Reduced Effect of Bromide on the Genotoxicity in Secondary Effluent

Jun 3, 2010 - ... China, Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, College of Environm...
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Environ. Sci. Technol. 2010, 44, 4924–4929

Reduced Effect of Bromide on the Genotoxicity in Secondary Effluent of a Municipal Wastewater Treatment Plant during Chlorination QIAN-YUAN WU,† YI LI,‡ H O N G - Y I N G H U , * ,† Y I N G - X U E S U N , † A N D FENG-YUN ZHAO§ Environmental Simulation and Pollution Control State Key Joint Laboratory, Department of Environmental Science and Engineering, Tsinghua University, Beijing 100084, PR China, Key Laboratory of Integrated Regulation and Resource Development on Shallow Lakes, Ministry of Education, College of Environment, Hohai University, Nanjing 210098, P.R. China, and College of Resource and Environment, Shaanxi University of Science & Technology, Xi’an 710021, PR China

Received May 20, 2009. Revised manuscript received May 21, 2010. Accepted May 21, 2010.

Chlorination of wastewater can form genotoxic, mutagenic, and/ or carcinogenic disinfection byproduct (DBPs). In this study, the effect of bromide on genotoxicity in secondary effluent of a municipal wastewater treatment plant during chlorination was evaluated by the SOS/umu test. The presence of bromide notably decreased the genotoxicity in secondary effluent during chlorination, especially under conditions of high ammonia concentration.Bromidesignificantlydecreasedtheconcentration of ofloxacin, a genotoxic chemical in secondary effluent, during chlorination with high concentration of ammonia, while genotoxic DBPs formation of humic acid and aromatic amino acids associated with bromide limitedly contributed to the changes of genotoxicity in secondary effluent under the conditions of this study. By fractionating dissolved organic matter (DOM) in the secondary effluent into different fractions, the fractions containing hydrophilic substances (HIS) and hydrophobic acids (HOA) contributed to the decrease in genotoxicity induced by bromide. Chlorination of HOA without bromide increased genotoxicity, while the addition of bromide decreased genotoxicity.

Introduction Water shortages have become serious in many countries. Therefore, wastewater reclamation and reuse have become almost essential for conserving and extending available water supplies (1). However, untreated wastewaters contain many types of pathogenic organisms (1). Therefore, wastewater must be thoroughly disinfected to prevent the transmission of disease. In most treatment facilities, chlorination is the most commonly used process in wastewater reclamation (1). Although it is highly effective at eliminating pathogens from wastewater, chlorine is also known to react with dissolved * Corresponding author phone: (+8610)6279-4005; fax: (+8610)62797265; e-mail: [email protected]. † Tsinghua University. ‡ Hohai University. § Shaanxi University of Science & Technology. 4924

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organic matter (DOM) to produce genotoxic, mutagenic, and/ or carcinogenic disinfection byproduct (DBPs) (2-4). Accordingly, some DBPs including total organic halogens, are listed as hazards in regulations of reclaimed water for indirect potable reuse (5). For chlorination of drinking water, the effect of bromide on the formation and speciation of DBPs is of concern (6, 7). Bromide naturally occurs in many source waters, with an observed range from undetectable to 2 mg/L (6, 8). Bromide can be oxidized by hypochlorous acid (HOCl) and monochloramine (NH2Cl) to various active bromine species, including hypobromous acid and monobromamine (9, 10), both of which are stronger halogenating agents than either HOCl or NH2Cl. These active bromine species can easily react with organic substances to form brominated DBPs, which pose even more significant health risks than do the chlorinated analogs (2). Recent studies have shown the emergence of a number of brominated DBPs, including tribromopyrrole, as serious drinking water contaminants (2, 7). At this point, a significant portion of the total organic bromine in drinking water remains uncharacterized (11, 12). Compared to drinking water, there is an even higher level of bromide in some wastewaters. For example, the effluents from the treatment plants in seaside areas, which use seawater for toilet flushing, can contain 20-30 mg/L bromide ion (13). In secondary effluent, there may be many more types of DOM, including soluble microbial products (14). Therefore, the reactions among chlorine, bromide, and DOM during chlorination of wastewater are more complex than drinking water chlorination. Only measuring the formation of the typical DBPs commonly associated with chlorination is not sufficient for evaluating the safety of wastewater if bromide is also present. Short-term genotoxicity tests are useful for monitoring the genotoxicity of DBPs and for evaluation of unidentified genotoxic chemicals (15, 16). Many studies have suggested that the effect of chlorination on genotoxicity of wastewater is very complex and is specific to the wastewater samples used (15, 16). For example, ammonia nitrogen (NH3-N) can induce the increase of genotoxicity during chlorination (15). Although bromide plays a very important role in the speciation of DBPs (17, 18), the effect of bromide concentration on genotoxicity of wastewaters during chlorination has not yet been studied. Therefore, the purpose of this study was to evaluate the effect of bromide on genotoxicity in secondary effluent during chlorination. An in vitro SOS/umu test by the genetically modified TA1535/pSK1002 strain of Salmonella typhimurium was used to evaluate the genotoxicity of wastewater samples. This strain allows measurement of the activation of the SOS repair response induced by exposure to genotoxic chemicals.

