Determination of heavy metals in the soils of tea plantations and in fresh and processed tea leaves: an evaluation of six digestion methods
© Rashid et al. 2016
Received: 27 August 2015
Accepted: 5 February 2016
Published: 18 February 2016
The aim of this study was to determine the levels of cadmium (Cd), chromium (Cr), lead (Pb), arsenic (As) and selenium (Se) in (1) fresh tea leaves, (2) processed (black) tea leaves and (3) soils from tea plantations originating from Bangladesh.
Graphite furnace atomic absorption spectrometry (GF-AAS) was used to evaluate six digestion methods, (1) nitric acid, (2) nitric acid overnight, (3) nitric acid–hydrogen peroxide, (4) nitric–perchloric acid, (5) sulfuric acid, and (6) dry ashing, to determine the most suitable digestion method for the determination of heavy metals in the samples.
The concentration ranges of Cd, Pb, As and Se in fresh tea leaves were from 0.03–0.13, 0.19–2.06 and 0.47–1.31 µg/g, respectively while processed tea contained heavy metals at different concentrations: Cd (0.04–0.16 µg/g), Cr (0.45–10.73 µg/g), Pb (0.07–1.03 µg/g), As (0.89–1.90 µg/g) and Se (0.21–10.79 µg/g). Moreover, the soil samples of tea plantations also showed a wide range of concentrations: Cd (0.11–0.45 µg/g), Pb (2.80–66.54 µg/g), As (0.78–4.49 µg/g), and Se content (0.03–0.99 µg/g). Method no. 2 provided sufficient time to digest the tea matrix and was the most efficient method for recovering Cd, Cr, Pb, As and Se. Methods 1 and 3 were also acceptable and can be relatively inexpensive, easy and fast. The heavy metal transfer factors in the investigated soil/tea samples decreased as follows: Cd > As > Se > Pb.
Overall, the present study gives current insights into the heavy metal levels both in soils and teas commonly consumed in Bangladesh.
KeywordsFresh tea Black tea Heavy metals Nitric acid Hydrogen peroxide Perchloric acid Dry ashing GF-AAS
Tea (Camellia sinensis L.) is one of the most popular nonalcoholic beverages, consumed by over two-thirds of the world’s population for its medicinal, refreshment and mild stimulant effects . Tea leaves contain polyphenols such as epigallocatechin 3‐gallate, which has many medicinal properties, including antioxidant , cholesterol-lowering , hepatoprotective  and anticancer activities . Moreover, its detoxifying properties are essential in the elimination of alcohol and toxins . However, considering that an estimated 18 billion cups of tea are consumed daily worldwide , its economic and social importance is unprecedented. In fact, tea has been reported to be valuable in the treatment and prevention of many diseases .
Ideally, tea should be free from contaminants such as heavy metals, which are toxic and harmful to the human body because of their non-biodegradable nature, long biological half-lives and persistent accumulation in different body parts . Tea is consumed in all of Bangladesh throughout the year, and Bangladesh is one of the leading tea producing and exporting countries in the world . In 2006, Bangladesh exported approximately 5 million kg of tea leaves, and this figure continues to increase even while the total local tea consumption in the country is reported to be 39 million kg .
Tea processing and packaging in Bangladesh is dependent on the type of tea, with a wide variety available in the country that is produced by different processing steps. However, the common steps involve the (1) hand plucking of tea leaves by the local farmers, (2) the weighing of tea leaves and (3) transportation to factories. Freshly plucked tea leaves are fragile, and as the first step in processing, the leaves are laid out to dry for several hours to allow them to “wither” as their moisture content decreases. The leaves are then rolled and oxidized, which alters their flavor and gives the processed tea its final appearance and color. The above steps are also known as Crush-Tear-Curl (CTC). The next step involves firing (final drying process), a process that is initiated once the tea leaves have dried. This is followed by visually sorting into various batches of similar sizes and color before being packaged and commercialized both nationally and internationally. For black tea, the leaves are rolled immediately after withering to quickly initiate the oxidation or fermentation processes. The leaves are then completely oxidized before they are dried, which is how they acquire their dark color and rich flavor.
