Photochemical Transformation of Nicotine in Wastewater Effluent

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Photochemical Transformation of Nicotine in Wastewater Effluent Lushi Lian, Shuwen Yan, Bo Yao, Shen-An Chan, and Weihua Song Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.7b03223 • Publication Date (Web): 25 Sep 2017 Downloaded from http://pubs.acs.org on September 26, 2017

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Environmental Science & Technology

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Photochemical Transformation of Nicotine in Wastewater Effluent

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3 Lushi Lian1, Shuwen Yan1, Bo Yao1, Shen-An Chan2, and Weihua Song1,*

4

5 6

1

Department of Environmental Science and Engineering, Fudan University, Shanghai, 200433, China

7

2

Agilent Technology, Inc., 1350 North Sichuan Road Shanghai, 200080, P. R. China

8 9 10 11

*Corresponding author: Tel.: +86-21-65642040 Email: [email protected]

12 13

Resubmitted to Environmental Science & Technology

1

ACS Paragon Plus Environment


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Abstract

15

Nicotine is a highly toxic tobacco alkaloid that is ubiquitous in wastewater effluent. For the

16

first time, we report the identification of the products and the pathways for the photodegradation of

17

nicotine in an effluent matrix under simulated solar irradiation. Nicotine was found to be degraded

18

by triplet state organic matter (3OM*), thus indicating that electron transfer is a preferred reaction

19

mechanism. Using the multivariate statistical strategies orthogonal projection to latent structures

20

discriminant analysis (OPLS-DA) and hierarchical clustering, 49 potential transformation products

21

(TPs) of nicotine were successfully extracted from the water matrix via high-resolution

22

ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry

23

(UHPLC-QTOF-MS). Overall, 30 TPs, including 4 groups of non-separated isomeric photo TPs,

24

were identified with various levels of confidence based on the MS2 information of standard

25

compounds and the isotope-labeling method (using rac-nicotine-2’,3’,3’-D3, rac-nicotine-13CD3, and

26

rac-nicotine-D4) under the air saturated condition. The pyrrolidine ring of nicotine was found to be

27

the reactive site under sunlight irradiation. Pseudooxynicotine was the main primary TP from

28

nicotine, with a maximum transformation ratio of 64%. Nicotinic acid, cotinine, 3’-hydroxycotinine

29

and myosmine were the final stable TPs after 72 h of solar irradiation, with yields of 13%, 3%, 5%

30

and 5%, respectively.

31 32 33 34

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35 36


TOC Art

37 38

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Introduction

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Cigarette smoking has well-documented negative impacts on human health. It is responsible for

41

90% of cases of lung cancer, 75% of cases of chronic bronchitis and emphysema, and 25% of cases

42

of ischemic heart disease.1-3 Nicotine is the principle alkaloid in cigarettes, and it is composed of a

43

pyridine ring and a pyrrolidine ring.4 The US Environmental Protection Agency (EPA) has included

44

nicotine in its Toxic Release Inventory since 1995,5 and the lethal oral dose for humans is

45

approximately 40-60 mg.6 The annual global consumption of nicotine can be estimated to be ∼ 5 ×

46

104 tons based on the fact that approximately 5.5 trillion cigarettes are produced globally each year7

47

and that the typical nicotine content of a cigarette is ∼8 mg.8 Most (93%) of nicotine existed in the

48

aqueous environment,9 and effluent from municipal wastewater treatment plants is one of primary

49

point sources. Nicotine has been reported to be presented at concentrations in the range of ng L-1 to

50

µg L-1.10,

51

wastewater contamination and for population size assessment.10, 11

11

Moreover, nicotine and its metabolites are used as chemical markers for domestic

52

Transformation pathways of nicotine are complex in the environment system, which include

53

chemical and biotic transformation reactions.12, 13 Transformation products (TPs) of nicotine have

54

gained substantial attention due to their potential environmental risk.14, 15 In aquatic environments,

55

nicotine can be biodegraded into more persistent and toxic TPs, such as cotinine and

56

4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,13,

57

considering the environmental risk of nicotine. In addition to biodegradation, solar-mediated

58

photodegradation might also be an important process for the attenuation of nicotine in surface

59

water.17 Previous studies have indicated that direct photodegradation of nicotine is slow, and indirect

60

photodegradation plays a key role in sunlit surface waters. In general, indirect photodegradation is

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associated with triplet states of organic matter (3OM*)18 and a series of reactive oxygen species

62

(ROS), such as singlet oxygen (1O2), hydroxyl radicals (HO•), superoxide anions (O2•-), and

16

which should be accounted for when

4


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carbonate radicals (CO3•-).19-21 The HO• and 1O2 reaction rate constants of nicotine were measured to

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be 1.08 × 109 M-1 s-1 and 3.38 × 106 M-1 s-1, respectively.12 Moreover, photochemical modeling

65

demonstrated that the reaction of nicotine with HO• would prevail over that with 1O2 in the surface

66

water transformation pathway. However, there is currently no mechanistic understanding of the

67

phototransformation of nicotine, especially in a complex water matrix. Obtaining such an

68

understanding is challenging for several reasons: 1) photodegradation is a complicated process that

69

involves a variety of ROS and 3OM*; 2) TPs always exhibit high variety; and 3) organic matter (OM)

70

interferes with TPs, thus making the identification process challenging. A number of studies have

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elucidated TPs from individual ROS reactions with micropollutants and proposed degradation

72

mechanisms, such as those involving HO•, 1O2, or model photosensitizers.22-24 However, there is a

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lack of information regarding TPs arising from 3OM*. Alternative technologies need to be developed

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to explore the photochemical transformation mechanism of micropollutants in a complex water

75

matrix.

76

High-resolution mass spectrometry (HRMS) can provide confirmative information in target

77

analysis mode and can aid in the discovery and elucidation of unknown analytes. Time-of-flight

78

(TOF) MS, a type of HRMS, has been widely employed in environmental fields to profile unknown

79

compounds,25-27 identify and confirm known target compounds and their degradation products.23, 28-30

80

Multivariate statistical strategies, such as principal component analysis (PCA) and orthogonal

81

projection to latent structures discriminant analysis (OPLS-DA), have been recently applied to

82

extract the promising TPs from the thousands of features recorded by liquid chromatography TOF

83

MS (LC-TOF-MS).31-33 However, qualitative information that supports the recognition and structural

84

elucidation of products other than precursors is still needed. MS2 spectra consisting of the fragments

85

are useful when identifying an unknown compound.34 Otherwise, in recent years, an isotope-labeling

86

method has been applied to investigate the biotransformation of organic compounds in a complex

87

matrix.35, 36 Moreover, site-specific labeled isotopes could be useful to elucidate the isomers of TPs37 5



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and could be helpful to confirm the reaction site.38 Therefore, in this study, we attempted to combine

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statistical analysis-assisted HRMS and isotope labeling methods to explore the photochemical

90

transformation of emerging contaminants in a complex water matrix.