Materials and Methods Water Samples. The wastewater samples used for the experiments were collected from the effluent of the anaerobic-anoxic-oxic process of a domestic wastewater treatment plant in Beijing twice (samples A and B). The samples were kept on ice, immediately delivered to the laboratory, and then filtered through glass fiber filters (0.7 µm) to eliminate suspended solids. Thereafter, 100-mL samples were taken for water quality analysis. The samples were adjusted to pH 2.0 with sulfuric acid and stored at 4 °C to minimize changes in the constituents. The characteristics of samples A and B are shown in Table S1 in the Supporting Information. The bromide concentration of samples A and B was below the method detection limit (0.1 mg/L). 10.1021/es100152j

 2010 American Chemical Society

Published on Web 06/03/2010

Water Quality Analysis. After filtration, NH3-N, dissolved organic carbon, pH, and bromide ion content of the sample were analyzed according to the standard methods (19). The concentration of NH3-N was determined by colorimetry using the phenate method. Concentration of dissolved organic carbon (DOC) was measured with a TOC analyzer (TOC-5000A, Shimadzu, Japan). Bromide ion concentration was measured by ion chromatography (Dionex ICS1000) using an anionic column (Dionex AS9-HC). Fractionation of Secondary Effluent Samples. DOM of the secondary effluent samples (5 L) was isolated into four fractions: hydrophilic substances (HIS), hydrophobic acids (HOA), hydrophobic bases (HOB), and hydrophobic neutral (HON). The fractionation was performed using XAD-8 resin columns (16 mm diameter and 20 cm high, 40 mL, Sigma, USA) as previously described (15, 20). Each fraction was adjusted to pH 7.0 ( 0.2 and sufficient ultrapure water (18.2 MΩ) was added to restore the volume to 5 L. The DOC recovery of the resin was about 98% under the conditions in this study. The fresh resin was previously solvent extracted with acetone and n-hexane for 6 days and then washed with HCl, NaOH, and ultrapure water solution for 3 days. After previous wash, the eluates from the fresh resin washed again with HCl, NaOH, ultrapure water, or methanol before fractionation were solvent-exchanged into DMSO and then the genotoxicity of the DMSO concentrates was found below the detection limit by the SOS/umu test. Chlorination. Chlorination experiments were conducted in 150-mL glass bottles with Teflon inner plugs. Before chlorination, the pH value of the samples was adjusted to 7.0 ( 0.2 with 1 M H2SO4 or 2 M NaOH solution. Secondary effluent samples or fractions of DOM (150 mL) were chlorinated with 10-30 mg/L of available chlorine for 30 min as previously described (15). All of the chemical reagents used were analytical or HPLC grade. Chlorine residuals were immediately measured by a N,N-Diethyl-p-phenylenediamine-ferrous ammonium sulfate titration (19). Concentration of Secondary Effluent Samples for Genotoxicity Analysis. Secondary effluent samples or fractions of DOM (140 mL) were acidified to pH 2.0 with 2 M H2SO4, and then passed through resin cartridges containing 1 g of CHP20P resin (Mitsubishi Chemical, Japan), which consisted of 75-150 µm poly(styrene-divinylbenzene), as previously described (15). Organics retained on the cartridge were eluted with acetone and completely dried under a nitrogen flow. The dry residues used for genotoxicity analysis were then dissolved in 140 µL of dimethylsulfoxide (DMSO) to obtain 1000-fold concentration (volume of secondary effluent/ volume extract). The dry residues for ofloxacin analysis were dissolved in 0.7 mL of liquid chromatography mobile phase (the mixture of 70% water (0.1% formic acid) and 30% methanol) to obtain 200-fold concentration, filtered through nylon syringe filters (0.45 µm), and then stored at -20 °C. Genotoxicity Assay. The genotoxicity of the concentrated secondary effluent samples and DOM fractions was evaluated with the SOS/umu test based on Salmonella typhimurium TA1535/pSK1002 without S9 activation according to ISO 13829 (21). In this assay, the β-galactosidase induced by genotoxic chemicals was applied to monitor overall genotoxicity. The DMSO solution of 4-nitroquinoline-N-oxide (4NQO) was used as the positive control. Based on the dose-response curves of 4-NQO and the concentrated sample, the genotoxicity of the sample was standardized to an equivalent 4-NQO concentration. The detail of genotoxicity assay was provided in Supporting Information Method S1. Ofloxacin Concentration Analysis. The ofloxacin concentration in secondary effluent samples was determined by ultra-performance liquid chromatography with tandem mass spectrometry detector (LC-MS/MS). The ofloxacin concen-

