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Occurrence of ochratoxin A in Rioja Alavesa wines
Food Chemistry 126 (2011) 302–305
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Occurrence of ochratoxin A in Rioja Alavesa wines Silvia Quintela ⇑, M. Carmen Villarán, Iratxe López de Armentia, Edurne Elejalde Fundación LEIA, C.D.T., Technological Park of Alava, Leonardo Da Vinci 11, 01510 Miñano, Spain
a r t i c l e
i n f o
Article history: Received 2 February 2010 Received in revised form 20 July 2010 Accepted 28 September 2010
Keywords: Ochratoxin A Rioja Alavesa Daily intake HPLC-fluorimetric Immunoaffinity column Wine
a b s t r a c t In this survey the influence of the geographical location and the kind of wine in the concentration of ochratoxin A (OTA) was studied. Forty percent of the Spanish wine market belongs to the Rioja Qualified Designation of Origen (DOCa Rioja) wines, which are already worldwide known. A total of 100 wines from the Rioja Alavesa (RA) production area of the DOCa Rioja were analysed, using immunoaffinity column clean-up and high-performance liquid-chromatography with fluorimetric detection (detection limit 0.002 lg/l). The presence of OTA was greater in wines produced in low rainfall and high temperature regions. The geographical location and kind of wine did not seem to have influence on the OTA concentration. The mean OTA concentration of RA wines was 0.035 lg/l and its contribution to mean daily OTA intake could be considered negligible, representing a 0.07% of the latest tolerable weekly intake (TWI) established in 2006 by EFSA. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Spain is the third country of the world with the highest wine production, behind Italy and France (OIV, 2009). According to the latest data of the Spanish Ministry of Environment and Rural and Marine Affairs, Spain has nowadays 67 of designations of origin (MARM, 2008b). The oldest one is the Rioja Qualified Designation of Origin (DOCa Rioja). The DOCa Rioja is leader of the Spanish market of quality wines, with near 40% of the total sales of these wines. It has great international prestige and it exports around 30% of wines production, mainly to the United Kingdom, Germany, Switzerland and the United States (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The Rioja Alavesa (RA) wines are produced in one of the production areas of the DOCa Rioja. In the area of RA is produced the 26% of the wine with DOCa Rioja. The economy of this region depends greatly on the wine activity. Thus, in order to preserve the position of the DOCa Rioja wines in the market, it is necessary to guarantee the quality and safety of these wines. The concern about food safety each day is greater, increasing the control of the content of different contaminants in food products, such the ochratoxin A (OTA), many pesticides, etc. One example, are the fungicides currently used to control the fungal diseases in the vineyards. Since there is a risk of transfering some undesirable contaminants from the grape to the wine, there is the need to ensure that such residues should not be found in wine at levels presenting an unacceptable risk to humans. In this ⇑ Corresponding author. Tel.: +34 945 29 81 44; fax: +34 945 29 82 17. E-mail address: [email protected] (S. Quintela). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.09.096
sense, strict regulations of fungicide use on grapes are being promoted. One of their consequences, is the need to develop new feasible and specific analytical methods adapted to the analysis requirements of the market. Several works have been presented already in this field, specially since 2009, although many research is still needed to undergo in the near future (González-Rodríguez, Cancho-Grande, & Simal-Gándara, 2009a,b; González-Rodríguez, Cancho-Grande, Torrado-Agrasar, Simal-Gándara, & Mazaira-Pérez, 2009; Rial Otero, Yagüe Ruiz, Cancho Grande, & Simal Gándara, 2002). Another contaminant of concern is the ochratoxin A (OTA). OTA is a mycotoxin produced by different species of fungi belonging to the genera Penicillium and Aspergillus. The systematic chemical nomenclature for OTA is (R)-N-[(5-chloro-3,4-dihydro-8-hydroxy-3methyl-1-oxo-1H-2-benzopyran-7-yl)-carbonyl]-L-phenylalanine. This mycotoxin has been detected in different foodstuffs such as cereals, related cereal products, coffee, wine and grape juice, but it has also been detected in animal products, such as pork meat (EFSA, 2006). The International Agency for Research on Cancer (IARC) has classified this mycotoxin in the category 2B, as ‘‘possibly carcinogenic to humans’’ (IARC, 1993). In addition, the OTA is known to be nephrotoxic, teratogenic, immunotoxic and possibly neurotoxic (EFSA, 2006). It is related also with an endemic kidney disease observed in the Balkans (Balkan Endemic Nephropathy and related Urinary Tract Tumours) (EFSA, 2006). After cereals, the wine is the second food with the greatest contribution in the daily intake of OTA by the population of EU (EC, 2002). It was detected in wine in 1996 by Zimmerli and Dick (Zimmerli & Dick, 1996). Since then, the number of studies about the content of OTA in wines around the world has been increased
S. Quintela et al. / Food Chemistry 126 (2011) 302–305
