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MICROCOPY RESOLUTION TEST CHART
NATIONAL BUREAU OF STANDARDS -1963
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A.
MASTER
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JUL ? 9 1967
RADIOSTRONTIUM UPTAKE IN BLOOD AND FLESH
CES ! CWS
IN BLUEGILLS (LEPOMIS MACROCHTRUS)
J. R. Reed and D. J. Nelson
HCN7.00 65
Radiation Ecology Section, Health Physics Division,
Oak Ridge National Laboratory, Oak Ridge, Tennessee
ABSTRACT
The rapid initial uptake of radiostrontium by bluegills (Lepomis macro-
chirus) was attributed to a quickly exchanged Sr pool in flesh and blood.
Two percent of the Sr pool in blood consisted of a component having a bio-
logical half-life of 2 hours. Another 1% of the blood Sr exchanged in 35
days and 97% was contained in a component with a long, undetermined biological
half-life. Strontium in blood contributed less than .0009% to the Sr in
flesh. At least three compartments of Sr metabolism were identified in
flesh. One percent of the Sr in flesh was turned over with a biological
half-life of about 2 hours while 9% was turned over with a half-life of 9
days. The remaining 90% of Sr in flesh had a long but undetermined half-
life.
Uptake of Sr in the quickly exchanged Sr pool was directly proportional
| to the Sr concentration in test solutions in the range from 0.3 to 300 pph
Sr. At 3000 and 30,000 ppb Sr, bluegills took up more Sr than at the lower
concentrations indicating an inability to discriminate against Sr at abnor-
mally high environmental concentrations.
Research sponsored by the U. S. Atomic Energy Commission under
26
contract with Union Carbide Corporation.
DISTRIBUTION OF THIS DOCUMENI ES UNLIMITED
UCN-0607
(a 0-46)
INTRODUCTION
The Sr contained in fish flesh is a small portion of the quantity in
3 the total fish body because of the relatively large accumulations in calcare-
ous tissues (Templeton and Brown 1964, Agnedal 1967, Nelson 1967). A similar
s distribution of 'sr in perch flesh and bone was observed by Ophel (1963).
Nevertheless, the small quantity of radiostrontiuni in flesh is important in
environmental transfers since this is the tissue normally eaten. Several
& previous studies included data concerning uptake by muscles and organs but
comparisons were difficult to make because of differences in methods and the
use of both marine and fresh-water species as test organisms. Data from
marine l'ish are not directiy comparable to those from fresh-water species
due to osmotic differences. Generally, there is a smaller concentration
factor of Sr by fish in salt water than in fresh water (Townsley 1967).
The role of soft tissues in the concentration and turnover of Sr has not
90
RSS
been defined clearly. Foroughs and Reid (1958) found that sr injected into
16 the blood of the euryhaline fish, Tilapia mossambica, was carried mainly by
the plasma. The Psr disappeared rapidly from the blood with less than 2% of
18 the injected dose remaining after 24 hours. Boroughs et al. (1956) reported
that more than 50% of the ysr injected intramuscularly in Tilapia was retained
after 20 days. Dark muscle retained less "Sr than light muscle, probably

due to a better blood supply and thus a faster turnover rate. The rate of
Sr uptake from fresh water was found to be linear with time for the various
tissues of the male guppy, Poecilia, white cloud mountain fish, Tanichythys,
24 and zebra fish, Danio (Rosenthal 1963). The rate of uptake was also found to
25 be a function of the concentration in the water at constant concentration while the
28 'biological half-life of Sr in muscle tissue was calculated to be about two years. Thi
Land
UCN-8867
.
.....
...
...
... ....-
is in contrast with a biological half-life of 12 to 48 minutes observed in
white crappies by Nelson (1967). These previously reported differences are
rather large. Hence, the objectives of this study were to determine: (1)
the quantitative importance of fish flesh and blood in the uptake and turn-
over of strontium and (2) the effect of different environmental Sr concentra-
tions on Sr uptake in fish flesh.
Several different types of experiments were used to clarify the
12
respective roles of fish flesh and blood in Sr uptake and turnover. The dif
ferent experiments were necessary because of the high concentrations and
rapid rate of deposition of Sr in scales and bone 'whic: literally masked Sr
uptake in flesh and other soft tissues. The biological half-life of Sr was
studied using both whole-body counting and dissection methods to delineate
the roles of individual tissues in Sr excretion.
