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Soil and Water Science Department, University of Florida, Gainesville, FL 32611-0290
* Corresponding author (lqma{at}ufl.edu)
Received for publication May 8, 2001.
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ABSTRACT |
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 TOPNOTES ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Abbreviations: BF, bioconcentration factor • CaMMA, calcium acid methanearsenate • DMA, dimethyl arsenic acid • MMA, monomethyl arsenic acid • NaDMA, sodium dimethylarsinic acid • NaMMA, sodium methylarsonic acid • TF, translocation factor
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INTRODUCTION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Current remediation methods for arsenic-contaminated soils include soil removal and washing, physical stabilization, and/or the use of chemical amendments, all of which are expensive and disruptive. Phytoextraction, the use of plants to remove contaminants from soils, is an emerging technology due to its cost-effectiveness and environmental friendliness (Cunningham et al., 1996; Brooks, 1998; Terry and Banuelos, 2000). Plant cultivation and harvesting are inexpensive processes compared with traditional engineering approaches involving intense soil manipulation, and minimize amounts of secondary waste generated compared with soil heaping, leaching, or washing. Furthermore, this technology creates minimal environmental disturbance.
Successful application of phytoextraction to arsenic-contaminated soils depends on many factors, among which are plant biomass and its arsenic concentration. Plants must be able to produce sufficient biomass while accumulating a high concentration of arsenic. In addition, phytoextraction species should be responsive to agricultural practices designed to enhance arsenic accumulation and to allow repeated planting and harvesting of arsenic-rich biomass. Furthermore, it is important to understand the availability and phytotoxicity of arsenic to the plant itself.
Arsenic is a nonessential element for plants. At higher concentrations, arsenic interferes with plant metabolic processes and can inhibit growth, often leading to death. Biomass production and yields of a variety of crops are reduced significantly at elevated arsenic concentrations (Carbonell-Barrachina et al., 1997), with application of only 50 mg As kg-1 to soil significantly decreasing the yields of barley (Hordeum vulgare L.) and ryegrass (Lolium perenne L.) (Jiang and Singh, 1994).
Arsenic availability to plants is greatly influenced by its forms in soil. Arsenic in soils can exist as the corresponding salts of arsenite [As(III)], arsenate [As(V)], monomethyl arsenic acid (MMA), and dimethyl arsenic acid (DMA). Different arsenic species have different solubilities and mobilities, and thus differing bioavailability to plants. In hydroponic conditions, the availability of arsenic to a marsh grass (Spartina alterniflora L.) followed the trend: DMA << MMA < As(V) < As(III) (Carbonell et al., 1998). Marin et al. (1992) reported that the order of arsenic availability to rice (Oryza sativa L.) was as follows: DMA < As(V) < MMA < As(III). Upon absorption, DMA is readily translocated to the plant shoot, whereas As(III), As(V), and MMA accumulated primarily in the roots upon uptake (Marin et al., 1992). In tomato (Lycopersicon esculentum Mill.) plants, both MMA and DMA had a greater upward translocation than arsenite and arsenate (Burlo et al., 1999). The presence of other ions also affected arsenic availability and phytotoxicity (Fowler, 1983).
Agricultural application of arsenicals has introduced many different kinds of arsenic compounds to the soil environment. Calcium arsenate [Ca3(AsO4)2] was used as insecticide from the 1800s through the 1960s. Currently arsenic acid (H3AsO4), sodium arsenate (Na3AsO4), sodium arsenite (NaAsO2), and DMA [(CH3)2AsO2H] are being used as defoliants, while DSMA (CH3AsO3Na2), MSMA (CH3AsOHNa), and MAA (CH3AsO3H2) are being used as herbicides (Onken and Hossner, 1996). It is well known that arsenic associations with Fe and Al control arsenic behavior in the soil (Rochette et al., 1998). These arsenicals may influence arsenic mobility and plant uptake though they are subjected to oxidation–reduction transformation in soils.