(Bellí, Marín, Duaigües, Ramos, & Sanchis, 2004; Berente et al., 2005; Blesa, Soriano, Moltó, & Mañes, 2004; Brera, Soriano, Debegnach, & Miraglia, 2005; Burdaspal & Legarda, 1999; Domijan & Peraica, 2005; Filali et al., 2001; Rosa, Magnoli§, Fraga, Dalcero§, & Santana, 2004; Shephard, Fabiani, Stockenström, Mshicileli, & Sewram, 2003; Visconti, Pascale, & Centonze, 1999). The Regulation (EC) No. 123/2005 of the European Commission sets the limit of Ochratoxin A for wine at 2 lg/l (EC, 2005). The main factors which have influence on the presence of OTA in the grapes are basically the meterological conditions, the sanitary status of the grapes (mould presence) and the conditions and grape cultivation practices. In terms of meteorological conditions, a relationship is usually found between the temperature and contamination severity, the frequent and concentration of this mycotoxin are higher in warmer regions (Czerwiecki, Wilczyn´ska, & Kwiecien´, 2005; Otteneder & Majerus, 2000; Pietri, Bertuzzi, Pallaroni, & Piva, 2001; Stefanaki, Foufa, Tsatsou-Dritsa, & Dais, 2003; Zimmerli and Dick, 1996). OTA and the fungi producers of this mycotoxin can be transferred from grapes to wine during winemaking. The different processes used in wine production can have a great influence on the final OTA concentration, for example the stage of maceration, where an increase in OTA content can be produced due of grape skins contact with must. On the contrary, another stages of the wine processing can reduce the OTA content, such is the wine ageing in bottle, although this effect is not appreciable on a short ageing of 12 months of storage (Grazioli, Fumi, & Silva, 2006). The Rioja Alavesa (RA) is situated in 42° North latitude, which corresponds to the temperate zone of Europe. Its climate is influenced by the Atlantic Ocean and the Mediterranean Sea, more or less mitigated by topographic factors that produce diverse climatic situations, appearing within this region major variations in rainfalls and temperatures among different areas. This region can be divided into four fitoclimatics areas: semiarid, dry, subhumid and humid. The 94% of vineyard hectares are situated in the semiarid and dry areas of RA (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The semiarid area is located to the south of RA and its altitude level is between 489–569 m above sea level, the mean annual rainfall is estimated to be around 502 mm and the mean annual temperature is 12.93 °C. The dry area it is a higher altitude (510–648 mm), its mean annual temperature is lower (around 12.51 °C) and the mean annual rainfall is sensibly higher (approximately 604 mm) (MARM, 2009). Almost 90% of the total wine commercialised is red wine. Depending on the different elaboration process, this means, the kind of ageing process (without, cask, bottles) or periods (months), the DOCa Rioja wine shows different categories: ‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ or ‘‘gran reserva’’ (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The aim of this work is to report the content of OTA in 100 commercial wines from the most relevant fitoclimatic areas of the Rioja Alavesa region. The results determine the influence of several variables, such as the kind of wine and the climatic conditions. The results have been compared with the limits established by the EC related directives and with the levels in other countries. Finally, the potential contribution to the dietary OTA exposure of consumers of these wines has been evaluated.
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A total of 100 wines were analysed. Most of the wines were provided generously by different wine cellars interested in the study and the rest were purchased from supermarkets. The wines correspond both to the semiarid area from (including the municipalities of Assa, Baños de Ebro, Elciego, Lapuebla de Labarca, Laserna, Móreda, Oyón and Villabuena de Álava) and to the dry area of the RA (including the municipalities of Barriobusto, Elvillar, Labastida, Laguardia, Lanciego, Leza, Navaridas, Páganos and Samaniego). The wines were produced between 1995 and 2008. The number of wine samples for each kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine, as well as white wine and rosé wine) was taken according to their importance in terms of commercialisation (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The total number of samples analysed in the semiarid area were: ‘‘joven’’ red wine (n = 16), ‘‘crianza’’ red wine (n = 21), ‘‘reserva’’ red wine (n = 10), ‘‘gran reserva’’ red wine (n = 1), white wine (n = 1) and rosé wine (n = 2). And in the dry area were: ‘‘joven’’ red wine (n = 16), ‘‘crianza’’ red wine (n = 20), ‘‘reserva’’ red wine (n = 8), ‘‘gran reserva’’ red wine (n = 2), white wine (n = 2) and rosé wine (n = 1). All the samples were maintained at 4 °C since their arrival at the laboratory to the time of analysis. 2.2. Reagents The OTA standard solution was purchased by Sigma–Aldrich (Seelze, Germany). The OTA concentration of the standard was determined accurately by UV spectroscopy at 333 nm and it was stored at 20 °C. Acetonitrile, methanol and glacial acetic acid (HPLC grade) were purchased from Panreac (Barcelona, Spain). Polyethylene glycol (PEG 8000) was obtained from Sigma–Aldrich (Steinheim, Germany), sodium chloride (ACS) and sodium hydrogencarbonate (ACS) from Panreac (Barcelona, Spain). OchraTest immunuoaffinity columns were purchased from Vicam (USA) and glass microfibre filters GF/A 47 mm from Whatman (England). 2.3. Apparatus and chromatographic conditions
2. Materials and methods
The chromatographic analysis was done with a HPLC equipment connected to a fluorescent spectrophotometer (kex = 333 nm y kem = 460 nm), both from Hewlett–Packard model 1100. The computer program used was a Agilent Chemstation for chromatographic analysis. The analytical column was a reversed-phase Synergi C12 (150 4.60 mm, 4 lm particles) (Phenomenex) preceded by guard cartridge AQ C18 (4 2 mm). Chromatographic separation was performed using isocratic elution with water–acetonitrile–acetic acid (99:99:2, v/v/v) at a flow rate of 1 ml/min. One hundred microliters of solution were injected. The calibration curve obtained by analysing OTA standard solutions at eight concentration levels (range: 0.004–7.242 lg/l) was linear (r2 = 0.9996). The precision and accuracy was tested by duplicate analyses on a red wine sample containing OTA at a concentration of 0.03, 1.5 and 7 lg/l. The coefficient of variation was less than 5%, demonstrating good repeatability of the HPLC analysis. The analytical recovery for OTA, determined by duplicate on a red wine sample spiked at a level of 0.3 and 3 lg/l was 99.2% and 101.8%, respectively. The limit of detection (S/N = 3:1) was 0.002 lg/l and the limit of quantitation (S/N = 10:1) was 0.004 lg/l.
2.1. Sampling design
2.4. Sample preparation
In order to study the main factors which affect the presence of OTA, for the sampling design variables such as RA area and kind of wine have been taken into account.
