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Translocation and Metabolism of
C14 -1-'abeled Tetranzine by Douglas-Fir,
Orchard Grass, and Blackberry
BY
M. A. RADWAN
Abstract, Absorption, translocation, and metabolism of C14-labeled tetramine were in­
vestigated in Douglas-fir, orchard grass, and blackberry. Applications were made to the
foliage and in nutrient culture solutions under controlled conditions, and the tracer was
followed in treated plants with autoradiography and counting techniques. C14-labeled tetra­
mine was absorbed from nutrient solution by roots of all three species. Transport occurred
via the transpiration stream, and translocation was slowest in Douglas-fir. After uptake by
roots and deposition in plant tissues, the tracer did not recirculate within the plants. Im­
mobility (nonsystemic properties) of the chemical was also demonstrated by lack of down­
ward movement after foliar applications. 04-labeled tetramine was metabolized in the
plants, and degradation was highest in orchard grass and lowest in Douglas-fir. Tetramine's
toxicity, possible hazards in its use, and its nonsystemic properties suggest that use of the
chemical to protect tree seedlings from animals does not look promising.
THE
sEARCH for a systemic chemical
with a potential for protecting forest tree
seedlings from animals led wildlife biolo­
gists to the toxic chemical, tetramethylene­
disulphotetramine (tetramine). Bioassay
studies, as well as pen and field tests, were
conducted to evaluate the chemical
(Spencer 1954, Kverno 1960). However,
these investigations were largely limited to
conifers, and methods used did not accur­
ately determine the chemical's mobility or
its metabolic fate in plants.
This paper presents studies of translo­
cation and metabolism of C14 uniformly
labeled tetramine (tetramine*)1 in three
plant species and discusses the results in
relation to possible use of the chemical for
animal damage control. A preliminary
account of the work has been reported
elsewhere (Radwan 1966).
1 Radioactive tetramine (2.02 mc/mM) was pre­
pared by Tracerlab, Waltham, Mass.
Reprinter1 from
Materials and Methods
Douglas-fir (Pseudotsuga menziesii (Mirb.)
Franco), orchard grass ( Dactylis glomer­
nta L.), and blackberry (Rubus ursinus
Cahm. & Schlecht.) were the test species.
These plants represented gymnosperms,
monocotyledonous angiosperms, and dico­
tyledonous angiosperms, respectively.
Growth of plants. Seed from individual
lots of Douglas-fir and orchard grass and
cuttings from one blackberry plant were
grown in sand until plants reached a
height of 3 to 5 inches. Plants, in pairs,
were then transferred to quart mason
jars, covered with aluminum foil and
containing Hoagland's nutrient solution 1
(Hoagland and Arnon 1950) with 3 ppm
of chelated iron, and placed in a plant
The author is Plant Physiologist, Pacific
Northwest Forest and Range Expt. Sta., Forest
Service, U.S. Dept. Agric., Portland, Ore. Manu­
script received Sept. 19, 1966.
FoRES'l' SCIENCE, Volume 1 3, Number 3, September, 1967
Purchased by the U. S. Forest Service for official use.
growth chamber under controlled condi­
tions. Temperatures in the chamber were
26 ° C during the day and 16 C at
night. Root zone temperature remained
constant at 19° C. Relative humidity
ranged from 70 to 90 percent, and
fluorescent-incandescent light at level of
plant leaves was controlled at 1,800 ft-c
on a 14-hr photoperiod. Solutions were
continuously aerated, pH was maintained
at 5.0, and cultures were allowed to accli­
matize for at least 10 days before treat­
ment.
o
Treatment of plants. Tetramine* was
used. The tracer was applied to the roots
via the nutrient solution at the rate of 17
/J-C in 2.0 mg tetramine* per jar contain­
ing 800 mi. Foliar applications were 0.42
/J-C each, in 50 �-tl of solution containing
5 0�-tg tetramine* and 0.1 percent "Tween
20" surfactant. Solution was applied with
a micropipette to one mature leaf of grass
and blackberry and to a group of mature
needles of Douglas-fir.
Effect of time on uptake by roots and
distribution of tetramine* in the three
species was determined in a time series.
Plants were removed from treatment so­
lutions and processed after 2, 6, 8, 24,
9 6, 168, 336, 504, and 588 hours.
