Volume 10, Number 2 (January 2023):4107-4117, doi:10.15243/jdmlm.2023.102.4107 ISSN: 2339-076X (p); 2502-2458 (e), www.jdmlm.ub.ac.id
Open Access 4107 Research Article
The benefit of the Arabidopsis halleri ssp. gemmifera root exudate in cadmium extraction from the cadmium contaminated soil
Agni Lili Ariyanti*, Mei-Fang Chien, Chihiro Inoue
Graduate School of Environmental Studies, Tohoku University, Aramaki Aza, Aoba 6-6-20, Sendai 980-8579, Japan
*corresponding author: email@example.com
Abstract Article history:
Received 10 September 2022 Accepted 21 October 2022 Published 1 January 2023
This study focuses on how to solve cadmium (Cd) contamination in soil because this contaminant decreases soil quality. Soil remediation using the hyperaccumulator plants is an optional process to solve soil contamination.
Arabidopsis halleri ssp. gemmifera (hereinafter referred to as A. halleri), is one of the candidate plants expected to be used for phytoremediation of Cd contaminated soil. The A. halleri promote solubilization of Cd in the soil directly or indirectly using its secreted root exudate. However, the effect of the metabolites profile of this plant to Cd uptake from contaminated soil is still unclear. The purpose of this study was to examine the contribution of root exudates of A. halleri to extract Cd in the soil. Cd-contaminated soil used in this study was a farmland soil containing 5.8 mg kg-1 of Cd and 648 mg kg-1 of Zn taken from Tome City, Miyagi Prefecture, Japan. Soil leaching tests were conducted using the solution containing the root exudates from A. halleri plant. The accelerating effect of root exudates of A. halleri on solubilization of Cd is fundamental information to construct the benefit of phytoremediation.
Arabidopsis halleri ssp.
cadmium phytoremediation root exudate soil leaching
To cite this article: Ariyanti, A.L., Chien, M.F. and Inoue, C. 2023. The benefit of the Arabidopsis halleri ssp. gemmifera root exudate in cadmium extraction from the cadmium contaminated soil. Journal of Degraded and Mining Lands Management 10(2):4107-4117, doi:10.15243/jdmlm.2023.102.4107.
Cadmium (Cd) is a highly toxic and unessential metal which a widespread main concern in food safety. The Cd contamination may interfere with the food chain and put human health at risk when the crops are exposed to this metal in the region, especially in Japan.
The main cause of Cd interference in the agricultural soil mainly comes from anthropogenic and industrialization activities, such as the usage of phosphate fertilizer and mining activity. The phosphate fertilizer contributed 0.75 mg kg-1 Cd contamination to the soil (Li et al., 2020). The phosphate fertilizer, for example, fluorapatite (Ca10(PO4)6F2), and tricalcium phosphate (Ca3(PO4)2) (Azzi et al., 2017) can exchange its calcium (Ca) bound with the Cd divalent in the raw material, and this can make Cd easier to enter the soil. The mining activity also can cause Cd contamination in the soil
because the Cd is not subject to recovery and is often discarded as the mine tailings, sludges, and wastewater. The mining activity contributes about 70% of the Cd input to the stream and the soil (Schaider et al., 2014).
The highest heavy metal contaminant in the soil is Cd (7%) compared with the other metal contaminant, such as nickel (Ni), arsenic (As), copper (Cu), mercury (Hg), lead (Pb), and chromium (Cr) (Wang et al., 2019). Various form of Cd in the soil affects the mobility of this metal by the plants. In parental soil, Cd is often found in sulfides, carbonates, and phosphorite forms. Usually, Cd is also found in water-soluble forms, such as Cd2+, CdCl+ and Cd(SO4)22- that makes it become the most mobile metal contaminant in nature (Kubier et al., 2019). The Cd contamination in the soil can be dangerous to living organisms. Cd caused the osteomalacia or well known
Open Access 4108 as “Itai-Itai Disease“ back in 20th century ago in
Toyama Prefecture, Japan (Robertsa, 2014; Wang et al., 2015).
Cd that interferes with the agricultural soil can cause chlorophyll damage (chlorosis), and growth retard in the plant (Manohar et al., 2012). Crops, rice, wheat, and legumes are the first targeted plant for Cd exposure. Cd in the soil can be transferred to the rice plant because it has the Cd transporters genes, for example, CAL1, OsNRAMP5, OsNRAMP1, OsIRT1, OsHMA3, and OsNAAT1. The OsNRAMP5 has an important role in Cd uptake in the root cells, especially when the availability of iron (Fe) as an essential nutrient is less than the availability of Cd (Hussain et al., 2020). Besides that, the external factors, such as pH in the rhizosphere, organic matter available, cation exchange capacity (CEC) in the soil, Fe and Mn oxide content, and clay content that form the soil texture were also affected the Cd uptake by the plants (Abedi and Mojiri, 2020). Therefore, a certain method to remove the Cd contamination in soil is needed to avoid the metal interfering with the food chain.
However, most of the soil treatments require high costs and produce abundant waste that needed further handling. There are so many physical and chemical treatments to eliminate Cd from the soil, for example, the use of zeolite, biochar, chemical amendments, or physical excavation to clean the soil (Khan et al., 2017; Wang et al., 2019; Hamid et al., 2020; Khum-in et al., 2020). Among all the in-situ and ex-situ soil remediation techniques, phytoremediation can be an effective alternative to solve the Cd contamination in the soil.
Recently, there were so many studies discussed the specific plants that have the ability to survive in the metals contaminated soil and accumulate the metals, such as Noccaea caerulescens which can uptake up to 81-fold Cd from the soil, barnyard grass (Echinochloa crus-galli) accumulated up to 6 mg kg-1 DW of Cd in its roots, and also Arabidopsis halleri subsp.
gemmifera (hereinafter referred to as A. halleri) that could accumulate Cd up to 5,641 mg kg-1 DW (Peng et al., 2017; He et al., 2021; Kudo et al., 2021). A. halleri is now wide-spread known hyperaccumulator, especially for Cd and Zn (Hokura et al., 2006; Kashem et al., 2007, 2010; Fukuda et al., 2020).
