v>EPA United States Environmental Protection Agency Environmental Monitoring and Support Laboratory P.O Box 15027 Las Vegas NV 89114 EPA-600/3-78-081 August 1978 Research and Development Ecological Research Series Plutonium Uptake by Plants Grown in Solution Culture ------- RESEARCH REPORTING SERIES Research reports of the Office of Research and Development. U.S. Environmental Protection Agency, have been grouped into nine series. These nine broad categories were established to facilitate further development and application of environmental technology. Elimination of traditional grouping was consciously planned to foster technology transfer and a maximim interface in related fields. The nine sereies are: 1. Environmental Health Effects Research 2. Environmental Protection Technology 3. Ecological Research 4. Environmental Monitoring 5. Socioeconomic Environmental Studies 6. Scientific and Technical Assessment Reports (STAR) 7. Interagency Energy—Environment Research and Development 8. "Special" Reports 9. Miscellaneous Reports This report has been assigned to the ECOLOGICAL RESEARCH series. This series describes research on the effects of pollution on humans.plant and animal species, and materials. Problems are assessed for their long-and short-term influences. Investiga- tions include formations, transport, and pathway studies to determine the fate of pollutants and their effects. This work provided the technical basis for setting standards to minimize undesirable changes in living organisms in the aquatic, terrestrial, and atmospheric environments. This document is available to the public through the National Technical Information Service, Springfield, Virginia 22161 ------- EPA-600/3-78-081 August 1978 PLUTONIUM UPTAKE BY PLANTS GROWN IN SOLUTION CULTURE By James C. McFarlane, Allan R. Batterman, and Kenneth W. Brown Monitoring Systems Research and Development Division Environmental Monitoring and Support Laboratory Las Vegas, Nevada 89114 U.S. ENVIRONMENTAL PROTECTION AGENCY OFFICE OF RESEARCH AND DEVELOPMENT ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY LAS VEGAS, NEVADA 89114 ------- DISCLAIMER This report has been reviewed by the Environmental Monitoring and Support Laboratory—Las Vegas, U.S. Environmental Protection Agency, and approved for publication. Mention of trade names or commercial products does not constitute endorsement or recommendation for use. ii ------- FOREWORD Protection of the environment requires effective regulatory actions which are based on sound technical and scientific information. This infor- mation must include the quantitative description and linking of pollutant sources, transport mechanisms, interactions, and resulting effects on man and his environment. Because o: the complexities involved, assessment of speci- fic pollutants in the environment requires a total systems approach which transcends the media of air, water, and land. The Environmental Monitoring and Support Laboratory — Las Vegas contributes to the formation and enhance- ment of a sound monitoring data base for exposure assessment through programs designed to: • develop and optimize systems and strategies for moni- toring pollutants and their impact on the environment • demonstrate new monitoring systems and technologies by applying them to fulfill special monitoring needs of the Agency's operating programs This report describes some of the characteristics of plutonium bonding to plant roots. The work was undertaken to learn about the plant-plutonium interactions which determine the contamination of plants, the possible use of plants as plutonium scavengers and their use as aquatic integrators for plutonium monitoring. This information will be of value to those concerned with monitoring plutonium in aquatic and marine environments. It will also be interesting to those studying the uptake and distribution of plutonium in plants. For further information on this subject contact the Pollutant Path- ways Branch of the Monitoring Systems Research and Development Division. r l George a. Morgan " Director Environmental Monitoring and Support Laboratory Las Vegas, Nevada iii ------- INTRODUCTION The distribution of plutonium (Pu) in terrestrial and aquatic environ- ments has resulted primarily from the fallout of nuclear debris. In 1968, Langham (1968) estimated the amount of Pu in the world environment from all sources was about 500 kilocuries; the primary source was fallout from nuclear atmospheric testing. Although most of the Pu has been distributed as world- wide fallout, the concentration resulting from this source is very low. Primary research emphasis has therefore been on the distribution of Pu from accidental releases and most work has emphasized terrestrial systems. Ac- cording to Noshkin (1972), "We know very little about the behavior of pluto- nium and relatively nothing of the behavior of other transuranics in the ocean