Food Control 20 (2009) 419422

Contents lists available at ScienceDirect

Food Control
journal homepage: www.elsevier.com/locate/foodcont

Screening of fresh fruit and vegetables for pesticide residues on Croatian market
Zorka Knezevic *, Maja Serdar
Environmental Health Service, Croatian National Institute of Public Health, Rockefellerova 7, 10000 Zagreb, Croatia

a r t i c l e

i n f o

Article history:
Received 6 February 2008
Received in revised form 4 July 2008
Accepted 11 July 2008

Keywords:
Pesticides
Fruit
Vegetables

a b s t r a c t
The aim of this study was to investigate pesticide residues in market foods in Croatia. A total of 240 samples of fresh fruit and vegetables from import and domestic production were analyzed. Pesticide resides
were determined by gas chromatography with mass selective detector (GCMSD). Sample extract was
cleaned up using gel permeation chromatography (GPC). In 66.7% of the samples no residues were found,
25.8% of samples contained pesticide residues at or below MRL, and 7.5% of samples contained pesticide
residues above MRL. Most frequently found pesticides were imazalil (found in 35 samples) and chlorpyrifos (found in 24 samples). The ndings of this study pointed to the following recommendations: the need
for a monitoring program for pesticide residues in food crops, especially imported food crops.
2008 Elsevier Ltd. All rights reserved.

1. Introduction
Fresh fruit and vegetables are an important part of a healthy
diet as they are a signicant source of vitamins and minerals. However, fresh fruits and vegetables can also be a source of noxious
toxic substances pesticides. Fruit and vegetables are traded
worldwide and the list of pesticides that might have been applied
in their agricultural production is usually not known (Stan, 2000).
Pesticides constitute a very important group of chemical compounds that have to be controlled due to their high toxicity and
their widespread use in agricultural practice for eld and post-harvest protection. The presence of pesticide residues in food is a direct result of pesticide use on crops. Over 1000 compounds may
be applied to agricultural crops in order to control undesirable
moulds, insects and weeds (Ortelli, Edder, & Corvi, 2006). The levels of pesticide residues in foodstuffs are generally legislated so as
to minimize the exposure of the consumer to harmful or unnecessary intakes of pesticides, to ensure the proper use of pesticides in
terms of granted authorization and registration (application rates
and pre-harvested intervals) and to permit the free circulation of
pesticide-treated products, as long as they comply with the xed
maximum residue level (MRLs). MRL for pesticide residues represents the maximum concentration of that residue (expressed
in mg/kg) that is legally permitted in specic food items. The
establishment of MRL is based on good agricultural practice data
on food derived from commodities. MRLs are not toxicological limits, but they must be toxicologically acceptable. Exceeded MRLs are
strong indicators of violations of good agricultural practices (Nasreddine & Parent-Massin, 2002).

* Corresponding author. Tel.: +385 1 4863 323; fax: +385 1 4683 907.
E-mail address: zorka.knezevic@hzjz.hr (Z. Knezevic).
0956-7135/$ - see front matter 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodcont.2008.07.014

The objective of this study was to investigate the presence of

pesticide residues (Table 1) in market foods (fruit and vegetables)
in Croatia. Samples were collected from June to December 2007.
Collected data are to be used as a reference point for future monitoring and taking preventive measures to minimize human health
risks.
2. Materials and methods
2.1. Materials
Reference materials were obtained from Riedel-de Han (Seelze,
Germany) or Supelco (Bellefonte, PA, USA) (see Table 1). Organic
solvents (residue analysis grade) for dissolving and extracting were
acetone, ethyl acetate and cyclohexane purchased from Merck
(Darmstadt, Germany). Stock standard solutions were prepared
by exact weighing of pesticide reference material and dissolution
in acetone. Working standard solutions were prepared by appropriate dilution with a mixture of ethyl acetate/cyclohexane (v/v,
1:1). Stock standard solutions and working standard solutions
were stored under refrigeration (4 C). Anhydrous sodium sulfate,
sodium chloride and sodium hydrogen carbonate (p.a.) were obtained from Kemika (Zagreb, Croatia).
2.2. Samples
A total of 240 samples of different kind of fresh fruit (oranges,
apples, peaches, pears and grapefruits) and vegetables (lettuce, tomato, cabbage, potato, onion and leek) were collected from supermarkets and markets of four Croatian cities Zagreb, Osijek, Rijeka
and Split. The samples taken included: 106 samples of fruits and
134 samples of vegetables. Samples were analyzed within 24 h