The analytical method was based on the UNE-EN 14133 (UNEEN 14133, 2004). A 10 ml volume of wine sample was diluted with 10 ml water solution containing PEG (1%) and NaHCO3 (5%), mixed
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S. Quintela et al. / Food Chemistry 126 (2011) 302–305
and filtered through Whatman GF/A glass microfibre filter. A 10 ml volume of diluted extract was cleaned up through an OchraTest immunoaffinity column at a continuous flow-rate of about 1 drop per s. The column was washed with a solution containing NaCl (2.5%) and NaHCO3 (0.5%) followed by Milli-Q water at a flow-rate of 1–2 drops per s. OTA was eluted with methanol. The eluate was evaporated to dryness under a nitrogen stream and redissolved with 250 ll in HPLC mobile phase. 2.5. Statistical methods When comparing the OTA positive samples and levels in the different RA areas and kind of wines, the H of Kruskal–Wallis non-parametric test for non-dependent samples was applied. A probability value of 0.05 has been used in order to determine the statistical significance, using the program SPSS Statistics 17.0. 3. Results and discussion Table 1 shows the OTA positive samples and concentration from different areas (semiarid and dry area) and kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine and white and rosé wine) from RA wines sampled in this survey. The statistical results obtained can be seen in Table 2. Altogether, OTA was found in the 57% of the total wine samples from both areas. All the RA wines showed a low level of OTA contamination ranged in positive wine samples from 0.004 to 0.179 lg/l with a mean concentration of 0.035 lg/l. The concentration of all the positive samples was below the maximum authorised level established in 2 lg/l for wine in the EU (EC, 2005). Thirty-six samples (71%) from the semiarid area of RA contained detectable amounts of OTA. The mean concentration of positive Table 1 OTA presence and concentration from different areas (semiarid and dry area) and kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine and white and rosé wine) from Rioja Alavesa wines. Kind of wine
Positives/ total
OTA mean concentration of positive samples (lg/ l)*
Range positive samples (lg/l)*
44 86 90 0
0.029 0.034 0.018 <0.002
0.014–0.056 0.004–0.144 0.004–0.042 –
100 50 71
0.013 0.007 0.028
– – 0.004–0.144
63 40 13 50
0.042 0.053 0.060 0.048
0.007–0.179 0.009–0.169 – –
50 0 43
0.015 <0.002 0.046
– – 0.007–0.179
17/32 26/41 10/18 1/3
53 63 56 33
0.037 0.040 0.022 0.048
0.007–0.179 0.004–0.169 0.004–0.060 –
2/3 1/3 57/100
67 33 57
0.014 0.007 0.035
0.013–0.015 – 0.004–0.179
Semiarid area of Rioja Alavesa ‘‘Joven’’ red wine 7/16 ‘‘Crianza’’ red wine 18/21 ‘‘Reserva’’ red wine 9/10 ‘‘Gran reserva’’ red 0/1 wine White wine 1/1 Rosé wine 1/2 Total wine 36/51 Dry area of Rioja Alavesa ‘‘Joven’’ red wine 10/16 ‘‘Crianza’’ red wine 8/20 ‘‘Reserva’’ red wine 1/8 ‘‘Gran reserva’’ red 1/2 wine White wine 1/2 Rosé wine 0/1 Total wine 21/49 Total Rioja Alavesa ‘‘Joven’’ red wine ‘‘Crianza’’ red wine ‘‘Reserva’’ red wine ‘‘Gran reserva’’ red wine White wine Rosé wine Total wine *
Positives (%)
Detection limit 0.002 lg/l. Quantitation limit 0.004 lg/l.
samples was 0.028 lg/l. The highest level of OTA was 0.144 lg/l and was found in a ‘‘crianza’’ red wine from 2001. Regarding to the dry area of RA, the 43% of the wine samples showed a OTA concentration ranging from 0.007 to 0.179 lg/l. The OTA mean concentration of the positive samples was 0.046 lg/l. The number of positive samples was higher in the semiarid area (71%) than in the dry area (43%), with a statistically significance (p < 0.05). The differences detected might be due to the fact that in the semiarid area there are less rainfalls and higher temperatures than in the dry area, so that it could favour the growth of OTA producing fungi. However, no significant differences (p > 0.05) were found in the OTA concentration. Several authors (Czerwiecki et al., 2005; Otteneder and Majerus, 2000; Zimmerli and Dick, 1996) have found an increase of OTA occurrence and concentration in wines from northern to southern countries. Others authors (Pietri et al., 2001; Stefanaki et al., 2003) have arrived to the same conclusion translated to different wine-growing areas in the same country (Italy and Greece, respectively). These results were in concordance with our results about a higher incidence of OTA (% of positives samples), but not about a higher OTA content in wines from southern areas. One reason that could explain the fact that no statistical differences have been found in the concentration of OTA between both areas (sermiarid and dry) from RA, could be the strict norms of control board of DOCa Rioja wine related to quality practices in the grape cultivation and the wine elaboration process. Moreover, no significant differences (p > 0.05) were found considering the different elaboration process or kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine, as well as white wine and rosé wine) in all the climatic areas studied. These results differ with those obtained by others authors (Otteneder and Majerus, 2000; Zimmerli and Dick, 1996) where the OTA concentration decrease from red to rosé to white wine. These results may be explained by good winemaking practice in the wines elaborated in RA. Currently, there are no surveys with such number of samples analysing the content of OTA in wine neither from Rioja Alavesa region nor from the total DOCa Rioja. Bellí et al. (2004) analysed 10 wines of each kind of wine from the DOCa Rioja: ‘‘joven’’, ‘‘crianza’’ and ‘‘reserva’’ red wine and white wine. In these study the exact area of the DOCa Rioja samples was not indicated. Our results differ basically from the ones of these authors related to the content of OTA of ‘‘joven’’ red wines. Whilst in our study the mean concentration of OTA for the ‘‘joven’’ red wine was 0.037 lg/l, in the above mentioned publication it was reported a mean concentration of 1.220 lg/l. According to the data of this study, the average concentration of OTA from RA analysed wine was 0.035 lg/l. According to the alimentary consume data of the Spanish Ministry of Environment and Rural and Marine Affairs, the designation of origen wine consumption in Spain from July 2007 to June 2008 is about to 24.22 ml/day per person (MARM, 2008a). Assuming that an average adult body weights 70 kg, the estimated daily intake of OTA could be 0.012 ng/kg bw per day (0.084 ng/kg bw per week). This value represents 0.07% of the tolerable weekly intake (TWI) established by the Scientific Panel on Contaminants in the Food Chain from the EFSA (European Food Safety Authority) in 2006 (120 ng/ kg bw per week) (EFSA, 2006). The contribution to OTA daily intake coming from the wines from RA studied were considerably smaller in comparison with the values obtained in others studies, for example the wines from Croatia (0.20–0.26 ng/kg bw per day) (Domijan and Peraica, 2005), Italy (0.90 and 1.36 ng/kg bw per day) (Brera et al., 2005), Greece (3.75 ng/kg bw per day) (Stefanaki et al., 2003), and others parts of Spain (0.15 ng/kg bw per day) (Blesa et al., 2004).
