1 Introduction

Low-cost chlorination is a primary approach to inactivate pathogens in secondary effluents during wastewater treatment processes. However, the chlorination of wastewater may induce new risks when the chlorinated wastewater effluent is discharged into the surface water. Residual chlorines, including hypochlorous acid and monochloramine, exhibit high acute toxicity to aquatic lives (Brungs 1973). Furthermore, chlorine also reacts with dissolved organic matter (DOM) in the wastewater to form disinfection by-products (DBPs) with acute toxicity, genotoxicity, and/or antiestrogenic activity (Krasner et al. 2009; Sun et al. 2009; Wu et al. 2009).

To reduce the acute toxicity of the residual chlorine, dechlorination with tetravalent sulfur, including SO2 and sulfite, is widely used after chlorination in wastewater treatment processes. During dechlorination, tetravalent sulfur not only reduces residual hypochlorous acid and monochloramine in wastewater but also transforms some of the DBPs into new DBPs. For example, after dechlorination, trichloroacetonitrile is reduced to lower-genotoxicity dichloroacetonitrile, and dibromoacetonitrile is transformed into bromoacetonitrile with similar genotoxicity (Croue and Reckhow 1989; Muellner et al. 2007). However, some DBPs in wastewater, including organic chloramines, remain during dechlorination (Helz and Nweke 1995). These different changes in DBPs indicate that the effects of dechlorination on DBPs are very complex.

Genotoxicity assays are considered valuable methods in water safety evaluation. The use of genotoxicity assays not only can quantify the genotoxicity of identified DBPs but also can evaluate genotoxic chemicals without prior information (Ohe et al. 2004). Using Ames test, Cheh et al. noted that dechlorination with sulfite can reduce the mutagenicity of drinking water induced by DBPs (Cheh et al. 1980). Morlay also noted that chlorinated humic substances exhibited mutagenicity, but after addition of sulfite, the mutagenicity decreased significantly (Morlay et al. 1991). Compared with drinking water and humic substances, the secondary wastewater effluent contains much more types of DOM, including non- or slowly biodegradable influent chemicals and soluble microbial products (Barker and Stuckey 1999). Some of the non- or slowly biodegradable influent chemicals are genotoxic and/or mutagenic. For example, the genotoxic chemical ofloxacin is frequently detected in the effluent of wastewater treatment plants at the same level (from 0.1 to 1 μg/L) as that in the influent (Shao et al. 2008). Furthermore, the soluble microbial products can react with chlorine to form some genotoxic DBPs (Wei et al. 2011). Therefore, reactions occurring during chlorination–dechlorination of secondary effluents and their genotoxic chemicals are more complex than those in drinking water and certain DBPs. However, studies concerning the effects of dechlorination on the genotoxicity of secondary effluents are still very limited.

The objective of this study was to evaluate the effect of dechlorination with sulfite addition on genotoxicity in secondary effluents, using an in vitro umu test. Changes in the genotoxicity of typical chemicals in secondary effluents during dechlorination were also evaluated.

2 Materials and methods

2.1 Water sample

A secondary effluent sample was collected from a domestic wastewater treatment plant that uses an anaerobic–anoxic–oxic process. The sample was kept at 4–10°C in ice and then immediately delivered to the laboratory. Thereafter, the sample was filtered through glass-fiber filters with a 0.7-μm pore size (Whatman GF/F). The sample was stored at 4°C at pH 2.0. The values of the ammonia nitrogen concentration, dissolved organic carbon, UV absorbance, and pH of the sample were 0.5 mg N/L, 7.4 mg C/L, 18.6 m−1, and 7.1, respectively.

2.2 Fractionation of secondary effluent sample

The fractionation of DOM in the secondary effluent sample was performed in XAD-8 resin to obtain hydrophilic substances (HIS), hydrophobic acids (HOA), hydrophobic bases, and hydrophobic neutral fractions based on previous methods (Leenheer 1981). The resin was previously solvent-extracted with acetone and n-hexane to remove the genotoxicity of the genotoxic chemicals adsorbed on the resins. The water quality characteristics of the fractions in the secondary effluent sample are shown in Table S1 of Electronic supplementary material.

