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A New Group of Disinfection Byproducts in Drinking Water: Trihalo-hydroxy-cyclopentene-diones Yang Pan, Wenbin Li, Aimin Li, Qing Zhou, Peng Shi, and Ying Wang Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.6b00798 • Publication Date (Web): 10 Jun 2016 Downloaded from http://pubs.acs.org on June 23, 2016
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State Key Laboratory of Pollution Control and Resource Reuse, School of the Environment, Nanjing University, Nanjing 210023, China S
Supporting Information
Abstract: We report the detection, synthesis, A new group of drinking water DBPs: trihalo-hydroxy-cyclopentene-diones SRNOM + Br + Cl / NH Cl trihalo-HCDs preparative isolation, structure characterization/identification, and formation of a new group of drinking water 100 180.9191.1 213.0 222.9 253.3258.9260.8 281.1 302.7306.8 339.1 347.0 353.2 379.2 0 m/z disinfection byproducts (DBPs) ― 180 200 220 240 260 280 300 320 340 360 380 precursor-to-Br molar ratio of 1:8 + Br tribromo-HCD 8 h, 20 ºC trihalo-hydroxy-cyclopentene-diones FTIR characterization Preparative isolation (trihalo-HCDs). With ultra performance liquid chromatography (UPLC)/electrospray ionization-triple quadruple mass spectrometry analyses (full scans, multiple reaction monitoring, and product ion scans) and high resolution mass spectrometry analyses (full scans), the new group of DBPs was identified with formulae and proposed with structures. However, due to lack of commercially available standard compounds, structure identification of this new group of DBPs was challenging. 2,4,6-Trihydroxybenzaldehyde was found to be a good precursor for the synthesis of the tribromo species (m/z 345/347/349/351) in the new group of DBPs by reacting with bromine at a 2,4,6-trihydroxybenzaldehyde-to-bromine molar ratio of 1:8. With UPLC/Photodiode Array analysis (simultaneous 2- and 3-dimensional operations), the new DBP was determined to have a maximum UV absorption at the wavelength of 280 nm. Through isolation with high performance liquid chromatography/UV-triggered collections followed by lyophilization, the pure standard of the new DBP was obtained. Characterized with Fourier Transformation Infrared Spectroscopy, the pure standard of the new DBP was finally identified to be tribromo-HCD, and thus the new group of DBPs was identified to be trihalo-HCDs. Based on the disclosed structure, formation pathways of tribromo-HCD through reactions of three different precursors and bromine were proposed and partially verified. Moreover, increasing the bromide level in source water shifted the formation of trihalo-HCDs from being more chlorinated to being more brominated; with the increasing contact time from 1 h to 5 d, the formation of trihalo-HCDs kept increasing in chloramination, whereas kept decreasing in chlorination; with the increasing pH from 6.0 to 8.5, the formation of trihalo-HCDs was decreased by ~ 80%. Notably, the concentrations of tribromo-HCD in eight Chinese tap water samples were from below detection limit to 0.53 µg/L. –
16 17 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38
O-
Br
O
O
O
O
Br Br
Br Br
CHO OH
2
2
OH
100
16.63
2.5e-1
5.22 8.33
0.0
18
O-
Cl
O
Br Cl
Cl Cl
HO
15
2
O-
O
O
O
2
Cl
24.72
29.12
20.00
40.00
Time
50
0 4000
693.4 606.3
14
O-
%
13
Water:
Yang Pan,* Wenbin Li, Aimin Li,* Qing Zhou, Peng Shi, and Ying Wang
Cl
12
Drinking
1752.3 1710.0
11
in
1604.0
10
Byproducts
3443.5
9
A New Group of Disinfection Trihalo-hydroxy-cyclopentene-diones
% Transmittance
1 2 3 4 5 6 7 8
Environmental Science & Technology
3000 2000 1000 Wavenumbers (1/cm)
1
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INTRODUCTION
40
Drinking water disinfection is regarded as a significant public health advance in the 20th
41
century,1 which has prevented numerous people from being infected by cholera, typhoid,
42
dysentery, etc.2,3 However, disinfection processes can unintentionally generate disinfection
43
byproducts (DBPs) through reactions of disinfectants (e.g., chlorine, chloramines, chlorine
44
dioxide, ozone, etc.), natural organic matter, anthropogenic contaminants, and halides.4
45
In the 40 years of research, around 600−700 halogenated DBPs have been reported in
46
drinking water,5−8 however, only several of them are under routine monitoring and regulation.9,10
47
A few toxicological and epidemiological studies have pointed out that it is unlikely for the
48
currently regulated DBPs to fully account for the toxicity effects and increased bladder cancer
49
risks resulted from consumption of chlorinated drinking water.4,11,12 Actually, a majority of total
50
organic halogen (TOX) produced in drinking water chlorination has not been well characterized,
51
and the percentages of unknown TOX produced in drinking water treated with other disinfectants
52
were even higher.13,14 Since the unknown part of TOX might contain substantial amounts of toxic
53
compounds as suggested by epidemiological studies,11,15 further exploration of unknown drinking
54
water halogenated DBPs should be of paramount importance.
55
Recently, halogenated DBPs with cyclic structures (including halogenated pyrroles,
56
benzoquinones, hydroxybenzaldehydes, hydroxybenzoic acids, and phenols) have been attracting
57
increasing concerns due to their higher cytotoxicity, genotoxicity, growth inhibition, and
58
developmental toxicity than those with chain structures.16−22 More recently, a group of highly
59
abundant halogenated DBPs was detected in drinking water and chlorinated ballast water, which
60
was tentatively proposed to be trihalo-furoic acids or their trihalo-hydroxy-cyclopentene-diones
61
(trihalo-HCDs) analogues.23−26 This new group of DBPs appeared to have high reactivity as they 2
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readily decomposed to trihalomethanes and haloacetic acids with an excess of free chlorine.24
63
Thus, they deserved great concern and might be toxicological important.25 However, due to
64
multiple isomers of their molecular formulae and lack of commercially available standard
65
compounds, structure characterization/identification of the new group of DBPs has been impeded.
