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Study of Clethodim Degradation and By-Product Formation in Chlorinated

Water by HPLC

Article  in  Chromatographia · August 2005

DOI: 10.1365/s10337-005-0592-x

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Study of Clethodim Degradation and
By-Product Formation in Chlorinated Water
by HPLC

2005, 62, 133–137

P. Sandı́n-España, J. O. Magrans, J. M. Garcı́a-Baudı́n&

Plant Protection Department, INIA, Ctra. de La Coruña, km. 7.5, 28040, Madrid, Spain; E-Mail: baudin@inia.es

Received: 3 January 2005 / Accepted: 12 May 2005

Online publication: 27 July 2005

At the same time, it is able to oxidize and

chlorinate various organic functional
Abstract groups. Hypochlorite and chloramines
Two of the most commonly used chlorinating agents for water disinfection, hypochlorite and are the most commonly employed chlo-
chloramines, were employed to investigate the degradation of clethodim in conditions simu- rination sources [3]. During treatment,
lating tap water treatment. The main clethodim degradation products were identified by using residues of pesticides may also undergo
liquid chromatography (LC) coupled with mass spectrometry (MS). The main degradation transformations that may produce
process was oxidation to sulfoxide and then to sulfone. Degradation half-life was calculated for chemicals more or less toxic than the
both parent clethodim and the first degradation product, clethodim sulfoxide. Whereas some parent substances [4, 5]. The rate of
other different minor by-products were identified when the degradation occurs with either degradation and the products obtained
sodium hypochlorite or chloramines, no other chlorinated by-products were found under the by either reagent may also vary consid-
conditions tested. erably.
Whereas the degradation of many
pesticides in chlorinated water has been
widely studied [6, 7], degradation of
Keywords cyclohexanedione herbicides has not.
Column liquid chromatography-mass spectrometry This family of herbicides are fatty acid
Clethodim and degradation products synthesis inhibitors that work by inhibi-
Water disinfection by chlorination tion of acetyl CoA carboxylase [8, 9].
Tap water They are used at low-dose rates as they
are biologically active at very low con-
centrations. Furthermore, their polar
character makes them easily leached to
Introduction permitted levels of contaminants in groundwater and so potentially contam-
drinking water supplies. For example, inate drinking water sources.
Pesticide contamination of drinking EU Water and Drinking Water directives In a previous study, the rapid
water is one route of human exposure to provide that no individual pesticide may degradation of the herbicide tepraloxydim
pesticides that causes major concern. exceed 0.1 lg L)1 and that the sum of all in chlorinated water was reported [10]. This
Usually, consumers’ intake of water is contaminants present in a drinking water caused us to further study the degradation
concentrated in few sources of public tap sample may not exceed 0.5 lg L)1 [1, 2]. of other cyclohexanedione herbicides in
water supply. Contamination of these Environmental contamination of ground chlorinated water to identify the main
sources by small amounts of pesticides and surface natural waters used for degradation processes and characterize
may result in long-term exposure to these abstraction is the main route for drinking degradation products. Clethodim, ((±)-2-
contaminants. Health risk, for prolonged water contamination by pesticides. [(E)-1-[(E)-3-chloroallyloxyimino] propyl]-5
exposure to very low levels of pesticide, is However, before entering the tap water [2-(ethylthio)propyl]-3-hydroxy-cyclohex-2-
not always covered by routine toxico- system, water is submitted to physical enone)) is applied at 60–240 g ha)1 and is
logical tests performed for regulatory and chemical treatments for disinfection, used for post-emergence control of annual
purposes. Therefore, regulation tends to commonly by chlorination. Chlorine is a and perennial grasses in a wide range of
be very conservative with respect to the potent oxidant that acts as a bactericide. broad-leaved crops, vegetable crops, trees

Original Chromatographia 2005, 62, August (No. 3/4) 133

DOI: 10.1365/s10337-005-0592-x
0009-5893/05/08
2005 Friedr. Vieweg & Sohn/GWV Fachverlage GmbH

