Distribution, accumulation, and potential risks of heavy metals in soil and tea leaves from geologically different plantations

Ecotoxicology and Environmental Safety 195 (2020) 110475

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Distribution, accumulation, and potential risks of heavy metals in soil and tea leaves from geologically different plantations

T

Jian Zhanga, Ruidong Yanga,∗, Yuncong C. Lib, Yishu Pengc, Xuefeng Wend, Xinran Nia a

College of Resource and Environmental Engineering, Guizhou University, Guiyang, 550025, China Department of Soil and Water Sciences, Tropical Research and Education Center, IFAS, University of Florida, Homestead, FL, 33031, USA c College of Tea Science, Guizhou University, Guiyang, 550025, China d College of Agriculture, Guizhou University, Guiyang, 550025, China b

A R T I C LE I N FO

A B S T R A C T

Keywords: Tea leaves Soil heavy metals Bioaccumulation factor Pearson correlation analysis Health risk assessment

Risk assessment regarding heavy metals in tea is crucial to ensure the health of tea customers. However, the effects of geological difference on distribution of heavy metals in soils and their accumulation in tea leaves remain unclear. This study aimed to estimate the impacts of geological difference on distribution of cadmium (Cd), lead (Pb), thallium (Tl), mercury (Hg), arsenic (As), antimony (Sb), chromium (Cr), nickel (Ni), and manganese (Mn) in soils and their accumulation in tea leaves, and further evaluate their health risks. 22 soils and corresponding young tea leaves (YTL) and old tea leaves (OTL), from geologically different plantations, were sampled and analyzed. Results showed that heavy metals concentrations in soils, derived from Permian limestone and Cambrian weakly mineralized dolomite, were obviously greater than those from Silurian clastic rock. The geological difference controlled the distribution of soil heavy metals to a large extent. Contents of Cd, Tl, and Mn in tea leaves mainly depended on their contents in soils. Soil Hg, Pb, As, and Sb contents may not be the only influencing factors for their respective accumulation in tea leaves. More attentions should be paid to soil acidification of tea plantations to ensure the tea quality security. Target hazard quotients (THQ) of Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni and hazard index (HI) via tea intake were below one, indicating no human health risk. The non-mineralized Silurian area was less at risk of heavy metals accumulation in tea leaves than the Cambrian metallogenic belt and the Permian Cd-enriched zone. This study could provide an important basis to understand and mitigate the potential risks of heavy metals in tea.

1. Introduction Heavy metals accumulation in soils not only reduces crop quality and production but also poses environmental and health risks. The consumption of foods and beverages is an important pathway for human exposure to toxic heavy metals (de Oliveira et al., 2018). Toxic heavy metals accumulated in different parts of human body can strongly damage the human health and cause carcinogenic, mutagenic, and teratogenic toxicity since they are nonbiodegradable (Shaheen et al., 2016). Tea, made from the leaves of Camellia sinensis, is one of the most frequently consumed beverages worldwide, mainly because of its pleasant aroma, flavor, and refreshing characteristics (Fung et al., 2009; Peng et al., 2018). Recent statistics estimated that the global area under tea cultivation is ~407 × 104 hm2, producing around 6 × 104 metric tons. China accounted for 54% and 41% of global tea acreage and production, respectively (Zhang et al., 2018b). Guizhou province ranks

number one in China for tea production, comprising a tea production area of 4.78 × 105 hm2 (Zhang et al., 2018b). Moreover, the teas produced in Guizhou province of China have been exported to 23 countries or regions (e.g., Russia, Germany, the United States, Myanmar, Morocco, and Belgium) worldwide. Several studies have reported that the concentrations and possible health risks of heavy metals in tea leaves, made tea, and tea infusions, such as lead (Pb), arsenic (As), chromium (Cr), cadmium (Cd), nickel (Ni), mercury (Hg), antimony (Sb), and thallium (Tl) (Li et al., 2015; de Oliveira et al., 2018; Zhang et al., 2018a, 2018b; Sun et al., 2019). These studies have shown that tea contains toxic heavy metals, and the long-term exposure may cause their accumulation in human body. Several researches have showed that the content of manganese (Mn) in tea leaves was extremely high (Wen et al., 2018; Peng et al., 2018; Zhang et al., 2018a), indicating that the tea plant, as the hyperaccumulator of Mn, could be used in phytoremediation for Mn

∗

Corresponding author. College of Resource and Environmental Engineering, Guizhou University, Huaxi district, Guiyang city, Guizhou province, 550025, China. E-mail addresses: [email protected] (J. Zhang), [email protected] (R. Yang), yunli@ufl.edu (Y.C. Li), [email protected] (Y. Peng), [email protected] (X. Wen), [email protected] (X. Ni). https://doi.org/10.1016/j.ecoenv.2020.110475 Received 29 October 2019; Received in revised form 8 February 2020; Accepted 11 March 2020 0147-6513/ © 2020 Elsevier Inc. All rights reserved.

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health of tea drinkers. The distribution and accumulation of heavy metals in soils and tea leaves from three differently geological background areas, Permian limestone distributed zone, Silurian clastic rock outcropped zone, and Cambrian hydrothermal mineralized belt, and potential human health risk of heavy metals through tea intake were investigated in this study. It was hypothesized that the effect of geological background difference may play an important role in impacting the distribution of heavy metals in cultivated soils and their accumulation in tea leaves under the acidic soil conditions. The objectives of this study were to: 1) quantify the concentrations of Cd, Pb, Tl, Hg, As, Sb, Cr, Ni, and Mn in tea leaves and corresponding cultivated soils from geologically different production zones; 2) analyze the influence of two factors (i.e., total concentrations of heavy metals in soil and soil pH) on metal accumulation in tea leaves; and 3) assess the human health risks of heavy metals (Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni) in tea leaves through tea intake. This study can provide an important reference and basis to understand and mitigate the potential risks of heavy metals in tea.