The metal fraction techniques can help to predict how is the chemical form of metal in soil. The chemical form of the metals in soil can be fractionated into five different forms by Tessier`s sequential extraction technique (Tessier et al., 1979; Alaboudi et al., 2020; Piri et al., 2020). This method has been widely used in many studies to determine the metal availability in the soil. The metal fractions are an ionic exchangeable, which is usually considered as an easy mobilized water-soluble form; carbonate-bound fraction, which is easily dissolved in weak acid conditions; iron and manganese-bound fraction, which is dissolved by reducing reagent; organic-matter bound
fraction, which is degraded in oxidizing condition, and the residual fraction which the metal content is in the most difficult form to be extracted (Piri et al., 2020).
The root exudate compounds are the representative information to determine plant response to the rhizosphere condition. Mostly, the plants secrete root exudates as secondary metabolites from photosynthesis products. In the case of the hyperaccumulator plants, some specific root exudate will show a survival feature in stress conditions. In Cd stress condition, hyperaccumulator plants secrete organic compounds to the rhizosphere to adapt and survive in a metal toxicity environment (Barceló and Poschenrieder, 2002; Luo et al., 2017; Guo et al., 2018). Commonly, in recent research progress using the chemicals, such as nitriloacetic acid, FeCl3, or another inositol phosphate (Bilgin and Tulun, 2016;
Xie et al., 2020; Marolt and Kolar, 2021). The leachability study of the A. halleri root exudate to extract Cd from the soil is still scarce. Moreover, Zn will play an important role as a Cd competitor to be extracted from the soil because the chemical property of both two metals is almost the same. This study focused on the ability of the A. halleri root exudate to extract Cd and Zn from the soil. The prominent information on the benefit of the root exudate will reveal its effectiveness in whole scale applications for feasible progressive phytoextraction management and technology to remediate Cd contamination in the soil.
Materials and Methods Plant culture
The seedlings of A. halleri grown in soil were purchased from Fujita Company. The seedlings were transferred to the hydroponic medium after washing the roots carefully using Milli-Q water. The seedling culture using 1/5x strength Hoagland solution as a hydroponic medium, 40 mL 5x strength solution was diluted into 1-liter Milli-Q water. The Hoagland solution's composition is represented in Tables 1 and 2 based on Hoagland and Arnon (1950) to provide sufficient nutrients in the hydroponic medium for the plants.
The seedlings were stored in the growth chamber (Biotron, NK system, Japan) with the condition of 25 ºC with 16 hours of light exposure, 22 ºC for eight hours of the dark cycle, and light intensity of 60%
based on photoperiodic lighting programs. The 1/5 Hoagland medium was changed once every week until the plant’s root length reached around 6-7 cm, and then culture plants were transferred into a 250 mL plastic bottle filled in with 200 mL of 1/5 Hoagland medium.
The plastic bottles were covered using aluminum foil and dark stone wool to darken the rhizosphere. The medium in the plastic bottle also changed once a week until one year old, and the plants were ready for further experiments (Wiyono et al., 2021). This experiment used a one-year old plant A. halleri. Five replications
Open Access 4109 were used in various treatments to analyze the
leachability of the secreted root exudate. Hoagland medium for every treatment was 200 mL in each hydroponic bottle. Three different treatments were: (1) 1/5 strength normal Hoagland, (2) 1/5 strength Hoagland with Cd 10 µM, and (3) 1/5 strength Hoagland with Zn 100 µM. The metal treatments were given for three days to induce the plants to secrete more root exudate compounds during the collection time. The root exudate was collected four times in Milli-Q water for six hours every three days had been cultured in Hoagland nutrient medium.
Table 1. Macronutrient components list.
100 x Strength Stock Solution
KNO3 81 g L-1
Ca(NO3)2 • 4H2O 95 g L-1
MgSO4 •7H2O 50 g L-1
NH4H2PO4 15.5 g L-1
Table 2. Micronutrient components list.
Components 1000 x Strength Stock Solution
Na-Fe-EDTA 20 g L-1
H3BO3 3 g L-1
MnSO4 • 4H2O 2 g L-1
ZnSO4 •7H2O 0.22 g L-1
CuSO4 • 4H2O 0.05 g L-1
Na2MoO4 • 2H2O 0.02 g L-1 Total Organic Content (TOC)
After one week of incubation in different treatments, the total carbon in the form of organic compounds to analyze the metabolites secreted from the plant roots to the medium as a response to the specific stress. TOC analysis was done using Total Organic Carbon Analyzer (TOC-L, Shimadzu, Japan). Ten milliliters root exudate samples were taken from root exudate stock and filtered it using 0.22 µm membrane filter.
The filtrate was introduced into a 15 mL centrifuge tube (Lu et al., 2009).
pH of the root exudate
After seven days of incubation in Hoagland treatments, the plant roots were washed thoroughly with Milli-Q water and placed in a centrifuge tube containing sterilized Milli-Q water for six hours to get the root exudate. The pH of the root exudate was recorded using a pH meter (LAQUAact pH meter D-71, Horiba, Japan).
X-Ray Fluorescence (XRF) spectrometry
The soil samples were taken from Tome City, Miyagi Prefecture, Japan, from the area in the agricultural field
where the farmland soils were contaminated by Cd.
The samples from three different spots were taken up to 15 cm depth from the surface. The samples were sieved with a 75 µm particle-size metal sieved to make homogenous size and dried for two days in an oven to eliminate microorganisms. The soil samples were ground using a vibrating sample mill (CTM TI-100) for ten minutes until it became a fine powder. After that, press it in a 3 cm plastic pellet using a hydraulic press with 50 kPa pressure for one minute, 150 kPa for 3 minutes, and 200 kPa for 5 minutes gradually (Li et al., 2020). The zinc and cadmium concentration in the soil was measured using XRF (Epsilon-5, PANanalytical, Japan) using the minor and cadmium method (Wamere, 2019).
The sequential extraction procedure was conducted based on the Tessier method, which was developed in 1979 with some adjustments (Tessier et al., 1979; Piri et al., 2020). The methods below were used to examine the five different fractions in the soil sample with three replications.
