Id ElMoueden et al.,Maghr. J. Pure &Appl. Sci.

, 1 N1 (2015) 11-17

Maghrebian Journal of Pure and Applied

Science
ISSN : 2458-715X
http://revues.imist.ma/?journal=mjpas&page=index

Disappearance of Azoxystrobin and difenoconazole in green beans

cultivated in Souss Massa valley (Morocco)
O. Id El Mouden1,M. Errami1,R. Salghi1*, H. Bouya1,S. Jodeh2,
I. Warad2, O. Hamed2, R. Touzani3
1

Laboratory of Applied Chemistry and Environment, ENSA, Universit Ibn Zohr,PO Box 1136, 80000 Agadir,
Morocco
2
Department of Chemistry, An-Najah National University, P. O. Box 7, Nablus, Palestine.
3
Laboratory of Applied Chemistry and Environment (URAC 18), Faculty of Sciences,University Mohammed
Premier, P. O. Box 4808, 60046 Oujda, Morocc
*Corresponding author: r.salghi@uiz.ac.ma ; Phone: 00212661145512
Received 14 Oct 2015, Revised 15 Nov 2015, Accepted 16 Nov 2015

Abstract
A study was undertakento evaluate the degradation behavior and residue levels of azoxystrobin and
difenoconazolein Belma green beans variety grown in an experimental plastic greenhouse. The measurements
were made over a 3 week period in which up to two successive treatments with azoxystrobin and a 5 week
period in which up to two successive treatments with difenoconazole were carried out.Residue levels of dicofol
and difenoconazolewere determined by Gas chromatography with electron capture detection (GC-ECD), liquidliquid extraction (LLE), solid phase extraction (SPE) and high-performance liquid chromatography (HPLC).
During the study, residue levels in the plantation ranged between 0.35 and 0.01 mg/kg for azoxystrobinand
between 0.25 and 0.01 mg/kg for difenoconazole.The residual concentrations after the preharvest intervals (PHI)
were below the legal limits.
Key words: Pesticide residues, azoxystrobin,Green Beans, greenhouse, Morocco.

1. Introduction and Experimental

Agriculture plays a major economic and social role in Morocco and largely contributes to efforts of
economic takeoff that the country has been undertaking for some decades. The Moroccan green
beanssector was chosen at the case study because of the increasing of private certification in this
sector. Souss Massa valley is the major region for green beans production in Morocco, exporting about
85% to the European Union (EU). Greens beans are the most important horticultural export product of
the country. In addition ,the sector has a particular social importance in that it offers income
possibilities to those with little access to land, and moreover provides possibilities of employment in
rural areas , where there tend to be few other alternative jobs.Development and productivity of green
beans system, however, are tightly linkedto pesticide use in Morocco. In fact, the use of pesticides,
mainly insecticides, remains one of the most important pest control measures for green beans plant
protection. Use of these chemicals in large amounts without consideration of potential human health
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Id ElMoueden et al.,Maghr. J. Pure &Appl. Sci., 1 N1 (2015) 11-17

hazards, resulting from pesticide residues accumulation in food and water [1,3].However, can be
dramatic.Among these chemicals, Azoxystrobin (methyl (E)-2-{2-[6-(2-cyanophenoxy)pyrimidin-4yloxy]phenyl}3-methoxyacrylate) a strobilurin fungicide, is a broad spectrum, systemic and
soilapplied fungicide . Azoxystrobin, rst-syntheticstrobilurin product launched by Syngenta, is now
registered for use on 84 different crops in 72 countries, representing over 400 crop/disease system [4].
The studies of the European Union carried out within the framework of studies 91/414/EEC of the
Council (European Commission 91/414/EEC, 1997), approved the registration of azoxystrobin in 1998
as an active substance of minimum purity of 930 g/kg which contains 25 g/kg of Z-isomer. It is the
mixture that does not cause any particular toxicological effects, with the Acceptable Daily Intake
(ADI) for humans of 0.1 mg/kg t.m/day [5,6].On the other hand,Difenoconazole (cis,trans-3-chloro-4[4-methyl-2-(1H-1,2,4-triazol-1-ylmethyl)-1,3-dioxolan-2-yl]phenyl-4-chloro phenyl ether) , is a
broad-spectrum 1,2,4-triazolefungicide used for the control of fungal diseases on fruits, vegetables,
cereals, and other eld crops [7,8]. The molecular structures of dicofol and difenoconazoleare given in
Fig. 1.The dissipation of the pesticides after their application depends on several factors, such as the
applied dose and formulation [9], application parameters, the number of applications, climatic
conditions, the species cultivated, physical phenomena, and chemical degradation [1017].

