Ultraviolet degradation of procymidone - Structural characterization by gas chromatography coupled with mass spectrometry and potential toxicity of photoproducts using in silico tests

Article abstract of DOI:10.1002/rcm.6598

RATIONALE Procymidone is a dicarboximide fungicide mainly used for vineyard protection but also for different crops. The structural elucidation of by-products arising from the UV-visible photodegradation of procymidone has been investigated by gas chromatography coupled with mass spectrometry. The potential toxicities of photoproducts were estimated by in silico tests. METHODS Aqueous solutions of procymidone were irradiated for up to 90 min in a self-made reactor equipped with a mercury lamp. Analyses were carried out on a gas chromatograph coupled with an ion trap mass spectrometer operated in electron ionization and methanol positive chemical ionization. Multistage collision-induced dissociation (CID) experiments were performed to establish dissociation pathways of ions. Toxicities of byproducts were estimated using the QSAR T.E.S.T. program. RESULTS Sixteen photoproducts were investigated. Chemical structures were proposed mainly based on the interpretation of multistage CID experiments, but also on their relative retention times and kinetics data. These structures enabled photodegradation pathways to be suggested. Only three photoproducts remain present after 90 min of irradiation. Among them, 3,5-dichloroaniline presents a predicted rat LD50 toxicity about ten times greater than that of procymidone. CONCLUSIONS 3,5-Dichloroaniline is the only photoproduct reported in previous articles. Eight by-products among the sixteen characterized might be as toxic, if not more, than procymidone itself considering the QSAR-predicted rat LD50. Copyright 2013 John Wiley & Sons, Ltd. Copyright

Full text of DOI:10.1002/rcm.6598

Research Article
Received: 25 February 2013
Revised: 9 April 2013
Accepted: 11 April 2013
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2013, 27, 1505–1516
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6598
Ultraviolet degradation of procymidone – structural
characterization by gas chromatography coupled with mass
spectrometry and potential toxicity of photoproducts using
in silico tests
Ahmad Rifai1,2, Yasmine Souissi1, Christophe Genty1, Carine Clavaguera1,
Sophie Bourcier1, Farouk Jaber2 and Stéphane Bouchonnet1
*
1Laboratoire des Mécanismes Réactionnels UMR-7651, Ecole Polytechnique, 91128 Palaiseau Cedex, France
2Laboratoire d’Analyse des Pesticides et des Polluants Organiques - Commission Libanaise de l’Énergie Atomique - Centre
National de Recherches Scientifiques - Beyrouth, Liban
RATIONALE: Procymidone is a dicarboximide fungicide mainly used for vineyard protection but also for different crops.
The structural elucidation of by-products arising from the UV-visible photodegradation of procymidone has been
investigated by gas chromatography coupled with mass spectrometry. The potential toxicities of photoproducts were
estimated by in silico tests.
METHODS: Aqueous solutions of procymidone were irradiated for up to 90 min in a self-made reactor equipped with a
mercury lamp. Analyses were carried out on a gas chromatograph coupled with an ion trap mass spectrometer operated
in electron ionization and methanol positive chemical ionization. Multistage collision-induced dissociation (CID)
experiments were performed to establish dissociation pathways of ions. Toxicities of byproducts were estimated using
the QSAR T.E.S.T. program.
RESULTS: Sixteen photoproducts were investigated. Chemical structures were proposed mainly based on the
interpretation of multistage CID experiments, but also on their relative retention times and kinetics data. These structures
enabled photodegradation pathways to be suggested. Only three photoproducts remain present after 90 min of
irradiation. Among them, 3,5-dichloroaniline presents a predicted rat LD50 toxicity about ten times greater than that
of procymidone.
CONCLUSIONS: 3,5-Dichloroaniline is the only photoproduct reported in previous articles. Eight by-products among
the sixteen characterized might be as toxic, if not more, than procymidone itself considering the QSAR-predicted rat
LD50. Copyright © 2013 John Wiley & Sons, Ltd.
Procymidone is a dicarboximide fungicide mainly used for
vineyard protection but also for strawberry, flowers and
various ornamental cultivations.[1,2] According to the
European Commission directive 2006/132/EC, all Member
States of the European Union (EU) should amend or
withdraw existing authorizations for plant protection
products containing procymidone as an active substance. In
2010, the EU set the Maximum Residue Level (MRL) of
procymidone in crops at 0.02 mg/kg.[3] Procymidone presents
anti-androgen toxicity and ability to induce developmental
and/or reproductive dysfunctions by interfering with the
endocrine system of exposed organisms[4–7] and is a member
of the endocrine disruptor family.[8] The United States
Environmental Protection Agency reported that procymidone
and its main metabolite, 3,5-dichloroaniline (3,5-DCA), are
carcinogenic products with dietary assessment.[9] Thus, the
detection of this pesticide and its metabolites in environmental
samples is a priority in quality control. The transformation of
pesticides in the environment is a highly complex process
affected by many physical and chemical factors.[10] According
to Burrows et al., most pesticides are resistant to chemical and/
or photochemical degradation under typical environmental
conditions and very little information is available regarding
their natural degradation processes, the quality, structure
and biological impact of their degradation products.[11]
Previous studies devoted to the degradation of procymidone
have been focused on photocatalytic degradation techniques.