2.3 Chlorination/dechlorination

The pH values of the secondary effluent sample and its fractions were adjusted back to 7.0 before chlorination and dechlorination. Thereafter, the water sample and its fractions (150 mL) were chlorinated at a chlorine dose of 10 mg Cl2/L for 30 min in 150-mL glass bottles, as previously described (Wang et al. 2007). The residual chlorine was immediately determined by N,N-diethyl-p-phenylenediamine–ferrous ammonium sulfate titration (APHA (American Public Health Association) et al. 1998). Afterward, a solution of Na2SO3 at 0–1.25 mole dose ratio to residual chlorine was added to the chlorinated samples for 5 min to remove the residual chlorine. The pH values of the samples were kept at 7.0 using phosphate buffer. Chlorination/dechlorination of synthetic solutions containing some typical chemicals was also performed under conditions similar to those in the experiments of secondary effluent sample.

2.4 Concentration of secondary effluent sample and synthetic solution

After chlorination or dechlorination, each sample (140 mL), acidified to pH 2.0, was pumped through a CHP20P resin cartridge containing 1 g poly(styrene-divinylbenzene) resin (75–150 μm, Mitsubishi Chemical, Japan) (Wang et al. 2007; Wu et al. 2010). Subsequently, acetone was used to elute the compounds bound to the resin. The acetone eluate was dried under nitrogen flow. Finally, the dry chemicals were dissolved in dimethylsulfoxide (DMSO) or methanol and then stored at −20°C for genotoxicity or ofloxacin evaluation.

2.5 Genotoxicity assay

The SOS/umu test was used to evaluate the β-galactosidase activity of Salmonella typhimurium TA1535/pSK1002 culture after exposure to the concentrated secondary effluent samples and synthetic solution to determine the genotoxicity of each sample. The test was done in triplicates, according to an ISO standard method (ISO 2000). In this assay, the negative and positive controls were culture medium and the DMSO solutions of 4-nitroquinoline-N-oxide (4-NQO), respectively. The dose–response curve of the concentrated sample was obtained and then converted to an equivalent 4-NQO concentration (Wu et al. 2010). The differences in the genotoxicity of the different samples were statistically significant when p < 0.05 according to the Holm-Sidak test. Statistical analysis was done using SigmaStat 3.5 software (Systat Software Inc., USA).

2.6 Ofloxacin concentration analysis

After concentration of secondary effluent sample and synthetic solutions, an ultra-performance liquid chromatograph with a tandem mass-spectrometry detector and a high-performance liquid chromatograph with a fluorescence detector were used to measure the ofloxacin concentrations in secondary effluent samples and synthetic solutions, as described in the literature (Peng et al. 2006; Lee et al. 2007).

2.7 Fluorescence spectroscopy

The excitation–emission matrix fluorescence spectra of the secondary effluent fractions were determined on a fluorescence spectrophotometer (Model: F-7000, Hitachi, Japan), according to the previous findings (Chen et al. 2003).

3 Results and discussion

3.1 Effects of dechlorination on secondary effluent genotoxicity

3.1.1 Effects of dechlorination on genotoxicity of secondary effluent after chlorination

The genotoxicity changes of the secondary effluent sample during chlorination–dechlorination are shown in Fig. 1. After chlorination, the dominant form of residual chlorine was free chlorine (data not shown), and the genotoxicity of the secondary effluent sample decreased from 13 to 5.9 μg 4-NQO/L (Fig. 1). This result is supported by the previous finding that chlorination with a low ammonia nitrogen concentration decreased the genotoxicity of the secondary effluent samples (Wang et al. 2007).

