United States
             Environmental Protection
             Agency
              Environmental Monitoring
              Systems Laboratory
              PO Box 15027
              Las Vegas NV 89114
EPA-600'3-80-030
February 1980
             Research and Development
v>EPA
Studies to Determine
the Absorption and
Excretion Dynamics
of Lead

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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 nine series. These nine 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 maximum interface in related fields. The nine series are:


       1.   Environmental Health Effects Research
       2.   Environmental Protection Technology
       3.   Ecological Research
       4.   Environmental Monitoring
       5.   Socioeconomic Environmental Studies
       6.   Scientific and Technical Assessment Reports (STAR)
       7.   Interagency Energy—Environment Research and Development
       8.   "Special" Reports
       9.   Miscellaneous Reports
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. Investiga-
tions include formations,  transport, and  pathway studies to determine the fate of
pollutants and their effects. This work provided 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 Information
Service, Springfield, Virginia  22161

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                                       EPA-600/3-80-030
                                       February  1980
    STUDIES TO DETERMINE THE ABSORPTION
       AND EXCRETION DYNAMICS OF LEAD
A. A. Mullen, R. E. Mosley, and Z. C. Nelson
        Exposure Assessment Division
Environmental Monitoring Systems Laboratory
          Las Vegas, Nevada  89114
    U.S. ENVIRONMENTAL PROTECTION AGENCY
     OFFICE OF RESEARCH AND DEVELOPMENT
ENVIRONMENTAL MONITORING SYSTEMS LABORATORY
          LAS VEGAS, NEVADA  89114

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                                  DISCLAIMER
    This report has been reviewed by the Environmental  Monitoring Systems
Laboratory, 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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                                   FOREWORD
    Protection of the environment requires effective regulatory actions based
on sound technical and scientific data.  The data must include the
quantitative description and linking of pollutant sources, transport
mechanisms, interactions, and resulting effects on man and his environment.
Because of the complexities involved, assessment of exposure to specific
pollutants in the environment requires a total  systems approach that
transcends the media of air, water, and land.  The Environmental  Monitoring
Systems Laboratory at Las Vegas contributes to  the formation and  enhancement
of a sound monitoring-data base for exposure assessment through programs
designed to:

         •  develop and optimize systems and strategies for moni-
            toring pollutants and their impact  on the environment

         t  demonstrate new monitoring systems  and technologies
            by applying them to fulfill special  monitoring needs
            of the Agency's operating programs

    This report summarizes information useful in assessing inorganic pollutant
exposure from pollutant-induced biological  responses.  The report deals
primarily with the absorption and excretion dynamics of lead in representative
biological monitors.  For further information contact the Exposure Assessment
Division.
                                                George B.  Morgan
                                                    Di rector
                                   Environmental  Monitoring  Systems  Laboratory
                                                    Las Vegas
                                     iii

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                                   ABSTRACT
    This is a summary report of studies designed to provide a basis for
developing a relatively rapid mammalian test system for lead, to provide
information on intestinal absorption, routes of excretion, and rates of
transfer to neonates, and to determine the usefulness of trace-element content
of feces, urine, blood, hair, and other tissues for estimating exposure.  As
rodents are endemic to most areas of interest, the laboratory rat was used as
the biological monitor.  As resident avian species are also readily available
in most areas of interest, a study was undertaken in conjunction with Dr.  F.
W. Edens et al. of North Carolina State University, Raleigh, N. C., to
determine if Japanese quail could function as reliable indicators to track the
movement of pollutants from source to receptor.

    Results indicate that 3 days following a single oral administration of
lead-210 to rats, about 0.2 percent of the dose was excreted in the urine  and
70 percent in the feces.  After 12 days the percentages had increased to about
0.3 and 90 percent respectively.  The main deposition sites were liver, 0.008
percent, and bone, 0.04 percent, of the oral dose.

    To ascertain the role of bile in the excretion of lead, the bile ducts of
young adult rats were ligated and comparisons of excretion rates were made
with rats having intact ducts.  During the 3 days following oral
administration  of lead-210, about 2 percent of the dose was accumulated in the
bile, whereas,  over the same time period, about 20 percent of an intravenous
dose was accumulated in bile.  This would indicate that biliary excretion  is a
main route for  removing absorbed lead.  Excretion curves for urine and feces
indicate the  primary difference between rats with intact bile ducts and those
with ligated bile ducts was the increased percentage of lead excreted in the
feces of intact rats.
                                                           *
    Bone was again found to be the main deposition site for lead, regardless
of the state of the bile duct.  Placental and milk transfer of stable lead
occurred in all animals.  Assessing exposure to lead by means of collecting
hair samples appeared to be a valid method provided the physiological state of
the animal was  considered.  During pregnancy and lactation, or rapid growth,
rats do not appear to incorporate as much lead in their hair as they do at
other periods of time.

    Using resident avian populations as reliable biological indicators of
increased lead  in the environment appears promising.  Dependent on the amount
of lead in the diet, lead concentrated in all portions of the quail egg, but
mainly in the shell.  Analysis of quail femur samples yielded similar results.

    This report covers a period from 1976 to 1979 and work was completed in
1979.

                                      iv

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                                   CONTENTS

                                                                          Page
Foreword	   iii
Abstract	    iv
Figures	    vl
Tables	   vii
Acknowledgment 	  viii
Introduction 	     1
Conclusions and Recommendations	     2
Experimental Procedures	     3
    Rodent monitoring	     3
         Excretion and tissue distribution 	     3
         Biliary excretion of lead 	     3
         Placental and milk transfer of stable lead	     4
         Analysis of lead in blood	     7
         Avian monitoring	     8

Results and Discussion 	     9
    Rodents	     9
         Excretion and tissue distribution in rats following
           a single oral  administration of lead-210	     9
         Excretion and tissue distribution of lead-210 following
           oral and intravenous administration to ligated and
           nonli gated rats	     9
         Placental and milk transfer of stable lead	    19
         Analysis of lead in blood	    24
    Birds	    26
References .	    28

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                                   FIGURES

Number                                                                    Page
  1  Mean Percent of Dose Excreted Daily in Urine of Rats
       Following a Single Oral Administration of Lead-210	   10

  2  Mean Percent of Dose Excreted Daily in Feces of Rats
       Following a Single Oral Administration of Lead-210	   11

  3  Mean Percent of Dose Excreted Daily in Urine of Rats
       with Ligated or  Intact Bile Ducts Following Either
       Oral or  Intravenous Administration of Lead-210	   13

  4  Mean Percent of Dose Excreted Daily in Feces of Rats
       with Ligated or  Intact Bile Ducts Following Either
       Oral or  Intravenous Administration of Lead-210	   14

  5  Micrograms of Equivalent Protoporphyrin per 100 ml
       of Blood from Rats Receiving Either Acute or Chronic
       Doses of Stable  Lead as the Acetate	   25
                                      vi

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                                    TABLES

Number                                                                   Page

  1   Treatment of Rats	    4

  2   Distribution of Offspring	    6

  3   Mean Percent of Administered Dose Recovered From Rats	   12

  4   Mean Percent of Dose to Rats Recovered per Organ	   16

  5   Mean Percent of Dose to Rats Recovered per Gram
        of Organ or Tissue	   17

  6   Placenta! Transfer of Lead in Rats	   20

  7   Mean Lead Content of Hair and Femur From Female and
        Young Rats at Various Times	   21

  8   Zinc Protoporphyrin Levels in the Blood of Rats
        and Offspring	   23

  9   Lead in Femurs From Japanese Quail Consuming Lead
        Acetate, From Hatching to Twelve Weeks of Age	   27

 10   Lead in Eggs From Japanese Quail  Consuming Lead
        Acetate, From Hatching to Twelve Weeks of Age	   27
                                     vi i

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                                ACKNOWLEDGMENT
    Since our Laboratory was interested in correlating the dose to
concentration values in the egg shells and femurs, a collection of Japanese
quail eggs and femurs were kindly sent to us by Dr. F- W.  Edens et al.  of
North Carolina State University, Raleigh, North Carolina,  for analysis.
                                     viii

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                                 INTRODUCTION


    A considerable body of information has been published on the environmental
aspects of lead (Barth et al., 1973; National Academy of Science 1972; Assoc.
Comm. Sci. Criteria Environ. Qual.  1973; Task Group on Metal  Toxicity 1976;
World Health Organization 1976; and U.S. EPA 1977).  The effects of lead on
laboratory animals have been extensively investigated for the purpose of
extrapolating this information to hazard evaluation in humans.   There is
relatively little information, however, on exposure/dose studies to develop
convenient biomonitors for assessing human exposure.

