Food Additives and Contaminants: Part B Vol. 3, No. 2, June 2010, 108–113
VIEW DATASET Nitrate in leafy vegetables, culinary herbs, and cucumber grown under cover in Estonia: content and intake T. Tammeab*, M. Reinikc, M. Roastoab, K. Merema¨eab and A. Kiisab a
Department of Food Hygiene of Institute of Veterinary Medicine and Animal Sciences, Kreutzwaldi 58A, 51014 Tartu, Estonia; Estonian University of Life Sciences, Estonia; cEstonian Health Protection Inspectorate, Tartu Laboratory, PK 272, 50002 Tartu, Estonia b
(Received 14 January 2010; final version received 23 February 2010) The content of nitrate in leafy vegetables, culinary herbs, and cucumber was determined during the years 2006–2008. All samples of Estonian origin, except white cabbage, were grown under cover. Seasonal differences in nitrate concentrations were observed in lettuce and spinach. Nitrate concentrations in lettuce were 22% and those in spinach were 24% higher in winter crops compared with samples collected in summer. The mean nitrate level was 3023 mg kg–1 for fresh lettuce and 2337 mg kg–1 for spinach. On average, 11.6% of fresh lettuce and spinach samples nitrate concentration exceeded the maximum level specified in European Commission Regulation No. 1881/2006. The mean levels were 999 mg kg–1 for imported iceberg lettuce and 1287 mg kg–1 for frozen spinach, which are below the maximum European Commission limits. Parsley, dill, basil, thyme, and rucola contained high concentrations of nitrate from mean levels of 2134 mg kg–1 for parsley up to 8150 mg kg–1 for rucola. Mean nitrate concentrations ranged from 382 to 1115 mg kg–1 for white cabbage and Chinese cabbage, respectively. The per capita mean daily intake of nitrates related to the consumption of leafy vegetables, culinary herbs, and cucumber for the whole Estonian population was 31.3 mg day–1, which comprised 14.2% of the acceptable daily intake (ADI). Keywords: high-performance liquid chromatography (HPLC); exposure assessment; nitrate; vegetables
Introduction Nitrate is a natural component of vegetables. Nitrate content is considered one of the most important factors characterizing vegetable quality. Both environmental and agrotechnical factors influence nitrate concentrations in plants. Typically, leafy vegetables contain higher nitrate concentrations compared with root and fruit vegetables. De Martin and Restani (2003) showed that leafy green vegetables accumulate nitrates, with concentrations reaching up to 6000 mg kg–1, depending on the biological properties of cultivars, the soil type, light intensity, air temperature, the duration of the growth period, harvesting time, and agricultural practice (Walker 1990; World Health Organization (WHO) 1995; Guadagnin et al. 2005; Merino et al. 2006; European Food Safety Authority (EFSA) 2008). Petersen and Stoltze (1999) reported that for leafy vegetables the nitrate content in the crops grown in the winter period was significantly higher compared with the summer crops. Similar results have been reported in a Brazilian study (Guadagnin et al. 2005) where the nitrate content in lettuce in the winter period was
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2010 Taylor & Francis DOI: 10.1080/19440041003725944 http://www.informaworld.com
one-third higher than in the summer period. Vegetables grown in heated glasshouses have higher nitrate contents than those grown in the open air during the same seasons, which is due to lower light intensity and high nitrogen mineralization (Gangolli et al. 1994). Similarly, hydroponically grown leafy vegetables have higher nitrate levels than conventionally grown vegetables (Guadagnin et al. 2005). Nitrate levels tend to be higher in samples obtained from Northern Europe compared with those grown in Mediterranean countries (Merino et al. 2006; EFSA 2008). Maximum limit concentrations of nitrates for certain vegetables are established by the Regulation of the European Commission (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs (European Commission 2006). Nitrate contents in leafy vegetables and culinary herbs grown under cover in Estonia were studied in different seasons during 2006–2008. The results for nitrate concentrations as well as for nitrate intake from the consumption of the investigated vegetables are reported in the present paper. Seasonal differences in
Food Additives and Contaminants: Part B nitrate content were observed for lettuce and spinach. In addition to leafy vegetables, the data on glasshousegrown cucumber as one the most widely consumed vegetable in Estonia were analysed.
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collaborative study of the method (Merino et al. 2000) and in performing inter-comparison tests.
Nitrate intake data for the whole population Materials and methods Vegetables A total of 131 samples of 16 different species of raw leafy vegetables, herbs and fresh cucumbers were analysed. From the analysed samples, 77% were of Estonian origin, whereas the species were common vegetables in an Estonian diet. All samples were collected at the retail level. Before analyses, the nonedible parts of the samples were removed. In the case of small vegetables, the whole sample was chopped. Large sample units, e.g. head cabbage, were cut vertically into quarters, but only one-quarter was used for analysis. During 2006–2008, the nitrate content of lettuce and spinach was determined in two different periods for each sampling year. The first sampling was performed in the summer period from April 1 to September 30, and the second sampling in the winter period from October 1 to March 31. Other vegetable samples were taken all year round. According to Commission Regulation (EC) No. 1881/2006, the maximum limit concentrations for spinach, depending on harvesting time, are 2500 mg kg–1 for the summer period and 3000 mg kg–1 for the winter period, and for frozen spinach was 2000 mg kg–1. Maximum limit concentrations for fresh lettuce are 3500 mg kg–1 for the summer period and 4500 mg kg–1 for the winter period. For iceberg-type lettuce grown under cover, the limit is 2500 mg kg–1. Concentration limits are not set for other vegetables, e.g. Chinese cabbage, celery, parsley, dill, etc.