Studies of tetramine* mobility within
plants were made in two ways. First,
tetramine* was applied to the foliage and
plants were harvested after 7 days; sec­
ond, plants were root treated with tetra­
mine*-6 hours for grass and blackberry
and 48 hours for Douglas-fir. Following
treatment, roots were washed and plants
were transferred to tetramine-free nutri­
ent solutions. Cultures of each species
were divided into two groups of two
cultures each. Seedling grass and blackber­
ry plants of the two groups were har­
vested after 7 and 14 days, respectively;
those of Douglas-fir were harvested at 30
and 60 days.
Blackberry plants were root treated for
48 hours to determine distribution of te­
266 I Forest Science
tramine* in flowering plants. One group
of plants showed flower buds at time of
treatment; the other group was treated
before flowering. Following treatment,
roots were washed and plants were trans­
ferred to tetramine-free nutrient solu­
tions. Plants were harvested when flower­
ing was complete.
At time indicated for each experiment,
roots were thoroughly washed in distilled
water and blotted with soft tissue.
Treated leaves were partly covered with
masking tape to prevent contamination
during further processing. Intact plants
were then quick-frozen with crushed solid
C02 and freeze-dried at -15° C (Pallas
and Crafts 1957).
Two lyophilized plants from each
treatment were divided into roots and
shoots. In the mobility experiment where
plants were root treated, shoots were fur­
ther divided into new and old growth.
Resulting parts were individally ground to
40 mesh in a Wiley mill and stored until
counted. Remaining two plants of each
treatment were used for autoradiography
but were divided and ground as before
after their autoradiograms were obtained.
Metabolism of tetramine* was studied
by ( 1) chromatography of extracts and
(2) collection of carbon dioxide.
1. Plants of the three species were root
treated for 7 and 28 days. Harvested
plants were washed, divided into roots and
shoots, and resulting parts were cut into
small sections and extracted with acetone
for 144 hours in Soxhlet apparatus. Ex­
tracts were concentrated and chromato­
graphed on Whatman No. 1 paper by the
ascending technique. The paper was pre­
.wa hed with 2N-HC1, distilled H 20,
and C2 H50H. Following spotting, the
paper was impregnated with 35-percent
formamide in methanol and developed in
chloroform saturated with formamide
hours
12
for
equilibration
after
(Heftmann 1961).
2. Plants of each of the three species
were root treated for 7 days, washed, and
transferred to tetramine-free solutions as
EFFECT OF TIME EXPOSURE ON TRANSLOCATION
OF TETRAMINE FROM ROOTS TO SHOOTS
0
=
::t
UJ
:::>
V>
V>
t=
10
0
:::t:
V> >c:::
Cl lJ_
0
::;:
(!)
c:::
UJ
0...
UJ
1:::>
:z: 120 110
100
90
80
Orchard grass
::E
c:::
UJ
0... 60
:z:
:::>
0
c..:> 50
40
Douglas-fir
EXPOSURE TIME
( hours )
FIGURE 1. Effect of time of esposure on translocation of tetramine* from roots to sl10ots of three plant species
grown in nutrient solution containing 2.5 ppm of the tracer.
before. Spaces between the plants and the
jar lid and between the lid and jar were
completely sealed with modeling clay to
prevent any escape of COz from the
nutrient solution. Plants were placed un­
der bell jars in the dark for 24 hours.
Carbon dioxide-free mr was passed
through the bell jars, and COz in the
outlet air was trapped in COa-free O.SN­
NaOH. Carbonate that formed was pre­
cipitated as BaC140s, and the precipitate
was filtered on filter paper disks, washed,
volume 13, number 3, 1967 I 267
)
/
FIGURE 2. Comparative uptake and distribution of tetramine* in orchard grass, blackberry,
and Douglas0/ir. Tracer was administered via nutrient solutions containing 17 w tetramine*
per 800 ml solution, and treatment was for 8 hours. Autoradiograms appear above the mounted
plants. Notice retmtion of activity by roots and concentration of tracer in older leaves and tips
qf grass blades.
and counted (Comar 1955). Cultures
with untreated plants served as check.
methods. The autoradiographic
method (Yamaguchi and Crafts 19 58)
was used as a qualitative estimate of C14•
In each case, plants were mounted on
paper and placed against Kodak no-screen
X-ray film for 4 weeks at room tempera­
ture. Paper chromatograms were also ex­
posed to film in a similar manner. Fol­
lowing exposure in each case, films were
developed by standard procedure, and re­
sulting autoradiograms were examined.
