Ecological Research Series
DISTRIBUTION  OF INGESTED AMERICIUM  IN
      CHICKENS  AND  TRANSPORT  TO EGGS
                      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-600/3-76-058
                                            May 1976
 DISTRIBUTION OF INGESTED AMERICIUM IN CHICKENS
              AND TRANSPORT TO EGGS
                         By
                   Anita A. Mullen
                   Stephen R. Lloyd
                   Robert E. Mosley
Monitoring Systems Research and Development Division
  Environmental Monitoring and Support Laboratory
                 Las Vegas, Nevada
  ENVIRONMENTAL MONITORING AND SUPPORT LABORATORY
         OFFICE OF RESEARCH AND DEVELOPMENT
        U.S. ENVIRONMENTAL PROTECTION AGENCY
              LAS VEGAS, NEVADA  89114

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                              DISCLAIMER

     This report has been reviewed by the Environmental Monitoring and Support
Laboratory-Las Vegas, U.S. Environmental Protection Agency, and approved for
publication.  Mention of trade names or commercial products does not constitute
endorsement or recommendation for use.
                                    ii

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                                  INTRODUCTION

      The necessity for obtaining further information concerning the fate of the
 transuranic nuclides in the environment has been well established.   However,
 little is known about the metabolism of americium in chickens  (Gdllus domesticus)
 and its subsequent transfer to eggs.   In the event that  farm areas  become contami-
 nated with americium, chickens allowed to forage around  such farmyards may ingest
 contaminated soil and feedstuffs.  This ingested americium may be metabolized and
 transferred to egg contents and edible body tissues, thereby causing contamina-
 tion of a portion of the human food chain.

      The absorption, distribution,  and excretion of the  actinide elements in
 biological systems are determined largely by their chemical characteristics,
 which are similar to those of the lanthanides (Durbin, 1973; Hamilton, 1948).
 Lanthanide fission products administered intramuscularly to laying  hens were
 found to be  distributed throughout the tissues with a major fraction concen-
 trated in the skeleton and a lesser fraction in the liver (Mraz et  al., 1964).
 Of the egg components, the yolk was found to contain amounts ranging from
 18.1 percent of the administered dose of lanthanum-140 to 1.8  percent of the
 administered dose of praseodymium-142 per four-egg composite sample.

      Several investigators have found indications that plutonium and americium
 may be metabolized in a manner similar to iron (Lindenbaum and Rosenthal, 1972;
 Taylor, 1972).  Transferrin appears to be the universal  transporting protein
 for plutonium (IV) in the blood plasma of all animals that have been studied.
 The main route of elimination of plutonium is through the gastrointestinal
 tract, via the bile and feces, which is also similar to  that for iron.  Laying
 hens require a large amount of iron in addition to their maintenance need,
 since the average egg yolk contains 1.1 mg of this element (Maynard and Loosli,
 1962).  If plutonium and americium are metabolized in a  manner similar to that
 reported for iron, then the egg yolk should contain these elements.

      This study was conducted to determine the extent to which the  relatively
 soluble americium citrate is absorbed and distributed in the edible tissues and
 egg components after oral administration to laying hens.


                                     SUMMARY

      Americium is one of the transuranic elements which, as a  daughter product
 of the radioactive decay of plutonium, may be found in the environment as a
 result of worldwide fallout or accidental contamination.  One  of the routes
 to man is through the food chain.  This study investigated the transport
 through one portion of the food chain, chickens and eggs.  The soluble citrate
 complex of americium-241 was orally administered to 20 white Leghorn laying
 hens daily for 2 weeks.  The yolks, whites, and shells from the eggs were
 analyzed for their americium content.  Yolk was the only egg fraction in which
-radioactivity was observed.  The americium-241 activity  in yolks reached a

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 maximum on the  14th  day  of  dosing  indicating  that equilibrium had not been
 reached and additional transfer  of americium  to yolk would have occurred if
 dosing had continued.  Analysis  of egg yolk yielded a two component excretion
 curve with biological half-times of 2.00  ± 0.18 days and greater than 33 days,
 indicated by the average concentration values of americium-241 in yolks laid
 after the maximum activity  was reached.

      The hens were serially sacrificed at 1,  10, and 20 days after the final
 administration  of americium-241.   Tissue  samples were collected and the
 americium content determined in  the edible portions and feathers of the hens.
 Americium was detected in most tissues at early times with the main concentra-
 tion in the liver and skeleton.  The highest  concentration per organ  (3.03 X
 10~3 percent of the  dose) occurred in the liver of hens sacrificed 10 days
 after final administration  of americium-241.