Method of analysis Concentrations of nitrates were determined by a highperformance liquid chromatography (HPLC) method based on Nordic Committee on Food Analysis (NMKL) method No. 165 (2000). Nitrates were extracted from the samples by hot water. Acetonitrile was added for removal of interfering substances. The filtered solution was injected onto a Shimadzu LC10 chromatograph; nitrates were separated by an Alltech C18 column and detected by a ultraviolet (UV) light detector at a wavelength of 205 nm. The limit of quantification was 5 mg kg –1; measurement uncertainty (U) at a concentration of 100 mg kg–1 was 12 mg kg–1 (k ¼ 2, normal). The mean recovery for nitrate in leafy vegetables was 98%. The method was accredited by the Estonian Accreditation Body. The laboratory has successfully participated in
Food consumption data obtained from the household consumption survey, composed by the government agency Statistics Estonia, were used to calculate the average intake for the whole population. The data include the monthly average amount of food products (grouped in detail) consumed by the average person during 2004–2005.
Results and discussion A total of 58 samples of fresh lettuce, fresh and frozen spinach, and iceberg lettuce were analysed. Seasonal differences in nitrate content were observed for lettuce and spinach cultivated under cover. The results are presented in Table 1. In addition, maximum permitted limit concentrations and the number of samples exceeding the European Commission limits are given. As shown, lettuce and spinach grown in the winter period had higher nitrate concentrations compared with the crops grown in summer. Mean nitrate contents were 3325 and 2584 mg kg–1 for the winter period, and 2720 and 2090 mg kg–1 for the summer period, respectively. Relatively high nitrate levels in winter crops (22% higher in lettuce and 24% in spinach compared with summer crops) reflect a general trend for higher nitrate levels in vegetables grown during winter, or under glass/plastic cover with less daylight and under lower temperatures. The maximum limits for nitrates were exceeded in 11.4% of lettuce samples and in 12.5% of spinach samples, while the nitrate levels in imported iceberg lettuce (mean ¼ 999 mg kg–1) and frozen spinach (mean ¼ 1287 mg kg–1) did not exceed the limits. The nitrate levels of frozen spinach were lower than those of fresh spinach, which is probably due to the loss of nitrate during the melting process. Besides lettuce and spinach, 73 samples other cultivars of leafy vegetables, herbs, and cucumbers grown under cover in Estonia were analysed. Comparative data on nitrate content of samples of Estonian origin, and nitrate contents detected in similar imported products are given in Table 2. Nitrate levels in the above-mentioned vegetables and herbs are not subject to maximum limits. The data presented in Table 2 reveal great variability in nitrate levels. High nitrate concentrations were detected in rucola, dill, basil, thyme, and parsley with mean values in the range of 2134 mg kg–1 (parsley) to 8150 mg kg–1 (rucola). Several-times lower levels were observed in spring onion (533 mg kg–1) and celery (660 mg kg–1). Chinese cabbage grown in Estonia contained significantly less nitrates compared with
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Table 1. Seasonal variation in nitrate content of lettuce and spinach grown under cover in Estonia: number of samples exceeding the maximum permitted levels. Nitrate content (mg kg–1 fresh weight) Vegetable commodity
Harvesting time
Fresh lettuce, Estonia Fresh spinach, Estonia Frozen spinach, imported Iceberg-type lettuce, imported
Summera Winterb Summera Winterb
Number of samples/number exceeding the permitted levels
Range
Mean
Median
Maximum permitted levels (MRLs)
1410–4000 2156–4795 340–3650 2508–2660 580–1460
2720 3325 2090 2584 1287
2600 3045 1310 2584 1390
3500 4500 2500 3000 2000
18/2 17/2 6/1 2/0 5/0
520–1690
999
1015
2500
10/0
Notes: aHarvested between 1 April and 30 September. b Harvested between 1 October and 31 March. MRL, maximum residue limit. Table 2. Comparison of nitrate contents of leafy vegetables without maximum residue limits (MRLs), fresh cucumber grown under cover in Estonia, and imported products. Nitrate content (mg kg–1 fresh weight) Vegetable Chinese cabbage, Estonia Chinese cabbage, imported Rucola, Estonia Celery, imported Parsley, Estonia Spring onion, Estonia Dill, Estonia Fresh basil, Estonia Thyme, Estonia Fresh cucumber, Estonia Fresh cucumber, imported White cabbagea, Estonia
Number of samples
Range
Mean
Median
6 4 2 4 7 4 6 2 1 12 7 18
212–1844 340–2236 7300–9000 256–1113 887–3590 99–870 2670–5290 4040–5350 4290 89–740 124–372 85–920
885 1344 8150 660 2134 533 3911 4695 4290 335 240 382
404 1228 8150 542 1880 580 3150 4695 4290 320 252 318
Note: aWhite cabbage was grown in the open air.
