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MICHieAN ST

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LIBRARY

Michigan State
University

 

 

1148819 |
)

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

This is to certify that the

thesis entitled -

A Thirty-Day Dosing Test to Assess the Toxicity
of Tungsten-Iron and Tungsten-Polymer Shot
in Game-Farm Mallards

presented by

Mary Elissa Kelly

has been accepted towards fulfillment
of the requirements for

M.S. degree in Animal Science

 

Major professor

Date April 16, 1997

 

0-7639 MS U is an Affirmative Action/Equal Opportunity Institution

 

PLACE IN RETURN BOX to remove this checkout from your record.
To AVOID FINES return on or before date due.

DATE DUE DATE DUE DATE DUE

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

MSU leAn Afflnnetive Action/Equal Opportunity lnetltuion
m

pure-m

A THIRTY-DAY DOSING TEST To ASSESS THE TOXICITY
OF TUNGSTEN-IRON AND TUNGSTEN-POLYMER SHOT IN GAME-FARM
MALLARDS
By

Mary Elissa Kelly

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Animal Science

1997

ABSTRACT
A THIRTY-DAY DOSING TEST TO ASSESS THE TOXICITY
OF TUNGSTEN-IRON AND TUNGSTEN-POLYMER SHOT IN GAME-FARM
MALLARDS
By

Mary Elissa Kelly

Groups of eight male and 8 female adult mallards each were dosed with 8 #4 steel
shot, 8 #4 lead shot, 8 BBS of tungsten-iron Shot, or 8 BBS of tungsten-polymer shot and
observed over a 30—day period. An additional 8 males and 8 females received no shot.
Mortality, changes in plasma chemistry and whole-blood parameters (Hb, Hct, and
ALAD), and histopathological lesions generally occurred in the lead-dosed mallards only.
Mild biliary stasis occurred in 5 tungsten-iron birds and 3 tungsten-polymer birds which
was considered unremarkable. The results of this study indicate that game-farm mallards
dosed with 8 BBS composed of tungsten-iron or tungsten-polymer were not adversely

affected during the course of the 30—day trial.

To my parents, Dr. Stephan A. and Susan L. Kelly

iii

ACKNOWLEDGEMENTS

I would first like to thank Dr. Steve Bursian, my major professor and friend, for
all of his support and guidance throughout my Masters program. I would also like to
thank the rest of my committee: Dr. Aulerich, for his advice and assistance during
necropsies, Dr. Balander for his assistance during blood collection and his good humor,
and Dr. Fitzgerald for his willingness and patience to help me during class and with the
histopathology.

I would also like to thank Debra Powell for her friendship and willingness to
help throughout my entire project and especially for her help with the statistics. I
would also like to thank Rachel Moreau for being my Study partner and friend.

I am very grateful for the hard work provided by Angelo Napolitano and his
crew out at the Poultry Teaching and Research Center. I would also like to extend
thanks to Cheryl Summer and her Dept. of Defense students for their assistance with
the birds during the study.

Finally, I would like to thank my family (Steve, Sue, Patrick, Theresa, Tammi,
and Andy) for their love and constant support throughout my college career. I would
expecially like to thank my father, Dr. Stephan A. Kelly, for being my positive and
hard-working role model and also my mother, Susan Kelly, for her patience and
confidence in my abilities.

iv

TABLE OF CONTENTS

List of Tables ............................................................................... vi
Introduction .................................................................................. 1
Objectives .................................................................................... 2
Literature Review ........................................................................... 3
Materials and Methods .................................................................... 17
Results
Mortality ........................................................................... 27
Clinical Signs ..................................................................... 27
Body Weights ..................................................................... 29
Hematocrit, Hemoglobin Concentration,
and ALAD Activity .............................................................. 29
Plasma Chemistries .............................................................. 33
Gross Pathology .................................................................. 36
Absolute Organ Weights ........................................................ 49
Organ Weights as a Percent of Body Weight ................................ 49
Histopathology of Kidney and Liver .......................................... 54
Tissue Metal Analysis ........................................................... 67
Shot Recovered and Percent Shot Erosion ................................... 7O

Discussion

Mortality .......................................................................... 75
Clinical Signs ..................................................................... 77
Body Weights ..................................................................... 78
Hematocrit, Hemoglobin Concentration,
and ALAD Activity .............................................................. 78
Plasma Chemistries .............................................................. 81
Gross Pathology .................................................................. 83
Organ Weights .................................................................... 84
Histopathology of Kidney and Liver .......................................... 84
Tissue Metal Analysis ........................................................... 86
Shot Recovered and Percent Shot Erosion ................................... 88
Conclusion .................................................................................. 90
References .................................................................................. 91

vi

10

11

LIST OF TABLES

High and low room temperatures and natural photoperiod
during the 30—day dosing test for candidate shot (16 January
1996 to 15 February 1996) .................................................... 19

The effect of treatment shot On percent mortality, time to death
(days), and percent weight lost at death of mallards on a 30—day
dosing test ........................................................................ 28

The effect of treatment shot on body weights (gm) of mallards on
a 30-day dosing test ............................................................. 30

The effect of treatment shot on whole-blood parameters of
male mallards on a 30-day dosing test ....................................... 31

The effect of treatment shot on whole-blood parameters of
female mallards on a 30—day dosing test ..................................... 32

The effect of treatment Shot on plasma chemistry parameters
of male mallards on day 15 of a 30—day dosing test ........................ 34

The effect of treatment shot on plasma chemistry parameters
of female mallards on day 15 of a 30-day dosing test ..................... 37

The effect of treatment shot on plasma chemistry parameters
of male mallards on day 30 of a 30-day dosing test ........................ 38

The effect of treatment shot on plasma chemistry parameters
of female mallards on day 30 of a 30-day dosing test ..................... 41

The gross necropsy observations from the effects of treatment shot
on male mallards on a 30-day dosing test for candidate shot ............. 43

The gross necropsy observations from the effects of treatment
shot on female mallards on a 30-day dosing test for candidate Shot ..... 46

vii

12

13

14

15

16

17

18

19

20

21

22

23

The effect of treatment shot on organ weights (gm) of male

mallards on a 30-day dosing test .............................................. 50
The effect of treatment Shot on organ weights (gm) of female
mallards on a 30-day dosing test .............................................. 51

The effect of treatment shot on organ weights expressed
as percent body weight of male mallards on a 30-day dosing test ....... 52

The effect of treatment Shot on organ weights expressed
as percent body weight of female mallards on a 30-day dosing test ..... 53

The histopathological effects of treatment shot on the liver and
kidneys of male mallards on a 30-day dosing test for candidate
shot ........... ‘ ..................................................................... .55

The histopathological effects of treatment Shot on the liver and
kidneys of female mallards on a 30-day dosing test for candidate
shot ................................................................................. 58

The severity of liver and kidney lesions induced by treatment shot
in male mallards on a 30-day dosing test .................................... 61

The severity of liver and kidney lesions induced by treatment shot
in female mallards on a 30—day dosing test .................................. 64

The effect of treatment shot on concentrations (mg/kg dry weight)
of iron, lead, tungsten, and molybdenum in the femur of mallards
on a 30-day dosing test ......................................................... 68

The effect of treatment shot on concentrations (mg/kg dry weight)
of iron, lead, tungsten, and molybdenum in the liver of mallards
on a 30-day dosing test ......................................................... 69

The effect of treatment shot on concentrations (mg/kg dry weight)
of iron, lead, tungsten, and molybdenum in the kidney of mallards
on a 30—day dosing test ......................................................... 71

The mean weight (gm) of individual Shot administered, the number

and mean weight (gm) of individual shot retrieved from each bird at
necropsy, and percent shot erosion during a 30-day dosing test ........ 72

viii

24 The mean weight (gm) of total Shot administered, the mean weight
(gm) of total shot retrieved from each bird at necropsy, and percent
Shot erosion during a 30-day dosing test ..................................... 74

Introduction

Lead poisoning affects every major species of waterfowl in North America and also
a wide range of other birds such as predatory Species. Within the United States, lead
poisoning is common in the mallard (Anas platyrhynchos), northern pintail (Anas acuta),
redhead (Aythya americana), scaup (Aythya marila), Canada goose (Branta canadensis),
snow goose(Anser caerulescens), and tundra swan (Olor columbianus). Friend (1987)
reported an estimated annual loss of approximately 2.0 million waterfowl from lead
poisoning. This estimate was based on a migratory fall flight of 100 million birds. Lead
poisoning has also been the cause of migratory bird mortality in other countries such as
Canada, Great Britain, Italy, and New Zealand (Friend, 1987).

Lead has been a major factor in the mortality of North American waterfowl since
the late 1800's (Cook and Trainer, 1996). Waterfowl mistakenly ingest spent lead Shotgun
pellets and lead fishing sinkers that have been deposited on the bottom of lakes, ponds, and
marshes as plant food items or grit. Mine wastes, paint pigments, bullets, and other less
common lead objects are also ingested by waterfowl. The problem of waterfowl poisoning
associated with spent lead shot is the fact that the pellets are usually retained in the gizzard
and ground to small lead particles which are then easily absorbed. If a large number of
shot have been consumed then acute lead poisoning usually occurs unless the bird
eliminates the Shot. However, chronic lead poisoning is more commonly seen and occurs
when a smaller number of shot is consumed and degrades over a longer period of time
(Friend, 1987).

Because of the increasing numbers of birds killed every year due to lead toxicosis,
lead shot was banned for waterfowl hunting in 1991 within the United States (Ringelman
et al., 1993). Also, a national ban on the use of lead for all migratory game bird hunting
will begin at the start of the 1997 season in Canada (Scheuhammer and Norris, 1995).
Currently, the only US. and Fish and Wildlife Service (U SFWS) approved substitute for

1

A

 

~

2

lead Shot is Steel shot (Ringelman :1 a1., 1993). However, a bismuth Shot has recently
received conditional approval (Ringelman et a. , 1993). While steel shot has generally
been accepted by hunters, there continues to be an effort to formulate a nontoxic shot with
lead's favorable ballistic characteristics.

In order for a candidate shot to be approved for use by the USFWS, it must first
undergo a variety of tests to establish that it's nontoxic to waterfowl and other impacted
species. A 3-tiered approval process has been designed by the USFW S for candidate Shot
and shot coatings with shot approval considered after each tier. With the advancement of
the next tier, shot testing becomes more demanding. Therefore, a candidate shot or Shot
coating found to be environmentally nontoxic could be granted approval at the first tier
thereby reducing time, expense, and burden on both applicant and the federal government
(Federal Register, 1996).

The present study is an acute toxicity test (short-term, 30- day acute toxicity test
using commercially available duck food) designed to assess the effects of short-term
periodic exposure of waterfowl to 2 different candidate shot, one composed of umgsten
(55 %) and iron (45 %) and the other composed of tungsten (95.5 %) and a polymer
(4.5 %). The protocol for this study was reviewed by the USFWS in 1995 and complies
with the general guidelines outlined in the amended test protocol for nontoxic shot approval
procedures for shot and shot coatings proposed by the USFWS in 1996.

Q] . I.

The overall objective of the 30-day dosing u'ial was to determine if exposure to the
2 different candidate shot, one composed of tungsten (55 %) and iron ( 45 %)(TI) and the
other composed of tungsten (95.5%) and a polymer (4.5%)(TP), caused any deleterious
effects in game farm mallards (Anas platyrhynchos). Toxicity of the candidate Shot was

assessed by:

3

1) Determination of hematocrit, hemoglobin and whole-blood delta-
aminolevulinic acid dehydratase activity values at day 15 and day 30 of the
trial.

2) Determination of plasma chemistry values at day 15 and day 30 of the trial.

3) Determination of changes in body weights and organ weights.

4) Determination of metal residue concentrations in the liver, kidney, and
femur.

5) Determination of gross and histological changes of selected tissues.

6) Determination of mortality.

Literatmkexim

The word lead (Pb) is derived from the Latin term plumbum. Thus, lead poisoning
often called "plumbism'. Lead is a soft, malleable heavy metal with a bright bluish tint.
It is found naturally at trace concentrations (15 ppm) but rarely occurs in its native form.
Instead, lead is usually found in the sulfide form in its chief ore, galena. Other common
inorganic salts are insoluble lead carbonate, lead sulfate, and lead chlorophosphate with
lead acetate a common soluble form (National Academy of Science, 1980b).

Lead once had a wide range of applications including lead shotshell and fishing
sinkers. In 1964, Bellrose estimated that the average hunter fired 5 shots for every duck
that was bagged. He also calculated a 12-gauge shell to contain about 280 pellets of # 6
shot. This amounts to approximately 1,400 pellets being deposited on waterfowl hunting
grounds for every duck killed. However, because the toxicity of spent lead shot to
waterfowl is so great it was banned for use in waterfowl hunting in 1991 in the United
States (Ringelman :1 31., 1993).

Other applications involving lead include storage batteries, gasoline, pigments,
ceramics, pesticides, plumbing, and crystal ware (Scheuhammer and Norris, 1995).

However, lead was recognized to be one of the major environmental pollutants in air, soil,

III II .III II IIII'C

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4

and water and thus the use of lead was either banned or severely reduced.

Birds exhibiting clinical signs from lead poisoning were reported as early as 1893-4
(Grinnell, 1901). Grinnell (1901) reported that hunters in the area were noticing wildfowl
to be sick with "croup" at Stephensen Lake in Galveston, Texas. Affected birds were
lethargic and had a change in tone of call. Yellowish fluid was also noted being eliminated
from the bill of the affected birds. Upon dissection of these sick birds, lead shot was found
in the gizzards. The inner membranes of the gizzards were cracked and decayed, and
yellowish in color. It was concluded from these examinations that the dissected birds had
died from chronic lead poisoning.

A case of sick wildfowl near Riverton, Kansas was reported in February of 1923
by Dr. Wetrnore. He described progressive paralysis of the legs and wings, ataxia, thin,
watery green-stained feces, and a greenish fluid discharge from the tongue and mouth
cavity. Post-martem observations included pale flesh color and distended gall bladder
(Phillips and Lincoln, 1930).

The first signs of lead exposure are often depression and anorexia. Birds then
become reluctant to fly when approached and may have a marked change in tone of call.
As the disease progresses, the wings are held in a ”roof—shaped” position which is often
followed by wing droop. There may be a fluid discharge from the bill, with swollen or
puffy heads in Canada geese from serum-like fluids which accumulate in the tissues ofthe
face. An abundance of bile-stained feces in areas used by waterfowl are also indicative of
lead poisoning (Friend, 1987).