Fig. 1
figure 1

Changes in genotoxicity of the secondary effluent sample after chlorination (10 mg Cl2/L) with and without dechlorination. Asterisks indicate that the genotoxicity of samples after chlorination with/without dechlorination was significantly different from that before chlorination (p < 0.05). Pound signs indicate that the genotoxicity of sample with dechlorination was significantly different from that without dechlorination (p < 0.05). Error bars represent the standard deviation based on triplicate analyses

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After dechlorination by adding the Na2SO3 solution, the genotoxicity of the chlorinated secondary effluent sample decreased to a significantly lower level than that before dechlorination (p < 0.05). This indicates that dechlorination reduced the genotoxicity of the secondary effluent sample after chlorination, in agreement with a similar observation in the literature concerning drinking water dechlorination (Cheh et al. 1980).

After dechlorination, the secondary effluent sample still exhibited genotoxicity, suggesting that some genotoxic chemicals were still present. This result concurs with previous findings that genotoxic DBPs, including 3-chloro-4-(dichloromethyl)-5-hydroxy-2(5H)-furanone, exhibited low reactivity toward sulfite (Croue and Reckhow 1989).

3.1.2 Effects of dechlorination on genotoxicity in secondary effluent with high ammonia nitrogen concentration after chlorination

Recently, chlorination at high ammonia nitrogen concentration has been reported to increase the genotoxicity of secondary effluent (Wang et al. 2007). Therefore, the changes in the genotoxicity of the chlorinated secondary effluent containing high ammonia nitrogen concentration during dechlorination were also evaluated. The secondary effluent sample was combined with NH4Cl to obtain an ammonia nitrogen concentration of 30 mg N/L, and then chlorinated (10 mg Cl2/L). Afterward, dechlorination was performed with Na2SO3 at the same mole dose as the residual chlorine (Fig. S1 in Electronic supplementary material).

After chlorination at high ammonia nitrogen concentration, the dominant residual chlorine was chloramine (data not shown), and the genotoxicity of the secondary effluent sample was slightly higher than the original value (Fig. S1 in Electronic supplementary material).

After dechlorination, the genotoxicity of the secondary effluent decreased to only 4% of that prior to dechlorination. This indicates that dechlorination reduced the genotoxicity of the chlorinated secondary effluent containing a high concentration of ammonia nitrogen. To demonstrate this finding, the changes in the genotoxicity of the chlorinated sample after dechlorination using different concentrations of Na2SO3 were investigated (Fig. 2). The genotoxicity of the secondary effluent also decreased significantly by increasing the Na2SO3 addition near the residual chlorine concentration (p < 0.05). This demonstrates the genotoxicity reduction by the Na2SO3 addition. The genotoxicity of the chlorinated secondary effluent may result from DBPs and residual genotoxic chemicals present in the secondary effluent before chlorination, so whether dechlorination decreased the level of genotoxic chemicals and resulted in the decrease of the genotoxicity should be of concern.

Fig. 2
figure 2

Changes in genotoxicity of the secondary effluent sample after adding a range of Na2SO3 doses following chlorination (10 mg Cl2/L) at high ammonia nitrogen concentration (30 mg N/L). Pound signs and error bars are as the same as those in Fig. 1

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3.1.3 Effects of Na2SO3 addition on genotoxicity in secondary effluent before chlorination

To evaluate the effects of Na2SO3 addition on genotoxic chemicals in secondary effluents before chlorination, different concentrations of Na2SO3 were directly added to the secondary effluent sample (pH 7.0). The doses of the Na2SO3 solutions were similar to those in dechlorination experiments as shown in Fig. 2. Thereafter, the genotoxicity was evaluated, as shown in Fig. 3. The genotoxicity of secondary effluent decreased with the increase in Na2SO3 addition. This demonstrates that Na2SO3 addition can significantly decrease the genotoxicity of the secondary effluent. Miller and Miller (1976) noted that most of the carcinogens are electrophiles. Whether the nucleophiles, including Na2SO3, can destroy the genotoxic chemicals in the secondary effluent is still unknown and requires further investigation.

