EXTERNAL SCIENTIFIC REPORT. Study on the influence of food processing on nitrate levels

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1 EXTERNAL SCIENTIFIC REPORT Study on the influence of food processing on nitrate levels in vegetables 1 Authors: Ksenija Ekart*, Alenka Hmelak Gorenjak*, Eneko Madorran*, Slavko Lapajne and Tomaž Langerholc (*contributed equally) University of Maribor ABSTRACT Nitrate is a naturally occurring compound that is part of the nitrogen cycle, as well as an approved food additive. Vegetables are the major source of nitrate in our nutrition, but nitrate content depends on type of vegetable as well as on environmental and agricultural factors. Apart from external factors, nitrate content of the vegetable can be influenced by kitchen processing techniques (PTs) used for preparing vegetables for food. In the present work we studied effects of common kitchen PTs on nitrate content in lettuce, spinach, potato, green bean, carrot, red beet, white cabbage, Chinese cabbage and courgette. Changes in the nitrate content between raw and processed samples were statistically evaluated where possible with respect to winter or summer time of harvest, agricultural cultivation and vegetable variety. In general, some kitchen PTs i.e boiling and washing decreased nitrate content irrespectively of the vegetable type. Blanching and purée decreased nitrate content during processing of all tested vegetables. In contrast, some techniques increased nitrate content i.e. deep frying, sauté and grilling. Time of harvest and agricultural cultivation system influenced nitrate content in raw vegetables, but were generally found less importantt for the changes during processing. Further research is needed for determination of vegetable varieties with the highest nitrate reduction during processing. University of Maribor, 2013 KEY WORDS vegetable, nitrate, food processing DISCLAIMER The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 1 Question No EFSA-Q Any enquiries related to this output should be addressed to contam@efsa.europa.eu Suggested citation: Ekart K, Hmelak Gorenjak A, Madorran E, Lapajne S and Langerholc T, Study on the influence of food processing on nitrate levels in vegetables. EFSA supporting publication 2013: EN-514, 150 pp. Available online: European Food Safety Authority, 2013

2 SUMMARY Nitrate is a naturally occurring relatively non-toxic compound. It is part of the nitrogen cycle, as well as an approved food additive. It accumulates in plants and higher levels of nitrate tend to be found in leaves whereas lower levels occur in seeds or tubers. Thus leafy crops such as lettuce and spinach generally have higher nitrate concentrations. Human exposure to nitrate is mainly through the consumption of vegetables and to a lesser extent water and other foods. Nitrate concentrations in vegetables can be influenced by both environmental and agricultural factors, such as soil moisture, type of soil, light intensity, temperature, fertilizers, variety and crop protection strategies. In addition to the above mentioned external factors, levels of nitrate can be influenced by storage time and storage conditions and food processing. The main objective of the present study was to study the effects of processing on nitrate content. Changes in the nitrate content during processing depend on the type of vegetables, the type of processing technique (PT) and the way of PT implementation. To exclude the impact of the implementation of PTs on nitrate levels, the PTs were performed by professional staff in large kitchens under controlled conditions. Therefore, changes in the nitrate levels during processing were only affected by the differences between samples (type of production, the season, the quality of vegetables). The study included 9 different vegetables: lettuce, spinach, white cabbage, Chinese cabbage, green bean, courgette, potato, carrot and red beet. The influence of the seasonal variations (summer/winter), farming variations (conventional, organic or integrated) were studied. Vegetables originated from Slovenia, Italy, Spain, the Netherlands, Belgium, France, Poland, Germany, Croatia, Bosnia and Herzegovina, Austria, Morocco and Egypt (all of them were mainly purchased from wholesalers present in Slovenia or local Slovenian farms). After processing, the aggregate sample was homogenized. The chilled sample of processed food was immediately frozen to -25 C, until samples were prepared for the analysis of nitrate content. The nitrate content in vegetables was determined by high performance liquid chromatography (HPLC) and ion exchange technique. Optimization of the methods, including validation, was carried out on intra-laboratory and inter-laboratory levels. During the time of the project the laboratory was successfully integrated into the Inter-laboratory Proficiency scheme Bipea ( Bureau Interprofessionnel d'etudes Analytiques ). Results obtained during this study on the influence of processing on nitrate content in different types of vegetables were statistically evaluated and a value of p<0.05 was considered as statistically significant. 2 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