66
As a result, their formation during various disinfection processes remained unclear.
67
With improving sensitivity, resolution, and separation efficiency, ultra performance liquid
68
chromatography (UPLC) becomes increasingly accepted by chromatographers/researchers.
69
Coupled by electrospray ionization-triple quadruple mass spectrometry (ESI-tqMS), the
70
UPLC/ESI-tqMS has been applied in detection of new DBPs in drinking water.23,24,27,28 Further
71
aided with high resolution MS, accurate molecular formulae of the detected new DBPs could be
72
assigned,25,26,29,30 which could provide important information for structure proposing. Since
73
standard compounds of some new DBPs with proposed structures are not commercially available,
74
they are required to be synthesized and isolated to a sufficient amount in the lab to facilitate
75
structure characterization/identification. Thus, the Waters AutoPurificationTM System should be
76
an ideal choice for preparative isolation of these new DBPs through high performance liquid
77
chromatography (HPLC)/UV-triggered collections, which can provide selective UV-directed
78
fraction collection from multiple samples automatically and reliably.
79
In this study, we report a new group of drinking water halogenated DBPs with cyclic
80
structures, trihalo-HCDs, with focuses on the following aspects: (1) detection, formula
81
identification, and structure proposing; (2) synthesis, preparative isolation, and structure
82
characterization/identification; (3) formation under various disinfection processes.
83 84
MATERIALS AND METHODS 3
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Materials. Suwannee River natural organic matter (SRNOM, 2R101N) was provided by the
86
International
87
1,2,4-trihydroxybenzene (99%) were provided by Sigma-Aldrich. 3,4,5-Trihydroxybenzaldehyde
88
(98%)
89
2,4,6-Trihydroxybenzaldehyde (>98%) was purchased from TCI. A NaOCl solution (~ 3000
90
mg/L as Cl2) was prepared by diluting a concentrated NaOCl solution (4.00−4.99% available
91
chlorine, reagent grade, Sigma-Aldrich) and titrated using a standard procedure.31 NH2Cl was
92
freshly prepared before use via a reaction between NaOCl and NH4Cl at an ammonia-to-chlorine
93
molar ratio of 1.25. Organic solvents (methyl tert-butyl ether, acetonitrile, and methanol) at
94
HPLC grade and other chemicals at reagent grade were all provided by Sigma-Aldrich and
95
Merck.
and
Humic
bromine
Substances
(99.5%)
Society.
were
1,2,3-Trihydroxybenzene
purchased
from
J&K
(97%)
and
Chemical.
96
Preparation of Simulated Drinking Water Samples and Collection of Real Tap Water Samples.
97
A series of simulated source water samples were prepared by adding SRNOM (3 mg/L as C),
98
NaHCO3 (90 mg/L as CaCO3), and different levels (0, 0.1, 0.4, 1.0, 2.0, and 4.0 mg/L as Br−) of
99
KBr in ultrapure water. For preparation of simulated drinking water samples, the simulated
100
source water samples were dosed with NH2Cl or NaOCl (5 mg/L as Cl2) at different pH values
101
(6.0, 6.5, 7.5, and 8.5), and kept without headspace in darkness at ~ 20 ºC for various contact
102
times (1, 24, and 120 h). The residual disinfectants in the samples were quenched with a 5%
103
excess of required amounts of Na2SO3.
104
As described later, a new group of DBPs was detected and identified to be trihalo-HCDs in
105
the simulated drinking water samples, and thus factors (including bromide level in source water,
106
disinfectant type, contact time, and pH) that may affect their formation during disinfection were
107
examined with batch experiments. A baseline simulated drinking water sample was defined as: a
108
1 L portion of the simulated source water sample containing 0.4 mg/L KBr as Br− (pH 7.5) was 4
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chloraminated by dosing 5 mg/L NH2Cl as Cl2 for 120 h. In each batch experiment, one factor
110
was changed at a time and all other factors were kept the same as those of the baseline sample.
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Moreover, eight real tap water samples (coded as A–H) were collected from drinking water
112
treatment plants in eight different cities of East China in May 2016. Upon collection of these
113
samples onsite, pH was measured to be from 7.2 to 7.7; disinfectant residuals were determined to
114
be all in the form of free chlorine at concentrations from 0.4 to 1.1 mg/L, which were
115
immediately quenched by 105% of the required amounts of Na2SO3. All the samples were put in
116
an ice bath during transportation by car to the lab, and kept refrigerated (~ 4 ºC) till pretreatment.
117
Pretreatment of Simulated Drinking Water Samples and Real Tap Water Samples.
118
Pretreatment of the simulated drinking water samples and the real tap water samples followed
119
previously reported steps.24 Generally, each 1 L of the water sample was added with diluted
120
H2SO4 (70% v/v) drop by drop with vigorous stirring to pH 0.5 and added with Na2SO4 till
121
saturation. Then, 100 mL methyl tert-butyl ether was mixed with the sample in a 2 L separation
122
funnel for extraction of organics. About 15 min later, the upper layer (~ 75 mL) was moved to a
123
250 mL flask and evaporated at 27 ºC to 1 mL. The 1 mL concentrate was dissolved in 10 mL
124
acetonitrile, which was further concentrated to 0.5 mL. Finally, the 0.5 mL solution was dosed
125
with 0.5 mL ultrapure water and all insoluble substances inside were removed by a 0.45 µm
126
filter.