and far less of the behavior and fate of these elements in fresh-water environments." The experiments presented here were conducted to provide information regarding Pu absorption and adsorption in plants rooted in solutions. Comparisons are made between terrestrial and aquatic root environments in regards to Pu uptake and deposition in foliar tissue. SUMMARY Plants grown in aquatic systems were shown to rapidly accumulate large amounts of plutonium, about 40% of which was removed by washing. Detergent removed debris, most of which consisted of particles larger than 0.8 microm- eters (ym). After removing a portion of the bound Pu by rinsing in DTPA, additional Pu was removed by a citric acid rinse. This implies that more than one type of Pu binding to plant roots exist, or that more than one chemical form of Pu are present. The high Pu concentration on plant roots did not facilitate uptake and translocation to aerial portions of the plant: discrimination ratios were similar to those typically found in terrestrial studies. Plants with filamentous root systems are suggested as possible scavengers for Pu in aquatic systems. It is recommended that pilot studies be conducted to evaluate the use of plants in accident cleanup technology. Consideration should be given to the use of plants as being the best samples to represent Pu contamination in marine and fresh water environments. ------- METHODS The uptake of Pu from solution cultures was measured for three different plants: Medicago, Lemna, and Typha. Medicago sativa (alfalfa) plants were grown in a glasshouse in hydroponic solution (Berry 1971); leaves and stems were cut when the plants were three-fourths flowering. Approximately 5 to 6 weeks were required between cuttings. At the time of each cutting, prior to Pu amendment, the roots were examined and dead roots were pruned. The plants were producing 30 to 40 grams (g) of oven-dried alfalfa per plant per cutting. The experiment consisted of two treatment groups. The acute treat- ments were accomplished by injecting 20 microcuries (yCi) of plutonium-238 nitrate [238Pu(N03)t+] in l.ON nitric acid (HN03) into each of four 7-liter containers filled with 1/2-strength Hoagland's nutrient solution. The solution was mixed, the pH was adjusted to 5.5 by addition of potassium hydroxide (KOH), and then the plants were returned to the solution. The chronic treatments were created by adding the same amount (20 yCi/pot) of Pu by continuous injection [0.25 milliliters per hour (ml/h)] over the entire 43-day growth period. The pH of the culture solution was tested weekly; it did not vary significantly from its original 5.5 and was therefore not ad- justed. The solution volume in each container was brought to a constant level daily by the addition of 1/4-strength nutrient solution. Samples were collected from the containers and from the Pu stock solution at 3-day inter- vals and analyzed for 238Pu content by liquid scintillation counting. At the termination of the chronic injection period, all plants were collected and leaves and roots analyzed for Pu. The roots were washed in a dilute non- ionic detergent and then rinsed in 0.01N HN03. Neutralized nitric acid digests of all tissues were analyzed for Pu concentration by liquid scintil- lation counting. Lemna minor (duckweed) was grown in 3 liters of 1/2-strength modified Hoagland's solution (Berry 1971) contained in each of eight 19-liter plastic containers. Constant agitation was accomplished by a magnetic stirring rod placed in the bottom of each container. Solution temperature was controlled to 25 ± 1 C by a heat-exchanging coil which circulated temperature-controlled water in each container from a temperature-control bath. The Hoagland's solution in each of four containers received 1.0 yCi of 238Pu in 1.0 ml of stock solution. The other four received a chronic exposure in which the same amount of 238Pu was added over a 30-day period. An approximation of Pu concentration in Lemna tissues was obtained by adding Lemna plants (approximately 0.5 g wet weight) to tared scintillation vials. Twelve ml of scintillation cocktail and 9 ml of H20 were then added. The solution was shaken, allowed to settle for 2 days, and counted on the liquid scintillation counter. An internal standard of 238Pu was then added to obtain the efficiency for each vial. Efficiencies determined in this ------- manner were 0.99 ± 0.02 and did not vary significantly from the standard prepared in distilled water. Although this analytical technique does not allow for the complete dissolution of the sample, all of the water and most of the organic components were dissolved into the scintillation cocktail. Typha angustifolia (cattail) was grown in the glasshouse in a recircu- lating hydroponic system. The system consisted of seven 19-liter polyethyl- ene containers connected with polyvinyl chloride (PVC) pipe 4.3 centimeters (cm) inside