    Avian species resident in the area being monitored have been considered  by
several investigators as possible indicators of lead pollution  (Ohi et al.,
1974; Tansy and Roth 1970).  Tansy and Roth, reporting on a small  number of
pigeons, showed that there was a substantial increase in the lead  content of
various organs and tissue from pigeons obtained in urban Philadelphia as
compared to those obtained in rural Pennsylvania.   Ohi obtained  a  large number
of pigeons from locations in both downtown and suburban Tokyo.   Determination
of lead levels in blood, femur, and kidney demonstrated highly significant
regional differences.  Other authors have reported the presence  of lead in
tissue and eggs from migratory bird populations (Lincer and McDuffie 1974;
Martin and Nickerson 1973).  However, little information is available on the
relationship of intake to tissue levels.

    Studies show the gastrointestinal tract to be  the main pathway for the
excretion of heavy metals (Witschi  1964; Castellino et al., 1964;  Cikrt 1972;
Stanley et al., 1971).  Three routes exist for transport of heavy  metals into
the intestinal lumen, namely ingestion, secretion  by the intestinal mucosa,  or
by the liver into the bile.  Some contradictory reports question whether the
bile or intestinal wall is the significant pathway for lead excretion
(Karhausen 1973).

    The present studies were undertaken in order to work out the problems
associated with determining the mammalian samples  to be collected, and with
their subsequent analyses to estimate dose and potential  hazard  to the
environment.

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                       CONCLUSIONS AND RECOMMENDATIONS


    Hair samples from rodents appears to be a valid medium for obtaining
information on ingested lead without having to sacrifice the animal.  Care must
be taken to wash the hair thoroughly before analysis.   It is also  important  to
ascertain the physiological state of the animal when selecting individuals to
represent the population as a whole.  Information on lead derived  from hair  or
bone samples of lactating animals cannot be considered representative because
metabolism is altered during lactation.

    Bile fluid appears to be the major excretory route of absorbed lead in
rats.  The most concentrated and earliest indicator of lead ingestion is the
bile.  Therefore, a quick test procedure for lead in bile would be useful for
field testing, and bile should always be sampled for early detection  of lead
absorption.

    Based on the results of the avian portion of the study, further
investigation into the feasibility of using egg shells of resident species for
determination of trace element pollutants would be advisable.

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                           EXPERIMENTAL PROCEDURES
RODENT MONITORING

Excretion and Tissue Distribution

    A preliminary range-finding study to determine the relatively long term
excretion and tissue distribution of lead by rats utilized adult, 100-day old,
268-gram (g) female Wistar rats.  Following fasting for 12 hours, the rats
each received a single oral  dose containing 4 microcuries (yCi)  of lead-210
(210Pb) as the nitrate plus  50 milligrams (mg) of stable lead nitrate per
kilogram (kg) of body weight.  The 210Pb content used in these studies was
obtained as lead nitrate [Pb (N03)2] in 3J^[ nitric acid (HN03) solution.  The
specific activity was 62.0 curies per gram (Ci/g) Pb and the purity was >99
percent.  The doses, aliquoted by volume measurements of known dilution, were
verified following administration by placing random doses in volumetric flasks
and counting aliquots of the doses.

    The rats were housed in  stainless steel metabolism cages and maintained on
commercial laboratory rat chow and deionized water.  Total urine and feces
excretions were collected daily, weighed, and analyzed by gamma  spectrometry.
Twelve days following dosing, the rats were sacrificed by anesthetic overdose,
and blood was removed by means of cardiac puncture.  Tissue samples were
removed, weighed, and placed in scintillation vials containing formaldehyde.
The 2iopb content was subsequently determined by counting the samples.

    All samples were counted for up to 40 minutes using a Nal(Tl) well crystal
connected to a single-channel analyzer.  Sample collection was discontinued
when the counting errors exceeded 30 percent.


Biliary Excretion of Lead

    To elucidate the mechanism by which lead is transferred to the intestine
by ligation of the bile duct of rats to measure the difference in the
excretion of lead by Ifgated and intact rats, two groups of 16 female Wistar
rats, 51-day-old young adults, weighing 150 g, were used.  A summary of rat
treatments is shown in Table 1.

    The common bile duct of  six animals in each group was ligated.  Food was
withheld for 12 hours prior  to surgery.  The rats were anesthetized with
Fluothane® and the bile duct was ligated at a point 5 or 6 millimeters (mm)
©Registered trademark

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                         TABLE 1.  TREATMENT OF RATS
No. of
 Rats
Condition of
 Bile Duct
  Method of
Administration
             Dose
  2

  4

  6
  2

  2

  6
  Li gated

  Intact

  Li gated


  Intact


  Li gated

  Intact
  Li gated


  Intact
   Control

   Control

   Oral


   Oral


   Control
   Control

   Intravenous


   Intravenous
None

None

4yCi  2iopb + 50 mg/kg Body Wgt
  Pb (No3)2

4yCi  2iopb + 50 mg/kg Body Wgt
  Pb (No3)2

None

None

2yCi  ziopb + 10 mg/kg Body Wgt
  Pb (No3)2

2yCi  2iopb + 10 mg/kg Body Wgt
  Pb (No3)2
below the hilus at the liver and just above where the pancreatic tissue no
longer  surrounded it.  A surgical procedure described by Lambert (1970) was
modified for  use in this procedure.  One hour after ligation of the bile duct,
when the animal had recovered  from the  anesthetic, 210Pb and stable lead were
administered  orally by means of a dosing needle and syringe or intravenously
via the tail  vein.

    The rats  were placed in stainless steel metabolism cages and total urine
and feces samples were collected every  12 hours.  Samples were collected in
the same manner as the preceding group.  Three days following administration
of the  radionuclide, the animals were weighed and sacrificed, and blood was
removed by cardiac puncture.   Tissue samples were collected and analyzed in
the same manner as in the  previous study.
Placental  and Milk Transfer of  Stable  Lead

     Fifteen  recently  impregnated  Wis.tar rats  were  obtained.  Samples of hair
and  blood  were collected  to determine  baseline  lead  levels.  On the ninth day
of gestation, all of  the  pregnant rats,  including  the controls, were weighed
and  orally dosed with 50  mg/kg  Pb (N03)2 in water  solution.  Thereafter all
drinking water supplied to  eight  of  these rats  contained 50 parts per million
(ppm) of lead as lead acetate.  The  remaining seven  pregnant rats received
deionized  water.  Daily records of water intake were kept for all animals.

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    A total of 112 offspring were born to 10 pregnant rats.  Of that total 67
were born to dams receiving chronic doses of lead as lead acetate, while 45
were born to dams receiving a single acute dose.  The average litter size for
dams receiving chronic lead was 11.2 with a standard deviation of ±2.3, while
that, of dams receiving a single chronic dose was 11.3 ± 1.5.  The distribution
of offspring is shown in Table 2.  At parturition, two animals from each
litter were sacrificed for whole body analysis of lead content.  The litters
were reduced to contain no more than six offspring.  Blood and hair samples
were collected from all dams at this time.

    Hair and blood were collected from the jdams and young upon weaning and at
monthly intervals.  Also upon weaning, two young from each litter were
sacrificed for whole body analysis of lead content.  Two young in each litter
received water containing lead and two received deionized water.   The dams
remained on their previous diet and hair and blood samples were taken at
monthly intervals from both dams and young.

    Following parturition and about 12 days after initial  dosing, four litters
(24 offspring) of the rats not receiving lead in water were cross-fostered
with lactating rats receiving lead daily.  Four litters from rats receiving
lead daily were cross-fostered with lactating rats not receiving  lead, and 36
remained with their natural mothers.  At weaning, 21 days following birth, 5
of the litters were placed on the same diet as the adults with water
containing 50 ppm lead as lead acetate available ad libitum, and  5 litters
received an identical diet but utilizing deionized water.