Tea safety has piqued great interest because contaminants threaten the life and health of humans, animals and the environment, leading to economic losses . The genetic and epigenetic effects of dietary heavy metals such as cadmium (Cd), chromium (Cr), lead (Pb), arsenic (As) and selenium (Se) in the human body are associated with an increased risk of different cancers . Prolonged consumption of heavy metals from food can lead to their accumulation in the kidney and liver, causing disruption of numerous biochemical processes and potentially causing cardiovascular, nervous, kidney and bone diseases .
Elemental analysis of a tea sample requires destruction of the organic fraction of the sample, leaving the heavy metals either in solution or in a form that is readily dissolved. Unfortunately, because of a large number of analytes and a variety of sample types, there is no universal sample preparation technique that meets all of the diverse requirements. Among the strategies for sample preparation, dilution, acid digestion and extraction are the most commonly considered [11–20]. Microwave digestion, wet digestion and dry ashing are commonly utilized for the total decomposition of organic matter in samples [11, 21, 22]. Apart from these techniques, ultrasound-assisted solubilisation/extraction sample preparation procedures were reported to be used for green and black tea samples .
Dry ashing consists of the ignition of organic compounds by air at atmospheric pressure and at relatively elevated temperatures (450–550 °C) in a muffle furnace. The resulting ash residues are dissolved in an appropriate acid. Wet digestion is used to oxidize the organic portion of samples or to extract elements from inorganic matrices by means of concentrated acids or mixtures there of . Compared to dry ashing, wet digestion may be performed with a wide variety of potential reagents. Although many types of acids, including hydrochloric acid (HCl), nitric acid (HNO3), sulfuric acid (H2SO4), perchloric acid (HClO4), and hydrogen peroxide (H2O2), are used to digest organic samples and soils [11, 25], it remains undetermined which type of acid/acid mixture is the most suitable.
In addition, little is known about the relative recovery of heavy metals from tea leaves, and there are no standard official methods in Bangladesh for the digestion of tea to determine heavy metals. Moreover, to our knowledge, there is limited data on the amount of heavy metals in fresh tea leaves, processed tea or soils from tea plantations in Bangladesh. Therefore, the aims of this study were (1) to determine the concentrations of common heavy metals such as Cd, Cr, Pb, As and Se in tea leaves and soils from tea plantations; (2) to report the degree of contamination and daily intake of toxic heavy metals via tea (3); to measure the interaction of heavy metal concentrations in fresh tea leaves, processed tea and soils from tea plantations by analyzing the transfer factor (TF); and (4) to evaluate six digestion methods using different acid combinations and recommend the most appropriate digestion method for determining the levels of five heavy metals in tea samples.
Chemicals and reagents
Heavy metal reference standards for Cd, Cr, Pb, As, and Se were purchased from Kanto Chemical (Tokyo, Japan). Digestion chemicals including HCl, HNO3, H2SO4, HClO4, and H2O2 were of analytical grade and were purchased from Merck (Darmstadt, Germany).
Description of study area
Collection and preservation of samples
Soils from tea plantations (n = 10) were randomly collected from locations similar to where the 10 fresh tea leaf samples were collected (from both Sylhet and Moulvibazar districts, Bangladesh). The soil samples (sandy clay loam) were collected (500 g each time) close (1–10 cm perimeter) to the tea plant by digging into the soil (1–5 cm depth). Some of the tea gardens were located near a highway (the closest was within 100 meters), and others were situated very far from the highway.
The collected samples were stored in clean, sterile polyethylene bags and were properly labeled. They were immediately sent to the laboratory of the Agrochemical and Environmental Research Division, Bangladesh Atomic Energy Commission, Dhaka, and were stored at −20 °C to reduce the risk of hydrolysis or oxidation prior to analysis.
