Identification of a new sulfonic acid metabolite of metolachlor in soil

Article abstract of DOI:10.1021/es9503600

An ethanesulfonic acid metabolite of metolachlor (metolachlor ESA) was identified in soil-sample extracts by negative-ion, fast-atom bombardment mass spectrometry (FAB-MS) and FAB tandem mass spectrometry (FAB-MS/MS). Production fragments from MS/MS analy

Full text of DOI:10.1021/es9503600

Environ. Sci. Technol. 1996, 30, 592-597
because alachlor ESAis relativelypersistent in surface waters
Identification of a New Sulfonic
Acid Metabolite of Metolachlor
in Soil
(3) and has high water solubility; thus, it is highly leachable
to the ground water. The occurrence of the sulfonic acid
metabolite ofalachlor in surface and ground waters suggests
that the formation of the metolachlor sulfonic acid me-
tabolite also might be important.
Both alachlor and metolachlor are detoxified rapidly by
nonsensitive plants via conjugation with glutathione and/
or homoglutathione (4). The glutathione conjugate of
alachlor further degrades to the sulfonic acid derivative as
a major metabolite. Although the sulfonic acid derivative
is reported to be a major soil metabolite of other chloro-
acetanilide herbicides, such as acetochlor (5), alachlor (6),
and propachlor (7), the presence ofthe metolachlor sulfonic
acid (metolachlor ESA) metabolite in soil, plants, or animals
has not been reported. The objectives of this study were
to identify and confirm the formation of metolachlor ESA
in soil under field conditions.
D . S . A G A , * , † E . M . T H U R M A N , †
M . E . Y O C K E L , †
L . R . Z I M M E R M A N , † A N D
T . D . W I L L I A M S ‡
U.S. Geological Survey, 4821 Quail Crest Place,
Lawrence, Kansas 66049, and Mass Spectrometry Laboratory,
University of Kansas, Lawrence, Kansas 66045
An ethanesulfonic acid metabolite of metolachlor
(metolachlor ESA) was identified in soil-sample extracts
by negative-ion, fast-atom bombardment mass
spectrometry (FAB-MS) and FAB tandem mass spec-
trometry (FAB-MS/MS). Product-ion fragments from
MS/MS analysis of the deprotonated molecular ion of
metolachlor ESA in the soil extract can be reconciled
with the structure of the synthesized standard. The
elemental compositions of the (M - H)- ions of the
metolachlor ESA standard and the soil-sample extracts
were confirmedbyhigh-resolutionmass spectrometry.
A dissipation study revealed that metolachlor ESA is
formed in soil under field conditions corresponding
to a decrease in the concentration of the parent
herbicide, metolachlor. The identification of the
sulfonated metabolite of metolachlor suggests that
the glutathione conjugation pathway is a common
detoxification pathway shared by chloroacetanilide
herbicides.
Experimental Procedures
Soil-Extraction Procedure. Soil samples were collected
from an experimental corn field near Topeka, KS, where
alachlor and metolachlor were applied as preemergent
herbicides at a rate of 1.5 kg/ ha (active ingredient). Soil
samples were extracted according to the method described
by Mills and Thurman (8), with slight modification. Briefly,
20 g of soil was extracted twice with a methanol (15 mL)/
water (5 mL) mixture at 75 °C for 30 min. The soil extracts
were combined and subjected to a solid-phase extraction
(SPE) procedure described by Aga and co-workers (9) using
a C-18 Sep-Pak cartridge (Waters, Milford, MA) to separate
the parent herbicides from their more polar metabolites.
In this procedure, the soil extracts were evaporated to less
than 10 mL to remove the methanol. Then, the remaining
aqueous extracts were passed through a preconditioned
C-18 resin by using an automated Millilab Workstation
(Waters-Millipore, Milford, MA). The C-18 resins were
eluted sequentially with 3 mL of ethyl acetate followed by
3 mL of methanol. The ethyl acetate fractions contained
the parent herbicides, which were analyzed by enzyme-
linked immunosorbent assay (ELISA). Random samples
were selected for confirmation by gas chromatography/
mass spectrometry (GC/ MS). The methanol fractions
contained the ESA metabolites and were analyzed by high-
performance liquid chromatography (HPLC).