Fig. 3
figure 3

Changes in genotoxicity of the secondary effluent sample after Na2SO3 addition (0–0.18 mmol/L). Asterisks indicate that the genotoxicity of samples after Na2SO3 addition was significantly different from that of the original sample (p < 0.05). Error bars are the same as in Fig. 1

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3.2 Effects of dechlorination on the genotoxicity of different DOM fractions with high ammonia nitrogen concentration

3.2.1 Effect of dechlorination on the genotoxicity of chlorinated DOM fractions after chlorination

The fractions containing HIS and HOA in the secondary effluent have been reported as the key fractions in the genotoxicity changes during chlorination (Wang et al. 2007; Wu et al. 2010). Therefore, HIS and HOA were chosen as typical fractions for study. These fractions, with added NH4Cl (30 mg N/L), were disinfected with 10 mg/L available chlorine for 30 min and then dechlorinated with different Na2SO3 concentrations, as shown in Fig. 4.

Fig. 4
figure 4

Changes in genotoxicity of the fractions of the secondary effluent sample after dechlorination with Na2SO3 following chlorination (10 mg Cl2/L) at high ammonia nitrogen concentration (30 mg N/L). HIS hydrophilic substances, HOA hydrophobic acids. Asterisks, pound signs, and error bars are the same as those in Fig. 1

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After chlorination under high ammonia nitrogen concentration, the genotoxicity of HIS and HOA was higher than those before chlorination. This suggests that the chlorination of HIS and HOA in the presence of ammonia nitrogen can form more genotoxic DBPs.

After dechlorination, the genotoxicity of HIS and HOA was significantly lower than those prior to dechlorination (p < 0.05). This demonstrates that dechlorination with Na2SO3 resulted in the reduction of genotoxicity in the secondary effluent sample and its fractions.

3.2.2 Effect of Na2SO3 addition on genotoxicity in DOM fractions before chlorination

In order to evaluate the effect of Na2SO3 addition on genotoxic chemicals in fractions before chlorination, Na2SO3 was directly added to HIS and HOA, and the changes in genotoxicity of the fractions during Na2SO3 addition are shown in Fig. S2. After Na2SO3 addition, the genotoxicity values of HIS and HOA were significantly lower than those prior to Na2SO3 addition (p < 0.05). This indicated that Na2SO3 addition can decrease genotoxic chemicals existing in the fractions before chlorination, which is in accordance with that during Na2SO3 addition of secondary effluent. Therefore, further investigation concerning changes of the genotoxic chemicals existing in secondary effluent before chlorination and their genotoxicity during Na2SO3 addition should be performed.

3.3 Effects of Na2SO3 addition on the genotoxicity of genotoxic chemicals in secondary effluent before chlorination

Many types of genotoxic chemicals are present in secondary effluents. For example, the secondary effluent sample used in this study contained genotoxic 0.5 μg/L ofloxacin. Ofloxacin is an important genotoxic/mutagenic chemical associated with the genotoxicity changes in secondary effluents induced by bromide (Isidori et al. 2005; Wu et al. 2010). Ohe and Nukaya (1996) noted that 1-nitropyrene contributed 1% of the genotoxicity in a river polluted by secondary effluent. Since Na2SO3 addition was found to reduce the genotoxicity of secondary effluent and its fractions, the genotoxicity in synthetic ofloxacin (100 μg/L nominal concentration) and 1-nitropyrene (100 μg/L nominal concentration) solutions during Na2SO3 addition was also evaluated (Fig. 5).