Absorbed lead is rapidly distributed to the soft tissues with the highest initial
concentrations detected in the liver and kidneys. Over a period of time, lead is
redistributed and accumulates in the bone (Goodman and Gilman, 1966). Coburn 91 a1.
(1951) conducted a study in adult mallards to obtain information on the absorption and

retention of lead. Soluble lead nitrate [Pb (N 03),] was administered to all test birds by

 

 

 

5

gavage into the gizzard. Doses of lead (3, 6, 8, and 12 mg / kg body weight) were given
each day with dose adjustments due to body weight variations made every 5 to 7 days. The
most significant increase in lead content was noted in the liver with lead poisoned birds
having hepatic lead concentrations which were 40 times those of the control birds.
Similarly, the Skeletal lead concentrations of the lead—dosed birds were 7 times greater
than the control concentrations. In a similar study lead was administered to game-farm
mallards in doses of 2, 4, or 8 #2 shot. Birds were kept on trial for 30 days. Lead residue
concentrations in the liver of lead dosed ducks varied from 50.7 to 84.4 ppm. Lead in
muscle ranged fiom 1.10 ppm to 1.31 ppm, with retention in bone ranging from 3.19 ppm
dry weight in the high-dosed ducks to 241 ppm in the low-dosed ducks (Sanderson er al.,
1992). Coburn et al (1951) also reported high levels of lead deposition in the bone, liver,
feathers, and soft tissues of lead-dosed adult mallard ducks.

Circulating lead combines with erythrocytes and causes increased fragility of red
blood cells which results in premature destruction and anemia (Osweiler et a1., 1976).
Lead can also interfere with the synthesis of heme. Lead blocks the metabolism of
aminolevulinic acid (delta-ALA) by inhibiting delta-aminolevulinic acid dehydratase
(ALAD) which causes abnormally high concentrations of delta-ALA in the urine and low
concentrations in the serum (Osweiler et 31., 1976). The detection of abnormal levels of
delta-ALA in the serum has been used extensively as a diagnostic tool in waterfowl
poisonings.

Bakalli gt a]. (1995) designed a study to determine the activity of erythrocyte
ALAD and the relationship between enzyme activity and tissue lead concentrations in
chickens, both during lead intake and after withdrawal of lead from the feed. Enzyme
activities in birds dosed with 50 ug lead / gm fwd were reduwd to 62% of control activity
within 24 hours and to 31% of control values after 7 days. Lead was then removed from
the feed and after 24 hours enzyme activity increased by 32% , and after 7 days activity

 

 

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6
was 90% of control activity. It should be noted that the chicken has been proposed as the

model for lead toxicity studies because this species is very sensitive with measurable
responses to low concentrations of lead after as little as 24 hours. Serum ALAD activity
in chickens may also be used to monitor lead in the environment. Pain and Rattner (1988)
reported that ALAD activities in adult black ducks dosed with 1 # 4 lead shot were
inhibited by 100% at day one post-dosing with ALAD activities being significantly lower
than control activity throughout the experiment (p=0.000 - 0.035).

Bates et a1. (1968) also reported severe lead-induced changes within the blood.
Eight # 6 lead pellets placed in the ventriculus of 10 adult mallard drakes for 25 days
caused maturation arrest of promegalobastic-like blood cells in the bone marrow. Coburn
eta]. (1951) reported a decrease in erythrocyte count during the first 10 to 14 days ofa
lead dosing trial in mallards. In some cases, erythrocyte counts recovered but when a
higher dose of lead was administered the red blood cell pattern changed to one
characteristic of acute poisoning. Changes in cell shape were also noted and included
dumbbell, bottle, oat, sickle, and tear-shaped forms.

The excretion of lead occurs slowly via the bile and urine. Urinary excretion of
inorganic lead under normal conditions is approximately 9% of the amount ingested
(Goodman and Gilman, 1966). The rate of urinary excretion depend on the duration of
exposure and not necessarily on the absolute body burden. Coburn er a1. (1951) dosed
adult mallard ducks with lead shot and reported an average excretion rate of 5 % of the lead
intake (range 1.4-8.2%).

Because elimination of lead from body tissues is slow, acute and chronic exposure
to high and low levels of lead, respectively, causes lead poisoning. The toxic dose for
wildfowl such as the mallard duck is 8-12 mg / kg body weight for a duration of 19-41
days(National Academy of Science, 1980b). Ducks have also been shown experimentally
to be susceptible to poisoning from consumption of marsh soil containing disintegrated lead

7

shot. Cook and Trainer (1966) exposed Canada geese to a variable number # 4 lead pellets
(2-100). Twenty-five or more pellets resulted in death within 10 days while fewer pellets
permitted survival for as long as 72 days. Pain and Rattner (1988) dosed adult black ducks
with 1 # 4 lead shot and reported 60% mortality at the end of 33 days. All birds that died
exhibited Signs of acute lead poisoning by day 3 post-dosage. Acute lead poisoning was
also reported by Grandy et a1. (1968) after 8 # 6 lead shot was administered to mallard
drakes. All 15 of the lead-dosed mallards died within 15 days of the study. The average
time of death after dosing was 8 days. These ducks lost an average of 22 % of their body
weight at death and had retained 5 to 8 shot (average 7.2).

Cook and Trainer (1966) dosed pairs of Canada geese with 5, 10, 25, and 100 #
4 lead pellets while single geese received 2 and 50 pellets. The first Signs of poisoning,
(anorexia and lethargy) occurred 5 days post-dosing. Green diarrhea was observed in one
bird as early as day 6, with 4 geese displaying swollen heads on day 6 or 7. Two geese
died on day 6 without evidence of lead poisoning. Birds dying from acute exposure lost
approximately 19 % of their body weight, while birds dying later in the trial lost 36% of
their body weight. The lethal dose of # 4 lead shot was determined to be 4 to 5 pellets for
Canada geese under the conditions of this study.

Lead toxicosis not only occur in waterfowl but also in other birds such as
mourning doves. Locke and Bagley (1967) collected a sick mourning dove in Maryland
which had 2 lead shot in its gizzard. It was extremely emaciated with a pronounced
"hatchet" breast that was devoid of any fat. Acid fast intranuclear inclusion bodies were
also found in the cells of the proximal convoluted tubules of the kidneys resembling those
described for mallards fed lead shot (Locke :1 a1. , 1967). However, the morphological
characteristics of the bodies appeared as individual granules, flakes, or clumps of granules
as opposed to the dumbbell shapes typically seen in the nuclei of kidney tubule cells of
mallards.

8

For areas where bird mortality is high from lead poisoning, two actions can reduce
the magnitude of mortality. One practice of control is denying birds the use of problem
areas and the other is the collection and proper disposal of dead and moribund birds. This
prevents raptures and other scavenger species from ingesting them. Other management
practices have also reduced a number of losses due to lead poisoning in site specific areas.
These practices include: (1) tillage of surface soil that has embedded lead so that shot is
not readily available; and (2) planting foodcrops other than corn and other grains that
intensify the toxicity of lead when ingested (Friend, 1987).

Medical treatment of lead poisoned birds is available, but generally not reasonable
in the field. However, endangered species or other birds of great importance can be
treated with lead-chelating chemicals. One such chemical is disodium or calcium
ethylenediaminetetraacetate (CaEDTA). Murase et a]. (1992) administered every 12 hours
1 ml of 6% CaEDTA in sterile water via the brachial vein to 27 wild geese suffering from
lead poisoning. These birds were treated until radiographic screening indicated that the
lead pellets were no longer in the gizzard. In addition to the CaEDTA treatment, birds
were also dosed orally with 1 gm of glucose, 0.2 gm of DL-methionine, 2 mg of
deoxycycline hydrochloride, 10 ml of vegetable juice, and 10 ml of water every 12 hours.
Sixteen of the treated birds died within 4 weeks with none recovering their appetite during
the treatment. However, 11 of the 27 birds did recover their appetite after 12-24 days of
treatment. The mean duration of the treatment period for these recovered birds was 31.1
days (range, 21-58 days).

Tungsten, the major component of the 2 candidate shot presently tested, is a
relatively rare element, occurring in the earth’s crust at concentrations averaging 5 ppm
(Standen, 1970). It is found in the form of tungstate ores such as wolframite [(Fe, Mn)
WOJ , scheelite (Ca WO,), ferberite (FeWO,) and hubnerite (MnWOJ. Major uses of

tungsten include incorporation into cutting and wear-resistant materials, mill products,

9

specialty Steels, tools, alloys, and chemicals. Tungsten has a molecular weight of 183.85,
specific gravity of 19.35, melting point of 3,410°C and boiling point of 5,660°C. Tungsten
metal is insoluble in aqueous solutions while forms such as sodium tungstate
(N a,WO,2H20) and ammonium paratungstate [(NH4)6W702,6H,O] are variably soluble
in water (Stokinger, 1978).

The tungstate ion (W 0,") is the most soluble and the most frequently occurring
form of the metal in biological systems. Radiotracer Studies utilizing this form of tungsten
have indicated relatively rapid absorption of the compound with most of it being eliminated
within a few days. For example, Wase (1956) reported that mice administered K,WO, (15
mg / kg) by intraperitoneal injection eliminated 78% of the dose via the feces after 24
hours and 98% after 96 hours. Ballou (1960) reported that 40% ofan orally administered
doseoflabeledmngsteninratswaseliminatedintheurineafieruhours, while 58% was
eliminatedviathefecesorremainedunabsorbedinthegut. Only 2% ofthedoseremained
in the tissue. Kaye (1968) administered labeled K,WO, to rats and reported that 17% of
the dose was present in the carcass 1 hour post-dosing which indicated rapid absorption
through the gastrointestinal tract into the systemic circulation. Twenty-four hours after
dosing, 40% of the compound had been eliminated via the urine and 20% via the feces.
At 72 hours post-dosing, 97% of the tungstate had been cleared from the body. Bell and
Sneed (1970) dosed swine with a tracer dose of (NH,),WO,by gavage or intravenously and
reported that most of the radioactivity was eliminated via the urine in 24 hours. In
contrast, these same authors reported that sheep administered a tracer dose of (NH4)2WO,
by capsule or by injection into the abomasum eliminated only 15 % of the dose.

Distribution of absorbed tungsten is limited to relatively few tissues. Kinard and
Aull (1945) fed rats tungsten as the metal (20,000 and 100,000 ppm), tungsten oxide
(1,000 ppm tungsten), sodium tungstate (1,000 ppm tungsten) or ammonium paratungstate
(5,000 ppm tungsten). They reported that the chief sites of deposition were bone and

10

spleen with smaller quantities found in the skin, kidney, and liver. This distribution
pattern was not dependent on the type of compound administered. Wase (1956) reported
that 8 hours after dosing mice with K,WO,, the highest concentrations of tungsten were
detected in the bone and gastrointestinal tract. Similarly, Kaye (1968) reported that bone
was the principle site of tungsten deposition in rats which were administered a tracer dose
of K,WO,. In the study conducted by Bell and Sneed (1970), the principle sites of tungsten
deposition in swine were, in descending order, kidney, bone, liver, and muscle, while in
sheep tungsten was found primarily in the kidney followed by the liver, bone and muscle,
respectively. Following inhalation of a radiolabeled tungsten oxide aerosol by dogs, the
highest concentrations of activity 165 days after exposure were in the lung and kidney with
smaller concentrations in bone, gall bladder, liver , and spleen. In terms of organ burden,
most of the activity was associated with bone (37% of the body burden), lung (31%),
kidney (15%), and liver (9.7%) (Aamodt, 1975).

Tungsten is eliminated in both the urine and feces, the predominant route
apparently being dependent on Species, type of tungsten compound, and the route of
administration. Wase (1956) reported that mice dosed intraperitoneally with K,WO,
eliminated 78-98 % of the compound via the feces from 24-96 hours post-dosing. Kaye
(1968) reported that 40% of an orally administered dose of K,WO, was eliminated in the
urine and 20% in the feces at 24 hours post-dosing. Dogs administered an intravenous
tracer dose of Na,WO, eliminated 91% of the tungsten via the urine (Aamodt, 1973).
Similarly, Bell and Sneed (1970) reported that most of a tracer dose of (NHJ,WO,
administered to swine either by intravenous injection or by gavage appeared in the urine
within 24 hours post-dosing. In the same study, sheep orally administered (N H,),WO,
excreted 44 % and 42 % of the radioactivity in the urine and feces, respectively, while
(N H,),WO, introduced into the abomasum resulted in 65 % being eliminated in the urine
and 17% in the feces.

11

The biological half-life of tungsten is relatively short, depending upon the tissue
being examined. Kaye (1968) reported that the half-life of orally administered K,WO, in
rats was approximately 10 hours for the initial fast component of the elimination curve.
In general, elimination of tungsten from soft tissue was rapid, but the half—life of tungsten
in the spleen was 44 days and that in bone was 1,100 days. Nell et al. (1980) reported an
hepatic half-life of 27 hours for N a,WO, injected intraperitoneally into broiler cockerels.

The toxicity of tungsten is dependent upon the solubility of the form administered,
with the soluble forms usually being considerably more toxic than the less soluble forms.
For example, Frederick and Bradley (1946) determined an LD,o for insoluble tungsten
metal powder injected intraperitoneally in the rat of 5,000 mg/kg body weight, whereas
when the soluble Na,WO, was injected subcutaneously, an LD,o of 140-160 mg tungsten/kg
body weight (223-255mg N a,WO,/kg body weight) was determined (Kinard and Van de
Erve, 1940). Pham-Huu-Chanh (1965) reported LD” values of 112 mg/kg body weight
and 79 mg/kg body weight when sodium trmgstate was administered by intraperitoneal
injection to rats and mice, respectively. However, there are exceptions to this relationship
between solubility and toxicity. Kinard and Van de Erve (1941) reported that diets
containing 5.0 % (50,000 ppm) tungsten as the relatively insoluble ammonium
paratungstate, 3.96% (39,600 ppm) tungsten as the insoluble mngstic oxide, or 2% (20,000
ppm) tungsten as the soluble sodium tungstate produced 100% mortality in rats while a diet
containing 2% tungsten as ammonium paratungstate resulted in 80% mortality. When rats
were fed diets containing 0.5 % (5,000 ppm) tungsten in different forms, tungstic oxide
caused 82% mortality, sodium tungstate caused 58 % mortality, and ammonium
paratungstate resulted in no deaths. Nell et a1. (1980) administered broiler cockerels
soluble sodium tungstate via daily injection at 5 mg tungsten fiom day l to day 11, 10 mg
from day 12 to day 21, and 20 mg from day 22 to day 35. Four of 40 birds died on trial
and all deaths occurred on day 29.

