A R T I C L E  I N F O		ABSTRACT
ORIGINAL ARTICLE		Introduction: In recent years, the contamination of food with heavy metals has received much attention. Plants can absorb metal pollutants through contaminated water, soil, and air. Materials and Methods: In the current study, accumulation of minerals in three types of soybeans was investigated by wet and dry digestion methods using ICP-OES technique. Thereafter, the metals' health risk was assessed by estimated daily intake, toxic hazard quotient (THQ), and hazard index (HI) values. Results: According to the results, the concentrations of Cr, Se, Ca, Fe, Mo, Mn, and Mg in soybean seeds were 0.034-170.88 mg/kg, 0.21-243.79 mg//kg, 2.50-33.37 mg/kg, 0.05-0.86 mg/kg, 0.071-203.57 mg/kg, 0-0.47 mg/kg, and 2.69-19.31 mg/kg, respectively. The ashing method had a better performance in determining Ca, Fe, Mo, Mn, and Mg concentrations than the wet digestion method. The THQ rates were below 1 for the three varieties of each mineral element, but the HI values of variety 2 and variety 3 were higher than 1 in both methods. Conclusion: Furthermore, continuous monitoring of the soybeans' mineral and heavy metal contents seems necessary.
Article History: Received: 10 February 2020 Accepted: 20 April 2020
*Corresponding Author: Elham Khalili Sadrabad Email: khalili.elham@gmail.com Tel: +989131946425
Keywords: Soybean, Digestion Method, ICP-OES, Risk Assessment.

