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IIIIIIIIIIIIIIIIIIIIIIIIII II IIIIIIII IIIIIIIZIIII

00177

This is to certify that the
thesis entitled

Chronic Dosing Study to Assess the Health and
Reproductive Effects of Tungsten-iron and
Tungsten-polymer Shot on Game-farm Mallards

presented by

Rachel R. Mitchell

has been accepted towards fulfillment
of the requirements for

M.S. Animal Science

degree in

 

 

V
Major professor

 

Date 5 May 1999

 

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

 

 

LIBRARY
Michigan State
University

 

 

 

 

PLACE IN RETURN Box to remove this checkout from your record.
TO AVOID FINE return on or before date due.
MAY BE RECALLED with earlier due date if requested.

 

I DATE DUE

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1M clam-mu

CHRONIC DOSING STUDY TO ASSESS THE HEALTH AND REPRODUCTIVE
EFFECTS OF TUNGSTEN-IRON AND TUNGSTEN-POLYMER SHOT ON GAME-
FARM MALLARDS
By

Rachel Rebecca Mitchell

A THESIS
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
MASTER OF SCIENCE
Department of Animal Science

1999

ABSTRACT

CHRONIC DOSING STUDY TO ASSESS THE HEALTH AND REPRODUCTIVE
EFFECTS OF TUNGSTEN-IRON AND TUNGSTEN-POLYMER SHOT ON GAME-
FARM MALLARDS
By

Rachel Rebecca Mitchell

Sixteen male and 16 female adult mallards were orally dosed with 8 #4 steel shot, 8 #4
tungsten-iron shot, or 8 #4 tungsten-polymer shot on days 0, 30, 60, 90, and 120 of a 150-
day trial. An additional 6 male and 6 female mallards received 8 #4 lead shot on day 0 of
the study. During the first 60 days of the trial, mallards were fed a nutritionally deficient
diet (shelled corn) and maintained in a cold environment. Ducks were then switched to
commercial layer ration for the subsequent 90 days during which reproductive
performance was examined. All lead—dosed ducks died by day 25 of the study, whereas no
ducks died in the other dosage groups. Lead-dosed mallards had significantly decreased
hematocrit, hemoglobin concentration and whole-blood delta aminolevulinic dehydratase
activity on day 7. Exposure to lead shot caused significant changes in a number of
plasma chemistry parameters compared to exposure to steel, tungsten-iron, or tungsten-
polymer shot at day 7. Mallards dosed with tungsten-iron or tungsten-polymer shot had
occasional significant differences in hematocrit and plasma chemistry values when
compared to steel-dosed mallards over the ISO-day period, but these values were within
the normal range reported for mallards and not considered to be indicative of deleterious

effects. Relative kidney, heart, brain and gizzard weights of lead-dosed ducks were

significantly greater in comparison to the relative weights of those organs of ducks in the
other 3 groups. Histological examination of kidneys and liver indicated renal nephrosis
and hepatocellular biliary stasis in the lead—dosed ducks. Significant liver hemosiderosis
was present in all steel- and tungsten-iron-dosed males examined, in 5 of 8 steel- and 3 of
8 tungsten-iron-dosed females examined, and in l tungsten-polymer—dosed male
examined. Concentrations of lead in the femur, gonads, kidneys, and liver were higher in
lead-dosed ducks than in ducks of the other 3 groups. Small amounts of tungsten were
detected in gonad and kidney samples from males and females, in femur samples from
males, and in liver samples from females dosed with tungsten-polymer shot. Higher
concentrations of tungsten were detected in femur, gonad, kidney, and liver samples from
tungsten-iron-dosed ducks. The rate of shot erosion was highest for tungsten-polymer
shot (99%), followed by tungsten-iron (72%), steel (55%), and lead (3 7%). There were no
significant differences in percent egg production, and percent fertility and hatchability of
eggs from tungsten-iron- and tungsten-polymer-dosed ducks when compared to steel-
dosed ducks. Egg weight and shell thickness of eggs from tungsten-iron-dosed ducks
were greater when compared to steel-dosed ducks. Concentrations of tungsten were
highest in the shell of eggs from tungsten-iron-dosed ducks than from the eggs of
tungsten-polymer-dosed ducks. There were no biological differences in percent
survivability and body weight of ducklings from tungsten-iron and tungsten-polymer
ducklings when compared to ducklings from steel-dosed ducks. The hematocrit of
ducklings from tungsten-iron-dosed ducks was slightly but significantly lower when
compared to ducklings from steel-dosed ducks. Relative kidney weight of ducklings from

tungsten-polymer-dosed ducks was significantly greater than relative kidney weight of

ducklings from steel-dosed ducks. Histological examination of duckling kidneys and
liver indicated no abnormalities. Tungsten was detected in 25%, 9%, and 13% of the
femur, kidney, and liver samples, respectively, from ducklings of the tungsten-iron and
tungsten-polymer groups. Results of this study indicated that tungsten-iron or tungsten-
polymer shot repeatedly administered to adult mallards did not adversely affect them or

the offspring they produced during the ISO-day trial.

To my husband, Andrew Thomas Mitchell
Where our world is just “ducky”!

ACKNOWLEDGMENTS

There are many people whom I wish to thank for their help, support, friendship, or
guidance during my master’s program. First, I would like to give a special thank you to
my major professor, Dr. Steven Bursian, who provided me with the guidance,
encouragement and support to obtain my goals during the 3 years I worked with him.
Most importantly I am very fortunate to have gained a fiiend and a confidant. In addition,
I would like to thank my committee members: Dr. Aulerich (my unofficial co-advisor)
for his assistance, great advice, good sense of humor and friendship; Dr. Balander for his
assistance, support, good sense of humor and fiiendship; Dr. Fitzgerald for his assistance,
patience during the necropsies and histology slides, good sense of humor and friendship;
and Dr. Giesy for his great advice, support, good sense of humor and friendship during
my master’s program. I hope to continue the relationship I have established with all of
my committee members!

Angelo Napolitano deserves a huge thank you for all his assistance during dosing
and blood collection during this project, for providing additional help from the staff at the
poultry farm, for his assistance when I was a teaching assistant and conducted
laboratories at the farm, and most importantly for his friendship. I would also like to
thank Debbie Powell for all of her assistance and support during my master’s program,
and most importantly for her friendship and great advice in dealing with life. In addition,
I would like to thank Mara Preisler and Doug Mashek for their willingness to help and for
being wonderful friends as we muddled through our master’s program together with a lot

of good times and good humor!!

vi

Finally, I would like to thank my family for trying to understand what I was doing
these past 3 years and providing me the support and courage to finally finish my college
career. I would like to personally thank my mother, Susan, and my father, Ron, for their
continual support and understanding, especially during my college career. Last but
certainly not least, I would like to thank my husband, Andrew, for his all of his love,
support, great advice and willingness to lend a shoulder to cry on and an ear to listen to
during the past 3 years. My love for him continues to grow with everyday we encounter

together, thank you!

vii

MW

List of Tables ......................................................................................... xi
Introduction ........................................................................................... 1
Objectives ............................................................................................. 2
Literature Review .................................................................................... 3
Materials and Methods ............................................................................. 14
Results ................................................................................................ 27
Adult Mortality ............................................................................. 27
Adult Clinical Signs ....................................................................... 27
Adult Body Weights ....................................................................... 29
Adult HCT, Hb Concentration,
and ALAD activity ......................................................................... 29
Adult Plasma Chemistries ................................................................. 33
Adult Gross Pathology .................................................................... 52
Adult Organ Weights ...................................................................... 62
Histopathology of Adult
Liver, Kidneys, and Gonads ............................................................... 67
Metal Residues in Tissues of Adults ..................................................... 67
Shot Recovery and Percent Shot Erosion ............................................... 84

Date First Egg was Laid and Number

of Days Required to Lay 21 Eggs ........................................................ 87
Percent Egg Production, Fertility, and Hatchability ................................... 87
Egg Weight and Shell Thickness ......................................................... 87

viii

Metal Residues in Egg Shell and Contents ............................................. 91

Survivability, Body Weight, and

Hematocrit of Ducklings .................................................................. 91
Duckling Organ Weights .................................................................. 91
Histopathology of Duckling Liver and Kidneys ....................................... 97
Metal Residues in Tissues of Ducklings ................................................ 97
Discussion .......................................................................................... 103
Adult Mortality ........................................................................... 103
Adult Clinical Signs ...................................................................... 104
Adult Body Weights ...................................................................... 105
Adult HCT, Hb Concentration,
and ALAD activity ........................................................................ 106
Adult Plasma Chemistries ............................................................... 107
Adult Gross Pathology ................................................................... 110
Adult Organ Weights ..................................................................... l 11
Histopathology of Adult
Gonads, Liver and Kidneys .............................................................. 112
Metal Residues in Tissues Of Adults ................................................... 113
Shot Recovery and Percent Shot Erosion ............................................. 115

Date First Egg was Laid and Number

of Days Required to Lay 21 Eggs ...................................................... 116
Percent Egg Production. Fertility. and Hatchability ................................. 117
Egg Weight and Shell Thickness ....................................................... 117
Metal Residues in Egg Shell and Contents ............................................ 118

Survivability, Body Weight. and

Hematocrit of Ducklings ................................................................. l 18 '

Duckling Organ Weights ................................................................ 1 l8
Histopathology of Duckling Liver and Kidneys ...................................... 119
Metal Residues in Tissues of Ducklings ............................................... 119
Conclusion .......................................................................................... 119
References .......................................................................................... 121

III—bk:

10

ll

12

13

WEE

Page
The effect of treatment shot on percent mortality, time to death
(days), and percent weight lost at death of mallards on a 150-day
dosing test ........................................................................ 28

The effect of treatment shot on body weight (gm) loss of mallards from
day 0 to day 30 Of a ISO-day dosing test ..................................... 30

The effect of treatment shot on body weight (gm) of mallards from
day 30 through day 60 of a ISO-day dosing test ............................. 31

The effect of treatment shot on body weight (gm) of mallards from
day 90 through day 150 of a ISO-day dosing test ........................... 31

The effect of treatment shot on whole-blood parameters of mallards
on day 7 of a ISO-day dosing test ............................................. 32

The effect of treatment shot on hematocrit of mallards from day 30
through day 60 of a ISO-day dosing test .................................... 34

The effect of treatment shot on hematocrit of male and female mallards
from day 90 through day 150 Of a ISO-day dosing test ..................... 34

