Environ. Sci. Technol. 2010, 44, 1112–1115
Effect of Mycorrhizal Fungi on the Phytoremediation of Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) PHILLIP L. THOMPSON* Department of Civil & Environmental Engineering, Seattle University, 901 12th Avenue - 525A ENGR, Seattle, Washington 98122 AUSTIN S. POLEBITSKI Department of Civil & Environmental Engineering, University of Washington, Seattle, Washington 98195
Received September 28, 2009. Revised manuscript received December 1, 2009. Accepted December 2, 2009.
The objective of this research was to decrease the bioaccumulationofRDX(hexahydro-1,3,5-trinitro-1,3,5-triazine)intheleaves of switchgrass (Panicum virgatum) and hybrid poplar trees (Populus deltoides × nigra, DN34) by exploiting the symbiotic relationship between these plants and mycorrhizal fungi. Prior to dosing with uniformly labeled 14C-RDX, plants were grown for three months to establish the symbiosis. Results showed that the presence of mycorrhizal fungi does not significantly decrease the concentration of RDX in leaf tissues for experimental periods of 30 days. Results also indicated that a radiolabeled volatile organic compound was emitted by both plant species. This is the first evidence that a whole plant can facilitate the transformation of RDX to a volatile organic chemical.
Introduction The explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is persistent in soil and groundwater throughout the world, over 60 years after its initial introduction into the environment. RDX may cause nervous system disorders, and the maximum contaminant level for drinking water has been estimated to be 0.1 mg/L (1). RDX has also been characterized as having both low water solubility (42 mg/L at 25 °C) and low volatility (2 × 10-11 dimensionless Henry’s constant) (2). For the past twenty years, researchers have studied the uptake of RDX by plants. Initial work focused on the possibility of food chain contamination by crops that had been exposed to RDX. Cataldo et al. (3) were the first to establish that RDX accumulated in the leaves of bush bean (Phaseolus vulgaris), wheat (Triticum aestivum L.), and blando brome (Bromus hordeaceus). In later work, RDX was shown to accumulate in the leaf tissues of a large percentage of the plant species tested at a contaminated site including black locust (Robinia pseudoacacia), red cedar (Juniperus virginiana), bromegrass (Bromus inermis), pigweed (Amaranthus sp.), reed canary grass (Phalaris arundinacea), Canadian goldenrod (Solidago canadesis), and ragweed (Ambrosia artemislilfolia) (4). Chen (5) also reported RDX accumulation of up to 440 mg/kg in the leaves of maize (Zea * Corresponding author e-mail:
[email protected]; phone: 206-296-5521. 1112
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mays), soybean (Glycien max.), sorghum (Sorghum Sudanese L.) and wheat (Triticum aestivum). In the mid-1990s, researchers and regulators began evaluating phytoremediation, the use of plants to restore RDX-contaminated soil and groundwater. Thompson et al. (6) studied the use of fast-growing hybrid poplar (Populus deltoides × nigra DN34) and found that about two-thirds of the accumlated RDX was translocated to leaf tissues and stored as RDX with very slow rates of degradation. Van Aken et al. (7) studied RDX-spiked aqueous solutions containing axenic poplar cell culture tissues (Populus deltoids × nigra DN34) and tissue extracts. These researchers confirmed that poplar tissues had the ability to transform RDX to MNX (hexahydro-1-nitroso-3,5-dinitro-1,3,5-triazine), DNX (hexahydro-1,3-dinitroso-5-nitro-1,3,5-triazine), formaldehyde, methanol, and carbon dioxide. Such transformations of RDX were proposed to be mediated by both the presence of light and intact plant cell structures. It was also suggested that the lack of 14C-CO2 detection in whole-plant studies was likely due to rapid carbon fixation into plant material. In related research, reed canary grass (Phalaris arundinacea) was grown in hydroponic solutions containing RDX (8). The leaves were excised, sealed in quartz cuvettes, and exposed to 2000 µmol/ m2-s of artificial light. Results indicated that photolysis could transform RDX to 4-nitro-2,4-diazabutanal and nitrous oxide. These researchers proposed that formaldehyde formation was likely based on its creation in irradiated solutions of aqueous RDX. Despite this ability for RDX to be transformed in chemically spiked laboratory systems, RDX persistence in the leaves of whole plants surveyed at munitions facilities has been documented (4) and is likely due to the continuous availability of RDX in the soil. Bioaccumulation