Z. Knezevic, M. Serdar / Food Control 20 (2009) 419422

Table 1 (continued)

Table 1
Selected pesticides, use and purity of reference materials

1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
13.
14.
15.
16.
17.
18.
19.
20.
21.
22.
23.
24.
25.
26.
27.
28.
29.
30.
31.
32.
33.
34.
35.
36.
37.
38.
39.
40.
41.
42.
43.
44.
45.
46.
47.
48.
49.
50.
51.
52.
53.
54.
55.
56.
57.
58.
59.
60.
61.
62.
63.
64.
65.
66.
67.
68.
69.
70.
71.
72.
73.
74.

Pesticide

Purity (%)

Obtained

Acephate
Allethrine
Azinphos-ethyl
Azinphos-methyl
Bifenthrin
Bioallethrin
Bromophos-ethyl
Bromophos-methyl
Brompropylate
Captan
Carbaryl
Chlordane cis
Chlordane trans
Chlormephos
Chlorfeninphos cis
Chlorfeninphos trans
Chlorothalonil
Chlorpropham
Chlorpyrifos
Chlorpyrifos-methyl
Coumaphos
Cyuthtin I
Cyuthtin II
Cyuthtin III
Cyuthtin IV
Cyhalothrin Lambda
Cypermethrin I
Cypermethrin II
Cypermethrin III
Cypermethrin IV
Cyphenothrin I
Cyphenothrin II
DDD-p,p
DDE-p,p
DDT-o,p
DDT-p,p
Deltamethrin
Demeton S
Demeton-S-Methyl
Demeton-S-Methyl-sulfon
Dialifos
Diazinon
Dichlouanid
Dichlorvos
Dicofol
Dimethoate
Endosulfane-alpha*
Endosulfane-beta*
Endosulfanesulfat
Ethion
Fenamiphos
Fenchlorphos
Fenitrothion
Fenprpathrin
Fenthione
Fonofos
HCB
HCH alpha
HCH beta
HCH delta
HCH gama (Lindan)
Heptachlorepoxid-egzo
Heptachlorepoxid-endo
Heptenophos
Imazalil
Iprodione
Isofenphos
Malaoxon
Malathion
Methamidophos
Methidathion
Metoksiklor
Mevinphos cis
Monocrotophos

96.0
95.3
99.9
98.5
99.5
99.5
99.3
99.8
99.2
99.4
99.8
98.4
98.4
99.2
97.2
97.2
99.2
99.9
99.5
99.7
99.7
98.3
98.3
98.3
98.3
99.7
96.7
96.7
96.7
96.7
97.2
97.2
99.2
99.5
99.1
98.2
99.8
99.0
95.4
95.4
99.6
98.9
98.8
99.4
97.6
99.4
70.0
30.0
97.7
97.9
99.5
99.0
95.4
98.4
96.8
98.6
99.6
99.7
98.1
99.7
99.8
99.7
99.9
97.1
99.8
99.9
98.9
96.3
97.3
98.4
96.0
98.8
98.6
99.9

Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Supelco
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Supelco
Supelco
Riedel-de
Supelco
Supelco
Riedel-de
Riedel-de
Supelco
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Supelco
Supelco
Riedel-de
Riedel-de
Riedel-de
Supelco
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de