The effect of treatment shot on plasma chemistry parameters of mallards
on day 7 of a ISO-day dosing test ............................................. 35

The effect of treatment shot on plasma chemistry parameters of male and
female mallards on day 7 of a 150-day dosing test ......................... 38

The effect of treatment shot on plasma chemistry parameters of mallards
from day 30 through day 60 of a ISO-day dosing test ...................... 41

The effect of treatment shot on plasma chemistry parameters of male and
female mallards from day 30 through day 60 of a ISO-day
dosing test ........................................................................ 45

The effect of treatment shot on plasma chemistry parameters of mallards
on day 30 and day 60 of a 150-day dosing test .............................. 47

The effect of treatment shot on plasma chemistry parameters of mallards
from day 90 through day 150 of a ISO-day dosing test .................... 48

xi

14

15

l6

17

18

19

20

21

22

23

24

25

26

27

The effect of treatment shot on plasma chemistry parameters of mallards
on days 90, 120 and 150 of a 150-day dosing test .......................... 53

The gross necropsy observations of the effect Of treatment shot
on male mallards on a ISO-day dosing test ................................. 54

The gross necropsy Observations of the effect of treatment shot
on female mallards on a ISO-day dosing test ............................... 58

The effect of treatment shot on organ weights (gm) of mallards on a 150-
day dosing test .................................................................. 63

The effect of treatment shot on organ weights (gm) of male and female
mallards on a ISO-day dosing test ............................................ 64

The effect of treatment Shot on organ weights expressed as percent body
weight of mallards on a ISO-day dosing test ................................ 65

The effect of treatment shot on organ weights expressed as percent body
weight of male and female mallards on a 150-day dosing test... ... ......66

The histopathological effects of treatment shot on the liver and
kidneys of male mallards on a ISO-day dosing test ........................ 68

The histopathological effects of treatment shot on the liver and kidneys
of female mallards on a 150-day dosing test ................................ 71

The severity of testis, liver and kidney lesions induced by treatment shot
in male mallards on a ISO-day dosing test .................................. 74

The severity of ovary, liver and kidney lesions induced by treatment shot
in female mallards on a ISO-day dosing test ................................ 76

The effect of treatment shot on concentration (mg/kg dry weight) of lead
in the femur Of mallards on a ISO-day dosing test ........................ 78

The effect of treatment shot on concentrations (mg/kg dry weight) of
iron and tungsten in the femur of male and female mallards on a ISO-day
dosing test ....................................................................... 79

The effect of treatment shot on concentrations (mg/kg dry weight) of

iron, lead, and tungsten in the gonads of male and female mallards on a
ISO-day dosing test ............................................................ 81

xii

28

29

30

31

32

33

34

35

36

37

38

39

40

41

The effect of treatment shot on concentrations (mg/kg dry weight) of
lead and tungsten in the kidneys of mallards on a ISO-day dosing
test ................................................................................. 82

The effect of treatment shot on concentrations (mg/kg dry weight) of
iron in the kidneys of male and female mallards on a ISO-day dosing
test ................................................................................. 83

The effect of treatment shot on concentration (mg/kg dry weight) of iron,
lead, and trmgsten in the liver of mallards on a ISO-day dosing
test ................................................................................. 85

Number of pellets recovered and percent erosion of shot in male and
female mallards on a ISO-day dosing test .................................... 86

The day the first egg was laid and the number of days required for
mallards to lay 21 eggs ......................................................... 88

The effect of treatment shot on egg production of mallards on a 150-
day dosing test and on fertility and hatchability of eggs .................... 89

The effect of treatment shot on weight (gm) and shell thickness (mm) of
eggs from mallards on a ISO-day dosing test ................................. 90

The effect of treatment shot on concentrations (mg/kg dry weight) of
iron, lead, and tungsten in the contents and shell of eggs from mallards

on a ISO-day dosing test ........................................................ 92
The effect of treatment shot on duckling survivability, body weight (gm)
from day 0 through day 14, and hematocrit on day 14 ...................... 93
The effect of treatment shot on organ weights (gm) of ducklings. . . . . . ...94

The effect Of treatment shot on liver, spleen and kidneys expressed as
percent body weight Of ducklings ............................................. 95

The effect of treatment shot on bursa, heart and brain expressed as
percent body weight of ducklings ............................................. 96

The histopathological effects of treatment shot on the liver and kidneys
of male ducklings ............................................................... 98

The histopathological effects of treatment shot on the liver and kidneys
of female ducklings .......................................................... 100

xiii

42

The effect of treatment shot on concentrations (mg/kg dry weight) of
iron, lead, and tungsten in tissues of ducklings ........................ 102

xiv

1' 5"..-

 

M99

In 1991, the United States banned the use of lead shot for waterfowl hunting
because of its toxic effects on waterfowl and other wildlife species upon ingestion. Steel
and bismuth shot are used as nontoxic alternatives to lead, but there has been a continual
effort to develop shot compositions that emulate the ballistic characteristics of lead.

In order for a candidate shot to receive permanent approval for use by the US.
Fish and Wildlife Service (U SFWS), it must undergo a variety of tests as documented in
USFWS 50 CFR Part 20.134, Migratory Bird Hunting: Nontoxic Shot Approval
Procedure (Federal Register, 1986) to establish that it is nontoxic to waterfowl and other
impacted species. The approval procedure is a 3-tiered approach. In Tier 1, the applicant
must provide statements of use, chemical characterization, volume of use of the material
requested to be approved, and samples of the candidate shot. In addition, the
toxicological data for the shot coating and/or shot pertaining to mammals, birds, fish,
amphibians, and reptiles should be summarized. The applicant must also provide
information on the environmental fate and transport of the shot and shot coatings. In Tier
2, providing that the results from the Tier 1 information is inconclusive, the applicant will
conduct a short-term (30—day) acute toxicity test using game-farm mallards provided a
diet of commercially available duck food. In Tier 3, a chronic toxicity test is to be
conducted. This test utilizes game-fann mallards fed a nutritionally-deficient diet of corn
and maintained in a cold environment for 60 days. Mallards are then switched to a
breeder diet and reproductive parameters are assessed for the subsequent 90 days. Shot
composed of tungsten-iron (55% tungsten and 45% iron) and tungsten-polymer (95.5%

ttmgsten and 4.5% of the polymer nylon 6) were given conditional approval for waterfowl

hunting by the USFWS based partly on the results of a 30-day acute toxicity trial utilizing
mallards (Tier 2 ) (Kelly et al., 1998).

The present study is a ISO-day dosing test designed to assess the effects of long-
term periodic exposure of waterfowl to 2 candidate shot types composed of 55% tungsten
and 45% iron, and 95.5% tungsten and 4.5% of the polymer nylon 6. The study was
conducted in 2 phases. The first phase consisted of maintaining mallards, dosed with
candidate shot every 30 days, on a nutritionally-deficient diet (shelled corn) in a
minimally heated environment with a constant photoperiod of 8 hours light: 16 hours dark
per day for 60 days. In the second phase of the study, the mallards were switched to a
commercial layer ration while dosing with candidate shot continued every 30 days, the
photoperiod was increased in increments to 18 hours light:6 hours dark, and reproductive
performance was assessed during the subsequent 90 days. The protocol for this study was
reviewed by the USFWS in 1997 and complies with the general guidelines outlined in the
amended test protocol for nontoxic shot approval procedures for shot and shot coatings

proposed by USFWS in 1996 (Tier 3).

Objectives
The overall objective of the ISO-day dosing trial was to determine if exposure to 2
candidate shot types, composed of 55% tungsten and 45% iron, or 95.5% tungsten and
4.5% of the polymer nylon 6, caused any deleterious effects in game-farm mallards.
Toxicity of candidate shot was assessed by:
1) Determination of hemoglobin (Hb) concentrations and whole-blood delta
aminolevulinic acid dehydratase (ALAD) activities on day 7 of the trial.

2) Determination of hematocrit (HCT) on days 7, 30, 60, 90, 120, and 150.

 

 

3) Determination of plasma chemistries on days 7, 30, 60, 90, 120 and 150.

4) Determination of egg production, fertility, hatchability, and duckling
survivability.

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

6) Determination of metal residue concentrations in the liver, kidneys, femur, and
gonads of adults, in the liver, kidneys, and femur of ducklings, and in the contents
and shell of eggs.

7) Determination of gross and histological changes in selected tissues.

8) Determination of mortality.
Literature Review

Lead is the most ubiquitous toxic metal and is detectable in practically all phases
of the inert environment and in all biological systems. This heavy, pliable metal has a
bright, bluish color and rarely occurs in the native form, but is usually found in nature as
its sulfide, the mineral galena. Because lead is toxic to most living things at high
concentrations and because there is no demonstrated biological need for it, the major
issue regarding lead is determining the dose at which it becomes toxic (Goyer, 1996).

Lead has been known to man for about 7000 years, and lead poisoning has
occurred for at least 2500 years (Eisler, 1988). Ancient Egyptians used lead in the
production Of paints, pottery glazing, weights, coins, net sinkers, piping, and cooking
utensils (Eisler, 1988). Later, Romans used lead in construction of water pipes, in
cosmetics, and even as a sweetner in the preparation of wines. The decline of the Roman
Empire may have been accelerated by endemic lead poisoning. This theory was later

supported by the high concentrations of lead found in the bones of Roman aristocrats

(Eisler, 1988). During the Middle Ages, there was considerable use of lead in paints,
weights, and in the preparation of stained glass windows for cathedrals. Later, following
the introduction of gunpowder, the need for a projectile made of malleable material
resulted in the production of lead shot and lead cannon balls. Today, domestic lead
consumption is 1.3 million tons annually, of which half is used in the production of
storage batteries and until recently, of gasoline antiknock compounds, specifically
tetraethylead and tetramethylead (Eisler, 198 8).

The traditional use of lead shot for waterfowl hunting has been the preferred metal
for centuries because of its widespread availability, low price, ease of manufacturing, and
chemical stability (Thomas, 1997). However, the primary source of lead poisoning in
wild waterfowl has been the ingestion of shotgun pellets. The amount of ingested lead
that will produce toxicosis and fatalities of waterfowl varies according to nutritional and
physiological conditions of birds. A single ingestion of 0.2-2.0 grams of lead shot may
prove acutely fatal to most waterfowl (Pain and Rattner, 1988; Rattner et al., 1989). Yet,
each year about 3000 tons of lead shot are deposited in the wetlands of North America by
waterfowl hunters alone (Thomas, 1997). Given that lead shot has been accumulating for
at least 200-300 years, and that it erodes slowly (Jorgensen and Willems, 1987), there is a
great risk that waterfowl will develop lead poisoning, both at present and in the future.
Other less common sources of lead poisoning in waterfowl include lead fishing sinkers,
mine wastes, paint pigments, bullets, and other lead objects that are swallowed.

