual WWTPs in the Kanto region of Japan. The characteristics of WWTPs are summarized in Table 1. In two WWTPs CAS was employed, and the other two WWTPs OD was employed. WWTP1, WWTP3 and WWTP4 are located in different adjoining local autonomies in Tochigi Prefecture. WWTP2 is located in Saitama Prefecture and is approximately 50 km away from the other WWTPs. All WWTPs mainly treated domestic wastewater. Activated sludge samples were taken from a return activated sludge channel or a sampling valve of return activated sludge pump on the morning of the experiment. The activated
sludge samples were carried to the laboratory which was situated next to WWTP1 within 1 or 2 h after the sampling, and were used for batch experiments immediately. To evaluate the removal efficiency of estrogens in WWTPs, influent to an aeration tank and effluent from a final settling tank were taken together.
2.2.
Three series of batch experiments were performed under the conditions shown in Table 2. The first series (Runs 1–8) was intended to examine the influence of the difference of activated sludge on the removal and degradation characteristics of E2 and E1, and was carried out using each activated sludge samples taken from four WWTPs under aerobic condition. The second series (Runs 9–12) was intended to examine the influence of aeration condition on the removal and degradation characteristics of E2 and E1, and was carried out under aerobic condition and anoxic condition, respectively. The third series (Run 13) was intended to examine the removal and degradation characteristic of EE2 under the condition of a low concentration of estrogen and the coexistence of organic substrates, and was carried out under aerobic condition. In both the second series and the third series, activated sludge samples taken from WWTP1 were used because of the convenience of sampling and the reduction of transport time of sample. Batch experiments were performed using the reactor shown in Fig. 1. The reactor had a volume of 20 L and was maintained at 20 C. An adequate amount of activated sludge sample was added to the reactor and diluted with tap water to a mixed liquor suspended solids (MLSS) concentration of approximately 2000 mg/L. At the beginning of each run, synthetic wastewater (peptone 6 g/L, beef extract 4 g/L, urea 1 g/L, NaCl 0.3 g/L, KH2PO4 1 g/L, KCl 0.14 g/L, CaCl2 0.14 g/L, MgSO4 0.1 g/L; approximately 10,000 mg/L as biochemical oxygen demand (BOD)) was added with the initial BOD of approximately 200 mg/L, and then the stock solution of target estrogen dissolved in methanol was spiked with the initial concentration of approximately 1000 ng/L. In aerobic condition
Table 2 – Experimental conditions of batch experiments. Series Run Activated sludge Spiked estrogen Aeration sample condition 1
Table 1 – Characteristics of wastewater treatment plants from which activated sludge samples were taken. Plant name Treatment Influent flow Population HRT SRT equivalent (h) (day) process (m3/d) (pe) WWTP1 WWTP2 WWTP3 WWTP4
CAS CAS OD OD
13,379 5,799 702 997
58,400 16,200 2,000 2,900
8.5 8.9 42 27
5.5 9.2 28 15
Influent flow and population equivalent are the annual average data in fiscal 2002. HRT and SRT are the data at the sampling day.
Batch experiment protocol
2
3
1 2 3 4 5 6 7 8
WWTP1 (CAS)
9 10 11 12
WWTP1 (CAS)
13
WWTP1 (CAS)
WWTP2 (CAS) WWTP3 (OD) WWTP4 (OD)
E2 E1 E2 E1 E2 E1 E2 E1
Aerobic
E2
Aerobic anoxic Aerobic anoxic
E1
EE2
Aerobic
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Activated sludge
Stainless tank (20L) M
measurement of LC/MS. Waters Alliance 2690 type high performance liquid chromatograph and Micromass Platform LCZ mass spectrometer (Waters Corporation) were used for analysis. Measured ion used in SIM mode detection by LC/MS analysis was m/z 271.4 for E2, m/z 269.4 for E1, m/z 274.4 for EE2 and m/z 269.4 for E2-d3. The minimum limits of detection (LOD) for E2, E1 and EE2 were 0.7 ng/L, respectively.