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S. Quintela et al. / Food Chemistry 126 (2011) 302–305 Table 2 H of Kruskal–Wallis test applied to the concentration and positive samples in Rioja Alavesa wines. KRUSKAL–WALLIS TEST Rioja Alavesa area
Test variable
Grouping variable
Chi-Square
Df
Asymp. Sig.
Semiarid area Dry area Total Rioja Alavesa Total Rioja Alavesa
Concentration (lg/l) Concentration (lg/l) Concentration (lg/l) Positive samples
Kind of wine Kind of wine Kind of wine Rioja Alavesa area
3.548 2.001 5.933 7.820
3 4 5 1
0.315 0.736 0.313 0.005
4. Conclusions The concentration detected in Rioja Alavesa wines analysed was below the limit established by the EU (2 lg/l) and its contribution to the tolerable daily intake could be considered as negligible. According to the obtained results, RA wines have much lower content in OTA than other European regions. Significant differences in the presence of OTA in different regions of RA has been found, being the highest in the area with lower mean rainfalls and higher mean temperature (semiarid area). However, in terms of concentration of OTA in wine, no significant differences have been found in terms of area in RA and elaboration process (kind of wine). Acknowledgements This work has been funded by the Agriculture and Fisheries Department of the Basque Government (AID-0486-05-C) and by the pre-doctoral grant of the Ikertu program of the Basque Government (year 05/06 and 06/07). We are also grateful to the wine cellars that kindly provided their wines for this survey. References Bellí, N., Marín, S., Duaigües, A., Ramos, A. J., & Sanchis, V. (2004). Ochratoxin A in wines, musts and grape juices from Spain. Journal of the Science of Food and Agriculture, 84, 591–594. Berente, B., Móricz, Á., -Otta, K. H., Záray, G., Lékó, L., & Rácz, L. (2005). Determination of ochratoxin A in Hungarian wines. Microchemical Journal, 79, 103–107. Blesa, J., Soriano, J. M., Moltó, J. C., & Mañes, J. (2004). Concentration of ochratoxin A in wines from supermarkets and stores of Valencian Community (Spain). Journal of Chromatography A, 1054, 397–401. Brera, C., Soriano, J. M., Debegnach, F., & Miraglia, M. (2005). Exposure assessment to ochratoxin A from the consumption of Italian and Hungarian wines. Microchemical Journal, 79, 109–113. Burdaspal, P. A., & Legarda, T. M. (1999). Ocratoxina A en vinos, mostos y zumos de uva elaborados en España y en otros países europeos. Alimentaria (Madrid), 299, 107–113. Consejo Regulador de la Denominación de Origen Calificada Rioja. Memoria; 2008. Czerwiecki, L., Wilczyn´ska, G., & Kwiecien´, A. (2005). Ochratoxin A: An improvement clean-up and HPLC method used to investigate wine and grape juice on the Polish market. Food Additives and Contaminants, 22(2), 158–162. Domijan, A. M., & Peraica, M. (2005). Ochratoxin A in wine. Archives of Industrial Hygiene and Toxicology, 56, 17–20. EC (2002). Reports on tasks for scientific cooperation. Report of experts participating in Task 3.2.7. Assessment of dietary intake of ochratoxin A by the population of EU Member States. Directorate-General Health and Consumer Protection; January 2002. EC (2005). Commission Regulation (EC) No. 123/2005 of 26 January 2005 amending regulation (EC) No. 466/2001 as regards ochratoxin A. Official Journal of the European Union, L25:3–5. EFSA (2006). Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. The EFSA Journal, 365, 1–56.
Filali, A., Ouammi, L., Betbeder, A. M., Baudrimont, I., Soulaymani, R., Benayada§, A., et al. (2001). Ochratoxin A in beverages from Morocco: A preliminary survey. Food Additives and Contaminants, 18(6), 565–568. González-Rodríguez, R. M., Cancho-Grande, B., & Simal-Gándara, J. (2009a). Efficacy of new commercial formulations to control downy mildew and dissipation of their active fungicides in wine after good agricultural practices. Journal of the Science of Food and Agriculture, 89, 2625–2635. González-Rodríguez, R. M., Cancho-Grande, B., & Simal-Gándara, J. (2009b). Multiresidue determination of 11 new fungicides in grapes and wines by liquid–liquid extraction/clean-up and programmable temperature vaporization injection with analyte protectans/gas chromatography/ion trap mass spectrometry. Journal of Chromatography A, 1216, 6033–6042. González-Rodríguez, R. M., Cancho-Grande, B., Torrado-Agrasar, A., Simal-Gándara, J., & Mazaira-Pérez, J. (2009). Evolution of tebuconazole residues through the winemaking process of Mencía grapes. Food Chemistry, 117, 529–537. Grazioli, B., Fumi, M. D., & Silva, A. (2006). The role of processing on ochratoxin A content in Italian must and wine: A study on naturally contaminated grapes. International Journal of Food Microbiology, 111, S93–S96. IARC (1993). IARC monographs on the evaluation of carcinogenic risks to humans. Some naturally occurring substances: food items and constituents, heterocyclic aromatic amines and mycotoxins. Summary of data reported and evaluation. IARC Science Publications, 56, 489–521. MARM (2008a). Ministry of Environment and Rural and Marine Affairs (MARM). Government of Spain. Food. Consumption and commercialisation. Food consumption panel. Available from:. MARM (2008b). Ministry of Environment and Rural and Marine Affairs (MARM). Government of Spain. Food. Protected Designations of Origen and Geographic Indications. Available from: . MARM (2009). Ministry of Environment and Rural and Marine Affairs (MARM). Government of Spain. Cartography and GIS. Agrarian data geographic. Information systems (ADGIS). Available from: htpp://sig.marm.es/siga. OIV (2009). Extract of the 2008 world vitiviniculture situation report, presented by Federico Castellucci, Director General of the OIV, in Zagreb, on 29 June 2009. International Organisation of Vine and Wine. Press release. Otteneder, H., & Majerus, P. (2000). Occurrence of ochratoxin A (OTA) in wines: Influence of the type of wine and its geographical origin. Food Additives and Contaminants, 17(9), 793–798. Pietri, A., Bertuzzi, T., Pallaroni, L., & Piva, G. (2001). Occurrence of ochratoxin A in Italian wines. Food Additives and Contaminants, 18(7), 647–654. Rial Otero, R., Yagüe Ruiz, C., Cancho Grande, B., & Simal Gándara, J. (2002). Solidphase microextraction–gas chromatographic–mass spectrometric method for the determination of the fungicides cyprodinil and fludioxonil in white wines. Journal of Chromatography A, 942, 41–52. Rosa, C. A. R., Magnoli§, C. E., Fraga, M. E., Dalcero§, A. M., & Santana, D. M. N. (2004). Occurrence of ochratoxin A in wine and grape juice