Characterization, DBPs formation, and mutagenicity of soluble microbial products (SMPs) in wastewater under simulated stressful conditions

Chemical Engineering Journal 279 (2015) 258–263

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Characterization, DBPs formation, and mutagenicity of soluble microbial products (SMPs) in wastewater under simulated stressful conditions Beibei Zhang a, Qiming Xian a,⇑, Jiping Zhu b, Aimin Li a, Tingting Gong a a b

State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, PR China Exposure and Biomonitoring Division, Health Canada, Ottawa, Ontario, Canada

h i g h l i g h t s  Stressful conditions contained more low molecular weight components of SMPs.  Stressful conditions had no effect on the species of disinfection by-products (DBPs).  Stressful conditions played a crucial role in the levels of DBPs.  Stressful conditions increased the mutagenicity of SMPs before and after chlorination.  Nitrogen-containing DBPs were one of the major contributors to mutagenicity.

a r t i c l e

i n f o

Article history: Received 21 January 2015 Received in revised form 10 May 2015 Accepted 11 May 2015 Available online 16 May 2015 Keywords: Soluble microbial products (SMPs) Stressful condition Disinfection by-products (DBPs) Mutagenicity

a b s t r a c t Soluble microbial products (SMPs) are an important group of components in activated sludge-treated wastewater effluents. Chlorination is the most widely used method for disinfecting wastewater effluents. During chlorination of wastewater effluents, SMPs may act as precursors to form disinfection by-products (DBPs), which may pose adverse impacts on the organisms in the receiving water body. In this study, SMPs were prepared by simulating activated sludge under different stressful conditions, including high ammonia content (HA), high salinity (HS), high levels of heavy metals (HM) and high temperature (HT), as well as a normal state (NS). The molecular weight (MW) distribution of SMPs was characterized using gel permeation chromatography (GPC). Chlorination of SMPs was conducted with sodium hypochlorite. Several species of DBPs were detected using gas chromatography–mass spectrometry (GC–MS). The mutagenicity of SMPs solutions and SMPs solutions treated with chlorination was also evaluated using the umu test. Compared with NS, stressful conditions were induced to produce more low MW components of SMPs. Stressful conditions, had no significant effect on the species of DBPs, but played a crucial role in the levels of DBPs produced in SMPs solutions during chlorination. Among the stressful conditions tested, HT and HS resulted in higher levels of both carbon-containing DBPs (C-DBPs) and nitrogen-containing DBPs (N-DBPs). Levels of C-DBPs and N-DBPs were lower under HM condition. HA stimulated the production of N-DBPs, but had no impact on the levels of C-DBPs. The mutagenicity of SMPs solutions was higher under the stressful conditions than that under NS condition in both before and after chlorination. For each SMPs solution, the mutagenicity of SMPs increased after the chlorination, except for SMPs solution under HM and NS conditions. Ó 2015 Elsevier B.V. All rights reserved.

1. Introduction Soluble microbial products (SMPs) are an important component in wastewater effluents resulting from the biological process, and consist of macromolecules and cellular debris including proteins, ⇑ Corresponding author. Tel./fax: +86 025 89680259. E-mail addresses: [email protected] (B. Zhang), [email protected] (Q. Xian), [email protected] (J. Zhu), [email protected] (A. Li), [email protected] (T. Gong). http://dx.doi.org/10.1016/j.cej.2015.05.039 1385-8947/Ó 2015 Elsevier B.V. All rights reserved.

polysaccharides, humic acids, and DNA [1,2]. SMPs comprise a majority of soluble organic material in the effluents and their presence, therefore, may not only degrade the quality of effluents [3], but also cause possible environmental hazard to the receiving water [4]. Moreover, they also pose a challenge in the reuse of wastewater, as some SMPs can cause membrane fouling [5,6], or act as precursors of disinfection by-products (DBPs) [7,8]. It has been suggested that the main fraction of SMPs that associated with DBPs formation are low molecular weight (MW) matters [9,10]. During a chlorination process, SMPs could generate