Since the first report of Grinnell (1894), the typical signs and lesions of lead
poisoning in waterfowl have been extensively documented in every North American

waterfowl flyway (Bellrose, 1959; Wobeser, 1981; Sanderson and Bellrose, 1986; Friend,

 

 

1987; Eisler, 1988). Waterfowl that are well advanced in lead intoxication usually
exhibit the following signs: varying degrees of emaciation (loss of up to 40% of the
original body weight, and a prominent keel bone), reduced activity with reluctance to fly,
lowered food intake, palsy (wing droop), bile staining of vent area, tendency to seek
isolation and cover, and loss of ability to stand, walk, or fly. The internal lesions
associated with lead poisoning in waterfowl include: lack of fat, atrophy of striated
muscle, excess fluid in pericardial sac, distended gallbladder, atrophied gizzard with
grinding pads hardened and bile stained, and anemia and paleness of the whole body. It
was the extent of the threat lead posed to waterfowl that led the United States government
to ban the use of lead shot for waterfowling in 1991.

Prior to the decision of the ban on lead shot for waterfowl hunting, there were 3
general options that were considered as potential solutions to the problem of lead shot
poisoning waterfowl: (l) manipulation of the habitat to reduce the availability and/or
toxicity of spent shot; (2) coating, plating, or otherwise altering lead shot pellets to reduce
toxicity; and (3) regulations prohibiting the use of lead shot, combined with the use of
alternative, nontoxic shot (Scheuhammer and Norris, 1995). Manipulation Of waterfowl
habitat required actions that were expensive, labor-intensive, of questionable
effectiveness, and inappropriate as general solutions to the lead shot problem. The
attempt to retain the ballistic qualities of lead, but to reduce its toxicity to waterfowl by
coating lead shot with other metals or nonmetallic materials, resulted in mortality of
waterfowl after ingestion of the modified shot types that was equal to or greater than
mortality caused by pure lead shot. The lack of success of the first 2 options led to the

search for affordable, nontoxic, ballistically-acceptable alternatives to lead.

Steel shot was found to be the preferred alternative to lead, considering its lack of
toxicity, ready availability, and relatively low cost (U .S. Department of the Interior,
1986). One of the major concerns surrounding the phase-out of lead shot has been that
the exclusive use of steel shot could lead to a dramatic increase in the proportion of game
birds injured but not killed by hunters (crippling rate). The ultimate effect might be that
increased losses of birds through crippling would surpass the number of birds saved by
the elimination of lead shot (Scheuhammer and Norris, 1995). For this reason, hunters
have been reluctant to accept the steel shot regulations. However, between 1950 and
1984, 16 published shooting tests comparing the effectiveness of lead and steel shot were
conducted in the United States. The results of these tests were equivocal: 3 of the tests
favored lead , 2 favored steel, 2 reported mixed results, and 8 showed no statistically
differences in crippling between the 2 shot types (Morehouse, 1992). It has been argued
that the crippling of waterfowl is a function of the skill of the shooter rather than the type
of ammunition used.

Since lead was banned from the North American marshlands in 1991, ammunition
companies have searched for ways to improve the performance of steel loads and to
emulate the ballistic characteristics of lead shot. Bismuth shot, the first nontoxic
alternative to steel shot, was the first nontoxic alternative to receive permanent approval
by the United States Fish and Wildlife Service in 1997 (Kelly et al., 1998). The second
nontoxic alternatives are tungsten-iron and tungsten-polymer shot, which received
conditional approval for use from the US. Fish and Wildlife Service in 1997 (Kelly et al.,

1998).

Tungsten 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) W04], scheelite (Ca W04), ferberite (FeWO4) and
hubnerite (MnWO4). Major uses of tungsten include incorporation into cutting and

wear-resistant materials, mill products, specialty steels, alloys, chemicals and tools.

Tungsten has a molecular weight of 183.85, specific gravity of 19.35, melting point of
3,4100 C and boiling point of 5,6600C. Tungsten metal is insoluble in aqueous solutions
while forms such as sodium tungstate (Na2W04-2H20) and ammonium paratungstate

[(NH4)6W7024.6H20] are variably soluble in water (Stokinger, 1978).

The tungstate ion (W042') 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

K2W04 (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% of an orally
administered dose of labeled tungsten in rats was eliminated in the urine after 24 hours
while 58% was eliminated via the feces or remained unabsorbed in the gut. Only 2% of

the dose remained in the tissue. Kaye (1968) administered labeled K2WO4 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 (NH4)2WO4 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)2WO4 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 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 K2WO4, 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 that were

administered a tracer dose of K2WO4. 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 K2WO4

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 K2WO4 was eliminated in the

urine and 20% in the feces at 24 hours post-dosing. Dogs administered an intravenous

tracer dose of Na2WO4 eliminated 91% of the tungsten via the urine (Aamodt, 1973).
Similarly, Bell and Sneed (1970) reported that most of a tracer dose of (NH4)2WO4

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 (NH4)2WO4

excreted 44% and 42% of the radioactivity in the urine and feces, respectively, while

(NH4)2WO4 introduced into the abomasum resulted in 65% being eliminated in the urine

and 17% in the feces.
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 K2WO4

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 a1. (1980) reported a

hepatic half-life of 27 hours for Na2WO4 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 LD50 for insoluble tungsten

metal powder injected intraperitoneally in the rat of 5,000 mg/kg body weight, whereas

when the soluble Na2W04 was injected subcutaneously, an LD50 of 140-160 mg
tungsten/kg body weight (223-255mg Na2W04/kg body weight) was determined (Kinard
and Van de Erve, 1940). Pham-Huu-Chanh (1965) reported LD50 values of 112 mg/kg

body weight and 79 mg/kg body weight when sodium tungstate 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 tungstic 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 al. ( 1980) administered broiler
cockerels soluble sodium tungstate via daily injection at 5 mg tungsten from day 1 to day
11, 10 mg from day 12 to day 21, and 20 mg from day 22 to day 35. Four of40 birds died
on trial and all deaths occurred on day 29.

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

10

summarized by Stokinger (1978). 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
provenn'iculus. Hemorrhages were also observed in the brain, heart, and kidney.

When mammals have been administered doses of tungsten compounds that do not
result in mortality, 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 (Higgens et al.,

1956; Teekell and Watts, 1959; Leach et al., 1962; Nell et al., 1980). The toxicity of

11

soluble tungsten compounds is determined by measuring xanthine dehydrogenase activity
in the liver. Xanthine dehyrdogenase is an enzyme involved in purine metabolism and in
the conversion of nitrogenous compounds to uric acid (Nell et al., 1980). In the following
studies, tungsten was shown to reduce xanthine dehydrogenase activity when fed to
breeder hens and chicks. Teekell and Watts (1959) fed chickens sodium tungstate at a
concentration of 250 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. Nell et a1. (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 xanthine dehydrogenase activities. Chicks fed
diets containing 1,000 ppm tungsten for 4 weeks had increased hepatic concentrations of

tungsten and decreased activities of xanthine dehydrogenase. In chicks either injected

12

 

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

In a study that served as the basis for the present test, Kelly et a1. (1998) dosed
mallards with 8 BBS of tungsten-iron or tungsten-polymer shot and monitored them for
30 days. All mallards survived the 30-day trial with a slight increase in body weight. No
statistical differences were observed in HCT, Hb concentrations, and ALAD activities in
the 2 tungsten shot-dosed groups when compared to control and steel-dosed groups.
Similarly, no changes were detected in selected plasma chemistry variables. The mallards
appeared normal at the time of necropsy on day 30 of the trial, and no changes were
detected in weights of organs. Five of 16 tungsten-iron-dosed ducks and 3 of 16
tungsten-polymer-dosed ducks manifested a mild hepatocellular biliary stasis, which was
not considered deleterious. This condition, however, was not observed in the control and
steel-dosed ducks. NO other histopathological lesions were noted. Tungsten residues
were detected in the femur, liver and kidneys of the tungsten-iron ducks. Concentrations
of tungsten only slightly above detection limits were detected in the femur and kidneys of
2 mallards dosed with tungsten-polymer shot. In a similar study, Ringelman et al. (1993)
closed mallards with 12-17 pellets of shot composed of 39% tungsten, 44.5% bismuth,
and 16.5% tin and monitored the ducks for the subsequent 32 days. Based on the lack of

effects on mortality, behavior, feed consumption, body weight gain, and blood parameters

13

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.

Nylon 6 is the other significant component of the ttmgsten-polymer shot,
comprising 4.5% of the total product. Nylon 6 is the commercially important
homopolymer of caprolactarn. Most completely polymerized materials are
physiologically inert, regardless of the toxicity of the monomer from which it’s made
(Peterson, 1977). Thus, few data exist relative to the toxicity of nylon 6 in animals. Most
of the toxicity studies that have been conducted relate to thermal degradation products
that are not relevant to the exposure Of wildlife to shot containing nylon. One animal
study reported in Montgomery (1982) indicated that nylon 6 fed to rats at a level of 25%
of the diet (250,000 ppm) for 2 weeks caused a slower rate of weight gain, presumably
due to the decrease in food consumption and feed efficiency. There were no anatomic
injuries attributable to the feeding of nylon 6 in this study. According to Montgomery
(1982), there are no known reports that attribute any metastatic carcinogenic potential to
nylon. No studies examining the effects of nylon 6 in avian species were found in the
literature.

Materials and Methods

Fifty-four male and 54 female S-month-old game-fann mallards (Anas
platyrhynchos) (hatched 28 July 1997) with plumage and body conformation that
resembled wild mallards were purchased from Whistling Wings, Inc. (Hanover, Illinois).
The ducks arrived by truck at the Michigan State University (MSU) Poultry Science

Research and Teaching Center (PSRTC) on 30 December 1997. The ducks were

14

ii

Sicu

removed from the transport cages and weighed, and the flight feathers were clipped. The
ducks were then randomly assigned as male-female pairs to individual cages.

