The nitrate content of fresh and cooked vegetables and their health-related risks

Background Vegetables are the most important source of nitrates in the human diet. During various processes in the body, nitrates are converted into nitrites, which causes various diseases, such as blue baby syndrome and cancer. This study aimed to determine the concentration of nitrates in several vegetable farms in Sanandaj city and to evaluate their health-related risks. Methods This descriptive cross-sectional study was conducted from October 2017 to July 2018. A total of 90 samples were taken from nine farms. Soil and water sampling was also carried out. All stages of sample preparation and extraction were carried out according to Food Standards 2–16721, and the nitrate measurements were performed using ion chromatography (Compact IC Plus 882 Model, Metrohm, Switzerland). A health risk assessment was performed using the non-carcinogenic risk assessment. Results This study’s results showed that the nitrate concertation in all vegetables was less than National Iranian Vegetable Nitrate Standard. Nitrate levels in leafy vegetables were higher than in root vegetables, and the root vegetables levels were higher than those in Fruit vegetable. The nitrate level in vegetables in autumn was higher than in spring. The cooking process reduced the raw vegetables’ nitrate content from 4.094% to 13.407%, while the frying process increased the vegetables’ nitrate content from 12.46% to 29.93%. The highest health risk level in raw, cooked and fried vegetables was parsley, parsley and beet leaves, respectively, and the lowest in all categories was tomatoes. Generally, the highest health risk was related to fried beet leaves, and the lowest was raw tomatoes. In addition, each of the abovementioned relationships between vegetables’ nitrate levels and the harvest season, type of processing procedure and type of vegetables was significant (p < 0.05). The irrigation water’s nitrate concentration in all fields was between 12.36 and 33.14 mg/l. The soil contained nitrate levels of between 4.35 and 9.7 mg/kg. Conclusion Based on this study, we can conclude that the amount of nitrates in raw vegetables was lower than the standard limit’s level and that this level does not cause health problems for consumers.

study's goal is to measure the amount of nitrates in vegetables that are cultivated in Sanandaj city, the effects of cooking and frying processes on nitrate concentration and, finally, the assessment of the health risks of nitrates in vegetables.

Materials and methods
This cross-sectional study was performed in 2017. Vegetables are classified into three groupsleafy, fruit vegetables, and root vegetables-in terms of growth location and vegetation shape. The statistical population consisted of vegetables beet leaves (B. vulgaris), parsley (P. crispum), basil (O. basilicum), lettuce (L. sativa), cabbage (B. oleracea), fenugreek (T. foenum-graecum), onions (A. cepa), radishes (R. raphanistrum), carrots (D. carota) and tomatoes (S. lycopersicum) cultivated on Sanandaj vegetable fields. Sanandaj is located on 35˚18 0 52@ N, 46˚59 0 32@ E. The height of this city is 1373.4 meters above sea level. Sanandaj with an average annual precipitation of 2.5 mm and the average temperature of 13.4˚C is classified as semi-arid regions [16]. According to previous studies, the soil texture of Sanandaj farms is loam-clay or sandyloam, and it contains 6-36% silt, 12-39% clay and 25-82% sand [17]. Most farmers used macro-fertilizer with NPK formula containing 15% nitrogen, 8% phosphorus, 15% potassium, and 1% zinc at a rate of 1-4 kg/m 3 [18]. Therefore, this type of fertilizer can increase soil and plant nitrate due to its high nitrogen content.