    Hair samples collected during this study were removed using electric
clippers.  The clipper heads were rinsed with dilute HN03, then with distilled
deionized water.  The hair was stored in HN03-washed polyethylene bottles.
The hair was washed using the procedure described by Clarke and Wilson (1974).
This consisted of washing the hair twice in deionized water, followed by two
detergent washes, and two deionized water rinses.  The hair was then soaked
twice in acetone, drained, and placed in a hot, saturated ethylene
diaminetetraacetic acid (EDTA) solution, and allowed to soak for  5 minutes.
The hair was then drained, rinsed in double-distilled deionized water, and
drained.  The EDTA soak was repeated and the rinsing procedure was repeated
twice.  The drained hair was covered and dried at room temperature.  The hair
was stored in HNO.-acfd washed, double-distilled deionized water-rinsed
polyethylene bottles until weighed and wet ashed..

    The tissue samples were removed using stainless steel  instruments,
weighed, and stored in acid-washed and rinsed polyethylene bottles, and stored
at 0°C or below until wet ashed.

    The samples were completely ashed in hot concentrated HNOa acid,  llltrex®
(redistilled, high purity nitric) acid was used to minimize error due to
introduced lead.  Borosilicate glassware was used throughout the  analysis.
The glassware was cleaned by being soaked overnight in 1:1 HMOs acid, rinsed
with water, and soaked overnight in distilled water.
©Registered trademark, Van Waters and Rogers Scientific Company.

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                                   TABLE 2.  DISTRIBUTION OF OFFSPRING
Treatment of Offspring

Number of Offspring


Cross-fostered to
lactating females
receiving lead
(lead in milk)

Not
cross-
fostered
Cross-fostered
to lactating
controls (no
lead in milk)
Weanlings
on water
containing
lead
Weanlings
on water
containing
no lead
Born to dams not receiving
 lead in water (controls):
        13                                          X              --
        12                         X*               -             -               X
        10                         X                —             —               X
Born to dams receiving
 lead in water:
11
9
8
14
13
12
10
X
x
x
X
X
x
x
X
X
X
X
X
X
X
   Each X represents one litter of rats.

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    Following digestion, the resulting solution was analyzed for lead by
atomic absorption spectrophotometry.  The sensitivity of the analysis was
about 0.11 micrograms per milliliter (yg/ml).  The results were checked by
using the method of standard additions.


Analysis of Lead in Blood

    In a search for alternate methods to determine lead uptake, the ZnP (zinc
protoporphyrin) Model 4000 Hematof1uorometer® was evaluated.  A preliminary
study was conducted to determine whether induced lead toxicity in laboratory
rats can be detected by the instrument, which is available commercially for
use in lead toxicity screening clinics.  Lead inhibits heme synthase, an
enzyme critical to the conversion of protoporphyrins to heme.   Lead toxicity
results in increased blood levels of protoporphyrins which combine with zinc
to form the fluorescent molecule, zinc protoporphyrin.  The Hematof1uorometer
is designed to detect zinc protoporphyrin (ZnP) by front surface illumination
of a drop of whole blood.  Fluorescent wavelengths emitted by  the ZnP
molecules are subsequently detected by a photomultipllier tube.  Because no
extractions or volume measurements are necessary, this technique is useful  for
field work.  Its application to other species of animals, however,  is
questionable since it has been tested only on human subjects.

    For purposes of variable dosing, 20 adult Wistar rats were divided into
three groups.  Group I consisted of eight females given an initial, one-time
dose of 50 mg/kg lead acetate.  Group II was comprised of eight females given
a daily dose of 10 mg/kg lead acetate.  Group III was made up  of four males
given an initial dose of 50 mg/kg lead acetate, followed by daily doses of
10 mg/kg.  The Teachaeetate was prepared from a reagent grade  chemical  and
nonsterile, deionized water at a concentration of 3 mg/ml.  Doses were
administered intraperitoneally with a 26-gauge needle.

    Before dosing a background sample was obtained from each animal.   The
animals were maintained on tap water and rat chow (ad libitum).  Each rat
inhabited a single cage in a constant temperature room with a  12-hour
light-dark cycle.

    Blood samples were collected weekly by cardiac  puncture.  For this
procedure the rats were anesthetized with 50 mg/kg  sodium pentobarbital.
Because of the lengthy recovery period from this drug, a barbituate,
Mikedimide®, was administered after the sample had  been obtained.  Blood  was
collected in heparinized 1-cubic-centimeter (cc) tuberculin syringes  and
analyzed on the same day.  Heparinized capillary tubes were filled from the
syringes for hematocrit determinations.  The Hematof1uorometer was  calibrated
according to hematocrit results before sample analysis.  The analytical
technique was that prescribed by the manufacturer's manual.
©Registered trademark, Environmental  Sciences Associates,  Inc.

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Avian Monitoring

    The experimental design relating to feeding and dosing of lead in avian
species has been reported by Edens et al. (1976).  The study conducted at
North Carolina State University, Raleigh, North Carolina, utilized Japanese
quail.  The quail received a nutritionally complete quail starter-grower diet
to which lead in the form of lead acetate was added in levels of 0.1, 10, or
100 ppm.  The amount of lead added was corrected for acetate moeity.  Quail
chicks consumed the experimental diet ad libitum from the time they were
hatched.

    When the quail were 6 weeks old, all experimental diets were changed to
consist of a quail layer ration with added lead.  The quail were sacrificed at
6 and 12 weeks of age and femurs were collected from both males and females.
Eggs were collected throughout the study and subsequently shipped to the
Environmental Monitoring Systems Laboratory.  Preparation of the egg and femur
samples for analysis consisted of weighing the sample and using a wet ashing
procedure as described in our study of rats.  Stable lead was determined by
atomic absorption spectrophotometry.
                                       8

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                            RESULTS AND DISCUSSION
RODENTS
Excretion and Tissue Distribution in Rats Following a Single Oral
Administration of Lead-210

    The average daily excretion percentages are shown in Figures 1 and 2.  The
peak excretion value occurred at the first collection 24 hours following
administration.  The mean percent of administered dose recovered in the urine
after three days was 0.160 with a standard deviation of ±0.088, and after 12
days it was 0.269 ± 0.168.  The percentage of the dose recovered in the feces
was 72.1 ± 13.0 and 89.0 ± 4.91 at 3 and 12 days respectively.  Twelve days
following oral administration, an average of 8.19 x 10~3 ± 4.62 x  10~3
percent of the dose was found in the liver, whereas bone contained 4.33 x
ID'2 ± 2.22 x lO-2 percent of the 210Pb.


Excretion and Tissue Distribution of Lead-210 Following Oral  and Intravenous
Administration to Li gated and Nonligated Rats

    As shown in Table 3, 3 days following a single oral administration of
210Pb as the nitrate, to rats having intact bile ducts, about 0.1  percent of
the lead had been excreted in the urine, while about 95 percent was excreted
in feces.  The amount of the dose recovered in the tissues was about 5
percent.  After a similar dose to rats with ligated bile ducts, about 0.1
percent was excreted in the urine and about 93 percent in the feces.  The
tissues maintained about 7 percent of the dose.  The difference between the
percentages found in the tissue indicates that about 2 percent of  the dose was
retained in the bile of the ligated rats.

    Following intravenous administration of 2iOpb as the nitrate,  rats with
ligated bile ducts excreted about 4 percent in the urine, while those with
intact bile ducts excreted about 6 percent.  Only 7 percent of the dose was
excreted via the feces of ligated rats as compared to 24 percent by intact
rats.  The amount of the dose recovered in the tissues was also different,
with 71 percent recovered in rats with intact bile ducts versus 88 percent in
rats with ligated bile ducts.  If the differences in these figures are caused
by lead in the bile, then as much as 20 percent of the 210Pb dose  would have
been accumulated in the bile during the 3 days following intravenous
administration; however, only 2 percent of the oral dose (or about 28 percent
of the lead absorbed) was accumulated in bile during the same time period.

