Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra

Article abstract of DOI:10.1007/s10646-018-1992-7

The possible involvement of the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra in the biodegradation of the insecticide chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP), in pure cultures and in plant surfaces (tomato fruits) was investigated. Higher biodegradation rates were observed as the concentration of chlorpyrifos and the inoculum of the microorganisms were increased, while the yeasts proved to be more active at 25 and 15 °C. The presence of glucose in the mineral nutrient medium, as an extra source of carbon, delayed the biodegradation by Rhodotorula glutinis, while Rhodotorula rubra proved to be more active. The detection and quantification of the parent compound and TCP was successfully achieved using a LC/MS/MS chromatographic system. The in vitro enzymatic assays applied suggested that esterases may be involved in the biodegradation of chlorpyrifos, a fact that was further enhanced after the addition of the synergists triphenyl phosphate, diethyl maleate and piperonyl butoxide in the biodegradation trials. The decrease of chlorpyrifos residues on tomato fruits confirmed the corresponding on pure cultures, resulting in the suggestion that the yeasts R. glutinis and R. rubra can possibly be used successfully for the removal or detoxification of chlorpyrifos residues on tomatoes.

Full text of DOI:10.1007/s10646-018-1992-7

Ecotoxicology
https://doi.org/10.1007/s10646-018-1992-7
Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the
epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra
E. D. Bempelou1 J. G. Vontas K. S. Liapis V. N. Ziogas2
2 ●
1 ●
●
Accepted: 8 October 2018
©
Springer Science+Business Media, LLC, part of Springer Nature 2018
Abstract
The possible involvement of the epiphytic yeasts Rhodotorula glutinis and Rhodotorula rubra in the biodegradation of the
insecticide chlorpyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP), in pure cultures and in plant surfaces (tomato
fruits) was investigated. Higher biodegradation rates were observed as the concentration of chlorpyrifos and the inoculum of
the microorganisms were increased, while the yeasts proved to be more active at 25 and 15 °C. The presence of glucose in
the mineral nutrient medium, as an extra source of carbon, delayed the biodegradation by Rhodotorula glutinis, while
Rhodotorula rubra proved to be more active. The detection and quantification of the parent compound and TCP was
successfully achieved using a LC/MS/MS chromatographic system. The in vitro enzymatic assays applied suggested that
esterases may be involved in the biodegradation of chlorpyrifos, a fact that was further enhanced after the addition of the
synergists triphenyl phosphate, diethyl maleate and piperonyl butoxide in the biodegradation trials. The decrease of
chlorpyrifos residues on tomato fruits confirmed the corresponding on pure cultures, resulting in the suggestion that the
yeasts R. glutinis and R. rubra can possibly be used successfully for the removal or detoxification of chlorpyrifos residues on
tomatoes.
Highlights
●
Biodegradation of chlorpyrifos by Rhodotorula glutinis and Rhodotorula rubra
●
●
Biodegradation of TCP by Rhodotorula glutinis and Rhodotorula rubra
Triphenyl phosphate inhibited the biodegradation of chlorpyrifos
●
●
●
Keywords Biodegradation Chlorpyrifos Epiphytic yeasts Synergists.
Introduction
Moraso 2000). Accordingly, increased interest has been given
to remove or detoxify pesticide residues from vegetables,
using environmental friendly approaches, such as micro-
organisms or recombinant enzyme systems.
The broad use of organophosphates has resulted in the con-
tamination of the environment. The excessive and frequent
application of pesticides has also resulted in high levels of
pesticide residues accumulated on vegetables, a matter that
poses a potential health risk to consumers (Bolognesi and
Chlorpyrifos, [Ο, O-diethyl O-[3,5,6-trichloro-2-pyr-
idinyl) phosphorothioate] is a broad spectrum organopho-
sphorus insecticide, also been used as acaricide and
termicide. It is a non systemic pesticide acting through
stomach or suffocation (Tomlin 2003). The main paths of its
degradation in the environment are photolysis in air (Racke
1993), hydrolysis in water (Meikle and Youngson 1978;
Chapman and Cole 1982 and McCall 1986a) and hydrolysis
and microbial degradation in soil (Getzin 1981a and Racke
et al. 1988). The half-life of chlorpyrifos in plants has been
measured from 1 to 9 days, mainly by evaporation and
secondary photolysis (Bauriedel 1986a). The processes by
which pesticides are transformed or degradated in
*
E. D. Bempelou
e.bempelou@bpi.gr
1
Department of Pesticides Control and Phytopharmacy, Pesticide
Residues Laboratory, Benaki Phytopathological Institute, 8 St.