3 Lettuce is a type of vegetable that is consumed fresh. The study included 60 samples of lettuce, butterhead type (Lactuca sativa var. capitata) and crisp lettuce (Lactuca sativaa var. crispa). Samples were from various areas in Italy, Slovenia, France and Bosnia and Herzegovina. The majority of the samples were grown outdoors by conventional farming. Nitrate levels in the samples of lettuce studied were on average 1862 mg/kg, with a 5 th percentile (P5) of 725 mg/kgg and a 95 th percentile (P95) of 2875 mg/kg. Lettuce, harvested in summer, with an average nitrate content of 1209 mg/kg, had statistically significantly lower nitrate content than lettuce harvested during winter, with an average nitrate value of 2164 mg/kg. During washing, the content of nitrate in lettuce was stastically significantly (p<0.05) reduced, by an average of -19%, but the effect was greater in winter (-24%) than in summer samples (-16%). Spinach is a type of vegetable that can be processed by washing, boiling, steaming, blanching, sauté and purée. It is commonly included in the diet of babies and children, who are the most vulnerable groups for methemoglobinemia. Most of the spinach samples originated from different areas of Italy (Abruzzo, Basilicata, Campania, Lombardia, Puglia, Veneto), with the addition of 4 summer samples originating from Germany. 90% of spinach samples were grown conventionally, few samples were of integrated as well as of organic cultivation. Only 5 samples were grown in greenhouses, the rest were grown outdoors. All the samples from greenhousess were collected in the winter period. Influence of processing on nitrate reduction was studied after washing, boiling, steaming, blanching, sauté, purée and some combinations of these PTs. In the literature, spinach is often referred to as the vegetable with the highest nitrate content. Average nitrate levels in all samples of unprocessed spinach analysed in our study amounted to1682 mg/kg, with a P5 of 495 mg/kg and a P95 of 3689 mg/kg. Comparison of nitrate content in spinach harvested in summer and winter time showed that winter spinach, with an average of 1601 mg/kg had lower nitrate content than the spinach harvested in summer, but the difference was statistically not significant. Most of the samples with higher nitrate content were grown in the Northern part of Italy. Due to a low number of spinach samples from organic and integrated cultivation included in our study, statistical comparison of nitrate levels between different types of cultivation was not performed. During processing of spinach there was a statistically significant (p<0.05) reduction in the levels of nitrate after the following techniques: boiling (-53%), blanching (-36%) and washing (-27%). Processing of washed samples by techniques mentioned above resulted in higher losses of nitrate. For the PTs steaming and sauté, the nitrate content in spinach increased; for the PT steaming by +7% (the difference was not statistically significant, p>0.05) and for the PT sauté by +25% (the difference was statistically significant; p<0.05). The increase in nitrate levels after both PTs can be probably attributed to the weight loss during processing. Washing before steaming but not before sauté also resulted in a lower nitrate content in the processed samples. Loss of nitrate during purée of spinach depended on the combination of PTs. Purée of washed + boiled spinach showed a statistically significant (p<0.05) reduction of the nitrate concentration by - 61% and purée of washed + sautéed spinach also statistically significantly (p< <0.05) reduced nitrate by -30%. Spinach purée is commonly used in baby food formulas; using boiled instead of steamed spinach for purée is recommended, since it results in the lower nitrate content. 3 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

