Tu & Ma: Arsenic Uptake by the Hyperaccumulator Ladder Brake

in plants, animals, and humans (Fowler, 1983). Remediation of arsenic-contaminated soils has thus become a Ladder brake (Pteris vittata L.) is a newly discovered arsenic major environmental issue. hyperaccumulator. No information is available about arsenic effects on ladder brake. This study determined the effects of different arsenic Current remediation methods for arsenic-contamiconcentrations (50 to 1000 mg kg 1 ) or forms (organic vs. inorganic nated soils include soil removal and washing, physical and arsenite vs. arsenate) applied to soils on growth and arsenic stabilization, and/or the use of chemical amendments, uptake by ladder brake. Young plants were grown in a greenhouse all of which are expensive and disruptive. Phytoextracfor 12 or 18 wk. Ladder brake was highly tolerant of arsenic and tion, the use of plants to remove contaminants from survived in soil containing up to 500 mg As kg 1. The fact that addition soils, is an emerging technology due to its cost-effectiveof arsenate up to 100 mg As kg 1 increased fern biomass by 64 to ness and environmental friendliness (Cunningham et 107%, coupled with higher arsenic concentration in younger fronds al., 1996; Brooks, 1998; Terry and Banuelos, 2000). Plant at low soil arsenic concentrations and older fronds at high soil arsenic concentrations, implies that arsenic may be beneficial for fern growth. cultivation and harvesting are inexpensive processes comAddition of 50 mg As kg 1 was best for fern growth and arsenic pared with traditional engineering approaches involvaccumulation, resulting in the highest fern biomass (3.9 g plant 1 ), ing intense soil manipulation, and minimize amounts of bioconcentration factor (up to 63), and translocation factor (up to secondary waste generated compared with soil heaping, 25). With an exception of FeAsO4 and AlAsO4, which had the lowest leaching, or washing. Furthermore, this technology creeffects due to their low solubility, little difference was observed among ates minimal environmental disturbance. other arsenic forms mainly because of arsenic conversion in soil. Successful application of phytoextraction to arsenicAboveground biomass was mostly responsible for accumulation of contaminated soils depends on many factors, among arsenic by plant (75–99%). Up to 26% of the added arsenic was removed by ladder brake, showing the high efficiency of ladder brake which are plant biomass and its arsenic concentration. in arsenic removal. The results suggest that ladder brake may be a Plants must be able to produce sufficient biomass while good candidate to remediate arsenic-contaminated soils. accumulating a high concentration of arsenic. In addition, phytoextraction species should be responsive to agricultural practices designed to enhance arsenic accuA contamination of soils from various anthromulation and to allow repeated planting and harvesting pogenic sources such as pesticides, fertilizers, of arsenic-rich biomass. Furthermore, it is important to wood preservatives, smelter wastes, and coal combusunderstand the availability and phytotoxicity of arsenic tion is of great environmental concern (Nriagu, 1994; to the plant itself. Smith et al., 1998). Severe arsenic contamination in soils Arsenic is a nonessential element for plants. At higher may cause a variety of problems such as loss of vegetaconcentrations, arsenic interferes with plant metabolic tion, ground water contamination, and arsenic toxicity processes and can inhibit growth, often leading to death. Biomass production and yields of a variety of crops are Soil and Water Science Department, University of Florida, Gainesreduced significantly at elevated arsenic concentrations ville, FL 32611-0290. C. Tu, present address: Department of Plant Pathology, North Carolina State University, Raleigh, NC 27695-7616. Approved for publication as Florida Agricultural Experiment Station Abbreviations: BF, bioconcentration factor; CaMMA, calcium acid Journal Series no. R-07998. Received 8 May 2001. *Corresponding methanearsenate; DMA, dimethyl arsenic acid; MMA, monomethyl author ([email protected]). arsenic acid; NaDMA, sodium dimethylarsinic acid; NaMMA, sodium methylarsonic acid; TF, translocation factor. Published in J. Environ. Qual. 31:641–647 (2002). 