Ecological Research Series
TRITIUM IN PLANTS AND SOIL
Environmental Monitoring and Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, Nevada 89114
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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 five series. These five 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 maximum interface in related fields.
The five series are:
1. Environmental Health Effects Research
2. Environmental Protection Technology
3. Ecological Research
4. Environmental Monitoring
5. Socioeconomic Environmental Studies
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. Investigations include formation, transport, and pathway studies to
determine the fate of pollutants and their effects. This work provides 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 Informa-
tion Service, Springfield, Virginia 22161.
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EPA-600/3-76-052
May 1976
TRITIUM IN PLANTS AND SOIL
by
J. C. McFarlane, W. F. Beckert, and K. 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 recommenda-
tion for use.
ii
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CONTENTS
Page
Introduction 1
Conclusions 3
Chemistry and Physics of Tritium and Tritiated Water 4
Tritiated Water in Soil 9
Tritium in Plants . 14
Routes of Entry 14
Transport in Plants 17
Isotope Fractionation in Plants 18
Tritium in Aquatic Plants 21
Effects 23
Pitfalls 27
Literature Cited 30
iii
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INTRODUCTION
The release and fate of radioactive elements in the
environment resulting from nuclear weapons testing and
the activities associated with the rapidly expanding nuclear
power industry are major public concerns. Tritium, the
only radioactive isotope of hydrogen, constitutes a significant
portion of the residual radioactivity being released during
these activities. Tritium is also a naturally occurring
radionuclide which originates in the atmosphere from the
action of certain components of the cosmic ray flux on
the nuclei of nitrogen and oxygen in air; however, this
results in a natural equilibrium concentration of only
16-35 picocuries per liter (pCi/1) in the hydrosphere.
Weaver et al. (1969) attributed about 4% of the total
estimated world tritium burden of 1.7 x 10 curies to the
natural equilibrium level; the remainder was primarily
ascribed to the atmospheric testing of nuclear weapons.
Since tritium decays with a half-life of 12.35 years, large
quantities of tritium produced by the atmospheric nuclear
weapons prior to the 1963 test ban treaty are slowly decaying
and the resultant concentrations in world waters and in
the atmosphere are decreasing. Except for tests by nations
not adhering to the treaty, the primary source of tritium
currently being released is the nuclear power industry.
Cowser et al. (1966) predicted that the world tritium burden
would continue to decrease until about 1995, at which time
it would start to increase due to the expanding production
and release by nuclear power related activities.
Tritium concentrations presently observed in the drinking
water of the United States vary from below 200 pCi/1 (the
detection limits of the Environmental Protection Agency
surveillance network) to 3,100 pCi/1 (Office of Radiation
Programs 1974). Localized variations are generally associated
with either nuclear activity or topography. For instance,
Mullins and Stein (1972) showed that higher levels occurred
in the mountainous regions of the western United States
than at lower elevations.
Tritium is released to the environment primarily as
tritiated water (HTO) and molecular tritium (HT, or Ta).
Once released, tritiated water is rapidly diluted by the
large volumes of water flowing in streams, rivers, and oceans,
and, when released via stacks, by dispersion in air. It has
been assumed that when elemental tritium is released it is
diluted by the whole atmosphere. However, there remains
some uncertainty as to its eventual fate in the atmosphere
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and to the relative significance of its contribution to
environmental tritium pollution. The large dilution capacities
of the hydrosphere and atmosphere reduce the current average
tritium concentration to very low levels, posing only a
potential threat for the distant future. Nevertheless,
tritium can be a problem on the local level.
Considerable research on the impact of environmental
tritium on man's existence has been reported and various
aspects have been reviewed. Outstanding publications on
environmental tritium include a review by Jacobs (1968)
on the physical and chemical aspects; another publication
by Elwood (1971) which extensively covers the pathways and
rates of tritium movement, the forms of incorporation, and
biotic turnover; and a collection of papers edited by Moghissi
and Carter (1973), which is based primarily on a conference
held in September 1971 in Las Vegas, Nevada, during which
an effort was made to evaluate all important aspects of
tritium.
This paper focuses on those aspects of tritium which
are specifically important for an understanding of the role
and behavior of this radionuclide in the plant-soil-water
system. Plant and soil literature was reviewed in light
of water potential terminology and wherever possible, an
effort was made to relate the studies carried out by agricultur-
ists to those conducted by radiation biologists. In addition
we have supplemented the review with the results of original
research where a particular point could be explained more
clearly by examples and where new data were required to
demonstrate a concept.
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CONCLUSIONS
Present knowledge of tritium movement in the environment
is clouded by several contradictions and by incomplete understand-
ing. The contradictions primarily arise in field studies
where fractionations are reported for the exclusion or
accumulation of tritium in plant materials. From controlled
studies, it has been concluded that fractionations do occur,
however, the maximum fractionation appears to be about 20%
between the free water and the organic constituents of plants.
Thus, even at its maximum, fractionation and exchange between
protium and tritium may account for some important errors
in analysis of plants and soils. These errors generally
occur in sampling and sample preparation and may result
in significant errors in interpretation of data.
Plants make up a large portion of the human diet and
therefore serve as an important vector of tritium to man.
Elevated tritium concentrations in crop plants will primarily
be the result of contamination of soil with tritiated water,
but the possibility of plant contamination from gaseous
releases is also important. The impact of elemental tritium
as a pollutant is incompletely understood. Recent data
shoxtfing a rapid conversion of elemental tritium (HT) to
tritiated water (HTO) in the environment of plants and soils
suggest that further study is needed to evaluate the impact
of this form of tritium as a pollutant.
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CHEMISTRY AND PHYSICS OF TRITIUM AND TRITIATED WATER
The three hydrogen isotopes protium (P)*, deuterium
(D), and tritium (T), have atomic weights of 1, 2, and 3,
respectively. Each has one proton and one electron, and
thus all have similar chemical properties, but since each
has a different mass, the free energies of the isotopes vary
and the corresponding reaction rates vary proportionately.
The melting and boiling points of the hydrogen isotopes are
listed in Table 1. The differences are accounted for by
the amount of molecular free energy each isotope possesses.
This decreases with increasing mass. Since the mass of HT
is the same as that of D2 , their boiling and melting points,
and other energy-related phenomena should be approximately
equal, although these data are not reported.
TABLE 1.
MELTING AND BOILING POINTS OF THE HYDROGEN
ISOTOPES
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and differs from protium only in mass. Deuterium studies
have therefore been useful in interpreting the results of
experiments with tritium. They have allowed discrimination
between mass effects and radiation effects to be elucidated.
Eisenberg and Kauzmann (1969) reported the bond angles
and lengths for D20, H20 and HDD as being very near equal
and pointed out that this was consistent with the Born-Oppenheimer
approximation, which predicts that the electronic structures
of molecules are independent of the masses of their nuclei.
Electronic and spatial characteristics largely determine
the chemical properties of an atom or molecule, therefore,
it would be expected that all reactions involving hydrogen
should be similar to those involving deuterium or tritium.