Exchangeable fraction (F1): One gram of the soil sample was mixed with 8 mL of 1 mol L-1 MgCl2 in 15 mL centrifuge tube at pH 7, and the mixed sample was shaken on the shaker (100 rpm) for one hour at 25 °C.
After that, the sample was centrifuged at 10,000 x g for ten minutes, and then decanted to collect the F1 supernatant. The residue was resuspended with 8 mL of Milli-Q water, centrifuged and decanted again, and then the supernatant was discharged. The washed residue was used for the next sequential extraction step.
Carbonate-bound fraction (F2): The residue obtained from the F1 process was added with 8 mL of 1 M NaOAc adjusted in pH 5 with HOAc and shaken at 100 rpm for five hours at 25 °C. Then the sample was centrifuged at 10,000 x g for ten minutes, and obtained the F2 supernatant. The residue was washed by centrifugation with Milli-Q water and used the residue for the next sequential extraction.
Iron and Manganese oxide-bound fraction (F3):
The residue obtained from F2 process was transferred in 50 mL centrifuge tubes and added with 20 mL of 0.04 M NH2OH • HCl in 25% acetic acid and shaken at 100 rpm for five to six hours at 95 °C. Then, the sample was centrifuged to obtain the F3 supernatant and washed by the same procedure as the previous step.
Organic matter-bound fraction (F4): The residue obtained from F3 process was mixed with 3 mL of 0.02 M HNO3 and 5 mL of 30% H2O2 (pH 2) and shaken for 2-3 hours at 85 °C. After cooling, 5 mL 3.2 M NH4OAc in 20% HNO3 was added, and the sample was diluted with Milli-Q water up to 20 mL and shaken for 6 hours. Then the F4 supernatant was obtained by centrifugation. The washed residue was also used for the next sequential extraction.
Open Access 4110 Residual fraction (F5): Ten milliliters of concentrated
HCl:HNO3 (3:1) solution was added carefully to the residue from F4 process. The digestion of the residue was conducted at 95 °C for two hours.
Lab-scale soil leaching
The solvents used to leach Cd and Zn from the soil were Milli-Q water and the root exudate solution which were collected from the plants with different rhizosphere treatments from 1/5 Hoagland solution, Cd10, and Zn100 treatment to see the different effects of extracting metals from the contaminated soil. A ten- milliliter solvent solution and 1-gram soil sample from Tome, Miyagi-Japan were mixed in 15 mL centrifuge tube. All leaching test tubes were put in a rotatory agitator (Taitec, Japan) and shaken at 200 rpm for 6 hours under 30 °C temperature. After 6 hours, the sample was centrifugated at 10,000 rpm for 6 minutes to separate the residue from the supernatant. The supernatant was transferred into new 15 mL centrifuge tubes and analyzed Cd and Zn concentrations by ICP- MS (Alaboudi et al., 2020).
Cd and Zn concentration extracted by the solvents in this study was analyzed using ICP-MS (Nexion 300-S, Perkin-Elmer Japan) using the method from Wiyono et al. (2021) with some adjustments. ICP-MS has become an essential method to analyze trace elements because of its significant sensitivity, accurate sensitivity, and its ability for isotope determinations.
The ICP-MS can attain limited detection limits with a low-range concentration in parts per billion (µg L-1) and parts per trillion (ng L-1), reach multi- element characters but allows only one to give precise detection, and give possibilities to measure isotope information of elements (Isaguirre et al., 2020). ICP- MS standard solution used for Cd was 1 µg L-1, 2 µg L-1, 50 µg L-1, and 100 µg L-1; for Zn were 1 µg L-1, 50 µg L-1, 100 µg L-1, and 200 µg L-1. All the samples were pre-treated before the analysis. Each sample is taken from the sample tube and placed in a 10 mL measuring flask. For cadmium treatment, 100 µL aliquot was added with 500 µL nitric acid and 10 µL of 1 µg L-1 indium solution (internal standard), added with Milli-Q water to be 10 mL. After mixing them well, the solution was poured into a 10 mL syringe and filtered by a 0.45 µm membrane filter. For zinc treatment, only needed 10 µL aliquot medium for ICP- MS pre-treatment.
The indium solution used as an internal standard to keep the instruments' stability and reduce the matrix interferences. Matrices that can cause interference with the ICP-MS can be acids other than nitric acid, organics, or solid matrices (Chang et al., 2020). The important thing is the internal standard chosen should not cause analyte contamination (Wilschefski and Baxter, 2019).
The plant culture was used in five replications, and the soil analysis was done using three replications, with the means of the standard error (SE). One-way ANOVA was conducted with p<0.05 for the significant test, and the Tukey-HSD method was done for the post hoc analysis. The graphs were produced using the OriginPro 2022 (OriginLab) software.
As shown in Figure 1, Cd was extracted from about 28.8 µg kg soil-1 when the Tome soil was leached by Milli-Q water. This percentage was almost the same as the leaching result using Hoagland solution, the Cd was extracted from 30 µg kg soil-1. The highest percentage of Cd extraction when using the root exudate solution without Cd or Zn treatment, it showed about 155 µg kg soil-1. The treatment given to the plant (Cd10 and Zn100) was not giving a significant difference (p>0.05) to the metal extraction in the soil leaching experiment (data not shown). The Zn was extracted from the Tome soil only about 245.2 µg kg soil-1 using the Milli-Q water in the soil leaching experiment. That percentage was increased when the soil sample was washed with the root exudate solution.
The Zn extraction percentage increased by about 2357.4 µg kg soil-1 using the root exudate from the control treatment and increased by about 2191 µg kg soil-1 when using the root exudate solution from Zn100 treatment, respectively.
The pH of the root exudate solutions that were measured after collecting in the Milli-Q water for six hours is shown in Figure 2. The initial pH of the Milli- Q water was 7.2 before being used for collecting the root exudate, and the pH decreased after collecting the root exudate. The root exudate solution pHs at 12 days were 6.55, 5.7, and 6.6 for the control solution, Cd10 treatment, and Zn100 treatment, respectively. The Cd10 treatment caused the collected root exudate to give the acidic condition of the root exudate solution.
The pH of the root exudate solution can represent the amount of organic carbon in the collected root exudate that is shown in the TOC analysis result.
