Identification of New Oxidation Products of Bezafibrate for Better

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Identification of new oxidation products of bezafibrate for better understanding of its toxicity evolution and oxidation mechanisms during ozonation Qian Sui, Wilhelm Gebhardt, Horst Friedrich Schröder, Wentao Zhao, Shuguang Lu, and Gang Yu Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b03548 • Publication Date (Web): 25 Jan 2017 Downloaded from http://pubs.acs.org on January 25, 2017

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Environmental Science & Technology

1

Identification of new oxidation products of

2

bezafibrate for better understanding of its toxicity

3

evolution and oxidation mechanisms during

4

ozonation

5

Qian Sui1,3, Wilhelm Gebhardt2, Horst Friedrich Schröder2 , Wentao Zhao4, Shuguang

6

Lu1, Gang Yu3,*

7 8

1

State Environmental Protection Key Laboratory of Environmental Risk Assessment

9

and Control on Chemical Process, School of Resources and Environmental

10

Engineering, East China University of Science and Technology, 200237, Shanghai,

11

China

12

2

RWTH Aachen University, Templergraben 55, D-52056 Aachen, Germany

13 14

Institute of Environmental Engineering, Environmental Analytical Laboratory,

3

Beijing Key Laboratory for Emerging Organic Contaminants Control, Tsinghua University, 100084, Beijing, China

15 16 17

4

State Key Laboratory of Pollution Control and Resource Reuse, College of

Environmental Science and Engineering, Tongji University, 200092, Shanghai, China

18

19

* Corresponding author

20

Email: [email protected]; Tel: 86-10-62794006

1

ACS Paragon Plus Environment


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21

Abstract

22

Bezafibrate (BF), a frequently detected pharmaceutical in the aquatic environment,

23

could be effectively removed by ozonation. However,

24

increased, suggesting the generation of toxic oxidation products (OPs). In this study,

25

eight OPs of BF ozonation were identified using a LTQ Orbitrap hybrid mass

26

spectrometer coupled with HPLC, and six of them have not been previously reported

27

during BF ozonation. Based on the abundant fragments and high assurance of accurate

28

molar mass, structure elucidation was comprehensively performed and discussed.

29

Hydroxylation, loss of methyl propionic acid group and Crigée mechanism were

30

observed as the oxidation mechanisms of BF ozonation. The toxicity of identified OPs

31

calculated by quantitative structure activity relationship indicated that three OPs were

32

probably more toxic than the precursor compound BF. This result tests together with

33

the evolution of identified OPs in the treated solutions, indicated that two OPs,

34

namely

35

N-(2,4-dihydroxyphenethyl)-4-chlorobenzamide, were the potential toxicity-causing

36

OPs during BF ozonation. To the best of our knowledge, this is the first attempt to

37

identify toxicity-causing OPs during the BF ozonation.

the toxicity of treated water

N-(3,4-dihydroxyphenethyl)-4-chlorobenzamide

2


and

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38


TOC

Potential toxicity-causing OPs O

OH

O

O

OH N H

N H

HO

Cl

Cl

100

Toxicity

60 10

BF remained 5

C/C0 (%) Relative inhibition (%)

40

0 2

4

6

8

OP-309-1

OH

Cl

OH

O

O

O OH

N H

O

NH

Cl

Cl

20

0 0

N H

80

15

O OH

HR-MSn C/C0 (%)

Relative inhibition (%)

20

O

O

OP-239

OP-291-1/2/3

ECOSAR

New OPs identified during BF ozonation

10

time (min)

O

O

Change of bezafibrate (BF) concentration and the toxicity during the ozonation process

OH COOH N H

Cl

O

O O

O

OH N H

OH Cl

OP-409

39

3


OP-393

COOH


40

Introduction

41

Due to their ubiquity in the aquatic environment and potential high and

42

increasing environmental impacts, pharmaceuticals and personal care products

43

(PPCPs), a group of emerging contaminants, have been recognized as an

44

environmental threat and were widely and intensively studied over the past decades.

45

Bezafibrate (BF), as one of these PPCPs, is used as a blood lipid level regulator. Due

46

to its large consumption and low degree of metabolic degradation in human (1), BF in

47

its original form has been frequently detected in wastewater, surface water,

48

groundwater, or even drinking water, and reached a concentration level of even µg/L

49

in some cases (2-4). The adverse effects of BF on the ecosystem have been reported

50

previously (5,6). For instance, Quinn et al. (2008) who investigated the teratogenic

51

potential of ten pharmaceuticals using Hydra attenuata regeneration assay, calculated

52

a predicted no-effect concentration (PNEC) value of 22.5 µg/L for BF (5). Although

53

measured environmental concentrations of BF predominantly were less than the

54

observed PNEC, the elimination of BF is still desirable especially from the drinking

55

water invoking the principle of precaution, considering that this pollutant is present as

56

mixture with others and also TPs.

