Persistence, fate, and metabolism of [14C]metalaxyl in typical Indian soils

Article abstract of DOI:10.1021/jf001181r

The biodegradation of ring-labeled [¹⁴C]metalaxyl in six Indian soils was examined. The total recovery of radioactivity from soil was 100 ± 6% of the applied radioactivity. Volatile organics and ¹⁴CO₂ were detected at lower levels. This suggests that neither mineralization nor volatilization is a major route of metalaxyl dissipation. The most rapid degradation of metalaxyl was observed in Bannimantap soil, in which the half-life of metalaxyl was 36 days. An inverse relationship was found when half-lives were plotted against microbial biomass and soil clay content. However, soil total organic carbon did not correlate with metalaxyl persistence. Five metabolites detected by thin-layer chromatography were more polar than metalaxyl.

Full text of DOI:10.1021/jf001181r

2352
J. Agric. Food Chem. 2001, 49, 2352−2358
P er sisten ce, F a te, a n d Meta bolism of [14C]Meta la xyl in Typ ica l
In d ia n Soils
Premasis Sukul† and Michael Spiteller*
Institute of Environmental Research, University of Dortmund, D-44221 Dortmund, Germany
The biodegradation of ring-labeled [14C]metalaxyl in six Indian soils was examined. The total recovery
of radioactivity from soil was 100 ( 6% of the applied radioactivity. Volatile organics and 14CO2
were detected at lower levels. This suggests that neither mineralization nor volatilization is a major
route of metalaxyl dissipation. The most rapid degradation of metalaxyl was observed in Banni-
mantap soil, in which the half-life of metalaxyl was 36 days. An inverse relationship was found
when half-lives were plotted against microbial biomass and soil clay content. However, soil total
organic carbon did not correlate with metalaxyl persistence. Five metabolites detected by thin-
layer chromatography were more polar than metalaxyl.
Keyw or d s: Metalaxyl; degradation; metabolism; distribution; soil
INTRODUCTION
mg was obtained from Novartis Crop Protection AG, Basel,
Switzerland. The radiochemical purity was 97.2% as shown
by HPLC.
Metalaxyl [N-(2,6-dimethylphenyl)-N-(methoxyacetyl)-
alanine methyl ester] is used to control diseases caused
by various fungi, such as late blight of potato caused
by Phytophthora infestans, downy mildew and white
rust of mustard and rapeseed caused by Peronospora
parasitica and Albugo candida, downy mildew of pearl
millet caused by Sclerospora graminicola, seed rot of
pea caused by Fusarium solani and Pythium ultimum,
and root and crown rot of capsicum caused by Phytoph-
thora capsici (1-6). It is a stable compound, resistant
to a wide range of pH, temperature, and light (7). These
properties lead to its easy use in agriculture. Due to its
broad spectrum of activities, metalaxyl is registered for
use in many countries worldwide, including the United
States, Europe, Australia, and India, on a variety of fruit
and vegetable crops. Upon spray or soil drench metal-
axyl is exposed to various biotic and abiotic factors,
which may affect its activity. There are few studies on
its photodegradation (8, 9), enhanced biodegradation
(10), and plant metabolism using cell suspension cul-
ture, etc. (11). However, a holistic approach toward
ascertaining the overall distribution pattern of metal-
axyl in soil has not been made so far, especially not with
Indian soils. This investigation examines the contribu-
tions of physicochemical and microbial processes on
metalaxyl mineralization and volatilization and quan-
titates extractable and bound metalaxyl residues from
six diverse Indian soils. In addition, the degradation
kinetics of metalaxyl in soil and the isolation of me-
tabolites of metalaxyl in these soils will be presented.
Soil. Six soils originating from India, representing different
physicochemical properties, were included in the present
study. The soil collection areas are shown in Figure 1. Two
batches of each soil sample were collected from a depth of 0-5
cm. One batch, shipped immediately by air freight to Germany
to maintain its original moisture status, is designated fresh
soil. The other batch, shipped by surface after air-drying, is
designated stored soil. It took nearly 4 months to reach
Germany. Different conditions, especially the moisture status
during transportation, were expected to affect the biological
activity of the soils. Prior to the laboratory experiments, the
soils were sieved to a maximum particle size of <2 mm. The
physical-chemical properties are shown in Table 1. None of
the soils investigated had been treated with metalaxyl in the
previous 5 years.
