Correlation between Selenium and Heavy Metal Content in Camellia Oleifera in Hainan, China

Eighteen forests of Camellia oleifera in Hainan province, China were selected, to measure the contents of selenium (Se), zinc (Zn), mercury (Hg), cadmium (Cd), chromium (Cr), lead (Pd) and arsenic (As) in soils, roots, leaves, and kernels, and analyze the correlation between the soil content and the corresponding content in the plant. It was found that the content of Se in soils ranged between 0.489 and 2.110 mg/kg, and higher than 1.0 mg/kg in 10 out of 18 forests. The average content of six heavy metals was low and fulfilled the requirements of soil environment for green-food production, except that the contents of Cr, Pb, and Cd exceeded standards in few regions. The content of Se in Camellia oleifera was relatively low. Only five regions showed detectable levels of Se in kernels, with the highest in Fuwen Town of Dingan county of 0.085 mg/kg. The contents of Zn, Cr, Pb, Cd, As, and Hg in roots of Camellia oleifera were higher than in leaves and kernels. The contents of Zn, Cr, and Pb were higher than that of Cd, As, and Hg. The As content in soil was significantly correlated with that in roots of Camellia oleifera. The soil Pd content was positively correlated with that in roots of Camellia oleifera. The Cr, Pd and Cd content in roots was positively correlated with that in leaves significantly. However, the Hg content in roots was positively correlated with that in kernels significantly. The Se content in roots of Camellia oleifera was negatively associated with that of the other six heavy metals, with a significant negative correlation between Se and As.


INTRODUCTION
Soil is an integral part of human ecological environment, and the foundation of agriculture and forestry production.Heavy metal pollution adversely affects the growth and development of plants and the quality of agricultural products [1].Studies on rice [2] and persimmon [3] suggested that Se not only reduced the uptake of heavy metals such as cadmium (Cd) and lead (Pd), but also exerted anti-cancer effect on the human body [4,5].Studies focused on selenium (Se) and the other heavy metals in the soils cultivating rubber and pepper found abundant Se in soil in the Hainan province [6][7][8].Camellia oleifera is an important and major source of edible oil in South China [9,10].It has a long history of planting and utilization in Hainan province.Also known as 'pomelo on the hill', Camellia oil from Hainan province is of high quality due to excellent production environment.Studies have been published on Camellia oleifera germplasm resources [11], varieties selection [12], ecological adaptability [13], and high yield cultivation techniques [14].However, the correlation between Se and heavy metal contents in soil and their corresponding contents in Camellia oleifera have yet to be reported.In the current study, 18 forests of Camellia oleifera from 11 major production counties in Hainan province were selected to measure the contents of selenium (Se), zinc (Zn), mercury (Hg), Cd, chromium (Cr), Pd and arsenic (As) in soils, roots, leaves, and kernels.The correlation between soil levels of Se and heavy metals and their corresponding contents in Camellia oleifera was analyzed in order to provide a scientific basis for pollution-free and Se-rich oil tea production in Hainan province, China.
Five mid-sized trees from each forest were selected as the samples.The samples of soil were collected by wooden spade one meter away from the sampling trees, at a depth of 0 to 20 cm.The soil samples were then mixed and stored up to 1 kg by quartation method.The slim roots of 5 g from each tree sample were taken after removing the surface soil.Forty mature leaves were collected from east, south, west, and north of each tree; four mid-sized fruits were collected as well.
Soil samples were naturally air-dried indoors and filtered through 0.147 mm nylon sieve.The plant samples were repeatedly cleaned by water and deionized water thrice to remove dirt and mud.The kernels were removed from the fruits.The leaves, roots and kernels were fixed at 105°C for 30 minutes and crushed after drying at 65°C to obtain a constant weight.

Determination of Se and Heavy Metals in Soil
The soil contents of Se, Hg, and As were measured according to the standard industrial methods in NYT1104-2006 (Chinese Standard, the same below), NY/T 1121.10-2006 and NY/T 1121.11-2006 using atomic fluorescence spectrophotometry (AFS-9120, Beijing Jitian Instruments Co., Ltd, China).The soil contents of Zn, Cr, Pb and Cd were measured according to the processing methods described in the national standard (GB/T 17141-1997) by inductively coupled plasma mass spectrometry (ICP-MS) (ELAN, DRC-e, PerkinElmer, US).

