Antiestrogenic Activities of

Environ. Sci. Technol. 2009, 43, 4940–4945

Effect of Chlorination on the Estrogenic/Antiestrogenic Activities of Biologically Treated Wastewater QIAN-YUAN WU, HONG-YING HU,* XIN ZHAO, AND YING-XUE SUN Environmental Simulation and Pollution Control State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China

Received December 3, 2008. Revised manuscript received April 29, 2009. Accepted May 4, 2009.

Chlorination is widely used in wastewater reclamation, however harmful disinfection byproducts (DBPs) may be formed during disinfection. These DBPs are considered as a potential and important source of endocrine-disruption. In this study,theeffectsofchlorinationonestrogenicandantiestrogenic activities in biologically treated wastewater were evaluated by yeast two-hybrid assay. For the first time, chlorination was found to increase the antiestrogenic activity of wastewater notably and decrease the estrogenic activity. By fractionating dissolved organic matter (DOM) in wastewater into different fractions, it was found that the polar compounds (PC) fraction ofDOMwasthekeyfractioninvolvedinincreasingantiestrogenic activity during chlorination of wastewater. Furthermore, fluorescence spectroscopy analysis on different fractions of soluble organic compounds in wastewater suggested that the PC fraction contained most of the aromatic amino acids and humic/fulvicacid,whichwerethendemonstratedastheprecursors of antiestrogenic DBPs through chlorination experiments of tryptophan, humic acid, and tannic acid.

Introduction With rapid economic development, the problem of water shortage is becoming more serious in many countries, including China. In recent years, wastewater reclamation and reuse has been considered to be a viable and attractive method to solve this problem (1). However, there are some types of harmful estrogenic/antiestrogenic chemicals in wastewater (2-5). The estrogenic/antiestrogenic chemicals, which mimic or antagonize the actions of steroid hormones, have been reported to affect reproduction and development of animals (2, 6, 7). Accordingly, some estrogenic/antiestrogenic chemicals such as nonylphenols were included in the list of priority substances in the field of water policy established by the European Union (8), and the fate of estrogenic/antiestrogenic chemicals in wastewater reclamation treatment process is now of a heightened concern (5, 9). Chlorination is widely used as a key process in wastewater reclamation to prevent the spread of harmful pathogens (1, 10). However, chlorine can react with organic substances to produce hazardous disinfection byproduct (DBPs) (11-16), which are probably associated with endocrine-disruptors (17). As a result, DBPs of drinking water have been recommended as one type of the highest priority mixtures for endocrine * Corresponding author phone: (+8610)6279-4005; fax: (+8610)62797265; e-mail: [email protected]. 4940

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disrupting screening and testing by the Endocrine Disruptor Screening and Testing Advisory Committee of USEPA (18). The fate of the well-known estrogenic chemicals during chlorination has been studied recently. The results of these studies suggest that chlorination can not only degrade these compounds but also produce halogenated derivatives with estrogenic/antiestrogenic activities (4, 19, 20). Furthermore, only a few studies suggest that chlorination might increase the estrogenic activity of drinking water (21). However, compared to drinking water, there are many more types of dissolved organic matter (DOM) in biologically treated wastewater, including soluble microbial products and unknown estrogenic/antiestrogenic chemicals (3, 22). Therefore the reactions between chlorine and DOM during chlorination of wastewater are more complex than the reactions during drinking water disinfection. But the related research concerning the effect of chlorination on wastewater estrogenic/ antiestrogenic activities is very limited. Only recently, antiestrogenic activity of chlorinated wastewater effluent was found to increase during storage in a shallow infiltration basin (9). However, the changes of estrogenic/antiestrogenic activities before and after chlorination have not yet been studied. The aim of this study was, therefore, to evaluate the effects of chlorination on estrogenic and antiestrogenic activities in biologically treated wastewater and also to investigate the potential precursors of DBPs with endocrine-disrupting activity. This was to be done by using the yeast two-hybrid assay which allows measurement of the estrogen receptormediated gene activation or inactivation after exposure to estrogenic/antiestrogenic chemicals.