Ap p lica tion of Meta la xyl in Soil. Ring-labeled [14C]-
metalaxyl (80 µg of active ingredient/100 g of soil on a dry
weight basis, which is equivalent to 109.6 kBq of radioactivity)
was mixed into the soils with a recommended commercial use
rate of 600 g of active ingredient/ha, assuming a soil depth of
5 cm and a soil density of 1.5 g/cm3. The moisture content was
adjusted to 60% of maximum water holding capacity. To avoid
any effects of the solvents upon the biological activity of the
soils, the calculated volumes of the application solution (1 mL
of a solution of metalaxyl in methanol) were initially dispensed
onto portions of ∼25 g of dry soil in porcelain dishes. The
treated subsamples of soils were thoroughly mixed with a
spatula until the solvent was completely evaporated (∼10 min)
and afterward added to the total mass of the corresponding
soils (1200 and 1800 g for fresh and stored soils, respectively).
Subsequently, the gross mass of each soil was mixed in a
tumbling mixer for 1 h. This was followed by aliquotation.
Batches of 100 g of soil each (relative to dry soil) were
incubated in the dark in Erlenmeyer flasks under controlled
temperature (20 ( 1 °C). The moisture content in each flask
was gravimetrically checked every 2 weeks. The flasks were
fitted with trap attachments to absorb volatile substances, CO2
[soda lime (NaOH in the form of self-indicating granules)], and
organic volatiles [PU foam (polyurethane foam in solid state)].
Each experiment was carried out in duplicate.
MATERIALS AND METHODS
Ch em ica ls. Metalaxyl, analytical standard grade (99%),
was obtained from Riedel-de Haen, Seelze, Germany, and
[phenyl-U-14C]metalaxyl with a specific activity of 1.37 MBq/
* Author to whom correspondence should be addressed
[telephone (++49 231 755 4081); e-mail spiteller@
infu.uni-dortmund.de
Soil Biologica l P r op er ty. The microbiological activity in
soils was determined with and without the addition of chemi-
cals at the beginning and end of the experiment with the help
of a method described by J oergensen (12). The application of
† Present address: Department of Agricultural Biochemis-
try, F/Ag., Bidhan Chandra Krishi Viswavidyalaya, Mohanpur,
Nadia 741252, India.
10.1021/jf001181r CCC: $20.00 © 2001 American Chemical Society
Published on Web 04/10/2001

Fate of [14C]Metalaxyl in Indian Soils
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2353
evaporator, the soil extracts were analyzed by HPLC using a
Shimadzu LC 10 AT equipped with a Raytest Ramona 2000
and a UV detector operated at 205 nm. A Macherey-Nagel
(250/8/4) C18 column was used. For the first 15 min, the mobile
phase (1 mL/min) was composed of 10% acetonitrile and 90%
water. For the next 5 min, the acetonitrile content was
increased to 90%.
The separation and identification of metalaxyl and its acid
metabolite were also confirmed by HPLC-MS/MS. The operat-
ing parameters were as follows: HPLC; Phenomenex Nucleosil
3 C18 100A (150 mm × 2 mm) column, mobile phase; solvent
A water (0.1% HCOOH) and solvent B acetonitrile (0.1%
HCOOH) with a gradient system (0-5 min 95% A and 5% B
and 5-35 min 80% A and 20% B), flow 250 µL/min. Fifty
microliters of sample were injected in methanol/water (1:1)
with 1% HCOOH (0.5 µL of crude soil extract was diluted with
50 µL of injection solvent). MS: TSQ 7000 (Finnigan MAT,
Bremen, Germany) equipped with an ESI and APCI source.
For ESI, the ionization voltage of 5 kV and transfer capillary
temperature of 220 °C were set. For APCI, a vaporizer
temperature of 450 °C and a transfer capillary temperature
of 230 °C were used. The ionization current and detector
voltage were set to 5 µA and 1.3 kV, respectively. For MRM
scans, a dwell time of 400 ms and a scanning time of 0.5 s
were applied.