has many undesirable ecological effects, such as the potential for food chain contamination or increased contaminant transport via leaf litter. The vast majority of phytoremediation research has neglected the effects of mycorrhizal fungi on experimental systems, yet over 98% of vascular plants have a natural symbiotic relationship with these fungi (9). Mycorrhizal fungi usually perform the function of providing nutrients to the plant that may be scarce within the soil such as phosphorus or nutritional metals such as zinc. For plants such as switchgrass (Panicum virgatum), mycorrhizal fungi may grow on the surface of the roots (endomycorrhizae), and with poplar trees (Populus sp.), mycorrhizal fungi species can penetrate the roots (ectomycorhizae). Schnabel and White examined the effect of mycorrhizal fungi on the uptake of a PCB (3-3′-44′-tetrachlorobiphenyl) (10) and aldrin (1,2,3,4,10,10-hexachloro-1,4,4a,5,8,8a-hexahydro-1,4:5,8-dimethanonaphthalene) (11) by poplar and willow trees and found positive correlations between chemical uptake and the presence of fungi. Due to the strong hydrophobicity of these compounds, translocation of the parent compounds and the formation of transformation products were insignificant. Mycorrhizal fungi are phylogenetically related to the white rot fungus (Phanerochaete chrysosporium) which has been shown to mineralize RDX (12, 13). The objective of this research was to measure RDX degradation by mycorrhizal fungi and subsequent bioaccumulation of RDX in hybrid poplar trees (Populus deltoids × nigra, DN34) and switchgrass (Panicum virgatum) by exploiting the naturally occurring symbiotic relationship between these plants and mycorrhizal fungi. 10.1021/es902950r
2010 American Chemical Society
Published on Web 12/29/2009
Experimental Section Chemicals. Uniformly labeled 14C-RDX having a purity of at least 99% and a specific activity of 44.1 mCi/mmole was purchased from Perkin-Elmer (Boston, MA). RDX was synthesized using fuming nitric acid and hexamethyldinitramine (Fisher Scientific, Houston, TX) (7). RDX purity was at least 99% pure when compared to standards purchased from Accustandard (New Haven, CT). All other chemicals were of reagent grade or better. Plant Materials. Poplar cuttings (Populus deltoides × nigra DN34) were purchased from Hybridpoplars.com (Grandview, WA) and switchgrass seeds (Panicum virgatum, Var. Alamo) were purchased from Pennington Seed, Inc. (Madison, GA). Poplar cuttings were trimmed to 24 cm and planted in 10cm deep and 15-cm top-diameter plastic pots containing 2200 g of sterilized play sand (Home Depot, Vinings, GA). Sand was used instead of soil because it was important to have low nutrient conditions for the fungi to grow. In addition, using sand helped to maximize uptake of RDX and minimize sorption competition with soil. Switchgrass seeds were planted in identical pots (25 g per pot) at a depth of approximately 1 cm. All plants were watered with an amended 50% Hoagland’s nutrient medium (14). To induce fungi growth, the nitrogen and phosphorus levels in the nutrient medium were lowered to 50 and 60 mg/L, respectively. Poplar trees were inoculated with BioGROW Blend (Mycorrhizal Applications, Grants Pass, OR) which is a proprietary liquid blend of ecotmycorrhizal fungi including Pisolithus tinctorius, Rhizopogon villosullus, Rhizopogon luteolus, Rhizopogon amylopogon, Rhizopogon fulvigleba, Scleroderma cepa, and Scleroderma citrini. The liquid inoculum was diluted to 2.5 mL per L of nutrient medium and applied to poplar cuttings daily for a period of 3 months prior to exposure to RDX. Switchgrass seeds were inoculated with BioGROW Micronized Endo (Mycorrhizal Applications) which is a proprietary powder of endomycorrhizal fungi containing Glomus intraradices, Glomus mosseae, and Glomus aggregatum spores. Switchgrass plants were grown for three months prior to dosing with RDX. Mycorrhizal colonization was determined by sending representative blind root samples to Mycorrhizal Applications. Ectomycorrhizae colonization percentages were determined by washing and removing adhering particles using forceps under a stereo dissecting microscope. Roots were subsampled in upper, middle, and lower positions. Rootlets were severed and mounted on glass slides in both lactophenol cotton blue and toludine blue and were examined by compound microscope. Rootlets were judged to be mycorrhizal if mantled by hyphae and Hartig nets typical of ectomycorrhizae. Endomycorrhizal colonization was determined using a grid intercept method where roots were first placed in capsules containing 10% KOH solution and incubated at 30 °C for 36 h. The capsules were then rinsed with three complete changes of tap water, incubated in 1% HCl for 30 min, and then transferred to a water bath of Tri Pan blue for 36 h. Roots from each capsule were then rinsed, chopped, and examined for endomycorrhizal colonization in a graduated Petri dish at 100 gridline crossings using a dissecting microscope. Environmental Conditions. After a 3-month growing period, plants were watered with a 10 microcurie spike of [U-14C]-RDX and then transferred to identical, 0.57 m3 (20 ft3) growing chambers. The chambers were constructed of 0.953-cm (3/8-in) Plexiglas (Laird Plastics, Seattle, WA) with dimensions of 91.4 cm (L), 55.9 cm (W), and 111.8 cm (H) (36 in. × 22 in. × 44 in.). Each chamber had a 29.2-cm (11.5in.) diameter access door and twelve 1-cm ports for air exchange. Two oil-free piston vacuum pumps (Gast Manu-
TABLE 1. Plants Inoculated with Mycorrhizal Fungi Had Significantly (p < 0.05) Higher Percentages of Root Colonization than Controls treatment
poplar mycorrhizal infection, %
switchgrass mycorrhizal infection, %
controls (n ) 7) fungi (n ) 7)
24 ( 12 56 ( 13
12 ( 5 34 ( 5
facturing Corporation, Benton Harbor, MI) provided 6 air exchanges per hour for each chamber. Each sample port was exhausted to a 2,4-dinitrophenylhydrazine (DNPH) cartridge followed in series by a flask containing 1 M sodium hydroxide for the capture of 14C-CO2. The DNPH cartridge was intended to trap formaldehyde (8). Daily photoperiods were 16 h long, and the average light intensity provided by 250-W metal halide bulbs (General Electric model MVR250/U, Cleveland, OH) was measured to be approximately 350 µmol/m2-s. Transpired water was replaced daily with nutrient medium containing 40 mg/L RDX for 30 days. At the end of each experiment, plant materials were dried, extracted, and analyzed for 14C using bio-oxidizer combustion and liquid scintillation counting. Analysis. After the 30-day experimental period, plants were removed from their pots and separated by tissue type (e.g., roots, stems, leaves). Plant leaves were subdivided for RDX extraction and [U-14C]-RDX mass balance analysis. Extraction was performed by first collecting all leaves from a plant and cutting them into uniform pieces. Next, 10 g fresh weight of this homogenized leaf sample was crushed with a mortar and pestle in the presence of liquid nitrogen. Ground tissues were placed in 30 mL of 1 N hydrochloric acid contained in 40-mL centrifuge tubes (5). The tubes were sonicated in a water bath at 25 °C for 24 h. Extracts were diluted 1:1 with acetonitrile and analyzed by HPLC. RDX was measured using an Agilent Technologies (Santa Clara, CA) model 1100 high-performance liquid chromatograph (HPLC) at an absorbance of 254 nm. A Supelco (Bellefonte, PA) LC-8 column was used with a mobile phase consisting of 20% 2-propanol and 80% HPLC-grade water flowing at 2 mL per min. RDX standards of 0.5, 1, 5, 10, 15, and 20 mg/L were used for calibration resulting in average correlation coefficients (R2) of 0.99. Mass balances were determined by combusting each type of plant tissue with an RJ Harvey OX-600 bio-oxidizer (RJ Harvey Instrument Corp, Tappan, NY). This was done by drying plant tissues for 72 h in an oven at 30 °C, after which up to 1 g of each plant tissue was combusted. Radioactivity was measured with a Beckman LS6500 (Fullerton, CA) using Bioscint scintillation cocktail (National Diagnostics, Atlanta, GA). Statistical differences between treatments were calculated using the Student’s two-tailed t test for significance.
Results and Discussion All plants were found to have some degree of mycorrhizal infection (Table 1), however there were significantly higher (p < 0.05) percentages of infection for plants that had been inoculated. While the infection of control plants could be interpreted as false positives, all data were used for this analysis. Others have used fungicides to avoid mycorrhizal infection of control plants (10) but this approach was not used in the present study due to concern for the effects of the fungicides on the uptake of RDX. The presence of mycorrhizal fungi significantly increased the biomass of poplar (p < 0.05) (Figure 1, Table 2), but there was no significant effect on the growth of switchgrass (Table 2). Mycorrhizal fungi did not significantly affect the leaf concentration of RDX in either plant species (Table 2). This VOL. 44, NO. 3, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY
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FIGURE 1. Poplars grown with mycorrhizal fungi (orange) had significantly more biomass than those without (green).