diameter, which were placed on stair-like supports. Each con- tainer was 2.5 cm lower than the preceding one and the overflow pipe emptied at the bottom of the adjacent container thus facilitating solution mixing. The solution was pumped from the seventh container to the first at a flow rate of 1.5 liters per minute. The amount of solution in the system was regulated by a float switch which actuated the pump in the solution reser- voir. By monitoring the solution level in the reservoir, the evapotranspi- ration rate of the system was determined. At harvest, there was approximately 18 kilograms of plant material distributed about equally between the six segments of this system. Roots made up 63% of the dry plant mass and leaves the remaining 37%. The root volume was estimated to be 2.4 liters per container. Plutonium nitrate (0.9 yCi) in 0.1N HN03 was injected into the hydro- ponic culture system at a constant rate over a 24-hour period. The delivery nozzle, a stainless steel 26-gauge tuberculin needle, yielded a nozzle velocity of 2.2 cm per minute (cm/min). This was rapid compared to ionic diffusion; therefore, no significant diffusional movement occurred up the nozzle. This precluded the possibility of Pu precipitation in the nozzle caused by a changing pH or contact with other ions present in the culture solution. Dispersion into the nutrient media was ensured by the continuous and rapid flow of culture solution past the nozzle (approximately 23 cm/min). Solution samples were periodically collected from each container and the solution analyzed for Pu concentration by liquid scintillation counting. A small plastic tube extending into the middle of each container was con- nected at the top to a large diameter hypodermic needle. At each sampling period, a syringe was attached to the needle and repeatedly filled and emptied to ensure a representative sample. Eight ml of solution were drawn into the syringe and dispensed directly into a scintillation vial containing 12 ml of scintillation cocktail (Lieberman and Moghissi, 1970). Using the total energy band of the liquid scintillation counter, backgrounds were on the order of 30 counts per minute (cpm); the efficiency ranged from 1.00 to 1.05. Radioactivity due to plutonium-238-contaminated plant debris in the culture solution was estimated by counting replicate solution samples before and after filtration through 0.8, 0.45, 0.22, and 0.01-micrometer (yra) filters. One month after the Pu addition, leaf and root samples were collected to evaluate the uptake and distribution of Pu in these tissues. Roots were cut off at the crown and placed into a cheesecloth basket. Leaves were divided into submerged leaves, old leaves, and new leaves. The leaf tissue was dried and weighed; subsamples were sent to Eberline Laboratories, Inc., ------- for 238PU analysis. The roots were washed in a series of four different solutions. This was accomplished by moving the cheesecloth container up arid down repeatedly in 4 liters of each solution for a period of 1 minute. The solutions were: distilled water in which 0.5 ml of a non-ionic detergent had been added, 0.01M DTPA (diethylenetriaminepentacetate), 0.01M citric acid, and 0.1M calcium chloride. Washings were repeated two or three times with fresh aliquots of the washing solution, and the roots were allowed to drain for 2 minutes between washes. The wash solutions were analyzed for 238Pu by liquid scintillation counting after filtering. The Pu activity on each filter was determined by counting the alpha particle associated with 238pu decay on a 2-inch zinc sulfide scintillation crystal attached to a 2-inch photomultiplier (Thrall et al., 1977). The calculated salt concentration of the nutrient culture was 0.008^1. This corresponded to an electrical conductivity (E.G.) of 3.7 millimhos (mmho). Electrical conductivity was monitored throughout the. growth of the Typha plants and kept constant by the addition of nutrient solution or distilled water. Make-up solution was added through the recirculating sys- tem and measurements of E.G. in each container showed that no nutrient gradient existed in the system. RESULTS Alfalfa plants growing in hydroponic solution were treated by adding 238Pu(N03)it to the root culture. The Pu concentration in the solution of the chronic exposure rose for the first 10 days to approximately 23 nCi/ml and remained at that concentration throughout the test period. The concen- tration of Pu in the acute exposure fell rapidly to an apparent equilibrium concentration of approximately 0.01 nCi/ml. Ten days after Pu exposure started and the plants were approximately two-thirds mature, the tops were cut and analyzed. Leaf concentrations were 0.10 ± 0.02 and 0.80 ± 0.40 nCi/g dry for the chronic and acute exposures, respectively. Thirty-three days later all plants were harvested and the leaf concentrations were then 28.0 ± 4.0 and 2.4 + 