The root exudate solution, after four times collections, was measured further the total organic carbon (TOC) concentration. As shown in Figure 3, the TOC detected in the root exudate solution from the control treatment was 0.5 mg h-1 g root DW-1, which was the lowest concentration compared to the root exudate solution collected from the Cd10 and Zn100 treatment. The TOC concentration from Cd10 treatment showed the highest concentration of about 2 mg h-1 g root DW-1. The TOC concentration of the root exudate solution from Zn100 treatment was less than the Cd10 treatment, about 1.55 mg h-1 g root DW-1. The concentration of the Cd and Zn in the soil are shown in Table 3.
Open Access 4111
MQ C Zn100
0 500 1000 1500 2000 2500 3000
Zn extracted concentration -1(µg kg soil) a
Zn Extraction From The Soil using The A. halleri Root Exudate Solution
MQ C Cd10
0 20 40 60 80 100 120 140 160 180 200
Cd extracted concentration (µg kg soil-1)
Cd Extraction From The Soil using The A. halleri Root Exudate Solution
Figure 1. The extraction of Cd and Zn from the Tome soil sample using the A. halleri root exudate solution after the plant cultured in Cd10 treatment and Zn100 treatment, compared with Milli-Q water and root exudate solution from Hoagland medium treatment as a control. Standard error bars represented the means of three replication, and the letters are the data significances between the different treatments based on ANOVA by HSD
Tukey test (p<0.05).
day 0 day 3 day 6 day 9 day 12 5.0
5.5 6.0 6.5 7.0 7.5
Root Exudate Collection Time
control Cd10 Zn100
pH of The Root Exudate Collection
Figure 2. The pH in the A. halleri root exudate solution recorded every after the plant cultured in Cd10 treatment and Zn100 treatment for seven days, compared with the root exudate solution from Hoagland medium treatment
as a control.
The upper part of the soil sample was taken about 15 cm from the soil surface, and the lower part of the soil was taken from 15 cm to 30 cm from the soil surface.
The Cd concentrations were 6x103 µg kg soil-1 for the upper part and the lower part of the soil. Meanwhile, the Zn concentrations were 6.48x105 µg kg soil-1 and 6.64x105 µg kg soil-1 for the upper part and the lower part of the soil, respectively. Those metal concentrations generally increased compared to the previous result measured in 2017. Figure 4 shows the estimation of Cd and Zn chemical forms in the Tome soil. The Cd chemical form in the exchangeable fraction, carbonate-bound fraction, Fe and Mn-bound fraction, organic-bound fraction, and residual fraction were 3.5, 0.7, 0.1, 0.7, and 0.16 mg kg soil-1,
respectively. Meanwhile, the Zn chemical form in the same sample were 49.3, 34.5, 29.6, 92.3, and 178.2 mg kg soil-1, respectively. The bioavailability is commonly related to the abundant amount of the exchangeable fraction of the metal in the soil, which is easy to be extracted in a water-based solution and in carbonate-bound fraction, which is easy to extract metal by decreasing the pH. In this study, the bioavailability of Zn in the soil was only 13%, but that of Cd was 67%. The metal speciation between Cd and Zn was significantly different for each fraction.
However, the result between Cd and Zn cannot have compared to each other because the metal concentration in the soil was quite different on the XRF result shown in the previous Table 3.
Open Access 4112 0.566
C Cd10 Zn100
0.0 0.5 1.0 1.5 2.0 2.5 3.0
TOC concentration (mg h-1 g root DW-1 )
Total Organic Carbon (TOC) Concentration in the Root Exudate Solution
Figure 3. The total organic carbon (TOC) concentration of the A. halleri root exudate solution collected four times for six hours after the plant cultured in Cd10 treatment and Zn100 treatment for three days, compared with
the root exudate solution from Hoagland medium treatment as a control. Standard error bars represented the means of three replication, and the letters are the data significances between the different treatments based on
ANOVA by HSD Tukey test (p<0.05).
Table 3. The X-Ray Fluorescence (XRF) result of the Tome soil sample.
Elements Upper part soil (2020) Lower part soil (2020) Previous study (Kudo, 2017) Cd 6x103 µg kg soil-1 6x103 µg kg soil-1 5x103 µg kg soil-1 Zn 6.48x105 µg kg soil-1 6.64x105 µg kg soil-1 6.01x105 µg kg soil-1
49.3 34.5 30
F1 F2 F3 F4 F5
0.00 0.05 0.10 0.15 0.20 4060 10080 120140 160180 200
b b b b
a a a
Metal Concentration (mg kg soil-1 )
Metal Speciation Concentration in The Tome Soil
Figure 4. The Cd and Zn speciation concentration of the Tome soil sample measured using the Tessier method.
The F1 = exchangeable fraction, F2 = carbonate-bound fraction, F3 = Fe and Mn oxide-bound fraction, F4 = organic matter-bound fraction, F5 = residual fraction. Standard error bars represented the means of three replication, and the letters are the data significances between the different treatments based on ANOVA by HSD
Tukey test (p<0.05).
Open Access 4113 Discussion
The role of the plant's root exudate is widely known since the 1980s because of its capability to mitigate the Cd contamination in the rhizosphere, both in natural habitat or under the hydroponic system for a wide range of gramineous plants (Mench and Martin, 1991;
Hinsinger et al., 2005). The plant's root exudate give contribution for Cd detoxification natural techniques to survive in metal contamination soil. The root exudate can enhance the solubility of the metal in the soil by chelating the Cd to form the Cd complex (Sidhu et al., 2019). The leachability of the A. halleri root exudate is shown in Figure 1. The water was not easily extracted the Cd from the Tome soil sample, only about sample 0.4% of the total Cd in the soil. Cd that was not easily removed from the soil using water because of its water-soluble form need the amendment to be extracted from the soil. The Cd extraction will be almost negligible without the amendment, so the secreted root exudate can help in enhancing the Cd extraction (Wang et al., 2018).