FIGURE 1. Changes in genotoxicity of secondary effluent samples A and B after chlorination (10 mg-Cl2/L) in the presence of different bromide concentrations. Asterisks indicate that the genotoxicity of chlorinated samples was significantly different from that of the original sample (p < 0.05). Pound signs indicate that the genotoxicity of samples after chlorination in the presence of bromide was significantly different from that without bromide addition (p < 0.05). Plus signs indicate that the sample did not exhibit the genotoxicity at 1000-fold. Error bars represent the standard deviation. tration in synthetic solution was measured by highperformance liquid chromatography with fluorescence detector. Details of ofloxacin analysis are provided in Supporting Information Method S2. Fluorescence Spectroscopy. Excitation emission matrix (EEM) fluorescence spectra of samples A and B during chlorination (10 mg-Cl2/L) in the presence of different concentrations bromide were recorded on a fluorescence spectrophotometer (F-7000, Hitachi, Japan). EEM spectra were obtained as described in the literature (22). The EEM spectra were divided into five regions, associated with humic/ fulvic acid-like, tyrosine-like, tryptophan-like, or soluble microbial byproduct-like organic compounds, according to the location of EEM peaks of many typical chemicals in wastewater or surface water as shown in Table S2 (22, 23). The spectra of the regions were quantitatively analyzed by the fluorescence regional integration (FRI) method (22). The Holm-Sidak test of SigmaStat 3.5 software (Systat Software Inc., USA) was used to evaluate the difference in fluorescence intensities (FI) of different samples (statistical significance at p < 0.05).

