Science of the Total Environment 473–474 (2014) 437–445

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Science of the Total Environment journal homepage: www.elsevier.com/locate/scitotenv

Oxidation of diclofenac by aqueous chlorine dioxide: Identification of major disinfection byproducts and toxicity evaluation Yingling Wang a,c, Haijin Liu a, Guoguang Liu a,b,⁎, Youhai Xie a a School of Environment, Henan Normal University, Henan Key laboratory for Environmental Pollution Control, Key Laboratory for Yellow River and Huaihe River Water Environment and Pollution Control, Ministry of Education, Xinxiang 453007, PR China b Faculty of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China c School of Basic Medicine, Xinxiang Medical University, Xinxiang 453003, PR China

H I G H L I G H T S

G R A P H I C A L

A B S T R A C T

• Diclofenac oxidation by ClO2 was investigated under simulated water treatment. • UPLC–MS and 1H NMR were performed to identify major disinfection byproducts. • Microtox bioassay was employed to evaluate acute toxicity of reaction solutions. • The intermediates were more toxic than parent compound.

a r t i c l e

i n f o

Article history: Received 4 August 2013 Received in revised form 11 December 2013 Accepted 11 December 2013 Available online 31 December 2013 Keywords: Diclofenac Chlorine dioxide Byproducts Mechanism Toxicity Water treatment

a b s t r a c t Diclofenac (DCF), a synthetic non-steroidal anti-inflammatory drug, is one of the most frequently detected pharmaceuticals in the aquatic environment. In this work, the mechanism and toxicity of DCF degradation by ClO2 under simulated water disinfection conditions were investigated. Experimental results indicate that rapid and significant oxidation of DCF occurred within the first few minutes; however, its mineralization process was longer than its degradation process. UPLC–MS and 1H NMR spectroscopy were performed to identify major disinfection byproducts that were generated in three tentative degradation routes. The two main routes were based on initial decarboxylation of DCF on the aliphatic chain and hydroxylation of the phenylacetic acid moiety at the C-4 position. Subsequently, the formed aldehyde intermediates were the starting point for further multistep degradation involving decarboxylation, hydroxylation, and oxidation reactions of C\N bond cleavage. The third route was based on transient preservation of chlorinated derivatives resulting from electrophilic attack by chlorine on the aromatic ring, which similarly underwent C\N bond cleavage. Microtox bioassay was employed to evaluate the cytotoxicity of solutions treated by ClO2. The formation of more toxic mid-byproducts during the ClO2 disinfection process poses a potential risk to consumers. © 2013 Elsevier B.V. All rights reserved.

⁎ Corresponding author at: Faculty of Environmental Science and Engineering, Guangdong University of Technology, Guangzhou 510006, PR China. Tel.: +86 373 3325971; fax: +86 373 3326335. E-mail address: [email protected] (G. Liu). 0048-9697/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.scitotenv.2013.12.056

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useful information regarding the safety of water treatment in the case of DCF micropollution.