127
UPLC/ESI-tqMS Analysis. An Acquity UPLC system coupled to a Xevo ESI-tqMS
128
(UPLC/ESI-tqMS, Waters) was employed for analysis of the pretreated samples. The sample
129
injection volume in the UPLC was 5 µL. An Acquity UPLC HSS T3 column (1.8 µm particle
130
size, 2.1×100 mm, Waters) was used for chromatographic separation. A gradient eluent applied at
131
0.4 mL/min was composed of water/methanol, and its composition (v/v) was linearly changed
132
from 95/5 to 10/90 in the initial 8 min, brought back to 95/5 within 0.1 min, and kept stable at 5
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95/5 for another 2.9 min. The parameters of MS were as follows: capillary voltage, 2.8 kV;
134
desolvation temperature, 400 ºC; source temperature, 110 ºC; desolvation gas flow, 800 L/h;
135
cone gas flow, 50 L/h; cone voltage, 20 V; collision energy, 30 eV; collision gas (Ar) flow, 0.25
136
mL/min.
137
High Resolution MS Analysis. To assign exact molecular formulae of the new group of DBPs,
138
a high resolution MS analysis was conducted with a Hybrid Ion Trap-Orbitrap mass spectrometer
139
(LTQ Orbitrap XL, Thermo Fisher Scientific) through accurate m/z value measurement. The
140
instrument with a heated electrospray interface was set in negative ionization mode, and the
141
parameters were set as: capillary voltage, −10 V; spray voltage, 4 kV; tube lens voltage, −40 V;
142
capillary temperature, 275 ºC; auxiliary gas, 15 au; sheath gas, 45 au; sweeping gas, 0 au.
143
Synthesis of the New DBP with m/z 345/347/349/351. Due to lack of the commercially
144
available standard compound, synthesis of the new DBP with m/z 345/347/349/351 was
145
performed in the lab via the reaction of “precursor + bromine” for its preparative isolation and
146
structure characterization/identification. Four precursors were selected for reaction with bromine,
147
including 1,2,3-trihydroxybenzene, 1,2,4-trihydroxybenzene, 3,4,5-trihydroxybenzaldehyde, and
148
2,4,6-trihydroxybenzaldehyde. A series of solutions containing 40 mg/L of a precursor
149
compound were dosed with bromine at precursor-to-bromine molar ratios of 1:2, 1:4, 1:6, 1:8,
150
1:12, and 1:20, and kept reaction in darkness at ~ 20 ºC for 8 h. The bromine residuals were
151
quenched with a 5% excess of required Na2SO3. Then, pretreatment of the reaction solutions was
152
conducted using the same method as that of the simulated drinking water samples.
153
UPLC/PDA Analysis of the New DBP with m/z 345/347/349/351. UPLC/Photodiode Array
154
(PDA) analysis of the new DBP was carried out with the UPLC system coupled to a Waters
155
Acquity PDA detector. Simultaneous 2- and 3-dimensional operations were applied at a scan 6
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wavelength range of 220 to 350 nm, suggesting that all UV absorbance values from 220 to 350
157
nm of the new DBP separated by the UPLC at a specific retention time (RT) were recorded. In
158
this way, the wavelength at which the new DBP had the maximum UV absorption was obtained.
159
Preparative Isolation and Structure Characterization/Identification of the New DBP with m/z
160
345/347/349/351. HPLC/UV-triggered preparative isolation of the new DBP was carried out using
161
the Waters AutoPurificationTM System with a Waters 2489 UV/Visible detector. A Waters Atlantis
162
T3 OBD Prep column (5 µm particle size, 19×100 mm) was used for the preparative HPLC
163
separation. To maintain selectivity and separation resolution in the preparative separation as
164
those achieved in the analytical analysis, inlet method of the preparative HPLC was migrated
165
from the analytical UPLC according to the approach provided by Waters Corporation.32 The
166
sample volume for each HPLC injection was 614 µL. The gradient eluent composed of
167
water/methanol (v/v) was applied at 11.8 mL/min, and the composition was changed linearly
168
from 95/5 to10/90 in the first 33.3 min, followed by a change to 95/5 within 0.5 min, and this
169
composition was kept by another 12 min for column re-equilibration. Elution of the new DBP
170
from the preparative column was monitored by UV detection and fraction collection was
171
triggered based on the UV absorbance value at the specific wavelength. The collected fractions
172
of the new DBP from multiple injections were combined to a total volume of 1 L in a single glass
173
vessel, which was analyzed with the UPLC/ESI-tqMS full scan to verify its purity and
174
subsequently reduced to dryness using lyophilization at 5 Pa and –50 ºC for 48 h. The obtained
175
pure standard was characterized by Fourier Transformation Infrared Spectroscopy (FTIR) to
176
determine its main functional groups and structure framework. FTIR analysis of the new DBP
177
was performed by an FTIR spectrometer (Nicolet Nexus 870) with omni sampler in ATR mode,
178
with a scan range between 4000 and 400 cm–1 for 32 scans (resolution: 4 cm−1).
