Accumulation and Distribution of Rare Earth Elements (REEs) and Non-REEs With Vetiver Grass in The Abandoned Ion-Absorbed Rare Earth Mine

Overexploitation of rare earth elements (REEs) has caused serious desertication and environmental pollution, and ecological restoration of mines has attracted increasing national attention. In this paper, experiments involved land plowing, organic fertilizer broadcasting and vetiver cultivation were carried out to repair abandoned ion-absorbed rare earth mines (REM). Toxic metals content and pH in mining soil, distribution and transportation of toxic metals in the soil – vetiver grass system were investigated in detail. Results revealed that the abandoned REM soil was weakly acidic (pH=4.09) and rich in various toxic metals composed of REEs (La, Ce, Nd, Y, Gd, Dy) of 657.57mg/kg and Non-REEs (Pb, Cu, Se, As, Cd) of 109.98mg/kg. The distribution pattern in vetiver grass illustrated that toxic metals accumulation was mainly concentrated in the roots instead of shoot, and then the cumulative concentration of REEs in roots were much greater than that of Non-REEs. Furthermore, vetiver grass exhibited preferential accumulation of Cd, Se and REEs during the absorption process (from soil to root) and preferential accumulation of Pb, Cu and As during the translocation process (from root to leaf). The adsorption behavior of toxic metals by vetiver was conrmed due to these observed irregular particles in the scanning electron microscopy.


Introduction
As a strategic resource, rare earth elements were widely used in various industries, such as clean energy (  However, restoration of ion-absorbed REM was extremely slow and di cult owing to the hostile growing conditions, such as lack of organic matter and toxic metals contamination (Pang et al 2003). Thus, the appropriate plant species selection to resist these unfavorable conditions was essential for rehabilitating abandoned rare earth mines.
Vetiver grass, as a fast-growing perennial C4 grass, possessed tall stem (1-2m) and extensive strong root system (up to 3-4m deep) (Vargas et al 2015). Moreover, vetiver was very popular in India due to its special economic value including its roots could be processed into volatile essential oil and leaves could be used as feeds for cattle, goats and horses (Chen et al 2020). Importantly, vetiver grass tolerated various adverse conditions, such as prolonged drought, extreme temperatures, acidity, alkalinity and high concentrations of toxic metals ( There were currently few researches that have been tested to evaluate the extraction capacity of REEs and Non-REEs using Vetiver grass from abandoned ion-absorbed rare earth mines. Therefore, the objectives of the present study were (1) to determine physical-chemical parameters and toxic metals content of contaminated sites, (2) to explore the feasibility analysis for vegetation restoration of rare earth mine, (3) to investigate toxic metals uptake ability of the roots and shoots of vetiver and (4) to assess the transportation of these toxic metals in the soil-vetiver grass system.

Study area
The experiments were conducted in the abandoned ion-adsorption rare earths mines, had a high background level Two abandoned heap leaching rare earth mines were selected for research in Dingnan County: REM-1 and REM-2, the site area of REM-1 and REM-2 were approximately 664.75m2 and 709.66m2, respectively. Two selected test sites had become exposed hillside and sandy soil land ll, and almost no plants can survive. The main reason was that ammonium sulfate was used to leach rare earth mines, resulting in a large amount of ammonia nitrogen and toxic metals remaining in the mines, which inhibited plant growth (Fig. 1a, c).

Planting experiment and sample preparation
The experiments land was evenly broadcasted with 75 g/m2 organic fertilizer at the soil surface, and then plowed to a depth of 80cm with excavator. Vetiver grass seedlings were purchased and each fresh plant sapling was pruned (the shoots were originally 15-25 cm high and the roots 5-15 cm long). The pretreated vetiver seedlings were planted at 50-60cm row spacing, 40-60cm cluster spacing and 6-10 tillers for each slip. All plants were watered with running water every other day to ensure high survival rate during the rst two months.
In each sampling site, the plants (root and shoot) were sampled at harvest and their corresponding rhizospheric soils was taken at different growth time (0, 36, 90, 164, 246d), and then REEs (La, Ce, Nd, Y, Gd, Dy) and Non-REEs (Pb, Cu, Se, As, Cd) content in samples were detected after drying and acid digestion. Before analysis, all the samples were stored at 4℃ to keep fresh.