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The goal of this research was to study the phototransformation, identify the photochemical TPs

92

and elucidate the degradation pathway of nicotine in a wastewater matrix by using

93

ultra-high-performance liquid chromatography quadrupole time-of-flight mass spectrometry

94

(UHPLC-QTOF-MS). Mathematical tools, OPLS-DA and hierarchical clustering, were applied to

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extract the potential TPs from the massive MS dataset through a comparison with control samples.

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Additionally, the isotope-labeling experiments and standard compounds, in addition to MS2

97

information, were employed to confirm the structure of the TPs. This study provides a potential

98

method for the unequivocal identification of the TPs of emerging contaminants in a complex effluent

99

matrix, particularly for 3OM*-induced photochemical transformation.

100 101

Material and Methods Materials.

Nicotine,

nicotinic

acid,

myosmine,

cotinine,

3’-hydroxycotinine

and

102

N-formylnornicotine were purchased from Alfa Aesar. Rac-nicotine-2’,3’,3’-D3, rac-nicotine-13CD3,

103

rac-nicotine-D4, pseudooxynicotine dihydrochloride, nornicotine and 4-oxo-4-(3-pyridyl)-butanal

104

were purchased from Toronto Research Chemicals (TRC). Furfuryl alcohol (FFA), furfural (FAD),

105

terephthalic acid (TA), aniline (ANL), HPLC-grade acetic acid and ammonium acetate were

106

purchased from Sigma-Aldrich. 2-Hydroxyterephthalic acid (2HTA) was synthesized according to

107

the literature.39 HPLC-grade acetonitrile and isopropanol were purchased from Fisher Scientific.

108

LC/MS-grade water was purchased from J&K Chemical. Stock solution of nicotine was prepared in

109

ultrapure water (DI-H2O) at 1000 mg L-1 and stored at 4 °C. Stock solutions of other compounds

110

were prepared in methanol at 100 mg L-1, and stored at -20 °C. All of the compounds were stable

111

under the storage conditions. The wastewater effluent in this study was collected from a municipal 6


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wastewater treatment plant (WWTP) located in Jiangsu Province, China. The detailed information of

113

the wastewater effluents could be found in Text S1 and Table S1 of Supporting information (SI).

114

Simulated Solar Irradiation Experiments. An Atlas solar simulator (Suntest XLS+) equipped

115

with a 1700 W xenon lamp and a solar filter was employed for all simulated solar irradiation

116

experiments. The absolute irradiance spectrum of the solar simulator was recorded by a spectrometer

117

(USB-4000, Ocean Optics Inc.) Natural sunlight was also measured on a summer day at Fudan

118

University in Shanghai, China for comparison (Figure S1, SI). All samples were placed in specially

119

made quartz containers, and aliquots were withdrawn and analyzed via UHPLC-QTOF-MS after

120

different doses of irradiation. TOC of the wastewater effluents using in the experiments were 5.0

121

mgC L-1. The initial concentration of nicotine in the effluent was 8.0 µM (1.3 mg L-1). The initial

122

concentration of the isotopic nicotine and standard compounds in the effluent used to confirm the

123

products was 5.0 mg L-1. For the O2-dependence experiments, argon (Ar) was purged slowly into the

124

reaction vessel (the flow rate is around 15 mL min-1) with varied time scale to achieve different

125

dissolved oxygen (DO) concentration. In order to eliminate the influence of sampling process, Ar

126

was post-purged after sampling, to maintain DO constant. The pH of the solution was determined to

127

be 7.51 by using a pH meter (Thermo Scientific) and kept constant during irradiation (within ± 0.1).

128

The control experiment was performed by photo-irradiating wastewater effluent without spiked

129

nicotine. All of the experiments were repeated in three times, and each sample was analyzed by

130

HRMS twice.

131

UHPLC-QTOF-MS Analysis. An Agilent 1290 UHPLC system was employed for separation.

132

Samples of 20 µL were loaded on a Waters Atlantis dC18 column (2.1 mm × 150 mm, 3.0 µm) with

133

the thermostat set to 30 °C and were separated with a gradient of 10 mM ammonium acetate with

134

0.01% acetic acid water and acetonitrile. The mobile phase flow rate was set to 0.3 mL min-1, the

135

gradient elution program was optimized and given in Table S2 of the SI.

7



136

A high-resolution Agilent 6540 QTOF-MS system with an Agilent Jet Stream (AJS)

137

electrospray ionization (ESI) source was used in both positive and negative modes. At the AJS ESI

138

interface, the sheath gas temperature was set to 400 °C, the sheath gas flow rate was set to 12 L min-1,

139

the drying gas temperature was set to 300 °C, the drying gas flow rate was set to 10 L min-1, and the

140

nebulizer was set to 60 psig. The capillary voltage of the ESI was set to 1000 V, the nozzle voltage

141

was set to 500 V, and the fragmentor was set to 125 V. TOF scanning was performed in both positive

142

and negative modes with a mass range of 50-3000 m/z, and the data acquisition rate was 2 spectra s-1.

143

Targeted MS/MS and auto MS/MS modes with various collision energies (5, 10, 15, and 30 eV) were

144

also employed to identify the potential products. These collision energies were chosen to ensure that

145

four diverse MS/MS spectra for per product can be obtained. Targeted MS/MS was conducted for the

146

TPs of which standards were available. The quantification of nicotine and transformation products

147

were performed by an UHPLC (Agilent 1290) coupled with a Triple Quadrupole MS system with an

148

ESI source (Agilent 6430), and the details are given in Text S2 and Table S3 of SI.

149

Statistical Analysis. The UHPLC-QTOF-MS data were first analyzed using Agilent Mass

150

Profinder software to remove the background noise and unrelated ions. All the features with

151

abundance > 1000 were extracted in this process. Then, the normalized peak area matrices (log 2)

152

were exported to SIMCA-P 13.0 (Umetrics) for OPLS-DA to reveal the differences between the

153

nicotine group and control group. Each OPLS-DA model was evaluated by the internal permutation

154

test. Finally, hierarchical clustering was performed using MeV 4.9.0 to identify the potential

155

photo-induced products of nicotine. The maxima normalized average peak area were exported for

156

clustering and Pearson correlation coefficient squared was used as the metric. Average linkage was

157

used as the clustering algorithm.