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  102,
  10-2
o
Q
(B
O

5
            24
48
96
120
 I
144
168
192
216
                                     Hours
      Figure 1.   Mean  percent  of dose excreted daily in urine of rats
            following  a  single oral administration of lead-210.
                                    10

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S
o
a
3?
24   48  72   96
120  144
 Hours
168
192
                                                216
240  264  288
  Figure 2.   Mean percent of dose excreted daily in feces of rats
        following a single oral administration of lead-210.
                                 11

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       TABLE 3.  MEAN PERCENT OF ADMINISTERED DOSE RECOVERED FROM RATS
 Measurement
                           Oral Administration
  Non-      Non-      Non-
Li gated   Li gated   Li gated   Li gated
I.V. Administration
   Non-
 Li gated   Li gated
Number of rats       12        12         6         6          66

Days after
 administration      12         3         3         3          33

Age in days         100       100        51        51         52        52

Urine:

 Mean              0.269     0.160     0.098     0.102      5.86      4.09
 Std. deviation    0.168     0.088     0.060     0.635      6.48      3.23
 Std. error        0.053     0.028     0.024     0.251      2.65      1.32

Feces:

 Mean             89.0      72.1      95.1      92.8        24.1      6.75
 Std. deviation    4.91     13.0       3.00      2.77        9.50     7.35
 Std. error        1.55      4.12      1.22      1.13        3.88     3.00

Tissue:
Mean
Std. devation
Std. error
4.79
2.97
1.21
7.14
2.77
1.13
70.6
13.0
5.31
88.2
9.14
3.73
    Cikrt (1972) found that 6.7 percent of intravenously administered 2iopb
was excreted into bile during the first 24 hours following administration, as
measured by cannulation of the bile duct of adult, 200-g rats, and collection
of the excreted bile.  Klaassen (1973) measured 1.0 ug/min/kg of lead in  bile,
2 hours after intravenous administration to rats.  A five times greater
affinity of lead for liver than for plasma was found.  Biliary excretion  is
temperature dependent, rising as the ambient temperature rises.  It was also
found by Klaassen that there were definite species differences in lead
excretion in bile.  Rabbits excreted less than one-half of the lead that  was
measured in the bile of rats, where dogs excreted less than one-fiftieth  that
of rats.

    Figures 3 and 4 show the mean excretion values for ligated and intact rats
following a single acute dose of lead nitrate.  In Figure 3 the excretion
values shown for urine after oral dosing were similar and therefore they  were
averaged for graphic representation.  The percentage of lead excreted in  the
urine appeared to be very nearly a constant value over the period of the
study.

                                      12

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      102.
   8
   o
  Q
   i
      10-
                                 • Urine I.V. Ligated
                                 • Urine I.V. Non-Ligated
                                 A Urine Oral Ligated and Non-Ligated
                      24
48
72
9*6
                            Hours
Figure 3.   Mean percent of dose  excreted daily in  urine of rats with
       ligated or intact bile ducts following either  oral  or
               intravenous administration of lead-210.
                                   13

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      in
      O
     Q
      i
         10=1
                                          Feces I.V. Ligated

                                          Feces I.V. Non-Ligated

                                          Feces Oral Ligated

                                          Feces Oral Non-Ligated
                     24
48
 I
72
96
 I
120
                                    Hours
Figure 4.   Mean percent  of dose excreted daily in feces  of rats with
       Iigated or intact bile ducts  following either  oral  or
               intravenous administration of lead-210.
                                   14

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    Excretion of lead via the feces, exhibited unique curves dependent on the
secretion of bile into the intestine.  The percentage of ingested lead
excreted in feces from rats having intact bile ducts peaked at 36 hours, then
decreased rapidly.  Lead in feces from rats with ligated bile ducts did not
peak until 50 hours after dosing.  These values seem to indicate that not only
did the ligation of the bile duct decrease the overall  percentage of lead
excreted, but it also appeared to increase the time that the ingested lead
remained in the gastrointestinal  tract.  The urine from ligated rats contained
a higher percentage of lead at 50 hours after dosing than did the intact rats.

    Figure 4 illustrates the excretion curve for the intravenously dosed rats.
The primary difference between the curves for the rats  having either ligated
or intact bile ducts was the increased percentages of lead excreted in the
feces of the intact rats, although the differences in the amount of lead
excreted became less pronounced in samples obtained more than 48 hours
following dosing.  The percentage of dose excreted by the intact rats
approached a peak 24 hours following dosing then dropped 12 hours later; this
drop also occurred in the ligated rats.  The peak value for the ligated rats
occurred 48 hours after dosing.  The urine curve for the intact rats appears
to be a typical  excretion curve following intravenous dosing.  The urine curve
for the ligated rats appears similar to that of an orally dosed animal.  This
may be due to the constant reabsorption of the bile by  the ligated animals and
slow excretion in the urine.

    As shown in Table 4, the average percentages of the dose recovered in the
various organs indicate that the  expected increase in liver concentration of
ligated rats occurred.  The amount of lead concentrated in the kidney of
ligated rats was higher than that of intact rats, following intravenous
administration.   The spleens of ligated rats contained  a greater amount of
lead than did the spleens of intact rats following oral  administration.

    Table 5 lists the percentages of lead recovered per gram of rat tissue or
compartment.  Following intravenous administration, the kidneys of ligated
rats still contain more lead per  gram than those of intact rats.   This may
indicate reabsorption of the ligated bile and excretion via the kidneys so the
amount of lead accumulated in the bile may be higher than is indicated from a
simple subtraction of tissue content.

    The action of bile in the absorption and excretion  of heavy metals appears
to be very complex.  Durbin (1972) reported that excretion of plutonium by
humans was by way of the bile and digestive juices, and that fecal  excretion
of plutonium was decreased by one-half or more in persons whose
gastrointestinal tracts were judged to be not normally  stimulated.   Ballou and
Hess (1972) found that 50 percent of the plutonium excreted into the perfused
intestine of the rat arrived by way of the bile during  the first hour after
plutonium injection.  After treatment with DTPA the proportion excreted in
bile was increased to 75 percent  and the bile plutonium concentration
increased 15 to 20 fold.  Barth and Mullen (1974) using an artificial  cow
rumen have found that the heavy metal transuranics are  dissolved by bile in
the duodenum.  Whether the heavy  metals became more soluble due to complex!ng
and whether the complex metal  ions are more or less available for reabsorption
was not definitely established.

                                      15

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         TABLE 4.   MEAN PERCENT OF DOSE TO RATS RECOVERED  PER  ORGAN
 Measurement
                         Oral  Administration
                    I.V.  Administration
                      Li gated
Non-ligated
Ligated
Non-ligated
Liver:
Mean
Std. deviation
Std. error
Spleen:
Mean
Std. deviation
Std. error
Kidney:
Mean
Std. deviation
Std. error
Heart:
Mean
Std. deviation
Std. error
Brain:
Mean
Std. deviation
Std. error
Skull:
Mean
Std. deviation
Std. error
Bone:
Mean
6.18 x 10-2
1.94 x 10" 2
7.93 x 10-3
6.41 x 10-3
5.06 x 10-3
2.26 x ID'3
3.70 x 10-2
1.43 x 10- 2
5.82 x 10-2
8.97 x 10'"
5.02 x 10-"
2.90 x 10-"
1,40 x 10-3
5.86 x 10' *»
4.14 x 10-"
1.58 x 10" 1
3.50 x 10-1
1.56 x 10- 1
9.70 x 10-1
3.05 x 10-2
3.47 x 10- 2
1.42 x 10-2
6.62 x 10-"
3.62 x 10-"
2.09 x 10-"
5.85 x 10-2
5.02 x 10'2
2.05 x lO'2
7.72 x ID'"
5.53 x 10-"
3.19 x 10-"
5.06 x 10-"
7.52 x ID'1*
3.76 x 10-"
9.16 x 10- 1
1.38
6.91 x 10- l
1.68 x 10- l
5.61
7.92
3.23
3.71 x ID'1
5.01 x 10-1
2.04 x 10- 1
3.11
2.29
9.34 x 10- 1
9.78 x ID" 3
8.44 x 10- 3
3.45 x 10- 3
2.20 x 10- 2
1.67 x ID" 2
6.83 x 10- 3
2.56
2.25
9.19 x 10- 1
1.36 x 10- l
1.97
2.13
8.70 x 10-1
5.46 x 10"1
1.16
4.74 x ID'1
1.13
1.28
5.23 x 10- l
5.71 x ID'3
4.22 x 10- 3
1.89 x 10- 3
1.57 x 10-2
1.16 x 10'2
4.74 x lO'3
1.92
1.77
7.21 x 10' l
1.23 x 10- l
    The rapid excretion of lead with the bile during the first 72 hours  after
dosing may decrease as the lead is more tightly bound to the hepatocytes.
Most lead carried in the blood is attached to the red blood cells (Goodman  and
Gilman 1970), therefore little is filtered when lower levels of lead are
present.  During exposure to high acute lead levels, a greater portion is
filtered, so that biliary excretion and urinary excretion is at first rapid,
then falls to a more steady state.  Castellino and Aloj (1964) found that
2iopb is rapidly distributed in the tissues of rats, the highest
concentrations being in the kidneys, liver, and bones.  Lead carried in  the
                                      16

