Kostić Kokić et al. 2025, Biologica Nyssana 16(2) 16 (2) December 2025: DOI: 10.46793/BiolNyss.16.2.1KK Effects of freezing vegetables on nitrate content and their health-related risks Original Article Ivana Kostić Kokić University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia ivana.chem@outlook.com (corresponding author) Tatjana Anđelković University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia Danica Bogdanović University of Niš, Faculty of Sciences and Mathematics, Department of Chemistry, Višegradska 33, 18000 Niš, Serbia Received: May 30, 2025 Revised: June 12, 2025 Accepted: July 30, 2025 Abstract: Vegetables are the major source of nitrate entry into the human body. The present study was conducted to investigate the influence of freezing on nitrate content in 15 species of vegetables from three commonly used groups: leafy, root and fruit vegetables. Determination of nitrate ions in sample extracts was performed using ion chromatography. The highest level of nitrate content before freezing was determined in samples of radish, while the lowest content of nitrate in raw vegetables was determined in samples of pepper. In raw samples of leafy celery and cucumber, nitrates were not detected. Results obtained by analyzing samples after freezing showed that this process has a different influence on the investigated samples. Some samples contained a higher level of nitrate after freezing, while others contained less. Part of the investigated vegetables showed an increased health risk if consumed in an amount of 400 g per day. Key words: nitrate, vegetables, freezing, health risk, ion chromatography Apstrakt: Uticaj zamrzavanja povrća na sadržaj nitrata i njihov zdravstveni rizik Povrće je glavni izvor unosa nitrata u ljudski organizam. Ovo istraživanje je sprovedeno sa ciljem ispitivanja uticaja zamrzavanja na sadržaj nitrata u 15 vrsta povrća iz tri najčešće korišćene grupe: lisnato, korenasto i voćno povrće. Određivanje nitratnih jona u ekstraktima uzoraka izvršeno je jonskom hromatografijom. Najviši nivo sadržaja nitrata pre zamrzavanja određen je u uzorcima rotkve, dok je najniži sadržaj nitrata u sirovom povrću određen u uzorcima paprike. U sirovim uzorcima lisnatog celera i krastavca nisu detektovani nitrati. Rezultati dobijeni analizom uzoraka nakon zamrzavanja pokazali su da ovaj proces ima različit uticaj na ispitivane uzorke. Pojedini uzorci su sadržali viši nivo nitrata nakon zamrzavanja, dok su drugi sadržali manji. Deo ispitivanog povrća pokazao je povećan zdravstveni rizik ako se konzumira u količini od 400 g dnevno. Ključne reči: nitrati, povrće, zamrzavanje, zdravstveni rizik, jonska hromatografija Introduction Vegetables are a rich source of minerals, vitamins, and biologically active compounds and play an important role in human nutrition (Prasad & Chetty, 2008). They are necessary for human wellbeing, and the World Health Organization (WHO) and Food and Agricultural Organization (FAO) have recommended a minimum intake of 400 g of fruits and vegetables per day to prevent chronic diseases (WHO, 2003; Qasemi et al., 2024). In recent years, rapid population growth, as well as industrialization, have increased need for more food production. This leads to extensive use of chemical fertilizers, which are nitrogen-based. In these conditions, vegetables can accumulate a notable amount of nitrate and nitrite (Kounnoun et al., 2024; Wang et al., 2024). Nitrogen uptake depends on many biological and environmental factors, including the composition of soil, light intensity, air temperature and moisture, duration of growth period, etc. (Bahadoran et al., 2016). Vegetables represent the major source of nitrate intake in the human diet, and it has been estimated that 75–80% of the total daily intake (TDI) comes from vegetables (Chang et al., 2013a and 2013b). Both inorganic compounds, nitrite (NO2 -) and nitrate (NO3 -), are involved in the nitrogen (N) cycle (Kyriacou et al., 2019). These nitrogen forms are present in edible plants produced endogenously as byproducts of the nitrate-nitrite-nitric oxide (NO) metabolic pathway in humans (Pinaffi-Langley © 2025 Kostić Kokić et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and build upon your work non-commercially under the same license as the original. et