Nitenpyram degradation in finished drinking water

Article abstract of DOI:10.1002/rcm.7581

Rationale: Neonicotinoid pesticides and their metabolites have been indicated as contributing factors in the decline of honey bee colonies. A thorough understanding of neonicotinoid toxicity requires knowledge of their metabolites and environmental breakdown products. This work investigated the rapid degradation of the neonicotinoid nitenpyram in finished drinking water. Methods: Nitenpyram reaction products were characterized using liquid chromatography/quadrupole time-of-flight mass spectrometry (LC/QTOFMS). A software algorithm that compared degraded and control samples was utilized to facilitate efficient data reduction. Fragmentation pathways for six reaction products and nitenpyram were proposed using predictive software and manual product ion analysis. Results: This study showed that nitenpyram degradation in unpreserved finished drinking water was likely the result of oxidation, hydrolysis and reaction with Cl₂. Structures for six nitenpyram reaction products were proposed that suggest the C9/C11 olefin as the key reactive site. Conclusions: Similarities between the identified nitenpyram reaction products and imidacloprid metabolites highlight the importance of this study, as the toxicity of neonicotinoids to pollinators has been linked to their metabolites. Based on the proposed reaction mechanisms, the identified nitenpyram reaction products in finished drinking water could also be present in the environment and water treatment facilities. As such, identifying these degradation products will aid in evaluating the environmental impact of neonicotinoid pesticides. Copyright

Full text of DOI:10.1002/rcm.7581

Research Article
Received: 2 February 2016
Revised: 7 April 2016
Accepted: 7 April 2016
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2016, 30, 1653–1661
(wileyonlinelibrary.com) DOI: 10.1002/rcm.7581
Nitenpyram degradation in finished drinking water
Matthew Noestheden1, Simon Roberts2 and Chunyan Hao3
*
1SCIEX, Ontario, Canada
2SCIEX, California, USA
3Ontario Ministry of the Environment and Climate Change, Ontario, Canada
RATIONALE: Neonicotinoid pesticides and their metabolites have been indicated as contributing factors in the decline of
honey bee colonies. A thorough understanding of neonicotinoid toxicity requires knowledge of their metabolites and
environmental breakdown products. This work investigated the rapid degradation of the neonicotinoid nitenpyram in
finished drinking water.
METHODS: Nitenpyram reaction products were characterized using liquid chromatography/quadrupole time-of-flight
mass spectrometry (LC/QTOFMS). A software algorithm that compared degraded and control samples was utilized to
facilitate efficient data reduction. Fragmentation pathways for six reaction products and nitenpyram were proposed using
predictive software and manual product ion analysis.
RESULTS: This study showed that nitenpyram degradation in unpreserved finished drinking water was likely the result of
oxidation, hydrolysis and reaction with Cl2. Structures for six nitenpyram reaction products were proposed that suggest
the C9/C11 olefin as the key reactive site.
CONCLUSIONS: Similarities between the identified nitenpyram reaction products and imidacloprid metabolites
highlight the importance of this study, as the toxicity of neonicotinoids to pollinators has been linked to their
metabolites. Based on the proposed reaction mechanisms, the identified nitenpyram reaction products in finished
drinking water could also be present in the environment and water treatment facilities. As such, identifying these
degradation products will aid in evaluating the environmental impact of neonicotinoid pesticides. Copyright © 2016
John Wiley & Sons, Ltd.
Neonicotinoids (NNIs) are neurotoxic pesticides that have
seen expanding use since the introduction of the first
generation NNI imidacloprid 30 years ago (Fig. 1).[1]
Currently, NNIs are involved in 80% of insecticidal seed
treatments and are the largest class of insecticides sold.[2]
Due to their low mammalian toxicity, NNIs have been deemed
relatively safe for the environment.[3] In recent years, however,
the widespread use of NNIs has been associated with a decline
in honeybee colonies.[2] Regulatory bodies in several countries
have restricted the use of certain NNIs while their impact
is assessed.[4]
Given their global use and potential impact on pollinators,
there has been a plethora of research on NNIs in recent years.