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TABLE 5.  MEAN PERCENT OF DOSE TO RATS RECOVERED PER GRAM OF ORGAN OR TISSUE
Measurement
Femur:
Mean
Std. deviation
Std. error
Skull:
Mean
Std. deviation
Std. error
Liver:
Mean
Std. deviation
Std. error
Spleen:
Mean
Std. deviation
Std. error
Kidney:
Mean
Std. deviation
Std. error
G.I. Track:
Mean
Std. deviation
Std. error
Muscle:
Mean
Std. deviation
Std. error
Oral Admini
Li gated

4.67 x 10-2
3.39 x 10-2
1.39 x lO-2

9.19 x 10- 2
7.23 x 10-2
3.23 x ID"2

5.79 x 10-3
3.49 x 10~3
1.43 x 10-3

3.44 x 10-3
2.09 x 10-3
1.21 x 10-3

2.85 x 10-2
2.01 x 10-2
8.21 x 10-3

2.66 x 10-1
8.71 x 10-2
3.56 x 10-2

4.39 x 10-4
4.58 x 10-^
2.05 x 10-^
strati on
Non-1 i gated

6.62 x lO-2
3.33 x lO-2
1.36 x lO-2

1.91 x 10'1
2.79 x 10-1
1.25 x 10'1

4.86 x 10-3
7.72 x 10" 3
3.15 x 10-3

1.35 x 10-3
1.28 x 10-3
7.40 x 10-t

3.61 x lO-2
3.23 x 10-2
1.32 x 10-2

1.55 x 10- 1
1.72 x 10-1
7.03 x 10-2

6.39 x 10-4
1.14 x 10- 3
5.68 x 10- 4
I.V. Admini
strati on
Li gated Non-1 igated

1.12
1.15
4.71 x 10-1

8.28 x 10-1
6.64 x 10- !
2.71 x lO'1

5.29 x 10- 1
6.20 x 10- l
2.53 x 10- l

6.15 x 10-i
8.69 x 10-1
3.55 x 10-1

1.93
1.48
6.06 x 10-1

1.92 x 10-1
2.18 x 10-1
8.90 x 10-2

2.90 x 10-2
2.41 x 10- 2
9.82 x 10- 3

1.10
9.50 x 10-1
3.88 x 10-1

5.48 x 10- 1
4.71 x 10-1
1.92 x 10- 1

1.78 x 10- :
1.84 x 10" 1
7.52 x 10- *

5.69 x 10-1
1.15
4.70 x 10-1

8.58 x 10-1
7.37 x 10-1
3.01 x 10-1

2.80 x 10-1
3.09 x 10-1
1.26 x 10-1

1.63 x 10-2
1.39 x 10- 2
5.69 x 10- 3
                                    17

-------
blood is deposited in bone as relatively insoluble tertiary lead phosphates
which are removed from bone tissue at a rather slow rate.  Neathery and Miller
(1972) found that most lead entering the systemic circulation by injection in
rats invades the reticuloendothelial system represented by bone marrow,
spleen, and liver.  In contrast, the lead entering the gut wall goes to the
bone and kidney of rabbits.  Blaxter (1950) found that in other species most
orally ingested lead is deposited in the skeleton.  Thus, lead may initially
be concentrated in bone until a possible threshold is reached and then
deposition is mainly in the kidney, where turnover rate is slow.

    As shown in Table 4, bone was the primary deposition site for lead in
rats. The skull and femurs were selected as representative of the skeleton of
the rat.  Data for total bone were calculated assuming 10 percent of the body
weight of the adult rat is represented by the skeleton (Sikov and Mahlum
1972).

    Holtzmann (1963) measured radium D (210Pb) concentration in bone and found
that it is higher in trabecular than cortical bone, although the skull
appeared to be the bone most representative of total skeleton.  Based on five
human subjects, the mean deviated by 16 percent.  Tibia, mandible, and joint
bone were lower than average and rib was higher (38 ± 34 percent above
average).  This agreed well with Kehoe (1961), who found that the
concentration of lead, after recent exposure, was often higher in the flat
bones than in the long bones.

    Holtzmann (1963) concluded that within a factor of 2, radium D is
uniformly distributed  in human skeletal mineral, and the total skeletal
content of radium D may be estimated with some assurance from measurements on
a  single bone.

    It  is interesting  to note, as shown in Table 5, that the percentages of
dose recovered in the  skull versus the femur were higher in orally dosed rats.
In  intravenously dosed animals the chemical form may be different from that of
lead absorbed through the  gut and a higher percentage could be deposited in
bone marrow.

    The total percent of 21OPb retained in the tissue was found to be about 5
percent in 51-day-old  rats, 3 days after an acute oral ingestion.  This agrees
with values found by other authors as noted by Karhausen (1973).  During the
same time period, 70 percent of an intravenously administered 21opb dose was
retained.

    The standard error was found to be relatively high in all measurements of
lead metabolism.  This has also been found to be true by other authors, as
noted by Di Ferranti and Bourdeau (1973) and seems to indicate that storage of
lead in different compartments is far from being consistent.  Absorption of
lead appears to be affected by dietary calcium, chemical form, Vitamin D
intake, diet composition,  load of lead and gastric acidity (Karhausen 1973).
                                      18

-------
Placental and Milk Transfer of Stable Lead

    The pregnant rat females receiving water containing 50 ppm of lead
ingested an average of about 30 ± 10 ml per day, approximately 1.5 mg of lead
per day, or a total of about 190 mg of lead prior to parturition.

    At parturition, 12 days following initial  dosing, a minimum of two
neonates per group, or enough to decrease the  rats litter size to six, were
sacrificed and the lead content of the whole body was determined.  The average
amount of lead recovered from the whole bodies of 19 neonates was 6 yg of lead
per neonate (see Table 6).  This is about 3 x  10-5 percent of the total  lead
ingested by the dams or about 4 x 10~2 percent of the lead absorbed.   An
average of one mg of lead per offspring or 6 x 10~3  percent of the dose, was
present in neonates from mothers who received  the same single dose (50 mg/kg)
of lead but did not ingest a daily lead supplement to their drinking  water.
The mean weight of neonates from rat dams ingesting lead daily was no
different from those of neonates born from dams ingesting a single dose of
lead and fell  within the mean weights, 5 to 6  g, for neonates of this breed
(Ralston Purina Company 1961).

    The lead content of the neonates may be different than it would have been
had the neonates been sacrificed immediately after birth.  In most cases the
dams gave birth during the night and the young were not removed until  the
following morning.  This introduced at least two possible errors.  First, the
young were suckling during this time, a maximum of 16 hours and excretion of
lead may have increased due to mobilization of lead in soft tissues shortly
after birth.  How much of this mobilized lead  is excreted and how much stored
in bone is not known.  Singh et al. (1976) showed that the kidney, liver,
heart, and brain of newborn rats contained very high amounts of lead  when the
young were sacrificed within half an hour after birth; a very significant
reduction occurred after 1 day with further reduction after 7 days, however,
blood levels remained almost identical to the  levels obtained 1 day after
birth.  No bone samples were taken so it is not known if increased deposition
occurred at this site or if the lead was secreted in the bile and excreted via
the feces.

    Other authors have found lead to be rapidly transported to the fetus.
(Hubermont et al., 1976; Schaller et al., 1976; McClain and Becker 1975;
Kostial and Momcilovic 1974; Green and Gruener 1974; and The Task Group  on
Metal Accumulation 1973).

    Green and Gruener (1974) found that lead transport was so rapid that the
fetus is in equilibrium with the mother rat 24 hours after injection,  although
other authors, Schaller et al. (1976) and McClain and Becker (1975),  indicate
that the placenta acts as a diffusion barrier  and limits the passage  of  lead
to the fetus since large maternal-fetal  concentration gradients existed.

