Human and Ecological Risk Assessment: An International

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Health risk assessment of heavy metals via dietary

intake of wheat in Golestan Province, Iran

Mojgan Nejabat, Hadi Kahe, Kobra Shirani, Payam Ghorbannezhad, Farzin

Hadizadeh & Gholamreza Karimi

To cite this article: Mojgan Nejabat, Hadi Kahe, Kobra Shirani, Payam Ghorbannezhad, Farzin
Hadizadeh & Gholamreza Karimi (2017): Health risk assessment of heavy metals via dietary intake
of wheat in Golestan Province, Iran, Human and Ecological Risk Assessment: An International
Journal, DOI: 10.1080/10807039.2017.1309265

To link to this article: http://dx.doi.org/10.1080/10807039.2017.1309265

Accepted author version posted online: 24

Apr 2017.

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Health risk assessment of heavy metals via dietary intake of wheat in Golestan Province, Iran

Mojgan Nejabat1, Hadi Kahe2, Kobra Shirani3,Payam Ghorbannezhad4, Farzin Hadizadeh1,*, Gholamreza

Karimi5,**

1
Departement of medicinal chemistry, Faculty of Pharmacy, Mashhad University of Medical Sciences,

Mashhad, Iran

2
Department of Chemistry, Faculty of Sciences, Ferdowsi University of Mashhad, Mashhad, Iran

3
Department of Pharmacodynamic and Toxicology, Faculty of Pharmacy, Mashhad University of Medical

Sciences, Mashhad, Iran

4
Cellulose and Paper Technology Department،Faculty of New Technologies, ShahidBeheshti University,

Tehran, Iran

5
Pharmaceutical Research Center, Pharmacy School, Mashhad University of Medical Sciences, Mashhad,

Iran

*
Corresponding authors: Department of medicinal chemistry, Faculty of Pharmacy, Mashhad

University of Medical Sciences, Mashhad, Iran, E Mail: hadizadehf@mums.ac.ir

**
Corresponding authors: Pharmaceutical Research Center, Pharmacy School, Mashhad University of

Medical Sciences, Mashhad, Iran, E Mail: karimig@mums.ac.ir

ABSTRACT

Concentration of five heavy metals including Lead (Pb), Cadmium (Cd), Copper (Cu), Iron (Fe) and

Zinc(Zn) in wheat collected from silages of Golestan Provice, Iran was determined using atomic

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absorption spectroscopy (AAS). Dry ashing method was applied for precise determination of the five

heavy metal contents. The concentrations of heavy metals were recorded as the following: Pb (0.013-

0.14), Cd (0.008 –0.031) by graphite furnace method and Cu (0.48- 6.2), Fe (58.50 -406.9), and Zn (3.41-

32.75) by flame method all in mg.kg1. The mean concentration of all the above-mentioned heavy metals

were (0.057± 0.003), (0.016± 0.005), (2.7± 0.17), (111.2± 21) and (5.7± 0.22) mg.kg1, respectively. The

level of heavy metals decreased in the order of Fe>Zn>Cu>Pb> Cd. Highest amounts was related to Fe

and the lowest amount to Cd. Concentration of heavy metals in all of the wheat samples were below the

permissible limits set by FAO/WHO and Iranian National Standard Organization and did not pose any

threat to the health of consumers

Keywords

Wheat, Heavy metals, Atomic absorption spectroscopy, Daily intake

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INTRODUCTION

Wheat is one of the most significant grains which plays a vital role in human food chain and similar to

rice, it is the staple and main food in many parts of the world (Doe and Awua et al. 2013). The

Cultivation of wheat dates back to antiquity, when its harvest was essential for human diet (Tejera et al.

2013). The greatest producers of wheat are China, the countries of European Union, India and the United

States of America (Araujo et al. 2008). Recently, food labeling legislation has highlighted the

requirement for determining the elemental composition information to consumers and health

professionals. Food safety has an important role in health policy. In this field, Food and Agriculture

Organization (FAO) and World Health Organization (WHO), European Commission (EC) and some

regulatory bodies of other countries strictly regulate the allowable concentrations or maximum permitted

concentrations of toxic heavy metals in foodstuffs (Bermudez and Jasan et al. 2011).