Soil and Plant Sample Analyses
All soil samples were sieved and dried to constant weight, and then underwent acid digestion with nitric acid (HNO 3 ), sulfuric acid (H 2 SO 4 ) and phosphoric acid (H 3 PO 4 ). The toxic metal content of soil sample was analyzed using atomic absorption spectrophotometry (AAS, AA6880, Shimadzu, Japan).
Freshly roots and shoots of Vetiver were washed with deionized water to remove any adhering soil particles, and then were oven-dried for 72 h at 70°C until it achieved a constant weight. The plant samples were dissolved with nitric acid (HNO 3 ), sulfuric acid (H 2 SO 4 ) and phosphoric acid (H 3 PO 4 ) after it was homogenized in a mortar and pestle for the elemental analysis using inductively coupled plasma mass spectrometry (ICP-MS, Aglient 7700X, America) (Ng et al 2016).
The crystal morphology of plant samples (roots and shoots) was analyzed by scanning electron microscope (SEM, JSM-7500F, JEOL Ltd, Tokyo, Japan).

Data Analyses
The biological concentration factor (BCF) and biological accumulation coe cient (BAC) were de ned as the ratio of toxic metals content in the roots and shoots to those in the soil, respectively. The translocation factor (TF) represented the ratio of toxic metals content in the above-ground part to those in the plant roots. Similarly, the percentage of metals uptake e cacy (MUE) were de ned as the ratio of toxic metals content in the above-ground part to those in plants (Ka l et al 2019). These indicators were used to assess the ability for toxic metals accumulation and translocation upwards in the soil -vetiver grass system.

REM soil analysis and vegetation restoration
The physico-chemical parameters and toxic metals content in REM-1 and REM-2 prior to vetiver planting were presented in Table 1. The results indicated that the unrepaired soil pH of REM-1 and REM-2 were 4.45 and 4.09, respectively. Moreover, the total concentrations of Non-REEs in two experiment soils were 109.98mg/kg and 107.51mg/kg, respectively. Noticeably, Pb content was higher than other Non-REEs.
The total soil REEs (542.78 and 657.57mg/kg) in abandoned rare earth mine, which might become the main source of REEs in the future, were much higher than the average levels (177 mg/kg) for the soils in China (Liang et al 2005). In addition, within sampling sites, the Ce content (431.56 and 463.64 mg/kg) in the rare earth mine was higher than that of any other rare earth element, probably because the parent material of rare earth mine for experiment in Dingnan County was primarily granite, rich in high-content Ce. Compared with other REEs, Ce was more di cult to leaching from the leaching of rare earth mines with ammonium sulfate, which caused its higher abundance in abandoned REM. Furthermore, the concentration of Y and La were 89.90 and 55.58 mg/kg, which were higher than that of Dy and Gd.
The selection of appropriate plant species was thought to be a key step to successful revegetation. In this study, it was very successful that vetiver grass was selected as a plant to restore abandoned REM (Fig. 1b, d). It was obvious that vetiver grass exhibited a strong tolerance to acidic soil and various toxic metals, and good growth ability, which indicated that it was feasible to achieve vegetation restoration and ecological restoration with vetiver grass in mining areas.

Metal concentrations and distribution pattern in REM soil
Remediation and degradation experiments of Non-REEs in REMs were estimated, and the results were shown in Fig. 2. Compared with the other elements (Cu, As, Se and Cd) content, the residual pb content in REM-1 and REM-2 was the highest, the concentrations of other metals in REM-1 and REM-2 were in the order Cu > As > Se > Cd. Besides, a signi cant reduction in the content of all Non-REEs with the growth time of vetiver grass in rare earth mines was observed during the experimental period. The main reason was that the vetiver grass roots continuously absorbed and extracted these toxic metals from the soil during the growth process.