2.4. Air pump Water-bath
Heater Spherical diffuser
Mixer
Fig. 1 – Schematic diagram of batch experimental apparatus.
experiments, the mixed liquor was stirred gently and aerated to maintain dissolved oxygen (DO) concentration of approximately 1.0–3.0 mg/L. In anoxic condition experiments, the mixed liquor was only stirred. The pH of the activated sludge samples ranged from 7.1 to 7.4, and it showed little change in all experiments. The mixed liquor was taken at just before the spike of estrogen, and at 5 min, 30 min, 1, 2 and 4 h after the spike of estrogen. Additionally, in only the third series of batch experiments, samples were taken at 8 and 24 h after the spike of estrogen. Estrogen concentrations were analyzed separately in the water phase and the solid phase. Total organic carbon (TOC) in the water phase was measured to examine the fate of organic substrates in batch experiments.
2.3.
Analysis of estrogens in water phase
E2, E1 and EE2 were analyzed simultaneously using a liquid chromatograph mass spectrometer (LC/MS). The analytical procedure was based on a method using a liquid chromatograph-tandem mass spectrometer described by Wastewater Examination Methods (JSWA, 2002) with minor modifications. The mixed liquor sample was centrifuged immediately at 1900xg for a few minutes, and the supernatant was filtered with a glass fiber filter of approximately a 1-mm pore size. In cases of influent sample or effluent sample, the sample was filtered as well as the supernatant of the mixed liquor. The filtrate was passed through a Sep-Pack plus C18 cartridge (Waters Corporation) after adding 50 ng of 17b-estradiol16,16,17-d3 (E2-d3) as an internal standard. The cartridge was dried with nitrogen gas, and estrogens were eluted with 20 mL of ethyl acetate/methanol (5:1, v/v). The dry residual was dissolved in 1 mL of hexane/dichloromethane (2:1, v/v), and then cleaned-up with a Sep-Pack plus Florisil cartridge (Waters Corporation). After washing with 5 mL of hexane/ dichloromethane (1:1, v/v), estrogens were eluted from the cartridge with 20 mL of acetone/ dichloromethane (1:2, v/v). The dry residual was dissolved in 1 mL of methanol, and then cleaned-up with a Sep-Pack plus NH2 cartridge (Waters Corporation). Estrogens were eluted from the cartridge with 10 mL of methanol. The dry residual was dissolved in 1 mL of methanol, and the supernatant was used as the sample for
Analysis of estrogens in sludge phase
The extraction procedure of estrogens from sludge samples followed the method described by Wastewater Examination Methods (JSWA, 2002). Fifty mL of mixed liquor sample was centrifuged at 1800xg for 15 min, and the supernatant was decanted. Forty mL of methanol/1 M acetate buffer (pH 5) (9:1; v/v) was added to the precipitate, and it was shaken for 30 min. After centrifugation at 1800xg for 15 min, the supernatant was collected. Moreover, 40 mL of methanol/1 M acetate buffer (pH 5) (9:1; v/v) was added to the precipitate, and it was shaken for 5 min. After centrifugation at 1800xg for 15 min, the supernatant was collected and mixed with the former supernatant. The mixture was then filtered through a glass fiber filter paper (GF/B). The filtrate was concentrated to below 10 mL using a rotary evaporator and ultra-pure water was added until total volume was approximately 200 mL. After homogenized by sonication, solid-phase extraction and LC/MS analysis were carried out in the same procedure as the water phase described above.
3.
Results and discussion
3.1.