    Results from the analysis of hair samples  are listed in Table 7.   Since
rats are born without hair the first samples were obtained at weaning, about
21 days after birth.  The transfer of lead from dam to young during lactation
has been previously established.  Green and Gruener (1974) found that signifi-
cant amounts of lead were transferred to the nursing rats even a week after a

                                      19

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                TABLE 6.   PLACENTAL TRANSFER OF  LEAD  IN  RATS

Number of
Neonates
Sacrificed
Experimental :**
6
5
2
2
2
2
Mean
Controls:
8
2
2
2
Mean
Mean Weight*
per Neonate
(9)
Mean S.D.

5.70 ± 0.46
5.93 ± 1.94
5.79 ± 0.65
5.93 ± 0.26
5.78 ± 0.87
6.00 ± 1.21
5.83 ± 0.49

5.99 ± 0.45
5.65 ± 0.50
5.68 ± 0.62
5.60 ± 0.59
5.69 ±0.37

ug of Lead*
per Gram
Whole Body

1.76 ± 0.60
0.88 ± 0.53
1.07 ± 0.18
0.84 ± 0.04
0.88 ± 0.13
0.85 ± 0.16
1.19 ± 0.59

Background
0.22 ± 0.28
0.20 ± 0.21
0.45 ± 0.18
0.22 ± 0.21

yg of Lead*
per Neonate


6.75 ± 4.80
5.20 ± 3.09
6.25 ± 1.80
5.00 ± 0.21
5.00 ± 0.45
5.00 ± 0.49
6.74 ± 3.16

Background
1.30 ± 1.70
1.14 ± 1.31
2.50 ± 0.71
1.24 ± 1.02
 * Standard deviations are shown for each mean value.
** Dams exposed to lead in drinking water mean ± one S.D.
single administration.  Kostial and Momcilovic in 1974 discovered that  during
pregnancy and lactation both lead-203 and calcium-47 were transferred in
substantial amounts from mother to fetuses and offspring.  The amount
transferred was related to the physiological state, as that amount was  higher
in late lactation.

    The results shown in Table 7 indicate that little lead was incorporated
into the hair of young rats prior to weaning, and with the relatively low lead
doses received by the animals in this study, the amount transferred to  milk
would be small.  Stanley et al. (1971) found that less than 0.02 percent  of  a
single oral dose of lead-203 was secreted in bovine milk over a 5-day period.
Neathery and Miller (1972) compiled the results of a number of investigations
into the secretion of lead into milk, all of which indicate that under  most
conditions, relatively little lead is secreted into milk.

    However, lead absorbtion is much higher in newborn rats.  Momcilovic  and
Kostial (1974) state that whole body retention of lead was higher in sucklings
compared to adults.  After an 8-day period there was still 85 percent of  an
injected dose remaining in the sucklings compared to 34 percent in the  adult
                                      20

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   TABLE 7.  MEAN* LEAD CONTENT OF HAIR AND FEMUR FROM FEMALE AND YOUNG RATS AT VARIOUS TIMES (pg/g)
Animals
3 dams
(a, b, c)
3 litters
(a, b, c)
2 dams
(d. e)
2 litters
.(d, e)
ro
"" 2 dams
(f, 9)
Hair
Treatment Weaning Ueaning
Parturition Weaning + 1 mo + 2 mo
Lead in drinking 20.4 ± 2.30 0.26 ± 0.02 15.2 ± 7.62 19.8 ± 4.30
water
Lead in milk and 0.36 ± 0.15 10.6 ± 3.0 11.7 ± 2.09
drinking water
Lead in drinking 9.72 ± 4.91 0.62 ± 0.26 12.8 ± 2.26 10.6 ± 3.70
water
Lead in milk only 0.24 ± 0.12 17.2 ± 1.89 10.6 ± 4.41
No lead exposure 1.12 ± 0.22 0.23 ± 0.09 0.20 ± 0.05 0.28 ± 0.02
Femur
Weaning
+ 2 mo
21.3 ± 5.92
13.1 ± 2.36
18.7 ± 6.79
13.4 ± 1.94
4.88 ± 1.14
2 litters       Lead in milk and                0.52 ± 0.25   11.2 ± 5.02    17.6  ± 6.31    20.0 ± 4.38
 (f, g, nursed   drinking water
 by dams h, i)

2 dams          Lead in drinking   9.80 ± 3.68  0.27 ± 0.21   19.6 ± 2.19    16.6  ± 1.98    20.5 ± 1.70
 (h, i)          water

2 litters       No lead exposure                0.60 ± 0.26   0.26 ± 0.13    0.32  ± 0.16    4.96 ± 2.03
 (h, i, nursed   from milk or
 by dams f, g)   water

Control  damNo lead exposure2.140.630.490.557.41

Control  litter  No lead exposure                0.34 ± 0.11   0.27 ± 0.10    0.26  ± 0.10    3.35 ± 1.20

* Standard deviations are shown for each mean value.                                  ~~~~

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rats.  Forbes and Reina (1972) investigating the effect of age on
gastrointestinal absorption of lead found that peak lead absorption,  89.7
percent, occurred at 20 days of age, decreasing to 16 percent at 89 days.   But
of the amount of lead absorbed, preferential deposition would probably take
place in the bone of the rapidly growing young with little being available  for
incorporation into hair.

    Kostial and Momcilovic (1974) also found that the transplacental  transport
of lead was eight times lower than that of calcium while the transmammary
transport of lead was only four times lower than calcium.  During lactation,
the retention of calcium-47 in femurs and teeth of the dams was 71 percent  and
50 percent lower than control females, and lead retention during the  same time
was 33 percent and 24 percent lower.  Therefore, during the stress of
lactation, lead was not as available for incorporation into the hair  of the
dams as it would be during other physiological states.

    This is confirmed in our study which shows the lowest amounts of  lead in
hair during lactation.  One month after weaning, the lead content of  hair from
both dams and young receiving lead had increased significantly.  The  hair of
offspring receiving milk containing lead but receiving no supplementary lead
following weaning contained elevated levels 1 month after weaning, but the
levels had started to drop after an additional month indicating that  there  was
a delay between increased ingestion of lead at the latter stages of lactation
and incorporation into hair.  Litters from control dams receiving milk from
lead-dosed foster dams gradually increased the amount incorporated in hair.

    Litters receiving lead only during fetal development did not show
significantly greater amounts of lead in their hair as compared to the hair of
control litters.

    Femurs collected 2 months after weaning showed the expected lead  levels
dependent on the exposure to lead.  Since all dams received some lead (the
control dams inadvertently received the initial oral dose of lead), the
concentration of lead at levels exceeding background in their femurs  and in
those of their offspring was unexpected.  The higher correlation of the amount
of lead in the femurs of the young in the cross-fostered groups to that of
their foster mothers rather than their natural mothers was unexpected.  The
correlation of lead in femurs of young remaining with their mothers was not as
close, even in the control groups.

    The results of the study indicate that analysis of hair for lead  is a
valid indication of past exposure but care would have to be taken as  the
uptake is affected by the physiological state of the animal.

    The lead content of blood samples from dams and offspring exhibited such
wide variability that treatment-related differences were absent.  Blood
samples were analyzed for zinc protoporphyrin content immediately after
collection using the ZnP Hematof1uorometer 4000.  The results are shown in
Table 8.  If one assumes the ZnP results are valid for rats (see section on
birds), it would appear that the equivalent protoporphyrin (see footnote to
Table 8) values for the weanlings were higher than those for any time after
weaning.  As the results from the one control litter follows this same trend,

                                      22

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                     TABLE  8.   ZINC  PROTOPORPHYRIN  LEVELS  IN THE BLOOD OF RATS AND OFFSPRING
ro
CO
ug EPP* per ml Blood

Animals

3 dams (a, b, c)
3 litters (a, b, c)

2 dams (d, e)
2 litters (d, e)
2 dams (f, g)
2 litters (f, g
nursed by dams h , i )
2 dams (h, i)
2 litters (h, i
nursed by dams f, g)
Control dam
Control litter

Treatment

Lead in drinking water
Lead in milk and
drinking water
Lead in drinking water
Lead in milk only
No lead exposure
Lead in milk and
drinking water
Lead in drinking water
No lead exposure from
milk or water
No lead exposure
No lead exposure
Dams 6 d
Pregnant
(background)
22 ± 6


23 ± 3

33 ± 18


18 ± 6


47


Weaning

20 ± 20
33 ± 13

12 ± 4
26 ± 6
30 ± 9
35 ± 22

25 ± 16
34 ± 9

25
29 ± 22
Weaning
+ 1 mo

23 ± 7
10 ± 6

16 ± I
13 ± 5
10 ± 5
11 ± 6

16 ± 1
15 ± 6

17
12 ± 5
Weaning
+ 2X mo
(sacrificed)
21 ± 8
10 ± 4

8 ± 0
8 ± 2
12 ± 2
10 ± 5

10 ± 4
10 ± 1

33
14 ± 12

     * EPP = equivalent protoporphyrin.   The instrument  measures  the  ratio between  zinc  protoporphyrin and
       hemoglobin in blood.  Consequently, the raw data  from  the  instrument  is  presented in ZnP  per  volume
       of red blood cells.  The instrument is calibrated to convert the  raw  reading electronically to units
       of equivalent yg per 100 milliliters of whole blood at a fixed hematocrit  to conform to the CDC
       definition of the Modified Piomelli Technique.