12

Clinical signs resulting from acute exposure of mammals to lethal or near-lethal
doses of the more toxic tungsten compounds via oral and parenteral routes have been
summarized by Stokinger (197 8). These include nervous prostration, diarrhea, and death
preceded by coma due to respiration paralysis. Clinical Signs reported by Nell et a1.
(1980) for chickens dying of exposure to soluble sodium tungstate included anorexia,
reduced weight gain, diarrhea, and labored breathing within an hour of death. On gross
examination of these birds, muscles and liver were dark red due to extensive hemorrhaging
and petechial hemorrhages were observed on the gizzard and proventriculus. Hemorrhages
were also observed in the brain, heart, and kidney.

When animals have been administered doses of tungsten compounds which do not
result in mortality (in excess of several thousand ppm), effects are often slight. Selle
(1942) injected male and female rats daily with 92 mg tungsten/kg body weight as sodium
tungstate and reported weight loss of 11 and 26% , respectively. No effects were noted
when the same dose was administered daily by oral gavage. Kinard and Van de Erve
(1941) reported that when growing rats were administered a diet containing 1,000 ppm
(0.1%) tungsten as tungstic oxide or sodium tungstate, or 5,000 ppm (0.5 %) tungsten as
ammonium paratungstate, the only effect observed was a similar and slight growth
depression after 70 days. Kinard and Van de Erve (1943) reported that feeding tungsten
metal to rats at concentrations of 25,000 ppm and 100,000 ppm for 70 days resulted in a
15 % decline in body weight gain of the females. Schroeder and Mitchner (1975)
administered 5 ppm sodium tungstate to rats via the drinking water throughout their
lifetime and reported a somewhat shortened lifespan in male rats (983 days vs 1,126 days
for controls).

As with mammals, studies in birds have indicated relatively few effects as a result
of exposure to moderate concentrations of soluble tungsten compounds (I-Iiggens et 31.,
1956; Teekell and Watts, 1959; Leach et al., 1962; Nell :1 a1., 1980). In most of these

13

studies, the effects of tungsten supplementation of the diet have been looked at in
conjunction with low dietary concentrations of molybdenum. Molybdenum is an essential
component of a number of enzymes important in avian as well as mammalian metabolism.
In particular, xanthine dehydrogenase (and the similar xanthine oxidase in mammals) is
involved in purine metabolism and the conversion of nitrogenous compounds to uric acid.
Because the tungstate ion, W0}, is isomorphic with the molybdate ion, M004", tungsten
antagonizes the normal metabolic action of molybdenum in its role as a metal carrier and
thus decreases the activity of xanthine oxidase/xanthine dehydrogenase (DeRenzo, 1954)
as well as other molybdenum-containing enzymes such as sulfite oxidase, aldehyde
oxidase, and nitrate reductase (Stokinger 1978).

Higgens gt fl. (1956) utilized dietary sodium tungstate to produce a molybdenum
deficiency in rats and chicks to examine the subsequent effects on xanthine oxidase and
xanthine dehydrogenase activity. Three to 4-week-old rats were fed a diet containing 4.5
ppm tungsten (as sodium tungstate) for 7 weeks. This diet provided a
tungsten:molybdenum ratio of 100:1 and resulted in reduced xanthine oxidase activity in
the liver and intestine as well as a decreased hepatic molybdenum concentration. Rats fed
Na,WO, in a 1,000:l or 2,000:1 ratio of tungsten:molybdenum grew normally and
oxidized xanthine to uric acid and allantoin despite the fact that no tissue examined had
detectable xanthine oxidase activity or molybdenum. In contrast, chicks fed diets
containing sodium tungstate which provided tungsten:molybdenum ratios of l,000:1 and
2,000:1 experienced depressed growth rates and 25% mortality after 5 weeks. All tissue
xanthine dehydrogenase activities and molybdenum concentrations were depleted and 50%
of the uric acid normally excreted by chicks was replaced by xanthine and hypoxanthine.
Addition of molybdenum to the diet reversed the effects of the 1.000;]
tungsten:molybdenum ratio.

Teekell and Watts (1959) fed chickens sodium tungstate at a concentration of 250

14

ppm for 10 days and then increased the concentration to 500 ppm for the subsequent 20
days. Incorporation of 250 ppm sodium tungstate had no effect on intestinal and hepatic
xanthine dehydrogenase activities, but increasing the dietary sodium tungstate
concentration to 500 ppm did cause a steady decline in enzyme activities. Egg production
by these hens and subsequent hatchability was not affected. However, those chicks
hatched from females fed the sodium tungstate-supplemented diet grew at a slower rate
than chicks from control females. Chicks fed a diet containing 500 ppm sodium tungstate
for 4 weeks had a slower rate of gain when compared to control chicks, yet tissue xanthine
dehydrogenase activities were not affected.

In a study by Leach et a1. (1962), the addition of 1,000 ppm tungsten (form not
specified) or more to the diets of chicks for 4 weeks resulted in depressed growth rates
while concentrations of 500 ppm tungsten or greater caused a marked decrease in hepatic
xanthine dehydrogenase activities. Addition of molybdenum to the diets caused a reversal
of the tungsten-induced decrease in xanthine dehydrogenase activities but only partially
reversed the growth inhibition resulting from 2,000 ppm dietary tungsten.

Nell et a. (1980) examined the effects of both injected and ingested sodium
tungstate on xanthine dehydrogenase activity in chicks. Cockerels receiving a single
intraperitoneal injection of 20 mg sodium tungstate had increased concentrations of hepatic
tungsten but there was no effect on hepatic molybdenum concentrations or xanthine
dehydrogenase activities. Chicks fed diets containing 1,000 ppm tungsten for 4 weeks had
increased hepatic concentrations of tungsten, reduced concentrations of molybdenum and
decreased activities of xanthine dehydrogenase. All of these effects were reversed by
supplementation of the diet with molybdenum. In chicks either injected intraperitoneally
with sodium tungstate at doses increasing from 5 to 10 to 20 mg at days 12 and 22 of a
35-day period or fed diets containing sodium tungstate at doses which increased from 150
to 600 ppm at day 22 of a 35-day period, mortality was associated with hepatic tungsten

15

concentrations of 25 ppm as well as decreases in liver molybdenum concentrations and
xanthine dehydrogenase activities. The decrease in tissue xanthine dehydrogenase
activities paralleled increases in plasma concentrations of uric acid, xanthine, and
hypoxanthine.

In a study similar to the present study, Ringelman et a1. (1993) dosed mallards with
12-17 pellets of Shot (equivalent in mass to 5 # 4 lead shot) composed of 39% tungsten,
44.5% bismuth, and 16.5% tin and monitored the birds for the subsequent 32 days. Based
on the lack of effects on mortality, behavior, feed consumption, body weight gain, and
blood parameters as well as the absence of gross and histological lesions, and no detectable
concentrations of tin and tungsten in the liver and kidney, these authors concluded that the
ingested candidate shot had no ill effects on the mallards over the 32-day period.

The other major component of one of the tungsten candidate shot in the present
study is iron (Fe). Iron is the fourth most abundant element of the earth's surface (5 %)
existing as hematite (F903), limonite (Fean-3HZO), magnetite (Fe30,), taconite, and
siderite [Fe(CrOz)2]. It has a molecular weight of 55.85, a specific gravity of 7.86, a
melting point of 1535°C and a boiling point of 2750°C. Iron metal is insouble in alkaline
solutions, alcohol, and ether (Stokinger, 1978).

Unlike tungsten, which is not essential for animals, iron is a required nutrient. Iron
is found primarily in the ferrous (Fe”) and ferric (Fe"3) states. Oral doses of iron salts
of both valence states are not necessarily toxic. However, iron salts, especially ferrous
salts, introduced directly into the bloodstream are highly and almost instantaneously toxic
(Stokinger, 197 8).

Iron is primarily absorbed in the small intestine as ferrous iron which is oxidized
to the ferric State in the blood plasma. Iron functions in the transport of oxygen and is
associated predominately with hemoglobin (70%) and the iron-storage proteins ferritin and

hemosiderin (26%), with lesser amounts in muscle myoglobin (3.5 %), and other iron-

16

containing enzymes distributed throughout the body (V enugopal and Luckey, 1978;
National Academy of Science, 1980a ). Iron is eliminated primarily via the bile, small
amounts may be ejected by way of Sloughed cells from duodenal villi as well as via the
sweat and urine. However, it should be noted that the overall rate of metabolism of
absorbed iron is very slow thereby limiting the actual amount of excreted iron. ‘

Clinical signs of acute iron toxicity occur in 5 phases subsequent to ingestiOn of the
iron compound (Ruka and Lovejoy, 1991). The first phase is apparent from 30 minutes
to 2 hours after the ingestion of large amounts of iron. Clinical signs are lethargy,
restlessness, hematemesis, abdominal pain, and bloody diarrhea. Iron can have a corrosive
effect on the gastrointestinal mucosa which could in turn cause severe hemorrhagic
necrosis with development of shock. However, iron that is absorbed through intact
mucosa may also cause shock. The second phase is a transitional period which appears as
a recovery period, but in fact progresses to the third phase. The third phase (2-12 hours
after phase 1) is characterized by the onset of shock, acidosis, cyanosis, and fever.
Acidosis is the result of hydrogen ion release from the conversion of ferric (Fe + 3) iron
to ferrous (Fe+2) iron and the accumulation of lactic and citric acids. The fourth phase
(2-4 days post-ingestion) involves the development of hepatic necrosis. This is
theoretically due to a direct toxic action of iron on mitochondria. The fifth and final phase
(2-4 weeks after ingestion) occurs with the onset of gastrointestinal obstruction which is
secondary to gastric or pyloric scarring and healed tissue. Histological evidence of acute
toxicity include vascular congestion of the gastrointestinal tract, liver, kidneys, heart,
lungs, spleen, brain, adrenals, and thymus. Smaller amounts of iron ingested over a longer
period of time can cause chronic symptoms which include hemorrhagic necrosis of the
gastrointestinal tract, hepatotoxicity, metabolic acidosis, increased blood-clotting time, and
elevation of plasma concentrations of serotonin and histamine (V enugopal and Luckey,

1978). McGhee er al. (1965) evaluated the effects of various concentrations of copper and

17

iron on growth, body weight gains, and survival of chickens. One hundred and twenty
birds were divided into 3 groups with 6 treatments per group. Each group had varied
amounts of copper and iron (CuSO,-5H20 and Fe SOflHzO, respectively) mixed into the
basal diets. Results indicated that when copper was held constant at 5 ppm and iron varied
from 50 to 1,600 ppm (Group 1), increasing levels of iron decreased average body weight.
Mortality was 10% at 200 ppm iron and 5 ppm copper. Group 3 had more noticeable
growth depressions at iron concentrations of 800 to 1,600 ppm and copper concentrations
of 80 to 160 ppm. Overall, iron suppressed growth at 4 weeks of age at concentrations
from 50 to 1,600 ppm when fed 5 ppm copper. However, no other toxic effects were seen
at 4 weeks of age.

Other signs of chronic iron toxicosis may involve decreased feed intake,
growth rate, and feed efficiency. The maximum tolerable level of iron in poultry is
reported to be 1,000 ppm with oral doses exceeding 1.5 mg / kg being considered
excessive and possibly leading to iron toxicosis (National Academy of Science, 1980a).
Materials and mm

Thirty-two male and 32 female 6-month-old game farm mallards (hatched 12 June
1995) with plumage and body conformation resembling wild mallards were purchased from
Whistling Wings, Inc. (Hanover, Illinois). The birds arrived by truck at the Michigan
State University Poultry Teaching and Research Center (PTRC) on 20 December 1995.
The birds were randomly removed from the transport cages, identified with a uniquely
numbered metal leg band, and weighed. Flight feathers were clipped and the birds were
assigned to individual cages.

Cages (0.914 m L x 0.914 m W x 0.457 m H) were constructed of vinyl-coated

wire (14 gauge, 2.54 cm mesh) and suspended 60.96 cm off the floor of an enclosed pole

18

ham-type building. Wood shavings were placed underneath the cages to absorb excreta
and water. Shavings were replaced on a weekly basis.

A gas brooder was utilized to keep the room temperature above 0°C. Room
temperature was continuously recorded on graph paper by a thermohydrograph. High and
low room temperatures during the 30-day test are presented in Table 1. Light was
provided by incandescent bulbs controlled by a timer such that lights went on at sunrise
and off at sunset. The timer was adjusted weekly to mimic within 15 minutes the natural
photoperiod appropriate for Lansing, Michigan (Table 1).

Each cage contained a food and water crock so that food and water were available
ad lihimm during the acclimation period (20 December 1995-15 January 1996) and the
30-day dosing trial (16 January-15 February 1996). The diet was a commercial pelleted
ration (Purina Duck Grower W/O; Batch #8858; crude protein 2 16.0% , crude fat 2
3.0%, crude fiber 5 5.0%, calcium 0.40-0.90%, phosphorus 20.55%, sodium chloride
0.20-0.70 %) and drinking water was obtained from a university well. The opened feed
bags were Stored in a covered container to keep them dry and pest-free. Water was
changed on a daily basis and feed was added to cleaned crocks as needed (usually every
other day).

The ducks were randomly assigned to 5 treatment groups (no-shot, Steel shot, lead
Shot, tungsten-iron Shot, and tungsten-polymer shot) with 16 ducks (8 males and 8
females) per group. The evening before the shot was to be administered, feed was
removed from the birds to facilitate dosing. On the first day of the 30—day dosing trial (16

January 1996), each bird was weighed and then dosed with shot. The birds in the steel and

19

 

 

 

 

Table 1. High and low room temperatures and natural photoperiod during the
30-day dosing test for candidate shot (16 January 1996 to 15 February
1996).
Date Temperature Photoperiod (EST)
High Low Sunrise Sunset
F° C° F° C°

1/16/96 48 9 40 4 0805 1730
1/17/96 56 13 49 9 0805 1732
1/18/96 63 17 56 13 0804 1733
1/19/96 60 16 41 5 0804 1734
1/20/96 49 9 40 4 0803 1735
1/21/96 47 8 44 7 0802 1737
1/22/96 53 12 44 7 0802 1738
1/23/96 54 12 51 11 0801 1739
1/24/96 53 12 44 7 0800 1740
1/25/96 47 8 40 4 0759 1742
1/26/96 53 12 40 4 0759 1743
1/27/96 42 6 36 2 0758 1744
1/28/96 46 8 40 4 0757 1746
1/29/96 45 7 36 2 0756 1747
1/30/96 46 8 36 2 0755 1748
1/31/96 53 12 34 1 0754 1750

 

 

20

 

Table 1 continued.