Introduction
Alachlor
[2-chloro-N-(2,6-diethylphenyl)-N-(methoxy-
methyl)acetamide] and metolachlor [2-chloro-N-(2-ethyl-
6-m ethylphen yl)-N-(2-m ethoxy-1-m ethylethyl)aceta-
mide] are structurally related chloroacetanilide herbicides
that are used extensively in the United States for the control
of some annual grasses and broadleaf weeds in crop and
noncrop areas. Although alachlor use is twice that of
metolachlor for corn and soybeans, metolachlor use has
increased substantially since 1980 (1). Recent pesticide-
monitoring studies revealed the occurrence of a sulfonic
acid metabolite of alachlor in ground water. The 2-[(2,6-
diethylphenyl)(methoxymethyl)amino]-2-oxoethanesulfon-
ic acid (alachlor ESA) was detected in samples collected
from rural private wells in the Midwest at concentrations
ranging from 1.2 to 74 µg/ L (2). This finding is important
Samples for fast-atom bombardment mass spectrometry
(FAB-MS) and tandem mass spectrometry (FAB-MS/ MS)
were prepared byusing the preceding SPEprocedure, except
that the methanol fractions were combined and concen-
trated by evaporation to 100 µL to facilitate detection and
confirmation by FAB-MS/ MS.
Identification of Metolachlor ESA by FAB-MS and FAB-
MS/MS. Negative-ion FAB analyses were conducted on a
Fisions/ VG AUTOSPEC-Q tandem mass spectrometer (Fi-
sions/ VG Analytical Ltd., Manchester, UK) of EBEqQ
configuration. FAB experiments were performed using a
cesium-ion gun operated at 20 keV of energy and 1 µA of
emission. Samples as methanol solutions were added to
a glycerol matrix on the FAB probe. Exact mass FAB
determinations were conducted at a resolving power of
10 000 using linear voltage scans. Spectra were acquired
* Author to whom correspondence should be addressed: e-mail
address: daga@qvarsa.er.usgs.gov; telephone: (913) 832-3561; fax:
(913) 832-3500.
† U.S. Geological Survey.
‡ University of Kansas.
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0013-936X/96/0930-0592$12.00/0
1996 Am erican Chem ical Society

A
B
FIGURE 1. FAB-MS/MS spectrum of the negatively charged molecular ion m/z 314 from the alachlor sulfonic acid standard (A) and the
soil-sample extract (B).
in continuum mode, and 10 scans were integrated for the
exact mass determination. Homologous fatty acid stan-
dards served as bracketing calibrant ions. FAB-MS/ MS was
conducted by using a linked scan at constant B/ E (10). The
collision gas was argon-adjusted to a pressure that attenu-
ated the precursor ion 80% in order to maximize product-
ion yield (11).
Synthesis of Alachlor ESA and Metolachlor ESA.
Alachlor ESA and metolachlor ESA were synthesized on the
basis ofthe procedure described byFeng (5). Either alachlor
or metolachlor was refluxed with excess sodium sulfite in
10% ethanol in water for 3-4 h or until the mixture became
homogeneous. Following acidification with sulfuric acid,
the product was extracted into methylene chloride. The
methylene chloride was evaporated, and the reaction
products were dissolved in hot ethanol. The hot ethanol
mixture was filtered and allowed to stand undisturbed for
recrystallization of the ESA. The white crystals that formed
were collected and washed several times with cold ethanol.
The chemicalstructures ofthe white crystals were confirmed
as alachlor ESA or metolachlor ESA by mass spectrometry.
Analysis of Metolachlor and Metolachlor ESA in Soil.
Concentrations of metolachlor in the ethyl acetate fractions
of the SPE eluate were measured by using ELISA kits
obtained from Idetek/ Quantix Systems (Sunnyvale, CA).
The ethyl acetate was first evaporated to dryness and then
reconstituted with 5 mL of water. Samples with concen-
trations greater than the highest calibration standard were
diluted first with distilled water until they were within the
linear range of the ELISA. The recommended procedure
indicated in the ELISA kits was followed.
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A
B
FIGURE 2. FAB-MS/MS spectrum of the negatively charged molecular ion m/z 328 from the metolachlor sulfonic acid standard (A) and the
soil-sample extract (B).
Concentrations of metolachlor ESA in the methanol
fractions were measured by HPLC using an HP Model 1090
series II liquid chromatograph with a photodiode array
detector (Hewlett-Packard, Palo Alto, CA). Areversed-phase
ODS-Hypersil column (Keystone Scientific, Bellefonte, PA)
with dimensions of 100 × 4.6 mm (3 µm particle size and
120 Åpore size) was used. The mobile phase was composed
of 40:60 methanol/ phosphate buffer (10 mM, pH 7.0) and
was run under isocratic mode at 1.2 mL/ min. The column
temperature was controlled and maintained at 60 °C.
ESA standard obtained from Monsanto Agricultural Co. (St.