Hustert and Moza studied the photochemical degradation of
procymidone and viniclozolin in water using a mercury
lamp with l ≥290 nm. This study established that the
addition of Fe2O3 and TiO2 catalyzes the photodegradation
of both compounds and that the presence of humic and fulvic
acids (naturally extracted by the water from the soil
constituents) increases the rate of photodegradation.
Monodechlorination, didechlorination and isomerization
are the major photo degradation pathways.[12] Schwack
* Correspondence to: S. Bouchonnet, Laboratoire des
Mécanismes Réactionnels UMR-7651, Ecole Polytechnique,
91128 Palaiseau Cedex, France.
E-mail: stephane.bouchonnet@polytechnique.edu
Rapid Commun. Mass Spectrom. 2013, 27, 1505–1516
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A. Rifai et al.
et al. studied the potential of procymidone to undergo
photo-induced processes in the presence of different
organic groups. Their objectives were to find an
explanation for the formation of bound residues in plant
cuticles in the presence of polymer molecules, waxes and
cutin. In isopropanol, cyclohexane and cyclohexene,
photodegradation led to substitution of a chlorine atom
by a solvent molecule.[13] Vanni and co-workers studied
the degradation of procymidone in agricultural land under
various pH and temperature conditions. They reported
that the total degradation of procymidone occurred at
alkaline pH and at high temperature in a few days; the
main metabolite detected in their studies was 3,5-DCA.[14]
The present study aims to identify photodegradation
products of procymidone in water and to assess their
potential toxicity. It is now well admitted that sunlight
absorbance in water may be significantly modified by the
presence of dissolved organic matter but we consider here
that photodegradation may start as soon as the aqueous
solution of herbicide is sprayed on leaves and fruits, under
intense sunlight irradiation. This study was first devoted
to the structural elucidation of photoproducts using
multiple-stage stage mass spectrometry coupled with gas
chromatography (GC/MSn). Structural elucidation of
photoproducts was performed on the basis of the collision-
induced dissociations observed in both electron ionization
and positive chemical ionization modes. Photolysis mechanisms
were also suggested to explain the formation of the
photoproducts formed from irradiated procymidone in water.
An approach of quantitative structure–activity relationship
(QSAR) was used to assess the potential toxicity effect of
procymidone and of its photodegradation products. For that
purpose we used a Toxicity Estimation Software Tool (T.E.S.T.),
a U.S. Environmental Protection Agency developed program,
to predict mutagenicity, oral rat lethal doses, developmental
toxicity as well as growth inhibition concentrations and lethal
doses of aquatic organisms.
was first prepared in ACN. This solution was then used to
prepare working solutions in water at 5.0 mg/L. This high
concentration was used for structural investigations on
photoproducts as multi-stage mass spectrometry experiments
require relatively high analyte concentrations. Aqueous
solutions at 0.5 mg/L were used for kinetic measurements.
Photolysis experiments
Photolysis experiments were carried out in a self-made
reactor equipped with a high-pressure mercury lamp (HPL-N
125 W/542 E27 SC; Phillips, Ivry-sur-Seine, France) delivering
radiation at wavelengths ranging from 200 to 650 nm. The
absorbance spectrum of procymidone displays an absorption
maximum between 200 and 208 nm.[15] According to the
manufacturer’s data, the incident radiation flux was 6200 lm.
The reactor consists of six quartz tubes of 120 mL arranged in
a circle around the lamp and immersed into a sonicator
(AL04-12, Advantage-Lab, Switzerland) filled with deionized
water. During experiments, the reactor was regularly cooled
by addition of ice to avoid uncontrolled heating of the
irradiated solutions and to maintain a constant temperature of
25ꢁ 3 ꢀC. For each experiment, 50 mL of a solution of
procymidone (see above) were used.
Sample preparation
After photolysis, 6 mL of solution were taken from the quartz
tube and transferred into a separating funnel. Methylene
chloride (6 mL) was added to the solution which was then
shaken and left to settle for 10 min. The organic phase was
collected and dried under a gentle nitrogen stream and the
dry residue was dissolved into 200 mL of methylene chloride
for GC/MS analysis.