      In a contaminated farmyard  where chickens are raised with equal likeli-
 hood of consuming equivalent proportions  of plutonium and americium, the
 americium would present  the greater hazard to those persons eating eggs and/
 or chicken liver from that  source.
                                 CONCLUSIONS

      Ingestion of the  relatively soluble citrate form of americium-241 by
 laying hens  resulted in  the  contamination of the egg yolks and the concentra-
 tion of americium-241  in the skeletons and livers of the hens.  The activity
 per egg yolk continued to increase  during the 14 days of americium ingestion,
 indicating that equilibrium  had  not occurred and that americium concentration
 may increase in yolks  for a  much longer period of time.  It appears that more
 americium-241 concentrates in egg yolk and chicken liver than plutonium-238
 when ingested in the relatively  soluble citrate form.  This concentration
 should be  recognized when contaminating events occur in the vicinity of a
 farming area.


                               RECOMMENDATIONS

      Although little hazard  to humans would be expected from ingesting americium-
 241 contaminated eggs  or chicken livers, the possibility of this  contamination
 must  be recognized in  the event  of  the contamination of farming areas.  Further
 study would  determine  the concentration of ingested americium oxide, the most
 probable form of released americium.  The biological availability of the
 organically  incorporated americium  should be investigated.  Knowing the genetic
 effects of americium on  chicks hatched from contaminated eggs and the concentra-
 tions of americium in  these  chicks  and its transfer ability to their eggs  after
 they  reach maturity would be of  additional help in evaluating the hazard of
 americium in the environment.
                          EXPERIMENTAL PROCEDURES

     Thirty white Leghorn laying hens were housed  in  stainless  steel animal
metabolism cages with a center partition  arranged  to  allow each hen to have
an individual cage area.  Excreta were collected in paper-lined trays beneath
                                     2

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the wire mesh.  The cages were cleaned daily to decrease airborne contamination
and to keep the eggs clean.

     The chickens were acclimated to their surroundings for several weeks prior
to the study.  During the study, the chickens were maintained on commercial
laying pellets and had access to water and oyster shell.

     The chickens used in this study were beginning their second laying cycle
and were about 2 years of age.
                            SOURCE PREPARATION

     The solution for dosing was prepared fresh daily from americium-241 (III)
nitrate and citrate buffer (pH 3.5 to preserve the monomeric state) and will
be referred to as americium citrate.  This preparation allows better comparison
with previous experiments in which plutonium solutions were prepared similarly.
                                  DOSING

     Each of the 20 hens was administered 2 microcuries of americium-241 citrate
orally by means of a dosing needle and syringe every day for 2 weeks.

     Ten control hens were caged randomly among the dosed chickens.   The control
hens did not receive a placebo, but were used only as biological samplers to
measure any cross-contamination.
                      SAMPLE COLLECTION AND STORAGE

     Eggs were collected daily.  After washing they were broken and the whites,
yolks, and shells were separated.  The separated fractions were weighed, placed
in counting containers, and refrigerated until analyzed.

     Six hens were sacrificed at 1 day, seven hens at 10 days, and seven hens
20 days after final dose administration.  After sacrifice and exsanquination,
tissue samples consisting of gizzard, heart, liver, and muscle were collected to
determine the amount of americium present in the edible tissues.  Feathers
were collected to ascertain if the concentration of americium occurred there-
in.  The feathers were washed with a commercial decontamination solution placed
in an ultrasonic generator prior to analysis to remove accumulated surface
contamination.
                             SAMPLE ANALYSIS

     The counting containers were covered with plastic film and turned film-
side down on the thin window crystal detector of a FIDLER instrument.  The
detector was housed in a standard shield and coupled to a RIDL single-channel
analyzer system with sealer readout.  This system was calibrated for various
sample types and weights.  Sample collection was stopped when the counting
error exceeded 30 percent.

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                             RESULTS AND  DISCUSSION

      In order to follow the possible mechanisms  for absorption of americium
 by chickens and its subsequent transfer  to  eggs,  it is necessary to review
 some of the physiology of digestion and  egg production.  The  following infor-
 mation is excerpted from P. D. Sturkie's (1970)  treatise on the subject.