Reaction rates are governed by molecular free energies which
in turn are functions of molecular masses; thus although
reactions are similar, slower reaction rates generally occur
with the heavier isotopes.
The properties of water which reflect average free energies
of solutions are termed colligative properties. These include
boiling and freezing temperatures and vapor and osmotic pres-
sures. The average free energy of a solution is primarily
determined by dissolved solutes, matrix properties of structural
materials, temperatures, and pressure. Materials dissolved
in water and structural material in contact with water are
termed hydrated, that is, surrounded by layers of water molecules.
The forces which cause this hydration restrict the free movement
of these water molecules and, as a consequence, the average
free energy of water in a system is reduced. This change
in average molecular free energy causes changes in the physical
properties of water. A treatise on how various factors affect
the average free energy, or the water potential in plants,
was written by Slatyer (1967) and is valuable in interpreting
the kinetics of tritiated water. In deuterated and tritiated
water, the effect of the HDD and HTO molecules on the average
free energy is similar to that of a solute, although for
a different reason. Because of increased mass, the free
energy of heavy water molecules is smaller than that of light
water molecules. Consequently, the addition of heavy water
to light water will result in an average free energy decrease
of the solution. This decrease is obviously proportional
to the concentration of the heavy water molecules. Physical
properties reported in the literature for some properties
of water altered by the presence of different hydrogen isotopes
are listed in Table 2.
In addition to other factors the evaporation rate of
a body of water is directly proportional to the average free
energy of the water molecules in the liquid phase; lowering
its average free energy by addition of a solute or deuterated
or tritiated water molecules results in a lower evaporation
rate. Evaporation rates are measured on the basis of molecular
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TABLE 2. BOILING POINTS, TRIPLE POINT TEMPERATURES AND
PRESSURES, AND MAXIMUM DENSITIES
Triple Point
Boiling , . Triple Point Pressure, Max. Density, Temp, of Max.
Point, °C(a) Temp., °CM cm Hg"aT g/cm3 ™ Density V^
H 0 100.00
D 0 101.42
T 0 101.51
0.010
3.82
4.49
0.458
0.502
0.492
0.999973
1.10589
1.2150
3.98
11.2
13.4
(a)
W. M. Jones (1968).
(b)
D. G. Jacobs (1968).
averages and are commonly expressed in milliliters per minute
(ml/min); however, evaporation does not proceed on im, average
basis but occurs on an individual molecular basis. The probabil-
ity of any molecule changing phases depends on its proximity
to a liquid-gas interface and on its vibrational activity.
In a mixture of heavy and light water molecules, the heavy
molecules have a smaller free energy than the lighter ones
and consequently their relative frequency of evaporation
is lower. The average or overall effect of heavy water molecules
on a solution is therefore the same as the addition of a
solute, that is, the overall water evaporation rate is decreased.
This occurs in direct proportion to the relative abundance
of the heavy molecules. But because the heavy molecule does
not affect the movement of the lighter ones, the lighter
molecules evaporate faster resulting in an enrichment of
the heavier molecules in the liquid phase. This type of
isotopic fractionation can be described by the fractionation
ratio. (FR), which is defined as
C2
FR = =-
Cl
where C, = concentration of heavy isotope in the primary
fraction (i.e., tritium concentration in the
liquid phase).
Ca = concentration of heavy isotopes in the secondary
fraction (i.e., tritium concentration in the vapor
phase).
For HTO the tritium concentration is directly proportional to tue
specific radioactivity in curies per milliliter ("•'/ml) which
can be used as Cj and Ca to calculate the FR. Since this term
is the ratio of two concentrations it is unitiess and can be
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expressed as a decimal fraction or a percent. It is emphasized
that the fractionation ratio describes the degree of fractiona-
tion and is independent of the original solute concentration
(e.g., HTO), whereas, the overall rate of evaporation is
directly dependent on the concentration of the heavy isotope.
Under environmental or typical experimental conditions,
the HTO concentration is so low that its influence on the
evaporation rate and on all other colligative properties
of the solution can be neglected. This has not always
been the case in deuterium studies, and the lack on consideration
of changes in water potential may have resulted in a misinterpr-
etation of experimental results.
00
00
1.25-
1.20-
1.15-
1.10-
1.05-
1.00-
10 20 30 40 50 60 70 80 90 100 110
TEMPERATURE, °C
FIGURE 1. Ratio of Vapor Pressures of
P20/T20 and P20/D20. Jones
(1968) as presented by Moghissi
et al. (1973a).
Moghissi et al. (1973a) pointed out that temperature
changes affect the vapor pressures of T20, D20, and H20 to
of vapor pressure of H.O/T,
a different degree. The ratios
and H,0/D,0 are presented in Figure 1. Both ratios
2 ' 2
1 ^J / ^J ^J Q, J^ ^£. \J ^ W* tj ^* *•* ** ^^ *"* ^- **• *" *** O ** *^ ^ ™"" * "^ — ^ — — ^ — ^ — —- — ^* ^•" ^^ •" ^* «* ^ ^"
with increasing temperature. There is also corresponding
decrease in the fractionation ratios with increasing temperatures
In addition, the water potential of heavy water is similarly
affected by temperature. In Table 3 are shown the calculated
water potentials of pure D20 at various temperatures. Since
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the water potential of pure free Ha 0 at a given temperature
is defined as zero, it is clear that this isotopic difference
in water potentials correspondingly decreases with increasing
temp erature.
TABLE 3. WATER POTENTIAL OF D,0 AT VARIOUS TEMPERATURES
Temperature
°C
Water Potential*
in Atmospheres
10
-193
20
-181
30
-166
40
-151
50
-137
60
-124
*Water potential calculated from the relationship
RT ln
w
w
where R - 0.082055 liter-atm. deg-1 mole 1t T = °K, e = vapor
pressure, e
molal volume.
_
vapor pressure of pure free water at T, V = partial
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TRITIATED WATER IN SOIL
Tritiated water applied to or accidentally deposited
on a soil surface moves downward as a pulse. Originally
there is a sharp boundary between contaminated and uncontaminated
layers but this is readily blurred by exchange with soil
water. Even in dry soils exchange of tritium with hydrogen
on various particle surfaces results in a gradient at the
forward edge. New water added to the soil surface pushes
the old water downward creating a contaminated water lense
(Figure 2). The rate and extent of downward movement depend
mainly on the structure, texture, and water content of the
soil. Agricultural research has been conducted for many
years to describe the influence of soil texture and structure
on the rate of water movement and on its availability to
plants. This vast reservoir of knowledge can be put to good
use in the study of the behavior of tritiated water in soil.
Unfortunately, agriculturists and radiobiologists have different
requirements and consequently, the data generally gathered
by each discipline are not wholly usable by the other. For
instance, agriculturists have been primarily concerned with
soil water in t ot o and have had little need to emphasize
the movement patterns of a particular pulse. However, the
study of tritium movement in soil involves just that because
only one portion of the water is of interest. Discussions
of soil-water content and soil-water relationships are neverthe-
less valuable for tritium studies. These discussions can
be found in many general texts, such as that by Black (1968),
and in books directed specifically at the soil-water relationships,
e.g., Nielsen et al. (1972).