Cd, Pb, Cu, Zn and Fe are the most common microelement pollutants in the environment. Cd and Pb are

nonessential elements in metabolic processes (Guerra and Trevizam et al. 2012).Cd as a toxic metal does

not have a role in biological systems in living organism, animals and plants(Hussain and Khan 2010). Cd

toxicity in man could lead to anemia, renal damage, bone disorder and cancer of lungs (Edward, and

Idowu et al. 2013). Its accumulation also leads in damaging the nerve cells (Saraf and Samant 2013). Pb

as another toxic element which interferes with the calcium metabolism and gets deposited in the bone.

Acute lead toxicity in humans leads to severe damage in the kidneys, liver, brain, reproductive system,

central nervous system, and sometimes causes death. Poisoning in small amounts of Pb causes anemia,

headache, and sore muscles and might be created a feeling of tiredness and irritability (Ahmed and

Mamun 2001). These elements have toxicity even at a very low concentration (Chirila and Canuta et al.

2009). Studies have shown that wheat more than Rye, Barley and Oats is able to absorb cadmium

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(Jafarnejadi and Homaee et al. 2012). Therefore, they can cause serious problems for the human health.

Hence, content of these toxic elements in wheat should be under control.

Unlike these toxic elements, Fe, Cu and Zn are essential metals for the development of life due to their

importance in many of the biochemical reactions which are specific in human (Škrbić and Čupić 2005).

Cu is an essential redox-active transition element that plays an important role in metabolic processes

(Parab 2016). Cu forms a component in many enzyme systems, such as cytochrome oxidase, lysyl

oxidase and an iron-oxidizing enzyme in blood. The observation of anemia in copper deficiency is

probably related to its role in facilitating iron absorption for incorporating of iron into hemoglobin

(Saraf and Samant 2013). Zn is an important component of many vital enzymes(Ihesinachi and Eresiya

2014). Zinc deficiency leads to coronary heart diseases and various metabolic disorders (Saraf &

Samant, 2013). Fe as another essential metal plays a vital role in human physiology mainly in the

formation of hemoglobin, oxygen and electron transport in human body (Ihesinachi and Eresiya 2014).

These elements are daily necessary in different quantities between milligrams and micrograms (Tejera

etal., 2013). However, if the level of these metals is higher than permissible limits, they leave toxic

effects on human health (Škrbić and Čupić 2005).

The focus of the current study was to determine the concentration of five heavy metals namely Pb, Cd,

Cu, Fe and Zn in wheat samples collected from silages of Golestan province, Iran based the analytical

data provided through atomic absorption spectroscopy technique.

MATERIALS AND METHODS

Sample collection

Wheat samples were collected from 35 silages in Golestan province, Iran. Wheat collection was

conducted in accordance with the Iranian National Standardization Organization (No.13535). Sampling

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was carried out in different parts of a silage and were mixed to obtain a homogeneous mixture. All

samples were collected and stored in polythene bags and brought to the laboratory for preparation and

analysis.

Reagents

Standard stock solutions of different metal ions at the concentration of 1000 mg.mL1 were used to

prepare working solutions after appropriate dilution. All the reagents were analytical grade and deionized

water was used in all dilution procedures. All glassware used was washed with liquid soap and was

soaked overnight in 10% (v/v) nitric acid to avoid metal contamination then rinsed with double distilled

water.

Apparatus

Slurry samples were analyzed by using a Perkin-Elmer model AAnalys 800 atomic absorption

spectrometer equipped with an electrothermal atomizer, Zeeman background correction, AS 800 auto-

sampler and a Hollow Cathode Lamp operated at (217 nm, 10 mA) for Pb and (228 nm, 4mA) for Cd

with a monochromator spectral band pass of 0.7 nm. Pyrolytic coated graphite tubes with a L'VOV

platform and 99.996% purity Argon were used. Cu, Zn and Fe was measured by flame atomic absorption

spectrophotometer equipped with a 100 mm air-acetylene burner head with deuterium background

correction The wavelengths of Zn, Cu and Fe were recorded 213.9, 324.8 and 248 nm, respectively.