Determination of Se and Heavy Metals in Plant
The content of Se in roots, leaves, and kernels of Camellia oleifera was measured according to the processing method described in the Chinese national standard GB/T 21729-2008.Zn, As, Cr, Hg, Pd and Cd were measured according to the processing method described in the national standards GB/T 5009.

Statistical Analysis
All measurements were performed in triplicate.Excel 2010 and SPSS 17.0 software were utilized for statistical analysis.Certain data and analysis were absent due to undetectable low content of metal elements.

Soil Contents of Se and Heavy Metals
It was found from 18 forests that Se content ranged between 0.480 and 2.110 mg/kg, which was high according to the classification method [3].The Se content exceeded 1.0 mg/kg in 10 forests such as in Penglai of Wenchang county.All the soil samples were acidic, and the heavy metal content was relatively low; the 'Soil Environmental Quality Standard' (GB, 15618 -1995) was used as a reference.The As and Hg contents of all the tested regions were lower than first-grade standard level.The Zn content was lower than first-grade standard level except of Penglai in Wenchang.The Cr content of all the tested regions was lower than the first-grade standard level except for Penglai in Wenchang county, Dongshan in Haikou county, and Longhu in Dingan county.Though Pb content of six counties such as Longhu in Dingan county exceeded first-grade standard level, all tested samples were lower than second-grade standard.The Cd content in all tested regions was lower than natural background, except in Jiaji of Qionghai county, which was higher than second-grade.All tested soil samples fulfilled the requirements of soil environmental quality for green food production using 'green food producing area environment quality' (NY/T 319 -2013) as reference, except soil Cr in Penglai of Wenchang county, Dongshan of Haikou county, and Longhu of Dingan county, soil Pb in Nankun of Tunchang county, and soil Cd in Jiaji of Qionghai county (Table 2).

Se and Heavy Metals Contents in Plant
As shown in Table 3, there was difference in all root samples obtained from 18 forests in terms of Se and heavy metals contents.The Se content in roots was relatively low,with the maximum of only 0.104 mg/kg being the highest in Heling farm and Jiale in Chengmai county, followed by Wuzhishan county.The Zn, Cr and Cd, Pb, As and Hg contents in roots were the highest in Nankun of Tunchang county, Jiale in Chengmai county, Changhao of Wuzhishan county, Dacheng of Danzhou county and Heling farm of Chengmai county, respectively.As shown in Table 5, Se content in kernels of Camellia oleifera was very low, only detectable in five regions and was highest in Fuwen of Dingan county.Zinc content in kernels was highest in Tongshi of Wuzhishan county, and undetectable in six regions including Penglai of Wenchang county.Cr content in kernels exceeded 1.0 mg/kg in seven regions such as Dongshan of Haikou county.Pd content in kernels was over 0.5 mg/kg in Dongshan of Haikou county and Zhongxin of Chengmai county.The contents of Cd, As and Hg were very low, undetectable in kernels of all the 18 forests.

Correlation of Se and Heavy Metals Between Soil and Roots, Leaves, and Kernels of Camellia oleifera
Significant positive correlation in As and Pb contents was observed between soil and roots of Camellia oleifera.However, significant negative correlation of Cd content between soil and roots of Camellia oleifera was observed.Significant positive correlation between soil and kernels was only found for Cd content; suggesting that only soil As and Pb content showed high biological availability for Camellia oleifera (Table 6).
To investigate the transferability of metal elements from roots to leaves and kernels, the correlation study was used to compare the contents of each element in different parts of Camellia oleifera (Table 7).It was found that there was significant positive correlation between roots and leaves in terms of Cr and Cd content; and there was positive correlation between roots and leaves in terms of Pd content.However, significant negative correlation was found between roots and leaves in terms of Hg and Se contents.In addition, there was significant positive correlation between roots and kernels with regard to Hg content; whereas, significant negative correlation was found between roots and kernels in terms of Cd content, implying that Cr and Cd are easily translocated from roots to leaves, and Hg from roots to kernels.

Correlation Between Se and Six Metal Elements in Roots and Leaves
The relationship between the roots and leaves regarding the Se content and 6 metal elements is shown in Table 8.There was negative correlation of content between Se and the other six metal elements in the roots of Camellia oleifera, and significant correlation was found between Se and As in roots.A positive correlation between Se and Cr content as well as Hg in leaves, and negative correlation between Se and Pd as well as Cd, suggested that Se reduced the absorption of heavy metals by roots, especially the absorption of As.