Water in soil can be regarded as being segmented into
different compartments, e.g., free water, capillary water,
etc. The vertical movement of a lense of tritiated water,
and the broadening which occurs with time, depend on a complicated
relationship between environmental conditions and various
water compartments. Each compartment has its special set
of characteristics which determine water movement through
and water behavior in it. In the simplest case, unobstructed
water percolation is possible primarily through the soil
free space. Movement rates of this "free water" are governed
by soil texture and structure. In many soils, free water
is also distributed throughout the soil by passage through
fractures, holes caused by insects, worms, reptiles, and
mammal activities, and through lumens created by decayed
plant or animal tissues. The rapid dispersion of tritj'ated
water through voids and irregularities in the soil structure
is often responsible for great and inconsistent variations
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10
DEPTH (cm)
FIGURE 2. Downward movement of HTO in soil.
Dilution of HTO at the wetting front
(•——•) of an oven-dried soil is caused
by exchange of tritium with protium in
colloidal water and protium on the soil
particles. After adding an equivalent
amount of water to the labeled pulse,
tritiated water lense is rather narrow
(a a). Diffusion causes this lense to
become wider with time.
10
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in a contamination depth profile. Investigations regarding
this fast irregular dispersion of tritiated water have been
conducted by various authors; Dixon and Peterson (1971),
Jordan et al. (1971), and Sasscer et al. (1973). A correction
for the water movement model of Sasscer et al. (1971) was
derived by Jordan et al. (1974).
Water held as a thin layer on the surface of soil particles
and in capillaries formed between soil particles and in decaying
organic material is termed capillary water and, as such,
constitutes a discrete compartment. Capillary water can
move via mass flow whenever a water potential difference
occurs in the capillary system as well as via molecular diffus-
ion. Both modes of movement cause a broadening of the contami-
nated water lense. Molecular diffusion is non-directional
and its extent depends primarily on the mean free path length
and on the temperature. Most studies on the diffusive movement
of water in soil systems have focused on vertical movements,
since horizontal diffusion within a uniformly contaminated
area is of no net consequence in terms of water availability.
Yet horizontal movement may also be responsible for loss
of tritium from a contaminated site to uncontaminated areas.
The sites of exchangeable cations on clay and organic
matter can be classified as a separate compartment for the
residence of tritium. Movement rates into and out of this
compartment depend on the number of exchange sites, on the
kinds and numbers of cations present, and on the soil water
content. The exchange of tritium in the free and capillary
water compartments with the cation exchange compartment is
relatively rapid; therefore, exchange reactions are usually
not important for the distribution pattern of tritium in
soil. However, under a combination of certain conditions,
e.g., when the tritium lense resides in the root zone, the
soil is relatively dry and transpiration demands are high,
this compartment can become significant. When Jordan et
al. (1974) tested a water movement model, corrections based
on tritium exchange with this compartment were required to
fit their experimental data to the theoretical model. This
was especially apparent towards the end of the growing season.
Tritium may also become an integral part of the clay
mycell and reside in the form of hydroxyl groups attached
mainly to aluminum and magnesium atoms. These hydroxyl groups
exchange slowly with water from other soil compartments.
This slow exchange rate renders the effect of this compartment
on movement of tritiated water through soil and its availability
to plants rather unimpo'rtant. Exchange in this compartment
may be a useful tool in clay minerology and in geophysical
studies since it allows the identification of specific reactions
and may be useful in identifying certain time-dependent inter-
actions. There have been studies regarding the possibility
of isotopic fractionation in the exchange of tritium and
11
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deuterium with the clay water compartment. Stewart (1967)
reported fractionation ratios as high as 3:1 for a type of
kaolinite clay, but in more recent studies he was unable
to repeat these observations (Stewart, 1973) and instead
showed the highest ratios were near one. Additional discussion
of exchange in this compartment can be found in clay minerology
texts and specifically in articles by Rabinowitz et al. (1973),
Helevy (1964), Corey (1968), and Coleman and McAuliffe (1955).
Soils contain varying amounts of organic material (humus),
most of which is derived from cellulose via degradation processes
This organic material contains exchangeable hydrogen which
is bound to oxygen (and to a much smaller degree, to nitrogen).
The amount of exchangeable hydrogen in this compartment depends
on the amount of organic material present, on its degradation
mode and on soil pH. These hydrogens exchange rapidly with
HTO, thus slowing the downward movement of a tritiated water
lense. However, soils high in organic material generally
occur in areas where precipitation and percolation rates
are high. Although in principle the interaction of tritiated
water with the soil organics compartment has an effect on
tritium movement in soil, it is relatively small in most
natural systems, due either to low organic content or high
percolation rates. However, additional information is desirable
to more fully clarify the significance of this soil compartment
to tritium movement in soil.
In addition to the effects of the various soil compartments
on movement of tritiated water in soils, other factors can
have a marked influence. When tritiated water is applied
to a soil surface, a rapid initial evaporation loss will
occur under most circumstances. The extent of this loss
depends on a number of factors such as climatic parameters,
soil composition and texture, kind and extent of plant cover,
etc.
Water loss via plant root absorption and transpiration
is of great importance in most terrestrial ecosystems. Plants
with a shallow root system such as many vegetables and cereals
absorb all of their water from the upper few centimeters
of the soil whereas other plants with tap roots may absorb
water at depths of 10 to 15 meters. Thus, movement of a
water lense to a meter below the surface may make this body
of water unavailable to some species, but optimize absorption
for others. Diversity in rooting patterns also results from
differences in plant age, vigor, soil type, climate, and
on the number and kinds of plants competing for space, water,
and nutrients. This great diversity makes it difficult to
predict the importance of plant transpiration on the fate
of a pulse of tritiated water. Although generalizations
are possible and valuable each situation must be evaluated
individually.
12
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The speed with which water moves from soil to plants
was illustrated in an experiment conducted by Woods and O'Neal
(1965). Tritiated water added to soil near an oak tree was
detected in the leaves after only four hours. We have observed
it in alfalfa and lettuce plants within minutes of application
to the soil. The residence time of water in plants is generally
short in comparison to its residence time in soil, notable
exceptions being certain desert plants (e.g., cacti). This
is a result of the large water-holding capacity of soil combined
with the large soil mass. Thus the residence time of tritium
in a soil system largely determines the exposure time, and
consequently the effect, on the rest of the ecological system.
The relatively short residence of tritium in mesophytic plants
growing on tritium contaminated soil is extended by the longer
residence of tritium in the soil. Root zones contain tritium
for varying lengths of time depending primarily upon rainfall,
soil characteristics, and on environmental parameters which
influence movement in and out of the various soil-water compart-
ments. There are several reports of particular value which
should be studied to obtain a broader understanding of the
interactions between soil and plants growing under different
climatic conditions. Among these are reports by Koranda
et al. (1967, 1968, 1969), who studied tritium movement in
desert soils following the Sedan detonation which dispersed
approximately one million curies of tritium into about six
million tons of earth. Koranda and Martin (1973) reported
on the movement of a pulse of tritiated water in an irrigated
corn field. Jordan et al. (1970) report on the movement
of tritiated water in a tropical rain forest and in (1974)
on its movement in a grassland community.