0.4 nCi/g dry for the chronic and acute exposures, respectively. In both 'the acute and chronic exposures more than 98% of the Pu in- jected was associated with the root tissue and less than 0.1% was found in the top of the plants. The ratio of Pu concentrations in the leaves to that in the roots after the final harvest was 3.0 x 10~"* and 1.7 x 10 "* for the chronic and acute exposures, respectively. Repeated .washings of the roots in weak detergent or in 0.01M HNOs released only very small amounts of Pu from the roots. This suggests that the Pu was bound to the root through chemical intermolecular forces other than surface adhesion. Following the acute injections of Pu into the Lemna containers the 238Pu concentration decreased exponentially over the first 10 days, reaching an apparent equilibrium concentration of approximately 10 pCi/ml. Pu ------- administered as a chronic injection rose to 25 pCi/ml during the first 5 days and remained at that concentration throughout the rest of the test. At the conclusion of the 33-day chronic exposure, the Lemna plants were collected from the surface of the treatment solutions with a fine-mesh plastic screen. The plants were placed into a cheesecloth envelope and washed sequentially in various solutions. The data in Table 1 show the concentration of Pu in the wash solutions. TABLE 1. PLUTONIUM-238 CONCENTRATION IN WASH SOLUTIONS IN WHICH LEMNA PLANTS WERE SEQUENTIALLY WASHED IN WATER 0.01M DTPA AND 0.01M CITRIC ACID WASHING SOLUTION H20 1 2 3 DTPA 1 2 CITRATE 1 2 ACUTE Pu ADDITION (pCi/ml) 14 ± 3 20 ± 3 18 ± 4 89 ± 3 27 ± 2 112 ± 3 44 ± 4 CHRONIC Pu ADDITION (pCi/ml) 3 ± 2 6 + 2 8 ± 2 90 ± 11 22 ± 2 48+8 27 ± 7 The initial washing in all cases was water containing a small amount of nonionic detergent. This was done in an effort to remove any particles adher- ing to the roots by surface tension. In both cases, the concentration found in the water was quite low compared to the concentration in the chelating agents. Filtration of these solutions through a 0.8-um filter removed only small amounts of the Pu from solution. The first washing with DTPA caused a large elution of Pu from the Lemna plants. The second washing released considerably smaller amounts of Pu; however, a subsequent washing with the citric acid solution released a large fraction of Pu from the tissues. While more Pu was removed from the acutely dosed plant tissues by the citrate washing, the pattern of Pu release was similar to that obtained in the chronically dosed plants. Comparison between washed and unwashed plants showed that 5 ± 3% of the original plutonium remained on the wnshed plants in ------- the acutely dosed treatment while 14 ± 6% remained on the chronically dosed plants. It is not presently apparent what caused the difference in binding. Prior to treating Typha. plants with Pu a similar system without plants was studied. Plutonium nitrate (50 yCi) was injected into the system at a constant rate over a 24-hour period. The peak concentration reached at the end of the injection was 425 pCi/ml. This represented 90% of the theoretical peak assuming that no precipitation or adsorption had occurred. Following a similar injection into the system containing Typha plants the peak concen- tration was only 52% of the theoretical. The difference was due to rapid adsorption of Pu to the plant roots. After the injection was completed the Pu concentration in both the Typha and control tests decreased with time finally reaching an apparent equilibrium of approximately 100 pCi/ml. Because of the roots, the initial decreasing rate of the ,Pu from the Typha test solution was significantly faster than in the control (Figure 1). However, after the first few days, the slopes were similar and the loss rates seemed to be independent of the presence of the Typha roots and submerged leaves. OCONTROL A TYPHA DAYS Figure 1. Plutonium loss from solution with and without Typha plants. f\ O Q ** J " "°Pu activities are expressed as a % of the peak concentration and day zero coincides with the termination of injection. 6 ------- Filtration of the control solution showed that about 90% of the Pu was associated with particles in the hydroponic solution. The results of the filter-filtrate tests are shown in Table 2. Most of the Pu labeled particles were larger than 0.8 ym and only about 10% of the Pu was either in solution or associated with particles smaller than 0.01 ym. In the Typha test Pu was not found in such abundance on free debris, but was mostly found on plant roots. TABLE 2. PLUTONIUM ACTIVITY ASSOCIATED WITH DIFFERENT-SIZED PARTICLES IN CULTURE TANKS WITHOUT PLANTS Plutonium-238 concentration remaining in solution after passing through various-sized filters. Activity expressed as percent of the unfiltered solution. Filter Pore Size No Filter 0.80 0.45 0.22 0.01 ym ym ym ym Serial Parallel Filtration Filtration 100 ± 5.0 100 30 ± 0.3 32 25 ± 0.3 21 11 ± 0.3 8 8 ± 1. ± 1. ± 1. ± 0. ± 0. 