Nitrate, nitrite, and lead contamination in leafy vegetables collected from local market sites of Go Vap district, Ho Chi Minh City

Cite this paper: Vietnam J. Chem., 2021, 59(1), 79-86 Article DOI: 10.1002/vjch.202000124 79 Wiley Online Library © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH Nitrate, nitrite, and lead contamination in leafy vegetables collected from local market sites of Go Vap district, Ho Chi Minh City Nguyen Quoc Thang1*, Nguyen Thi Mai Tho1, Nguyen Thi Kim Phuong2,3* 1Chemical Engineering Faculty, Industrial University of Ho Chi Minh City, 12 Nguyen Van Bao, Go Vap, Ho Chi Minh City 70000, Viet Nam 2Hochiminh City Institute of Resources Geography, Vietnam Academy of Science and Technology, 01 Mac Dinh Chi, District 1, Ho Chi Minh City 70000, Viet Nam 3Graduate University of Science and Technology, Vietnam Academy of Science and Technology, 18 Hoang Quoc Viet, Cau Giay, Hanoi 10000, Viet Nam Submitted July 20, 2020; Accepted August 18, 2020 Abstract Lead, nitrate, and nitrite are among the major contaminants of vegetables. These concentrations appraise the quality characteristic of vegetables. Governments and regulators has control the level of nitrate, nitrite, and lead in vegetables to protect the human health. The present study was initiated to investigate the levels of these contaminants in five leafy vegetables in local market (Brassica juncea, Brassica integrifolia, Lactuca sativa, Ipomoea aquatica, and Nasturtium officinale). The vegetable species can be listed by decreasing nitrate content as follows: Lactuca sativa > Nasturtium officinale > Ipomoea aquatica > Brassica integrifolia > Brassica juncea. The nitrite content in leafy vegetables was significantly lower than nitrate content. Among observed vegetables, highest concentration of lead is in Ipomoea aquatica (0.200±0.011 mg/L), whereas lead content in Lactuca sativa and Nasturtium officinale were not detected. In addition, the Pb levels in the leafy vegetables were not correlated with nitrate concentration. Based on the results of our investigation, the approximate daily intake (DI) of NO3–, NO2– and Provisional Tolerable Weekly Intake (PTWI) of lead were assessed to human health in consuming the observed leafy vegetables. Keywords. Nitrate, nitrite, lead, leafy vegetables. 1. INTRODUCTION Nitrate and nitrite are the natural form of nitrogen. They are an integral part of the nitrogen cycle in the environment. Nitrate is formed from fertilizers, decaying plants and other organic residues. Nitrate is also used as a food additive, which are a preservative and antimicrobial agent.[1] Vegetables are the part of the essential diet in the human lifestyle which were the rich sources of vitamins, minerals, and fibers, and also have beneficial antioxidative effects. Their consumption is increasing gradually, particularly among the urban community. Vegetables depend on nitrate for nutrition and biological function.[2,3] However, plant crops especially tend to absorb nitrate more than required.[4,5] Therefore, these vegetables are also the main route for nitrate to enter and accumulate in the human body which compared to the other route of entries for its consumed by humans every day.[6] High amount of nitrate in vegetables is a worldwide problem. The nitrate toxicity had been identified in several human health hazards. Nitrate is reduced into nitrite and reacted with amines and amides to nitrosamines and nitrosamides.[7] The toxic effect of nitrite in the body is that it can oxidize ferrous in hemoglobin to ferric to produce methemoglobin, thus, reduce the ability of hemoglobin to carry oxygen.[8] Lead (Pb) is highly toxic metal and it have no function in the physiological processes of living organisms.[9] The main target for lead toxicity in human is the nervous system and may also cause weakness in fingers, wrists, or ankles. Exposing to lead at high levels can severely damage the brain and kidneys.[10] Therefore, the importance of studies about nitrate, nitrite and lead bioaccumulation in leafy vegetables, which represents a valuable source of food for humans, in the context of environmental pollution, seems to be questionable. Vietnam Journal of Chemistry Nguyen Quoc Thang et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 80 2. MATERIALS AND METHODS 2.1. Chemicals - Lead stock standard solution (1000 mg/L), sodium nitrate, sodium nitrite, zinc (powder), glucose, borax, oxalic acid, hydrogen peroxide, and nitric acid were purchased from Merck, Darmstadt, Germany. - N,N-dimethyl-1-naphthylamine, sulfanilic acid, acetic acid, and sodium acetate were purchased from Sigma-Aldrich, Singapore. 