4 Chinese cabbage is a type of vegetable that can be consumed fresh or stir-fried. The study included samples of Chinese cabbage, mainly from Austria, others from Germany, Hungary, Poland, Slovenia, Spain and the Netherlands. All samples were grown outside, of which 18 were grown in summer time and others in winter. Most samples were grown by conventional farming and 12 by integrated farming. Nitrate levels in the samples of Chinese cabbage studied were on average 838 mg/kg, with a P5 of 199 mg/ /kg and a P95 of 1886 mg/kg. Chinese cabbage harvested in summer had significantly higher nitrate content than Chinese cabbage harvested during winter time. Chinese cabbage farmed conventionally had statistically significantly lower nitrate content than the samples farmed integratedly. Washing of Chinese cabbage was the only PT which decreased the nitrate content. Application of stir-frying and washing + stir-fryincontent. to Chinese cabbage resulted with a variable effect on the nitrate The nitrate content in white cabbage was determined in raw white cabbage (blank samples), fermented and boiled fermented samples. All three forms of white cabbage consumption are common. The study includedd 25 samples of white cabbage, of which 22 samples were harvested and fermented by large growers and producers of sauerkraut in northeastern Slovenia, with the production of sauerkraut greater than 40 tons per year. 3 samples were fermented on small farms, that produce sauerkraut only for domestic needs. Most of the samples were of integrated cultivation, four samples were from organic cultivation. All samples were grown outdoors. White cabbage belongs to the group of Brassica vegetables, with a mediumm nitrate content. An average nitrate content of 374 mg/kg (P5 of 149 mg/kg and P95 of 691 mg/kg) was observed. During fermentation the nitrate content in white cabbage was not statistically significantly altered (p>0.05). As for other types of vegetables, nitrate levels during boiling decreased. After boiling of fermented cabbage, nitrate in cabbage statistically significantly (p<0.05) decreased by -22%. Nitrate content after boiling of fermented cabbage also decreased by -24% in comparison with initial nitrate content in cabbage before fermentation. Courgette is a type of vegetable that can be consumed fresh, boiled or grilled. The study included samples of courgette from Italy, Morocco and Spain. All samples were grown outside, of which 12 were grown in winter time and 13 were grown in summer by conventional farming. Nitrate levels in the samples of courgette were on average 618 mg/kg, with a P5 of 146 mg/kg and a P95 of 1208 mg/kg. Courgette harvested in summer, with an average nitrate content of 527 mg/kg, had no statistically significantly lower nitrate content than courgette samples harvested in winter. Difference was observed when comparing the influence of harvesting time on the reduction rate of nitrate levels after boiling and washing + boiling. Both PTs decreased the nitrate levels statistically significantly (-35% and -31%, respectively). In contrast, grilling and washing + grilling statistically significantly increased nitrate content in courgette samples (+49% and +44%, respectively). Washing was the only PT where no statistically significant effect on the nitrate levels was measured. The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors. 4

5 Green bean is a type of vegetable that can be consumed boiled, steamed, blanched or heated with a microwave. The study included samples of green bean from Egypt, Italy, Spain and mostly from Morocco. Most of the samples were grown outside (18) and 9 were grown in a greenhouse. All samples were grown by conventional farming, of which 12 were grown during winter time and 15 samples were grown in summer. Nitrate levels in the samples of green beans studied were on average 365 mg/kg. Although the study included green beans from various parts of Europe and North Africa, with different climatic conditions and different types of production, there was no statistically significant difference in the levels of nitrate between summer and winter samples. Green bean grown outdoor had an average nitrate content of 301 mg/kg, and it was statistically significantly lower than in the green bean samples grown in a greenhouse (493 mg/kg). Since greenhouse dissipates light, the obtained results are in accordance with the reports of higher nitrate levels during low light conditions. Green bean is a vegetable with fibrous structure that helps to keep the shape and structure of the vegetable during processing. This could be a reason for our observation, that PTs had a limited effect on the changes of nitrate content during processing. However, washing, boiling, blanching, washing + boiling and washing + blanching statistically significantly decreasedd the nitrate content. Potato is a type of vegetablee that can be consumed boiled, steamed, blanched, baked, deep-fried, puréed or heated with a microwave. The study included samples of potato from Austria, Belgium, Egypt, France, Germany, Italy, Spain and mostly from Slovenia. All sampless were grown outside. 35 samples were grown by conventional farming, 20 by integrated farming and 3 by organic farming. Since many potato varieties containing highly different nitrate levels were analysed, no comparison was made between type of farming or growing season. Nitrate levels in the sampless of potato studied were on average 182 mg/kg. The study included potato from various parts of Europe and North Africa grown under different climatic conditions and different types of production. From the results on changes in nitrate content after processing it was concluded, that potato variety could be one of the important factors we included 26 varieties into our study. PTs which included boiling were the only PTs that showed a clear tendency to decrease the nitrate content. In the case of deep-frying, a clear tendency to increase the nitrate content was observed. The rest of the PTs had variable effects. The number of samples should be increased for combinations of each variety and PT to get a more reliable statistical evaluation of the results. Carrot is a type of vegetable that can be consumed fresh, peeled or boiled. The study included samples of carrot from Italy, Austria, the Netherlands, Poland and Slovenia. All samples were grown outside, except one. 18 samples were harvested in summer and 7 during winter. Most of the samples were grown by conventional (16), 2 by integrated and 7 by organic farming. 5 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