57

Ozonation has been proven to be one of the most promising treatment

58

technologies either as tertiary treatment process after biological wastewater treatment

59

or as an advanced process in drinking water treatment plants (DWTPs). Typically, an

60

ozone dose of 0.4 mg/L and contact time of 4 min is sufficient for an effective

61

disinfection (7), and an ozone dose of 2.5 mg/L was found to be highly effective for

62

PPCPs removal in 20 DWTPs from diverse locations across the United States (8).

63

However, although the concentrations of target PPCPs were dramatically reduced in

64

many cases, low mineralization rates of PPCPs determined as dissolved organic 4


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carbon (DOC) could be observed, suggesting the transformation of target compounds

66

into other oxidation products (OPs). Some PPCPs, such as chlorophene (9) or

67

tetracycline (10), generated OPs that were less toxic than their parent compounds,

68

resulting in a decrease of toxicity. However, for many other PPCPs, O3 treated

69

solutions were more harmful for aquatic organisms than the original ones, thus

70

challenging the application of ozonation process, especially in the DWTPs.

71

Consequently, the identification of toxicity-causing OPs generated during an O3

72

treatment process is important.

73

Several studies have identified the OPs of various PPCPs generated by O3

74

treatment, such as carbamazepine (11,12), diclofenac (13), clofibric acid (14),

75

metoprolol (15), tetracycline (10), etc. However, OPs responsible for the increased

76

toxicity remained unidentified, because even if an ecotoxicity assessment was

77

conducted, it was mainly performed on the reaction mixtures produced, and therefore

78

could not be used to determine the toxicity of individual OPs.

79

In recent years, several studies have shed some light on the identification of

80

toxicity-causing OPs during the ozonation. One of the approaches was to conduct the

81

toxicity test directly using selected pure OP samples, either purchased or collected as

82

fractions of HPLC separations (16,17). The collection and concentration of OPs,

83

however, are labor-intensive, and most of them, especially the intermediates, are not

84

easy to be purchased or synthesized.

85

As an impressive alternative, quantitative structure activity relationship (QSAR)

86

analysis can be used to estimate the toxicity of OPs identified during O3 treatment of

87

PPCPs (18-20). For instance, Kuang et al. (2013) and Tay et al. (2015) applied the

88

Ecological Structure Activity Relationships Class Program (ECOSAR), using

89

available data to estimate and predict acute and chronic toxicity of chemicals such as 5



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trimethoprim and ofloxacin as well as their OPs, respectively (18,20). For

91

phenazone-type pharmaceuticals and metabolites, the toxicity estimation QSAR and

92

EPI SuiteTM software were used to assess the ecotoxic potential of OPs characterized

93

by structure applying high resolution (HR) mass (MS) and multiple tandem mass

94

(MSn) spectrometry (19,21).

95

Similarly to other PPCPs, BF was reported to be effectively removed by

96

ozonation (22-26). However, an increase of acute toxicity during its ozonation was

97

observed (23,27). Several OPs of BF during the ozonation process have been

98

identified applying the HPLC-single stage mass spectrometry (MS) technique

99

(HPLC-MS) (23) or HPLC-UV-DAD (25), but not all the compounds could be

100

identified due to the limitations of single MS and UV-DAD detectors. In addition, as

101

the toxicity of identified OPs was not investigated in such studies, it remained unclear

102

whether the identified OPs were responsible for the elevated toxicity. Consequently,

103

more efforts should be made to identify OPs, especially the toxicity-causing OPs, of

104

BF during the ozone treatment with more supporting evidences.

105

In this study, major OPs of BF ozonation were identified,

Six of which have

106

not been previously reported. Structure elucidation of the OPs was accomplished

107

using an LTQ Orbitrap hybrid mass spectrometer coupled on line with HPLC. The

108

application of HR-MS and -MSn spectrometry provided abundant fragments and high

109

assurance of correct molecular mass information about unknown compounds using

110

their multiple stage MS fragment ions for structural elucidation. The combination of

111

experimental evidence and QSAR analysis was employed, for the first time, to

112

distinguish the toxicity-causing OPs of BF during its ozonation process. The findings

113

can help better understanding the transformation of BF and reducing the potential risk

114

caused by its OPs during the ozonation process in the real DWTPs. 6


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Experimental

116

Materials. Detailed information about chemicals and bacteria applied for

117

luminescence inhibition tests used in the experiments is described in the

118

supplementary material.

119

Ozonation experiments. Semi-continuous experiments were carried out in a 350 mL

120

reactor containing BF solution (25 µM) prepared by Milli-Q water to study the change

121

of BF concentration and acute toxicity during the ozonation. Instead of tap water or

122

raw water collected in a DWTP, Milli-Q water was used to exclude the influence of

123

coexisting compounds in the water that might have effects on the interpretation of the

124

mass spectra. O3 was injected at a flow rate of 0.225 mg min-1. The temperature was

125

maintained at 20±2°C. Samples were taken at defined time and purged immediately

126

by helium to eliminate unreacted O3 prior to quantification by HPLC-UV or

127

assessment for acute toxicity.