Bluegills (Lepomis macrochirus Rafinesque) were selected for the study
on the basis of previous work with the closely related white crappie. They
were kept easily in the laboratory and were readily available from a small
pond at the Oak Ridge National Laboratory. Fish were obtained with hoop nets
which selected for larger individuals ranging in total length from 13 to 19
cm. The fish were held in large tanks of flowing spring water (12-16° C) for
at least 10 days since there was a two-fold difference between the stable Sr
er
"
18
...
w
.
m
-
;*-de
concentrations of the pond water and the spring. These differences were re-
.
flected in the flesh values for Sr in fish from the two localities. It was
assumed that the Sr in flesh would equilibrate with Sr in spring water in 7
-t
..."
to 10 days.
In the uptake studies bluegills were subjected to waters with various
Wow
.
26
stable Sr concentrations ranging from 0.3 ppb to 3 x 10* ppb. Experimental
t
mer.171.
h
ir
UCN-0067
(3 666)
--
a va en AW N
| containers consisted of plastic utility tubs 45 cm in diameter at the top
and 22 cm deep. Strontium chloride was added to a large volume of spring
water to raise the concentration of Sr to 3 x 104 ppb and from it serial
dilutions were made with spring water for experiments at lower Sr concentra-
| tions. The test solutions containing Sr concentrations less than 30 ppb were
| diluted with distilled water and received Ca and Mg amendments to bring the
concentrations of these cations to the same level as that in spring water.
Solutions were analyzed chemically for Sr es a check on the accuracy of the
9 | Sr dilutions. Nine liters of water were put in each tub and a polyethylene
was
cover was fitted to prevent contamination from external sources. The water
was aerated continuously. For the experiments testing concentrations of Sr
12 | lower than the spring water (less than 30 ppb), carrier-free sr was used
es a tracer; in other ceses high specific activity °?sr was used. The activ-
14 ity of each tub was adjusted to approximately 200 pCi/ml.
Prior to immersion in the test waters, the fish were allowed to equili-
brate for 24 hrs. in similar tubs which contained stable Sr at the test con-
centration but lacked the radioisotope. Usually three individuals were used
per tub. They remained in the isotope for 24 hrs., then were removed, weighea,
measured, bled by severing the caudal fin, and frozen. After the fish were
removed, a water sample was taken to determine the exact radioactivity of
the test water. Flesh samples were obtained by scaling the fish and dissect-
ing the dorsal musculature, which in bluegills is free from ribs and epipleural
bones. The flesh and blood were processed to obtain wet wt., dry wt., and
ash wt. (at 500°c) and they dissolved in 0.1 N HCI for chemical analysis.
Stable chemical analyses were performed by the Analytical Chemistry Division
LEGAL NOTICE
of Oak Ridge National. Laboratory.
This report was prepared as an account of Government sponsored work. Neither the United
States, nor the Commission, nor any person acung on behalf of the Commission:
A. Makes any warranty or representation, expressed or implied, with respect to the accu-
racy, completeness, or usefulness of the information contained in this report, or that the use
of any information, apparaius, method, or process disclosed in this report may not infringe
privately owned rights; or
B. Assumes any liabilities with respect to the use of, or for daniages resulting from the
use of any information, apparatus, method, or proce88 disclosed in this report.
As used in the above, person acting on behalf of the Commission includes any em-
ployee or contractor of the Commission, or employee of such contractor, to the extent that
auch employee or contractor of the Commisslon, or employee of such contractor prepares,
dløseminates, or provides access to any information pursuant lo his employment or contract
with the Commission, or his emplo: ment with such contractor.
WCN8667
(a 0.88)
Another aspect of the uptake study was concerned with the response of
bluegills to a continuous radiostrontium exposure for 35 days. A large stain-
less steel kettle containing 190 liters of spring water (30 ppb Sr) served
as an experimental container. The initial activity was 800 pci/ml of carrier
free "Ysr. Eighteen bluegills were placed in the kettle. Three fish were
removed randomly on each of six occasions, after 1, 2, 4, 8, 16 and 35 days
in the test solution. All were bled and samples of flesh taken from each.
