Chelate-induced accumulation of rare earth elements in plants grown on soil and ash-based growing media

Rare earth elements (REE) have a wide range of applications. They are used, among others, in industry, agriculture, modern technologies or so-called “green” technologies. As a result of their increasingly common use, they might be released into the environment in the form of waste. Due to the mining sector associated with the extraction of rare metals, their impact on the environment and soil pollution is increasing (Lima, Ottosen, 2021). The waste can be a secondary source of their transfer to the environment.

REE accumulation in soil can potentially create a risk of toxic effects on living organisms or even humans in certain cases. It has been shown that REE can negatively affect the level of brain intelligence, which can ultimately result in memory loss. REE can also enter the placenta and blood during pregnancy, which can lead to birth defects (Wu et. al., 2013; Adeel et al., 2019).

According to the International Union of Pure and Applied Chemistry (IUPAC), REE are a group of 15 lanthanides with atomic numbers from 57 to 71 and 2 scandiums with atomic numbers 21 and 39, namely lanthanum (La), cerium (Ce), praseodymium (Pr), neodymium (Nd), promethium (Pm), samarium (Sm), europium (Eu), gadolinium (Gd), terbium (Tb), dysprosium (Dy), holmium (Ho), erbium (Er), thulium (Tm), ytterbium (Yb), lutetium (Lu), scandium (Sc) and yttrium (Y) (Tao et al., 2022). REE are not as rare in the Earth’s crust as their name suggests. The most abundant element is Ce, since its average concentration is about 66.5 mg kg⁻¹. However, REE deposits are scattered in the Earth’s crust in low concentrations (Dinh et al., 2022). Natural resources of REE are limited so these elements are treated as critical raw materials. Therefore there is an increasing interest in recovery of metals, including REE, from industrial waste such as ash or REE enriched soils.

In the case of plants, REE are accumulated in the order root > leaf > stem > flower > fruit. However, near polluted areas, the metal content on the leaf surface is higher due to dust deposition (Yin, 2021). Phytoextraction is a process where plants extract contaminants from soil or water, then transport and store them in aboveground tissues. The most effective plants for this purpose are hyperaccumulators. These are plant species that can accumulate larger amounts of metals in their aboveground parts without toxic effects on them. For REE hyperaccumulators, the threshold concentration is 100 to 1000 mg kg⁻¹ (Dinh et al., 2022; Deepika, Haritash, 2023). This is an environmentally friendly and inexpensive method, useful in soil remediation, but the main limitation is that it is inefficient. Therefore, ways to improve the method are being sought (Zhang et al., 2024). Adding chelates in phytoremediation methods can accelerate metal removal from soil or waste. Chelating agents act as a chemical bond, which results in the formation of metal chelate complexes. These chelates contribute to the increased solubility and plant availability of metals (Wu et al., 2013; Salifu et al., 2024). It has been shown that chelators can increase the rate of absorption and translocation of metals by up to 45% from roots to aboveground plant parts in the process of phytoremediation. Both synthetic and natural compounds can be used as chelators. The synthetic ones include: ethylenediaminetetraacetic acid (EDTA) and nitrilotriacetic acid (NTA), and the natural ones: histidine, citric acid, malic acid and citrate (Beiyuan et al., 2021; Zulkernain et al., 2023). Chelators such as EDTA or amino-acids (AA) act by increasing the bioavailability of heavy metals and rare earth elements. Low molecular weight organic compounds, including malic acid, citric acid, histidine and citrates, are secreted by roots into the soil and then act as chelating agents enabling the release, translocation and accumulation of heavy metals (Rabbani et al., 2024). Chelator-induced solubility of metals can pose an environmental risk related to leaching of metals to groundwater. Therefore, it would be justified to perform such induced phytoextraction under controlled conditions, for example in containers without leakage of soil solution. Such a technology would enable use of constructed substrates, produced on a basis of industrial waste combined with some organic materials to improve plant growth conditions.

In order to fill some knowledge gaps, the aim of the study was to demonstrate the differences between the use of two doses of chelators (5 mM and 10 mM): CA, EGTA, and EDTA on three selected plant species which grew on two substrates. The plant species were used: Achillea millefolium L., Trifolium pretense L., Dryopteris erythrosora (D. C. Eaton) Kuntze). In this study, the following REE representatives were measured in plant tissues: lanthanum (La), cerium (Ce), europium (Eu), gadolinium (Gd). The substrates were used based on: soil with increased REE content and power plant ash.