imported vegetables with mean levels of 885 and 1344 mg kg–1, respectively (Table 2). At the retail level, mostly cucumbers grown under cover, both domestic and imported, are available in Estonia (Table 2). Nitrate levels tend to be higher in domestic cucumbers compared with imported products, with mean values of 335 and 240 mg kg–1, respectively. White cabbage is the major leafy vegetable consumed in Estonia. It is usually grown in fields without cover, whereas the average nitrate concentration was 382 mg kg–1 for domestic cabbage samples. Studies about the influence of seasonal variation of nitrate contents in leafy vegetables have been carried out in several other countries (Table 3), for instance, in Belgium (Dejonckheere et al. 1994) and Finland (Penttila¨ 1995). The results of both of these studies clearly showed that nitrate contents in leafy vegetables were higher in winter. The concentration of nitrates in
lettuce was 44% higher during winter compared with the summer period in Belgium and twice higher in the winter compared with the autumn in Finland (Table 3). Some studies (Ysart et al. 1999; Chung et al. 2003) demonstrated an opposite, trend as shown in Table 3. The concentration of nitrate in lettuce was 1.8 times higher in the summer period in the UK compared with the winter period. Santamaria et al. (1999) reported that the nitrate content in spinach was higher in autumn/winter compared with spring, while other vegetable species had no significant differences in nitrate content through the seasons. The disparity in data may be due to variable environmental conditions (i.e. the length of exposure to sunlight, cultivation methods, etc.). Light intensity is widely recognized as an important determinant of the nitrate level in vegetables (Bottex et al. 2008). Nitrate concentrations in leafy vegetables, herbs, and cucumber grown under
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Food Additives and Contaminants: Part B
Table 3. Comparison of mean seasonal nitrate content of selected leafy vegetables and cucumber cultivated in different seasons and countries. Nitrate concentration (mg kg–1 fresh weight) Belgium Vegetable Lettuce Spinach Parsley Celery leaves Cabbage Cauliflower Chinese cabbage Cucumber
Finland
United Kingdom
Korea
Iran
Summer
Winter
Summer
Autumn
Summer
Winter
Summer
Winter
Spring
Winter
2219 2108 2488 2728
3205 2548 2813 3503
1050
2443
1568 1639
887 1272
2728 4814
1933 3334
812
412
488 87
94 57
722
730
2601 748 374 887 504 –
2288 887 359 2169 428 559
964
1081
2009 180
1291 267
813
999
363
322
Note: For Belgium, see Dejonckheere et al. (1994); Finland, Penttila¨ (1995); UK, Ysart et al. (1999); Korea, Chung et al. (2003); and Iran, Shahlaei et al. (2007).
cover in Estonia are comparatively high due to the two major factors: light and temperature conditions. Almost all Estonian leafy vegetables are grown under glass, which means limited daylight and a relatively low temperature. The mean nitrate levels of leafy vegetables and cucumber varied widely depending on the countries of growth, as shown in Table 4, indicating that white and Chinese cabbage and celery grown in China contain several times higher concentrations of nitrates compared with the products imported from other countries. It is quite evident that the figures obtained from the present study, regarding mean nitrate levels of leafy vegetables cultivated in Estonia, are in line with those obtained from similar Danish and Finnish studies (Penttila¨ 1995; Petersen and Stoltze 1999).
Nitrate intake The mean daily nitrate consumption of leafy vegetable and cucumber is demonstrated in Table 4, accompanied by similar data from other countries. The overall mean nitrate contents in raw vegetables were calculated using the mean data obtained from the analysis of samples of vegetables grown in Estonia as well as imported products (frozen spinach and iceberg lettuce). According to the statistics, the per-capita consumption of different leafy vegetables and herbs is low in Estonia – only 5.9 g day–1, with the exception of white head cabbage (38.5 g day–1). The overall consumption of leafy vegetables and herbs is 44.4 g day–1. In the present study, the dietary exposure to nitrate from raw leafy vegetables was estimated to be 24.3 mg day–1 for the average consumer. The results revealed that from all leafy vegetables white cabbage was the main source of nitrate intake in Estonia. Based on the results of the present study, leafy vegetables and herbs comprise 11% of the acceptable daily intake
(ADI) for nitrates. China and the UK have the highest levels of consumption of leafy vegetables totalling 127 and 87.5 g day–1, respectively (Table 4). In Sweden, the mean estimated nitrate intake from leafy vegetables was 18–21 mg day–1 (Merino et al. 2006). Daily per-capita consumption of fresh cucumber is 23 g in Estonia (Table 4), which is the highest in Europe (EFSA 2008). According to Zhong et al. (2002), the Chinese consume even higher amounts of cucumbers (53 g day–1). Since nitrate levels in cucumber are relatively low, the daily intake is 3.2% of the ADI and the consumption of 100 g of cucumber per day comprises 13.8% of the ADI in Estonia. The overall nitrate intake for Estonian consumer from vegetables is 58 mg day–1 (Tamme et al. 2006), whereas it mostly originates from root and fruit vegetables, as calculated based on the consumption survey of Statistics Estonia. Low nitrate levels in vegetables are probably associated with the relatively low use of fertilizers in Estonia. Chinese consumer’s nitrate intake is 312.2 mg day–1 per person as an average, which exceeds the recommended maximum daily dose: ADI ¼ 222 mg day–1 per 60 kg body weight, which is 140% (Zhong et al. 2002). China is followed by Belgium with 63.4 mg day–1 per person (Dejonckheere et al. 1994), and the UK with 45 mg day–1 per person (Ysart et al. 1999).