al., 2025). Nitrate can easily be reduced in the mouth and stomach to nitrite, which can react with secondary amines and then form cancerogenic N-nitrosamines. Also, nitrite has a high affinity for haemoglobin in the blood and can combine vigorously to form methemoglobinemia, known as blue baby syndrome (Kiani et al., 2022). Analysis of nitrate in vegetables is certainly necessary to assess the exposure to nitrate for adverse or beneficial effects (Yu et al., 2018). According to the scientific risk assessment on nitrate in vegetables, requested by the European Commission and adopted by the European Food Safety Authority (EFSA), the FAO/ WHO Expert Committee on Food Additives set the acceptable daily intake of 3.7 mg nitrate/kg body weight (FAO/WHO, 2003, 2005a, and 2005b). Numerous methods have been recommended for determining nitrate in fresh vegetables, but three techniques have been validated by international standards: ISO 6635, Ion chromatography (IC) and Cadmium column reduction (Hasheminasab et al., 2025). The aims of this research were to determine the amount of nitrate in different types of vegetables that are commonly represented in the human diet in Serbia, the effects of the freezing process on nitrate concentration and the assessment of the health risks of nitrates in vegetables. Materials and Methods Sampling This study was performed during the summer in 2022. Vegetables are classified into three groups: root vegetables, leafy, and fruit vegetables. The population consisted of carrot (Daucus carota sub- sp. sativus (Hoffm.) Schübl. & G.Martens), radish (Raphanus sativus L.), celery (Apium graveolens L.), parsley (Petroselinum crispum (Mill.) Fuss), parsnip (Pastinaca sativa L.), lettuce (Lactuca sa- tiva L.), chard (Beta vulgaris L.), spinach (Spinacia oleracea L.), parsley leaves, celery leaves, tomato (Solanum lycopersicum L.), bell pepper (Capsicum annuum L.), blue tomato, zucchini (Cucurbita pepo L.), and cucumber (Cucumis sativus L.) cultivated on Niš vegetable fields. All samples were purchased on the local Open market. Chemicals and instruments All chemicals used (sodium tetraborate acid, potassium hexacyanoferrate (III) for Carrez I solution and zinc acetate dihydrate for Carrez II solution) were purchased from Sigma Aldrich (Germany). All solutions were prepared using ultra- deionized water. Whatman quantitative filter paper (No. 41) and membrane filters with 0.22 µm pore size were purchased from Merck (Germany). An analytical balance (ABT 100-5M, Kern, Germany) with precision at ±0.00001 g was used to measure chemicals and samples. Ultra-deionized water was obtained using the Smart2Pure system (Thermo Scientific, USA). Nitrate determination in extracts was done using an ion chromatograph (Dionex Aquion, Thermo Scientific, USA). Sample preparation Five samples of all the mentioned vegetables were prepared for analysis. At first, non-edible parts were removed, and vegetables were washed with tap water. Then, vegetables were washed with distilled water, followed by deionized water. The vegetables were dried at room temperature for 24 h. One half of each vegetable was put in polypropylene storage bags, separately, and samples were stored in a freezer for 45 days at -20 °C. A 10 g portion of each vegetable was homogenized by a mixer (Bosh, Germany). From each homogenised sample, three subsamples of 1.0 g were weighed out by analytical balance and placed into a 100 mL glass. Next, 1.25 mL of disodium tetraborate (Na2B4O7x10H2O) solution and 25 mL of hot ultrapure deionized water (80 °C) were added. The beaker was placed into a boiling water bath for 15 min. After this time, the beaker was removed from the water bath and 0.5 mL of Carrez (I) solution was added and the solution was shaken. Then, 0.5 mL of Carrez (II) solution was added, and it was shaken once more. This solution was allowed to cool down to room temperature. After cooling, the solution was transferred into a 25 mL volumetric flask. The beaker was rinsed with ultra deionized water, and the rinse water was transferred to the same volumetric flask. The ultrapure water was added to fill up the volumetric flask. Solutions were filtered through the Whatman No. 41. Then, the obtained filtrates were filtered through a 0.22 µm cellulose filter prior to