158

Results and Discussion

8


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Identification of the Potential TPs of Nicotine in Wastewater Effluent. UHPLC-QTOF-MS

160

was employed to explore the photochemical transformation of nicotine in the effluent. Wastewater

161

effluents consist of a variety of compounds, resulting in a high background. As shown in Figure S2

162

of SI, the decay of nicotine could be observed, but it is impossible to distinguish the products directly

163

from the total ion chromatography. Thus, the extraction of the compound features from the

164

background is of great necessity. Applying a recursive feature extraction algorithm (Agilent

165

Profinder 8.0), 932 features in the positive mode and 1027 features in the negative mode were

166

extracted from the irradiated effluents spiked with 8.0 µM nicotine. The extracted MS features table

167

are shown in the excel file of SI. These compounds with the TPs resulted in the difficulty of

168

identification. The variations in the chemical profiles between the effluents alone and the

169

nicotine-spiked effluents were examined via supervised multivariate OPLS-DA at various simulated

170

solar irradiation times, as shown in Figure 1a and b. The OPLS-DA scatter plots demonstrated

171

clearly time trends and significant separation of the effluent alone from the nicotine-spiked samples

172

was achieved for both positive and negative modes. It indicated that the TPs of nicotine in the

173

effluent could be separated from the effluent background. A permutation test (with 100 iterations) in

174

the corresponding PLS-DA model was performed to further evaluate the quality of the OPLS-DA

175

model. The R2 and Q2 values were greater than the original points in the permutation test, and the

176

regression of the Q2 points intersected the vertical axis below zero (Figure S3), demonstrating that

177

the OPLS-DA model was statistically valid. The key features responsible for the discrimination

178

between nicotine-spiked samples and effluent samples were ranked. A total of 55 discriminatory

179

features in the two groups (with and without the nicotine) were then filtered out based on a minimum

180

variable importance for the projection (VIP) value of 1.0 for the positive mode. The histogram of

181

VIP values of all features in positive mode was shown in Figure S4.

182

(Insert Figure 1)

9



183

The 55 discriminatory features consisted of the nicotine TPs and the nicotine-impacted TPs

184

generated from OM. To further extract the photo-induced TPs of nicotine, hierarchical clustering was

185

performed to analyze the 55 features. Hierarchical clustering is a data-mining approach that has been

186

widely applied in metabolomics, proteomics and genomics studies.40-42 Figure 1c shows the

187

hierarchical clustering result of the 55 features in the nicotine-spiked samples and the control effluent

188

as a function of irradiation time. The promising photo-induced TPs of nicotine should be present or

189

increase only in the nicotine samples after irradiation. Based on this principle, 49 features were

190

extracted as promising photo-induced TPs of nicotine. Among them, 30 TPs presented as detectable

191

levels (S/N >10) and details were shown in Table 1. The remaining 23 features were discarded due to

192

the uncorrelated chemical formula with nicotine or low signal intensities (S/N 5 mg L-1), the role of O2 mainly presents as quenching 3OM* to

252

reduce the degradation rates of nicotine. At low concentrations of DO (< 5 mg L-1), O2 mainly

253

presents as oxidant, participating in the oxidation of nicotine.

OM* was the primary reactive species. However, the degradation rates of nicotine decreased when

254

Proposed Mechanism for the Photochemical Transformation of Nicotine in air saturated

255

conditions. Nicotine comprises a pyrrolidine and pyridine ring, and both moieties react with 1O2 and

256

HO•.12, 49 However, as confirmed by isotope-labeled nicotine-D4, the pyridine moiety remains intact 12


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257

during the photochemical transformation. This observation illustrates that the pyrrolidine ring is the

258

reactive site of nicotine under simulated solar irradiation. As mentioned previously, the removal of

259

nicotine in the D2O solvent was slightly slower than that in H2O, and the kinetic solvent isotope

260

effect (H/D KSIE) was calculated to be 1.5, indicating that H/D-transfer involved in the 3OM* with

261

protonated (H+/D+) nicotine. The primary reactions involving 3OM* are redox reactions, with 3OM*

262

mainly acting as an oxidant.18 It has been well documented that the reaction between 3OM* and

263

amines and phenols proceeds by an initial rapid charge-transfer from the N/O-electrons of the

264

amines/phenols to 3OM*, followed by the 1,2-H shift and the formation of C-centered radicals.50-52

265

The C-centered radicals from nicotine consist of primary, secondary, and tertiary C-centered radicals,

266

which are referred to as intermediates I, II and III (Scheme 1A). They were hypothesized to further

267

react with O2 to form the corresponding peroxyl radicals, denoted as intermediates IV, V and VI,

268

respectively, in Scheme 1A. Bimolecular reaction and intramolecular hydrogen abstraction were

269

proposed to be two important reactions of peroxyl radical.53 As shown in Scheme 1B/C, the tetroxide

270

intermediates, which are formed via the combination of peroxyl radicals with α-hydrogen (primary

271

and secondary C-centered radicals), mainly generate alcohols and aldehydes or ketones via the

272

Russell or/and Bennett reactions.54, 55 Regarding nicotine, the degradation of a series of peroxyl

273

radical has been proposed and further decayed to 30 TPs at detectable levels afterwards (Table 1) via

274

varied pathways, as described below. All identified TPs were further verified via comparison with the

275

isotope-labeled

276

compound-spiked experiments and the MS2 spectra of standard compounds. Schymanski et al.

277

proposed a widely accepted level system for communicating identification confidence in

278

HRMS-based identification depending on the amount of information available.34, 56 The levels of

279

identification confidence of all TPs were listed in Table 1. The structures and chemical identifiers of

280

isotope-labeled nicotine and standard compounds are shown in Figure S9 and Table S5. The structure

nicotine-spiked

experiments

(nicotine-D3

or

13


nicotine-13CD3)

or

standard


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281

elucidation evidence, including extracted-ion chromatographs and MS2 spectra of the TPs can be

282

found in Text S9 of the SI. (Insert Scheme 1 and Table 1)

283 284

Reaction Pathway I: Transformation of Intermediate (X). As shown in Scheme 2, the

285

hydrogen at position C2’ has the lowest hemolytic dissociation enthalpies (BDEs) of C-H bonds in

286

nicotine due to the conjugated effects from the pyridine moiety.49 Intermediate (VII) has been

287

assumed to be a major transient in the HO•-induced oxidation of nicotine.49 The intermediate X

288

would react with other primary or secondary peroxyl radicals and undergo the Russell reaction to

289

form P178a and pseudooxynicotine (reactions 1 and 2 in Scheme 3) with an observed m/z of 179.1177,

290

which was verified with standard compound. The corresponding MS2 result of pseudooxynicotine is