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TRITIUM IN PLANTS
ROUTES OF ENTRY
Water enters plants primarily through the roots. Plant
exposure to tritium therefore depends mainly on the concentra-
tion of HTO in the soil and the position of the contaminated
water lense relative to the plant roots. Under some conditions
the foliar absorption of tritium may also become important.
Of particular concern are plants growing in areas with aerial
levels of tritiated water vapor or elemental tritium gas
(HT), such as may exist in the vicinity of some nuclear facili-
ties.
The exchange and incorporation of liquid HTO into plant
leaves was demonstrated by Vaadia and Waisel (1963). They
exposed the leaves of Allepo pine (Pinus halepensis) and
the common sunflower (Helianthus annua) to tritiated water
for various periods and showed that, under conditions of
no transpirational water loss, a rapid accumulation of HTO
occurred in both species. Transpiring plants, in an environ-
ment of tritiated water vapor, accumulate tritium at a much
slower rate (Koranda and Martin 1973), (Cline 1953), and
(Aronoff and Choi 1963).
The results of an experiment designed to measure foliar
absorption of tritiated water by leaves and its subsequent
translocation to the roots are shown in Figure 3. Alfalfa
plants growing in sealed hydroponic containers were intro-
duced into a growth chamber which was maintained at a constant
HTO vapor concentration of 110 nanocuries per milliliter.
Lights remained on continuously during the first 72 hours
but were turned off the entire fourth day. Samples of the
root nutrient solutions were collected periodically during
the experiment and analyzed for tritium. Every 24 hours
distilled water was added to each hydroponic container to
replenish the water transpired by the plants. These dilutions
account for the observed minima of the tritium solutions
as illustrated in Figure 3. At the conclusion of the first
portion of this experiment (lights on) the tritium concentrations
in the nutrient solutions had increased from zero to about
40 picocuries per milliliter (pCi/ml) (0.07% of the steady
state atmospheric level). On the fourth day of the experiment
the lights were deliberately left off and the transpiration
rate decreased from about 4 X 10~3 milliliters per minute
per gram (ml/min/g) of fresh leaf tissue to about one-tenth
that value. In the light (high transpiration rates) basipetal
translocation of water vapor absorbed by the leaves was accounted
14
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120-
8tH
40-
LIGHT
DARK
24
I
48
72
HOURS
96
120
FIGURE 3. Tritium concentration in the hydroponic
solution of alfalfa plants. The route
of tritium contamination to the solution
was by foliar absorption of HTO vapor.
for by a net movement of 0.8 milliliters per day (ml/day).
Obviously, foliar absorption and water loss from roots were
much greater than this value but because of water mixing
between phloem and xylem the net transport was small. When
the lights were out and transpiration rate decreased, the
rate of basipetal movement was increased to 2.4 ml/day.
Under these conditions the foliar absorption rate was decreased
because of increased stomatal resistance. But because of
decreased dilution coincident with slower rates of water
movement in the xylem, the resultant basipetal movement was
increased by a factor of 3. At the termination of the 24-
hour dark period, the extractable free water of the r.oots
and leaves was found to contain 521 and 39,431 pCi/ml, respectively.
These values correspond to 0.5% and 35% of the tritium concentra-
tion present in the vapor phase. The concentration gradient
from the leaves to the roots is thought to be due to dilution
via exchange with water moving upward in the xylem. In the
dark, when less water was moving upwards, less dilution occurred,
a consequence, an
and as
in the hydroponic solutions was
from these data that even under
increased rate of
observed.
conditions
HTO accumulation
It is evident
of high relative
humidity (85%), moderate transpiration rates, and continuous
HTO vapor exposure, plants do not reach an equilibrium with
the HTO in the atmosphere, and contamination of the roots
and soil via foliar absorption would be very slow. It is
also clear that under conditions of low net transpirational
15
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water loss, that the leaves do accumulate tritium from contami-
nated water vapor. Since the leaf water is used as a photosyn-
thetic substrate the photosynthate will naturally be contamina-
ted to a level dependent on the leaf concentration. As pointed
out, this may be determined by the soil water or water vapor
depending on the environmental conditions and the type of
plant involved.
Under most conditions, transpiring plants which are
contaminated with HTO (regardless of the contamination route)
rapidly lose tritium from the free water in the tissue, as
was demonstrated by Koranda and Martin (1973). They found
that both woody and herbaceous plants which had absorbed
HTO from the gas phase lost tritium quickly with the residence
half-times varying between 20 and 70 minutes, depending on
differences in water turnover rates. The plant with the
largest transpiration rate obviously had the shortest residence
half-time.
Recent experimentation by Kline and Steward (1974) regard-
ing foliar tritium uptake and loss from atmospheric HTO have
yielded some interesting rates. Their conclusion aptly summa-
rizes the importance of the foliar contamination of trans-
piring plants.
"In the event of accidental HTO release to
the atmosphere near ground, rapid contamination
of vegetation could be expected. When the
atmospheric source is dissipated, rapid
decontamination could also be expected during
periods of normal plant transpiration but not
at night or other times when transpiration
flow is not taking place."
Under certain conditions, e.g., near nuclear facilities
or in the vicinity of natural gas wells which had been stimula-
ted by nuclear explosions, plants might be exposed to significant
levels of HT or tritiated methane (CH,T). McFarlane (1976)
3 ^^
has shown that when plants were exposed to HT they rapidly
incorporated tritium into the free water and the organic
material of the plants. The significance of this observation
and the rate of the conversion of HT to HTO are presently
under investigation. Mason et al. (1971, 1973) determined
in the field and in the laboratory that plants incorporate
tritium into the plant free-water and into the organic phase
when exposed to CH3T. Their data indicate that in transpiring
plants the incorporation of tritium from CH3T into the leaf
free-water reaches steady state in about 18 minutes. They
concluded that the deposition velocity of tritiated methane
was only about 4% of that of HTO vapor.
In summary, root absorption of HTO is the most important
pathway of tritium entry into plants. Tritiated water vapor
appears to be of consequence only if it occurs at very high
16
-------
X
levels persistent over a long period of time in conjunction
with high humidity which results in low or zero net transpira-
tion, or if it condenses in the form of dew to become a compon-
ent of the soil water. The foliar contamination of plants
by CH3T, although an observed fact, appears to be relatively
unimportant because of the infrequence of release and the
relatively low incorporation. The consequence of plant exposures
to HT is not yet fully understood. It is known, however,
that large quantities of HT are released during nuclear fuel
reprocessing operations, and the foreseeable increase of
nuclear facilities demands a through examination of the result-
ing increased exposure of plants to HT.