1. Introduction Pharmaceutical and personal care products (PPCPs) have been found to pollute a wide range of aquatic environments, including surface water, ground water and drinking water (Yang et al., 2011). The formation of harmful disinfection byproducts (DBPs) could be a serious threat to human health during drinking water treatment resulting from the reaction of disinfectants with PPCPs. Therefore, research on PPCPs removal as well as byproducts formation is critically important to address the safety concerns of water treatment, since some byproducts may be of similar or even higher toxicity compared with their parent compounds (Calza et al., 2006; Tian et al., 2010). Diclofenac (DCF), a synthetic non-steroidal anti-inflammatory drug, is one of the most commonly used pain killers, largely used clinically as the sodium salt. The chemical structure of DCF is shown in Fig. 1. The global consumption of DCF is estimated to be 940 tons per year, with a defined daily dose of 100 mg (Fent et al., 2006; Radjenovic et al., 2007; Vieno et al., 2007; Zhang et al., 2008). Approximately 65% of the dosage is excreted through urine, mainly as hydroxylated metabolites conjugated to glucuronides after enterohepatic circulation (Zhang et al., 2008; Kenny et al., 2004). However, the actual amount of metabolites in the feces is still not clear. DCF is generally removed by only about 30% in conventional sewage treatment plants (STPs) (Suárez et al., 2008). Because of its low biodegradability (Joss et al., 2006) and its limited sorption properties onto activated sludge (Ternes et al., 2004), DCF has been detected in STP-effluent and surface water up to 4.7 μg L−1 and 1.2 μg L−1, respectively, and even in groundwater and tap water at concentrations up to 380 ng L− 1 and below 10 ng L−1, respectively (Aguinaco et al., 2012; Heberer, 2002). DCF is considered to be one of the most relevant compounds in terms of ecotoxicity and persistence in the environment. It will be a serious threat to public health if drinking water sources are contaminated by DCF. Chlorine dioxide is a powerful disinfectant that has comparable biocidal efficacy, but less pH dependence and DBP formation potential, compared with free chlorine (Hey et al., 2012a; Lim et al., 2010). There are approximately 700–900 public water systems using ClO2 to treat potable water in the world (Wang et al., 2010b). As a highly selective oxidant (E0 = 0.936 V), ClO2 can remove or oxidize many chemical contaminants including pharmaceutical drugs (Hey et al., 2012b; Huber et al., 2005). In view of the increasing use of ClO2 in water treatment, it is of great interest to gain information about the reaction of ClO2 with commonly consumed pharmaceutical drugs that have been detected in groundwater, because some of the resulting products can have an adverse biological effect (Tian et al., 2010). The objectives of this work were to: (1) investigate the DCF degradation by ClO2 under simulated water treatment conditions; (2) identify the degradation byproducts with ultra performance liquid chromatography/ mass spectrometry (UPLC/MS) and 1H nuclear magnetic resonance (NMR); (3) propose the reaction mechanism for DCF degradation based on the identified byproducts; and (4) evaluate the cytotoxicity of the reaction solutions using the Microtox test. The results would clarify the fate and behavior of DCF during water disinfection with ClO2, thus providing

O HO

H N

Cl

Cl Fig. 1. Chemical structure of diclofenac.

2. Materials and methods 2.1. Chemicals and bacteria DCF, 2-[(2,6-dichlorophenyl) amino] benzeneacetic acid, sodium salt (98% purity), was purchased from J&K Chemical Co. Ltd. (Beijing, China). Sodium chlorite (90% purity) was obtained from Tianjin Guangfu Chemical Reagents Co. Ltd. (Tianjin, China). The pure stock solution of ClO2 (2.79 g L−1) was prepared from gaseous ClO2 by slowly adding dilute H2SO4 to NaClO2 solution, according to Eq. (1). Impurities such as chlorine were removed from the gas stream of N2 by a NaClO2 scrubber and the gaseous ClO2 was passed into ultra-pure water and stored in a brown bottle at 4 °C in a refrigerator (APHA, 2005). HPLC-grade reagents (acetonitrile, methanol, ethanol, etc.) and dimethyl sulfoxide (DMSO-d6) were obtained from Suqian Guoda Chemical Reagents Co. Ltd. (Jiangsu, China). Other reagents (Na2S2O3, NaCl, H2SO4, etc.) were of analytical grade and used without further purification. Freeze-dried Vibrio (V.) fischeri bacteria were obtained from Nanjing CAS Kuake Technology Co. Ltd. (Nanjing, China). Ultra-pure water from a Milli-Q apparatus (Millipore, USA) was used for preparing all aqueous solutions. 5NaClO2 þ 2H2 SO4 →2Na2 SO4 þ 4ClO2 þ NaCl þ 2H2 O:

ð1Þ

2.2. DCF degradation experiments Degradation experiments of DCF by ClO2 were performed in 250 mL closed round-bottom flasks on a collector-type magnetic stirrer in the dark. To prevent DCF from hydrolyzing, the aqueous reaction solution of DCF (250.00 mL) was freshly prepared by spiking 2.50 mL of its stock solution (1.00 g L−1) to reach a concentration of 10.00 mg L−1. Preliminary experiments to determine the stability of drugs were done with initial DCF concentration (10.00 mg L−1), and different dosages of ClO2 stock solution (0.25–5.00 mL) were then added to initiate the reaction at room temperature, according to the factorial experiment design. After 5 min of reaction, 2.00 mL of the mixture solution was taken out from the round-bottom flask using a pipette and the oxidant residues were immediately quenched with the pre-added Na2S2O3 solution. Samples were immediately analyzed using UPLC to determine the degradation efficiency (η) by the following Eq. (2): η¼