128

Batch experiments were conducted to obtain samples at different times to

129

identify the OPs formed at low ozone doses. Aqueous O3 stock solutions, prepared by

130

sparging O3 gas into iced Milli-Q water and quantified spectrophotometrically using

131

molar extinction coefficient (260 nm) = 3300 M-1 cm-1 (13), were added to 100 mL of

132

BF solution (25 µM), reaching an initial O3 concentration of approximately 10 µM.

133

The low temperature provide by the iced Milli-Q water leads to an increased and

134

stable solubility of ozone in the stock solution. Therefore, the required amount of

135

ozone stock solution added in the experiment can be reduced, and the ozone dosage in

136

different batches of experiments can be relatively stable. Periodical samples (0 to 10

137

min), were taken and stripped with helium gas to remove residue O3 prior to mass

138

spectrometric analysis.

139

In all the ozonation experiments, no radical scavengers (e.g., t-BuOH) were 7



140

added into the reaction mixtures, therefore the contribution of ozone and OH radicals

141

could not be identified separately.

142

Determination and identification of BF and its OPs. Concentrations of BF in

143

semi-continuous experiments were quantified using HPLC-UV. The detailed

144

description about its quantification by HPLC-UV is provided in the supplementary

145

information.

146

For identification and semi-quantitative determination of OPs, an LTQ Orbitrap

147

hybrid mass spectrometer coupled with HPLC was employed. The working conditions

148

of HPLC and MS are shown in supplementary information. The extracted ion

149

chromatograms (EIC) of the HPLC separated mixtures allowed to recognize the

150

potential OPs generated during ozone treatment. The peak areas of OPs in the EICs

151

showing increase and decrease of compounds over the reaction time were compared

152

and quantified to make a semi-quantitative determination of OPs, as no standards

153

were available.

154

To propose the chemical structure of each OP observed in their EICs, MSn (n = 2,

155

3) analysis was conducted by syringe infusion. The LTQ Orbitrap was operated in

156

collision induced dissociation (CID) and/or higher energy collisional dissociation

157

(HCD) mode to acquire the high resolution, high mass accuracy, full scan MSn spectra.

158

The LTQ isolation width was set to 1,000 mmu, the activation Q value was 0.25, the

159

activation time was 30 ms, and the maximum fill time was 100 ms. Normalized

160

collision energy was optimized for the most significant spectra. The samples collected

161

at 10 min in the batch experiment were directly injected into the MS in the flow

162

injection analysis (FIA) mode recording MS and MSn scans (n=2,3) to obtain more

163

information about the recognized OPs.

8


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Determination and calculation of toxicity. The acute toxicity of untreated and

165

ozone-treated BF solutions was determined according to a procedure described in a

166

standard method protocol of the Institute of Soil Science, Chinese Academy of

167

Sciences (28). The inhibition of light emission of luminescent bacteria

168

(Photobacterium phosphoreum T3) was measured after an incubation period of 15

169

min at room temperature (20℃–25℃). All experiments were performed at least in

170

duplicate proving that relative deviation did not exceed 15%.

171

QSAR were used to assess the ecotoxicological potential of identified OPs with

172

the help of the ECOSAR program in combination with the EPIWIN software. The

173

software uses the structural information about compounds of interest and provides

174

LC50 values for fish (96 h) and for daphnid (48 h) as well as EC50 values (96 or 144 h)

175

for green algae besides the values for chronic toxicity.

176

Results and discussion

177

Evolution of BF concentration and toxicity. The treatment of BF in aqueous solution

178

with O3 was found to be effective in its elimination. The removal efficiency of BF

179

reached more than 80% after 10 min. However, the relative inhibition of luminescent

180

bacteria was significantly increased during the ozonation, as shown in Figure 1.

181

Increased toxicity of BF solution after ozonation was also observed in (23) and (27).

182

Nevertheless, in their study, after a rapid increase in the initial phase, the toxicity

183

gradually decreased in the subsequence, probable due to the much higher ozone doses

184

applied. The finding in the present study indicated that at lower ozone doses, which

185

are more commonly applied in DWTPs, the toxicity of the treated water might be

186

higher than the untreated water, highlighted the requirement to identify the generated

187

OPs with higher toxic potential than the parent compound. 9



188

OPs identification. Solutions obtained at different reaction time in the batch

189

experiments were analyzed by HPLC-high resolution (HR)-MSn under high resolution,

190

high mass accuracy conditions. The EICs of BF and its OPs before and after ozone

191

treatment are shown in Figure S1a and S1b, respectively. After ozonation, the peak

192

area of the ion trace of the negatively charged parent compound ([BF]-) was

193

significantly reduced (Figure S1a and S1b). New signals, corresponding to the various

194

OPs can be observed in selected EICs displayed in Figure S1b. In these EICs recorded

195

by HPLC-HR-MSn at m/z=290.01399-290.10101 and m/z=307.98530-308.15291,

196

respectively, more than one signal with same exact mass (290.0573-290.0578 and

197

308.0315-308.0317) could be observed, showing the occurrence of several isomers.