Activity in the ground tissue and in the
BaC140a was quantitatively determined
with a thin-window gas-flow Geiger­
MUller tube and Tracerlab "Versa/Mat­
ic" scaler. In some cases, activity was
determined by counting directly on the
paper of the chromatograms containing
Assay
268 I Forest Science
the spots. All counts were appropriately
corrected for background and selfabsorption.
Results and Discussion
Effect of time on uptake and distribution.
The quantitative count data, summarized
in Figure 1, were confirmed by autoradiog­
raphy. Only plants treated for 8 hours
and their autoradiograms are presented
(Fig. 2).
The tracer was translocated into the
tops at different rates in the different
species. In 2 hours (Fig. 1 ), Douglas-fir
translocated a very small amount of the
tracer to the shoots; blackberry and grass,
respectively, moved two and six times as
much labeled material. 0 nly after 24
hours did appreciable activity appear in
shoots of Douglas-fir. This slow translo­
cation in Douglas-fir was probably due to
its characteristic limited root system (less
absorbing area), smaller area of transpir­
ing foliage, and lack of vessels compared
with the other species. These characteris­
tics probably restricted uptake and trans­
port in the xylem.
As absorption and translocation contin­
ued, the level of activity in the tops in­
creased in all three species until a plateau
was reached during the last two exposure
periods of the time series. At this time,
Douglas-fir showed the slowest absorption
and translocation, and blackberry exceeded
grass in moving the tracer to the tops.
Rapid translocation in blackberry was
probably due to greater transpiration, as
transpirin foliage increased more rapidly
than that of grass.
Examination of the autoradiograms
showed that the tracer acquired from the
nutrient solutions was retained in roots of
all seedlings to a much higher concentra­
tion than was moved into the tops. It was
not determined, however, whether activity
was from absorption on root surfaces or
accumulation within the roots.
The autoradiograms also showed ac­
cumulation of activity in older leaves and
in the older parts of those leaves (Figs. 2
and 5) in areas where transpiration was
presumably rapid. This observation sug­
gests that the tracer was transported up­
ward in the transpiration stream, and
retranslocation out of older leaves via the
phloem did not occur.
M ability within plants. Tetramine* was
applied to the roots in nutrient solutions
and to the foliage. In each case, move­
ment of the tracer was determined fol­
lowing growth of treated plants in tetra­
mine-free solutions.
The mobility of the tracer within the
three species following root applications
is shown in Table 1. Initially, some
movement of activity occurred in the new
growth of all test species. This activity
did not appear to be derived from residual
tracer in the old growth. Old growth of
Douglas-fir and blackberry showed only a
small decrease in activity; that of grass
showed a considerable decrease, but this
was probably due to rapid metabolism of
the tracer (see later) and not to move­
ment into new growth. This suggests that
activity in new growth may have resulted
from transport of excess tracer in or on
the roots.
As growth continued in tetramine-free
solutions, old growth contained less activi­
ty due to continued dilution by growth
TABLE 1. D;stri!mt;on of act!Vztv in shoots of the three plant species immediately after
root treatment and followin[[ two periods of growth in tetramine-free solutions.
Old growth1
Plant species
Immediately
following
treatment
-----
Douglas-fir
Blackberry
Orchard grass
11,026± 190
5,389±129
9,575±150
After first
gmwth period
without
tett·amine
New growthl
After second
growth period
without
tetramine
After first
After second
growth period growth period
without
without
tetramine
tetramine
Count.< per minute per gram dry s!zoot tisstte2
3,224±165
5,103±130
10,249±1R6
2,426±120
5,202±119
3,976± 108
1,583± 75
2,193± 90
3,585± 96
-------
541±79
780±70
775±60
1 Treatments were 48 hours for Douglas-fir and 6 hours for blackberry and grass, First growth periods were
30 days for Douglas-fir and7 days for blackberry and grass. Second growth periods were an additional30 days
fo1· Douglas-fir and another7 days for blackberry and grass.
