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Screening of fresh fruit and vegetables for pesticide residues on Croatian market
Food Control 20 (2009) 419–422
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 Knezˇevic´ *, 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 (GC–MSD). 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 findings of this study pointed to the following recommendations: the need for a monitoring program for pesticide residues in food crops, especially imported food crops. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Fresh fruit and vegetables are an important part of a healthy diet as they are a significant 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 field 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 fixed maximum residue level (MRLs). MRL for pesticide residues represents the maximum concentration of that residue (expressed in mg/kg) that is legally permitted in specific 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: [email protected] (Z. Knezˇevic´). 0956-7135/$ - see front matter Ó 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 Haën (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. Knezˇevic´, M. Serdar / Food Control 20 (2009) 419–422 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 Cyfluthtin I Cyfluthtin II Cyfluthtin III Cyfluthtin 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 Dichlofluanid 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 Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
Haën
Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
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 Tolylfluanid Transfluthrin 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 Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
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. Sufficient 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. Knezˇevic´, M. Serdar / Food Control 20 (2009) 419–422
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 30–60 min), the upper organic phase was collected and exactly 100 ml of the organic phase was measured out in a graduated cylinder and filtered through a glass wool plug layered with approx. 50 g sodium sulfate in a funnel. The filtrate was collected in a 500 ml round-bottomed flask 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 filtrate 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 filtered through a membrane filter, 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 acetate–cyclohexane (1:1, v/ v) at a flow-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 film 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 finally increased to 300 °C at 4 °C min 1 (hold for 5 min) (Arrebola, Martínez Vidal, Mateu-Sánchez, & Álvarez-Castellón, 2003). The carrier gas was helium. Flow rate was constant flow 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 Tolylfluanid Iprodione Chlorothalonil Viclozolin Procymidone Chlorpyrifos Chlorpyrifos-methyl Azinphos-methyl Methidathion Malathion Fenthione Endosulfane beta Diazinon Tetramethrin Chlorpropham
Fruits
Vegetables
Number of positive samples
Min–Max (mg/kg)
Number of positive samples
Min–Max (mg/kg)
35 17 8 – – – – 24 4 1 3 1 1 – 4 3 –
0.08–27.9 0.12–10.62 0.06–10.74 – – – – 0.03–0.67 0.03–0.14 0.32 0.13–0.33 0.07 0.05 – 0.06–0.08 0.05–0.82 –
– – 1 2 1 3 1 1 – – – – – 2 – – 6
– – 0.07 10.57–11.57 0.51 2.79–3.27 3.58 0.37 – – – – – 0.01–0.04 – – 0.7–3.6
Z. Knezˇevic´, M. Serdar / Food Control 20 (2009) 419–422
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., Martínez Vidal, J. L., Mateu-Sánchez, M., & Álvarez-Castellón, F. J. (2003). Determination of 81 multiclass pesticides in fresh foodstuffs by a single injection analysis using gas chromatography-chemical ionization and electron ionization tandem mass spectrometry. Analytica Chimica Acta, 484, 167–180. DFG Method S19 – Modular multiple analytical method for determination of pesticide residues in foodstuffs LMBG 00.00 34 S19. Narodne novine. (1998). Pravilnik o nacˇinu uzimanja uzoraka odnosno o metodama za obavljanje analiza i superanaliza namirnica i predmeta opc´e uporabe. N.N.58/98. Nasreddine, L., & Parent-Massin, D. (2002). Food contamination by metals and pesticides in the European Union. Should we worry? Toxicology Letters, 127, 29–41. Ortelli, D., Edder, P., & Corvi, C. (2006). Multiresidue analysis of 74 pesticides in fruit and vegetables by liquid chromatography-electrospray-tandem mass spectrometry. Anayitica Chimica Acta, 520, 33–45. Stan, H. J. (2000). Pesticide residue analysis in foodstuffs applying capillary gas chromatography with mass spectrometric detection State-of-the-art use of modified DFG-multimethod S19 and automated data evaluation. Journal of Chromatography A, 892, 347–377.