6 Nitrate levels in the samples of carrot were on average 290 mg/kg, with a P5 of 5 mg/kg and a P95 of 1506 mg/kg. Carrot samples harvested in summer, with an average nitrate content of 71 mg/kg, had lower nitrate content than carrot samples harvested during winter, with an average nitrate content of 855 mg/kg. Conventionally grown samples had lower nitrate levels than samples grown by organic farming. No statistically significant influence on the nitrate contentt was observed during processing of carrot. Red beet is a type of vegetable that is usually prepared by a single combination of techniques: washing, boiling, peeling and slicing. 37 samples in total were analysed. 222 samples were from Slovenia, 8 samples from the Netherlands, 4 samples from Italy and 3 samples from Poland. All samples of red beet were grown outdoors and harvested in summer, but sampled in October and May. 15 samples were produced by conventional, 14 by integrated and 8 samples by organic farming. The average nitrate content in all samples of red beet was 2816 mg/kg, with a P5 of 335 mg/kg and a P95 of 5313 mg/ /kg. Nitrate content in red beet samples was studied after boiling of washed, unpeeled beet. The reduction of nitrate during processing was statistically significant (p<0.05) and amounted to -10%. Based on the results of the measured nitrate content in blank and processed vegetables, it can be concluded that washing and washing in combination with boiling decreased nitrate content irrespective of the type of vegetables. In addition, blanching decreased nitrate content in all tested vegetables (spinach, potato and green bean), the same was observed for purée (spinach and potato). Some techniques increased nitrate content, but were tested in combination with one type of vegetable: grilling of corgette, sauté of spinach and deep-frying of potato. As for other techniques (heating with a microwave, steaming, stir frying), ambiguous or no statistically significant effects on nitrate content have been determined. Statistically significant differences between summer and winter samples on the nitrate changes during processing were observed for some PTs in the case of lettuce and courgette, but not for spinach. For other vetegable types it was not possible to reliably evaluate effects due to the small number of samples. Type of vegetable cultivation (conventional, integrated, organic) was not found statistically significant for nitrate changes during processing for any of the vegetables, or it was not possible to reliably evaluate the effects due to the small number of samples included in our study. 6 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

7 TABLE OF CONTENTS Abstract... 1 Key words... 1 Summary... 2 Background as provided by EFSA Terms of reference as provided by EFSA Introduction and objectives Legislation on nitrate and acceptable daily intake Acceptable daily intake Nitrate accumulation in vegetables Biological factors Enviromental and agricultural factors Storage and processing Nitrate content in vegetables Objectives Material and methods General informationn Sample preparation Preparation of aggregate sample Preparation of laboratory sample Chemical analysis Method 1. Cheng and Tsang (1988) Method 2. CEN-prEN Part 4 (1998) (CEN-Part4) Validation Comparison of the Analytical Method 1 (according to Cheng and Tsang) and Analytical Method 2 (CEN Part 4) Statistical analysis Results of the effects of processing on nitrate content for each group of vegetables Lettuce Information about the samples Nitrate contentt Conclusions Spinach Information about the samples Nitrate contentt Conclusions Chinese cabbage Information about the samples Nitrate contentt Conclusions White cabbage Information about the samples Nitrate contentt The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

8 Conclusions Courgette Information about the samples Nitrate contentt Conclusions Green bean Information about the samples Nitrate contentt Conclusions Potato Information about the samples Nitrate contentt Conclusions Carrot Information about the samples Nitrate contentt Conclusions Red beet Information about the samples Nitrate contentt Conclusions Conclusions Recommendations References Acknowledgement Appendix A. Schemes of sample processing A.1. Lettuce A.2. Spinach A.3. Chinese cabbage A.4. White cabbage A.5. Courgette A.6. Green bean A.7. Potato A.8. Carrot A.9. Red beet Appendix B: Analytical and statistical data (XLS data set tables) Abbreviations The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