Historically, no arsenic-hyperaccumulating plants were reported due to arsenic phytotoxicity. However, an arsenic-hyperaccumulating plant, ladder brake, was recently discovered. This plant accumulates large amounts of arsenic from soils, with arsenic concentrations in its aboveground biomass as high as 2.3% when grown in an arsenic-amended soil (500 mg As kg-1) and 0.7% when grown in an arsenic-contaminated soil (38.9 mg As kg-1) from a former chromium–copper–arsenic (CCA) wood preservation site (Ma et al., 2001). It also has the potential to produce large plant biomass (Jones, 1987). However, no information is available about the effect of soil arsenic on biomass production and arsenic uptake and distribution of ladder brake.
The objective of this study was to examine the growth and arsenic uptake and accumulation by ladder brake in soils amended with different arsenic concentrations and various arsenic compounds. Results should provide critical information regarding ladder brake's ability to tolerate and extract arsenic from soil and to translocate arsenic to its aboveground biomass, shedding further light on its applicability for remediating arsenic-contaminated soils.
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MATERIALS AND METHODS |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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Preparation of Arsenic Chemicals
The arsenic chemicals AlAsO4·2H2O, Ca3(AsO4)2·14H2O, and FeAsO4·2H2O were each synthesized in the laboratory (Hess and Blanchar, 1976), since they are not available from commercial sources. They were washed free of salts and verified with X-ray diffraction and total chemical analysis. The chemicals (reagent grade) NaAsO2, Na2HAsO4, K2HAsO4, sodium dimethylarsinic acid (NaDMA), sodium methylarsonic acid (NaMMA), and calcium acid methanearsenate (CaMMA) were purchased commercially (Sigma Chemical Co., St. Louis, MO; Zeneca Agricultural Products, Wilmington, DE; Amchem Products, Ambler, PA).
Soil Sampling
The soil used in this study was collected from central Florida. It is classified as Grossarenic Paleudult (sandy, siliceous, hyperthermic). The soil pH was measured using a 1:1 soil to water ratio. Cation exchange capacity (CEC) was determined by an ammonium acetate method (Thomas, 1982); organic matter content by the Walkley–Black method (Nelson and Sommers, 1982); and particle size by the pipette method (Day, 1965). Total soil phosphorus was digested using USEPA Method 3051, and water-soluble phosphorus was extracted with deionized water at a 2:20 soil to solution ratio. Phosphorus concentrations in solution were measured on a PerkinElmer (Norwalk, CT) ELAN 6000 ICP–MS unit. Selected physical and chemical properties of the soil are presented in Table 1.
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Soil (1.5 kg) was thoroughly mixed with arsenic solution and 1.5 g of Osmocote extended time-release fertilizer (Scotts-Sierra Horticultural Products Co., Marysville, OH) was added as base fertilizer. The amount applied for N, P, and K nutrients was thus 180, 60, and 120 mg kg-1, respectively. Treated soil was then placed in a 2.5-L plastic pot. Each treatment was replicated four times. After 1 wk of incubation, one ladder brake plant was transplanted into each pot. Plants were grown in a greenhouse for 12 and 18 wk for Part I and Part II, respectively. The greenhouse temperature ranged from 14 to 30°C, and average photosynthetically active radiation was 825 µmol m-2 s-1. The plants were watered daily as needed. After harvesting, plants were washed free of soil with tap water and then rinsed with 0.1 mol L-1 HCl solution followed by several rinses with deionized distilled water. The plants were then separated into aboveground biomass, which was further separated into young, mature, and old fronds based on their ages, and belowground (roots including rhizomes) biomass. Biomass was measured on a dry-weight basis (after 3 d at 65°C). Dry plant samples were ground to a fine powder before analysis, and soil samples were taken from the pots both before plant transfer and after harvest, air-dried, and sieved for analytical use.
Determination of Arsenic in Plant and Soil
Plant (approximately 0.1000–0.5000 g) and soil (approximately 1.000 g) samples were weighed into a 120-mL Teflon pressure digestion vessel, mixed with 10 mL of concentrated trace-metals grade nitric acid, and digested using USEPA Method 3051 on a CEM (Matthews, NC) MDS-2000 microwave sample preparation system. After cooling, the sample solution was filtered through Whatman (Maidstone, UK) no. 42 filter paper and diluted to a volume of 100 mL. For soil water-soluble arsenic, a 2-g soil sample was shaken with 20 mL deionized water for 30 min. The suspension was then filtered through Whatman no. 42 filter paper. Determination of aqueous arsenic concentration was performed using a graphite furnace atomic absorption spectrophotometer (PerkinElmer SIMMA 6000). Results were expressed as a mean of four replicates, with standard error. Analysis of variance was performed using SAS software (SAS Institute, 1987). The Tukey procedure was used for mean separation.
