Page 22 - Volume 6, Issue 2, July 2013

© Benaki Phytopathological Institute
Bempelou et al. 70
respiratory system as well as may cause skin
and eye irritation (Gallo and Lawryk, 1991).
The contamination of aquatic and terres-
trial ecosystems with diazinon, even though
no longer registered for use in the Europe-
an Union (Desicion 2007/393/EC), has in-
creased the public concern to establish an
efficient and safe method to detoxify di-
azinon residues from contaminated envi-
ronments. Generally, the microorganisms
able to degrade diazinon belong to the ge-
nus Streptomyces, Arthrobacter, Flavobacter
and Pseudomonas. Gunner and Zuckerman
first reported (1968) the synergistic action
of the bacteria Arthrobacter sp. and Strep-
tomyces sp. in the degradation of diazinon.
A year later (1969), Sethunathan and Mac-
Rae observed that Streptomyces sp., isolat-
ed from rice fields, consumed diazinon only
on the presence of glucose. Sethunathan
and Pathak (1972) also reported the degra-
dation of diazinon in water, rhizosphere and
soil of rice fields by Arthrobacter sp. and Fla-
vobacter sp. The degradation of diazinon,
as a sole source of carbon, by Pseudomonas
was mediated by hydrolase activity accord-
ing to Rosenberg and Alexander (1979) and
Barik and Munnecke (1982). Flavobacterium
sp. ATCC27551 was responsible for the hy-
drolysis of diazinon and its transformation
to 2-isopropyl-4-methyl-6-hydroxypyrymi-
dine (IMHP) and carbon dioxide (Adhya et al.,
1981; Sethunathan, 1989) with the biodegra-
dation to be attributed to the enzymatic sys-
tem of phosphorotriesterases. Arthrobacter
sp. and Enterobacter strain B14 (used for the
bioremediation of soil contaminated with
chlorpyrifos) and Alcaligenes faecalis DSP3
also degraded diazinon (Ohshiro et al., 1996).
Cycon et al. (2009) reported that the bacteria
Pseudomonas sp., Serratia liquefacienes and
Serratia mascencens depleted diazinon in the
presence of glucose.
In the present study, the capability of the
epiphytic yeasts Rh. glutinis and Rh. rubra to
degrade diazinon was examined, aiming to
explore the feasibility of using these natu-
rally present microorganisms to detoxify or-
ganophosphate residues and improve food
safety.
Materials and Methods
Chemicals and reagents
Analytical standards of diazinon (97.5%)
and piperonyl butoxide (92.5%) were pur-
chased from Dr Ehrenstofer (Augsburg,
Germany). The metabolite 2-isopropyl-6-
methyl-4-pyridinol (IMP) (99.1%), triphe-
nyl phosphate (99.5%) and diethyl maleate
(95%) were obtained from ChemService
(West Chester, UK). Ethyl acetate, acetone,
dichloromethane, acetonitrile and hexane
all of pesticide residues grade, methanol
and water of LC-MS grade and petroleum
ether (40-60 oC) of analytical reagent grade,
were all obtained from Lab Scan (Dub-
lin, Ireland). Stock solutions of 1000 μg/mL
and 10000 μg/mL were made in methanol
or acetonitrile (enzymatic assays) and were
stored at –20o C. Working solutions were pre-
pared in order to be used in analytical pro-
cedures of the study. Formic acid purchased
from Sigma (Greece) was also used. The re-
agents used in the enzymatic assays were
p-Nitrophenyl acetate (PNPA), a-naphthyl
acetate, b-naphthyl acetate and 1-chloro-
2,4-benzene (CDNB).
Chromatographic analysis and valida-
tion of analytical methods
A Varian [2x prostar 210 (LC) and 1200 L
(quadrupole MS/MS)] LC-MS/MS was used
for the determination of diazinon and its
metabolite 2-isopropyl-6-methyl-4-pyrimi-
dinol (IMP) with a Varian Polaris C18-A col-
umn (5 cm length, 2 mm internal dimension
and 5 μm particle size) at ambient temper-
ature (25 ± 4oC). Elution solvents were the
mixtures of methanol / water supplement-
ed with 1 mM HCOONH 4 (10/90) (Solvent A)
and methanol / water supplemented with 1
mM HCOONH4 (90/10) (Solvent B). A flow of
0.25 mL/min and an injection volume of 5
μL (full loop) were applied. The elution pro-
gram used was gradient, starting with 90%
of solvent A and 10% of solvent B, reaching
the 100% of solvent B at 14 min, remaining
there for 6min and returning to its first con-
stitution at 25 min. The mass spectrome-
try multiplier was set at 1500 V voltage, ion