642 J. ENVIRON. QUAL., VOL. 31, MARCH–APRIL 2002 (Carbonell-Barrachina et al., 1997), with application of MATERIALS AND METHODS only 50 mg As kg 1 to soil significantly decreasing the Fern Propagation yields of barley (Hordeum vulgare L.) and ryegrass (LolLadder brake plants were propagated from spores (Jones, ium perenne L.) (Jiang and Singh, 1994). 1987). After germination, young ferns were grown in a seedbed Arsenic availability to plants is greatly influenced by until they achieved two or three fronds and a height of 3 to its forms in soil. Arsenic in soils can exist as the corre4 cm. Thereafter, they were transferred into 4-inch (10.16-cm) sponding salts of arsenite [As(III)], arsenate [As(V)], plastic pots filled with potting mixture and allowed to grow monomethyl arsenic acid (MMA), and dimethyl arsenic until they had five or six fronds approximately 10 cm in height acid (DMA). Different arsenic species have different prior to experimental use. solubilities and mobilities, and thus differing bioavailability to plants. In hydroponic conditions, the availabilPreparation of Arsenic Chemicals ity of arsenic to a marsh grass (Spartina alterniflora L.) The arsenic chemicals AlAsO4·2H2O, Ca3(AsO4 )2·14H2O, followed the trend: DMA MMA As(V) As(III) and FeAsO4·2H2O were each synthesized in the laboratory (Carbonell et al., 1998). Marin et al. (1992) reported (Hess and Blanchar, 1976), since they are not available from that the order of arsenic availability to rice (Oryza sativa commercial sources. They were washed free of salts and veriL.) was as follows: DMA As(V) MMA As(III). fied with X-ray diffraction and total chemical analysis. The Upon absorption, DMA is readily translocated to the chemicals (reagent grade) NaAsO2, Na2HAsO4, K2HAsO4, soplant shoot, whereas As(III), As(V), and MMA accudium dimethylarsinic acid (NaDMA), sodium methylarsonic acid (NaMMA), and calcium acid methanearsenate (Camulated primarily in the roots upon uptake (Marin et MMA) were purchased commercially (Sigma Chemical Co., al., 1992). In tomato (Lycopersicon esculentum Mill.) St. Louis, MO; Zeneca Agricultural Products, Wilmington, plants, both MMA and DMA had a greater upward DE; Amchem Products, Ambler, PA). translocation than arsenite and arsenate (Burlo et al., 1999). The presence of other ions also affected arsenic Soil Sampling availability and phytotoxicity (Fowler, 1983). Agricultural application of arsenicals has introduced The soil used in this study was collected from central Florida. It is classified as Grossarenic Paleudult (sandy, siliceous, many different kinds of arsenic compounds to the soil hyperthermic). The soil pH was measured using a 1:1 soil to environment. Calcium arsenate [Ca3(AsO4 )2] was used as water ratio. Cation exchange capacity (CEC) was determined insecticide from the 1800s through the 1960s. Currently by an ammonium acetate method (Thomas, 1982); organic arsenic acid (H3AsO4 ), sodium arsenate (Na3AsO4 ), somatter content by the Walkley–Black method (Nelson and dium arsenite (NaAsO2 ), and DMA [(CH3 )2AsO2H] are Sommers, 1982); and particle size by the pipette method (Day, being used as defoliants, while DSMA (CH3AsO3Na2 ), 1965). Total soil phosphorus was digested using USEPA MSMA (CH3AsOHNa), and MAA (CH3AsO3H2 ) are Method 3051, and water-soluble phosphorus was extracted being used as herbicides (Onken and Hossner, 1996). with deionized water at a 2:20 soil to solution ratio. Phosphorus It is well known that arsenic associations with Fe and concentrations in solution were measured on a PerkinElmer Al control arsenic behavior in the soil (Rochette et al., (Norwalk, CT) ELAN 6000 ICP–MS unit. Selected physical and chemical properties of the soil are presented in Table 1. 