Conclusions The present investigation examined nitrate levels in leafy vegetables, culinary herbs, and cucumber grown under cover. Seasonal differences in nitrate concentrations were observed for lettuce and spinach. Nitrate concentrations were higher in winter compared with the summer period – by 22% in lettuce and by 24% in spinach. Nitrate concentrations exceeded the permitted limits in 11.6% of the investigated lettuce
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T. Tamme et al. Table 4. Comparison of mean nitrate content and intake from selected leafy vegetables and cucumber grown under cover in Estonia with those grown in other countries.
Vegetable Lettuce
Spinach
Cauliflower and broccoli Chinese cabbage Celery
White cabbagea
Cucumber
Country
Consumption of vegetables (g person day–1)
Mean nitrate (NO3) concentration (mg kg–1 fresh weight)
NO3 intake (mg person per day–1)
Estoniab Finlandc Italyd Belgiume Denmarkf UKg Estoniab Finlandc Italyd Belgiume Denmarkf UKg Estoniab Italyd UKg Estoniab Denmarkf Chinah Estoniab Italyd Belgiume Chinah Estoniab Finlandc Italyd Denmarkf UKg Chinah Estoniab Finlandc Italyd Belgiume Chinah
2.0 3.4 17.1 19 3.0 9.9 0.5 1.3 3.1 4 1.3 15.5 1.0 8.8 43.2 1.4 0.9 54 1.0 2.3 17 28 38.5 5.5 1.7 4 18.9 45 23 5.6 2.7 5 53
2348 1835 832 1910 2631 1051 1987 1747 1845 1594 1231 1631 314 420 73 1115 1058 2120 2056 1678 1223 4600 382 607 400 333 338 1530 303 240 79 344 170
4.7 6.2 14.2 36.3 8.9 10.4 1.0 2.3 5.7 6.4 1.7 25 0.3 3.7 3.1 1.5 1.0 114.5 2.1 3.9 20.7 128.8 14.7 3.3 0.68 1.5 6.4 68.9 7.0 1.3 0.2 1.7 9.1
Notes: aWhite cabbage was grown in the open air. b Present study. c Penttila¨ (1995). d Santamaria et al. (1999). e Dejonckheere et al.’s (1994) calculated intake was based on NO3 content after pretreatment, e.g. washing, peeling, etc. These treatments result in the reduction of the nitrate content. f Petersen and Stolze (1999). g Ysart et al. (1999). h Zhong et al. (2002).
and spinach samples. Besides lettuce and spinach, there are many other leafy vegetables and culinary herbs for which the maximum nitrate limits have not been established. In these plants nitrate contents differed to a large extent, from 533 mg kg–1 in spring onion to 4695 mg kg–1 in basil. The highest mean nitrate levels (8150 mg kg–1) were determined in rucola. The average intake of nitrates from raw leafy vegetables and culinary herbs by an Estonian consumer was 24.3 mg day–1 and for fresh cucumber was 7 mg day–1, calculated on the basis of 60 kg body weight, which made up 11% and 3.2% of the acceptable daily intake (ADI), respectively.
References Bottex B, Dorne JLCM, Carlander D, Benford D, Przyrembel H, Heppner C, Kleiner J, Cockburn A. 2008. Risk–benefit health assessment of food – food fortification and nitrate in vegetables. Trends Food Sci Tech. 19:113–119. Chung SY, Kim JS, Kim M, Hong MK, Lee JO, Kim CM, Song IS. 2003. Survey of nitrate and nitrite contents of vegetables grown in Korea. Food Addit Contam. 20(7):621–628. De Martin S, Restani P. 2003. Determination of nitrates by a novel ion chromatographic method: occurrence in leafy vegetables (organic and conventional) and exposure
Food Additives and Contaminants: Part B assessment for Italian consumers. Food Addit Contam. 20(9):787–792. Dejonckheere W, Streubaut W, Drieghe S, Verstraeten R, Braeckman H. 1994. Nitrate in food commodities of vegetable origin and the total diet in Belgium, 1992–1993. Microbiologie-Aliments-Nutrition. 12:359–370. European Commission. 2006. Commission Regulation (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs. Available from: http://eur-lex.europa.eu/LexUriServ/site/en/oj/2006/ l_364/l_36420061220en00050024.pdf/ European Food Safety Authority (EFSA). 2008. Nitrate in vegetables Scientific Opinion of the Panel on Contaminants in the Food Chain. EFSA J. 689:1–79. Gangolli SD, van den Brandt PA, Feron VJ, Janzowsky C, Koeman JH, Speijers GJ, Spiegelhalder B, Walker R, Wisnok JS. 1994. Nitrate, nitrite and N-nitroso compounds. Eur J Pharmacol. 292:1–38. Guadagnin SG, Rath S, Reyes FGR. 2005. Evaluation of the nitrate content in leaf vegetables produced through different agricultural systems. Food Addit Contam. 22(12):1203–1208. Merino L, Darnerud PO, Edberg U, Aman P, Castillo MDP. 2006. Levels of nitrate in Swedish lettuce and spinach over the past 10 years. Food Addit Contam. 23(12):1283–1289. Merino L, Edberg U, Fuchs G, A˚man P. 2000. Liquid chromatographic determination of residual nitrite/nitrate in foods: NMKL Collaborative Study. J AOAC Int. 83:365–375. Nordic Committee on Food Analysis (NMKL). 2000. Nitrite and nitrate. Nitrite and/or nitrate in foodstuffs by ion chromatography. Oslo (Norway): National Veterinary Institute, No. 165. Penttila¨ P-L. (1995). Estimation of food additive and pesticide intakes by means of stepwise method. Doctoral thesis, University of Turku.