contaminated soil (Li et al., 2019b). Numerous studies have been performed to investigate the concentration levels of heavy metals in tea from many countries or regions worldwide, such as Sri Lankan (Pourramezani et al., 2019), Italy (Barone et al., 2016), Ghanaian (Nkansah et al., 2016), Brazil (Milani et al., 2016), India (Fung et al., 2009), and China (Wen et al., 2018; Peng et al., 2018). Several studies have shown that there were the phenomena of the distinctly exceeding the standard limits for heavy metals in tea leaves from some areas seriously affected by human activities (Wen et al., 2018; Huang et al., 2018). However, to our knowledge, it is absent for a comparative study on the distribution, accumulation, and health risks of heavy metals in soils and tea leaves from different geological background areas in Guizhou province, China. Anthropogenic activities, e.g., mining (Yaylali-Abanuz and Tuysuz, 2009), traffic emissions (Huang et al., 2018), machining and building industries (Wen et al., 2018), and fertilization (Atafar et al., 2010), can cause soils heavy metals accumulation and pollution. However, in some regions where impact and disturbance of human activities may be little, high concentrations of geogenic heavy metals can also play an important role in giving rise to soil heavy metals contamination, especially in carbonate rock distributed area and hydrothermal mineralized belt. Several studies have showed that heavy metals contents in the soil derived from carbonate rock were higher than these in other soils derived from clastic and intermediate-acid rocks in the karst region, southwest China (Tu et al., 2011; Jia et al., 2020; Qu et al., 2020). The soils formed by carbonate rock are widely distributed in the karst region of southwest China, thus the high levels of heavy metals may cause hazardous effects on the health of living organisms. Moreover, several studies have revealed that the contents of heavy metals in soils from mineralized areas were higher than those from non-mineralized areas (Ngure and Kinuthia, 2020; Sungur et al., 2020). These studies showed that geogenic heavy metals may play an important role in forming the soil heavy metal pollution and influencing crop quality. Unfortunately, the distribution and potential risks of heavy metals in agro-products, especially tea leaves, from carbonate rock distributed area and hydrothermal mineralized belt in eastern Guizhou of China remain unclear. Several studies have indicated that tea plantation soils gradually became increasingly acidified with the increase in age of plantation after tea plants were cultivated (Li et al., 2016; Yang et al., 2018; Yan et al., 2018). The soil acidification of tea plantations may be attributed to the application of nitrogen fertilizers (Yang et al., 2018; Li et al., 2019b), the leaching and depletion of base cations in soils (Yang et al., 2018), the organic acids (oxalic acid, malic acid, succinic acid, etc.) secretion from the roots of tea plants, and the decomposition of litter of tea plants. Old tea plantations exhibit decreased soil pH, which not only makes the loss of nutrients (nitrogen and phosphorus), but also contributes to elevated concentrations of bioavailable water-soluble and exchangeable metals, enhanced transport of these metals, and intensified accumulation of metals in tea plants; this may increase the human health risks of heavy metals in tea leaves. The accumulation of heavy metals in tea plants depends primarily on the physiological properties and the metal absorption mechanism of tea plant, the chemical speciation of metals in soil, and the physicochemical properties of the soil (Zhang and Fang, 2007; Li et al., 2017; Zhang et al., 2018a). Several studies have showed that the negative correlation was observed between soil pH and concentrations of heavy metals in tea plants (Yaylali-Abanuz and Tuysuz, 2009; Wen et al., 2018; Zhang et al., 2018a). Therefore, the total concentrations of heavy metals in soils may also play an important role in regulating their contents in tea leaves because of the surroundings of acidic soil. In detail, a prior study showed that tea plants cultivated in soils derived from mineralized rocks can exhibit high concentrations of toxic heavy metals and some symptoms of toxicity (Yaylali-Abanuz and Tuysuz, 2009), enhancing the health risk of heavy metals in tea. Thus, it is important to investigate tea security in metallic anomalous zones and hydrothermal metallogenic belts to mitigate the risks of heavy metals and protect the

2. Materials and methods 2.1. Study area The study area is located in the eastern region of Guizhou province (southwest China). The study area has a humid monsoonal climate typical of the middle subtropical zone, characterized by adequate sunshine and two seasons, a warm and humid season and a long frost-free mild season. The mean annual temperature is approximately 16.8 °C and the average annual precipitation is ~1200 mm. The frost-free period is approximately 303 d and the average annual sunshine hours is ~1233 h. The main soil types in Tangshan (TS) and Wude (WD) are sandy yellow soils derived from the weathering of sandstone, siltstone, and mudstone, while in Longtang (LT) and Pingshan (PS), the main soil types are yellow soils containing clay from the weathering of limestone and dolomite. The most common tea cultivars are Funding small-leaf tea and local moss tea. The lithology of the study area is as follows: limestone of the upper Permian Wujiaping and Changxing Formations (P3w + c) in LT, siltstone and mudstone of the Silurian Hanjiadian Group (S2-3h) in TS, and siltstone and mudstone of the Silurian Hanjiadian Group (S2-3h) and Shiniulan Formation (S2sn) in WD. The outcropped strata in PS are of the Cambrian Qingxudong (Є1q) and Gaotai (Є2g) Formations and the lithology is weakly mineralized dolomite (Fig. 1). 2.2. Collection and pre-treatment of samples With consideration for the distribution of tea plantations in the study area, 22 samples of young and old tea leaves, and corresponding soil samples (0–20 cm depth), were collected from the LT, TS, WD, and PS regions during the tea harvest season of April 2018. The number of sampling sites obtained from each region was as follows: 7, 4, 5, and 6 from LT, TS, WD, and PS, respectively (Fig. 1). Each sample comprised five subsamples from within an ~100 m2 area; each sample comprised 0.25 kg tea leaves and ~0.5 kg soil. Specifically, young tea leaves (YTL; one bud and two expanded leaves) and perennial old tea leaves (OTL) were plucked by hand at random from five subsites at each site (within an ~100 m2 area). Meanwhile, the corresponding soils were sampled from the non-rhizospheric area within the projection area of the canopy of tea plant (~30 cm from the trunk bottom of the tree) at each subsite using a hand hoe. Fresh tea leaves were rinsed with running water and then deionized (DI) water, three times each, and were then dried in a drying oven at 60 °C until a steady weight was recorded. The dried tea leaves were then triturated using a grinder, sieved through a 0.075 mm nylon sifter, and stored in a sealed bag until analysis. Soils were dried in the same oven at 40 °C until a constant weight was recorded. They were then 2

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Fig. 1. Geological map of the 22 sampling sites across the four tea plantations in the eastern region of Guizhou, China: Longtang (LT), Tangshan (TS), Wude (WD), and Pingshan (PS) plantations.

2% (v/v) HNO3 solution and then purged with DI water prior to use. According to the NY/T 1377–2007 standard, 10.0 g of soil was mixed with 25 mL DI water and then stirred; the solution was set aside to rest for 30 min before analysis. The pH values of supernatant were analyzed with an acidimeter (PHSJ-4F, Shanghai INESA Scientific Instrument Co., Ltd., China). Blanks, duplicates, and certified referenced materials (CRM) were assayed using the above-mentioned consistent procedure. The CRM one (GL03) for tea leaves was an internal standard material made from leaves of eucalyptus sampled near the ALS Minerals laboratory in Brisbane, Queensland, Australia. The CRM two (OREAS-45e) for soils was self-developed using lateritic soil collected from the southern Murchison of Western Australia. The recovery rates of the CRMs ranged from 89% to 111% for tea leaves and from 91% to 107% for soil samples. The standard deviation (SD) of duplicates varied from 0 to 13% for tea leaves and from 0% to 5% for soils. In addition, the assayed values of all elements in the blanks were less than the limit of detection (LOD) of the matched elements.

triturated and sieved through a 200-mesh nylon sifter (0.075 mm) and stored in sealed bags for pH and metals analyses. 2.3. Analysis of soil and tea leaves The concentrations of heavy metals in the tea leaves and soil samples were determined at the ALS Minerals laboratories (Guangzhou, China) using an ICP-AES (Vista-MPX, Agilent, CA, USA) and an ICP-MS (7700x, Agilent, CA, USA). The ICP-MS and ICP-AES running conditions described by Zhang et al. (2018a, 2018b) were utilized in this assay. 1.0 g of tea powder and 5 mL of concentrated nitric acid were transferred to a digestion container and were digested at room temperature for ~8 h before heating for ~3 h on a preheated hot plate (150 °C) under a fume hood. The remaining solid crystal was dissolved and then transferred into a volumetric flask after cooling; the final volume was precisely adjusted to 25 mL using 2% hydrochloric acid. Every soil sample was digested in two methods. For the first one, 0.25 g of soil was digested by a concentrated acid mixture (a ratio of 1:2.5:2:2.5 for the HClO4:HNO3:HF:HCl) in an oven at ~190 °C for 48 h, cooled to room temperature, heated on a preheated hot plate (150 °C) to get rid of excess acid until crystalline solid was formed, and then diluted to a steady volume (12.5 mL) with 2% hydrochloric acid. For the second one, 0.5 g of soil sample was dissolved in aqua regia method (1:3, v/v: conc. HNO3 and conc. HCl) in an oven at ~190 °C for 48 h and was then placed and heated on a preheated hot plate (150 °C) under a fume hood until white fumes appeared and crystalline solid was formed. If required, more aqua regia was added into the digestion container to achieve complete digestion of soil sample. Afterwards, the crystalline solid was dissolved and precisely adjusted to a stable volume (12.5 mL) with DI water. All materials were infiltrated for 24 h with a