B. Zhang et al. / Chemical Engineering Journal 279 (2015) 258–263

both carbon-containing DBPs (C-DBPs) and nitrogen-containing DBPs (N-DBPs) [11,12]. In general, C-DBPs are formed in higher concentrations than N-DBPs. However, N-DBPs are more genotoxic than C-DBPs [13]. Up to now, mutagenicity of nearly 100 DBPs has been determined. However, few mutagenicity data are available for the DBP mixture in effluents. Both the umu and Ames tests are used to determine the mutagenicity of DBPs [14,15], and the results show a consistency of 90% between the two assays [16]. The umu test has however been more widely used for determining mutagenicity of drinking water and effluent from wastewater treatment plants owing to its simplicity, convenience, and rapidity [17,18]. The species and levels of DBPs resulting from SMPs during disinfection are greatly influenced by the composition of SMPs in the wastewater [19,20]. The formation of SMPs is also closely related to the conditions of wastewater [7,21]. However, characterization of SMPs including molecular weight (MW) distribution of SMPs, formation of DBPs during chlorination processes and the mutagenicity of SMPs before and after chlorination are still not well studied, especially under certain stressful conditions of the wastewater. These stressful conditions, such as high ammonia content (HA), high salinity (HS), high level of heavy metal (HM) and high temperature (HT) are common wastewater effluents in treatment process. For example, HA is present in chemical, metallurgy and circuit boards etching wastewater; HS in picking, cheese manufacturing and tanning factories wastewater; HM in electroplating and tannery wastewater [20] and HT in industrial and mining enterprises, thermal power plant and cooling water wastewater [22]. The purposes of this study were (1) to characterize the SMPs under a number of simulated stressful conditions, (2) to evaluate the formation of DBPs resulting from chlorination of SMPs under these stressful conditions, and (3) finally to examine the mutagenicity of SMPs before and after chlorination under each of these conditions. 2. Materials and methods 2.1. Batch experiments and preparation of SMPs Activated sludge was collected from an aeration tank in a municipal wastewater treatment plant, and used as inoculums for the reactor. Five series of batch experiments (one normal state (NS) and four simulated stressful conditions of HA, HS, HM and HT) were conducted in this study. The cultured activated sludge was added into five 5-L reactors filled with synthetic wastewater to a final biomass concentration of about 2000 mg/L to create NS wastewater. The synthetic wastewater contained the following substances (in mg per L): glucose (800), (NH4)2SO4 (189), KH2PO4 (35), CaCl2 (0.37), MgSO4 (5.07), MnCl2 (0.27), ZnSO4 (0.44), FeCl3 (1.45), CuSO4 (0.39), CoCl2 (0.42), Na2MoO4 (1.26), NaBr (0.26) [5]. (NH4)2SO4, NaCl and CrCl3 were added into one of the NS wastewater samples to create simulated stressful condition of HA (500 mg/L ammonia nitrogen (NH+4-N)), HS (5% NaCl) and HM (50 mg/L CrCl3) solutions, respectively. HT was the NS samples conducted at elevated temperature of 45 °C. Each reactor was incubated for 6 h at 25 °C followed by a precipitation time of 30 min. Supernatant was then collected and filtered through a 0.45 lm filter paper. The filtrate was defined as SMPs [11]. 2.2. Water quality measurements Dissolved organic carbon (DOC) was measured using a TOC analyzer (TOC-VCH, Shimadzu, Japan). Total nitrogen (TN), NH+4-N,