9 Table 1: Maximum levels for nitrate as laid down in Commission Regulation (EC) No 1258/ Table 2: Classification of vegetables according to nitrate content (mg/kg fresh weight) (Santamaria, 2006) Table 3: Summary of the differences between conventional, integrated and organic systems (Agra CEAS, 2002) Table 4: PT combinations for each vegetable type Table 5: Criteria for Method Table 6: Data for Recovery test (Method 1) Table 7: Statistical parameters for the analysis of nitrate inter-laboratory comparison Table 8: Criteria for Method 2 (CEN Part 4) Table 9: Data for Recovery test Table 10: Statistical parameters for the analysis of nitrate inter-laboratory comparison Table 11: Comparison of the analytical methods Table 12: General information on lettuce samples Table 13: Comparison of nitrate content in lettuce harvested during a different time of the year 37 Table 14: Comparison of nitrate content in lettuce for different cultivation types Table 15: Nitrate contentt in different lettuce varieties Table 16: Nitrate contentt in blank and processed samples of lettuce (mg/kg) Table 17: Changes in the nitrate content (%) in processed samples of lettuce with regard to blank samples Table 18: Influence of harvesting time (summer/winter) on the nitrate levels in lettuce after washing (PT 01) (%) Table 19: General information on spinach samples Table 20: Comparison of nitrate content in spinach harvested in a different time of the year.. 41 Table 21: Nitrate contentt in blank and processed samples of spinach (mg/kg) Table 22: Changes in the nitrate content (%) in processed samples of spinach with regard to blank samples Table 23: General information on Chinese cabbage samples Table 24: Comparison of nitrate content in Chinese cabbage harvested in a different time of the year 48 Table 25: Comparison of nitrate content in Chinese cabbage for different cultivation types Table 26: Nitrate contentt in blank and processed samples of Chinese cabbage (mg/kg) Table 27: Changes in the nitrate content (%) in processed samples of Chinese cabbage with regard to blank samples Table 28: General information on white cabbage samples Table 29: Comparison of nitrate content in white cabbage for different types of cultivation Table 30: Nitrate contentt in different white cabbage varieties Table 31: Nitrate contentt in blank and processed samples of white cabbage (mg/kg) Table 32: Changes in the nitrate content (%) in processed samples of white cabbage with regard to blank samples Table 33: General information on courgette samples Table 34: Comparison of nitrate content in courgette harvested in a different time of the year 58 9 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

10 Table 35: Nitrate contentt in blank and processed samples of courgette (mg/kg) Table 36: Changes in the nitrate content (%) in processed samples of courgette with regard to blank samples Table 37: Influence of harvesting time (summer/winter) on the nitrate levels in courgette after washing 61 Table 38: Influence of harvesting time (summer/winter) on the nitrate levels in courgette after boiling (%) Table 39: Influence of harvesting time (summer/winter) on the nitrate levels in courgette after washing + boiling (%) Table 40: Influence of harvesting time (summer/winter) on the nitrate levels in courgette after grilling (%) Table 41: Influence of harvesting time (summer/winter) on the nitrate levels in courgette after washing + grilling (%) Table 42: General information on green bean samples Table 43: Comparison of nitrate content in green bean for different production types Table 44: Nitrate contentt in blank and processed samples of green beans (mg/kg) Table 45: Changes in the nitrate content (%) in processed samples of green beans with regard to blank samples Table 46: General information on potato samples Table 47: Comparison of nitrate content in potato harvested in a different time of the year Table 48: Comparison of nitrate content in potato for different cultivation types Table 49: Nitrate contentt in blank and processed samples of potato (mg/kg) Table 50: Changes in the nitrate content (%) in processed samples of potato with regard to blank samples Table 51: General information on carrot samples Table 52: Nitrate contentt in carrot samples (mg/kg) Table 53: Nitrate contentt in blank and processed samples of carrot (mg/kg) Table 54: Changes in the nitrate content (%) in processed samples of carrot with regard to blank samples Table 55: General information on red beet samples Table 56: Comparison of nitrate content in red beet harvested in a different time of the year. 81 Table 57: Comparison of nitrate content in red beet for different cultivation types Table 58: Nitrate contentt in blank and processed samples of red beet (mg/kg) in a different time of sampling Table 59: Changes in the nitrate content (%) in processed samples of red beet with regard to blank samples sampled in a different time of the year The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