GC and MS operating conditions
Analyses were carried out on
a Varian 450GC gas
chromatograph equipped with a CP8400 autosampler and
coupled with a VARIAN 240MS ion trap mass spectrometer
operated in the internal ionization mode. A 60-m-long ’VF-
Xms’ capillary column (VARIAN) with an i.d. of 0.25 mm
and a film thickness of 0.25 mm was used. For all analysis,
2 mL of solution were injected automatically in the splitless
mode at a rate of 50 mL s–1. The injector temperature was
set to 280 ꢀC and high-purity helium (99.999 %) was used
as carrier gas at a constant flow of 1.4 mL min–1. The
column oven temperature was programmed from an initial
temperature of 50 ꢀC held during 1 min, increased to
280 ꢀC at 15 ꢀC/min, held for 5 min at 280 ꢀC, then
increased at 10 ꢀC/min to a final temperature of 300 ꢀC,
which was held for 20 min, for a total analysis duration
of 43 min. The manifold, the ion-trap electrodes and the
transfer line temperatures were respectively held at 100,
200 and 280 ꢀC. The mass spectrometer was calibrated
with the ions resulting from electron ionization (EI) of
perfluorotributylamine. Chemical ionization (CI) experiments
were performed using methanol as the reagent. Spectra were
recorded using the automatic gain control (AGC) function with
target values fixed at 20 000 and 5000 for EI and CI,
respectively. The filament emission current was set at 10 mA in
EI and 50 mA in CI. The electron multiplier voltage was
automatically optimized at 2200 V for a gain value of 105. Full
EXPERIMENTAL
Chemicals and reagents
Procymidone (99% purity) was purchased from Sigma
Aldrich (St Quentin Fallavier, France). The chemical structure
of procymidone is shown in Fig. 1. Chromatographic grade
solvents (99.99% purity), methylene chloride and acetonitrile
(ACN), were also purchased from Sigma Aldrich.
Considering theꢀ poor solubility of procymidone in water
(4.5 mg/L at 25 C), a solution of procymidone at 100 mg/L
Figure 1. Chemical structure of procymidone and nature of
the first excited state (CC2 level).
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UV degradation of procymidone
scan mass spectra were acquired recording ions from m/z 50 to
500 at a scan rate of 0.33 s/scan, each spectrum resulting
from ion acquisitions on 5 microscans. In multi-stage mass
spectrometry (MSn) experiments, precursor ions were
stored with a Paul stability parameter (qz) of 0.30 and
fragmented by collision-induced dissociation (CID) with
activation energies ranging from 0.30 to 0.80 V in the
resonant excitation mode.
RESULTS AND DISCUSSION
Characterization of the photoproducts of procymidone
A series of experiments was performed with eight irradiation
times ranging from 0 to 90 min: 0, 5, 10, 15, 20, 40, 60 and
90 min. The comparison of the corresponding chromatograms,
recorded in the EI mode, showed that the relative abundances
of photoproducts vary significantly according to the irradiation
time. The relative abundances of photoproducts are plotted as a
function of the irradiation time in Fig. 2. A precise quantitation
of photoproducts was not possible given the lack of standards.
Thus abundances were just estimated by integrating
chromatographic peaks on the total ion current (TIC); they are
expressed in Fig. 2 in an arbitrary unit proportional to the
TIC. The abundance of procymidone was not plotted
because it was out of scale. Disappearance of procymidone
follows a pseudo-exponential type curve. Only 5% of the
initial amount of procymidone is still detected after 60 min
of irradiation. Procymidone is no longer detected after
90 min of irradiation. Among the 16 photoproducts detected,
13 are formed as soon as irradiation begins; their con-
centrations reach maximum values for photolysis times
around 10 min before decreasing to reach trace amounts after
40 min of irradiation. The three other compounds, referred as
1, 2 and 7 (see numbering in Table 1), are also detected as
soon as irradiation begins; their concentrations rapidly
increase during the first 30 min and go on increasing at a
Quantum chemistry calculations
Geometry optimizations were performed at the DFT/
B3LYP-D3 level, including the new implementation of
dispersion corrections[16,17] in the TURBOMOLE 6.4
package.[18] The minima were characterized by vibrational
frequency calculations. All the calculations were carried out
with the polarized valence-triple-zeta TZVPP basis set. The
most energetically stable geometry of the molecule (Fig. 1)
was used for the calculation of the electronic excited states
by both the CC2 coupled cluster model and the time-
dependent density functional theory (TD-DFT) using the
B3LYP-D3 functional. The CC2 equations are an
approximation to the coupled cluster singles and doubles
(CCSD) ones.[19] This method is considered as an accurate
tool for the investigation of the excited states of molecules.[20]
Regarding the nature and the transition energies, both
methods provide similar results that will be described
in the Results section (see Supplementary Table S1,
Supporting Information).