2.2. Vegetables sampling Five species of vegetable were collected random from three local markets from Go Vap district, Ho Chi Minh city (Go Vap, Duong Quang Ham, and Xom Thuoc market). Table 1: Scientific name, number of samples and number of individuals for each sample in each local market Scientific name Picture of vegetables No. of analysis sample No. of individual per one sample Brassica juncea 30 10 Brassica integrifolia 30 10 Lactuca sativa 30 10 Ipomoea aquatica 30 10 Nasturtium officinale 30 10 Samples were directly taken from vegetable sellers at the markets. Only healthy and undamaged samples were collected. Immediately after sampling, the samples were taken to the laboratory in polyethylene bags using an ice box. As soon as arrived in the laboratory, vegetable samples were treated by removing inedible parts, then washed under milli-Q water and drained vegetables by drying in room temperature. Prior to analysis, the drained vegetables samples were prepared by mixing and grounding homogenously. The composite sample was analyzed nitrate, nitrite and lead. 2.3. Nitrate and nitrite analysis About 10 g of the composite sample was weighed into a 250 mL flask. 2.5 mL saturated borax solution, 50 mL hot milli-Q water were added to it and the flask heated on a water bath for 25 minutes at 80 °C with occasional shaking. After treatment, the sample was allowed to cool to room temperature. The flask was filled up to the mark with milli-Q water. The extract solution was filtered through 0.45 µm Millipore filter and then analyzed the nitrate and nitrite. After adding NH4Cl to 10 mL filtered solution, the obtained mixture was shaken until the solid was dissolved totally. The nitrate in the treatment sample was reduced to nitrite using the mixture of Zn powder and glucose. Under acidic condition, nitrite reacted with sulfanilic acid to form a diazo compound. A diazo compound couples with N, N- dimethyl-1-naphthylamine to form a purple azo-dye. The color solution was diluted with milli-Q water to 50 mL. The intensity of colored compound was quantified using UV-Vis GENESYS 20 absorption at a wavelength of 520 nm to determination of total concentration of nitrate in the sample. The method had a limit of detection (LOD) of 0.35 mg total NO3- /kg and assay recovery was approximately 89.6 %. Relative standard deviation (RSD%) was approximately 3.81 %. Total nitrate concentration in vegetables (mg/kg) = C x 50 x 250 m x 10 where: - m: Weight of samples (g), - C: Concentration of total nitrate in the sample as from calibration graph (mg/L). Nitrite concentration in samples was determined using the same method which did not reduce nitrate to nitrite. Nitrate concentration in samples (mg/kg) = Total nitrate concentration - Nitrite concentration. Vietnam Journal of Chemistry Nitrate, nitrite, and lead contamination © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 81 2.4. Lead analysis Prior to determination, 5 g of composite sample were subjected to preliminary mineralization in presence of 15 mL of HNO3 (65 %) in the closed vessel. The sample was then heated to 85 ºC for 30 min until a clear liquid was obtained and then diluted with deionized water to 25 mL. The solution was assayed for concentration of Pb using inductively coupled plasma - optical emission spectrometry (Optima 2100 DV, Perkin Elmer). Various conditions of ICP-OES parameters such as wavelength, linear range, linear regression, LOD, limit of quantification (LOQ), assay recovery, and RSD were tested to establish the optimum condition. The optimal condition of ICP-OES parameters were presented in table 2. Table 2: ICP-OES parameters No. Parameters Optimal condition 1 Wavelength 220.353 nm 2 RF Power 1300 W 3 Plasma flow 15 L/min 4 Nebulizer flow 0.8 L/min 5 Auxiliary flow 0.2 L/min 6 Analysis mode Axial 7 Nebulizer pump 1.5 mL/min 8 Acid HNO3 2 % 9 Linear range 0.01-22 mg/L 10 Linear regression y = 10781x – 302.09 11 LOD 9.4 µg/L 12 LOQ 31.5 µg/L 13 Assay recovery 95.0 % 14 RSD% 0.29 % Lead concentration was calculated as follows: Lead concentration in vegetables (mg/kg) = 𝐂 𝐱 𝟐𝟓 𝐦 Where: m: Weight of samples (g), C: Concentration of lead in the sample as from calibration graph (mg/L). 2.5. Statistical analysis The statistical significance of the obtained results was analyzed using the Statistical Package for Social Sciences (SPSS) version 22.0. Data were expressed by mean  SD. The significance of differences was established with the one-way analysis of variance (ANOVA), assuming that the significance level p is less than 0.05. 