However, there is still a lack of monitoring data evaluating the
fate and stability of NNIs in environmental waters.[5] This
study started out looking at the stability of NNIs in surface,
ground and finished drinking water to support an initiative by
the Government of Ontario to enhance pollinator health and
reduce the use of NNIs.[6,7] During stability characterization
it was observed that nitenpyram degraded rapidly in
unpreserved (without added sodium thiosulfate) finished
drinking water while other NNIs were stable, including the
structurally similar imidacloprid, (Supplementary Fig. S1,
Supporting Information). This was surprising given the
photolytic and pH stability (< pH 9) of nitenpyram.[5]
Studies on NNI metabolites have shown appreciable levels
of toxicity.[1,5] While the majority of this data came from the study
of imidacloprid metabolites in bees, prudence suggests that an
understanding of potential environmental breakdown products
of other NNIs is required to ensure continued pollinator
health.[2,8,9] Indeed, such metabolite characterizations are
becoming more important given the increasing use of reclaimed
water and biosolids on food crops, which may result in the
uptake of agrochemicals and their metabolites into plants.[10,11]
To understand the rapid loss of nitenpyram in unpreserved
finished drinking water an investigation was carried out to
identify nitenpyram degradation products using detailed
fragmentation analysis. To conduct this investigation a liquid
chromatography/quadrupole time-of-flight mass spectrometry
(LC/QTOFMS) method was used to acquire high-resolution,
accurate mass precursor and product ion data.
EXPERIMENTAL
Reagents and sample preparation
* Correspondence to: C. Y. Hao, Laboratory Services Branch,
Ontario Ministry of the Environment and Climate Change,
125 Resources Road, Toronto, Ontario M9P 3V6, Canada.
E-mail: chunyan.hao@ontario.ca
The following chemicals were purchased from Sigma Aldrich
(Ontario, Canada) and used as received: nitenpyram,
imidacloprid, acetamiprid, thiacloprid, flonicamid, clothianidin,
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Copyright © 2016 John Wiley & Sons, Ltd.

M. Noestheden, S. Roberts and C. Hao
Figure 1. Structures of commonly used neonicotinoid (NNI) pesticides. The IUPAC numbering
of nitenpyram atoms is shown to aid the reader during fragmentation analysis (vide infra).
thiomethoxam, dinotefuran, acetamiprid-d3, clothianidin-d3,
in MeOH. Chromatographic separation was achieved using
a flow-gradient and non-linear mobile phase gradient
(Supplementary Table S2, Supporting Information).
imidacloprid-d4, thiamethoxam-d3, sodium thiosulfate (NaS2O3)
and ACS-grade formic acid. HPLC-grade solvents were
purchased from Caledon Labs (Ontario, Canada). High-purity
water was produced by passing reverse osmosis water
through a Barnstead NANOpure™ water purification system
(Ontario, Canada). Ten milliliter solution dropper bottles for
NaS2O3 were purchased from ACP Chemicals Inc (Quebec,
Canada).
NNI and isotope-labelled (acetamiprid-d3, clothianidin-d3,
imidacloprid-d4 and thiamethoxam-d3) NNI working solutions
were prepared in MeOH and stored at 2–8°C for up to 6 months
(Supplementary Table S1, Supporting Information). Preservative
solution (25% NaS2O3) was prepared in dropper bottles using
high-purity water.
Mass spectrometry
LC/QqQ data were acquired with electrospray ionization (ESI)
on a QTRAP® 5500 LC/MS/MS system operating in positive
mode (SCIEX). The Scheduled MRM™ (sMRM) algorithm
contained in the Analyst® 1.6.2 software was utilized for data
acquisition, with a target cycle time of 1000 ms and a 60 s
retention time window. The sMRM and QqQ MS source
parameters are summarized in Supplementary Table S3
(Supporting Information).