High and low room temperatures and natural photoperiod
during the 30-day dosing test for candidate shot (16 January

1996 to 15 February 1996).

 

 

 

Date Temperature Photoperiod (EST)
High Low Sunrise Sunset
F ° C ° F° C °
2/1/96 44 7 26 -3 0753 1751
2/2/96 45 7 39 4 0752 1752
2/3/96 47 8 36 2 0751 1754
2/4/96 45 7 38 3 0750 1755
2/5/96 46 8 29 -2 0748 1756
2/6/96 49 9 40 4 0747 1758
2/7/96 51 11 43 6 0746 1759
2/8/96 57 14 51 11 0745 1800
2/9/96 70 21 52 1 1 0744 1801
2/10/96 59 15 40 4 0742 1803
2/1 1196 55 13 39 4 0741 1804
2/12/96 44 7 35 2 0740 1805
2/13/96 47 8 39 4 0738 1807
2/ 14/96 53 12 49 9 0737 1808
2/15/96 53 12 40 4 0736 1809

 

 

21

lead groups received 8 #4 pellets and the birds in the tungsten-iron and tungsten-polymer
group received 8 BBS. Prior to dosing, individual shot or BBS were weighed and placed
in groups of 8 into individual glass vials which were identified according to shot type, cage
number, and the bird’s individual band number. At dosing, each vial containing Shot or
BBS was matched to the appropriate bird. Pellets were introduced into the proventriculus
by inserting a latex tube (0.953 cm inner diameter) 21.60 cm through the esophagus into
the proventriculus. A plastic funnel was attached to the end of the tube so that the pellets
or BBS could be flusbd down the tube with approximately 5 ml of water. Between birds,
the tube and funnel were placed in a bucket of water to keep the tube moist for easy
insertion. Control ducks were sham-dosed identically to treatment birds except for the
presence of shot. All birds were observed twice daily for assessment of general
well-being. Any clinical signs including, but not limited to, inappetence, apparent weight
loss, ataxia, lethargy, and discolored excreta were noted in the daily log. Screens
suspended underneath each cage were cleaned on a daily basis and the excreta was
examined for any expelled Shot or BBS. In the event expelled shot or BBS was recovered,
it was to be rinsed off and placed back into the appropriate glass vial for subsequent
weighing. Presence or absence of expelled shot on each screen was recorded daily. In
addition to these observations, feed and water were checked daily and the room
temperature was recorded at each entry.

On day 7 of the trial (23 January 1996), the ducks were transported (10 birds/crate)
to the Michigan State University Large Animal Veterinary Clinic (4 miles fi'om the PTRC)

for fluoroscopy by radiologist Dr. Russell Stickle to determine retention of shot. Control

22

birds were also fluoroscoped to insure that no shot had been ingested prior to arrival at
Michigan State University. All birds were manually immobilized on their side on the
examination table and slowly rotated by hand until the greatest number of shot could be
observed on the viewing screen. At this point, a radiograph identified by the last 3
numbers of the bird’s leg band was taken.

Blood samples were collected on days 15 and 30 (31 January and 15 February
1996) of the trial after the birds were weighed. Birds were held manually on an
examination table on their backs with one wing extended. A 21-gauge, 3.81 cm
Vacutainer“ multiple sample needle was inserted into the brachial vein and blood was
sequentially collected into 1 3-ml Vacutainer ° tube containing liquid EDTA (lavender top)
and 2 3-ml Vacutainer ' tubes containing sodium heparin (green top). Each tube was
labeled with the bird’s band number, treatment, and the date of collection.

The lavender top tube and one green top tube from each bird were gently rotated
for 1 minute and then refrigerated until all blood samples were collected over a 6-hour
period. Since birds were bled in order of their cage number rather than by treatment,
blood samples from all treatments were collected throughout the day. When blood
collection was completed, the samples were packed in Styrofoam coolers containing U-Tek'
polyfoam refi'igerant packs and shipped by overnight express to the Division of
Comparative Pathology of the University of Miami, Miami, Florida. These blood samples
were processed upon receipt for determination of hematocrit (I-ICI'), and hemoglobin (Hb)
concentration (lavender top tube) and red blood cell delta-aminolevulinic acid dehydratase

(ALAD) activity (green top tube).

23

The other green top tube from each bird was used for separation of plasma from
whole blood. Tubes were transported in groups of 12 to a laboratory adjacent to the
building containing the birds and spun in a centrifuge at 50 x g for 5 minutes. Plasma was
removed from the Vacutainer' tube by a glass Pasteur pipet and transferred to an
identically labeled 1—dram glass vial. Plasma vials were stored in a Styrofoam cooler
containing dry ice until all plasma samples had been collected. Vials were then transferred
to an ultracold freezer (-72°C) until shipping at the end of the 30-day trial. Plasma samples
were sent on dry ice by overnight express to the Division of Comparative Pathology,
University of Miami, for determination of albumin, albumin/ globulin ratio, alkaline
phosphatase activity, amylase activity, aspartate aminotransferase activity, blood urea
nitrogen, blood urea nitrogen/creatine ratio, calcium, chloride, cholesterol, C02, creatine
phosphokinase activity, creatinine, direct bilirubin, total bilirubin, gamma glutamyl
transpeptidase activity, glucose, lactate dehydrogenase activity, phosphorus, potassium,
sodium, total protein, triglycerides, and uric acid.

Within 1 hour of arrival of the whole-blood samples at the University of Miami,
the tubes were unpacked, separated by tube type, and arranged sequentially by the bird’s
band number on each tube. Tubes were then assigmd a second number (l,2,3,etc.). The
quality of each sample was grossly examined and noted on the log-in worksheet. Lavender
top tubes were at room temperature prior to determination of Hb and HCI‘. Green top
tubes were Stored at 4°C for 3 hours prior to analysis for ALAD.

Hemoglobin was determined by removing 100 pl of whole blood from the lavender

top tubes by an automatic pipet and placing it in a plastic 96-well microtiter plate. Fifty

24

pl of lysis solution (Hematall LA-Hgb, Fisher Scientific) was added to each well. The
solutions were then mixed by automatic pipet for 10 . seconds. After incubation at - room
temperature for 1 minute, the plate was centrifuged at 1,200 rpm for 10 minutes to pellet
red blood cell nuclei and other debris. The supernatant was removed and hemoglobin was
measured using a Leica hemoglobinometer. Hemoglobin was quantitated as g/dL (x 1.5
for dilution factor). The hematocrit was determined by drawing approximately 50 pl of
blood from the lavender top tube by capillary action into a microhematocrit tube. Tubes
were then sealed at one end using hematocrit clay and centrifuged in a standard hematocrit
centrifuge (IEC MB microhematocrit centrifuge) for 5 minutes. HCT was determined by
a manual hematocrit reader (IEC microcapillary reader #2201). ALAD activity (expressed
in ALAD units) was measured according to the prowdures of Burch and Siegel (1971) and
Dieter and Finley (197 9). Samples, which were run in triplicate, were brought to room
temperature after 1 hour of rocking on a Standard tube rocker. Twenty pl of blood from
the green top tube was placed in a 96-well microtiter plate and 130 pl Triton-X and 100
pl ALA-S were added to each well and mixed by automatic pipet. One hundred pl of this
solution was removed and then incubated in a 96—well format at 37°C for 1 hour. For a
control, an additional 100 pl aliquot was mixed with 100 pl trichloroacetic acid. The
sample was then centrifuged for 10 minutes at 1,200 rpm. After centrifugation, 100 pl of
the supernatant was removed for later measurement. The incubated mixture went through
the same procedure (addition of trichloroacetic acid and subsequent centrifugation). One
hundred pl of Ehrlich's reagent was then added to the experimental and control

supematants. After a 13 minute room temperature incubation, the plates were read at 555

25

nm using a Molecular Devices plate reader. Means for each determination were calculated
and control absorbance was subtracted. Samples with a standard deviation greater than
10% were repeated. Final ALAD calculations were done as a function of HCT where
ALAD units of activity equal (Corrected Absorbance x 12,500)/HCT.

Upon arrival at the University of Miami, frozen plasma samples were quickly
arranged sequentially by the bird’s band number on each tube, assigned a second number
(1,2,3 etc.), and placed in a freezer until analysis. Twenty-five samples were thawed at
a time and assessioned 1 hour prior to analysis. The quality of each sample was grossly
examined and noted in the log-in work sheet. Samples were analyzed using a Kodak 750
chemistry analyzer. Control sera samples were run daily prior to analysis of test samples
to maintain a check on instrument calibration. Plasma chemistry analyses were performed
over a 7-day period.

On day 30 of the trial, birds which had not died were weighed, bled as previously
described, killed by cervical dislocation, and subjected to necropsy in a laboratory adjacent
to the building housing the ducks. The necropsy procedure included a complete gross
examination of all body cavities and organs by Dr. Scott Fitzgerald, diplomate of the
American College of Veterinary Pathologists (ACV P). Gizzards were opened for
inspection of cracked and discolored mucosa and retention of shot. Shot were counted and
placed back into the appropriate glass vial for subsequent cleaning and weighing for
determination of shot erosion. The femur, spleen, heart, liver, kidneys, brain, and testes
were removed and weighed. Small samples of the liver and kidneys from each bird were

placed in labeled plastic containers containing formalin for subsequent histopathological

26

examination by Dr. Scott Fitzgerald. lesions in the liver and kidney were scored on a 4-
point scale where 0=normal and 3 = severe. The femur and the remaining portions of the
liver and kidneys were placed in individual labeled plastic bags and frozen on dry ice until
all tissues were obtained. The 3 tissues from each bird were then placed in a larger labeled
plastic bag and packed in a Styrofoam cooler containing dry ice. The cooler was then
placed in a chest freezer (-72°C) until it was picked up by a representative of Anatech
Laboratories on 19 February 1996. The cooler was repacked with dry ice and transported
by car to the Anatech facility in Ludington, MI. Tissues were stored frozen until sample
preparation and analysis. Sample preparation/digestion was performed according to EPA
procedures for iron, tungsten, molybdenum (by Inductively Coupled Argon Emission
Plasma Spectroscopy or ICP) and lead (by Graphite Furnace Atomic Absorption or
GFAA). Blanks, calibration verification, duplicate analysis, and spike analysis were
performed and processed with the quality control (QC) package.

All statistical analyses were performed on the SAS Statistical package (version 6.22;
Cary NC). Significance was set at p < 0.05. Males and females were analyzed
separately. Body and organ weights from those birds dying prior to the end of the trial
were excluded from statistical analysis. Data from blood samples which had clotted were
also removed from the analysis.

Qualitative data (mortality and histopathological lesions) were analyzed using the
Pearson Chi-Square Test. Quantitative data (plasma and whole-blood parameters, body
weights, organ weights, tissue metal concentrations, and percent shot erosion) were

analyzed using one-way analysis of variance (AN OVA). When statistically significant

27

differences were detected in the overall AN OVA, Tukey’s Studentized Range Test was

used to determine differences among groups.

Results

Mortality

The effect of treatment shot on mortality is presented in Table 2. Eight of 16
(50%) mallards dosed with lead shot died during the course of the 30-day Surdy. Of the
8 birds dying, 5 (62.5%) were males and 3 (37.5%) were females. The average time to
death was 17.6 days for males and 15.3 days for females with a range of 10 to 25 days for
both sexes. The average weight loss of those birds dying was slightly over 30%. N o
ducks in the other 4 treatment groups (no-shot, steel, tungsten-iron and tungsten-polymer)
died during the 30-day trial.

Clinical Signs

The leadodosed birds were the only ones which had obvious clinical signs during
the course of the 30—day trial. Green excreta was the first abnormality noted, being
apparent in 50% of the males and females within 24 hours of dosing. By 4 days
post-dosing, all lead-dosed birds were eliminating green excreta. Of the 5 males which
died, the 3 which died the earliest (days 11, 15, and 17) had no other clinical signs. The
2 males which died on days 20 and 25 developed progressive ataxia from 2 to 4 days prior
todeath. The3 survivingmalesappearednormalduringthelast9days ofthetrial. Two
of the 3 lead-dosed females which died (days 10 and 15) had no clinical signs other than
green excreta prior to death. The female which died on day 21 appeared ataxic the day

before it died. Of the 5 females which survived the 30—day trial, 2 appeared to be normal

28

 

 

 

 

 

 

Table 2. The effect of treatment shot on percent mortality, time to death (days),
and percent weight lost at death of mallards on a 30—day dosing test‘.
% Mortality Time to Death % Weight
Lost at Death
Males
No-Shot - - -
Steel - - -
Lead 62.5 17.6 31.2
(5/ 8) (1 1-25) (22.4-37.9)
Tungsten-Iron - - -
Tungsten-Polymer - - «-
Females
N o-Shot - - -
Steel - - -
Lead 37.5 15.3 32.7
(3/8) (1021) (17.1-46.9)
Tungsten-Iron - - -
Tungsten-Polymer - - -

 

 

' Numbers in parentheses represent number of birds dying/number of birds per
group for percent mortality, range of time to death, and range of percent weight
lost.

 

 

29

by day 15 with the exception of an occasional appearance of green excreta. The other 3
females had varying degrees of ataxia which became increasingly severe and then the
condition seemed to improve somewhat near the end of the Study.

Body Weights

The effects of treatment shot on body weights of mallards surviving the 30—day test
period are presented in Table 3. Significant differences in male body weights were
detected at day 15. Lead male body weights were significantly lower than no-shot and
tungsten-polymer body weights, but similar to steel and tungsten-iron body weights. There
were no significant differences in body weight at days 0 and 30 for either sex. During the
30-day test period, birds receiving no Shot gained a Slight amount of weight (5.8 % for
males and 2.8 % for females) as did those birds receiving tungsten-iron (5 .0% for males
and 0.7 % for females) and tungsten-polymer (3.7% for males and 3.8 % for females).
Steel-dosed males also gained a small amount of weight (2.2%) while the females
maintained the same weight. Lead—dosed males and females that survived the 30day trial
lost approximately 6.0% of their body weight.