       Digestive organs of chickens  are similar to those of mammals except that
 birds have a gizzard and a crop.  The crop  is formed as a dilatation of the
 esophagus and is a storage organ.   The proventriculus of the  bird is comparable
 to the simple glandular stomach of  mammals.  The  gizzard is a highly special-
 ized organ for grinding, as the bird has no teeth.

       Rate of passage of food through the alimentary canal is influenced by the
 consistency, hardness, and water content of the  food and the  amount consumed.
 If the crop is full, it requires 10 to 18 hours  for all of the food to leave
 the crop.  In experiments with dye-impregnated food, it was indicated that
 the dye can be detected in the feces within 24 hours of administration.  When
 radioactive barium was administered to chickens,  it appeared  that approximate-
 ly half the activity was excreted in 4 to 5 hours.

       Although information about digestion  is meager, the evidence indicates
 that little digestion occurs in the mouth or the  crop.  Enzymes contained
 in pure intestinal juice have not been determined, but mixed  intestinal
 juice contains pancreatic juice and proteases in  an alkaline  medium.

       The pH of the contents of digestive organs  in the chicken is: crop, 4.5;
 proventriculus, 4.4; gizzard, 2.6;  duodenum, 5.7-6.01; jejunum, 5.8-5.9;
 ileum, 6.3-6.4; rectum or colon,  6.3; cecum, 5.7.  In live birds, the large
 intestine was slightly alkaline (7.1).   The acidity of avian  bile (5.88 in
 chickens) may, in part, account for the  lower pH in the alimentary tract of birds
 as compared to mammals.  The secretion rate of bile in white  Leghorn chickens is
 about 1 milliter per hour,  or about 9.5  milliter/kilogram of  body weight per day.
 The role of bile in avian digestion has  not been  fully studied, but it is presumed
 that it aids in the absorption of fats by its emulsifying action and activating
 effects on pancreatic lipases.  Little fat  is absorbed until  the third segment
 of small intestine is reached, where 42  to  60 percent  (depending on type) is
 absorbed.   Most of the remaining fat (90 to 98 percent) is absorbed in the
 fourth segment.  Little fat is absorbed  in  the cecum or large intestine.

       The liver has many functions  other than its digestive action.  There  are
 pronounced sex differences  in blood levels  of plasma proteins, lipids, and
 calcium of birds.   These differences are due primarily to the effect of  the
 female sex hormone on the liver in  the formation and retention of these
 substances.   The clearance  rate of  liver, as measured by its  ability to
 extract sodium bromsulphthalein (BSP) from  blood and excrete  it  in  the bile,
 is markedly influenced by estrogen  and,  as  expected, the clearance  rate  is
 greater in laying  hens.

       The  development of an egg starts in the ovary, where  the yolk portion is
 formed.  Here there are many ova, each enclosed  in a follicle.   The yolk is
 deposited  in  concentric layers during growth and maturation,  and these  rings
may vary in color,  depending on the amount  of yellow pigment,  or  xanthophyll,
                                     4

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in the food.  When the process is complete, the follicle bursts and the yolk,
surrounded by a membrane, passes into the oviduct.  This process may take from
7 to 10 days.  As the yolk passes into the magnum, the albumin of the egg is
secreted around the ova.  This process takes approximately 3 hours; then the
egg passes into the isthmus, where the inner and outer shell membranes are formed
in about an hour and a quarter, before the water is added through the semi-
permeable shell membrane to the albumin and where the egg shell is deposited.
The egg remains in the shell gland approximately 18 to 20 hours, then passes
to the vagina for laying (P. D. Sturkie's, 1970).

     There are many problems inherent in the interpretation of the data derived
from studies of this nature.  Not only do the hens vary in the length of time
they take to produce an egg, but the production of eggs is variable, with some
hens laying an egg a day for weeks while others lay sporadically.  Thus, a
large range of activity can be anticipated in an egg laid at any given time
after ingestion of a radionuclide.  Considering the above variables, the range
of the data shown in Table 1 would be expected.
       TABLE 1.  TRANSFER OF ORALLY ADMINISTERED AMERICIUM TO EGG YOLK
Total
Dose
(yci)
Time to
Peak
(days)
Peak Activity
Range
(pCi/18-g yolk)
Peak Activity
Average
(pCl/18-R yolk)
Half-time
(T% days)
   Am      26.7        14        60-2,900             870       (1) 2.00±0.18
                                                                (2) >33
     The transfer of americium to the egg yolks is presented in Figure 1.  Each
point represents the average activity per yolk in eggs laid during that time
period.  The americium activity increased over the dosing period with the maxi-
mum, 0.00326 percent of the total administered activity, occurring in eggs laid
after the fourteenth dose.  Since no obvious plateau was evident, additional
doses might have resulted in a higher activity.  The americium activity in the
egg yolks decreased with a half-time of 2.00 ± 0.18 days followed by a second
component of greater than 33 days.  Americium was not detected in the white or
shell of the eggs, nor in any portion of the eggs from the control hens.