Materials and Methods Water Samples. Undisinfected wastewater samples used in this study were collected from the effluent of two different domestic wastewater treatment plants which used anaerobicanoxic-oxic process in one plant (sample A and C) and anoxic-oxic process in the other (sample B) as the main treatment methods. The samples were immediately delivered to the laboratory, filtered through glass fiber filters (0.7 µm) to eliminate suspended solids, and stored at 4 °C within 2 h for 20 days to minimize changes in the constituents. In each experiment, the estrogenic/antiestrogenic activities of wastewater after stored a certain time before and after chlorination were evaluated at the same time. Water Quality Analysis. The ammonia nitrogen, dissolved organic carbon, and pH of the sample were analyzed according to standard methods within 24 h after sampling (23). Concentration of ammonia nitrogen (CNH3-N) was determined by colorimetry using the phenate method. Concentration of dissolved organic carbon (CDOC) was detected with a TOC analyzer (model TOC-5000A, Shimadzu, Japan). The values of CNH3-N, CDOC, and pH of the samples used range 0.1-46.4 mg/L, 10.8-56.5 mg/L and 7.2-8.2, respectively. Chlorination. Chlorination experiments were conducted in 300 mL glass bottles with Teflon inner plugs. Wastewater samples (280 mL) were chlorinated with 2-50 mg/L of available chlorine for 30 min as previously described (16). All of the chemical reagents used were of an analytical or HPLC purity. Concentration of Wastewater Samples. Wastewater samples (280 mL) were acidified to pH 2.0 with 2 M H2SO4, and passed through OASIS HLB resin cartridges (Waters Corporation, America), which were previously washed with 10 mL of methanol and 10 mL of ultrapure water. The 10.1021/es8034329 CCC: $40.75

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cartridges were then dried under a flow of air. Retained organics on the cartridge were eluted with methanol (10 mL) to obtain polar compounds (PC), followed by dichloromethane (10 mL) to yield midpolar compounds (MPC), and finally by hexane (10 mL) to obtain nonpolar compounds (NPC). Thereafter, 5 mL of the fractions were mixed to obtain the whole adsorbed organics of wastewater. Seventy five percent of the mixture and fractions were dried under a nitrogen flow. The dry residues were dissolved in 105 µL of dimethylsulfoxide (DMSO) to obtain 1000-fold concentration (volume of wastewater/volume extract) for estrogenic/ antiestrogenic activity assay. For fluorescence spectroscopy analysis, the rest of the mixture and fractions were evaporated to near dryness and dissolved with ultrapure water to obtain 3.5-fold concentration. Estrogenic Activity Assay. The estrogenic activity of the concentrated samples was evaluated with the yeast twohybrid assay based on yeast cells (Saccharomyces cerevisiae Y190) which contained the rat estrogen receptor ERR and the coactivator TIF2 (24). In this assay, the β-galactosidase induced by estrogenic chemicals was used to monitor the estrogenic activity. The assay was conducted as follows: The yeast cells were preincubated overnight at 30 °C. The 100 µL overnight yeast culture and 20 µL DMSO solution containing the samples were added to 400 µL of fresh medium and incubated for 4 h at 30 °C. After incubation, 150 µL of yeast culture was fractioned for the absorbance at 595 nm. The residual culture (370 µL) was collected by centrifugation and then digested by incubation with 1 g/L Zymolyase 20T at 37 °C for 15 min. The enzymatic reaction was started by addition of 4 g/L 2-nitrophenyl-β-D-galactoside (ONPG) at 30 °C and then stopped by addition of 1 M Na2CO3 after 30 min. Thereafter, the solution was centrifuged and 150 µL of supernatant liquid was taken for the absorbance at 415 and 570 nm. The absorbance at 415, 570, and 595 nm was converted to β-galactosidase activity according to the equation described in the literature (24). The DMSO solutions with different concentrations of 17βestradiol (E2) were used as positive controls to obtain the dose-response curve of E2. The estrogenic activity of the sample was standardized to an equivalent E2 concentration and then divided by concentration fold to obtain the estrogenic activity value of the sample before concentration. Antiestrogenic Activity Assay. The ability of the concentrated sample to inhibit β-galactosidase activity of E2 was measured to determine the antiestrogenic activity of the concentrated sample according to the yeast two-hybrid assay (25, 26). In this assay, besides 150 µL of yeast culture, 20 µL of DMSO solution containing the concentrated sample and additional E2 was also added to 400 µL of fresh medium. The final concentration of additional E2 was 0.77 µg/L which gave a 40% submaximal ER agonist response in the absence of antiestrogenic chemicals. After 4 h incubation, the β-galactosidase activity of yeast culture with the sample and E2 was determined with three replicates and then converted to percentage inhibition of the concentrated sample to the β-galactosidase induction according to eq 1, as follows: Ig(%) )