1998). These arsenicals may influence arsenic mobility and plant uptake though they are subjected to oxidaGreenhouse Experiment tion–reduction transformation in soils. Historically, no arsenic-hyperaccumulating plants The experiment consisted of two parts. For Part I, the soil were reported due to arsenic phytotoxicity. However, was amended with arsenic at different concentrations (0, 50, an arsenic-hyperaccumulating plant, ladder brake, was 100, 200, 500, or 1000 mg As kg 1 as K2HAsO4 ) to examine the effect of different arsenic concentrations on ladder brake. recently discovered. This plant accumulates large The potassium salt of arsenic was selected to avoid sodium amounts of arsenic from soils, with arsenic concentraharm to plant. For Part II, the soil was amended with different tions in its aboveground biomass as high as 2.3% when arsenic compounds at the rate of 50 mg As kg 1 as inorganic grown in an arsenic-amended soil (500 mg As kg 1 ) and arsenicals [AlAsO4·2H2O, Ca3(AsO4 )2·14H2O, FeAsO4·2H2O, 0.7% when grown in an arsenic-contaminated soil (38.9 Na2HAsO4, NaAsO2, K2HAsO4] or organic arsenicals (Namg As kg 1 ) from a former chromium–copper–arsenic MMA, NaDMA, and CaMMA). (CCA) wood preservation site (Ma et al., 2001). It also Soil (1.5 kg) was thoroughly mixed with arsenic solution has the potential to produce large plant biomass (Jones, and 1.5 g of Osmocote extended time-release fertilizer (Scotts1987). However, no information is available about the Sierra Horticultural Products Co., Marysville, OH) was added effect of soil arsenic on biomass production and arsenic as base fertilizer. The amount applied for N, P, and K nutrients was thus 180, 60, and 120 mg kg , respectively. Treated soil uptake and distribution of ladder brake. was then placed in a 2.5-L plastic pot. Each treatment was The objective of this study was to examine the growth replicated four times. After 1 wk of incubation, one ladder and arsenic uptake and accumulation by ladder brake brake plant was transplanted into each pot. Plants were grown in soils amended with different arsenic concentrations in a greenhouse for 12 and 18 wk for Part I and Part II, and various arsenic compounds. Results should provide respectively. The greenhouse temperature ranged from 14 to critical information regarding ladder brake’s ability to 30 C, and average photosynthetically active radiation was 825 tolerate and extract arsenic from soil and to translocate mol m 2 s . The plants were watered daily as needed. After arsenic to its aboveground biomass, shedding further harvesting, plants were washed free of soil with tap water and light on its applicability for remediating arsenic-contamthen rinsed with 0.1 mol L 1 HCl solution followed by several rinses with deionized distilled water. The plants were then inated soils. TU & MA: ARSENIC UPTAKE BY THE HYPERACCUMULATOR LADDER BRAKE 643 Table 2. Dry biomass of ladder brake after 12 wk of growth in a Table 1. Selected properties of soil used in this study. soil amended with arsenic† of varying concentrations. Property This soil Total soil As Fronds Roots pH (1:1 soil to water ratio) 7.3 Organic matter content, g kg 1 11.0 mg As kg 1 g plant 1 CEC†, cmolc kg 1 4.4 0.69 (control) 1.4b‡ 1.0a Total P, mg kg 1 645 50 2.9cd 1.0a Water-soluble P, mg kg 1 3.0 100 2.3c 1.2a Total As, mg kg 1 0.69 200 1.2ab 0.8a Water-soluble As, mg kg 1 0.02 500 0.5a 0.3a Sand, g kg 1 882 Silt, g kg 1 91 † Arsenic was added as K2KAsO4. Clay, g kg 1 27 ‡ All results are the means of four replicates. Values followed by the same letter in a column are not significantly different (p 0.05). † Cation exchange capacity of soil. whereas addition at 200 mg As kg 1 had little effect on separated into aboveground biomass, which was further sepabiomass yield. The addition of 500 mg As kg 1 reduced rated into young, mature, and old fronds based on their ages, aboveground biomass by 64%, which is a common sympand belowground (roots including rhizomes) biomass. Biotom of arsenic phytotoxicity (Kabata-Pendias and Penmass was measured on a dry-weight basis (after 3 d at 65 C). dias, 1991). Compared with typical plants, ladder brake Dry plant samples were ground to a fine powder before analysis, and soil samples were taken from the pots both before is thus much more tolerant to arsenic levels, up to 200 plant transfer and after harvest, air-dried, and sieved for anamg As kg 1 in a sandy soil. lytical use. Growth stimulation of ladder