Quantitative Measurement of Toxic Metals and Assessment of Health Risk in Plant-based Food from Markazi Province of Iran

The aim of current study is to measurement and investigate the toxic metals levels in plant-based food collected in Markazi province and human health risk by using inductively coupled plasma - optical emission spectrometry (ICP-OES). The levels of arsenic (As) and cadmium (Cd) in all samples were lower than LOD, while level of Cd in potato samples were lower than permitted limit of European commission (EC). The highest mean of toxic metals were observed for lead (Pb) in legume samples (562.17 µg kg − 1 ). Mercury (Hg) and Pb levels in all samples were higher than LOD, while Pb level in wheat samples were lower than of EC. The rank order of Hg and Pb levels in all samples based on target hazard quotient (THQ) value was wheat > potato > legume. The 95% THQ index of Hg and Pb for adults in wheat samples were 2.59E + 00 and 7.19E-01, in potato samples were 2.07E-01 and 1.64E-01; in legume samples were1.41E-01 and 6.61E-02respectively, while in the case of children, the 95% THQ index of Hg and Pb in wheat samples were 8.90E + 00 and 2.44E + 00; in potato samples were 1.17E + 00 and 5.81E-01; in legume samples were4.77E-01 and 2.20E-01 respectively. The high hazard index values were estimated, indicating a high health risk from consumption of wheat and potato. also the concentration of Cd in potato samples was less than permitted limit of EC. Pb had the highest mean of toxic metals in all plant food samples. The concentrations of Hg and Pb in all samples (legume, wheat, potatoes) of Markazi Province were lower than LOD, and also the concentration of Pb in wheat samples was lower than permitted limit of EC. Wheat was identied as the most important source of toxic metal exposure among children and adults due to its high consumption compared to potatoes and legumes. The results of MCS showed exposed population including adults and children from Markazi Province are at the considerable health risk (HQ > 1 and CR > 1E-4), by ingestion of wheat and potato samples. Therefore, further research to identify the potential sources of contamination with toxic metals and possible control or corrective strategies and continuous monitoring of plant-based food are recommended. Also farmers, craftsmans and others people in the community should be aware of the dangers of food exposed to heavy metals in the province, which can reduce the level of toxic metals in these foods by providing guidelines and action.


Introduction
Plant-based foods contain plant-derived substances, such as cereals, legumes, kernel seed, vegetables and fruits. Plant-based foods are an important part of the human daily diet that can be contaminated with toxic metals such as Hg, Cd, Pb and As, therefore plant-based foods are considered a serious concern for human health (Stefanović et al. 2008) (Dodangeh et al. 2018). The contamination of plant-based food with toxic metals can be related to either natural resources or human activities such as industrial processing, mining, waste disposal and application of wastewater as well as sewage sludge to irrigation of cultivated plant-based food or even application of fertilizers (especially phosphate type) and pesticides (Tadesse et Shariatifar et al. 2017). It is worthy to note that the atmospheric deposition could be accounted as the primary sources of soil, and water contaminations and consequently further contaminations in the food chain . In this regard, plants can absorb toxic metals from the soil, water and air and furthermore concentrate them in different parts such as roots, leaves, and grains (Qu et al. 2012).
Toxic metals as the non-essential compounds for the human body could cause several adverse health effects, even in trace amounts (Yılmaz et al. 2010). Due to their non-degradable nature, they could biologically accumulate in different tissues (kidney, liver, bone, and brain) after their intake through diet as well as inhalation pathways (Bjørklund et al. 2017). For instance, the consumption of contaminated food with Cd can cause bone pain, cardiovascular diseases, and kidney damages (Cheng et al. 2017). Pb can accumulate in the bone and can cause gastrointestinal colitis, hypertension, kidney damages, leukemia, brain dysfunctions, and thrombotic illnesses in human (Fang et al. 2014).
Hg is one of the major pollutants that can cause to brain problems such as hearing, vision and tactile disorders ( In this context, the International Agency for Research on Cancer (IARC) categorized Pb as (group 2B) and Cd as (group 1) a human carcinogenic compound (Cancer 1993). As may cause cancer in human in various organs including the lung, skin, blood and skeleton. (Sarkar and Paul 2016).
The investigation of toxic metals (Cd and Pb) were carried out in the word (all over it) by different analytical methods (Vinas et al. 2000;Salah et al. 2013). Among several method to determine the trace metals in the food samples, the ICP-OES, ame atomic absorption spectrometry (FAAS), and graphite furnace atomic absorption spectrometry (GF-AAS) are the original techniques. Due to saving the time, speci city and more sensitivity of ICP-OES, these technique, it is regarded as better method (Karimi et al. 2012). In various study, agricultural soil is generally considered to be one of the main sources and receptors for heavy metals. However, in Markazi province, the presence of heavy metals in the soil may be from various sources such as industrial activities, lead and zinc mines and fuel combustion, greenhouse gas emissions and municipal waste disposal.
Excessive entry of heavy metals and synthetic chemicals into the studied soils may lead to deterioration of the soil biology, thereby altering the physicochemical properties of the soil and causing other environmental problems. Increasing the pollution of air, water and soil caused by highway tra c, mines, various manufacturing industries and industrial waste is a serious problem that negatively affects the public health (Qu et  The sample size was based on the agricultural elds. In this regard, the samples were taken in January 2019 from agricultural products produced in the Markazi Province. A total of 120 samples were examined (60 samples of legume (chickpea (n = 20), lentil (n = 20) and bean (n = 20), 30 samples of potato and 30 samples of wheat were collected). The experiments were conducted in three levels of replication. After coding of samples based on the type and sampling site. They were put in polyethylene (PE) bags. The bean, potato samples were thoroughly washed with water after transfer to the laboratory in order to remove possible contamination.