6 2 3 9 2 Control* 100 99 100 99 100 ± 1 ± 0 ± 0 ± 0 ± 0 .3 .6 .4 .0 .0 ± = 1 standard deviation. * = Control consisted of sterilized culture solution in which no algae growth occurred and no dust was allowed. At harvest the plants were separated into several subsamples for anal- ysis. The plutonium-238 concentration in each of these subsamples is shown in Table 3. The designation "young leaves" refers to those leaves newly emerged; they were approximately half the length of the most fully matured leaves. Five-centimeter samples were taken 15 cm from the terminal end of the leaf. The submerged leaf samples consisted of cross sections of all leaf tissue, approximately 5 cm long. Young roots were those newly developed and still white; all other root material was considered to be old. The evapotranspiration rate of the entire Typha system was determined throughout the Pu study. Water loss rates increased as the plants became larger. During the last week of the study, the water loss rates were 58 ± 5 milliliters per minute (ml/min) in the light and 8 ± 2 ml/min in the dark. Based on leaf area, transpirational water losses from young and old leaves were not significantly different. This was determined by the measure- ment of diffusive resistance to water vapor loss which was 16.8 ± 1.5 and 17.0 ± 20 seconds per cm on a clear day at noon for old and young leaves. ------- TABLE 3. PLUTONIUM-238 DISTRIBUTION IN VARIOUS TYPHA TISSUES 30 DAYS AFTER CONTAMINATING THE HYDROPONIC CULTURE WITH PLUTONIUM NITRATE Tissue Young leaves Old leaves Submerged leaves Young roots Old roots nCi/g* 1.5 ± 0.5 0.5 ± 0.2 22 ± 8 596 ± 199 2389 ± 714 * = Concentration is based on tissue dry weight. ± = 1 standard deviation. The difference between Pu concentration in young and old leaves (Table 3) is opposite in direction to that expected if deposition were a passive phenomenon associated with water movement. The young leaves developed sub- sequent to Pu treatment, and because of their size and age the total amount of water which passed through the xylem system was significantly smaller than through the older leaves. Also, the high Pu concentrations during and immediately after Pu injection were concurrent with old leaf transpiration, but occurred at a time when young leaves were embryonic. A calculation using the total dry weight of the leaves and the transpiration rates indi- cated that 60 ml of water per gram of dry leaf matter per day passed through the leaf. Considering that the Pu reached a peak concentration in solution of 4.5 nCi/ml and decreased to an average of 0.1 nCi/ml during the last 10 days of this experiment, it is obvious that Pu uptake was not coincident with the passive transport of water. Therefore, higher Pu concentrations found in the young leaves are thought to represent the continued presence of Pu throughout growth and development of leaf tissue. This also suggests that Pu accumulation in leaves may be associated with the movement of organ- ics transported in the phloem. Submerged Typha leaves are held together in a sheath and only the outer cuticles of the outer leaves are in actual con- tact with the water. Therefore, the high activity on the submerged leaves is thought to represent only surface contamination of the outer sheath and it is lower than the root activity because of the difference in the surface area to mass ratio. The young roots developed after the Pu addition and were therefore exposed to lower levels than the old roots which were present at the begin- ning of the experiment. This accounts for the difference in Pu content. The large variability associated with these means represents a lack of pre- cision in separating the two root types. ------- Samples were collected from the bottom of alternate pots with a large syringe and an agitator next to the bottom of the pot. This was done in an effort to ascertain differences in particle-size distribution in the debris found in the settlings. Aliquots of each solution were filtered through 0.8, 0.45, and 0.22-ym filters. The data from these tests are pre- sented in Figures 2 and 3. 15- 10- 020—1 015-1 010— NON FILTERED SOLUTION FILTERED SOLUTION 005 — \ \ \ \ \ \ \ 0 45u 0 22u POT SEQUENCE Figure 2. Plutonium contaminated sediments in alternate buckets. Confidence brackets represent ± standard deviation. Figure 3. Plutonium in solution after sediments were removed by fil- tration. This is an expansion and separation of the components which made up the line shown in Figure 2 (filtered solution). A pronounced gradient occurred in the Pu concentration of debris in the direction of solution flow. A similar gradient was not observed in the con- trol tests where no plants were present in the test solution. This gradient is thought to Indicate an attachment of Pu to root surfaces and subsequent sloughing off of organic debris which