A0 −Ax  100% A0

ð2Þ

where A0 is the initial peak area of 10.00 mg L−1 DCF, and Ax is the peak area of remaining DCF in reaction solution. 2.3. Identification of degradation products In order to demonstrate the reaction mechanism of DCF, the intermediate products are of great value. The concentrations and degradation products of DCF were determined through a Waters ACQUITY TQD UPLC/MS system (Waters Corporation) equipped with a BEH-C18 column (100 × 2.1 mm, 5 μm). A mixture of 70% HPLC-grade acetonitrile and 30% Milli-Q water (containing 1% acetic acid) was used as the eluent at a flow rate of 0.3 μL min−1. A 5 μL of sample was injected by using an autosampling device, with the detection wavelength set at 276 nm. The eluent from the chromatographic column enters the UV–vis detector, followed by the electrospray ionization (ESI) interface and then the mass analyzer. MS analyses were performed using a Waters TQ detector equipped with an ESI source. Data acquisition was performed in the negative ion mode, and the optimized parameters were as follows: source temperature, 120 °C; desolvation temperature, 350 °C; capillary voltage, 3.0 kV; cone voltage, 28 V; desolvation gas flow, 650 L h−1 and cone gas

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flow, 50 L h−1. Argon (99.99%) was used as the collision gas, and the argon pressure in the collision cell was maintained at 50 kPa. Full scan spectra were recorded in range from 100 to 400. All of the data were acquired and processed using MassLynx 4.1 software. The reaction solutions were concentrated at 45 °C using vacuum rotary evaporation by means of constantly adding ethanol, and then vacuum dried. Subsequently obtained solid mixtures were isolated by thin-layer chromatography (TLC) with silica gel G plate (Shandong Jiangyou Silica Gel Development Co. Ltd, 0.4–0.5 mm, 20 × 20 cm) and dichloromethane–methanol (30:1, v/v) as a developing solvent. The structures of the separated products were determined by means of 1H NMR. 1H (400.13 MHz) NMR spectra of the products were recorded on a Bruker AV-400 Fourier transform NMR spectrometer (Bruker, Fallanden, Switzerland) equipped with a 5 mm 1H/broadband gradient probe at 25 °C. The proton chemical shifts (δH) were referenced to residual solvent, DMSO-d6 at 2.54 ppm. The patterns of peaks were reported as singlet (s), doublet (d), triplet (t), or double doublet (dd). In order to facilitate reading and understanding, abbreviated names for the byproducts (P1–P9) are used. 2.4. Toxicity measurements The samples were collected after 5 min of ClO2 treatment and immediately quenched with Na2S2O3, then the toxicity of samples and initial DCF solution was examined by a Microtox Model DXY-2 Toxicity Analyzer that measures the ability of the byproducts to inhibit the bioluminescence of the bacterium V. fischeri (Marco-Urrea et al., 2010; Calza et al., 2006). Freeze-dried bacteria pellets were reconstituted by adding 1.00 mL of diluent (2.5% NaCl). Briefly, samples were tested in medium containing 3.0% NaCl, toxicity data were recorded on 15 min exposure of 10 L reconstituted bacteria solution to every sample at 25 °C. The inhibition of the luminescence, compared with a blank control to give the percentage of inhibition (I %), was calculated based on Eq. (3), where Ix and I0 are the luminosity of sample solution and blank solution without DCF and ClO2, respectively. The initial DCF concentration was 10.00 mg L−1. I% ¼

I0 −Ix  100%: I0

ð3Þ

2.5. Other analyses ClO2 stock solution was standardized by the iodometric method just before application (Wang et al., 2010a). Solution pH and temperature were simultaneously measured by a Mettler Toledo Delta 320 pH meter.

