EPA 680/4-75-006
JUNE 1975
Environmental Monitoring Series
TRITIUM FRACTIONATION IN PLANTS
NATIONAL ENVIRONMENTAL RESEARCH CENTER
OFFICE OF RESEARCH AND DEVELOPMENT
U.S. ENVIRONMENTAL PROTECTION AGENCY
LAS VEGAS. NEVADA 89114
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EPA-680/4-75-006
June 1975
TRITIUM FRACTIONATION IN PLANTS
by
J. C. McFarlane
Monitoring Systems Research and Development Laboratory
National Environmental Research Center
U.S. Environmental Protection Agency
Las Vegas, Nevada 89114
Program Element No. 1FA083
ROAP 21AMI
National Environmental Research Center
Office of Research and Development
U.S. ENVIRONMENTAL PROTECTION AGENCY
Las Vegas, Nevada 89114
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ABSTRACT
Alfalfa plants were hydroponically grown in environmental
growth chambers in which they were continuously exposed to tritium
throughout growth. All segments of the environment were in equi-
librium with respect to the specific activity of tritium. The
tritium content in plant organic matter was about 22 percent
lower than in the plant free water or rooting solution. Under
conditions of low transpiration, there was a higher concentration
(about 1.8 percent) of tritium in the leaves than in the stems
and rooting solution. This is thought to represent the result
of fractionation during transpiration.
iii
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ACKNOWLEDGMENT
I wish to express appreciation to Harry Hop for operation
and maintenance of the growth chambers and oxidation of plant
material. His professional approach and technical expertise con-
tributed greatly to the success of these experiments.
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INTRODUCTION
In 1931, Urey, Brickwedde, and Murphy (16) demonstrated
the existence of deuterium by separating it from ordinary hydro-
gen and analyzing it in a mass spectrometer. This discovery
was due to different masses which resulted in the separation or
fractionation of the two isotopes. The importance of this mass
difference, and the possibility of fractionation of hydrogen
isotopes in biological systems has been of concern since that
time. The thermodynamic basis for isotopic fractionation was
presented by Urey and Rittenberg (15) only two years after its
discovery. Additional considerations of isotope fractionation
have been detailed by several authors including Bigeleisen (3, 4),
Wiberg (20), Salomon (12), and Weston (19). Because water is
the most abundant hydrogen containing molecule and is essential
to all living organisms, it has been the object of most fractiona-
tion studies. In plants, water functions as a reactant, tempera-
ture regulator, and transport medium. Fractionation may be ex-
pected at several sites, not particularly dependent on its position
or function, but rather on the type of reaction, degree of com-
partmentation, and on the source of energy causing the reaction.
In general, the mass flow of water is unaffected by isotope dif-
ferences since the energy for transport is remote from the mole-
cules being moved. This does not eliminate the possibility of
observing fractionation along a water transport continuum in
soil or plants. But if observed, it is clear that fractionation
must have resulted from reactions such as exchange or dilution.
When reactions depend on the molecular-free energy of the hydro-
gen-containing species, fractionation is always possible. This
occurs in chemical reactions and other processes such as exchange
and evaporation which are the result of molecular diffusion.
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When fractionation occurs between molecules containing
tritium or deuterium, and those with normal hydrogen, there are
always two results: (1) the newly formed product is depleted
in the heavier isotope, and (2) the substrate is enriched. If
the substrate pool is large or is rapidly being replenished,
the effect of isotopic fractionation can be observed by separating
and analyzing both segments. But if the substrate is formed
at the same rate as the product, such as in a chain of reactions,
the concentration of the heavy isotope in the substrate increases
until a steady state is reached and an equilibrium concentration
is achieved. The existence of this steady state fractionation
is difficult to observe because the size of the substrate pool
is often so small that it precludes isolation, and because the
net or overall fractionation is very small.
It was shown by Stewart (14) that a characteristic fractiona-
tion occurs in the exchange of hydroxide ions from water to specific
clays. None was observed in water percolation. Although this
fractionation may be important in identifying the age of soil
water where the flow rate is slow, it is generally concluded
(Zinunermann, 22) that, in an agricultural setting, fractionation
in soil water has an insignificant impact in terms of tritium
availability to plants.
Reports of hydrogen isotope fractionation in plants started
in 1934 (Washburn and Smith, 17) only three years after the first
isolation of deuterium. Since then, many reports have been pub-
lished giving evidence of hydrogen isotopic fractionation in
plants. Reports of Washburn and Smith (17) and Zimmerman et al.
(21) indicate that no fractionation occurs during transpiration;
however, Wershaw e£ aL, (18) reported finding that deuterium
was greatly enriched in the leaves of trees with respect to the
water in the xylem and phloem. Most studies have indicated the
existence of an isotopic fractionation against the heavy hydrogen
isotopes being incorporated into the organic molecules of plants.