2.4. Health risk assessment Mn is a necessary nutrient element for both humans and tea plants; therefore, the health risk associated with Mn was not considered. The health risks to tea consumers as a result of long-term exposure to Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni through tea intake were assessed based on the estimated daily intake (EDI), target hazard quotient (THQ), and hazard index (HI) (Zhang et al., 2018a). The calculation formulas of EDI, THQ, and HI are as follows: EDIi = (Ci × IR × TRi)/(BW × THQi = EDIi/RfDi 3

1000)

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soil formation. Overall, the results showed that the contents of heavy metals in soils, derived from the carbonate distributed areas (Permian limestone distributed zone and Cambrian mineralized dolomite outcropped belt), were obviously greater than those from the Silurian clastic rock outcropped areas. The pH values in all soil samples varied from 3.6 to 5.2, with a mean of 4.3, and 22.7% of soil pH values in our study area were lower than 4.0. The result reflected extreme acidity for these tea plantation soils and were lower than the mean values reported for the Lishui district (5.04, n = 74), Nanjing, China (Wen et al., 2018) and the major tea production regions (4.8, n = 26) in Anhui province, China (Peng et al., 2018). Similar values (4.29, n = 13) were observed in the Pu'an plantation in Guizhou, China in our previous study (Zhang et al., 2018a). Therefore, it was serious for the soil acidification occurred in tea plantations in eastern Guizhou of China.

HI = THQ1 + THQ2 +··· + THQn where Ci is metal i concentration in tea leaves (mg kg−1), IR is the ingestion rate of tea leaves (11.4 g person−1 d−1) (Peng et al., 2018), TRi is the transfer rate of metal i from tea leaves into tea infusion (Zhang et al., 2018a), BW is body weight, namely 70 kg for adults (de Oliveira., 2018), RfDi (mg kg−1 bw d−1) is the oral reference dose for metal i regulated by the U.S. Environmental Protection Agency (US EPA), THQi is the target hazard quotient of metal i, and HI is the total health risk related to the toxic metals (n = 8). The RfD values for Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni were 5.0 × 10−4, 1.5 × 10−3, 1.0 × 10−5, 3.0 × 10−4, 3.0 × 10−4, 4.0 × 10−4, 1.5, and 0.02 mg kg−1 bw d−1, respectively (US EPA , 2019; Cai et al., 2019). 2.5. Statistical analyses

3.2. Heavy metals contents in tea leaves

One-way analysis of variance (ANOVA) was used to test the significance of differences in heavy metals concentrations in soils and tea leaves from different regions. These data analyses were performed with SPSS software, version 25.0 (IBM Corporation, Armonk, New York, USA). SPSS software was also used to conduct the Pearson correlation analysis (p < 0.05 or p < 0.01) between the concentrations of heavy metals in tea leaves and the soil properties (total metal concentration and pH value) and between each element in soils.

The mean concentrations of Cd, Pb, Tl, Hg, As, Sb, Cr, Ni, and Mn in the YTL and OTL from the four plantations (LT, TS, WD, and PS) in eastern Guizhou of China are presented in Fig. 3. The result showed that the concentrations of Cd, Pb, Hg, As, and Cr in all tea samples were lower than the corresponding thresholds set by the World Health Organization (WHO) and the Chinese national food safety standard for maximum levels of contaminants in foods (GB 2762–2017 and NY 659–2003) (de Oliveira et al., 2018; Zhang et al., 2018a). In addition, the concentrations of Cd and Pb in all YTL samples were lower than the European Union (EU) standards (1 and 3 mg kg−1 for Cd and Pb, respectively), but the Pb concentration in one OTL sample (3.04 mg kg−1; site 20, PS plantation) was above the EU limit (Zhang et al., 2018b). Moreover, in terms of metallic concentrations, the distribution of Tl and Mn in the YTL were consistent, showing a decreased rank of PS > LT > TS > WD. For the contents of Pb and Ni in the YTL, the similar distribution pattern (i.e., TS > LT > WD > PS) was observed. Further, the concentration of Hg in YTL was the highest for the WD plantation (0.008 mg kg−1), while those of the other three plantations were similar, with the mean contents of 0.006 mg kg−1. The Cd contents in the YTL showed the reduced order of LT > WD > TS > PS. For As, Sb, and Cr in YTL, their concentrations were the greatest for the TS plantation. In terms of OTL, the distribution of Pb, Tl, and Cr contents were consistent, which showed that PS plantation was the greatest. Besides, the concentrations of Cd, Ni, and Mn in OTL showed LT plantation was the highest. For Sb and As contents in OTL, the TS plantation was the greatest, being consistent with their contents in YTL. The Hg contents in OTL displayed a reduced order of WD (0.143 mg kg−1) > TS (0.116 mg kg−1) > PS (0.109 mg kg−1) > LT (0.108 mg kg−1), indicating that WD plantation was the greatest as that of the YTL. Comparatively, the contents of Cd, Pb, Tl, Hg, As, Sb, Cr, and Mn in OTL were distinctly greater than those in YTL, whereas the Ni content in tea leaves exhibited the opposite distribution pattern.

3. Results 3.1. Heavy metals contents and pH in soil The mean total concentrations of Cd, Pb, Tl, Hg, As, Sb, Cr, Ni, and Mn in soils from the eastern region of Guizhou, China were 0.33, 40.9, 0.39, 0.19, 26.3, 2.0, 66, 31.1, and 700 mg kg−1 (n = 22), respectively. Further, 41%, 14%, and 23% of soil samples exceeded the risk screening values (RSV) for Cd, Pb, and As in agricultural soils in China (pH < 5.5), respectively (MEEPRC, 2018). A total of 22 surface soil samples were classified into four groups based on the geographical and lithological differences in the soil parent materials, namely soil derived from Permian limestone, soil derived from Cambrian mineralized dolomite, and soil derived from Silurian clastic rock. The average total concentrations of heavy metals in soils from the four tea plantations (LT, TS, WD, and PS) in eastern region of Guizhou, China are presented in Fig. 2. It can be seen that the mean concentration of Cd (0.6 mg kg−1) in the soil from LT plantation was significantly greater than those from the three other plantations (0.26 for TS, 0.16 for WD, and 0.19 mg kg−1 for PS) (p < 0.05). Moreover, the average total concentrations of Pb (72.9 mg kg−1), Tl (0.67 mg kg−1), Hg (0.39 mg kg−1), As (58.0 mg kg−1), and Sb (3.28 mg kg−1) in soils from PS plantation were significantly greater than those of the soils from LT, TS, and WD plantations (p < 0.05). To be specific, the contents of Pb, Tl, Hg, As, and Sb in soils were 29.4, 0.32, 0.13, 19.9, and 1.69 mg kg−1 for LT plantation, 26.2, 0.26, 0.12, 10.3, and 1.55 mg kg−1 for TS plantation, and 30.4, 0.25, 0.08, 9.9, and 1.19 mg kg−1 for WD plantation, respectively. Furthermore, the similar distribution of Cr and Mn in soils were observed, which showed that LT and PS plantations were higher than TS and WD plantations. In detail, the average total Mn contents in the soils from these four plantations showed a decreased order of PS (852 mg kg−1) > LT (798 mg kg−1) > WD (570 mg kg−1) > TS (463 mg kg−1). For the LT, PS, TS, and WD plantations, mean Cr concentrations in soils were 70, 72, 55, and 61 mg kg−1, respectively. However, the contents of Ni in soils displayed a different distribution pattern and ranked as PS > WD > LT > TS. Further, except for Cr and Ni, a great range of concentrations were observed for the other heavy metals and their coefficient of variation (CV) values exceeded 47%, reflecting that these heavy metals were subjected to distinct geochemical migration and leach in the processes of weathering and