P r ep a r a tion of Com p a r ison Com p ou n d : Meta la xyl
Acid Meta bolite [N-(2,6-Dim eth ylp h en yl)-N-(m eth oxy-
a cetyl)a la n in e]. Metalaxyl (100 mg) was added to 5 mL of 2
N KOH, and the mixture was refluxed (110-115 °C) for 1 h
and then cooled to ambient temperature. Seven milliliters of
2 N HCl were added to the reaction mixture, wich was kept
in a freezer overnight. The resulting white crystals were
collected and washed with ice-cold H2O.
F igu r e 1. Illustration map of India showing soil collection
areas.
the active ingredient and the subsequent incubation for the
soil samples used for the determination of microbial biomass
(Cmic) were performed similarly as described under Application
of Metalaxyl in Soil.
Recover y, Extr a ction , a n d Qu a n tita tive Deter m in a -
tion of Ra d ioa ctivity. The actual amount of radioactivity
applied was determined by combustion of four aliquots (1 g
each) of the soil immediately after the treatment in an oxidizer
(Biological Oxidizer OX 500, R. J . Harvey Instrument Corp.),
and the released 14CO2 was measured by liquid scintillation
counting (LSC). The distribution of the test substance was
found to be homogeneous. This result served as a reference
value for the total mass value. Prior to the opening of an
incubation vessel, volatile compounds possibly present in the
headspace of the vessel were purged into the trap attachment
by means of a gentle stream of air (20 min). Soil samples were
exhaustively extracted three times at room temperature on a
mechanical shaker in a centrifuge beaker for 1 h each. The
first extraction was done with 100 mL of methanol. The second
and third extractions were made with 100 mL of methanol/
water (80:20) followed by ultrasonic vibration (Branson Soni-
fier) for 5 min. After each extraction step, the extracts were
separated from soil solids by centrifugation (Beckman J 2-HS,
Beckman) for 20 min (9600g) and pooled. The extract was
analyzed for radioactivity content by LSC (Beckman LS 6500)
using Rotiscint Eco plus (Zinsser Analytic) and for metabolites
by thin-layer chromatography (TLC). The PU foam of each trap
was extracted twice with 30 mL of ethyl acetate. For the
determination of 14CO2, sodalime (10 g) of the trap was
dissolved with 18% HCl (60 mL) in a suitable apparatus. The
liberated 14CO2 was absorbed by a series of three vials
containing 15 mL of ice-cooled cocktail of Oxysolve C-400
(Zinsser Analytic), and the radioactivity was subsequently
measured by LSC. The soil-bound residue was freeze-dried,
homogenized in a mortar, and radioassayed by combustion
followed by LSC.
Qu a n tita tive Deter m in a tion of Meta la xyl a n d Its
Meta bolites in Soil by Ch r om a togr a p h ic Meth od s. The
degradation of metalaxyl and the formation of its metabolites
were monitored by using TLC. The radioactive components in
the extracts were separated and quantified by TLC using silica
gel plates on aluminum sheets (silica gel 60 F254, Merck,
Darmstadt, Germany) and a mixed solvent system of ethyl
acetate, propanol-2, and water (65:23:12). A 1 mL aliquot was
directly taken from the pooled extract and spotted on the plates
as bands by using an automatic plate spotter (Linomat IV,
Camag). The distribution of the radioactive zones on the TLC
plates was measured by using a Bio-Imaging Analyzer (BAS
1000, Fuji Co.). Radioactive regions on the measured tracks
were quantified with the software package TINA (Raytest).
Selected samples were also subjected to high-performance
liquid chromatography (HPLC) to confirm the results obtained
by TLC. After reduction of the volume to 3 mL on a rotary
RESULTS AND DISCUSSION
Ra d ioa ctivity Distr ibu tion . On the basis of the
LSC data a radioactivity balance was established for
each vessel in stored and fresh soils at each sampling
interval (Tables 2 and 3). The total recovery of radio-
activity was 100 ( 6% of the applied radioactivity. The
total amount of extractable radioactivity declined while
the percentage of nonextracted radioactivity increased
simultaneously. Volatile organics and 14CO2 were not
detected in higher amounts in either stored or fresh soil
at any sampling interval, which indicates that neither
mineralization nor volatilization is a major route of
metalaxyl dissipation in the case of the studied soils.