AzoxystrobinDifenoconazole
Figure 1. Molecular structures of fungicides studied.
The objective of this work was to evaluate the degradation behavior and residue levels of azoxystrobin
and difenoconazole in green beans in a plastic greenhouse using the typical horticultural practices of
this type of crop (i.e. multiple pesticide applications and short preharvest intervals).
2.Field trial.
Field experiments were conducted in a plastic greenhouse with an area of 500 m 2 located in Souss
Massa (Agadir, Morocco). The planting density (variety Belma) was 10600 plants/ha. The planting
density (variety Belma) was 12000 plants/ha. In the case of field experiments, a random block scheme
was employed. Each block contained 25 plants in a single row, and tests were carried out in triplicates.
Tested blocks were partitioned and isolated from one another by leaving two untreated rows as guard
rows. Azoxystrobin and difenoconazole were sprayed at doses equivalent to 50 cc/hl,using a hand
operated Knapsack sprayer (Table1).
Table 1. Details of pesticides and doses used.
Activeingredient Commercialformulation

Dose

Pre-

MLR(mg/kg)

(cc/hl)

harvestinterval(days)

Azoxystrobin

Ortiva250g/L

50

Difenoconazole

Score 250g/L

50

14

Residue levels of pesticide were determined in green beans during three in which up to two successive
treatments with azoxystrobin were carried out and a five week period in which to two successive
treatments with difenoconazole were applied to the different plantations (treatments I and II ), with
intervals of 0, 2, 4, 7, 12, 15 and 21 days for difenoconazole and 0, 1, 3, 7, 8 , 9, 10, 11 and 12 days for
azoxystrobin.Analysis samples of difenoconazole were collected at 0, 2, 4, 7, 12 and 15 days after
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Id ElMoueden et al.,Maghr. J. Pure &Appl. Sci., 1 N1 (2015) 11-17