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nitrite nitrogen (NO2 -N) and nitrate nitrogen (NO3 -N) were determined using a DR2800 analyzer (HACH, USA). Dissolved organic nitrogen (DON) was calculated by subtracting values of NO2 -N, NO3 -N and NH+4-N from the TN value. 2.3. MW distribution of SMPs MW distribution of SMPs was determined using gel permeation chromatography (GPC) (1515, Waters Inc., USA) with UV detector at wave 254 nm. Phosphate buffer solution (pH = 7.4) was used as eluent at a flow rate of 1.0 mL/min. The injection volume was 100 lL and the measurement was carried out at 35 °C. Polystyrene sulfonates (PSS 1690, 4800, 7540, 15,450 with retention time about 12.12, 10.17, 9.90 and 9.47 min, respectively) were used for the calibration of apparent molecular weight (AMW), and the peaks after 9.47 min were regarded as the low MW matters. 2.4. Chlorination of SMPs SMPs were chlorinated by adding sodium hypochlorite solution (NaClO, 5%). Samples of 200 mL each were prepared in the glass bottles with Teflon inner plugs. NaClO solution was added to all the samples according to a Cl2/DOC ratio of 2:1. A phosphate buffer solution was added to the samples to maintain the pH at 7.0. Chlorination was conducted in darkness at 25 °C for 48 h. The residual chlorine was then quenched using Na2S2O3. 2.5. Analysis of DBPs and total organic halogen (TOX) THMs1 (TCM, BDCM, DBCM, TBM), CS (1,1,1-TCE, CTC, TCE, DBE, PCE, DBC), HKs (1,1-DCP; 1,1,1-TCP) and N-DBPs (DCAN, TCAN, BCAN, DBAN, TCNM) were measured according to EPA Method 551.1. HAAs (MCAA, MBAA, BCAA, BDCAA, CDBAA, DBAA, DCAA, TCAA) were analyzed according to EPA Method 552.3. All DBPs were detected using a gas chromatography–mass spectrometer (GC–MS) (Thermo Polaris Q, USA). Total organic halogen, (TOX, measured as chloride ion) was analyzed by a total organic halogens analyzer (TE XPLOTER, Netherlands) according to the national standard method of China [23]. Briefly, the sample for TOX analysis was first acidified to pH 1.5–2.0 and then enriched through adsorption onto activated carbon sorbent. The activated carbon sorbent was then placed in a quartz sample boat and introduced into the combustion chamber of a SYS 100000Xplorer TOX Analyser and combusted in the presence of oxygen for 15 min at 1000 °C. The hydrogen halide gases produced were dried through H2SO4 and collected in titration cell, then analyzed by electrolysis electrode and measuring electrode. 2.6. Mutagenicity analyses SMPs samples were concentrated for mutagenicity tests based on the method of Wang [24]. Briefly, a sample of SMPs or chlorinated SMPs was acidified to pH 2 with HCl and then passed through a HLB cartridge (Oasis, Waters, USA) that had previously been washed with 10 mL of methanol and 10 mL of deionized 1 Abbreviations: THMs (trihalomethanes): TCM, trichloromethane; BDCM, bromodichloromethane; DBCM, dibromochloromethane; TBM, tribromomethane. CSs (chlorinated solvents): 1,1,1-TCE, 1,1,1-trichloroethane; CTC, carbon tetrachloride; TCE, trichloroethylene; DBE, 1,2-dibromoethane; PCE, tetrachloroethylene; DBC, 1,2-dibromo-3-chloropropane. HKs (haloketones): 1,1-DCP, 1,1-dichloro-2-propanone; 1,1,1-TCP, 1,1,1-trichloro-2-propanone. N-DBP: DCAN, dichloroacetonitrile; TCAN, trichloroacetonitrile; BCAN, bromochloroacetonitrile; DBAN, dibromoacetonitrile; TCNM, trichloronitromethane. HAAs (haloacetic acids): MCAA, monochloroacetic acid; MBAA, monobromoacetic acid; BCAA, bromochloroacetic acid; BDCAA, bromodichloroacetic acid; CDBAA, chlorodibromoacetic acid; DBAA, dibromoacetic acid; DCAA, dichloroacetic acid; TCAA, trichloroacetic acid.