TRANSPORT IN PLANTS
Plant-water relations have been the subject of extensive
investigations and elaborate mathematical models have been
developed to describe both water stress and water movement
in plants (Slatyer, 1967). Under mass flow conditions (i.e.,
transpirational water movement through the xylem) HTO moves
at the same rate as H20. In diffusional flow, (i.e., evaporat-
ion or intracellular movement), HTO moves at a slower rate
than H20 due to its larger mass. As previously indicated,
these differences are so slight and water movement in plants
so dependent on mass flow, that diffusional differences are
inconsequential and are generally considered unimportant
in describing tritium movement in plants and soil. Therefore,
the net movement of tritiated water can best be described
by systems developed to identify and define water movement.
More than 99% of the water entering most plants moves
from the root through the xylem system to the stems and leaves,
where it is rapidly lost to the atmosphere via stomatal openings
(Lieth, 1963). The residence time for water in plants varies
between minutes and days, depending on parameters such as
species, size, age, plant morphological features, soil water
availability and atmospheric conditions. Also constant exchange
with metabolic water causes dilution and a fairly rapid loss
of HTO from plants which have had an acute exposure. Following
imbibition and equilibration by soybean seedlings exposed
to HTO, it was shown that the tritium depletion rate from
the free water component of the seedlings was exponential.
Twenty-four hours after tritiated water in the rooting zone
was exchanged with H20, only 1% of the original tritium concen-
tration remained in the plant. After 120 hours, less than
0.01% of the original concentration was present (Vig and
McFarlane, 1975).
Investigations using HTO as a tracer in soil-plant studies
have illustrated the rapid movement of water in these systems.
For example, Woods and O'Neal (1965) observed tritium in
oak leaves 4 hours after an addition of HTO was made to the
soil. Lewis and Burgy (1964) also experimenting with oak
17
-------
found tritium in the leaves after a soil application 55 feet
from the base of the tree. In another investigation it has
been observed that water movement occurs primarily in the
outermost vessels of the xylem (Wray and Richardson, 1964).
ISOTOPE FRACTIONATION IN PLANTS
It was previously pointed out that there are no qualitative
differences in the chemical and physical behavior of the
three hydrogen isotopes. However, differences in the mass
of protium, deuterium and tritium result in reaction rate
differences which are dependent on the amount of free energy
in the molecules containing them. Under identical conditions,
heavier molecules have lower velocities and thus experience
fewer collisions and also have lower vibrational energy and
consequently experience a smaller chance of dissociation.
Differences in rates result in fractionation. Significant
fractionation of hydrogen isotopes in biological systems
can occur in phase changes such as the evaporation of water
and in chemical reactions like the synthesis of chemical
compounds in plants and animals.
Since fractionation occurs during evaporation of H 0/HTO
mixtures, it may seem puzzling that there is no agreement
in the data indicating fractionation in plant transpiration
(Washburn and Smith, 1934; Zimmerman et al., 1967). It can
be rationalized that, if fractionation occurs during transpira-
tion (in the evaporation step), then the concentration of
HTO would increase at and near the evaporative surface.
This increasing concentration of HTO at the evaporative surface
would cause a concomitant increase in the amount of HTO evapora-
ted (which is proportional to the concentration) until a
steady state is reached wherein the loss of tritium would
be exactly equal to the supply of tritium. Under conditions
of rapid water loss, the accumulation of HTO would be restricted
to the evaporative surface and very little diffusion of enriched
HTO into the surrounding leaf tissue would be expected.
As a consequence, the overall enrichment of tritium would
be very small. Under slow transpirative conditions, tritiated
water enriched at the evaporative surface would be expected
to diffuse into surrounding tissue, thus raising the overall
tritium concentration in the leaves to a higher level. Diffusive
movement away from the evaporative site and mass flow of
tritium in the phloem may result in increased concentrations
in the nontranspiring tissues. Consequently, the transpiration
rate, not only determines the rate of buildup of HTO at the
surface, but also the amount of HTO present in the leaves
and other organs. This rationale has led to the hypothesis
that fractionation of water may only be evident under certain
conditions, i.e., low transpiration. Fractionation is not
normally observed because even though evaporation is a diffusive
phenomena (subject to fractionation), the supply of water
to the evaporative site is by mass flow (no fractionation)
18
-------
which entails a more rapid velocity of water molecules moving
toward the evaporation site than the diffusive movement
of HTO molecules moving in the opposite direction.
'To test this theory, we grew alfalfa plants in hydroponic
solutions containing HTO in closed environmental simulators .
Only the humidity in the simulators was altered to vary the
rate of transpiration. The plants were exposed to 8 hours
of constant humidity before sampling to ensure that equilibrium
had been obtained. The leaves were separated from the stems,
and water was extracted from the respective tissues. The
tritium concentration in the extracted water was determined
by liquid scintillation counting. The results which are
listed in Table 4 indicate a significantly higher tritium
concentration in the water of the leaves collected under
slow transpiration conditions. This slightly higher level
is thought to be evident because of diffusion of HTO-enriched
water away from the evaporative surfaces into the surrounding
mesophyll tissue. Despite this fractionation at the evaporative
surface, under most experimental or environmental conditions
any fractionation occurring in the process of transpiration
would be so small as to make its contribution to biological
processes insignificant.
TABLE 4. TRITIUM CONCENTRATION IN FREE WATER OF ALFALFA
STEMS AND LEAVES UNDER CONDITIONS OF SLOW AND
RAPID TRANSPIRATION
nCi/ml
Leaves
Stems
Hydroponic Solution
SLOW TRANSPIRATION
(High humidity > 70%)
77.2 + 0.6*
75.4 + 0.5
75.0+0.6
RAPID TRANSPIRATION
Low humidity > 25%)
75.4 + 0.5
75.8 + 0.5
75.1 + 0-5
* Significantly different from other means at the 0.05 level
.based on paired T tests.
+ The 95% confidence interval
The thermodynamic basis for discrimination among the
hydrogen isotopes in the synthesis of organic molecules was
established by Urey and Rittenberg (1933) and expanded by
Bigeleisen (1949) and Waterfield et al. (1968). The observations
of fractionation of tritium between supply (free water) and
incorporation in the organic molecules in plants were reviewed
by Bruner (1973) and Weston (1973). These reviews list examples
19
-------
of both concentration of and discrimination against tritium
in organic incorporation. Since tritium is heavier than
protium, it is difficult to imagine a condition where it
would be incorporated at a faster rate than protium. In
reactions such as the elimination of hydrogen from a particular
site (e.g., in metabolic processes), the heavier isotopes
are transferred at lower rates than hydrogen. As a result,
an elevated tritium level could develop in certain fractions.
Subsequent synthetic reactions based on these fractions as
substrates could then lead to compounds with a higher tritium
content than the bulk water supply.
Smith and Epstein (1970) demonstrated that all plants
tested gave a similar fractionation ratio between protium
and deuterium where the source of water was common to all
species. They also showed that although very little discrimina-
tion against deuterium occurred at the cell membrane, the
photosynthetic splitting and incorporation of the water fragments
into organic compounds resulted in a fractionation ratio
of 0.958, or in other words, there was a discrimination of
4.2%. An additional fractionation of about 9.2% occurred
between carbohydrate and lipid. The overall fractionation
from water to lipid ranged from about 13% to 17%. Fat synthesis
was previously singled out by Bokhoven and Theeuwen (1956)
as a process resulting in a considerable isotope effect.