UE2 - UX × 100 UE2

(1)

where Ig represents the inhibition of the concentrated sample to β-galactosidase inducted by E2, UE2 is the β-galactosidase activity of E2, and Ux is the observed β-galactosidase activity of E2 and the concentrated sample. Toxicity Assay. Because toxic chemicals can also inhibit the growth of yeast cells to result in the β-galactosidase activity inhibition, the toxicity of the sample was evaluated by the value of the absorbance at 595 nm (OD595) after 4 h incubation of yeast culture during the measurement of antiestrogenic

FIGURE 1. Changes in estrogenic activity of wastewater samples A-C after chlorination (10 mg-Cl2/L). Asterisks represent the estrogenic activity of samples which was below method detection limit (1 ng-E2 equivalents/L). Error bars represent the standard deviation based on triplicate analyses. activity (24, 26). The value of OD595 was converted to percentage inhibition of the concentrated sample to yeast growth according to eq 2, as follows: Ic(%) )

ODE2 - ODx × 100 ODE2

(2)

where Ic represents the inhibition of the sample to growth of yeast cell, ODE2 is the absorbance after 4 h incubation with E2 (0.77 µg/L final concentration), and ODx is the absorbance after incubation with E2 and the concentrated sample. The sample is assessed as toxic when the value of ODx is significantly less than that of ODE2 (p < 0.05). Fluorescence Spectroscopy. Fluorescence spectra of concentrated sample C and its fractions before chlorination at 3.5-fold concentration were recorded on a fluorescence spectrophotometer (model F-7000, Hitachi, Japan). Threedimensional spectra were obtained as previously described (16). All contour maps were plotted using the same scale range of fluorescence intensities and number of contours.

Results and Discussion Effect of Chlorination on Wastewater Estrogenic Activity. The changes in estrogenic activity of sample A-C before and after disinfection with 10 mg/L of available chlorine were investigated (Figure 1). It can be seen that the estrogenic activity of the samples before disinfection ranged 3-6 ng-E2 equivalents/L. After chlorination, the estrogenic activity of all samples was less than 50% of the original. Since chlorination could reduce some estrogenic chemicals such as E2 (19, 20), it is possible for some estrogenic chemicals of wastewater to produce less estrogenic byproduct after chlorination and thus result in a decrease of estrogenic activity. Effect of Chlorination on Estrogenic Activity of Different DOM Fractions. To discover the main precursors causing the changes in wastewater estrogenic activity, sample C disinfected with different chlorine dosages was isolated into three fractions, and thereafter the estrogenic activity of these fractions was measured (Figure 2). It can be clearly seen that the changes in estrogenic activity of sample C were consistent with the previous result in this paper. For the three fractions from sample C before chlorination, PC and MPC exhibited significant estrogenic activity, while the estrogenic activity of NPC was below the detected limit (1 ng-E2 equivalents/ L). After chlorination, the estrogenic activity of both PC and MPC decreased noticeably, while no estrogenic activity was detected in NPC. VOL. 43, NO. 13, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. Changes in estrogenic activity of sample C and its fractions after chlorination with different chlorine dosages. SC, sample C; PC, polar compounds; MPC, midpolar compounds; NPC, nonpolar compounds. Error bars represent the standard deviation based on triplicate analyses.

FIGURE 3. Changes in antiestrogenic activity and toxicity of sample C at 250-fold concentration after chlorination with different chlorine dosages. Error bars represent the standard deviation based on triplicate analyses. We also found that the sum of estrogenic activity of three fractions before chlorination was higher than that of the whole sample C, while all of the fractions did not have toxic effects (growth inhibition) to yeast cells (data no shown here). It is possible that some compounds in the sample may have antiestrogenic activity. It is also possible that macromolecules in water, including humic/fulvic acid, adsorb the estrogenic chemicals and mask the estrogenic activity in the yeast bioassay analysis (27-29). These phenomena suggest that the decrease of estrogenic activity may result from formation of antiestrogenic byproduct and/or reduction of estrogenic chemicals. Effect of Chlorination on Wastewater Antiestrogenic Activity. The effect of chlorination on the changes in antiestrogenic activity and toxicity of sample C is presented in Figure 3. Sample C before and after chlorination showed inhibition of β-galactosidase induction, but not inhibition of yeast cell growth. This phenomenon suggests that the β-galactosidase activity inhibition of the wastewater sample results from the antiestrogenic action. This suggestion was supported by the fact that the sum of estrogenic activity of three fractions before chlorination was higher than that of the whole wastewater sample. It has been reported that wastewater exhibited antiestrogenic activity, while the nature of the inhibitory compound was still elusive (3). The antiestrogenic activity of wastewater in the bioassay analysis may result from antiestrogenic chemicals (3) and/or macromolecules, which can adsorb the additional E2 (27-30). 4942