Sample preparation

First, samples were dried in an oven during 24 hours to remove the humidity of the wheat flour at 100 C.

Dried samples were then ground into a fine powder by using acid washed pestle. Based on the method

reported by AOAC, 10 g of sample was weighted in porcelain capsules. The sample was incinerated in an

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oven under a temperature that was gradually increased in 50ºC every 60 minutes time until the desired

final temperature was reached to 450 ºC and was left to stand overnight. The resultant ashes were

subjected to another digestion with some drops of 65% concentrated HNO3 in order to help oxidation of

the organic matter that could last, then the rests of nitric was removed through evaporation by means of a

hot plate. After that, it was introduced again into the oven proceed with ashing at 450 C for 1--2 h or

longer until a white/gray or slightly colored product was obtained. Then ash samples were exposed to 5

ml HCl (6M) on a water bath. Residue was dissolved in 10 ml HNO3 (1%) up to a volume of 50 ml. Also,

a sample blank was prepared in the same way as products. Concentrations of heavy metals (Pb, Cd, Cu,

Fe, Zn) were determined using atomic absorption spectrometry (Lars Jorhem 2000).

Validation of method

Samples were spiked with various concentrations of heavy metals for the recovery repeatability tests. The

achieved recovery for all examined microelements was in the range of 70–110%, with a relative standard

deviation less than 20%. This validation also included testing of linearityand limit of detection (LOD) that

was calculated as 3 times the standard deviation of 10 measurements in blank matrix.

Tolerable daily intake (TDI)

The tolerable daily intake (TDI), recommended by the Joint FAO/WHO Expert Committee on Food

Additives (JECFA), show appropriate safe exposure levels and is used to estimate the amount of

contaminants, ingested over a lifetime without appreciable risk. Based on JECFA, the tolerable daily

intakes of metals in this study through wheat consumption has shown in Table 2 (Chamannejadian and

Sayyad et al 2013)

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Estimated daily intake (EDI)

The daily intake of metals depends on both the metal concentration in food and the daily food

consumption. In addition, the body weight can influence the tolerance of pollutants. The estimated

daily intake (EDI) is a concept introduced to take into account these factors. The EDI was calculated

as the following equation (1):

EF × ED× FIR×C
EDI = (1)
WAB ×TA
Where EF is exposure frequency (365 days/year); ED is the exposure duration (70 years), equivalent

to the average lifetime; FIR is the food ingestion rate (334 g/person/day); C is the metal concentration

in wheat (μg/g1); WAB is the average body weight (70 kg) and TA is the averaging exposure time for

non-carcinogens (365 days/ year x ED) (Qian et al., 2010).

Statistical analysis

The results were reported as mean ± standard deviation. Statistical analyses were carried out using SPSS

software and data were analyzed by ANOVA.

RESULTS

Heavy metal concentrations in different wheat samples were presented in table 1. In all cases, highest

concentration of heavy metal related to Fe, whereas the lowest amount associated to Cd. The content of

each element follows the sequence Fe>Zn>Cu>Pb>Cd in analyzed of wheat.

(Table 1)

The results of each heavy metal concentration are discussed as the following:

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Cadmium (Cd)

Cadmium is a heavy metal with a high toxicity even at trace level. Therefore,the level of this element in

food is a world-wide concern for the sanitary authorities who are trying to reduce the concentration of

cadmium in food (Ambedkar & Muniyan 2012). The Cd concentration in our study was varied from0.008

to 0.031mg.kg1 in wheat samples collected from Golestan province, Iran and the mean total content of

cadmium was (0.016± 0.005) mg.kg1. The superimposed calibration curves indicates linearity of the

methods, with excellent values of correlation coefficient 0.9981.The average amount of Cd in the wheat

samplesindicated that its value is lowerthan the maximum permissible limit of 0.2 mg/kg by FAO/WHO

(2001) and 0.03 mg.kg1 by Iranian National Standard Organization. (INSO 2013).