Smith and Epstein (13) showed that in marsh plants a 4.4 percent
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fractionation occurred in the formation of organic compounds.
In addition, fractionation of 9.2 percent occurred between car-
bohydrates and lipids. An apparent exception was demonstrated
by Helvey (6) when he showed that honey had a higher deuterium
content than the apparent water source. Aronoff and Choi (1)
showed that sugar synthesis in green plants used a substrate
which was in rapid equilibrium with water, but that hydrogen
from some other sources not in equilibrium with water was also
used. This resulted in the water having a higher tritium content
than the sugars in short term experiments.
A literature review by Bruner (5) contains lists of data
from studies comparing the tritium content in various segments
of plants and animals. Some data support the idea of discrimina-
tion against heavy hydrogen, some suggest preferential accumulation,
and some report no fractionation. Thus, as a result of the con-
flicting general summaries reported, there is considerable confusion
in the literature regarding hydrogen isotope fractionation in
biological systems.
Difficulties in sample preparation and experimental design
have made a complete understanding of fractionation difficult.
Problems which are most often overlooked in plant research, and
which are most often the source of serious errors, are caused by
failing to understand the rapid exchange which can occur between
tritium in water, either in solution or vapor, and the OH groups,
especially on carbohydrates (Lang and Mason, 8). This may partially
account for the lack of agreement in the literature regarding
the extent and nature of hydrogen Isotope fractionation. Field
studies are especially plagued by another problem: the interac-
tion of time on the interpretation of results. It is tempting to
analyze a plant or animal for tritium existing in various com-
partments and assign differences to isotopic fractionation.
However, it is obvious, but sometimes forgotten, that compart-
mentation is based on both a physical and time separation.
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The purpose of this work was to carefully Investigate the
fractionation of tritium from protium in the formation of organic
molecules in plants grown in a controlled, uniformly tritiated
environment. Also, the experiment was designed to measure any
fractionation which may occur during transpiration and absorption.
METHODS
Alfalfa plants (Medicago sativa) were grown in an environ-
mental simulation chamber from seedlings to mature plants. The
chamber was designed to eliminate air leakage (Hill, 7) and
thus allowed the maintenance of a uniformly tritiated environment
throughout growth. Radiant energy was produced by cool white
fluorescent and incandescent lamps and yielded a quantrum flux
2
of 325 uE/m in the wave band from 400-700 nm at the level of
the plant containers. The temperature was maintained at 25 ±
0.5°C during the day and 20 ± 0.5°C at night. Relative humidity
was 70 ± 4 percent. Carbon dioxide was monitored continuously
and maintained at 350 ± 15 PPM by automatic metered injections
of C02. Humidity was controlled by the temperature of a cold
radiator and the condensate was collected and reused to fill
the hydroponic containers. Thus, the water moved in a cycle
through the plants into the air, and returned to the rooting
medium. The tritium concentration of the water was monitored
periodically, and when additional water was added to replace
water lost in harvesting, it was made up at the same concentration
of tritium. Once every two months, the buckets containing the
hydroponic solution were drained and cleaned to remove organic
debris and refilled with fresh hydroponic solution (Berry, 2).
In the interim, the nutrient content of the solution was main-
tained by measuring pH and electrical conductivity and adding
water or nutrients as needed.
The alfalfa plants used in these studies were grown in sup-
port of another project (Moghissi £t aU 11) and were only
4
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sampled for fractionation evaluation after reaching maturity
from the previous harvest in an undisturbed chamber (about five
weeks).
When harvested, plants were immediately placed into benzene.
Water was extracted by heating the benzene to form a water/benzene
azeotrope, and was collected by cooling the distillate (Moghissi
£t al., 11). This collection system has been shown to be free of
isotopic discrimination which is an extremely important considera-
tion not true with many drying systems (McFarlane j2£ al., 9).
The plants were dried of benzene in desiccators under vacuum
with great care being exercised to eliminate contact with the
ambient environment. This precaution was taken to eliminate the
possibility of hydrogen exchange from water vapor and cellulose
OH groups. When dry, the plants were transferred to a stainless
steel container (Parr bomb) ignited in an 02 atmosphere of 500 psi.
The water of combustion was collected by bleeding the air from
the bomb slowly through a liquid nitrogen trap. Water samples
were analyzed by liquid scintillation counting (Moghissi, 10).
RESULTS AND DISCUSSION
The concentrations of tritium in plant-free water, gross
organic structure, and in the nutrient solution are shown in
Table I. These results have been normalized to reflect the con-
centration of the hydroponic solution as being 100 percent.
