Technical paper Pesticide Residues in Cauliflower, Eggplant, Endive, Lettuce, Pepper, Potato and Wheat of the Slovene Origin Found in 2009 V V Helena Basa Cesnik, Spela Velikonja Bolta and Ana Gregorcic Agricultural Institute of Slovenia, Central Laboratories, Hacquetova 17, SI-1000 Ljubljana, Slovenia * Corresponding author: E-mail: helena.basa@kis.si Received: 14-02-2010 Abstract In the year 2009, 170 cauliflower, eggplant, endive, lettuce, pepper, potato and wheat samples from Slovene producers were analysed for pesticide residues. The samples were analysed for the presence of 214 different active compounds using three analytical methods. MRL exceedances have not been observed, which is better than the results obtained from the monitoring of pesticide residues in the products of plant origin in the European Union, Norway, Iceland and Liechtenstein for the years 2004 to 2006. We have observed that MRL exceedances in Slovenia have been reduced in recent times. We assume that the farmers have learned how to use PPP safely in accordance with good agricultural practice. Keywords: GC/MS, LC/MS/MS, pesticides, plant protection products 1. Introduction Monitoring of plant protection product (PPP) residues in agricultural products of Slovene producers allows to control the correct use of PPPs in accordance with the good agricultural practice applied in conventional, integrated and ecological production, and designation of origin of the residues. In 2009, inspectors were sampling cauliflower, eggplant, endive, lettuce, pepper, potato and wheat. For the monitoring purposes, lettuce and potato are sampled each year while the other agricultural products are sampled every three years. The samples were taken randomly in eight production areas in Slovenia: Celje, Koper, Kranj, Nova Gorica, Novo mesto, Murska Sobota, Maribor, and Ljubljana. Agricultural products were taken directly in the field or in the storehouses after the expiration of pre-har-vest interval of the PPPs. For the monitoring purposes, laboratories need quick and reliable methods that enable simultaneous determination of a wide spectrum of active substances. The methods mainly use three types of solvents for extraction: ethylacetate,1-4 acetonitrile (method also known as QuEC-hERS method)5,6 or acetone.7-9 Our laboratory used acetone because of its low toxicity, high volatility and miscibi-lity with water in plant matrices. For better extraction of active substances we added petroleumether and dichloro-metane to acetone.10,11 For the determination of extracted active substances, laboratories mainly use gas chromato-graphy coupled to various detectors: flame ionisation detector (FID), electron capture detector (ECD), nitrogen phosphor detector (NPD), flame photometric detector (FPD) or as in our case mass spectrometer (MS) which is the only one to enable unequivocal qualitative and quantitative detection of active substances. In case of thermally labile compounds liquid chromatography coupled to UV, fluorescence detector or, as in our case, to MS is used, which is again, the only one to enable unequivocal qualitative and quantitative detection of active substances. This paper presents the results of the 2009 Slovene monitoring of vegetables and cereals and the comparison with the previous monitoring results of the same agricultural products in Slovenia (period 2001-2008) and EU, Norway, Iceland and Liechtenstein (period 2001-2006). In spite of the enhanced analytical capabilities of the laboratory (better equipment that enables lower limits of quantification, larger number of active substances sought) no Maximum Residue Levels (MRLs) were exceeded in the samples analysed in 2009. In comparison with the years 2001-2004, when a large number of potato samples violated dithiocarbamate MRLs, we can conclude that farmers' proper use of PPPs is today's reality. 