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RESULTS AND DISCUSSION |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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500 mg As kg-1 as arsenate and 50 mg As kg-1 as sodium dimethylarsonate. Three days after transplanting, arsenic toxicity was observed in fronds of ladder brake in the 1000 mg As kg-1 treatment (approximately 200 mg As kg-1 water-soluble arsenic). These fronds had dark brown coloration and necrosis at the leaf tips and margins, and plants were dead after 1 wk. In the treatment with 500 mg As kg-1, the symptoms of arsenic toxicity appeared in the old fronds of ladder brake after 2 wk, but the plants survived throughout the study. Addition of 50 mg As kg-1 as sodium dimethylarsonate killed the plants within 4 d of transplanting. Dimethylarsinic acid, a herbicide, apparently was more readily translocated to the shoot than inorganic arsenicals or MMA, and thus was more phytotoxic (Carbonell-Barrachina et al., 1998). Based on common arsenical application rates of 6 to 12 kg As ha-1, arsenic contents in soil would be increased by only 3 to 6 mg As kg-1 (O'Neill, 1995), which could be 10 to 20 times lower than the rate of 50 mg As kg-1 used in our experiment. Arsenite has been considered at least twice as phytotoxic as arsenate either foliarly or root-applied (Sachs and Michael, 1971). At 50 mg As kg-1, such a difference was not observed for ladder brake, that is, there was no significant difference in biomass production between treatments with Na2HAsO4 or NaAsO2 (Table 3). This may be due to the conversion of arsenite to arsenate in soil (Smith et al., 1998). However, the differences in plant effects was reported between arsenite and arsenate for up to one year after entering to soil (Jiang and Singh, 1994).
Arsenic Distribution in Ladder Brake
An artificially contaminated soil was used in this experiment. It is expected that arsenic availability in this soil would be greater than that of "real-world" soils. One week after amending the soil with 50 to 500 mg As kg-1 as K2HAsO4, 11.6 to 17.8% of the arsenic remained water soluble (Table 4). One week after amending the soil with 50 mg kg-1 of differing arsenic forms, water-soluble arsenic varied greatly (Table 5), with <1% of the arsenic being water soluble for AlAsO4 and FeAsO4, and 10.0 to 31.6% for the remaining forms. In contrast, water-soluble arsenic in soils from a number of arsenic-contaminated mine sites was <0.02% (Porter and Peterson, 1977). At the end of the experiment, water-soluble arsenic in all treatments was reduced due both to aging effects (Alexander, 1995) and arsenic uptake by ladder brake (Tables 4 and 5). However, soil drying may change the extractability of arsenic. This issue has been observed for other elements, for example Al, Ca, Fe, K, Mg, Mn, and P (Bartlett and James, 1980).