The experiment was repeated three times with four replicates
at each harvest. Although the hydroponic solutions were slightly
different between harvest dates, the coefficient of variation of
the replicated hydroponic solution was less than 0.7 percent in
all cases. Records of the tritium concentration in the hydroponic
solution were kept throughout the growth. The concentration
did not vary more than ± 1 percent from the time of harvesting
the last crop until harvesting the experimental samples. Using
all 12 sets of data, paired T tests showed significant differences
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Table I. RELATIVE TRITIUM CONCENTRATIONS IN PLANTS GROWN IN
A CONSTANT AND UNIFORMLY CONTAMINATED ENVIRONMENT*
Hydroponic Solution Plant Free H_0 Water from Oxidation
of Organic Hydrogen
nCi/ml nCl/ml nCi/ml
100.0 ± 0.2* 97.4 ± 0.6 77.5 ± 1.9
* Based on paired T tests of 12 replicates, the mean values
are significantly different from each other at the 0.001 level.
± The 95% confidence interval.
+ Normalized to hydroponic solution.
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between the concentration of tritium in the free water and organic
portion at the .001 level.
The fractionation between the free water and the organic
constituents of the plant is extremely difficult to evaluate in
the field and in most experimental conditions because the plant
water is rapidly exchanged with the water source. Exchange of
water in the atmosphere with plants also compounds the interpre-
tation of fractionation data. In these experiments where the
environment was carefully controlled to eliminate these variables,
the tritium fractionation between free water and organics was
about 22 percent.
Fractionation in evaporation is familiar to all who have
tried to collect water from plants and soil by any of the evapora-
tion methods. It may therefore be surprising to find that no
fractionation has been consistently reported in plant transpiration.
Transpiration is a diffusive phenomenon dependent upon molecular
free energy of the water molecule. Thus, it falls into the cate-
gory of reactions subject to fractionation. The supply of water
to the evaporative surface is by mass flow and is not expected
to be subject to fractionation. In this situation, fractiona-
tion would only be evident if diffusion of heavy water away from
the evaporative site was sufficiently rapid to increase the con-
centration of tritium in the surrounding tissue. When transpira-
tion is rapid, the mass flow of water toward the evaporation
site is so fast that it counteracts the diffusive movement of
tritiated water away from the site. Thus, under conditions of
rapid transpiration, fractionation would not be evident. The
data in Table II show that when all conditions remained constant
except the rate of transpiration, which was decreased by increas-
ing the humidity, there was an apparent fractionation between
the water in the stem and leaves of alfalfa plants. This was
thought to be evident because of a decrease in the ratio of
evaporation to diffusion away from the site.
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Table II. TRITIUM CONCENTRATION IN FREE WATER OF ALFALFA
STEMS AND LEAVES UNDER CONDITIONS OF SLOW AND
RAPID TRANSPIRATION
Leaves
Stems
Hydroponic
Solution
SLOW TRANSPIRATION
(High Relative
Humidity j> 70%)
nCi/ml
77.2 ± 0.6f
75.4 ± 0.5
75.0 ± 0.6
RAPID TRANSPIRATION
(Low Relative
Humidity < 25%)
nCi/ml
75.4 ± 0.5
75.8 ± 0.5
75.1 ± 0.5
t Significantly different from other means at the .05 level
based on paired T tests.
± The 95% confidence interval.
8
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It was concluded from experimentation in a controlled environ-
ment that tritium was fractionated at several sites in plants.
Discrimination against tritiated water entering plants was not
evident but, under conditions of slow transpiration, a small
fractionation could be detected in the leaves. Discrimination
against tritium in the formation of organic compounds was about
twenty-two percent.
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LITERATURE CITED
1. Aronoff, S. and I. C. S. Choi (1963): "Specific Activity
of Photosynthetic Sugars in Soybean Leaves Equilibrated
with Tritiated Water." Archives of Blochem and Biophysics.
102:159-160.
2. Berry, W. L. (1971): "Evaluation of Phosphorous Nutrient
Status in Seedling Lettuce." J. Amer. Soc. Hort. Sci.
93(3):341-344.
3. Bigeleisen, J. (1949): "The Validity of the Use of Tracers
to Follow Chemical Reactions." Science 110:14-16.
4. Bigeleisen, J. (1965): "Chemistry of Isotopes." Science
147(3657): 463-471.
5. Bruner, H. D. (1973): "Distribution of Tritium Between
the Hydrosphere and Invertebrates." Tritium edited by A.
A. Moghissi and M. W. Carter. CONF 710809 p. 303-317.
Messenger Graphics, Phoenix, AZ and Las Vegas, NV.
6. Helvey, T. C. (1953): "The Natural Concentration of Deuterium
in Honey." Science 117:276-277.
7. Hill, A. C. (1967): "A Special Purpose Plant Environmental
Chamber for Air Pollution Studies." J. Air Pollution Con-
trol Association 17(11); 743-748.