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Petersen A, Stoltze S. 1999. Nitrate and nitrite in vegetables on the Danish market: content and intake. Food Addit Contam. 16(7):291–299. Santamaria P. 2006. Nitrate in vegetables: toxicity, content, intake and EC regulation. J Sci Food Agric. 86:10–17. Santamaria P, Elia A, Serio F, Todaro E. 1999. A survey of nitrate and oxalate content in fresh vegetables. J Sci Food Agric. 79:1882–1888. Shahlaei A, Ansari NA, Dehkordie FS. 2007. Evaluation of nitrate and nitrite content of Iran southern (Ahwaz) vegetables during winter and spring of 2006. Asian J Plant Sci. 6(8):1197–1203. Tamme T, Reinik M, Roasto M, Juhkam K, Tenno T, Kiis A. 2006. Nitrates and nitrites in vegetables and vegetable-based products and their intakes by the Estonian population. Food Addit Contam. 23(4): 355–361. Umar AS, Iqbal M, Abrol YP. 2007. Are nitrate concentrations in leafy vegetables within safe limits? Curr Sci. 92(3):355–360. Walker R. 1990. Nitrates, nitrites and N-nitroso compounds: a review of the occurrence in food and diet and diet and the toxicological implications. Food Addit Contam. 7(6):717–786. World Health Organization (WHO). 1995. Evaluation of certain food additives and contaminants. Joint FAO/ WHO Expert Committee on Food Additives (JECFA). WHO Technical Report. 859:29–35. Ysart G, Miller P, Barrett G, Farrington D, Lawrance P, Harrison N. 1999. Dietary exposures to nitrate in UK. Food Addit Contam. 16(12):521–532. Zhong W, Hu C, Wang M. 2002. Nitrate and nitrite in vegetables from north China: content and intake. Food Addit Contam. 19(12):1125–1129.
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VIEW DATASET Nitrate in leafy vegetables, culinary herbs, and cucumber grown under cover in Estonia: content and intake T. Tammeab*, M. Reinikc, M. Roastoab, K. Merema¨eab and A. Kiisab a
Department of Food Hygiene of Institute of Veterinary Medicine and Animal Sciences, Kreutzwaldi 58A, 51014 Tartu, Estonia; Estonian University of Life Sciences, Estonia; cEstonian Health Protection Inspectorate, Tartu Laboratory, PK 272, 50002 Tartu, Estonia b
(Received 14 January 2010; final version received 23 February 2010) The content of nitrate in leafy vegetables, culinary herbs, and cucumber was determined during the years 2006–2008. All samples of Estonian origin, except white cabbage, were grown under cover. Seasonal differences in nitrate concentrations were observed in lettuce and spinach. Nitrate concentrations in lettuce were 22% and those in spinach were 24% higher in winter crops compared with samples collected in summer. The mean nitrate level was 3023 mg kg–1 for fresh lettuce and 2337 mg kg–1 for spinach. On average, 11.6% of fresh lettuce and spinach samples nitrate concentration exceeded the maximum level specified in European Commission Regulation No. 1881/2006. The mean levels were 999 mg kg–1 for imported iceberg lettuce and 1287 mg kg–1 for frozen spinach, which are below the maximum European Commission limits. Parsley, dill, basil, thyme, and rucola contained high concentrations of nitrate from mean levels of 2134 mg kg–1 for parsley up to 8150 mg kg–1 for rucola. Mean nitrate concentrations ranged from 382 to 1115 mg kg–1 for white cabbage and Chinese cabbage, respectively. The per capita mean daily intake of nitrates related to the consumption of leafy vegetables, culinary herbs, and cucumber for the whole Estonian population was 31.3 mg day–1, which comprised 14.2% of the acceptable daily intake (ADI). Keywords: high-performance liquid chromatography (HPLC); exposure assessment; nitrate; vegetables
Introduction Nitrate is a natural component of vegetables. Nitrate content is considered one of the most important factors characterizing vegetable quality. Both environmental and agrotechnical factors influence nitrate concentrations in plants. Typically, leafy vegetables contain higher nitrate concentrations compared with root and fruit vegetables. De Martin and Restani (2003) showed that leafy green vegetables accumulate nitrates, with concentrations reaching up to 6000 mg kg–1, depending on the biological properties of cultivars, the soil type, light intensity, air temperature, the duration of the growth period, harvesting time, and agricultural practice (Walker 1990; World Health Organization (WHO) 1995; Guadagnin et al. 2005; Merino et al. 2006; European Food Safety Authority (EFSA) 2008). Petersen and Stoltze (1999) reported that for leafy vegetables the nitrate content in the crops grown in the winter period was significantly higher compared with the summer crops. Similar results have been reported in a Brazilian study (Guadagnin et al. 2005) where the nitrate content in lettuce in the winter period was
*Corresponding author. Email: [email protected] ISSN 1939–3210 print/ISSN 1939–3229 online ß 2010 Taylor & Francis DOI: 10.1080/19440041003725944 http://www.informaworld.com
one-third higher than in the summer period. Vegetables grown in heated glasshouses have higher nitrate contents than those grown in the open air during the same seasons, which is due to lower light intensity and high nitrogen mineralization (Gangolli et al. 1994). Similarly, hydroponically grown leafy vegetables have higher nitrate levels than conventionally grown vegetables (Guadagnin et al. 2005). Nitrate levels tend to be higher in samples obtained from Northern Europe compared with those grown in Mediterranean countries (Merino et al. 2006; EFSA 2008). Maximum limit concentrations of nitrates for certain vegetables are established by the Regulation of the European Commission (EC) No. 1881/2006 of 19 December 2006 setting maximum levels for certain contaminants in foodstuffs (European Commission 2006). Nitrate contents in leafy vegetables and culinary herbs grown under cover in Estonia were studied in different seasons during 2006–2008. The results for nitrate concentrations as well as for nitrate intake from the consumption of the investigated vegetables are reported in the present paper. Seasonal differences in
Food Additives and Contaminants: Part B nitrate content were observed for lettuce and spinach. In addition to leafy vegetables, the data on glasshousegrown cucumber as one the most widely consumed vegetable in Estonia were analysed.