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3. Results and discussion 3.1. UPLC–MS analysis Fig. 2 represents an example of total ion chromatograms (TIC) of DCF oxidized for 5 min by 16.74 mg L−1 ClO2 at 25 °C. In addition to the DCF peak, four primary peaks of degradation products were observed. The peaks at 4.61, 4.24, 3.98, 3.54 and 3.44 min were assigned to P1, DCF, P2, P3 and P4, respectively, elaborated in Fig. 3. Moreover, added to these primary products, the other five chromatographic peaks were also noted in Fig. 2. Mass spectra in the negative mode and chemical structure of DCF and suggested degradation products were shown in Figs. 3 and 4. DCF (C14H11Cl2NO2): the MS spectrum in Fig. 3(a) shows a deprotonated molecular ion of DCF at m/z 294 and one major fragment ion at m/z 250 (−44), which is due to the loss of CO2 in the ionization source (Hu et al., 2011). For this compound the isotopic distribution (i.e. presence of ions at m/z 296 and 252 with abundance about 2/3 of molecular ion) is characteristic of the presence of two chlorine atoms in their chemical structure, which will be considered in identifying the intermediates formed from the DCF degradation. Product P1 (C14H10Cl3NO2, chloro-DCF): the MS spectrum of chloroDCF in Fig. 3(b) shows a deprotonated molecular ion at m/z 328 and an additional ion fragment at m/z 284 (−44) which could correspond to the loss of CO2, following a fragmentation pathway similar to that of DCF (Soufan et al., 2012). Due to a mass difference of + 34 between the molecular ion of chloro-DCF and DCF, as well as the presence of an additional chlorine atom in the chemical structure of DCF, a chlorinated derivative of DCF was proposed. In addition, the isotopic distribution (i.e. presence of ions at m/z 330 (+2) and 332 (+4) with about equivalent and 1/3 abundance of molecular ion, respectively) further confirmed the presence of three chlorine atoms in the chemical structure of this compound. Product P2 (C13H8Cl3NO, chloro-decarboxyl-DCF): a deprotonated molecular ion at m/z 298 is expected for this product in the MS spectrum in Fig. 3(c) obtained in the negative mode. Due to similar isotopic distribution in Fig. 3(b), this compound contains three chlorine atoms in the chemical structure. Added to molecular ion, one major fragment ion at m/z 262 (−36) was observed which could result from HCl loss in the ionization source (Quintana et al., 2010). Product P3 (C14H11Cl2NO3, hydroxyl-DCF): the MS spectrum in Fig. 3(d) shows a major ion at m/z 310 and a minor ion at m/z 266. Accordingly, a deprotonated molecular ion at m/z 310 would give fragment ions at m/z 266 (−44) which could come from CO2 loss. Similar to DCF, the isotopic distribution (i.e. presence of ions at m/z 312 (+2) and 268 (+ 2) with abundance about 2/3 of molecular ion) is also

Fig. 2. UPLC–MS (TIC 50–400 uma) chromatogram of DCF oxidized for 5 min by ClO2.

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Fig. 3. Mass spectra and chemical structures of DCF and P1–P4.

characteristic of the presence of two chlorine atoms in the chemical structure. Due to one more oxygen atom than DCF, the compound is probably produced through an •OH radical attack on the aromatic ring (Huguet et al., 2013), yielding a monohydroxylated derivative, which is further oxidized into the corresponding aldehyde compounds (Othman et al., 2000; Vogna et al., 2004). Product P4 (C13H9Cl2NO, decarboxyl-DCF): a deprotonated molecular ion at m/z 264 and its fragment ion m/z 228 (−36) is clearly observed in Fig. 3(e). Their isotopic distribution (i.e. parent ion at m/z 266 with abundance about 2/3 of molecular ion and fragment ion at m/z 230 with abundance about 1/3 of molecular ion) shows the presence of two chlorine atoms and one chlorine atom of their structure, respectively. Moreover, no fragment peak of CO2 loss was shown and one major fragment ion at m/z 228 (− 34) which should correspond to chlorine loss suggests that P4 resulted from the decarboxylation of the phenylacetic acid group of DCF directly to aldehyde group (Bartels et al., 2007; PerezEstrada et al., 2005). Further, some other products, such as P5–P9, were also constituents of the ClO2 oxidation process in which one chlorine atom or hydrogen

atom was replaced by an •OH group. The corresponding MS peaks of these byproducts were identified in the negative mode, as shown in Fig. 4. However, failure to detect the 1H NMR of P5–P9 in Table 1 indicated that these compounds were not sufficiently isolated from the reaction mixtures by TLC. 3.2. 1H NMR analysis The reaction mixtures were isolated by preparative TLC and characterized by 1H NMR analysis in order to confirm their structures. The proton chemical shifts (δH, ppm) of DCF and P1–P4 listed in Table 1 were obtained from the products dissolved in DMSO-d6 by using residual solvent as internal standard. In brackets indicate δH values for DCF and P3 in CD3OD, described previously (Marco Urrea et al., 2010). As can be seen in Table 1 (DCF), the aromatic protons of the phenylacetic acid moiety (assigned atom number (δH (ppm)): 2 (6.22d), 3 (7.02t), 4 (6.71t), 5 (7.06d) and the \CH2\ protons (3.38s)) could be observed, respectively, while the protons of the dichlorobenzene ring (3′ (7.43d), 4′ (6.90t), 5′ (7.43d)) and the bridging

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441

Fig. 4. Mass spectra and chemical structures of P5–P9.