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FIGURE 4. Changes in antiestrogenic activity of sample C and its fractions at 250-fold concentration after chlorination with differing chlorine dosages: SC, sample C; PC, polar compounds; MPC, midpolar compounds; NPC, nonpolar compounds. Error bars represent the standard deviation based on triplicate analyses. Both the fates of the estrogenic and antiestrogenic chemicals in wastewater reuse process and the target organs and dose-response relationship of these chemicals are complex. Therefore, the effects of both the estrogenic and antiestrogenic chemicals in wastewater on health and ecological risk are complex and should be of concern. The β-galactosidase activity inhibition of chlorinated sample C increased from 50% to 80% with the addition of chlorine disinfectant (Figure 3), suggesting that chlorination can increase the antiestrogenic activity of wastewater notably. This suggestion was supported by the dose-response curves of antiestrogenic activity for sample C before and after chlorination (10 mg-Cl2/L) as shown in Figure S1. The results clearly demonstrate that wastewater exhibits inhibition of β-galactosidase induction, and this inhibition increases after chlorination, while none of the concentrated samples did not have toxic effects (growth inhibition) to yeast cells (data not shown here). Effect of Chlorination on Antiestrogenic Activity of Different DOM Fractions. To discover the main precursors causing the changes in wastewater antiestrogenic activity, sample C disinfected with different chlorine dosages was fractionated into three fractions, and then antiestrogenic activity and toxicity of these fractions were measured. For sample C and its fractions, no notable toxicity (growth inhibition) was found (data no shown here), suggesting that the β-galactosidase activity inhibition of the sample and its fractions is due to the antiestrogenic action (Figure 4). As shown in Figure 4, the changes in antiestrogenic activity for sample C with different chlorine doses were in accordance with the results mentioned above in this study. Among the three fractions before chlorination, PC exhibited significant antiestrogenic activity, while no inhibition of β-galactosidase activity was detected in MPC and NPC. After chlorination, the inhibition of β-galactosidase activity in PC increased from 33% to 79% with increasing chlorine dose, which was close to that of the whole wastewater sample, while no antiestrogenic activity was detected in MPC and NPC. This reveals that the PC fraction plays an important role in increasing antiestrogenic activity during chlorination. Because it is hard to consider how chlorination can enhance adsorption capacity of macromolecules, the increase of antiestrogenic activity is more likely due to formation of antiestrogenic DBPs. Further efforts on antiestrogenic activity evaluation and chemical identification of chlorinated wastewater should be performed. In the past few years, much progress has been made in understanding the mechanisms of antiestrogenic chemicals.

