TECHNICAL REPORT DATA

(Please read Instructions on the reverse before completing)

74, REPORT NO, 2.
EPA-600/3-78-081

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

lg. 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

71, 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

citric acid rinse.

to aerial portions of the plant:
typically found in terrestrial studies.

Plants grown in aquatic systems were shown to rapidly accumulate large amounts
of plutonium, about 40% of which was removed by washing.
debris, most of which consisted of particles larger than 0.8 um.
a portion of the bound Pu by rinsing in DTPA additional Pu was removed by a
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
discrimination ratios were similar to those
Plants with filamentous root systems are
suggested as possible scavengers for Pu in aquatic systems.

Detergent removed
After removing

ia KEY WORDS AND DOCUMENT ANALYSIS

ia. DESCRIPTORS Ib. IDENTIFIERS/OPEN ENDED TERMS |e. COSATI Field/Group
Pollution 13B
Radiobiology O6R
Plutonium 18B

8. DISTRIBUTION STATEMENT.

19. SECURITY CLASS (This Report)

21. NO. OF PAGES:

UNCLASSIFIED 20
RELEASE TO PUBLIC 20. SECURITY CLASS (This page) 22. PRICE
UNCLASSIFIED

EPA Form 2220-1 (Rev. 4-77) PREVIOUS EDITION IS OBSOLETE



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.

20

wf \

aCi/mt

2 ACi/mi

10—4

NON FILTERED
SOLUTION

>—___—_...
0.05—4 —

FILTERED SOLUTION

T T qT
H 4 Hi ? 1 3 5 1
POT SEQUENCE
Figure 2. Plutonium contaminated Figure 3. Plutonium in solution
sediments in alternate buckets. after sediments were removed by fil-
Confidence brackets represent + tration. This is an expansion and
standard deviation. 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


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 o7 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.

Sage Il f

Director
Environmental Monitoring and Support Laboratory
Las Vegas, Nevada

iii


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° to 10 © with most of the numbers being approximately 10 7.
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 10° to 10° 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 4 are obtained. These are
similar in magnitude to the more conventional calculation (activity of
plants + 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, Cation exchange

13


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 ACUTE Pu CHRONIC Pu
SOLUTION ADDITION (pCi/ml) ADDITION (pCi/ml)
H20 1 14 +3 342

2 20 +3 642

3 1844 82
DIPA 1 89 + 3 90 +11

2 2742 22 +2
CITRATE 1 112 +3 48 +8

2 4444 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-ym 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 washed plants in

5


METHODS

The uptake of Pu from solution cultures was measured for three different
plants: Medicago, Lemma, 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 (uCi) of plutonium-238
nitrate [238pu(NO3),] in 1.0N nitric acid (HNO3) 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 uCi/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 HNO3. 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 41°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 uCi of 29®Pu 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 238py was then added
to obtain the efficiency for each vial. Efficiencies determined in this

2


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 (um). 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.


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. Devel. (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

YY US. Government Printing Office: 1978 786-258/2013


for 238py analysis. The roots were washed in a series of four different
solutions. This was accomplished by moving the cheesecloth containec up and
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
238py 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 0.008M.
This corresponded to an electrical conductivity (E.C.) 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,C. in each container showed that no nutrient
gradient existed in the system.

RESULTS

Alfalfa plants growing in hydroponic solution were treated by adding
238Pu(NO3)y 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 + &.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 19" for
the chronic and acute exposures, respectively. Repeated washings of the
roots in weak detergent or in 9.01M HNO; 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
238p, concentration decreased exponentially over the first 10 days. reaching
an apparent equilibrium concentration of approximately 10 pCi/ml. Pu



United States Environm ental Monitoring EPA-600/3-78-081
Environmen ital Protection and Support Laboratory August 1978
Agency P.O. Box 15027

Las Vegas NV 89114

Research and Development

SEPA Ecological
Research Series

Plutonium Uptake
by Plants Grown in
Solution Culture

yy»



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 um 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 Serial Parallel

Size Filtration Filtration Control*
No Filter 100 + 5.0 100 + 1.6 100 + 1.3
0.80 um 30 + 0.3 © 32 4 1.2 99 + 0.6
0.45 um 25 + 0.3 21+ 1.3 100 + 0.4
0.22 um 11 +0.3 8+0.9 99 + 0.0
0.01 um _- 8+ 0.2 100 + 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.

7


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.

oO CONTROL
ATYPHA
100

2

3

=

= 75

=

Fe

o

z

o

© 50

=

4

ra

325 a

DAYS

Figure 1. Plutonium loss from solution with and without Typha plants.
238py activities are expressed as a % of the peak concentration
and day zero coincides with the termination of injection.

6


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 HNO3 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 0.0l-micrometer (ym)
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.,

3


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


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-ym filters in the upstream samples.

But apparently, all or most of the debris was larger than 0.22 um,, 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-um 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 waa 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
C(mCi 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 jn 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 ef the particulate Pu removed from these root
samples in all of the washingg represented about 10% of the total Pu removed.
Ninety-five percent of this particulate 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 Py 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 wag 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 calcium presuma—
bly releasing Pu from these binding sites. The data in Figure 6 suggest
that calcium chloride has little or no effect in removing addlitional Pu
from the root tissue; subsequent washings with DTPA and citric acid gave

10


results very similar to those experiments without CaClo.

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 %

Activity Removed from . Container
Roots Roots by Washing Leaves Solution Surfaces
97.8 + 1.8 42.3 + 13.7 1.2 41.0 0.9 40.5 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 10° 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


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


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 1s available to the public through the National Technical tnformation
Service, Springfield, Virginia 22161


TABLE 3. PLUTONIUM-238 DISTRIBUTION IN VARIOUS TYPHA TISSUES 30 DAYS
AFTER CONTAMINATING THE HYDROPONIC CULTURE
WITH PLUTONIUM NITRATE

Tissue nCi/g*
Young leaves 1.5 + 0.5
Old leaves 0.5 + 0.2
Submerged leaves 2248
Young roots 596 + 199
Old roots 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.


76

50:
.
°
°
« =x.
a4
=
3
2
30:
20:
10:
WATER CITRATE DIPA
1 2 1 2 3041 2 3
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.

50:

>
eo

nCisqm ROOT

e

20.

+

WATER CaCl, DPTA
1 2 1 2 301 2

CITRATE

3

il

70

60:

wn
So

nCi/qgm, ROOT
So

30:
20.
10: Tr
i
WATER OTPA CITRATE
1 2 1 203 ~71 2.3
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.

Figure 6. Plutonium in wash solutions
in which Typha roots were sequentially
rinsed. Washing order was: water,
CaClj, DTPA, citric acid. Concentra-
tion is expressed on the basis of

nCi per gram of root being washed.