Cages (0.914 m L x 0.914 m W x 0.457 m H) were constructed from vinyl-coated
wire (14 gauge, 2.54 cm mesh) and suspended 61.0 cm from the floor in an enclosed pole
barn-type building. Wood shavings were placed underneath the cages to absorb excreta
and water. Shavings were replaced every two weeks.

A gas brooder was utilized to keep the room temperature above 0°C. Room
temperature and humidity were monitored by an LCD digital thermometer/hygrometer
that displayed the current temperature/humidity in addition to the high and low
temperature/humidity readings during the previous 24-hour period.

Incandescent bulbs controlled by a timer provided light. Lights were maintained
at 8 hours light: 16 hours dark during the 26-day acclimation period (30 December 1997 -—
25 January 1998).

Food and water were available Q m during the acclimation period. The diet
during the acclimation period was a commercial pelleted ration (Purina Duck Grower
W/O, St. Louis, Missouri; Batch #8858; crude protein 3 16.0%, lysine 2 0.63%,
methionine 2 0.30%, crude fat 3 3.0%, crude fiber 5 5.0%, calcium 0.40-0.90%,
phosphorus _>_ 0.55%, sodium chloride 0.20-0.70%). Water was obtained from a
university well. Crocks containing drinking water were replenished twice daily and feed

was added to the feed crocks as needed (usually every other day).

Each cage contained a nest box consisting of a S-gallon plastic pail that was

secured in a horizontal position in a rear comer of the cage. Attached to the bottom front

15

of the pail was a 5.08 cm high vinyl-coated wire fence to prevent eggs from rolling out of
the nest box. A rubber mat was placed inside the pail to provide a cushion for the eggs

and to facilitate cleaning of the nest boxes, which was done on a weekly basis.

On 26 January 1998 (day 0), ducks were randomly assigned to 4 treatment groups
and identified by metal leg bands (size 14; National Band and Tag CO., Newport,
Kentucky) bearing a unique number and color-coded by treatment. The treatment groups
were a lead group (6 males and 6 females receiving 8 pellets of #4 lead shot on day 0), a
steel group (16 males and 16 females receiving 8 pellets of #4 steel shot on days 0, 30,
60, 90, and 120), a tungsten-iron group (16 males and 16 females receiving 8 #4 tungsten-
iron shot composed of 55% tungsten and 45% iron on days 0, 30, 60, 90, and 120), and a
tungsten-polymer group (16 males and 16 females receiving 8 #4 tungsten-polymer shot
composed of 95.5% tungsten and 4.5% nylon on days 0, 30, 60, 90, and 120). Each cage
was identified with a color-coded card bearing the cage number, the pair’s individual
band numbers, and the treatment. For record keeping purposes, the ducks were identified
by a 4-digit number. The first 2 digits designated the treatment (10 = lead, 20 = steel, 30
= tungsten-iron, 40 = tungsten-polymer), and the last 2 digits were the duck’s individual
band number.

Mallards were switched to a shelled corn diet on day 0. Each duck was weighed
and dosed with the appropriate shot. Prior to closing, pellets were weighed and placed in
groups of 8 into individual plastic vials that were identified by the duck’s 4-digit number,

cage number, treatment, sex, and day of dosing. Pellets were introduced into the

16

 

proventriculus by means of a funnel and a 21.60 cm latex tube through the esophagus.
Approximately 5 mls of water helped to flush the pellets into the proventriculus.

All ducks 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. Any duck that died before
day 150 was weighed and taken to MSU’s Animal Health Diagnostic Laboratory for
necropsy as described below. In addition to these observations, feed and water were
checked twice daily and the room temperature/humidity was recorded at each entry during
the ISO-day period. Photoperiod was maintained at 8 hours light: 16 hours dark for the
duration of the 60-day phase of the ISO-day trial.

On day 7 (2 February 1998), blood was collected from the brachial vein Of each
duck using a 22 gauge needle. Blood was placed into 2 microhematocrit capillary tubes
(75 x 1.2 mm), 1 2-ml Vacutainer tube (Becton Dickinson, Franklin Lakes, New Jersey)
containing EDTA (lavender stopper) and 2 2-ml Vacutainer tubes containing sodium
heparin (green stopper). Each Vacutainer tube was labeled with the duck’s 4 digit
number, cage number, treatment, sex and the date of collection.

The microhematocrit capillary tubes were sealed and transported to a small
laboratory adjacent to the building where the ducks were housed. Tubes were centrifuged
in an IEC MB microhematocrit centrifuge (International Equipment Co., Boston,
Massachusetts) and hematocrits were measured with an [EC MB microcapillary reader.

The Vacutainer tube containing EDTA and l Vacutainer tube containing sodium
heparin from each duck were gently rotated for 1 minute and then refiigerated until all

blood samples were collected over a 4-hour period. Since mallards were bled in order of

17

their cage number rather than by treatment, blood samples from all 4 treatments were
collected throughout the period. When blood collection was completed, samples were
packed unfiozen in coolers containing U-Tek polyfoam refrigerant packs (Polyfoam
Packers, Wheeling, Illinois) and shipped by overnight express to the Division of
Comparative Pathology at the University of Miami, Miami, Florida.

The second Vacutainer tube containing sodium heparin from each duck was used
for separation of plasma from whole-blood. Refrigerated tubes were transported to the
Toxicology Laboratory in Anthony Hall ( 4 miles from the PSTRC) and spun in a GLC-4
General Laboratory centrifuge (Sorvall Instruments, Newtown, Connecticut) at 50 x g for
5 minutes. Plasma was removed from the Vacutainer tube by a glass Pasteur pipet and
transferred to a labeled l-dram glass vial. Plasma vials were stored in a cooler containing
dry ice until all plasma samples had been collected. Vials were then transferred to an
ultracold freezer (-72°C) until shipping the next day. Plasma samples were sent on dry
ice by overnight express to the Division of Comparative Pathology, University of Miami,
Miami, Florida.

Within 1 hour of arrival of the whole-blood and plasma samples at the University
of Miami, the tubes and vials were unpacked, separated by container type, and arranged
sequentially by the ducks’ 4 digit numbers. Tubes and vials were then assigned a second
number (l,2,3,etc.). The quality of each sample was grossly examined and noted on the
log-in worksheet. EDTA-containing tubes were at room temperature prior to
determination of Hb concentration. Tubes containing sodium heparin were stored at 4°C
for 3 hours prior to analysis of ALAD activity. Plasma samples were kept frozen prior to

determining plasma chemistries.

l8

 

 

Hemoglobin was determined by removing 100 pl whole-blood from the
Vacutainer tube containing EDTA and placing it in a plastic 96-well microtiter plate.
Fifty pl of lysis solution (ammonium chloride) was added to each well and the solutions
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 (Buffalo, New York). Hemoglobin was quantitated as
g/dL (x 1.5 for dilution factor). ALAD (expressed in ALAD units) was measured
according to the protocol of Burch and Siegel (1971) and Dieter and Finley (1979).
ALAD units equal (corrected absorbance x 12,500)/I-ICT. Plasma samples were analyzed
using a Johnson and Johnson 700XR automated analyzer (Rochester, New York).
Control sera samples were run daily prior to analysis to maintain a check on instrument
calibration.

On day 9 (4 February 1998), half of the mallards in each treatment group, and on
day 11 (6 February 1998), the remaining ducks in each treatment group were transported
(12 ducks/crate) to the MSU Large Animal Veterinary Clinic for fluoroscopy by
radiologist Dr. Russell Stickle to determine retention of shot. All ducks 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 monitor. Each radiograph was
identified by the duck’s 4-digit number.

On days 30 (25 February 1998) and 60 (27 March 1998), mallards were weighed
and redosed with 8 pellets of their respective shot. Blood was collected from all ducks

for HCT determination and from 8 males and 8 females in each treatment group for

19

 

determination of plasma chemistries. Hematocrits were determined at MSU and the
Division of Comparative Pathology, University of Miami, Miami, Florida assessed
plasma chemistries, as described above. F luoroscopies were performed as previously
described on days 37 (4 March 1998) and 39 (6 March 1998), and on days 70 (6 April

1998) and 72 (8 April 1998).

On day 61 (28 March 1998), all surviving mallards were switched to a
commercial layer ration (Mazuri Waterfowl Breeder, Brentwood, Missouri; Batch #5640;
crude protein 2. 17.0%, crude fat 2 2.5 %, crude fiber 5 6.0 %, ash S 10.0%, added
minerals S 5.2%) for the next 90 days (reproduction trial). Photoperiod was increased on
a weekly basis over 6 weeks beginning on 21 April 1998 and ending on 1 June 1998 to
achieve 18 hours light:6 hours dark. Ducks were weighed and redosed with 8 pellets of
the appropriate shot, and blood samples taken for HCT and plasma chemistries on days
90 (27 April 1998) and 120 (26 May 1998). Mallards were fluoroscoped on days 100 (6

May 1998) and 102 (8 May 1998) and on day 130 (5 June 1998).

When egg laying began, cages were checked twice daily and all eggs were
collected from each pair throughout the 90-day reproduction phase. Eggs were removed,
dated, identified by the respective hen’s 4-digit number and sequential egg number,
weighed, and held for up to 1 week in a cooler at temperatures between 55°- 60°F with

75% relative humidity.

The 11th egg laid by each female was used for determination of shell thickness and

for elemental analysis of shell and contents. Measurements of shell thickness were taken

20

 

at 6 locations (2 on the pointed end, 2 on the blunt end and 2 on the equator) on each egg
with an Ames 25 ME Digimatic Outside Micrometer (Waltham, Massachusetts) and the 6
measurements were averaged. Shells were stored at room temperature in individually
labeled plastic bags and the contents were stored in individually labeled I-Chem jars

(Nalge, New Castle, Delaware) in a freezer (-4°C).