The evolution of a low level of 14CO2 (0.4-1.5%) from
the ring-labeled metalaxyl suggests that the aromatic
moiety of metalaxyl was not degraded. A much higher
14CO2 evolution rate (2.1-11.3%) may occur in the
enhanced biodegradation of metalaxyl (10).
Soil Biologica l P r op er ty. The microbial activity of
the test soil is particularly an important factor to study
the degradation and metabolism of agrochemicals in
soils (13-15). The microbial biomasses of the stored and
fresh soils used in this study are shown in Table 4. In
all soils, a small decline in microbial biomass was
observed at the end of the experiment. The same trend
was also observed in the control soil without metalaxyl
application. This may be attributed to the influence of
the incubation period and incubation conditions rather
than to the effect of metalaxyl. During the experimental
period, the microbial populations in these soil samples
were not supplied with exogenous food or energy. With
the passage of time, a similar phenomenon of decreasing
microbial biomass in soil was also demonstrated with
different pesticides (16). However, there are many
studies reporting the favorable effect of pesticides on
the growth and activities of microorganisms in soil (17-

2354 J. Agric. Food Chem., Vol. 49, No. 5, 2001
Sukul and Spiteller
Ta ble 1. P h ysica l-Ch em ica l Ch a r a cter istics of Test Soils
soil
Bannimantap
Canning
CFTRI farm
Coochbehar
15.5
12.5
72.0
6.9
12.4
2.1
113.7
10.0
1.5
Mohanpur
42.5
34.0
23.5
8.2
7.6
0.9
37.2
10.9
2.2
Purulia
texture (%)
clay
silt
sand
11.7
42.0
16.4
19.4
12.4
68.2
5.8
5.3
0.8
31.4
9.0
1.3
13.6
74.7
7.4
15.5
42.5
6.7
6.3
1.4
13.7
2.7
2.6
18.2
65.4
6.9
41.0
2.3
pH (H2O)
org C (g/kg)
total N (g/kg)
available N (mg/kg)
available P2O5 (mg/kg) nd
total Fe (%)
14.0
1.3
nda
nd
nd
1.5
1.6
free Fe2O3 (%)
CEC (mol/kg)
exch Ca (mol/kg)
exch Mg (mol/kg)
max water holding
capacity (%)
0.1
0.3
9.2
7.6
1.3
0.1
0.1
10.0
4.2
1.7
41.5
0.1
11.0
7.5
1.2
38.3
0.2
7.4
2.5
0.7
11.9
12.0
2.7
20.3
19.5
3.2
37.3
45.2
44.9
26.8
origin
Mysore, Karnataka, Canning, WB, Mysore, Karnataka, Coochbehar, WB, Mohanpur, WB, Purulia, WB,
India India India India India India
a
nd, not determined.
