For soil remediation initiatives, it is important to characterize both the chemical and physical parameters of the soil. Composition (e.g., nutrients, organic and inorganic materials), and soil mixture may all influence how the contaminant will behave. In general, in can be concluded that the chemistry of metal interaction with soil matrix is central to the phytoremediation concept. While the soil used in this study was high in phosphorus, potassium, and magnesium, these were nonetheless essential plant nutrients. Results of the soil analysis by Mississippi State University Soil Testing Laboratory are summarized in Table 1
, and showed that the parameters of the soil used in this study were well within limits for our objectives.
Solubility Test and Chelate Selection
Results of ICP-OES analyses revealed that among the chelates tested (EDTA, EGTA, and HAc), EDTA was the most effective in solubilizing soil-bound Pb (Fig. 1
). Lead concentrations in soil solution increased with extraction time and remained constant 6 to 7 days after chelate amendment.
After 6 days, Pb concentration in soil solution of the EDTA-treated soil was 10.53 μg Pb/mL, as compared to 1.057, 0.047, and 0.048 μg Pb/mL for EGTA, HAc, and H2
O, respectively. These findings are consistent with those reported by Shen and his colleagues [25
], who found that the Pb-concentration in soil solution of the EDTA-treated soil was 42-fold higher than that of the control soil, and that citric acid application to the soil produced only a small increase in the Pb concentration of soil solution and used by itself was much less effective than other chelates used.
We hypothesized that the efficacy of EDTA in solubilizing Pb from the soil may be related to the high binding capacity of EDTA for Pb as shown in previous studies [3
]. It may also be presumed that EGTA and HAc are more rapidly degraded than EDTA. Our aim in the solubility study was not only to utilize a Pb-specific chelate, but to also apply it in a time-efficient manner so that we could harvest the plant during its peak phytoextraction period. In a practical field application, this would reduce the likelihood of herbivores eating the contaminated plants, as well as limit the risk of water pollution due to chelates and/or chelate-metal complexes migrating from the soil.
: Lead taken up by plants is usually increased with Pb concentrations in soil [26
]. Lead concentrations in root tissues were highest at 2000 mg Pb/kg, with EDTA alone or in combination with HAc (Fig. 2
Also, Pb concentrations were higher for day 6 than for day 7. When no chelates were applied, shoot Pb concentrations slightly increased with increasing levels of soil-applied Pb. This could be due to Pb binding to ion exchangeable sites on the cell wall and extracellular deposition mainly in the form of Pb carbonates deposited on the cell wall as previously demonstrated [28
]. Lead being a soft Lewis acid, forms a strong covalent bond not only with the soil, but with plant tissues as well [20
]. It is believed that since the xylem cell walls have a high cation exchange capacity, the upward movement of metal cations are severely retarded [10
In the absence of chelates Pb concentration in shoots of coffeeweed plants grown at 1000 and 2000 mg Pb/kg were minimal (Fig. 3
). With the addition of chelates alone or in combination with HAc, translocation significantly increased in day 0 and day 6. With the addition of chelates, it appeared that not only did the roots absorb more lead, but the metal was translocated to the shoots, facilitating some of the desirable characteristics of a hyperaccumulator, such as a high metal uptake by the roots, and translocation of the metal from the root to the above ground shoots. By day 7, however, there was not only a decline in the translocation of Pb to the shoots, but the decline was significantly lower in the 2000 mg Pb/kg treatments as compared to 1000 mg Pb/kg treatments.
Our results are comparable with experiments by other investigators who have reported that bringing the Pb into solution with a chelating agent, not only makes more Pb bioavailable for root uptake [10
], but also moves the Pb that is sequestered in the xylem cell wall upwards and into the shoots. Blaylock [3
] demonstrated with Indian mustard (Brassica juncea
(L.), and Huang et al. [20
] demonstrated with peas (Pisum sativum
L.) and corn (Zea mays
L.) that the addition of EDTA to Pb-contaminated soil increased the shoot Pb concentrations by 300-fold, 111-fold, and 57-fold, respectively. Transport across root cellular membrane is an important process which initiates metal absorption into plant tissues. Several studies have shown that sequestration in root vacuole may prevent the translocation of some metals from root to shoot [30
] whereas in hyperaccumulating plants, the mechanism of vacuolar sequestration may be disabled, allowing metal translocation and hyperaccumulation in leaves [30
]. Therefore, it is generally agreed that the ability of plants to move the Pb upwards into the shoots varies much more than their ability to accumulate metals in the roots [29
: The capacity of plants to remove contaminants from the soil is a function of biomass per unit area and concentration of the contaminant in the plants [32
]. Lead is not considered to be an essential element for plant growth and development, rather Pb inhibits growth, reduces photosynthesis (by inhibiting enzymes unique to photosynthesis), interferes with cell division and respiration, reduces water absorption and transpiration, accelerates abscission or defoliation and pigmentation, and reduces chlorophyll and adenosine triphosphate (ATP) synthesis [33
]. At maturity, metal-enriched aboveground biomass is harvested and a fraction of soil metal contamination removed [29
Our results revealed that root biomass was not significantly different across the treatments. However, roots that were grown in 0 mg Pb/kg soil had the lowest biomass, followed by treatments of 1000 mg Pb/kg soil. The highest root biomass was seen in treatments of 2000 mg Pb/kg soil (Table 2
). Shoot biomass followed the trend of root biomass. For each harvesting period (0, 6, and 7 days) shoots that were grown in the highest lead treatments (2000 mg Pb/kg soil) alone or in combination with chelates had the highest biomass (Table 3
). It is known that metal phytotoxicity causes stress to the plant resulting in a reduction in biomass and eventual death (in some cases). However, we observed no discernible phytotoxic symptoms in neither roots nor shoots. We concluded from this study and from earlier studies [24
] that Sesbania
may be tolerant to Pb-EDTA complex. Vassil et al. [21
] demonstrated with Indian mustard that EDTA appears to chelate Pb outside of the plant, and then the soluble Pb-EDTA complex is transported through the plant, via the xylem, and accumulates in the leaves. Further, they found that toxicity symptoms in Indian mustard exposed to Pb and EDTA were strongly correlated with the presence of free protonated EDTA in solution.
Another explanation for the apparent metal tolerance seen in Sesbania
may be the presence of natural metal-binding peptides. Cunningham and Ow [2
] described the presence of specific high-affinity ligands as one of the metal-resistant mechanisms existing in some plants. These ligands, known as phytochelatins and metallothioneins, are reported to make the metal less toxic to the plant [36
]. We are not certain whether this resistance mechanism may exist in Sesbania
, however, corollary studies regarding the relationship between Pb uptake and phytochelatin synthesis in coffeeweed are being investigated in our laboratory.
Results of this study indicated that EDTA can be applied to selected Pb-contaminated soils in a time-efficient manner so that plants can be harvested during their peak phytoextractive period, thereby limiting the likelihood of exposure to herbivores as well as reducing the risk of water pollution due to chelates and /or chelate-metal complexes migrating from the soil.