All eggs, except the 11th egg, were set on a weekly basis and incubated with their
blunt end up in a Petersime poultry incubator (Gettysburg, OH) for up to 30 days.
Conditions in the incubator were standard for commercial Operations, 99.0-99.5°F with
wet bulb readings of 83-85°F to yield approximately 60% relative humidity. Eggs were
automatically rotated every 2 hours. Embryo fertility was determined by candling eggs on
incubation days 7, l4 and 21, and infertile eggs were removed. On incubation day 22,
embryo viability was assessed with an embryo viability detector (EVD) that was provided
by USFWS. The EVD detects vibrations within the egg and changes the vibrations to
sound waves that can be heard in headphones attached to the EVD (Mineau and Pedrosa,
1986). All viable eggs were then transferred to pedigree hatching baskets that were
placed in a Sure-pip hatcher (Agro Environmental Systems Inc., Dallas, Georgia) 4 days
prior to hatching. The temperature in the hatcher was maintained at 99.0°F with a wet-
bulb reading Of 89.0°F to yield approximately 70% relative humidity. Eggs were kept in
the hatcher until hatching or until day 30 of incubation. Eggs not hatching were identified
as shell-less, cracked, dead non-pipped, live non-pipped, dead pipped or live pipped. The
eggs were then Opened, examined for deformities, and the approximate age of embryos at

death was determined.

21

Ducklings were removed from the incubator (within 18 hours after hatching),
weighed, and identified with a Swiftak identification tag (Heartland Animal Health, Inc.,
Fair Play, Missouri). They were housed in heated floor pens (3.05 In W x 2.29 m L) with
water and starter mash (Purina Duck Starter W/O, Batch #8855; crude protein 2 20.0%,
lysine 2 0.95%, methionine Z 0.40%, crude fat 2: 3.0%, crude fiber S 6.0%, calcium 0.60-
1.10%, phosphorus 2 0.60%, sodium chloride 0.20-0.70%) being provided @ l_ibi_tum.
Water was available in Plasson waterers and feed was placed in metal feeders that were
refilled at least twice daily. Shavings were placed on the floor to absorb excreta and
water and were replaced on a weekly basis.

At 14 days of age, each duckling was weighed, and blood was collected from the
brachial vein into microhematocrit capillary tubes (32 x 0.8 mm ) for determination of
HCT. Tubes were sealed and centrifuged in a Drummond Scientific microhematocrit
centrifuge (Broomall, Pennsylvania). Hematocrits were measured with a Drummond
Scientific microcapillary reader. Ducklings from eggs number l-10 and 12-21 from each
hen, if available, were euthanized by cervical dislocation and necropsied. The brain,
heart, liver, spleen, kidneys, and bursa were removed for weighing. The gonads were
examined to determine sex. Small samples Of the liver and kidneys were placed in
individually labeled plastic vials containing a 10% formalin-saline solution for
subsequent histopathological examination. Additionally, the right femur and the
remaining portions of the liver and kidneys from each necropsied duckling were placed in
individually labeled plastic bags and frozen for subsequent elemental analysis.

On day 150 of the trial, all surviving adult mallards were weighed, bled as

previously described, killed by cervical dislocation, and subjected to necropsy. The

22

necropsy procedure included a complete gross examination of all body cavities and
organs by Dr. Scott Fitzgerald, board-certified veterinary pathologist. Gizzards were
opened for inspection of cracked and discolored mucosa and retention of shot. Shot were
counted and placed into individually labeled plastic vials for subsequent cleaning and
weighing for determination of shot erosion. The brain, gizzard, heart, liver, spleen,
kidneys, testes/ovary were removed and weighed. Small samples of the liver, kidneys
and testes/ovary from each duck were placed in labeled glass jars containing a 10%
formalin-saline solution for subsequent histopathological examination. The right femur
and remaining portions of the liver, kidneys, and testes/ovary were placed in individually
labeled plastic bags and frozen for subsequent elemental analysis.

Histological examination of tissues was performed by Dr. Scott Fitzgerald, board-
certified veterinary pathologist. Liver and kidney samples from 8 male and 8 female
ducklings in each treatment (excluding lead) were assessed as were liver, kidney, and
ovary/testes samples from 8 male and 8 female adult mallards from the steel, tungsten-
iron and tungsten-polymer groups and from the 6 males and 6 females in the lead group.
Tissues for microscopic examination were fixed in 10% formalin and embedded in
paraffin. Tissue sections were trimmed to 8 microns and stained with hematoxylin and
eosin. Selected liver sections from steel-, tungsten-iron- and tungsten-polymer—dosed
mallards were stained with Prussian blue for determination of iron pigment (Mallory,
1942)

Elemental analysis of tissues was performed by CT&E Environmental Services
(Ludington, Michigan). Frozen samples were transported by ground courier from MSU

to Ludington. All tissues were stored frozen until sample preparation and analysis.

23

Samples analyzed included: individual liver, kidney, femur, and gonad samples from the
12 leadvdosed adults; individual liver samples from 8 adult males and 8 adult females in
the steel, tungsten-iron and tungsten-polymer groups and individual testis samples from 8
adult males in the steel, ttmgsten-iron and tungsten-polymer groups; 16 pooled kidney
and femur samples (8 male and 8 female), each consisting of tissues from 2 adult males
or 2 adult females in the steel, tungsten-iron and tungsten-polymer groups and 8 pooled
ovary samples from 2 adult females in the steel, tungsten-iron and tungsten-polymer
groups; the shell and contents of the 11th egg from each hen, if available; 16 pooled liver,
kidney, and femur samples (8 male and 8 female), each consisting of tissues from 3 male
or 3 female ducklings from the same hen, in the steel, tungsten-iron, and tungsten-
polymer groups. Tissues were digested using EPA method 200.3 (U .8. Environmental
Protection Agency, 1991). Iron and tungsten were analyzed by Inductively Coupled
Argon Emission Plasma Spectroscopy (ICAP) following EPA method SW-836 Method
6010, revision 2.0 (U .S. Environmental Protection Agency, 1996) and lead was analyzed
by Graphite Furnace Atomic Absorption (GFAA) based on EPA method SW-846 Method
7421 (U .S. Environmental Protection Agency, 1986). A matrix spike was prepared and
analyzed with each digestion batch. When the matrix spike recoveries were outside of
quality control acceptance criteria, an analytical spike or post-digestion spike was
performed. All matrix spike and/or analytical spike recoveries were within quality
control acceptance criteria with the exception of tungsten in batch 8829 that yielded
recoveries of 70% and 72% for the matrix spike and analytical spike, respectively.
Selected tissues from lead-dosed and steel-dosed ducks were re-analyzed because

tungsten was reported in those tissues. Upon reanalysis, tungsten was not detected in any

24

of the samples in question, with the exception of 3 kidney samples from lead-dosed adults
and 2 kidney samples from steel—dosed adults. There was not a sufficient amount of
material left to reanalyze these 5 samples. Average percent recovery of iron, tungsten,
and lead were 100%, 92%, and 97%, respectively.

All statistical analyses were performed using SAS® software (SAS, 1997). Adult
body weights, plasma chemistries, and hematocrits were analyzed by analysis of variance
(ANOVA) involving the factors treatment and sex, with repeated measurements on
animals, when applicable, over a third factor, days. SAS® PROC MIXED was used to
model a first-order autoregressive correlation structure for repeated measurements over
days within animals, as residuals involving measurements taken at adjacent time periods
are likely to be more correlated than measurements taken further apart in time (Gill,
1990). Body weights were analyzed based on the status of the ducks on the specific days
of measurement. Body weights were analyzed separately over three different time points
due to differences in status over these periods. First, body weights of ducks from the
lead-dosed group were extrapolated to day 30 since all of the lead-dosed ducks died by
day 25. Body weight difference from day 0 to day 30 was then analyzed with mean
weight differences compared among the 4 treatment groups. Second, at day 60, the body
weights of adult ducks in the steel, tungsten-iron and tungsten-polymer groups were
analyzed at this single time point because none of the ducks were yet reproductively
active. Finally, adult body weights were analyzed over the time period that ducks were
reproductively active (days 90, 120. and 150). Hematocrits and plasma chemistries of

adult ducks in the steel, tungsten-iron and tungsten-polymer groups were analyzed over

25

the time period that ducks were not yet reproductively active (days 30 and 60) and over
the time period that they were reproductively active (days 90, 120, and 150).

Hematocrit, Hb concentration, ALAD activity and plasma chemistries for all 4
treatment groups at day 7 were analyzed under a two-way AN OVA model involving the
factors treatment and sex. Duckling body weights and hematocrits, adult and duckling
organ weights, concentrations of metal residues in adult and duckling tissues, and percent
shot erosion were also analyzed under a two-way AN OVA model.

Egg production, hatchability, fertility, egg weights, eggshell thickness,
concentrations of metal residues in egg shell and egg contents, and duckling survivability
were analyzed under a one-way AN OVA model involving the factor treatment.

Residual plots were used to check for homogeneity of variance and for aberrant
values. Residual plots for plasma chemistry parameters at days 7, 30, 60, 90, 120, and 150
and adult elemental analysis indicated aberrant values, therefore, those data were log
transformed to normalize data. The reported means and 95% confidence intervals for
treatment means of plasma chemistries and adult elemental analysis were back (anti-log)
transformed to the scale of observation. Percent shot erosion, adult and duckling relative
organ weights, egg production, hatchability, fertility, and duckling survivability were
percentage data subjected to arcsine, square root transformation prior to Statistical
analysis. The reported means and 95% confidence intervals for treatment means of
percent shot erosion, adult and duckling relative organ weights, egg production,
hatchability, and fertility were back [(sin(x))2] transformed to the scale of Observation.

Treatment group means were reported as the least square mean plus or minus the

standard error. Since variability was homogenous across days, all standard error

26

computations were based on a pooled estimate of residual variance. Therefore, the
standard errors of means for a particular parameter were the same unless the sample sizes
were not equal. Treatment means were reported separately for each sex, and/or day, if
treatment by sex and/or treatment by day interactions, respectively, were statistically
significant. Otherwise, reported treatment means and differences were based on pooling
information over the sexes and/or days. To control for experimental error rates, a Tukey
adjustment was used to test comparisons between means based on the total number of
pairwise comparisons. Differences between treatment group means were statistically

significant based on a Type I error rate of 0.05.
Results

Adult Mortality

All mallards dosed with lead shot died within the first 25 days of the 150-day trial
(Table l). The average time to death was 16.7 days for males and 11.0 days for females
with a range of 9 to 25 days for both sexes. The average weight loss Of those mallards
dying was 61%. No ducks in the steel-, tungsten-iron-, or tungsten-polymer-dosed groups
died during the ISO-day trial.
Adult Clinical Signs

Lead-dosed mallards were the only ducks that had obvious clinical signs during

the trial. All of them had green-stained excreta within 24 hours of dosing. By day 5, all

27

 

Table l. The effect of treatment shot on percent mortality, time to death (days), and
percent weight lost at death of mallards on a ISO-day dosing test“.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment % Mortality Time to death % Weight loss at death
Males

Steel - - -
Lead 100 16.7 i 5.25 60.3 i 4.93
(6/6) (9-25) (55.0-69.0)

Tungsten-iron - - -

Tungsten-polymer - - -

Females

Steel - - -
Lead 100 11.0 i 0.26 61.6 i 7.84
(6/6) (9-12) (54.0-72.7)

 

Tungsten-iron

 

Tungsten-polymer

 

 

 

 

 

‘ Data presented as mean : standard error of the mean. Numbers in parentheses

represent number of birds dying/number of birds per group for % mortality, range for
time to death, and range for % weight loss at death.