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water. 100 mL of each sample was loaded on the cartridge at a flow rate of about 5 mL/min, and then the cartridge was dried under a flow of air and eluted with 5 mL of methanol, 5 mL of acetone and 5 mL of acetonitrile in sequence. The eluate was reduced to a small volume by rotary evaporation and then dried under a nitrogen flow. The residue was dissolved with 2 mL of dimethyl sulfoxide (DMSO) and stored in the dark at 20 °C prior to the umu tests. The analysis of umu test was performed with Salmonella typhimurium TA1535/pSK1002 without S9 activation based on ISO 13829 [25]. In our study, the dose of the samples and reagents was 0.8 times of that suggested in the ISO 13829 protocol to avoid overflow from the 96-well microplate. 4-Nitroquinoline-N-oxide (4-NQO) and DMSO were used as a positive and a negative control, respectively. The mutagenicity of 4-NQO with different concentrations was measured concurrently in each microplate to obtain dose–effect curves. The mutagenicity of each sample was standardized to an equivalent 4-NQO concentration. 2.7. QA/QC Duplicate SMPs samples were taken from the reactor for the experiments of water quality measurement and MW distribution of SMPs. Chlorination of SMPs, TOX detection and mutagenicity analysis were conducted in triplicate to ensure the reproducibility and reliability of the results. Blank water samples from the reactor without the biomass and substrate additions were used to determine the background DBPs formation. The internal standards, 2-bromofluorobenzene (for THMs) and 1,2,3-trichloropropane (for HAAs), were used for quantification. The recoveries of DBPs ranged from 82.9% to 98.8% and the detection limits of DBPs ranged from 0.06 to 1.69 lg/L. 3. Results and discussion

the growth of nitrifying bacteria and denitrifying bacteria, as well as the nitrification reaction were inhibited under HA, HS and HT conditions [32], thus resulting in high levels of inorganic nitrogen and DON. The concentration of inorganic nitrogen under HM condition was almost the same with that under NS condition, while the concentration of DON was significantly lower, implying that heavy metal might favor the growth of anaerobic microorganisms and promote the degradation of DON. 3.2. MW distribution of SMPs The results of MW distribution of SMPs are presented in Fig. 1 and Table 2. Different from previous studies [33,34], SMPs under different conditions showed neither a large proportion of high MW organic compounds (MW > 15,000, retention time up to 9.47 min in Fig. 1) nor showed an obvious bimodal pattern. Compared with NS condition, SMPs under stressful conditions contained more low MW components and fewer high MW components. The reason maybe that in order to relieve the adverse effects of high ammonia, hypertonic, heavy metal and high temperature, microbial cells widened the hydration layers around the cell and produced a large amount of low MW matters [35]. Low MW components of SMPs usually consist of hydrophilic carboxyl, hydroxyl and amino groups such as amino acid, polycarboxylate-type humic acid-like organics and hydrophobic protein-like and amino acid-like matters, while high MW components of SMPs contain hydrophilic polysaccharides, hydrophobic humic matters and decomposed and lysed debris of microbial cells [7,35]. In this study, the high MW fraction of SMPs decreased 50% under HT and HS conditions compared with NS treatment (Table 2). This is different from the observation in a previous study, in which the high MW organics of SMPs were enhanced with high temperature owing to the decay and lysis of microbial cells [7]. 3.3. DBPs and TOX concentrations of SMPs after chlorination

3.1. Water quality of SMPs The water quality of the SMPs samples are presented in Table 1. Among them, DOC and DON are two key parameters since DOC and DON are important precursors of C-DBPs and N-DBPs, respectively [26,27]. Compared with NS condition, DOC concentrations were 1.2 times, 2.6 times, 1.8 times and 8.3 times higher under the HA, HS, HM and HT conditions, respectively. This might be attributed to two reasons: first, the inhibitory effect of stressful conditions might lead to the plasmolysis and aggravate cell lysis. The release of intracellular constituents, such as polysaccharides and proteins after cell lysis contributed to DOC [3,28]. Second, stressful conditions might also stimulate extracellular polymeric substances (EPS) production [3,29,30], and the slough or hydrolysis of EPS could contribute to DOC [20,31]. Compared with NS condition, the concentrations of inorganic nitrogen (NH+4-N, NO3 -N and NO2 -N) and DON were both higher under HA, HS and HT conditions. The results were expected since