settled to the bottom of the pot. This pattern indicates a preferential and rapid accumulation of Pu on the ------- first plant surface available which occurred during the treatment. By com- paring activities of non-filtered and filtered solutions, it is obvious that most of the Pu in these samples was associated with particles. In Figure 3 the concentration of Pu remaining in solution after filtering through various-size pores is depicted. Again, the gradient decreased in the same direction as the solution flow. There was a greater abundance of parti- cles which passed the 0.8-ym and 0.45-um filters in the upstream samples. But apparently, all or most of the debris was larger than 0.22 ym,, and the activities passing through filters of this size probably represent soluble Pu. When harvested, the Typha roots were placed in a cheesecloth basket and serially rinsed in various solutions. Aliquots of these rinse solutions were filtered through 0.8-ym filters, and the filters and filtrates were both analyzed for 238pu concentration. After the first water wash, 94 ± 2% of the Pu in the rinse sample was collected on the filter. After the second water wash, this had decreased to 60% and in the third washing, the filter- able Pu was down to 2%. For the first two washings, summation of the activ- ity on the filter plus that in the filtrate did not correlate well with the solution counted directly. This is attributed to self-absorption of the alpha particle in the samples with a thick layer of debris on the filter. Extremely good correlation existed in the samples subsequent to the water wash. For instance, in the citric acid solution the comparison coefficient [(nCi on the filter plus nCi in the filtrate) * (nCi in unfiltered solution)] was 1.00 ± 0.05. Thus, scintillation counting is a reliable way of deter- mining Pu concentrations in solution without particulates. Plutonium concentration in the wash solutions after sequential washings are shown in Figures 4, 5, and 6. Each figure represents a different order of washing. The total amount pf the particulate Pu removed from these root samples in all of the washinga represented about 10% of the total Pu removed. Ninety-five percent of this paniculate matter was removed by the first rinsing in water. Because the filterable fraction represents physical dis- lodgement of plant debris and the quantity was small, except in the first detergent washings, only the PU concentration in the filtrate is presented in Figure 4, 5, and 6 as an indication of Pu release from plant surfaces. When the washing order was water, citric acid, DTPA (Figure 4), there was maximum release of Pu into the first chelating solution. The subsequent washings each contained smaller amounts of Pu and appear to represent an ordered progression heading toward complete elution. When the order was changed to water, DTPA, citric acid, the initial release of Pu into DTPA was similar to that in Figure 4 for citric acid. The second and third washings in DTPA decreased in Pu concentration more rapidly than when citric acid was used first. Subsequent washings in citric acid released additional amounts of Pu into the solution. Calcium chloride washes were added before elution with the two chelating agents in order to saturate the cation exchange sites with ca.lcium presuma- bly releasing Pu from these binding sites. The data in Figure 6 suggest that calcium chloride has little or no effect in removing additional Pu from the root tissue; subsequent washings with DTPA and citric acid gave 10 ------- 70—1 60— 50— o o SO— 2 fl- WATER CITRATE OTPA 121231 23 Figure 4. Plutonium in wash solu- tions in which Typha roots were sequentially rinsed. Washing order was: water, citric acid, DTPA. Concentration is expressed on the basis of nCi per gram of root being washed. SO- WATER DTPA CITRATE 121 231 23 Figure 5. Plutonium in wash solutions in which Typha roots were sequentially rinsed. Washing order was: water, DTPA, citric acid. Concentration is expressed on the basis of nCi per gram of root being washed. o o oe E40— 30— 20— WATER CaCI2 1 2 DPTA 1 2 3 CITRATE 1 2 3 Figure 6. Plutonium in wash solutions in which Typha roots were sequentially rinsed. Washing order was: water, CaCl2, DTPA, citric acid. Concentra- tion is expressed on the basis of nCi per gram of root being washed. 