Removal efficiencies of estrogens in WWTPs
The concentrations and the removal efficiencies of estrogens in four WWTPs which activated sludge samples used in this study were taken are summarized in Table 3. Removal efficiencies of effluent samples below the LOD were calculated by using the value of LOD (i.e., 0.5 ng/L) for effluent concentrations. EE2 was not detected in all samples. The removal efficiencies of E2 and E1 in WWTP1 employed CAS were clearly inferior to those in the other WWTPs. The effluent concentrations of E2 were below the LOD except for WWTP1. The effluent concentration of E1 in WWTP1 was higher than the influent concentration although the other WWTPs showed a decrease of E1 between the influent and the effluent. Similar
Table 3 – - Estrogen concentrations and removal efficiencies of estrogens in wastewater treatment plants. Plant name Treatment Influent Effluent Removal process (ng/L) (ng/L) efficiency (%)
WWTP1 WWTP2 WWTP3 WWTP4
CAS CAS OD OD
E2
E1
11 12 8.9 5.8
66 68 21 40
E2
E1
E2
E1
1.9 80 <0.5 22 <0.5 9.0 <0.5 0.6
83 98 97 95
21 68 57 99
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increases of E1 during the biological treatment process were reported previously (Baronti et al., 2000; Servos et al., 2005; Hashimoto et al., 2007). Overall, the effluent concentrations of estrogens tended to be high in the CAS plants operated at lower SRT than the OD plants operated at higher SRT. This tendency was consistent with the previous studies reported (Kreuzinger et al., 2004; Clara et al., 2005; Johnson et al., 2005; Servos et al., 2005; Hashimoto et al., 2007).
3.2. Influence of difference of sludge to removal and degradation characteristics of E2 and E1 The concentration profiles of estrogens in the first series of batch experiments (Runs 1–8) conducted using the activated sludge samples taken from four WWTPs spiked with E2 or E1 are shown in Fig. 2. Estrogen concentrations immediately after the spike of estrogen (time at 0 min) were estimated to be the sum of the measured estrogen concentrations just before the spike of target estrogen and the spiked estrogen concentration (1000 ng/L) because the estrogen concentration of synthetic wastewater used for batch experiments was below the LOD. The measurement results of estrogen concentrations were corrected by the volumes of the supernatant and the precipitate, and expressed with the estrogen concentrations in each of the water phase and the sludge phase per 1 L of the mixed liquor. The behaviors of E2 in the batch experiments spiked with E2 (Runs 1, 3, 5 and 7) showed the same tendency regardless of the difference of activated sludge sample. E2 was immediately removed from the water phase after the spike of E2, and approximately 94–98% of spiked E2 was removed from the water phase within 5 min. The concentrations of E1 increased both in the water phase and the sludge phase after 5 min, thereby the conversion from E2 to E1 was observed, as well as in the previous studies (Ternes et al., 1999; Shi et al., 2004). However, the total amount of E2 and E1 both in the water phase and the sludge phase at 5 min after the spike of E2 was lower than the amount of spiked E2. Thus, it was considered that E1 was further degraded immediately after the conversion from E2. More than 99% of spiked E2 was removed from the water phase after 0.5–1.0 h. However, the tendency to remain as mainly E1 in the sludge phase was observed, and this tendency was especially prominent in the activated sludge samples of CAS plants (Runs 1 and 3) in which the effluent concentrations of E1 were significantly high. In contrast, the behaviors of E1 in the batch experiments spiked with E1 (Runs 2, 4, 6 and 8) were different between the activated sludge samples. In Runs 6 and 8, which were conducted using the activated sludge samples taken from OD plants, the behaviors of E1 showed the same tendency of the behavior of E2 in the batch experiments with spiked E2 mentioned above. E1 was immediately removed from the water phase, approximately 84–96% of spiked E1 was removed within 5 min, and more than 99% of spiked E1 was removed after 1 h. On the other hand, in Run 2, which was conducted using an activated sludge sample from WWTP1 which employed CAS and was observed the negative removal of E1, approximately 40% of spiked E1 remained in the water phase and approximately 20% of spiked E1 remained in the sludge phase after 5 min. A clear decrease of E1 both in the water