nitrogen proton (8 (10.28s)) appear in the case of DCF sodium which are generally consistent with those described previously (Hüsch et al., 2011; Marco Urrea et al., 2010; Viegas et al., 2011). Table 1 (P1–P4) also showed the 1H NMR assignments of degradation products. The dichlorophenyl ring has not been modified if compared with DCF, and it showed proton chemical shifts very similar to DCF, which implied a lack of direct bond involvement of the dichlorophenyl ring with ClO2. For the ring initially bonded to the acetylic chain, several changes have taken place. Comparing the δH values of P1 and P3 to DCF, the substitution in position 4 was confirmed, corresponding to a chlorine

atom or hydroxyl group (Vogna et al., 2004). Moreover, the δH at 9.89 ppm and 9.38 ppm from P1 and P3, respectively, which exhibited upfield Δδppm values, are signals of carboxyl group, then the compounds P1 and P3 were gradually transformed into P2 and P4 which possessed the aldehyde group additively resonating at 9.29, 9.35 ppm. The phenylacetate group protons of P2 and P4 generally showed larger shifts than those protons of P1 and P3, due to the influence of CHOsubstitution on the δH values of benzene ring protons. In contrast, the phenylacetate group played an important role during the reaction of DCF with ClO2.

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Table 1 1 H NMR chemical shifts (δH) of DCF and P1–P4. Compound

DCF

P1

P2

P3

P4

6.25d 7.15d

6.61d 7.39d

6.15d/[6.39d] 7.15d/[6.56dd]

7.18d 3.65s 10.42s 9.89s

7.66s

7.30t/[6.73d] 3.61s/[3.72s] 9.90s 9.38s 9.12s 7.56d/[7.36d] 6.91t/[6.98t] 7.56d/[7.36d]

Structure

δH

2 3 4 5 7 8 9 10 3′ 4′ 5′

6.22d/[6.43d] 7.02t/[7.09t] 6.71t/[6.93t] 7.06d/[7.24d] 3.38s/[3.77s] 10.28s

7.43d/[7.42d] 6.90t/[7.09t] 7.43d/[7.42d]

Too small 9.29s

7.48d 6.84t 7.48d

7.42d 6.82t 7.42d

O -

O

Cl

H N

decarboxylation

Cl DCF: m/z=294 hydroxylation

chlorination O HO O

O HO OH

OH

O

or HO

Cl

H N

Cl

HO

OH

hydroxylation

P1: m/z=328

Cl

H N

H N

HO

Cl

P8: m/z=167

Cl

CHO

HO

Cl

P3: m/z=310

CHO

Cl

HO

Cl

Cl

H N

CHO

Cl

P2: m/z=298

OH

P4: m/z=264

OH Cl

HO

OH

Cl

CHO Cl HO

OH P6: m/z=193

Cl

Cl

P5: m/z=280

Cl

H N

P7: m/z=177

opened ring structures

Fig. 5. Reaction mechanism of DCF degradation by ClO2.

P9: m/z=121

6.53d 7.49t 7.02t 7.81d 10.24s 9.35s 7.45d 6.90t 7.45d

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3.3. Reaction mechanism The mechanism for DCF degradation by ClO2 was proposed on the basis of the identified byproducts and the evolution of their concentrations throughout the reaction, as shown in Fig. 5. According to our results and the literature data, the transformations on the phenylacetic acid moiety proceeded through three tentative routes simultaneously: (i) chlorination reaction with the expected nucleophilic attack of chlorine giving chloro-DCF (P1) formation (Quintana et al., 2010; Soufan et al., 2012); (ii) hydroxylation reaction of DCF and P1 promoted by • OH leading to hydroxyl-DCF (P3) formation, and (iii) decarboxylation reaction resulting with the formation of decarboxy-DCF (P4) as the primary degradation route of DCF with ClO2. Later, the transformation went on with the loss of CO and H2 of chloro-DCF, leading to the formation of chloro-decarboxy-DCF (P2). All of the maintain the 2, 6-dichloroaniline moiety, suggests that of the two NH-bearing positions, the C-1′ position is not activated by •OH attack, as previously reported (Vogna et al., 2004). As we could see from Figs. 4 and 5, hydroxyl-decarboxy-DCF (P5) was most likely generated from P3 through the decarboxylation