11 BACKGROUND AS PROVIDEDD BY EFSA Nitrate is a naturally occurring compound that is part of the nitrogen cycle, as well as an approved food additive. It plays an important role in the nutrition and function of plants. Nitrate is an important component of vegetables due to its potential for accumulation; this can be affected by a number of biotic and abiotic factors. Higher levels of nitrate tend to be found in leaves whereas lower levels occur in seeds or tubers. Thus leafy crops such as lettuce and spinach generally have higher nitrate concentrations. Human exposure to nitrate is mainly throughh the consumption of vegetables, and to a lesser extent water and other foods (EFSA, 2008). Nitrate concentrations in vegetables can be influenced by both environmental and agricultural factors, such as soil moisture, light intensity, temperature, fertilizers, variety and crop protection strategies. In addition, levels of nitrate can be influenced by storage time and storage conditions and food processing (e.g. washing, peeling, blanching, steaming, boiling). The EFSA s Panel on contaminants in the food chain (CONTAM) carried out a risk assessment on nitrate in vegetables in 2008 and a risk assessment on the possible health risks for infants and young children from the presence of nitrate in leafy vegetables in In the risk assessment of 2008, the CONTAM Panel described the available information regarding the influencee of storage and food processing on nitrate levels and concluded that there is a paucity of published data in this area. Therefore, the Panel recommended to perform research on the factors that influence nitrate and nitrite concentrations and alterations during production, storage and processing. The European Food Safety Authority (EFSA) wishes to outsource a study on the influence of various food processing techniques (PTs) on the concentrations of nitrate in vegetables. This study could possibly serve as supporting information to the CONTAM panel for refining its exposure assessment for nitrate in vegetables for adults and for nitrate in leafy vegetables for infants and young children. TERMS OF REFERENCE AS PROVIDED BY EFSA The project aims at obtaining data on the influence of processing on nitrate levels in vegetables. The study should be done with vegetables which are relevant based on their nitrate content and consumption frequency and amount (e.g. lettuce, spinach, Chinese cabbage, courgette, potato). This grant was awarded by EFSA to: Beneficiary: Grant title: University of Maribor Study on the influence of food processing on nitrate levels in vegetables. Grant number: GP/EFSA/CONTAM/2012/01 11 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

12 1. Introduction and objectives 1.1. Legislation on nitrate and acceptable daily intake Nitrate is widely distributed in nature. It can be found in soil, surface and underground water as well as in biomass. High level of nitrate can be accumulated in plants - vegetables. The actual content of consumed nitrate in vegetables depends on many different factors. There may be a significant difference in the level of nitrate between the same type of a vegetable which grew at two different areas or during a different period of time. The amount of nitrate consumed with vegetables also depends on dietary habits and preparation of the vegetable (Pennington, 1998; Thomson et al., 2007) and can reach up to 85% of the total intake (WHO, 1995). Nitrate per se is relatively non- via nitrite to toxic. It is discussed in toxicology due to its metabolic products, which are converted N-nitroso compounds (Gangolli et al., 1994). However, the first legal restrictions regarding nitrate levels were due to their levell in drinking water (in Europe in 1975 by Commission Directive (EC) No 75/440/EEC 2 ) in order to prevent methaemoglobinaemia from nitrate-contaminated water in infants (Knobelock et al., 2000; Fan and Steinbers, 1996). The content of nitrate in vegetable, however, became legally limited 16 years later. Several authors (McKnight et al., 1997; L Hirondel, 2001; Lundberg, 2008; Ying and Hofseth, 2007; Hord et al., 2009; Ralt, 2009) emphasized positive physiological effects of metabolic products of nitrate and consequently advocate a high concentration of nitrate in vegetable. The first international evaluation of the risks associated with the ingestion of nitrate and nitrite was conducted by the Joint FAO/WHO Expert Committee on Food Additives (JECFA) in 1961 (WHO, 1962). Maximum levels for nitrate in vegetables were first set in the EU in 1997 by Commission Regulation (EC) No 194/ Since then, the Regulation has been amended several times, the last time in 2011 by Commission Regulation (EC) No 1258/ (adapted from Hmelak Gorenjak and Cencič, 2013). The Regulation on nitrate applies for the following food commodities: fresh spinach, preserved deep-frozen or frozen spinach, fresh lettuce (glasshouse and open-grown lettuce), iceberg-type lettuce and processed cereal-based foods and baby foods for infants and young children. Maximum levels are expressed as mg nitrate kg -1 fresh weight (Table 1). Different limits for nitrate concentration in spinach and lettuce are defined depending on the season of cultivation. Higher levels of nitrate are permitted for produce grown in the winter months than in summer. 2 Commission Directive (EC) No 75/440/EEC of June 1975 concerning the quality of surface water intended for the abstraction of drinking water in the Member States. OJ L 194, , p Commission Regulation (EU) No 194/1997 of 31 January 1997 setting maximum levels for certain contaminants in foodstuffs. OJ L 31, , p Commission Regulation (EU) No 1258/2011 of 2 December 2011 amending Regulation (EC) No 1881/2006 as regards maximum levels for nitrates in foodstuffs. OJ L 320, , p The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