chromatographic analysis. A dilution with ultrapure water was necessary due to high concentrations of ions. The final filtrate was diluted to one twenty- fifth to match the dilution for analysis on an ion chromatograph. Each of the five filtrate samples was separated into a replication for nitrate analysis. Instrumentation The three replications were analysed for nitrate concentrations as follows. Volume of 5 mL of each filtrate sample was placed into the Dionex AS 50 model autosampler vials. Each of the samples was loaded automatically into the ion chromatograph. Separation was achieved using a Dionex IonPac AS22 column (4×250 mm) with the guard column Dionex IonPac AG22 (4×50 mm). The ion chromatograph BIOLOGICA NYSSANA ● 16 (2) December 2025: Kostić Kokić et al. ● Effects of freezing vegetables on nitrate content and their health-related risks BIOLOGICA NYSSANA ● 16 (2) December 2025: Kostić Kokić et al. ● Effects of freezing vegetables on nitrate content and their health-related risks used a mix of 4.5 mM sodium carbonate and 1.4 mM sodium bicarbonate as an eluent at a flow rate of 1.2 mL/min. The concentration of nitrate anions in samples was detected by the Dionex AERS 500, Carbonate, 4 mm, conductivity detector. Dionex Seven Anion Standard (Product No. 056933) was used for preparing standard solutions. The nitrate concentration values were calculated automatically based on a previously made processing method using Chromeleon 7 software. Human health risk assessment Non-cancerogenic risk assessment was performed by the Target Hazard Quotient (THQ) method provided by the US Environmental Protection Agency (EPA) (USEPA, 2001). The estimated daily intake (EDI) was calculated using Equation 1, and then THQ was calculated using Equation 2. (1) (2) Where EF is exposure frequency (365 days/year), ED is exposure duration (70 years), IRF is average daily intake (g/day), C is concentration in vegetable (mg/kg), LT is average length of life (70 years for non-cancerogenic effect), BW is average body weight (70 kg) and RFD is chronic referent dose for contaminant (3.7 mg/kg of body weight per day). According to the report provided by Milešević et al. (2024), the vegetable consumption in Serbia is 312 g/day. A one-way ANOVA test was used to examine the significance relationship between vegetable nitrate levels in raw and frozen samples. The difference between nitrate levels obtained from raw and frozen vegetables was compared to a critical value to determine if the difference is significant. The post-hoc test, Tukey’s test, was performed, and the test compares the difference between each pair of mean values with appropriate adjustment for the multiple testing. Values of HSD (honest significant difference) for each pair were computed by the Origin© program. Comparing was performed in case p<0.05. Results and discussion In total, 15 species of vegetables were examined. Vegetables are divided into three categories: leafy, root, and fruit vegetables. Nitrate concentrations were determined using ion chromatography, based on a previously defined processing method. Calibration curve showed a good correlation coefficient, 0.99899, in the concentration range 0.25 to 10 mg/L (Fig. 1). Fig. 1. Calibration curve for nitrate ion at 0.25–10 mg/L concentration range BIOLOGICA NYSSANA ● 16 (2) December 2025: Kostić Kokić et al. ● Effects of freezing vegetables on nitrate content and their health-related risks Figs. 2 and 3 present the chromatograms obtained after analyzing the standard solution and the extract of the radish sample after freezing. mg/kg, while the lowest nitrate level was in parsley, 315.56 mg/kg. In the same vegetable category, the highest level of nitrates in frozen samples was determined in samples of radish, too (2335.90 mg/kg), as well as the lowest in parsley (126.61 mg/kg). In the category of leafy vegetables, the highest nitrate level in raw samples was determined in lettuce (851.42 mg/kg), as well as in frozen samples (865.52 mg/kg), while in raw samples of celery, nitrates were not detected. The lowest level of nitrate in frozen samples from the category of leafy vegetables was determined in spinach (173.68 mg/kg), while in raw samples, it was detected in samples of parsley (269.37 mg/kg). In the last category, fruit