DICUSSION
Se-enriched soil is widely distributed in Hainan Island of China.Se content was high in the soils of Haikou and Chengmai counties [15].It has been confirmed in the current study that Se content ranged between 0.489 and 2.110 mg/kg in 18 forests of Camellia oleifera, which is higher than that in the soil of rice paddy fields [15].The soil Se content was higher than 1.0 mg/kg in 10 forests out of 18.However, there was no significant correlation between the soil and roots of Camellia oleifera with respect to Se content.The Se content in leaves and kernels was also low, probably due to acidic soil conditions and impaired biological availability of Se [16].Our findings suggest that blindly supplementing Se fertilizer in the nutrient management of  Camellia oleifera should be avoided.Instead, alkaline fertilizer to promote the activation of Se and improve its bioavailability in soil [17], or screen for suitable Camellia oleifera varieties should be recommended to produce effective and economical Se-enriched camellia oil [2,18].The average content of heavy metals in soil was low in Camellia oleifera forests of Hainan province and fulfilled the requirements for green-food production.However, the heavy metal content in soil and Camellia trees greatly varied geographically with different cultivation methods, which might be attributed to diverse soil characteristics and use of chemical fertilizers [19].Therefore, instead of chemical fertilizers containing heavy metals, ecofriendly cultivation using manures [20] and forest intercropping [21] should be adopted.
The content of heavy metals in roots was higher than that in leaves and kernels, suggesting that Camellia restricts the translocation of heavy metals from roots to leaves and kernels [22], which is the key to successful utilization of seeds of Camellia oleifera.The mechanism of heavy metal distribution in the plant following absorption from soils should be further elucidated.Also, production processes that limit heavy metal transfer from roots to kernels should be adopted.Variations in heavy metal accumulations by different organs of plants are a consequence of species and climate diversity [23], which is also known as heavy metal enrichment coefficient.

CONCLUSION
In the current study, the enrichment coefficient was relatively higher for Zn, Cr and Pb by the roots of Camellia oleifera, which was consistent with the study based on tropical trees such as Casuarina equisetifolia [24].Significant positive and negative correlations were found between soil and roots with regard to As and Pb contents, respectively, implying that As and Pb are easily activated and absorbed by Camellia oleifera, since they are associated with high utilization efficiency of phosphate [25].Therefore, it is necessary to further analyze the relationship between essential elements and heavy metal activation in Camellia [26].A significant positive correlation between the roots and leaves in terms of Cr and Cd contents was observed.A significant positive correlation between roots and kernels in terms of Hg content suggested that the transportation of Hg into fruits was relatively easier.It is thus essential to control the Hg content in soil to reduce the risk of Hg absorption.Se and six heavy metals were in negative correlation in roots, especially Se and As, implying that strengthing of Se enrichment effectively reduced the accumulation of heavy metals [27].Therefore, in addition to increasing Se bioactivity in soil, Se supplementation through leaves is recommended to regulate heavy metal distribution in fruits [28], after carrying out additional testing and analysis.

Table 4 ,
Se content in leaves of Camellia oleifera was the highest in Heling farm of Chengmai county, followed by Jiale of Chengmai county; and it was the lowest

Table 5 . Se and six heavy metals in Camellia oleifera Kernels in Hainan (mg•kg -1 ).
in Dacheng of Danzhou county and Huishan of Qionghai county.Cr content in leaves was the highest in Longhu of Dingan county, followed by Fuwen of Dingan county.Pd, Cd, and Hg contents in the leaves were the highest in Heling farm of Chengmai county, Xishui of Baisha county, and Zhongxing of Chengmai county, respectively.The Cd content was undetectable in leaves in four counties including the Heling farm of Chengmai county; the content of Hg was undetectable in leaves in three counties which include Penglai of Wenchang county.The Zn and As contents in leaves from all 18 counties were undetectable.

Table 6 . Coefficients of Se and heavy metal elements in soils and different parts of Camellia oleifera in Hainan.
*indicates correlated at P<0.05 level Certain data are absent due to undetactable content of Zn and Se in leaves and As and Se in kernels in some areas.Similar conditions apply in the following tables.