brake by arsenic was further confirmed in the experiment using varying arsenic compounds. At 50 mg As kg 1, all arsenic forms Determination of Arsenic in Plant and Soil increased the aboveground biomass of ladder brake by Plant (approximately 0.1000–0.5000 g) and soil (approxi7 to 24% except ferric arsenate (Table 3). This suggests mately 1.000 g) samples were weighed into a 120-mL Teflon that the stimulatory effect on growth of ladder brake pressure digestion vessel, mixed with 10 mL of concentrated observed at 50 and 100 mg As kg 1 as K2HAsO4 was trace-metals grade nitric acid, and digested using USEPA a result of arsenic, but not the accompanying cation, Method 3051 on a CEM (Matthews, NC) MDS-2000 microwave sample preparation system. After cooling, the sample potassium (Table 2). There is no evidence that arsenic solution was filtered through Whatman (Maidstone, UK) no. is essential for plant growth, although growth stimula42 filter paper and diluted to a volume of 100 mL. For soil tion at low arsenic concentrations in soils ( 25 mg As water-soluble arsenic, a 2-g soil sample was shaken with 20 kg 1 ) has been reported, especially for tolerant crops mL deionized water for 30 min. The suspension was then fil(Adriano, 1986). Unlike aboveground biomass, arsenic tered through Whatman no. 42 filter paper. Determination of additions at differing concentrations and forms had little aqueous arsenic concentration was performed using a graphite effect on root biomass (Tables 2 and 3). furnace atomic absorption spectrophotometer (PerkinElmer Though ladder brake is highly tolerant of arsenic, it SIMMA 6000). Results were expressed as a mean of four replisuffered arsenic toxicity at 500 mg As kg 1 as arsenate cates, with standard error. Analysis of variance was performed and 50 mg As kg 1 as sodium dimethylarsonate. Three using SAS software (SAS Institute, 1987). The Tukey procedure was used for mean separation. 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). RESULTS AND DISCUSSION These fronds had dark brown coloration and necrosis Biomass Production and Arsenic Toxicity at the leaf tips and margins, and plants were dead after 1 wk. In the treatment with 500 mg As kg 1, the sympFor a hyperaccumulating plant to be used successfully toms of arsenic toxicity appeared in the old fronds of in remediating arsenic-contaminated sites, it should ladder brake after 2 wk, but the plants survived throughhave sufficient biomass along with efficient extraction out the study. Addition of 50 mg As kg 1 as sodium of arsenic from the soil. The effects of arsenic concentrations on biomass and phytotoxicity for ladder brake Table 3. Dry biomass of ladder brake after 18 wk of growth in a were determined in this greenhouse experiment. The biosoil amended with arsenic in varying forms at 50 mg kg 1. mass of ladder brake was separated into aboveground Arsenic forms Fronds Roots (fronds) and belowground (roots including rhizomes) g plant 1 material. Arsenic is generally considered phytotoxic and Control (0.69 mg As kg 1 ) 10.7ab† 3.3a is expected to negatively affect plant growth (KabataFeAsO4 9.9a 3.1a Pendias and Pendias, 1991). Sheppard (1992) concluded CaMMA‡ 11.6bc 3.6a NaAsO2 12.5cd 2.8a that the mean arsenic toxicity threshold for plants is 40 NaMMA‡ 12.7cd 3.2a and 200 mg As kg 1 in sandy and clayey soils, respecAlAsO4 12.2cd 3.8a tively. The yields of barley and ryegrass were signifiNa2HAsO4 13.1d 2.9a K2HAsO4 13.1d 4.2a cantly reduced by addition of 50 mg As kg 1 to soil Ca3(AsO4 )2 13.3d 3.8a (Jiang and Singh, 1994). Ladder brake, however, be† All results are the means of four replicates. Values followed by the same haved differently (Table 2). Addition of arsenic at 50 or letter in a column are not significantly different (p 0.05). 100 mg As kg 1 significantly increased its aboveground ‡ CaMMA, calcium acid methanearsenate; NaMMA, sodium methylarsonic acid. biomass (107 and 64% greater than the control), 644 J. ENVIRON. QUAL., VOL. 31, MARCH–APRIL 2002 Table 4. Arsenic concentration in soil and ladder brake as affected by soil arsenic concentrations. Water-soluble As Fronds Total soil As Initial Final Roots Young Mature Old