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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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Photochemical Transformation of Nicotine in Wastewater Effluent
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3 Lushi Lian1, Shuwen Yan1, Bo Yao1, Shen-An Chan2, and Weihua Song1,*
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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
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Abstract
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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.
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Introduction
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Cigarette smoking has well-documented negative impacts on human health. It is responsible for
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90% of cases of lung cancer, 75% of cases of chronic bronchitis and emphysema, and 25% of cases
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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
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nicotine in its Toxic Release Inventory since 1995,5 and the lethal oral dose for humans is
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approximately 40-60 mg.6 The annual global consumption of nicotine can be estimated to be ∼ 5 ×
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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,
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nicotine can be biodegraded into more persistent and toxic TPs, such as cotinine and
56
4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone,13,
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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
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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
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which should be accounted for when
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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
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water transformation pathway. However, there is currently no mechanistic understanding of the
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phototransformation of nicotine, especially in a complex water matrix. Obtaining such an
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understanding is challenging for several reasons: 1) photodegradation is a complicated process that
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involves a variety of ROS and 3OM*; 2) TPs always exhibit high variety; and 3) organic matter (OM)
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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
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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.
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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
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(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
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Multivariate statistical strategies, such as principal component analysis (PCA) and orthogonal
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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
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method has been applied to investigate the biotransformation of organic compounds in a complex
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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
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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
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and elucidate the degradation pathway of nicotine in a wastewater matrix by using
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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
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method for the unequivocal identification of the TPs of emerging contaminants in a complex effluent
99
matrix, particularly for 3OM*-induced photochemical transformation.
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Material and Methods Materials.
Nicotine,
nicotinic
acid,
myosmine,
cotinine,
3’-hydroxycotinine
and
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N-formylnornicotine were purchased from Alfa Aesar. Rac-nicotine-2’,3’,3’-D3, rac-nicotine-13CD3,
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rac-nicotine-D4, pseudooxynicotine dihydrochloride, nornicotine and 4-oxo-4-(3-pyridyl)-butanal
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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.
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LC/MS-grade water was purchased from J&K Chemical. Stock solution of nicotine was prepared in
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ultrapure water (DI-H2O) at 1000 mg L-1 and stored at 4 °C. Stock solutions of other compounds
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were prepared in methanol at 100 mg L-1, and stored at -20 °C. All of the compounds were stable
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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
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the wastewater effluents could be found in Text S1 and Table S1 of Supporting information (SI).
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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
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University in Shanghai, China for comparison (Figure S1, SI). All samples were placed in specially
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made quartz containers, and aliquots were withdrawn and analyzed via UHPLC-QTOF-MS after
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different doses of irradiation. TOC of the wastewater effluents using in the experiments were 5.0
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mgC L-1. The initial concentration of nicotine in the effluent was 8.0 µM (1.3 mg L-1). The initial
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concentration of the isotopic nicotine and standard compounds in the effluent used to confirm the
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products was 5.0 mg L-1. For the O2-dependence experiments, argon (Ar) was purged slowly into the
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reaction vessel (the flow rate is around 15 mL min-1) with varied time scale to achieve different
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dissolved oxygen (DO) concentration. In order to eliminate the influence of sampling process, Ar
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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).
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The control experiment was performed by photo-irradiating wastewater effluent without spiked
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nicotine. All of the experiments were repeated in three times, and each sample was analyzed by
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HRMS twice.
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UHPLC-QTOF-MS Analysis. An Agilent 1290 UHPLC system was employed for separation.
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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
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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.
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A high-resolution Agilent 6540 QTOF-MS system with an Agilent Jet Stream (AJS)
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electrospray ionization (ESI) source was used in both positive and negative modes. At the AJS ESI
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interface, the sheath gas temperature was set to 400 °C, the sheath gas flow rate was set to 12 L min-1,
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the drying gas temperature was set to 300 °C, the drying gas flow rate was set to 10 L min-1, and the
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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.
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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.
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Statistical Analysis. The UHPLC-QTOF-MS data were first analyzed using Agilent Mass
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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)
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were exported to SIMCA-P 13.0 (Umetrics) for OPLS-DA to reveal the differences between the
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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
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used as the clustering algorithm.
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Results and Discussion
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Identification of the Potential TPs of Nicotine in Wastewater Effluent. UHPLC-QTOF-MS
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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
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Profinder 8.0), 932 features in the positive mode and 1027 features in the negative mode were
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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
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nicotine-spiked effluents were examined via supervised multivariate OPLS-DA at various simulated
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solar irradiation times, as shown in Figure 1a and b. The OPLS-DA scatter plots demonstrated
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clearly time trends and significant separation of the effluent alone from the nicotine-spiked samples
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was achieved for both positive and negative modes. It indicated that the TPs of nicotine in the
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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.