The same authors could also distinguish synthetic and fermented
alcohol by the lower deuterium content of the latter due
to the combined discrimination effects against deuterium
in various metabolic processes.
Very high fractionations have been reported by some
authors. For instance, Weinberger and Porter (1953) measured
tritium incorporation in Chlorella pyrenoidosa and reported
that the specific tritium activity in this aquatic plant
species was only 47% of the specific activity of the water
pool. Later, the same authors (1954) reported fractionation
between various organic fractions. Their method employed
extracting components using methanol and ether and isolating
cells by repeated resuspensions in water. Unfortunately
this method allowed ample opportunity for tritium to exchange
with the hydrogen in the solvents. Consequently, fractionations
which they reported are probably unrealistically high.
In an attempt to evaluate the extent of fractionation
possible in plants, McFarlane (1976b) grew alfalfa plants
in a sealed growth chamber. In these experiments great care
was taken to assure that equilibrium conditions persisted
throughout the growing period. It was shown in these experiments
that a discrimination of 22% occurred against the incorporation
of tritium into the total organic complex which made up the
leaves and stems.
20
-------
It is evident from the reported results that the extent
of fractionation in plants and its importance as a possible
means of concentrating or reducing tritium concentrations
is small. Therefore, future research should be directed
not to merely observe and document fractionation between
water and plants, but rather to determine the sites and the
extent of fractionation.
TRITIUM IN AQUATIC PLANTS
In aquatic systems the retention and turnover of tritium
in vegetation depend on the kind and duration of exposure,
which in turn depend on the type of insult (chronic or acute)
and the nature of the water body. For instance, in a large
turbulent river contaminated by a single pulse of tritium,
rapid dilution will occur and incorporation of tritium into
aquatic plants will be restricted to a relatively short period.
Consequently, the exposed plants would have a very short
accumulation period followed by a retention period which
would depend primarily on the morphological characteristics
of the plants. On the other hand, a continuous release of
tritium into a lake or other relatively stable body with
a slow water turnover rate would result in a steady tritium
accumulation by plants in equilibrium with the water.
Most information dealing with tritium in aquatic systems
focuses on incorporation into aquatic animals; only a few
published reports are concerned with the uptake by aquatic
plants. Weinberger and Porter (1954) reported discrimination
against tritium incorporation in the biomass formation of
Chlorella pyreno idosa grown in tritiated water. However,
these results should be regarded with caution, as explained
previously. Cohen and Kneip (1973) determined the tritium
concentrations in water, sediments, and aquatic plants downstream
from a nuclear reactor. Their data showed that "In the aquatic
environment, there is significant incorporation and retention
of tritium in the bound state of bottom sediment and biota."
Tritium concentrations when compared to the concentrations
in the contaminated water were, on the average, 10 times
as high in sediments, 4 times as high in fish, and 3 times
as high in rooted aquatic plants. Their conclusion was that
plants may derive much of their hydrogen from the organic
sediments on the river bottom. Unfortunately, they did not
account for the impact of changing ambient tritium concentrations
during different periods of time or at different locations
in the river.
On the Nevada Test Site, Typha and Potamogeton samples
were collected in Raines Pond by Brown (1971). This pond
was formed by water seepage originating near an underground
nuclear detonation, and contains a relatively high amount
of tritium. Analysis of the two plant species showed a higher
tritium activity in the free water and organic bound fractions
21
-------
of both species than in the pond water. Although it would
be tempting to assign this difference to isotopic fractionation,
no valid interpretation of these results can be made without
more details of the history, the water turnover rate and
the contamination source. It is this temptation to draw
conclusions based on incomplete data that has particularly
plagued aquatic-tritium studies. The authors can find no
compelling evidence in the literature of any large tritium
fractionation in aquatic systems. To the contrary, it appears
clear that free water fractionation does not exist in aquatic
plants. However, fractionation against organic incorporation
in the range of 10% or 20% would be expected.
22
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EFFECTS
The effects on plants of tritium incorporation can be
caused by the mass difference, the beta particle emission,
or the properties of the decay product, helium-3, which differ
vastly from the properties of hydrogen.
Deuterium is the ideal tool to study the role of mass
difference because it differs from protium and tritium only
in mass and is not radioactive. As far back as 1937, deuterium
was reported as retarding respiration in yeast (Taylor and
Harvey, 1937). Germination of seeds and spores and the growth
of Chlorella were shown to be inhibited in proportion to
the concentration of D20 in the culture solution by Crumley
and Meyer (1950) and Moses et al. (1958). Weinberger and
Porter (1954) observed Chlorella cell enlargement in the
presence of D20, and additional evidence by Bennet et al .
(1958) pointed out that this can occur in the absence of
cell division. They concluded that D20 was only inhibiting
the cell division process. They also observed that cells
could be acclimated to D20 by gradually increasing its concentra-
tion in the culture medium. Blake et al. (1968) suggested
that the effect of deuterium on embryo development was ameliora-
ted by the presence of large hydrogen-containing food reserves
in the seed. The same researchers (Crane et al . 1969) later
separated the embryos from various seeds and showed that
D20 inhibited all species in the same manner.
Biological effects caused by deuterated water molecules
are probably associated with a change in the average free
energy of metabolic water. Calculations of water potentials
from the vapor pressures reported by Jones (1968) give the
values listed in Table 3. It is obvious that putting tissue
into pure D20 would cause a tremendous osmotic shock, i.e.,
at 25°C it would be comparable to immersing the tissue sample
into a solution more concentrated than 7M NaCl. Since the
water potential (¥„) equals the sum of the component potentials,
the potentials of mixtures of D20 (or HTO) with water are
proportional to the abundance of D20 (or KTO) ;
where ¥j and ¥, represent the water potentials and a1 and a2
represent the fractional portion of the solution volume
represented by H20 and D20 (or HTO), respectively.
23
-------
When HTO is added to H20, it is clear that the water potential
of the solution is
¥ = ¥ HTOa
w
since by definition *H20 = °
Thus, when evaluating experiments conducted with high levels
of D20 (or HTO), considerable attention should be given to
the water potential gradient imposed by the treatment. Unfortu-
nately, this aspect has generally been overlooked. Available
literature indicates that at concentrations of D20 less than
1%, effects are not generally evident. This seems to support
the idea expressed above and may mean that insults do not
take place, or on the contrary may mean that our detection
systems are not sensitive enough to observe their manifestations.
In contrast to most deuterium studies, the concentrations
of tritium found in the environment, and in most experiments,
are such a small fraction of the total water content that
the contribution of tritium to altering the average molecular
free energy is generally insignificant and can be ignored.