13 Table 1: Maximum levels for nitrate as laid down in Commission Regulation (EC) No 1258/2011 Fresh spinach (Spinacia oleracea) Preserved, deep frozen or frozen spinach Fresh Lettuce (Lactuca sativaa L.) (protected and open grown lettuce), excluding Iceberg type lettuce Maximum nitrate levels (mg/kg) Harvested 1 st October to 31 st March Lettuce grown under cover Lettuce grown in the open air Harvested 1 st April to 30 th September Lettuce grown under cover Lettuce grown in the open air Iceberg type lettuce Rucola (Eruca sativa, Diplotaxis sp., Brassica tenuifolia, Sisymbrium tenuifolium) Processed cereal based foods and baby foods for infants and young children Lettuce grown under cover Lettuce grown in the open air Harvested 1 st October to 31 st March 7000 Harvested 1 st April to 30 th September Acceptable daily intake The Scientific Committee for Food reviewed the toxicological effects of nitrate and nitrite and established an Acceptable Daily Intake (ADI) of mg/kg b.w. for nitrate (equivalent to 222 mg nitrate per day for a 60 kg adult) in 1990 (European Commission, 1992). It also retained the ADI in 1995 and derived an ADI of mg/kg b.w. for nitrite (European Commission, 1997). The JECFA completed its most recent review in 2002 and reconfirmed an ADI of mg/kg b.w. for nitrate and set an ADI of mg/kg b.w. for nitrite (FAO/WHO, 2003). Assessments of the intake of nitrate and nitrite should include sources other than food additives, such as vegetables and drinking water (FAO/WHO, 2003). Foods should be analysed as ready to consume to account for losses of chemicals over time and during food storage, preparation and cooking (Thomson et al., 2007). 13 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

14 1.3. Nitrate accumulation in vegetables Most vegetables can accumulate large amounts of nitrate. Nitrate concentrations in vegetables depend on the biological properties of the plant culture, (day)light intensity, type of soil, temperature, humidity, frequency of plants in the field, plant maturity, vegetation period, harvesting time, size of the vegetable unit, storage time and source of nitrogen (Tamme et al., 2006). Factors influencing the concentrationn of nitrate in plants are: a) biological, b) environmental/agricultural and c) storage and processing Biological factors Most of the nitrate accumulates in the mesophyll cells of the leaves; fruits and seeds have low nitrate levels because nitrate is exclusively transported by the xylem (Blan-Zandstracan be listed in the decreasing order of the nitrate content as follows: 1989; Pate, 1980). The vegetable organs petiole > leaf > stem > root > inflorescence > tuber > bulb > fruit > seed (Santamaria, 2001) Enviromental and agricultural factors Both environmental and agricultural factors can influence the nitrate concentrations in vegetables. The former include: soil moisture, light intensity and temperature, nitrogen fertilizers, variety and crop protection strategies (EFSA, 2008). Most of the studies suggest that the imbalance between nitrate uptake and reduction should be the fundamental factor (Du et al., 2007) ) Storage and processing Temperature might have a significant effect on the changes in the nitrate content in vegetables, particularly during ambient temperature storage. Nitrate levels in four vegetables dropped significantly at ambient temperature, while nitrite levels increased dramatically from the fourth day of storage. However, refrigerated storage caused nitrate and nitrite levels in the vegetables to be largely unaffected during the following 7 days (Chung et al., 2004) and there was no significant change in nitrate or nitrite content of spinach, beet, carrot, parsley-root, celery or potato during frozen storage for up to 12 weeks (Schuster and Lee, 1987). 14 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