Louis, MO) and of the metolachlor ESA synthesized in the
laboratory. FAB-MS/ MS spectra of precursor ions (m/z 314
and 328) derived from standard samples (Figures 1A and
2A) and soil extracts (Figures 1B and 2B) revealed char-
acteristic fragment ions consistent with the structures of
the alachlor ESA and metolachlor ESA. Figure 3 is a
structural interpretation of the fragmentation observed for
the two suspected sulfonic acid metabolites. The product
ions that are common to both metabolites are m/z 80 [SO3],
94 [CH2SO3], and 121 [COCH2SO3 - H]. Their source is
indicated in Figure 3A,B. A distinguishing ion unique for
alachlor ESA is m/z 270, which represents the removal of
the methoxymethyl group. Similarly, m/z 256 results from
the analogous removal of the 2-methoxy-1-methylethyl
group in metolachlor ESA. The m/z 256 ion in metolachlor
Results and Discussion
Analysis of the soil extracts by negative-ion FAB-MS showed
the presence of the (M - H)- ions of alachlor ESA (m/z 314)
and metolachlor ESA (m/z 328). The mass spectra of the
soil extracts were compared with the spectra of the alachlor
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TABLE 1
A
B
Negative-Ion FAB Exact Mass Determinations
ion
form ula
calculated standard m assa soil-sam ple m assa
m ass
314 C14H20NO5S 314.1062 314.1060 ( 0.6
314.1057 ( 1.6
328.1234 ( 4.6
328
C15H22NO5S 328.1219 328.1222 ( 0.9
a
Mass m easurem ent error with respect to the calculated m ass,
expressed as parts per m illion.
FIGURE 3. Structural interpretation of the fragmentation of alachlor
ESA and metolachlor ESA by FAB-MS/MS. (A) Alachlor sulfonic
acid, molecular weight 314. (B) Metolachlor sulfonic acid, molecular
weight 328.
ESA is noticeably less abundant than the equivalent m/z
270 ion in alachlor ESA. This may be attributed to the
more favorable cleavage of the C-N bond in alachlor ESA,
where C is adjacent to an O atom (12). On the other hand,
the C-N bond in the methoxymethylethyl group of me-
tolachlor ESA is one carbon away from the O atom, resulting
in less favorable cleavage of the C-N bond, as is evident
in the lower abundance of the m/z 256 ion. The favored
cleavage of the C-C bond adjacent to the O atom in the
metolachlor ESA molecule results in the formation of m/z
282. The other prominent ions observed in both ESA
metabolites is the loss of one (M - 16) or two (M - 32)
oxygen atoms in the sulfonate group. These ions are the
m/z 298 and 282 in alachlor ESA and the equivalent m/z 312
and 296 in metolachlor ESA. There was significant ion
current in ions adjacent to the (M - H)- ion in the crude
soil extracts. This gave rise to a suite of precursor ions
transmitted from the samples and, thus, a busy B/ E-linked
scan spectrum (Figures 1B and 2B). Precursor-ion resolu-
tion is generally modest using B/ E linked scans (10). This
can be observed in the clear natural abundance isotope
clusters associated with the major product ions in the MS/
MS spectra of the standards. The agreement between the
fragmentation pattern of the authentic ESA standards and
the suspected ESA metabolites in the soil extracts is strong
evidence of the identification of metolachlor ESA and
alachlor ESA in the samples.
Soilextracts and standard-derived anions were subjected
to exact mass determinations. The (M - H)- ions derived
from the soil extracts and standard samples have the same
mass units within experimental error and matched the
calculated formula mass of the sulfonic acid metabolites
within less than 5 ppm error (Table 1). The exact mass and
MS/ MS results indicate that both alachlor ESA and me-
tolachlor ESA were present in the soil samples.
Additional evidence of the formation of the metolachlor
ESA metabolite in the metolachlor-treated soil is provided
by the HPLC chromatograms in Figure 4. The peak at 5.947
min in Figure 4A (soil extract) matched the retention time
of the metolachlor ESA standard peak (5.945 min) in Figure
4B. The UV spectrum of the unknown peak at 5.947 min
in the soil extract also gives a 100% match with the UV
spectrum of the metolachlor ESA standard.
FIGURE 4. HPLC chromatograms of the soil extract (A) and ESA
standards (B) and comparison of the UV spectrum of the unknown
peak in the soil extract with that of metolachlor ESA.
The evidence that metolachlor ESA is formed under the
same field conditions in which alachlor ESA is formed is
important in several aspects. First, the formation of
metolachlor ESA indicates that both chloroacetanilide
herbicides follow a common metabolic pathway in soil.