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RESULTS AND DISCUSSION
181
Detection, Formula Identification, and Structure Proposing of A New Group of Drinking
182
Water DBPs. Figure 1a shows the UPLC/ESI-tqMS full scan chromatogram of the baseline
183
simulated drinking water sample. The peaks detected from RT 0.5 to 1.0 min and from RT 7.8 to
184
8.5 min should correspond to three dihalo-acetic acids (i.e., dichloroacetic acid,
185
chlorobromoacetic
186
(2,6-dibromo-4-chlorophenol and 2,4,6-tribromophenol), respectively (Figure S1).28 Notably,
187
several dominant peaks were partially overlapped and detected from RT 2.7 to 3.1 min. In their
188
corresponding full scan spectrum, four major ion clusters m/z 213/215/217/219 (isotopic
189
abundance
190
301/303/305/307 (isotopic abundance ratio: 3:7:5:1), and 345/347/349/351 (isotopic abundance
191
ratio: 1:3:3:1) were observed (Figure 1b). The isotopic abundance ratios of the four ion clusters
192
suggested that they should contain 3 Cl, 2 Cl 1 Br, 1 Cl 2 Br, and 3 Br in their structures,
193
respectively. As displayed in Figure 1c−f, RTs of the four ion clusters were 2.74, 2.87, 2.90, and
194
2.98 min, respectively, suggesting that they were not aliphatic (since RTs of aliphatic compounds
195
should be in the domain of 0–2.5 min under the aforementioned UPLC settings).23 In the product
196
ion scan spectra of ion cluster m/z 345/347/349/351 (Figure 1g−j): a loss of 28 indicated the
197
presence of a carbonyl group; a loss of 44 indicated that a CO2 fraction could be lost from the
198
molecular ion. The fragmentation pathways of ion clusters m/z 213/215/217/219,
199
257/259/261/263, and 301/303/305/307 in their product ion scans were similar to that of ion
200
cluster m/z 345/347/349/351. Based on the closely related m/z values, isotopic abundance ratios,
201
RTs, and fragmentation pathways of the four ion clusters, we believed that they should belong to
202
a group of DBP analogues with the same main structure and different combinations of Cl and Br
ratio:
acid,
and
3:3:1:0.1),
dibromoacetic
257/259/261/263
acid)
(isotopic
and
two
abundance
trihalo-phenols
ratio:
9:15:7:1),
8
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substitutions. In a recent study, compounds with m/z 257/259/261/263, 301/303/305/307, and
204
345/347/349/351 were detected in a chlorinated simulated drinking water sample, and the one
205
with m/z 345/347/349/351 was suspected to be tribromo-HCD.24 More recently, Gonsior et al.
206
reported the detection of compounds with m/z 213/215/217/219, 257/259/261/263, and
207
301/303/305/307 (proposed to be either trihalo-furoic acids or trihalo-HCDs) in a Swedish
208
chlorinated drinking water,25 and a highly abundant compound with m/z 345/347/349/351
209
(proposed to be tribromo-HCD) in chlorinated ballast water.26 However, due to lack of
210
commercially available standard compounds, structure identification of this new group of DBPs
211
remained to be an unsettled problem in these previous studies.
212
Formulae identification was an important step towards revealing the structures of the new
213
group of DBPs, which was accomplished by a high resolution MS analysis of the four ion
214
clusters for obtaining their accurate m/z values. As displayed in Figure 2, the accurate m/z values
215
of the four ion clusters were determined to be 212.8914/214.8884/216.8854/219.1017,
216
256.8408/258.8384/260.8357/262.8327,
217
344.7394/346.7374/348.7354/350.7332, respectively. Calculated with a built-in formula
218
predictor software (m/z value shift within ± 1 ppm), the corresponding molecular ion formulae of
219
the four ion clusters were C5O3Cl3−, C5O3Cl2Br−, C5O3ClBr2−, and C5O3Br3−, with m/z value
220
shifts of 0.65, 0.56, 0.58, and 0.19 ppm, respectively.
221
300.7903/302.7881/304.7858/306.7832,
and
According to the RTs, fragmentation pathways, and molecular formulae of the new group of
222
DBPs,
their
possible
structures
could
only be
trihalo-furoic
acids,
trihalo-HCDs,
223
trihalo-methylfurandiones, trihalo-hydroxypyranones, or trihalo-cyclopentane-triones.
224
Synthesis of the New DBP with m/z 345/347/349/351. For the purpose of structure
225
identification, the standard compound of the new DBP with m/z 345/347/349/351 was aimed to 9
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be
227
trichloro-5-methoxy-4-cyclopentene-1,3-dione
228
3,5-dimethoxy-4-hydroxybenzaldehyde with NaOCl; Zhai et al.24 reported that the compound
229
with
230
1,2,3-trihydroxybenzene/1,2,4-trihydroxybenzene with bromine. Based on the above reaction
231
pathways,
232
3,4,5-trihydroxybenzaldehyde, and 2,4,6-trihydroxybenzaldehyde) were selected for reacting
233
with bromine at various precursor-to-bromine molar ratios to optimize synthesis conditions of
234
the new DBP. As illustrated in Figure 3a−f, among the four precursors, 1,2,3-trihydroxybenzene,
235
3,4,5-trihydroxybenzaldehyde, and 2,4,6-trihydroxybenzaldehyde could react with bromine to
236
generate the same compound as that (the new DBP with m/z 345/347/349/351) formed in the
237
simulated
238
2,4,6-trihydroxybenzaldehyde
239
2,4,6-trihydroxybenzaldehyde was selected to react with bromine for the synthesis of the new
240
DBP. As shown in Figure 3g−l, with the increasing bromine dose in the reaction of
241
“2,4,6-trihydroxybenzaldehyde
242
345/347/349/351 (detected at UPLC RT 3.65 min) exhibited a trend of first increasing and then
243
decreasing, peaking at a 2,4,6-trihydroxybenzaldehyde-to-bromine molar ratio of 1:8; moreover,
244
at the molar ratio of 1:8, the peak at UPLC RT 3.65 min contained the pure compound of the new
245
DBP with m/z 345/347/349/351 (impurities were hardly detected). Accordingly, the pretreated
246
reaction
247
2,4,6-trihydroxybenzaldehyde-to-bromine molar ratio of 1:8 was used for preparative isolation of
248
the new DBP with m/z 345/347/349/351.
m/z
in
the
lab.