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100 mg As kg-1, arsenic concentrations in ladder brake fronds increased linearly with soil arsenic (r2 0.977), with slopes ranging from 41.7 to 70.2, depending on frond age. At arsenic concentrations 100 mg As kg-1, arsenic accumulation in the fronds decreased, possibly due to restricted upward arsenic translocation to fronds owing to toxic levels of arsenic in the roots (Table 4). This was also reflected by biomass reduction for ladder brake fronds (Table 2) (Carbonell-Barrachina et al., 1997). Arsenic toxicity in bean (Phaseolus vulgaris L.) was also found to be directly proportional to root arsenic concentration (O'Neill, 1995). Several reports on the linear relationship between arsenic content of vegetation and soil arsenic concentrations suggested that plants take up arsenic passively in conjunction with water flow (Kabata-Pendias and Pendias, 1991). It is possible that ladder brake takes up arsenic passively at soil arsenic concentration 100 mg As kg-1, whereas a different mechanism may apply at higher arsenic levels. Arsenic concentrations in soils, especially water-soluble arsenic, significantly affected distribution in fronds of differing ages (young, mature, and old) (Tables 4 and 5). The effects noted can be divided into two patterns. At low water-soluble arsenic levels in soils (<0.5 mg As kg-1), arsenic concentrations in the fronds increased from old to young, whereas at moderate to high water-soluble arsenic levels in soils (>0.5 mg As kg-1), arsenic concentrations in the fronds increased from young to old. This behavior is similar to that of nutrients, especially phosphorus. Concentrations of mobile plant nutrients are typically higher in younger leaves than in older ones when soil nutrient levels are low, because such nutrients are preferentially supplied to actively growing parts of the plant (Mengel and Kirkby, 1987). At low levels, arsenic appears to be taken up by ladder brake much like a nutrient, since more arsenic was observed in the young fronds (Tables 4 and 5). When adequate levels of arsenic were present in soils, arsenic was probably translocated to all fronds with little discrimination, leading to greater concentrations in older fronds since they have been receiving arsenic for a longer time. Our results are consistent with what has been in the literature. Arsenic concentrations in Douglas fir [Pseudotsuga menziesii (Mirb.) Franco] biomass were greater in recent growth than in older growth when arsenic concentrations in soils were relatively low, ranging from 3 to 330 mg As kg-1 (Warren et al., 1968). On the other hand, arsenic concentrations in bent grass (Agrostis capillaris L.) plants collected from highly contaminated soils were greater in older leaves (Porter and Peterson, 1975). Arsenic concentrations increased from 150 mg As kg-1 for young leaves to 1100 mg As kg-1 for old leaves at total soil arsenic levels of 8500 to 26500 mg As kg-1. As older leaves abscised, this process could be considered a means of detoxification to assist the removal of arsenic from the plant. The greater arsenic concentration observed in young fronds of ladder brake at 500 mg As kg-1 (Table 4) was possibly due to arsenic toxicity, and appeared related to a significant reduction in frond biomass (Table 2).
Arsenic Bioconcentration and Translocation Factors
Tissue arsenic concentrations alone may not be a good indicator for comparing arsenic uptake by plants from soils because tissue concentrations do not take into account soil arsenic concentration. The bioconcentration factor (BF), which is defined as the ratio of arsenic concentrations in plant tissue to those in soil, can be used to compare the effectiveness of the plant in concentrating arsenic from soil into its biomass. Overall, arsenic concentrations in ladder brake biomass, especially aboveground biomass, were considerably higher than those in soils, indicating significant arsenic bioconcentration (Tables 6 and 7). Although other plants reportedly take up large amounts of arsenic from contaminated soil, they are not generally arsenic hyperaccumulators like ladder brake. Bent grass plants collected from a number of arsenic-contaminated soils contained up to 3470 mg As kg-1 in aboveground biomass (Porter and Peterson, 1977). However, arsenic concentrations in the corresponding soil were much greater (26500 mg As kg-1), that is, the plant was unable to hyperaccumulate arsenic from that soil.
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The effects of different arsenic forms on the BF and TF of ladder brake were more complicated due to differences in arsenic solubility (Tables 5 and 7). The least water-soluble forms tested were FeAsO4 and AlAsO4, whereas the organic arsenic forms NaMMA and CaMMA were the most water soluble. The BFs of ladder brake fronds increased with an increase in water-soluble arsenic, that is, the BF increased from 3.4 for FeAsO4 to 9.2 for AlAsO4 (Table 7) as water-soluble arsenic increased from 0.15 to 0.48 mg As kg-1 (Table 5). As water-soluble arsenic concentrations increased to >0.5 mg As kg-1, arsenic BFs increased significantly to 41.3 to 54.9. The addition of arsenic at low levels (<50 mg As kg-1) increased concentrations in the fronds as reflected by an increases in BF values. Among arsenate forms with comparable solubility (K, Na, and Ca), Ca was more effective in increasing arsenic concentrations in fronds. It appeared that arsenic forms (arsenite vs. arsenate, and organic vs. inorganic arsenic) had little effect on arsenic concentrations in ladder brake fronds, with no clear trends being observed. This is possibly because all arsenic species could have transformed to As (V) during the experiment (18 wk) due to chemical oxidation–reduction and microbial transformations. Carbonell et al. (1998) reported that DMA, MMA, As(III), and As(V) were stable for only 4 d with respect to oxidation–reduction, methylation–demethylation reactions in hydroponics.