Ta ble 2. Distr ibu tion of Ra d ioa ctivity in Differ en t Soils (Stor ed ) a fter Ap p lica tion of [14C]Meta la xyl a t P er iod ica l
In ter va ls
% recovered radioactivity
intact metalaxyl remaining (%)
in extractable radioactivity
recovered radioactivity
as % of applied dose
soil/day
org extract
soil-bound residue
14CO2
org volatiles
Canning
0
1
3
7
14
30
60
90
93.9
90.1
90.0
75.2
66.5
61.7
48.3
42.3
40.3
101.6
101.2
104.9
102.1
99.7
102.5
101.7
101.6
106.4
93.9
91.8
94.7
83.8
78.9
80.8
78.9
78.1
72.2
7.7
9.4
<0.01
<0.01
<0.01
<0.01
<0.01
0.2
0.5
0.7
1.5
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
10.2
18.3
20.8
21.5
22.3
22.8
32.7
120
Coochbehar
0
1
3
7
14
30
60
90
101.2
99.8
97.1
94.7
94.8
88.9
87.5
86.2
83.3
104.9
103.3
105.6
104.9
106.3
99.8
100.4
99.4
96.5
101.5
98.2
97.3
94.8
95.6
88.8
88.3
87.2
83.7
3.4
5.1
8.3
<0.01
<0.01
<0.01
0.3
<0.01
<0.01
<0.01
<0.01
0.1
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
9.8
10.7
11.0
12.1
12.2
12.7
120
Mohanpur
0
1
3
88.1
88.6
90.0
87.7
84.7
81.0
71.3
60.9
57.9
97.0
98.1
100.3
99.7
87.9
88.6
90.6
88.5
88.0
86.9
81.5
80.5
77.2
9.1
9.5
9.7
11.2
11.2
17.5
19.9
23.7
24.3
<0.01
<0.01
<0.01
<0.01
0.1
0.2
0.2
0.3
0.4
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
7
14
30
60
90
120
Purulia
0
1
3
7
14
30
60
90
120
99.3
104.6
101.6
104.5
101.9
98.2
98.4
100.3
95.9
89.3
86.2
83.0
81.7
79.2
101.6
101.7
102.2
103.8
100.2
100.7
97.4
98.2
98.0
97.4
95.8
88.9
87.2
84.9
82.5
81.1
3.4
3.7
4.8
<0.01
<0.01
<0.01
0.1
<0.01
<0.01
<0.01
0.1
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
7.9
11.3
13.5
12.5
12.7
15.9
95.3
97.1
0.1
20). Therefore, the present experiment clearly shows the
limitations of soil degradation studies under standard-
ized laboratory conditions using microbially depleted
soils due to drying and storage. Therefore, it may be
unrealistic to extrapolate laboratory results to the
natural environment.
time. Interestingly, a higher bound residue quantity was
found under fresh than under stored conditions of the
same soil (Tables 2 and 3). This might be explained
through microbial activity in soil. More microbial activ-
ity means more microbial exudates, which generate a
cementing effect on the pesticides with soil matrix by
rendering their biounavailable. Microbes are also re-
sponsible for the conversion of parent organic com-
Bou n d Resid u es. In both stored and fresh soils, the
amount of bound residues gradually increases with

Fate of [14C]Metalaxyl in Indian Soils
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2355
Ta ble 3. Distr ibu tion of Ra d ioa ctivity in Differ en t Soils (F r esh ) a fter Ap p lica tion of [14C]m eta la xyl a t P er iod ica l
In ter va ls
% recovered radioactivity
intact metalaxyl remaining (%) recovered radioactivity
soil/day
in extractable radioativity
as % of applied dose
org extract
soil-bound residue
14CO2
org volatiles
Bannimantap
0
7
14
30
60
90
96.4
84.6
74.2
55.8
25.8
18.9
99.4
98.0
97.3
100.3
99.0
99.4
95.6
91.7
87.9
83.1
68.0
66.0
3.8
6.3
9.0
16.5
30.2
32.4
<0.01
<0.01
0.4
0.7
0.8
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
1.0
CFTRI Farm
0
7
14
30
60
90
98.6
88.7
78.7
75.8
58.9
56.3
101.9
99.8
95.9
99.1
96.9
98.7
98.0
88.7
80.5
79.6
64.4
63.0
3.9
10.9
15.1
19.1
32.0
35.0
<0.01
0.2
0.3
0.4
0.5
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
0.7
Coochbehar
0
7
14
30
75
90
97.7
94.8
84.9
79.7
78.7
77.0
102.8
102.9
103.1
103.6
99.6
97.5
95.4
94.7
94.4
86.9
85.8
5.3
7.4
8.1
8.8
12.2
14.7
<0.01
0.1
0.3
0.4
0.5
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
101.1
0.6
Mohanpur
0
7
87.5
73.3
49.9
41.3
39.8
21.4
97.7
100.8
99.2
97.5
94.0
96.6
87.1
82.5
76.1
73.9
61.5