The water leachability was compared with the root exudate from the control treatment. Using the root exudate solution from the control treatment could slightly enhance the Cd extraction became 0.5% of the total Cd in the soil. Furthermore, the result from the control treatment was then compared to the root exudate solution from the Cd10 treatment. The result showed that the excess Cd treatment in the hydroponic medium could enhance the Cd extraction by up to 3%, because the toxicity of Cd10 treatment could change the physiological function of the photosynthesis product, which resulted in the abundant of the secondary metabolites as survival techniques (Dresler et al., 2014). Those secondary metabolites could further enhance the Cd solubilization.
As a comparison, Zn extraction from the soil was also conducted. However, the Zn solubilization was rather less than the Cd solubilization. The Zn solubilization was almost negligible because it can only be extracted 0.03% from the soil. This percentage was increased when using the secreted A. halleri root exudate solution. The Zn was extracted about 0.4%
and 0.3% using the root exudate from the control treatment and Zn100 treatment, respectively. The Zn extraction was less than the Cd extraction, so it might be because the Milli-Q water was not feasible enough to breakdown the complex between Zn with the soil matrix, and this result was related to the result of Moon et al. (2012), which the soil washing using distilled water shown limited Zn extraction compared with various acid washing as extraction agents.
The acidic extraction agents are commonly related to the acidic pH condition. The pH condition in the root exudate solution in Figure 2 shows acidic pH after being collected four times before being used to wash the Tome soil sample. The more the acidic pH of the root exudate solution, the more also the ability of the root exudate solution to extract Cd from the soil.
The rhizosphere pH was often shown to have a positive result on Cd extraction from soil to be easy to accumulate in the plants (Liu et al., 2015; He et al., 2017). The Cd was easily extracted about 44.3-78.1%
and Zn 29.2-59.4% from the soil in pH 2 condition and the extractability was decreased when the pH was increased as well as in this study (Li et al., 2019). The metal extractability process under low pH can change the Cd ions into the cationic state, so the hydrogen ions can change their function into the adsorption sites for Cd ions and enhance the metal extraction in the soil (Zhai et al., 2018).
The pH condition is often related to the TOC concentration secreted in the root exudate collected from the A. halleri under excess Cd and Zn treatment compared with the control treatment. In Figure 3, the TOC concentration measured from four times root exudate collection after Cd10 treatment showed the highest concentration compared to the Zn100 treatment and the control treatment without the metal addition in the nutrient medium. This result was related to the Sahito et al. (2022) study that the TOC result in acidic condition was 300-589 mg h−1 kg FW−1 from Cd treatment in pH between 6-4.9 of the Rhizobium rhizogenes-mediated root proliferation in Sedum alfredii roots exudates. The TOC concentration was significantly different between the control, Cd10 treatment, and Zn100 treatment (p<0.05), which means the excess metal treatments given to the hydroponic nutrient medium were able to induce the plants to secrete more organic compound collected in the root exudate solutions.
The ability of the organic compounds in the root exudate to extract the metal in Figure 1 was compared to the metal concentration in the soil sample analyzed by the XRF, as shown in Table 3. The Cd and Zn concentration in the Tome soil increased in 2020 compared to the soil sample measured in 2017. The Cd contamination in the soil increased because of the weathering, and its mobilization was affected mainly by the pH and oxic-bound. The distribution of Cd in the soil mainly occur in free (Cd2+) or complexed compound with Cl- and (SO4)22-, which are easy to mobilize because of their water-soluble forms, especially in aerobic soil (Kubier et al., 2019). The Cd behavior and ability to be extracted from the soil were related to the Cd chemical form. The more the bioavailability of the Cd, the easier also the Cd to be extracted from the contaminated soil. Commonly, the bioavailability is related to the ionic exchangeable water-soluble form of metal available in the soil sample, which can be easily transported by the plants (Peralta-Videa et al., 2009). The metal bioavailability depends on several factors, such as weather and pH.
Heavy rainfall in nature, especially in farmland, increases the metal leaching in the soil and decreases the pH, which can increase the Cd bioavailability (Kubier et al., 2019; Luo et al., 2019). The Cd chemical form concentration calculated from metal fractionation
Open Access 4114 in the soil sample using the Tessier method shown in
Figure 5, there were about 67%, 13%, 3%, 14%, 3% of the exchangeable fraction, carbonate-bound fraction, Fe and Mn oxide-bound fraction, organic-bound fraction, and residual fraction, respectively. The Zn
bioavailability is fewer than the Cd. The Zn chemical form was 13%, 9%, 8%, 24%, and 47% of the exchangeable fraction, carbonate-bound fraction, Fe and Mn oxide-bound fraction, organic-bound fraction, and residual fraction, respectively.
9.04% 11.46% 8.15%
Exchangeable Carbonate-bound Fe & Mn-bound Organic bound Residual
Zn Chemical Form in the Tome Soil Sample
Exchangeable Carbonate-bound Fe & Mn-bound Organic bound Residual
Cd Chemical Form in the Tome Soil Sample
Figure 5. The percentage of Cd and Zn speciation in the Tome soil sample measured using the Tessier method.
The Zn bioavailability overall was less than the Cd.
This might be happened because of the higher competitive stabilization of Zn than the Cd with PO4
from phosphate fertilizer usage in the Tome farmland, absorbed in the soil matrix that caused the immobilization of Zn was higher than the Cd. The metal-phosphate formation of Zn causing the low solubility (logKsp) was -63.1 for the Zn5(PO4)3OH
compared to the Cd −32.6 for the Cd3(PO4)2 in Andrunik et al. (2020) study which similar with the result in this study. The low solubility also caused the less leachability of the Zn (1-17%) compared to the Cd (11%-32%), while in this study, the leachability of the Zn exchangeable fraction was 13%, and the Cd was 67%. The same pattern also shown for the carbonate- bound fraction of Zn was fewer than the Cd in
Open Access 4115 Figure 5. The A. halleri root exudate solution from
excess metal treatment can change the soil properties, and the complexes between the metal and the soil matrix bind in the Tome soil to be easily extracted.