3.3. Bioaccumulation factors The migration and accumulation of heavy metals from soil to the edible parts of plants were the main route of entry of heavy metals into the food chain (Sharma et al., 2018). Different metals accumulate in plants at different rates depending on various factors (e.g., cultivars, physicochemical properties of the soil, phytoavailability of the heavy metals in the soil) (Sharma et al., 2018). Bioaccumulation factors (BAFs) were calculated to evaluate the accumulation capacities of heavy metals in tea leaves. BAF was calculated as the ratio of the metallic content in the plant to that in the soil; a BAF greater than one indicates the significant accumulation of heavy metals in the plant from the soil (Pang et al., 2017). The calculated results showed that 63.6% of YTL samples had a BAF > 1 for Mn and that all YTL samples had a 4

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Fig. 2. Concentrations of Cd, Pb, Tl, Hg, As, Sb, Cr, Ni, and Mn in soils (mg kg−1) from the Longtang (LT), Tangshan (TS), Wude (WD), and Pingshan (PS) tea plantations in eastern Guizhou province, China. The concentrations of heavy metals in soils significantly differed between each region when different lowercases existed. The significance level (α) was set as 0.05.

BAF < 1 for Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni. The BAF analysis revealed that 9.1%, 27.3%, and 100% of OTL samples had BAFs > 1 for Cd, Hg, and Mn, respectively, indicating that tea leaves acted as an important hyperaccumulator of Mn, as reported in the prior studies (Li et al., 2017; Li et al., 2019a). Furthermore, in descending order, the BAFs of the nine heavy metals in the tea leaves were as follows: Mn (1.40) > Ni (0.44) > Cd (0.32) > Hg (0.054) > Tl (0.053) > Sb (0.016) > Cr (0.015) > Pb (0.008) > As (0.004) for YTL and Mn (6.12) > Hg (0.98) > Cd (0.53) > Ni (0.32) > Tl (0.18) > Sb (0.064) > Cr (0.025) > Pb (0.06) > As (0.016) for OTL. Moreover, the BAF of Ni for the YTL was greater than that for the OTL, while the BAFs of the rest eight heavy metals (Cd, Pb, Tl, Hg, As, Sb, Cr, and Mn) indicated that the OTL was higher than the YTL, indicating that leaf-age effect existed for the accumulation of the same heavy metal in tea leaves.

of Cd, Pb, Tl, and Mn in the OTL and those in the soil, with correlation coefficients of 0.587 (p < 0.01, n = 22), 0.573 (p < 0.05, n = 22), 0.507 (p < 0.01, n = 22), and 0.49 (p < 0.05, n = 22), respectively. On the contrary, there were negative correlations between the OTL and the soil in terms of the concentrations of Ni, As, and Hg. Moreover, concentrations of heavy metals in both the YTL and the OTL were negatively correlated with soil pH values on the exception of Hg and Sb in the YTL. In terms of the YTL and OTL, the negative correlation coefficients between tea leaves and soil pH, for both Mn and Tl, were the smallest. To be specific, the correlation coefficients of Mn and Tl were −0.463 (p < 0.05, n = 22) and −0.584 (p < 0.01, n = 22) for the YTL, respectively, while for the OTL their correlation coefficients were −0.355 and −0.479 (p < 0.05, n = 22), respectively.

3.4. Effects of soil metals and pH on metals in tea leaves

Heavy metals in tea leaves are not completely leached into tea infusions. Thus, the transfer rate (TR) of metals from tea leaves into infusions should be considered (Zhang et al., 2018a). The leaching experiment of heavy metals from tea leaves into infusions was not performed in the present study; thus, reported TR values were used to evaluate EDI in this study. The TR values were 14.18% for Cd, 7.11% for Pb, 33.1% for Tl, 45.2% for Hg, 23.83% for As, 11.78% for Sb, 11.45% for Cr, and 67.71% for Ni (Nookabkaew et al., 2006; Zhao, 2016; Zhang et al., 2018a). The calculated EDI values (mg kg−1 bw d−1) of heavy metals through tea infusion intake are listed in Table 1. It can be seen that the average EDI values (mg kg−1 bw d−1) of Cd, Pb, Tl, Hg, As, Sb, and Cr for the OTL infusion were greater than those for the YTL infusion. By contrast, the average EDI of Ni via OTL infusion intake was lower than that via the YTL infusion intake. This can be

3.5. Estimated daily intake

The Pearson correlation coefficients (R) between concentrations of heavy metals in tea leaves (YTL and OTL) and total concentrations of heavy metals and pH values in growing soils indicated that concentrations of heavy metals in soil and soil pH had obvious influences on the concentrations of heavy metals in tea leaves (Fig. 4). The contents of Cd, Mn, and Tl in the YTL were positively correlated with the corresponding heavy metals in the soil, respectively (Cd, R = 0.582, p < 0.01; Mn, R = 0.396; Tl, R = 0.324). However, the concentrations of Cr, Sb, Hg, As, and Pb in the YTL and those of corresponding metals in the soil were negatively correlated, respectively. In particular, Pb displayed a significant level (R = −0.458, p < 0.01, n = 22). Moreover, positive correlations were observed between concentrations 5

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Fig. 3. Concentrations of Cd, Pb, Tl, Hg, As, Sb, Cr, Ni, and Mn in young tea leaves (YTL) and old tea leaves (OTL) (mg kg−1) from the Longtang (LT), Tangshan (TS), Wude (WD), and Pingshan (PS) plantations in the eastern region of Guizhou province, China. The different lowercases and uppercases indicated that concentrations of heavy metals in YTL and OTL significantly differed between each region, respectively (p < 0.05).

ascribed to the obviously higher concentrations of Cd, Pb, Tl, Hg, As, Sb, and Cr and the lower content of Ni in OTL compared to these in YTL. Moreover, the EDI of Cd in tea infusions, for both the YTL and the OTL,

was greater for the LT plantation relative to the TS, WD, and PS plantations. This may be attributed to the natural high background concentration of Cd in soil from the LT plantation and the easy