Fig. 1. Proposed degradation pathway of clethodim in chlorinated water
and vines. There are only a few studies
published on the degradation of this her-
bicide and none them address its degrada-
tion in chlorinated water. Photolytic and
acid catalyzed degradation of clethodim
has been reported to yield a number of
transformation products not fully charac-
terized [11, 12].
In the present paper, the results of an
investigation undertaken to study the
reaction kinetics of clethodim with
sodium hypochlorite and chloramines in
water are reported. Identification and char-
acterization of the resulting by-products was
performed to allow a degradation route
of this herbicide during water treatment
to be proposed. This should allow
refinement of the assessment of this her-
bicide with respect to potential drinking
water contamination.
Experimental
Chemicals and Reagents
Clethodim (Fig. 1a) ((±)-2-[(E)-1-[(E)-
3-chloroallyloxyimino]propyl]-5-[2-(eth-
ylthio)propyl]-3-hydroxy-cyclohex-2-en-
one)), (98% purity) was obtained from
ChemService (West Chester, PA, USA).
Stock standard solutions were prepared
by weighing and dissolving clethodim in
the minimum amount of acetonitrile fol-
lowed by water addition up to volume.
Standard solutions were stored at 4  C
in darkness and were used to prepare
more dilute standard solutions for cali-
bration. Stability of standard solutions
under these conditions was checked and
demonstrated for at least two months.
Acetonitrile and water used in liquid
chromatography and degradation studies
134
were of HPLC grade from Lab-scan
(Stillorgan, Co. Dublin, Ireland), analyt-
ical grade formic acid (98%) and sodium
hypochlorite (10%) were from Panreac
(Barcelona, Spain).
Equipment
For the kinetic experiments, HPLC with
a UV detector was used to measure
clethodim concentrations. The HPLC-
UV instrument consists in two pumps
(System Gold 126 Solvent Module), a
UV detector (166 Detector; UV: 200 to
290 nm) and an autosampler (System
Gold 508). Chromatographic data were
collected through a PC with System
Gold Software package (Beckman
Coulter Instruments, Inc., Fullerton,
CA 92634–3100). Compounds were
separated using a 4 lm Nova-Pak C18
column (150 · 3.9 mm, 60 Å; Waters
Corporation, Milford, Massachusetts).
To identify and characterize degrada-
tion and reaction products HPLC-ESI–MS
was used. The HPLC instrument HP
1100MSD (Agilent Technologies, Inc.,
Palo Alto, CA, USA) included a binary
solvent system, autosampler a diode array
detector and a mass detector with quad-
rupole analyzer. A 5 lm Zorbax C)18
column (50 · 2 mm; Agilent Technologies,
Madrid, Spain) was employed for chro-
matographic separation.
Kinetic Experiments
Sodium hypochlorite solutions (0.14 mM)
employed in degradation studies were
prepared from the commercial stock
solution and checked by iodometric
Chromatographia 2005, 62, August (No. 3/4)
titration. Chloramines solution (0.14 mM)
was prepared by the appropriate mixture
of sodium hypochlorite/ammonium
chloride (1:1.1, molmol)1). A small molar
excess of ammonium chloride was added
to ensure the complete formation of
chloramines [13–15]. All reactions inves-
tigated were carried out under a con-
trolled temperature of 25 ± 2  C
The reaction between clethodim and
the disinfectant solutions was studied
using a disinfectant to herbicide ratio of
10:1 (mol mol)1). Separate vials with
clethodim solution (0.014 mM, 500 lL)
were prepared. Aliquots of disinfectants
(0.14 mM, 500 lL) were added to each of
these samples. Finally, the reaction in
each vial was quenched with sodium
thiosulfate (100 lL, 10 mM) at scheduled
time intervals.
Blank samples containing only cle-
thodim (0.014 mM) were used to ensure
that no photodegradation or hydrolysis
occur during the experiment. Also, sam-
ples of clethodim (0.014 mM) and
sodium thiosulfate (10 mM) were tested
to ensure that no matrix effect was caused
by this quenching agent. Kinetic and
control samples were analysed by HPLC-
UV.
Analysis of Samples
Sample aliquots of 50 lL were
injected into the HPLC-UV system.
The mobile-phase was 60:39.9:0.1 aceto-
nitrile:water:formic acid at a flow rate of
1 mL min)1. The detector was set at the
maximum absorbance wavelength of cle-
thodim (255 nm). Under these conditions,
retention time of clethodim was 7.1 min,
2.2 min for the Z,E-isomer (Fig. 1a) and
1.6 min for clethodim sulfoxide (Fig. 1a¢).
Concentrations of clethodim were deter-
mined by measuring peak areas and com-
parison with a calibration curve (n = 4,
r2 = 0.997).
Statistics and Calculations
Pseudo-first-order kinetics was assumed
to calculate the corresponding degrada-
tion rate constant (k):
C ¼ C0 e
kt
where Co is the initial concentration of
clethodim (mg L)1), C is the concentra-
tion of clethodim (mg L)1) at a given
Original
time t (s or min) and k is the first-order
degradation rate constant (min)1 or s)1).
Half-life, t1/2 (min or s), can be calculated
from k using the following equation:
t1=2 ¼ ln 2=k
A Statgraphics Plus Software program