Commonly, the root exudate solution contained various metabolites from photosynthesis, such as amino acids, organic acids, and sugar compounds which can have the ability to change the soil structure, thus, can easily extract Cd from the soil. As in addition, since the root exudates were collected in the Milli-Q water, so the water content can also help the soil to decrease the yield stress by about 54% and 87% using the root exudate from chia seed and maize plants, respectively (Naveed et al., 2017). The root exudate, as a photosynthetic product secreted to the rhizosphere, has a correlated relationship with the rhizosphere microbial, this process affected the modification of soil structure. However, the proportion of the chemical compound in the root exudate solutions was different depending on many external factors, such as the soil stress in the rhizosphere, the plant species, root exudate collection method which were also affected the microorganism in the rhizosphere ( Naveed et al., 2017; Canarini et al., 2019;
Rolfe et al., 2019).
The result in this study might not be separated from the influence of the chemical form of metal in the soil, for which the Cd extracted was the most mobile fraction, and this process might not be separated from the influence of rhizosphere microorganism (Zhao et al., 2021). Moreover, we suggest further study discuss about the root microorganism induced by the root exudate compounds secreted from the A. halleri.
Root exudate solution collected four times from A.
halleri ssp. gemmifera which showed the acidic pH, appeared to have abundant organic carbon shown in TOC result, and this root exudate showed potential leachability more than 50% of the Cd from the Tome soil. The leachability of the metal from the soil is related to the chemical fraction of the metal in the soil.
The more the metal bioavailability, which is water- soluble that makes the metals easier to solubilize, the more also the metal contaminant can be extracted from the soil. The acidic condition given in the root exudate solution made the metal extraction process was also easier because it can provide more hydrogen ions for metal adsorption sites.
This research was supported by the Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 19H01158 (Grant-in-Aid for Scientific Research (A). The author Agni Lili Ariyanti was supported by Pioneering Research Support Project of Japan Science and Technology (JST) and scholarship from Asahi Glass Foundation. We also express our gratitude to Dr. Noboyuki Kitajima (Chief
Engineer of Fujita Co., Ltd.) for providing the A. halleri ssp.
gemmifera. A part of this article was presented at the 2nd International Conference on Environment, Socio-Economic, and Health Impacts of Degraded and Mining Lands, 30-31th August 2022, Universitas Brawijaya, Malang, Indonesia.
Abedi, T. and Mojiri, A. 2020. Cadmium uptake by wheat (Triticum aestivum L.): an overview. Plants 9(4):1-14, doi:10.3390/plants9040500.
Alaboudi, K.A., Ahmed, B. and Brodie, G. 2020. Soil washing technology for removing heavy metals from a contaminated soil: a case study. Polish Journal of Environmental Studies 29(2):1029-1036, doi:10.15244/pjoes/104655.
Andrunik, M., Wołowiec, M., Wojnarski, D., Zelek-Pogudz, S. and Bajda, T. 2020. Transformation of Pb, Cd, and Zn minerals using phosphates. Minerals 10(4):342, doi:10.3390/min10040342.
Azzi, V., Kanso, A., Kazpard, V., Kobeissi, A., Lartiges, B.
and El Samrani, A. 2017. Lactuca sativa growth in compacted and non-compacted semi-arid alkaline soil under phosphate fertilizer treatment and cadmium contamination. Soil and Tillage Research 165:1-10, doi:10.1016/j.still.2016.07.014.
Barceló, J. and Poschenrieder, C. 2002. Fast root growth responses, root exudates, and internal detoxification as clues to the mechanisms of aluminium toxicity and resistance: a review. Environmental and Experimental Botany 48(1):75-92, doi:10.1016/S0098- 8472(02)00013-8.
Bilgin, M. and Tulun, S. 2016. Removal of heavy metals (Cu, Cd and Zn) from contaminated soils using EDTA and FeCl3. Global Nest Journal 18(1):98-107, doi:10.30955/gnj.001732.
Canarini, A., Kaiser, C., Merchant, A., Richter, A. and Wanek, W. 2019. Root exudation of primary metabolites: mechanisms and their roles in plant responses to environmental stimuli. Frontiers in Plant Science 10:157, doi:10.3389/fpls.2019.00157.
Chang, H.F., Wang, S.L., Lee, D.C., Hsiao, S.S.Y., Hashimoto, Y. and Yeh, K.C. 2020. Assessment of indium toxicity to the model plant Arabidopsis. Journal of Hazardous Materials 387(October 2019):121983, doi:10.1016/j.jhazmat.2019.121983.
Dresler, S., Hanaka, A., Bednarek, W. and Maksymiec, W.
2014. Accumulation of low-molecular-weight organic acids in roots and leaf segments of Zea mays plants treated with cadmium and copper. Acta Physiologiae Plantarum 36(6):1565-1575, doi:10.1007/s11738-014- 1532-x.
Fukuda, N., Kitajima, N., Terada, Y., Abe, T., Nakai, I. and Hokura, A. 2020. Visible cellular distribution of cadmium and zinc in the hyperaccumulator: Arabidopsis halleri ssp. gemmifera determined by 2-D X-ray fluorescence imaging using high-energy synchrotron radiation. Metallomics 12(2):193-203, doi:10.1039/c9mt00243j.
Guo, S.H., Hu, N., Li, Q.S., Yang, P., Wang, L.L., Xu, Z. M., Chen, H.J., He, B.Y. and Zeng, E.Y. 2018. Response of edible amaranth cultivar to salt stress led to Cd mobilization in rhizosphere soil: A metabolomic analysis. Environmental Pollution 241:422-431, doi:10.1016/j.envpol.2018.05.018.
Open Access 4116 Hamid, Y., Tang, L., Hussain, B., Usman, M., Lin, Q.,
Rashid, M.S., He, Z. and Yang, X. 2020. Organic soil additives for the remediation of cadmium contaminated soils and their impact on the soil-plant system: a review.
Science of the Total Environment 707:136121, doi:10.1016/j.scitotenv.2019.136121.
He, S., Yang, X., He, Z. and Baligar, V.C. 2017.
Morphological and physiological responses of plants to cadmium toxicity: a review. Pedosphere 27(3):421-438, doi:10.1016/S1002-0160(17)60339-4.
He, W., Long, A., Zhang, C., Cao, M. and Luo, J. 2021. Mass balance of metals during the phytoremediation process using Noccaea caerulescens: a pot study. Environmental Science and Pollution Research 28(7):8476-8485, doi:10.1007/s11356-020-11216-x.