     These results would appear to agree in general with published data on the
metabolism of iron.  In an investigation to determine the metabolism of manga-
nese and iron by poultry  (Panic, 1970), it was determined that measurable
quantities of iron-59 were found only in the egg yolk.  An iron-59 concentra-
tion of 1.90 percent of the dose per yolk appeared in eggs laid 51 hours after
oral administration, with the peak concentration of 2.80 percent per yolk
occurring 6.1 days after initial ingestion.  It was found that iron is trans-
ported from the blood to the yolk by a protein or proteins deposited in yolks
only.  Furthermore, the iron is more firmly bound to the protein phosvitin
than to transferrin.  Phosvitin appears in hen plasma during the laying
period and is deposited only in the yolk.

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Figure 1.
               XlOA


Average concentration of americium-241 in egg yolks from chickens

ingesting 2 microcuries/day for 14 days

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     Americium was absorbed by the hens with tissue concentrations occurring
mainly in the liver and skeleton (see Table 2).  These results agree with
those of Taylor (1962) studying the metabolism of americium in rats.

     In previous studies conducted with plutonium, it was found that chickens
absorbed a very small percentage of plutonium-238 ingested as the relatively
insoluble dioxide.  This small percentage of absorption and transfer to egg
yolk appears to present little hazard to humans consuming the eggs.   Laying
hens absorbed a somewhat higher percentage of the nuclide after ingesting a
more soluble form of plutonium-238 than that absorbed from ingestion of the
dioxide-form.  Also, a large percentage of the absorbed plutonium was trans-
ferred to the yolk of the eggs.  In older laying hens ingesting either form
of plutonium-238, plutonium does not appear to concentrate in tissue in
amounts large enough  to constitute a hazard when human consumption is limited
to normal quantities.  This may not be true of the livers of younger poultry
raised solely for food although little hazard could be expected.

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    TABLE  2.  ACTIVITY  IN TISSUE FROM CHICKENS INGESTING 2 yCi AMERICIUM-241
             CITRATE EACH DAY FOR 14 DAYS  (AVERAGE AND STANDARD DEVIATION)
Tissue
Gizzard
w/o lining
Liver
Gall Bladder
w/bile
Heart
Muscle
Fat
Long bone
Sternum
Rib
Blood
Days After
Final Ingestion
1 (6)*
10 (7)
20(7)
1
10
20
1
10
20
1
10
20
1
10
20
1
10
20
1
10
20
1
10
20
1
10
20
1
10
20
% Dose/g
9-08 2.96X10"!?
8.67 4.45X10
2.48 1.71X10
3.36 3.11X10";!
8.00 2.28X10 ,
7.47 8.58X10
4.27 2.21X10"^
6.53 9.28X10
6.85 2.60X10
4.27 0.76X10"*?
8.73 8.67X10~£
5.68 3.80X10
1O Q A C f\ YT f\
. /.O U * j UA.JLU —
7.01 3.80X10~.L
6.59 2.29X10
3.23 3.02X10"!?
3.53 1.43X10 ;
2.63 2.69X10
1.94 1.15X10":?
8.35 6.26X10 J?
4.19 5.47X10
7.49 2.74X10"^?
1.26 0.76X10":*
6.42 6.93X10
8.80 4.27X10"^?
1.98 2.55X10 ,
5.77 5.63X10
7.00 5.39X10"^
4.19 5.43X10"^
1.05 0.46X10
Mean Weight
Total Organ, g
24.9
21.7
21.6
41.9
37.9
42.4
2.6
2.0
1.0
8.4
6.6
6.2






% Dose/organ
2 . 26X10"'?
1.88X10
5.30X10
1.41X10";*
3.03X10 .
3.16X10
1.11X10"^
1.31X10 ,
7.20X10
3.58X10~^
5.76X10
3.55X10






*Number of samples.