TABLE 2. Mycorrhizal Fungi Significantly Increased Poplar Height and Biomass but Did Not Decrease RDX Concentrations in Leaf Tissues for Either Poplar or Switchgrass treatment
height, cm
dry biomass, g
leaf RDX, mg/kg DW
poplar controls (n ) 9) fungi (n ) 9)
38.2 ( 2.3* 54.3 ( 10.3*
21.5 ( 2.3* 33.1 ( 7.9*
1233 ( 122 1144 ( 128
switchgrass controls (n ) 12) fungi (n ) 12)
76.5 ( 4.5 78.0 ( 9.5
35.1 ( 4.6 36.6 ( 3.0
956 ( 188 1086 ( 167
* Iindicates significantly different at p < 0.05.
FIGURE 2. Fate of 14C-related-RDX uptake by switchgrass was statistically the same for plants with and without mycorrhizal fungi. was either due to the recalcitrance of RDX under aerobic conditions or insufficient contact time between the fungi and RDX during uptake. RDX concentrations in the leaf tissues averaged approximately 1000 mg/kg dry weight which is very similar to values reported for other plants (4–6, 15). The leaf concentrations of RDX in poplar were also comparable to those previously reported (6). On average, switchgrass and poplar were able to extract approximately 40 mg of RDX per plant during the 30-day chemical exposure. 14 C-RDX mass balances for switchgrass averaged 95.0 ( 23.0% with a range of 65-119%. 14C-RDX mass balances for poplar were not completed due to a problem with the biological oxidizer; however the fate of 14C-RDX in poplar was not expected to be different from previously reported results (6). Figure 2 illustrates that approximately 60% of the applied label was translocated to leaf tissues which is 1114
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consistent with studies conducted with other plant species (3, 5, 6). The presence of fungi had no significant effect on the mass of radiolabel remaining in the sand, however it is possible that some portion of the radiolabel was transformed within the sand. Since the purpose of this study was to focus on the concentration of RDX in leaf tissues, future experiments are needed to ascertain the effect of mycorrhizal fungi on the chemical fate of RDX in soil. For the first time, these experiments revealed that plant systems could facilitate the transformation of RDX into a volatile organic chemical. This was confirmed by attaching DNPH organic resin traps to the air-outflow ports on the plant grow chambers. The traps were placed on chambers that contained either switchgrass or poplar and on control chambers that had pots with plants which had above-ground tissues excised after dosing with [14C]-RDX. The analysis of the column extracts with liquid scintillation counting showed the presence of radiolabel in the plant chambers was ten times the background of the control chambers. Since no radiolabel was detected from the control chamber, the volatile chemical must have been produced via the intact plant and not from root-fungi transformations. This volatile fraction represented approximately 0.25% of the total radiolabel uptaken, and the identity of the volatile chemical is unknown. However, since others have trapped formaldehyde with DNPH columns during photolysis experiments with canary grass leaves containing RDX (8), it is possible that the 14C detected in our DNPH columns was also formaldehyde that was generated by the same photolytic mechanism. Further research is required to ascertain the identity of this compound(s). No 14C was detected in the NaOH traps so it was assumed that if 14C-CO2 had been produced, it was quickly fixed by the plants. The results from these studies demonstrated that mycorrhizal fungi did not significantly reduce the accumulation of RDX in leaf tissues. If RDX bioaccumulation is to be controlled, another approach may involve the use of plants that have been genetically engineered to express RDXdegrading genes that have been isolated from bacteria or fungi. Attempts to express an RDX-degrading cytochrome P450 from Rhodococcus rhodochrous in thale cress plants (Arabidopsis thaliana) have resulted in 2-fold decreases in leaf tissue concentrations of RDX relative to wild-type plants, however the engineered plants still had leaf RDX levels as high as 500 mg/kg dry weight (15). If phytoremediation is used for RDX contamination, careful management of leaf
litter and of herbivorous animal populations will be required. Phytoremediation of RDX contaminated soils may also be limited by the rate of cleanup to acceptable regulatory standards which has been estimated to take decades (16).
Acknowledgments This work was supported by National Science Foundation Award BES-0216066. Any opinions, findings, and conclusions or recommendations expressed in this material are those of the author(s) and do not necessarily reflect the views of the National Science Foundation. We thank Dr. Craig Just for assistance with RDX synthesis.
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