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(Insert Figure 1)
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The 55 discriminatory features consisted of the nicotine TPs and the nicotine-impacted TPs
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generated from OM. To further extract the photo-induced TPs of nicotine, hierarchical clustering was
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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
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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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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
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or
standard
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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,
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the
formation
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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
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P178a
and
pseudooxynicotine.
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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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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
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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)
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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
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[NIC]t = [NIC]0e-kNIC t
428
[TP]t =
429
k1 [NIC]0 k2 - kNIC
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(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
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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
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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
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(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.
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504
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Brenton, J. D.; Ylstra, B.; Caldas, C. High-resolution aCGH and expression profiling identifies a novel genomic subtype of ER negative breast cancer. Genome Biol. 2007, 8 (10), R215. (43) Rosario-Ortiz, F. L.; Canonica, S. Probe Compounds to Assess the Photochemical Activity of Dissolved Organic Matter. Environ. Sci. Technol. 2016, 50 (23), 12532-12547. (44) Canonica, S.; Kohn, T.; Mac, M.; Real, F. J.; Wirz, J.; Von Gunten, U. Photosensitizer method to determine rate constants for the reaction of carbonate radical with organic compounds. Environ. Sci. Technol. 2005, 39 (23), 9182-9188. (45) Parker, K. M.; Mitch, W. A. Halogen radicals contribute to photooxidation in coastal and estuarine waters. Proc. Natl. Acad. Sci. U. S. A. 2016, 113 (21), 5868-5873. (46) Mopper, K.; Znou, X. Hydroxyl radical photoproduction in the sea and its potential impact on marine processes. Science 1990, 250 (4981), 661-664. (47) Brigante, M.; Minella, M.; Mailhot, G.; Maurino, V.; Minero, C.; Vione, D. Formation and reactivity of the dichloride radical (Cl2•-) in surface waters: A modelling approach. Chemosphere 2014, 95, 464-469. (48) Golanoski, K. S.; Fang, S.; Del Vecchio, R.; Blough, N. V. Investigating the mechanism of phenol photooxidation by humic substances. Environ. Sci. Technol. 2012, 46 (7), 3912-3920. (49) Kosno, K.; Janik, I.; Celuch, M.; Mirkowski, J.; Kisała, J.; Pogocki, D. The role of pH in the mechanism of HO· radical induced oxidation of nicotine. Isr. J. Chem. 2014, 54 (3), 302-315. (50) Cohen, S. G.; Davis, G. A.; Clark, W. D. K. Photoreduction of π-π* triplets by amines, 2-naphthaldehyde, and 2-acetonaphthone. J. Am. Chem. Soc. 1972, 94 (3), 869-874. (51) Chen, Y.; Hu, C.; Hu, X.; Qu, J. Indirect photodegradation of amine drugs in aqueous solution under simulated sunlight. Environ. Sci. Technol. 2009, 43 (8), 2760-2765. (52) Canonica, S.; Jans, U.; Stemmler, K.; Hoigné, J. Transformation kinetics of phenols in water: Photosensitization by dissolved natural organic material and aromatic ketones. Environ. Sci. Technol. 1995, 29 (7), 1822-1831. (53) Schuchmann, M. N.; Von Sonntag, C. Hydroxyl radical-induced oxidation of diethyl ether in oxygenated aqueous solution. A product and pulse radiolysis study. J. Phys. Chem. 1982, 86 (11), 1995-2000. (54) Russell, G. A. Deuterium-isotope effects in the autoxidation of aralkyl hydrocarbons: Mechanism of the interaction of peroxy radicals. J. Am. Chem. Soc. 1957, 79 (14), 3871-3877. (55) Bothe, E.; Schulte-Frohlinde, D. The bimolecular decay of the α-hydroxymethylperoxyl radicals in aqueous solution. Z. Naturforsch., B: Chem. Sci. 1978, 33 (7), 786-788. (56) Schymanski, E. L.; Jeon, J.; Gulde, R.; Fenner, K.; Ruff, M.; Singer, H. P.; Hollender, J. Identifying Small Molecules via High Resolution Mass Spectrometry: Communicating Confidence. Environ. Sci. Technol. 2014, 48 (4), 2097-2098. (57) Bothe, E.; Behrens, G.; Schulte-Frohlinde, D. Mechanism of the first order decay of 2-hydroxy-propyl-2-peroxyl radicals and of O2·- formation in aqueous solution. Z. Naturforsch., B: Chem. Sci. 1977, 32 (8), 886-889. (58) Chen, B.; Wang, L.; Gao, S. Recent advances in aerobic oxidation of alcohols and amines to imines. ACS Catal. 2015, 5 (10), 5851-5876. 27
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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
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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.
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ln ([nicotine]/[nicotine]0)
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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.
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PAproduct/PAinitial NIC.
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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
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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
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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.
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734 735 736
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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
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739
Scheme 2. Proposed first step of 3OM* oxidation mechanism for nicotine under air saturated condition.
740 741
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742
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743
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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.
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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
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#
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
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752
753 754
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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.
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120 ± 10