2. Experimental Samples were analysed for the content of selected active substances. In 2009, residues of 214 different compounds were determined using three different methods: 1. Multiresidual GC/MS method for the determination of 100 compounds: acephate, acrinathrin, aldrin, azinphos-methyl, azoxystrobin, bifenthrin, boscalid, bromopropy-late, bupirimate, captan, carbaryl, carbofuran, carboxin, chloridazon, chlorothalonil, chlorpropham, chlorpyrip-hos, chlorpyriphos-methyl, clomazone, cyhalotrin-lambda, cypermethrin, cyproconazole, cyprodinil, dazo-met, DDT, deltamethrin, desmethylpirimicarb, diazinon, dichlofluanid, dichlorvos, dimethachlor, dimethoate, di-niconazole, diphenylamine, endosulfan, endrin, esfenva-lerate, fenamidone, fenbuconazole, fenitrothion, fent-hion, fenvalerate, flonicamid, fludioxonil, fluquincona-zole, folpet, HCH-alpha, HCH-betha, HCH-deltha, hep-tachlor, heptenophos, hexachlorobenzene, imazalil, in-doxacarb, iprodione, kresoxim-methyl, lindane, malat-hion, mecarbam, metalaxyl, metalaxyl-M, metconazole, methacrifos, methamidophos, methidathion, metrafeno-ne, metribuzin, myclobutanil, omethoate, oxadixyl, oxy-demeton-methyl, parathion, parathion-methyl, pencona-zole, permethrin, phorate, phosalone, pirimicarb, piri-miphos-methyl, procymidone, profenofos, propargite, propyzamide, pyridaphenthion, pyrimethanil, quinalp-hos, quinoclamine, quinoxyfen, spiroxamine, tebucona-zole, tetraconazole, tetradifon, thiabendazole, tolclofos-methyl, tolylfluanid, triadimefon, triadimenol, triazop-hos, trifloxystrobin and vinclozolin. Extraction was performed by the mixture of acetone, petroleumether and dichlorometane, clean-up by gel permeation chromato-graphy and determination by GC/MS.10,11 2. GC/MS method for the determination of dithiocarbama-te group: maneb, mankozeb, metiram, propineb and zi-neb, the sum is expressed as carbon disulfide. Samples were heated in a two-phase system isooctane/stannous (II) chloride in diluted hydrochloric acid. The produced carbon disulfide was dissolved in the organic phase (isooctane) and determined by GC/MS.11,12 3. Multiresidual LC/MS/MS method for the determination of 113 compounds: 2,4-D, acetamiprid, aldicarb, aldi-carb sulfone, aldicarb sulfoxide, amidosulfuron, ami-trole, azinphos-ethyl, beflubutamid, benalaxyl, bena-laxyl-M, bentazon, bitertanol, bromoxynil, buprofezin, carbendazim, carbosulfan, chlortoluron, clofentezine, clopyralid, clothianidin, cyazofamid, cycloxydim, cymoxanil, cyromazine, demeton-S-methyl sulphone, desmedipham, dichloprop-P, difenoconazole, diflufeni-can, dimethenamid-P, dimethomorph, epoxiconazole, ethofumesate, famoxadone, fenarimol, fenazaquin, fen-hexamid, fenoxaprop-P-ethyl, fenoxycarb, fenpropidin, fenpropimorf, fenpyroximate, fenthion sulfone, fent-hion sulfoxide, fipronil, florasulam, fluazifop-P-butyl, fluazinam, flufenacet, fluorochloridone, fluroxypyr, flusilazole, flutriafol, foramsulfuron, hexaconazole, hexythiazox, imidacloprid, iodosulfuron-methyl-so-dium, iprovalicarb, isoproturon, isoxaflutole, linuron, lufenuron, malaoxon, mandipropamid, MCPA, metami-tron, metazachlor, methiocarb, methiocarb sulfone, methiocarb sulfoxide, methomyl, methoxyfenozide, metosulam, monocrotophos, napropamide, nicosulfu-ron, oxamyl, paraoxon-methyl, pendimethalin, phen-medipham, phorate sulfone, phorate sulfoxide, phoxim, prochloraz, propamocarb, propaquizafop, propiconazo-le, prosulfocarb, prosulfuron, pymetrozine, pyraclostro-bin, pyrazophos, pyridate, rimsulfuron, spinosad, spiro-diclofen, tebufenozide, teflubenzuron, terbuthylazine, thiacloprid, thiamethoxam, thifensulfuron-methyl, thio-dicarb, thiophanate-methyl, triasulfuron, tribenuron-methyl, trichlorfon, trifluralin, triflusulfuron-methyl, trinexapac-ethyl, zoxamide. Extraction was performed by mixture of acetone, petroleumether and dichlorome-tane, clean-up by gel permeation chromatography and determination by LC/MS/MS.1315 Limits of quantification (LOQs) of all active substances determined were in the range of 0.003 to 1 mg/kg. The trueness of methods is verified from recoveries which had to be from 70% to 120% and by participation in the inter-laboratory proficiency testing schemes: BIPEA (Bureau interprofessionnel d'etudes analytiques) and CRL European Proficiency Test 10. Table 1: List of vegetable and cereal samples analysed in 2009, and distribution of sample locations among individual production areas Area Cauliflower Eggplant Agricultural product Endive Lettuce Pepper Potatoes Wheat Sum Celje 2 0 3 4 3 5 1 18 Koper 2 2 1 3 2 2 0 12 Kranj 1 1 3 0 1 18 1 25 Ljubljana 5 4 9 6 4 9 2 39 Maribor 3 1 4 4 3 8 4 27 Murska Sobota 0 0 1 2 2 3 1 9 Nova Gorica 1 1 3 2 3 1 10 21 Novo mesto 3 0 4 2 3 6 1 19 Sum 17 9 28 23 21 52 20 170 In January 2005, a range of analyses covering pesticide residues were accredited by the French accreditation body COFRAC. 