Page 24 - Volume 6, Issue 2, July 2013
Basic HTML Version
© Benaki Phytopathological Institute
Bempelou
et al.
72
eppedonrf tube (1.5 mL) and centrifuged in
5000 rpm for 5 min. The supernatant was re-
moved and 1 mL of sodium phosphate buf-
fer solution 100 mM, pH 7, was added in the
tube. Following the same procedure, the fi-
nal sediment was redissolved in 0.5 mL sodi-
um phosphate buffer solution 100 mM, pH
7.2, supplemented with 0.2% Triton.
The activity of esterases and glutathi-
one-S-transferases (GSTs) was determined
spectrophotometrically on an UVmax mic-
totitre plate reader, as previously described
(Vontas
et al
., 2001). A-naphthyl and b-naph-
thyl acetate were used as substrates for es-
terases and for glutathione-S-transferases
(GSTs) the corresponding 1-chloro-2,4-din-
trobenzene (CDNB). All treatments were car-
ried out in triplicate.
Protein was assayed by using the Bio-
Rad protein assay kit (Bio-Rad, Hemel Hemp-
stead, Herts, UK) with BSA as a standard pro-
tein (Bradford
et al
., 1976).
Inhibition studies were conducted by
incubation of the homogenates for 10 min
with diazinon concentrations of 23, 46, 59,
66 and 79 μM in the presence of acetonitrile.
The remaining activity was determined in
triplicate as in the standard assays men-
tioned above and expressed as units (U) per
mg of proteins (IU = μmol of substrate hy-
drolyzed per minute).
Addition of synergists in the biodegra-
dation trials
10μg/mL of the synergists triphenyl
phosphate (inhibitor of esterases) piperonyl
butoxide (inhibitor of monoxygonases and
esterases) and diethyl maleate (inhibitor of
monoxygonases and glutathione- S- trans-
ferases) were added (one in a time) in the
biodegradation trials and the biodegrada-
tion procedure was studied. Previously, the
sensitivity of the two yeasts in the synergists
was studied. 250 mL flasks containing 50
mL mineral salts medium (NH
4
NO
3
, KH
2
PO
4
,
Na
2
SO
4
, KCl, MgSO
4
.
7H
2
O, CaCl
2
, ZnSO
4
.
7H
2
O,
FeSO
4
.
7H
2
O and Na
2
MbO
4
.
7H
2
O) were inoc-
ulated with 10
6
cells/mL under sterile con-
ditions and supplemented with triphenyl
phosphate, piperonyl butoxide and diethyl
maleate (one in a time) at concentrations 1,
5, 10 and 15 μg/mL and incubated in an or-
bital incubator at 25
o
C and 150 rpm.
Biodegradation on tomato fruits
In order to estimate the activity of the
yeasts to biodegrade diazinon in plant sur-
faces, trials were conducted in tomato fruits
of Noa variety. Fruits were washed and dis-
infected with an ethanolic solution 90% and
sprayed with 10
8
cells/mL of yeast and one
day later with 2 mg/kg of diazinon. Control
samples were treated only with the insecti-
cide. All treatments were in triplicate. Fruits
were stored in an incubator at 20
o
C with
high relative humidity and 12 hours photo-
period. Samplings occurred in 5 days and 20
days. Each sample, constituting of 5 fruits,
was homogenated and then analyzed as de-
scribed above.
Results and Discussion
Chromatographic determination and
validation results
The qualitative and quantitative deter-
mination of diazinon and its metabolite IMP
was successfully achieved in the LC-MS/MS
chromatographic system (Figure 1) with the
technique of multiple reaction monitoring
(MRM), using the ions 305.1>169.1 m/z and
305.1>96.6 m/z for diazinon and 153>84 m/z
and 153>70 m/z for IMP, applying electro-
spray ionization in positive mode ESI (+), col-
lision energy at 31V for diazinon transitions
and 22 for the transitions of IMP. The corre-
sponding values in capillary were 21 V and
76 V. Confirmation was based on the crite-
ria of retention time and ion abundance of
qualitative and quantitative ions accord-
ing to the European guideline SANCO 2011/
12495 and the method was found to be ef-
fective for the extraction of the tested com-
pounds.
The method was validated by assess-
ing the basic parameters such as sensitivi-
ty, mean recovery (as a measure of trueness)
and repeatability (as a measure of precision).
The transitions 305.1>169.1 m/z and 153>84