treatment I (samples I+0, I+2, I+4, I+7, I+12 and I+15); at 0, 2, 4, 7, 12, 15 and 21 days after treatment
II (samples II+0, II+2, II+4, II+7, II+12, II+15 and II+21).
Analysis samples of azoxystrobin were collected at 0, 1, 3, 7 and 8 days after treatment I (samples
I+0, I+1, I+3, I+7 and I+8); at 0, 1, 3, 7, 8 , 9, 10, 11 and 12 days after treatment II (samples
II+0, II+1,I I+3,I I+7, II+8,I I+9,I I+10,I I+11 and II+12).The daily maximum/minimum
temperatures inside the greenhouse throughout the study were 14 and 25C, whereas the daily
mediumrelative humidity was 90 %. The irrigation flow was 40 m3/ha.
2.1. Chemicals
Acetone, ethyl acetate, dichloromethane, n-hexane, diethyl ether and anhydrous sodiumsulphate
(pesticide residue grade) were obtained from Panreac (Barcelona, Spain). Florisiladsorbent (1630
mesh) was obtained from Sigma-Aldrich (St. Louis, MO, USA).Standards of Azoxystrobin and
difenoconazolewere obtained from Dr. Ehrenstorfer (Augsburg, Germany) with purity higher than
98%. Stock standard solutions of Azoxystrobin and difenoconazole were prepared in acetone. Standard
solutions for gas chromatographic (GC) analysis were prepared by suitable dilution of the stock
standard solutions with n-hexane.
2.2. Apparatus and Chromatography
The gas chromatograph was a Hewlett-Packard model 6890 (Palo Alto, CA, USA) equipped with a
split/split less injection port, auto sampler, a micro electron-capture detector (ECD) and an HP-5
fused-silica capillary GC column (25 m length, 0.32 mm internal diameter and 0.52 mm film
thickness).The chromatographic conditions were as follows: detector temperature, 300C; injector
temperature, 250C; temperature programming from 80 C to 250 C (15 C min -1), carrier gas
(helium) flow rate, 2.6 mL/min; makeup gas (nitrogen) flow rate, 60 mL.min-1; injection volume, 1 mL
and splitless time, 0.1 min. Data were acquired and the equipment was controlled using HP
(PaloAlto,CA,USA) Chem-Station software, which was run under Microsoft Windows NT on an HP
compatible personal computer.The High-performance liquid chromatography (HPLC) model HP
1050equipped with fluorescence detector mode HP 1046, stainless steel columnC18 (250 mm length
and 4.6 mm internal diameter),The mobile phase was a mixture of methanol and solution tampon
(60%/40%), flow rate, 0.8 mL/min and the sample volume injected for analysis was 50 m1.
2.3. Extraction Procedure
In total, the greenhouse samples consisted of 30 pieces of peach a taken at random according to the
method described by the DGCCRF [18]. Immediately after picking, the greenhouse samples were
placed into polyethylene bags, stored at 24C in a portable cooler and transported to the laboratory.
For the extraction of pesticides from green beans samples, themethod was adapted from Charles and
Raymond[19] .For each 50 g of the sample ground using a food processor (Type, model blender), 150
ml of acetonewas added and the mixture was homogenised for 2minutes and after mixed during 2
hours. The mixturewas filtered with glass wool. After filtration, the acetone residues were partitioned
with saturated aqueous sodium chloride (30 ml) and dichloromethane (70 ml) in a separating funnel.
The extraction was repeated with other 70 ml of dichloromethane and dried over anhydrous sodium
sulphate. The dichloromethane fraction was collected and evaporated on a rotatory vacuum evaporator
at 40C and the residues were dissolved in an acetone-hexane (1:9) mixture (10 ml). For clean-up, 1 ml
of the extract was passed through a florisil column previously conditioned with 5 ml of acetone/diethyl
ether (6:4) and 5 ml of diethyl ether.The pesticide residues were eluted with acetone/diethyl ether (6:4)
(4 ml). All samples wereextracted separately and analysed by gas chromatography six-times.
3.Results and discussion
3.1. Performance of the analytical method
To evaluate the efficiency of the analytical procedures a recovery assay was employed. The method was
validated by determining the limits of quantification (LOQ), recovery percentages and coefficient of variation.
Analytical standards of azoxystrobinand difenoconazole were purchased from Dr. EhrenstorferGmbH

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Id ElMoueden et al.,Maghr. J. Pure &Appl. Sci., 1 N1 (2015) 11-17

(Augsburg, Germany). Each standard was dissolved in acetone (50 mL) to obtain stock solutions
ofapproximately 200 mg L-1, which were stored light protected at 4C until further use. The freshly working
standardsolutions were obtained by dilutions with n-hexane. The linear dynamic range, precision (as relative
standard deviation) values for determination of azoxystrobin and difenoconazoleare reported in Table 2.

Table 2. Limit of quantification, average recovery and coefficient of variation values for azoxystrobin
and difenoconazole pesticides.
Analyte

LOQ
-1

(mg kg )

Regressionequation

R2

Y=a X+ b

Average

RSD

Recovery (%)

(%)

Azoxystrobin

0.01

Y=3.09 e+004 X + 1.08 e+0004

0.9994

108

1.34

Difenoconazole

0.01

Y=3.47 e+004 X + 7.49 e+0004

0.9992

102

1.89

a: slope; b: intercept; R: regression coefficient; LOQ: limit of quantification, RSD: relative standard
deviation (n=5).
Linear range: Individual calibration graphs were run with mixtures of azoxystrobin and difenoconazole at
concentrations in the range 10 to 200 ng.mL-1. Each solution was injected five times. The linear range, intercept
and slope of the curve are given in Table 1along with the regression coefficient for each pesticide.
Sensitivity: The LODs calculated in this way were 0.001 mg.mL-1 and 0.004 mg.mL-1 for difenoconazole and
azoxystrobin, respectively. The limits of quantification (LOQ) were0.001 mg.mL-1and 0.013mg.mL1
fordifenoconazole and azoxystrobin, respectively.
Precision: Untreated samples were fortified by the addition of an intermediate pesticide mixture solution.
Samples were allowed to equilibrate for 2 h prior to extraction and were processed according to the procedure
described above. The precision values for the method, expressed as relative standard deviation (RSD), were 1.34
and 1.89 % (n =5) for azoxystrobin and difenoconazole, respectively.