Hinsinger, P., Gobran, G.R., Gregory, P.J. and Wenzel, W.W. 2005. Rhizosphere geometry and heterogeneity arising from root-mediated physical and chemical processes. New Phytologist 168(2):293-303, doi:10.1111/j.1469-8137.2005.01512.x.
Hokura, A., Onuma, R., Kitajima, N., Terada, Y., Saito, H., Abe, T., Yoshida, S. and Nakai, I. 2006. 2-D X-ray fluorescence imaging of cadmium hyperaccumulating plants by using high-energy synchrotron radiation X-ray microbeam. Chemistry Letters 35(11):1246-1247, doi:10.1246/cl.2006.1246.
Hussain, B., Li, J., Ma, Y., Tahir, N. and Ullah, A. 2020.
Effects of Fe and Mn cations on Cd uptake by rice plant in hydroponic culture experiment. PLoS ONE 15(12 December):1-15, doi:10.1371/journal.pone.0243174.
Isaguirre, A.C., Moyano, M.F., Gil, R.A. and Moglia, M.M.
2020. A novel and simple method for elements determination in aerobiological samples by Inductively Coupled Plasma Mass Spectrometry (ICP-MS) analysis.
Water, Air, and Soil Pollution 231(2), doi:10.1007/s11270-020-4416-2.
Kashem, M.A., Singh, B.R., Kubota, H., Nagashima, R.S., Kitajima, N., Kondo, T. and Kawai, S. 2007. Assessing the potential of Arabidopsis halleri ssp. gemmifera as a new cadmium hyperaccumulator grown in hydroponics.
Canadian Journal of Plant Science 87(3):499-502, doi:10.4141/CJPS06058.
Kashem, M.A., Singh, B.R., Kubota, H., Sugawara, R., Kitajima, N., Kondo, T. and Kawai, S. 2010. Zinc tolerance and uptake by Arabidopsis halleri ssp.
gemmifera grown in nutrient solution. Environmental Science and Pollution Research 17(5):1174-1176, doi:10.1007/s11356-009-0193-6.
Khan, M.A., Khan, S., Khan, A. and Alam, M. 2017. Soil contamination with cadmium, consequences and remediation using organic amendments. Science of the Total Environment 601-602:1591-1605, doi:10.1016/j.scitotenv.2017.06.030.
Khum-in, V., Suk-in, J., In-ai, P., Piaowan, K., Phaimisap, Y., Supanpaiboon, W. and Phenrat, T. 2020. Combining biochar and zerovalent iron (BZVI) as a paddy field soil amendment for heavy cadmium (Cd) contamination decreases Cd but increases zinc and iron concentrations in rice grains: a field-scale evaluation. Process Safety and Environmental Protection 141:222-233, doi:10.1016/j.psep.2020.05.008.
Kubier, A., Wilkin, R.T. and Pichler, T. 2019. Cadmium in soils and groundwater: a review. Applied Geochemistry 108(July), doi:10.1016/j.apgeochem.2019.104388.
Kudo, H., Inoue, C. and Sugawara, K. 2021. Effects of growth stage and cd chemical form on Cd and Zn
accumulation in Arabidopsis halleri ssp. gemmifera.
International Journal of Environmental Research and Public Health 18(8), doi:10.3390/ijerph18084214.
Li, H., Yang, Z., Dai, M., Diao, X., Dai, S., Fang, T. and Dong, X. 2020. Input of Cd from agriculture phosphate fertilizer application in China during 2006-2016. Science of the Total Environment 698:134149, doi:10.1016/j.scitotenv.2019.134149.
Li, X., Yu, Z., Xu, J., Pan, Y., Bo, W., Liu, B., Zhang, P., Bai, J. and Zhang, Q. 2020. The technique of high- pressure powder pressing with polyester film covering for XRF of geochemical samples. X-Ray Spectrometry (July 2019), doi:10.1002/xrs.3147.
Li, Y., Liao, X. and Li, W. 2019. Combined sieving and washing of multi-metal-contaminated soils using remediation equipment: a pilot-scale demonstration.
Journal of Cleaner Production 212:81-89, doi:10.1016/j.jclepro.2018.11.294.
Liu, K., Lv, J., He, W., Zhang, H., Cao, Y. and Dai, Y. 2015.
Major factors influencing cadmium uptake from the soil into wheat plants. Ecotoxicology and Environmental Safety 113:207-213, doi:10.1016/j.ecoenv.2014.12.005.
Lu, Y., Zhou, Y., Nakai, S., Hosomi, M., Zhang, H., Kronzucker, H.J. and Shi, W. 2014. Stimulation of nitrogen removal in the rhizosphere of aquatic duckweed by root exudate components. Planta 239(3):591-603, doi:10.1007/s00425-013-1998-6.
Luo, P., Xiao, X., Han, X., Ma, Y., Sun, X., Jiang, J. and Wang, H. 2019. Application of different single extraction procedures for assessing the bioavailability of heavy metal(loid)s in soils from overlapped areas of farmland and coal resources. Environmental Science and Pollution Research 26(15):14932-14942, doi:10.1007/s11356-019-04833-8.
Luo, Q., Wang, S., Sun, L.N. and Wang, H. 2017. Metabolic profiling of root exudates from two ecotypes of Sedum alfredii treated with Pb based on GC-MS. Scientific Reports 7(November 2016):1-9, doi:10.1038/srep39878.
Manohar, M., Shigaki, T. and Shigaki, L. 2012. Past, present and future approaches for reducing cadmium content.
United States Department of Agriculture / Agricultural
Research August 2015:5-33,
Marolt, G. and Kolar, M. 2021. Analytical methods for determination of phytic acid and other inositol phosphates: a review. Molecules 26(1), doi:10.3390/MOLECULES26010174.
Mench, M. and Martin, E. 1991. Mobilization of cadmium and other metals from two soils by root exudates of Zea mays L., Nicotiana tabacum L. and Nicotiana rustica L.
Plant and Soil 132(2):187-196,
Moon, D.H., Lee, J.R., Wazne, M. and Park, J.H. 2012.
Assessment of soil washing for Zn contaminated soils using various washing solutions. Journal of Industrial and Engineering Chemistry 18(2):822-825, doi:10.1016/j.jiec.2011.11.137.