198

The OPs were named in accordance to their molecular masses and isomers were

199

differentiated by numbers according to their increasing retention time (tr).

200

Accurate mass determinations yielded by the HR-MS allowed to calculate the

201

chemical formulas of different OPs. Besides, signals of an isotopic compound with an

202

m/z ratio of +2 Da and a peak intensity of around 1:1/3 in the ion patterns facilitated

203

the recognition that this compound contains one chlorine atom. All BF degradation

204

compounds generated by ozonation, shown in the mass spectra, provided this isotopic

205

pattern proving the presence of one chlorine atom in the molecules of all these OPs.

206

The tr, m/z ratios, ring-double-bond equivalents (RDB), proposed chemical formulas

207

and calculated accurate masses of BF and its OPs are compiled in Table 1.

208

As shown in Table 1, eight OPs with five different m/z ratios were recognized in

209

this study. Six OPs, namely OP-291-1, OP-291-2, OP-291-3, OP-239, OP-309-1,

210

OP-309-2, were first reported, to the best of our knowledge, while OP-409 and

211

OP-393 were reported previously as main intermediates during the ozonation of BF

212

(23). 10


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213

Molecular structure determination of six new OPs. To elucidate the structure of OPs

214

observed in their EICs, MS2 and MS3 product ion experiments were conducted for

215

each OP. The obtained spectrum, the information of elemental composition, as well as

216

the knowledge of the potential reaction mechanisms under O3-treatment finally led to

217

the molecular structure determination of six new OPs observed.

218

The molecular structure of BF allows the attack of O3 and/or •OH radicals in

219

several positions. Three isomers of OP-291 by O3 treatment were generated from this

220

attack combined with an in-parallel neutral loss fragment (H-C(CH3)2COOH)

221

generated from the 2-methylpropanoic acid moiety resulting from the cleavage of an

222

aryloxy-carbon bond. Although benzene rings are susceptible to electrophilic attack

223

by electrophilic agents, such as O3 and/or •OH radicals, the presence of product ion

224

m/z 154.0063 (Figure S2), the typical chlorine containing fragment of BF, implies that

225

the 4-chlorobenzoyl moiety remained unchanged, excluding the hydroxylation on

226

4-chlorobenzoyl. In fact, the electron withdrawing effect of the electronegative

227

chlorine substituent in the 4-chlorobenzoyl moiety of BF resulted in a lower reactivity

228

for the attack of O3 and/or •OH radicals at this end of BF molecule. On the contrary,

229

the 2-oxy-2-methylpropanoic acid moiety (-O-C(CH3)2COOH), serving as an electron

230

donating substituent at the other side of BF molecule increased the electron density,

231

and facilitates the O3 attack (29). As the para position of the phenoxy substituent is

232

not available for substitution, hydroxylation can only be facilitated either in the ortho

233

or meta position. However, as shown in the EIC of OP-291 (Figure S1b), three peaks

234

of isomers can be observed, proving that a hydroxylation nevertheless must had taken

235

place in other position of the ozonation product than the phenoxy moiety. Rao et al.

236

(2010) found that during the ozonation of linuron, hydroxylation happened at the

237

methyl group neighboring the amide group (30). Similar results were reported for N, 11



238

N-diethyl-meta-toluamide during its reaction with •OH radicals (31). Therefore, it

239

could be put forward that the hydroxyl group here might be generated at the

240

methylene (-CH2-) group neighboring the amide group. The MS2 spectrum of

241

OP-291-(1-3) with the three proposed structures is shown in Figure S2. It should be

242

mentioned that to propose the chemical structure of each OP, the samples were

243

directly injected into the MS in the flow injection analysis (FIA) mode. The target

244

ions observed in the EICs were selected, and underwent collision induced dissociation

245

(CID) and/or higher energy collisional dissociation (HCD), and the recorded full scan

246

MSn (n=2,3) provided information for the molecular structure determination of OPs.

247

Therefore, the isomers could not be differentiated by their retention times during the

248

MSn (n=2,3) analysis.

249

The information obtained from MS2 spectrum of OP-239 (Figure S3) was limited.

250

Nevertheless, according to the chemical formula C11H11O3NCl and a RDB of 7.5 for

251

OP-239, it could be suggested that two double bonds in the phenoxy moiety of BF

252

were attacked by O3 following the Crigée mechanism. As a result of the stepwise

253

breakdown of the phenoxy moiety, a further oxidization generating two aldehyde

254

functional groups took place. This is in accordance with the result of the TiO2

255

catalyzed advanced oxidation of BF reported in Ref. 30.

256

Three oxidation steps were involved in the formation of OP-309: 1) The loss of

257

methyl propionic acid group, 2) ring-opening following the Crigée mechanism and

258

decarboxylation, and 3) hydroxylation.