Computer-aided toxicity prediction
The Toxicity Estimation Software Tool (T.E.S.T.) is an Envi-
ronmental Protection Agency online available computerized
predictive system with Quantitative Structure Activity
Relationships (QSAR) mathematical models.[21] T.E.S.T. has a
variety of toxicity endpoints used to predict chemical toxicity
values from their physical properties such as molecular
structure. The T.E.S.T. model uses a simple linear function of
molecular descriptors such as the octanol-water partition
coefficient, molecular weight or the number of benzene rings
(see Eqn. (1)):
Toxicity ¼ ax1 þ bx2 þ c
(1)
where x1 and x2 are independent descriptor variables and
a, b, and c are fitted parameters. Models for assessing
toxicity solely from molecular structure are based on
information-rich structural descriptors that quantify
transport, bulk, and electronic attributes of a chemical
structure. Besides molecular weight, the QSAR model
employs size-corrected E-values for quantification of
molecular bulk. The size-corrected E-values are computed
from
a
rescaled count of valence electrons.[22]
Electrotopological state values (E-values), as numerical
quantifiers of molecular structure, encode information
about the electron content (valence, sigma, pi and lone-
pair), topology, and environment of an atom, or a group
of atoms, in a molecule.[22] The predicted toxicity is
estimated by taking an average of the predicted toxicities
from the above QSAR methods, provided the predictions
are within the respective applicability domains.
Figure 2. Abundances of photoproducts (arbitrary unit) as a
function of the irradiation time (refer to Table 1 for the
numbering of compounds). Abundances of major compounds
(1, 2, 3, 5, 9, 11 and 14) are plotted at the top of the figure;
abundances of minor compounds (4, 6, 7, 8, 10, 12, 13, 15 and
16) are plotted at the bottom of the figure.
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A. Rifai et al.
Table 1. Molecular weights measured, retention times, relative abundances after 15 min of irradiation and main MSn
transitions recorded in GC/MS with both CI and EI ionization modes for photolysis products of procymidone. Suggested
structures based on mass spectra interpretation are given in the right-hand column
Molecular
weight
Retention time
(min)
Relative
Main transitions in
Main transitions in
Chemical structure
proposed
measured
abundancea
CI-MSn (m/z)
EI-MSn (m/z)
1
2
3
4
187
161
231
285
17.00
20.27
25.45
28.80
11.5
10.3
12.6
1.7
188 ! 160 (-28)
188 ! 153 (-35)
153 ! 125 (-28)
125 ! 90 (-35)
187 ! 159 (-28)
159 ! 124 (-35)
124 ! 97 (-27)
162 ! 127 (-35)
127 ! 126 (-1)
126 ! 100 (-26)
161 !134 (-27)
134 ! 99 (-35)
161 !126 (-35)
232 ! 162 (-70)
162 ! 127 (-35)
231 !187 (-44)
187 ! 152 (-35)
231 !161 (-70)
161 !126 (-35)
286 ! 258 (-28)
258 ! 230 (-28)
230 ! 202 (-28)
285 ! 257 (-28)
257 ! 187 (-70)
187 ! 152 (-35)
187 ! 145 (-42)
145 ! 110 (-35)
5
6
273
283
29.71
30.26
5.9
2.3
274 ! 256 (-18)
256 ! 228 (-28)
274 ! 246 (-28)
246 ! 218 (-28)
273 ! 245 (-28)
245 ! 161 (-84)
161 !126 (-35)
284 ! 186 (-98)
284 ! 256 (-28)
256 ! 238 (-18)
238 ! 221 (-28)
221 !181 (-40)
181 !165 (-18)
283 ! 255 (-28)
255 ! 254 (-1)
254 ! 226 (-28)
226 ! 191 (-35)
7
8
287
271
30.42
30.54
1.4
1.9
287 ! 259 (-28)
259 ! 189 (-70)
189 ! 161 (-28)
161 !126 (-35)
288 ! 270 (-18)
270 ! 242 (-28)
242 ! 240 (-2)
271 ! 256 (-15)
256 ! 228 (-28)
228 ! 200 (-28)
200 ! 265 (-35)
272 ! 256 (-16)
272 ! 244 (-28)
9
283
30.99
5.6
284 ! 256 (-28)
256 ! 241 (-15)
241 ! 203 (-38)
203 ! 185 (-18)
203 ! 158 (-45)
158 ! 130 (-28)
283 ! 268 (-15)
268 ! 240 (-28)
240 ! 205 (-35)
283 ! 241 (-42)
241 ! 213 (-28)
213 ! 178 (-35)
(Continues)
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UV degradation of procymidone
Table 1. (Continued)