Fig. 4. Pearson correlation analysis between concentrations of heavy metals in young tea leaves (YTL) and old tea leaves (OTL) and soil properties, including total concentrations of heavy metals and pH values. Analyses were conducted using SPSS 25.0 software. Correlations are significant at p < 0.05 (*) or p < 0.01 (**). 6

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Table 1 Estimated daily intake (EDI) (mg kg−1 bw d−1) of Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni for adults associated with the consumption of infusions of young tea leaves (YTL) and old tea leaves (OTL) from the Longtang (LT), Tangshan (TS), Wude (WD), and Pingshan (PS) plantations in eastern Guizhou province, China. Region and leaf type

Cd

Pb

Tl

Hg

As

Sb

Cr

Ni

LT-YTL TS-YTL WD-YTL PS-YTL Mean-YTL

3.14E-06 1.38E-06 1.52E-06 1.15E-06 1.80E-06

3.58E-06 3.83E-06 2.59E-06 2.41E-06 3.10E-06

1.18E-06 8.22E-07 6.04E-07 1.23E-06 9.59E-07

4.73E-07 4.60E-07 5.59E-07 4.54E-07 4.87E-07

1.92E-06 2.82E-06 2.00E-06 1.58E-06 2.08E-06

5.18E-07 6.00E-07 4.91E-07 4.60E-07 5.17E-07

1.02E-05 3.60E-05 1.69E-05 1.05E-05 1.84E-05

1.40E-03 1.45E-03 1.37E-03 1.14E-03 1.34E-03

LT-OTL TS-OTL WD-OTL PS-OTL Mean-OTL

3.39E-06 2.41E-06 2.70E-06 2.18E-06 2.67E-06

2.46E-05 2.15E-05 2.14E-05 2.80E-05 2.39E-05

3.43E-06 4.10E-06 1.57E-06 4.83E-06 3.48E-06

7.93E-06 8.54E-06 1.05E-05 8.00E-06 8.75E-06

9.46E-06 1.19E-05 8.39E-06 8.19E-06 9.48E-06

1.97E-06 2.33E-06 2.07E-06 2.11E-06 2.12E-06

2.71E-05 3.45E-05 2.04E-05 3.74E-05 2.99E-05

1.33E-03 8.44E-04 1.09E-03 4.82E-04 9.38E-04

The mean reflects the average value of the four plantations.

accumulation capacity of Cd in tea leaves. The EDIs of Tl for the YTL and OTL infusions, and of Pb for the OTL infusion, were greater for the PS plantation than those for the LT, TS, and WD plantations. This may be related to the greater contents of Pb in OTL and of Tl in YTL and OTL from PS plantation. In term of Hg, the EDIs for WD plantation were the highest through YTL and OTL infusion intake because of the high contents of Hg in tea leaves from WD. Moreover, the EDI of Pb through YTL infusions intake was greater for TS plantation compared to LT, WD, and PS plantations. The reason can be that the Pb content in YTL from the TS plantation was higher relative to the LT, WD, and PS plantations. Also, for EDIs of As and Sb, the similar patterns can be obtained via YTL and OTL infusions intake, showing the TS plantation was the greatest. Additionally, it should be noted that the mean daily intake dosage of Pb (2.8 × 10−4) in a prior study (Peng et al., 2018) was reported to be two magnitudes greater than that (3.1× 10−6) for the YTL infusion in this study. The reason may be that the study by Peng et al. (2018) performed a health risk assessment without considering the TRs of metals from tea leaves into infusions. Fig. 5. The calculated target hazard quotients (THQ) of Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni and the accumulative hazard indexes (HI) for adults associated with the consumption of infusions of young tea leaves (YTL) and old tea leaves (OTL) from the Longtang (LT), Tangshan (TS), Wude (WD), and Pingshan (PS) plantations in eastern Guizhou province, China.

3.6. Health risks and comprehensive risk of metals The calculated THQs of Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni and accumulative HI values for YTL and OTL infusions from the four plantations (LT, TS, WD, and PS) in eastern Guizhou of China are presented in Fig. 5. For both the YTL and OTL infusion, THQ of individual metal and accumulative HI values for Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni were all less than one, suggesting that the eight heavy metals will not result in adverse health effects for adults via daily tea intake, which made the tea from eastern Guizhou of China safe to the resident. The similar results have also been reported by Peng et al. (2018) and Zhang et al. (2018a), showing no health risk to human from heavy metals exposure via tea intake. In detail, the mean THQs of each metal associated with consuming YTL infusion were ranked as Tl (0.096) > Ni (0.067) > As (6.9 × 10−3) > Cd (3.6 × 10−3) > Pb (2.1 × 10−3) > Hg (1.62 × 10−3) > Sb (1.29 × 10−3) > Cr (1.23 × 10−5). This result was different from the reported order of THQ values, i.e., Tl (0.476) > As (0.041) > Sb (0.0053) > Cd (0.0021) > Pb (0.0011), associated with drinking the Tieguanyin tea infusion from Anxi city of Fujian province of China (Sun et al., 2019). However, for the OTL infusion, the average THQs of heavy metals were in the following order: Tl (0.348) > Ni (0.067) > As (0.032) > Hg (0.029) > Pb (0.016) > Cd (5.34 × 10−3) > Sb (5.3 × 10−3) > Cr (1.99 × 10−5), showing a difference from that of the YTL infusion, especially for Hg, Pb and Cd. This may be because atmospheric Hg and Pb can be accumulated in old tea leaves by the stomata of leaf tissue of tea plant. Additionally, the THQs of Cd, Pb, Tl, Hg, As, Sb, and Cr via the OTL infusion intake were greater than those via the YTL infusion intake for the same region, owing to their higher

concentrations in the OTL, whereas the THQ of Ni showed an inverse pattern. Furthermore, the THQ of Tl was distinctly higher than those of the other metals for both YTL and OTL, which could be related to the extremely low RfD value of Tl (1.0 × 10−5 mg kg−1 bw d−1). Comparatively, the average THQs of Tl in YTL (0.096) and OTL (0.349) infusions from eastern Guizhou of China were lower than that of the Tieguanyin tea infusion (0.476) from Anxi, Fujian of China (Sun et al., 2019). This can be ascribed to the high concentration of Tl in Tieguanyin tea leaves and its great leaching capacity from tea leaves into tea infusion. In terms of the YTL infusion, HI values were ranked as LT > PS > TS > WD, indicating that tea leaves from the Silurian clastic rock distributed regions (TS and WD) were lower at risks of heavy metals than those from the Permian limestone outcropped region (LT) and the Cambrian mineralized dolomite distributed area (PS). This could be related to the low concentrations of heavy metals in tea leaves from TS and WD. In addition, HI values for the OTL infusions displayed in the following order: PS > TS > LT > WD. The reason could be that the Tl concentration in the OTL from the TS plantation was higher and that the greater contribution rate of THQ of Tl (75.2%) to the HI than those of the other heavy metals in the TS plantation. The finding highlighted the human health risk of Tl in tea infusions; more attention 7