Gong
345/347/349/351
four
solution
of
water
was
the
synthesis
be
compared
proved
bromine”,
to
generated
the
a
higher
formation
“2,4,6-trihydroxybenzaldehyde
of
+
by
reacting
1,2,4-trihydroxybenzene,
with
have
of
reacting
(1,2,3-trihydroxybenzene,
sample;
+
reported by
could
precursors
drinking
et
al.33
226
249
synthesized
Page 10 of 27
the
new
bromine”
other
precursors,
yield.
Therefore,
DBP with
prepared
at
m/z
the
Preparative Isolation and Structure Characterization/Identification of the New DBP with m/z 10
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345/347/349/351. Under the optimized condition, a reaction solution was prepared and pretreated
251
for isolation of the new DBP with m/z 345/347/349/351 (Figure 4a). Prior to HPLC/UV-triggered
252
preparative isolation, the new DBP was determined to have a maximum UV absorption at the UV
253
wavelength of 280 nm by UPLC/PDA analysis (simultaneous 2- and 3-dimensional operations)
254
(Figure 4b). When analyzing the pretreated reaction solution of “2,4,6-trihydroxybenzaldehyde +
255
bromine” with the UPLC/PDA at 280 nm, a very intensive peak corresponding to the new DBP
256
with m/z 345/347/349/351 was detected at RT 3.37 min (Figure 4c). For larger-scale isolation of
257
the new DBP with m/z 345/347/349/351, inlet method migration from the analytical UPLC to the
258
preparative HPLC was performed. As can be seen in Figure 4d, by applying the migrated inlet
259
method, acceptable and reproducible preparative separations were achieved by the Waters
260
Atlantis T3 OBD Prep column, and the obtained HPLC/UV chromatogram was very similar to
261
the UPLC/PDA chromatogram (Figure 4c) in terms of selectivity and resolution (regardless of
262
scales). Fraction collection was set up using FractionLynxTM Application Manager and the peak
263
corresponding to the new DBP with m/z 345/347/349/351 (at HPLC RT 16.63 min) was collected
264
(triggered by UV absorbance). After multiple HPLC injections, the collected fractions from all
265
separations were combined in a vessel to a final volume of 1 L, and the purity of the combined
266
solution was verified by the UPLC/ESI-tqMS full scan (Figure 4e). Then, the solution was
267
lyophilized to dryness under 5 Pa and –50 ºC for 48 h, and ~ 10 mg yellow solids of the new
268
DBP suitable for the FTIR characterization were obtained.
269
Figure 5 displays the FTIR spectrum of the new DBP. The broad peak at 3443.5 cm–1 should
270
be assigned to stretching vibration of O–H, suggesting that this DBP contains a hydroxyl group.
271
Two dominant characteristic peaks at 1710.0 and 1604.0 cm–1 should correspond to stretching
272
vibrations of C=O, indicating that this DBP contains two carbonyl groups. The peak at 1752.3 11
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273
cm–1 should correspond to stretching vibration of C=C. Several peaks corresponding to
274
stretching vibrations of C–Br were detected at wavenumbers between 600 to 700 cm–1.
275
According to above structural information provided by the FTIR spectrum, the previously
276
proposed structures of trihalo-furoic acids, trihalo-methylfurandiones, trihalo-hydroxypyranones,
277
and trihalo-cyclopentane-triones were excluded, and the new DBP with m/z 345/347/349/351
278
was finally identified to be tribromo-HCD. Logically, new DBPs with m/z 213/215/217/219,
279
257/259/261/263, and 301/303/305/307 should be trichloro-HCD, dichlorobromo-HCD, and
280
chlorodibromo-HCD, respectively.
281
Formation of the New Group of DBPs. Based on the disclosed structure, formation pathways
282
of tribromo-HCD through reactions of the three precursors (1,2,3-trihydroxybenzene,
283
3,4,5-trihydroxybenzaldehyde, and 2,4,6-trihydroxybenzaldehyde) and bromine were proposed
284
(shown in Figure 6), which were partially verified through the successful detection of proposed
285
intermediate compounds I, II, IV, VI, VII, VIII, and X using the UPLC/ESI-tqMS MRM
286
(displayed in Figure S2). Proposed intermediate compounds III, V, and IX were not detected
287
since they were unstable and might be quickly decarboxlyated/hydrolyzed. As illustrated in
288
Figure 6, the formation reactions started with bromine substitutions on the benzene rings of these
289
precursors, followed by multistep oxidation and hydrolysis. Since the 3- and 5-positions of
290
2,4,6-trihydroxybenzaldehyde were activated by both hydroxyl and aldehyde groups, bromine
291
substitutions
292
1,2,3-trihydroxybenzene and 3,4,5-trihydroxybenzaldehyde. Thus, the highest tribromo-HCD
293
yield was achieved by the reaction of 2,4,6-trihydroxybenzaldehyde and bromine, as
294
demonstrated in Figure 3.
295
on
2,4,6-trihydroxybenzaldehyde
were
much
easier
than
those
on
We further studied factors that may affect the formation of trihalo-HCDs during drinking 12
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296
water disinfection, including source water bromide level, disinfectant type, contact time, and pH.