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 Knezˇevic´ *, 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 (GC–MSD). 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 findings of this study pointed to the following recommendations: the need for a monitoring program for pesticide residues in food crops, especially imported food crops. Ó 2008 Elsevier Ltd. All rights reserved.
1. Introduction Fresh fruit and vegetables are an important part of a healthy diet as they are a significant 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 field 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 fixed maximum residue level (MRLs). MRL for pesticide residues represents the maximum concentration of that residue (expressed in mg/kg) that is legally permitted in specific 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: [email protected] (Z. Knezˇevic´). 0956-7135/$ - see front matter Ó 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 Haën (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. Knezˇevic´, M. Serdar / Food Control 20 (2009) 419–422 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 Cyfluthtin I Cyfluthtin II Cyfluthtin III Cyfluthtin 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 Dichlofluanid 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 Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
Haën
Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
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 Tolylfluanid Transfluthrin 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 Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën Haën
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. Sufficient 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. Knezˇevic´, M. Serdar / Food Control 20 (2009) 419–422
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 30–60 min), the upper organic phase was collected and exactly 100 ml of the organic phase was measured out in a graduated cylinder and filtered through a glass wool plug layered with approx. 50 g sodium sulfate in a funnel. The filtrate was collected in a 500 ml round-bottomed flask 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 filtrate 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 filtered through a membrane filter, 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 acetate–cyclohexane (1:1, v/ v) at a flow-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 film 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 finally increased to 300 °C at 4 °C min 1 (hold for 5 min) (Arrebola, Martínez Vidal, Mateu-Sánchez, & Álvarez-Castellón, 2003). The carrier gas was helium. Flow rate was constant flow 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 Tolylfluanid Iprodione Chlorothalonil Viclozolin Procymidone Chlorpyrifos Chlorpyrifos-methyl Azinphos-methyl Methidathion Malathion Fenthione Endosulfane beta Diazinon Tetramethrin Chlorpropham
Fruits
Vegetables
Number of positive samples
Min–Max (mg/kg)
Number of positive samples
Min–Max (mg/kg)
35 17 8 – – – – 24 4 1 3 1 1 – 4 3 –
0.08–27.9 0.12–10.62 0.06–10.74 – – – – 0.03–0.67 0.03–0.14 0.32 0.13–0.33 0.07 0.05 – 0.06–0.08 0.05–0.82 –
– – 1 2 1 3 1 1 – – – – – 2 – – 6
– – 0.07 10.57–11.57 0.51 2.79–3.27 3.58 0.37 – – – – – 0.01–0.04 – – 0.7–3.6
Z. Knezˇevic´, M. Serdar / Food Control 20 (2009) 419–422
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., Martínez Vidal, J. L., Mateu-Sánchez, M., & Álvarez-Castellón, F. J. (2003). Determination of 81 multiclass pesticides in fresh foodstuffs by a single injection analysis using gas chromatography-chemical ionization and electron ionization tandem mass spectrometry. Analytica Chimica Acta, 484, 167–180. DFG Method S19 – Modular multiple analytical method for determination of pesticide residues in foodstuffs LMBG 00.00 34 S19. Narodne novine. (1998). Pravilnik o nacˇinu uzimanja uzoraka odnosno o metodama za obavljanje analiza i superanaliza namirnica i predmeta opc´e uporabe. N.N.58/98. Nasreddine, L., & Parent-Massin, D. (2002). Food contamination by metals and pesticides in the European Union. Should we worry? Toxicology Letters, 127, 29–41. Ortelli, D., Edder, P., & Corvi, C. (2006). Multiresidue analysis of 74 pesticides in fruit and vegetables by liquid chromatography-electrospray-tandem mass spectrometry. Anayitica Chimica Acta, 520, 33–45. Stan, H. J. (2000). Pesticide residue analysis in foodstuffs applying capillary gas chromatography with mass spectrometric detection State-of-the-art use of modified DFG-multimethod S19 and automated data evaluation. Journal of Chromatography A, 892, 347–377.