Chemical reagents
All chemicals (nitric acid 65%, perchloric acid, sulfuric acid, and hydrogen peroxide 70%) and standard stock solutions of toxic metals with analytical grade (purity > 99%) were purchased from Merck (Darmstadt, Germany). Double-deionized water was used in all dilutions.

Sample preparation
In this study closed vessel, acid decomposition in the microwave oven system (Milestone Ethos D closed vessel microwave system with a maximum power of 1400 W, and the maximum pressure in Te on vessels -100 bar) was used to minimize the effects of the organic matrix as well as to prevent the possibility of sample contamination and loss of analyte. The plant-based food samples were washed with distilled water and dried at 105 °C for 48 h. The dried samples were ground, then homogenized using an agate pestle and sorted in glass bottles until analysis. All of the glass containers used were cleaned by means of soaking, overnight in a 10% nitric acid solution, and then rinsed with deionized water (Türkmen and Dura 2016; Mendil et al. 2004). Triplicate plant-based food samples (0.25 g) were digested with 9 mL of nitric acid (65%) and 1 mL of hydrogen peroxide (70%) in the microwave digestion system for 30 min at a maximum temperature of 300 °C. The residue was then diluted to 10 mL with deionized water in a 10 mL volumetric ask. A blank digest was conduct in the same way (

Condition of Instrument
All prepared samples (triplicate) were analyzed by the aid of an ICP-OES (Spectro Arcos, SPECTRO, Germany) with Torch type of ared end EOP Torch 2.5 mm. The functioning optimum parameters were: RF generator (1400 W), argon gas grade 6 was used for plasma, nebulizer, and auxiliary gas. The gas ow of plasma, auxiliary, and nebulizer were 14.5, 0.9 and 0.85 (L/min), respectively. Afterward, initial stabilization time, time of rinse, time of sample uptake was 240 seconds total and 45 seconds for pre ush. Also, the time between replicate analysis and time of delay was zero.
The analysis was a 3-time replicate and the frequency (resonance frequency) of the generator of RF was 27.12 MHz. The type of solid-state, detector and spray chamber were cyclonic, CCD and Modi ed Lichte, respectively. The type of pump of sample delivery was four-channel, softwarecontrolled; peristaltic pump enables exact sample ows. The Prewash pump speed was 60 rpm (for 15seconds), 30 rpm (for 30seconds) and Prewash time was 45 seconds, and nally, the pump speed of sample injection was 30 rpm.

Validation of the analytical method
The validation of the analytical method for the quantitative analysis of elements present in toxic metals and its aqueous extracts was performed by evaluating selectivity, working and linear ranges, limit of detection (LOD), limit of quanti cation (LOQ), repeatability and reproducibility In this study, "C" is the toxic metals concentration (mg kg − 1 ); "IRi" ingestion rate was set as (legume 19 g day − 1 , wheat 320 g day − 1 and potato 58 g The actual TTHQ act in the both A and B zones was also calculated by using Eq. 3 : If THQ > 1 value, the exposed population is at considerable health risk, but if TTHQ ≤ 1, the health risk is not likely (Dadar et

Statistical analysis
The statistical analysis was conducted with SPSS v.24 using analysis of variance (ANOVA) and Chi-square tests. All experiments were performed in triplicate. The considered signi cant level was p < 0.05.