LC/QTOF data were acquired on a SCIEX TripleTOF® 5600
system using the Analyst® TF 1.7 software (SCIEX). Data was
collected using positive mode ESI and two acquisition
workflows. The first utilized the information-dependent
acquisition (IDA) algorithm of the Analyst® TF 1.7 software
(Supplementary Table S4, Supporting Information). The IDA
algorithm parameters were set to collect product ion spectra
from four precursor ions that were identified in each full scan
TOFMS spectrum. Dynamic background subtraction was used
and product ion spectra were collected with a collision energy
of 30 15 V. Isotopes within 6 Da of a precursor ion the IDA
algorithm identified were ignored. The second acquisition
method used dedicated product ion scanning at collision energies
of 10, 20 and 40 V. QTOFMS source parameters are summarized
in Supplementary Table S4 (Supporting Information).
Stability study samples were prepared by fortifying
surface, ground and finished drinking water with NNI
working solution and isotope-labelled NNI working solution
to
a final nitenpyram concentration of 50 ng/L and
100 ng/L, respectively, in 50 mL. One drop of a 25% NaS2O3
solution was added to a second finished drinking water
sample prior to fortification. At each time point samples
were prepared for analysis by adding 800 μL of fortified
water sample to 100 μL of MeOH and 100 μL of internal
standard stock solution (1000 ng/L). Stability samples were
held at 2–8°C, with analytical samples prepared at 0, 7, 14,
21 and 28 days.
To assess nitenpyram degradation products, 200 μL of
MeOH was added to 800 μL of finished drinking water. This
sample was the control to aid in data processing (vide infra).
Analytical samples were prepared by adding 200 μL of
nitenpyram working solution to 800 μL of tap water. The final
concentration of nitenpyram was 200 μg/mL.
The QTOF mass spectrometer was mass calibrated at the start
of each day and approximately every 2 h during active use and
was calibrated from m/z 146.1176 to 922.0098 (eight unique ions).
Product ion scans were calibrated from m/z 58.0651 to
315.1623 (eight unique ions).
Chromatography
Data processing
LC/QqQ chromatography for NNI stability evaluation was
performed on
a
Shimadzu Prominence LC20 system.
Unknown compound identification was performed using a
non-targeted screening work-flow in the MasterView™
software (version 1.1; SCIEX, USA). This work-flow utilized
a naïve peak-finding algorithm that permitted comparison to
a control sample. To focus this study on higher responding
and, assuming similar response factors, higher abundance
degradation products, only candidate precursor ions present
at >5× the response in a control sample were evaluated. This
threshold represents an empirical assessment of mass spectral
LC/QTOF chromatography was performed on a SCIEX
ExionLC™ AD system equipped with a binary pump,
degasser, autosampler (50 μL sample loop) and column heater
(SCIEX, USA). Chromatographic parameters on both LC
systems were adapted from Hao et al.[6] The changes made
include a 50 μL injection volume, a column temperature of
30°C, an aqueous mobile phase of 0.05% formic acid +10%
MeOH and an organic mobile phase of 0.05% formic acid
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Copyright © 2016 John Wiley & Sons, Ltd.
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Nitenpyram degradation in finished drinking water
responses for the control and degradation samples. For candidate
ions that passed this filter, extracted ion chromatograms
(EICs) were generated using an extraction window of 5 mDa
and a retention time tolerance of 30 s. An EIC intensity
threshold of >100 counts and a signal-to-noise ratio > 5 were
applied as data quality filters. The remaining candidate ions
were processed using a formula finding algorithm, with
C50H200N10O10S5Cl3 as the maximum permitted elemental
compositions. If no suitable formula could be generated from
within those elemental limitations the candidate ion was
discarded. Proposed empirical formulae were assessed for
precursor (<5 ppm) and production ion (<10 ppm) mass
accuracy. As well, the isotope ratio difference was calculated
by comparing the isotopic ratio of the proposed empirical
precursor formulae to the experimental data. A final data
review was accomplished by manually comparing the
generated empirical formulae and product ion spectra to the
nitenpyram parent formula and fragmentation pattern.