FIGURE 5. Fluorescence spectroscopy of sample C and its fractions before chlorination at 3.5-fold concentration: SC, sample C; PC, polar compounds; MPC, midpolar compounds; NPC, nonpolar compounds; Flu1, tyrosine-like aromatic protein; Flu2, tryptophan-like, aromatic protein; Flu3 and 4, soluble microbial byproduct-like; Flu 5, humic/fulvic acid-like. The antagonists such as raloxifene and 4-hydroxytamoxifen can competitively bind to ER and result in blocking gene expression (31). The antagonists can also promote interaction between ER and corepressors, which repress ER-mediated gene induction, and result in a portion of antiestrogenic activity (31). In our study, antiestrogenic activity of DBPs may arise from the mechanisms such as competitive binding, but not promotion of corepressors interacting with ER because there are not any corepressors in the yeast cell. These mechanisms indicate that the antiestrogenic DBPs may block the natural estrogen action. Exposure to DBPs in drinking water such as trihalomethanes was reported to probably affect ovarian functions (32). Therefore, further research on the risk and mechanism of antiestrogenic DBPs in chlorinated reclaimed water is required. In the MPC fraction, no antiestrogenic activity was detected during chlorination (Figure 4), while estrogenic activity was found to decrease significantly (Figure 2). This phenomenon indicates that some estrogenic chemicals of MPC may be reduced during chlorination. Fluorescence Spectroscopy of Different DOM Fractions. In this study, different fractions showed different changes in the antiestrogenic activity during chlorination. Thus, an excitation emission matrix (EEM) fluorescence spectroscopy was used to characterize the chemical structures of the fractions. The EEM spectra for the sample C and its fraction before chlorination are shown in Figure 5. It can be seen that there were different peaks for sample C and its different fractions. The position of Exmax/Emmax and the intensity at Exmax/Emmax for these peaks are shown in Table S1. These EEM peaks are associated with humic/fulvic acid-like, tyrosine-like, tryptophan-like, or soluble microbial byproduct-like organic compounds according to a location of EEM peaks of many typical chemicals in wastewater or surface water (33).

It was found that the PC fraction contained an amount of humic/fulvic acid and aromatic protein similar to that of the whole wastewater sample, while MPC and NPC contained little. Since the PC fraction played an important role in increasing antiestrogenic activity, it is necessary to find whether aromatic amino acids and humic/fulvic acid can produce antiestrogenic byproduct during chlorination. Furthermore, since macromolecules including humic/ fulvic acid were reported to exhibit antiestrogenic activity and mask the estrogenic activity in the bioassay (29, 30), it is possible that the distribution of humic/fulvic acid in the fractions results in the phenomena that PC exhibited the most antiestrogenic activity and MPC presented the most estrogenic activity. Antiestrogenic Activity of Typical Precursors after Chlorination. Typical aromatic precursor solutions were prepared by dissolving tryptophan (Trp), humic acid (HA), or tannic acid (TA) in ultrapure water at a concentration of 20 mg/L. After the solutions were chlorinated with 30 mg/L available chlorine for 30 min, the antiestrogenic activity and toxicity (growth inhibition) were measured. HA and TA displayed obvious inhibition on β-galactosidase activity, but not yeast cell growth (Figure 6). After chlorination, the β-galactosidase activity inhibition caused by all solutions increased notably with ranges which were obviously greater than those of yeast growth inhibition. This phenomenon was partially supported by the dose-response curves of antiestrogenic activity for Trp (20 mg/L) before and after chlorination (Figure S2). It was found that chlorinated Trp exhibited obvious inhibition of β-galactosidase induction, while the concentrated samples did not have toxic effects (growth inhibition) to yeast cells (data no shown here). These results indicate that Trp, HA, and TA display obvious antiestrogenic activity after chlorination, demonstrating that antiestrogenic byproduct can be formed during chlorination of aromatic amino acid and humic/fulvic acid. Further work on antiesVOL. 43, NO. 13, 2009 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 6. Changes in the antiestrogenic activity and toxicity of typical chemicals at 500-fold concentration after chlorination (30 mg-Cl2/L): Trp, tryptophan; HA, humic acid; TA, tannic acid. Error bars represent the standard deviation based on triplicate analyses. trogenic activity evaluation and chemical identification of chlorinated byproduct of amino acids and humic/fulvic acid should be performed. Furthermore, amino acid chlorination is of great concern at points of in vivo oxidation. In vivo, hypochlorite generated by phagocyte cells reacts with a number of biological targets including amino acids, peptides, and proteins (34). The chlorination of amino acids in vivo is linked to some diseases such as atherosclerosis (35). Our findings concerning the chlorinated amino acid with antiestrogenic activity suggest that more work will be required to determine whether amino acids chlorination in vivo produce antiestrogenic DBPs. Also investigation of the relationship between antiestrogenic DBPs in vivo and the diseases is needed.

Acknowledgments This study was funded by National Science Fund for Distinguished Young Scholars of China (50825801) and National Hightech R&D Program (863 Program) (2008AA062502). We thank Prof. Nishikawa for providing the yeast cells.

Supporting Information Available One table and two figures present detailed information on the EEM peaks of the samples and dose-response of the samples and tryptophan. This material is available free of charge via the Internet at http://pubs.acs.org.

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