Enhanced Uptake of Cadmium by Native Plant (Artemisia princeps var. orientalis) Using Ethylenediaminetetraacetic Acid

MATERIALS AND METHODS

Hydroponic experiment: Rhizomes of A. princeps were field-collected and cultivated for 30 days in a sand bed irrigated with the modified Epstein hydroponic solution. Plant seedlings were transplanted into a hydroponic solution in a reaction vessel and therein equilibrated for 10 days. The reaction vessels that contained the dialysis tube and hydroponic solution were prepared. The dialysis tubes were filled with 25 mL of hydroponic solution and Cd-contaminated soils (both field collected and artificially contaminated with Cd nitrate, refer to soil sample section). Total Cd concentrations in both soils were 20 mg kg^-1 (Ok et al., 2004a).

Table 1:	Physical and chemical properties of the soil used in the hydroponic experiment
1Organic matter; 2Field moisture holding capacity

One mM of EDTA, oxalate or sulfate were added separately into the reaction vessels, followed by transplanting the seedlings into the reaction vessels. After growing the seedlings for 48 h, phytoavailability of Cd by Artemisia princeps was determined by analysing Cd contents in roots and shoots. The harvested plant seedlings were rinsed carefully in distilled water, dried at 80°C and weighed. The Cd concentration in the plant was then determined using ICP-AES (JY 138 Ultrace, JOBIN YVON) after a digestion with the H₂SO₄-HNO₃-H₂O₂ solution. The calibration standards were prepared using the standard solution which was certified by the supplier. Five calibration standards and blank solution were used to calibrate the ICP-AES. A linear calibration curve was obtained after calibration. If the correlation coefficient R² was less than 0.999, the equipment was re-calibrated to ensure the accuracy of results. All the instrumental conditions were optimized for the maximum sensitivity as indicated by the manufacturer’s manual.

Soil sample: Soil samples used in hydroponic experiments were artificially contaminated with the CdCl₂ solution to a final concentration of 20 mg kg^-1 of Cd and were spiked as follows: 400 mg L^-1 of Cd with the CdCl₂ stock solution was prepared and exactly 2.0 kg of soil was placed in a rectangular vessel made of low density polyethylene (LDPE). Fifty milliliters of prepared stock solution was uniformly sprayed on the soil, thoroughly mixed with a plastic rod after 5 min of contact time and air-dried. These steps were repeated until the resulting Cd concentration of spiked soil was 20 mg kg^-1. The soil used in this study showed very similar characteristics with typical Korean soils (Table 1): a relatively low pH was found in the soil with a value of 5.12 as compared to the average Korean soil pH of 5.7. According to the USDA classification, the soil was classified as sandy soil. The low organic matter content in the soil was attributed to lack of vegetation resulting from poor soil condition for plant growth (low soil pH, unbalanced nutrition and a high sand fraction). This condition also contributed to low cation exchange capacity (CEC) (4.05 cmol_c kg^-1).

RESULTS AND DISCUSSION

The Artemisia is one of the largest genera in the family, Compositae, found in Korea and it is a common weed but an important medicinal plant used as a styptic in the central part of Korea (Yun and Choi, 2003).

Table 2:	The bioaccumulation factors for Cd accumulating plants reported in the literature in the Republic of Korea

Some species are aromatic and used as herbs, spices and folk medicines. The bioconcentration factors for the Cd accumulating plants reported in the Republic of Korea are summarized in Table 2. Among the plants, the bioaccumulation factor was high in the Artemisia princeps var. orientalis with 5.72 for Cd in its shoot. In a previous research, Ok et al. (2003) reported that Artemisia princeps var. orientalis could be used as a possible phytoremediator to clean up the Cd contaminated soils. The authors showed that in a sequential extraction of the Cd contaminated soil, where Artemisia princeps was grown, 2.4 mg kg^-1 (below 7%) of Cd existed as an exchangeable fraction. But, A. princeps took up over 50 mg kg^-1 of Cd in its harvestable part and was also grown vigorously in the metal contaminated areas. It is also known to have a strong resistance to water stress and grows well under various pH regimes and soil textural conditions (Kim, 2003). Thus, further experiments were performed to evaluate Artemisia princeps for phytoextraction of Cd contaminated soils.