291

presented in S9.18 and illustrates three major fragments with m/z values of 148.0757, 106.0287 and

292

78.0338. The fragment with m/z 148.0757 indicates the loss of CH3NH2 (M-31), and the m/z

293

106.0287 is a fragment of nicotinaldehyde. The remaining fragment was assigned to the pyridine ring

294

moiety. The proposed tetroxide intermediate process (Russell reaction) for pseudooxynicotine was

295

further verified by a concentration dependence experiment. As illustrated in Figure S10, the

296

transformation ratio of pseudooxynicotine increased with increasing initial concentrations of nicotine,

297

suggesting the bimolecular combination of peroxyl radicals occurs. Pseudooxynicotine can be easily

298

photo-oxidized, as shown in Figure 3a. The phototransformation of the pseudooxynicotine standard

299

was

300

4-hydroxy-1-(3-pyridyl)-1-butanone (HPB), with an m/z value of 166.0860, was observed. The MS2

301

of HPB yielded the fragment of 148.0757, demonstrating the presence of one hydroxyl group (M-18).

302

The levels of the remaining fragments of 120.0444, 109.0522, 108.0444 and 80.0495 increased with

303

increasing collision energy. Particularly, both the m/z values of 109.0522 and 108.0444 were the

304

predominant values at 30 eV (S9.13), consistently with the fact that HPB contains a pyridine moiety

305

and a carbonyl group at the C2’ position. The hydroxyl group at HPB can be further oxidized by

also

performed

under

similar

irradiation

conditions,

14


and

the

formation

of

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306

3

OM* to the corresponding aldehyde,57 thus generating 4-oxo-(3-pyridyl)butanal. Moreover,

307

4-oxo-(3-pyridyl)butanal can also be generated from pseudooxynicotine through the imine

308

intermediate (reactions 558 and 659). A standard compound was employed to verify the formation of

309

4-oxo-(3-pyridyl)butanal, and the MS2 result is shown in S9.12 of the SI. Figure 3c illustrates that

310

this aldehyde was not stable under irradiation, and the oxidation of the aldehyde group yielded a

311

corresponding keto acid. The decarboxylation of the keto acid produced another major product, P135.

312

A process involving an excited-state intramolecular proton transfer would be a promising mechanism

313

for the decarboxylation reaction.60 Figure 3c shows that P135 is relatively stable and slowly decays

314

after 48 h of irradiation. In addition, keto-enol tautomerism can occur at the aldehyde group of

315

4-oxo-(3-pyridyl)butanal as reaction 9,61 and the subsequent oxidation of the enol intermediate

316

yielded P181. Fragments with m/z values of 164.0706 (M-18) and 146.0600 (M-36) indicated the

317

presence of at least two hydroxyl groups (S9.22). Therefore, the structure of P181 is proposed and

318

shown in Scheme 3. The photodegradation of pseudooxynicotine also obviously gave rise to

319

N-methylnicotinamide and nicotinamide, which have been identified in the fermentation of

320

nicotine.62 The nicotinamide could be further oxidized to nicotinic acid (reaction 13), and the

321

mechanism from amide to acid has been previously reported.63 The MS2 result revealed that the

322

primary fragments of N-methylnicotinamide included nicotinaldehyde (m/z 108.0444) and a pyridine

323

ring moiety (m/z 80.0495), as shown in S9.5 of the SI. (Insert Scheme 2)

324 325

In addition to the Russell reaction, the peroxyl radical intermediate X could further lose HO2• 24

326

and yield N-methylmyosmine, with an observed m/z of 161.1074 (reaction 14 of Scheme 3).

327

N-methylmyosmine has also been observed to be an important product of both the chemical and

328

biological degradation of nicotine.16, 49, 62, 64 Figure 3a illustrates that N-methylmyosmine was not

329

stable and photodegraded rapidly. N-methylmyosmine could spontaneously undergo ring

330

tautomerism

and

hydrolysis

to

hydroxylated

nicotine

15


P178a

and

pseudooxynicotine.


331

Pseudooxynicotine has been previously reported to be a predominant biodegradation product of

332

N-methylmyosmine.16 However, N-methylmyosmine was also expected to undergo the electron

333

transfer, followed by the 1,2-H shift to form a C5’-centered radical (reaction 16). Then, the

334

C5’-centered radical reacted with O2 to form a peroxyl radical. As mentioned previously, the Russell

335

reaction and Bennett reaction lead to the formation of an alcohol (P176) and a ketone (P174) (reaction

336

17 and 18). The alcohol can be oxidized to the ketone, as previously reported.57 Furthermore, the

337

hydroxylated ketone P190 was formed by an attack of ROS on the C4’ of the ketone. Previous

338

investigations have established that the photo-oxidation of tertiary amines with a methyl group can

339

lead to demethylated amine, namely the “methylene shuttle” (reaction 21 and 22).65 In this study, the

340

demethylated product of N-methylmyosmine (P146a) was observed at the m/z of 147.0916 and is

341

illustrated in Figure 3a. P146a would either tautomerize to myosmine or be oxidized by ROS (3OM*

342

and HO•) to yield the N-oxide product P162a, as postulated elsewhere.66

343

(Insert Figure 3)

344

Reaction Pathway II: Transformation of Intermediate (XI). Scheme 3 presents the proposed

345

reaction pathway of intermediate (XI). Similar to the transformation of intermediate (X), the

346

dehydrogenation product P160 (with an m/z value of 161.1073) was one of the major products. P160

347

and N-methylmyosmine are isomers, which cannot be separated by the chromatographic method

348

used in this study. Thus, the nicotine-D3 experiments were performed to verify the TPs. The three

349

deuterium product (with an m/z value of 164.1264) was observed in the nicotine-D3 experiments,

350

further supporting the formation of P160 (S9.9). The MS2 fragment with an m/z of 161.1073 was a

351

mixture of N-methylmyosmine and P160. The observed m/z of 159.0917 indicated the

352

dehydrogenation of P160 and N-methylmyosmine, and the corresponding demethylation structure was

353

evident as the fragment with an m/z of 145.0760. Furthermore, the fragment with an m/z of 84.0808

354

confirmed the structure of the dehydrogenated pyrrolidine ring. The remaining fragments were also

355

observed in the MS2 of nicotine. P160 decayed rapidly after 8 h of irradiation (Figure 3b). P160 further 16


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356

underwent the methylene shuttle reaction and yielded the demethylation product P146b, which

357

produced P146c via tautomerism. P162b accumulated via the hydroxylation of P146b in a reaction similar

358

to reaction 23. Considering the presence of hydrogen at the C5’ position, the bimolecular reaction of

359

the peroxyl radical produced P178b and cotinine, with observed m/z values of 179.1179 and 177.1020,

360

respectively. P178b was verified by the corresponding tri-deuterated compound with the observed m/z

361

of 182.1367 in the nicotine-D3 experiments (S9.20). The fragment ions observed (m/z values of

362

132.0808, 130.0651, and 117.0573) in the MS2 spectrum of P178b were the same as those observed in

363

the spectrum for nicotine. Cotinine was verified via the standard compound, as shown in S9.15. The

364

lactam structure, with an m/z of 98.0600, was observed as a product ion in the MS2 spectrum.