Delta street, Kifissia 14561, Greece
2
Laboratory of Pesticide Science, Agricultural University of
Athens, Iera Odos 75, Athens 11855, Greece

E. D. Bempelou et al.
environmental compartments and the factors that modulate
the kinetics are critical from both pest control efficacy and
non target organism toxicity points. Both abiotic and
microbial transformation of chlorpyrifos has been found to
contribute significantly to its degradation. Microorganisms
possess the unique ability to completely mineralize many
aliphatic, aromatic and heterocyclic compounds (Bhagobaty
et al. 2007).
Generally, the microorganisms able to degrade chlorpyrifos
are bacteria and fungi. Very few works have been reported on
the degradation of 3,5,6-trichloro-2-pyridinol (TCP). To date the
degradation of the chlorpyrifos has been reported by several
bacteria belonging to the genera Bacillus, Lactobacilus, Strep-
tococcus, Pseudomonas, Micrococcus, Flavobacterium, Arthro-
bacter; Enterobacter, Alcaligenes, Sphingomonas, Paracoccus,
Brucella, Klebsiella, Serratia, Sphingobacterium, Cupriavidus,
and also by Ochrobactrum and Achromobacter (Jones and
Hastings 1981; Ivashina 1986; Shaker et al. 1988; Havens and
Rase 1991; Guha et al. 1997; Mallick et al. 1999; Singh et al.
phyllosphere of strawberry plants. Both strains had been
characterized by the protocols of classical microbiology,
according to their macro-morphology (colony color,
appearance, texture, no pigment), micro-morphology (cell
shape, budding) and strain physiology (fermentation in
different sugars, starch formation and assimilation growth).
Cultures were developed in mineral salts medium
(MSM) containing in g/L: 12 NH NO , 8 KH PO , 2
4
3
2
4
.
Na SO , 4 KCl, 1 MgSO 7 H O, 0.5 CaCl , 0.26
2 4 4 2 2
.
.
4
.
ZnSO 7H O, FeSO 7 H O and Na MbO 7 H O in 250 mL
4
2
2
2
4
2
Erlenmeyer flasks. The pH was adjusted to 7.2 and cultures
were incubated in an orbital incubator at 25 °C and 150 rpm.
Chlorpyrifos exposure
Cultures of yeasts were supplemented with chlorpyrifos and
afterwards different parameters, namely yeast inoculum
density, chlorpyrifos exposure, temperature of incubation
and presence of glucose in the MSM as an additional source
of carbon, were examined regarding their potential influ-
ence in the biodegradation of the insecticide, The effect of
the yeast inoculum density on the degradation was exam-
2004; Yang et al. 2005; Li et al. 2007; Xu et al. 2008, Lakshmi
et al. 2008; Anwar et al. 2009; Abraham and Silambarasan 2013;
Lu 2013; Abraham and Silambarasan 2016 and Akbar and
Sultan 2016). Regarding fungi, Trichoderma, Verticillium,
Aspergillus, Ganoderma and Cladosporium have been up to
now proved to be able to breakdown chlorpyrifos (Ivashina
2
4
6
ined by incubation of 10 , 10 and 10 cells/mL of each
yeast in 250 mL flasks containing 50 mL MSM and 10 μg/
mL chlorpyrifos. To examine the effect of chlorpyrifos
exposure on the biodegradation rates, experiments were
conducted in 250 mL flasks containing 50 mL MSM sup-
plemented with chlorpyrifos at concentrations of 10 and
25 μg/mL. The influence of temperature was tested by the
conduction of degradation trials in 250 mL flasks containing
50 mL of MSM supplemented with 10 μg/mL chlorpyrifos
at 15, 25 and 35 °C, respectively.
1986; Fang et al. 2008, Silambarasan and Abraham 2013, 2014
and Chen et al. 2012), while Lal and Lal (1987) have studied the
possible degradation of chlorpyrifos (1-10 ppm) by the yeast
Saccharomyces cerevisiae, which is the only reference on yeasts.
In the present study, the capability of the epiphytic yeasts
Rhodotorula glutinis and Rhodotorula rubra to degrade
chlorpyrifos and its metabolite TCP in pure cultures and
tomato fruits was investigated. This is the first report on the
biodegradation of chlorpyrifos and TCP by those micro-
organisms. Furthermore, this is a first report of investigating
their bio-degradative action using synergists as well as
examining the phenomenon in plant surfaces. The ultimate
objective of this study was to explore the feasibility of using
these microorganisms, which, are naturally present in fruits
and vegetables, to detoxify chlorpyrifos residues from the
plant surface and furthermore to improve food safety.