15 Nitrate levels in potato can also be decreased by 18 to 40 % after preliminary processing methods (washing, peeling and rinsing) (Mozolewski and Smoczynski, 2004). Thermal processing of potato tubers with different heating methods (boiling, microwave, steaming, and deep-frying) caused a decrease in nitrate content by 16 62%. Generally, about 45% of the total nitrate in the vegetables can be reduced by pickling and 75% reduced by cooking (Du et al., 2007). Handling, storage, processing including washing, peeling and cooking, can significantly reduce the amount of nitrate in vegetables. This holds true for vegetables eaten cooked, such as potato, spinach and cabbage. For vegetables eaten raw only handling and storage would impact nitrate levels (Hmelak Gorenjak and Cencič, 2013). The nitrate content in vegetables is controlled on the national basis with all-year inspection in most of the European countries. However, information on the raw nitrate levels is not enough. Most of the vegetables are not consumed raw but rather further processed and nitrate can increase / decrease during these procedures. Moreover, digestion as well as absorption and bioavailability of nitrate in the intestine demand furtherr attention. Information on these changes is relatively scarce in the literature. The aim of our project was to obtain new data on nitrate content changes during culinary treatment of vegetables thatt are frequently consumed. It should be emphasized, that data on processed vegetables that are most frequently consumed by babies and small children are of special importance due to more severe complications that may arise in this particularly vulnerable population group. Originality of the project is also demonstrated by inclusion of vegetables that are used as a substitute for fresh vegetables (red beet, white cabbage) in the continental traditional cuisine Nitrate content in vegetables Nitrate-accumulating vegetables belong to the families of Brassicaceae (rocket, radish, and mustard), Chenopodiaceae (red beet, Swiss mangold, spinach) and Amarantaceae; in addition, Asteraceae (lettuce) and Apiaceae (celery, parsley) include species with high nitrate contents (Table 2) (Santamaria, 2006). Nitrate content can also vary within species, cultivars and even genotypes with different ploidy (Blon- Zandstra, 1989). A call for detailed information on nitrate concentrations in individual vegetable samples was issued by the European Commission to EU Member States in November In total, EFSA received analytical results from 20 Member States and Norway covering the period from 2000 to There was a large variation in the median concentration of nitrate in different vegetables from 1 mg/kg (peas and Brussels sprouts) to 4800 mg/kg (rucola) (EFSA, 2008). 15 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.

16 The lowest nitrate concentrations were observed in brassica vegetables (from 24 mg/kg in Brussels sprouts to 987 mg/kg in kohlrabi), bulb vegetables (from 69 mg/kg in garlic to 164 mg/kg in onions) and fruiting vegetables (from 43 mg/kg in tomato to 894 mg/kg in pumpkin). The nitrate concentrations in legumes were generally fairly low, with the exception of French beans (756 mg/kg) and string beans (618 mg/kg). The mean value for stem vegetables was higher than for fruiting vegetables rhubarbb showed particularly high values (the mean values were just above 3000 mg/kg) (EFSA, 2008). Although the median concentration of nitrate for the roots and tuber group was low with 152 mg/kg, the median values for the different product groups ranged from 12 to 1256 mg/kg. Potato and carrot are both major components in the diet of the population in different countries and their medians were just above 100 mg/kg. Red beet, on the other hand, was almost ten times higher with a median of 1100 mg/kg (EFSA, 2008). Leafy vegetables are one of the vegetables with the highest nitrate accumulation. The median for spinach (785 mg/kg) was well below the maximum level with 5% of the samples exceeding 3000 mg/kg. Rucola had the highest median concentration of nitrate (4800 mg/kg), followed by amaranth and Lamb s lettuce. Butterhead lettuce, a common lettuce vegetable, had a median nitrate concentration just below mg/kg (EFSA, 2008). Hmelak Gorenjak et al. (2012) observed in the same period a high variation between the nitrate content in each lettuce variety; conventionally cultivated lettuce with a mean nitrate content of 1298 mg/kg had a higher nitrate content than organically cultivated lettuce with a mean value of 890 mg/kg. Table 2: Classification of vegetables according to nitrate content (mg/kg fresh weight) (Santamaria, 2006) Very low, <200 Low, 200 to <500 Middle, 500 to <1000 High, 1000 to <2500 Very high, >2500 Vegetable varieties Artichoke, asparagus, broad bean, eggplant, garlic, onion, green bean, mushroom, pea, pepper, potato, summer squash, sweet potato, tomato, watermelon Broccoli, carrot, cauliflower, cucumber, pumpkin, chicory Cabbage, dill, turnip, savoy cabbage Celeriac, Chinese cabbage, endive, fennel, kohlrabi, leek, parsley Celery, cress, chervil, lettuce, red beet, spinach, rocket (rucola) 1.8. Objectives The objectives of this projects were to provide information on : - Nitrate content in blank as well as processed vegetables originatingg from conventional, integrated and organic farming, 16 The present document has been produced and adopted by the bodies identified above as author(s). In accordance with Article 36 of Regulation (EC) No 178/2002, this task has been carried out exclusively by the author(s) in the context of a grant agreement between the European Food Safety Authority and the author(s). The present document is published complying with the transparency principle to which the Authority is subject. It cannot be considered as an output adopted by the Authority. The European Food Safety Authority reserves its rights, view and position as regards the issues addressed and the conclusions reached in the present document, without prejudice to the rights of the authors.