365

Cotinine is an important product of nicotine identified during ozonation,67 HO• oxidation,12 and

366

biodegradation.8 It was more stable than nicotine, as demonstrated by its accumulation during

367

irradiation, as shown in Figure 3b. The photochemical transformation of cotinine was investigated by

368

irradiating the standard compound to elucidate the TPs originated from cotinine. The methylene

369

shuttle reaction led to the product norcotinine, an analog of N-methylmyosmine. 3’-Hydroxycotinine

370

has been reported to be an oxidation product of cotinine,12 which was also observed in this study.

371

However, as shown in Figure 3b, the degradation of cotinine is quite slow; meanwhile the formation

372

of 3’-hydroxycotinine is significant. This result suggested another formation pathway of

373

3’-hydroxycotinine exists. We hypothesized that 3’-hydroxycotinine could be formed through the

374

degradation of P160 via a dihydroxylated intermediate (reaction 33). This process was proposed by

375

previous investigations.22,

376

3’-hydroxycotinine, and the MS2 data in S9.24 shows fragment with m/z values of 134.0600,

377

106.0651 and 80.0495. The major ion, which represents the pyridine moiety, was found at an m/z of

378

80.0495 at 30 eV. The fragments with m/z values of 134.0600 and 106.0651 resulted from the loss of

379

C2H5NO and C3H5NO2, respectively. Both cotinine and 3’-hydroxycotinine could undergo

68, 69

The standard compound was used to confirm the formation of

17



380

dehydrogenation and hydrolysis to yield products P192a (reaction 38 and 39) and P208 (reaction 35 and

381

36), respectively.

382

Reaction Pathway III: Transformation of Intermediate (XII). Similar to (XI), intermediate

383

(XII) underwent the Bennett reaction of the tetroxide intermediate, forming the aldehyde product,

384

N-formylnornicotine (reaction 40).55 In addition, intermediate (XII) underwent the dehydrogenation

385

reaction, and subsequently, the imine intermediate hydrolyzed to N-formylnornicotine, as shown in

386

reaction 42. The formation of N-formylnornicotine was confirmed using the standard compound, as

387

shown in S9.16. It was dehydrogenated and further hydrolyzed to yield the ring opening product

388

(P192b) analog to nicotine/cotinine, as mentioned above. The demethylation product nornicotine, with

389

an observed m/z of 149.1073, appeared to be another major product of nicotine. This reaction

390

mechanism is referred to as the methylene shuttle65 and is an analog to the aforementioned

391

demethylation of N-methylmyosmine and cotinine. S9.8 demonstrate the standard compound result

392

and the MS2 spectra. The majority of the fragments were also found in the fragments from nicotine,

393

indicating the similar structure of nornicotine with respect to nicotine. The fragment with an m/z of

394

70.0651 further confirmed the demethylated pyrrolidine structure. The accumulation of nornicotine

395

was observed in the initial 12 h of irradiation, after the fast decomposition (Figure 3c). The

396

decomposition of nornicotine yielded P162b via the Bennett reaction mechanism.55 In the other

397

pathway, nornicotine underwent dehydrogenation, leading to myosmine and P146b. Myosmine was

398

more stable than P146b under irradiation, as shown in the results in Figure 3b and c. The subsequent

399

transformation of P146b resulted in the formation of HPB via an N-oxide and oxazirane intermediate,

400

an analog to the peroxidation of myosmine.66 Further reactions of HPB have been discussed in

401

pathway I, generating 4-oxo-4-(3-pyridyl)butanal, P181, keto acid and P135. This pathway was

402

confirmed by the nicotine-D3 experiments with the observed three deuterium products.

403

(Insert Scheme 3)

18


Page 18 of 39

Page 19 of 39


404

Influence of O2 on the Phototransformation of Nicotine. As discussed above, O2 plays a dual

405

role in the photochemical transformation of nicotine. Under the anaerobic condition (DO < 0.05 mg

406

L-1), the photodegradation rate of nicotine was about two-fold lower than the one under the air

407

saturated condition (DO = 8.5 mg L-1). The TPs at anaerobic condition and low DO condition (DO =

408

3.5 mg L-1) were analyzed using the above protocol and cross compared with that under air saturated

409

condition. The time trends of TPs under these two conditions were demonstrated in Figure S11 and

410

S12. Two unique TPs were identified at the anaerobic condition as demonstrated in S9.28 and S9.29

411

of SI. One of the unique TPs was suggested to be the dimer of nicotine with the m/z value of

412

323.2230, P322. The dimerization of carbon radical was shown in Scheme 1 (D). P320 with m/z

413

321.2074 was the other unique product, knowing as the loss of two hydrogen from P322. Moreover,

414

most of TPs, which have been observed at air saturated condition, could not be detected, excluding

415

P160 or N-methylmyosmine, pseudooxynicotine and nicotinic acid. They could be introduced via

416

trace amount of oxygen. Otherwise, the detectable TPs at DO concentration 3.5 mg L-1 were same as

417

the TPs at DO concentration 8.5 mg L-1, whereas the dimers were not detected, as shown in Figure

418

S12. These results further verified that O2 participate the photodegradation of nicotine and the

419

formation of TPs in Scheme 3.

420

Kinetics of TPs of Nicotine. Seven commercially available products, including

421

pseudooxynicotine, nornicotine, cotinine, 3’-hydroxycotinine, nicotinic acid, myosmine and

422

4-oxo-4-(3-pyridyl)butanal, were selected to quantify the photochemical transformation of nicotine.

423

Although the standard compound of N-formylnornicotine is available, the yield of this TP was quite

424

low (< 0.08%). Thus, the kinetic study of N-formylnornicotine is excluded. First-order models (a)

425

and (b) were applied to evaluate the formation rates and transformation rates of the seven products

426

by using MATLAB R2015b. The model has been successfully applied to investigate the TPs of

427

carbamazepine.70

19



[NIC]t = [NIC]0e-kNIC t

428

[TP]t =

429

k1 [NIC]0 k2 - kNIC

Page 20 of 39

(a)

ቀe-kNICt - e-k2t ቁ + [TP]0 e-k2t

(b)

430

where [NIC]0 is the initial concentration of nicotine, [NIC]t is the concentration of nicotine at time t,

431

and kNIC is the pseudo-first-order reaction rate constant of nicotine. [TP]t is the TP concentration at

432

time t, [TP]0 is the initial concentration of TP ([TP]0 = 0 in this study), k1 is the pseudo first-order

433

formation rate of the TPs from nicotine, and k2 is the pseudo first-order transformation rate of the

434

TPs under irradiation.