Flesh samples were processed as above and a 4 m.). aliquot of the dissolved
flesh was removed from each sample and dried on a planchette to be counted.
The blood was spread on planchettes immediately and counted when dry. A cor-
WOS
rection was made in the calculation of sample activities for the inclusion
of 90sr-Sy in 89sr tracer solutiuns.
The biological half-life of Sr in bluegills was measured by using ºsr
as a tracer.and counting the whole fish in a small-animai, whole-body counter
Fish were tagged by immersion for 10 minutes in spring water contai ning ºsr
with en activity of 4 x 204 pC1/m2. Then they were rinsed for one minute and
counted at one-minute intervals. Fresh water was pumped through the assembly
to remove excreted "sr and to provide a supply of oxygenated water. Influ-
ent water was directed toward the head of the fish and the flow through the
system was approximately 2 liters/minute. Three additional fish were counted
at 5-minute intervals for two hours and then counted once daily for 30 minutes.
Specific activities or radiostrontium-to-tota) strontium ratios were used
VS
to determine whether the Sr in flesh and blood was in equilibrium with that
in water. At equilibrium the specific activities in water and fish tissue
will be equal, while at nonequilibrium conditions the specific activity in-
dicates the proportion of Sr in tissue which was exchanged. Concentration
UCN.8667
6.0 )
factors, or the amount of radiostrontium in fish flesh or blood relative to
the amount in the test water, were formulated in some cases for ease of cal-
mo
culation.
RESULTS AND DISCUSSION
Uptake and Turnover of Radiostrontium by Flesh and Blood
The blood incorporated radiostrontivă rapidly, reaching about two-thirds
of the maximum activity level in 24 hrs. (Fig. 1). The concentration factor
rose from 0.124 at one day to 0.194 at 35 days. A second component of the
blood uptake curve was very long but sufficient data were not available for
accurate extrapolation. On the basis of stable Sr concentration factors in
the blood in fish from natural waters, radiostrontium concentration factors
of between 3,26 and 6.23 should be reached (Table I).
The specific activity (uuci 59s:/ụg x 10-3 sr) in the blood did not
change appreciably in the test period, rising from 0.47 at one day to 0.74 at
16 | 35 days. The specific activity expected at equilibrium was 24.1. In the
rapidly exchanged component of the soft tissues, blood accounted for 2% of
the activity in 24 hours and in the longer component, for 3% of the activity
19 in 35 days. Since blood represents about 3% of the body weight in fish, the
radiostrontium present in the blood was a very small fracüion of that present
in the whole fish. The blood was & compartment which repidly exchanged
radiostrontium but which comprised very little of the total Sr in flesh.
The greatest rate of 59sr uptake by the flesh occurred in the first
four days. The second component of the uptake curve was faster for flesh
than for blood (Fig. 1) and the maximum concentration factor reached after
26 | 35 days was 0.121. Extrapolation or the second component indicates that
WCN.8807
(3 3.68)
--

approximately 72 days are necessary for bluegill flesh to reach a concentra-
tion factor of one, which may be expected from stable Sr analyses (Table I).
The specific activity in the flesh rose from 0.28 in one day to 2.4 in
35 days, while again the expected specific activity ratio was 24.1 (uuci sr/
mg x 10-3 Sr). Approximately 1% of the activity in the soft tissues was
present in the flesh in 24 hours increasing to 10% in 35 days. Thus, the short
components measured for flesh and blood represented only 1% of the activity
in the soft tissues in 24 hor 's while the longer components represented som
of the activity in 35 days. The remaining activity (90%) probably was in a
compartment which did not readily exchange strontium.
'
The turnover of strontium in bluegills was studied in a series of excre-
tion experiments. Various combinations of live and dead fish were tagged
| with Sr and counted in a whole-body counter to study short-term excretion
patterns. The results showed a rapid rate of ºr loss during the initial 40
minutes (Fig. 2). The rate slowed somewhat in those fish which were tagged
while alive, whereas those tagged when dead declined steadily. The curve for
fish tagged alive but counted dead was similar to that for fish tagged alive
and counted alive. This indicated that Sr was incorporated into the body
of the live fish, but that early losses were due to physical processes rathe
19
than biological excretion. This physical loss, attributed to the fiushing
action of the water, occurred despite the rinsing of fish in uncontaminated
water prior to placing there in the excretion chamber.