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agents, can adsorb heavy metals in soils, resulting in decreased heavy metals fractions in the soil solution owing to the strong binding capacities of the heavy metals with Fe oxides and Al oxides (Shen et al., 2020; Qu et al., 2020). It was reported that the weathered soil derived from carbonate rock has a high content of physical clay (< 0.002 mm) (Tu et al., 2011). Further, the adsorptive mechanism may be electrostatic attraction between the negatively-charged colloidal fine particles (Fe oxides and Al oxides) in the clay mineral and heavy metal cations with positive charges in soil solutions. Moreover, the previous studies also indicated that soils derived from carbonate rock had the high cation exchange capacity (CEC) (Tu et al., 2011), which showed that the cations exchange could also contribute to the adsorption of heavy metals by clay minerals such as montmorillonite (Yao et al., 2014). Additionally, the soils derived from carbonate rock may be have high contents of soil organic matter (SOM) (Tu et al., 2011; Jia et al., 2020), which can be further increase the adsorption capacities of heavy metals by SOM via the complexation and chelation with organo-functional groups. On the contrary, the soils derived from clastic rock have low background levels of heavy metals, low physical clay (Fe oxides and Al oxides), and low SOM, which can decrease and weaken the enrichment of heavy metals in the soils. On the whole, in some areas weakly influenced by human activity, the difference of geological background controlled the distribution of soil heavy metals to a large extent.

should be focused on the health risk of Tl from tea infusions given its high risk identified here. 4. Discussion 4.1. Geological difference effect on metals in soil According to the results, the contents of heavy metals in the soil derived from carbonate rock were distinctly greater than those from clastic rock. This may be caused by the high contents of heavy metals in carbonate rock, clay minerals formation, and the secondary enrichment of heavy metals in the process of the carbonate rock weathering and soil formation. The previous study showed that the background levels of heavy metals in carbonate rocks were obviously higher the crustal abundance values (Wang et al., 2019), especially Cd and As in limestone. The carbonate rocks with high heavy metallic contents can form the soil parent materials with the higher heavy metallic contents than the bedrock through the weathering. Moreover, the heavy metals accumulation in the topsoil depends on the further weathering of the soil parent material to a great extent, though there were the external inputs. Similarly, many studies have indicated that heavy metals concentrations were high in the soils that were formed through the weathering of limestone and dolomite (Quezada-Hinojosa et al., 2009; Rambeau et al., 2010; Qu et al., 2020). Further, our data showed that Cd in soils was significantly positively correlated with Ca (R = 0.57, n = 22, p < 0.01) and P (R = 0.52, n = 22, p < 0.05) in soils, respectively (Table S1). Thus, it can be speculated that the content of Cd in soil derived from limestone could be higher than that from clastic rock. Indeed, the current results also showed the similar distribution characteristics (Fig. 2). As per the above-mentioned correlation results, it can be speculated that the precipitation through forming cadmium phosphate (Cd3(PO4)2) may be the predominant mechanism of Cd enrichment in soils from LT plantation (Bolan et al., 2003). Moreover, it does not preclude the precipitate formation of mixed Ca–Cd phosphate (Bolan et al., 2003). Therefore, the high Cd content in the soil from LT plantation can be caused by the high natural background level of Cd in limestone and the secondary enrichment of Cd in the process of limestone weathering and soil formation. Moreover, the contents of Pb, Tl, Hg, As, and Sb showed consistent distribution patterns, indicating that their concentrations in soils from PS plantation were the highest. The eastern region of Guizhou, China contained the most famous Pb–Zn metallogenic belt in the world, which is derived from the Cambrian Qingxudong Formation (Ye et al., 2012). Similarly, a study by Lin et al. (2019) reported that Pb concentrations in soils around a lead/zinc mining area ranged from 31–326 mg kg−1. Additionally, the mineralized rock and its weathered soil displayed congruent patterns for some harmful elements (e.g., As, Sb, and Hg), showing that mineralized rocks can facilitate the release of heavy metals to weathered soils and result in environmental concerns (Yin et al., 2020). Therefore, it can be speculated that the high concentrations of Pb, Tl, Hg, As, and Sb in the soils from PS plantation may be caused by the mineralization of the regional strata, Cambrian Qingxudong and Gaotai Formations, and the secondary enrichment of heavy metals in the process of mineralized carbonate rocks weathering and soil formation. In the process of weathering, the basic cations and alkaline-earth metals were leached, causing the decrease of K, Na, Ca, and Mg concentrations in soils, whereas Fe and Al were highly enriched in the weathered soil (Jia et al., 2020). Hence, it can be speculated that the enrichment mechanism of heavy metals in soils derived from carbonate rock may be the adsorption of clay minerals (Fe oxides and Al oxides). Further, except for Cd, the heavy metals in the soils were significantly positively correlated to Fe and Al in soils, indicating that the enrichment of heavy metals may be caused by the adsorption by Fe oxides and Al oxides (Table S1). Similarly, in naturally occurring heavy metals enriched soils, Fe oxides and Al oxides, as the major micro-aggregating

4.2. Soil acidification in tea plantations The result showed that the soils from tea plantation were extremely acidic. Tea plant is a typical acid-like crop, the optimum pH range is between 4.0 and 5.5 for the normal growth of tea plant. Naturally, due to the specific physiological action of the tea plant, soil acidification occurs in tea plantations with an annual rate of 0.071 (Yang et al., 2018). Moreover, it is inevitable for the fertilization in tea plantations to ensure the yield of tea leaves. Thus, it can be speculated that the dominant mechanism of soil acidification in tea plantation may be from the following aspects: 1) The secretion of organic acids (e.g., oxalic acid, malic acid, and succinic acid) from the roots of the tea plant caused the soil acidification. 2) As an economic crop, from which leaf organs are harvested, tea plant requires more nitrogen fertilizer to ensure the sufficient production. Therefore, the nitrification of ammonium of nitrogen fertilizer in soil can produced H+ and accelerate the soil acidification of tea plantation (Yemane et al., 2008). 3) The long-term application of nitrogen fertilizer promoted the accumulation of exchangeable Al3+, and the hydrolysis of Al3+ further produced H+, which facilitated the soil acidification of tea plantation. It was reported that soil acidification can occur in the depth of 0–200 cm in soil profiles from tea plantation owing to the cultivation of tea plants and the excessive application of chemical fertilizers (Yan et al., 2018). Soil acidification of tea plantation can cause the leaching of nutrient-based ions (K+, Na+, Ca2+, and Mg2+), reducing soil nutrients and accelerating the loss of nitrogen and phosphorus (Yan et al., 2018). Further, heavy metals in acidic soils have relatively high availabilities and migration capacities (Liao et al., 2016), enhancing environmental and health risks. Therefore, soil acidification of tea plantations is needed more attentions and control to ensure the tea quality security. 4.3. Heavy metals in tea leaves and their BAFs The contents of all harmful elements in YTL met the WHO's and Chinese standard limits, indicating that the quality of YTL from eastern Guizhou of China was safe only considering heavy metals. The presence of Pb concentration in OTL exceeding the EU limit was observed. Accordingly, only YTL are recommended for tea export destined for the EU market. The contents distributions of Cd, Tl, and Mn in all tea samples were consistent with their distributions in soils. On the contrary, the contents of Pb, Hg, As, Sb, Cr, and Ni in all tea leaves 8

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2005; Połeć-Pawlak et al., 2005). In detail, the in-situ analytical result of micro-particle induced X-ray emission indicated that the largest concentrations both of Mn and of Ca occurred in the epidermis and oxalate crystals in tea leaves, thus, the possible enrichment mechanism of Mn in tea leaves is the chelation with oxalate through replacing Ca to form Mn-oxalate and to be immobilized and stabilized (Pongrac et al., 2020). Therefore, more researches are needed to elucidate the enrichment mechanism of Mn in tea leaves, especially the old tea leaves.