marketed in Rio de Janeiro, Brazil. Food Additives and Contaminants, 21(4), 358–364. Shephard, G. S., Fabiani, A., Stockenström, S., Mshicileli, N., & Sewram, V. (2003). Quantitation of ochratoxin A in South African wines. Journal of Agricultural and Food Chemistry, 51, 1102–1106. Stefanaki, I., Foufa, E., Tsatsou-Dritsa, A., & Dais, P. (2003). Ochratoxin A concentrations in Greek domestic wines and dried vine fruits. Food Additives and Contaminants, 20(1), 74–83. UNE-EN 14133 (2004). Productos alimenticios. Determinación de ocratoxina A en vino y cerveza. Método mediante cromatografía líquida de altas características (HPLC) y purificación por columna de inmunoafinidad. Visconti, A., Pascale, M., & Centonze, G. (1999). Determination of ochratoxin A in wine by means of immunoaffinity column clean-up and high-performance liquid chromatography. Journal of Chromatography A, 864, 89–101. Zimmerli, B., & Dick, R. (1996). Ochratoxin A in table wine and grape-juice. occurrence and risk assessment. Food Additives and Contaminants, 13(6), 655–668.
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Occurrence of ochratoxin A in Rioja Alavesa wines Silvia Quintela ⇑, M. Carmen Villarán, Iratxe López de Armentia, Edurne Elejalde Fundación LEIA, C.D.T., Technological Park of Alava, Leonardo Da Vinci 11, 01510 Miñano, Spain
a r t i c l e
i n f o
Article history: Received 2 February 2010 Received in revised form 20 July 2010 Accepted 28 September 2010
Keywords: Ochratoxin A Rioja Alavesa Daily intake HPLC-fluorimetric Immunoaffinity column Wine
a b s t r a c t In this survey the influence of the geographical location and the kind of wine in the concentration of ochratoxin A (OTA) was studied. Forty percent of the Spanish wine market belongs to the Rioja Qualified Designation of Origen (DOCa Rioja) wines, which are already worldwide known. A total of 100 wines from the Rioja Alavesa (RA) production area of the DOCa Rioja were analysed, using immunoaffinity column clean-up and high-performance liquid-chromatography with fluorimetric detection (detection limit 0.002 lg/l). The presence of OTA was greater in wines produced in low rainfall and high temperature regions. The geographical location and kind of wine did not seem to have influence on the OTA concentration. The mean OTA concentration of RA wines was 0.035 lg/l and its contribution to mean daily OTA intake could be considered negligible, representing a 0.07% of the latest tolerable weekly intake (TWI) established in 2006 by EFSA. Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction Spain is the third country of the world with the highest wine production, behind Italy and France (OIV, 2009). According to the latest data of the Spanish Ministry of Environment and Rural and Marine Affairs, Spain has nowadays 67 of designations of origin (MARM, 2008b). The oldest one is the Rioja Qualified Designation of Origin (DOCa Rioja). The DOCa Rioja is leader of the Spanish market of quality wines, with near 40% of the total sales of these wines. It has great international prestige and it exports around 30% of wines production, mainly to the United Kingdom, Germany, Switzerland and the United States (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The Rioja Alavesa (RA) wines are produced in one of the production areas of the DOCa Rioja. In the area of RA is produced the 26% of the wine with DOCa Rioja. The economy of this region depends greatly on the wine activity. Thus, in order to preserve the position of the DOCa Rioja wines in the market, it is necessary to guarantee the quality and safety of these wines. The concern about food safety each day is greater, increasing the control of the content of different contaminants in food products, such the ochratoxin A (OTA), many pesticides, etc. One example, are the fungicides currently used to control the fungal diseases in the vineyards. Since there is a risk of transfering some undesirable contaminants from the grape to the wine, there is the need to ensure that such residues should not be found in wine at levels presenting an unacceptable risk to humans. In this ⇑ Corresponding author. Tel.: +34 945 29 81 44; fax: +34 945 29 82 17. E-mail address: [email protected] (S. Quintela). 0308-8146/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.foodchem.2010.09.096
sense, strict regulations of fungicide use on grapes are being promoted. One of their consequences, is the need to develop new feasible and specific analytical methods adapted to the analysis requirements of the market. Several works have been presented already in this field, specially since 2009, although many research is still needed to undergo in the near future (González-Rodríguez, Cancho-Grande, & Simal-Gándara, 2009a,b; González-Rodríguez, Cancho-Grande, Torrado-Agrasar, Simal-Gándara, & Mazaira-Pérez, 2009; Rial Otero, Yagüe Ruiz, Cancho Grande, & Simal Gándara, 2002). Another contaminant of concern is the ochratoxin A (OTA). OTA is a mycotoxin produced by different species of fungi belonging to the genera Penicillium and Aspergillus. The systematic chemical nomenclature for OTA is (R)-N-[(5-chloro-3,4-dihydro-8-hydroxy-3methyl-1-oxo-1H-2-benzopyran-7-yl)-carbonyl]-L-phenylalanine. This mycotoxin has been detected in different foodstuffs such as cereals, related cereal products, coffee, wine and grape juice, but it has also been detected in animal products, such as pork meat (EFSA, 2006). The International Agency for Research on Cancer (IARC) has classified this mycotoxin in the category 2B, as ‘‘possibly carcinogenic to humans’’ (IARC, 1993). In addition, the OTA is known to be nephrotoxic, teratogenic, immunotoxic and possibly neurotoxic (EFSA, 2006). It is related also with an endemic kidney disease observed in the Balkans (Balkan Endemic Nephropathy and related Urinary Tract Tumours) (EFSA, 2006). After cereals, the wine is the second food with the greatest contribution in the daily intake of OTA by the population of EU (EC, 2002). It was detected in wine in 1996 by Zimmerli and Dick (Zimmerli & Dick, 1996). Since then, the number of studies about the content of OTA in wines around the world has been increased