259

The fragment ions at m/z = 290.0259 (fragment ②) and m/z = 280.0378

260

(fragment ③) in the MS2 spectrum (Figure 2a), which differed from OP-309 by

261

18.0068 and 27.9949 Da, were the result of either a dehydration or a decarbonylation 12


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262

from OP-309, respectively. The fragment ion at m/z = 262.0274 (fragment ⑤), then,

263

vice versa, may result from both types of reaction, decarbonylation or dehydration of

264

the fragment ions ② or ③, respectively (Figure 2d). Based on the three fragments

265

(②, ③ and ⑤), we propose that a) the hydroxyl group was introduced next to an

266

extractable proton, and b) an aldehyde moiety was located at the opposite point to the

267

4-chlorobenzoyl moiety of OP-309.

268

In the MS3 spectrum of the product ion at m/z = 280.0378 (Figure 2b) the

269

fragment ion at m/z = 154,0013 (fragment ⑦) and the fragment ion at m/z = 125,0245

270

(fragment ⑧), resulted from the cleavage of a nitrogen-carbon bond, confirmed the

271

exact position of the hydroxyl group in fragment ③(m/z = 280.0378). In the MS3

272

spectrum of the product ion ⑤ (m/z=262.0274, Figure 2c), the presence of the

273

fragment ion at m/z = 234.0324 (fragment ⑨), which differed from fragment ⑤ by ∆

274

m/z = 27.9950 Da, confirmed the loss of CO in the CID-process.

275

As shown in Figure S1b, two isomers at tr = 6.2 min and tr = 8.0 min are present

276

in the EIC with the same ion masses. With the help of information obtained, however,

277

we could only identify the structure of one isomeric compound. This compound with

278

a tr of 6.2 min is presented in Figure 2d as compound ① (OP-309-1). The low

279

concentration and the broad peak profile of the other isomer with a tr of 8.0 min did

280

not allow the identification by multiple stage CID. Therefore further efforts should be

281

done to determine the structure of the second isomeric ozonation product OP-309-2.

282

Molecular structure confirmation of two previously reported OPs. Although OP-409

283

and OP-393 were previously reported as OPs of BF during ozonation by HPLC-MS

284

(23) or HPLC-UV-DAD (25), their molecular structures were proposed mainly based 13



285

on the knowledge of the potential reaction mechanisms, and should be confirmed with

286

more supporting evidences.

287

OP-409, which differs from [BF]- ion by three additional oxygen atoms and has

288

the unchanged RDB, should be the oxidation product of hydroxylation. Dantas et al.

289

(2007) also observed this compound, however as no conclusive indications could be

290

found for the exact positions of the hydroxyl groups in the molecule applying the

291

HPLC-single stage-MS technique, it was speculated that the hydroxylation took place

292

at both aromatic rings, the 4-chlorobenzoyl moiety and phenoxy moiety (23).

293

Lambropoulou et al. (2008) also detected an OP with the same molecular mass as a

294

photoproduct of BF (29). Different from the suggestions given by Dantas et al. (2007),

295

it was proposed by Lambropoulou et al. (2008) after LC-TOF-MS analysis that all

296

three hydroxyl groups were introduced into the phenoxy moiety. Data from our study,

297

based on information obtained by the FIA-HR-MS2 and -MS3 data, however, proved

298

that the hydroxylation occurred at the phenoxy moiety as well as at the

299

4-chlorobenzoyl moiety but also at the -CH2- group neighboring the amide group.

300

In the MS2 spectrum of the ion at m/z = 408.0847 in Figure S4a, three fragments

301

with relative intensities >40% were observed, among which the fragment at m/z =

302

322.0483 (fragment ③), as one of three relevant product ions of OP-409 losing the

303

methyl propionic acid group, has the highest intensity. To confirm propositions of the

304

hydroxylation sites, the MS3 product ion spectrum of fragment ③ was generated

305

(Figure S4b). The occurrence of a fragment at m/z = 123.0453 (fragment ⑥) in the

306

MS3 spectrum confirmed that only one hydroxyl group was generated at the phenoxy

307

moiety. The other product ion at m/z = 167.0318 (fragment ⑤) in the MS3 spectrum

308

provided proof that hydroxylation might not only occur at both aromatic ring systems 14


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309

in the BF-molecule, but also at the methylene group (-CH2-) neighbouring the amide

310

group. This assumption was strongly supported by the presence of fragment ion at m/z

311

= 304.0383 (fragment ④) in the MS2 spectrum which can be formed as a product of

312

fragment ③ after a loss of water as neutral leaving group. The third hydroxylation

313

occurred at the 4-chlorobenzoyl moiety, despite its lower reactivity towards an attack

314

by O3 or •OH radicals. In the MS2 spectra of all OPs with unchanged 4-chlorobenzoyl

315

moieties, the signal of characteristic fragment ion at m/z = 154.0013 could be

316

observed. In the MS2 and MS3 spectra of OP-409, however, this characteristic

317

fragment could not be observed. The absence of this very stable chlorine containing

318

characteristic fragment ion in the product ion spectra of OP-409 confirmed that the

319

structure of 4-chlorobenzoyl moiety in this OP had changed during ozonation. The

320

proposed fragmentation pathway for OP-409 is presented in Figure S4c.