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                               REFERENCES

Durbin, P. W.  Uranium and Plutonium—Transplutonic Elements.  H. C. Hodge,
     J. N. Stannard, and J. B. Hursh, eds.  Springer-Verlag, New York.
     740 pp. 1973

Hamilton, J. G.  "The metabolic properties of the fission products and
     Actinide elements."  Rev. Mod. Phys. _2lD:718.  1948

Lindenbaum, A. and M. W. Rosenthal.  "Deposition patterns and toxicity of
     plutonium and americium in liver."  Health Phys. 22:597.  1972

Maynard, L. A. and J. K. Loosli.  Animal Nutrition.  McGraw Hill Book Co.,
     Inc., New York.  449 pp. 1962

Mraz, F. R., P. L. Wright, J. M. Ferguson, and B. L. Anderson.  "Fission
     product metabolism in hens and transference to eggs."  Health Phys.
     JLO:777.  1964

Panic, B.  "Manganese and iron metabolism studies in poultry and swine."
     Mineral Studies With Isotopes in Domestic Animals.  STI/PUB-293,
     CONF-700966.  81 pp.  1970

Sturkie, P. D.  Duke's Physiology of Domestic Animals, J. J. Swenson, ed.
     Cornel University Press, Ithaca, New York.  526 pp.  1970

Taylor, D. M.  "Some aspects of the comparative metabolism of plutonium
     and americium in rats."  Health Phys. 8.:673.  1962

Taylor, D. M.  "Interactions between transuranium elements and the components
     of cells and tissues."  Health Phys. 22:575.  1972

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                                    TECHNICAL REPORT DATA
                            (Please read Instructions on the reverse before completing)
 1. REPORT NO.
 EPA-600/3-76-058
             3. RECIPIENT'S ACCESSION-NO.
 4. TITLE AND SUBTITLE
 DISTRIBUTION OF INGESTED AMERICIUM IN CHICKENS AND
 TRANSPORT  TO EGGS
             5. REPORT DATE
              May 1976
             6. PERFORMING ORGANIZATION CODE
 7. AUTHOR(S)
 Anita A. Mullen,  Stephen R. Lloyd,  and
 Robert  E.  Mosley
                                                           8. PERFORMING ORGANIZATION REPORT NO.
 9. PERFORMING ORGANIZATION NAME AND ADDRESS
 Environmental Monitoring and  Support  Laboratory-LV
 U.S.  Environmental Protection Agency
 P. 0. Box 15027
 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  FY75
                                                            14. SPONSORING AGENCY CODE
                                                             EPA-ORD
                                                             Office of  Health & Ecological
 15. SUPPLEMENTARY NOTES
 16. ABSTRACT
 The soluble  citrate complex of americium-241 was orally administered  to  20 white
 Leghorn laying  hens daily for 2 weeks.   The yolks,  whites, and shells from the
 eggs were analyzed for their americium  content.   Yolk was the only egg fraction in
 which radioactivity was observed.  The  americium-241 activity in yolks reached a
 maximum on the  14th day of dosing.  Biological half-times of 2.00 ± 0.18 days and
 greater than 33 days were indicated by  the  average  concentration values  of
 americium-241 in yolks laid after the maximum activity was reached.               /

 The hens were serially sacrificed at 1,  10,  and 20  days after the final  admini-
 stration of  americium-241.  Tissue samples  were collected and the americium
 content determined in the edible portions and feathers of the hens.   Americium
 was detected in most tissues shortly after  dosing;  the main concentrations were
 found in the liver and the skeleton.  The highest concentration per organ (3.03 X
 10~3 percent of the dose) occurred in the liver of  the hens sacrificed 10 days
 after final administration of americium-241.
 7.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
                                              b.lDENTIFIERS/OPEN ENDED TERMS
                           c.  COSATI Field/Group
Americium
 'hickens
 Itrates
 Radioactive Isotopes
 ladiation Dosage
 Americium in Chickens
 Citrate Complex
 Distribution and Trans-
   port
 Exposure/Dose Assessment
   06C
   06R
   07B
   07C
   07E
   18B
 8. DISTRIBUTION STATEMENT
 RELEASE  TO PUBLIC
19. SECURITY CLASS (ThisReport)
  UNCLASSIFIED	
21. NO. OF PAGES

   17	
                                               20. SECURITY CLASS (This page)
                                                UNCLASSIFIED
                                                                          22. PRICE
EPA Form 2220-1 (9-73)
                                                                              691- 220-1976

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