RESULTS AND DISCUSSION
NNI stability in finished drinking water
To support a government action plan in Ontario, Canada, aimed
at enhancing pollinator health and reducing the use of NNIs, an
LC/MS/MS method was developed to assess environmental
waters and finished drinking water. This analysis included eight
NNIs: acetamiprid, imidacloprid, nitenpyram, thiacloprid,
clothianidin, thiamethoxam, dinotefuran and flonicamid (Fig. 1).
During development of this method it was observed that
nitenpyram was stable in surface and ground waters, but
completely disappeared shortly after fortifying unpreserved
finished drinking water (Fig. 2). This was quite surprising as
none of the structurally related NNIs (e.g., imidacloprid)
displayed similar behavior (Supplementary Fig. S1, Supporting
Information). Stabilization of nitenpyram in finished drinking
water was achieved by adding NaS2O3 as a dechlorinating agent,
which extended nitenpyram stability through 4 weeks (Fig. 2).
Fragmentation analysis was performed manually and with the
aid of a fragmentation prediction tool contained in the PeakView™
version 2.2 software (SCIEX). Proposed primary fragmentation
pathways for nitenpyram were labelled P1 → P9 and for reaction
products were labelled according to their assigned numbers
(e.g., Deg_01 = D1–1, etc.). Proposed higher order fragments
were assigned a lower-case letter designation (e.g., D1–1a).
Identification of nitenpyram degradation products
To investigate the rapid loss of nitenpyram, a study was
conducted to identify degradation products in unpreserved
finished drinking water. Such work is important not only
Figure 2. Nitenpyram is stable in surface (A) and ground waters (B).
Degradation of nitenpyram in unpreserved finished drinking water (C). The
nitenpyram response remains after fortification in finished drinking water
with the addition of a NaS2O3 (D). Chromatograms are shown with quantifier
(blue) and qualifier (pink) MRM transitions overlaid. Nitenpyram is stable for
4 weeks in NaS2O3-preserved finished drinking water (bottom).
Rapid Commun. Mass Spectrom. 2016, 30, 1653–1661
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M. Noestheden, S. Roberts and C. Hao
for understanding how nitenpyram degrades in finished
drinking water, but may also provide insight into the
mechanism of NNI degradation in the environment and water
treatment facilities. As a starting point, nitenpyram was
fortified into finished drinking water and analyzed by
data, with values <10 indicating good agreement. All of the
proposed empirical formulae show excellent agreement with
the theoretical isotope ratios (Table 1).
For TOFMS data the accurate mass was calculated based on
the proposed empirical formulae, with the identified reaction
product list showing < 1 ppm mass accuracy (Table 1). For
product ion mass accuracy, the MasterView™ software used
for data processing contained an embedded feature that used
the assigned precursor formula to interrogate the acquired
product ion spectra and suggest plausible formulae. The
software then averaged the suggested product ion formulae
mass accuracy. While the proposed product ion formulae cannot
be used to blindly deduce reaction product structures, the
average mass accuracy does provide an additional level of
confidence in the proposed empirical precursor formulae. The
average product ion mass error calculated using this approach
was < 7 ppm for the identified reaction products (Table 1).
LC/QTOFMS
identification of reaction products an information-dependent
acquisition algorithm was utilized in tandem with
6 h after fortification. To aid in the
a
software package that permits comparative analysis to a
control sample. For this study the control sample was
finished drinking water fortified with MeOH. This approach
maximized the information obtained from a single injection
and ensured that the percentage of relevant compounds
identified was high.