321

For OP-393, which was reported in (23) and (27) during BF ozonation or

322

photocatalytic degradation, respectively, our findings confirmed that it follows the

323

Crigée mechanism (32), which led via ring-opening by O3 cycloaddition to the

324

formation of two aldehyde moieties.

325

Oxidation pathways. MSn identification of OPs provided the useful information to

326

elucidate the oxidation pathways of BF during its ozonation. Although the real

327

concentrations of OPs could not be determined due to the unavailability of chemical

328

standards, the formation as well as the partial abatement of OPs recognizable by their

329

peak areas in their EICs could be determined.

330

According to the normalized peak area versus reaction time of each OP, the

331

compounds OP-291-1, OP-393 and OP-309-2, first increased in concentration before

332

they decreased partly (Figure S5b). They might be the precursors of other OPs 15



333

observed in the reaction mixture. The oxidation pathway of BF during its ozonation is

334

put forward in Figure 3. The oxidation mechanisms involved were: a) hydroxylation,

335

which was the reaction mechanism to form OP-291 and OP-409, b) loss of methyl

336

propionic acid group, leading to the formation of OP-291, OP-239 and OP-309 and c)

337

ring opening following the Crigée mechanism with the formation of OP-393.

338

Toxicity estimation and identification of toxicity-causing OPs. Toxicity of BF and its

339

OPs was calculated based on QSAR using ECOSAR program. This program can

340

screen and predict the aquatic toxicity of chemicals based on the similarity of

341

structure to chemicals for which the aquatic toxicity has been previously reported, and

342

therefore is widely used to predict acute and chronic toxicity of compounds observed

343

during water treatment processes (18, 21). The results based on fish, daphnid and

344

green algae, by ECOSAR reflected the general toxicity level of the target compounds,

345

and should be comparable to the results of luminescence inhibition assay, which was

346

used to exhibit the toxicity of BF and its OPs in this study. Furthermore, the results

347

obtained by ECOSAR were found to be comparable with the ones observed in

348

luminescent bacterium assay (33). In our study, since standards of OPs are not

349

commercially available, QSAR acts as a good alternative to experimentally derived

350

toxicity data for fish, daphnia and algae, and to link the toxicity results obtained by

351

luminescent bacterium test. As illustrated in Table 2, the predicted toxicity of most

352

OPs was lower than that of BF, with the exceptions of OP-291-1/2/3. Especially, the

353

calculated LC50/EC50/ChV values of OP-291-2 and OP-291-3 were approximately 2-6

354

times higher than BF toxicity for algae (acute and chronic toxicity), daphnid (acute

355

and chronic toxicity) and fish (chronic toxicity), indicating that these OPs were

356

probably more harmful than their precursor compound BF.

357

Although we could not directly show the evolution of identified OPs and toxicity 16


Page 16 of 31

Page 17 of 31


358

in one figure due to the different methods adopted in the two experiments, we still can

359

compare the formation of identified OPs and the toxicity patterns of the ozonated

360

solutions under similar ozone exposure, to help identifying which OPs might be

361

responsible for the increased toxicity. According to the normalized peak area versus

362

reaction time of each OP (Figure S5), the eight OPs observed could be divided into

363

two groups. The normalized peak areas of OP-291-2, OP-291-3, OP-409, OP-239 and

364

OP-309-1 increased continuously during the reaction time, consistent with the

365

increased acute toxicity pattern in the semi-continuous experiments under the similar

366

O3 exposure (Figure 1). However, the normalized peak areas of OP-291-1, OP-393

367

and OP-309-2, first increased until the reaction time of 5 min in the batch experiment,

368

and decreased afterwards. The different patterns of OP concentrations and acute

369

toxicity observed indicated that the latter three OPs, OP-291-1, OP-393 and OP-309-2,

370

could be excluded from the candidates of toxicity-causing OPs. Therefore, together

371

with the results of ECOSAR, we suggested that among the identified OPs, OP-291-2

372

and

373

N-(2,4-dihydroxyphenethyl)-4-chlorobenzamide, respectively, could be regarded as

374

potential toxicity-causing OPs during the BF ozonation.

OP-291-3,

namely

N-(3,4-dihydroxyphenethyl)-4-chlorobenzamide

and

375

Nevertheless, it should be noted that other OPs, which were not identified under

376

the given experimental conditions, might also contribute to the increased toxicity.

377

According to the EICs, the peak areas of the three isomer of OP-291 were 40-100

378

times smaller than that of BF in the solution before the ozonation. The low abundance

379

of identified potential toxicity-causing OPs (OP-291-2 and OP-291-3) indicated their

380

concentrations might be much lower than initial BF, although they could not be

381

quantified due to the lack of standards. However, the results obtained by ECOSAR

382

showed that the toxicity of OP-291-2 and OP-291-3 was only 2-6 times higher than 17



383

BF. Therefore, we could not exclude the possibility that there are other unknown OPs

384

which are also responsible for the elevated toxicity.