Use
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han

Han

Han
Han
Han
Han
Han
Han
Han
Han
Han
Han

Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han

I
I
A,I
A,I
A,I
I
I
I
I,A
F
I
I
I
I
A,I
A,I
F
H,PR
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
I
A,I
I
F
A,I
A
A,I
I
I
I
A,I
I,N
I
I
I
I
I
F
I
I
I
I
I
I
I
F
F
I
A,I
A,I
A,I
I
I
A,I
A

75.
76.
77.
78.
79.
80.
81.
82.
83.
84.
85.
86.
87.
88.
89.
90.
91.
92.
93.
94.
95.
96.
97.
98.
99.
100.

Pesticide

Purity (%)

Obtained

Omethoate
Paraoxon
Parathion
Parathion-methyl
Permethrin-cis
Permetrin-trans
Phenothrin cis
Phenothrin trans
Phorate
Phosalone
Pirimicarb
Pirimiphos-ethyl
Pitimiphos-methyl
Procymidone
Pyrazophos
Resmethrin cis
Resmethrin trans
Tetrachlovinphos
Tetramethrin cis
Tetramethrin trans
Tolclofos-methyl
Thiabendazole
Tolyluanid
Transuthrin
Trichlorfon
Vinclozolin

98.4
97.2
98.0
99.8
98.0
98.0
94.9
94.9
95.7
99.3
99.0
99.8
99.9
98.7
99.8
94.3
94.3
97.9
98.4
98.4
99.2
99.9
99.6
99.7
97.8
99.6

Riedel-de
Riedel-de
Supelco
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de
Riedel-de

Use
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han
Han

A,I
A,I
I
I
I
I
I
I
I
A,I
I
I
I
I
F
I
I
I
I
I
F
F
F
I
I
F

A: acaride; F: fungicide; H: herbicide; I: insecticide; N: nematicid; PR: plant growth

regulator.
*
Reference material of endosulfane alpha and beta is a mixture of 70% endosulfane
alpha and 30% endosulfane beta.

stored at 4 C until the analysis. All samples were collected according to the method of taking representative sample (Narodne novine, 58/98). Samples origin is shown in Fig. 1.
2.3. Sample preparation
2.3.1. Sample preparation of material with a water content exceeding
70 g/100 g and a fat content below 2.5 g/100 g
Samples prepared according to this procedure are: apple (water
content 85%), peach (water content 90%), pear (water content 85%),
lettuce (water content 95%), tomato (water content 95%), cabbage
(water content 90%), potato (water content 80%), onion (water content 85%) and leek (water content 85%).
Samples were homogenized in a food cutter and subsamples of
50 g (ms) of the material having a water content of x g/100 g
weighted into a glass jar. Sufcient amount of water was then
added to adjust the total water presence to 100 g. The amount of
water (mw) to be added is calculated as follows: mw = 100
100
86

90

Number of samples

420

80
70

Fruits

61

Vegetables

60
50
40
30

25

22

20

26
20

10
0
Domestic

Import

Fig. 1. Origin of analyzed samples.