Using this work-flow 20 potential reaction products
were identified in nitenpyram-fortified finished drinking
water (Table 1). Since the comparative analysis used by the
MasterView™ software was used, it was known that all 20
compounds were present in the fortified finished drinking
water at >5× the response observed in the control sample. This
provided strong evidence that the compounds identified were
all related to nitenpyram. However, to increase confidence in
the identifications and aid in the assignment of empirical
formulae mass accuracy (TOFMS and MS/MS), theoretical
isotopic distribution and MS/MS fragmentation analysis were
also evaluated. Such data quality objectives are consistent with
common publishing requirements for unknown identification
using LC/MS/MS.[12,13]
Nitenpyram fragmentation analysis
With high confidence in the empirical precursor ion formulae,
the product ion spectra were interrogated to assign plausible
reaction product structures based on predicted ionization
and fragmentation characteristics. Rather than view each
reaction product separately, a thorough understanding of
nitenpyram fragmentation was pursued first (Fig. 3). This step
was critical since diagnostic fragment ions from nitenpyram
would guide the proposal of structures for the identified
reaction products.
The product ion spectrum of nitenpyram was feature rich,
with ten major components (>30% of the base peak) and a
plethora of minor product ions. However, since the information
dependent acquisition algorithm used a collision energy of
The predicted isotope ratio was very useful for this study
given the presence of chlorine in the parent and proposed
reaction products (vide infra). The isotope ratio difference
provided a metric for evaluating how the theoretical isotope
ratio of a proposed formula matched with the experimental
Table 1 Proposed empirical formulae, retention times (RT) and area responses for nitenpyram reaction products identified in
unpreserved finished drinking water
Empirical
formula
Exact
TOFMS
MS/MS
Isotope ratio
difference
Name
RT (min)
mass (Da) Adduct error (ppm) error (ppm)
Area
Nitenpyram
Deg_01
Deg_02
Deg_03
Deg_04
Deg_05
Deg_06
Deg_07
Deg_08
Deg_09
Deg_10
Deg_11
Deg_12
Deg_13
Deg_14
Deg_15
Deg_16
Deg_17
Deg_18
Deg_19
5.08
3.31
9.44
3.75
7.58
8.64
7.32
5.61
4.76
4.58
8.79
6.04
8.42
10.72
7.41
8.83
8.58
11.85
10.23
11.30
C11H15ClN4O2
C8H11ClN2
270.0884
170.0611
199.0512
211.0876
227.0825
257.0567
282.0884
282.0884
284.1040
284.1040
284.1040
284.1040
295.0643
301.0829
304.0494
381.0759
401.1102
409.1072
506.1600
539.1451
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
+H
0.7
0.3
0.2
0.6
0.9
0.8
−0.9
0.9
0.5
0.4
0.8
0.8
0.9
0.8
0.9
−0.6
1.0
0.8
0.5
−0.1
6.8
3.4
6.5
2.4
2.8
3.6
3.0
3.9
5.5
6.2
3.7
3.1
2.8
3.7
1.7
1.8
3.7
1.5
3.1
1.0
9.6
1.0
1.1
1.2
1.3
1.7
0.9
2.0
2.5
2.0
4.1
0.7
0.2
2.0
1.3
0.7
4.4
2.6
5.3
5.0
1,504,245
107,935
2,451
70,936
12,232
161,312
4,181
157,568
2,539
2,751
C8H10ClN3O
C10H14ClN3
C10H14ClN3O
C10H12ClN3O3
C12H15ClN4O2
C12H15ClN4O2
C12H17ClN4O2
C12H17ClN4O2
C12H17ClN4O2
C12H17ClN4O2
C14H15Cl2N3
39,021
31,224
11,375
41,687
53,831
7,716
C12H16ClN3O4
C11H14Cl2N4O2
C16H17Cl2N5O2
C15H20ClN5O6
C18H21Cl2N5O2
C23H28Cl2N6O3
C22H27Cl2N7O5
27,919
39,050
1,988
9,882
Mass error and Isotope ratio difference data are reported as determined by the MasterView™ software.