Results and Discussion Effect of Bromide on Secondary Effluent Genotoxicity. To investigate the effect of bromide on genotoxicity during chlorination, NaBr was added to samples A and B to obtain a bromide concentration of 10 mg/L. After the samples (with and without added NaBr) were chlorinated with 10 mg/L of chlorine for 30 min, the genotoxicity was measured, as shown in Figure 1. The genotoxicity of the samples before chlorination ranged 22-32 µg-4NQO/L. After chlorination, the genotoxicity of both samples was less than 20% of the original (p < 0.05) (Figure 1). This suggested that chlorination did clearly decrease the genotoxicity of samples A and B, in agreement with previously published findings (15, 24). The dominant residual chlorine was free chlorine (data not shown here). Since free chlorine can reduce some genotoxic chemicals, including nitrofurazone, and frazolidone (25), it is possible for some genotoxic chemicals of secondary effluent to generate less genotoxic byproduct after chlorination and this would explain the observed decrease in genotoxicity. It is interesting to note that after chlorination, the genotoxicity of sample B with added bromide was significantly lower than that without added bromide (p < 0.05). VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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Bromide therefore promoted a decrease in genotoxicity in sample B during chlorination. During chlorination, bromide can be oxidized to form hypobromous acid and thus react with some of DOM in secondary effluent to form brominated DBPs (17, 18), which are much more genotoxic and cytotoxic than their chlorinated analogs (2, 26). Hua et al. (11) also found that with bromide addition, total organic bromine formation of natural water after chlorination increased and total organic chlorine formation decreased, while there was no significant change in the overall total organic halogen concentration. Meanwhile, some of the DOM in secondary effluent were genotoxic and/or mutagenic themselves. For example, the sample B in this study contained 350 ng/L ofloxacin, a genotoxic chemical. Ofloxacin was often reported in the influent and effluent of wastewater treatment plants, with an observed range from 0.1 to 1 µg/L (27, 28). Therefore, it is necessary to find whether bromide could decrease or increase the genotoxicity of these genotoxic chemicals during chlorination. To evaluate the effect of bromide on genotoxic chemicals during chlorination, several solutions of genotoxic chemicals were prepared by dissolving ofloxacin and 1-nitropyrene in ultrapure water at the nominal concentration of 100 µg/L. Thereafter, NaBr was added to each solution to obtain a bromide concentration of 10 mg/L. After the samples (with and without added NaBr) were chlorinated with 10 mg/L of chlorine for 30 min, the genotoxicity was measured, as shown in Figure S3. These chemicals before chlorination exhibited strong genotoxicity. It is interesting to note that the genotoxicity of ofloxacin (96 µg/L final concentration) was about 7200 µg4NQO/L, which is similar to previous findings based on the results of SOS/umu test, SOS-Chromotest, and Ames assay (29, 30). The ofloxacin concentration in sample B was 350 ng/L and the genotoxicity of sample B was 32 µg-4NQO/L. Therefore, ofloxacin contributed importantly to the genotoxicity of secondary effluent. After chlorination without bromide addition, the ofloxacin concentration of synthetic solution decreased from 96 to 0.2 µg/L (Figure S4) and the synthetic solution did not exhibit genotoxicity in the test with 0.056-2-fold (Figure S3). The dominant residual chlorine was free chlorine (data not shown here). This indicated that free chlorine can decrease ofloxacin into less genotoxic byproduct, in agreement with the above finding that free chlorine decreased the genotoxicity in secondary effluent. Further research concerning the decrease mechanism of ofloxacin and its genotoxicity during chlorination is still needed. However, the genotoxicity of 1-nitropyrene was significantly higher than the original (p < 0.05), suggesting that more genotoxic byproduct can be formed during chlorination. After chlorination with bromide addition, the concentration of synthetic ofloxacin solution and its genotoxicity were also below the detected limited, not statistically significantly different from those without bromide addition (Figures S3-S4). Since free chlorine efficiently decreased ofloxacin, it is hard to evaluate the effect of bromide on ofloxacin during chlorination. Further study concerning effect of bromide on ofloxacin is necessary. After chlorination, the genotoxicity of 1-nitropyrene with added bromide was about 114% of that without added bromide, suggesting that bromide can result in the formation of more genotoxic byproduct during chlorination of 1-nitropyrene. The different genotoxicity changes of these genotoxic chemicals during chlorination suggested that the genotoxicity changes during chlorination was influenced by species and amounts of the genotoxic chemicals in secondary effluent. Under the conditions of this study, the genotoxicity of ofloxacin was much higher than that of 1-nitropyrene at the same concentration. The genotoxicity changes of ofloxacin 4926