-------
it is questionable if this was a result of the lead ingestion.  On the other
hand, if the EPP value for the control dam is correct then this animal was
either exhibiting clinical lead poisoning as interpreted for humans (>40
yg/100 ml blood, U.S. Public Health Service 1971) or was anemic.  Unfortu-
nately the hematocrit for this blood sample was not obtained.  However, the
subsequent collection indicated a low hematocrit reading, which would indicate
anemia during pregnancy and lactation.  The blood collected from this animal
after the young were weaned and one month after weaning showed that the
hematocrit was normal based on the mean normal packed cell volumes listed by
Schalm in 1965.

    The EPP values of offspring receiving lead from either placental transfer,
milk or water ingestion did not indicate a significant difference from the
control offspring.  The amount of lead actually available to the offspring was
evidently too small to affect the ZnP values as determined by the ZnP
Hematof1uorometer 4000.   (See also following section.)

    It was hoped that the placental transfer study could be repeated using
lead-poisoned dams and bucks from our own breeding colony but lack of
personnel precluded repeating the study.


Analysis of lead in Blood

    Figure 5 shows the fluctuation in zinc protoporphyrin levels over the
experimental period of 50 days.  All data points reflect mean values for the
animals sampled.  Mean background levels for each group were all equal to or
less than 10 yg EPP/100 ml blood.  No appreciable increases in ZnP levels, were
noted for 12 days.

    Within 20 days Group  I showed an increase of 15 yg EPP/100 ml of blood.
The zinc protoporphyrins  then maintained a plateau for 18 days, followed by a
rise to 20 yg EPP/100 ml  of blood above background levels.  At the conclusion
of the study ZnP levels were still elevated 15 yg EPP/100 ml of blood over
baseline.

    For sampling purposes, Group II was divided into two subgroups of four,
which were sampled on separate days.  This procedure was also used on Group I
and the data reflect a homogeneous group.  The data for Group II, however,
reveals separate levels of ZnP for each subgroup.  While each subgroup reached
an initial increase of approximately 20 yg EPP/100 ml of blood above
background within 14 days, their subsequent courses diverged.  One subgroup
continued to rise to 50 yg EPP/100 ml of blood above background, at which
point the animals died.   The second subgroup showed a slight rise (5 yg
EPP/100 ml of blood) and, after day 32 had decreasing levels of ZnP.
Nevertheless, at the experiment's conclusion, subgroup II showed ZnP levels
that were 20 yg EPP/100 ml of blood above background.

    Group III showed a 20 yg EPP/100 ml of blood rise in 18 days.  A plateau
followed for 15 days, at  which time there was another rise to 28 yg EPP/100 ml
of blood above background.
                                      24

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ro




•O
§
CO
•^
o
E
o
o
T-
0.
Q.
UJ
O


.060-
.055-
.050-
.045-
.040-

.035-
.030-
.025-
.020-
.015-
.010-1
a
.005-
                          • 50 mg/kg Initial Dose
                          • 10 mg/kg Daily Dose (Group A)
                          • 10 mg/kg Daily Dose (Group B)
                          A 50 mg/kg Initial Dose—
                           10 mg/kg Daily Dose
                         ® Common Point
                                i
                               10
15
20
25
Days
30
35
40
45
 I
50
55
       Figure 5.  Micrograms of equivalent protoporphyrin per 100 ml  of blood from rats receiving  either
                            acute or chronic  doses of stable lead as the acetate.

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    From the data presented here it appears that the Hematof1uorometer is able
to detect lead in the rat, delivered in toxic doses, in a period of 12 to 18
days.  The rise in zinc protoporphyrins of approximately 20 yg EPP/100 ml of
whole blood is not so dramatic that it would be of significance without
background levels with which to compare it.  Of value is the fact that the
measured effect has considerable longevity, although not as long as in humans.
Zinc protoporphyrins, once formed, bind to the cellular membranes of red blood
cells.  Thus, the effect may be measured for the life of the red blood cell
(60 to 70 days, Schalm 1965).  The 2-week lag time between dosing and effect
is presumably due to the time required for heme synthase enzyme inhibition to
be translated into detectable levels of zinc protoporphyrins.

    Large hematocrit fluctuations were observed in the rats.  Lead is a known
etiologic agent of anemia, but the possibility exists that frequent blood
collection also contributed to this phenomenon.  It is important to note that
the Hematof1uorometer yields a value based on the ratio of zinc
protoporphyrins to hemoglobin within calibration standards established for
humans.  Thus, anemia should not affect the validity of these results in
humans but could do so if the hematocrit values for the animal in question
fell below standards supplied by the manufacturer.

    Erthropoietic porphyria produces a porphyrin with different absorption and
fluorescence maxima than zinc protoporphyrins and thus the Hematof1uorometer
will not confuse the two.  It must be kept in mind, however, that the
porphyrin produced by iron deficiency is identical to that produced by lead
toxicity.

    From the small amount of data gathered here it would appear that there are
no appreciable differences between male and female rats with respect to zinc
protoporphyrin detection of lead toxicity.


BIRDS

    The mean lead content in six eggs from each dose group is shown in Table
9.  The lead was concentrated mainly in the shell, but also occurred in the
white and yolk portions of the quail eggs.  The concentration of lead in all
portions of the egg was dependent on the amount of lead in the diet of the
laying hens.  As it was not known precisely how much lead the hens ingested, a
percentage of dose value could not be calculated, so it is not known if there
is a maximum amount of lead deposited in eggs regardless of the availability.

    Femurs from five quail in each dose group were averaged to obtain the mean
values listed in Table 10.  The femurs show definite correlation with the
amount of lead consumed by the quail.  These results agree with those obtained
by Ohi et al. (1974), Tansy and Roth (1970), and Getz et al. (1977), who found
a correlation between lead in femurs of birds and their location of residence.

    It would appear that resident avian populations may provide a reliable
biological indicator of an increase of lead in the environment of a specific
area.  Further work must be done to determine the minimal time between
exposure to lead and detection of increased content in eggs or femurs.

                                      26

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                     TABLE 9.  LEAD IN FEMURS FROM JAPANESE QUAIL CONSUMING LEAD ACETATE,
                                       FROM HATCHING TO 12 WEEKS OF AGE
ro
Lead in Feed yg Pb per g shell y




TABLE 10.
Lead in Feed
Consumed
1 ppm
10 ppm
100 ppm
Control
Consumed Mean S.D.
1 ppm 2.95 0.836
10 ppm 78.8 37.6
100 ppm 1,930 1,050
Control 2.17 1.74
S.D. = standard deviation
S.E. = standard error
LEAD IN EGGS FROM JAPANESE QUAIL
yg Pb per g shell
Mean S.D. S.E.
0.688 0.249 0.102
1.26 0.676 0.302
2.87 1.00 0.408
0.346 0.266 0.109
S.E. Mean
0.374 1.03
15.4 30.3
471 741
0.780 1.16

CONSUMING LEAD ACETATE
yg Pb per shell
Mean S.D.
0.591 0.189 0
0.794 0.375 0
2.07 0.581 0
0.242 0.191 0
g Pb per shell
S.D. S.E.
0.283 0.127
17.4 7.11
625 297
0.660 0.295