Digestion of samples
Digestion of tea samples
Before sample digestion, the tea leaves were freeze-dried at −50 °C at 100 Pa for 24 h. They were then crushed using a sterile mortar and pestle and sieved (particle size <100 µm) at room temperature. Finally, 1 g of tea leaves was used for digestion (refer to the six digestion methods described below).
Digestion of soil samples
Soil samples were oven dried at 60 °C for 24 h before being ground into a fine powder using a sterile mortar and pestle. The samples (2.5 g) were transferred into a crucible before being mixed with 10 mL of aqua regia, which consisted of HCl:HNO3 (3:1). The mixture was the digested on a hot plate at 95 °C for 1 h and was allowed to cool to room temperature. The sample was then diluted to 50 mL using deionized distilled water and was left to settle overnight . The supernatant was filtered through Whatman No. 42 filter paper and (<0.45 µm) Millipore filter paper, (Merck Millipore, Darmstadt, Germany) prior to analysis by graphite furnace atomic absorption spectrometry (GF-AAS).
Method 1 (HNO3 digestion)
Based on the method previously described by Huang et al.  and Narin et al. , the sample (1 g) was placed in a 50 mL crucible before the addition of 10 mL of concentrated HNO3. The sample was heated on a hot plate until the solution became semi-dry. This was followed by the addition of 10 mL of concentrated HNO3. The solution was kept on a hot plate for 1 h to allow the formation of a clear suspension. After the sample was semi-dried, it was cooled and filtered through Whatman No. 42 filter paper. It was then transferred to a 50 mL volumetric flask by adding deionized distilled water to the mark [27, 29] before GF-AAS analysis.
Method 2 (HNO3 overnight digestion)
Concentrated HNO3 (10 mL) was added to the sample (1 g) and allowed to stand overnight at room temperature. The sample was then heated on a hot plate until the solution became clear and semi-dried. The solution was then cooled and filtered through Whatman No. 42 filter paper. It was then transferred quantitatively to a 50 mL volumetric flask by adding deionized distilled water . Finally, the solution was analyzed using GF-AAS.
Method 3 (HNO3–H2O2 digestion)
In this method, the sample (1 g) was weighed into a 50 mL crucible and treated with 10 mL of concentrated HNO3. The solution was placed on a hot plate for 30–45 min to allow for oxidation. After cooling, 4 mL of H2O2 (20 %) was added, and the solution was reheated on a hot plate until the digest became clear and semi-dried. After cooling, the suspension was filtered into a 50 mL volumetric flask and diluted with deionized distilled water to the mark  before GF-AAS analysis.
Method 4 (HNO3–HClO4 digestion)
Approximately 1 g of sample was placed in a 50 mL crucible before the addition of 10 mL of concentrated HNO3. The mixture was placed on a hot plate for 30–45 min to allow for oxidation. After cooling, 5 mL of HClO4 (70 %) was added, and the mixture was reheated on a hot plate until the digest became clear and semi-dried. Then, the sample was cooled and filtered through Whatman No. 42 filter paper before being quantitatively transferred to a 50 mL volumetric flask by adding deionized distilled water [29, 30]. Finally, the solution was analyzed using GF-AAS.
Method 5 (H2SO4 digestion)
The sample (1 g) was placed in a 50 mL crucible followed by the addition of 7 mL of concentrated H2SO4. The mixture was allowed to stand for 30 min at room temperature. Approximately 7 mL of H2O2 (30 %) was added to the crucible, and the sample was reheated on the hot plate for 40 min. Thereafter, 1 mL of H2O2 (30 %) was added until the digest appeared clear upon cooling. Then, deionized distilled water was added to bring the final sample volume to 50 mL. The solution was filtered through Whatman No. 42 filter paper  and then analyzed using GF-AAS.