Finally, simultaneously with all the above parameters the
influence of glucose in the MSM, as an extra source of
carbon, was also investigated. Additional 250 mL flasks
containing 50 mL MSM supplemented with glucose under
each time experimental conditions tested were incubated
and the influence of this factor was also estimated.
Controls were also used for the comparison of the
obtained results. Uninoculated MSM with the same con-
centration of chlorpyrifos were used as controls. All treat-
ments were in triplicate. Flasks were incubated in an orbital
incubator at 25 °C and 150 rpm. Samplings took place
immediately after treatments and at time intervals of 1-day.
Aliquots of cultures were taken aseptically and analyzed for
chlorpyrifos residues as described below.
Materials and methods
Isolation of the yeasts and maintenance of pure
cultures
Extraction of chlorpyrifos and TCP from pure
cultures and tomato fruits
The microorganisms examined in the present study are the
epiphytic yeasts Rhodotorula glutinis and Rhodotorula
rubra. Both strains were isolated epiphytically from culti-
vated plants. Rhodotorula glutinis was isolated from tomato
plants phyllosphere and Rhodotorula rubra from the
Two different sample preparation techniques were applied
for the extraction of chlorpyrifos and TCP from pure cul-
tures and tomato fruits.

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula. . .
Regarding pure cultures, subsamples of 5 mL of MSM
Chromatographic analysis
(
50 mL) were taken aseptically and filtered through a
Whatman No 2 filter above a separating funnel of a 100 mL.
Liquid–liquid extraction was carried out with 20 mL hexane
for three times. Extracts were passed through anhydrous
sodium sulfate and collected in a flat bottom flask (100 mL)
and evaporated to dryness on a rotary evaporator. The dry
residue was diluted to 2 mL methanol: water (30:70) and
filtered through a 0.2 μm film filter to be further analyzed in
the LC/MS/MS chromatographic system.
A Varian [2x prostar 210 (LC) and 1200 L (quadrupole MS/
MS)] LC/MS/MS was used for the determination of chlor-
pyrifos and its metabolite 3,5,6-trichloro-2-pyridinol (TCP)
with a Varian Polaris C18-A (5 cm length, i.d.2 mm and
5 μm p.s) at ambient temperature (25 ± 4 °C). Elution sol-
vents were the mixtures of methanol/water supplemented
with 1 mM HCOONH (10/90) (Solvent A) and methanol/
4
water supplemented with 1 mM HCOONH (90/10) (Sol-
4
Regarding tomato fruits, an existing method (Bilthoven
996) was used for the sample processing of tomato fruits,
according to which 30 mL of acetone were added in an
aliquot of 15 g of the homogenated sample in a 250 mL
PTFE centrifuge bottle (Nalgene, Rochester, NY, USA) and
stirred for 1 min in an ultra-turrax homogenizer at
vent B). A flow of 0.25 mL/min and an injection volume of
5 μL (full loop) were applied. The elution program used was
gradient, starting with 90% of solvent A and 10% of solvent
B, reaching the 100% of solvent B at 14 min and remaining
there for 6 min and returning to its first constitution at
25 min. The MS detector was set at 1500 V voltage, ion
1
1
5000 rpm. 30 mL of dichloromethane and 30 mL of pet-
source to 50 °C and drying gas (N ) to 19 psi pressure and
2
roleum ether (40–60 °C) were added following by a new
stirring for 1 min. The sample was centrifuged at 4000 rpm
for 2 min. 25 mL of the supernatant were evaporated to
dryness on a water bath, at 65–70 °C and afterwards 5 mL
of 2,2,4-trimethyl pentane/ toluene (90/10) were added. The
extract was placed in an ultrasonic bath for 30 s, filtered
through a 0.2 μm film filter and transferred in a vial with
Teflon septa, to be further analyzed in the LC/MS/MS
chromatographic system.
250 °C temperature. The electrospray ionization mode was
in positive mode for the determination of chlorpyrifos and
in negative for TCP, using nitrogen as nebulizer gas at
50 psi in both cases. The needle voltage was 5000 V in
ESI(+) and 4500 V in ESI(-), while the spray shield voltage
was ± 600 V, respectively.
Method validation
The analytical methods were validated in accordance
with the requirements of EU (SANΤΕ 2015/11945).
Different known concentrations of chlorpyrifos and
TCP were fortified in the mineral salts medium (0.05,
0.1 and 0.5 μg/mL) and of chlorpyrifos in tomato fruits
Chromatographic determination of chlorpyrifos and
3.3,5,6-trichloro-2-pyridinol method validation
Chemicals and reagents
(
0.05, 0.1 and 0.5 μg/mL) at least in 5 replicates for
The analytical standards of chlorpyrifos (98.4%) and
piperonyl butoxide (92.5%) were obtained purchased from
Dr Ehrenstofer (Augsburg, Germany). The corresponding of
both substrates.