[16] was used to obtain non-linear least-
squares regression fit for clethodim deg-
radation data to the first-order model.
By-products Identification
and Characterization
For HPLC-ESI-MS analysis, two chro-
matographic conditions were employed.
Initially, the mobile-phase 60:39.9:0.1
acetonitrile:water:formic acid was used to
characterize peaks previously identified
by HPLC-UV analysis. A second method
was developed in order to identify deg-
radation products less polar than cletho-
dim that eluted very early in the
chromatogram. In this case, the mobile-
phase was 30:69.9:0.1 acetonitrile:water:
formic acid. The flow rate was set to
0.2 mL min)1 in both cases to account for
the shorter column. The sample injection
volume was 20 lL.
Electrospray conditions were set as
follows: drying gas flow (nitrogen) 10 mL
min)1; drying gas temperature 335  C;
nebulizer pressure 2590 mmHg; capillary
voltage 4000 V. The ESI-MS spectrome-
ter was operated in the positive ion mode.
Mass spectrometer conditions were opti-
mised by ramping fragmentor value to
obtain maximum ionic efficiency of
molecular ion of 100 V. MS chromato-
grams were obtained with a mass range of
50–2000 amu and a scan rate of 1.65 s
cycle)1.
Results and Discussion
Degradation Rate of Clethodim
in Chlorinated Water with
Hypochlorite and Chloramines
Degradation of clethodim in water con-
taining either sodium hypochlorite or
chloramines was investigated. The 10:1
molar excess of disinfectant represents a
realistic worst case for a low dose her-
bicide such as clethodim and ensures
pseudo first-order kinetic conditions,
since concentration of chlorine may be
considered constant during all the deg-
Original
Fig. 2. Reaction curves of clethodim and clethodim sulfoxide with disinfectants hypochorite and
chloramines. Molar ratio clethodim:disinfectants was 1:10. Concentration of clethodim was
0.014 mM, 500 lL. Reactions were quenched with sodium thiosulfate 10 mM, 100 lL at scheduled
time intervals. Tª = 25 ± 2  C. (a) Degradation curve of clethodim sulfoxide with hypochlorite.
(b) Degradation curve of clethodim and formation curve of clethodim sulfoxide with chloramines.
(c) Formation and degradation curve of clethodim sulfoxide with chloramines. Symbols: d,
clethodim; ¤, clethodim sulfoxide;—, non-linear regression fitted curve.
Fig. 3. (a) Blank sample chromatogram of clethodim. Chromatographic conditions: column,
Zorbax C18 (50 · 2 mm · 5 lm); mobile-phase, 60:39.9:0.1 acetonitrile:water:formic acid; flow
rate, 0.2 mL min-1. (b) Degradated sample of clethodim with hypochlorite (t = 11 s). Peaks at 8.56
and 8.84 min represent the two pairs of enantiomers of clethodim sulfoxide, RR+SS and SR+SR.
Chromatographic conditions: column, Zorbax C-18 (50 · 2 mm · 5 lm); mobile-phase, 30:69.9:0.1
acetonitrile:water:formic acid; flow rate, 0.2 mL min-1
radation experiments. Initial mass con- was also clethodim sulfoxide. Subsequent
centration of clethodim was 5 mg L)1 degradation of this compound was fol-
(0.014 mM). This high concentration lowed and the half-life calculated. In this
was selected to perform precise analyti- case clethodim sulfoxide also degrades
cal measurements for detection and slower than with hypochlorite (t1/2 = 9.3
identification of by-products generated ± 0.6 h, r2 = 0.991), (Fig. 2c).
during the degradation. The fast degradation of clethodim in
Clethodim degrades very fast with chlorinated water (either with hypochlo-
hypochlorite (t1/2 < 1 s). This degrada- rite or chloramines) practically precludes
tion gives rise to a single by-product that any possible exposure of consumers to
was identified as clethodim sulfoxide. this compound when tap water is sub-
Experiments were continued in order to jected to a chlorine treatment. However,
follow clethodim sulfoxide degradation. it is not possible to ensure complete
Non-linear regression fitting of data destruction of the reaction product cle-
points from the maximum concentration thodim sulfoxide before the distribution
attained by clethodim sulfoxide to simple point when chloramines are used for
first order kinetics allowed estimation of disinfection due to the slower degrada-
its degradation half life (t1/2 = 4.4 ± 0.4 s, tion rate.
r2 = 0.991) (Fig. 2a). The degradation of
sulfoxide yielded several degradation
products that were further investigated
by HPLC-MS. Identification of Main
In the presence of chloramines, deg- Degradation By-products
radation of clethodim was slower than
with hypochlorite (t1/2 = 8 ± 0.4 min, A blank sample, freshly prepared with
r2= 0.996), (Fig. 2b). This is in agree- analytical grade clethodim, was analysed
ment with the stronger oxidizing poten- using the first chromatographic method.
tial of hypochlorite versus chloramines The main chromatographic peak (reten-
[5]. The main reaction product in this case tion time 6.37 min) with an m/z 360
Chromatographia 2005, 62, August (No. 3/4) 135
Table 1. Fragmentation of clethodim, clethodim sulfoxide and clethodim sulfone