Fig. 5
figure 5

Changes in genotoxicity of synthetic ofloxacin and 1-nitropyrene solutions (100 μg/L nominal concentration) after Na2SO3 addition. Asterisks and error bars are the same as those in Figs. 3 and 1, respectively. Plus sign indicates that the sample did not exhibit the genotoxicity at twofold

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Before Na2SO3 addition, the genotoxicity values of ofloxacin (100 μg/L) and 1-nitropyrene (100 μg/L) were 7,177 and 25 μg 4-NQO/L, respectively. After Na2SO3 addition, the ofloxacin solution did not exhibit the genotoxicity at test concentration folds (Fig. 5). Furthermore, the residual ofloxacin concentration of the ofloxacin solution notably decreased below the detection limit (Fig. S3). This indicates that Na2SO3 addition decreased the levels of some genotoxic chemicals and their genotoxicity in the secondary effluent sample. However, the less genotoxic compound which ofloxacin was converted to remained unknown. Further research concerning conversion mechanism of ofloxacin by Na2SO3 is required.

The genotoxicity of 1-nitropyrene after Na2SO3 addition was similar to that before addition. This suggests that Na2SO3 addition did not reduce the genotoxicity of some genotoxic chemicals such as 1-nitropyrene and resulted in the phenomenon that the secondary effluent still exhibited genotoxicity after Na2SO3 addition, as shown in Fig. 3.

3.4 Effect of Na2SO3 addition on genotoxicity of DBPs

Certain DBPs and genotoxicity induced by chlorination are associated with some DOMs such as humic acid and aromatic proteins/amino acids in secondary effluent and drinking water (Xie and Reckhow 1993; Wang et al. 2007; Yang et al. 2008). In this study, these chemicals were found in the HIS and HOA of the secondary effluent sample, according to the fluorescence spectra of the samples (Fig. S4). Therefore, the effect of dechlorination on the genotoxicity of chlorinated aromatic amino acids was evaluated. In this study, the chlorination (35 mg Cl2/L) of synthetic tryptophan solution (30 mg/L) in the presence of ammonia nitrogen (30 mg N/L) was done to form genotoxic DBPs. Thereafter, Na2SO3 was added in the chlorinated tryptophan solution (Fig. 6).

Fig. 6
figure 6

Changes in genotoxicity of tryptophan after dechlorination with Na2SO3 following chlorination (10 mg Cl2/L) at high ammonia nitrogen concentration (30 mg N/L). Asterisks, pounds signs, and error bars are the same as those in Fig. 1. Plus sign indicates that the sample did not exhibit the genotoxicity at 1,000-fold

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After chlorination, the tryptophan solution exhibited significant genotoxicity, demonstrating that the chlorination of tryptophan can produce genotoxic DBPs. After dechlorination, the genotoxicity of the tryptophan solution significantly decreased to 22% lower than before dechlorination (p < 0.05), indicating that dechlorination can decrease the DBP-induced genotoxicity of the secondary effluent. This finding is similar to that observed in the chlorination–dechlorination of typical precursors, including humic acids (Morlay et al. 1991).

4 Conclusions

Dechlorination with Na2SO3 addition notably decreased the genotoxicity of the secondary effluent after chlorination, especially in the presence of high ammonia nitrogen concentrations (30 mg N/L). Addition of Na2SO3 was found to significantly decrease the genotoxicity of the secondary effluent before chlorination. This suggests that Na2SO3 addition can remove genotoxic chemicals from the secondary effluent and thus decrease the genotoxicity. This was further demonstrated by dechlorination experiments using ofloxacin, a typical genotoxic chemical in secondary effluents.

The secondary effluent was separated into different fractions. The genotoxicity in HIS and HOA increased after chlorination with ammonia nitrogen addition (30 mg N/L), suggesting that the chlorination of DOM can form genotoxic DBPs. Addition of Na2SO3 was also found to decrease the genotoxicity of HIS and HOA.

Aromatic proteins or amino acids were found in HOA, based on the fluorescence spectra of HOA. After chlorination, the tryptophan solution exhibited genotoxicity, suggesting that the chlorination of tryptophan formed genotoxic DBPs. By adding the Na2SO3 solution to the chlorinated tryptophan, dechlorination was found to decrease the genotoxicity induced by DBPs.