© Benaki Phytopathological Institute
Degradation of methomyl residues in tomato and soil
57
was no rainfall during the experiment.
Sampling procedures and storage
Samples of pre marked tomato fruits of
the same ripening stage and size were har-
vested at random from each replicate of the
treated and control plots separately at regu-
lar time intervals on 0 (2 hour after spraying),
1, 2, 4, 7, 10, 13, and 17 days after methomyl
application. Soil samples were collected at
the same times with the tomato samples,
by obtaining a 2 kg of soil for each replicate.
Immediately after collecting, samples were
transported at 4ºC and in darkness in la-
belled polyethylene bags to the laboratory,
where they were processed. The tomatoes
were homogenised in a food processor and
the homogenate of each sample was placed
in polyethylene containers and frozen at -18
°C until analysis. Soil samples were air-dried
at 20 °C, sieved to obtain sub-samples and
were placed in polyethylene bags and fro-
zen at -18 °C until analysis.
Extraction and clean up
A sub-sample of 10 g was extracted from
each tomato or soil sample with 10 ml eth-
yl acetate in the presence of 10 g anhy-
drous sodium sulphate by homogenization
followed by centrifugation at 3800 rpm for
10 min. An aliquot of 4 ml of the superna-
tant was drowning in a 15 ml polypropyl-
ene tube containing 100 mg of the clean-
up agent PSA and 20 mg GCB. The mixture
was shaken vigorously and centrifuged at
3800 rpm for 5 min. 2ml of the supernatant
was taken, evaporated to dryness, the res-
idue was re-dissolved in 2 ml of methanol,
filtered through a 0.2 μm PTFE syringe filter
and then directly measured by HPLC-DAD.
HPLC analysis
Final analysis of methomyl residues was
done by HPLC. The chromatographic col-
umn was C
18
Zorbax XDE (250 mm x 4.6 mm,
5 μm film thickness). The column was kept
at room temperature. Flow rate of mobile
phase (methanol/water = 90/10 v/v) was 1
ml/min., and injection volume was 20 μl. De-
tection wavelength for detection of meth-
omyl was set at 254 nm. The retention time
of methomyl was 5.7 min. The residues in the
field incurred samples were tentatively iden-
tified by comparing the retention times (RTs)
of the sample peaks with that of the injected
standard. The chromatographic apparatus
was controlled by Chemstation software.
Method validation
The recovery experiment were carried
out on fresh untreated tomato and soil by
fortifying the samples (10 g) with methom-
yl at three concentration levels (i.e. LOQ, 5
x LOQ and 25 x LOQ). The fortified samples
were processed as previously described and
analyzed by HPLC to evaluate the accuracy
and the precision of the analytical proce-
dure. Recovery test was replicated five times
for each fortification level. The calibration
curve for methomyl was obtained by plot-
ting the peak area against the concentration
of the corresponding calibration standard.
The limit of detection (LOD) of methomyl
was determined as the lowest concentra-
tion giving response of three times the stan-
dard deviation of the base line noise defined
from the analysis of three control samples.
The limit of quantification (LOQ) was deter-
mined as the lowest concentration of a giv-
en response that could be quantified with
relative standard deviation lower than 20%.
Statistical Analysis
The dissipation kinetics of methomyl in
tomato and soil were determined by plot-
ting the residue concentration against time
and the maximum squares of correlation co-
efficient found were used to determine the
equations of best fit curves. For all the sam-
ples studied, exponential relationships were
found to apply, corresponding to the first
order rate equation. Confirmation of the
first order kinetics was further made graph-
ically from the equation of
C
t
=C
0
e
-kt
,
where
Ct
represents the concentration of the pes-
ticide residue at the time of
t
,
C
0
represents
the initial deposits after application and k is
the degradation rate constant in days
-1
. The
half-life (
t
1/2
) was calculated from the k value
for each experiment, being
t
1/2
=ln 2/k
.