Lead(Pb)

lead is a toxic metal and non-essentialelement for human body(Khan et al. 2011). The level of this

element should be measured even at trace amount.Our results demonstrated that the level of Pb ranged

from 0.013 to 0.14 mg. kg1 in wheat samples. Also, mean total content was 0.057 ± 0.003 mg. kg1 and

calibration curves of Pb showed an excellent values of correlation coefficient 0.9973. The average

amount of Pb indicated that its value was lower than the maximum permissible limit of 0.3 mg. kg1 by

FAO/WHO and 0.15 mg. kg1 by Iranian National Standard Organization. (INSO 2013).

Copper (Cu)

Copper has a physiological function in the organism and is necessary in different quantities between

milligrams andmicrograms daily (Tejera et al. 2013).In our study, concentration of Cu was in the range

of 0.48 to 6.2 mg. kg1 and mean total content was2.7 ± 0.17 mg. kg1. Calibration curve of Cu showed

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an excellent values for correlation coefficient of 0.9978. The FAO/WHO maximum limit for Cu

concentration is 73.3 mg. kg 1 which referring to our results, it was lower than the recommended limit.

Zinc (Zn)

Zinc is an essential metal for all organisms and has an important role in metabolism, growth, development

and general well-being. The content of this heavy metal in mg. kg1 dry weight was in the range from3.41

to32.75 mg.kg1. The mean concentrations of Zn in wheat grain was 5.7± 0.22mg.kg1. The modeling of

Zn indicated a linearity equation in atomic absorption spectrometry with values of correlation coefficient

0.9989. The FAO/WHO maximum limit for Zn concentration is 99.4 mg.kg1and the content of all the

wheat samples was lower than permission limit by FAO/WHO.

Iron (Fe)

Iron is a necessary element in human diet andplays a significant role in metabolic processes(Ihesinachi

and Eresiya 2014).The results showed that Fe has highest concentration of heavy metals in wheat

samples. The maximum and minimum levels of Fe was rangedbetween 58.50- 406.9 mg.kg1. The mean

total content of Fe was 111.2 ± 21 mg.kg1.The content of Fe in the present study was below the value

proposed by FAO/WHO.

DISCUSSION

In the present study, the mean concentrations of five heavy metal were determined in wheat samples and

the results showed that they were below the maximum limits set by WHO/FAO and Iranian legislation for

food sources. The most frequently occurring pattern for wheat was Fe>Zn>Cu>Pb>Cd. The mean of

elements contents in wheat grain were in fair agreement with those reported by various recent surveys

conducted in different countries. The determination of cadmium in six wheat flour samples acquired in

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supermarkets of different Brazilian cities indicated that the cadmium level varied between (0.008.9±0.5)

and (0.013±2) mg.kg −1(Araujo et al. 2008). In another study the level of some heavy metals such as Cu,

Zn, Cd and Pb in different areas of industrial cities in Southeast China were investigated that they were in

the range of (2.43-6.83), (12.06-80.33), (0.006-0.179) and (0.017-1.158) mg. Kg1, respectively.

However, in 1, 6 and 10 wheat samples, Zn, Pb and Cd concentrations were above the permissible limits

of Chinese standard, respectively. similar to our study, concentrations of these elements decreased in the

order of Zn> Cu>Pb>Cd (Huang and Zhou 2008).

Evaluation the level of the heavy metals from two kinds of commercial brands of wheat flours sold in

Calabar-Nigeria showed that the Fe (0.040 mg.kg1) has the highest concentration in Dangote flour

followed by Zn (0.019 mg.kg1), Cu (0.016 mg.kg1) and Cd (0.002 mg.kg1) with the distribution pattern

of Fe> Zn>Cu >Cd. Therefore, the concentration of these heavy metal were within acceptable range

(Edem Christopher and Iniama 2009).

Cadmium in wheat grain from the Swedish long-term soil fertility experiments (n = 120; year 1995–2003)

showed that the level of Cd was between (0.007 – 0.229 mg.kg1), pb (<0.04 -0.16), Cu (1.28 = 6.91) and

Zn (14 -72) mg.Kg1(Kirchmann and Mattsson 2009).

