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PRESENCE OF OCHRATOXIN A IN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED ON GRAPES

PRESENCE OF OCHRATOXIN A IN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED ON GRAPES. R.Lo Curto, F. Vilasi, T. M. Pellicanò, P.Munafò, G. Dugo. Milano 15-16 ottobre 2002. MYCOTOXINS.

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PRESENCE OF OCHRATOXIN A IN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED ON GRAPES

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  1. PRESENCE OF OCHRATOXIN AIN EXPERIMENTAL WINES RELATED TO CHEMICAL AND NATURAL TREATMENTS EMPLOYED ON GRAPES R.Lo Curto, F. Vilasi, T. M. Pellicanò, P.Munafò, G. Dugo Milano 15-16 ottobre 2002

  2. MYCOTOXINS Their presence is depending upon factors such as fungal strain, climate and geographical conditions, cultivation technique and foodstuff conservation. • Class of highly toxic chemical compounds produced, under particular environmental conditions, by several moulds developing in many foodstuffs. • May occur in various vegetal products as cereals, dried fruits, coffee bends, cocoa and common beverages as beer and wine. • Among mycotoxins very important are the ochratoxins which include a group of metabolites, showing similar chemical structures, produced by strains of the genus Aspergillus (A. ochraceus) and Penicillium (P. ferrucosum) Milano 15-16 ottobre 2002

  3. OCHRATOXIN Term indicates a group of metabolites, having similar chemical structure, produced by fungi of the genus Aspergillus ochraceus and Penicillium ferrucosum. The most studied compound, both for its diffusion and toxicological importance, is Ochratoxin A, chemically R-N-[(5-chloro-3,4-dihydro-8-hydroxy-3-methyl-1-oxo-1H-2benzopyran-7-yl)carbonyl]-phenylalanine. Milano 15-16 ottobre 2002

  4. OCHRATOXIN A Highly toxic, it caused severe animal and human intoxications (“Porcine nephropathy” and “Balcan Endemic nephropathy”) OTA exhibits nephrotoxic and teratogen activites and moreover suppressive actions on immune system, causing a diminution of immune globulin level and of other humoral factors both in mice and chicken and a reduction of cell immune responses Its presence at levels ranging 0,1- 40 ng\ml in biologic fluids demonstrates that about 50% of European countries, enclosed Italy, are exposed to this contamination Milano 15-16 ottobre 2002

  5. LEGISLATION WHO/FAO/JECFA proposes that Provisional Tolerable Weekly Intake (PTWI) value, on renal damage basis, is 100 ng\Kg body weight • Regulation 466/2001 EEC sets limits in food products • Regulation 472/2002 EEC (modification of Regulation 466/2001 EEC) • European Commission 2001 defines acceptable sampling and analysis methods Milano 15-16 ottobre 2002

  6. TOLERABLE LIMITS (mg/Kg) OF OCHRATOXIN A IN SOME FOOD (472/2002 EEC) Milano 15-16 ottobre 2002

  7. OCHRATOXIN A AND WINE • Wine is a product widely consumed and it may represents, after cereals, a major source of daily OTA intake for people. • Codex Alimentarius Commission suggest that 15% of the total intake of this toxin is due to wine consumption • Some Authors report a high toxin concentration (up to 7.0 ng\ml) with considerable level of contamination (incidence up to 92%) in red wine produced in southern regions of Europe and in North Africa. • Other Authors report OTA contamination levels ranging 3,9% for white wines to 16,6% for red wines • Generally, red wines contain a greater amount of OTA than the white or rosè ones. These differences can be attributed to climatic factors, grape cultivation and storage conditions apart from wine-making Milano 15-16 ottobre 2002

  8. A reversed-phase HPLC method was utilized for Ocrhratoxin A (OTA) determination in 23 white and red wine samples produced in the year 2000 in three experimental vineyards, situated in three different Italian regions and treated with different pesticides • Analytical methods utilized to detect OTA include commercial immunoaffinity columns (Visconti et al.) and a HPLC system equipped with a RF detector. The method sensibility was 0.01 ng /ml-1 Milano 15-16 ottobre 2002