Concentrations of C-DBPs and N-DBPs in SMPs after chlorination under various conditions are presented in Figs. 2(A)–(D) and 3, respectively. After chlorination, several major C-DBPs including four THMs (TCM, BDCM, DBCM and TBM, Fig. 2A), one CS (CTC, Fig. 2B), five HAAs (BDCAA, TCAA, DCAA, MCAA and BCAA, Fig. 2C) and two HKs (TCP and DCP, Fig. 2D) were detected.

Table 2 Molecular weight (MW) distribution of SMPs. MW (%)

NS

HA

HS

HM

HT

Low MW (%)a High MW (%)

70 30

75 25

86 14

74 26

84 16

NS = normal state, HA = high ammonia content, HS = high salinity, HM = high level of heavy metal, HT = high temperature. a Low MW is defined as components eluted after 9.47 min (corresponding to MW = 15,000) and High MW before 9.47 min on gel permeation chromatography (Fig. 1).

Table 1 Selected water quality parameters of SMPs (mg/L). Conditions

DOC

NS HA HS HM HT

23.0 ± 26.6 ± 60.7 ± 40.6 ± 190.0 ±

NH+4-N

TN 2.5a 1.3 2.8 2.9 1.4

7.6 ± 290 ± 21.6 ± 3.8 ± 33.2 ±

0.4 7 1.7 0.3 2.1

0.041 ± 240 ± 0.161 ± 0.061 ± 19.372 ±

0.003 14 0.007 0.007 0.932

NO3 -N

NO2 -N

0.99 ± 1.38 ± 4.98 ± 1.00 ± 2.59 ±

0.019 ± 0.017 ± 0.181 ± 0.011 ± 0.030 ±

0.07 0.08 0.18 0.03 0.02

DON 0.002 0.003 0.014 0.003 0.004

6.5 ± 48.6 ± 16.3 ± 2.7 ± 11.2 ±

0.3 7.2 1.5 0.2 1.2

DOC = dissolved organic carbon; TN = total nitrogen; NH+4-N = ammonia nitrogen; NO3 -N = nitrate nitrogen; NO2 -N = nitrite nitrogen; DON = dissolved organic nitrogen; NS = normal state, HA = high ammonia content, HS = high salinity, HM = high level of heavy metal, HT = high temperature. a Values are presented as mean ± standard deviation (based on duplicate analyses).

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42 NS HA HS HM HT

Response (mV)

39 36 33 30

27 0.0 2.5 5.0 7.5 10.0 12.5 15.0 17.5 20.0 Retention time (min) Fig. 1. Molecular weight distribution of SMPs under various conditions. (NS = normal state, HA = high ammonia content, HS = high salinity, HM = high level of heavy metal, HT = high temperature.)

While the species of C-DBPs formed were almost the same under different conditions, the levels of each of them were different under different conditions. Under HT condition, SMPs generated the most C-DBPs after chlorination, with the total amount of THMs, CSs, HAAs and HKs approximately 6.0 times, 3.2 times, 4.2 times and 3.4 times of NS, respectively. HS condition also promoted the formation of C-DBPs except for HKs. There are two possible reasons: first, higher DOC concentrations were found under HT and HS conditions (Table 1), which could provide more organic precursors for the formation of C-DBPs. Second, the high levels of organic compounds with low MW (Fig. 1 and Table 2) might also help the formation

A

TCM

BDCM

DBCM

TBM

8 7

(μ g/L )

6

80 60

CSs

THMs

(μ g/L )