3. RESULTS AND DISCUSSION 3.1. Content of the nitrate form in leafy vegetables The nitrate concentration in observed leafy vegetables from three local markets of Go Vap district, Ho Chi Minh City, during April 2019 was presented in table 3. In this study, the highest and lowest nitrate concentration in vegetables was Lactuca sativa and Brassica juncea, respectively. These results were correlation with other research. Dang Tran Trung et al. (2018) reported that nitrate concentration in Brassica juncea, Lactuca sativa, and Ipomoea aquatica were 548±44, 1420±57, and 652±11 mg/kg of fresh mass, respectively.[11] Similarly, Nguyen Thi Lan Huong (2013) documented that nitrate level in Ipomoea aquatica, Brassica juncea, and Brassica integrifolia after 9 days of fertilized were 613.09, 587.08, and 580.73 mg/kg of fresh mass, respectively.[12] Table 3: Nitrate content in leafy vegetables from local market Leafy vegetable Conc. of NO3- (mg/kg of fresh mass) in leafy vegetable from local market*1 The ML value of nitrate (mg/kg of fresh mass)*2 Go Vap Duong Quang Ham Xom Thuoc Brassica juncea 679.8±34.5 687.2±30.9 712.0±16.8 500 Brassica integrifolia 671.4±25.9 702.6 ± 25.1 733.7±33.1 500 Lactuca sativa 1634.5±31.6 1458.1±23.6 1547.2±25.2 1500 Ipomoea aquatica 960.2±47.4 932.1±52.4 1008.9±44.4 600 Nasturtium officinale 1358.6±34.6 1440.7±28.7 1261.9±28.8 1500 *1Each value represents the mean ± SD of six replicates. *2The safe limit value set by the VMARD[13] In 2008, the Vietnamese Ministry of Agriculture and Rural Development (VMARD) brought out a Regulation to set the maximum limits (ML) for nitrate in vegetable. Inside, the maximum levels (limits) for nitrate (mg/kg fresh matter) in leaf vegetable were shown in table 3. As seen in table 3, Vietnam Journal of Chemistry Nguyen Quoc Thang et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 82 three leafy vegetables in this study (Brassica juncea, Brassica integrifolia, Ipomoea aquatica) were significantly higher than safe limit value set by the VMARD. The nitrate level in Brassica juncea was approximately from 1.36 to 1.42-fold higher than ML level of nitrate. In the case of Brassica integrifolia, nitrate concentration was about from 1.36 to 1.47-fold higher than the safe limit value. For the Ipomoea aquatica, nitrate concentration was higher than the safe limit about 1.55 to 1.68-fold. In the case of Lactuca sativa, nitrate amount was roughly from 0.97 to 1.09-fold comparative the ML level. Finally, nitrate concentration in Nasturtium officinale were slightly lower from 0.84 to 0.96-fold compared to the ML level. The concentrations of nitrate in leafy vegetables depend on a range of factors including season, light, temperature, growing conditions, fertilizer use, and storage of the crop.[14] The primary nitrate source for plant uptake is nitrogen fertilization which is the major increase of nitrates in edible crops. In general, the yields will be increased in increasing nitrogen fertilizers but the nitrate content in leafy vegetables also increase when higher rates of nitrogenous fertilizers were applied. Because nitrogen requirement of matures leafy vegetables decrease, so that it is not surprising that excessive utilizer of nitrate based-fertilizers at late stages of vegetative growth have a strong impact on nitrate accumulation in leafy vegetables. For this reason, reducing nitrate concentration in plants is to deprive them of nitrogen for few weeks before harvesting, during which process nitrates in the plant are removed from the vacuoles and the plant stores organic compounds to replace the declining osmoticum.[15] Concentration of nitrate in vegetables is also dependent on vegetable species such as the families Amaranthaceae, Apiaceae, Asteraceae, and Brassicaceae.[16] The nitrate accumulation is influenced by the age of plant wherefore the younger leaves accumulate less nitrate than the older leaves.[17] The mature outer leaves of lettuce heads were found to contain concentration of nitrates, that the nitrate concentration could be measured up to five times higher than that of the inner leaves.[18] Also, young rocket leaves accumulate nitrates lower than older leaves.[19] In fact, the farmer can harvested leafy vegetables as baby greens to reduce nitrate concentration.[20] 3.2. Content of the nitrite form in leafy vegetables Table 4 showed the concentration of nitrite of selected vegetables from Go Vap district, during April 2019. Table 4: Content of nitrite in leafy vegetables from local market Leafy vegetable Conc. of NO2- (mg/kg of fresh mass) in leafy vegetable from local market * Average (mg/kg of fresh mass) Go Vap Duong Quang Ham Xom Thuoc Brassica juncea 54.9±5.1 62.5±3.0 69.7±6.6 62.4±7.9 Brassica integrifolia 56.4±4.9 65.2±6.0 74.8±5.0 65.5±9.2 Lactuca sativa 88.4±5.8 101.8±6.7 72.3±5.7 87.5±13.6 Ipomoea aquatica 75.8±4.4 64.0±4.7 71.7±6.0 70.5±7.0 Nasturtium officinale 66.2±4.8 63.9±5.8 73.0±5.9 67.7±6.6 * Mean ± SD (n = 6). The content of nitrite in vegetables was significant lower than nitrate concentration. This observation is consistent with the results reported in the literature.