Method 6 (dry ashing)
Initially, 1 g of sample was placed in a crucible on a hot plate at 100–150 °C for 1 h. It was transferred to a muffle furnace set at 480 °C. After 4 h, the sample was removed from the furnace and cooled. Then, 2 mL of 5 M HNO3 was added, and the sample was evaporated to dryness on a hot plate. The sample was placed in a cool furnace and reheated to 400 °C for 15 min before being removed, cooled and moistened with four drops of deionized distilled water. Then, 2 mL of concentrated HCl was added, and the sample was evaporated to dryness before the addition of 2M HCl (2 mL). The solution was filtered through Whatman No. 42 filter paper and <0.45 µm Millipore filter paper and then quantitatively transferred to a 25 mL volumetric flask by adding deionized distilled water [29, 30].
Operating parameters for the GF-AAS analysis of heavy metals
Detection limit (µg/g)
Slit width (nm)
Lamp current (mA)
Gas flow (L/min)
Temperature programming of GF-AAS for the analysis of Cd, Cr, Pb, As and Se in tea leaves and soil samples
Cd temperature °C, hold time (s)
Cr temperature °C, hold time (s)
Pb temperature °C, hold time (s)
As temperature °C, hold time (s)
Se temperature °C, hold time (s)
Calibration curves for Cd, Cr, Pb, As and Se were prepared at seven different concentrations (0.0, 0.1, 1.0, 5.0, 10.0, 20.0 and 40.0 µg/L).
Determination of the transfer factor (TF)
Results and discussion
Heavy metal contents in fresh tea leaves
Heavy metal contents in fresh tea leaves (FTL)
Mean ± SD (µg/g)
0.07 ± 0.0001
0.05 ± 0.0005
1.84 ± 0.0001
0.03 ± 0.0008
0.31 ± 0.0001
1.66 ± 0.0062
0.90 ± 0.0024
0.08 ± 0.0064
0.40 ± 0.0003
0.13 ± 0.0038
1.14 ± 0.0005
2.06 ± 0.0056
1.28 ± 0.0004
0.07 ± 0.0040
0.05 ± 0.0003
1.17 ± 0.0013
0.78 ± 0.0015
0.09 ± 0.0015
0.09 ± 0.0001
1.45 ± 0.0054
1.31 ± 0.0098
0.11 ± 0.0056
0.22 ± 0.0006
1.48 ± 0.0237
0.06 ± 0.0025
0.46 ± 0.0004
1.81 ± 0.0064
0.47 ± 0.0004
0.13 ± 0.0041
0.44 ± 0.0022
0.77 ± 0.0002
0.12 ± 0.0036
0.19 ± 0.0001
0.85 ± 0.0075
Level of Cd, Pb, As and Se (µg/g) in tea leaves from various countries
The mean Pb concentration in all of the fresh tea leaves investigated was 0.27 ± 0.35 µg/g (Fig. 3), which is lower than the WHO-recommended limit of 0.30 µg/g . This is also lower than the Pb content of tea leaves from Turkey (17.90 ± 7.10 µg/g)  as well as tea leaves from India (1.86 ± 0.04 µg/g), China (1.49 ± 0.03 µg/g) and Japan (1.55 ± 0.03 µg/g), but is slightly higher than that from Italy (0.23 ± 0.01 µg/g) . Pb is a cumulative toxin that can primarily affect the blood, nervous system and kidneys. If present in high concentrations, Pb inhibits red blood cell formation, which can result in anemia .
The mean As concentration in fresh tea leaves was 1.21 ± 0.74 µg/g (Fig. 4), which is higher than the WHO-recommended limit (0.10 µg/g)  and higher than that of green tea from China (0.28 µg/g) , Thailand (0.013 µg/g) , Canada (0.04 µg/g)  and Japan (0.00 µg/g). A potential source of As is the high amount of As present in the soils of the studied tea plantations. As is toxic to humans, especially in its methylated forms produced by glutathione s-transferase (GST), As III methyltransferase (AS3MT) and S-adenosyl methionine (SAM). These enzymes can compete with DNA methyltransferase (DNMT) for DNA methylation, hence indirectly inhibiting DNA methyltransferase and inducing the reactivation of silenced tumor suppressor genes (Mishra et al. 2009). Chronic toxicity from high exposure to inorganic As is associated with arsenicosis, melanosis, keratoses of the skin and cancer .