2 Averages of four replications and means are followed by the standard error.
volume 13,"number 3,..1967 I 269
B
A
•
FIGURE 3. Autoradiograms of Douglas-firs slwwing redistribution of tetramine*. Seedlings
were treated (see Fig. 2 caption) for 18 hours before tliey were transferred to tetramine.jree
solution and allowed to grow for 30 days (A) and for 60 days (B). In eacl1 autoradiogram, a
is image of new growth and b is image of old growth.
I
•
\
\
- ---
\
..
\.
/
FIGURE 4. Comparative movement of tetramine* from leaves of orclwrd grass, blackberry, and
Douglas-fir. Leaves were treated for 7 days with 50 JJ.g tetramine* in 50 JJ.I of acetone solution.
Upper section shows autoradiograms of treated plants slwwn in lower section, and arrows
indicate treated leaves and tl1eir images.
270 I Forest Science
A
FIGURE 5. Autoradiograms of blackberry plants showing distribution of tetramine* in the flowers.
Plants were treated (see Fig. 2 caption) for 48 l1011rs before IIIey were transferred to teframine-free
solution. Autoradiograms in A and B are from plants treated before and after flowering, respectively.
Arrows point to images of tl1e flowers.
.and degradation of the tracer. In the
meantime, new growth showed consider­
able decrease in activity (Table 1), and
images on the autoradiograms became
barely visible (Fig. 3).
Tetramine*, therefore, is nonsystemic
(immobile) in the test species. Once tetra­
mine* is deposited in the tissues, recircula­
tion does not appear to occur.
Very limited movement out of treated
leaves occurred (Fig. 4). Grass showed a
typical apoplastic movement. Similar lim­
ited movements probably occurred in the
other two species, but detection of the
tracer was not possible because of the
shorter leaves of the species and the
masking tape covering treatment areas.
Apoplastic movement toward the
treated-leaf tip in direction of the transpi­
ration stream and absence of basipetal
movement present additional evidence of
immobility of tetramine* in the plants.
Foliar applications of tetramine, there­
fore, would not result in appreciable pene­
tration and distribution within treated
plants. Furthermore, tetramine in its trans­
location pattern resembles the urea and
triazine herbicides, certain surfactants,
and the mineral elements calcium, mag­
nesium, and strontium (Crafts and Ya­
maguchi 1964)
.
Distribution in flowering plants. Appreci­
able activity appeared in flowers of black­
berry plants which were treated after
flowers had formed. The tracer was
present in all parts of the flower, especial­
ly the calyx (Fig. 5B). Flowers formed
after treatment, however, showed only a
trace of activity (Fig. SA).
Availability of tetramine to the roots
during flowering, therefore, is essential if
considerable amounts are to appear in
flowers. Activity acquired before flower­
ing is not available for redistribution and
depositon in new flowers because of the
chemical's immobility.
Metabolism in plants. Figure 6 shows
autoradiograms of the acetone extracts
obtained from plants treated with tetra­
mine* at 7 and 28 days. Extracts rep­
resented only 60 to 80 percent of the
volume 13, number 3, 1967 1 271
'
'
'
•
-t
1'1. •
i+
•
FIGURE 6 . Autoradiograms of paper c!Jromatograms developed ascendingly in formamide-saturated chloro­
form. E.\'tracts shown in A and B are from plants treated (see Fig. 2 caption) for 7 and 28 days, respectively.
In eacl1 case, upper section shows autoradiograms of shoot e.\'tracts and lower section shows those of roots.
Spotting from left to riglit: grass e ·tract, blackberry e.\'lract, Douglas-fir extract, tetramine*, Douglas-fir
e.\'tract plus tetramine*, blackbeny e.\'tract plus tetramine*, and grass e.\'tract plus tetramine*.
total activity, since some activity always
remained chemically bound to plant res­
idues, suggesting the presence of acetone
insoluble metabolites.