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Figure 3. Nitenpyram fragmentation analysis (top) and the nitenpyram product ion spectrum (bottom)
that was acquired with a collision energy (CE) of 35 15 V. Proposed primary fragments are denoted
P1 → P9. Higher order fragments are denoted by a lower case letter (e.g., P3a).
30 15 V the spectrum was a complex mixture of primary and
higher-order product ions that was difficult to interpret (Fig. 3).
To facilitate thorough characterization dedicated product ion
scans were acquired at discrete collision energies (10, 20, 30,
40 V; Supplementary Fig. S2, Supporting Information).
The first product ions identified were a series initiating from
loss and decomposition of the nitro group. P1 and P2 represent
the neutral and radical loss of NO2, respectively, with the
neutral loss to the carbocation dominating at higher collision
energies. It was proposed that P1a forms from P1 via the
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M. Noestheden, S. Roberts and C. Hao
neutral loss of ethylene, also at higher collision energies. P1
and P2 showed secondary and tertiary fragmentation
products that terminated with the neutral loss of HCl.
The P3 fragmentation series started from the breakdown of
the nitro group via sequential loss of hydroxide from the
protonated nitenpyram.[14,15] It is proposed that P3a loses
neutral HCN to give the carbocation P3b. Note that P1b and
P3b are structural isomers and it is not clear if one or both of
these fragmentation products are formed.
The product ions at m/z 171.0675 (P4) and 169.0518 (P5) likely
originate from a pyridine-protonated nitenpyram (see Fig. 1 for
numbering of nitenpyram atoms). The more abundant P5 could
form via a six-membered transition state that leads to imine
formation, with the loss of NO2 and acetylenamine providing
an entropic driving force. Alternatively, a five-membered
transition state with proton transfer between N2 and N3 could
yield P4. The high mass accuracy for these (and other) product
ion assignments was attributed to their low relative response
(<2% of the base peak). It should be noted that the above
proposals do not preclude alternate structures/mechanisms.
Regardless, the conservation of these product ions (and
analogues) throughout the observed nitenpyram reaction
products underscores their diagnostic potential.
Assessing the smaller m/z product ions (P7–P9) started with
m/z126.0096, which was proposed as a homolytic fragmentation
from N2-protonated nitenpyram (P7). The same product ion
was observed for the fragmentation of the structurally related
NNI imidacloprid and its metabolites.[16]
An understanding of the other small m/z nitenpyram
product ions was obtained via a thorough characterization of
Deg_01 (Fig. 4). The empirical formula for Deg_01 suggested
it was formed by cleavage between N2 and C9 to give the free
secondary amine (P4 in Fig. 3). The product ion spectrum for
Deg_01 was information-rich under m/z 130, with the majority
Figure 4. Deg_01 fragmentation analysis (top) and the Deg_01 product ion spectrum (bottom)
that was acquired with a CE of 35 15 V. Detailed analyses of the fragmentation pathways of
Deg_01 were used in the proposal of nitenpyram reaction products. Proposed primary
fragments are denoted D1–1 → D1–4. Higher order fragments are denoted by a lower case letter
(e.g., D1–3a).
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Nitenpyram degradation in finished drinking water
of the observed product ions shared with nitenpyram. However,
the lower overall complexity of the Deg_01 spectrum (relative to
nitenpyram) permitted the assignment of detailed fragmentation
patterns with increased confidence. It was postulated that an
understanding of these small m/z product ions would prove
useful based on potentially diagnostic shared product ions
observed for nitenpyram and its reaction products (vide infra).
The key fragments for Deg_01 were D1–1, D1–2 and D1–3,
which differ nominally by 1 Da. Similar to P7 of nitenpyram
(Fig. 3), these three product ions were observed for the
imidacloprid and its metabolites.[16] These product ions
resulted from different fragmentation mechanisms between
C6 and N2 of Deg_01. The D1–1 carbocation (m/z 126.0105)
forms through neutral loss of ethylamine and permits
isomerization to a tropylium-like intermediate (Fig. 4). This is a
well-established mechanism for the fragmentation of benzyl
cations and their derivatives.[15,17] Following isomerization two
different product ion pathways were proposed. The first
involved the sequential neutral loss of HCl (D1–1a) and ethylene
(D1–1b), with the latter characteristic of tropylium fragmentation.