The Se content of all investigated fresh tea leaves was 0.64 ± 0.50 µg/g (Fig. 4), and the WHO-recommended limit and contents of Japanese sencha green tea, Japanese jasmine tea, Chinese pai mu tan tea and Chinese gunpowder tea were 0.125, 0.092, 0.089, 0.075 and 0.070 µg/g, respectively . Se can lead to selenosis if taken in doses exceeding 400 µg per day . Symptoms of selenosis include a garlic odor of the breath, gastrointestinal disorders, hair loss, sloughing of nails, fatigue, irritability and neurological damage. Extreme cases of selenosis can result in cirrhosis of the liver, pulmonary edema and death .
Heavy metal contents in black tea
Heavy metal contents in processed tea leaves (PTL, black tea)
Mean ± SD (µg/g)
0.16 ± 0.0013
9.31 ± 0.0493
0.27 ± 0.0008
1.90 ± 0.0006
1.44 ± 0.0038
0.10 ± 0.0031
7.03 ± 0.0156
0.70 ± 0.0004
0.12 ± 0.0023
10.73 ± 0.0348
0.40 ± 0.0009
1.17 ± 0.0153
0.80 ± 0.0002
0.04 ± 0.0030
2.10 ± 0.0004
0.07 ± 0.0021
1.40 ± 0.0036
0.76 ± 0.0023
0.06 ± 0.0031
1.71 ± 0.0032
0.31 ± 0.0008
0.89 ± 0.0017
0.11 ± 0.0030
0.45 ± 0.0026
0.22 ± 0.0006
1.78 ± 0.0066
10.79 ± 0.0065
0.05 ± 0.0001
2.75 ± 0.0086
0.66 ± 0.0002
1.02 ± 0.0030
0.44 ± 0.0003
0.07 ± 0.0004
1.19 ± 0.0084
1.03 ± 0.0011
1.16 ± 0.0006
0.05 ± 0.0035
0.72 ± 0.0006
1.00 ± 0.0060
1.89 ± 0.0101
0.07 ± 0.0020
0.54 ± 0.0049
1.30 ± 0.0004
0.21 ± 0.0016
The mean concentration of Cd in black tea (0.08 ± 0.04 µg/g) (Fig. 3) was lower than the World Health Organization (WHO)-recommended limit of 0.10 µg/g , but higher than that reported in black tea from Canada (0.026 µg/g) , Thailand (0.0071 µg/g)  and Turkey (0.0100 µg/g) . However, its level was lower than that reported in India (0.8900 µg/g) , Nigeria (0.1200 µg/g) and Saudi Arabia (0.9890 µg/g) . Moreover, our result was also lower than Cd content of black teas from Turkey (2.30 ± 0.40 µg/g) . In a previous study, the concentration of Cd was 0.03 µg/g  which is slightly lower than that of our findings. In another study, the presence of some trace elements (Cu, Ni, Mn and Zn) in three commercially available tea from Bangladesh were analyzed . Nevertheless, they were different from that of the current investigation.
Cr was detected in rather high amounts in black tea (3.581 ± 3.941 µg/g), but it was not detected in fresh tea leaves or tea plantation soils (Fig. 3). Its level is higher than the recommended limit for Cr by the WHO of 0.05 µg/mL . Moreover, Cr concentrations in black tea from India, China, Sri Lanka and Turkey were reported at 0.371, 0.155, 0.050 and 3.000 µg/g [39, 44], respectively. It is plausible that Cr contamination occurred during the fermentation process, which is one of the important processing steps of black tea in Bangladesh. In particular, it may occur during the CTC rolling steps involved in the production of black tea. However, this finding is lower than the previously reported Cr concentration (32.87 µg/g) in some tea samples from Bangladesh  which may be contributed to the different types of tea samples used as well as variance in the type of soil in the tea garden.