Molecular
weight
Retention time
Relative
Main transitions in
Main transitions in
Chemical structure
proposed
measured
(min)
abundancea
CI-MSn (m/z)
EI-MSn (m/z)
10
299
31.39
5.5
300 ! 258 (-42)
258 ! 240 (-18)
258 ! 230 (-28)
230 ! 186 (-44)
299 ! 282 (-17)
299 ! 257 (-42)
257 ! 256 (-1)
256 ! 221 (-35)
11
12
283
315
31.57
31.73
15.3
5.3
284 ! 256 (-28)
256 ! 238 (-18)
256 ! 228 (-28)
228 ! 193 (-35)
193 ! 178 (-15)
193 ! 158 (-35)
283 ! 268 (-15)
268 ! 240 (-28)
240 ! 205 (-35)
315 ! 300 (-15)
300 ! 272 (-28)
272 ! 244 (-28)
315 ! 298 (-17)
298 ! 270 (-28)
270 ! 242 (-28)
316 ! 298 (-18)
298 ! 270 (-28)
270 ! 242 (-28)
13
14
315
283
31.77
31.97
5.3
9.7
315 ! 300 (-15)
300 ! 272 (-28)
272 ! 244 (-28)
315 ! 298 (-17)
298 ! 270 (-28)
270 ! 242 (-28)
283 ! 268 (-15)
268 ! 240 (-28)
240 ! 205 (-35)
283 ! 256 (-27)
256 ! 228 (-28)
228 ! 193 (-35)
316 ! 298 (-18)
298 ! 270 (-28)
270 ! 242 (-28)
284 ! 256 (-28)
256 ! 238 (-18)
238 ! 203 (-35)
203 ! 167 (-36)
15
16
283
281
32.46
33.90
0.5
5.2
284 ! 256 (-28)
256 ! 238 (-18)
238 ! 223 (-15)
238 ! 200 (-38)
200 ! 169 (-31)
200 ! 154 (-46)
283 ! 256 (-27)
256 ! 228 (-28)
228 ! 193 (-35)
281 ! 253 (-28)
253 ! 252 (-1)
252 ! 217 (-35)
281 ! 229 (-52)
229 ! 201 (-28)
201 ! 200 (-1)
200 ! 165 (-35)
282 ! 264 (-18)
264 ! 229 (-35)
a% of the total of all the product abundances.
slower rate until the end of the experiment; 1, 2 and 7 are the
only by-products remaining in significant amounts in water
after 90 min of irradiation of procymidone.
All degradation products were detected with a significant
abundance (≥2%) in the solution corresponding to an
irradiation time of 15 min. This solution was then chosen to
perform MSn experiments on all the photoproducts. The
corresponding chromatogram is displayed in Fig. 3. Peaks
that are not numbered correspond to compounds that are
detected in the non-irradiated reference solution of
procymidone (corresponding to t = 0), which has undergone
the same sample preparation process. Only the compounds
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A. Rifai et al.
Figure 3. Chromatogram (EI mode) of an aqueous solution of procymidone at
5 mg/L irradiated for 15 min. Non-numbered peaks correspond to compounds
also detected in the non-irradiated solution.
detected with a signal-to-noise ratio higher than 10 were
investigated. few articles report the analysis of
to the NIST spectral database so that no particular
structural investigation was carried out.[28] Compounds 6,
9, 11, 14 and 15 have the same nominal molecular weight
(MW = 283 amu for the major isotopomer including two
35Cl atoms) as procymidone and were assumed to be
isomers of the latter. A mechanism has been suggested
(see Fig. 4) to rationalize isomerization of procymidone
under irradiation. The TD-DFT and CC2 calculations
reveal that the first excited state of procymidone
corresponds to a triplet spin state with an excitation from
the dichlorophenyl group to the two C = O groups (Fig. 1).
Consequently, it can be considered that the oxygen atoms
constitute the most favorable ionization sites of
procymidone. In the gas phase, electron ionization of a
carbonyl function is followed by the heterolytic cleavage
of the bond(s) in alpha; this is referred as alpha cleavage
by mass spectrometrists.[29] We postulated that the same
alpha cleavage occurs in aqueous solution and is
accompanied by the aperture of the strained three-
centered ring. This leads to a distonic ion displayed in
the form of three mesomeric structures referred as a, b
and c at the left-hand side of Fig. 4. Like in mass
spectrometry, we considered that the reactivity induced
by the radical should be greater than that induced by the
charge, the latter being furthermore stabilized by
surrounding water molecules in the present experiment.