The excretion curves
23 of fish, which were tagged alive show evidence of a "biological sink" which
prevented "Sr 103ses since the majority of the isotope was bound in the sinkl.
25 | Fish which were tagged when dead lost sr rapidly and lacked the "sink"
aspect in the excretion curve.
UCN-8867
(2 0.66)
An experiment lasting several days was performed to determine if the
flesh was actually losing sr via excretion. The initial phase was like
that described above. After two hours the l'ish still retained 70% of the
initial activity (Fig. 2). However, after one day the loss of activity was
breater and after seven days of excretion only 50% of the initial whole body
activity remained. At that point, the curve flattened and the value for 14
days was also 50%. Rosenthal (1963) stated that for Sºsr in Poecilia , the
first rapidly disappearing component with a biological half-life of eight
days was due to losses from viscera. Current experiments with bluegills em-
ploying dissections immediately following a short tag with Sr and after
two hours of excretion indicate that blood is also an important factor in
early losses of activity and may comprise a portion of the losses attributei
to viscera. The biood lost 75% of its initial activity in two hours. The
15
He attributed this to turnover of all tissues in the body except viscers.
The lack of an intermediate component was thought due to a very slow turnover
in the flesh. Rosenthal (1963) calculated a biological half-life of two
years for po sr in muscie tissue. A similar biological half-life for Osr in
muscle tissue was calculated for Tilapia mossambica by Boroughs et al. (1956).
Results from dissections of bluegills as described above for blood excretion
showed that the flesh lost 46% of its initial activity in two hours. Nelson
(1967) reported a biological half-life of 12 to 48 minutes for "sr in flesh
of white crappies.
The bluegill excretion curve was not unlike those described above (Fig.
25 2). The initial rapid component of two hours was due to blood and flesh
26 excretion and, to some extent, physical losses. A slower component of
UCN8867
(3 6.68)

two hours to mine days represented the long blood component and viscera and
muscle excretion. The very long third component reached at approximately
nine days may be attributed to flesh excretion and possibly to turnover of
Sr in connective tissue. On the basis of precious estimates of the flesh
5
turnover of radiostrontium and from the current study, it appears that there are
6
at least three
compartments in flesh excretion.
.
An indication of possible binding sites for radiostrontium in fish flesh
was given by micro-autoradiographs of bone and muscle of the rudd, Scardi.nius
erythrophthalms by Foreman and Bidwell (1959). In the autoradiographis there
appeared blackened areas within the flesh which may have been connective
tissue. Such tissue could have been responsible for the slower flesh compon-
ent in the excretion curve, whereas the rapid component was probably due to
the flesh turnover itself. The long component described by. Rosenthal (1963)
and Boroughs et al. (1956) and found in bluegills in this study, may also have
been attributable in part to flesh excretion although no separatsion of this
component was attempted.
Uptake Responses to Different Environmental Sr Concentrations
Studies of the uptake responses for Sr over environmental Sr concentra-
tions ranging from 0.3 to 30,000 ppb were completed before the 35-day uptake
experiment was initiated. On the basis of previous work (Nelson 1.967) the
interpretation was made that the entire pool of Sr in soft tissues turned
over with a biological half-life of less than one hour. This proved to be a
false assumption. Since comparisons of uptake at different stable Sr concen-
trations were made on a uniform 24-hour exposure time, these results should be
UCN-0807
0.00)
related only to the quickly. exchanged fraction of Sr in flesh and blood.
Concentration factors of radiostrontium (Fig. 3) indicate that about one
percent of the Sr in flesh was exchanged in 24 hours.
The concentration factor for radiostrontium was virtually constant with-
in the range from 0.3 to 30,000 ppb stable Sr in the test solutions (Fig. 3).