The second pathway involved loss of neutral HCN (D1–1c),
likely in a mechanism analogous to the loss of neutral ethylene.
D1–1c then appeared to yield product ions corresponding to
the neutral loss of HCl (D1–1d) and ethylene (D1–1e).
shift of m/z 34 for all of the proposed nitenpyram product ions
in Fig. 3 (Supplementary Fig. S3, Supporting Information).
Proposed structures D14–7–D14–9 provided strong evidence
that the second chlorine was added to the pyridine ring and
D14–6 confirmed that the modification was not on the
C9/C11 olefin. While no product ions were observed that
definitively localized the position of the second chlorine on the
pyridine ring, D14–7b suggested that it was on C2 or C3. This
assignment was based on the proposed neutral loss of HCN that
mechanistically involved C5 and the fact that C1 and C4 did not
have hydrogens to lose.
Deg_16 corresponded to the net addition C5H2ClN to
nitenpyram, which represented the addition of chloropyridine.
As with Deg_14, Deg_16 possessed an MS/MS spectrum with
similar features to nitenpyram, although this time a nominal
shift ofm/z 111 was observed for all oftheproposed nitenpyram
product ions in Fig. 3 (Supplementary Fig. S4, Supporting
Information). To aid in the identification of diagnostic product
ions for Deg_16, the monoisotopic mass and the strong A + 2
isotope resulting from the presence of two chlorines were
subjected to MS/MS scanning. Analyzing the A + 2 isotope
clearly showed when fragmentation involved the loss of one
chlorine, as the resulting m/z was a doublet due to the loss
of either 35Cl or 37Cl during fragmentation. Product ions
D16–5 and D16–7–D16–9 demonstrated that the aliphatic
tertiary amine and chloropyridine were, respectively,
unchanged from nitenpyram. This meant the modification
was present on the olefinic side of N2. All of the analogous
product ions associated with fragmentation of the nitro group
of nitenpyram were observed for Deg_16 (D16–1–D16–3). As
well, D16–3c and D16–3d were proposed as product ions that
were not observed for nitenpyram. It was postulated that these
product ions were formed as a result of the addition of
chloropyridine at C10. Further supporting this were the
nitenpyram product ions analogous to D16–1b/3b. For
nitenpyram they were proposed as structural isomers, but for
Deg_16 they were clearly differentiated due to the proposed
substitution of chloropyridine at C10.
D1–2 and D1–3 resulted from fragmentation of the
pyridine-protonated Deg_01. Moving beyond the site of
protonation, D1–2 and D1–3 are the homo- and heterolytic
cleavage products, respectively, between C6 and N2. D1–2 and
D1–3 fragment further via neutral loss of HCl to give D1–2a
and D1–3a. Neutral loss of the alkylamine side chain was
proposed as the origin of product ion D1–4. Following from these
proposed fragmentation pathways for Deg_01, fragmentation
pathways P7–P9 were assigned for nitenpyram (Fig. 3).
Proposed structures of nitenpyram degradation products
Using the detailed product ion analysis of nitenpyram and
Deg_01, a review of the reaction products in Table 1 was
conducted and structures were proposed for a total of six reaction
products that had strong MS/MS spectral support (Fig. 5).
The empirical formula for Deg_14 suggested the substitution
of chlorine for hydrogen (Table 1). This formula was supported
by the MS/MS spectrum for Deg_14, which displayed a nominal
Closely related to Deg_16 was Deg_18, which shared a
number of product ions and displayed several that were
nominally shifted by m/z 28 from those observed for Deg_16
(Supplementary Fig. S5, Supporting Information). The
empirical formula and observed product ions suggested the
Figure 5. Structures of the nitenpyram reaction products identified in
unpreserved finished drinking water. Only those reaction products
that had strong MS/MS spectral support were discussed herein.