297
In a UPLC/ESI-tqMS MRM chromatograph, the peak area of a DBP of a simulated drinking
298
water sample is positively correlated to its concentration.18
299
As displayed in Figure 7a, with the increasing bromide level from 0 to 4.0 mg/L, peak areas
300
of the four new DBPs exhibited diverse trends: trichloro-HCD kept decreasing; dichlorobromo-
301
and chlorodibromo-HCDs first increased and then decreased (reached maximum at the bromide
302
concentration of 0.4 mg/L); tribromo-HCD kept increasing. Moreover, as the bromide
303
concentration increased, initially the most dominant species was trichloro-HCD, which was
304
gradually taken over by the mixed-chlorobromo species, and finally tribromo-HCD became the
305
most abundant one. All these indicate that, increasing the bromide concentration shifted the
306
formation of trihalo-HCDs from being more chlorinated to being more brominated. Similar shifts
307
within a group of DBPs have been reported for trihalomethanes, haloacetic acids, and phenolic
308
halogenated DBPs,28,34,35 however, it is the first time that such kind of shift was proved to exist in
309
trihalo-HCDs. Figure 7b compares the formation of trihalo-HCDs in chloramination and
310
chlorination at contact times from 1 to 120 h. Basically, with the increasing contact time, the
311
peak areas of trihalo-HCDs kept increasing in chloramination, whereas kept decreasing in
312
chlorination. The formation of trihalo-HCDs was higher in chlorination than that in
313
chloramination with a short contact time (i.e., 1 h), whereas it became lower in chlorination than
314
that in chloramination as contact time further increased. This is because that, in chlorination,
315
trihalo-HCDs could be generated very quickly due to the strong oxidation ability of HOCl/OCl–,
316
however, the formed trihalo-HCDs were not stable and could be decomposed during prolonged
317
chlorination; in chloramination, although less trihalo-HCDs were produced with a relatively
318
short contact time, their accumulation with the increasing contact time was allowed since NH2Cl 13
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319
was a mild oxidant. As shown in Figure 7c, with the increasing pH from 6.0 to 8.5, peak areas of
320
trihalo-HCDs experienced a dramatic decrease by ~ 80%. The decrease of trihalo-HCDs with the
321
increasing pH might result from base-catalyzed hydrolysis, which matched previous findings that
322
most halogenated DBPs appeared to favor hydrolysis in alkaline conditions.36−38
323
Notably, occurrence of trihalo-HCDs was investigated in the real tap water samples A–H.
324
As shown in Figure S3, trichloro-HCD was the most prevalent species (detected in all the tap
325
water samples), whereas dichlorobromo-, chlorodibromo-, and tribromo-HCDs were detected in
326
six, seven, and five of the eight tap water samples, respectively. With the synthesized standard
327
compound, tribromo-HCD was further quantified in these tap water samples and the baseline
328
simulated drinking water sample using the standard addition method.28,38 The concentrations of
329
tribromo-HCD were from below detection limit to 0.53 µg/L in the real tap water samples and
330
3.94 µg/L in the baseline simulated drinking water sample. According to Figure 7, the
331
concentrations of tribromo-HCD should be even higher in simulated chloraminated drinking
332
water samples with higher bromide concentrations, longer contact times, and lower pH. Such
333
high levels of tribromo-HCD formed in the simulated drinking water samples and real tap water
334
samples suggested that, compared with other emerging drinking water halogenated DBPs with
335
cyclic structures (e.g., halogenated benzoquinones, hydroxybenzaldehydes, hydroxybenzoic
336
acids, and phenols), trihalo-HCDs might occur in drinking water at more abundant levels. 28,37
337
The
successful
detection,
synthesis,
preparative
isolation,
and
structure
338
characterization/identification of trihalo-HCDs provided a novel and effective approach for
339
exploring new DBPs (especially those without commercially available standards) in drinking
340
water. With UPLC/ESI-tqMS and high resolution MS analyses, an exact formula of a new DBP
341
could be assigned. Through optimization of synthesis condition, UPLC/PDA analysis, 14
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342
HPLC/UV-triggered preparative isolation, and lyophilization, a pure standard of the new DBP
343
could be obtained, which makes its structure characterization/identification possible by other
344
analytical techniques such as FTIR. Our findings also suggest that chloramination of source
345
water containing higher levels of bromide under less alkaline condition may enhance the
346
formation of trihalo-HCDs (especially the more brominated ones). Regarding the cyclic
347
structures and the high abundance in drinking water of trihalo-HCDs, further evaluation of their
348
toxicity (e.g., cytotoxicity, genotoxicity, developmental toxicity, etc.) should be important for
349
understanding their adverse health effects on human.
350 351
AUTHOR INFORMATION
352
Corresponding Authors
353
*(Pan, Y.) Phone: +86-25-83592903; fax: +86-25-83592903; e-mail:
[email protected].
354
*(Li, A.) Phone: +86-25-89680576; fax: +86-25-86269876; email:
[email protected].
355
Notes
356
The authors declare no competing financial interest.
357 358
ACKNOWLEDGEMENTS
359
We thank National Natural Science Foundation of China (Grants 51408296, 51290282,
360
51438008) and Natural Science Foundation of Jiangsu Province, China (Grant BK20140607) for
361
providing financial supports to this study. We also thank Prof. Xiangru Zhang from the Hong
362
Kong University of Science and Technology for his useful suggestions.