phase and the sludge phase was not observed within 2 h. After 4 h, approximately 6% of spiked E1 remained in the water phase, and approximately 15% of spiked E1 remained in the sludge phase. In Run 4, which was conducted using an activated sludge sample from WWTP2, more than 99% of spiked E1 was removed from the water phase within in 1 h, like the activated sludge samples taken from the OD plants, but approximately 60% of spiked E1 remained in the water phase and the sludge phase after 5 min, and approximately 30% remained after 30 min. Thus, E1 tended to remain in the activated sludge of the CAS plants as compared with the OD plants because both the removal rate from the water phase and the degradation rate in the sludge phase were slower in the activated sludge of the CAS plants than those of the OD plants. This tendency of the behavior of E1 in the batch experiments agreed with the measurement result of effluent concentrations presented above. It is considered that the removal of estrogens in the activated sludge process is influenced by various factors. Li et al. (2005) suggested that the removal of E2 was strongly dependent on the influence on the influent concentrations of E2, MLVSS and the temperature from the results of aerobic batch experiments using activated sludge samples spiked with E2. The first series of batch experiments in this study was conducted under the same conditions, except for the activated sludge sample. Therefore, the difference in the removal efficiencies of estrogens observed in the first series of batch experiments is considered to be caused by the differences of activated sludge, particularly in between the CAS plants at lower SRT and the OD plants at higher SRT. The SRT indicates the mean retention time of the microorganisms in a reactor. The SRT is related to the growth rate of microorganisms, and only microorganisms which have growth rates more than this time can be detained and enriched in the reactor. According to this definition, high SRT allows the enrichment of slowly growing bacteria and the establishment of a more diverse microbial population as compared with low SRT (Kreuzinger et al., 2004; Clara et al., 2005). Furthermore, the SRT could influence not only the microbial population but also the physical properties of floc particles which would have an important effect on their affinity as sorbents for such compounds as estrogen (Johnson et al., 2000). The results of the first series of batch experiments suggest that the higher SRT is preferable to establish activated sludge adapted to degrade estrogens and tends to lower the effluent concentrations of estrogens.
3.3. Influence of aeration conditions to removal and degradation characteristics of E2 and E1 The concentration profiles of estrogens in the second series of batch experiments (Runs 9–12) conducted under the aerobic condition or the anoxic condition are shown in Fig. 3. Under the aerobic condition, both E2 (Run 9) and E1 (Run 11) were immediately removed from the water phase, and more than 99% of spiked estrogen was removed from the water phase and the estrogen concentrations in the sludge phase decreased to the same level as the initial concentrations before spiked with estrogen within 30 min. On the other hand, under the anoxic conditions, approximately 20% of spiked E2
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Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
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1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before 0min 5min 30min 1hr spiked
Time
1,400 1,200 1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
2hr
4hr
1,400 1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
Time
1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before spiked 0min 5min 30min 1hr
Time
4hr
1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
(6) Run 6 (WWTP3(OD), spiked E1)
Estrogen concentration [ng/L]
(5) Run 5 (WWTP3(OD), spiked E2)
Estrogen concentration [ng/L]
2hr
Time
1,200
2hr
4hr
1,200 1,000 800 600 400 200 0
before 0min 5min 30min 1hr spiked
Time
2hr
4hr
Time
(7) Run 7 (WWTP4(OD), spiked E2) E2 in water phase
4hr
(4) Run 4 (WWTP2(CAS), spiked E1)
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
(3) Run 3 (WWTP2(CAS), spiked E2)
0
2hr
Time
1,200
0
4hr
(2) Run 2 (WWTP1(CAS), spiked E1)
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
(1) Run 1 (WWTP1(CAS), spiked E2)
0
2hr
Time
E1 in water phase
(8) Run 8 (WWTP4(OD), spiked E1) E2 in sludge phase
E1 in sludge phase
Fig. 2 – Concentration profiles of estrogens in the first series of batch experiments (Runs 1–8) conducted using the activated sludge samples taken from four WWTPs under aerobic condition.