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(Bellí, Marín, Duaigües, Ramos, & Sanchis, 2004; Berente et al., 2005; Blesa, Soriano, Moltó, & Mañes, 2004; Brera, Soriano, Debegnach, & Miraglia, 2005; Burdaspal & Legarda, 1999; Domijan & Peraica, 2005; Filali et al., 2001; Rosa, Magnoli§, Fraga, Dalcero§, & Santana, 2004; Shephard, Fabiani, Stockenström, Mshicileli, & Sewram, 2003; Visconti, Pascale, & Centonze, 1999). The Regulation (EC) No. 123/2005 of the European Commission sets the limit of Ochratoxin A for wine at 2 lg/l (EC, 2005). The main factors which have influence on the presence of OTA in the grapes are basically the meterological conditions, the sanitary status of the grapes (mould presence) and the conditions and grape cultivation practices. In terms of meteorological conditions, a relationship is usually found between the temperature and contamination severity, the frequent and concentration of this mycotoxin are higher in warmer regions (Czerwiecki, Wilczyn´ska, & Kwiecien´, 2005; Otteneder & Majerus, 2000; Pietri, Bertuzzi, Pallaroni, & Piva, 2001; Stefanaki, Foufa, Tsatsou-Dritsa, & Dais, 2003; Zimmerli and Dick, 1996). OTA and the fungi producers of this mycotoxin can be transferred from grapes to wine during winemaking. The different processes used in wine production can have a great influence on the final OTA concentration, for example the stage of maceration, where an increase in OTA content can be produced due of grape skins contact with must. On the contrary, another stages of the wine processing can reduce the OTA content, such is the wine ageing in bottle, although this effect is not appreciable on a short ageing of 12 months of storage (Grazioli, Fumi, & Silva, 2006). The Rioja Alavesa (RA) is situated in 42° North latitude, which corresponds to the temperate zone of Europe. Its climate is influenced by the Atlantic Ocean and the Mediterranean Sea, more or less mitigated by topographic factors that produce diverse climatic situations, appearing within this region major variations in rainfalls and temperatures among different areas. This region can be divided into four fitoclimatics areas: semiarid, dry, subhumid and humid. The 94% of vineyard hectares are situated in the semiarid and dry areas of RA (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The semiarid area is located to the south of RA and its altitude level is between 489–569 m above sea level, the mean annual rainfall is estimated to be around 502 mm and the mean annual temperature is 12.93 °C. The dry area it is a higher altitude (510–648 mm), its mean annual temperature is lower (around 12.51 °C) and the mean annual rainfall is sensibly higher (approximately 604 mm) (MARM, 2009). Almost 90% of the total wine commercialised is red wine. Depending on the different elaboration process, this means, the kind of ageing process (without, cask, bottles) or periods (months), the DOCa Rioja wine shows different categories: ‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ or ‘‘gran reserva’’ (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The aim of this work is to report the content of OTA in 100 commercial wines from the most relevant fitoclimatic areas of the Rioja Alavesa region. The results determine the influence of several variables, such as the kind of wine and the climatic conditions. The results have been compared with the limits established by the EC related directives and with the levels in other countries. Finally, the potential contribution to the dietary OTA exposure of consumers of these wines has been evaluated.
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A total of 100 wines were analysed. Most of the wines were provided generously by different wine cellars interested in the study and the rest were purchased from supermarkets. The wines correspond both to the semiarid area from (including the municipalities of Assa, Baños de Ebro, Elciego, Lapuebla de Labarca, Laserna, Móreda, Oyón and Villabuena de Álava) and to the dry area of the RA (including the municipalities of Barriobusto, Elvillar, Labastida, Laguardia, Lanciego, Leza, Navaridas, Páganos and Samaniego). The wines were produced between 1995 and 2008. The number of wine samples for each kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine, as well as white wine and rosé wine) was taken according to their importance in terms of commercialisation (Consejo Regulador de la Denominación de Origen Calificada Rioja, 2008). The total number of samples analysed in the semiarid area were: ‘‘joven’’ red wine (n = 16), ‘‘crianza’’ red wine (n = 21), ‘‘reserva’’ red wine (n = 10), ‘‘gran reserva’’ red wine (n = 1), white wine (n = 1) and rosé wine (n = 2). And in the dry area were: ‘‘joven’’ red wine (n = 16), ‘‘crianza’’ red wine (n = 20), ‘‘reserva’’ red wine (n = 8), ‘‘gran reserva’’ red wine (n = 2), white wine (n = 2) and rosé wine (n = 1). All the samples were maintained at 4 °C since their arrival at the laboratory to the time of analysis. 2.2. Reagents The OTA standard solution was purchased by Sigma–Aldrich (Seelze, Germany). The OTA concentration of the standard was determined accurately by UV spectroscopy at 333 nm and it was stored at 20 °C. Acetonitrile, methanol and glacial acetic acid (HPLC grade) were purchased from Panreac (Barcelona, Spain). Polyethylene glycol (PEG 8000) was obtained from Sigma–Aldrich (Steinheim, Germany), sodium chloride (ACS) and sodium hydrogencarbonate (ACS) from Panreac (Barcelona, Spain). OchraTest immunuoaffinity columns were purchased from Vicam (USA) and glass microfibre filters GF/A 47 mm from Whatman (England). 2.3. Apparatus and chromatographic conditions
2. Materials and methods
The chromatographic analysis was done with a HPLC equipment connected to a fluorescent spectrophotometer (kex = 333 nm y kem = 460 nm), both from Hewlett–Packard model 1100. The computer program used was a Agilent Chemstation for chromatographic analysis. The analytical column was a reversed-phase Synergi C12 (150 4.60 mm, 4 lm particles) (Phenomenex) preceded by guard cartridge AQ C18 (4 2 mm). Chromatographic separation was performed using isocratic elution with water–acetonitrile–acetic acid (99:99:2, v/v/v) at a flow rate of 1 ml/min. One hundred microliters of solution were injected. The calibration curve obtained by analysing OTA standard solutions at eight concentration levels (range: 0.004–7.242 lg/l) was linear (r2 = 0.9996). The precision and accuracy was tested by duplicate analyses on a red wine sample containing OTA at a concentration of 0.03, 1.5 and 7 lg/l. The coefficient of variation was less than 5%, demonstrating good repeatability of the HPLC analysis. The analytical recovery for OTA, determined by duplicate on a red wine sample spiked at a level of 0.3 and 3 lg/l was 99.2% and 101.8%, respectively. The limit of detection (S/N = 3:1) was 0.002 lg/l and the limit of quantitation (S/N = 10:1) was 0.004 lg/l.