Unknown

Z. Knezevic, M. Serdar / Food Control 20 (2009) 419422

ms
x/100. Next 100 ml acetone was added and the mixture

homogenized for 2 min with a homogenizer (Politron PT-MR3100, Kinematica AG, Switzerland). Sodium chloride (17.5 g) and
50 ml mixture of cyclohexane/ethyl-acetate, 1:1 (v/v) were added
to homogenate and homogenize it again for 1 min. After the phases
was clearly separated (after 3060 min), the upper organic phase
was collected and exactly 100 ml of the organic phase was measured out in a graduated cylinder and ltered through a glass wool
plug layered with approx. 50 g sodium sulfate in a funnel. The ltrate was collected in a 500 ml round-bottomed ask and the graduated cylinder and funnel were rinsed four times each with approx.
10 ml mixture of cyclohexane/ethyl-acetate, 1:1 (v/v). Combined
ltrate and rinsings were concentrated using the rotary evaporator
(Heidolph Laborota, Germany) at bath temperature 40 C. The almost dried residues (not completely dried) were re-dissolved with
7.5 ml ethyl acetate. Afterwards, approx. 2.5 g salt mixture
(Na2SO4, anhydrous + NaCl, 1:1 (w/w)) was added in the solution
and 7.5 ml cyclohexane. Solution was ltered through a membrane
lter, 0.45 lm pore size, 25 mm dia (Minisart RC 25, Sartorius AG,
Germany). (For details see Modular Multiple Analytical Method for
Determination of Pesticide Residues in Foodstuffs LMBG 00.00 34
S19.)
2.3.2. Sample preparation of material with a water content exceeding
70 g/100 g and a fat content below 2.5 g/100 g and a high acid
Samples prepared according to this procedure are: orange
(water content 85%) and grapefruit (water content 90%).
Samples were homogenized in a food cutter and subsamples of
50 g (ms) of the material having a water content of x g/100 g and
high acid content weighted into a glass jar. It is necessary prior
extraction to adjust the pH value of the test material to approx. 7
(using pH indicator paper) by adding small portions of sodium
hydrogen carbonate. Extraction procedure is described in Section
2.3.1. (For details see Modular Multiple Analytical Method for
Determination of Pesticide Residues in Foodstuffs LMBG 00.00 34
S19.)
2.4. Clean-up
The extract solution derived from extraction module E1 and E3
is cleaned up by gel permeation chromatography. A GPC system
(GPC Ultra LC Tech GmbH Dorfen, Germany) consisting of a 26 position sample rack, isocratic HPLC pump, vacuum pump a
(400  25 mm I.D.) GPC glass column packed with Bio-Beads SX-

421

3, a 5 ml sample loop. Eluent: ethyl acetatecyclohexane (1:1, v/

v) at a ow-rate of 5 ml/min.
2.5. Analysis
An Agilent Technologies (Paolo Alto, CA, USA) 6890N GC-MSD
system equipped with autosampler, split/splitless injector with
electronic pressure control and mass selective detector (MSD) series 5975B Inert was used. Separation was performed on HP-5MS
capillary column (30 m  0.25 mm I.D.  0.25 lm lm thickness).
Column temperature program began at 70 C (hold 3.5 min), after
which it was ramped to 150 C at 50 C min 1, then to 180 C at
5 C min 1, then to 183 C at 1 C min 1 (hold for 4 min), then
raised to 191 C at 1 C min 1 (hold for 4 min), then ramped to
205 C at 1 C min 1, and nally increased to 300 C at 4 C min 1
(hold for 5 min) (Arrebola, Martnez Vidal, Mateu-Snchez, &
lvarez-Castelln, 2003). The carrier gas was helium. Flow rate
was constant ow mode at 1 ml min 1. Splitless injection of 1 ll
volume was carried out at 260 C with the purge valve on 2 min.
The liner used was a splitless single trapper, no glass wool, deactivated from Agilent (Paolo Alto, CA, USA). Transfer line temperature
was 300 C. Ion energy for electron impact (EI) was always 70 eV.
Calibration of the mass spectrometer was done weekly. Mass
detection was performed in the single ion monitoring (SIM) mode
(with consideration of the relative intensities of selected ions).
3. Results
A total 240 samples were analyzed 106 samples of fruit (orange, apple, peache, pear and grapefruit) and 134 samples of vegetables (lettuce, tomato, cabbage, potato, onion, leek). In the
analyzed samples 17 different pesticides were found. Frequency,
concentration and identity of pesticides found in the analyzed
samples are outlined in Table 2.
Of the 240 samples analyzed, in 160 (66.7%) no pesticide
residues were detected. 62 (25.8%) samples contained pesticide
residues at or below MRLs established by either the Croatian Legislation or the European Union (EU). 18 samples (7.5%) contained
pesticide residues above MRL. MRL values were exceeded most often in oranges. All samples of cabbage, onion and leek were residue-free. The total number of commodities containing residues
above MRL is presented in Fig. 2. The highest concentration of pesticide residue was 27.9 mg/kg of imazalil. The occurrence of pesticide residues in fruit and vegetables samples are shown in Fig. 3.