[21,22] The highest nitrites were found in Lactuca sativa, with a mean concentration of 87.5±13.6 mg/kg. Although vegetables contained a minor amount of nitrite, however, if they are stored incorrectly, this amount can be increased significantly by microbiological reduction of nitrate. This is especially a problem for leafy vegetables, because it may be difficult to remove completely soil adhering to them.[23] Hence, vegetables that accumulate high nitrate levels may contain higher amounts of nitrite. Nitrite may react with amines or amides in living organisms to produce N-nitroso compounds.[7] These compounds cause carcinogenic effects in more than 40 animal species, including human.[24] About 177/209 nitrosamines and 79/86 nitrosamides have been shown to be carcinogenic in a variety of species.[25] Higher nitrate/nitrite intake cause an increased risk of thyroid cancers.[26] The high nitrate Vietnam Journal of Chemistry Nitrate, nitrite, and lead contamination © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 83 consumption is associated with increased risk of cancers in urinary bladder, esophagus, nasopharynx and prostate, colon and oral cancers.[24,27,28] 3.3. Content of the lead in leafy vegetables The Pb accumulation in leafy vegetable, during April, 2019 were shown in table 5. It could be observed that Pb level in all of the observed samples, regardless of location is lower than the health criteria level of lead set by the VMARD (0.3 mg/kg fresh mass). In this study, the lead concentrations in Ipomoea aquatica varied high significantly among the five leafy vegetable species. The lead level in Lactuca sativa and Nasturtium officinale were lower than the limit of detection of the method (0.032 mg/kg). The contamination of heavy metal in vegetables may be due to soil pollution, contaminated water, fertilizers, industrial emissions, the harvesting process, storage, species, [29,30] Heavy metals can be absorbed by leafy vegetables through contaminated soil and irrigation water sources. Furthermore, it has an ability to absorb the metals deposited on plant surfaces exposed to the polluted environments.[31] Table 5: Content of lead in leafy vegetables from local market Leafy vegetable Conc. of Pb (mg/kg of fresh mass) in leafy vegetable from local market *1 The ML value of nitrate (mg/kg of fresh mass)*2 Go Vap Duong Quang Ham Xom Thuoc Brassica juncea 0.122±0.014 0.126±0.010 0.135±0.007 0.3 Brassica integrifolia 0.108±0.009 0.105±0.011 0.109±0.011 Lactuca sativa < 0.032 < 0.032 < 0.032 Ipomoea aquatica 0.205±0.010 0.191±0.005 0.204±0.010 Nasturtium officinale < 0.032 < 0.032 < 0.032 *1Mean ± SD (n = 6). *2The safe limit value set by VMARD.[13] 3.4. Relationship between leafy vegetable Pb concentrations (mg/kg fresh mass) and nitrate concentration No correlation was observed between Pb and nitrate concentrations (figure 1). The results of the present study showed that nitrate concentration had different lead accumulation in five leafy vegetables. This identified the difference from mechanic of the absorbed of nitrate and lead in these crops. Figure 1: Relationship between leafy vegetable Pb concentrations (mg/kg fresh mass) and nitrate concentration: (a) Brassica juncea, (b) Brassica integrifolia and (c) Ipomoea aquatica In the case of lead, it is probably transported in the vegetable by the form of compounds or chelate complexes. Lead influences to mobilizing function of root exudates.[32] IRT1, a gene that encodes an Fe2+ transporter presumably involved in Fe acquisition in nongrass plants, was recently cloned in Arabidopsis.[33] The physiological role of IRT1 is to uptake the iron from the rhizosphere across the plasma membrane into the root epidermal cell layer. It is facilitate the transport of heavy metals in the y = 2077.4x + 479.28 R² = 0.3251 600 650 700 750 800 0.080 0.105 0.130 N it ra te c o n c. ( m g /k g ) Lead conc. (mg/kg) b) y = 2904.7x + 389.3 R² = 0.3302 800 850 900 950 1000 1050 1100 1150 0.180 0.200 0.220 N it ra te c o n c. ( m g /k g ) Lead conc. (mg/kg) c)y = 1475.7x + 504.49 R² = 0.321 600 650 700 750 0.090 0.115 0.140 N it ra te c o n c. ( m g /k g ) Lead conc. (mg/kg) a) Vietnam Journal of Chemistry Nguyen Quoc Thang et al. © 2021 Vietnam Academy of Science and Technology, Hanoi & Wiley-VCH GmbH www.vjc.wiley-vch.de 84 form of divalent cations such as Cd2+, Pb2+, and Zn2+.[34] Nitrate is found in cell vacuoles and it is transported in the xylem. The role of xylem carries the water and nutrients from the roots to the leaves. This means leaf crops have fairly large nitrate concentrations. 3.5. Evaluation of adverse