3.2. Evaluation of the decrease in azoxystrobin and difenoconazole residues in greenhouse green
beans samples
Azoxystrobin and difenoconazole residue levels determined in all of the green beans samples analyzed during
the study are indicated in Tables 3 and 4. Residues levels in green beans samples ranged between 0.35 and 0.01
mg kg-1 for azoxystrobin and 0.25 and 0.01 mg. kg-1for difenoconazole.Typical chromatograms from the
analysis of (a) the blank of green beans, (b) the analysis of the difenoconazle sample (II+4). (c) Chromatogram
from the analysis of the azoxystrobin sample( I+1).

3.3. Azoxystrobin
After the rst treatment, residue of azoxystrobin (0.15 mg/kg) was determined in green beans (table 2). Eight
days after the application, azoxystrobin residue was 0.01 mg/kg; therefore, at PHI (7days) the residue was
considerably below the EU MRL (3.0 mg/kg). Before the second application, the residue level on the fruit was
0.01 mg/kg, and after, it increased to 0.35 mg/kg. At harvest time, the residue was 0.01 mg/kg, while 7 days
after application (PHI) it was below the EU MRL.

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Id ElMoueden et al.,Maghr. J. Pure &Appl. Sci., 1 N1 (2015) 11-17

Figure 2. (a) Chromatogram from the analysis of a blank green beans sample. (b) Chromatogram from the
analysis of the difenoconazle sample II+4. (c) Chromatogram from the analysis of the azoxystrobin sample
I+1.

3.4. Difenoconazole
This fungicide had an initial residue of 0.12 mg/kg. Our results show that 15 days after the rst application,
which correspond to the PHI, difenoconazole residue was minor to the EU MRL (1.0 mg/kg). Similar
behaviourwas obtained after the second application, with an initial residue of 0.25 mg/kg.
Table 3. Difenoconazole residue levels, determined in in green beans samples analyzed during the study.
Day

Samplescollected

Concentration (mg/kg)

I+0

0.12 0.01

I+2

0.11 0.01

I+4

0.09 0.01

I+7

0.06 0.02

12

I+12

0.03 0.01

15

I+15

0.01 0.02

II+0

0.25 0.03

II+2

0.23 0.02

II+4

0.21 0.02

II+7

0.17 0.01

12

II+12

0.12 0.01

15

II+15

0.07 0.01

21

II+21

0.02 0.05

Treatment

II

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Id ElMoueden et al.,Maghr. J. Pure &Appl. Sci., 1 N1 (2015) 11-17

Table 4. Azoxystrobin residue levels, determined in in green beans samples analyzed during the study.
Day

Samplescollected

Concentration (mg/kg)

I+0

0.15 0.01

I+1

0.13 0.01

I+3

0.07 0.01

I+7

0.02 0.01

I+8

0.01 0.00

II+0

0.35 0.05

II+1

0.32 0.03

II+3

0.27 0.02

II+7

0.13 0.02

II+8

0.08 0.01

II+9

0.05 0.01

10

II+10

0.03 0.01

11

II+11

0.02 0.02

12

II+12

0.01 0.00

Treatment

II

4. CONCLUSION
We have studied the pesticide residue levels in green beans cultivated in souss masa valley (Morocco) after
multiple applications of azoxystrobin and difenoconazole. The results obtained showed thatthepreharvest
intervals established by Morocco law for these pesticides are correct because, after two applications at the
maximum recommended doses, the residue level was below the EU MRL for fungicides studied. The different
degradation in eld of theses pesticides is probably due to the different chemical structures of the compounds .

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