Hematocrit, Hemoglobin Concentration, and ALAD Activity

The effects of treatment shot on hematocrit, hemoglobin concentration, and ALAD
activity at day 15 and day 30 of the dosing test are presented in Tables 4 (males) and 5
(females). When blood samples were taken on day 15 of the trial, it was apparent that the
blood taken from a mrmber of the lead-dosed birds had a fluorescent-like cast and was less
viscous. These observations were substantiated by both hematocrit and hemoglobin

concentrations. In lead-dosed males, hematocrit concentrations were Significantly lower

30

 

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33

compared to steel, tungsten-iron, and tungsten-polymer birds and hemoglobin
concentrations were significantly lower compared to no-shot, steel, and tungsten-polymer
birds. Lead-dosed females had significantly lower hematocrit's and hemoglobin
concentrations when compared to the other 4 groups. The no-shot, steel, tungsten-iron,
and tungsten—polymer-dosed birds were not significantly different from one another in
hematocrits and hemoglobin concentrations. In addition to the lead-induced changes in
hematocrit and hemoglobin concentration, red blood cell ALAD activity was significantly
depressed at day 15 in the lead-dosed females when compared to the other 4 groups.
While the 15 day ALAD activity for lead-dosed males was numerically lower than the
activities for the other 4 groups, it was not significant. By day 30 of the trial, there were
no significant differences in any of the whole-blood parameters between the 5 treatment
groups.

Plasma Chemistries

The effects of steel, lead, tungsten-iron and tungsten-polymer shot on day 15
plasma Chemistries for males are presented in Table 6. Lead shot resulted in a significant
increase in plasma glucose concentration when compared to the other 4 dose groups. The
albumin/globulin ratio was significantly increased in both the lead and tungsten-polymer
birds when compared to no-shot and steel-dosed birds. The plasma activities of the hepatic
enzymes alanine aminotransferase, aspartate aminotransferase, lactate dehydrogenase, and
creatine phosphokinase were all significantly elevated in lead-dosed males at day 15 when
compared to activities in the other 4 groups. There were no significant differences in

plasma chemistry parameters between the no-shot, steel, tungsten-iron, and

34

 

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36

tungsten-polymer males at day 15 with the exception of the elevated albumin! globulin ratio
in the tungsten-poylmer birds.

The effects of steel, lead, tungsten-iron and tungsten-polymer shot on day 15
plasma Chemistries for females are presented in Table 7. Exposure of female mallards to
lead shot caused a significant decrease in plasma sodium concentration compared to the no-
shot, tungsten-iron, and tungsten-polymer birds and a significant decrease in chloride
concentration when compared to the other 4 treatment groups. Plasma creatinine
concentrations were significantly higher in the leaddosed females when compared to the
no-shot, steel and tungsten-polymer groups. The activities of alanine aminotransferase and
lactate dehydrogenase were significantly higher in lead-dosed females when compared to
values for the other 4 treatment groups.

By day 30, there were no differences in male plasma parameters (Table 8). In the
lead-dosed females, chloride concentrations continued to be depressed when compared to
tungsten-polymer birds. In addition, the concentration of carbon dioxide was significantly
higher in lead-dosed females at day 30 when compared to tungsten-polymer birds.
Alanine aminotransferase continued to be significantly increased in the lead-dosed females
when compared to steel and tungsten-iron-dosed females (Table 9).

Gross Pathology

A summary of gross necropsy observations is presented in Tables 10 (males) and
11 (females). Those lead-dosed birds that died on trial were characterized by either having
gizzards with discolored mucosa] lining and multiple linear erosions (3 males and 2

females) or the birds were severely emaciated with little breast muscle (2 males and l

37

 

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53...... 2938.2 2.2.38.2 £938.: .832 .2 5...»... 22.28
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43

 

 

 

Table 10 . The gross necropsy observations from the effects of treatment shot on
male mallards on a 30-day dosing test for candidate shot.
Duck ID Treatment Days on Trial Observation(s)‘I
4671 1 l N o-Shot 30 Normal
467119 No-Shot 30 Fatty Liver
467128 No-Shot 30 Normal
467135 No-Shot 30 Normal
467155 No-Shot 30 Normal
467163 No-Shot 30 Normal
467172 No-Shot 30 Normal
467180 No—Shot 30 Normal
467107 Steel 30 Normal
4671 15 Steel 30 Normal
467124 Steel 30 Normal
467141 Steel 30 Normal
467151 Steel 30 Normal
467159 Steel 30 _ Normal
467168 Steel 30 Normal
467187 Steel 30 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald.

 

 

44

 

Table 10 continued. The gross necropsy observations from the effects of treatment
shot on male mallards on a 30-day dosing test for candidate
shot.

 

Duck ID Treatment Days on Trial Observation(s)‘

 

467109 Lead 25 Discolored mucosal lining of the gizzard
with multiple linear erosions

467117 Lead 11 Discolored mucosal lining of the
gizzard with linear erosions

467126 Lead 30 Normal
467133 Lead 30 Severe breast muscle atrophy
467153 Lead 30 Normal

467161 Lead 17 Prominent keel bone with no
subcutaneous or abdominal fat
(emaciation)

Moderately thickened koilin layer of the
gizzard
Small liver (1/2 to 3/4 normal)

467170 Lead 20 Prominent keel bone (emaciation)
Multifocal, moderate thickening of the
koilin layer of the gizzard
Small liver (3/4 normal) with multiple 1

m white foci scattered throughout the
capsular and cut surfaces

467178 Lead 15 Discolored mucosal lining of the gizzard
with multiple linear erosions

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald, or in the case of
467170, by Dr. R.M. Fulton, board-certified avian diagnostician.

 

 

45

 

Table 10 continued. The gross necropsy observations from the effects of treatment
shot on male mallards on a 30-day dosing test for candidate

 

 

shot.
Duck ID Treatment Days on Trial Observation(s)‘
467103 Tungsten-Iron 30 Normal
467113 Tungsten-Iron 30 Normal
467132 Tungsten-Iron 30 Normal
467139 Tungsten-Iron 30 Normal
467147 Tungsten-Iron 30 Normal
467157 Tungsten-Iron 30 Normal
5417 Tungsten-Iron 30 Normal
467185 Tungsten-Iron 30 Normal
467101 Tungsten-Polymer 30 Normal
467121 Tungsten-Polymer 30 Normal
467130 Tungsten-Polymer 30 Normal
467137 Tungsten-Polymer 30 Normal
467145 Tungsten-Polymer 30 Normal
467165 Tungsten-Polymer 30 Normal
467174 Tungsten-Polymer 30 Normal
467182 Tungsten-Polymer 30 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald.

 

 

45

 

 

 

Table 11 . The gross necropsy obervations from the effects of treatment shot on
female mallards on a 30-day dosing test for candidate shot.
Duck ID Treatment Days on Trial Observation(s)‘
4671 12 No-Shot 30 Normal
467120 No-Shot 30 Moderate fatty liver
467127 No-Shot 30 Moderate fatty liver
467136 No-Shot 30 Normal
467156 No—Shot 30 Normal
467164 No-Shot 30 Fatty liver
467171 No-Shot 30 Normal
467179 No-Shot 30 Normal
467108 Steel 30 Normal
4671 16 Steel 30 Normal
467123 Steel 30 Normal
467142 Steel 30 Normal
467152 Steel 30 Normal
467160 Steel 30 Normal
5418 Steel 30 Normal
467186 Steel 30 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald.

 

 

47

 

Table 11 continued. The gross necropsy observations from the effects of treatment
shot on female mallards on a 30-day dosing test for candidate

 

 

shot.
Duck ID Treatment Days on Observation(s)‘
Trial

4671 10 Lead 30 Normal
5421 Lead 10 Discolored, cracked mucosal lining of the

gizzard with linear erosions
467125 Lead 30 Normal
467134 Lead 30 Fatty liver
467154 Lead 30 Normal
467162 Lead 30 Moderately emaciated
467169 Lead 21 Emaciated

Air sac granuloma
467177 Lead 15 Discolored mucosal lining of the gizzard

with linear erosions

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald.

 

 

48

 

Table 11 continued. The gross necropsy observations from the effects of treatment
shot on female mallards on a 30-day dosing test for candidate

 

 

shot.
Duck ID Treatment Days on Trial Observation(s)'
467106 Tungsten-Iron 30 Normal
4671 14 Tungsten-Iron 30 Normal
467131 Tungsten-Iron 30 Normal
467140 Tungsten-Iron 30 Normal
467150 Tungsten-Iron 30 Normal
467158 Tungsten-Iron 30 Normal
5419 Tungsten-Iron 30 Normal
467184 Tungsten-Iron 30 Normal
467102 Tungsten-Polymer 30 Normal
467122 Tungsten-Polymer 30 Normal
467129 Tungsten-Polymer 30 Normal
467138 Tungsten-Polymer 30 Normal
5436 Tungsten-Polymer 30 Normal
467166 Tungsten-Polymer 30 Normal
467173 Tungsten-Polymer 30 Normal
467181 Tungsten-Polymer 30 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald.

 

 

49

female). With 2 exceptions, gizzard erosion occurred in those birds dying during the first
half of the trial and emaciation was characteristic of those birds dying between days 16-25.
Of the 8 surviving lead-dosed birds, 1 male and 1 female were emaciated at the time of
necropsy, 1 female had a fatty liver while the other 5 birds (2 males and 3 females)
appeared normal. Four no-shot birds (1 male and 3 females) had moderately fatty livers
while all birds in the steel, tungsten-iron and tungsten-polymer shot groups appeared
normal.

Absolute Organ Weights

The effects of steel, lead, tungsten-iron, and tungsten-polymer shot on absolute
organ weights of male and female mallards are presented in Table 12 and 13. There were
no significant differences between treatments in male organ weights while lead-dosed
female liver weight was significantly higher when compared to the average liver weight
of steel-dosed females.

Organ Weights as a Percent of Body Weight

The effects of treatment shot on male and female organ weights expressed as a
percent of body weight are presented in Table 14 and 15. The only change noted within
males was an increase in relative kidney weights in lead-dosed birds when compared to
relative kidney weights of no-shot and tungsten-iron males. In females, relative bean
weights of the lead-dosed birds were significantly higher when compared to the no-shot
and tungsten-polymer birds whereas relative kidney weights of lead-dosed females were
significantly greater when compared to the tungsten-iron and tungsten-polymer birds.

Relative liver weights were significantly higher in lead-dosed females when compared to

50

 

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54

the steel, tungsten-iron and tungsten-polymer groups.

Histopathology of Kidney and Liver

The effects of treatment shot on liver and kidney pathology are presented in Tables
16 (males) and 17 (females). Lesion scores for the liver and kidney are presented in
Tables 18 (males) and 19 (females). Five male lead-dosed birds developed renal nephrosis
ranging from mild to severe with accompanying mild to severe hepatic biliary stasis. All
of these birds died during the course of the trial (days 11-25). The remaining 3 lead-dosed
males had normal kidneys and mild to moderate hepatic biliary stasis. These birds
survived to the end of the 30—day trial. Two tungsten-iron—dosed males and 3
tungsten-polymer-dosed males developed mild liver biliary stasis. The severity of liver
lesions in the lead—dosed males was significant when compared to the other 4 groups while
there were no statistical differences in the severity of kidney lesions. Three lead-dosed
females had renal nephrosis ranging from mild to moderate with accompanying moderate
to severe hepatic biliary stasis. These 3 females died on days 10, 15, and 21 of the trial.
Three of the 8 lead-dosed females developed liver biliary stasis only (mild to severe) and
they survived the duration of the 30—day trial. Two lead-dosed females had normal liver
and kidney tissues. Three tungsten-iron females developed mild biliary stasis with no
kidney involvement while the tungsten-polymer females were unaffected. The severity of
liver lesions in the lead-dosed females was significant when compared to the other 4
groups. There were no statistical differences in the severity of kidney lesions. Throughout
all 5 groups, diffuse hepatocellular vacuolation was apparent, but this condition was judged

not to be treatment related.

55

 

 

 

Table 16 . The histopathological effects of treatment shot on the liver and kidneys

of male mallards on a 30-day dosing test for candidate shot.
Duck ID Trt‘ AHDL" Observation(s)c

Code

4671 11 NS 467-111 Moderate, diffuse hepatocellular vacuolation
467119 NS 196881-14 Severe, diffuse hepatocellular vacuolation
467128 NS 169881-23 Normal
467135 NS 196881-30 Moderate, diffuse hepatocellular vacuolation
467155 NS 196881-45 Normal
467163 NS 196881-52 Mild, diffuse hepatocellular vacuolation
467172 NS 196881-58 Moderate, diffuse hepatocellular vacuolation
467180 NS 196881-62 Normal
467107 S 196881-5 Moderate, diffuse hepatocellular vacuolation
467115 S 196881-12 Moderate, diffuse hepatocellular vacuolation
467124 8 169881-19 Moderate, diffuse hepatocellular vacuolation
467141 8 196881-30 Normal
467151 S 196881-41 Normal
467159 8 196881-49 Mild, diffuse hepatocellular vacuolation
467168 S 196881-56 Normal
467187 S 196881—68 Mild, diffuse hepatocellular vacuolation

 

 

‘ NS refers to no—shot and S refers to steel.
” AHDL refers to Michigan State University's Animal Health Diagnostic

Laboratory where the tissues were prepared and assessed for histopathological

changes.

° Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald.

 

 

56

 

Table 16 continued.

The histopathological effects of treatment shot on the liver and
kidneys of male mallards on a 30-day dosing test for candidate shot.

 

Duck ID

Trt‘ AHDL”Code

Observation(s)c

 

467109

467117

467126

467133

467153

467161

467170

467178

L

196210

195237-1

196881-21

196881-28

196881-43

195403-1

195549-2

195403-1

Diffuse, acute proximal (mild)and distal (severe)
convoluted renal tubule necrosis with pyknosis,
vacuolation,and inclusions

Moderate, diffuse hepatocellular and cholangial biliary
stasis with mild extramedullary hematopoiesis, 25 days
on trial

Mild, diffuse acute proximal and distal renal tubular
necrosis with pyknosis and rare inclusions

Severe, diffuse hepatocellular and cholangial biliary stasis,
11 days on trial

Kidney-normal
Moderate, diffuse hepatocellular vacuolation
Mild hepatocellular biliary stasis

Kidney-normal
Mild, diffuse hepatocellular vacuolation
Moderate hepatocellular and cholangial biliary stasis

Kidney-normal
Mild, diffuse hepatocellular vacuolation
Mild hepatocellular and cholangial biliary stasis

Moderate proximal and distal renal tubule necrosis with
pyknosis, vacuolation

Mild hepatocellular and cholangial biliary stasis, 17 days

on trial

Mild proximal renal tubule necrosis with inclusions
Moderate hepatocellular and cholangial biliary stasis with
multifocal hepatic abssesses, 20 days on trial

Moderate proximal and distal renal tubule necrosis with
pyknosis, vacuolation, and inclusions

Moderate hepatocellular and cholangial biliary stasis, 15
days on trial

 

 

' L refers to lead.