 

28

 

lead-dosed mallards had marked tail and wing droop. Prior to death, ducks were
emaciated, lethargic and ataxic.
Adult Body Weights

By day 30 of the trial, lead-dosed ducks lost approximately 530 grams of body
weight, while steel-, tungsten-iron-, and tungsten-polymer-dosed ducks lost 73 to 79
grams of body weight (Table 2). Because the lead-dosed ducks died by day 25, body
weights of all lead-dosed ducks were extrapolated to day 30 based on body weight taken
at time of death to allow for statistical comparison.

From day 30 through day 60, there were no treatment by sex interaction or
treatment by day interaction, thus data were combined for both sexes over days 30 and 60.
There were no significant differences in body weight between the steel, tungsten-iron and
tungsten-polymer groups (Table 3). From day 90 through day 150, there were no
treatment by sex or treatment by day interactions, thus data were combined for both sexes
over days 90, 120 and 150. Mallards in the tungsten-polymer group had significantly
greater body weight when compared to ducks in the steel-dosed group (Table 4).

Adult HCT, Hb Concentration, ALAD Activity

Lead-dosed mallards had significantly lower HCT, Hb concentration, and whole-
blood ALAD activity at day 7 when compared to mallards in the steel, tungsten-iron, and
tungsten-polymer groups. In contrast, ducks in the tungsten-polymer group had
significantly higher ALAD activity than mallards in the other 3 treatment groups (Table

5). From day 30 to day 60, there were no significant differences in HCT between ducks

29

 

Table 2. The effect of treatment shot on body weight (gm) loss of mallards from day 0
to day 30 of a 150-day dosing test”.

 

 

 

 

 

Treatment Body weight lossb
Steel «73.3A
Lead -527.5‘B
Tungsten-iron -78.6A
Tungsten-polymer -77.5A

 

 

 

' Data presented as mean of body weight loss. Sample size is 32 for
all groups except lead, which is 12. Means with different superscripts are
significantly different within the column (p < 0.5).

b Body weights at day 30 for lead-dosed ducks only were derived by linear
extrapolation.

 

30

 

 

Table 3. The effect of treatment shot on body weight (gm) of
mallards from day 30 through day 60 of a ISO-day dosing

 

 

 

 

test'.
Treatment Body weight
Steel 966.1 1 18.70
Tungsten-iron 1024.1 1 18.70
Tungsten-polymer 1017.0 i 18.70

 

 

' Data presented as mean : standard error Of the mean. Sample size

 

is 32 for all groups.

 

 

 

Table 4. The effect of treatment shot on body weight (gm) of
mallards from day 90 through day 150 of a ISO-day

 

 

 

 

dosing test’.
Treatment Body weight
Steel 1122.1 : 19.03"
Tungsten-iron 1 172.3 : 19.06’5‘B
Tungsten-polymer 1204.5 : 18.78B

 

 

‘ Data presented as mean 3: standard error of the mean. Sample size

is 32 for all groups. Means with different superscripts are
significantly different within the column (p < 0.5).

 

 

 

31

 

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in the steel, tungsten-iron, and nmgsten-polymer groups (Table 6). Between day 90 and
day 150, there was a significant treatment by sex interaction for HCT. There were no
significant differences between HCT of steel-, tungsten-iron-, and tungsten-polymer-
dosed males, but HCT of tungsten-polymer-dosed females was significantly lower than
HCT of steel- and tungsten-iron—dosed females (Table 7).
Adult Plasma Chemistries

There were a number of significant differences in plasma chemistry parameters at
day 7 (Tables 8 and 9). Sodium concentration in lead-dosed mallards was significantly
lower when compared to the other 3 groups and significantly lower in the tungsten-
polymer-dosed mallards compared to steel-dosed ducks. Conversely, potassium
concentration in lead-dosed ducks was higher when compared to tungsten-iron-dosed
mallards. Concentrations of blood urea nitrogen and creatinine were significantly higher
in lead-dosed ducks compared to the other 3 groups. The blood urea nitrogen/creatinine
ratio was significantly lower in lead-dosed ducks compared to the other 3 groups. Total
protein and albumin concentrations were significantly lower in lead-dosed ducks when
compared to the other 3 groups. Albumin/globulin ratio, concentrations Of total bilirubin
and uric acid, and activities of alanine aminotransferase, aspartate aminotransferase, and
lactate dehydrogenase were all significantly higher in lead-dosed ducks when compared
to steel-, tungsten-iron- and tungsten-polymer-dosed groups. Phosphorus concentration
was significantly higher in lead-dosed ducks as compared to steel- and tungsten-iron-
dosed groups and significantly higher in tungsten-polymer—dosed ducks as compared to

the tungsten-iron-dosed ducks. Alkaline phosphatase activity was significantly lower in

33

 

Table 6. The effect of treatment shot on hematocrit of mallards from day 30
through day 60 of a ISO-day dosing test“.

 

 

 

 

Treatment Hematocrit
Steel 50.8 i 0.45
Tungsten-iron 51.1 i 0.45
Tungsten-polymer 50.5 i 0.45

 

 

’ Data presented as mean : standard error. Sample size is 32 for all groups.

Hematocrit is expressed as percentage of packed red blood cell
volume. Means with different superscripts are significantly different within

the column (p < 0.5).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Table 7. The effect of treatment shot on hematocrit of male and female mallards
from day 90 through day 150 of a 150-day dosing test”.
Treatment Hematocrit
Males
Steel 47.8 i 0.77
Tungsten-iron 47.9 i 0.77
Tungsten-polymer 47.2 i 0.77
Females
Steel 43.5 : 0.77A
Tungsten-iron 44.0 : 0.78A
Tungsten-polymer 40.4 i 0.78B
‘ Data presented as mean : standard error. Sample size is 16 for all groups.
Hematocrit is expressed as percentage Of packed red blood cell
volume. Means with different superscripts are significantly different within
the column (p < 0.5).

 

 

 

34

 

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lead-dosed ducks when compared to ducks in the steel, tungsten-iron, and tungsten-
polymer groups. Lead-dosed ducks had significantly lower triglyceride concentration
when compared to the other 3 groups.

There was a significant treatment by sex interaction for calcium and chloride
concentrations and creatinine phosphokinase and amylase activities at day 7 (Table 9).
Lead-dosed males had significantly lower chloride concentration when compared to the
other 3 groups. Calcium concentration in lead-dosed males was significantly lower
compared to steel- and tungsten-polymer-dosed males and tungsten-iron—dosed males had
significantly lower calcium concentration than steel-dosed males. Lead-dosed males had
significantly higher creatinine phosphokinase activity compared to the other 3 groups.
Amylase activity in lead-dosed males was significantly lower compared to steel-and
tungsten-polymer-dosed males. Lead-dosed females had significantly lower chloride
concentration when compared to steel-, tungsten-iron, and tungsten-polymer-dosed
females and tungsten-polymer-dosed females had significantly lower chloride
concentrations than steel-dosed females. Calcium concentration was significantly lower
and creatinine phosphokinase activity was significantly higher in lead-dosed females
when compared to the other 3 groups. Amylase activity in lead-dosed females was
significantly lower when compared to the other 3 dose groups and tungsten-iron-dosed
females had significantly higher amylase activity than tungsten-polymer-dosed females.

There were no significant treatment by day or treatment by sex interactions for
most of the plasma chemistry parameters between days 30 and 60 (Table 10). Tungsten-

iron- and tungsten-polymer-dosed mallards had significantly lower carbon dioxide

40

 

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43

 

concentration when compared to the steel-dosed group. Tungsten-polymer-dosed ducks
had significantly higher aspartate aminotransferase activity than steel-dosed ducks.
Glucose, sodium, chloride, total protein, and phosphorus concentrations in tungsten-
polymer-dosed ducks were significantly higher than in tungsten-iron-dosed ducks.

There was a significant treatment by sex interaction for total bilirubin
concentration and lactate dehydrogenase, amylase, and gamma glutarnyl transpeptidase
activities from day 30 through day 60 (Table 11). Tungsten-iron-dosed males had
slightly, but significantly higher total bilirubin concentration then steel- and tungsten-
polymer-dosed ducks. Lactate dehydrogenase activity in male tungsten-iron-dosed ducks
was significantly lower when compared to steel-dosed males. Amylase activity in
nmgsten-iron-dosed males was significantly lower when compared to steel-dosed males
and was significantly higher in tungsten-iron-dosed females when compared to steel- and
tungsten-polymer-dosed females. Gamma glutamyl transpeptidase activity in nmgsten-
iron-dosed males was significantly higher when compared to steel- and tungsten-polymer-
dosed males.

There was a significant treatment by day interaction for blood urea
nitrogen/creatinine ratio and uric acid concentration (Table 12) from day 30 through day
60. At day 30, the blood urea nitrogen/creatinine ratio was significantly higher and uric
acid concentration was significantly lower in tungsten-iron-dosed ducks compared to
tungsten-polymer—dosed ducks. At day 60, the blood urea nitrogen/creatinine ratio was
significantly higher in tungsten-polymer-dosed ducks when compared to the steel-dosed
group.

From day 90 through day 150, there were a few differences in plasma chemistry

44

 

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51

parameters (Table 13). Total protein and cholesterol concentrations were significantly
higher in tungsten-polymer—dosed ducks when compared to the tungsten-iron-dosed
group. There was a significant treatment by day interaction for potassium and blood urea
nitrogen/creatinine ratio (Table 14). At day 90, blood urea nitrogen/creatinine ratio was
significantly higher in tungsten-polymer-dosed ducks as compared to the other 2 groups.
At day 150, potassium concentration was significantly higher in tungsten-iron-dosed
ducks when compared to ducks in the steel-dosed group.