All chemicals used (include potassium hexacyanoferrate (III), zinc acetate dihydrate, sodium tetraborate and acetic acid glacial) were purchased from Sigma Aldrich at 99% purity and were used without any additional process. Whatman quantitative filter paper Grade 41, Membrane Filter (0.22 and 0.45 μm pore size) purchased from Merck Germany. With a standard deviation of 2.1 mg/kg and a precision of 0.2 and a 95% confidence level, 90 samples were selected at nine periods from 10 vegetable farms in autumn and spring (harvesting seasons). From each farm, ten samples (each 1 kg) of all the mentioned vegetables prepared, at first, non-edible parts were removed, and then the vegetables were washed first with tap water followed by distilled water. The vegetables were dried at 60˚C using an oven. The time required for drying each type of vegetable depends on their texture. This time took about 12 and 24 hours for the thin-textured vegetables (such as lettuce, cabbage, fenugreek, radish leaves, beet leaves, parsley and basil) and thick-textured vegetables (such as radishes, tomatoes, onion bulb and carrots), respectively. The moisture content of each vegetable was obtained from the difference between fresh and dry weight [19,20]. An electric mill was used to grind the vegetables. Because the nitrates in the vegetables is sensitive to light and temperature and it decomposes, dried samples were stored in a freezer at a temperature of -18˚C to maintain a longer storage time until the tests [20]. To accomplish the frying process, the required amount of soybean oil (Hengameh™), according to the taste of the people in the study area (about 100 ml) was poured into a stainless steel pan. When the oil temperature reached 190˚C, the chopped vegetables were fried for 15 minutes. A stainless steel pot was used for the cooking process. Half of the pot was filled with distilled water (400 ml), and the water in the pot was raised to 90˚C. The volume of water in all samples was consistent. After 15 minutes, the cooked vegetables passed through a filter and then were ground into powder after dehydrating and drying. All stages of sample preparation and extraction were carried out according to Food Standards 16721-2, and the nitrate measurements were performed using ion chromatography (Compact IC Plus 882 Model, Metrohm, Switzerland). Briefly, 10 g of dried vegetable powder was placed in a volumetric flask then 400 ml of hot water was added and kept in Bain Marie for about 15 minutes. The solution was then cooled to room temperature and filtered using Whatman quantitative filter paper Grade 41. Finally, 40 ml of filtered solution was taken to determine nitrate concentration. Carrez reagents were used to remove the interfering compounds. To prevent damage to the ion chromatography column and reduce the interference effect of the vegetable dye, the final samples were diluted ten times and filtered using syringe filter (0.45 μm and 0.22 μm). Nitrate concentration experiment was repeated three times in each sample and their mean was presented.
For risk assessment, the nitrate concentration in each type of vegetable and the amount of vegetable consumed by each person during the day were suggested as the basis of the calculation. Then, risk potential and risk indicator were determined for the health risk assessment [15]. It should be noted that the amount of consumption of each vegetable in the Iranian food basket is not specified separately and people use different types of vegetables depending on their tastes. Therefore, the health risk assessment for each vegetable was performed separately. In this study, the risk of non-carcinogenic effects was evaluated by calculating the Non-Carcinogenic Hazard Quotient (NHQ). For a single compound, the NHQ is obtained from Eq 1 where CDI is the chronic daily intake of chemical in mg/kg of body weight per day, and RFD is the chronic reference dose for the contaminant, which is 3.65 mg/kg of body weight per day (Eq 2) [21].
Note that C is the nitrate concentration in vegetable tissue (mg/kg), BW is body weight (for adults of~70 kg), ED is exposure duration (70 years), EF is exposure frequency (365 days per year), IRF is the average daily intake of vegetables based on the type of vegetable (g/day) and LT refers to the average length of life-70 years for non-carcinogenic effects. The amount of CDI for each kind of vegetable is calculated and, given the specified amount of nitrate RFD, the amount of health risk was obtained for all vegetable types.
To measure soil nitrate levels, sampling from three points at each farm was carried out at depths from 0 to 30 cm, and three samples from each farm were combined. About 1 g of the sample was dissolved in 100 ml of distilled water and filtered using Whatman filter paper No. 42 before being filtered using a Mixed Cellulose Esters Membrane filter. The nitrate amount in the filtered solution was measured using ion chromatography [22]. To measure water nitrate levels, sampling was performed from irrigation water. The water sample was put into polyethylene containers and filtered with Whatman filter paper No. 42, and then nitrate levels were measured using ion chromatography.

Statistical analysis
SPSS software was used for data analysis. For quantitative variables mean, maximum, minimum and standard deviation and for qualitative data frequencies and percentages were calculated, respectively. To evaluate the normality of the quantitative data, a Kolmogorov-Smirnov test was used before analysis. The significance of the relationship between different parameters was considered with regard to an error rate of 5% and a 95% confidence. A oneway ANOVA test was used to examine the significance relationship between vegetable nitrate levels and vegetable type (leafy, fruit and root vegetables), in which vegetables were classified into 10 groups based on type. The significance of the relationship between the vegetables' nitrate levels and the sampling period was investigated using t-tests. Based on the sampling period, vegetables were evaluated in two groups. Pearson correlation tests were used to study the significant difference between the nitrate levels of raw and cooked vegetables. A Pearson correlation test was used to study the significant difference between the nitrate levels of raw and fried vegetables.

Results
In total, 10 species of vegetables, including beets, basil, lettuce, parsley, cabbage, fenugreek, onion bulbs, radishes, carrots and tomatoes, were examined. Of all these types of vegetables, nine samples were taken from nine different farms. Vegetables were divided into three categories, including leafy vegetables (beet leaf, basil, lettuce, parsley, cabbage, fenugreek and onion bulbs), root vegetables (radishes and carrots) and fruit vegetables (tomatoes). Table 1 shows the moisture content and nitrate levels of vegetables, water and soil in all samples, and Table 2 shows the above parameters based on vegetable categories.