49.5
10.6
18.2
23.0
23.5
32.1
46.5
<0.01
0.1
0.1
0.1
0.4
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
14
30
75
90
Purulia
0
0.6
95.8
91.0
85.1
79.8
65.5
57.5
101.8
102.1
101.8
99.0
97.0
101.8
95.8
93.0
92.5
88.6
73.8
73.7
6.0
8.8
8.9
10.0
23.0
27.8
<0.01
0.3
0.4
0.4
0.2
<0.01
<0.01
<0.01
<0.01
<0.01
<0.01
7
14
30
75
90
0.3
Ta ble 4. Micr obia l P r op er ties of Soils In vestiga ted
Ta ble 5. Regr ession Equ a tion s, Cor r ela tion Coefficien ts,
a n d Ca lcu la ted Ha lf-Lives for Degr a d a tion Kin etics of
Meta la xyl in Differ en t Soils
microbial biomass (mg of Cmic/kg of soil)
soil
start
end
soil
regression eq
Fresh Soil
Bannimantap Y ) 1.87 - 0.0083X 0.9845
r2
cald t1/2 (days)
Stored Soil
Canning
288
104
252
96
241
83
210
71
36
116
301
58
Coochbehar
Mohanpur
Purulia
CFTRI farm
Coochbehar
Mohanpur
Purulia
Y ) 1.88 - 0.0026X 0.9297
0.7339
Y ) 1.74 - 0.001X
Y ) 1.64 - 0.0052X 0.8149
Fresh Soil
1364
1031
634
1316
Y ) 1.75 - 0.0022X 0.9842
137
Bannimantap
CFTRI Farm
Coochbehar
Mohanpur
Purulia
1330
992
590
1250
920
Stored Soil
Canning
Y ) 1.83 - 0.0031X 0.8980
Y ) 1.86 - 0.0006X 0.8539
Y ) 1.84 - 0.0017X 0.9871
Y ) 1.89 - 0.0008X 0.8372
97
502
177
376
Coochbehar
Mohanpur
Purulia
978
pounds into reactive intermediates, which cannot be
extracted with normal organic solvents (21). However,
bound residues may also be generated by adsorption.
Metalaxyl seemed to be preferentially adsorbed on soil
mineral surfaces (22). A xenobiotic substance may also
undergo chemical reactions with humic acid precursors,
lignin, and polysaccharides present in soil and thus be
incorporated into the polymeric structure without being
degraded (23-26). The spaces between the sheets of clay
minerals can also incorporate pesticides, as well as
natural products derived from them (27). However, as
far as we know, there is not any knowledge about the
nature and characterization of metalaxyl bound residue.
This should be investigated.
time, and the half-lives were calculated by means of the
formula t1/2 ) ln 2/K. The straight-line regression
equations with correlation coefficient values and the
half-lives of different soils under different conditions are
presented in Table 5. In general, greater persistence of
metalaxyl was found in stored soil than in fresh soil.
However, in stored Canning soil metalaxyl persistence
was comparatively less (calculated t1/2 ) 97 days). This
may be attributed to the presence of a microbial popula-
tion, although it was low (241 mg of Cmic/kg of soil, Table
4), which might have a high potential to degrade
metalaxyl. The aerobic soil metabolism half-life of
metalaxyl was determined to be ∼40 days (28). How-
ever, there was no evidence of metalaxyl biodegradation
in sandy loam soil even after 10 weeks (29). A half-life
in the range of 69-159 days was observed in a soil
degradation study of metalaxyl (30). In the present
study, different calculated half-lives in different soils
Dissip a tion of Meta la xyl. Intact metalaxyl residues
remaining in extractable radioactivity in stored and
fresh soils are separately presented in Tables 2 and 3.
Log residues of intact metalaxyl were plotted against

2356 J. Agric. Food Chem., Vol. 49, No. 5, 2001
Sukul and Spiteller
Ta ble 6. Meta bolites Gen er a ted in Soils (F r esh ) a t
P er iod ica l In ter va ls (P er cen t)a
metab 1 metab 2 metab 3 metab 4 metab 5
Rf ) 0.70 Rf ) 0.36 Rf ) 0.31 Rf ) 0.29 Rf ) 0.10
soil/day
Bannimantap
0
7
14
30
60
6.7
12.7
24.2
38.2
44.5
0.4
1.0
1.7
2.4
2.6
90
CFTRI farm
0
-
7
14
30
60
0.7
1.9
2.3
3.8
4.6
0.2
0.4
0.4
0.6
0.4
F igu r e 2. Correlation between microbial biomass and half-
life of metalaxyl in stored and fresh soils.