The Pb concentration in black tea was 0.438 ± 0.328 µg/g (Fig. 3), which is higher than the WHO recommended limit of 0.30 µg/g . Moreover, our findings are also similar to the previously reported concentration of Pb (0.34 µg/g)  in tea samples from Bangladesh but is higher than those reported for Nigeria (0.330 µg/g) , Egypt (0.395 µg/g) and Thailand (0.0237 µg/g) , but lower than that in Turkey (2.500 µg/g) , Iran (2.915 µg/g), Saudi Arabia (1.250 µg/g), China (3.270 µg/g), Pakistan (2.500 µg/g) and India (0.810 µg/g) .
The mean As concentration in black tea was 1.162 ± 0.524 µg/g (Fig. 4, which was higher than the WHO-recommended limit (0.10 µg/g) , as well as higher than in Thailand (0.00084 µg/g) and China (0.280 µg/g) . However, it was lower than that reported in Nigeria (2.220 µg/g) . The Se content in black tea from Bangladesh was higher (1.633 ± 3.280 µg/g) (Fig. 4) than that reported in black tea from Nigeria , India, China and Sri Lanka , which were 0.520, 0.070, 0.087 and 0.050 µg/g, respectively.
Heavy metal contents in soils from tea plantations
Heavy metal contents in soils from tea plantations (STP)
Mean ± SD (µg/g)
0.16 ± 0.0013
66.54 ± 0.5200
0.15 ± 0.0042
10.65 ± 0.0120
2.75 ± 0.0013
0.34 ± 0.0019
63.63 ± 4.2400
1.13 ± 0.0041
0.24 ± 0.0016
8.65 ± 0.0420
4.49 ± 0.0128
0.22 ± 0.0021
11.86 ± 0.0660
1.79 ± 0.0017
0.45 ± 0.0015
6.90 ± 0.2000
2.32 ± 0.0022
0.16 ± 0.0038
2.80 ± 0.0100
1.08 ± 0.0117
0.03 ± 0.0003
0.23 ± 0.0015
9.48 ± 0.0260
3.03 ± 0.0004
0.16 ± 0.0011
3.60 ± 0.0350
0.99 ± 0.0000
0.11 ± 0.0059
10.22 ± 0.4400
0.78 ± 0.0157
0.81 ± 0.0032
Similar to the findings for fresh tea leaves, Cr was not detected in the tea garden soil samples (Fig. 4). However, Cr has been reported in agricultural soils in the United States (48.5 µg/g) , India (1.23 µg/g)  and Kunshan, China (87.73 µg/g) . Low concentrations of Cd (mean 0.222 ± 0.103 µg/g) were observed in all investigated soils from the tea plantations samples (Fig. 3). These levels were lower than that previously reported in U.S. agricultural soils (13.5 µg/g) , but higher than in Indian agricultural soils (0.05 µg/g)  and soil from Kunshan in China (0.20 µg/g) .
Because of the toxicological importance of Pb, many studies have investigated the levels of this element in soil from several countries. Among all of the soil samples investigated, STP-1 had the highest Pb concentration (66.54 ± 0.520 µg/g) potentially because of its location, which was adjacent to a highway. Overall, the mean level of Pb in the tea plantation soil samples was 19.43 ± 24.25 µg/g (Fig. 3). This is higher than that reported for agricultural soils in India (2.82 µg/g)  but lower than agricultural soils in the U.S. (55.00 µg/g)  and Kunshan, China (30.48 µg/g) .