363 364
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365 366 367 368 369 370 371 372
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468
0
2.50 O
5.00
O- Cl
Cl Cl
Cl
O
Cl
O O Br
1.41
0
3.51
2.50
349 345 351
3.05
0.90 1.45
0
2.50
300
350
m/z
5.535.88
7.47 8.07 9.31 10.15
5.00
7.50
10.00
7.50
347 79
266
143
240
187
345
0 −79 (79Br)
−79 (79Br)
−81 (81Br)
−81 (81Br)
−28 (CO)
268
81
10.00
270
145
189
305 −81 (81Br)
5.00
7.50
3.66
2.50
270
100 (j)
10.00
2.98 1.37
349 347
240 267
0 4.96
2.50
100 (f)
268
−81 (81Br)
1.30
0
−81 (81Br)
−44 (CO2)
2.90
100 (e)
−79 (79Br)
−79 (79Br) −28 (CO)
100 (i)
4.94 5.78 7.35
5.00
−81 (81Br)
−44 (CO2)
379
344 345 347 301
238 237
100 (h)
2.87
100 (d) %
O
−44 (CO2)
%
%
200 250 2.74 100 (c) 2.61
%
Br
%
% 0
%
O O Br
303 257 305 261 301 213 225 307 249 267 281
181
469 470 471 472 473 474
Br
345
−28 (CO)
187
0
O-
O- Br
143
79
Time
10.00
259
100 (b)
0
7.50
O- Cl
O Br
−44 (CO2)
%
6.67
4.54
−79 (79Br)
266 265
100 (g)
%
%
3.01
0.82
−79 (79Br)
8.48 8.37 7.88
100 (a)
81
5.14 6.32 7.22 8.18
5.00
7.50
10.00
Time
0
50
100
−28 (CO)
189 145
150
200
242 269
250
351 350 271 307 323 353
300
350
m/z 400
Figure 1. (a) UPLC/ESI-tqMS full scan chromatogram of the baseline simulated drinking water sample; (b) UPLC/ESI-tqMS full scan spectrum from RT 2.7 to 3.1 min of the baseline simulated drinking water sample; (c−f) UPLC/ESI-tqMS MRM chromatograms of ion clusters m/z 213/215/217/219, 257/259/261/263, 301/303/305/307, and 345/347/349/351, respectively; (g−j) UPLC/ESI-tqMS product ion scan spectra of ion cluster m/z 345/347/349/351.
20
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475 Relative Abundance Relative Abundance
100 50 40
(a)
C5O3Cl3− 218.8303 m/z shift: 0.65 ppm
214.8344 214.8884
212.8914
30 218.8710
20 216.8854 10
213.0764
215.1283
Relative Abundance
214
100
258.7321 256.8408
80 60
Relative Abundance
C5O3Cl2Br− m/z shift: 0.56 ppm
258.9241 260.8357
40 261.0761 20 256.9450
100
262.8327
259.8420
258
260
(c)
262
302.7881
C5O3ClBr2− m/z shift: 0.58 ppm
80
304.7858 60 300.7903 301.2378
40 20
302.9140 300
Relative Abundance
218
258.8384
256
302
(d)
304.9118 306.7832
304
346.7374
100
306
348.7354
C5O3Br3− m/z shift: 0.19 ppm
80 60 40
344.7394
20
350.7332
345.0970 344
476 477 478 479 480
216
(b)
219.1017
217.8356
346
347.7407 348 m/z
349.1283 350
Figure 2. (a−d) High resolution MS spectra of ion clusters m/z 213/215/217/219, 257/259/261/263, 301/303/305/307, and 345/347/349/351, respectively, in the baseline simulated drinking water sample.
21
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481
100 (c)
5.00
7.50 347
%
345 0 344 346
2.50
100 (d)
5.00 345
2.89 0 344
0
2.50
346
5.00
348
7.50
350
5.00
1.58
3.67 4.91
6.23
2.50
5.00
0.85
6.25
2.50
5.00
%
%
1.65
2.50
5.00
7.50
0
10.00
3.12
0
8.68200
400
m/z 600
10.00 349 345 351
0
8.67200
7.50
m/z 600
400
10.00
3.333.68
2.50
5.00
100 (l)
203
0
5.61 6.23
8.74200
7.50
349 400
m/z 600
10.00
100
%
%
%
100 (f)
349 345 351
100
0.83
8.26
m/z 600
400
10.00
100
4.93 5.60
1.55 2.44
100 (k) 5.96 6.55
8.66200
7.50
3.65
2.98 3.23
0
7.50
5.59
0.85
0
10.00
311 309 313 349
100
m/z 352
100 (e)
0
5.63 6.24
m/z 600
400
10.00
100
2.42
100 (j) 351
7.50
4.04
2.50
0
10.00 349
347
100
m/z 352
350
7.50 %
%
351 348
5.00
0.85
100 (i)
349
200 8.70
%
0
2.36
0
10.00
%
100
3.04
%
%
%
2.50
m/z 352
350
347 0
4.95
351
348
311
5.61
2.50
100 (h)
347
100
2.51
0.85
0
10.00 349
100
4.96
%
2.71
m/z 352
350
7.50
345 0 344 346
0
348
4.05
%
5.00
%
100 (b)
346
%
2.50
351
%
%
% 0
100 (g)
345
0 344
3.14
349
%
347
100
%
100 (a)
0
482 483 484 485 486 487 488 489 490 491 492 493 494
0.86 1.72
2.50
5.97 6.53
5.00
8.27
7.50
10.00
Time
0
0.65 1.77
205
3.37
2.50
0
8.68200
4.82 5.67
5.00
7.50
347 400
10.00
m/z 600
Time
Figure 3. (a−d) UPLC/ESI-tqMS MRM (345→79, 347→79/81, 349→79/81, 351→81) chromatograms and spectra (in dashed line boxes) of the pretreated reaction solutions of “1,2,3-trihydroxybenzene + bromine”, “1,2,4-trihydroxybenzene + bromine”, “3,4,5-trihydroxybenzaldehyde + bromine”, and “2,4,6-trihydroxybenzaldehyde + bromine”, respectively; (e,f) UPLC/ESI-tqMS MRM (345→79, 347→79/81, 349→79/81, 351→81) chromatograms of the baseline simulated drinking water sample and the baseline sample spiked with the pretreated reaction solution of “2,4,6-trihydroxybenzaldehyde + bromine”, respectively; (g−l) UPLC/ESI-tqMS full scan chromatograms and spectra (peak centered at RT 3.6 min, in dashed line boxes) of the pretreated “2,4,6-trihydroxybenzaldehyde + bromine” reaction solutions prepared at 2,4,6-trihydroxybenzaldehyde-to-bromine molar ratios of 1:2, 1:4, 1:6, 1:8, 1:12, and 1:20, respectively. The y-axes of charts (a−d), (e,f), and (g−l) are on the same scales, respectively.