Naveed, M., Brown, L.K., Raffan, A.C., George, T.S., Bengough, A.G., Roose, T., Sinclair, I., Koebernick, N., Cooper, L., Hackett, C.A. and Hallett, P.D. 2017. Plant exudates may stabilize or weaken soil depending on species, origin and time. European Journal of Soil Science 68(6):806-816, doi:10.1111/ejss.12487.
Peng, Q., Chen, W., Wu, L. and Bai, L. 2017. The uptake, accumulation, and toxic effects of cadmium in barnyard grass (Echinochloa crus-galli). Polish Journal of
Open Access 4117 Environmental Studies 26(2):779-784,
Peralta-Videa, J.R., Lopez, M.L., Narayan, M., Saupe, G.
and Gardea-Torresdey, J. 2009. The biochemistry of environmental heavy metal uptake by plants:
implications for the food chain. International Journal of Biochemistry and Cell Biology 41(8-9):1665-1677, doi:10.1016/j.biocel.2009.03.005.
Piri, M., Sepehr, E., Samadi, A., Farhadi, K.H. and Alizadeh, M. 2020. Contaminated soil amendment by diatomite:
chemical fractions of zinc, lead, copper and cadmium.
International Journal of Environmental Science and Technology 18(5):1191-1200, doi:10.1007/s13762-020- 02872-0.
Robertsa, T.L. 2014. Cadmium and phosphorous fertilizers:
the issues and the science. Procedia Engineering 83:52- 59, doi:10.1016/j.proeng.2014.09.012.
Rolfe, S.A., Griffiths, J. and Ton, J. 2019. Crying out for help with root exudates: adaptive mechanisms by which stressed plants assemble health-promoting soil microbiomes. Current Opinion in Microbiology 49:73- 82, doi:10.1016/j.mib.2019.10.003.
Sahito, Z.A., Zehra, A., Chen, S., Yu, S., Tang, L., Ali, Z., Hamza, S., Irfan, M., Abbas, T., He, Z. and Yang, X.
2022. Rhizobium rhizogenes-mediated root proliferation in Cd/Zn hyperaccumulator Sedum alfredii and its effects on plant growth promotion, root exudates and metal uptake efficiency. Journal of Hazardous Materials 424(PB):127442, doi:10.1016/j.jhazmat.2021.127442.
Schaider, L.A., Senn, D.B., Estes, E.R., Brabander, D.J. and Shine, J.P. 2014. Sources and fates of heavy metals in a mining-impacted stream: Temporal variability and the role of iron oxides. Science of the Total Environment 490:456-466, doi:10.1016/j.scitotenv.2014.04.126.
Sidhu, G.P.S., Bali, A.S. and Bhardwaj, R. 2019. Role of organic acids in mitigating cadmium toxicity in plants.
Cadmium Tolerance in Plants: Agronomic, Molecular, Signaling, and Omic Approaches 2019:255-279, doi:10.1016/B978-0-12-815794-7.00010-2.
Tessier, A., Campbell, P.G.C. and Bisson, M. 1979.
Sequential extraction procedure for the speciation of particulate trace metals. Analytical Chemistry 51(7):844- 851, doi:10.1021/ac50043a017.
Wamere, L.K. 2019. Heavy Metals Pollution using XRF Spectrometry- A Case Study of Kilimapesa Gold Mines Processing Plant, Narok County. Thesis, University of Nairobi.
Wang, D., Zhang, G., Dai, Z., Zhou, L., Bian, P., Zheng, K., Wu, Z. and Cai, D. 2018. Sandwich-like nanosystem for simultaneous removal of Cr(VI) and Cd(II) from water and soil. ACS Applied Materials and Interfaces 10(21):18316-18326, doi:10.1021/acsami.8b03379.
Wang, L., Cui, X., Cheng, H., Chen, F., Wang, J., Zhao, X., Lin, C. and Pu, X. 2015. A review of soil cadmium contamination in China including a health risk assessment. Environmental Science and Pollution Research 22(21):16441-16452, doi:10.1007/s11356- 015-5273-1.
Wang, P., Chen, H., Kopittke, P.M. and Zhao, F. J. 2019.
Cadmium contamination in agricultural soils of China and the impact on food safety. Environmental Pollution 249:1038-1048, doi:10.1016/j.envpol.2019.03.063.
Wang, Y.M., Tang, D.D., Zhang, X.H., Uchimiya, M., Yuan, X.Y., Li, M. and Chen, Y.Z. 2019. Effects of soil amendments on cadmium transfer along the lettuce-snail food chain: Influence of chemical speciation. Science of the Total Environment 649(1):801-807, doi:10.1016/j.scitotenv.2018.08.323.
Wilschefski, S. and Baxter, M. 2019. Inductively Coupled Plasma Mass Spectrometry: introduction to analytical aspects. Clinical Biochemist Reviews 40(3):115-133, doi:10.33176/aacb-19-00024.
Wiyono, C.D.A.P., Inoue, C. and Chien, M.F. 2021. HMA4 and IRT3 as indicators accounting for different responses to Cd and Zn by hyperaccumulator Arabidopsis halleri ssp. gemmifera. Plant Stress 2, doi:10.1016/j.stress.2021.100042.
Xie, X., Yang, S., Liu, H., Pi, K. and Wang, Y. 2020. The behavior of cadmium leaching from contaminated soil by nitrilotriacetic acid: implication for Cd-contaminated soil remediation. Water, Air, and Soil Pollution 231(4), doi:10.1007/s11270-020-04545-7.
Zhai, X., Li, Z., Huang, B., Luo, N., Huang, M., Zhang, Q.
and Zeng, G. 2018. Remediation of multiple heavy metal-contaminated soil through the combination of soil washing and in situ immobilization. Science of the Total
Zhao, M., Zhao, J., Yuan, J., Hale, L., Wen, T., Huang, Q., Vivanco, J.M., Zhou, J., Kowalchuk, G.A. and Shen, Q.
2021. Root exudates drive soil-microbe-nutrient feedbacks in response to plant growth. Plant Cell and Environment 44(2):613-628, doi:10.1111/pce.13928.