11 ------- results very similar to those experiments without CaCl2• At the termination of this experiment the plastic containers were sampled and analyzed for plutonium-238 content. A budget of Pu residence was then constructed and is presented in Table 4. The largest sink for Pu deposition was the plant roots. Approximately half of this contamination could be removed by washing in various solutions as previously detailed. The other segments of the system accounted for only small portions of the whole and are not considered to be important Pu sinks. TABLE 4. PLUTONIUM DISTRIBUTION IN TYPHA EXPERIMENT 30 DAYS AFTER INJECTING PLUTONIUM NITRATE INTO THE HYDROPONIC SOLUTION EXPRESSED IN % Roots 97.8 ± 1.8 Activity Removed from Roots by Washing 42.3 ± 13.7 Leaves Solution 1.2 ± 1.0 0.9 ± 0.5 Container Surfaces 1.0 ± = 1 standard deviation. DISCUSSION In this study, three plant species were grown in hydroponic solution contaminated with Pu. In all cases, Pu accumulated on the submerged plant tissues after acute or chronic exposure. A strong concentration gradient persisted between the submerged plant material and the solution. The ratio of root activity (expressed in nCi/g) to the solution activity (nCi/ml) was approximately 106 indicating a strong affinity of plant tissue to plutonium. When Hodge et al. (1974) studied the accumulation of fallout Pu on the Giant Brown algae off the coast of California they found that the concentra- tion on the blades of the kelp far exceeded the concentration found in the ocean. The older blades were approximately four times as contaminated as the young blades, indicating that the length of time of contact with the contaminated seawater was important in the accumulation of Pu by this organism. This accumulation of Pu on kelp was suggested by those authors as useful in monitoring Pu concentrations in seawater. From our studies, it is clear that submerged plant tissues in general accumulate Pu. There- fore, we suggest that submerged plant tissues in any aquatic ecosystem, including fresh water, will accumulate Pu and should be considered as an appropriate monitor for Pu contamination. This strong affinity of Pu to plants might even be utilized to scavenge Pu from contaminated waterways. Plants such as water hyacinth (Eichornia crassipes) which have an extensive filamentous root system should be considered as possible candidates for such a task. 12 ------- Absorption and translocation of Pu to the aerial portion of emergent aquatic species were observed to be quite low. In soil studies ratios of the Pu concentration in foliar plant tissue divided by the concentration in the rooting media (discrimination ratios) have generally been reported to_ vary between 10 3 to 10 6 with most of the numbers being approximately 10 ^. Using the concentration of Pu in the media (in this case the hydroponic cul- ture solution) and the Pu concentration in the Typha leaves to calculate this ratio, the values were 103 to 10s depending on leaf age. These are clearly different than the plant:soil ratios typically reported in the literature. This example points to the importance of specifying the experi- mental procedures carefully in association with discussions of supposedly comparable discrimination ratios. If discrimination ratios are recalculated in an unconventional wav by using the concentration of Pu on the root compared to the concentration in the aerial portions of the plants, ratios of 10 k are obtained. These are similar in magnitude to the more conventional calculation (activity of plants T activity in soil) found in many soil experiments. These data seem to imply that Pu attached to the surface of the root is generally unavailable for absorption and translocation by the plant, but is only as available to uptake as is Pu in soil. It also suggests that in terms of absorption into plant tissue, close physical contact with Pu may not be of any particular value in some cases. When Jacobson and Overstreet (1948) grew barley in an aerated clay slurry, they found a positive correlation between the amount of Pu absorbed by the roots and the percent which was translocated to the leaves. They concluded that Pu fixation on the clay and roots was primarily a surface phenomenon. Although a positive correlation between root and leaf content also existed in our experiments, the large discrimination against absorption and translocation makes us believe that uptake was primarily determined by something other than the proximity of Pu to the roots. In solution culture, as in soil, solubilization or complexing of Pu to a form suitable for absorption is probably associated with exudates from roots. When contaminated plant tissues were washed with weak detergent, no significant amount of soluble Pu was released into the solution. Since detergent would release particles held by the van der Waal forces of adhe- sion, these results imply that Pu was not attached to the submerged plant tissues by adhesion, but by some stronger bond. Roots typically slough off old cells and undoubtedly this is the primary source of Pu-labeled particu- late matter found in sediments and washings. When roots were washed in a calcium chloride solution, no detectable quantities of Pu were removed into the solution. Calcium ions are high in the lyotropic exchange series and used in high concentrations would replace most other cations at the exchange sites on the roots. Francis (1973) reviewed the current literature regarding Pu movement