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before 0min 5min 30min 1hr spiked
Time
1,200 1,000 800 600 400 200 before 0min 5min 30min 1hr spiked
2hr
1,200 1,000 800 600 400 200 0
4hr
before 0min 5min 30min 1hr spiked
Time
2hr
4hr
Time
(3) Run 11 (aerobic condition, spiked E1) E2 in water phase
4hr
(2) Run 10 (anoxic condition, spiked E2)
Estrogen concentration [ng/L]
Estrogen concentration [ng/L]
(1) Run 9 (aerobic condition, spiked E2)
0
2hr
Time
(4) Run 12 (anoxic condition, spiked E1)
E1 in water phase
E2 in sludge phase
E1 in sludge phase
Fig. 3 – Concentration profiles of estrogens in the second series of batch experiments (Runs 9–12) conducted under aerobic condition or anoxic condition.
condition, it assumed that the conversion between E2 and E1 was repeated and both E2 and E1 were gradually decreased, and this conversion may be one of the reasons for the difference of removal rates of estrogens between aerobic condition and anoxic condition.
80
60
TOC [mg/L]
(Run 10) or spiked E1 (Run 12) remained in the water phase and the sludge phase after 4 h. Compared with the aerobic condition, both the removal of estrogens from the water phase and the degradation of estrogens in the sludge phase were slower under the anoxic condition. The aerobic condition is considered to be favorable for the effective removal of estrogens in the wastewater treatment process. The concentration profile of TOC in the second series of batch experiments is shown in Fig. 4. The removal efficiency of TOC in anoxic condition was inferior to that in aerobic condition, and the removal rate of TOC in anoxic condition (5.3–5.7 mg/(L h)) was about one third of that in aerobic condition (14–16 mg/(L h)) The difference of the removal efficiency of organic substrate may be related to the difference of the removal efficiency of estrogens between aerobic condition and anoxic condition because the co-metabolism is considered to be important for the biodegradation of micropollutants (Langford and Lester, 2003). In Run 10 in which E2 was spiked, due to the increase of E1 that was observed both in the water phase and the sludge phase, the conversion from E2 to E1 appeared to occur under the anoxic condition as well as under the aerobic condition. In Run 12 in which E1 was spiked, due to the increase of E2 that was observed both in the water phase and the sludge phase, the reduction from E1 to E2 appeared to occur under the anoxic condition (Joss et al., 2004). Therefore, under the anoxic
40
Run9 (E2,aerobic) 20
Run10 (E2,anoxic) Run11 (E1,aerobic) Run12 (E1,anoxic)
0 0
1
2
3
4
Time [hr] Fig. 4 – Concentration profile of TOC in the second series of batch experiments (Runs 9–12) conducted under aerobic condition or anoxic condition.
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3.4. Removal and degradation characteristic of EE2 under coexistence of organic substances The concentration profile of EE2 in the aerobic batch experiment (Run 13) conducted using the activated sludge sample taken from WWTP1 spiked with EE2 is shown in Fig. 5. The behavior of EE2 was different from the behaviors of E2 and E1. As presented above, both E2 and E1 were removed from the water phase and degraded in the sludge phase immediately after the estrogen was spiked. On the other hand, approximately 50% of spiked EE2 was removed from the water phase to the sludge phase after 5 min, but the total amount of EE2 in the water phase and in the sludge phase was rarely different from the initial spiked amount of EE2. Until 2 h after the spike of EE2, the amount of EE2 in both the water phase and the sludge phase showed little change and the clear degradation of EE2 was not observed. After 8 h, the amount of EE2 in both the water phase and the sludge phase decreased, and approximately 85% of the initial spiked EE2 was degraded, and then the concentrations of EE2 in both the water phase and the sludge phase became below the LOD after 24 h. Thus, the removal and degradation of EE2 showed the lag phase which neither E2 nor E1 showed, but EE2 was finally removed and degraded. The previous batch experiments (Ternes et al., 1999; Vader et al., 2000; Shi et al., 2004) have shown that EE2 is much more resistant as compared with E2 and E1. Ternes et al. (1999) reported that E2 at the initial concentration of 1 mg/L could be degraded using activated sludge taken from a WWTP over a period of 1–3 h, but EE2 at the initial concentration both of 1 mg/L and 1 mg/L was degraded to only 20% within 48 h and 24 h, respectively. Shi et al. (2004) showed that the degradation of estrogens during the batch experiment which used nitrifying activated sludge at the initial concentration of 1 mg/L obeyed first-order reaction kinetics with the degradation rate constants of 1.3 h1 for E2, 0.056 h1 for E1 and 0.035 h1 for EE2. On the other hand, it has been reported that EE2 is removed effectively during wastewater treatment process as a result of previous surveys conducted in actual WWTPs. For example, Baronti et al. (2000) reported that the