Rapid Commun. Mass Spectrom. 2016, 30, 1653–1661
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M. Noestheden, S. Roberts and C. Hao
net addition of C2H4 to Deg_16, with the modification
proposed at C11 (Fig. 5). The presence of D18–4, D18–5 and
D18–7–D18–9 again provided evidence that the modification
was on the olefinic side of N2. In tandem with all of the
analogous product ions shared with Deg_16, strong support
for modification at C10 was obtained by comparing D16–3a/b
to the predicted structures for Deg_18. If C10 was modified as
proposed, then the formation of analogous product ions would
be expected to differ from Deg_16, which was the observed
behavior of Deg_18. There was an m/z value that suggested a
product ion similar to D16–3a, but the experimental m/z was
off by one hydrogen. If, as proposed, C10 of Deg_16 was
modified by the addition of ethylene, then D18–3f was the
plausible product ion that would follow a nitro fragmentation
similar to D18–3. The absence of product ions analogous to
D16–3b provided strong support for the modification of C10.
Starting from the proposed D18–3f it would not be favorable
to form a product ion analogous to D16–3b, as there was not a
strong leaving group present to facilitate this mechanism.
Deg_03 and Deg_04 are related structures that followed from
oxidation of the C9/C11 olefin (Fig. 5). The product ion spectra
and empirical formula of Deg_04 supported the presence of an
unmodified benzyl chloropyridine (D4–1–D4–4; Supplementary
Fig. S6, Supporting Information). After evaluating the proposed
empirical formula and the plausible reactivity of nitenpyram it
was deduced that C9 of the nitenpyram olefin was fully oxidized
to the urea derivative Deg_04 (Fig. 5). This assignment was
strongly supported by D4–7/8. Also supporting the proposed
Deg_04 structure was the ratio of D4–6:D4–9. For nitenpyram
this ratio favoured D4–9 (P5, Fig. 3), while, for Deg_04, it was
reversed with a preference for D4–6 due to the loss of the more
favourable leaving group (methyl isocyanate). It was also
viewed as diagnostic that the ratio of D4–1:D4–2:D4–3
favoured D4–3, whereas D4–1 (P7, Fig. 3) was favoured for
nitenpyram. This likely occurs due to the formation of the urea
functional group and the loss of the strongly electron-
withdrawing nitro group. The resulting decreased basicity of
N2/N3 would make the pyridine nitrogen (relatively) more
basic and. Thus, more likely to be the site of proton-adduct
formation.
oxidation/hydrolysis and reaction with Cl2 lead to the
observed rapid loss of nitenpyram in unpreserved finished
drinking water. For instance, Deg_14 clearly suggests a direct role
for Cl2, while Deg_03/04 strongly suggests oxidative/hydrolytic
mechanisms.
CONCLUSIONS
While not exhaustive in the characterization of the observed
degradation products, the results presented herein show that
nitenpyram degradation in unpreserved finished drinking
water is likely mediated by a combination of oxidation/
hydrolysis and reaction with Cl2. Structures for a variety of
reaction products were proposed that all point to the
C9/C11 olefin as the key labile site. The proposed reaction
products are consistent with reported metabolites of the
structurally related NNI, imidacloprid.[2,19] Such similarities
highlight the importance of identifying these reaction
products given that the toxicity of NNIs to pollinators has been
linked to NNI metabolites.[5] Based on the proposed
degradation mechanisms, the identified nitenpyram reaction
products in finished drinking water could also be present in
aquatic environments and water treatment facilities. Thus,
identifying these degradation products will aid in evaluating
the overall risks/impact of NNIs to pollinators.
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SUPPORTING INFORMATION
Additional supporting information may be found in the online
version of this article at the publisher’s website.
Rapid Commun. Mass Spectrom. 2016, 30, 1653–1661
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