In aqueous solution, the radical can react with a water
molecule to abstract a hydrogen atom or a hydroxyl
A
procymidone using LC/MS coupling with an ESI source in
positive mode.[14,23,24] We did not succeed in detecting
procymidone in LC/MS whatever the device used: HPLC/
Q-TOF and nanoLC/FT-ICR. Nevertheless, the comparison
of the total ion currents of the photoproducts in the
chromatograms of the irradiated solutions with that of
procymidone in the reference solution suggests that more
than 90% of products are detected by GC/MS.
Nominal molecular weights of the photoproducts of
procymidone were easily determined. For each compound,
the heavier ion (often also the most abundant one) in the CI
mass spectrum was first assumed to correspond to the MH+
pseudo-molecular ion. This assumption was systematically
confirmed by the presence of an ion at m/z = M (assumed to
correspond to M+.) in significant abundance in the EI
spectrum. Molecular weights are reported in Table 1.
According to literature data devoted to the photolysis of
chlorinated compounds, the loss of the chlorine atom under
irradiation often constitutes the first step of the degradation
process.[25–27] In the case of procymidone, the isotopic
distributions observed for M.+ and MH+ ions of photoproducts
show that chlorine atoms were not removed by photolysis in
any case.
Compounds 1 and 2 were identified as 1,3-dichloro-
5-isocyanatobenzene and 3,5-dichlorobenzenamine,
respectively, with probabilities greater than 95% according
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UV degradation of procymidone
Figure 4. Mechanisms proposed to explain the isomerization of procymidone under irradiation in water.
radical. Both cases were considered from each mesomeric
form to provide six structures that may likely cyclize to
lead to carbocations better stabilized by inductive and
mesomeric effects. The cyclized structures are referred as
a1, a2, b1, b2, c1 and c2 in Fig. 4. In pure water, those six
even-numbered electron cations may each react with a
water molecule by capturing either a hydride H– or a
hydroxyl anion OH–. Considering both hypotheses leads
to the twelve structures displayed at the right-hand side
of Fig. 4. a11 and a12 result respectively from capture by
a1 of H– and of OH–. The same notation is used for
structures b11, b12, c11, etc.
They are six photoproducts detected with the same
nominal molecular weight (283 amu) as procymidone and
with close retention times (ranging from 30.26 to 32.46 min)
around that of procymidone (30.8 min). Figure 4 shows that
six compounds with the same nominal mass (283) as
procymidone (and thus assumed to be isomers of the latter)
can be formed according to the mechanisms suggested: water
loss from a12 leads to three isomers, water loss from a21
provides two isomers (one of which is common with one
issued from a12), water loss from b12 gives the only five-
membered ring isomer and water loss from c12 leads to two
tautomeric forms of isomers issued from a12. Compounds 6,
9, 11, 14 and 15 were assumed to correspond to these five
isomers. Their chemical structures were elucidated on the
basis of the MSn experiments performed in the EI mode. For
the molecular ion of each structure, the charge has been
assumed to be carried by one of the oxygen atoms according
to the results of quantum chemistry calculations (see above).
As shown in Fig. 5, alpha cleavage leads to distonic ions
whose reactivity is largely driven by the radical. 6 is the only
structure for which the alpha cleavage from M.+ leads to a
tertiary radical stable enough to permit monoxide elimination
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A. Rifai et al.
Figure 5. Dissociation pathways in EI-MSn of compounds 6, 9, 11, 14 and 15.
prior to radical elimination. 9 corresponds to the only structure
that can eliminate CH3. and CH2 = CH-CH3 following electron
ionization (transitions m/z 283 ! 268 and m/z 283 ! 241 in
Table 1). 11 corresponds to the structure that can only eliminate
latter, by a mechanism analogous to that proposed for the
formation of 4 from ionized procymidone (Fig. 5). EI-MSn
and CI-MSn transitions recorded for 7 are rationalized in
Supplementary Fig. S2 (see Supporting Information).
Concerning compound 3 (MW 231, eluted at 25.45 min), the
losses of 70 amu in CI-MS2 and 44 amu in EI-MS2 suggest that
a C3H7CO group is bound to the nitrogen atom. The chemical
structure including an isopropyl group displayed in Table 1
for 3 may be formed after photoionization of 4, as proposed
in Fig. 6. Kinetics data show that the formation of 7 and 3 is
accompanied by the disappearance of 4 (see above); this
strongly supports the hypothesis according to which 3 and
7 would result from photoionization of 4. EI-MSn and CI-
CH3 from M.+ (transition m/z 283 ! m/z 268 in Table 1). 14
.