" AHDL refers to Michigan State University's Animal Health Diagnostic Laboratory
where the tissues were prepared and assessed for histopathological changes.
° Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald.

 

 

57

 

Table 16 continued. The histopathological effects of treatment shot on the liver and
kidneys of male mallards on a 30-day dosing test for candidate

 

 

shot.
Duck ID Trt' AHDL” Observation(s)c
Code
467103 TI 196881-3 Mild, periportal hepatocellular vacuolation
467113 TI 196881-10 Normal
467132 TI 169881-27 Moderate, diffuse hepatocellular vacuolation
467139 TI 196881-34 Moderate, diffuse hepatocellular vacuolation
467147 TI 196881-39 Mild hepatocullular vacuolation
467157 TI 196881-47 Mild, diffuse hepatocellular biliary stasis
5417 TI 196881-60 Mild hepatocellular vacuolation
Mild hepatocellular and cholangial biliary stasis
467185 TI 196881-66 Normal
467101 TP 196881-1 Moderate, diffuse hepatocellular vacuolation
467121 TP 196881-16 Mild, diffuse hepatocellular vacuolation
467130 TP 196881-25 Moderate, diffuse hepatocellular vacuolation
467137 TP 196881-32 Moderate, diffuse hepatocellular vacuolation
467145 TP 196881-38 Moderate hepatocullular vacuolation
Mild hepatocellular biliary stasis
467165 TP 196881-54 Mild hepatocellular biliary stasis
467174 TP 196881-60 Normal
467182 TP 196881-64 Mild, hepatocellular biliary stasis

 

 

TI refers to tungsten-iron and TP refers to tungsten-polymer.

" AHDL refers to Michigan State University's Animal Health Diagnostic
laboratory where the tissues were prepared and assessed for histopathological
changes.

° Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald.

 

 

58

 

 

 

Table 17 . The histopathological effects of treatment shot on the liver and kidneys

of female mallards on a 30-day dosing test for candidate shot.
Duck ID Trt“ AHDL” Observation(s)c

Code

467112 NS 196881-9 Moderate, diffuse hepatocellular vacuolation
467120 NS 196881-15 Moderate, diffuse hepatocellular vacuolation
467127 NS 196881-22 Severe, diffuse hepatocellular vacuolation
467136 NS 196881-31 Mild hepatocellularivacuolation
467156 NS 196881-46 Moderate, diffuse hepatocellular vacuolation
467164 NS 196881-53 Moderate, diffuse hepatocellular vacuolation
467171 N S 196881-57 Mild, diffuse hepatocellular vacuolation
467179 NS 196881-61 Moderate, diffuse hepatocullular vacuolation
467108 S 196881-6 Mild, diffuse hepatocellular vacuolation
467116 8 196881-13 Mild, diffuse hepatocellular vacuolation
467123 S 169881-18 Moderate, diffuse hepatocellular vacuolation
467142 S 196881-37 Normal
467152 8 196881-42 Normal
467160 S 196881-50 Moderate, diffuse hepatocellular vacuolation
5418 S 196881-70 Mild, diffuse hepatocellular vacuolation
467186 S 196881-67 Mild, diffuse hepatocellular vacuolation

 

 

N 8 refers to no-shot and S refers to steel.
" AHDL refers to Michigan State University's Animal Health Diagnostic

Laboratory where the tissues were prepared and assessed for histopathological

changes.

° Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald.

 

 

59

 

Table 17 continued. The histopathological effects of treatment shot on the liver and
kidneys of female mallards on a 30-day dosing test for candidate shot.

 

 

Duck ID Trt‘ AHDL” Observation(s)°
Code
4671 10 L 196881-7 Kidney-normal

Moderate, diffuse hepatocellular vacuolation
Moderate hepatocellular and cholangial biliary stasis

5421 L 195237-2 Diffuse proximal (mild) and distal (moderate) renal
tubule necrosis with pyknosis and inclusions
Severe hepatocellular and cholangial biliary stasis
with mild extramedullary hematopoiesis, 10 days on
trial

467125 L 196881-20 Kidney-normal
Moderate, diffuse hepatocellular vacuolation
Mild hepatocellular and cholangial biliary stasis

467134 L 196881-29 Kidney-normal
Severe, diffuse hepatocellular vacuolation
467154 L 196881-44 Kidney-normal

Moderate, diffuse hepatocellular vacuolation
Moderate hepatocellular and cholangial biliary stasis

467162 L 195881-51 Kidney-normal
Mild, diffuse hepatocellular vacuolation
467169 L 195785 Moderate proximal and distal renal tubule necrosis

with pyknosis and inclusions
Moderate, diffuse hepatocellular vacuolation
Moderate hepatocellular and cholangial biliary stasis
Air sacculitis/histocytic, 21 days on trial

467177 L 195403-2 Mild proximal and distal renal tubule necrosis with
pyknosis
Mild, diffuse hepatocellular vacuolation
Severe hepatocellular and cholangial biliary stasis,
15 days on trial

 

 

‘ L refers to lead.

" AHDL refers to Michigan State University's Animal Health Diagnostic Laboratory
where the tissues were prepared and assessed for histopathological changes.

° Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald.

 

 

6O

 

Table 17 continued. The histopathological effects of treatment shot on the liver and
kidneys of female mallards on a 30-day dosing test for

 

 

candidate shot.
Duck ID Trt’ AHDL” Observation(s)c
Code
467106 TI 196881-4 Moderate, diffuse hepatocellular vacuolation
467114 TI 196881-11 Moderate, diffuse hepatocellular vacuolation
Mild, diffuse hepatocellular biliary stasis
467131 TI 169881-26 Mild, diffuse hepatocellular vacuolation
Mild hepatocellular biliary stasis
467140 TI 196881-35 Mild, diffuse hepatocellular vacuolation
467150 TI 196881-40 Moderate, diffuse hepatocullular vacuolation
Mild hepatocellular biliary stasis
467158 TI 196881-48 Mild, diffuse hepatocellular vacuolation
5419 TI 196881-71 Moderate, diffuse hepatocellular vacuolation
467184 TI 196881-65 Normal
467102 TP 196881-2 Moderate, diffuse hepatocellular vacuolation
467122 TP 196881-17 Moderate, diffuse hepatocellular vacuolation
467129 TP 169881-24 Moderate, diffuse hepatocellular vacuolation
467138 TP 196881-33 Moderate, diffuse hepatocellular vacuolation
5436 TP 196881-72 Normal
467166 TP 196881-55 Normal
467173 TP 196881-59 Normal
467181 TP 196881-63 Moderate, diffuse hepatocellular vacuolation

 

 

' TI refers to tungsten-iron and TP refers to tungsten-polymer.

b AHDL refers to Michigan State University's Animal Health Diagnostic
Laboratory where the tissues were prepared and assessed for histopathological
changes.

° Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald.

 

 

61

 

Table 18. The severity of liver and kidney lesions induced by treatment shot in
male mallards on a 30-day dosing test.

 

 

Duck ID Treatment Days on Trial Renal Hepatic Biliary
N ephrosis' Stasis'
467111 No-Shot 30 0 0
467119 No-Shot 30 0 0
467128 No-Shot 30 0 0
467135 No-Shot 30 0 0
467155 No-Shot 30 0 0
467163 No-Shot 30 0 0
467172 No-Shot 30 0 0
467180 No-Shot 30 0 0
467107 Steel 30 0 0
467115 Steel 30 0 0
467124 Steel 30 0 0
467141 Steel 30 0 0
467151 Steel 30 0 0
467159 Steel 30 0 0
467168 Steel 30 0 0
467187 Steel 30 0 0

 

 

' Lesion scores: 0, normal; +, mild; + +, moderate; + ++, severe.

 

 

62

 

Table 18 continued. The severity of liver and kidney lesions induced by treatment
shot in male mallards on a 30-day dosing test.

 

 

Duck ID Treatment Days on Trial Renal Hepatic Biliary

Nephrosisa Stasis‘
467117 Lead 11 + + + +
467178 Lead 15 + + + +
467161 Lead 17 + + +
467170 Lead 20 + + +
467109 Lead 25 + + + + +
467126 Lead 30 0 +
467133 Lead 30 0 + +
467153 Lead 30 0

 

 

‘ Lesion scores: 0, normal; +, mild; + +, moderate; + + +, severe.

 

 

63

 

Table 18 continued. The severity of liver and kidney lesions induced by treatment
shot in male mallards on a 30-day dosing test.

 

 

Duck ID Treatment Days on Renal Hepatic Biliary
Trial Nephrosis' Stasis'

467103 Tungsten-Iron 30 I 0 0
4671 13 Tungsten-Iron 30 0 0
467132 Tungsten-Iron 30 0 0
467139 Tungsten-Iron 30 0 0
467147 Tungsten-Iron 30 0 0
467157 Tungsten-Iron 30 0 +
5417 Tungsten-Iron 30 0 +
467185 Tungsten-Iron 30 0 0
467101 Tungsten-Polymer 30 0 0
467121 Tungsten-Polymer 30 0 0
467130 Tungsten-Polymer 30 0 0
467137 Tungsten-Polymer 30 0 0
467145 Tungsten-Polymer 30 0 +
467165 Tungsten-Polymer 30 0 +
467174 Tungsten-Polymer 30 0 0

’ 467182 Tungsten-Polymer 30 0 +

 

 

' Lesion scores: 0, normal; +, mild; + +, moderate; + + +, severe.

 

 

64

 

Table 19. The severity of liver and kidney lesions induced by treatment shot in
female mallards on a 30-day dosing test.

 

 

Duck ID Treatment Days on Renal Hepatic Biliary
Trial Nephrosis'I Stasis'
467112 No—Shot 30 0 0
467120 No-Shot 30 0 0
467127 N o-Shot 30 0 0
467136 N o-Shot 30 0 0
467156 No-Shot 30 0 0
467 164 N o-Shot 30 0 0
467171 No-Shot 30 0 0
467179 No-Shot 30 0 0
467108 Steel 30 0 0
4671 16 Steel 30 0 0
467123 Steel 30 0 0
467142 Steel 30 0 0
467152 Steel 30 0 0
467160 Steel 30 0 0
5418 Steel 30 0 0
467186 Steel 30 0 0

 

 

' Lesion scores: 0, normal; +, mild; + +, moderate; + + +, severe.

 

 

 

65

 

Table 19 continued. The severity of liver and kidney lesions induced by treatment
shot in female mallards on a 30-day dosing test.

 

 

Duck ID Treatment Days on Trial Renal Hepatic Biliary
Nephrosis‘ Stasis'
5421 Lead 10 + + + + +
467177 Lead 15 + + + +
467169 Lead 21 + + + +
4671 10 Lead 30 0 + +
467125 Lead 30 0 +
467134 Lead 30 0 0
467154 Lead 30 0 + +
467162 Lead 30 0 0

 

 

' Lesion scores: 0, normal; +, mild; + +, moderate; + + +, severe.

 

 

 

66

 

Table 19 continued. The severity of liver and kidney lesions induced by treatment
shot in female mallards on a 30-day dosing test.

 

 

Duck Treatment Days on Renal Hepatic Biliary
ID Trial N ephrosis‘ Stasis'
467106 Tungsten-Iron 30 0 0
4671 14 Tungsten-Iron 30 0

467131 Tungsten-Iron 30 0 +
467140 Tungsten-Iron 30 0 0
467150 Tungsten-Iron 30 0 +
467158 Tungsten-Iron 30 0 0
5419 Tungsten-Iron 30 0 0
467184 Tungsten-Iron 30 0 0
467102 Tungsten-Polymer 30 0 0
467122 Tungsten-Polymer 30 0 0
467129 Tungsten-Polymer 30 0 0
467138 Tungsten-Polymer 30 0 0
5436 Tungsten-Polymer 30 0 0
467166 Tungsten-Polymer 30 0 0
467173 Tungsten-Polymer 30 0 0
467181 Tungsten-Polymer 30 0 0

 

 

‘ Lesion scores: 0, normal; +, mild; + +, moderate; + + +, severe.

 

 

67

Tissue Metal Analysis

The concentrations of iron, lead, tungsten, and molybdenum in the femur of male
and female mallards are presented in Table 20. Lead-dosed males had significantly higher
concentrations of iron in the femur when compared to the no-shot, tungsten-iron and
tungsten-polymer birds and significantly higher concentrations of lead than the other 4
groups. Tungsten was detected in the femur of tungsten-iron males only. In females,
femur lead concentration was significantly higher in the lead-dosed group when compared
to the other 4 treatment groups. Tungsten was detected in both tungsten-iron and tungsten-
polymer birds with the concentration in tungsten-iron birds being significantly higher
compared to the tungsten-polymer group. Molybdenum was not detected in the femur of
any bird.

The concentrations of iron, lead, tungsten, and molybdenum in the liver of male
and female mallards are presented in Table 21. Hepatic iron concentrations were
significantly higher in the lead-dosed males when compared to the no-shot, steel, and
tungsten-polymer birds and tungsten-iron birds had significantly higher liver iron
concentrations compared to no-shot males. Hepatic lead concentrations were significantly
higher in the males receiving lead shot compared to the other 4 groups. Lead-dosed males
had significantly higher concentrations of molybdenum when compared to no-shot, steel,
and tungsten-polymer birds and tungsten-iron males had a higher molybdenum
concentration than no-shot males. Tungsten was detected only in the liver of tungsten-iron
males. In females, hepatic iron concentrations were significantly elevated in the lead-dosed

birds when compared to the no-shot birds and hepatic lead concentrations were significant

68

 

 

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70

higher in the lead-dosed females when compared to birds in the other 4 groups. As with
the males, tungsten was detected only in the liver of tungsten-iron-dosed females.

The concentrations of iron, lead, tungsten, and molybdenum in the kidneys of male
and female mallards are presented in Table 22. Renal lead concentrations were
significantly elevated in lead-dosed males compared to the other 4 groups while tungsten
was significantly higher in the kidneys of tungsten-iron males and females compared to
tungsten-polymer males and females. The concentrations of renal lead and tungsten were
similarly elevated in the lead-dosed and tungsten-polymer-dosed females, respectively.
In addition, female mallards dosed with steel had significantly higher kidney concentrations
of iron when compared to the no-shot, lead, and tungsten-polymer birds while
molybdenum concentrations were highest in the steel-dosed birds, intermediate in the
no-shot and tungsten-polymer groups, and lowest in the lead-dosed and tungsten-iron—
dosed birds.