Since the concentration factors remained constant over the wide range of stable
Sr concentrations, the readily exchanged Sr faction was accumulating Sr in

| direct proportion to that in the environment. Results of the stable Sr ...
analyses (Fig. 3, Table II) indicated that concentration factors of stable
Sr were inversely proportional to the Sr concentration in test waters, except
at the two highest environmental Sr concentration, 3000 and 30,000 ppb. Other-
12 | wise, the inverse relationship showed that concentration factors calculated
on the basis of stable Sr concentrations were highly dependent upon the en-
vironmental Sr concen'iration. The levels of stable Sr in the flesh of
the experimental fish did not change significartly. These results showed
that the quickly exchanged fraction of Sr in bluegill flesh wes a small pro-
portion of the total Sr in flesh.
At 3000 and 30,000 ppb Sr concentrations the fish flesh contained approxi-
mately 2 and 20 times, respectively, as much Sr as those fish at the lower
.
1
environmental concentrations (Table II). These results suggested the lack of
21 an ability by bluegills to discriminate against the uptake of Sr from waters
containing abnormally high concentrations. At environmental concentrations of
23 300 ppb and below, the fish had similar concentrations of Sr in flesh which
24 ranged from 37 to 65 ppb. The constancy of these flesh values was due, in
part, to a lack of sufficient time for test fish to cquilibrate with the test
solutions. However, at these lower concentrations it was evident that active
UCN-6667

regulation of the Sr content in flesh occurred ant that it did not occur
at the two higher concentrations.
The specific activity in flesh (Fig. 3) reflected the relative constancy
of Sr and stable Sr concentrations in fish at all concentrations except
30,000 ppb. At the 30,000 ppb Sr concentration, the specific activity was
reduced because of a diluent effect of stable Sr which might be expected at
exceedingly high concentrations.
Strontium Relationships in Flesh and Blood
Strontium concentrations in fish flesh and blood are a minor fraction of
the total quantity in fish. Flesh and blood appeared to have short components
in both uptake and excretion experiments. However, the flesh contribution to
these quickly exchanged components is of greater significance. Blood is
about threa percent of the body weight of bluegills and the 35-day uptake
experiment demonstrated that 2% of the blood Sr was exchanged in one day and
3% was exchanged at 35 days. The remaining 97% of the Sr in blood consists
of a component with a long biological half-life. Since blood-rich organs -
such as the kidney, heart, liver and spleen contain most of the blood, the
contribution of blood Sr to the turnover of Sr in flesh is minor (less than
.0003%)
The three separate experiments showed the presence of a quickly exchanged
Sr fraction in flesh. The specific activity at the end of one day in the
35-day uptake study was about 1% of the expected equilibrium value and the
24-hour experiments also suggested about 1% of the Sr in flesh was exchanged
in this time. The excretion studies showed the biological half-life of this
26 | Praction was approximately two hours. The 35-day experiment and the excretion
UCN-0337
17. 0.88
.
.
.
study also showed an additional nine percent of the Sr in flesh was turned
over with a half-life of nine days. Since five biological half-lives are
required for exchange reactions to reach 97% of equilibrium values, the nine
| percent value for Sr exchanged within 35 days is slightly low. Nevertheless,
about 90% of the Sr in flesh is in a component having a long biological
half-life.
These experiments have clarified our knowledge regarding quantitative
aspects of Sr uptake and turnover by flesh and blood of bluegills. Also, an
additional application of specific activities was demonstrated in determining
the fraction of Sr in flesh and blond which was turned over in short-term
experiments
· ACKNOWLEDGEMENTS
The authors thank N. A. Griffith for assistance in collecting fish and
in the preparation of samples for analysis.
.
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.
20
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UCN-8867
13 6.68)
FIGURE CAPTIONS
Sr in Bluegill Flesh and Blood During
FIGURE 1. Concentration Factors of
a 35-day Exposure Period.
FIGURE 2. Comparative Whole-Body Excretion of sr in Bluegills Which Had
Been Tagged Either While Living or After Having been killed by
Freezing. The initial excretion in the left portion of the figure
is included within the dotted box in the right portion of the fig-
ure. Note difference in time scales.
FIGURE 3. Concentration Factors of Stable Sr and
sr and Specific Activities
Sr in Biluegill Flesh at the End of a 24-Hour Uptake Experiment.