Adult Gross Pathology

All lead-dosed mallards had severe atrophy of breast muscle with minimal
subcutaneous or abdominal fat with the exception of 1 female, which had moderate breast
muscle atrophy (Tables 15 and 16). Three lead-dosed males and females had
discoloration of the mucosal lining of the gizzard. The vent areas of 2 male and 1 female
lead-dosed mallards were stained with bile and 1 male and 2 female lead-dosed ducks had
enlarged gallbladders. One lead-dosed female had urate crystals surrounding the heart,
while 1 lead-dosed male had a focal area of the liver with a firm, gray covering on the
subcapsular surface.

During the 90-day reproduction phase (day 60 to day 150), there were 2 steel-
dosed and 3-tungsten-polymer-dosed females that did not lay any eggs. It was noted that
l steel-dosed female had a small egg blocking the lumen of the magnum and the other
steel-dosed duck had obstructing scar tissue in the oviduct. Of the 3 tungsten-polymer-

dosed females that did not lay eggs. 2 had egg yolk peritonitis while the third female

 

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53

 

Table 15. The gross necropsy observations of the efi‘ect of treatment shot
on male mallards on a ISO-day dosmflest.

 

 

ID# Treatment Days on Trial Observation(sia
2001 Steel 150 Normal
2003 Steel 1 50 Normal
2005 Steel 1 50 Normal
2007 Steel 1 50 Normal
2009 Steel 1 50 Normal
201 1 Steel 150 Normal
2013 Steel 150 Normal
201 5 Steel 150 Normal
2017 Steel 1 50 Normal
2019 Steel 1 50 Normal
202 1 Steel 1 50 Normal
2023 Steel 1 50 Normal
2025 Steel 1 50 Normal
2027 Steel 1 50 Normal
2029 Steel 1 50 Normal
203 1 Steel 150 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified

veterinary pathologist

 

54

 

 

Table 15 continued. The gross necropsy observations of the efi'ect of treatment shot
on male mallards on a ISO-day dosing test.

 

 

ID# Treatment Days on Trial Observation(s)a

1001 Lead 9 Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat
Discolored mucosal lining of the gizzard
Enlarged gallbladder

1003 Lead 25 Severe breast muscle atrophy with minimal

subcutaneous or abdominal fat

1005 Lead 16 Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat
Discolored mucosal lining of the gizzard

1007 Lead 21 Vent area stained with bile
Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat
Focal area of the liver with a firm gray covering
on the subcapsular surface

1009 Lead 15 Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat

1011 Lead 14 Vent area stained with bile
Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat
Mild, discolored mucosal lining of the gizzard

 

 

" Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified
veterinary pathologist

 

55

 

 

 

Table 15 continued. The gross necropsy observations of the effect of treatment shot
on male mallards on a 150.day dosing test.

 

 

ID# Treatment Days on Trial Observation(s)‘
3001 Tungsten-iron 150 Normal
3003 Tungsten-iron 150 Normal
3005 Tungsten-iron 1 50 Normal
3007 Tungsten-iron 1 50 Normal
3009 Tungsten-iron 1 50 Normal
301 1 Tungsten-iron 150 Normal
3013 Tungsten-iron l 50 Normal
3015 Tungsten-iron 150 Normal
3017 Tungsten-iron 150 Normal
3019 Tungsten-iron 1 50 Normal
3021 Tungsten-iron 1 50 Normal
3023 Tungsten-iron l 50 Normal
3025 Tungsten-iron 1 50 Normal
3027 Tungsten-iron 1 50 Normal
3029 Tungsten-iron 1 50 Normal
3031 Tungsten-iron l 50 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified
veterinary pathologist

 

56

 

 

Table 15 continued. The gross necropsy observations of the effect of treatment shot
on male mallards on a ISO-day dosing test.

 

 

ID# Treatment Days on Trial Observation(s)'I
4001 Tungsten-polymer 150 Normal
4003 Tungsten-polymer 1 50 Normal
4005 Tungsten-polymer 1 50 Normal
4007 Tungsten-polymer 1 50 Normal
4009 Tungsten-polymer 1 50 Normal
401 1 Tungsten-polymer 150 Normal
4013 Tungsten-polymer 1 50 Normal
401 5 Tungsten-polymer l 50 Normal
4017 Tungsten-polymer 1 50 Normal
4019 Tungsten-polymer 1 50 Normal
4021 Tungsten-polymer l 50 Normal
4023 Tungsten—polymer l 50 Normal
4025 Tungsten-polymer l 50 Normal
4027 Tungsten-polymer 1 50 Normal
4029 Tungsten-polymer 1 50 Normal
4031 Tungsten-polymer l 50 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified

veterinary pathologist

 

57

 

 

 

 

Table 16. The gross necropsy observations of the effect of treatment shot
on female mallards on a ISO-day dosing test.
ID# Treatment Days on Trial Observation(s)'
2002 Steel 150 Normal
2004 Steel 150 Small egg blocking lumen of the magnum
2006 Steel 150 Normal
2008 Steel 1 50 Normal
2010 Steel 150 Normal
2012 Steel 1 50 Normal
2014 Steel 1 50 Normal
2016 Steel 1 50 Normal
2018 Steel 1 50 Normal
2020 Steel 1 50 Normal
2022 Steel 1 50 Normal
2024 Steel 1 50 Normal
2026 Steel 1 50 Normal
2028 Steel 150 Obstructing scar tissue in the oviduct
2030 Steel 1 50 Normal
2032 Steel 1 50 Normal

 

 

' Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified
veterinary pathologist

 

58

 

 

Table 16 continued. The gross necropsy observations of the effect of treatment shot
on female mallards on a ISO-day dosing test.

 

ID# Treatment

Days on Trial

Observation(s)'

 

1 002

1004

1 006

1008

1010

1012

Lead

Lead

Lead

Lead

Lead

Lead

12

12

10

12

11

Vent area stained with bile

Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat

Mild, discolored mucosal lining of the gizzard

Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat

Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat

Discolored mucosal lining of the gizzard
Enlarged gallbladder

Moderate breast muscle atrophy
Enlarged gallbladder
Urate crystals surrounding the heart

Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat

Discolored mucosal lining across entire surface
of the gizzard

Severe breast muscle atrophy with minimal
subcutaneous or abdominal fat

 

 

‘ Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified
veterinary pathologist

 

59

 

 

Table 16 continued. The gross necropsy observations of the effect of treatment shot
on female mallards on a ISO-day dosing test.

 

 

ID# Treatment Days on Trial Observation(sL‘I
3002 Tungsten-iron 1 50 Normal
3004 Tungsten-iron 1 50 Normal
3006 Tungsten-iron 150 Normal
3008 Tungsten-iron 1 50 Normal
3010 Tungsten-iron 150 Normal
3012 Tungsten-iron 150 Normal
3014 Tungsten-iron 150 Normal
3016 Tungsten-iron 150 Normal
3018 Tungsten-iron 1 50 Normal
3020 Tungsten-iron 1 5 0 Normal
3022 Tungsten-iron 1 50 Normal
3024 Tungsten-iron 1 50 Normal
3026 Tungsten-iron l 50 Normal
3028 Tungsten-iron l 50 Normal
3030 Tungsten-iron 1 50 Normal
3032 Tungsten-iron 150 Normal '

 

 

a Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified
veterinary pathologist

 

60

 

 

Table 16 continued. The gross necropsy observations of the effect of treatment shot
on female mallards on a ISO-day dosinLtest.

 

 

ID# Treatment Days on Trial Observation(s)a
4002 Tungsten-polymer 150 Normal
4004 Tungsten-polymer 1 50 Normal
4006 Tungsten-polymer 1 50 Normal
4008 Tungsten-polymer 150 Fatty liver
4010 Tungsten-polymer l 5 0 Normal
4012 Tungsten-polymer 150 Egg yolk peritonitis
4014 Tungsten-polymer l 50 Normal
4016 Tungsten-polymer 150 Normal
401 8 Tungsten-polymer 1 50 Normal
4020 Tungsten-polymer 1 50 Normal
4022 Tungsten-polymer 1 50 Normal
4024 Tungsten-polymer 150 Egg yolk peritonitis
4026 Tungsten-polymer 1 50 Normal
4028 Tungsten-polymer 1 50 Normal
4030 Tungsten-polymer 150 Focal area of liver with fibrous tag
4032 Tungsten-polymer 1 50 Normal

 

 

‘ Gross necropsy observations performed by Dr. Scott Fitzgerald, board-certified
veterinary pathologist

 

61

 

appeared normal. All other ducks in the steel-, tungsten-iron- and tungsten-polymer-dosed
groups appeared normal except for 1 tungsten-polymer female that had a fibrous tag on a
focal area of the liver.

Adult Organ Weights

Spleen and heart weights of lead-dosed mallards were significantly lower when
compared to the other 3 groups (Table 17). There was a significant treatment by sex
interaction for liver and gonad weights (Table 18). Testes weight of lead-dosed males
and liver weight of lead-dosed females were significantly lower when compared to the
steel-, tungsten-iron-, and tungsten-polymer-dosed groups.

When organ weight was expressed as a percent of body weight, the relative
weights of kidneys, heart, brain and gizzard of lead-dosed mallards were significantly
higher when compared to the other 3 groups (Table 19). Relative spleen weight of lead-
dosed ducks was significantly lower than relative spleen weights of steel-dosed and
tungsten-iron-dosed mallards and relative spleen weight of tungsten-iron-dosed ducks
was significantly higher compared to tungsten-polymer-dosed ducks. There was a
significant treatment by sex interaction for relative liver weights (Table 20). Relative
liver weight of lead-dosed males was significantly higher compared to the other 3 groups.
Relative testis and ovary weights were analyzed separately because of the anatomical
difference (Table 20). Testis weight of lead-dosed males and ovary weight of lead-dosed

females were significantly lower when compared to the other 3 groups.