High biomass growth or the ability of plants to accumulate higher concentrations of metals without toxic effects on the plant are the conditions that a plant must meet in order to be effective in phytoremediation or phytomining. When using phytoremediation techniques, it is important to ensure that the plant used is capable of creating a sufficient biomass. Plants that accumulate metals are harvested and then subjected to a pyrolysis to recover the metals. It has been proven that lanthanides (especially La and Ce) can stimulate the growth of certain plant species. For example, by promoting nitrogen metabolism and other metabolic pathways (He et al., 2022). In this experiment the largest amount of biomass was obtained for T. pratense. This plant is usually grown as a fodder plant due to its high biomass growth and high protein content. Higher biomass was collected for plants grown on substrate 1 compared to substrate 2. This could have been due to stimulation of growth after prior adding REE in easily soluble form and overall better growth conditions than present in substrate 2 (Grčman et al., 2001).

Evaluating direct effects of the applied chelates on plant growth, it is difficult to draw general conclusions. The effects was rather plant specific. Literature search provides ambiguous information on EDTA impact on plant growth since both toxic and protective effects have been reported (Saleem et al., 2020). Our data suggests that the doses of chelates applied (5 and 10 mM) are in general not harmful to the tested plant species, however the sensitivity of plants can be diverse.

Soil pH directly affects plant development by determining the availability of nutrients and metal toxicity to plants. Adequate availability of macronutrients for plants occurs in the range of pH 6–7 (Remigio et al., 2020). In the experiment, the pH of the substrates ranged from 5.5 to 7.6. In the studies conducted by Cao et al. (2001) it was shown that the release of La, Ce and Gd gradually increases with decreasing pH.

In accordance with this theory, it was observed that plants accumulated higher amounts of REE in substrate 1, which had a lower pH compared to substrate 2. Some plants prefer more acidic environments and may contribute to lower soil pH due to the release of organic acids into the substrate or through root exudates as observed in the case of the fern D. erythrosora (Shan et al., 2003).

Two substrates were used in the experiment: one consisting of soil (substrate 1) and the other whose main component was ash (substrate 2). Due to the global use of coal-based energy, environmental problems are increasing, including the creation of areas where fly ash (FA) landfills are located. FA waste disposal is characterized by a high degree of absorption of water, energy and land surface. It is also the cause of pollution associated with the atmosphere and water bodies. FA, compared to soil, has a lower water retention capacity, so it can cause water stress more quickly (Yadav et al., 2021).

This may result in slower plant growth and reduced biomass production. In this experiment, plants growing on substrate 2 had lower biomass compared to plants growing on substrate 1, however the differences can be of complex character, involving overall physical and chemical growth conditions, direct effects of chelates and chelate-driven availabilities of nutrients and micronutrients.

For substrate 1, the REE content in plant tissues ranged from 0.31 to 63.60 mg kg⁻¹, while for substrate 2, the REE ranged from 0.01 to 0.94 mg kg⁻¹. The determined REE content was higher in substrate 1 than in substrate 2.

There were substantial differences between levels of REE in aboveground and underground parts between the substrate 1 and substrate 2. Addition of REE salts have created much larger pool of plant available REE in soil. It is commonly observed that elements added to soil as salts or salt solutions are in short-term much more easily absorbed by plants as those present in soil minerals or sorbed by soil components (Dong et al., 2021). Both in control and chelate treated substrates the greatest ability to bioaccumulate REE showed T. pretense.

Most of the REE were accumulated in underground parts and not transferred further to aboveground parts. According to Ramos et al. (2016) this is because REE absorbed by roots encounter an apoplastic barrier on their way to the xylem. This results in difficulties during the translocation of lanthanides to other plant organs. As a result, the order of REE accumulation in plant tissues is as follows: roots > stems > leaves > flowers > fruits > seeds. Similarly, in the study of Lihong et al. (1999) showed that the application of EDTA increased REE bioaccumulation in wheat (Triticum aestivum L.) seedlings, metal both in roots and tops (stem and leaves). However, still higher REE content was measured in roots than in tops. It is assumed that in the chemically assisted phytoremediation method by adding chelating substances the content of metals taken up by the plant increases. In this study, the use of chelators: CA, EGTA and EDTA had a small influence of the studies plants. In the studies of Ozaki et al. (2000) after the addition of chelating reagents: NTA, lactic acid and succinic acid to the medium containing Y, Ce, Pm, Eu, Gd, Lu and Yb content in D. erythrosora increased compared to the control. Nawaz et al. (2022) investigated the usefulness of Brassica napus in phytoremediation of Ni using two chelators: 10 mM CA and 1.5 mM EDTA. The studies showed greater metal accumulation by EDTA-treated plants as compared to CA. Additionally, it was shown that the addition of chelators alleviated the toxic effects of Ni on canola. Other studies using chelating agents show that enrichment of soil with histidine, malic and citric acids increased the concentration of light REE in the natural REE hyperaccumulator Dicropteris dichotoma by 21–78%, as compared to the control (Shan et al., 2003).