2.1. Sampling design
2.4. Sample preparation
In order to study the main factors which affect the presence of OTA, for the sampling design variables such as RA area and kind of wine have been taken into account.
The analytical method was based on the UNE-EN 14133 (UNEEN 14133, 2004). A 10 ml volume of wine sample was diluted with 10 ml water solution containing PEG (1%) and NaHCO3 (5%), mixed
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and filtered through Whatman GF/A glass microfibre filter. A 10 ml volume of diluted extract was cleaned up through an OchraTest immunoaffinity column at a continuous flow-rate of about 1 drop per s. The column was washed with a solution containing NaCl (2.5%) and NaHCO3 (0.5%) followed by Milli-Q water at a flow-rate of 1–2 drops per s. OTA was eluted with methanol. The eluate was evaporated to dryness under a nitrogen stream and redissolved with 250 ll in HPLC mobile phase. 2.5. Statistical methods When comparing the OTA positive samples and levels in the different RA areas and kind of wines, the H of Kruskal–Wallis non-parametric test for non-dependent samples was applied. A probability value of 0.05 has been used in order to determine the statistical significance, using the program SPSS Statistics 17.0. 3. Results and discussion Table 1 shows the OTA positive samples and concentration from different areas (semiarid and dry area) and kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine and white and rosé wine) from RA wines sampled in this survey. The statistical results obtained can be seen in Table 2. Altogether, OTA was found in the 57% of the total wine samples from both areas. All the RA wines showed a low level of OTA contamination ranged in positive wine samples from 0.004 to 0.179 lg/l with a mean concentration of 0.035 lg/l. The concentration of all the positive samples was below the maximum authorised level established in 2 lg/l for wine in the EU (EC, 2005). Thirty-six samples (71%) from the semiarid area of RA contained detectable amounts of OTA. The mean concentration of positive Table 1 OTA presence and concentration from different areas (semiarid and dry area) and kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine and white and rosé wine) from Rioja Alavesa wines. Kind of wine
Positives/ total
OTA mean concentration of positive samples (lg/ l)*
Range positive samples (lg/l)*
44 86 90 0
0.029 0.034 0.018 <0.002
0.014–0.056 0.004–0.144 0.004–0.042 –
100 50 71
0.013 0.007 0.028
– – 0.004–0.144
63 40 13 50
0.042 0.053 0.060 0.048
0.007–0.179 0.009–0.169 – –
50 0 43
0.015 <0.002 0.046
– – 0.007–0.179
17/32 26/41 10/18 1/3
53 63 56 33
0.037 0.040 0.022 0.048
0.007–0.179 0.004–0.169 0.004–0.060 –
2/3 1/3 57/100
67 33 57
0.014 0.007 0.035
0.013–0.015 – 0.004–0.179
Semiarid area of Rioja Alavesa ‘‘Joven’’ red wine 7/16 ‘‘Crianza’’ red wine 18/21 ‘‘Reserva’’ red wine 9/10 ‘‘Gran reserva’’ red 0/1 wine White wine 1/1 Rosé wine 1/2 Total wine 36/51 Dry area of Rioja Alavesa ‘‘Joven’’ red wine 10/16 ‘‘Crianza’’ red wine 8/20 ‘‘Reserva’’ red wine 1/8 ‘‘Gran reserva’’ red 1/2 wine White wine 1/2 Rosé wine 0/1 Total wine 21/49 Total Rioja Alavesa ‘‘Joven’’ red wine ‘‘Crianza’’ red wine ‘‘Reserva’’ red wine ‘‘Gran reserva’’ red wine White wine Rosé wine Total wine *
Positives (%)
Detection limit 0.002 lg/l. Quantitation limit 0.004 lg/l.