UCN-8867
(3 6.68)
60
ORNL-DWG 67-4338

50
40
• BLOOD
O FLESH
30
TIME (days)
20
20
10
o
0.005 L
CONCENTRATION FACTOR 89 Sr IA STANDARD ERROR
.-
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PERCENT INITIAL ACTIVITY
NS3
TAGGED SODEAD FISH
ALIVE LIVE FISH
CA FROZEN
THAWED
TAGGED
TAGGED
DEAD
COUNTED
A FROZEN
TAGGED
COUNTED
UULI |
0 10 20 30
TIME (min)
40
1
2
7
8
9
10
3 4 5 6
TIME (days)
Fig. 3
ORNL-DWG 67-4340

Sr
Huc/g FISH FLESH
i juc/mi H2O
Sr yg X100% FISH FLESHE
Sr ug x 10-Om H2O H
85 sr uuc /FLESH
Srug x 10 % FLESH
HE
N=6,52 STANDARD ERRORS
SPECIFIC ACTIVITY
CONCENTRATION FACTOR IN BLUEGILL FLESH
101
108
100 101 10² 103
ENVIRONMENTAL Sr CONCENTRATION
.
ht
TABLE I. Stable strontium in bluegill flesh and blood
og en
No. of
Fish
met 2 standard Proro e
Concentration
Factor
ppb + 2 Standard Errors
Blood
Pond
3.26
196 + 62
187 + 50
Spring
u
6.23
Flesh
Pond
M
1+
0.75
vu
Spring
1+
1.17
+
Water
Pond
Spring
TABLE II. Strontium in bluegill flesh from waters
with different Sr concentrations.
Water
Sr ppb
No Fish
Analyzed
Flesh
Sr ppb
30,000
3,000
2017 + 230(a)
132 + 36
57 + 16
37 + 9.4
44 + 12.2
36.7 + 2.6
(a) mean + 2 standard errors
16
LITERATURE CITED
Agnedal, P.O. 1967. Calcium and strontium in Swedish waters and fish and
accumulation of Yºsr. pp. 879-896. In Radioecological concentration
Processes. Aberg, B. and F.P. Hungate (eds.). Pergamon Press. Oxford
and New York. 2040 p.
Boroughs, H. and D. F. Reid. 1958. The role of the blood in the transporta-
tion of strontium-90 - yttrium-90 in teleost fish. Biol. Bull. 115(1):
64-73.
S. J. Townsley and R.W. Hiatt: 1956. The metabolism of
radionuclides by marine organisms. I. The uptake, accumulation and loss
of strontium-89 by fishes. Biol. Bull. 111: 336-351.
Foreman, E. E, and K.W.E.Bidwell. 1959. A biological investigation of the
fate of strontium-90 in a disused filter bed. UKAEA IGR-27 (RD/W). 16p.
Nelson, D.J. 1967. The prediction of Sr uptake in fish using data on
specific activities and biological half-lives. p. 843-851. In Radio-
Pergamon Press, Oxford and New York. 2040 p.
Ophel, I. L. 1963. The fate of radiostrontium in a freshwater community.
pp. 213-216. In Radioecology. Schultz, V. and A. W. Klement, Jr. (eds.).
Reinhold, New York. 746 p.
__and J. M. Judd. 1967. Experimental studies of radiostrontium
accumulation by freshwater fish from food and water. p. 859-866. In
Radioecological concentration Processes. Aberg, B. and F. P. Hungate
(eds.). Pergamon Press, Oxford and New York. 1040 p.
Rosenthal, H.L. 1963. Uptake, turnover and transport of bone seeking elements
i n lishes. Ann. New York Acad. Sci. 109: 278-293.
UCN-6667
la 6.00)
17

Templeton, W. L. and V. M. Brown. 1964. The relationship between the con-
centrations of calcium, strontium and strontium-90 in wild brown trout,
Salmo trutta L., and the concentrations of the stable elements in come
waters of the United Kingdon, and the implications in radiological
health studies. Int. J. Air Wat. Poll. 8: 49-75.
Townsley, S. J. 1967. The concentration of Yusr and other nuclides of
strontium in fish. pp. 867-878. In Radioecological Concentration
Processes. Aberg, B. and F. P. Fungate (eds.). Pergamon Press, Oxford
and New York. 1040 p.
UCN-002
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