vegetables, the highest level was determined in raw samples of zucchini (2578.33 mg/kg), as well as in frozen samples (1309.76 mg/kg), but in raw samples of cucumber, nitrates were not detected. In frozen samples from fruit vegetables, nitrate level was not detected in tomato, but the lowest determined concentration was in samples of pepper (43.64 mg/kg). In 10 of 15 vegetables, the nitrate concentrations in frozen samples were lower than in raw samples. Results of Tukey’s test showed that there is a significant difference in ni- trate level between all raw and fro- zen samples, except in the samples of lettuce. The average nitrate level in all raw vegetables was 928.18 mg/kg in raw samples, with the lowest and highest nitrate levels in vegetables being 96.40 mg/kg in pepper and 3406.66 mg/kg in radish. In case of frozen vegetables, the average nitrate level was 592.80 mg/g and the lowest and highest nitrate levels were found in the same vegetables, 43.64 mg/kg in pepper and 2335.90 mg/kg in radish. The mean level of nitrate in lettuce samples was lower than values reported in literature (Eichholzer & Gutzwiller, 1998; Chung et al., 2011; Ziarati & Arbabi-Bidgoli, 2014; Yu et al., 2018). The amount of nitrate in raw tomato was higher than the results reported in some other studies (Zhou et al., 2000; Ali Fig. 2. Chromatograph of standard solution at nitrate concentration 2.5 mg/L Fig. 3. Chromatogram of frozen radish sample The present study found that vegetable nitrate concentrations were variable. Tab. 1 shows the nitrate levels (mg/kg) in all investigated samples of vegetables, raw and frozen. The highest nitrate level in the raw sample of the root vegetables category was in samples of radish with an average of 3406.66 BIOLOGICA NYSSANA ● 16 (2) December 2025: Kostić Kokić et al. ● Effects of freezing vegetables on nitrate content and their health-related risks et al., 2021). In another study in Mashhad (Zendehbad et al., 2022) reported mean nitrate levels in carrot and cucumber, 355.88 and 221.36 mg/kg, respectively, and these levels were lower for carrot and higher for cucumber. In a study by Bahadoran et al. (2016), nitrate concentrations in radish and celery were significantly higher than concentrations determined in our study (6250 mg/kg and 2610 mg/ kg, respectively). Also, investigation of nitrate concentration in samples of chard showed that the obtained results are lower compared to some studies which reported 1690 and 1728 mg/kg (EFSA, 2008; Roila et al., 2018) and for parsley 526 mg/kg (Salehzadeh et al., 2020). The average amount of any kind of vegetable in the Serbian food basket is not clear. According to data in the literature, 58 g/day of leafy vegetables and 68 g/day of some fruit vegetables, and 39 g/day of root vegetables are consumed from some household baskets (Salehzadeh et al., 2020). Nitrate risks in case of consuming 58, 68, and 39 g/day are presented in Tab. 2 and Tab. 3. Tab. 2 reveals that the highest health Table 1. Nitrate content in each type of investigated vegetable Vegetable Nitrate content (mg/kg) Raw Frozen Root Carrot 918.87±4.11a 127.57±1.56b Radish 3406.66±7.41a 2335.90±8.70b Celery 421.56±14.52a 831.78±3.39b Parsley 315.56±5.73a 126.61±2.83b Parsnip 887.26±1.50a 0b Leafy Lettuce 851.42±4.70a 865.52±8.89a Chard 647.53±6.23a 266.64±2.06b Spinach 468.29±6.35a 173.68±3.09b Parsley 269.37±3.28a 258.24±1.09b Celery 0a 274.93±2.26b Fruit Tomato 579.74±2.28a 0b Pepper 96.40±3.20a 43.64±2.08b Blue tomato 625.39±4.34a 838.19±3.33b Zucchini 2578.33±7.94a 1309.76±3.49b Cucumber 0a 253.89±5.69b Table 2. Health risk of nitrates in raw vegetables based on the food basket Vegetable EDI for raw vegetables Health risk (THQ) Carrot 0.5119 0.1403 Radish 1.8980 0.5200 Celery 0.2349 0.0643 Parsley 0.1758 0.0482 Parsnip 0.4943 0.1354 Lettuce 0.7055 0.1933 Chard 0.5365 0.1470 Spinach 0.3880 0.1063 Parsley 0.2232 0.0611 Celery 0.0000 0.0000 Tomato 0.5632 0.1543 Pepper 0.0936 0.0257 Blue tomato 0.6075 0.1664 Zucchini 2.5047 0.6862 Cucumber 0.0000 0.0000 Values with the same letter (a-b) within a row are not statistically significantly different at the p<0.05 level Table 3. Health risk of nitrates in frozen vegetables based on the food basket Vegetable EDI for frozen vegetables Health risk (THQ) Carrot 0.0711 0.0195 