Results
The wavelengths applied for determination of the elements concentration, based on baseline signals and their interferences at selected lines observed experimentally during the measurements, are presented in Table 1. respectively. The recoveries all the studied elements were 94% and 105% (Table 1). Table 2 illustrates toxic metals content in legume, wheat, and potato samples Markazy province as well as permissible levels for toxic metals. The concentration of As and Cd in plant-based food was lower than the detection limit (LOD), except for Cd in some samples of potato which they were lower than permissible limit of EC. Therefore, there is no need to estimate their non-carcinogenic risk in the population. Table 3 shows the estimated THQ for toxic metals (Hg, Cd and Pb) exposure through food products (legume, potato, and wheat) consumption by adults and children in Markazi province, respectively.   Table 3 and Figure. 2. The results of CR indexes for adults and children due to consumption of toxic metals content in plant-based food samples by MCS is depicted in Figure. 3. The validity of the data in this study was determined by reference to known, elements concentrations. The samples were experimented through the same sample processes. Percentage of standard elements detected at acceptable level, 95%.

Toxic metals concentration
The The results demonstrated that the rank order of food products based on THQ was wheat > potato > legume among adults and children population (Table 3).
Non-carcinogenic risk of wheat was found to higher than potatoes and legumes due to high consumption rate of wheat (320 g/day). As illustrated in Table 2, Markazi province had a hazard index for non-carcinogenic health risks, which can be correlated with the higher concentration of toxic metals in all tested samples. Children because of lower body weight and more intakes of toxic metals in each kilogram of the body weight while compared with adults are exposed to higher risk. The calculated THQ of children was approximately 3times higher than adults in Markazi province as expected ( Figure. (Table 3). In Markazi province is a considerable non-carcinogenic risk (THQ > 1) for the children due to ingestion wheat and potatoes. When TTHQ ≤ 1, risk is improbable, but when TTHQ > 1 shows the probability of adverse effects, when TTHQ > 10, the adverse health effects in exposed population is high for chronic or even acute adverse effects. As Fig. 1 represents, Hg is main contributors in total THQ, whiles the other toxic metals estimated to have less than 20 percent contribution.
Total non-carcinogenic risk from the plant-based food exceeded the safety limit in Markazi Province (TTHQ > 1); for the exposed population, Hg and Pb were indicated as the most evident pollutant leading to non-carcinogenic risk regarding the wheat and potatoes. Because the high daily consumption of wheat and persistent nature of toxic metals during food processing and bioaccumulation of them in the body of human (Stefanović et al. 2008), the associated safety considering the toxic metals concentration is highly concern.
In various study, agricultural soil is generally considered to be one of the main sources and receptors for toxic metals. However, in Markazi Province, the presence of toxic metals in the soil may be from various sources such as industrial activities, Pb and Zn mines and fuel combustion, greenhouse gas emissions and municipal waste disposal.
Excessive entry of heavy metals and synthetic chemicals into the studied soils may lead to deterioration of the soil biology, thereby altering the physicochemical properties of the soil and causing other environmental problems. Increasing air, water and soil pollution caused by tra c jams, mines, various manufacturing industries and industrial waste is a serious problem that negatively affects the public health ( Although the urban structure of this province seems to be advanced, but despite the advanced urban facilities, the main problems caused by air, water and soil pollution of the area may cause pollution in crop base products of the Markazi Province. Plant base products are the main foodstuffs used in many countries around the world, and pollution in most parts of the world has been reported due to soil, groundwater and air. With regard to the increase the level of heavy metals in agricultural soils and their uptake in plant base products, may be a serious health problem has arisen in some parts of the this Province.

Conclusion
The results showed that the concentration of As and Cd in all samples (legumes, wheat, potatoes) were lower than LOD, and also the concentration of Cd in potato samples was less than permitted limit of EC. Pb had the highest mean of toxic metals in all plant food samples. The concentrations of Hg and Pb in all samples (legume, wheat, potatoes) of Markazi Province were lower than LOD, and also the concentration of Pb in wheat samples was lower than permitted limit of EC. Wheat was identi ed as the most important source of toxic metal exposure among children and adults due to its high consumption compared to potatoes and legumes. The results of MCS showed exposed population including adults and children from Markazi Province are at the considerable health risk (HQ > 1 and CR > 1E-4), by ingestion of wheat and potato samples. Therefore, further research to identify the potential sources of contamination with toxic metals and possible control or corrective strategies and continuous monitoring of plant-based food are recommended. Also farmers, craftsmans and others people in the community should be aware of the dangers of food exposed to heavy metals in the province, which can reduce the level of toxic metals in these foods by providing guidelines and action.