90
Coochbehar
0
7
14
30
75
0.8
3.9
2.3
3.4
4.9
1.4
1.4
2.2
90
Mohanpur
0
7
14
30
75
9.2
25.0
23.6
22.1
26.2
90
Purulia
0
7
14
30
75
0.8
3.4
3.2
0.5
1.4
0.6
1.5
F igu r e 3. Correlation between soil clay content and half-life
of metalaxyl in stored and fresh soils.
7.5
9.6
90
0.7
1.5
0.6
a
under different conditions highlight the differences in
the degradation rates, which can be attributed to the
differences in physicochemical properties of soils, the
presence of microbial population in varying degrees,
variability in activity among particular components of
a microbial population taking part in the degradation
of metalaxyl, and the high potential of specific soils to
degrade metalaxyl. Although the present study does not
encounter the identification of specific microbial strains
being responsible for the degradation of metalaxyl, it
needs to be investigated.
In any of the soils, an initial fast degradation and a
decrease at the later stage were observed. This might
be a reflection of the higher microbial biomass at the
beginning and the low biomass at the end of the
experiment. Moreover, the selected soils seem to be
extremely sensitive to microbial characters.
Cor r ela tion betw een Soil P r op er ties a n d Meta -
la xyl P er sisten ce. An attempt was made to correlate
the half-life values (Table 5) of metalaxyl in stored and
fresh soils and the soil properties (Tables 1 and 4). An
inverse relationship (Figure 2) was found when half-
lives were plotted against Cmic in fresh (r2 ) 0.9532) and
stored (r2 ) 0.8893) soils, suggesting a possible microbial
degradation of metalaxyl in soils. From former experi-
ments (10, 31, 32) it is a well-known fact that a wide
range of fungi, bacteria, and actinomycetes seem to take
part in the degradion process of metalaxyl. A correlation
was also found between the soil clay content and the
half-lives of metalaxyl in stored soils (r2 ) 0.9344;
Figure 3). Metalaxyl seems to be preferentially adsorbed
on soil mineral surfaces (22, 33). A possibility of the
trapping of metalaxyl in the intralattice structure of clay
components may not be ruled out, either (27). Thus, a
higher clay content may lead to a higher amount of
Values are presented in percent of extracted radioactivity.
bound residues of the chemical. As in the present study,
the half-life of metalaxyl was calculated on the basis of
its extractable form; an inverse relationship was found
between the soil clay content and the half-life values.
However, the effect of the clay content in the dissipation
of metalaxyl is not evidenced in fresh soil (r2 ) 0.0929).
Fresh soils contained a high amount of Cmic. So it may
be suggested that, in the absence of sufficient microbial
population, soil clay plays a vital part in attenuating
metalaxyl in soil; otherwise, microbes are responsible
for the metalaxyl dissipation. Soil organic carbon did
not correlate with metalaxyl dissipation in the present
study.
Meta bolism . In the course of the experiments, few
radioactive metabolites were detected along with the
unchanged metalaxyl. In almost all soils, except for
stored Coochbehar soil, two metabolites were found
(metabolite 2, Rf ) 0.36, and metabolite 4, Rf ) 0.29;
Tables 6and 7), which were generated at various degrees
at different intervals. However, in stored Coochbehar
soil no metabolites were detected. This confirms the
higher persistence of metalaxyl in stored Coochbehar
soil, as has also been observed in the kinetic study of
metalaxyl dissipation. In fresh Purulia soil three me-
tabolites (Rf ) 0.70, 0.31, and 0.10) were also detected.