Both water-soluble arsenic and accompanying cations affected arsenic TF values for ladder brake. Generally speaking, arsenic TF values increased with an increase in water-soluble arsenic in soils regardless of arsenic forms with r2 = 0.70 (Table 7). For a given arsenic form at comparable water-soluble arsenic levels, Ca enhanced arsenic translocation from roots to fronds [i.e, Na2HAsO4 vs. Ca3(AsO4)2 and NaMMA vs. CaMMA]. Compared with arsenate (Na2HAsO4), arsenite (NaAsO2) resulted in less arsenic translocation from roots to fronds, that is, more arsenic was stored in the roots (Table 7). However, no difference in arsenic translocation by ladder brake was observed between organic (NaMMA and CaMMA) and inorganic [Na2HAsO4 and Ca3(AsO4)2] arsenate.
Phytoextraction Capacity of Ladder Brake
Application of a hyperaccumulator is determined by its ability to extract contaminants from a soil, which depends on both plant biomass and an arsenic concentration in that biomass. Phytoextraction capacity may take both factors into consideration, since it yields total arsenic accumulation in the biomass (Tables 8 and 9). Most of the arsenic taken up by ladder brake was concentrated in its aboveground biomass, ranging from 75 to 98%. Increasing soil arsenic concentrations from 100 to 500 mg As kg-1 resulted in lower arsenic extraction by aboveground biomass, however (Table 8). Also, compared with other forms, FeAsO4 and AlAsO4 showed lower arsenic extraction by the aboveground biomass of ladder brake (i.e., 75 to 82% vs. 95 to 98%). However, increases in soil arsenic concentrations up to 100 mg As kg-1 actually increased arsenic extraction by ladder brake. Among the four arsenic concentrations tested, 100 mg As kg-1 resulted in the greatest arsenic accumulation by ladder brake into aboveground biomass with 13.8 mg plant-1, accounting for about 10% of initial soil arsenic (Table 8). This demonstrated this plant's effectiveness as an arsenic accumulator from contaminated soils. Although arsenic concentrations in ladder brake biomass increased significantly with increasing arsenic concentrations in soils (Table 4), reduction in fern biomass (Table 2) resulted in lower arsenic phytoextraction at the highest soil arsenic levels (Table 8).
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This experiment clearly demonstrated the effectiveness of ladder brake in taking up large amounts of arsenic from a contaminated soil and in translocating it to aboveground biomass. Large biomass is necessary for efficient removal of arsenic from soil. It was also seen that concentrations of 50 to 100 mg As kg-1 significantly increased the plant's biomass and associated bioconcentration factors and translocation factors for ladder brake, demonstrating a stimulation by arsenic of plant growth, arsenic uptake, and translocation. However, an arsenic concentration of 500 mg As kg-1 resulted in a significant reduction in plant biomass. At low soil arsenic levels, arsenic was concentrated in the younger fronds, whereas at moderate to high levels, arsenic was concentrated more in older fronds. Solubility of arsenic was more important than was arsenic form in determining the uptake and accumulation capacities of arsenic by ladder brake. This can be seen in a comparison of uptake from MMA compared with arsenate in the presence of identical cations, along with poor accumulation in the presence of the relatively insoluble Fe and Al arsenate forms. Of the arsenic concentrations considered in this study, 50 mg As kg-1 yielded the highest amount of arsenic removal into aboveground biomass as a percentage of soil concentration. Based on the presented findings it can be concluded that ladder brake has great potential to remediate and revegetate arsenic-contaminated soils.
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ACKNOWLEDGMENTS |
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NOTES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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REFERENCES |
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TOPNOTESABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTS AND DISCUSSIONREFERENCES |
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of varying concentrations.