  9. WINE SAMPLES • 23 wine samples (16 white and 7 red), collected in the year 2000 from three experimental vineyards situated in the countries “Campania” (Avellino), “Sicily” (Catania) and “Tuscany” (Grosseto), were analyzed. Milano 15-16 ottobre 2002

  10. WINE CHARACTERISTICS AND PESTICIDE TREATMENTS • White wines were produced in Sicily and Campania. • Sicilian wines were produced from 15-20 years old plants, grown up on Etna (300 m asl, S. Venerina, Catania, Italy), in a vulcanic soil. Vines were grafted with Inzolia and Carricante varieties in 1:1 ratio. • Wines from Campania were produced from 25 years old plants grown up on Montefredane hills (700 m asl, Avellino, Italy), in a clayey soil. Vines were grafted with Fiano d’Avellino variety. • Red wines were produced in Tuscany from 25 years old plants, cultivated in Maremma Toscana coast (100 m asl, Grosseto, Italy), on a calcareous soil. Vines were grafted with Sangiovese variety, Morellino clone. Vinification process, started within 24 hours after grape harvesting. Milano 15-16 ottobre 2002

  11. Wine samples Pesticide and treatments utilized during grape ripening Sicily 1 1 Sulphur 80 Pb 6 Quinoxyfen250 SC Sicily 3 1 Sulphur 80 Pb 6 Fenarimol 12 SC Sicily 4 1 Sulphur 80 Pb 6 Azoxystrobin 250 SC Sicily 5 1 Dinocap 350 EC 6 Penconazole 100 EC Sicily 8 1 Sulphur 80 Pb 6 Sulphur 80 Pb Sicily 9 1 Sulphur 80 Pb 6 Dinocap350EC Sicily 10 1 Sulphur Powder 6 Sulphur Powder Sicily 11 1 Water 6 Water Pesticide treatments on “Inzolia” and “Carricante”(Sicily)variety grapes Milano 15-16 ottobre 2002

  12. Wine samples Pesticide and treatments utilized during grape ripening Campania 1 2 Sulphur 80 Pb 6 Quinoxyfen 250 SC Campania 2 2 Sulphur 80 Pb 6 Fenarimol 12 SC Campania 3 2 Sulphur 80 Pb 6 Azoxystrobin 250 SC Campania 4 2 Sulphur 50 PS 6 Sulphur 50 PS Campania 5 2 Dinocap350EC 6 Penconazole 100 EC Campania 6 2 Sulphur 80 Pb 6 Dinocap 350 EC Campania 7 2 Water 6 Water Campania 8 2 Sulphur 80 Pb 6 Sulphur 80 Pb Pesticide treatments on “Fiano di Avellino”(Campania)variety grapes Milano 15-16 ottobre 2002

  13. Wine samples Pesticide and treatments utilized during grape ripening Tuscany 1 2 Sulphur 80 Pb 9Quinoxyfen 250 SC Tuscany 2 2 Sulphur 80 Pb 9 Fenarimol 12 SC Tuscany 3 2 Sulphur 80 Pb 9 Azoxystrobin 250 SC Tuscany 4 2 Dinocap 350 EC 9 Penconazole 100 EC Tuscany 5 2 Sulphur Powder 9 Sulphur Powder Tuscany 6 2 Sulphur 80 BWG 9 Sulphur 80 PBWG Tuscany 7 2 Dinocap 350 EC 6 Sulphur + 3 Quinoxyfen 250 SC Pesticide treatments on “Sangiovese”(Tuscany)variety grapes Milano 15-16 ottobre 2002

  14. STANDARDS • Standard of OTA, sodium chloride, polyethylene glycol (Peg 8000), sodium hydrogencarbonate, glacial acetic acid and toluene were obtained from Fluka (Milano-Italy) • Immunoaffinity columns were purchased from Vicam (Waterton, MA, USA) • Acetonitrile, methanol and water (HPLC grade) were supplied from Carlo Erba Reagents (Milano-Italy) Milano 15-16 ottobre 2002