Tetramine* separated into two spots
with RF values of 0.40 and 0.75. The
compound with the higher RF amounted
to approximately 20 percent of the total
activity and was considered an impurity,
although no qualifying tests were per­
formed. This impurity is apparently com­
mon in all tetramine preparations, smce
the oral toxicity2 and the infrared spec­
trum3 of the labeled tetramine were
found to be identical with those of unla­
beled tetramine used by the Bureau of
2 Determined by W. E. Dodge, U.S. Bureau of
Sport Fisheries and Wildlife, Olympia, Wash.
3
Determined by Tracerlab, Waltham, Mass.
272 I Forest Science
Sport Fisheries and Wildlife m bioassay
tests.
Extracts from the 7-day treatments
showed some metabolic transformations
from the original tetramine*. These
changes were much more noticeable with
shoot than with root extracts, indicating
that tetramine* was probably metabolized
in the shoots. In shoots of all test species,
activities in the upper spot increased 2 7 to
69 percent at the expense of the decrease
shown by the lower tetramine* spot (Fig.
6A), Species, however, were different in
their ability to metabolize tetramine*.
Thus, degradation was highest in grass,
intermediate in blackberry, and lowest in
Douglas-fir.
In 28 days (Fig. 6B), essentially the
same results were obtained. However,
root and shoot extracts of grass and, to a
lesser extent, those of blackberry showed a
third spot, as yet unidentified, with an
Rre value of 0.96. Activity of this spot
was highest in grass and averaged 1 7
percent of the total activity.
Carbon dioxide collections from the
three species showed that C1402 was lib­
erated during the experiment. Activities
of the BaC140a collected from Douglas­
fir, blackberry, and grass were 0. 7 , 2.1,
and 6.1 counts per minute per milligram
carbonate per gram of shoot dry weight.
These data support those collected from
chromatography experiments and show
that, although the test species metabolized
tetramine*, they differed in their ability to
degrade the chemical.
Conclusions
Tetramine has been suggested for treating
tree seedlings to control damage by hares
and rabbits (Kverno 1960). At present,
suggested methods of application involves
use of the chemical on tree seedlings in
the field (in planting hole or root spray)
or in the nursery (root spray 1 year
before outplanting). Results obtained in
this study indicate serious limitations to
such usages, although complete evaluation
should await further study. Applications
of tetramine in the field would make the
chemical available to associated plant spe­
cies such as blackberries and grass. Al­
though tetramine would be less stable in
these species, the plants would remain
toxic and hazardous as long as a supply of
the chemical is available in the root zone
or if metabolites were as toxic, or possibly
more so than the parent compound.
Treatment of seedlings in the nursery, on
the other hand, would protect existing
growth only because of the chemical's
nonsystemic properties. Field applications,
therefore, would be necessary if new
growth were to be protected. This, also,
would be costly and hazardous.
Literature Cited
CoMAR, C. L. 1955. Radioisotopes in biology and
agriculture. McGraw-Hill, Inc., New York.
481 pp.
CRAFTs, A. S., and S. YAMAGUCHI. 1964 . The
autoradiography of plant materials. Calif.
Agric. Expt. Sta. Manual 35 . 143 pp.
HEFTMANN, ERICH (ed.). 1961 . Chromatography.
Reinhold Publishing Corp., New York. 753 pp.
HoAGLAND, D. R., and D. I. ARNON. 1950. The
water-culture method for growing plants with­
out soil. Calif. Agric. Expt. Sta. Circ. 347. 32 pp.
KvERNO, NELSON B. 1960 . The problems in the use
of systemic rodenticides. Soc. Amer. For. Proc.
1959:97-98.
PALLAs, J. E., JR., and A. S. CRAFTS. 1957. Critical
preparation of plant material for autoradiog­
raphy. Science 125:192-193 .
M. A. 1966. Absorption and distribution
of C14-labeled tetramine in relation to its possible
use in animal damage control. Pacif. Nthwest.
For. Range Expt. Sta. U.S. For. Serv. Res.
Pap. PNW-34. 16 pp.
RADWAN,
DoNALD A. 1954. Rodents and direct
J. For. 52:824-826.
YAMAGUCHI, S., and A. S. CRAFTS. 1958. Auto­
radiographic method for studying absorption
and translocation of herbicides using C14-labeled
compounds. Hilgardia 28:161-191 .
SPENCER,
seeding.
vol11m.e 13, number 3, 1967 I 273