The concentrations of As ranged from 0.78 to 4.49 µg/g. The highest As level was 4.49 µg/g in STG-4, but As was not detected in STP-1 or STP-9. The mean concentration of As was 1.74 ± 1.429 µg/g (Fig. 4), which is lower than that reported in Kunshan, China (8.15 µg/g) . Among all of the investigated soil samples, the mean Se concentrations in STP-1, STP-2, STP-3, STP-4, STP-5, STP-6 and STP-8 were below the detection limit. Low Se contents (mean 0.18 ± 0.398 µg/g) (Fig. 4) have also been reported in soils from garlic (0.026 µg/g), radish (0.028 µg/g), carrot (0.011 µg/g) and orchard grass (0.069 µg/g) plantations . In comparison, higher Se concentrations were detected in the soils of oilseed rape (0.316 µg/g), white clover (0.211 µg/g), red clover (0.223 µg/g) and English plantain (0.277 µg/g) plantations . These higher Se concentrations may be attributed to fertilizer (sodium selenite) use in tea plantations. High levels of heavy metals such as Se and As can potentially be easily transported to the tea leaves through the roots of the plant from contaminated soils. In addition, the acidic nature of tea garden soils can increase the extraction of As and hence the detected As concentration.
Heavy metal transfer from soils to tea leaves in Bangladesh
Transfer factors of heavy metals from tea plantation soils of tea leaves
Recovery analysis (n = 2) of heavy metals and LODs of investigated methods for method validation
Percentage, LOD (µg/g)
Method 1 (HNO3)
Method 2 (HNO3 overnight)
Method 3 (HNO3 and H2O2)
Method 4 (HNO3 –HClO4)
Method 5 (H2SO4)
Method 6 (Dry ashing)
Method 1 (destruction with HNO3) and Method 3 (digestion using HNO3 and H2O2) yielded acceptable recoveries of Cd, Cr, Pb, As and Se. However, only 70.70 % of Pb was recovered by Method 1, which is below the acceptable limit. Therefore, Methods 1 and 3 could also be used as relatively inexpensive, simple and rapid substitutes. Method 6 (Dry ashing) is not recommended because of the high cost incurred due to the requirement of a muffle furnace. Method 4 (HNO3–HClO4 procedure) is also not recommended because HClO4 is potentially hazardous during digestion. This method also yielded poor recoveries. For all procedures, recovery of Cd was significantly higher, while recovery of Pb was relatively lower. The likely reason for the lower recovery of Pb is the effect of the acidic pH used during sample digestion, which does not favor sample extraction.
Six digestion methods followed by GF-AAS have been successfully optimized in the present study. An overnight digestion with nitric acid (method no. 2) offered adequate time to digest the tea matrix and was the most efficient method for recovering Cd, Cr, Pb, As and Se. Moreover, Methods 1 and 3 were also satisfactory, relatively cheap, simple and fast. Method no. 5 is not recommended for the digestion of tea samples while method no. 6 was expensive. Cd, Pb, As and Se were detected in fresh tea leaves, but Cr was not detected. The concentrations of As were high in both fresh and black tea, while the concentration of Pb and Cr in black tea was higher than the recommended level set by the WHO. The soil from tea plantations was contaminated with As and Se, levels of which were at times higher than the WHO recommendation. High levels of heavy metals can easily be transported to tea leaves through the roots of tea plants. However, Cr was not detected in the soil samples. The trend in heavy metal TFs in the investigated tea samples was Cd > As > Se > Pb. An overnight digestion with HNO3 was the most efficient digestion method for recovering heavy metals.
HR and ZF conducted the experiments, analyzed the data, and wrote the manuscript. AZC, KA and LB designed the experiments and supervised the work. MM analyzed the data and wrote the manuscript while SHG critically revised the manuscript. All authors read and approved the final manuscript.
The authors would like to acknowledge IAEA for financial assistance under IAEA Research Contact no. 15052/R2 and Universiti Sains Malaysia Research University Team (RUT) Grant (1001/PPSP/853005). We would also like to thank Bangladesh Atomic Energy Commission for providing laboratory facilities to carry out the study and Abdullah-Al-Masud Mazumder, Botanist of Bangladesh Forest Research Institute, for authenticating the tea leaf samples.
The authors declare that they have no competing interests.
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