.
corresponds to the structure that can eliminate CH3 and CH2 =
CH. from M.+ (transitions m/z 283 ! 268 and m/z 283 ! 256 in
Table 1). Finally, 15 corresponds to the structure that can
only eliminate CH2 = CH. from M.+ (transition m/z
283 ! 256 in Table 1). It is to be noted that Cl. losses are
not displayed in Fig. 4 because they result from homolytic
cleavages under high activation energies that do not
contribute to distinguish isomers in the present case.
Compound 4 (285 amu, see Table 1) could correspond to
one of the structures a11, b11 or c11, the latest corresponding
to the enolic form of a11, in Fig. 4. The fact that only one
isomer is detected rather suggests the symmetrical structure
displayed in Table 1 whose formation is easily explained by
consecutive captures of a hydrogen atom and a hydride ion
by ionized procymidone, according to the mechanism
detailed in Fig. 6. This hypothesis is supported by the fact that
the symmetrical structure proposed for 4 is the only one
allowing the transitions observed in CI-MSn (m/z 286 ! 258
230 ! 202); the corresponding mechanisms are given in
Supplementary Fig. S1 (see Supporting Information). These
three consecutive losses of 28 amu in CI-MSn are not possible
from a11, b11 nor c11. The formation of compound 7 (287 amu,
see Table 1) can be rationalized by consecutive additions of a
hydrogen atom and a hydride ion on 4 after ionization of the
MSn transitions recorded for
3
are rationalized in
Supplementary Fig. S2 (see Supporting Information). The
dissociation pathways of 3 in EI-MSn lead to ions at m/z 187
and 161 which correspond respectively to the ionized
compounds 2 and 1.
Compound 5 has a molecular weight of 273 amu; its
formation likely results from HO. addition on ionized
procymidone followed by CO elimination and hydride
addition, as displayed in Fig. 7. Figure 6 shows that three
isomers with molecular weight of 273 amu can be formed
from structures a, b and c (see Fig. 4) according to such a
mechanism. The consecutive transitions m/z 273 ! 245 ! 161
in CI-MS3 suggest that the detected isomer is that issued from
structure b in Fig. 7, based on the dissociation pathways
proposed in Supplementary Fig. S3 (see Supporting
Information). Compound 8 has a molecular weight of
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Copyright © 2013 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 1505–1516

UV degradation of procymidone
Figure 6. Mechanisms proposed to explain the formation of compounds 3, 4 and 7 from irradiated
procymidone in water.
271 amu; its formation likely results from HO. addition on
ionized procymidone followed by CO elimination and proton
elimination, as displayed in Fig. 7. Five isomers with a
nominal mass of 271 can be formed from structures a, b and
c according to such a mechanism. The only isomer allowing
consecutive losses of a methyl radical and two carbon
monoxide molecules in EI-MS4 (m/z 271 ! 256 ! 228 ! 200)
is one of those issued from structure a in Fig. 7 (see Table 1).
Dissociation pathways suggested to explain the recorded
dehydrogenation of procymidone. A mechanism involving
consecutive losses of H. and H+ from ionized procymidone
has been proposed in Fig. 8. Mechanisms corresponding to
the EI-MSn and CI-MSn transitions observed for 16 are
proposed in Supplementary Fig. S7 (see Supporting Information).
Toxicity prediction
In order to evaluate the toxicity of procymidone and its
photodegradation products, a toxicity simulation using in
silico tools was performed. We chose in this study only to
report the significant results, i.e. oral rat LD50 toxicity (Fig. 9).
We observed that the procymidone and all its identified
photodegradation products exhibit a developmental toxicity.
Procymidone was reported in the literature as being a
competitive antagonist to the androgen binding to their
receptor causing thereby development in male offspring.[30]
The effects on reproduction and the induction of testicular
tumors in a long-term rat study can be explained by the
effects of procymidone on the endocrine system.[31] A recent
study stated that this dicarboximide fungicide reduces
weights of the prostate and seminal vesicle.[32] Thus the
procymidone developmental toxicity prediction through the
QSAR simulation was coherent with the literature data. It is
to be noticed that all degradation products generated through
procymidone photolysis kept the 3,5-dichloroaniline (3,5-
DCA) on the molecule skeleton. The U. S. Environmental
EI-MSn and CI-MSn transitions for
8 are given in
Supplementary Fig. S4 (see Supporting Information).
Compound 10 (MW 299 amu) was detected at 31.39 min.
