Evaluation of heavy metal concentration in imported black tea in Iran and consumer risk assessments

Abstract Tea grows in the contaminated soils, absorbs the heavy metals, and enters them into the human food chain. The concentrations of Pb, Cd, Cu, As, and Hg of the imported black tea leaves to Hormozgan Province were evaluated by atomic absorption spectrometer. Then, the Hazard Quotient (HQ) and Hazard Index (HI) levels of heavy metal intakes were calculated to estimate the health hazard for consumers. The Pb, Cd, Cu, As, and Hg concentrations in the Sri Lankan and Indian blank tea were 0.14, 0.017, 11.29, 0.057, 0.0076 mg/kg, and 0.21, 0.02, 14.56, 0.067, 0.01 mg/kg, respectively. It was found that except for As concentration in Indian black tea were higher than Sri Lankan black tea. The HQ and HI levels of all studied metals were less than one, but they were higher in Indian black tea compared with the Sri Lankan black tea. The HI of Indian and Sri Lankan black tea samples were 0.061 and 0.048, respectively, which indicated no significant health hazard for tea consumers. The results showed that the consumption of the studied tea could not have any risk of heavy metal exposure.

human body which could cause adverse effects on human health (Nkansah et al., 2016;Zhang et al., 2018). World Health Organization introduced arsenic (As), lead (Pb), mercury (Hg), and cadmium (Cd) among 10 chemicals which considered as major public health concerns (RoyChowdhury, Datta, & Sarkar, 2018). The presence of excess amounts of heavy metals such as Pb, As, Cu, Cd, and Hg in tea can increase the concentration of toxic metals in the human body.
The excessive intake of these elements by humans leads to poisoning and causes various complications, such a renal failure, methemoglobinemia, liver cirrhosis, and impairment in the formation of red blood cells (Escott-Stump, . It has been estimated that about 50 percent of black tea in Iran market were imported from different countries. Regarding the habit of drinking tea among Iranian, evaluation of the heavy metal concentration and their maximum allowable levels in tea as a basic part of health risk assessment could be effective on the protection of human health (Cao, Qiao, Zhang, & Chen, 2010). So, the objective of the present study was the evaluation of heavy metal (Pb, Cd, Cu, As, and Hg) concentration in black tea leaves imported to Iran from India and Sri Lankan at 2017 by atomic absorption spectrometry. Consequently, the health risk of these heavy metals in Iranian tea consumers was assessed by Hazard Quotient (HQ) and Hazard Index (HI) methods, which show the potential noncarcinogenic effects of individual and multiple metals, respectively (Zhang et al., 2018).

| Apparatus
All glasswares were washed and rinsed by distilled water to remove any particles. Then, they were soaked and kept overnight in 10% (v/v) nitric acid (Merck). After that, the glasswares were washed with distilled water, rinsed, and dried before use (Sadrabad, Boroujeni, & Heydari, 2018). The atomic absorption spectrometer (SavantAA) was used to determine the concentration of heavy metals. The operational characteristics for measurement of each heavy metal are mentioned in Table 1.

| Sampling
The sampling was done according to Institute of Standards and Industrial Research of Iran (ISIRI, 2008). A total of 122 black tea samples were randomly collected from local market of Hormozgan, Iran. The origin of black tea samples was from India and Sri Lanka.

| Sample digestion and analysis
For determination of Pb, Cu, and Cd, the dry digestion was done by burning 5 g of each tea powder sample by an electric furnace at 450 ± 5°C for 8 hr. The residuals were solved in 5 ml 37% (v/v) hydrochloric acid (Merck) and diluted by deionized distilled water to reach 50 ml volume. The Pb and Cd contents of samples were analyzed using graphite furnace atomic absorption spectrometer (GFAAS, SavantAA GBC). The Cu content of samples was evaluated by flame atomic absorption spectrophotometer (ISIRI, 2007).
For As determination, 5 g of each sample was mixed with MgNO 3 and 6H 2 O 8MgO and 32% nitric acid (Merck) to a ratio of 20:5, then, the mixture was put on hotplate (100 ± 5°C) until it was evaporated.
Then, the mixture was put into electric furnace (425 ± 25°C) for 12 hr. The heating by electric furnace (400°C) was continued for 2 to 3 hr along with adding 5 ml nitric acid (65%). The dried samples were treated by distilled water and mixture of potassium iodide and ascorbic acid in HCl (6M), in a ratio of 1:5:5 ml. The obtained solution was filtered through 0.45 µm membrane filter and diluted to 25 ml using HCl (6M). The As content of samples were measured by hydride generation atomic absorption spectrophotometer (ISIRI, 2013).
In order to measure Hg, 5g of the powdered samples were weighted. Then, nitric acid (7M), sulfuric acid (9M), and sodium molybdate (2%) in a proportion of 20:25:1 ml were added to each sample. The mixture was heated by electrical heater for 1 hr. The heating was continued by adding 10 ml of distilled water. Then, the digested samples were filtered and reached 100 ml by sulfuric acid solution (0.5M). The cold vapor atomic absorption spectrophotometer was used for the determination of Hg of samples.