samples was 0.028 lg/l. The highest level of OTA was 0.144 lg/l and was found in a ‘‘crianza’’ red wine from 2001. Regarding to the dry area of RA, the 43% of the wine samples showed a OTA concentration ranging from 0.007 to 0.179 lg/l. The OTA mean concentration of the positive samples was 0.046 lg/l. The number of positive samples was higher in the semiarid area (71%) than in the dry area (43%), with a statistically significance (p < 0.05). The differences detected might be due to the fact that in the semiarid area there are less rainfalls and higher temperatures than in the dry area, so that it could favour the growth of OTA producing fungi. However, no significant differences (p > 0.05) were found in the OTA concentration. Several authors (Czerwiecki et al., 2005; Otteneder and Majerus, 2000; Zimmerli and Dick, 1996) have found an increase of OTA occurrence and concentration in wines from northern to southern countries. Others authors (Pietri et al., 2001; Stefanaki et al., 2003) have arrived to the same conclusion translated to different wine-growing areas in the same country (Italy and Greece, respectively). These results were in concordance with our results about a higher incidence of OTA (% of positives samples), but not about a higher OTA content in wines from southern areas. One reason that could explain the fact that no statistical differences have been found in the concentration of OTA between both areas (sermiarid and dry) from RA, could be the strict norms of control board of DOCa Rioja wine related to quality practices in the grape cultivation and the wine elaboration process. Moreover, no significant differences (p > 0.05) were found considering the different elaboration process or kind of wine (‘‘joven’’, ‘‘crianza’’, ‘‘reserva’’ and ‘‘gran reserva’’ red wine, as well as white wine and rosé wine) in all the climatic areas studied. These results differ with those obtained by others authors (Otteneder and Majerus, 2000; Zimmerli and Dick, 1996) where the OTA concentration decrease from red to rosé to white wine. These results may be explained by good winemaking practice in the wines elaborated in RA. Currently, there are no surveys with such number of samples analysing the content of OTA in wine neither from Rioja Alavesa region nor from the total DOCa Rioja. Bellí et al. (2004) analysed 10 wines of each kind of wine from the DOCa Rioja: ‘‘joven’’, ‘‘crianza’’ and ‘‘reserva’’ red wine and white wine. In these study the exact area of the DOCa Rioja samples was not indicated. Our results differ basically from the ones of these authors related to the content of OTA of ‘‘joven’’ red wines. Whilst in our study the mean concentration of OTA for the ‘‘joven’’ red wine was 0.037 lg/l, in the above mentioned publication it was reported a mean concentration of 1.220 lg/l. According to the data of this study, the average concentration of OTA from RA analysed wine was 0.035 lg/l. According to the alimentary consume data of the Spanish Ministry of Environment and Rural and Marine Affairs, the designation of origen wine consumption in Spain from July 2007 to June 2008 is about to 24.22 ml/day per person (MARM, 2008a). Assuming that an average adult body weights 70 kg, the estimated daily intake of OTA could be 0.012 ng/kg bw per day (0.084 ng/kg bw per week). This value represents 0.07% of the tolerable weekly intake (TWI) established by the Scientific Panel on Contaminants in the Food Chain from the EFSA (European Food Safety Authority) in 2006 (120 ng/ kg bw per week) (EFSA, 2006). The contribution to OTA daily intake coming from the wines from RA studied were considerably smaller in comparison with the values obtained in others studies, for example the wines from Croatia (0.20–0.26 ng/kg bw per day) (Domijan and Peraica, 2005), Italy (0.90 and 1.36 ng/kg bw per day) (Brera et al., 2005), Greece (3.75 ng/kg bw per day) (Stefanaki et al., 2003), and others parts of Spain (0.15 ng/kg bw per day) (Blesa et al., 2004).
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S. Quintela et al. / Food Chemistry 126 (2011) 302–305 Table 2 H of Kruskal–Wallis test applied to the concentration and positive samples in Rioja Alavesa wines. KRUSKAL–WALLIS TEST Rioja Alavesa area
Test variable
Grouping variable
Chi-Square
Df
Asymp. Sig.
Semiarid area Dry area Total Rioja Alavesa Total Rioja Alavesa
Concentration (lg/l) Concentration (lg/l) Concentration (lg/l) Positive samples
Kind of wine Kind of wine Kind of wine Rioja Alavesa area
3.548 2.001 5.933 7.820
3 4 5 1
0.315 0.736 0.313 0.005
4. Conclusions The concentration detected in Rioja Alavesa wines analysed was below the limit established by the EU (2 lg/l) and its contribution to the tolerable daily intake could be considered as negligible. According to the obtained results, RA wines have much lower content in OTA than other European regions. Significant differences in the presence of OTA in different regions of RA has been found, being the highest in the area with lower mean rainfalls and higher mean temperature (semiarid area). However, in terms of concentration of OTA in wine, no significant differences have been found in terms of area in RA and elaboration process (kind of wine). Acknowledgements This work has been funded by the Agriculture and Fisheries Department of the Basque Government (AID-0486-05-C) and by the pre-doctoral grant of the Ikertu program of the Basque Government (year 05/06 and 06/07). We are also grateful to the wine cellars that kindly provided their wines for this survey. References Bellí, N., Marín, S., Duaigües, A., Ramos, A. J., & Sanchis, V. (2004). Ochratoxin A in wines, musts and grape juices from Spain. Journal of the Science of Food and Agriculture, 84, 591–594. Berente, B., Móricz, Á., -Otta, K. H., Záray, G., Lékó, L., & Rácz, L. (2005). Determination of ochratoxin A in Hungarian wines. Microchemical Journal, 79, 103–107. Blesa, J., Soriano, J. M., Moltó, J. C., & Mañes, J. (2004). Concentration of ochratoxin A in wines from supermarkets and stores of Valencian Community (Spain). Journal of Chromatography A, 1054, 397–401. Brera, C., Soriano, J. M., Debegnach, F., & Miraglia, M. (2005). Exposure assessment to ochratoxin A from the consumption of Italian and Hungarian wines. Microchemical Journal, 79, 109–113. Burdaspal, P. A., & Legarda, T. M. (1999). Ocratoxina A en vinos, mostos y zumos de uva elaborados en España y en otros países europeos. Alimentaria (Madrid), 299, 107–113. Consejo Regulador de la Denominación de Origen Calificada Rioja. Memoria; 2008. Czerwiecki, L., Wilczyn´ska, G., & Kwiecien´, A. (2005). Ochratoxin A: An improvement clean-up and HPLC method used to investigate wine and grape juice on the Polish market. Food Additives and Contaminants, 22(2), 158–162. Domijan, A. M., & Peraica, M. (2005). Ochratoxin A in wine. Archives of Industrial Hygiene and Toxicology, 56, 17–20. EC (2002). Reports on tasks for scientific cooperation. Report of experts participating in Task 3.2.7. Assessment of dietary intake of ochratoxin A by the population of EU Member States. Directorate-General Health and Consumer Protection; January 2002. EC (2005). Commission Regulation (EC) No. 123/2005 of 26 January 2005 amending regulation (EC) No. 466/2001 as regards ochratoxin A. Official Journal of the European Union, L25:3–5. EFSA (2006). Opinion of the scientific panel on contaminants in the food chain on a request from the commission related to ochratoxin A in food. The EFSA Journal, 365, 1–56.
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