displayed different distribution patterns with the variation of their total concentrations in soils. Therefore, the accumulation of Cd, Tl, and Mn in tea leaves could depend on their total concentrations in soils to a large extent, whereas for the contents of Pb, Hg, As, Sb, Cr, and Ni in tea leaves, their total contents in soils could be not the only influencing factor. Further, this may be also because tea leaves have different physiological absorption mechanisms for different heavy metals (Pang et al., 2017; Zhang et al., 2018a; Sun et al., 2019). The concentrations of Cd, Pb, Tl, Hg, As, Sb, Cr, and Mn in the OTL were distinctly greater than those in the YTL, and their BAFs also displayed the consistent trend, whereas, both the concentration of Ni and its BAF showed that the YTL was higher than the OTL, as reported by Peng et al. (2018). The reason may be that the YTL and OTL have different physiological requirements and uptake mechanisms in terms of the same heavy metal. Similarly, it has been reported that some nonnutritional elements (e.g., Hg, As, Cd, Cr, and Pb) tended to accumulate more in old leaves than in young leaves (Zhang et al., 2018a). Moreover, it was reported that the actual mobilities of elements from old to young plant parts via the phloem is element-dependent (Xu et al., 2006). Heavy metals in plants may not be reallocated and remobilized, unlike some nutrients (e.g., N, P, K, and Zn) with high mobility in plant tissues. Therefore, the older the leaves of tea plants, the more the heavy metals in the leaves accumulated. Additionally, the BAF of Ni in the YTL was higher than that in the OTL, indicating that Ni can be an essential nutrient element for the metabolism of tea plant owing to the vigorous metabolism of young leaves. A prior study (Neumann and Chamel, 1986) showed that Ni in pea plants with a comparative mobility greater than that of rubidium (Rb). Thus, Ni in tea plants could be also readily remobilized out of the old leaves to the developing tissues (e.g., new leaves and buds) via the phloem because the concentration of Rb in YTL was obviously higher than that in OTL (Fig. S1). In addition, the concentrations of Mn were 3784 and 852 mg kg−1 (n = 22) for OTL and YTL, respectively, which was consistent with previous reports (Karak et al., 2017; Wen et al., 2018; Zhang et al., 2018a), showing the high content of Mn in tea leaves. It is clear that Mn easily accumulates in tea leaves; the average BAFs were 1.4 for YTL and 6.1 for OTL in this study. Similarly, our previous study reported that the BAFs of Mn in YTL and OTL from Pu'an in Guizhou of China were 3.9 and 12.5, respectively (Zhang et al., 2018a). In terms of Mn enrichment in tea leaves, it can be speculated that the mechanism of Mn enrichment by the old tea leaves may be ascribed to the following aspects: 1) It can be attributed to the extremely acidic soil in tea plantations (pH = 4.30) since the migration ability of Mn is found to be strong in acidic soil, especially pH value below 4.5. The concentration of available Mn in soil is reported to prominently increase with the decrease in soil pH (Ishibashi et al., 2004; Zhang et al., 2018a); this promotes the greater absorption of Mn by the root of tea plants. In detail, the extremely acidic condition (pH < 4.5) can facilitate the spontaneous rearrangement of the valence electrons in hausmannite and manganite in soil to generate Mn2+ and birnessite (Ishibashi et al., 2004). 2) Tea plant has a great translocation factor of Mn, which facilitates the mobility of Mn from the roots to the aboveground parts and leaves tissue under the transpiration stream (Memon et al., 1981; Ruan et al., 2000). 3) Once Mn reached the old tea leaves, it could not be readily remobilized through the phloem to other organs (e.g., young leaf, shoot, and new branch), as reported by Xu et al. (2006) on the hyperaccumulator plant (Phytolacca acinosa) of Mn. 4) The high accumulation ability of Mn in old tea leaves can be attributed to the roles of genes expression responsible for the distribution, absorption, translocation, transformation of Mn in tea plants (Li et al., 2017; Li et al., 2019a). 5) The complexation and chelation of Mn by organo-functional groups in OTL could reduce its remobilization from old tea leaves to other tissues. Additionally, heavy metals in plants could be mainly distributed in the cell wall and cellsap owing to the compartmentalization, complexed with phytochemicals (phytochelatin, metallothionein, organic acids, etc.), and lost their activities and re-mobilization abilities (Chen et al.,

4.4. Effect of soil properties on metals in tea leaves The results showed that the concentrations of Cd, Tl, and Mn in YTL and OTL depended on their concentrations in the soil to a great extent, respectively. Yet, Pb in OTL and YTL can be affected by soil Pb, with different degrees, and the Pb in OTL can be sourced from the growing soil, while Pb in the YTL can be more influenced by the source of atmospheric Pb. Combined with the regional distribution of Pb contents in YTL, the TS plantation was the highest. The TS plantation is located near to Shiqian city of Guizhou province of China with a distance to the city less than 500 m. The urbanization effect (population, vehicle exhaust, machine and construction industries, burning coal, etc.) may produce the atmospheric deposit including Pb. The Pb in the atmospheric dust-fall was deposited on the surface of the YTL and entered the YTL through the stomata of leaf tissue. Also, some studies indicated that Pb contamination in tea leaves may not only originate from the soils, but also result from anthropogenic sources (e.g., metal industry, chemical fertilizer, and transportation) in some rapidly developing regions (e.g., Jiangsu and Zhejiang provinces in China) (Shi et al., 2010; Wen et al., 2018). Based on the source tracing model of the two endmembers of Pb isotopes (soil parent material and vehicle exhaust), a previous study showed that 76% of the Pb in young tea leaves came from vehicle exhaust, while for the Pb in old tea leaves, the contribution rate of the vehicle exhaust source accounted for 58% (Sun et al., 2020). Therefore, the prominent source of the Pb in tea leaves may not be the soil parent material but the vehicle exhaust. This can be the main reason that explained the significantly negative correlation for Pb contents between in the YTL and in the soil. Moreover, Hg in tea leaves was negatively correlated with total Hg in the soils. The possible reasons may be the following two aspects: 1) The absorbed Hg by the plant through the soil-to-plant pathway was mainly localized to the root system, and the extremely low Hg was translocated from the root system to the aboveground parts of the plant (Wang and Greger, 2004; Stamenkovic and Gustin, 2009). 2) The atmospheric Hg, as a predominant source, is incorporated into the leaf tissue of terrestrial plants via the stomatal and non-stomatal pathways (Stamenkovic and Gustin, 2009; Cui et al., 2014). Also, in the case of As, a negative correlation was obtained between soil and tea leaf. The reason may be that the As taken up by the root of tea plant is localized to the root system that acted as a buffer and defense and that only small amount is translocated to the aboveground parts of tea plant (Shi et al., 2008). Additionally, the As absorbed by the root of tea plants derived mainly from the phytoavailable As in the soil, rather than the total As in the soil. A previous study has shown that under the acidic conditions of tea plantation, the phytoavailable As in soil was extremely low (Karak et al., 2011). The concentrations of heavy metals in both the YTL and the OTL were negatively correlated with the soil pH of tea plantation except for Hg and Sb in the YTL. The reason can be that heavy metals in acidic soils have more phytoavailable fractions and higher migration capacities, and are more readily taken up by the root of tea plant. Also, several studies have showed that both the availabilities of heavy metals in soils and their concentrations in tea leaves were reduced with the increases in soil pH values (Wen et al., 2018; Zhang et al., 2018a). Although the tea plant is a perennial leaf-harvested crop that grows best in acidic soils, with an optimum soil pH between 4.0 and 5.5, unlike many other crops (Yang et al., 2018), 22.7% of soil pH values in this 9