Cadmium was not detected in any of the wheat samples that purchased from Ambo market, Ethiopia

(Tegegne 2015). Determination the content of twelve heavy metals in the two types of whole wheat flour

among50 samples which coming from a wheat flour industry in Canary Islands were illustrated that the

content of micro elements was in the following order: Fe > Zn >Mn> Cu > Cr > Ni in both of the

analyzed types of wheat flour. Samples were safe for toxic elements including Pb and Cd (Tejera et al.

2013). Trace Metals mean in Swedish market from (1987-2009) for Zn, Cu, Fe were reported (6.43-11.2),

(1.45- 2.01) and (9.66- 14.0) mg.kg1 in household and biodynamic wheat flour, respectively. The level of

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Pb was below the LOD of 0.013 mg.kg1 in both kinds of wheat flour (Jorhem and Sundström 2013). A

survey was carried out with the aim of assessing the level of some toxic and essential in wheat grown in

Serbia. The results showed that the mean of all samples (on a dry weight basis) were 29 mg.kg1 for Cd,

137 mg/kg-1 for Pb, 4.1 mg/kg1 for Cu, 24 mg.kg1 for Znand 53 mg.kg1 for Fe. The most frequently

occurring pattern for wheat was Fe >Mn> Zn > Cu >Pb> As > Cd > Hg. The levels of Cd and Pb were

below maximum limits set by Serbian legislation (Škrbić and Čupić 2005).

Estimated daily intake (EDI)

The EDIs of heavy metals for Iranian people via consumption of wheat are presented in Table 2. The

trends of EDIs for heavy metals in wheat were in the order of Fe > Zn >Cu>Cd>Pb.

(Table 2)

The comparison of EDI with the respective TDI for Pb, Cd, Cu, Fe, and Zn are shown in Table 2. It

was assumed that the Iranian population consumes the Golestan wheat, and the EDIs that were

calculated from Eq. (1) is based on heavy metal levels from the wheat samples. The EDI of Pb, Cd,

Cu, Fe and Zn by the Iranian population were 0.27, 0.075, 12.69, 522.64 and 26.79 μg/ kg/bw/per

day, respectively. Thus, EDI values of Pb, Cd, Cu, Fe, and Zn in the present study are much lower in

compare to TDA values. It shows that there is no health risk to Pb, Cd, Cu, Fe, and Zn by the wheat

intake. However, several other dietary and non-dietary factors may also affect heavy metals daily

intake by Iranian consumers. Separate studies would be needed to investigate the other possible

routes.

CONCLUSION

In this study, the monitoring of heavy metals in wheat samples was performed for avoiding unnecessary

exposures. The levels of heavy metals in all of wheat samples were within acceptable range and do not

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pose any threat to the health of consumers. It is important to note that the calculated EDIs in this study

were only obtained through wheat consumption, and intake through other dietaries would probably

increase the EDI values.

The results of this study provide valuable information about the heavy metals contents in wheat, the main

substance of bread, and the staple food of Iranian citizens.

ACKNOLEDGMENT

The authors are thankful to Asrae Shargh laboratory for their help in the process of analysis and Mashhad

University of Medical Sciences for financial support.

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Table1. Mean± SD, recovery and limit of detection (LOD) of heavy metals concentrations from 35

silages of Golestan province, Iran

Heavy Metals Mean ± SD* Recovery LOD LOQ FAO/WHO Safe limit NSO Safe limit
mg.kg1

(% mg.kg1 mg.kg mg.kg1 mg.kg1

[mean±SD])

Cd 0.016 ±0.005 93.05 ± 1.25 0.0007 0.002 0.2 0.03

Pb 0.057±0.003 92.1 ± 10.6 0.002 0.005 0.3 0.15

Cu 2.7 ±0.17 109.6 ± 1.72 0.057 2.0 73.3 -

Fe 111.2 ± 21 73.7 ± 2.16 1.4 5.0 425.5 -

Zn 5.7±0.22 86.1±2.18 0.56 2.0 99.4 -

*
(Mean ±standard deviation)

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Table 2 Estimated daily intake (EDI) of heavy metals for Iranian people via consumption of wheat.

Type of metal Pb Cd Cu Fe Zn

EDI (μg/day kg bw) 0.2679 0.0752 12.69 522.64 26.79

TDI (μg/day kg bw) 3.5 0.8 50 800 30-100

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