For example, it can be shown by using the specific activity
of tritium (Ci/g), Avogadro's number (molecules/mole), and
the molecular weights of HTO and H20 (g/mole) that a solution
of 1 microcurie per milliliter (yCi/ml) (which is much higher
than environmental levels, but representative of some experimental
concentrations) contributes only one molecule per 1.83 X 1011
molecules of H20. Thus the significant effects of tritium
on biological systems are probably not due to mass differences,
but are rather due to the beta radiation resulting from decay
and/or due to the decay product.
The effect of ionizing radiation (x-rays) on bean roots
has been one of the classical demonstrations of radiation
effects. Many outstanding works on this subject were authored
by Gray, Thoday, Read, and Scholes in the British Journal
o_f Radiology from 1942 to 1952. Their work showed that the
apical meristem of the root was radiosensitive and was therefore
a good system to study relative biological effectiveness.
Spaulding et al. (1956) repeated Gray's techniques and compared
the effects of x-rays to the damage found in plants exposed
to beta radiation from HTO. They immersed bean roots in
HTO and calculated the dose from diffusion time and the specific
activity of the treatment solution. Exposure times were
from 1 to 4 hours an.d no consideration was given to residual
tritium which became part of the organic molecules. Their
calculations showed a relative biological effectiveness of
1.0 + 0.06 according to the equation:
- effect of B dose
'
effect of x-ray dose
24
-------
They concluded that in their system, beta radiation derived
from the decay of tritium in the plant water had the same
effect both quantitatively and qualitatively as did 175
kilovolt peak x-rays applied externally.
In the process of preparing tracers by exposing soybeans
to carbon-14 labeled carbon dioxide and HTO, Chorney et al.
(1965) observed effects on the growth rate and gross morphology
of the soybean plants. Somatic aberrations caused by tritium
resulted in characteristic bulbous enlargements at the nodes
and below the terminal influorescence and the leaves were mottled
just as in other radiation treatments. These observations were
assigned to a calculated accumulated dose of 1,000 rads which
resulted from growing the plants in culture solution containing
37.5 yCi/ml of tritium. The differences in growth rates between
the treated plants and the controls were reported, but these
data are of a questionable value since in the hermetically sealed
growth chambers the carbon dioxide (C02) concentrations were
allowed to fluctuate between 50 and 1,000 parts per million
(ppm) while the control plants were grown in atmospheric levels
of C02. Seeds from the plants exposed to tritium contained
17.6 {iCi of tritium per gram and, when stored for 44 days and
then germinated, a pigment abnormality was observed in the primary
leaves .
The continued culturing (18 months ) of alfalfa plants
in a closed environmental simulator containing 300 nanocuries
per milliliter (nCi/ml) of tritium as HTO revealed neither detect-
able morphological damage nor alteration of any physiological
parameter of the plants (McFarlane 1975). However, using an
extremely sensitive indicator of somatic alteration, Vig and
McFarlane (1975) have shown genetic effects in soybean plants
when the seeds were germinated in water containing as little
as 10 nCi/ml of tritium.
When a beta particle leaves an atom, a resilient energy
is imposed on the atom, called recoil energy. In the decay
of 32P this energy is sufficient to cause bond breakage. In
tritium decay, the maximum recoil energy is too small to have
much effect on chemical bonds (Woodward, 1970). The decay product
of tritium is helium-3 which is a noble gas with vastly different
chemical properties than its parent. The replacement of the
newly formed helium with stable hydrogen has been thought to
occur with sufficient ease to cause little or no effect. This
logic has led to the assignment of all damage caused by tritium
in biological systems, whether somatic or genetic, to the effect
of the radiated 3 megaelectron volts beta particle. One experiment
which argues against this rationale was conducted by Funk and
Person (1969). They showed that decay of tritium in the 5 position
of cytosine resulted in a specific mutation. This specificity
argues in favor of assigning the effect to transmutation of
tritium to helium which causes interruption of the coding sequence
at a particular point. When helium is formed, it immediately
25
-------
leaves the site formerly occupied by the tritium atom, and
the atom formerly bound to the tritium atom becomes a free
radical. Free radicals are usually highly reactive, unstable
species which can stabilize by combining with another free
radical or by intramolecular rearrangement. This is often
accompanied by the elimination of an atom or molecular fragment,
and the formation of a double bond in the parent molecule.
Therefore, it should be remembered that the possibilities
of damage other than direct radiation damage have largely
been ruled out by hypothesis and not by test.
Radiation doses from environmental tritium are very
low. The Federal Radiation Council cites 170 millirems per
year as the recommended maximum dose from all sources except
medical radiation and natural background for the human population,
This conservative figure is designed to be below the dose
which could cause any damage to man. Using a quality factor
of 1.0, the recommended maximum dose from tritium by man
would be 170 millirads if tritium were the only source of
radiation. Near the Humboldt Bay Pacific Gas and Electric
nuclear power reactor, plant samples were collected which
contained up to 3.8 nanocuries per liter of tritium in the
extractable plant water. If this were a chronic contamination
level, the plant would have an absorbed dose of 0.00044 millirads
per year. Compared to the 170 millirads suggested as safe
for man, this seems to be an insignificant dose. Compared
to the 1,000 rads used by Spaulding et al. (1956) in their
experiments, even the highest doses which were observed in
plants near that reactor were miniscule. This is not an
endorsement of the hypothesis that no effects would occur,
but, if present, it is probable that they would not be observed
because of their infrequence.
26
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PITFALLS
Many pitfalls await the investigator studying tritium
in biological systems. Experimental errors are generally
associated with the isotopic fractionation effect, and most
commonly occur during the steps of sample collection, storage,
and preparation for analyses. It has already been pointed
out that the tritium bound to oxygen as in water, alcohols,
organic acids, carbohydrates, etc., exchanges with hydrogen
bound to oxygen. Therefore, if a tritium-containing plant
sample is in contact with moist air at any time between the
time of collection and the time of tritium analysis, sufficient
exchange with atmospheric water vapor may occur to invalidate
the analysis. This is even more critical when the samples
are in contact with solvents which contain exchangeable hydrogen.
Not only is the plant water affected but tritium atoms incorpora-
ted into organic molecules as hydroxyls are also readily
exchanged (Lang and Mason, I960). Under appropriate conditions
this could amount to a tritium loss of up to 30% from the
organic plant material.
Tritiated water is often collected from plant, animal,
and soil samples by freeze-drying or other methods of vacuum
extraction. The fractionation between HTO and H20 occurring
during this step under some conditions may account for large
errors. Because the degree of fractionation is inversely
proportional to the temperature of the evaporating solution,
the error is larger when the sample is chilled or frozen.
Even without intentional freezing, sample temperatures always
decrease during vacuum vaporization unless the heat loss
due to the heat of vaporization is compensated for by heating
the sample. Figure 4 shows the temperature fluctuations
of plant and soil samples during vacuum vaporization extraction
at an ambient air temperature of approximately 25°C. Water
in both kinds of samples quickly froze when the vacuum was
applied (0.1 millimeters of mercury); then the temperature
gradually increased until the samples were completely dry.
