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

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                  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

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                                                 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

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                                 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

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                                  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

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                               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.

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                                  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

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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.,

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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

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 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

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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

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     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.

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  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.

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      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

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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

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                                              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

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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

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      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

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 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

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                                   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

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