  15. HPLC Shimadzu: Controller System SCL-10A 1 Pump LC-10A Detector RF-10AXL: (lex=330 nm, lem=460 nm) Injector Rheodyne: loop da 20 ml Reversed-phase Supelco Column C18 (5mm 150 x 4.6 mm) HPLC/RF ANALYSIS Milano 15-16 ottobre 2002

  16. HPLC/RF ANALYSIS Analyses were run at room temperature in isocratic conditions with a mobile phase compose of water / acetonitrile / acetic acid 99:99:2 v/v/v at a flow rate of 1 ml/min. OTA quantification is made by measuring peak areas at OTA retention time and by comparing them with calibration curve. A wine sample was added with OTA to confirm the OTA identification standard. The sensibility test of method was of 0,01 ng/ml of mycotoxin. Milano 15-16 ottobre 2002

  17. HPLC chromatogram of standard OTA Milano 15-16 ottobre 2002

  18. Wine Samples Ochratoxin A (mg L-1) Sicily 1 n. d. Sicily 3 0.02 Sicily 4 0.01 Sicily 5 0.02 Sicily 8 0.02 Sicily 9 0.03 Sicily 10 0.01 Sicily 11 traces Tuscany 1 0.22 Tuscany 2 0.11 Tuscany 3 0.07 Tuscany 4 0.24 Tuscany 5 0.71 Tuscany 6 2.00 Tuscany 7 0.10 Concentration of OTA in wine samples Milano 15-16 ottobre 2002

  19. Results • No OTA traces were found in white wine sample from Campania • In six samples from Sicily OTA traces were found; (0.03 ng\ml for sample treated with dinocap). The sample treated with quinoxifen and the sample treated with water were found not contaminated with OTA • Values of OTA found in red wines show that all samples are contaminated • The wine produced with grapes treated with sulphur 80 PBWG only, showed a Ochratoxin A content (2.00 ng\ml) higher than the other samples, followed by the sample obtained from grapes treated with powdered sulphur (0.71 ng\ml). The concentration of OTA in the other samples spanned from 0.07 ng\mg in sample treated with “azoxystrobin” to 0.24 ng\mg in sample treated with “dinocap and penconazole”. The values of OTA found, are comparable with those reported in literature on micotoxins presence in red wines. Milano 15-16 ottobre 2002

  20. CONCLUSION Values of OTA found in wine samples show that mycotoxins more frequently detected in red than in white wines The OTA levels reported in this work are comparable with those reported in literature on mycotoxin presence in red wine Moreover the content of Ochratoxin A in the wines can be considered an index of pesticides effectiveness on parasitic fungi. The experimental vineyards located in Campania seem not to be subjected to pollution from OTA-producing fungi. The experimental Sicilian cultivars seem, on the contrary, more easily subjected to OTA contamination since, of eight wine samples, six were found contaminated. Values of OTA found in red wines show that experimental vineyards located in Tuscany are strongly subject to infection from OTA-producing fungi. Synthetic pesticides can reduce OTA concentration from 96.5% in the sample treated with azoxystrobin, to 88% in the sample treated with dinocap and penconazole. Since OTA is strictly related to the growth of some toxigenic fungi on grapes, the different Ochratoxin A content in wine samples can be also considered an efficiency test of the pesticides used. Milano 15-16 ottobre 2002

  21. CONCLUSION The experimental vineyards located in Campania seem not to be subjected to pollution from OTA-producing fungi. The experimental Sicilian cultivars seem, on the contrary, more easily subjected to OTA contamination since, of eight wine samples, six were found contaminated. Values of OTA found in red wines show that experimental vineyards located in Tuscany are strongly subject to infection from OTA-producing fungi. Synthetic pesticides can reduce OTA concentration from 96.5% in the sample treated with azoxystrobin, to 88% in the sample treated with dinocap and penconazole. Since OTA is strictly related to the growth of some toxigenic fungi on grapes, the different Ochratoxin A content in wine samples can be also considered an efficiency test of the pesticides used. Milano 15-16 ottobre 2002

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