All of the metabolites are more polar than metalaxyl
(Rf ) 0.78). In most soils the main metabolite found was
metabolite 2, and an increasing trend in its formation
was noticeable during the experiment. In Bannimantap
soil the formation of this metabolite was maximum,
44.5% of total extractable radioactivity after 90 days of
incubation. However, in fresh Purulia soil the main

Fate of [14C]Metalaxyl in Indian Soils
J. Agric. Food Chem., Vol. 49, No. 5, 2001 2357
Ta ble 7. Meta bolites Gen er a ted in Soils (Stor ed ) a t
P er iod ica l In ter va ls (P er cen t)a
metab 2
metab 4
soil/day
Rf ) 0.36
Rf - 0.29
Canning
0
1
3
0.9
4.3
7
14
30
60
90
120
9.2
0.2
0.3
0.9
1.7
1.5
1.8
14.1
22.6
37.1
44.0
44.3
Coochbehar
no metabolite detected
Mohanpur
0
1
3
7
1.1
2.4
5.4
11.4
23.1
23.8
14
30
60
90
120
Purulia
0
0.2
0.5
0.6
1.0
1.0
1
3
7
14
30
60
90
120
0.5
0.7
0.8
0.9
0.8
0.2
0.3
0.6
F igu r e 4. Daughter ion spectra of protonated metalaxyl (280)
and protonated metalaxyl acid metabolite (266) at 20 eV
collision energy and 1.5 mbar collision cell pressure from
Bannimantap 90 day soil sample.
a
Values are presented in percent of extracted radioactivity.
metabolite was metabolite 1 (Rf ) 0.70), which accounts
for 9.6% of total extractable radioactivity after 90 days
of incubation. During the initial periods of incubation,
it was not observed but appeared after 75 days of
incubation.
The accumulation of metabolites combined with low
levels of 14CO2 evolution provides good evidence for a
degradation process involving the transformation rather
than the mineralization of metalaxyl. The attempt was
made to identify the metabolites spectroscopically.
Metabolite 2 was identified as metalaxyl acid metabo-
lite. MS data were in good agreement with those of the
synthesized one. The daughter ion spectra of protonated
metalaxyl and its acid metabolite and their fragmenta-
tion scheme from the Bannimantap 90 day soil sample
are presented in Figures 4 and 5. The fragmentation of
metalaxyl, as well as its acid metabolite, preferentially
yields fragment ions, which keep the right part of the
molecule intact and lead to the same m/z values (248,
220, and 192). Additional fragments result from second-
ary fragmentation pathways: 160 ) 192 - 32 (-MeOH),
148 ) 220 - 72 (elimination of the right part as ketene,
-C3H4O2). The fragment ion with m/z 206 (parent m/z
266) is probably due to a rearrangement of the original
molecule. The amounts of other metabolites were so
small that no spectral data could be obtained. In former
reports (10, 28) the acid metabolite of metalaxyl was
found to be the main metabolite in the soil study.
Con clu sion . Our study highlights the high potential
of specific Indian soils to degrade metalaxyl. A signifi-
cant effect of microbial biomass on metalaxyl transfor-
mation and formation of bound residues is noticed. The
major metabolite is the metalaxyl acid. Metalaxyl and
its acid metabolite have a tendency to migrate to deeper
soil horizons with a potential to contaminate ground
F igu r e 5. Primary fragmentation pathways of metalaxyl (R
) Me) and its acid metabolite (R ) H).
water, particularly in soils with low organic matter and
clay content (34). It has been noticed that, on average,
Indian soils contain low amounts of organic matter, and
metalaxyl is extensively used in India on a variety of
fruit and vegetable crops due to its broad spectrum
activity. Therefore, precautions should be taken for the
continuous application of metalaxyl to crops. If use of
metalaxyl is greatly increased, the risk of occurrence
in ground water must be assessed, as by monitoring
studies in the most vulnerable areas in main use
regions.
ACKNOWLEDGMENT
We thank A. Achermann, Novartis Crop Protection
AG, Bassel, Switzerland, for providing radiolabeled
metalaxyl standard. We deeply appreciate the help
rendered by Dr. T. Pfeifer, University of Dortmund, in
recording the mass spectral data.

2358 J. Agric. Food Chem., Vol. 49, No. 5, 2001
Sukul and Spiteller
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Received for review September 27, 2000. Revised manuscript
received J anuary 30, 2001. Accepted J anuary 31, 2001. This
study was funded as part of a research grant to the first author
from the Alexander von Humboldt Foundation, Germany.
J F001181R