Translocation factor (TF) is a parameter indicating the efficiency of a plant in transferring metals from roots to shoots. In order for a plant to be classified as most useful in phytomining techniques, the ratio of metal content in shoots to roots should be greater than 1. If TF is lower than 1, metal accumulation is predominant at the root level (Takarina, Pin, 2017).

In general, the effect of chelates on REE accumulation was plant-specific. D. erythrosora that accumulated more REE then other species, also differently responded to the chelates tested. Both in case of substrate 1 and 2. So independently on the level of soluble REE in a substrate, 10 Mm CA stimulated REE accumulation most efficiently for this plant. This can be treated as a positive result due to lower potential toxicity of CA than that represented by EDTA or EGTA. In study Ibrahim (2023) examines how citric acid affects the phytoextraction capabilities of pumpkin (Cucurbita pepo L.) in soils contaminated with heavy metals. The application of citric acid significantly enhances plant growth, biomass, and the uptake of heavy metals, suggesting its potential as an effective agent in phytoremediation strategies.

The differences between plant species in TF values were greater than the differences between the chelates used. Our TF data indicate that translocation from underground to aboveground parts for D. erythrosora exceeded the other species in this respect. The highest differences between D. erythrosora in terms of TF index values can be seen for substrate 2.

TF factor higher than 1 was obtained in the experiment only for two plant species: D. erythrosora and A. millefolium growing in substrate 2. The highest TF was obtained for D. erythrosora using 10 mM CA. It was proven that the REE concentration in soils and wastes depends on the parent substrate material. To determine the transfer of REE from the substrate to the plants, the bioaccumulation factor (BCF) was calculated. In the experiment, the BCF index value was not higher than 1.

Different effects of chelates on REE bioconcentration index were observed for the plant species, which confirms differences in physiology of REE uptake and transport between those diverse plants. D. erythrosora responded with increased BCF for all elements to addition of 10 mM doses of CA, EDTA and EGTA. This indicates the potential for further enhancement of REE bioaccumulation by the fern through optimisation of chelate dosing and application strategy.

The results of the study showed that the addition of chelates can contribute to increased REE accumulation in plant tissues but to small extent. In addition, the use of plants in phytoremediation techniques is one of the limitations, because the uptake and accumulation of REE by plants depends on, among others, environmental factors or the plant species and its morphology. Therefore, it is necessary to search for suitable plants that will be able to accumulate REE in their aboveground parts and chelating compounds and their appropriate doses in order to enhance the bioaccumulation process.

From a practical point of view, D. erythrosora can be considered the most suitable species for use in chelate-assisted REE phytoextraction, despite the lowest total biomass than other plants tested. However, the significantly higher REE content in the aboveground parts and the highest REE translocation to the aboveground parts, expressed by the TF index, suggest that further process optimization should include this plant.

The fern positively responded to chelate addition with increased biomass. This observation combined with the recorded translocation factor above 1, characteristic for intensive transfer of REE from roots to shoots, indicates a potential for the REE bioaccumulation enhancement, as compared to that observed in our study. It seems that there is room for enhancing effectiveness of the entire process through optimisation of ash substrate chemical composition and optimal combination of chelates, substrates and plant species. We can assume that the assisted bioaccumulation process would be more efficient in case of ashes richer in REE. It seems that further research shall focus on tailor-made combinations of chelates and ash substrate compositions (for example additions of organic or pH affecting materials to ashes) for specific characteristics of pre-selected ashes. Since various REE respond to chelates differently, further optimisation can be achieved by selecting optimal chelates and their doses depending on the chemical composition of the ash. TF of fern in many cases increased after chelate addition, therefore it can be assumed that certain modification of the substrate, for example lowering pH, in order to create better growth conditions and greater REE phytoavailability, would greatly enhance the REE amounts transferred from the growing media to plant tissues.