4000 3000 2000 1000

of more C-DBPs, because low MW compounds prevailed in SMPs were the main contributors for the DBPs formation [21]. The total amounts of C-DBPs under HA condition was similar to that of NS. This result was expected as there was no significant difference of DOC concentration between HA and NS condition (Table 1). Under the HM condition, the total amount of C-DBPs was smaller than that of NS despite the fact that the DOC concentration under HM condition was twice the amount in NS condition. The result might be explained that chlorine is a strong oxidizing agent and might react with trivalent chromium to form toxic hexavalent chromium first before DOC resulting in less chlorine being available to form C-DBPs [36]. Moreover, the major low MW matters under HM condition were hydrophilic rather than hydrophobic matters [7], which were not the precursors of THMs and HAAs [37]. Although C-DBPs were present in relatively high concentrations, N-DBPs should not be ignored because they are more toxic than C-DBPs [13]. Three N-DBPs (DCAN, TCAN and TCNM) were formed in SMPs treated with chlorination under different conditions (Fig. 3). While the amount of TCAN was almost constant (1.6–1.9 lg/L), the amount of DCAN and TCNM varied greatly under different conditions (DCAN: 1.3–8.2 lg/L; TCNM: 0.2– 3.1 lg/L). Compared with NS condition, the formation of N-DBPs was higher under HA, HS and HT conditions, but was lower under HM condition. For example, the amount of TCNM was 10 times higher under the HA condition than that under NS condition, owing to the high ammonia content under HA condition. Hypochlorite can react with ammonia first and convert to chloramine disinfection, thus significantly increasing the amount of N-DBPs [38]. HT and HS condition also promoted the formation of N-DBPs, probably due to the large proportion of SMPs with low MW, which could produce higher amount of N-DBPs [39].

40 20 0

NS

HA

HS

HM

3 2

0

HT

C

BCAA

MCAA

DCAA

TCAA

160

NS

HA

HS

HM

HT

TCP

D

DCP

BDCAA (μ g/L )

400

HKs

(μ g/L ) HAAs

4

200

600

200

0

5

1

1200 1100

CTC

B

120 80 40

NS

HA

HS

HM

HT

0

NS

HA

HS

HM

HT

Fig. 2. (A)–(D) C-DBPs of SMPs under various conditions. Error bars represent the standard deviation based on triplicate analyses. (NS = normal state, HA = high ammonia content, HS = high salinity, HM = high level of heavy metal, HT = high temperature.)

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16 14 12

TCAN

1200

DCAN

1000

10 8 6 4

NS

HA

HS

HM

Fig. 3. N-DBPs of SMPs under various conditions. Error bars represent the standard deviation based on triplicate analyses. (NS = normal state, HA = high ammonia content, HS = high salinity, HM = high level of heavy metal, HT = high temperature.)

30

b

400

a a

b

NA

a

HA

HS

a

a b

HM

HT

Fig. 5. Mutagenicity of SMPs before and after chlorination under various conditions. Error bars represent the standard deviation based on triplicate analyses. Means with the different letter were significantly different (p < 0.05, One Way Anova test by Duncan).

of a previous report [40], in which no correlation between DON and N-DBPs was observed.

25

TOX (mg/L)

b

600

0

HT

after chlorination

800

200

2 0

b

before chlorination

ng 4-NQO/L

N-DBPs (μ g/L )