385

Environmental implications. In this study, high resolution HPLC-MSn demonstrated

386

superiority of structure determination of unknown OPs, and QSAR was found to be

387

feasible and reliable to predict the toxicity of OPs with identified molecular structure.

388

As most OPs of PPCPs could not be purchased or easily synthesized, the methodology

389

adopted in present study could provide useful information for other studies in which

390

increased toxicity was observed during the oxidation processes of other PPCPs.

391

Besides, our study also indicated that toxicity assessment should be taken into

392

consideration in water treatment processes when the operational conditions, such as

393

the ozone doses and contact time or the type of ozonation process, were designed,

394

with the purpose of eventually reducing the potential risk posed by the emerging

395

contaminants to ecosystem and humans.

396

Acknowledgements

397

This research was partly supported by the National Natural Science Foundation

398

(21577033, 51208199, 51408425), the Fundamental Research Funds for the Central

399

Universities (22A201514057), Beijing Key Laboratory for Emerging Organic

400

Contaminants Control, the Foundation of The State Key Laboratory of Pollution

401

Control and Resource Reuse, China (PCRRG 11017), and Specialized Research Fund

402

for the Doctoral Program of Higher Education of China (20130072120033).

403

Analytical support in detection and elucidation of ozonation products by the staff of

404

the Environmental Analytical Laboratory of the Institute of Environmental

405

Engineering of RWTH Aachen University is also greatly appreciated. In addition, we

406

also thank Dr. Yunho Lee from Gwangju Institute of Science and Technology, Korea,

407

for the revision of this manuscript. 18


Page 18 of 31

Page 19 of 31


408

Tables

409

Table 1. Retention time (tr), accurate ion masses recorded and calculated theoretical

410

masses of

411

bond equivalent (RDB) values of BF and its OP-ions

412

Table 2. Toxicity calculations for BF and its OPs after O3 treatment using ECOSAR

413

program

35

Cl isomeric APCI--ions, proposed chemical formulas and ring-double

414

19



Page 20 of 31

415

Table 1. Retention time (tr), accurate ion masses recorded and calculated theoretical

416

masses of 35Cl isomeric APCI--ions, proposed chemical formulas and ring-double

417

bond equivalent (RDB) values of BF and its OP-ions

Compound

Retenion time tr [min]

[M-H]- accurate ion mass recorded [u]

Chemical formula of compound

RDB

BF

11.7

360.0989

C19H20O4NCl

10.5

361.1081

OP-291-1 b

4.4

290.0575

OP-291-2 b

6.1

290.0573

C15H14O3NCl

9.5

291.0662

OP-291-3 b

8.5

290.0578

OP-239 b

5.9

238.0264

C11H10O3NCl

7.5

239.0349

OP-309-1 b

6.2

308.0315

OP-309-2 b

C14H12O5NCl

9.5

309.0404

8.0

308.0317

OP-409

8.7

408.0847

C19H20O7NCl

10.5

409.0928

OP-393

10.4

392.0890

C19H20O6NCl

10.5

393.0979

Theoretical mass a [u]

418

a

Monoisotopic masses: 1H, 12C, 14N, 16O and 35Cl.

419

b

Compounds that highlighted in bold refer to new OPs identified in this study.

420

20


Page 21 of 31


421

Table 2. Toxicity results for BF and its OPs after O3 treatment using ECOSAR

422

program Acute toxicity [mg/L]

Chronic toxicity (ChV) [mg/L]

Proposed Structure Fish Daphnid Algae Fish Daphnid Algae (LC50) (LC50) (EC50) O COOH

BF

N H

O

2.8

2.0

3.5

0.17

0.34

1.4

10

1.4

2.6

0.15

0.34

0.83

3.5

0.87

0.58 0.05

0.10

0.35

3.5

0.87

0.58 0.05

0.10

0.35

151

655

39

1.0

66

16

259

607

70

1.7

61

26

195

22

55

2.7

6.4

13

7.2

1269

150

0.42

96

93

Cl

OH

O

OH

OP-291-1

N H Cl O OH

OP-291-2

N H OH

Cl O

OH

OP-291-3

N H HO

Cl

O

O

OP-239

O

N H Cl

OH

O

O

N H

OP-309-1/2 Cl

O O O

OH COOH N H

OP-409

O HO

Cl OH

O O O

OP-393

O

COOH

N H Cl

423

The predicted toxicity values are classified according to the system established by the

424

Globally Harmonized System of Classification and Labelling of Chemicals (GHS)

425

(34):

426

Not harmful: LC50/EC50/ChV>100;

427

Toxic: 10≥LC50/EC50/ChV>1;

Harmful: 100≥LC50/EC50/ChV>10; Very toxic: LC50/EC50/ChV≤1.