Complexes of toxic metals due to organic acids or EDTA facilitate metal accumulation in the plant. This fact is very important in developing the strategies for phytoextraction since chelation can maximize metal accumulation in the shoot as well as in the root. For this reason, chelating agents were added as chemical ameliorants in another experiment to increase the Cd desorption from the soil and to facilitate Cd accumulation in the plant. Oxalic acid was selected among organic acids as the previous research has shown that Artemisia princeps released oxalic acid as a major source of organic acids. The contrasting samples of soil for the Cd availability were prepared to compare the Cd accumulation between two treatments: One is the soil artificially contaminated with Cd and the other is the soil naturally contaminated with Cd. The physical and chemical properties of soils (texture, organic matter, CEC and fertility status of soil) were not significantly different (Ok et al., 2004b). Table 3 showed that the EDTA (1 mM) was more effective than oxalate or sulfate in mobilizing Cd in the contaminated soil into labile forms, resulting also in enhanced translocation of Cd from roots into shoots. There was no significant difference in Cd contents in shoots between the treatments of Cd contaminated soils and the artificial Cd solution.

Table 3:	Cadmium concentrations in Artemisia princeps var. orientalis after growing for 48 h in a hydroponic culture (Means with the same letters are not significantly different by Tukey’s studentized range test; SE: Standard Error)

Fig. 1:	Rate of Cd uptake from the Cd-contaminated soils by mugwort (Artemisia princeps var. orientalis) after growing for 48 h in a hydroponic culture (FCS: Field Contaminated Soil; FCS + EDTA: FCS treated with 1 mM EDTA; ACS: artificially contaminated soil: and ACS + EDTA: ACS treated with 1 mM MEDTA)

These results were supported by the Cd contents in the roots, as revealed the lower Cd in the root in the EDTA treatment. Oxalate and sulfate had little effect on the Cd availability by Artemisia princeps. Figure 1 showed that the availability of Cd was lower in the field contaminated soils than the artificially contaminated soils (Ok et al., 2004b). The EDTA increased the Cd availability in both soils with similar increments. Wallace et al. (1974) reported that the metal-EDTA complexes formed in the soil could increase metal solubility and promote diffusion and thus increase the potential for plant uptake. Blaylock et al. (1997) also showed that EDTA was the most efficient increasing water-soluble Pb concentration. However, Halvorson and Lindsay (1977) found that chelates in the soil lowered metal toxicity and uptake. Moreover, application of EDTA to the soil has been observed to lead to decreases in the uptake of cadmium by plants (Wolterbeek et al., 1988). This apparent contradiction in the results of the studies reported by different researchers may be attributable to phytotoxicity of metals or EDTA. These results indicate that the species of Cd not the total content govern the Cd availability and EDTA could be used as the soil treatment agent enhancing phytoextraction of cadmium from the contaminated soil (Blaylock et al., 1997; Chen et al., 2000).

There was no significant difference of the Cd content in the shoot among the treatments of the Cd contaminated soil and the artificial Cd solution. These results were supported by the Cd contents in the root, as revealed by the lower Cd in the root for the EDTA treatment. Oxalate had little effect of Artemisia princeps on Cd availability. Recently, several similar studies on Pb accumulation in the plant in the presence of ¹⁴C-labeled EDTA revealed that both Pb and EDTA were present in the shoot, suggesting that Pb was absorbed and transferred as a Pb-EDTA complex (Sarret et al., 2000). A physiological basis of uptake of the Pb-EDTA complex and particularly possibility for this negatively charged molecule to cross the membrane is still not clear. However, Vassil et al. (2003) suggested that EDTA could damage the membrane of the root cells by chelating Zn and Ca cations that stabilize this membrane, thus allowing a free equilibrium between the soil solution and the xylem sap. Until now, no evidence has been found that favors the hypothesis of uptake of the Pb-EDTA^2- complex or of Pb²⁺ and EDTA^4-.