109
collaborative study of the method (Merino et al. 2000) and in performing inter-comparison tests.
Nitrate intake data for the whole population Materials and methods Vegetables A total of 131 samples of 16 different species of raw leafy vegetables, herbs and fresh cucumbers were analysed. From the analysed samples, 77% were of Estonian origin, whereas the species were common vegetables in an Estonian diet. All samples were collected at the retail level. Before analyses, the nonedible parts of the samples were removed. In the case of small vegetables, the whole sample was chopped. Large sample units, e.g. head cabbage, were cut vertically into quarters, but only one-quarter was used for analysis. During 2006–2008, the nitrate content of lettuce and spinach was determined in two different periods for each sampling year. The first sampling was performed in the summer period from April 1 to September 30, and the second sampling in the winter period from October 1 to March 31. Other vegetable samples were taken all year round. According to Commission Regulation (EC) No. 1881/2006, the maximum limit concentrations for spinach, depending on harvesting time, are 2500 mg kg–1 for the summer period and 3000 mg kg–1 for the winter period, and for frozen spinach was 2000 mg kg–1. Maximum limit concentrations for fresh lettuce are 3500 mg kg–1 for the summer period and 4500 mg kg–1 for the winter period. For iceberg-type lettuce grown under cover, the limit is 2500 mg kg–1. Concentration limits are not set for other vegetables, e.g. Chinese cabbage, celery, parsley, dill, etc.
Method of analysis Concentrations of nitrates were determined by a highperformance liquid chromatography (HPLC) method based on Nordic Committee on Food Analysis (NMKL) method No. 165 (2000). Nitrates were extracted from the samples by hot water. Acetonitrile was added for removal of interfering substances. The filtered solution was injected onto a Shimadzu LC10 chromatograph; nitrates were separated by an Alltech C18 column and detected by a ultraviolet (UV) light detector at a wavelength of 205 nm. The limit of quantification was 5 mg kg –1; measurement uncertainty (U) at a concentration of 100 mg kg–1 was 12 mg kg–1 (k ¼ 2, normal). The mean recovery for nitrate in leafy vegetables was 98%. The method was accredited by the Estonian Accreditation Body. The laboratory has successfully participated in
Food consumption data obtained from the household consumption survey, composed by the government agency Statistics Estonia, were used to calculate the average intake for the whole population. The data include the monthly average amount of food products (grouped in detail) consumed by the average person during 2004–2005.
Results and discussion A total of 58 samples of fresh lettuce, fresh and frozen spinach, and iceberg lettuce were analysed. Seasonal differences in nitrate content were observed for lettuce and spinach cultivated under cover. The results are presented in Table 1. In addition, maximum permitted limit concentrations and the number of samples exceeding the European Commission limits are given. As shown, lettuce and spinach grown in the winter period had higher nitrate concentrations compared with the crops grown in summer. Mean nitrate contents were 3325 and 2584 mg kg–1 for the winter period, and 2720 and 2090 mg kg–1 for the summer period, respectively. Relatively high nitrate levels in winter crops (22% higher in lettuce and 24% in spinach compared with summer crops) reflect a general trend for higher nitrate levels in vegetables grown during winter, or under glass/plastic cover with less daylight and under lower temperatures. The maximum limits for nitrates were exceeded in 11.4% of lettuce samples and in 12.5% of spinach samples, while the nitrate levels in imported iceberg lettuce (mean ¼ 999 mg kg–1) and frozen spinach (mean ¼ 1287 mg kg–1) did not exceed the limits. The nitrate levels of frozen spinach were lower than those of fresh spinach, which is probably due to the loss of nitrate during the melting process. Besides lettuce and spinach, 73 samples other cultivars of leafy vegetables, herbs, and cucumbers grown under cover in Estonia were analysed. Comparative data on nitrate content of samples of Estonian origin, and nitrate contents detected in similar imported products are given in Table 2. Nitrate levels in the above-mentioned vegetables and herbs are not subject to maximum limits. The data presented in Table 2 reveal great variability in nitrate levels. High nitrate concentrations were detected in rucola, dill, basil, thyme, and parsley with mean values in the range of 2134 mg kg–1 (parsley) to 8150 mg kg–1 (rucola). Several-times lower levels were observed in spring onion (533 mg kg–1) and celery (660 mg kg–1). Chinese cabbage grown in Estonia contained significantly less nitrates compared with
