PILOT STUDIES COMPARING ABOVE AND BELOW-GROUND EFFECTS OF RADIONUCLIDE ACCUMULATION IN SELECTED SPECIES

PILOT STUDIES COMPARING ABOVE AND BELOW-GROUND EFFECTS OF RADIONUCLIDE ACCUMULATION IN SELECTED SPECIES

James A. Entry^a, Lidia S. Watrud^b, Paul Rygiewicz^b, and William H. Emmingham^c

^aDepartment of Agronomy and Soils, 202 Funchess Hall, Auburn University, AL 36849-5412; ^bTerrestrial Ecology Branch, US Environmental Protection Agency, Environmental Research Laboratory, 200 SW 35th St., Corvallis, OR 97333; and ^cDepartment of Forest Science, College of Forestry, Oregon State University, Corvallis, OR 97331

SUMMARY

Ecosystems throughout the world have been contaminated with radionuclides by aboveground nuclear testing, nuclear reactor accidents and nuclear power generation. Radioisotopes characteristic of nuclear fission, such as ¹³⁷Cs and ⁹⁰Sr, are released into the environment and become more concentrated as they move up the food chain often becoming human health hazards. Natural environmental processes will redistribute long lived radionuclides that are released into the environment among soil, plants and wildlife. Numerous studies have shown that ¹³⁷Cs and ⁹⁰Sr are not removed from the top 0.4 meters of soil even under high rainfall and migration rate from the top few centimeters of soil is slow. The top 0.4 meters is the area of the soil where plant roots are actively accumulating large amount of elements from the soil. Removal of these radionuclides from contaminated soils by plants would provide a reliable and economical method of remediation.

We tested the ability of fast growing perrenial plants to accumulate and remove ¹³⁷Cs and ⁹⁰Sr from contaminated growth medium in growth chamber or greenhouse condtions. Ponderosa pine seedlings accumulated 6.3 % and 1.5 % of ¹³⁷Cs and ⁹⁰Sr, respectively, while Monterey pine seedlings accumulated 4.5 % and 8.3 % of ¹³⁷Cs and ⁹⁰Sr from the contaminated growth medium in 30 days. Ponderosa and Monterey pine seedlings inoculated with ectomycorrhizal fungi accumulated substantially more ¹³⁷Cs and ⁹⁰Sr from the growth medium. Eucalyptus seedlings removed 31.0 % of the ¹³⁷Cs and 11.3 % of the ⁹⁰Sr in a contaminated growth medium in 30 days. Alamo switchgrass accumulated 43.6 % and 36.2 % of the total amount of ⁹⁰Sr and ¹³⁷Cs added to growth medium after 5 monthly harvests. Duration of exposure correlated curvilinearly with accumulation of both ⁹⁰Sr and ¹³⁷Cs by plants in all experiments (r² ranged from 0.75 to 0.98) As concentration of both ¹³⁷Cs and ⁹⁰Sr in the growth medium increased, accumulation of both radionuclides increased and correlated curvilinearly in all plant species (r²ranged from 0.87 to 0.99). Ponderosa and Monterey pine bioconcentration ratios for ⁹⁰Sr averaged 28.0 and 27.0 without ectomycorrhizal associations; with ectomycorrhizal associations Ponderosa pine ⁹⁰Sr bioconcentration ratios ranged from 102.0 to 144.0 and Monterey pine ⁹⁰Sr bioconcentration ratios ranged from 88.0 to 133.0. Without mycorrhizal associations Eucalyptus seedling bioconcentration ratios for ¹³⁷Cs and ⁹⁰Sr averaged 54.0 and 13.0 respectively. Alamo switchgrass bioconcentration ratios Of ¹³⁷Cs averaged from 35.3 in July to 3.2 in October; ⁹⁰Sr bioconcentration ratios averaged from 27.0 in June to 19.9 in September.