1400

TCNM

20

3.4. Mutagenicity of SMPs

15

The mutagenicity of SMPs before and after chlorination under different conditions were presented in Fig. 5. The mutagenicity of SMPs before chlorination was 267, 405, 360 and 451 ng 4-NQO/L for HA, HS, HM and HT condition, respectively, which was higher than the level (258 ng 4-NQO/L) under NS condition. The reason may be that stressful conditions inhibited the microbial metabolic activity and aggravated the production of toxic cell lysis organic products during the biological treatment process [43], thus resulting in a higher mutagenicity of SMPs under stressful conditions. The mutagenicity of SMPs significantly decreased after chlorination under NS and HM conditions. This may be because chlorine could oxidize some toxic organic matters and convert them into less toxic substances [24]. However, the mutagenicity of SMPs increased after chlorination under HA, HS and HT conditions, probably due to the increased levels of N-DBPs in these SMPs after chlorination (Fig. 3), which are highly toxic [13]. Compared with NS condition, the mutagenicity of SMPs after chlorination were higher under stressful conditions. For HA, HS and HT conditions, the results were in accordance with the results of DBPs and TOX. For HM condition, although the concentrations of DBPs and TOX were almost the same or even less than NS condition, more toxic cell lysis organic products were produced during the biological treatment process and they were also concentrated on the HLB cartridge and contributed to the mutagenicity of SMPs. The correlations between the mutagenicity of SMPs after chlorination and the concentrations of DBPs (C-DBPs and N-DBPs) as well as TOX were analyzed using Pearson’s correlation test. A significant correlation was observed between N-DBPs and mutagenicity (P < 0.05), implying that N-DBPs were one of the major contributors to the mutagenicity.

10 5 0

NS

HA

HS

HM

HT

Fig. 4. Total organic halogen (TOX) of SMPs under various conditions. Error bars represent the standard deviation based on triplicate analyses. (NS = normal state, HA = high ammonia content, HS = high salinity, HM = high level of heavy metal, HT = high temperature.)

The amount of N-DBPs generated under HT condition was only 2.5 times of that under NS, much less than the increase of C-DBPs, indicating that SMPs under HT condition contained more precursors of C-DBPs than ones of N-DBPs. According to previous studies [40,41], the amount of N-DBPs formed in the chlorination process is related to the DON levels in SMPs samples. Therefore, higher N-DBPs observed under HA, HS and HT conditions may be due to high levels of DON under these conditions as well (Table 1). This could be confirmed indirectly by the low production of N-DBPs under HM condition, in which the DON level was only half of that in NS condition. TOX is an important collective parameter which indicates the overall formation of halogenated DBPs [42]. The concentrations of TOX in SMPs under different conditions were shown in Fig. 4. Similar to C-DBPs and N-DBPs, TOX concentrations were higher under other stressful conditions compared with NS, except for HM condition. These results indicated that HA, HS and HT condition promoted the formation of halogenated DBPs. Correlations among DOC, C-DBPs, N-DBPs, DON and TOX were examined using Pearson’s correlation test. There were statistically significant correlations between DOC and C-DBPs, between DOC and TOX, and between TOX and C-DBPs (P < 0.05), implying that the amount of C-DBPs and TOX could be predicted by the levels of DOC in SMPs. The amount of C-DBPs could also be predicted by TOX in SMPs. There was however, no significant correlation between DON and N-DBPs (P > 0.05). This finding is similar to that

4. Conclusions Overall, four stressful conditions (HA, HS, HM and HT) affected the MW distribution of SMPs, concentrations of DBPs and the mutagenicity of SMPs before and after chlorination. In particular, stressful conditions might induce the formation of SMPs with low MW. Stressful conditions had no significant effect on DBPs species, but showed significant effects on the levels of each DBP. Compared with NS condition, HT and HS conditions promoted