428

21



429

Figures

430

Figure 1. BF concentration and acute toxicity assessed by luminescence inhibition test

431

during the ozonation process

432

Figure 2. Negative MS2 (a) product ion spectrum of OP-309 with m/z = 308.0327 and

433

negative MS3 product ion spectra of selected product ions with (b) m/z = 280.0378

434

and (c) m/z = 262.0274). (d) fragmentation pathways of OP-309 proposed from results

435

obtained by MS2 and MS3 negative product ion spectra. Structural formulas and

436

chemical formulas shown in the pathways are neutrals ([m/z]- + H+) with

437

monoisotopic masses (1H, 12C, 14N, 16O and 35Cl).

438

Figure 3. Proposed oxidation pathways of BF during the ozonation (Compounds in

439

the different boxes display different isomers)

440 441

22


Page 22 of 31

Page 23 of 31


442 100 80

15

60 10 40 5

C/C0 (%)

Relative inhibition (%)

20

20

C/C0 (%) Relative inhibition (%)

0

0 0

443

2

4

6

8

10

time (min)

444

Figure 1. BF concentration and acute toxicity assessed by luminescence inhibition test

445

during the ozonation process

446

23



Page 24 of 31

④ 276.0010

(a)

276.0010

100

④ 90 80

Relative Abundance

70

③ ③

60

280.0378 280.0378 50

⑦ ⑦ 154.0013

40 30

⑧ ⑧ 125.0244 125.0244

20

⑤ ⑤ 262.0274

⑥ ⑥

154.0013

② ② 290.0259①① 308.0327 308.0327

262.0274

226.0274 226.0274

290.0259

10 0 80

100

120

140

160

180

200 m/z

447

(b)

220

240

260

280

300

280.0378 280.0379

100

③ ③ 90 80

Relative Abundance

70 60 50 40 30

⑧ ⑧

⑤ ⑤

125.0244 125.0245

262.0274 262.0275

⑦ ⑦ 154.0013 153.9870

20 10 0 80

100

120

140

160

180

448

200

220

240

260

280

300

m/z

(c)

234.0324 234.0324

100

⑨

90

Relative Abundance

80 70

⑤ ⑤

60

262.0274 262.0270

50 40 30 20 10 0 80

100

120

140

160

180

449

200

220

m/z

24


240

260

280

300

Page 25 of 31


O

O

(d)

① NH HOOC Cl O

OH

O

O

NH

Cl

O O

m/z=308.0327 C14H 12O5NCl

-18.0068 -H2 O

②

-27.9949 -CO

③

O

O

-32.0317 -CH4 O

OH

O

NH

O

④

O O

NH

Cl

N

Cl

O

Cl O

O

O

-27.9985 -CO

-155.0134 -C7 H 6ONCl

-18.0104 -H2 O

⑤

m/z=276.0010 C13 H8 O4NCl

m/z=280.0378 C13H 12O4NCl

m/z=290.0259 C14H 10O 4NCl

O

O

⑧

O

-54.0104 -C3 H2 O

⑥

O

OH

O

O

NH

NH

Cl

Cl O

O

m/z=262.0274 C13H 10O 3NCl -27.9950 -CO

⑨

O

m/z=226.0274 C10 H10O3NCl

m/z=125.0244 C6 H6 O3 -108.0261 -C6H 4O 2 O

O

-72.0261 -C3H 4 O2 O

⑦

NH2

NH

Cl

Cl

m/z=234.0324 C12H 10O 2NCl

m/z=154.0013 C7 H6 ONCl

450 451

Figure 2. Negative MS2 (a) product ion spectrum of OP-309 with m/z = 308.0327 and

452

negative MS3 product ion spectra of selected product ions with (b) m/z = 280.0378

453

and (c) m/z = 262.0274). (d) fragmentation pathways of OP-309 proposed from results

454

obtained by MS2 and MS3 negative product ion spectra. Structural formulas and

455

chemical formulas shown in the pathways are neutrals ([m/z]- + H+) with

456

monoisotopic masses (1H, 12C, 14N, 16O and 35Cl). 25



Page 26 of 31

O

O

COOH N H

O

OH

O

N H

OH

Cl

COOH N H

O OH

Cl

Cl

O

OH

COOH

O OH

OH

OP-409

OH COOH N H

O

Cl

OH

O

O

O OH

OH

Cl

Cl O

N H HO

OH

Cl

OP-291-1/2/3

COOH N H

OH

N H

N H

O

Cl

BF OH

O

O

N H

O

Cl

O O

O O

O

COOH

N H

O

O Cl

OP-393

?

NH

O

Cl

OP-309-1/2 OP-239

457 458

Figure 3. Proposed oxidation pathways of BF during the ozonation (Compounds in the different boxes display different isomers, the question

459

mark indicates the structure of one of the isomer of OP-309 is not determined) 26


Page 27 of 31


460

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Identification of New Oxidation Products of Bezafibrate for Better

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