Key words: Plant accumulation, radionuclide contamination, ¹³⁷Cs, ⁹⁰Sr

INTRODUCTION

Radionuclide contamination of ecosystems throughout the world has resulted from nuclear bombing (Mahara, 1993), aboveground nuclear testing (Robison and Stone, 1992) and nuclear reactor accidents (Paasikallo, 1984; Nifontova, 1989). Upon release into the environment, ¹³⁷Cs and ⁹⁰Sr become more concentrated as they move up the food chain and often exceed human health standards (FAO, 1964; Garner, 1970; Abbott and Rood, 1994). Even under prolonged high rainfall, appreciable quantities of ¹³⁷Cs and ⁹⁰Sr are unlikely to be leached from soil (Kirk and Staunton, 1989). Several studies have shown that plants can sequester ¹³⁷C s and ⁹⁰Sr from soil and accumulate these radioisotopes in their tissue (FAO, 1962; Pinder et al., 1984; Salt et al., 1992). Certain plant species could be good choices for removing ¹³⁷Cs and ⁹⁰Sr from contaminated soil because of their rapid growth rates, the ability to sequester large amounts of radionuclides from soil, nonpalatability to herbivores, and ability to survive and grow in a wide range of geographic conditions (Entry et al., 1995).

Ponderosa pine (Pinus ponderosa), Monterey pine (Pinus radiata) and Eucalyptus (Eucalyptus tereticornis) are fast growing tree species that are adaptable to a wide range of geographic conditions. Switchgrass (Panicum virgatum L.), is a perennial C⁴ species native to North America that has produced the highest biomass yields of any grass tested. Previous research has identified the Alamo variety of switchgrass as a prime candidate for biomass production because of its unusually fast growth rate, ability to grow in soils with low nitrogen concentrations, and its low production costs (Sladden et al., 1991). Plants would remove radioisotopes from the soil and immobilize them in plant tissue. Once the grass is established, harvesting operations could periodically remove above-ground portions and radioisotopes could be recovered and concentrated by burning plant tissue at from temperatures from 500 to 600C or by microbial decomposition (Entry et al., 1995). Radionuclides would be concentrated in ash and could be properly disposed. We tested the ability of Eucalyptus, Monterey pine, Ponderosa pine, and Alamo switchgrass to remove ¹³⁷Cs and ⁹⁰Sr from plant growth media soil to determine the possibility of using these plants to remediate contaminated soils.

MATERIALS AND METHODS

The above plants were tested for their ability to accumulate and remove ¹³⁷Cs and ⁹⁰Sr from peat or sand in growth chamber or greenhouse conditions. In the first study we tested the ability of Monterey and Ponderosa pines to remove ¹³⁷Cs and ⁹⁰Sr from a peat growth medium. In the second study we tested the ability of Eucalyptus seedlings to remove ¹³⁷Cs and ⁹⁰Sr from a peat-vermiculite growth medium. In the third study we tested the ability of Alamo switchgrass to remove ¹³⁷Cs and ⁹⁰Sr from a sand growth medium in five separate cuttings. In each study, three separate experiments were conducted. All experiments were arranged in a completely randomized design. The first experiment tested the efficacy of the plant to accumulate ¹³⁷Cs and ⁹⁰Sr and remove these radionuclides from a contaminated peat growing medium. The second experiment was to determine the duration of plant growth in peat on accumulation of ¹³⁷Cs and ⁹⁰Sr. The third experiment was to determine how radionuclide concentrations in peat affect plant accumulation of ¹³⁷Cs and ⁹⁰Sr (Entry et al., 1993; 1994;

Entry and Emmingham, 1995).