Introduction
Trace elements or essential micronutrients (manganese, iron, copper, zinc, cobalt, and selenium) are metals with different biochemical functions in living beings, but their excess amounts can cause toxicity ¹. Exposure to trace elements causes acute and chronic symptoms, such as cardiovascular disease, impaired fertility, and disorders in nervous and immune system. Transmission of metals to food chain has been considered as a crucial problem during the last decade ². Trace elements can be absorbed by plants through soils, fertilizer, air, and industrial activities ¹. Metals can be accumulated in crops grown in contaminated soils, which pose a risk to human health ³. One of the most wide spread crops in the world is soybean (Glycine max) ⁴. Considering the nutritional ingredients of soybeans, such as protein (40%), oils (20%), carbohydrates (35%), and essential elements, soybeans can be considered as an inexpensive and good source of protein, dietary fiber, and isoflavones, especially in developing countries ^5-8. To determine the metal content in foods, different analytical devices were proposed including atomic absorption spectrometry, inductively coupled plasma optical emission spectrometry (ICP-OES), and inductively coupled plasma mass spectrometry (ICP-MS) ^{1, 9-10}. Prior to metal measurements, sample decomposition is necessary ¹. Wet ashing, dry ashing, and microwave digestion are among the most usual methods for sample decomposition ^{9, 11}. In wet ashing, a mixture of acids with or without oxidants (H₂O₂) was used along with heating in open or closed vessels ^11-12. This method needs constant monitoring and its applicability depends on the type of food ¹³. Dry ashing is a convenient method in which thermal decomposition of samples occurs in thermal furnace at temperatures of 450 to 550 ^°C. Sample preparation is completed by dissolving the ash residues in diluted acids ¹³. Although dry ashing is not suitable for volatile metals and pyrolytic organic materials
(which is resistance to thermal decomposition), it is simpler, safer, and more applicable for large quantities than wet digestion ^13-14. The dried ash samples are completely free of organic matter and are suitable for determining low metal concentrations ¹³.
Consumption of foods contaminated with different metals can contribute to health problems. The potential health risk of metals can be estimated by Target Hazard Quotient (THQ) and Hazard Index (HI) for individual and multiple metals, respectively ¹⁵. Thus, providing information about metal concentration of foods and the health risk evaluation of Iranian soybean consumers seem necessary in this area. In the present study, the THQ and HI of different metals were estimated after investigating the Cr, Mo, Mn, Mg, Se, Ca, and Fe contents of soybeans by two methods of digestion.
Materials and Methods
Reagents
All chemicals were of analytical reagent grade (Merck, Germany), all glass wares were soaked overnight in 10% HNO₃, and rinsed with distilled de-ionized water for three times ¹⁶.
Sample preparation
Three soybean cultivars were purchased from different local markets in Iran. The samples were transported to the laboratory. After cleaning and rinsing, the soybeans were milled with stainless steel blender ¹⁷.
Sample digestion
In dry digestion, one gram of each sample was placed in a crucible furnace until the temperature of 450 ^°C was reached slowly. After 16 hours, the white ash residue was treated with 10 ml HNO₃ (10% v/v). The filtered solution was transferred to 25 ml volumetric flask and diluted by distilled de-ionized water ¹⁶.
In wet digestion, the powdered soybean samples (1 g) were digested with a mixture of HNO₃ (65%), HCl (37%), and H₂O₂ (30%) in ratios of 5:2:1. The mixture was put on a hot plate (60 ^°C) until the digestion was completed ¹⁷.
The digested samples were filtered and diluted to 25 ml with distilled de-ionized water and injected to Inductively Coupled Plasma-Atomic Emission Spectrometry (ICP-AES, SPECTRO GENESIS model). The multi element calibration standard was also used as the standard solution.
Health risk assessment
Regarding the Estimated Daily Intake (EDIs), the concentration of metals in soybean is considered important to estimate the daily intakes. The following equation was used to determine the EDI ^{15, 18}.
EDI= (C×CR)/BW
Where, C is the metal concentration (µg/kg), CR is the average daily consumption (which is evaluated 1.36 g/person/day for an Iranian adult), and BW is the body weight (which is considered 65 kg for an Iranian adult) ¹⁸.
The Target Hazard Quotient (THQ), is determined as the ratio of metal dose to a reference dose (RfD). The proportion of less than 1 represents that the population is exposed to adverse effect of these pollutants ¹⁸.
THQ = EDI/RfD
The reference dose (RfD) for Cr, Fe, Mn, Se, Ca, Mo, and Mg were 1.5, 0.7, 0.014, 0.005, 0.001, 0.005, and 0.14 mg/kg/day, respectively ¹⁹. To assess the non-carcinogenic effects of
more than one metal, the Hazard Index (HI) was used ⁴.
HI=∑THQ= THQ₁+THQ₂+THQ₃+…+THQ_n
Results
The concentrations of metals by two digestion methods are presented in Table 1. It was found that Ca, Fe, Mn, and Mg concentration of dry digestion was estimated higher than that of wet digestion. The highest Cr concentration was found in variety 2 of wet digestion (170.88 mg/kg). The lowest Se and Mo were determined in variety 1 of dry digestion with concentration of 0.21 and 0.071 mg/kg, respectively. The concentrations of Cr, Se, Ca, Fe, Mo, Mn, and Mg in soybean seeds ranged from 0.034 to 170.88 mg/kg, 0.21 to 243.79 mg/kg, 2.50 to 33.37 mg/kg, 0.05 to 0.86 mg/kg, 0.071 to 203.57 mg/kg, 0 to 0.47 mg/kg, and 2.69 to 19.31 mg/kg, respectively.

Wet digestion			Dry digestion			Elements
Variety 3	variety 2	variety 1	variety 3	variety 2	variety 1
33.36d	170.88a	163.41b	51.65c	25.56e	0.034f	Cr
243.79a	171.19c	189.25b	190.56b	40.42d	0.21e	Se
8.06c	16.67b	2.50d	30.14a	33.37a	19.11b	Ca
0.20b	0.86a	0.05b	0.91a	0.81a	0.26b	Fe
33.66d	109.16c	9.33e	160.21b	203.57a	0.071f	Mo
0.04cd	0.33ab	NDd	0.47a	0.25bc	0.072cd	Mn
2.69d	8.77c	4.16d	19.31a	16.5b	15.41b	Mg

Health risk assessment
The estimated dietary intakes and target hazard quotients of the mineral elements are given in Tables 2 and 3, respectively. The EDIs of the mineral elements different pattern in different varieties and methods. According to EDIs, only Fe of all samples was lower than the RfD value. Since the EDIs of Ca, Se, Mo, Mn, and Mg were higher than RfD in most samples, attention should be paid in soybean consumption.
According to Table 3, the THQ of individual mineral elements for three varieties were below 1, but the HI values of Variety 2 and Variety 3 were higher than 1 in both methods. Samples in wet digestion showed higher HI than dry digestion method.