8. Lang, A. R. G. and S. G. Mason (1960): "Tritium Exchange
Between Cellulose and Water: Accessibility Measurements
and Effects of Cyclic Drying." Can. J. Chem. 38:373-387.
9. McFarlane, J. C., W. Beckert, and K. W. Brown (1975): "Tritium
in Plants and Soil." Unpublished data.
10. Moghissi, A. A., E. W. Bretthauer, and E. H. Compton (1973):
"Separation of Water from Biological and Environmental Samples
for Tritium Analysis." Anal. Chem. 45:1565-1566.
11. Moghissi, A. A., R. E. Stanley, J. C. McFarlane, E. W.
Bretthauer, R. G. Patzer, and S. R. Lloyd (1974): "Biological
Concentration of Tritium." Proceedings, Fifth International
Congress of Radiation Research, July 1974, Seattle Washington,
U.S.A.
10
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12. Salomon, M. (1966): "Isotope Effects in Mixtures of Liquid
H20 and T20." Can. J. Chem. 44:689-694.
13. Smith, B. N. and S. Epstein (1970): "Biogeochemistry of
the Stable Isotopes of Hydrogen and Carbon in Salt Marsh
Biota." Plant Physiol. 46:738-742.
14. Stewart, G. L. (1965): "Fractionation of Tritium and
Deuterium in Soil Water." Isotope Techniques in the Hydro-
logic Cycle, edited by G. E. Stout. Nat. Aca. of Sci. Nat.
Res. Council Pub. 1488:159-168.
15. Urey, H. C. and D. Rittenberg (1933): "Some Thermodynamic
Properties of the H1 H2 Molecules and Compounds Containing
the H2 Atom. J. Chem. Phys. 1:137-143.
16. Urey, H. C., F. G. Brickwedde, and G. M. Murphy (1932):
"A Hydrogen Isotope of Mass 2." Phys. Rev. 39(1):164-165.
17. Washburn, E. W. and E. R. Smith (1934): "The Isotopic
Fractionation of Water by Physiological Processes."
Science 79:188-189.
18. Wershaw, R. L., I Friedman, S. J. Heller, and P. A. Frank
(1969): "Hydrogen Isotopic Fractionation of Water Passing
Through Trees." Advances in Organic Geochemistry. Proc.
Third International Conference, p. 55-67.
19. Weston, R. E., Jr. (1973): "Kinetic and Equilibrium Isotope
Effects of Tritium Substitution." Tritium Edited by
A. A. Moghissi and M. W. Carter, p. 289-303.
20. Wiberg, K. B. (1955): "The Deuterium Isotope Effect."
Chem. Rev. 55:713-743.
21. Zimmermann, U., D. Ehhalt, and K. 0. Muennich (1967):
"Soil-Water Movement and Evapotranspiration: Changes
in the Isotopic Composition of the Water." Isotop.
Hydrol., Proc symp. Vienna 1966, pp 567-584.
11
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-680/4-75-006
2.
3. RECIPIENT'S ACCESSIOI*NO.
4. TITLE AND SUBTITLE
Tritium Fractionation in Plants
5. REPORT DATE
June 1975
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J. C. McFarlane
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORG \NIZATION NAME AND ADDRESS
Monitoring Systems Research and Development Laboratory
National Environmental Research Center
P. 0. Box 15027
Las Vegas, Nevada 89114
10. PROGRAM ELEMENT NO.
1FA083
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Office of Research and Development
U.S. Environmental Protection Agency
Washington, DC 20460
13. TYPE OF REPORT AND PERIOD COVERED
Progress Report
14. SPONSORING AGENCY CODE
15. SUPPLEMENTARY NOTES
16. ABSTRACT
Alfalfa plants were hydroponically grown in environmental growth chambers in
which they were continuously exposed to tritium throughout growth. All segments
of the environment were in equilibrium with respect to the specific activity of
tritium. The tritium content in plant organic matter was about 22 percent lower
than in the plant free water or rooting solution. Under conditions of low
transpiration, there was a higher concentration (about 1.8) percent of tritium
in the leaves than in the stems and rooting solution. This is thought to represent
the result of fractionation during transpiration.
17.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS
COS AT I Field/Group
Tritium
Pollutant Pathways
Fractionation
Plant Physiology
Plant Biology
Radiochemistry
Radioactivity
Pollutant Pathways
Hydroponics
06 03
07 05
14 07
18 02, 08
13. DISTRIBUTION STATEMEN1
Release unlimited
19. SECURITY CLASS (ThisReport)
Unclassified
21. NO. OF PAGES
16
20. SECURITY CLASS (Thispage)
Unclassified
22. PRICE
EPA Form 2220-1 (9-73)
GPO 693-739/42
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