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studies of other heavy metals exposure pathways and digestion models in vitro for humans are needed.

study area were lower than 4.0. Therefore, it was suggested that controlling soil pH and preventing excessive acidification is necessary to avoid the extreme migration of metals from soils into tea plants. On the other hand, a prior study indicated that the SOM effect on the enhancement of bioavailable Pb in soil was higher than that of soil pH, showing that SOM played an important role in influencing the bioavailability of Pb (Jin et al., 2005). On the contrary, several studies have showed the SOM can immobilize heavy metals and reduce their bioavailabilities via the complexation and chelation (Meier et al., 2019; Huang et al., 2019; El-Naggar et al., 2020). Therefore, more researches are needed to quantify the effect of SOM on bio-availability of heavy metals in the soil and their accumulation in tea leaves. Overall, the difference of soil heavy metal contents may not be the only factor affecting heavy metals accumulation in tea leaves, on the contrary, the heavy metals accumulation in tea leaves could be affected by multiple factors.

5. Conclusions This study revealed that 41%, 14%, and 23% of soils in eastern Guizhou, China, have higher concentrations of Cd, Pb, and As than the RSVs for soil contamination of agricultural land. More attention should be paid to soil acidification in tea plantations since 22.7% of soil pH values were lower than 4.0 in the studied area. The concentrations of Cd, Pb, Hg, As, and Cr in all tea samples met the WHO's and Chinese standard limits. The accumulation ability of tea was the strongest for Mn and the BAF of Mn in OTL was 6.1. Leaf-age effect facilitated the higher contents of Cd, Pb, Tl, Hg, As, Sb, Cr, and Mn and the lower of Ni in tea leaves. High contents of Cd, Tl, and Mn in soils promoted the increase in their contents in tea leaves, respectively. Heavy metals concentrations of in both the YTL and the OTL increased with the decrease in soil pH except for Hg and Sb in the YTL. Exposure of eight heavy metals (Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni) do not cause human health risks via tea infusion intake. Furthermore, there was less of the risk associated with tea leaves from the Silurian clastic rock outcropped area (WD) compared to tea leaves from the Permian Cd-enriched zone (LT) and the Cambrian mineralized region (PS). This study could provide an important basis to screen low-risky areas regarding heavy metals for the cultivation plan of tea plants.

4.5. Health risk assessment The calculated result of HI values for Cd, Pb, Tl, Hg, As, Sb, Cr, and Ni showed that these eight heavy metals may not cause adverse health effects for adults through the YTL and OTL infusion intake. The THQ and HI values were used to estimate possible non-carcinogenic effects of heavy metals on human health via the consumption of fruits, vegetables, and beverages (Shaheen et al., 2016; de Oliveira et al., 2018; Peng et al., 2018). A previous study showed that the health risks of heavy metals in tea were lower than those in rice and vegetables (Huang et al., 2018). Also, the HI values for exposure to As, Cd, Cr, and Pb in three types of teas (green, black, and oolong) in Taiwan were also less than one, indicating no risk to human health (Shen and Chen, 2008). Although the health risk of heavy metals ingestion via tea intake was relatively small, there are other pathways that can play an important role in heavy metals exposure to humans (Hadayat et al., 2018). Hence, more comprehensive investigation for dietary structure and heavy metal contents in food are needed for health risk assessment. Moreover, the uncertainty of the results of the present assessment may exist, to some extent. Firstly, the tea infusion intake was considered as the only pathway for heavy metals exposure to humans. Other sources of heavy metals in the dietary structure, such as drinking water, vegetables, fruits, and grains, may play important roles in forming the probable health risks. A prior study indicated that the health risks caused by heavy metals depended on crop species, which showed that solanaceous fruiting vegetables exhibited the lowest risk, whereas grain (e.g., rice, corn, and wheat) posed the highest (Zheng et al., 2020). Secondly, heavy metals that entered the human body are not all taken up by the gastrointestinal tract of humans. A previous study showed that the bioavailable heavy metals absorbed accounts for ~40% of the amount in human body (Laparra et al., 2005). Moreover, the heavy metal bioavailability to humans are impacted by many factors (individual lifestyle, food types, the dose taken up by the human digestive system, etc.). The bioavailability of heavy metals may be different according to meals and mealtimes (Zheng et al., 2020). Additionally, the results of this study were based on the contents of heavy metals in unprocessed dried leaves of tea plants. During the actual tea processing, the made tea after processing may be contaminated by the heavy metals from the tea-made machine. For example, the levels of Cr and Ni in processed tea were chiefly increased from the rollers at the stage of cutting, while Fe and Cu significantly increased after rolling by the rotor vane (Zhang et al., 2018). Furthermore, there are differences for the potential health risks of heavy metals to different age groups of exposed receptors. In detail, the THQs of heavy metals showed a decreased order of toddlers > children > adults > teens (Karimyan et al., 2020). Moreover, the same transfer rates of specified heavy metals were used to calculate the EDI for the YTL and OTL infusion, but there may be difference from the actual situation. Thus, the results of the present assessment should be considered as preliminary; further

CRediT authorship contribution statement Jian Zhang: Investigation, Visualization, Writing - original draft. Ruidong Yang: Conceptualization, Methodology. Yuncong C. Li: Writing - review & editing. Yishu Peng: Software. Xuefeng Wen: Formal analysis. Xinran Ni: Investigation. Declaration of competing interest All of the authors declare no conflict of interest. Acknowledgements This research was funded by the National Natural Science Foundation of China (No. 41463009), the Innovation Group Major Research Project of Education Department, Guizhou Province, China (No. KY[2016]024), the Construction Project of the First-Class Discipline (Ecology) in Guizhou Province, China (No. GNYL[2017] 007), the Science and Technology Fund of Guizhou Province, China (No. J[2014]2072), and the Graduate Innovation Foundation Project ofEducation Department, Guizhou Province, China (No. QJH-YJSCXJH2018-049). The first author thanks his supervisor, Prof. Ruidong Yang and the First-Class Discipline (Ecology) in Guizhou Province for financial assistance to support his Visiting Scholarship in University of Florida, United States and Dr. Ashok Kumar Alva (former Research Leader, United States Department of Agriculture, Agricultural Research Service, Prosser, WA) for giving constructive revision suggestions and comments on this manuscript. Appendix A. Supplementary data Supplementary data to this article can be found online at https:// doi.org/10.1016/j.ecoenv.2020.110475. References Atafar, Z., Mesdaghinia, A., Nouri, J., Homaee, M., Yunesian, M., Ahmadimoghaddam, M., Mahvi, A.H., 2010. Effect of fertilizer application on soil heavy metal concentration. Environ. Monit. Assess. 160 (1), 83. Barone, G., Giacominelli-Stuffler, R., Storelli, M.M., 2016. Evaluation of trace metal and

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