As shown in Table 2, the highest amount of moisture was found in tomato with a mean of 95%, and the lowest was from root vegetables, with an average of 90%. Also, the highest nitrate level was obtained in leafy vegetables with a mean of 5190 mg/kg in dry vegetables and the lowest in fruit vegetables with a mean of 3332 mg/kg in dry vegetables.
According to Fig 1, the highest moisture content was recorded in tomatoes with a mean of 95%, and the lowest was for cabbage with an average of 88.22%.
Based on Table 3, the highest and lowest nitrate levels (ppm) were in beet leaves and tomatoes, with an average of 75.99 ppm and 33.32 ppm, respectively. Results showed that the average nitrate level in leafy vegetables was 5190 mg/kg of dry weight, in root vegetables was 3709 mg/kg of dry weight and in fruit vegetables was 3332 mg/kg of dry weight.
As shown in Table 4, the nitrate concertation in all vegetables was less than National Iranian Vegetable Nitrate Standard.
As shown in Table 5, the vegetable nitrate concertation in the fall, which averaged 6200 mg/ kg in dry vegetables, was higher than in the spring, which had an average of 3217 mg/kg.
As shown in Table 6, the relationship between the vegetable nitrate concertation in species and vegetable type and sampling season was significant (p < 0.05).
As shown in Table 7, the nitrate concertation in cooked vegetables was lower than that in raw vegetables, and the relationship between the cooking process and the nitrate level of vegetables was significant inverted (p < 0.05), showing that cooking the vegetables decreased the nitrate levels. Table 8 demonstrates that the nitrate level in fried vegetables was higher than in raw vegetables, and the relationship between frying vegetables and vegetable nitrate levels was significant (p < 0.05). The frying process significantly increased the nitrate level in vegetables by 12.46% to 29.93%. Table 9 reveals that the highest health risk in raw vegetables can be ascribed to parsley at 0.1277, and the lowest is in tomatoes at 0.0465. The highest health risk was in cooked parsley with a level of 0.1248, and the lowest was in tomatoes at 0.0447. Also, the highest health risk in the fried vegetables was in beet leaves at 0.1298, and the lowest was ascribed to tomatoes (0.0524).

Discussion
In this study, we found that the average moisture content in all vegetables was 91.17%. The lowest moisture was 85%, and the highest was 98%. The average nitrate level in all vegetables was 4708.91 mg/kg in dry vegetables, with the lowest and highest nitrate levels in vegetables being 527 mg/kg in dry vegetables and 15394 mg/kg in dry vegetables, respectively. Our findings are consistent with those of Pourmoghim and colleagues, who determined that the average moisture content of all samples was 90%, with the highest moisture being 85.94% and the lowest being 81.34%. Thus, the small differences in moisture levels can be explained byamong other things-weather conditions, irrigation conditions and moisture levels (32). In a study conducted by Shahbazzadegan et al., the highest and lowest concentrations of vegetable nitrates were 35075 and 299 mg/kg of dry weight, respectively, and the reason for this difference is the variety of the examined vegetables, the difference in weather conditions, the soil, the fertilizers used and the cultivation frequency [26]. We also found that the irrigation water nitrate concentration on farms was between 14.31 mg/l and 36.12 mg/l. Moreover, the nitrate levels in the soil were between 4.35 and 9.57 mg/ kg, which is less than the critical levels (20 to 22 mg/kg, according to various studies) on all the farms sampled (33). According to a study by Mehrabi et al., the water nitrate levels on farms was 2.53 to 69.7 ppm, and the soil nitrate content ranged from 4 to 33 mg/kg. Another study carried out in the Tehran suburbs showed that soil nitrate concentrations at a depth of 2.5 m were more than 40 mg/kg. A comparison of this study's results with Mehrabi et al. and other studies showed that the nitrate concentration in the soil is quite variable and mainly relates to the application of fertilizer; however, the type of fertilizer and the rate and time of fertilization can cause changes in the nitrate concentration in the soil. The findings of this study showed that the nitrate concentration in mg/kg of dry weight in different studied vegetables is ordered as follows: leafy > root > fruit. This shows that there is a downward trend in the amount of nitrates in leafy, root and Fruit vegetable, respectively. These findings are consistent with a study conducted by Shahbazzadegan and colleagues, where the nitrate concentration in leafy vegetables was higher than in the root and root vegetables [26]. In a study carried out by Table 10. Health risks of nitrates in raw, cooked and fried vegetables in accordance with