Enzymatic assays–synergists
Enzymatic assays
3
,5,6-trichloro-2-pyridinol (TCP) (99.1%), triphenyl phos-
phate (99.5%) and diethyl maleate (95%) were purchased
from ChemService (West Chester, UK). Regarding solvents
used, they were all obtained from Lab Scan (Dublin, Ire-
land). Acetone, dichloromethane, acetonitrile and hexane
were of pesticide residues grade, while methanol and water
of LC/MS grade and petroleum ether (40–60 °C) of analy-
tical reagent grade. Formic acid purchased from Sigma
1 mL of liquid culture was added in an eppendorf tube
(1.5 mL) and centrifuged in 5000 rpm for 5 min. The
supernatant was removed and 1 mL of sodium phosphate
buffer solution 100 mM, pH 7, was added in the tube.
Following the same procedure, the final sediment was
redissolved in 0.5 mL sodium phosphate buffer solution
100 mM, pH 7.2, supplemented with 0.2% Triton. The
activity of esterases and glutathione-S-transferases (GSTs)
was determined spectrophotometrically on an UVmax
microtite plate reader, as previously described (Vontas et al.
2001). A-naphthyl and b-naphthyl acetate were used as
substrates for esterases and for glutathione-S-transferases
(GSTs) the corresponding was 1-chloro-2,4-dintrobenzene
(CDNB) (Roditakis et al. 2009). All treatments were carried
out in triplicate.
(
Greece) was also used. p-Nitrophenyl acetate (PNPA), a-
naphthyl acetate, b-naphthyl acetate and 1-chloro-2,4-ben-
zene (CDNB) were the reagents used in the enzymatic
assays.
Stock solutions of 1000 and 10000 μg/mL of each ana-
lyte were made in methanol (residue analysis) or acetonitrile
(
enzymatic assays) and were stored in –20 °C. Composite
working solutions were prepared and also stored at −20 °C.
These stock solutions were used during the study for pre-
paring the working standard solutions.

E. D. Bempelou et al.
Protein was assayed by using the Bio-Rad protein assay
kit (Bio-Rad, Hemel Hempstead, Herts, UK) with BSA as a
standard protein (Bradford 1976).
Inhibition studies were conducted by incubation of the
homogenates for 10 min with chlorpyrifos concentrations of
Results
Chromatographic determination and validation
results
1
4.25, 28.5, 42.75 and 57 μM in the presence of acetonitrile.
The qualitative and quantitative determination (Fig. 1) of
the two analytes was successfully achieved in the LC/MS/
MS chromatographic system with the technique of multiple
reactions monitoring (MRM). Electrospray ionization in
positive mode ESI (+) was applied for the determination of
chlorpyrifos, using the ions 350 > 198 m/z and 352 > 200 m/
z, while negative mode ESI (-) was considered as better for
TCP with the ion transitions of 198 > 198 m/z and 196 >
196 m/z. Confirmation was based on the criteria of retention
time and ion abundance of qualitative and quantitative ions
according to EU Guidelines.
The analytical methods were validated by assessing the
basic parameters such as sensitivity, mean recovery (as a
measure of trueness) and repeatability (as a measure of
precision). After the fortification of the MSM with chlor-
pyrifos and its metabolite TCP, the obtained recoveries
ranged from 78.6 to 95.3%, values which were accepted
(SANTE/11945/2015) (Table 1). Relative standard devia-
tions (RSDs) values ranged from 6.4 to 13%, all lower to
20%, also accepted. Validation results of tomato fruits with
chlorpyrifos gave an average recovery of 91.16% and a
mean RSD of 8.13% (Table 1). The limit of quantification
(LOQ) was set for both substrates in the lowest validation
level, 0.05 μg/mL and the limit of detection (LOD) was
calculated to be 0.02 μg/mL (Table 1). The methods were
found to be effective for the extraction of the tested com-
pounds and the above results indicate their efficiency for the
determination of chlorpyrifos and its metabolite TCP from
the mineral salts medium and tomato fruits and ensure the
accuracy of the biodegradation trials results.
The remaining activity was determined in triplicate as in the
standard assays mentioned above and expressed as units (U)
per mg of proteins (IU = μmol of substrate hydrolyzed per
minute). The IC50 value was calculated for each yeast, as the
concentration of chlorpyrifos needed to inhibit half (50%)
of the maximum activity of enzymes (esterases and glu-
tathione-S-transferases).