Radish 1.3014 0.3566 Celery 0.4634 0.1270 Parsley 0.0705 0.0193 Parsnip 0.0000 0.0000 Lettuce 0.7171 0.1965 Chard 0.2209 0.0605 Spinach 0.1439 0.0394 Parsley 0.2140 0.0586 Celery 0.2278 0.0624 Tomato 0.0000 0.0000 Pepper 0.0424 0.0116 Blue tomato 0.8142 0.2231 Zucchini 1.2723 0.3486 Cucumber 0.2466 0.0676 Conclusion In this study, nitrate concentration in vegetables and the effect of freezing on nitrate levels were investigated. Also, the health risk assessment was done. The obtained results indicate that the average nitrate concentration in root vegetables is the highest, then in fruit vegetables, and the lowest nitrate concentration is determined in leafy vegetables. The results of this study indicate that the freezing process reduces the nitrate amount in all investigated root vegetables except celery. In a group of leafy vegetables, nitrate concentration after freezing and storing was higher in samples of lettuce and celery, while in a group of fruit vegetables, it was the case in samples of blue tomato and cucumber. The health risk is directly related to the nitrate concentrations in vegetables. Therefore, the highest average health risk showed root vegetables, then fruit vegetables, and at the end, the lowest health risk showed leafy vegetables. It should be emphasized that consuming vegetables is only one way to intake nitrate. It means that if the value of health risk is lower than 1, it cannot alone indicate a healthy level of nitrate intake. Other nitrate sources, such as other foodstuffs and drinking water, should be considered to determine the nitrate health risk. Therefore, the risk in raw vegetables can be ascribed to zucchini at 0.6862, and the lowest health risk in pepper at 0.0257, excluding leaf celery and cucumber in which samples nitrates were not detected in raw samples. Also, considering the data from Tab. 3, the highest health risk in frozen vegetables can be ascribed to radish at 0.3566. The lowest health risk in frozen samples was obtained for pepper, as in raw vegetables, at 0.0116. In frozen samples, nitrates were not detected in tomato and parsnip. The obtained results showed that health risk values for most of the investigated samples are far below the maximum allowed value (THQ<1). But the much higher nitrate risk of consuming vegetables is in the case of the consumption of 400 g/day, which results are presented in Tab. 4 and Tab. 5. In some cases, the health risk is very close to the maximum permissible health risk (raw tomato), while in some cases it is higher than 1. Increased health risk, in case of consuming 400 g/day, was obtained for the following raw samples: carrot, radish, parsnip, lettuce, chard, and zucchini, while in the group of frozen samples, those were: radish, root celery, lettuce, blue tomato, and zucchini. In this study, health risk regarding nitrates in vegetables decreases in the following order: root vegetables, fruit vegetables, and leafy vegetables, respectively. BIOLOGICA NYSSANA ● 16 (2) December 2025: Kostić Kokić et al. ● Effects of freezing vegetables on nitrate content and their health-related risks Table 4. Health risk of nitrates in raw vegetables in accordance with the WHO standard (400 g of vegetable daily intake) Table 5. Health risk of nitrates in frozen vegetables in accordance with the WHO standard (400 g of vegetable daily intake) Vegetable EDI for raw vegetables (400 g) Health risk (THQ) Carrot 5.2507 1.4385 Radish 19.4666 5.3333 Celery 2.4089 0.6600 Parsley 1.8032 0.4940 Parsnip 5.0701 1.3891 Lettuce 4.8653 1.3329 Chard 3.7002 1.0137 Spinach 2.6759 0.7331 Parsley 1.5393 0.4217 Celery 0.0000 0.0000 Tomato 3.3128 0.9076 Pepper 0.5509 0.1509 Blue tomato 3.5737 0.9791 Zucchini 14.7333 4.0365 Cucumber 0.0000 0.0000 Vegetable EDI for frozen vegetables (400 g) Health risk (THQ) Carrot 0.7290 0.1997 Radish 13.3480 3.6570 Celery 4.7530 1.3022 Parsley 0.7235 0.1982 Parsnip 0.0000 0.0000 Lettuce 4.9458 1.3550 Chard 1.5237 0.4174 Spinach 0.9925 0.2719 Parsley 1.4757 0.4043 Celery 1.5710 0.4304 Tomato 0.0000 0.0000 Pepper 0.2494 0.0683 Blue tomato 4.7897 1.3122 Zucchini 7.4843 2.0505 Cucumber 1.4508 0.3975 control of possible contamination sources and the minimization of vegetable nitrate concentration are recommended. 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