435

Figure 4a exhibits the formation of the selected TPs in addition to the degradation of nicotine

436

under irradiation. Pseudooxynicotine, nornicotine and 4-oxo-(3-pyridyl)butanal exhibited an increase

437

followed by a rapidly decreasing trend during the irradiation process. Otherwise, cotinine,

438

3’-hydroxycotinine, nicotinic acid and myosmine were all observed to be stable TPs from nicotine

439

that increased with increasing time. Table 2 lists the modeled formation rates and transformation

440

rates of the selected products. Pseudooxynicotine was demonstrated to be the primary product with

441

the fastest formation rate among the seven quantified products. Moreover, the model indicates that

442

pseudooxynicotine would be more reactive than nicotine in the effluent (k2 = 1.9 × kNIC), despite its

443

high concentration after 4 h of irradiation. Nornicotine was hypothesized to be an important TP in

444

pathway III, and exhibited a transformation rate of 0.03 h-1. 4-Oxo-4-(3-pyridyl)butanal was

445

demonstrated to be another unstable product among the quantified products, of which transformation

446

rate was modeled to be 0.12 h-1. Among the four stable TPs, nicotinic acid exhibited the highest

447

formation rates (0.007 h-1) and yields (13%) after 72 h of irradiation. The transformation ratio of the

448

products from nicotine was calculated using equation (c).

449

Transformation ratio ሺ%ሻ =

[TP]t ି [TP]0

[NIC]0 ି [NIC]t

×100

(c)

450

Figure 4b shows the transformation ratios of the seven selected products, meaning the

451

percentage of TPs formed from the degraded nicotine. The transformation ratio indicated the mass 20


Page 21 of 39


452

balance of nicotine and its TPs during irradiation. The total transformation ratio of selected TPs at 10

453

min irradiation time was 14%, indicating other important primary TPs. N-methylmyosmine and P160

454

would be responsible for the initial transformed nicotine referring to the proposed pathway (Scheme

455

3) and the semiquantified result (Figure 2). After 24 h of irradiation, the total transformation ratio

456

tended to be stable at ~27%, suggesting the importance of other ultimate TPs (such as HPB,

457

N-methylnicotinamide).

458

Pseudooxynicotine was demonstrated to be the primary product of nicotine at irradiation time of

459

1-2 h. In total, 64% degraded nicotine transformed to pseudooxynicotine after 2 h of irradiation. As

460

pseudooxynicotine was hypothesized to be a primary TP in pathway I, this result indicated that this

461

pathway was the primary phototransformation pathway of nicotine. Nornicotine, the primary TPs in

462

pathway III, exhibited a maximal transformation ratio of 8% at 6 h of irradiation. Cotinine and

463

3’-hydroxycotinine, as two major urinary metabolites of nicotine, have been widely detected in

464

wastewater, and they have been used as chemical markers for domestic wastewater.11,

465

products continuously accumulated during the solar irradiation of nicotine, as shown in Figure 4b.

466

The transformation ratios in the time frame of 72 h of irradiation were calculated to be 3% for

467

cotinine and 5% for 3’-hydroxycotinine. Myosmine and 4-oxo-4-(3-pyridyl)butanal are also two

468

important products of nicotine, with maximum transformation ratios of 6 % and 4%, respectively.

469

Nicotinic acid was observed to be one of the predominant stable ultimate products of nicotine, with a

470

transformation ratio of 13% after 72 h of irradiation.

471

Environmental Significance

71

Both

472

In this study, the photochemical transformation pathways of nicotine were investigated. The

473

combined method of statistical analysis-assisted HRMS and isotope-labeling experiments has been

474

employed to extract and identify the TPs of nicotine in the complicated matrix of wastewater

475

effluents. 30 distinct products of nicotine were identified, including four groups of non-separated 21



476

isomeric products. 3OM* is expected to be the main reactive species involved in the degradation of

477

nicotine under solar irradiation. The reactive site is significantly different in the photodegradation

478

and biodegradation process of nicotine. Pyrrolidine ring is the only photo reactive site of nicotine,

479

while previous studies suggested the pyridine moiety is bioactive.72 Pathway I is the main expected

480

phototransformation processes, and N-methylmyosmine is the major initial TP of nicotine,

481

considering its further TP (pseudooxynicotine) exhibited high transformation ratio (64%). 26% of the

482

nicotine ultimately phototransformed to cotinine, 3’-hydroxycotinine, myosmine and nicotinic acid

483

after 24 h of irradiation. Nicotinic acid was the primary ultimate product in the high transformation

484

ratio (13%). These compounds would contribute to the environmental risk posed by nicotine, for

485

example, cotinine is a potential teratogen14 and myosmine can cause DNA damage.73 Whereas

486

negative effects of nicotine on aquatic organisms have received great attentions from environmental

487

scientists,14, 15 much less concerns are given for the TPs investigated here. Possible ecotoxicological

488

risks associated with these compounds, should be examined in the future.

489

Acknowledgements

490

We acknowledge partial support from the Natural Science Foundation of China (21422702,

491

21377030, 21677039 and 21607026), and the China Postdoctoral Science Foundation

492

(2016M590321). W. S. also acknowledges support from the Professors of Special Appointment

493

(Eastern Scholar) at the Shanghai Institution of Higher Learning. We are grateful for the reviewers’

494

valuable insight and suggestions.

495

Supporting Information

496

Additional information as noted in the text. Figure S1 to S12, Table S1 to S5, an excel file

497

showing the extracted MS features by Mass Profinder, and additional texts describing the wastewater

498

effluents information (Text S1), the quantification method of nicotine and the transformation

499

products (Text S2), the determination of the steady state concentration of ROS (HO•, 1O2 and CO3•-) 22


Page 22 of 39

Page 23 of 39


500

(Text S3, S5, and S8), the estimation of the contribution of ROS to the degradation of nicotine (Text

501

S4), the bimolecular reaction rate constants of ROS with nicotine (1O2 and CO3•-) (Text S6 and S7),

502

structure elucidation based on the UHPLC-qTOF-MS/MS data (Text S9) and the concentration

503

dependence experiment (Text S10) are provided.