the FAO/WHO standard (400 g vegetable daily intake). Pirsaheb et al., the average nitrate concentration in leafy vegetables was higher than in root vegetables, and it was the lowest in Fruit vegetable [30]. A study conducted by Jafari et al. found that the average nitrate concentration in leafy vegetables was higher than than in root vegetables [31]. Several studies have demonstrated that the nitrate concentration in vegetables is related to different factors, such as the biological properties of the plant, light intensity, soil type, temperature, moisture, plant density/seed, plant maturity, growth period, harvest time, plant unit size, storage time and nitrogen source [23,32]. Moreover, it has been shown that nitrates are formed in leaf mesophilic cells; fruits and seeds have low nitrate levels, and nitrates are exclusively transported by xylem, which occurs mainly in leaves [33,34]. It was thus found that the nitrate concentration in vegetables adheres to the following order: leaf > stem > root > inflorescence > gland > fruit > grain [35]. According to Fig 1, [26]. We further found that the average nitrate level of beet leaves was 439 mg/kg in fresh vegetables, while the nitrate level of beet leaves varied in other studies and was reported at between 93.4 and 1250 mg/kg [24]. Furthermore, the highest nitrate level in raw vegetables was seen in beet leaves (with an average of 7599 mg/kg), and the lowest nitrate amount was seen in tomatoes (with an average of 3332 mg/kg). However, in a study by Taisser et al., the highest nitrate levels were related to spinach and beet leaves [36]. In this study, the nitrate content seen in all vegetables was lower than the standard level [27]. The nitrate concentration in vegetables is determined via different factors, such as the biological properties of the plant, light intensity, soil type, temperature, moisture, plant density/seed, plant maturity, growth period, harvest time, plant size, storage time and nitrogen source [23,32]. In addition, we found a significant relationship between vegetable nitrate levels and vegetable categories, vegetable type and sampling season (p < 0.05). Based on studies by Pourmoghim et al., Tabatabaee et al. and Ierna, nitrate levels were higher in leafy, root and Fruit vegetable, respectively [29,37,38]. Moreover, the nitrate levels in vegetables in the fall were higher than that in the spring.

Consumption type Nitrate level in vegetables (mg/kg) Vegetable consumption (g/day) Daily chronic absorption of contaminants Health risk
This correlation has also been reported by other researchers. For instance, Pourmoghim et al. showed that the nitrate concentration in tomatoes and potatoes in winter was higher than in the summer [37]. Shahlaei et al. indicated a significant relationship between the nitrate levels of vegetables and the sampling season and the type and species of vegetable [28]. Brkić et al. showed that the nitrate levels of vegetables in the fall were higher than in the spring [39]. This difference can be attributed to various factors, such as the duration and intensity of light radiation, soil and weather temperature, moisture and plant age [8,[39][40][41]. Extreme temperatures reduce the nitrate levels of plants through the assimilation process. Since there are more cloudy days in the fall than in the spring and the air temperature is lower, this results in lower nitrate assimilation during the fall, so the vegetables have higher nitrate levels [42,43].
We also found that the nitrate levels in cooked vegetables were lower than those in raw vegetables. The cooking process significantly reduced the nitrate levels in vegetables from 4.26 to 15.48%, and the relationship between raw vegetable nitrate content and the amount of cooked vegetables was significant (p < 0.05). Cooking changing the amount of nitrates in vegetables has also been reported by other researchers. Prasad et al. reported that the cooking process reduced the nitrate content of vegetables by 47 to 56% [10]. Sadeghi et al. showed that the cooking process caused a slight increase in nitrate levels of some vegetable categories and a slight decrease in nitrate levels in others [44].
In justifying the role of cooking processes in reducing nitrate levels, it is important to note that nitrates have a high tendency to dissolve in water, and when vegetables are immersed in water, the nitrates tend to move along the diffusion gradient from being more concentrated (inside the vegetable) to being less concentrated (inside the water in which the vegetable is located). Increasing temperature and time will facilitate the diffusion process and the movement of nitrates from the inside the vegetable into the distilled water, and finally, more nitrates will be released from the vegetable into the water, which will reduce the nitrate levels in the vegetable [10,45].