Based on the reactions in Fig. 4, five isomers with molecular
weight of 299 amu can be easily formed from photo-ionized
procymidone. The CI-MSn transitions, especially m/z
300 ! 258 and m/z 258 ! 240 (consecutive losses of CH2 = CO
and H2O) allowed a conclusion in favor of the structure
proposed in Table 1 (one of the structures issued from a22 in
Fig. 4), according to the dissociation pathways provided in
Supplementary Fig. S5 (see Supporting Information).
Compounds 12 and 13 have the same molecular weight of
315 amu, which, compared with the MW of procymidone
(283 amu), may likely correspond to the addition of two
hydroxyl groups and the loss of H2. Figure 4 displays three
isomers with MW 317 amu resulting from the addition of
two hydroxyl groups on procymidone: a22, b22 and c22.
Among them, only b22 can easily eliminate H2 through
oxidation of an alcohol function into an aldehyde one.
Compounds 12 and 13 provide exactly the same transitions
in EI-MSn and CI-MSn experiments (see Table 1) and have
very close retention times (31.73 and 31.77 min, respectively).
This led us to consider 12 and 13 as two diastereoisomers of
b22 (see Fig. 4) whose formation under irradiation would be
explained by the mechanism suggested in Fig. 8. The fact that
both compounds are formed in equal ratios is in good
agreement with this mechanism. The EI-MSn and CI-MSn
transitions perfectly match the structures proposed in Table 1
for 12 and 13; the corresponding dissociation pathways are
displayed in Supplementary Fig. S6 (see Supporting Information).
Compound 16 (281 amu) was assumed to result from
Protection Agency treats 3,5-DCA as
a cancer-causing
chemical based on the carcinogenicity of the structurally
related compound p-chloroaniline.[33] This metabolite was
reported as the final product of the degradation metabolic
pathway of dicarboximidic fungicides.[14] It also was assigned
as more toxic and persistent than one of its parent compounds:
vinclozolin. In particular, toxicity of 3,5-DCA has been shown
to be related to anti-androgenic activity.[34] The hypothesis of
developmental toxicity potential related to the 3,5-DCA
common part of all degradation products is then postulated.
Procymidone is of low acute toxicity by the oral route. Its
rat and mice oral LD50 was reported in the literature to be
>5000 mg/kg.[31] The displayed value of oral rat LD50 by
Rapid Commun. Mass Spectrom. 2013, 27, 1505–1516 Copyright © 2013 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

A. Rifai et al.
Cl
MW 273
N
Cl
O
Cl
Cl
OH
Cl
-
H
O
O
N
Cl
.
H
H2
O
R
O
+
+
N
+
Cl
- H
N
Cl
N
- CO
O
O
.
+
MW 271
O
OH
+
- H
a
OH
OH
Cl
N
Cl
O
MW 271
OH
Cl
N
Cl
Cl
Cl
O
-
H
O
O
MW 273
OH
R
.
H2O
+
.
H
+
N
Cl
N
N
Cl
- CO
Cl
+
O
O
O
+
- H
OH
OH
b
MW 283
N
Cl
MW 300
MW 272
O
MW 271
OH
Cl
MW 273
N
Cl
HOO
Cl
-
H
Cl
Cl
O
O
N
Cl
+
- H
.
H2
O
R
+
HOO
H
+
N
Cl
N
- CO
N
Cl
HOO
MW 271
O
+
HOO
+
- H
.
Cl
c
N
Cl
HOO
MW 271
Figure 7. Mechanisms proposed for the formation of compounds with molecular
weights of 271 and 273 amu from irradiated procymidone in water (structures a, b
and c are mesomeric forms resulting from the aperture of the five-membered ring
of ionized procymidone, see Fig. 3).
the simulation software cannot be taken into consideration
because the prediction does not represent an external
prediction as procymidone was present in the training
set. We observed a low acute toxicity as this endpoint
with values which varied for the several degradation
products between 1000 and approximately 6000 mg/kg
except for compounds 2 and 5 for which those values
were estimated at 302 and 590 mg/kg, respectively, is
thus considered as moderately toxic. Compound 2, the
3,5-DCA, was reported also in the literature as of
moderate acute toxicity in mice in coherence with the
QSAR-predicted rat LD50.[35] Based on the overall effects
on renal function and morphology, Lo et al.[36] found that
3,5-DCA is the most nephrotoxic of the dichloroanilines
and that it can cause acute kidney toxicity in rats which
may justify the predicted LD50.
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Copyright © 2013 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 1505–1516

UV degradation of procymidone
Figure 8. Mechanisms proposed to explain the formation of compounds 12, 13 and 16 from irradiated
procymidone in water.
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Copyright © 2013 John Wiley & Sons, Ltd. Rapid Commun. Mass Spectrom. 2013, 27, 1505–1516