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FIGURE 2. Changes in genotoxicity and residual chlorine of sample A under condition of high ammonia concentration (30.0 mg-N/L) after chlorination (10 mg-Cl2/L) in the presence of a range of bromide concentrations. Pound signs and error bars are the same as those in Figure 1. during chlorination were also higher than those of 1-nitropyrene, suggesting that ofloxacin might play a more important role involving the genotoxicity changes during chlorination compared to 1-nitropyrene. Effect of Bromide on Genotoxicity in Secondary Effluent with High NH3-N Concentration. Wang et al. (15) reported that ammonia can induce an increase in genotoxicity of secondary effluent during chlorination. Therefore, the effect of bromide on genotoxicity in secondary effluent containing a high concentration of ammonia during chlorination was also evaluated. NH4Cl was added to samples A and B to obtain a NH3-N concentration of 30.0 mg/L, and then chlorination in the presence of different concentrations of bromide was performed (Figure S5). Without bromide addition, the genotoxicity of the sample A increased to 132% of the original value after chlorination under high NH3-N concentration (p < 0.05). The genotoxicity of the sample B decreased, but the range of the decrease was much lower than that seen in the absence of ammonia addition (Figure 1). These changes in genotoxicity during chlorination suggested that chlorination in the presence of high levels of NH3-N may increase the genotoxicity in secondary effluent, as previously described (15). In secondary effluent, ammonia can react immediately with free chlorine and form chloramines due to its high reaction rate coefficient (Table S3). This was partly supported by the fact that the dominant residual chlorine form was chloramines, but not free chlorine. Chloramines can react with some types of DOM to form much more genotoxic DBPs, including N-nitrosodimethylamine and iodoacetic acids (3, 26). Furthermore, compared to free chlorine, chloramines react with DOM much more slowly due to their lower reaction rate coefficients (Table S3). This implies that chloramines may cause less reduction in the levels of genotoxic chemicals in secondary effluent than chlorine does. After chlorination, samples with high NH3-N concentration in the presence of bromide had genotoxicity levels of only 6-9% of that prior to chlorination (p < 0.05). Bromide therefore decreased the genotoxicity of the chlorinated secondary effluent. This suggestion was supported by the reduction in genotoxicity in sample A following additions of different concentrations of bromide, as shown in Figure 2. The dose-response curves of sample A with different concentrations of bromide are also shown in Figure S6. The genotoxicity of sample A presented a nearly logarithmic decrease with increasing bromide concentration (p < 0.05). After chlorination, the genotoxicity of the sample with 2.5 mg/L bromide was only 32% of that without bromide addition. This demonstrated that bromide can significantly

FIGURE 3. Changes in genotoxicity of humic acid, tyrosine, and tryptophan (11.6 mg/L DOC nominal concentration) under condition of high ammonia concentration (30.0 mg-N/L) after chlorination (10 mg-Cl2/L) in the presence of different bromide concentrations. Asterisks, pound signs, plus signs, and error bars are the same as those in Figure 1. decrease the genotoxicity of secondary effluent during chlorination. In secondary effluent having a high concentration of NH3-N, chloramines were formed first. In the presence of bromide, the chloramines then oxidized bromide to various active bromine species including monobromamine and bromochloramine. This occurred because the rate coefficient of chloramine formation was 3 orders of magnitude greater than active bromine formation (Table S3) (9). However, the reaction rate coefficients of active bromine reacting with DOM were 4 orders of magnitude greater than that of chloramine (Table S3) (9), which suggests that the active bromine can react with DOM much faster than chloramines and free chlorine. The changes in EEM spectra for samples A and B (with high NH3-N concentration) during chlorination were measured. Representative EEM spectra and their fluorescence intensity (FI) of sample A are shown in Figures S7 and S8. The EEM spectra indicated that sample A contained humic/ fulvic acid and aromatic protein-like organic compounds. Bromide was found to induce a decrease in FI in secondary effluent during chlorination (Figure S8). This effect of bromide most likely reflects a change in the chemical structure of DOM during chlorination. This also partly supports the above supposition that the active bromine can react with DOM much faster than chloramines do. Effect of Bromide on Genotoxicity in Typical Chemicals with High NH3-N Concentration. In this study, fluorescence spectra of secondary effluent samples exhibited the presence of humic/fulvic acid and aromatic protein-like organic compounds, which have been reported as important precursors of DBPs (6, 15). Thus, the effect of bromide on genotoxicity of these precursors during chlorination with high NH3-N concentration was evaluated. Typical precursor solutions were prepared by dissolving humic acid, tyrosine, and tryptophan in ultrapure water at a DOC value of 11.6 mg/L, which was the same as that of sample A. NH4Cl was added to each solution to obtain a NH3-N concentration of 30.0 mg/L, and then chlorination of each solution in the presence of different concentrations of bromide was performed (Figure 3). The dominant residual chlorine form was chloramines (data not shown here). After chlorination, the genotoxicity of humic acid and tyrosine with bromide addition was significantly higher than that without bromide addition, suggesting that bromide can induce the formation of more genotoxic DBPs during the chlorination of these typical precursors. This is similar to the previously described finding concerning drinking water disinfection (31). However, the increase of genotoxicity in these precursors induced by bromide only ranged about