Two-way ANOVA was employed, with the studied
condition (control or treatment) and the concentration of
chlorpyrifos (0, 14.25, 28.5, 42,75 and 57 μΜ) been the two
factors examined. Data analysis was conducted using the
statistical package JMP (SAS Institute 2008).
Addition of synergists in the biodegradation trials
The synergists triphenyl phosphate (inhibitor of esterases),
piperonyl butoxide (inhibitor of monoxygonases and
esterases) and diethyl maleate (inhibitor of monoxygonases
and glutathione-S-transferases) were used in the biode-
gradation trials. 10 μg/mL of each synergist (one in a time)
were added in the biodegradation flasks and the biode-
gradation procedure was studied.
The sensitivity of the two yeasts in the synergists had
been previously examined as follows. 250 mL flasks con-
taining 50 mL MSM were inoculated with 10 cells/mL
under sterile conditions, 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 orbital incubator at 25 °C and 150 rpm. As observed,
none of the compounds showed adverse effects in the
growth of the yeasts.
6
Biodegradation of chlorpyrifos by Rhodotorula
glutinis and Rhodotorula rubra
Biodegradation on tomato fruits
Effect of yeast inoculum density on biodegradation
As a first approach to estimate the possible bio-degradative
activity of the yeasts in plant surfaces, trials were conducted
in tomatoes. Groups of 5 tomato fruits (Noa variety) were
washed and disinfected with an ethanolic solution 90%,
2
When 10 cells/mL of the yeasts R. glutinis and R. rubra
were supplemented in the MSM, no biodegradation was
observed. However, in the inoculum density of 10 cells/mL
and especially when the MSM was non-glucose-supple-
mented, strong indications were provided regarding the
ability of the two yeasts to degrade chlorpyrifos. These
indications were confirmed in the case of incubation of each
4
8
sprayed with 10 cells/mL of yeast (one in a time) and
sprayed with chlorpyrifos at 2 mg/kg 1 day later. Equivalent
groups of tomato fruits were treated only with the insecti-
cide and were used as control samples. All treatments were
in triplicate. All treatments were stored in an incubator at
6
culture with an initial inoculums density of 10 cells/mL,
2
0 °C, with high relative humidity and 12 h photoperiod.
where chlorpyrifos was depleted. The metabolite TCP was
detected as a degradation product on day 1 or 2 of incu-
bation with its concentration to reach a maximum on day 5
Samples were collected at 5 and 30 days, homogenated and
then analyzed as described above.

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula. . .
Fig. 1 Determination of
chlorpyrifos and its metabolite
3
,5,6-trichloro-2-pyridinol
(
TCP) in the LC/MS/MS
chromatographic system, during
a time period of 12 days, by
changing the ionization mode
for each analyte. Application of
ESI (+) for chlorpyrifos and
with ESI (+) for TCP
Table 1 Mean recoveries (R, %)
and relative standard deviations
Compound
Mineral salts medium (n = 5)
Tomato fruits (n = 5)
(
RSD, %) for the determination
C (μg/mL) Mean recovery (%) RSD (%) C (μg/mL) Mean recovery (%) RSD (%)
of chlorpyrifos and 3,5,6-
trichloro-2-pyridinol (TCP) in
mineral salts medium and
chlorpyrifos in tomato fruits, in
Chlorpyrifos 0.05
78.6
84.4
88.7
82.3
89.6
95.3
13
0.05
0.1
0.5
–
84.4
92.4
96.7
–
10.7
8.3
5.4
–
0
0
.1
.5
10.4
9.3
5
replicates (n) at 3 fortification
levels in the LC-MS/MS
chromatographic system
TCP
0.05
6.4
0
0
.1
.5
7.8
–
–
–
11.2
–
–
–
or 6 (respectively for each yeast) and then started to
decrease (Fig. 2). Hydrolysis percentages of chlorpyrifos
were less than 5% in all cases.
degraders of chlorpyrifos in a different way. As shown in
Fig. 2, under the presence of glucose R. glutinis did not
consume chlorpyrifos during the first 4 days of the trials.