Wet digestion			Dry digestion			EDI Elements
Variety 3	variety 2	variety 1	variety 3	variety 2	variety 1
0.534794	1.080677	3.41904	3.575335	0.697994	0.000711	Cr
0.845711	3.987102	3.959692	3.581822	5.100837	0.004394	Se
0.698203	0.630622	0.052308	0.348788	0.16864	0.39984	Ca
0.016948	0.01904	0.001046	0.017994	0.004185	0.00544	Fe
4.259311	3.352086	0.195212	2.283963	0.704271	0.001486	Mo
0.005231	0.009834	--------	0.006905	0.000837	0.001506	Mn
0.345231	0.404025	0.08704	0.183495	0.056283	0.322425	Mg

 
The HI value or additive effects of contaminants in 2 varieties of soybean were higher than 1 by both wet and dry digestion methods. The variety 2 in wet digestion method had the highest HI value (2.10) followed by variety 3 of wet digestion (1.72). The lowest HI value was reported in variety 1 in the dry digestion method.

Wet digestion			Dry digestion			THQ Elements
variety 3	variety 2	variety 1	variety 3	variety 2	variety 1
3.57 × 10-4	7.2 × 10-4	2.27 × 10-3	2.38 × 10-3	4.65 × 10-4	4.74 × 10-7	Cr
1.69 × 10-1	7.97 × 10-1	7.91 × 10-1	7.16 × 10-1	1.02	8.78 × 10-4	Se
6.98 × 10-4	6.30 × 10-1	5.23 × 10-2	3.48 × 10-1	1.68 × 10-1	3.99 × 10-1	Ca
2.42 × 10-5	2.72 × 10-5	1.49 × 10-6	2.57 × 10-5	5.97 × 10-6	7.77 × 10-6	Fe
8.51 × 10-1	6.70 × 10-1	3.90 × 10-2	4.56 × 10-1	1.40 × 10-1	2.97 × 10-4	Mo
3.73 × 10-3	7.02 × 10-3	---------	4.93 × 10-3	5.97 × 10-4	1.07 × 10-3	Mn
2.46 × 10-3	2.88 × 10-3	6.22 × 10-4	1.31 × 10-3	4.02 × 10-4	2.30 × 10-3	Mg
1.72	2.10	0.88	1.53	1.33	0.40	HI