| Health hazards estimation
The Hazard Quotient (HQ) of each metal was evaluated for the determination of noncarcinogenic health risks in Iranian tea consumer.
The HQ less than 1 shows no significant risk of noncarcinogenic effects in consumers (Zhang et al., 2018). The HQ is calculated by equation described by Cao et al as follows (Equations 1 and 2) (Cao et al., 2010).
where ADD is the average daily intake of metal (μg/kg/day) and RFD is the daily intake reference dose. According to Environmental Protection Agency (EPA), RfDs for Cd, Cu, and As are 1, 40, and 0.3 μg kg −1 day −1 , respectively (Li, Fu, Achal, & Liu, 2015). The rate of RfD was not calculated by US-EPA for Hg and Pb. In this study, the provisional tolerable weekly intake (PTWI) given by WHO was used for Hg (5 μg kg −1 week −1 ) and Pb (25 μg kg −1 week −1 ), respectively (Cao et al., 2010).

In above Equation 2, C is the mean concentration of metals and IR
is the consumption rate which was considered as 6 g/day according to the study was done by (Naghipour, Amouei, Dadashi, & Zazouli, 2016). Furthermore, BW is the average weight of consumers, which was estimated 65 kg.
The Hazard Index (HI) could calculate the overall or interactive effects of exposure to two or more contaminants (Cao et al., 2010;Zhang et al., 2018). Therefore, total noncarcinogenic health risk of multiple contaminants is estimated as bellow (Equation 3) (Cao et al., 2010).

| Data analysis
The data were analyzed by the SPSS16 software. Mann-Whitney test was used to determine the abnormal information, and the onesample t test was applied to compare the quantitative values.

| Heavy metal concentration
According to Table 2, Pb, Cd, Cu, As, and Hg were 0.14, 0.017, 11.29, 0.057, and 0.0076 (μg/g) in Sri Lankan tea, respectively, which were less than the acceptable limit. The results (Table 2) show that the level of Pb, Cd, Cu, As, and Hg was 0.21, 0.02, 14.56, 0.067, and 0.01 (μg/g) in Indian tea, respectively. It was indicated that the total amount of metals in Indian tea samples was lower than the acceptable limit. The results of the statistical analysis indicated that Pb, Cd, Cu, and Hg were significantly higher in Indian tea than the Sri Lankan tea samples. However, no significant difference was observed between the Sri Lankan and Indian tea with regard to As.
According to Table 3, the correlation coefficients among the concentrations of metals in black tea samples are given. The metal-tometal correlation was not significant for all samples, and each metal was independent of the other elements, except Cu, Pb, and Cd. In other words, Cu had positive correlation with Pb and Cd.

| Estimation of HQ and HI
The result of Hazard Quotient and Hazard Index are presented in