The consumption of chlorpyrifos in this day proved to be
no-significant different from the control (t-test, t(2) = 0.44,
P < 0.05) and therefore, it is assumed that the degradation
actually started on day 5. Later on, the yeast decreased the
insecticide rapidly and finally depleted it (day 7). TCP was
detected on day 3. On the contrary in the flask without
Effect of presence of glucose on mineral salts liquid
medium
The addition of glucose in the MSM, as an extra source of
carbon, effected the action of Rhodotorula yeasts as

E. D. Bempelou et al.
Fig. 2 Biodegradation of chlorpyrifos by the yeasts Rhodotorula
glutinis and Rhodotorula rubra in MSM supplemented with 10 μg/mL
chlorpyrifos and 10 cells/mL inoculum. Production of the metabolite
3,5,6-trichloro-2-pyridinol (TCP). Each value is the mean of three
replicates with error bars representing the standard deviation of the
mean
6
glucose degradation was observed from the beginning of the
trial; chlorpyrifos was depleted up to day 6, while TCP was
detected from day 2.
flasks without glucose in the MSM were 1.62 and 4.76 μg/
mL/day.
On the other hand, R. rubra depleted chlorpyrifos in all
cases presenting higher biodegradation rates in the con-
centration of 25 μg/mL (Fig. 3). In flasks supplemented with
glucose the rate of 2.22 μg/mL/day (10 μg/mL chlorpyrifos)
increased to 5.5 μg/mL/day (25 μg/mL chlorpyrifos). The
corresponding values in the flasks without glucose in the
MSM were 1.94 and 4.8 μg/mL/day. As it was observed the
rate of biodegradation of chlorpyrifos by the two yeasts was
proportionally increased with their exposure in the
insecticide.
On the contrary, R. rubra showed similar action with or
without the addition of glucose in the nutrient medium as an
extra source of carbon. Both mediums gave positive bio-
degradation results from the beginning of the trials. The
medium supplemented with glucose gave actually a higher
biodegradation rate of 2.1 μg/mL/day comparing to 1.38 μg/
mL/day on the medium without glucose. In both mediums
TCP was detected on day 1.
Effect of chlorpyrifos concentration on biodegradation
Effect of temperature on biodegradation of chlorpyrifos
Biodegradation trials of chlorpyrifos with different con-
centrations (10 and 25 μg/mL) of the insecticide were con-
ducted in the MSM at 25 °C. Both yeasts were proved to be
capable degraders of chlorpyrifos. Rhodotorula glutinis
consumed fully 25 μg/mL chlorpyrifos at 10 days when the
MSM was supplemented with glucose and at 6 days in the
case of the non- glucose-supplemented MSM. As it was
observed, R. glutinis maintained the delayed action under
the presence of glucose. Biodegradation was faster and
higher rates were obtained when the yeast was incubated
with 25 μg/mL chlorpyrifos in both media (Fig. 3). In flasks
supplemented with glucose the rate of 1.24 μg/mL/day
Both yeasts showed similar action when they were incubated
under different conditions of temperature(15, 25 and 35 °C).
At 35 °C no degradation was observed after 7 days of
incubation, while the degradation rates in 25 and 15 °C were
2.6 and 1.8 μg/mL/day for Rhodotorula glutinis and 1.88
and 1.52 μg/mL/day for Rhodotorula rubra, respectively.
The results are considered acceptable regarding that Rho-
dotorula yeasts belong to phychrophyllic microorganisms.
Production of metabolite 3,5,6-trichloro-2-pyridinol
(
(
10 μg/mL chlorpyrifos) increased to 2.74 μg/mL/day
25 μg/mL chlorpyrifos). The corresponding values in the
The metabolite 3,5,6-trichloro-2-pyridinol (TCP) was pro-
duced as a degradation product from both yeasts (Fig. 2).

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula. . .
Fig. 3 Biodegradation of chlorpyrifos by the yeasts Rhodotorula glutinis and Rhodotorula rubra in MSM supplemented with 10 or 25 μg/mL
6
chlorpyrifos and 10 cells/mL inoculum
for R. rubra at concentration of 57 μM. The IC50 (half
maximal inhibition concentration- IC ) values calculated
5
0
were 43 μΜ for R. glutinis and 42 μΜ for R. rubra. On the
other hand, chlorpyrifos did not reduce the activity of GSTs
at the concentrations tested and under assay conditions
described (Fig. 5). These results provide an indication that
esterases mediate the observed biodegradation of chlor-
pyrifos by the two yeasts of the present study.