Seeds were surface sterilized, then placed on Melin-Norkrans agar and incubated at 22C for 2 weeks to detect any microorganisms growing on the seed coat. Non-contaminated , pre-germinated seeds were then placed in a 3.0 cm diameter x 30.0 cm long test tubes containing , 165 ml of 1: 1 (v:v) spaghum peat moss: perlite mixtures. Alamo Switchgrass was grown in acid washed sand. Forty five ml of nutrient solution (Ingestad, 1979) was added to each test tube. The tubes with media were capped with 50 ml beakers and autoclaved for 60 min. After tubes cooled for 48 hr, a sterilized, pre-germinated seed was placed in each one. Seedlings were grown in the tube for 3 months in a growth chamber maintained at 22C. In studies 1 and 2, tree seedlings were grown in a growth chamber that had 180 ± 20 µmol s^-1 m^-2PAR with 16 hr photoperiods (Entry et al., 1993; 1994; Entry and Emmingham, 1995). Tree seedlings were not watered until radionuclides were added. In the third study Alamo switchgrass was grown in a temperature controlled greenhouse that had approximately 400-700 ± 50 µmol s^-1 m^-2 PAR with 14 -16 hr photoperiods. Grass was watered twice weekly with 100 ml of distilled deionized water. After 3 months either 2,717 Bq ¹³⁷Cs or 5798 Bq ⁹⁰Sr was added to each test tube in 1 ml water and dispersed throughout the growth medium by an additional 5 ml distilled delonized water. Seedlings were grown for an additional 30 days. At harvest, seedlings were removed from the test tubes and separated into root and shoot tissues. Root tissue exposed to ¹³⁷Cs was washed in deionized distilled water and then 1.0 M KCl; root tissue exposed to ⁹⁰Sr was washed in deionized distilled water and then 1.0 M CaCl₂ to remove radionuclides adhering to root surfaces. All roots were then rewashed twice in deionized distilled water. (Entry et al., 1993; 1994; Entry and Emmingham, 1995).

Radionuclide Counts. All root and shoot tissue was dried at 80C for 48 hr and weighed. All roots and shoots were counted separately. Mean values for replicate counts for both radioisotopes were compared with known activity standards to determine dpm/cpm ratios. Tissues containing ¹³⁷Cs were placed in a 10 ml plastic counting vial and counted in a 7.62 x 7.62 cm Val (TL) well detector coupled with a single analyzer adjusted to record counts at the 661.65 keV gamma energy region. Background ¹³⁷Cs was determined by averaging the results of six 100 minute counts of blank vials obtained by subtracting this background value from the sample values. The lower limit of detection was calculated at the 95 % confidence level from the system background described above. Tissues containing ⁹⁰Sr were placed in 20 ml scintillation vials and ashed for 6 h at 525C ± 5C. Residue was resuspended in 1 ml 3 M HCl and 17 ml of Biosafe II scintillation cocktail (Research Products International Corp., Mt Prospect, IL) was added. Vials were then shaken and stored in the dark for 48 h.

Radioisotope counts were taken for 10 min at 1.0 meV on a Beckman LS 7000 autoscintillation counter. (Entry et al., 1993; 1994; Entry and Emmingham, 1995).

Calculations. Calculations were performed as follows: the amount of radionuclide removed was calculated by multiplying the Bq of radionuclide g^-1 in five separate 1,000 g samples from each container each month of the above-ground plant tissue by the total weight of harvested tissue. Percentage uptake of radioisotope from soil was determined by dividing the amount of radioisotope measured in seedling tissue by the amount of radioisotope placed in each container or test tube, and then multiplying by 100. The bioconcentration ratio was calculated as Bq radioisotope g^-1 in plant tissue divided by Bq radioisotope in peat or sand growth medium.

Statistical Analysis. All data were normally distributed and subjected to a one-way analysis of variance (ANOVA; Kirk, 1982). Differences among treatment means were considered to be significant at Wë3ÀFðVòÐufÇFð 0.05 using the Least Square Means test.

RESULTS

In the first experiment, Ponderosa pine seedlings accumulated 6.3 % and 1.5 % of ¹³⁷Cs and ⁹⁰Sr, respectively, while Monterey pine seedlings accumulated 4.5 % and 8.3 % of ¹³⁷Cs and ⁹⁰Sr ftom the contaminated growth medium in 30 days. In the second experiment Ponderosa and Monterey pine seedlings accumulated significantly more ¹³⁷Cs and ⁹⁰Sr from the growth medium when inoculated with ectomycorrhizal fungi. In the third experiment, Eucalyptus seedlings removed 31.0 % of the ¹³⁷Cs and 11.3 % of the ⁹⁰Sr in a contaminated growth medium in 30 days. Alamo switchgrass accumulated 43.6% and 36.2 % of the total amount of ⁹⁰Sr and ¹³⁷Cs added to the sand growth medium after 5 monthly harvests.