| D ISCUSS I ON
Previous studies indicated that the excessive levels of heavy metals in tea and their absorption in the human body can cause poisoning and resulted in several problems . Due to the role of heavy metal on human health, the concentration of heavy metals such as Pb, Cu, Hg, As, and Cd was evaluated in two samples of the imported tea in Hormozgan Province. Then, the noncarcinogenic effects of individual and multiple metals were determined by the HQ and HI, respectively. The mean concentration of heavy metals in Indian and Sri Lankan black tea showed the following order: Cu > Pb > As > Cd > Hg. Peng et al. showed the mean of Cu in tea was higher than those of Pb and Cd, which is consistent with the results of the present study . The results showed that the concentration of Pb, Cd, Cu, and Hg in Indian tea samples were significantly higher than those in Sri Lankan tea.
However, no significant difference was observed between the Sri Lankan tea and Indian tea in terms of As.
(3) HI = HQ 1 + HQ 2 + HQ 3 +…+HQ n TA B L E 2 Average heavy metals of lead, cadmium, copper, arsenic, and mercury (μg/g) in Indian and Sri Lankan tea samples Qin and Chen reported that the mean concentration of Pb in tea samples of the Chinese market was 1.32 mg/kg, which was higher than the allowable limit of the Chinese Ministry of Health (0.3 µg/g) in some samples (Qin & Chen, 2007 (Karimzadeh et al., 2013). Nasri reported that the concentrations of Pb, Cd, Cu, and As in different tea samples were all in the range of 0.049-10.12, 0.016-0.123, 3.05-37.41, and 0.0431-0.287 mg/kg, respectively. The maximum concentration of Cu in the current study was higher than the results of Nasri et al (Nasri, Amini, & Mohammadi, 2017). The study by Ansari et al. showed that the average Cu in cultivated black tea was 29.3 mg/ kg (Ansari, Norbaksh, & Daneshmand, 2007). Naithani and Kakkar (2005) indicated that the concentration of Cu in tea was 11.1 µg/g, which is less than the concentrations in Sri Lankan and Indian tea samples of the current study (Naithani & Kakkar, 2005). Studies conducted in Japan, Turkey, and Saudi Arabia on heavy metals in tea represented that the concentrations of Cu were 27.7, 24.8, and 18.1 mg/kg, respectively, which are higher than the Cu concentration of the current study (Ashraf & Mian, 2008;Matsuura, Hokura, Katsuki, Itoh, & Haraguchi, 2001;Narin, Colak, Turkoglu, Soylak, & Dogan, 2004). Naghipour et al. (2016)  These differences in results are due to the difference in cultivation region, soil conditions, height of the land from sea, rainfall, and other environmental conditions (Barone et al., 2016;Karimzadeh et al., 2013). However, the use of phosphate fertilizers and fungicides in agriculture may also contaminate herbal foods with heavy metals. These materials often contain significant amounts of toxic elements such as Hg, Cd, Pb, and Cu (Ferreira et al., 2015;Karak & Bhagat, 2010;Soliman, 2016). The concentration of heavy metals in tea plant is greatly caused by the presence of factors such as combination of metals in soil, the plant species, the pH of the growth environment, and the genetic characteristics of the tea plant (Foy, Chaney, & White, 1978). Although, high amounts of heavy metals in tea can be attributed to dust particles during the production process as well as contaminations during the packaging process (Ashraf & Mian, 2008 India ADD 1.9 × 10 -3 1.8 × 10 -3 9.2 × 10 -4 1.34 6.1 × 10 -3 HQ 5.3 × 10 -3 1.9 × 10 -3 1.2 × 10 -3 3.36 × 10 -2 2 × 10 -2 HI ꞊ ∑ HQ 6.1 × 10 -2 Sri Lanka ADD 1.2 × 10 -2 1.5 × 10 -3 7 × 10 -4 1.04 5.2 × 10 -3 HQ 3.5 × 10 -3 1.5 × 10 -3 9.8 × 10 -4 2.6 × 10 -2 1.7 × 10 -2 HI ꞊ ∑ HQ 4.8 × 10 -2 2010). As mentioned previously, HI values of less than one indicated that the sample had no health risk. The values of HI in Indian and Sri Lankan tea samples of the present study were 0.061 and 0.048, respectively. In the study by Cao et al. (2010), the amounts of HI in Puerh tea were 0.17 and 0.29 among the consumers of Kunming and Puerh cities, respectively, which are higher than the amounts of HI in the present study. In another study by Zhang et al. (2018) on fresh and mature tea leaves, the amount of HI in fresh leaves was less than one which was consistent with the present study. The HI of five metals of the present study in Indian and Sri Lankan black tea samples indicated no significant health hazards for Iranian consumers.

| CON CLUS ION
The findings of the current study indicated that heavy metal concentrations in tea samples were at allowable level. The amounts of HQ and HI were less than one in all studied metals. However, comparison of the HQ values between the Indian and Sri Lankan tea samples showed that the Indian tea had higher HQ values.
However, it was showed that the consumption of the Indian and Sri Lankan tea samples is not harmful with regard to the presence of Cd, Pb, Cu, Hg, and As. Considering that Hormozgan Province is placed in a special location and the major imports of the country, it is recommended that imported tea would be controlled, traced, and checked to ensure the health of consumers and provide their psychological and economic security. The active, real, continuous, and reliable monitoring of the administrative organizations will ensure the safety of consumers.

ACK N OWLED G EM ENTS
The authors acknowledge the cooperation and assistance of the laboratory department of Food Hygiene and Safety of the School of Public Health of Yazd, Shahid Sadoughi University of Medical Sciences.

CO N FLI C T S O F I NTE R E S T
The authors declare no conflict of interest.

E TH I C A L A PPROVA L
The current study does not involve any human or animal testing.