170 of the vegetable and cereal samples presented in Table 1 were analysed in 2009. 3. Results and Discussion From the samples analysed in 2009, the number and portion of samples where residues were not found and number and portion of samples lower or equal to MRLs are presented in Table 2. In cauliflower, dithiocar-bamates were the only active substance found. Some substances that are naturally present in cauliflower give responses for dithiocarbamates. So the question arises whether cauliflower was really treated with plant protection products containing active substances from the dithiocar-bamate group. Active substances that were found in agricultural products analysed in 2009 are presented in Table 3. Multiple residues by matrices are presented in Table 4. Detailed results of PPP residues found in 2009 are presented in Table 5. Active substances not registered in the Republic of Slovenia were found in cauliflower (in cauliflower are naturally present compounds which give responses for dit-hiocarbamates) and endive (chlorothalonil, dithiocarba- mates).16 Active substances not allowed in the integrated production in the Republic of Slovenia were found in endive (cyprodinil, fludioxonil, dithiocarbamates).17,18 Active substances not allowed in the ecological production in the Republic of Slovenia were not found. In 142 samples (83.5%) out of 170 samples residues were not found, in 28 samples (16.5%) residues were lo- 16.5 % 83.5 % □ sample portion « A «M ^ ° ft £ S Z a M X « J§ o o t!S ^ ° ft £ S Z a u S a « ■a S u I I .-H o a\ ^^ o O o o 1 1 (M 1 \o 1 (N O o O O o o O o I I ^ o o t-^ o o o cM cric (N C^ O I 00 o o 00 CO in o 1 1 (N o 1 1 CO O r- o O CO o o o o o o CGC C co ^O I to o c4 O O o o O >0 O Oco O (N O O O co O O O OI O ^SiN O o C ^ o o o o o o >0 o o o o o o odo o o o o es Pi S = TTO^ - Sw ^ " ft ž s § s « S s - O Z § 1 es S s ®'-S tS oi-e^ - Sw ^ ° "S J= s Z C3 § 1 « S S o ^ ®°-S»s — Sw ^ ^ O, J= S Z C3 u S (S t« ■fi S O I CD CD Cf O ta ^ 13 ^ CD I O co <3 CO C ^ ef o o ■iS i ¡3 Ojh» -Q-S-O UQi^ 3 -O O I I* g la es S s » o^ - Sw ^ " ft J= S ^ « s f « S s - Sw ^ " ft J= S Z C3 s a « S S \o © ,—v ®'-S t® oi-e^ - Sw ^ ° "S ž s Z « § 1 « S S ©S.» — Sw ^ ^ ft J= S Z C3 u S (S t« ■fi S I I I ooo CDCDCD CD CD CDOOCD oc4o o o CD CD CDCDCD CDcoco COOCD CDCDCD i ¡3 OjH» -Q-S-O UQÎ^ only: in 2001 (20.0% of potato samples), in 2002 (40% of potato samples), in 2003 (37.1% of potato samples) and in 2004 (8.2% of potato samples). Over the years we have observed that MRL exceedances are lower, which suggests that the farmers have learned to use PPP safely, in accordance with good agricultural practice. The results are presented in Table 10. The results of monitoring during the years 20012006 for lettuce, potato, cauliflower, pepper, eggplants and wheat in the EU countries and in Norway, Iceland and Liechtenstein19 are presented in Table 11. Endive was not sampled on the EU level. Lettuce in EU, Norway, Iceland and Liechtenstein in 2001 had less exceedances than lettuce in Slovenia in the same year. But in 2004, lettuce had no exceedances in Slovenia while in EU, Norway, Iceland and Liechtenstein it kept the same level of exceedances as in 2001. The same is valid for potato. Potato in EU, Norway, Iceland and Liechtenstein in 2002 had much less excee-dances than potato in Slovenia in the same year. But in 2005, potato had no exceedances in Slovenia while in EU, Norway, Iceland and Liechtenstein it kept the same level of exceedances as in 2002. Cauliflower, pepper and eggplants in the years 2003 and 2006 and wheat in 2006 had some exceedances in EU, Norway, Iceland and Liechtenstein while the same agricultural products in Slovenia had none. 4. Conclusions Levels of pesticide residues in cauliflower, eggplant, endive, lettuce, pepper, potato and wheat in Slovenia in 2009 do not give any cause for alarm. 