in soils and, from observations that inorganic salts did not cause desorption of Pu from soils, concluded that Pu may form an anion com- plex with soil. Cummings and Bankert (1971) showed a lack of correlation between plant uptake of Pu and the cation exchange capacity of the substrate soil. Taken together, those authors' conclusions lead to speculation that root complexing of Pu may be due to anion bonding. Alternately, it seems possible that Pu with a large positive charge may immediately develop bonds in the plant material more stable than those formed by Ca"1"4". Cation exchange 13 ------- sites on plant roots appear to be mainly carboxyl groups of the pectic sub- stances (Keller and Deuel, 1957). No drastic change in potential for cation exchange is evident upon the death of root cells, therefore Pu remains attached to those cells when they were sloughed off. When the alfalfa roots were washed in 0.01N nitric acid, no detectable Pu was released in this solution. Release of Pu from roots was obtained, however, in response to DTPA and to citric acid. Serial rinsing in DTPA released smaller and smaller amounts of Pu from the roots. However, subse- quent rinsing in citrate caused a release of additional Pu from the roots. This may represent two forms of binding of Pu to plant roots or perhaps the existence of more than one chemical form of Pu. REFERENCES Berry, W. L. (1971) Evaluation of phosphorous nutrient status in seedling lettuce. J. Amer. Soc. Hort. Sci. 93(3):341-344. Cummings, S. L., and L. Bankert. (1971) The uptake of cerium-155, prome- thium-147, and plutonium-238 by oat plants from soil. Radiolog. Health Data and Rep. 12:83-85. Francis, C. W. (1973) Plutonium mobility in soil and uptake in plants - A review. J. Environ. Qual. _2_(1) :67-70. Hodge, J. V. , F. L. Hoffman, and T. R. Folsom. (1974) Rapid accumulation of plutonium and polonium on giant brown algae. Health Physics 27:29-35. Jacobson, L., and R. Overstreet. (1948) The uptake by plants of plutonium and some products of nuclear fission absorbed on soil colloids. Soil Science .65_:129-134. Keller, P., and H. Deuel. (1957) Kationenaustauschkapazitat und pektigehalt von pflanzewurzeln. Zeilsckr. Pflanzenernahr., Dung., Bodenk. 79:119- 131. Langham, W. T. (1968) Bureau of Radiological Health, PHS, Seminar Paper Number 002. Lieberman, R., and A. A. Moghissi. (1970) Low-level counting by liquid scintillation II. Application of emulsion in tritium measurement. Int. J. Appl. Radiat. Isotopes 21:319-327. Noshkin, V. E. (1972) Ecological aspects of plutonium dissemination in aquatic environments. Health Physics 22:537-549. Thrall, J. E., W. Schause, and R. W. Coulter. (1977) A new zinc sulfide scintillation counter for alpha analysis. Unpublished data. 14 U.S. Government Printing Office: 1978 786-258/2013 ------- TECHNICAL REPORT DATA (Please read Instructions on the reverse before completing) 1. REPORT NO. EPA-600/3-78-081 2. 3. RECIPIENT'S ACCESSION NO. 4. TITLE AND SUBTITLE PLUTONIUM UPTAKE BY PLANTS GROWN IN SOLUTION CULTURE 5. REPORT DATE August 1978 6. PERFORMING ORGANIZATION CODE 7. AUTHOR(S) James C. McFarlane, Allan R. Batterman, and Kenneth W. Brown 8. PERFORMING ORGANIZATION REPORT NO. 9. PERFORMING ORGANIZATION NAME AND ADDRESS Environmental Monitoring and Support Laboratory Office of Research and Development U.S. Environmental Protection Agency Las Vegas, Nevada 89114 10. PROGRAM ELEMENT NO. 1FA628 11. CONTRACT/GRANT NO. 12. SPONSORING AGENCY NAME AND ADDRESS U.S. Environmental Protection Agency—Las Vegas, NV Office of Research and Development Environmental Monitoring and Support Laboratory Las Vegas, Nevada 89114 13. TYPE OF REPORT AND PERIOD COVERED Final 14. SPONSORING AGENCY CODE EPA/600/07 15. SUPPLEMENTARY NOTES 16. ABSTRACT Plants grown in aquatic systems were shown to rapidly accumulate large amounts of plutonium, about 40% of which was removed by washing. Detergent removed debris, most of which consisted of particles larger than 0.8 ym. After removing a portion of the bound Pu by rinsing in DTPA additional Pu was removed by a citric acid rinse. This implies that more than one type of Pu binding to plant roots exists or that more than one chemical form of Pu was present. The high Pu concentration on plant roots did not facilitate uptake and translocation to aerial portions of the plant: discrimination ratios were similar to those typically found in terrestrial studies. Plants with filamentous root systems are suggested as possible scavengers for Pu in aquatic systems. 17. KEY WORDS AND DOCUMENT ANALYSIS DESCRIPTORS b.IDENTIFIERS/OPEN ENDED TERMS COSATl l-ield/Group Pollution Radiobiology Plutonium 13B 06R 18B 18. DISTRIBUTION STATEMENT RELEASE TO PUBLIC 19. SECURITY CLASS (This Report) UNCLASSIFIED 21. NO. OF PAGES 20 20. SECURITY CLASS (Thispage) UNCLASSIFIED 22. PRICE A02 EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION is OBSOLETE ------- |