FIGURE 4. Changes in genotoxicity of ofloxacin and 1-nitropyrene (100 µg/L nominal concentration) under condition of high ammonia concentration (30.0 mg-N/L) after chlorination (10 mg-Cl2/L) in the presence of different bromide concentrations. Plus signs indicate that the ofloxacin solution did not exhibit the genotoxicity at 2-fold. Asterisks, pound signs, and error bars are the same as those in Figure 1. 0.4-2.2 µg-4NQO/L, which was much lower than the decrease of genotoxicity in sample A and B containing similar concentration of DOC. This indicated that genotoxic DBPs formation of typical precursors associated with bromide limitedly contributed to the changes of genotoxicity in secondary effluent under the conditions of this study. To evaluate the effect of bromide on genotoxic chemicals during chlorination under high concentration of NH3-N, NH4Cl was added to the synthetic solutions of ofloxacin and 1-nitropyrene (100 µg/L nominal concentration) to obtain the NH3-N concentration of 30.0 mg/L. Thereafter, chlorination of each solution in the presence of different concentrations of bromide was performed (Figure 4). The dominant residual chlorine form was chloramines (data not shown here). It is interesting to note that after chlorination with high concentration of NH3-N, the genotoxicity and residual ofloxacin concentration in the presence of bromide were significantly lower than those without bromide addition (Figure 4, Figure S9). This was supported by the finding on the reduction of ofloxacin in sample B obtained from secondary effluent during chlorination (Figure S10). After chlorination, the ofloxacin concentration in sample B without bromide addition decreased from 350 to 230 ng/L, while the ofloxacin concentration in the presence of bromide decreased to 12 ng/L. These reductions of ofloxacin concentration in secondary effluent and synthetic solution demonstrated that bromide can significantly accelerate the decrease of some genotoxic chemicals during chlorination and result in the decrease of genotoxicity. Furthermore, after chlorination with high concentration of NH3-N, the genotoxicity of 1-nitropyrene in the presence of bromide was significantly higher than that without bromide addition, suggesting that the presence of bromide might also accelerate the removal of 1-nitropyrene and induce the formation of genotoxic DBPs during chlorination. The changes of genotoxicity in ofloxacin, 1-nitropyrene, humic acid, and aromatic amino acids indicated that the effect of bromide on DOM in secondary effluent is very complex. On one hand, bromide can accelerate the decrease of some genotoxic chemicals, including ofloxacin, and thus decrease the genotoxicity of secondary effluent; on the other hand, bromide can also induce the formation of more genotoxic DBPs during chlorination of DBPs precursors and some genotoxic chemicals, including humic acid and 1-nitropyrene, and result in the increase of genotoxicity as the finding concerning drinking water disinfection (31). The VOL. 44, NO. 13, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 5. Changes in genotoxicity of sample B and its fractions in the presence of ammonia nitrogen (30.0 mg-N/L) after chlorination (10 mg-Cl2/L) in the presence of different bromide concentrations. Asterisks, pound signs, plus signs, and error bars are the same as those in Figure 1. species, genotoxicity, and chlorination mechanism of both genotoxic chemicals and DBPs precursors in secondary effluent influence the genotoxicity changes induced by bromide. Since different biotoxicity tests have different mechanisms and end points, it is necessary to conduct further studies on genotoxicity/mutagenicity evaluation and biotoxicity test based chemical identification of chloraminated secondary effluent in the presence of bromide with different biotoxicity tests, including Ames test. Effect of Chlorination on Genotoxicity of Different DOM Fractions in the Presence of High NH3-N Concentration. To discover the main precursors causing the changes in secondary effluent genotoxicity, sample B was separated into four fractions (HIS, HOA, HOB, and HON). These fractions were combined with NH4Cl (30.0 mg-N/L nominal concentration) and disinfected with 10 mg/L available chlorine in the presence of different concentrations of bromide. The resulting genotoxicity of the fractions was evaluated and is shown in Figure 5. Prior to chlorination, the genotoxicity of HIS and HOA was higher than that of HON and HOB. Furthermore, HIS and HOA were dominant in the DOM of secondary effluent according to DOC and UV254 values (Table S4). Therefore, HIS and HOA might play key roles in the changes in genotoxicity during chlorination. After chlorination, the genotoxicity of HIS and HOA in the presence of bromide was significantly lower than that after chlorination without bromide addition (p < 0.05). The changes in genotoxicity of HIS and HOA were much greater than those of HON and HOB, demonstrating that HIS and HOA were the primary components of the DOM involved in the decrease in genotoxicity during chlorination. It is interesting to note that following chlorination, the genotoxicity of HOA added with bromide slightly decreased, while its genotoxicity in the absence of bromide significantly increased (p < 0.05). These differences suggest that chlorination of HOA can form more genotoxic byproducts, while the presence of bromide may result in the reduction of DBPs during chlorination. Effect of Chlorination on Genotoxicity of the HOA Fraction in the Presence of High NH3-N Concentration. To investigate the effect of bromide on the genotoxicity of DBPs in secondary effluent, the HOA fraction in sample B was combined with NH4Cl (30.0 mg-N/L nominal concentration) and disinfected by 10-30 mg/L available chlorine (with and without added NaBr). The genotoxicity of the HOA fractions is shown in Figure 6. After chlorination, the 4928