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495 5.64 3.41
0.83 2.42
0
6.26
4.45
2.50
5.00
AU
5.96 0.77
10.00 280 281
220
5.0 (b)
0.0
7.07 8.67
7.50
5.63
2.39 3.37
2.50
5.00
AU
%
100 (a) 1.56
4.0e-3
341
2.0e-3 0.0
250
7.50
300
nm 350
10.00
AU
1.0e-2 (c) 3.37
0.77 2.39
0.0
2.50
5.0e-1 (d)
5.00
8.558.91
7.50
10.00
Time
AU
16.63
24.72
8.33
0.0
5.97
4.41
10.00
20.00
30.00
40.00 347
100
100 (e)
%
3.13
%
0
Time
345 351 200 8.77
400
m/z 600
0.74 2.11
0
496 497 498 499 500 501 502 503 504 505 506
2.50
5.00
7.50
10.00
Time
Figure 4. (a) UPLC/ESI-tqMS full scan spectrum of the pretreated reaction solution of “2,4,6-trihydroxybenzaldehyde + bromine”; (b) UPLC/PDA simultaneous 2- and3-dimensional scan (wavelength range: 220 to 350 nm) chromatogram and spectrum (peak at RT 3.37 min, in a dashed line box) of the pretreated reaction solution of “2,4,6-trihydroxybenzaldehyde + bromine”; (c) UPLC/PDA 2-dimensional scan (wavelength: 280 nm) chromatogram of the pretreated reaction solution of “2,4,6-trihydroxybenzaldehyde + bromine”; (d) HPLC/UV (wavelength: 280 nm) chromatogram of the pretreated reaction solution of “2,4,6-trihydroxybenzaldehyde + bromine”; (e) UPLC/ESI-tqMS full scan chromatogram and spectrum (peak at RT 3.13 min, in a dashed line box) of the isolated fractions.
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507 100
0 4000
508 509 510
1710.0
20
3000
2000 Wavenumbers (1/cm)
606.3 498.2
816.0 693.4
1604.0
1752.3
40
1372.2 1240.1 1122.2 1077.7
60 3443.5
%Transmittance
80
1000
Figure 5. FTIR spectrum of the new DBP with m/z 345/347/349/351.
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Environmental Science & Technology
511 O
OH OH HOBr HO
OH HOBr HO
Br
Br
Br
HOBr
HO
OH
Br
OH
OH OH
HOBr
HO
CHO OH
HOBr
HO
OH
512 513 514 515 516
OH
Br H2O
Br
Br
HO
OH
O
OH
(Ⅴ)
(Ⅵ)
CHO HOOC Br OH H O HO OH HOBr HO 2
Br
Br OH (Ⅷ)
O OH Br OH –HCO2H Br O Br O Br
Br
Br O (Ⅸ)
OH O Br
(Ⅲ)
HOOC Br Br Br
(Ⅳ)
HO
Br
(Ⅱ)
CHO Br
HO
Br O
(Ⅰ)
CHO
OH HOBr HO
HOBr
OH HO
OH
Br
Br OH
Br
OH H2O HO
Br OH (Ⅶ)
Br OH (Ⅹ)
Figure 6. Proposed formation pathways of tribromo-HCD through reactions of the three precursors (1,2,3-trihydroxybenzene, 3,4,5-trihydroxybenzaldehyde, and 2,4,6-trihydroxybenzaldehyde) and bromine.
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517 4
10
(a)
3
Peak area
10
2
10
1
10
0
10
0 10
Peak area
10
0.1 0.4 1.0 2.0 Bromide concentration (mg/L)
4.0
4
(b) Chloramination
Chlorination
3
10
2
10
1
10
0
1
24
120 1 Contact time (h)
24
120
4
10
(c)
3
Peak area
10
2
10
1
10
0
10
6.0
518 519 520 521 522
6.5
trichloro-HCD chlorodibromo-HCD
pH
7.5
8.5 dichlorobromo-HCD tribromo-HCD
Figure 7. Peak areas (in log scales) of trihalo-HCDs in the UPLC/ESI-tqMS MRM chromatograms of the simulated drinking water samples prepared with different (a) source water bromide levels, (b) disinfectant types and contact times, and (c) pH values.
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Environmental Science & Technology
An Author-created Table of Contents Graphic (TOC Art) A new group of drinking water DBPs: trihalo-hydroxy-cyclopentene-diones SRNOM + Br– + Cl2 / NH2Cl Cl
O-
Cl
%
180.9191.1 213.0 222.9
0 180
200 HO
220 CHO OH
253.3
240
260
O
281.1
302.7 306.8
280
300
339.1 347.0 353.2 379.2
320
precursor-to-Br2 molar ratio of 1:8 8 h, 20 ºC
+ Br2
O Br Br
Br Br
258.9 260.8
100
O-
Br
O
O
Br Cl
Cl Cl
trihalo-HCDs O-
Cl
O
O
O
O
O-
340
m/z 380
360
tribromo-HCD
OH
0.0
524
24.72 29.12
20.00
40.00
Time
0 4000
693.4 606.3
50
1752.3 1710.0
5.22 8.33
FTIR characterization 1604.0
2.5e-1
% Transmittance
100
16.63
3443.5
Preparative isolation
3000 2000 1000 Wavenumbers (1/cm)
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