Chemical m/z Fragments

Clethodim 360 [MH]+
268 [M - OCH2CH=CHCl]
240 [M - OCH2CH=CHCl - CH2CH3]
206 [M - OCH2CH=CHCl - SCH2CH3]
164 [M - OCH2CH=CHCl - CH2CH(CH3)SCH2CH3]
Clethodim sulfoxide 376 [MH]+
298 [M - SOCH2CH3]
268 [M - OCH2CH=CHCl - O]
206 [M - OCH2CH=CHCl - SOCH2CH3]
164 [M - OCH2CH=CHCl - CH2CH(CH3)SOCH2CH3]
Clethodim sulfone 391 [MH]+
298 [M - SO2CH2CH3]
284 [M - OCH2CH=CHCl - O]
206 [M - OCH2CH=CHCl - SO2CH2CH3]
164 [M - OCH2CH=CHCl - CH2CH(CH3)SO2CH2CH3]

corresponds to clethodim (calculated
mass for [MH]+ = 360) (Fig. 3a, Ta-
ble 1). Another minor peak (4%) is ob-
served in the chromatogram (retention
time 1.75 min) that was assigned to the
Z,E isomer of clethodim at the oxime
ether double bond (Fig. 1a¢). The amount
of this isomer was constant in the blank
samples during all the period covered by
the degradation experiments. Whereas
most of the cyclohexanodione herbicides
are marketed as the E,E-isomer at the
oxime ether double bond, it has been
stated that some of them may equilibrate
with the Z,E isomer in a polar medium
such as water [10, 11]. In a separate
experiment, we found that isomerization
of clethodim to Z,E-isomer proceeded
slowly in water at room temperature (40
% after two months).Therefore, the small
amount of this isomer found in the blank
solutions is probably already present as
an impurity in the clethodim analytical
sample.
Samples from different reaction time
in the degradation experiments were
selected for HPLC-MS analysis to iden-
tify and characterize the main degrada-
tion or reaction products. These samples
were selected taking into account the re-
sults of the kinetic study. For the degra-
dation with hypochlorite two samples
were chosen corresponding to reactions
quenched at 1.5 s and 11 s in the kinetic
experiment. For the degradation with
chloramines, samples quenched at
210 min and 420 min were investigated.
As stated above, degradation of cle-
thodim in hypochlorite chlorinated water
proceeds almost immediately to yield a
single reaction product m/z 376. This
corresponds to the molecular weight of
clethodim plus an oxygen atom. Detailed
examination of the main fragments in the
MS spectra allowed unequivocal identifi-
cation of this compound as clethodim
sulfoxide (Fig. 1b) (Table 1). This com-
pound has also been reported to be the
main metabolite of clethodim in the
environment [17]. It is easily produced
through the oxidation of the sulfur atom.
A similar oxidation has been previously
reported for other thioethers [7].
In clethodim sulfoxide, a new chiral
centre is generated due to the pyramidal
arrangement of the atoms bound to the
sulfur in the sulfoxide group. Since a
neighbouring carbon atom is also chiral,
four stereroisomers (two pairs of enanti-
omers) are produced: RR, SS and SR,
RS. With the second chromatographic
method the diasteriomers were clearly
separated in two equal peaks, containing
a pair of enantiomers each (Fig. 3b).
Oxidation of the sulfur atom proceeds
without any stereogenic control in an
achiral medium. Consequently, similar
amounts for each of the isomers
(RR+SS, SR+SR) result from this
reaction. The Z,E isomer of clethodim
also degrades to the corresponding Z,E
isomer of clethodim sulfoxide (Fig 1b¢)
and it has been detected with the MS
detector as a minor peak into the chro-
matograms of the initial phases of the
degradation (sample t = 1.5 s; retention
time 1.9 min).
At later reaction time (sample t =
11 s), degradation of clethodim sulfoxide
and appearance of new peaks in the
chromatogram are observed. The most
important of these peaks (retention time