Fig. 4 Biodegradation of the metabolite 3,5,6-trichloro-2-pyridinol
TCP) by the yeasts Rhodotorula glutinis and Rhodotorula rubra in
MSM supplemented with 5 TCP μg/mL chlorpyrifos and 10 cells/mL
inoculum. Each value is the mean of three replicates with error bars
representing the standard deviation of the mean
Synergists
(
6
As a first step the potential inhibition of three tested
synergists in the development of the cultivations of the
yeasts was tested by incubating with 1, 5, 10 and 15 μg/mL
of triphenyl phosphate, diethyl maleate and piperonyl but-
oxide (one in a time) and no significant inhibition (< 5%)
was observed. Therefore 10 μg/mL of each enzymatic
inhibitor was added in the 250 mL flasks of the biode-
gradation trials. As it was observed after 10 days of incu-
bation, diethyl maleate did not affect the biodegradation of
chlorpyrifos by R. glutinis and R. rubra and similar results
were produced after the addition of piperonyl butoxide. On
the contrary, triphenyl phosphate substantially inhibited the
biodegradation rate of chlorpyrifos. After the end of the
experiment 97.5 and 97.2% of the initial concentration of
chlorpyrifos was recovered respectively for each yeast.
These results enforced the above preliminary statements
that esterases are involved in the biodegradation of chlor-
pyrifos by the two epiphytic yeasts.
TCP was determined in all flasks of biodegradation. It was
also observed that TCP levels increased proportionally to
the concentration of chlorpyrifos added in the MSM. As
shown TCP levels were increasing up to a maximum (day 5
to 7) and then started to decrease, a fact that gave indica-
tions of its possibly degradation by the yeasts. The above
was confirmed by the results of biodegradation trials with
the addition of TCP (5 μg/mL) in the MSM as a sole source
of carbon. The initial concentration of TCP was decreased
but not depleted (Fig. 4).
Enzymatic assays–Synergists
Enzymatic assays
As observed, the activity of esterases of both yeasts was
significantly reduced by the addition of chlorpyrifos in the
homogenates (F = 330,38; df = 1; P < 0.05), (Fig. 5). Fur-
thermore, statistical analysis of the results showed a sig-
nificant difference between the response of the control
Biodegradation of chlorpyrifos on tomato fruits
Primary indications of biodegradation of chlorpyrifos by the
yeasts R. glutinis and R. rubra can be derived as shown in
Table 2 The decrease of chlorpyrifos in the treated tomatoes
was significant different for both yeasts to the correspond-
ing of the control 30 days after the application (Rhodotorula
glutinis: t-test, t(2) = 9.379, P < 0.05 and Rhodotorula
rubra: t-test, t(2) = 75.44, P < 0.05). The initial
(
homogenate) over the 5 concentrations of chlorpyrifos
added compare to the response of the treatment (homo-
genate+chlorpyrifos) over the 5 concentrations of chlor-
pyrifos added (F = 38.19; df = 4; P < 0.05).The maximum
inhibition observed was of 55.7% for R. glutinis and 65.7%

E. D. Bempelou et al.
Fig. 5 Effect of chlorpyrifos on glutathione-S-transferases (gsts) and esterases activity of Rhodotorula glutinins and Rhodotorula rubra. Each
value is the mean of three replicates ± relative standard deviation
6
Table 2 Residues of chlorpyrifos (mg/kg) on tomato fruits sprayed with 2 mg/kg chlorpyrifos and 10 cells/mL of the yeasts Rhodotorula glutinis
and Rhodotorula rubra
Days
Concentration of chlorpyrifos C (mg/kg)
Rhodotorula glutinis
Rhodotorula rubra
Control
Application
% of the control in the flask
Control
Application
% of the control in the flask
5
1.75 ± 4.23
1.02 ± 8.39
1.47 ± 0.89
0.22 ± 2.01
84
1.75 ± 3.22
1.02 ± 0.59
1.31 ± 1.9
0.08 ± 2.5
89
3
0
21.6
7.8
Control are the fruits sprayed only with chlorpyrifos and application the fruits sprayed with chlorpyrifos and the inoculum. Each value is the mean
of three replicates ± relative standard deviation
concentration of chlorpyrifos was decreased up to 78.4% of
the control when R. glutinis was added, and up to 92.2% in
the case of R. rubra.
analytical methods for the determination of chlorpyrifos and
TCP are based on HPLC/UV (Abu-Qare and Abou-Donia
2001) or GC/MS after derivatization (Koch and Angerer
2001). Sancho et al. (2000) reported the simultaneous
determination of the insecticide and its metabolite by LC/
MS/MS using different analytical columns and different
mobile phases for their separation, a technique much more
time and reagent consuming.
Discussion
The biodegradation of the insecticide chlorpyrifos by the
epiphytic yeasts Rhodotorula glutinis and Rhodotorula
rubra was shown in the present report. Both organisms are
naturally occurring in plants and are not pathogens.
The approach followed to examine biodegradation was
based on the incubation of each yeast in MSM supple-
mented with chlorpyrifos under different experimental
conditions.