23



504

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664 665 666 667 668 669 670 671 672 673 674 675 676 677 678 679 680 681 682 683 684 685 686 687 688 689 690 691 692 693 694 695 696 697 698 699 700 701 702 703

(59) Dash, A. C.; Dash, B.; Mahapatra, P. K.; Patra, M. Hydrolysis of imines. Part 2. Kinetics and mechanism of hydrolysis of N-salicylidene-2-aminopyridine in the presence and absence of copper(II) ion. A study of the catalytic effect of some mixed-ligand complexes of copper(II). J. Chem. Soc. Dalton Trans. 1983, 1503-1509. (60) Ding, L.; Chen, X.; Fang, W.-H. Ultrafast asynchronous concerted excited-state intramolecular proton transfer and photodecarboxylation of ο-acetylphenylacetic acid explored by combined CASPT2 and CASSCF studies. Org. Lett. 2009, 11 (7), 1495-1498. (61) Chiang, Y.; Kresge, A. J.; Popik, V. V.; Schepp, N. P. The mandelic acid keto−enol system in aqueous solution. generation of the enol by hydration of phenylhydroxyketene and phenylcarboxycarbene. J. Am. Chem. Soc. 1997, 119 (42), 10203-10212. (62) Frankenburg, W. G.; Gottscho, A. M. The chemistry of tobacco fermentation. I: Conversion of the alkaloids. B. The Formation of oxynicotine. J. Am. Chem. Soc. 1955, 77 (21), 5728-5730. (63) Le Lacheur, R. M.; Glaze, W. H. Reactions of ozone and hydroxyl radicals with serine. Environ. Sci. Technol. 1996, 30 (4), 1072-1080. (64) Craig, J. C.; Mary, N. Y.; Goldman, N. L.; Wolf, L. Tertiary amine oxide rearrangements. III. the mechanism of the demethylation of nicotine. J. Am. Chem. Soc. 1964, 86 (18), 3866-3869. (65) Gan, H.; Zhao, X.; Whitten, D. G. Amine photoredox reactions: a photoinduced methylene shuttle initiated via two-electron oxidation of a tertiary amine by anthraquinone. J. Am. Chem. Soc. 1991, 113 (24), 9409-9411. (66) Zwickenpflug, W.; Tyroller, S. Reaction of the tobacco alkaloid myosmine with hydrogen peroxide. Chem. Res. Toxicol. 2006, 19 (1), 150-155. (67) Destaillats, H.; Singer, B. C.; Lee, S. K.; Gundel, L. A. Effect of ozone on nicotine desorption from model surfaces: evidence for heterogeneous chemistry. Environ. Sci. Technol. 2006, 40 (6), 1799-1805. (68) Infante, G. A.; Jirathana, P.; Fendler, E. J.; Fendler, J. H. Radiolysis of pyrimidines in aqueous solutions. Part 2.-Product formation in the interaction of eaq-, ·H, ·OH and Cl2·- with uracil. J. Chem. Soc., Faraday Trans 1 1974, 70, 1162-1170. (69) Smith, K. C.; Hays, J. E. The response of uracil-2-14C to X-irradiation under nitrogen and oxygen and to treatment with ascorbic acid. Radiat. Res. 1968, 33 (1), 129-141. (70) De Laurentiis, E.; Chiron, S.; Kouras-Hadef, S.; Richard, C.; Minella, M.; Maurino, V.; Minero, C.; Vione, D. Photochemical fate of carbamazepine in surface freshwaters: Laboratory measures and modeling. Environ. Sci. Technol. 2012, 46 (15), 8164-8173. (71) Benotti, M. J.; Brownawell, B. J. Distributions of pharmaceuticals in an urban estuary during both dry- and wet-weather conditions. Environ. Sci. Technol. 2007, 41 (16), 5795-5802. (72) Gong, X.; Ma, G.; Duan, Y.; Zhu, D.; Chen, Y.; Zhang, K.-Q.; Yang, J. Biodegradation and metabolic pathway of nicotine in Rhodococcus sp. Y22. World J. Microbiol. Biotechnol. 2016, 32 (11), 188. (73) Kleinsasser, N. H.; Wallner, B. C.; Harréus, U. A.; Zwickenpflug, W.; Richter, E. Genotoxic effects of myosmine in human lymphocytes and upper aerodigestive tract epithelial cells. Toxicology 2003, 192 (2-3), 171-177. 28


Page 28 of 39

Page 29 of 39


704

705 706 707 708 709 710 711 712 713 714

Figure 1. OPLS-DA score plots of the profiling of the wastewater effluent (without spiked nicotine, blue triangles) and the nicotine-spiked effluent (red circles) under various doses of irradiation. The analyses were performed on the 932 and 1027 features extracted using Mass Profinder. (a) Positive mode (R2 (cum) = 0.98, Q2 (cum) = 0.98), (b) negative mode (R2 (cum) = 0.79, Q2 (cum) = 0.73), (c) hierarchical clustering of compound abundance in the effluent and nicotine-spiked effluent in the positive mode based on the 55 VIP > 1.0 compounds derived from OPLS-DA, in which the Pearson correlation coefficient squared was used as the metric. [nicotine]0 = 1.3 mg L-1, [DOC] = 5.0 mgC L-1, [DO] = 8.5 mg L-1.

29


ln ([nicotine]/[nicotine]0)


Page 30 of 39

0

-1

(a)

-2

DI-H2O

-3

Effluent Effluent + D2O

-4

Effluent + Isopropanol

0

6

12

18

24

time (h) 715

kNIC (h-1)

0.6

(b)

0.4

0.2

0.0 0

2

4

6

8

Dissolved oxgen concentration (mg/L) 716 717 718 719 720 721

Figure 2. Photodegradation of nicotine in ultrapure water and the wastewater effluent. (a) Photodegradation of nicotine in wastewater effluents under various condition; (b) pseudo first order reaction rate of nicotine under various dissolved oxygen concentration condition. [nicotine]0 = 1.3 mg L-1, [DOC] = 5.0 mgC L-1.

30



PAproduct/PAinitial NIC.

Page 31 of 39

N-methylmyosmine Pseudooxynicotine Nicotinic acid N-methylnicotinamide Nicotinamide P178a P174

(a) 0.6

0.4 0.2

P146a

P190

P162a

P176

0.0 0

20

40

60

80

Time (h) PAproduct/PAinitial NIC.

722

0.6 (b)

P160

Cotinine

P146b/c

P162b

3'-Hydroxycotinine Norcotinine P178b P192a P208

0.4

0.2

0.0 0

20

40

60

80

Time (h) PAproduct/PAinitial NIC.