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the formation of both C-DBPs and N-DBPs, while HM condition reduced the levels of C-DBPs and N-DBPs. HA condition had no significant impact on C-DBPs, but it resulted in the highest level of N-DBPs. The mutagenicity of SMPs under stressful conditions was higher than that under NS condition. After chlorination, the mutagenicity of SMPs decreased under NS and HM conditions, while it significantly increased under HA, HS and HT conditions. Additionally, compared with NS condition, the mutagenicity of SMPs after chlorination were higher under stressful conditions. There were significant correlations between DOC and C-DBPs, between DOC and TOX, between TOX and C-DBPs, and between N-DBPs and mutagenicity (p < 0.05). In this study, the mutagenicity of effluent under different stressful conditions suggested that the characteristics of influent and DBPs of chlorinated SMPs should be strictly controlled during wastewater treatment process. A better understanding of the mechanisms will enable the development of new strategies to reduce detrimental effects of SMPs on the ecotoxicity of effluents. Acknowledgments The authors gratefully acknowledge financial support from the National Natural Science Foundation of China (No. 20777032), Natural Science Foundation of Jiangsu Province (Nos. BK2011032, BK20131271), and National Hightech R&D Program of China (Grant No. 2013AA06A309). References [1] J.L. Liu, X.Y. Li, Biodegradation and biotransformation of wastewater organics as precursors of disinfection byproducts in water, Chemosphere 81 (2010) 1075–1083. [2] E.C. Wert, F.L. Rosario-Ortiz, D.D. Drury, S.A. Snyder, Formation of oxidation by products from ozonation of wastewater, Water Res. 41 (2007) 1481–1490. [3] Y. Li, A.M. Li, J. Xu, W.W. Li, Formation of soluble microbial products (SMP) by activated sludge at various salinities, Biodegradation 24 (2013) 69–78. [4] B.H. Zheng, Y. Zhang, Q. Fu, Environmental problems and countermeasures for city drinking water sources of China, Water Ind. Mark 10 (2007) 31–35. [5] S. Liang, C. Liu, L.F. Song, Soluble microbial products in membrane bioreactor operation: behaviors, characteristics, and fouling potential, Water Res. 41 (2007) 95–101. [6] Y.X. Shen, W.T. Zhao, K. Xiang, X. Huang, A systematic insight into fouling propensity of soluble microbial products in membrane bioreactors based on hydrophobic interaction and size exclusion, J. Membr. Sci. 346 (2010) 187– 193. [7] Z.P. Wang, T. Zhang, Characterization of soluble microbial products (SMP) under stressful conditions, Water Res. 44 (2010) 5499–5509. [8] H. Zhang, J.H. Qu, H.J. Liu, X. Zhao, Characterization of isolated fractions of dissolved organic matter from sewage treatment plant and the related disinfection by-products formation potential, J. Hazard. Mater. 164 (2009) 1433–1438. [9] E.E. Chang, P.C. Chang, Y.W. Ko, W.H. Lan, Characteristics of organic precursors and their relationship with disinfection by-products, Chemosphere 44 (2001) 1231–1236. [10] B.J. Ni, J.Z. Raymond, F. Fang, W.M. Xie, G.P. Sheng, H.Q. Yu, Fractionating soluble microbial products in the activated sludge process, Water Res. 44 (2010) 2292–2302. [11] Y.Y. Wei, Y. Liu, Y. Zhang, R.H. Dai, X. Liu, J.J. Wu, Q. Zhang, Influence of soluble microbial products (SMP) on wastewater disinfection byproducts: trihalomethanes and haloacetic acid species from the chlorination of SMP, Environ. Sci. Pollut. Res. 18 (2011) 46–50. [12] Z.H. Fan, S. Gong, X. Xu, X.H. Zhang, Y. Zhang, X. Yu, Characterization, DBPs formation, and mutagenicity of different organic matter fractions in two source waters, Int. J. Hyg. Environ. Health 217 (2014) 300–306. [13] M.J. Plewa, M. Muellner, S.D. Richardson, F.S. Cafasanok, K.M. Bueeener, Y.T. Woo, A.B. Mckague, E.D. Wagner, Occurrence, synthesis, and mammalian cell cytotoxicity and genotoxicity of haloacetamides: an emerging class of nitrogenous drinking water disinfection byproducts, Environ. Sci. Technol. 42 (2008) 955–961. [14] S. Caillet, S. Lessard, G. Lamoureux, M. Lacroix, Umu test applied for screening natural antimutagenic agents, Food Chem. 124 (2011) 1699–1707. [15] S. Gartiser, C. Hafner, K. Kronenberger-Schäfer, O. Happel, C. Trautwein, K. Küm-merer, Approach for detecting mutagenicity of biodegraded and

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