Exposure to ¹³⁷Cs and ⁹⁰Sr had no apparent effect on shoot biomass production by Ponderosa and Monterey pine seedlings; both mycorrhizal and nom-nycorrhizal seedlings allocated 61 % and 72 % percent of their total biomass to shoot production. After I month Ponderosa pine seedlings stored 55 % of the ¹³⁷Cs and 69 % of the ⁹⁰Sr in shoot tissues while Monterey pine seedlings stored 52 % of the ¹³⁷Cs and 88 % of the ⁹⁰Sr in shoot tissues. Eucalyptus seedlings allocated 86 % of the biomass to shoot tissues. Eucalyptus seedlings stored 50 % of the ¹³⁷Cs and 70 % of the ⁹⁰Sr in shoot tissues. The above and below-ground portion of Ponderosa pine seedlings, Monterey pine seedlings, Eucalyptus seedlings, and Alamo switchgrass exposed to ¹³⁷Cs or ⁹⁰Sr did not differ with regard to shoot or root biomass over the course of the experiments. After 5 months, Alamo switchgrass root biomass was almost twice as much as the above-ground biomass in plants receiving ¹³⁷Cs or ⁹⁰Sr treatment. Concentration of both ¹³⁷Cs and ⁹⁰Sr found in root tissue was substantially less than that found

in shoot tissues. After the first two harvests concentration of ¹³⁷Cs in Alamo switchgrass tissue and the amount of ¹³⁷ Cs removed from the growth medium declined with each successive harvest. Alamo switchgrass receiving ⁹⁰Sr had a higher concentration of ⁹⁰Sr in above-ground tissue and more ⁹⁰Sr removed from growth medium at the first two harvests than the last three harvests. Duration of exposure correlated curvilinearly with accumulation of both ⁹⁰Sr and ¹³⁷Cs by plants in all experiments (r² ranged from 0.75 to 0.98) As concentration of both ¹³⁷Cs and ⁹⁰Sr in the growth medium increased, accumulation of both radionuclides increased and correlated curvilinearly in all plant species (r²ranged from 0.87 to 0. 99).

Ponderosa and Monterey pine bioconcentration ratios for ⁹⁰Sr averaged 28.0 and 27.0 without ectomycorrhizal associations; with ectomycorrhizal associations Ponderosa pine ⁹⁰Sr bioconcentration ratios ranged from 102.0 to 144.0 and Monterey pine ⁹⁰Sr bioconcentration ratios ranged from 88.0 to 133.0. Without mycorrhizal associations Eucalyptus seedling bioconcentration ratios for ¹³⁷Cs and ⁹⁰Sr averaged 54.0 and 13.0 respectively. Alamo switchgrass bioconcentration ratios of ¹³⁷Cs averaged from 35.3 in July to 3.2 in October; ⁹⁰Sr bioconcentration ratios averaged from 27.0 in June to 19.9 in September.

DISCUSSION

Alamo switchgrass, Ponderosa pine, Monterey pine and Eucalyptus seedlings removed large substantial amounts of ¹³⁷Cs and ⁹⁰Sr in short periods of time in laboratory tests. These results support the concept that selected plant species and associated mycorrhizal fungi may be useful in removing radionuclides from contaminated soils. Radionuclides in the aboveground portions can then be harvested and processed for recovery. To ensure the environmental safety of using non-indigenous, genetically selected or modified plants and associated microflora such as mycorrhizal fungi in habitat restoration efforts, several types of ecological effects studies are proposed. Initially, these studies will examine the effects of radionuclide uptake in plants on i. plant productivity and nutrient status, and ii. changes in the establishment and functioning of symbiotic relationships with mycorrhizal fungi and the potential for phytotoxicity to the accumulating plant species. Subsequent studies to address toxicity to pollinating and herbivorous non-target organisms are also suggested.

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