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FIGURE 6. Changes in genotoxicity of hydrophobic acids of sample B under condition of high ammonia concentration (30.0 mg-N/L) after chlorination (10-30 mg-Cl2/L) in the presence of different bromide concentrations. Asterisks, pound signs, and error bars are the same as those in Figure 1. genotoxicity of HOA in the absence of bromide significantly increased to 2.5-4.4 times than that observed before chlorination (p < 0.05), demonstrating that chlorination of HOA can produce genotoxic DBPs with the presence of NH3-N. It was also notable that after chlorination, the genotoxicity of HOA in the presence of bromide notably decreased to 50-70% of original (p < 0.05). These results suggest that the presence of bromide can decrease the genotoxicity of secondary effluent induced by DBPs.

Acknowledgments This study was funded by National Science Fund for Distinguished Young Scholars of China (50825801) and NationalHigh-techR&DProgram(863Program)(2008AA062502). We thank Prof. Oda for providing the Salmonella typhimurium strain.

Supporting Information Available Two methods, four tables, and ten figures present detailed information on the genotoxicity assay, ofloxacin concentration analysis, water quality, chemical description of the regions in EEMs, stoichiometric coefficients in chlorine/ monochloramine-DOM reactions, ofloxacin concentration changes, genotoxicity changes, and fluorescence spectra changes. This material is available free of charge via the Internet at http://pubs.acs.org.

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