723

Nornicotine Myosmine 4-Oxo-4-(3-pyridyl)butanal N-formylnornicotine HPB Keto acid P135 P181 P192b

(c) 0.5 0.4 0.3 0.2 0.1 0.0 0

724 725 726 727 728

20

40

Time (h)

60

80

Figure 3. Reaction profiles of TPs as a function of irradiation time. [nicotine]0 = 1.3 mg L-1, [DOC] = 5.0 mgC L-1, [DO] = 8.5 mg L-1. (a) Products in pathway I; (b) products in pathway II; (c) products in pathway (III). (Note: HPB, 4-oxo-4-(3-pyridyl)butanal, P181, keto acid and P135 were included in (c), while they are products in both pathway I and III.) 31



Nicotine (µ µM)

6 4

1.5 1.0 0.5

0

0.0 80

20

40 Time (h)

729

Transformation ratio (%)

2.0

2

0

100

60

Pseudooxynicotine Nornicotine Nicotinic acid 3'-Hydroxycotinine Cotinine

(b)

80

Myosmine

60

4-Oxo-(3-pyridyl)butanal

40 20 0 0.2 0.5 1

730 731 732 733

(a)

Products (µ µM)

Nicotine Pseudooxynicotine Nornicotine Nicotinic acid 3'-Hydroxycotinine Cotinine Myosmine 4-Oxo-(3-pyridyl)butanal

8

Page 32 of 39

2

4

6

8

12 15 24 36 48 60 72

Time (h) Figure 4. Quantitative results regarding 7 selected TPs of nicotine. (a) Time profiles of nicotine and the 7 TPs under irradiation. (b) Transformation ratios of the 7 TPs at different irradiation times. [nicotine]0 = 1.3 mg L-1, [DOC] = 5.0 mgC L-1 , [DO] = 8.5 mg L-1.

32


Page 33 of 39

734 735 736


Scheme 1. Proposed primary oxidation mechanism for C-centered radicals. (A) Proposed formation mechanism of peroxyl radical; (B) proposed combination of two peroxyl radicals with α-hydrogen; (C) proposed combination of one peroxyl radical with α-hydrogen and one tertiary peroxyl radical; (D) proposed dimerization of C-centered radicals under anaerobic condition.

737 738

33



739

Scheme 2. Proposed first step of 3OM* oxidation mechanism for nicotine under air saturated condition.

740 741

34


Page 34 of 39

Page 35 of 39


35

742



743

36


Page 36 of 39

Page 37 of 39


744

745 746 747 748

Scheme 3. Degradation products and proposed reaction pathway for the photodegradation of nicotine in wastewater. Products in red have available corresponding standard compounds. The intermediates are shown in the square brackets.

37



749 750

Table 1. General information regarding the identified products of the photodegradation of nicotine. C6H6N2O

Retention time (min) 4.16

m/z (theor) 123.0913

m/z (measd) 123.0912

Mass Error (ppm) -0.8

Nicotinic acid

C6H5NO2

2.23

124.0394

124.0392

-1.6

1

3

P135

C8H9NO

5.33

136.0757

136.0755

-1.5

2b

4

N-methylnicotinamide

C7H8N2O

4.67

137.0710

137.0706

-2.9

2b

5

Myosmine

C9H10N2

6.41

147.0917

147.0916

-0.7

1

6

P146a

C9H10N2

6.41

147.0917

147.0916

-0.7

2b

7

P146b

C9H10N2

6.07

147.0917

147.0915

-1.4

3

8

P146c

C9H10N2

6.07

147.0917

147.0915

-1.4

3

9

Nornicotine

C9H12N2

4.45

149.1073

149.1070

-2.0

1

10

P160

C10H12N2

4.80

161.1073

161.1074

0.6

2b

11

N-methylmyosmine

C10H12N2

4.80

161.1073

161.1074

0.6

2b

12

P162a

C9H10N2O

5.03

163.0866

163.0865

-0.6

3

13

P162b

C9H10N2O

4.71

163.0866

163.0863

-1.8

3

14

Norcotinine

C9H10N2O

5.03

163.0866

163.0865

-0.6

3

C9H9NO2

5.68

164.0706

164.0707

0.6

1

No.

Compound Name

Formula

1

Nicotinamide

2

15

751

Page 38 of 39

#

4-Oxo-4-(3-pyridyl)bu tanal

Levels 3

16

HPB

C9H11N2O

5.32

166.0863

166.0860

-1.8

2b

17

P174

C10H10N2O

5.35

175.0866

175.0861

-2.9

3

18

Cotinine

C10H12N2O

5.49

177.1023

177.1020

-1.7

1

19

P176

C10H12N2O

4.13

177.1023

177.1020

-1.7

3

20

N-formylnornicotine

C10H12N2O

5.67

177.1023

177.1019

-2.3

1

21

Pseudooxynicotine

C10H14N2O

4.91

179.1179

179.1177

-1.1

1

22

P178a

C10H14N2O

4.34

179.1179

179.1180

0.6

3

23

P178b

C10H14N2O

5.34

179.1179

179.1179

0

3

24

Keto acid

C9H9NO3

4.45

180.0655

180.0652

-1.7

2b

25

P181

C9H11NO3

3.31

182.0812

182.0806

-3.3

2b

26

P190

C10H10N2O2

5.39

191.0815

191.0814

-0.5

3

27

3’-Hydroxycotinine

C10H12N2O2

4.78

193.0972

193.0970

-1.0

1

28

P192a

C10H12N2O2

5.06

193.0972

193.0970

-1.0

3

29

P192b

C10H12N2O2

4.60

193.0972

193.0969

-1.6

3

30

P208

C10H12N2O3

3.93

209.0921

209.0917

-1.9

2b

31

#

P322

C20H26N4

7.63

323.2230

323.2234

1.2

3

32

#

P320

C20H24N4

7.60

321.2074

321.2070

1.3

3

: Products detected only under anaerobic condition (DO concentration < 0.05 mg L-1) 38


Page 39 of 39

752

753 754


Table 2. Kinetic modeling results regarding the 7 selected products. Product k1 (× 10-3 h-1) k2 (× 10-3 h-1) Pseudooxynicotine

150 ± 20

310 ± 40

Nornicotine

10 ± 1

30 ± 3

Cotinine

3±1

-

3’-Hydroxycotinine

7±1

-

Nicotinic acid

7±1

-

Myosmine

5±1

-

4-Oxo-4-(3-pyridyl)butanal

7±2

k1: The rate constant of product formation from nicotine. k2: The rate constant of product transformation.

39


120 ± 10

Photochemical Transformation of Nicotine in Wastewater Effluent

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