Table 2
Pesticides concentration ranges found in the samples
Pesticide type

Fungicide

Insecticide

Other

Chemical name

Imazalil
Thiabendazole
Tolyluanid
Iprodione
Chlorothalonil
Viclozolin
Procymidone
Chlorpyrifos
Chlorpyrifos-methyl
Azinphos-methyl
Methidathion
Malathion
Fenthione
Endosulfane beta
Diazinon
Tetramethrin
Chlorpropham

Fruits

Vegetables

Number of positive samples

MinMax (mg/kg)

Number of positive samples

MinMax (mg/kg)

35
17
8

24
4
1
3
1
1

4
3

0.0827.9
0.1210.62
0.0610.74

0.030.67
0.030.14
0.32
0.130.33
0.07
0.05

0.060.08
0.050.82

1
2
1
3
1
1

0.07
10.5711.57
0.51
2.793.27
3.58
0.37

0.010.04

0.73.6

Z. Knezevic, M. Serdar / Food Control 20 (2009) 419422

Number of samples

422

10
9
8
7
6
5
4
3
2
1
0

28% ( 5 samples)

Oranges

Apple

Lettuce

Peach

22% (4 samples)

Potato

Commodity

50% (9 samples)

Domestic

Fig. 2. Number of samples containing residues above MRL.

Import

Unknown

Fig. 5. Origin of samples containing residues above MRL.

140

118

Number of samples

120
100

Fruits
Vegetables

80
60

50
42

40
20

12

14
4

0
No reidues found Residues at or below

residue 19 samples (30.65%) contained two pesticide residue.

The combination of two fungicides or one fungicide and one insecticide was frequent. Nine samples (14.52%) contained three pesticide residues. Total samples contained two fungicides and one
insecticide, and all were oranges. More than one pesticide residues
were found in 18 samples of fruits and only 1 sample of vegetables.
Occasionally, residues of pesticides which are not approved for
use on a particular crop or food in general were found. In three
samples of apples tetramethrin was found in the concentrations
of 0.05, 0.06 and 0.82 mg/kg, which was probably due to storage
contamination. One sample of lettuce contained 0.51 mg/kg of
chlorothalonil, and one sample of potato 0.37 mg/kg of
chlorpyrifos.

Residues above MRL

MRL found

4. Conclusions
Fig. 3. Occurrence of pesticide residues in fruit and vegetables samples.

13% (8 samples)

31% (19
samples)

The present study shows a high incidence rate of pesticide residues (mostly fungicides and insecticides) in analyzed samples,
especially fruits. The most critical commodity is oranges. The contamination level of pesticide residues could be considered a possible public health problem. The results also emphasize the need for
regular monitoring of a greater number of samples for pesticide
residues, especially imported samples.
References

56%(35 samples)
Domestic

Import

Unknown

Fig. 4. Origin of samples containing residues at or below MRL.

The occurrence of pesticide residues in samples according to origin

of fruit or vegetable is shown in Fig. 4, and the number of samples
containing residues above MRL according to origin of samples is
shown in Fig 5. In terms of co-occurrence of pesticide residues,
28 samples (45.2% of positive samples) contained more the one

Arrebola, F. J., Martnez Vidal, J. L., Mateu-Snchez, M., & lvarez-Castelln, F. J.

(2003). Determination of 81 multiclass pesticides in fresh foodstuffs by a single
injection analysis using gas chromatography-chemical ionization and electron
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