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Table 1. Seasonal variation in nitrate content of lettuce and spinach grown under cover in Estonia: number of samples exceeding the maximum permitted levels. Nitrate content (mg kg–1 fresh weight) Vegetable commodity
Harvesting time
Fresh lettuce, Estonia Fresh spinach, Estonia Frozen spinach, imported Iceberg-type lettuce, imported
Summera Winterb Summera Winterb
Number of samples/number exceeding the permitted levels
Range
Mean
Median
Maximum permitted levels (MRLs)
1410–4000 2156–4795 340–3650 2508–2660 580–1460
2720 3325 2090 2584 1287
2600 3045 1310 2584 1390
3500 4500 2500 3000 2000
18/2 17/2 6/1 2/0 5/0
520–1690
999
1015
2500
10/0
Notes: aHarvested between 1 April and 30 September. b Harvested between 1 October and 31 March. MRL, maximum residue limit. Table 2. Comparison of nitrate contents of leafy vegetables without maximum residue limits (MRLs), fresh cucumber grown under cover in Estonia, and imported products. Nitrate content (mg kg–1 fresh weight) Vegetable Chinese cabbage, Estonia Chinese cabbage, imported Rucola, Estonia Celery, imported Parsley, Estonia Spring onion, Estonia Dill, Estonia Fresh basil, Estonia Thyme, Estonia Fresh cucumber, Estonia Fresh cucumber, imported White cabbagea, Estonia
Number of samples
Range
Mean
Median
6 4 2 4 7 4 6 2 1 12 7 18
212–1844 340–2236 7300–9000 256–1113 887–3590 99–870 2670–5290 4040–5350 4290 89–740 124–372 85–920
885 1344 8150 660 2134 533 3911 4695 4290 335 240 382
404 1228 8150 542 1880 580 3150 4695 4290 320 252 318
Note: aWhite cabbage was grown in the open air.
imported vegetables with mean levels of 885 and 1344 mg kg–1, respectively (Table 2). At the retail level, mostly cucumbers grown under cover, both domestic and imported, are available in Estonia (Table 2). Nitrate levels tend to be higher in domestic cucumbers compared with imported products, with mean values of 335 and 240 mg kg–1, respectively. White cabbage is the major leafy vegetable consumed in Estonia. It is usually grown in fields without cover, whereas the average nitrate concentration was 382 mg kg–1 for domestic cabbage samples. Studies about the influence of seasonal variation of nitrate contents in leafy vegetables have been carried out in several other countries (Table 3), for instance, in Belgium (Dejonckheere et al. 1994) and Finland (Penttila¨ 1995). The results of both of these studies clearly showed that nitrate contents in leafy vegetables were higher in winter. The concentration of nitrates in
lettuce was 44% higher during winter compared with the summer period in Belgium and twice higher in the winter compared with the autumn in Finland (Table 3). Some studies (Ysart et al. 1999; Chung et al. 2003) demonstrated an opposite, trend as shown in Table 3. The concentration of nitrate in lettuce was 1.8 times higher in the summer period in the UK compared with the winter period. Santamaria et al. (1999) reported that the nitrate content in spinach was higher in autumn/winter compared with spring, while other vegetable species had no significant differences in nitrate content through the seasons. The disparity in data may be due to variable environmental conditions (i.e. the length of exposure to sunlight, cultivation methods, etc.). Light intensity is widely recognized as an important determinant of the nitrate level in vegetables (Bottex et al. 2008). Nitrate concentrations in leafy vegetables, herbs, and cucumber grown under
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Food Additives and Contaminants: Part B
Table 3. Comparison of mean seasonal nitrate content of selected leafy vegetables and cucumber cultivated in different seasons and countries. Nitrate concentration (mg kg–1 fresh weight) Belgium Vegetable Lettuce Spinach Parsley Celery leaves Cabbage Cauliflower Chinese cabbage Cucumber
Finland
United Kingdom
Korea
Iran
Summer
Winter
Summer
Autumn
Summer
Winter
Summer
Winter
Spring
Winter
2219 2108 2488 2728
3205 2548 2813 3503
1050
2443
1568 1639
887 1272
2728 4814
1933 3334
812
412
488 87
94 57
722
730
2601 748 374 887 504 –
2288 887 359 2169 428 559
964
1081
2009 180
1291 267
813
999
363
322
Note: For Belgium, see Dejonckheere et al. (1994); Finland, Penttila¨ (1995); UK, Ysart et al. (1999); Korea, Chung et al. (2003); and Iran, Shahlaei et al. (2007).
cover in Estonia are comparatively high due to the two major factors: light and temperature conditions. Almost all Estonian leafy vegetables are grown under glass, which means limited daylight and a relatively low temperature. The mean nitrate levels of leafy vegetables and cucumber varied widely depending on the countries of growth, as shown in Table 4, indicating that white and Chinese cabbage and celery grown in China contain several times higher concentrations of nitrates compared with the products imported from other countries. It is quite evident that the figures obtained from the present study, regarding mean nitrate levels of leafy vegetables cultivated in Estonia, are in line with those obtained from similar Danish and Finnish studies (Penttila¨ 1995; Petersen and Stoltze 1999).