2.16 min, m/z 392) has been assigned to
clethodim sulfone (Fig. 1c) based on the
interpretation of the mass spectrum (cal-
culated mass for [MH]+ = 392;
(Table 1)). As oxidation of sulfoxide to
sulfone implies the loss of the asymmetry
around the sulphur atom, clethodim sul-
fone has only the chiral centre corre-
sponding to the carbon atom and it is a
racemic mixture of R and S isomers. With
the non-chiral column employed for
chromatographic separation, only one
chromatographic peak is observed for
this compound (Fig. 3b).
When the samples from the degrada-
tion experiments with chloramines were
analysed, the same by-products, cletho-
dim sulfoxide and clethodim sulfone,
were identified. This indicates that the
degradation route is mainly the same
with the two reagents. However, new
minor peaks (retention times 6.2 min and
7.4 min) with the same m/z as that of
clethodim sulfone (m/z 392) appear in
these chromatograms. The mass spectra
for these compounds do not show the m/z
206 characteristic of clethodim sulfone.
The main fragment for these compounds
is m/z 222, which corresponds to the loss
of the oxime and the sulfoxide group
(calculated mass for [M-91-78] =
222 amu). An additional fragment is
produced by the additional loss of
17 amu (m/z 205). This pattern suggests
the introduction of a hydroxyl group in
the sulfoxide molecule, giving rise to the
corresponding isomers. According to the
fragments observed for these compounds,
the hydroxyl group will result from the
oxidation of one of the free positions in
the cyclohexanodione ring. However,
with the information obtained from the
mass spectrum it is not possible to
determine its exact position within the
ring.
Some other minor by-products
(retention time 0.8 min, m/z 338; reten-
tion time 1.1 min, m/z 286) were ob-
served both in the hypochlorite and the
chloramines experiments but were not

136 Chromatographia 2005, 62, August (No. 3/4) Original

 definitively characterized. However, none
of the corresponding molecular peaks of
these minor products have a mass/charge
ratio and the isotopic relative abundance
pattern that would be expected for a
compound containing a new chlorine
atom [18]. This indicates that clethodim
will not give rise to more chlorinated by-
products in chlorinated water, at least no
as major degradation products. This is
an important feature since polychlori-
nated compounds are usually related to
long-term toxicological effects [19].
Conclusions
It has been demonstrated that water
chlorination for disinfection purposes
degrades completely any possible residue
of the herbicide clethodim. This degra-
dation is very rapid when chlorination is
produced by sodium hypochlorite and
rapid when chloramines are used, giving
rise in both cases to the corresponding
clethodim sulfoxide. This compound is
subsequently degraded to clethodim sul-
fone and other minor by-products.
Whereas some minor degradation prod-
ucts remain unidentified, none of the
major degradation products of clethodim
contain more chlorine atoms than the
parent compound which represents a
Original
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positive aspect of this compound with
respect to consumers safety.
Acknowledgements
We are grateful to Marı́a Jesús Vicente
Arana from the SIDI Mass Service
(Universidad Autónoma de Madrid) for
the skilful performance of the HPLC/MS
experiments and its helpful support in the
interpretation of the results.
This work has been financed by grant
RTA 01-034 from INIA. P.S-E and has
been supported by an INIA pre-doctoral
fellowship.
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