Shot Recovered and Percent Shot Erosion

The initial and final weights of ueatment shot (on a pellet basis) administered to
mallards and the resulting erosion rates are presented in Table 23. Fluoroscopy of the
birds on day 7 of the trial indicated that all birds receiving shot had retained the 8 pellets
administered on day 1 within the gizzard with the exception of 1 steel-dosed bird which
had 6 pellets . Most of the shot administered (at least 75 %) was recovered from the
gizzard at the time of necropsy regardless of shot type. Only 1 bird, a lead-dosed female,
had no shot present at necropsy. No shot was found in the excreta of any of the birds

which was examined daily during the course of the trial. Steel shot had the least amount

71

 

 

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73

of erosion (29.7 % for males and 35.3 % for females) while tungsten-polymer shot had the
most erosion (85 % and 76% , respectively, for males and females). In males, the erosion
of tungsten-iron shot was significantly greater than steel shot, but significantly less than
tungsten-polymer shot while the erosion of lead shot was similar to both steel and tungsten-
iron. In females, there was no significant difference in erosion between lead, tungsten-
iron, and tungsten-polymer shot which all eroded to a greater extent than steel shot. If
erosion of lead shot is considered separately in those birds which died during the trial from
those which survived the trial, then percent erosion of shot retrieved from birds dying was
37.0% for lead-dosed males and 30.3 % for lead-dosed females compared to 41.8% for
surviving males and 83.0% for surviving females.

Shot erosion rates when the total weight of shot administered per bird is compared
to the total weight of shot retrieved, regardless of pellet number are presented on Table 24.
When expressed in this manner, erosion rate was highest for tungsten-polymer shot in the
males (86%) followed by lead (54%), tungsten-iron (49%), and steel (37%). These values
were statistically different when tungsten-polymer was compared with the other 3 treatment
groups. In the females, tungsten-polymer shot had the highest erosion rate (7 6%) which
is comparable with the lead (64%) and tungsten-iron erosion rate (64%), and significantly
greater than the erosion rate of the steel shot (37 %). When the erosion rate of lead shot
in birds dying on trial was compared to the erosion rate in birds surviving the trial, the
erosion of lead shot was considerably more in males surviving the trial (83 %) compared
to those dying (37%) as it was in females (84% vs 30%).

The steel shot recovered maintained their original round shape while most of the

 

 

 

 

Table 24. The mean weight (gm) of total shot administered, the mean weight of
total shot retrieved from each bird at necropsy , and percent shot
erosion during a 30-day dosing test“.

Total Shot Wt. at Total Shot Wt. at Percent Shot
Day 0 Necropsy Erosion

Males

No Shot - - -

Steel 1.19781000518 0.753110.07029 372215.765"

Lead 157591000732 072111014724 542219.327"

Tungsten-Iron 424131000722 114441009625 494412.266B

Tungsten-Polymer 438981002536 060981012029 861012.759A

 

Females

 

No Shot

Steel

Lead
Tungsten-Iron

Tungsten-Polymer

1.193410.00295
155961000593
423581006256
4.431510.01796

015001004022
056871017660
154491012915
1.0849 10.27588

37.16:l:3.353B
6358111315A
635313.049“
756316.190A

 

 

' Data presented as mean1standard error of the mean. On day 0, male and
female mallards were dosed with 8 pellets of the appropriate shot. Values
reported reflect the average total weight of all pellets administered per bird in
each treatment. Sample size is 8 birds for each parameter. Means with different
superscripts are significantly different within the column (ps0.05).

 

 

75

lead pellets recovered were flattened and oval in shape. The tungsten-polymer shot were
flattened and disk-like in appearance while the tungsten-iron shot were round with small
dimples on the surface.

D . .

Mortality

In the present study, only lead-dosed birds died during the course of the 30—day trial
(Table 2). Eight #4 lead shot resulted in 50% mortality (62.5% for males, 37.5% for
females) within 10-25 days of dosing (average of 16.8 days). Those birds dying lost an
average of 30% of their body weight (Ill-46.9%). These results are generally similar
to those reported in other studies involving lead-dosed waterfowl, although differences in
shot size, number of shot administered, diet, and environmental conditions preclude direct
comparison. It should be pointed out that temperatures were somewhat low, compared to
a normal housing temperature of 13°C, during the course of the trial (the average low
temperature was 9°C, Table 1) which can be considered an additional stress to the dosed
birds. It is thus significant that only lead-dosed birds died and not those birds administered
the steel and tungsten shot.

Pain and Rattner (1988) administered 1 #4 lead shot to pen-reared black ducks and
reported 60% mortality (4 of 5 males and 2 of 5 females) between 4 to 6 days post-dosing.
While mortality in this study was thought to be uncommonly high based on the dose, the
authors felt that a number of factors including lack of acclimatization, high ambient
temperatures (376°C), and frequent handling may have been responsible. Body weight

loss was no greater in the lead-dosed birds than in the control birds.

76

In a subsequent study, Rattner 91 a1. (1989) administered 1 #4 lead shot to pen-
reared and wild black ducks and to game-farm and wild mallards maintained on pelleted
feed during the winter. In contrast to the previous study, no mortalities were reported
during the first 14 days, although transient signs of lead toxicity were noted. Birds were
then dosed with either 2 or 4 #4 lead shot. Twenty-eight days after receiving the second
dose, the wild mallards experienced 25 % mortality and by 49 days post—dosing there was
40% mortality among the wild black ducks and 45% among the wild mallards. When this
study was repeated in the summer, a single black duck died 21 days after receiving 1 #4
lead shot followed by 4 #4 lead shot.

Sanderson and associates (1992) dosed mallards with 2, 4, or 8 #2 lead shot or 4
#2 lead shot plus 4 #2 bismuth shot and maintained the birds up to 30 days on a diet of
shelled corn. In this study, mortality was 95 % with only 2 ducks (2 #2 lead shot)
surviving. The average survival times were 19.1, 15.0, 12.6, and 14.4 days for birds
receiving 2, 4, and 8 #2 lead shot, and the lead/bismuth combination, respectively.
Average body weight loss was 42.2% with a range of 16-56%. The high mortality in the
latter study was probably due in part to the shelled corn diet. Diet has been reported to
influence the severity of lead poisoning in waterfowl (Kendall :1 31., 1996).

In the present study, none of the birds dosed with tungsten-iron or tungsten-
polymer shot died. In a similar toxicity study in which mallards were closed with 12-17
pellets (an average of 1.03 gm which is equivalent to 5 #4 lead shot) composed of 39.05 %
tungsten, 44.49% bismuth, and 16.46% tin, no mortalities were reported during the 32-day

trial (Ringelman :1 3.1., 1993). In the latter study, birds received approximately 0.4 gm

77

tungsten while in the present study, birds were dosed with approximately either 4.2 gm
tungsten (4.43 gm of TP shot x 95.5% tungsten) or 2.3 gm tungsten (4.24 gm of TI shot
x 55 % tungsten).

Tungsten has been reported to cause mortality in birds. Nell gt a1. (1980)
administered broiler cockerels sodium tungstate by injection at 5 mg tungsten from day 1
to day 11, 10 mg from day 12to day 21, and20mg fromday 22 to day 35. They reported
that 4 of 10 birds died on day 29 of the trial. The total quantity of nmgsten administered
to the birds over the 35-day period was 0.44 gm. If an average erosion rate of 80 % for
the tungsten-polymer shot is used (Tables 21 and 22), then the birds in the present study
were exposed to approximately 3 .4 gm tungsten. However, if an average erosion rate of
56% for the tungsten-iron shot is used, then the birds were exposed to approximately 1.3
gm tungsten. In the chicken study, however; the tungsten was in a soluble form
administered by injection to relatively small birds, all of which would enhance toxicity.

Clinical Signs

Birds receiving lead shot were the only ones which had obvious clinical signs.
These signs (green excreta and, in some cases, ataxia) are typical of birds intoxicated with
lead (Friend, 1987; Pain and Rattner, 1988; Rattner :1 al., 1989). Lead-dosed ducks
which survived the trial appeared relatively normal at time of necropsy.

Ducks dosed with tungsten-iron and tungsten-polymer shot in the present study
appeared normal throughout the 30-day trial which agrees with results reported by
Ringelman gt a1. (1993) for mallards dosed with tungsten-bismuth—tin shot. Clinical signs

of acute tungsten poisoning reported for mammals include nervous prostration, diarrhea

78

and death preceded by coma due to respiratory paralysis (Stokinger, 1978). Clinical signs
for birds administered tungsten include anorexia, reduced weight gain, diarrhea and
labored breathing within an hour of death (Nell et 31., 1980).

Body Weights

Body weights of birds surviving the 30-day trial changed little. No—shot, steel,
tungsten-iron and tungsten-polymer-dosed birds gained a slight amount of weight or stayed
approximately the same (04 to 5.8 %) while lead-dosed ducks lost approximately 6% of
their body weight (Table 3).

Pain and Rattner ( 1988) reported that black ducks which survived dosing with 1 #4
lead shot were similar in body weight to the control birds at the end of the trial. Sanderson
et a1. (1992) indicated that the 2 mallards which survived dosing with 2 #2 lead pellets lost
16% of their body weight at the end ofthe 30—day trial compared to a 4% body weight loss
for the non-dosed controls. Mallards dosed with 12 to 17 pellets of tungsten-bismuth-tin
shot gained a similar amount of body weight as controls in the study by Ringelman et a1.
(1993).

Hematocrit, Hemoglobin Concentration, and ALAD Activity

Depressions in hematocrit, hemoglobin concentration and delta-aminolevulinic acid
dehydratase (ALAD) activity are all indicators of lead toxicity. Lead interacts with the
erythrocyte which results in increased fragility of the membrane, thus shortening the
lifespan of the erythrocyte. Additionally, lead inhibits ALAD, a key enzyme in the
synthesis of heme which is an integral component of hemoglobin. The combined effect of

lead on erythrocyte lifespan and heme synthesis results in lead-induced anemia which

79

results in decreased hematocrit and hemoglobin (Goyer, 1996).

In the present study, lead-dosed males alive at day 15 had a significantly lower
hematocrit (packed erythrocyte volume) concentration than steel, tungsten-iron and
tungsten-polymer birds and hemoglobin concentration than the no-shot, steel and tungsten-
polymer-dosed birds (Table 4). In the lead-dosed females alive at day 15 , hematocrit and
hemoglobin concentration were also significantly lower when compared to the other 4
groups (Table 5). ALAD activity was numerically lower in the lead-dosed males at day
15 (63 % of average activity in no-shot and steel-dosed birds) but individual variation
precluded statistical significance (Table 4). In the females, the lead-dosed birds had
significantly lower ALAD activities when compared to the other 4 treannent groups (58%
of average activity in no-shot and steel-dosed birds, Table 5). At the end of the 30-day
trial, there were no statistical differences in these whole-blood parameters in either sex.

It should be pointed out that blood samples were not obtained from those birds
dying on trial unless they were alive at day 15. It is likely that hematocrit, hemoglobin
concentration, and ALAD activity were depressed in those birds at time of death. It is
somewhat surprising that activities in lead-dosed birds were not inhibited to a greater
extent. A possible explanation could be inadequate preservation of the blood sample. In
the present study, blood samples used for ALAD analysis were kept cool during storage
and shipping rather than being frozen. It is possible that this resulted in lower than normal
acitivity for all samples, but it would not account for only moderate inhibition of ALAD
activity in lead-dosed birds. Another possibility is that of the 6 male and 6 female lead-

dosed birds bled on day 15, the 3 males and 5 females that survived the 30~day trial may

80

not have experienced lead toxicosis which would account for the relatively high ALAD
values as well as the lack of an effect on hematocrit and hemoglobin concentration at day
30.

Pain and Rattner (1988) reported that hematocrit and hemoglobin concentration
were significantly depressed in black ducks administered 1 #4 lead shot within 6 days of
dosing but recovery was apparent by 30 days post—dosing. ALAD activity was inhibited
by 100% at 1 day post-dosing, increased between 3-9 days post-dosing (approximately
70% inhibition) and then declined again until the end of the 30—day study. In a subsequent
study, Rattner gt a. (1989) reported no change in hematocrit and a transient decrease in
ALAD activity (> 90%) over 14 days in black ducks and mallards dosed with 1 #4 lead
shot. Birds were then re-dosed with either 2 or 4 #4 lead shot and observed for an
additional 4 weeks. ALAD activity continued to be inhibited by more than 90%.

In the study by Sanderson et a1. (1992) where mallards were dosed with either 2,
4 or 8 #2 lead shot or 4 #2 lead shot plus 4 #2 bismuth shot, hematocrit was significantly
depressed in those birds dying (by 36.4% from dosing to the last time they were bled).
In the 2 lead-dosed birds which survived the trial, hematocrits had returned to pre-test
values.

Bakalli gt a]. (1995) reported that blood ALAD activity was quickly and
significantly depressed in broiler chicks administered 50 ppm lead acetate via the feed for
42 days (38 % inhibition within 24 hours, 69% inhibition after 7 days). However, 24 hours
after the birds were placed on clean feed, ALAD activity significantly increased by 32%

and by 7 days on clean feed, ALAD activity was near normal (90% of control).

8 1
In the present study, birds receiving the tungsten-iron or ttmgsten-polymer shot did
not have hematocrits, hemoglobin concentrations or ALAD activities which were
significantly different than values for the no-shot and steel-dosed birds at 15 and 30 days
post-dosing. These results agree with those reported by Ringelman et a1. (1993) who
dosed mallards with 12-17 pellets (equivalent in mass to 5 # 4 lead shot) of shot composed
of tungsten, bismuth, and tin in that hematocrit and hemoglobin concentrations were

unaffected over the 32- day trial. ALAD activity was not assessed in the latter trial.

Plasma Chemistries

The plasma values reported in the present study are reasonably close to values
reported for mallards in other studies (Fairbrother et al., 1990; Ringelman et 31., 1993)
considering the influence of sex, age, reproductive status, and environmental conditions.
The administration of lead shot caused a number of changes'in 15-day plasma chemistry
values in both males and females (Tables 6 and 7).