83.5% samples examined did not contain any residues and exceeding maximum residue levels were not found. For comparison, the results of national monitoring in the same agricultural products performed in Slovenia in 2001-2008 are presented. MRL exceedances were found only in lettuce in 2001, 2002, 2007 and 2008 and in potato in 2001-2004. Also, the results of national monitoring performed in the EU countries and in Norway, Iceland and Liechtenstein in 2001-2006 are presented.19 MRL exceedances for potato in EU, Norway, Iceland and Liechtenstein are not so high as for potato in Slovenia, but there are some MRL exceedances in EU, Norway, Iceland and Liechtenstein in cauliflower, pepper, eggplants and wheat while in Slovenia there are none. 5. Acknowledgements The authors thank those who contributed to the work: Mateja Fortuna and co-workers at the Central Laboratories of Agricultural Institute of Slovenia. For financial support we express our thanks to the Inspectorate of the Republic of Slovenia for Agriculture, Forestry and Food, MAFF. Table 8: Plant protection product residues obtained in 2003 and 2006 in cauliflower. 2003 2006 Active substance No. of Portion Range No. of Portion Range samples (%) (mg/kg) samples (%) (mg/kg) Difenoconazole n.a. 1 9.1 0.01 Dithiocarbamates 1 10.0 0.98 10 90.9 0.05-0.41 n.a. means not analysed Table 9: Plant protection product residues obtained in 2003 and 2006 in pepper. 2003 2006 Active substance No. of Portion Range No. of Portion Range samples (%) (mg/kg) samples (%) (mg/kg) Difenoconazole n.a. 1 6.3 0.04 Chlorothalonil 0 0.0 - 1 6.3 0.04 Dithiocarbamates 1 6.7 0.14 1 6.3 0.14 Imidacloprid n.a. 2 12.5 0.01 n.a. means not analysed Table l0a: MRL exceedances in 2001 to 2008. Matrix 200l Active substance No. of samples Matrix 2002 Active substance No. of samples 2003 Matrix Active substance No. of samples Lettuce Dimethoate 1 Lettuce Lettuce Dithiocarbamates 1 Potato Potato Dithiocarbamates 6 Metalaxyl 1 Dithiocarbamates 12 Potato Dithiocarbamates 13 Table l0b: MRL exceedances in 2001 to 2008. 2004 2007 2008 . ... No. of No. of No. of Matrix Active substance , Matrix Active substance , Matrix Active substance , samples samples samples Potato Dithiocarbamates 5 Lettuce Chlorothalonil 1 Lettuce Pendimethalin 1 Table 11: Results of the EU, Norway, Iceland and Liechtenstein monitoring in 2001-2006. Lettuce Potato Cauliflower Pepper Eggplants Wheat 200l 2004 2002 2005 2003 2006 2003 2006 2003 2006 2003 2006 No residues (%) 47 49 89 74 82 78 60 55 80 63 78 73 Residues < MRL (%) 49 48 10 25 17 20 34 42 18 33 22 27 Rresidues > MRL (%) 3.9 3.3 1 1.2 1 1.6 6 3.5 3 4.3 0 0.1 6. References 1. Berrada H., Fernández M., Ruiz M. J., Moltó J. C., Mañes J., Food Addit. 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Gregorčič, Acta chim. slov., 2006, 53, 100-104. 13. R. Bossi, K. V. Vejrup, B. B. Mogensen, A. H. Asman, J. Chromatogr. A, 2002, 957, 27-36. 14. D. Ortelli, P. Edder, C. Corvi, Anal. chim. Acta, 2004, 520, 33-45. 15. S. J. Lehotay, A. de Kok, M. Hiemstra, P. Bodengraven, J. AOAC Int., 2005, 88, 595-614. 16. FITO-INFO, Informacijski sistem za varstvo rastlin. Taken in 2009 from http://spletni2.furs.gov.si/FFS/REGSR/index. htm 17. Tehnolo{ka navodila za integrirano pridelavo zelenjave za leto 2009, MAFF, 2009, edited by Tomaž Džuban. 18. Tehnolo{ka navodila za integrirano pridelavo polj{~in za leto 2009, MAFF, 2009, edited by Tomaž Džuban. 19. Monitoring of pesticide residues in products of plant origin in the European union, Norway, Iceland and Liechtenstein, 2003, 2004, 2005, 2006, 2007,2008. Taken 02.02.2010 from http://ec.europa.eu/food/fvo/specialreports/pesticides_index _en.htm Povzetek V letu 2009 smo analizirali ostanke pesticidov v 170 vzorcih cvetače, jajčevcev, endivije, solate, paprike, krompirja in pšenice slovenskih pridelovalcev. Vzorce smo analizirali na prisotnost 214 različnih aktivnih spojin s tremi analitskimi metodami. Preseženih maksimalno dovoljenih količin ostankov nismo določili, kar je boljše od rezultatov monitoringa ostankov pesticidov v rastlinskih proizvodih v Evropski skupnosti, Norveški, Islandiji in Liechtensteinu v letih od 2004 do 2006. Opazili smo, da je zadnje čase v Sloveniji manj preseženih MRL-jev. Domnevamo, da so se kmetovalci naučili varno uporabljati fitofarmacevtska sredstva, v skladu z dobro kmetijsko prakso.