EE2 concentration [ng/L]
3.5. Removal rate constants of estrogens in batch experiments It is generally known that the removal of organic substances in the water phase during the activated sludge process can be described by a pseudo-first-order reaction as given in Eq. (1). Previous studies which conducted batch experiments using activated sludge spiked estrogen have been reported that the removal of estrogen in the water phase can be described by a pseudo-first-order reaction as well as organic substances (Layton et al., 2000; Shi et al., 2004; Li et al., 2005). (1)
where C is the concentration of the target substance in the water phase (mg/L), k is the first-order-rate constant (h1) and t is the reaction time (h). Rearranging Eq. (1), the following equation is obtained, C ¼ kt (2) ln C0
1,000 800 600 400 200 0
median concentrations of EE2 in the influents and the effluents of six WWTPs in Rome, Italy, were 3.0 ng/L and 0.4 ng/L respectively, and the average removal rate of EE2 was 85%. In the previous studies mentioned above, the initial spiked concentrations of EE2 ranged from a few 10 mg/L to a few mg/L which were extremely higher than the concentrations of EE2 detected in wastewater, and EE2 was spiked into activated sludge as the sole energy source and/or the sole carbon source although many organic substrates coexist with estrogens in wastewater. Therefore, the previous batch experiments may underestimate the degradation ability of EE2 in actual activated sludge treatment process. It is difficult to validate the recovery efficiencies of estrogens from the sludge phase because of the sorption of estrogens on sludge, so that we could not validate them directly. In Run 13, as shown in Fig. 5, the total amount of EE2 until 2 h after the spike of EE2 was about the same as the initial spiked amount of EE2. It was considered that EE2 detected in Run 13 was derived from the initial spiked EE2, because EE2 was not detected in both the activated sludge sample and the synthetic wastewater. Therefore, we considered that EE2 was almost recovered from both the water phase and the sludge phase, and it seems that the recovery of other estrogens is similar to EE2 because of their relatively similar physicochemical properties. To increase reliability of the results in this study, further experiments will be required.
dC ¼ kC dt
1,200
579
n.d.
n.d.
before spiked 0min 5min 30min 1hr
2hr
4hr
8hr
24hr
Time EE2 in water phase
EE2 in sludge phase
Fig. 5 – Concentration profile of EE2 in the aerobic batch experiment (Run 13). ‘‘n.d.’’ means ‘‘not detectable’’, a.k.a. below the detection limit.
where C0 is the initial concentrations of the target substance in the water phase (mg/L). This equation suggests that a plot of ln(C/C0) versus t yields a straight line and the first-order rate constant k is obtained from a slope of the straight line. The concentration profiles of TOC and estrogen in the first series of batch experiments (Runs 1–8) plotted in the manner of ln(C/C0) versus t are shown in Fig. 6. As shown in Fig. 6 (1) and (3), in the removal of TOC, the linear relationships between ln(C/C0) and t were observed at all experiments, although the first-order rate constants k were different between activated sludge samples. Therefore, the removal of organic substance
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0
0 Run 1 (WWTP1) Run 3 (WWTP2) Run 5 (WWTP3) Run 7 (WWTP4)
Ln(C/C0) [-]
Ln(C/C0) [-]
-2 -1
Run 1 (WWTP1) Run 3 (WWTP2) Run 5 (WWTP3) Run 7 (WWTP4)
-2
-4
-6
-3
-8
0
1
2
3
4
0
1
2
t [hr]
(1) TOC (Runs spiked E2)
(2) E2 concentration Runs spiked E2
0
4
0 Run 2 (WWTP1) Run 4 (WWTP2) Run 6 (WWTP3) Run 8 (WWTP4)
Ln(C/C0) [-]
-2
Ln(C/C0) [-]
3
t [hr]
-1
Run 2 (WWTP1) Run 4 (WWTP2) Run 6 (WWTP3) Run 8 (WWTP4)
-2
-4
-6
-8
-3
0
1
2
3
4
0
1
2
3
4
t [hr]
t [hr]
(3) TOC (Runs spiked E1)
(4) E1 concentration (Runs spiked E1)
Fig. 6 – Description of the concentration profiles of TOC and estrogen in the first series of batch experiment (Runs 1–8) by the first-order rate expression.