, FROM HATCHING TO 12 WEEKS OF AGE
yg Pb per g yg Pb per g
S.E. Egg White Egg Yolk
.770 0.051 0.162
.168 0.083 0.274
.237 0.767 0.873
.078 0.030 0.130
     S.D. = standard deviation
     S.E. = standard error

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                                  REFERENCES


Associate Committee on Scientific Criteria for Environmental  Quality Lead in
    the Canadian Environment.  Ottowa Nat. Res. Coun. of Canada.  Pub.  No. BY
    73-7 (ES) 1973

Ballou, J. E. and J. 0. Hess.  "Biliary plutonium excretion in the rat."
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Barth, D., A. Berlin, R. Engel, P. Recht, and J. Smeets (eds).  Environmental
    Health Aspects of Lead.  EUR 5004 d-e-f.  Proceedings of International
    Symposium, Amsterdam, 1972.  Pub. CEC, Directorate General for
    Dissemination of Knowledge CID, Luxembourg.  May 1973

Barth, J., and A. A. Mullen.  "In vitro Plutonium Studies Using the Artificial
    Rumen and Simulated Abomasal and Intestinal Fluids."  pp. 143-150.   In:
    The Dynamics of Plutonium in Desert Environments.  NVO-142.  P. B.  Dunaway
    and M. G. White (eds.)  July 1974

Blaxter, K. L.  "Lead as a nutritional hazard to farm livestock.  III.  Factors
    influencing the distribution of lead in the tissues."  J.. Comp. Pathol.
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Castellino, N., and S. Aloj.  "Kinetics of the distribution and excretion of
    lead in the rat."  Brit. ^. Industr. Med. 21:308.  1964

Cikrt, M.  "Biliary excretion of 203Hg, 61fCu, 52Mn, and 210Pb in the rat."
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Clarke, A. N., and D. J. Wilson.  "Preparation of hair for lead analysis."
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DeFerranti, E., and P. Bourdeau.  "Metabolism and Distribution of Radioactive
    and Stable Lead in Man."  pp. 357-363.  In:  Environmental Health Aspects
    of Lead.  EUR 5004 .d-e-f.  D. Barth, A. Berlin, R. Engel, P. Recht, and J.
    Smeets (eds.)  May 1973

Durbin, P. W.  "Plutonium in Man:  A New Look at the Old Data."  in:
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Edens, F. W., E. Benton, S. J. Bursian, and G. W. Morgan.  "Effect of dietary
    lead on reproductive performance in Japanese quail."  Toxicol. Appl.
    Pharmacol. 38:307-314.  1976


                                      28

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Forbes, G. B., and J. C. Reina.  "Effect of age on gastrointestinal absorption
    (Fe, Sr, Pb) in the rat."  J_. Nutr. 102:647-652.  1972

Getz, L. L., L. B. Best, and M. Prather.  "Lead in urban and rural song
    birds."  Environ. Pollut. 12.  1977

Goodman, L. S., and A. Gilman.  The Pharmacologic Basis of Therapeutics.  The
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Green, M., and N. Gruener.  "Transfer of lad via the placenta and milk."  Res.
    Commun. Chem. Pathol. Pharmacol. 8_(4): 735-738.  1974

Holtzmann. R. B.  "Measurement of the natural contents of RaD (Pb210) and RaF
    (Pu21°) in human bone—estimates of whole-body burdens."  Health Phys.
    9.: 385-400.  1963

Hubermont, G., J. Buchert, H. Rolls, and R.  Lauwerys.   "Effect of short-term
    administration of lead to pregnant rats."  Toxicology 5(3):279-384.  1976

Karhausen, L.  "Intestinal Lead Absorption."  pp. 427-437.  In:   Environmental
    Health Aspects of Lead.  EUR 5004 d-e-f.  D. Barth, A. Berlin, R. Engel,
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Klaassen, C. D.  "Biliary excretion of Pb in rats, rabbits, and dogs."
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Kehoe, R. A.  "The metabolism of lead in man in health and disease."  The
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Kostial, K., and B. Momcilovic.  "Transport  of lead-203 and calcium-47 from
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Lambert, R.  Surgery of the Digestive System in the Rat.  Charles C. Thomas,
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Lincer, J. L., and B. McDuffie.  "Heavy Metal Residues in the Eggs of Wild
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Momcilovic, B., and K. Kostial.  "Kinetics of lead retention and  distribution
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                                      29

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Neathery, M. W., and W. J. Miller.  "Metabolism and toxicology of cadmium,
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    1972                                      ~  	  	  ~

Ohi, G., H. Seki, K. Akiyama, and H. Yagyu.  "The pigeon, a sensor of lead
    pollution."  Bull. Environ. Cont. and Toxic. 12.  1974

Ralston Purina Company.  "Manual for Laboratory Animal Care."  Educational
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    1961

Schaller, K. R., R. Schiele, D. Weltle, C. Krause, and H. Valentin.  ."The
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Schalm, 0. W.  Veterinary Hermatology.  Pub. Lea and Febiger, Philadelpia,
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Sikov, M. R., and D. D. Mahlum.  "Plutonium in the developing animal."  Health
    Phys. 22:707-712.  June 1972

Singh, N. P., I. S Thind, L. F. Vitale, and M. Pawlou.  "Lead content of
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Stanley, R. E., A. A. Mullen, and E. W. Bretthauer.  "Transfer of milk of
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Tansy, M. F., and R. P. Roth.   "Pigeons:  A new role in air pollution."  J_.
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                                      30

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                                    TECHNICAL REPORT DATA
                             (Please read Instructions on the reverse before completing)
 1. REPORT NO.

 EPA-600/3-80-030
                              2.*
              3. RECIPIENT'S ACCESSION NO.
4. TITLE AND SUBTITLE

STUDIES TO DETERMINE  THE ABSORPTION AND  EXCRETION
DYNAMICS OF LEAD
              5. REPORT DATE

                February 1980
              6. PERFORMING ORGANIZATION CODE
7. AUTHOR(S)

Anita A. Mullen,  Robert E. Mosley, and  Zachary C. Nelson
                                                            8. PERFORMING ORGANIZATION REPORT NO
9. PERFORMING ORGANIZATION NAME AND ADDRESS
Environmental Monitoring Systems Laboratory
Office of Research  and Development
U.S.  Environmental  Protection Agency
Las Vegas, Nevada   89114
              10. PROGRAM ELEMENT NO.

                1  HP 626	
              11. CONTRACT/GRANT NO.
                N/A
 12. SPONSORING AGENCY NAME AND ADDRESS
 U.S. Environmental  Protection Agency—Las  Vegas, NV
 Office of Research  and Development
 Environmental Monitoring Systems Laboratory
 Las Vegas, Nevada   89114
              13. TYPE OF REPORT AND PERIOD COVERED
               Summary 1976 -  1979	
              14. SPONSORING AGENCY CODE
                EPA/600/07
 15. SUPPLEMENTARY NOTES
 16. ABSTRACT
     The studies were  designed to provide  a  basis for developing  a  relatively
 rapid mammalian test  system for lead, to  provide information on  intestinal  absorption,
 routes of excretion,  and rates of transfer  to  neonates, and to determine the usefulness
 of trace-element content of feces, urine, blood, hair, and other tissues for estimating
 exposure.  As rodents are endemic to most areas of interest, the laboratory rat was
 used as the biological  monitor.  As resident avian species are also  readily available
 in most areas of interest, a study was undertaken to determine if  Japanese quail could
 function as reliable  indicators to track the movement of pollutants  from source to
 receptor.
17.
                                KEY WORDS AND DOCUMENT ANALYSIS
                  DESCRIPTORS
b.IDENTIFIERS/OPEN ENDED TERMS  C.  COSATI Field/Group
  Animal  physiology
  Biological indicators
  Environmental biology
  Radiobiology
  Trace elements
  Biological accumulation
  Tissue concentration
  Concentration in  eggs
  Biliary excretion
  Placental transfer
  06F
  07E
  57Z
 18. DISTRIBUTION STATEMEN I
  RELEASE TO PUBLIC
                                               19. SECURITY CLASS (ThisReport)
                                                  UNCLASSIFIED
                                                                          21.
                              NO. OF PAGES
                                 40
2O. SECURITY CLASS (This page)
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22. PRICE
EPA Form 2220-1 (Rev. 4-77)   PREVIOUS EDITION is OBSOLETE
4U.S. GOVERNMENT PRINTING OFFICE: 1980-683-282/2228

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