The variation on the levels of chlorpyrifos and its
metabolite TCP during time were determined by LC/MS/
MS. The measurements obtained are considered indis-
putable, since they are based on the fully successful vali-
dation of the analytical method performed and the optimum
detection and separation of both analytes on the chroma-
tographic system. The determination of the two compounds
was achieved in the LC/M/MS system using different
ionization mode for each compound. However, the simul-
taneous detection of the two analytes in one chromatogram
by changing the ionization mode of the electrospray was
managed, a technique not been reported yet. Up to now the
Biodegradation proved to be dependent on the inoculum
density of each yeast, as almost negligible biodegradation of
2
4
the insecticide occurred from low inoculums (10 and 10
cells/mL), a fact that has also been reported for Sphingo-
monas sp. strain DSP-2, Enterobacter strain-B14 (Li et al.
2007; Yang et al. 2006; Singh et al. 2004). Another para-
meter tested on biodegradation was the possibly different
action of the microorganisms when the MSM was supple-
mented with an additional source of carbon, glucose. As
shown, under the presence of glucose Rhodotorula glutinis
showed delayed bio-degradative activity. In agreement with
our results Muncnerova and Augustin (1995) and Bempelou
et al. (2013) reported the degradation of benzoate and dia-
zinon, respectively, by R. glutinis only after the consump-
tion of glucose in the medium. The difference in the
degradation rate of chlorpyrifos depending on the nutrient
medium has also been reported for Enterobacter strain B-
14, which delayed the consumption of chlorpyrifos under
the presence of glucose or succinic acid (Singh et al. 2004).

Biodegradation of chlorpyrifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodotorula. . .
According to Ruiz-Amil et al. (1965); Torrontegui et al.
1966); Fernadez et al. (1967) and Medrano et al. (1969) the
by the by the epiphytic yeasts R. glutinis and R. rubra can
be made.
Finally, biodegradation was also investigated on plant
(
presence of glucose in the nutrient medium prevents the
assimilation or the metabolism of another substance from
the cells of the yeast until the depletion of the levels of
glucose. On the contrary, in the case of Rhodotorula rubra,
the addition of glucose in the nutrient medium did not affect
its ability to degrade chlorpyrifos. This has also been
reported by Xu et al. (2008) during the degradation of
chlorpyrifos by the bacterium Paracoccus sp. strain TRP.
As far as the effect of chlorpyrifos concentration on bio-
degradation is concerned, as it was observed the higher the
concentration it was, the more active the yeasts were.
Similar observations have been made by Yang et al. (2005)
and Fang et al. (2008) while studying the biodegradation of
chlorpyrifos by the bacterium Alcaligenes faecalis DSP3
and the fungi Vericillium sp. DSP, respectively.
surfaces and in particular in tomato fruits and the results
measured in the LC/MS/MS system were in line with our
in vitro biodegradation study, providing a primary indica-
tion of the phenomenon in plant surfaces. It should also be
mentioned that the trials were conducted in harvested
tomato fruits and therefore are considered as a worst-case
scenario since the reaction between the microorganism and
the insecticide was studied under completely controlled
conditions without the interferences of other factors (bio-
logical, environmental) which might contribute to the dis-
appearance of chlorpyrifos from the plant surfaces. Our
findings indicate that the epiphytic yeasts R. glutinis and R.
rubra could possibly be used successfully for the removal
or detoxification of chlorpyrifos, residues in/on vegetables.
The degradation product identified in all trials was the
metabolite TCP whose concentration was increased up to a
maximum and then started decreasing since R. glutnis and
R. rubra proved also to be able to consume TCP as well.
Chlorpyrifos-oxon, 3,5,6-trichloro-2-methoxypyridine and
Acknowledgements Our study entitled “Biodegradation of chlorpyr-
ifos and 3,5,6-trichloro-2-pyridinol by the epiphytic yeasts Rhodo-
torula glutinis and Rhodotorula rubra”, was not funded.
Compliance with ethical standards
2
-chloro-6-hydroxypyridine have also been identified as
degradation product of chlorpyrifos (Singh et al. 2006; Yu
et al. 2006), but were not detected in the present study. The
ability of biodegradation of TCP has also been reported for
Sphingomonas sp. strain DSP-2 (Li et al. 2007), Alcaligenes
faecalis strain DSP-3 (Yang et al. 2005), Paracoccus sp.
strain TRP (Xu et al. 2008), Verticillium sp. DSP (Fang
et al. 2008), Bacillus pumilis strain C2A1 (Anwar et al.
Conflict of interest The authors declare that they have no no conflict of
interest.
Ethical approval This article does not contain any studies with human
participants or animals performed by any of the authors.
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