Feng (5) and Lamoureux and Rusness (7) have proposed
glutathione conjugation as an initial pathway of acetochlor
and propachlor metabolism in soil, resulting in the forma-
tion of sulfonic acid metabolites. The evidence obtained
from this research suggests that alachlor and metolachlor
undergo the same glutathione conjugation as a metabolic
pathway in soil, which ultimately results in the substitution
of chlorine with a sulfonic acid group as shown in Figure
5. It is thought that conjugation with glutathione is a
common mechanism utilized byliving organisms to detoxify
electrophilic xenobiotics such as pesticides, drugs, and other
chemical pollutants (13). An understanding of the forma-
tion and final metabolism of the sulfonic acid metabolites
of chloroacetanilide herbicides is necessary if glutathione
conjugation is to be evaluated as a detoxification process
or if the fate of pesticides is to be considered. Secondly,
because the sulfonic acid metabolites have high water
solubility, they are expected to leach through the soil. A
study on the mobility and transport of these metabolites,
therefore, would become an important contribution to the
understanding ofthe fate and transport ofchloroacetanilide
herbicides in the environment.
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FIGURE 5. Formation of ESA metabolites of alachlor and metolachlor via glutathione (GSH) conjugation.
approximately 25 days based on the equation described by
Hurle and Walker (15) for first-order kinetics. A corre-
sponding rapid increase in metolachlor ESA was observed
for the first 5 weeks after application. The highest
concentration of metolachlor ESA, which was observed
between 12 and 13 weeks after application, occurred after
harvest and reached an average of 120 µg/ kg. It is possible
that corn plants took up the ESA metabolite and then
released it back into the soil after harvest, resulting in a
considerable increase in metolachlor ESA concentration in
the soil. A similar observation has been reported for
propachlor ESA (7), which was taken up by soybean plants
grown in soil treated with propachlor.
The initial rapid formation of metolachlor ESA indicates
that sulfonation is an important detoxification process for
metolachlor in soil. It has been previously reported that
microbial dechlorination of metolachlor by mixed and
isolated microbialcultures results in the formation ofsulfur-
containing metolachlor derivatives as intermediate and end
products (16, 17). However, in those laboratory studies
the sulfonic acid metabolite was not identified as one of
the metabolic products. Under field conditions, where the
natural microbial community exists, formation of the
sulfonated metabolite of metolachlor appears to be an
important product of microbial activity. The gradual
disappearance of the metolachlor ESA could either be due
to its leaching through the unsaturated zone because of its
high water solubility or due to further degradation in the
topsoil. This could be an important area of research that
needs to be addressed further.
FIGURE 6. Disappearance of metolachlor and formation of meto-
lachlor sulfonic acid metabolite in soil from an experimental field
plot.
To determine the rate of formation of metolachlor ESA
relative to the disappearance of the parent herbicide, soil
samples were collected from a metolachlor-treated corn
field. Sampling was performed weekly for the first month
and then at 2 week intervals thereafter until the 19th week.
Then, samples were collected again 300 days after herbicide
application to determine the persistence of the ESA
metabolite in the topsoil. The soil consisted of a Eudora
siltloam, with a pH ranging from 6.8 to 7.8 and an organic
carbon content of 0.45% (top 15 cm). Figure 6 shows the
average concentrations ofmetolachlor and metolachlor ESA
in soil (0-15 cm depth) with respect to the number of days
after application. Each point in the graph represents the
average of three soil samples analyzed in duplicate. The
concentrations observed varied greatly at each sample
location with time, resulting in coefficients of variation
ranging from 23%to 110%. This is attributed to the difficulty
of applying herbicides uniformly due to variations in wind
velocity and oscillations of the sprayer booms during
herbicide application. Similar problems have been en-
countered by other researchers when measuring pesticide
contents under field conditions (14). However, for the
purpose of this study, the data obtained are useful in
showing the trend in the formation of the metolachlor ESA
in soil with respect to the dissipation of metolachlor under
natural field conditions.
Because of increasing public concern about the quality
of drinking water, renewed pesticide-monitoring programs
are being implemented, and the toxicological significance
of extensively used herbicides is receiving closer examina-
tion. Increased knowledge about the degradation of
herbicides and the dissipation of their metabolites in the
environment could include consideration of herbicide
metabolites as part of the basis for the establishment of
health advisories and water-quality regulations.
Acknowledgments
The authors thank Mr. Robert Drake and Ms. Homigol
Biesiada of the University of Kansas Mass Spectrometry
The concentration of metolachlor in soil declined
exponentially with time, with a disappearance half-life of
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chased with the aid of a National Institutes of Health Grant
S10 RRO 6294-01 (T.D.W.) and the University of Kansas.
The metolachlor ELISA kits were gifts from Idetek/ Quantix
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in this article is for identification purposes only and does
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Received for review May 26, 1995. Revised manuscript re-
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ES9503600
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