Nitrate intake The mean daily nitrate consumption of leafy vegetable and cucumber is demonstrated in Table 4, accompanied by similar data from other countries. The overall mean nitrate contents in raw vegetables were calculated using the mean data obtained from the analysis of samples of vegetables grown in Estonia as well as imported products (frozen spinach and iceberg lettuce). According to the statistics, the per-capita consumption of different leafy vegetables and herbs is low in Estonia – only 5.9 g day–1, with the exception of white head cabbage (38.5 g day–1). The overall consumption of leafy vegetables and herbs is 44.4 g day–1. In the present study, the dietary exposure to nitrate from raw leafy vegetables was estimated to be 24.3 mg day–1 for the average consumer. The results revealed that from all leafy vegetables white cabbage was the main source of nitrate intake in Estonia. Based on the results of the present study, leafy vegetables and herbs comprise 11% of the acceptable daily intake
(ADI) for nitrates. China and the UK have the highest levels of consumption of leafy vegetables totalling 127 and 87.5 g day–1, respectively (Table 4). In Sweden, the mean estimated nitrate intake from leafy vegetables was 18–21 mg day–1 (Merino et al. 2006). Daily per-capita consumption of fresh cucumber is 23 g in Estonia (Table 4), which is the highest in Europe (EFSA 2008). According to Zhong et al. (2002), the Chinese consume even higher amounts of cucumbers (53 g day–1). Since nitrate levels in cucumber are relatively low, the daily intake is 3.2% of the ADI and the consumption of 100 g of cucumber per day comprises 13.8% of the ADI in Estonia. The overall nitrate intake for Estonian consumer from vegetables is 58 mg day–1 (Tamme et al. 2006), whereas it mostly originates from root and fruit vegetables, as calculated based on the consumption survey of Statistics Estonia. Low nitrate levels in vegetables are probably associated with the relatively low use of fertilizers in Estonia. Chinese consumer’s nitrate intake is 312.2 mg day–1 per person as an average, which exceeds the recommended maximum daily dose: ADI ¼ 222 mg day–1 per 60 kg body weight, which is 140% (Zhong et al. 2002). China is followed by Belgium with 63.4 mg day–1 per person (Dejonckheere et al. 1994), and the UK with 45 mg day–1 per person (Ysart et al. 1999).
Conclusions The present investigation examined nitrate levels in leafy vegetables, culinary herbs, and cucumber grown under cover. Seasonal differences in nitrate concentrations were observed for lettuce and spinach. Nitrate concentrations were higher in winter compared with the summer period – by 22% in lettuce and by 24% in spinach. Nitrate concentrations exceeded the permitted limits in 11.6% of the investigated lettuce
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T. Tamme et al. Table 4. Comparison of mean nitrate content and intake from selected leafy vegetables and cucumber grown under cover in Estonia with those grown in other countries.
Vegetable Lettuce
Spinach
Cauliflower and broccoli Chinese cabbage Celery
White cabbagea
Cucumber
Country
Consumption of vegetables (g person day–1)
Mean nitrate (NO3) concentration (mg kg–1 fresh weight)
NO3 intake (mg person per day–1)
Estoniab Finlandc Italyd Belgiume Denmarkf UKg Estoniab Finlandc Italyd Belgiume Denmarkf UKg Estoniab Italyd UKg Estoniab Denmarkf Chinah Estoniab Italyd Belgiume Chinah Estoniab Finlandc Italyd Denmarkf UKg Chinah Estoniab Finlandc Italyd Belgiume Chinah
2.0 3.4 17.1 19 3.0 9.9 0.5 1.3 3.1 4 1.3 15.5 1.0 8.8 43.2 1.4 0.9 54 1.0 2.3 17 28 38.5 5.5 1.7 4 18.9 45 23 5.6 2.7 5 53
2348 1835 832 1910 2631 1051 1987 1747 1845 1594 1231 1631 314 420 73 1115 1058 2120 2056 1678 1223 4600 382 607 400 333 338 1530 303 240 79 344 170
4.7 6.2 14.2 36.3 8.9 10.4 1.0 2.3 5.7 6.4 1.7 25 0.3 3.7 3.1 1.5 1.0 114.5 2.1 3.9 20.7 128.8 14.7 3.3 0.68 1.5 6.4 68.9 7.0 1.3 0.2 1.7 9.1
Notes: aWhite cabbage was grown in the open air. b Present study. c Penttila¨ (1995). d Santamaria et al. (1999). e Dejonckheere et al.’s (1994) calculated intake was based on NO3 content after pretreatment, e.g. washing, peeling, etc. These treatments result in the reduction of the nitrate content. f Petersen and Stolze (1999). g Ysart et al. (1999). h Zhong et al. (2002).
and spinach samples. Besides lettuce and spinach, there are many other leafy vegetables and culinary herbs for which the maximum nitrate limits have not been established. In these plants nitrate contents differed to a large extent, from 533 mg kg–1 in spring onion to 4695 mg kg–1 in basil. The highest mean nitrate levels (8150 mg kg–1) were determined in rucola. The average intake of nitrates from raw leafy vegetables and culinary herbs by an Estonian consumer was 24.3 mg day–1 and for fresh cucumber was 7 mg day–1, calculated on the basis of 60 kg body weight, which made up 11% and 3.2% of the acceptable daily intake (ADI), respectively.
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