In the lead-dosed males, glucose concentration was approximately 17 % higher
when compared to the other 3 groups. While March 91 al. (1976) have reported a slight
hyperglycemia in lead-poisoned geese, the increase reported here is not considered to be
biologically relevant. The albumin/globulin ratio in lead-dosed birds and tungsten-polymer
birds was elevated by approximately 15 % when compared to the no-shot and steel-shot
dosed males. While statistically significant, this increase is probably not biologically
relevant. Plasma uric acid levels for all 5 groups remained within the range of normal
values (Campbell and Coles, 1986). In terms of uric acid, it is significant that there were

no changes noted in the tungsten-iron or tungsten-polymer-dosed birds in that high levels

82

of tungsten will inhibit xanthine oxidase (e. g. Higgens et al., 1956; Teekell and Watts,
1959; Leach et 31., 1962; Nell at 3.1., 1980). Xanthine oxidase normally oxidizes xanthine
to uric acid. When xanthine oxidase was inhibited in chicks fed diets high in tungsten,
increased plasma concentrations of uric acid and xanthine plus hypoxanthine were apparent
(Nell :1 a1. , 1980). The plasma activities of alanine aminotransferase, aspartate
aminotransferase, and lactate dehydrogenase were significantly elevated in the lead-dosed
males whencomparedtotheother4groups. Whileanincreaseintheplasmavalue ofone
of these enzymes would not necessarily be a useful diagnostic tool, the fact that all three
are elevated in the lead-dosed birds would strongly suggest significant liver damage
(Campbell and Coles, 1986). There was also a dramatic increase in creatine
phosphokinase activity (20-fold) in the lead-dosed males compared to the other 4 groups.
Increases in the serum activity of this enzyme have been associated with lead toxicity
(Campbell and Coles, 1986).

Changes in plasma chemistry values for lead-dosed females at day 15 included
decreases in sodium and chloride and increases in creatinine and the activities of alanine
aminotransferase and lactate dehydrogenase compared to one or a combination of the other
4 groups. The decrease in plasma sodium and chloride concentrations could be indicative
of renal damage (Coles, 1986), but the drop was less than 8% when compared to the other
4 groups. The increase in creatinine could also reflect kidney damage, but Fairbrother :1
a1. (1990) discount the use of this parameter as a diagnostic tool in birds. The increased
activity of alanine aminotransferase (4.0 fold) and lactate dehydrogenase (2.2 fold) are

suggestive of lead-induced hepatic damage (Campbell and Coles, 1986).

83

There were no significant differences in plasma parameters between the no-shot,
steel, tungsten-iron and tungsten-polymer birds at 15 days post-dosing with the exception
of an increased albumin/ globulin ratio in tungsten-polymer males. These results agree with
Ringelman et al. (1993) who reported that the administration of shot composed of tungsten-
bismuth-tin had no effect on plasma chemistry variables in mallards over the 32-day test
period.

At 30 days post.dosing, there were few changes in plasma parameters and those
changes were only associated with the females (Tables 8 and 9). Lead-dosed females at
day 30 still had a low chloride concentration and a high concentration of carbon dioxide
compared to tungsten-polymer birds. Alanine aminotransferase acitivities were quite
variable, but were significantly higher in lead-dosed females compared to the steel and
tungsten-iron birds. These differences were not considered to be necessarily indicative
of lead toxicity. In general, those lead-dosed birds which survived the 30-day trial had
relatively normal plasma values and whole blood parameters when compared to the other
4 groups.

Gross Pathology

Several birds (5 of 16) in the lead-dosed group exhibited discoloration and erosions
in the lining of the gizzard (Tables 10 and 11). This effect has been previously described
in both naturally occurring and experimentally-induced cases of lead toxicosis (Slauson and
Cooper, 1990; Alden and Frith, 1991; Popp and Cattley, 1991). No birds in the other 4
groups exhibited gross lesions within their gizzards. Fatty liver was noted grossly in birds

in both the no-shot and lead-dosed groups and was therefore not considered a significant

84

lesion. Fatty liver is considered a non-specific change, as it may result from hepatocellular
damage (ie. toxicosis), mobilization of internal fat stores (ie. inadequate energy intake),
or a variety of other metabolic conditions (Slauson and Cooper, 1990). The lack of gross
changes in the tungsten-iron and tungsten-polymer birds agrees with results reported by
Ringelman :1 a1. (1993) for mallards dosed with tungsten-bismuth—tin shot.

Organ Weights

Organ weights of birds surviving the 30—day trial (Table 12 and 13) were expressed
as a percent of body weights (Table 14 and 15) to correct for any differences that might
be due to the size and growth of the bird. When expressed on a relative basis, lead—dosed
males had higher relative kidney weights compared to no-shot and tungsten-iron males and
lead-dosed females had increased relative heart ( vs. no-shot and tungsten-polymer) and
kidney weights (vs tungsten-iron and tungsten-polymer birds) and increased relative liver
weights (vs steel, umgsten-iron and tungsten-polymer birds). Sanderson :1 d- (1992)
reported no differences in absolute liver weights in lead-dosed mallards when compared
to control birds except for ducks dosed with 2 #2 lead pellets which had significantly
lighter livers. These authors commented, however, that liver weights of lead-poisoned
waterfowl are difficult to evaluate because the organ can be enlarged or atrophied.

Histopathology of Kidney and Liver

Microscopic renal lesions were found only in birds from the lead-treated group, and
only in those birds which died spontaneously prior to the conclusion of the trial. These
renal lesions were characterized by acute tubular necrosis (nephrosis), and were

accompanied in most cases by variable numbers of eosinophilic intranuclear inclusions

85

within tubular epithelial cells. These changes have been previously reported to be
associated with lead toxicoses in many different animal species (Alden and Frith, 1991).
Since renal lesions were not found in any of the 8 lead-treated birds at the termination of
the trial, it appears that toxic nephrosis is an acute or subacute effect of lead toxicosis that
occurred while the birds were actively absorbing lead from their gizzard. Whether the
tubular necrosis was due to direct toxic effects of lead on the tubular epithelium, or was
mediated through hemoglobin released during periods of intravascular hemolysis, or a
combination of the two is unknown, as both are recognized to produce this lesion (Alden
and Frith, 1991). The red blood cell parameters did indicate anemia at day 15 of the study
which may have been due to lead-induced intravascular hemolysis. The absence of renal
lesions in either the steel, tungsten-iron or tungsten-polymer dosed birds suggests either
that these metals are non-toxic to the renal tubular epithelium, or that these substances
were not absorbed in sufficient quantities to produce renal tubular toxicity.

The hepatic histologic lesions were divided into two distinct groups; non-specific
fatty accumulation and more significant biliary stasis. Intrahepatocellular fatty vacuolation
was present in over 50% of the birds in each of the 5 experimental groups. As previously
discussed, fatty accumulation can be due to a variety of causes and was judged an
incidental finding in this study. The accumulation of bile within hepatocytes or within
canaliculi is also somewhat non-specific, as it may occur due to obstruction of bile ducts,
or primary hepatocellular disfunction (Popp and Cattley, 1991). In this study, there was
no evidence of cholelithiasis or other obstructive biliary disease, and so biliary stasis was

considered evidence of hepatocellular dysfunction. The degree of biliary stasis was

86

graded, and statistical analysis indicated that only the lead-dosed group had significant
elevated amounts of biliary stasis, although several individual birds in the tungsten-iron
and tungsten-polymer group also exhibited mild biliary stasis. None of the birds in the no-
shot and steel-dosed groups had biliary stasis. There was good correlation between the
morphologic appearance of biliary stasis, and the elevated plasma hepatic enzyme activities
(indicating hepatocellular damage) at day 15 in the lead-dosed group. The hepatic biliary
stasis was considered to be a morphologic indicator of hepatocellular damage in this study,
and that significant evidence of this damage was present only in the lead-dosed group.
However, since biliary stasis was observed in some of the tungsten-iron and tungsten-
polymer birds, but not the no-shot and steel—dosed birds, it is apparent that the
experimental shot is inducing a pathological condition, however slight, that is not found
in the control birds.

Tissue Metal Analysis

Birds dosed with lead shot tended to have elevated iron concentrations in the femur
liver, andkidney (Tables 20-22). Femur iron concentrations were significantly higher in
the lead-dosed males compared to the no-shot, tungsten-iron and tungsten-polymer birds,
liver iron concentrations were higher in lead-dosed males compared to no-shot, steel, and
tungsten-polymer birds and liver iron concentrations were higher in lead-dosed females
compared to no-shot females. Tungsten-iron males had higher liver iron concentrations
than did the no-shot birds. In the kidney, steel and tungsten-polymer-dosed fenmles had
significantly higher concentrations of iron when compared to the no-shot and lead-dosed

birds with steel also significantly higher than tungsten-polymer females. The high levels

87

of iron in the liver of lead—dosed birds agrees with results reported by Sanderson et a1.
(1992) who indicated that the concentrations of iron in the liver and muscle of lead-dosed
mallards was higher when compared to birds not dosed with lead. They attributed the
increase to a lead-induced interference of heme synthesis which in turn caused an
accumulation of iron in the liver and muscle.

All treatment groups, including the no-shot birds, had detectable concentrations of
lead in the femur, liver, and kidney (Tables 20-22). However, concentrations of lead in
the birds dosed with lead shot were increased by a factor of 10 when compared to the other
4 groups. Sanderson et al. (1992) reported concentrations of lead in the femurs of all
mallards on trial regardless of treatment with concentrations in the birds receiving lead shot
the highest. Lead was not detected in the liver and muscle of control birds and birds dosed
with steel shot in the latter study.

Tungsten was detected in the femur and kidney of tungsten-polymer-dosed birds at
concentrations slightly above detection limits and in the femur, liver, and kidney of
tungsten-iron birds (Tables 18-20). The bone, liver, and kidney are principle sites of
tungsten disposition in a number of different species (Kinard and Aull, 1945; Wase, 1956;
Kaye, 1968; Bell and Smd, 1970; Aamodt, 1975) with the primary site apparently being
species-dependent. In the present study, the highest concentrations of tungsten were
detected in the liver. In contrast, Ringelman at al. (1993) who dosed mallards with
trmgsten bismuth-tin shot did not detect tungsten in the liver and kidney, the only tissues
examined. Tungsten was detected in the kidneys of both tungsten-iron and tungsten-

polymer males and females tissues. Tungsten residues in the kidney of male and female

88

tungsten-ironodosed birds were both significantly higher than residues found in the
tungsten-polymer-dosed birds.

Molybdenum concentrations were assessed in the present study because it has been
reported that administration of tungsten can cause a decrease in tissue molybdenum
concentrations (Higgens :1 a1., 1956; Nell at d. , 1980). Of the 3 tissues examined,
molybdenum was detected in the liver and kidney. In the liver, the no-shot birds had the
lowest concentrations of molybdenum (significant when males were compared to their lead
counterparts) while the lead-dosed birds had the highest concentrations (significant in males
when compared to no-shot, steel, and tungsten-polymer birds). In the kidney, male no-
shot birds had the highest concentration of molybdenum although there were no significant
differences between the 5 groups. Steel—dosed females had the highest concentration of
renal molybdenum, with no-shot and tungsten-polymer birds containing intermediate
concentrations, and lead-dosed and tungsten-iron birds having the lowest concentrations
of renal molybdenum. Based on these results it would seem that assessing molybdenum
concentration as an indication of tungsten exposure is not warranted.

Shot Recovered and Percent Shot Erosion

Fluoroscopy of the birds on day 7 of the trial indicated that each dosed bird had 8
pellets present in the gizzard with the exception of 1 steel-dosed bird which had 6 pellets.
During the course of the trial, the excreta was checked on a daily basis and there was no
evidence of shot passage. It is possible, however, that since 100% of the pellets were not
recovered at necropsy, some may have been eliminated from the bird and not detected in

the excreta.

89

Males dosed with steel shot retained an average of 89 % of the pellets while females
retained 98%. Recovery of lead shot was 85 % in the males and 75 % in the females, the
latter being the lowest percent recovery. One hundred percent of the trmgsten—iron pellets
and 94 % of the tungsten-polymer pellets administered were recovered in the males at
necropsy while the recovery rate in tungsten-iron and tungsten-polymer females was 93 %
and 98%, respectively (Table 23). These recovery rates are similar to those reported by
Sanderson :1 a1. (1992) for lead and iron shot and higher than the retention rate of the
bismuth shot. Ringelman :1 al. (1993) indicated that by 11 days post-dosing, only 44%
of the tungsten-bismuth-tin shot had been retained.

If shot erosion is based on the average weight of the pellets actually recovered, then
the erosion of the steel shot was significantly less than erosion of tungsten-iron and
tungsten-polymer in the males and significantly less than both lead and tungsten-polymer
in the females (Table 23). The erosion rate of steel, lead, and tungsten-iron shot in
females is greater than the erosion rates of the same shot in males. If shot erosion is based
on the total weight of the pellets recovered (this would assume that a non-recovered pellet
had eroded completely), the erosion rate of tungsten-polymer shot in males is significantly
greater than that of steel, lead, and tungsten-iron shot. However, in the females erosion
rates of tungsten-polymer shot is only significantly greater when compared to steel shot
(Table 24).

Steel pellets recovered at necropsy retained their original round shape. It was
sometimes difficult to find the lead, tungsten-iron and tungsten-polymer shot in the gizzard

contents because of their smaller size and, in the case of the lead and tungsten-polymer

9 0

shot, their flattened disk-like appearance.
Conclusions

Male and female mallards administered 8 BB size tungsten-iron and tungsten-
polymer shot and maintained over a 30—day period did not experience any adverse effects
based on the parameters examined. All birds survived the 30-day trial with a slight
increase in body weight. There were no significant differences in hematocrit, hemoglobin
concentration, and ALAD activity in the 2 candidate groups when compared to no-shot and
steel (control) groups. Similarly, there were no changes in the 25 plasma chemistry
parameters except for the elevated albumin/ globulin ratio in tungsten-polymer males on day
15. The birds appeared normal at the time of necropsy on day 30 ofthe trial and there
were no changes in organ weights. Five of 16 tungsten-iron birds and only 3 of 16
tungsten-polymer birds had mild hepatocellular biliary stasis which was not considered to
be deleterious. However, this condition was not observed in the no-shot and steel-dosed
birds. No other histopathological lesions were noted. Tungsten was detected in the
femur, liver, and kidney of the tungsten-iron birds and at concentrations only slightly
above detection limits in the femur of tungsten-polymer males and in the kidneys of
tungsten-polymer-dosed males and females. Even though the erosion rate of tungsten-
polymer shot was greater than tungsten-iron shot, the erosion rate was only significant
when compared in male birds. Therefore, the results of this study indicate that game farm
mallards dosed with either 8 BBs composed of trmgsten-iron or tungsten-polymer were not
adversely affected during the course of the 30-day trial. However, if the two experimental

shot were to be compared tungsten-polymer would be the better shot due to its apparent

 

91

low body tissue deposition, low occurrence of biliary stasis, and greater erosion pattern.
It seems the tungsten-polymer shot is more environmental friendly than the tungsten-iron

shot.

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