Toxic metals concentrations and distribution pattern in vetiver grass
Extraction experiments of Non-REEs (Pb, Cu, As, Se, and Cd) and REEs (La, Ce, Nd, Y, Gd and Dy) from REM with vetiver grass were analyzed, and the results were shown in Fig. 3. The concentrations of Non-REEs and REEs in roots were consistent with those in the initial soil, but those in the shoot were slightly different at harvest. This indicated that the adsorption capacity of vetiver was closely related to the metal content in the contaminated soils, which was similar to other research conclusions (Liang et al 2005). In addition, toxic metals concentration in the tissue of vetiver grass decreased in the order of root shoot, which showed that vetiver grass accumulated large amounts of metals in the roots and restricted their translocation to the shoots. These ndings boldly speculated that when vetiver grass was used for the rehabilitation of sites contaminated with these metals, their roots can be centralized processing through harvested, while shoots can be safely harvested to feed animals due to these toxic metals were almost concentrated in roots, not in shoots.
As can be seen from Fig. 3, Among the Non-REEs (Pb, Cu, As, Se, and Cd) studied in the experiment, the concentration of Pb in root was far more than that of other Non-REEs, nevertheless, Cu content in the stems was the largest. Similarly, Unlike the stems, the REEs contents in the roots were extremely large, especially Ce (100.1mg/kg in REM-1) and Y (101.1mg/kg in REM-2), which revealed that vetiver grass was a hyperaccumulator plants for REEs.
The distribution and transportation of toxic metal in the soilvetiver grass system In order to study the accumulation and translocation of Non-REEs and REEs in the soil -vetiver grass system, the biological concentration factor (BCF), biological accumulation coe cient (BAC), translocation factor (TF) and percentage of metal uptake e cacy (MUE) were used to evaluate the absorption capacity of vetiver for Non-REEs and REEs (Fig. 4).
Obviously, the BCF value of Non-REEs and REEs were signi cantly higher than BAC value, indicating that these toxic metals absorbed by vetiver were mostly retained in the roots. Similarly, according to the lower TF value (Fig. 4b), it was found that Cd, Se and REEs performed extremely poor translocation capability from root to shoot,which indicated that the accumulation of these metals by vetiver mainly depend on the roots instead of shoot. In addition, different BCF and TF value in the Fig. 4 indicated that vetiver grass had selective absorption and translocation of Non-REEs and REEs in the soil -vetiver system. Figure 4a showed that vetiver grass roots had a strong accumulation capacity for Cd, Se and REEs (except Ce) due to their BCF value exceeding 1. On the contrary, the Pb, Cu and As accumulation by vetiver relies on the joint action of the root and stem of vetiver, due to their high TF value, the MUE value of these metals just con rmed this view.

SEM analysis
To observe clearly the surface topography of vetiver roots and shoot, the SEM micrograph of vetiver roots and shoot obtained at initial and harvest were shown in Fig. 5. As can be seen from the SEM micrograph of vetiver roots at planting (A) and harvest (B), the initial root had a smoother surface with larger gaps, while the harvested root surface was rough and rich in a lot of irregular particles. These observed irregular particles might be composed of Non-REEs and REEs extracted by vetiver grass from REM. In addition, the SEM micrograph of the vetiver shoots at planting (C) and harvest (D) showed noticeable differences in morphology. Figure 5C showed that the vetiver shoots at planting have su cient moisture and abundant stomata, on the contrary, those at harvest were shriveled and had fewer stomata (Fig. 5D). FA Melato (Melato et al 2016) reported that excessive concentration of toxic metals in contaminated soils disturb the physiological system, including transpiration and respiration, which resulting in the reduction of water content and stomata in the plant. These changes in the plant stems in this study were in agreement with the results of previous studies (Singh et al 2004).

Conclusions
In this paper, the experimental soil was weakly acidic (4.09) and rich in various toxic metals composed of REEs (657.57mg/kg) and Non-REEs (109.98mg/kg) with the highest content of Ce. After 246 days of remediation with vetiver grass, Non-REEs concentrations in REM soil have decreased, at the same time, all toxic metals accumulation in vetiver grass was mainly concentrated in the roots instead of shoot, which indicated that the ecological restoration technology with vetiver was effective in repairing abandoned REM. In addition, vetiver grass was considered to be an effective REEs phyto-stabilizer, owing to the high considerably REEs accumulation in its roots.
Furthermore, vetiver grass exhibited preferential accumulation of Cd, Se and REEs during the absorption process (from soil to root) and preferential accumulation of Pb, Cu and As during the translocation process (from root to leaf).
SEM micrograph results revealed that Non-REEs and REEs in the soil were absorbed and extracted mainly depended on vetiver roots. Besides, excessive toxic metals reduced the water content and the number of stomata in shoot, which might disturb the physiological system.

Declarations Authors declarations
Ethics approval and consent to participate Not applicable  Page 12/12 a soil-root BCF for toxic metals of REM-1 and REM-2 b root-shoot TF for toxic metals of REM-1 and REM-2 c soilshoot BAC for toxic metals of REM-1 and REM-2 d metal uptake e cacy for toxic metals of REM-1 and REM-2 Figure 5 SEM micrographs of vetiver roots at planting (A) and harvest (B); the vetiver shoots at planting (C) and harvest (D)