Our study also indicates that the nitrate level in fried vegetables was higher than in raw vegetables. The frying process significantly increased the nitrate levels in vegetables. Our findings are consistent with other studies that found that the frying process increases the nitrate level of vegetables [1,10,46]. Studies have shown that increasing the concentration of vegetable nitrates after frying can be related to the reduction of the vegetable's mass, which ultimately results in the condensation of nitrates in the sample and the amount of oil consumed. This can be explained by the fact that during the frying process, vegetable nitrates remain at the same level, but the volume or weight of the vegetable is reduced; this causes the fixed amount of nitrates to be crowded into a lower volume. Nevertheless, the type of oil used for frying may be effective in reducing the amount of nitrates in the vegetable; that is, the oil itself may contain nitrates. This hypothesis has been well documented in that the roots of immature plants, such as soybeans, have specific bacteria that stabilize elemental nitrogen to ammonia in plants. The ammonia is converted into nitrates through the nitrification process and by other microorganisms in soy plants, and because soybean oil was used in this study, the oil itself could contain nitrates and may have contaminated the fried vegetables [10,47].
The average amount of any kind of vegetable in Iran's food basket is clear. Accordingly, 58 g/day of leafy vegetables (leeks, parsley, beet leaves, etc.); 7 g/day of peas and beans; 68 g/day of potatoes and tomatoes; and 39 g/day of radishes, garlic and bulbs are consumed from the food basket of Iranian households [15]. According to the WHO, the average vegetable consumption per person is 400 g/day [24]. Therefore, the health risk for each individual vegetable is calculated based on the amount of consumption in the food basket of Iranian households and based on the 400 g/day intake. In this study, the highest health risks regarding nitrates in vegetables are fried vegetables, raw vegetables and cooked vegetables, respectively. Nitrate risks from consuming vegetables is high regarding the consumption of 400 g/day and, in some cases, is very close to the maximum permissible health risk. The highest health risk was related to fried beet leaves at 0.8948, and the least risk was from raw tomatoes with a level of 0.2607. In other studies, such transparency on the health risks of nitrates in vegetables has not been discussed. In a study conducted in Korea in 2013, the health risk was estimated for 400 g/day. According to the study, the daily intake of nitrates per unit of body weight for each of the vegetables was less than 1. Due to the low daily intake of nitrates compared to the maximum permissible daily intake, the health risk in all cases was lower than 1, meaning that the health risk was lower than the standard limit. In this study, the highest and lowest health risks were related for spinach and bean sprouts with a daily intake of 0.235 and 0.00 mg/kg/day, respectively. Many of the vegetables used in this research were not studied, but considering that the highest health risk was related to vegetables with the highest nitrate concentrations, the lowest health risk was related to vegetables with the lowest nitrate concentrations; therefore, the health risk is directly related to the nitrate concentrations in vegetables, and the results of that study are consistent with this study [48].
In another study by Gruszecka et al., the acceptable daily intake of nitrate (ADI) was considered to be 3.7 mg/kg body weight and the NHQ formula was used to assess health risk. The health risk of consuming several vegetables, including carrots and tomatoes, was far below the permitted level (HQ<1). Also, the health risk for some other vegetables, such as beets and cabbage, was higher than carrots and tomatoes, but the health risk for these vegetables was also below the standard (HQ<1), and in some cases the health risk of vegetables consumption was close to the limit of acceptable risk value [49]. Therefore, the results of this research are similar to those of the present study. It should be emphasized that consuming vegetables is only one way to intake nitrate. Thus a health risk of less than 1 in this condition cannot alone indicate a healthy level of nitrate intake. Therefore, other sources of nitrate such as drinking water and other foodstuff should be considered to determine the health risk of nitrate.

Conclusion
In this study, nitrate concentration in vegetables, the effect of home cooking processes on nitrate reduction and assessment of health risks of nitrate in vegetables for consumers were investigated. The obtained research results indicate that, nitrate concentrations were higher in tomato and onion bulb but lower in other vegetables. Nitrate concentration in leafy vegetables was higher than root and fruit vegetables and these values were higher in autumn than in spring. The results of this study indicate that the cooking process reduces vegetable nitrate levels and lowers the health risk of eating raw vegetables, while the frying process increases the nitrate level in vegetables and thus increases their health risk. The highest health risk level in raw, cooked and fried vegetables was related to parsley, parsley and beet leaves, respectively, and the lowest in all categories was tomatoes. Generally, the highest health risk was related to fried beet leaves, and the lowest was raw tomatoes. Therefore, it is necessary to consume fewer fried vegetables, and most vegetables should be consumed raw. Also, based on this study, the consumption of cooked vegetables is not problematic. In most cases, the nitrate levels in raw vegetables in Sanandaj is acceptable (lower than the standard). Considering the high benefits of vegetables, their consumption in terms of nitrates is unlikely to be problematic.