The water was collected from these samples in 5-ml increments
and each increment was analyzed for tritium content. Figure
5 shows the relationship between the tritium activity in
each increment and the total amount of moisture removed.
From these data, correction factors could be used to calcu-
late the true tritium concentration of the samples. However,
this procedure is risky when high accuracy is desired, since
sample type and size, rate of water vaporization, and temperature
all" independently influence the degree of fractionation.
Using a vacuum extraction system, the only way to assure
an accurate analysis of extractable water appears to be by
i
27
-------
p 30
co 20
UJ
10
0
10
C9
0 10 20 30 40 50 60 70 80 90 100
% MOISTURE EXTRACTED
FIGURE 4. Temperature of a lOOg soil sample (....)
and a 150g plant sample ( ) during
vacuum extraction of water (McFarlane
1975c).
3120-
10 20 30 40 50 60 70 80 90 100
X MOISTURE EXTRACTED
FIGURE 5. Tritium in water samples collected in
5ml increments from soil (....) and plant
( ) samples by vacuum extraction
(McFarlane 1975c).
120-
0 10
-i
40
20 30 40 50 60 70
% MOISTURE EXTRACTED
FIGURE 6. Fractionation of tritium in the water
extracted from plants (— ) and the
calculated concentration remaining in the
plant tissue (....) (McFarlane 1975c).
collecting 100% of the tissue water.
Even this may result in errors,
if organic tritium is part of the
desired data. In Figure 6 it is
shown that the tritium concentration
of the free water remaining in
the sample increases during the
extraction up to the concentration
of the last fraction. This may
be 30% higher than the original
tritium concentration in the free
plant water. Since the tritium
in water can readily exchange with
up to 30% of the hydrogen atoms
in the organic fraction, an artificial
tritium enrichment (up to 9%) of
the organic fraction could occur.
Obviously the tritium content of
the collected free plant water
would be lowered by the amount
transferred to the organic fraction.
A preferred method for critical
work is based on the formation
of an azeotropic mixture between
the free sample water and a suitable
organic solvent such as toluene
or benzene. An excess of dry solvent
is added to the sample, the azeotrope
is recovered by distillation and
the tritiated water separated from
the solvent upon cooling. This
method is reportedly less subject
to isotopic fractionation (Cline,
1953; Moghissi et al. 1973b).
Another type of error which
is also based on isotopic fractionation
became apparent during experiments
with HTO conducted in an environmental
simulation chamber. The chamber
humidity was controlled by a coil
cooled to 10°C and the condensate
dripping from the coil was analyzed
for tritium as an indicator of
the HTO concentration in the chamber
air (Table 5, Column B). In a
control experiment, air from the
chamber was passed through a molecular
sieve tap and the water extracted
by azeotropic distillation; another
air sample was passed through freeze
traps and the water pooled for
28
-------
analysis. Both control experiments gave identical results
and are believed to be the true values for tritiated water
vapor (Table 5, Column A). In a third part of the test,
chamber air was passed through a tap water-cooled condenser
(cooling water temperature approximately 20°C) for partial
condensation followed by freeze traps which collected the
remaining water vapor (Table 5, Column C and D). The data
show that isotopic fractionation occurred whenever partial
condensation occurred. Variations of the relative humidity
in the chamber did not reveal any particular trend in the
analytical^results suggesting that the condenser temperature
(10° or 20 C) were more important than the condensation
rate. The difference between Column B and C is qualitatively
in line with the expected temperature effect on the vapor
pressure ratio of H20/T20 as illustrated in Figure 1.
TABLE 5. TRITIATED WATER VAPOR COLLECTED BY DIFFERENT METHODS
EXPRESSED AS PERCENT OF THE CONTROL VALUE
Cold Water Condenser
Relative Humidity Control Cold Water Freeze Trap
Humidity Control Equipment Condenser Following
ftj . Xtt (c) (D
35%
55%
90%
100
100
100
114 + 2
113 + 4
118 + 3
111 + 1
108 + 2
107 + 2
95 + 1
90 + 9
96 + 4
(a) Control Water collected by freeze trap and by molecular sieve (both, metliods
identical results). Arbitrarily set at 100%.
(b) Condensed water was collected from the humidity control equipment 0-0 CJ..
(c) Air was drawn past a cold water condenser (20 C) and then through
(d) a freeze trap.
+ Standard deviation of replicate analysis
These examples show that the failure to consider and apply all
of the appropriate experimental controls and a failure to take into
account all of the possibilities of isotopic fractionation can easily
cause errors of up to 30% in tritium research.
29
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LITERATURE CITED
Aronoff, S. and Choi, Ivan C. "Specific Activity of Photo-
synthetic Sugars in Soybean Leaves Equilibrated with Tritiated
Water," Arch. Biochem. Biophys, 102, pp 159-160 (1963).
Bennett, E. L., Calvin, M., Holm-Hansen, 0., Hughes, A. M.,
Lonberg-Holm, K. K., Moses, V., Tobbert, B. M., Effect of
Deuterium Oxide (Heavy Water) on Biological Systems, UCRL
3981, pp 199-202 (1958).
Bigeleisen, J., "The Validity of the Use of Tracers to Follow
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TECHNICAL REPORT DATA
(Please read Instructions on the reverse before completing)
1. REPORT NO.
EPA-600/3-76-052
3. RECIPIENT'S ACCESSION-NO.
4. TITLE AND SUBTITLE
TRITIUM IN PLANTS AND SOIL
5. REPORT DATE
May 1976
6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)
J. C. McFarlane, W. F. Beckert, and K. W. Brown
8. PERFORMING ORGANIZATION REPORT NO.
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring & Support Laboratory
Office of Research and Development
U.S. Environmental Protection Agency
Las Vegas, NV 89114
10. PROGRAM ELEMENT NO.
1FA628
11. CONTRACT/GRANT NO.
12. SPONSORING AGENCY NAME AND ADDRESS
Same as Above.
13. TYPE OF REPORT AND PERIOD COVERED
Final
14. SPONSORING AGENCY CODE
EPA-ORD, Office of Health
& Ecological Effects
15. SUPPLEMENTARY NOTES
16. ABSTRACT
This is a review of literature regarding the fate and consequences of
tritium in plants and soils. The kinetics of tritium in plants and soils
was reviewed in light of water potential terminology, and some original
research data are enclosed to illustrate specific concepts. The review
cites 70 articles.
7.
KEY WORDS AND DOCUMENT ANALYSIS
DESCRIPTORS
b.lDENTIFIERS/OPEN ENDED TERMS C. COSATI Field/Group
Fractionation
Tritium
Hydrogen
Plant physiology
Soil physics
Radioactive isotopes
Tritium in plants and
soils
Isotopic fractionation
Pollutant pathways
Q6C
08M
18B
8 DISTRIBUTION STATEMENT
RELEASE TO PUBLIC
19. SECURITY CLASS (ThisReport)'
UNCLASSIFIED
21. NO. OF PAGES
40
20. SECURITY CLASS (Thispage)
UNCLASSIFIED
22. PRICE
EPA Form 2220-1 (9-73)
*GPO 691-306—1976
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