1.             Demirel S, Tuzen M, Saracoglu S, et al. Evaluation of various digestion procedures for trace element contents of some food materials. J Hazard Mater. 2008;152(3): 1020-6.
2.             Bakircioglu D, Kurtulus YB, Ucar G. Determination of some traces metal levels in cheese samples packaged in plastic and tin containers by ICP-OES after dry, wet and microwave digestion. Food Chem Toxicol. 2011;49(1):202-7.
3.             Zhao Y, Fang X, Mu Y, et al. Metal pollution (Cd, Pb, Zn, and As) in agricultural soils and soybean, glycine max, in southern China. Bull Environ Contam Toxicol. 2014;92(4):427-32.
4.             Salazar MJ, Rodriguez JH, Nieto GL, et al. Effects of heavy metal concentrations (Cd, Zn and Pb) in agricultural soils near different emission sources on quality, accumulation and food safety in soybean [Glycine max (L.) Merrill]. J Hazard Mater. 2012;233:244-53.
5.             Barbosa JTP, Santos CM, Peralva VN, et al. Microwave-assisted diluted acid digestion for trace elements analysis of edible soybean products. Food Chem. 2015;175:212-7.
6.             Cai TD, Chang KC, Shih MC, et al. Comparison of bench and production scale methods for making soymilk and tofu from 13 soybean varieties. Int Food Res J. 1997;30(9):659-68.
7.             Lavado RS, Porcelli CA, Alvarez R. Nutrient and heavy metal concentration and distribution in corn, soybean and wheat as affected by different tillage systems in the Argentine Pampas. Soil Tillage Res. 2001;62(1–2):55-60.
8.             Khalili Sadrabad E, Moshtaghi Boroujeni H, Fallah Mehrjardi AA, et al. Evaluation of Zinc, Cadmium, and Nickel transition from soy milk to soy cheese. Toloo-e-behdasht. 2018;16(6):103-19.
9.             Soylak M, Tuzen M, Narin I, et al. Comparison of microwave, dry and wet digestion procedures for the determination of trace metal. J Food Drug Anal. 2004;12(3): 254-8.
10. Williams CB, Wittmann TG, McSweeney T, et al. Dry ashing and microwave-induced plasma optical emission spectrometry as a fast and cost-effective strategy for trace element analysis. Microchem J. 2017;132:15-9.
11. Lavilla I, Filgueiras A, Bendicho C. Comparison of digestion methods for determination of trace and minor metals in
    plant samples. J Agric Food Chem. 1999; 47(12): 5072-7.
12. Zachariadis G, Stratis J, Kaniou I, et al. Critical comparison of wet and dry digestion procedures for trace metal analysis of meat and fish tissues. Mikrochim Acta. 1995;119(3-4):191-8.
13. Andrade Korn MDG, da Boa Morte ES, Batista dos Santos DCM, et al. Sample preparation for the determination of metals in food samples using spectroanalytical methods—a review. Appl Spectrosc Rev. 2008;43(2):67-92.
14. Enders A, Lehmann J. Comparison of wet-digestion and dry-ashing methods for total elemental analysis of biochar. Commun Soil Sci Plant Anal. 2012;43(7): 1042-52.
15. Pourramezani F, Mohajeri FA, Salmani MH, et al. Evaluation of heavy metal concentration in imported black tea in Iran and consumer
    risk assessments. Food Sci Nutr. 2019;7(12): 4021-6.
16. Altundag H, Tuzen M. Comparison of dry, wet and microwave digestion methods for the multi element determination in some dried fruit samples by ICP-OES. Food Chem Toxicol. 2011;49(11):2800-7.
17. Khalili Sadrabad E, Moshtaghi Boroujeni H, Heydari A. Heavy metal accumulation in soybeans cultivated in Iran, 2015-2016. Field Exch. 2018;3(1):27-32.
18. Zhuang P, Zhi-An L, Bi Z, et al. Heavy metal contamination in soil and soybean near the Dabaoshan Mine, South China. Pedosphere. 2013; 23(3):298-304.
19. Chauhan G, Chauhan U. Human health risk assessment of heavy metals via dietary intake of vegetables grown in wastewater irrigated area of Rewa, India. International Journal of Scientific and Research Publications. 2014;4(9):1-9.
20. Zhou H, Zeng M, Zhou X, et al. Assessment of heavy metal contamination and bioaccumulation in soybean plants from mining and smelting areas of southern Hunan Province, China. Environ Toxicol Chem. 2013; 32(12): 2719-27.
21. Jayakumar K, Jaleel CA. Uptake and accumulation of cobalt in plants: a study based on exogenous cobalt in soybean. Botany Research International. 2009;2(4):310-4.
22. Gonçalves Jr AC, Nacke H, Schwantes D, et al. Heavy metal contamination in brazilian agricultural soils due to application of fertilizers.  Environmental risk assessment of soil contamination: IntechOpen. 2014:105-35.
23. Akinyele I, Shokunbi O. Comparative analysis of dry ashing and wet digestion methods for the determination of trace and heavy metals in food samples. Food Chem. 2015;173:682-4.
24. Saracoglu S, Saygi KO, Uluozlu OD, et al. Determination of trace element contents of baby foods from Turkey. Food Chem. 2007;105(1): 280-5.
25. Zhang T, Xu W, Lin X, et al. Assessment of heavy metals pollution of soybean grains in North Anhui of China. Sci Total Environ. 2019; 646:914-22.