was considered to follow the first-order-rate reaction given in Eq. (1). On the other hand, in the removal of estrogens as shown in Fig. 6 (2) and (4), the slopes between ln(C/C0) and t within 5 min immediately after the spike of estrogen were significantly steep when compared with those 5 min later. Thus, the concentration profiles of estrogens plotted in the manner of ln(C/C0) and t did not exhibit linear features, and the removal of estrogens was not considered to follow the first-order-rate reaction unlike the previous studies. In actual wastewater, estrogens exist at extremely low concentrations of ng/L level and many other substances coexist at much higher concentrations of mg/L level. On the other hand, estrogen was spiked into activated sludge with synthetic wastewater in this study, but estrogen was spiked into activated sludge as the sole energy and carbon source in the previous studies (Layton et al., 2000; Shi et al., 2004; Li et al., 2005). Furthermore, the initial concentrations of estrogens in these previous studies ranged from a few 10 mg/L to a few mg/L, and they were very high compared with the concentrations of estrogens observed in wastewater. Therefore, it is considered that the batch experiments in the previous studies mentioned above were conducted on the condition which distinctly differed from the actual condition in wastewater treatment processes, and these results would not accurately
reflect the actual reaction in activated sludge. From the results in this study, we assume that estrogens are immediately removed from the water phase to the sludge phase by adsorption at the first step of the reaction, and then the further removal of estrogens remaining in the water phase progress gradually with the degradation of estrogens in the sludge phase. However, the initial concentrations of estrogens in this study, i.e., 1000 ng/L, were about two orders of magnitude higher than the concentrations of estrogens observed in wastewater, although those were relatively lower than the initial concentrations of estrogens in the previous works. Further study is required about the mechanism of the removal and degradation of estrogens by activated sludge under the really similar conditions in actual wastewater treatment processes. The results of our study will give significant suggestions to future studies.
4.
Conclusions
The removal and degradation characteristics of estrogens by activated sludge were investigated by a series of the batch experiments using activated sludge samples taken from four
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actual WWTPs and synthetic wastewater spiked with estrogen. The conclusions are summarized as follows: 1. The removal and degradation characteristics in the aerobic batch experiments agreed with the actual removal efficiencies in the WWTPs from which the activated sludge samples were taken. E2 and E1 were immediately removed from the water phase and then further degraded in the sludge phase by the activated sludge samples of the OD plants which operated at higher SRT and showed the effective removal of estrogens. On the other hand, the removal and degradation of E1 were slower and E1 tended to remain in the activate sludge samples of the CAS plants which operated at lower SRT and showed higher effluent concentrations of E1. The higher SRT was considered to be preferable to establish the activated sludge adapted to degrade estrogens. 2. Under anoxic conditions, both E2 and E1 remained both in the water phase and the sludge phase after 4 h. The anoxic condition was considered to be not favorable for the effective removal of estrogens in wastewater treatment. 3. The removal and degradation of EE2 showed the lag phase during 2 h after the spike of EE2 which neither E2 nor E1 showed, but EE2 was finally removed and degraded completely after 24 h. 4. The large part of the spiked estrogen was immediately removed from the water phase to the sludge phase by adsorption within 5 min after the spike, and then estrogen was further removed gradually 5 min later. The removal of estrogens in the water phase did not follow the first-orderrate reaction.
Acknowledgement The authors thank the local authorities for kindly providing activated sludge samples and wastewater samples.
references
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