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reaction in the aliphatic chain, meanwhile degradation of P2 involving • OH radical attack at a halogenated site also led to the formation of P5 by replacing one chlorine atom with an \OH group, further dehydroxylation step of P5 could also increase the amount of P4 in another pathway. P4 and P5 would trigger an alternate C\N cleavage route leading to P6, P7 and P9, resulting from •OH radical attack. Similarly, hydroxylation on the phenylacetic acid group of DCF, P1 or P3 would then induce fission of the C\N bond leading to P8. Subsequent cleavage of the C\C bond would yield other opened ring structures in small amounts, suggesting that ClO2 degradation is an effective way for complete mineralization of DCF in the aquatic environment. In agreement with the profiles of the intermediates, fast formation of chlorinated derivatives (P1 and P2) was observed at the beginning of the reaction and almost completely disappeared just before the disappearance of DCF seen from Fig. 6. Regarding the P3 and P4, stoichiometric increases accumulated in the solution along with the increase of ClO2 and showed quite good stability, even in the presence of an excess of ClO2. Then P3 and P4 decreased gradually in the higher concentrations of ClO2. As treated under the overdoses of ClO2, the degradation process could provide the cleavage of the aromatic rings and DCF

Fig. 6. UPLC spectra (in integrogram) and % inhibition of V. fischeri bioluminescence (in bars) of DCF oxidized for 5 min by different dosages of ClO2 stock solution.

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could be further oxidized to other unidentified aliphatic compounds; complete mineralization could be accomplished in a high excess of ClO2 (Pera Titus et al., 2004; Martínez et al., 2011). 3.4. Toxicity assessment Toxicity is an important factor for assessing the safety of drinking water. Since the byproducts may be more toxic than DCF, it is necessary to assess the toxicity of the DCF-polluted water treated by ClO2. The toxicity of the water samples was evaluated by monitoring changes in the natural emission of the luminescent bacteria V. fischeri when challenged with toxic compounds. Because the ClO2 residues in the samples had already quenched with Na2S2O3 before Microtox test, the toxicity of ClO2 had no interference on the toxicity of DCF degradation samples. Twelve samples (10.00 mg L−1 DCF) submitted to degradation process at different concentrations of ClO2 were analyzed to estimate the percentage of inhibition of each sample. The initial toxicity of DCF solution showed an inhibition rate of 15.7% (15 min of incubation) in Fig. 6(a) according to the above Eq. (3). This value in Fig. 6(b) increased to 23.2% under the condition of 21.68% DCF degradation rate based on the Eq. (2) mentioned before. Subsequently, the overall toxicity quickly increased and reached a maximum inhibition rate of 57.8% at the disappearance of DCF in Fig. 6(h). These observations clearly demonstrated that during the degradation process, compounds more toxic than DCF were formed between the degradation rates of 0% and 100%. The toxicity changes were primarily attributed to the variation in concentrations of the intermediates. Above all, the highest toxicity observed was in accordance with the maximum abundances of P3 and P4 generated (Méndez Arriaga et al., 2008). Chloroderivatives (P1, P2) would not significantly affect the overall toxicity because of their low concentration, though they are also more toxic than DCF, which has been reported previously (Calza et al., 2006; Wang et al., 2005). The inhibition rate decreased along with the increase of an excess of ClO2, however, it remained still higher than the parent compound. It should be pointed out that synergistic effects among the byproducts might also strengthen the toxicity of the solution (Scheurell et al., 2009). Afterwards, the toxicity decreased gradually, reaching a much lower value (2.4%) in Fig. 6(l) showing the efficiency of detoxification of the disinfection process. In general, these results were in accordance with our previous observation of disappearance of both DCF and its byproducts. The reaction mechanism suggested, although with a few similarities, differs from the literature previously reported. As to the photolysis process, DCF is mainly transformed into carbazole derivatives due to the initial photocyclization (Eriksson et al., 2010). Under the ozonization (Coelho et al., 2009) and H2O2/UV (Vogna et al., 2004), DCF has similar, but not identical, reaction routes resulting in hydroxylated intermediates and C\N cleavage products, but the formation of aldehyde derivatives is rarely reported. With the chlorination of DCF, a similar electrophilic substitution on the aromatic ring leading to chlorinated derivatives is observed (Soufan et al., 2012), however, subsequent degradation pathways are quite different. In a word, DCF transformation follows various routes, depending on the treatment applied. 4. Conclusions ClO2 treatment is an efficient method for DCF degradation, bringing about rapid degradation of DCF in aqueous solution within the first few minutes. Because of the presence of intermediates during the reaction, mineralization process of DCF was longer than its degradation process. P1–P4 comprised the primary products that were simultaneously generated during DCF degradation through three types of reaction pathways: chlorination, hydroxylation, and decarboxylation. By using excess ClO2, five byproducts from further multistep degradation, P5–P9, were produced.

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