62

 

 

 

 

 

 

 

 

 

 

 

 

 

 

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66

Histopathology of Adult Liver, Kidneys, and Gonads

All lead-dosed mallards had kidney nephrosis ranging from mild to moderate with
the exception of 1 female that had a normal kidney. All lead-dosed mallards had mild to
moderate liver biliary stasis (Tables 21-24). Mallards dosed with steel, tungsten-iron, or
tungsten-polymer shot that were examined had normal kidneys and no indication of liver
biliary stasis. However, all of the steel- and tungsten-iron-dosed males examined as well
as 1 tungsten-polymer-dosed male had liver hemosiderosis ranging from mild to
moderate. Five of 8 steel-dosed females and 3 of 8 tungsten-iron-dosed females that were
examined had liver hemosiderosis ranging from mild to moderate. Diffuse hepatocellular
vacuolation was apparent in ducks of all 4 groups, but this condition was judged not to be
treatment related. The testes and ovary in the lead-dosed mallards were inactive, while
these tissues in steel- tungsten-iron- and tungsten-polymer-dosed ducks that were
examined appeared normal.
Metal Residues in Tissues of Adults

Lead-dosed mallards had a significantly higher concentration of lead in the femur
when compared to the steel-, tungsten-iron- and tungsten-polymer-dosed groups (Table
25). There was a significant treatment by sex interaction for iron and tungsten
concentrations in the femur (Table 26). Iron concentrations in the femur samples of lead-
and tungsten-polymer-dosed female mallards were significantly lower when compared to
steel- and tungsten-iron-dosed females. Tungsten was detected in all of the femur samples
from the tungsten-iron-dosed males and females and in 5 of 8 samples from the tungsten-
polymer-dosed females. Tungsten-iron-dosed females had a significantly higher femur

tungsten concentration compared to tungsten-polymer-dosed females.

67

 

 

 

Table 21. The histopathological effects of treatment shot on the liver and kidneys of
male mallards on a ISO-day dosing test.
ID# Treatment Observation(s)‘
2001 Steel Mild hemosiderosis
2003 Steel Mild hemosiderosis
2013 Steel Mild hemosiderosis
2015 Steel Mild hemosiderosis
2021 Steel Mild hemosiderosis
2023 Steel Moderate hemosiderosis
2025 Steel Mild hemosiderosis
2027 Steel Mild hemosiderosis

 

 

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

board-certified veterinary pathologist

 

68

 

 

Table 21 continued.

The histopathological effects of treatment shot on the liver and
kidneys of male mallards on a ISO-day dosig test.

 

ID#

Treatment

Observation(s)ll

 

1001

1003

1005

1007

1009

1011

Lead

Lead

Lead

Lead

Lead

Lead

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

Mild, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse hepatocellular vacuolation

Moderate, diffuse acute proximal convoluted tubule
epitheliular necrosis with pyknosis

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse hepatocellular vacuolation

Focal, hepatocellular parenchymal necrosis
Moderate, diffuse acute proximal convoluted tubule
epitheliular necrosis with pyknosis

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

Mild, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse hepatocellular vacuolation

Moderate, diffuse acute proximal convoluted tubule
epitheliular necrosis with pyknosis

 

 

Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald,
board-certified veterinary pathologist

 

69

 

 

 

Table 21 continued. The histopathological effects of treatment shot on the liver and
kidneys of male mallards on a ISO-day dosing test.

 

 

ID# Treatment Observation(s)'

3005 Tungsten-iron Mild hemosiderosis
3007 Tungsten-iron Moderate hemosiderosis
3009 Tungsten-iron Mild hemosiderosis
3011 Tungsten-iron Mild hemosiderosis
3017 Tungsten-iron Mild hemosiderosis
3019 Tungsten-iron Mild hemosiderosis
3029 Tungsten-iron Mild hemosiderosis
3031 Tungsten-iron Mild hemosiderosis
4001 Tungsten-polymer Normal

4003 Tungsten-polymer Mild, diffuse hepatocellular vacuolation
4013 Tungsten-polymer Normal

4015 Tungsten-polymer Normal

4021 Tungsten-polymer Normal

4023 Tungsten-polymer Normal

4025 Tungsten-polymer Mild hemosiderosis
4027 Tungsten-polymer Normal

 

 

l

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

board-certified veterinary pathologist

70

 

EFF:

 

 

 

Table 22. The histopathological effects of treatment shot on the liver and kidneys of
female mallards on a ISO-day dosing test.

 

 

ID# Treatment Observation(s)‘I

2004 Steel Mild, diffuse hepatocellular vacuolation
2006 Steel Mild hemosiderosis

2018 Steel Mild hemosiderosis

2014 Steel Mild hemosiderosis

2018 Steel Mild, diffuse hepatocellular vacuolation
2020 Steel Normal

2028 Steel Moderate hemosiderosis

Mild, diffuse hepatocellular vacuolation

2030 Steel Mild hemosiderosis
Mild, diffuse hepatocellular vacuolation

 

 

Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald,
board-certified veterinary pathologist

 

71

 

 

Table 22 continued.

The histopathological effects of treatment shot on the liver and
kidneys of female mallards on a ISO-day dosing test.

 

ID#

Treatment

Observation(§)'I

 

1002

1004

l 006

1008

1010

1012

Lead

Lead

Lead

Lead

Lead

Lead

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis
Kidney - normal

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Moderate, diffuse hepatocellular vacuolation

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

Mild, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse hepatocellular vacuolation

Multifocal hepatocellular necrosis

Moderate, diffuse acute proximal convoluted tubule
epitheliular necrosis with pyknosis

Mild, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

Moderate, diffuse acute hepatocellular and cholangial
biliary stasis

Mild, diffuse hepatocellular vacuolation

Mild, diffuse acute degeneration and vacuolation of
proximal convoluted tubule epithelium

 

 

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

board-certified veterinary pathologist

 

72

 

 

 

 

Table 22 continued The histopathological effects of treatment shot on the liver and
kidneys of female mallards on a ISO-day dosing test.

ID# Treatment Observation(.s)a

3002 Tungsten-iron Normal

3004 Tungsten-iron Mild hemosiderosis

3014 Tungsten-iron Mild, diffuse hepatocellular vacuolation

3016 Tungsten-iron Mild, diffuse hepatocellular vacuolation

3022 Tungsten-iron Moderate hemosiderosis

3024 Tungsten-iron Normal

3026 Tungsten-iron Mild hemosiderosis

3028 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

4010 Tungsten-polymer Normal

4012 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

4014 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

4016 Tungsten-polymer Normal

401 8 Tungsten-polymer Normal

4024 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation

4028 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

4030 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

 

 

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

board-certified veterinary pathologist

 

73

 

 

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77

 

Table 25. The effect of treatment shot on concentration (mg/kg dry weight) of
lead in the femur of mallards on a ISO-day dosing test“.

 

 

 

 

 

 

Treatment Lead
Steel 4.5A
(2.66 - 7.47)
Lead 311.3B
(171.48 - 564.87)
Tungsten-iron 4.9]r
(2.94 - 8.25)
Tungsten-polymer 4. 1 A
(2.46 - 6.91)

 

 

a Data presented as means (95% confidence intervals). Sample size is 16 for

all groups except for lead, which is 12. Means with different superscripts are
significantly different within the column (p < 0.5).

 

78

 

 

dosing test“.

Table 26. The effect of treatment shot on concentrations (mg/kg dry weight) of iron
and tungsten in the femur of male and female mallards on a ISO-day

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Iron Tungsten
Males
Steel 96.2 ND
(80.53 - 115.00)
Lead 90.8 ND
(73.94 - 111.58)
Tungsten-iron 88.9 23.5
(74.37 - 106.20)
Tungsten-polymer 81.0 ND
(67.78 - 96.78)
Females
Steel 220.5A ND
(181.28 - 268.00)
Lead 91.1B ND
(72.70 - 114.13)
Tungsten-iron 162.7A 34.0A
(133.85 - 197.79) (28.60 - 40.33)
Tungsten-polymer 99.013 5. 1B
(81.41 - 120.30) (4.12 - 6.36)
[3]

 

 

a Data presented as means (95% confidence intervals). Sample size is 8 for

all groups except for lead, which is 6. Means with different superscripts are

significantly different within the column (p < 0.5). Numbers in brackets refer to
the number of pooled samples having a tissue concentration below detection limits.
ND refers to not detected. Tungsten detection limit is 3 mg/kg dry weight.

 

 

79

IN 5".

Iron concentrations in the testes were significantly higher in lead-dosed males
compared to the other 3 groups (Table 27). Lead was detected in the testes of 1 of 8
tungsten-polymer-dosed males. Tungsten was detected in the testes of 5 of 8 tungsten-
iron-dosed males and 2 of 8 nmgsten-polymer-dosed males. Tungsten-polymer-dosed
female mallards had significantly lower concentrations of iron in the ovary when
compared to steel-dosed females. Lead-dosed females had a significantly higher
concentration of lead in the ovary when compared to the other 3 groups. Lead was
detected in the ovary of l of 8 samples from steel-dosed females, 7 of 8 samples from
lead-dosed females, and 2 of 8 samples from tungsten-polymer-dosed females. Tungsten
was present in 6 of 8 ovary samples from tungsten-iron-dosed females and in 1 of 8
samples from tungsten-polymer-dosed females.

Lead-dosed mallards had a significantly higher concentration of lead in the
kidneys when compared to the other 3 groups (Table 28). Lead was detected in the
kidney of 4 of 16 samples from steel-dosed mallards, 3 of 16 samples from tungsten-iron-
dosed ducks, and 5 of 16 samples from tungsten-polymer-dosed ducks. Tungsten was
detected in 2 of 16 kidney samples from the steel-dosed group, 3 of 12 kidney samples
from the lead-dosed ducks, 13 of 16 kidney samples from the tungsten-iron-dosed ducks,
and 7 of 16 kidney samples from the tungsten-polymer-dosed ducks. Tungsten-iron-
dosed ducks had a significantly higher concentration of tungsten when compared to the
other 3 groups.

There was a significant treatment by sex interaction for iron concentration in the
kidney samples (Table 29). Lead-dosed and tungsten-polymer-dosed males had

significantly lower concentrations of iron in the kidneys when compared to steel-dosed

8O

 

Table 27. The effect of treatment shot on concentrations (mg/kg dry weight) of iron,
lead, and tungsten in the gonads of male and female mallards on a ISO-day

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

dosing test'.
Treatment Iron Lead Tungsten
Males
Steel 58.5“ 4.3 ND
(45.11 - 75.80) (0.10 — 189.92)
Lead 2225.7B 12.2 ND
(167.27 - 304.60) (2.59 - 57.16)
Tungsten-iron 563‘ ND 5.6
(43.40 - 72.94) (3.13 - 9.99)
[31
Tungsten-polymer 67.8A 0.5 7.0
(52.27 - 87.85) (0.01 - 22.09) (2.80 - 17.53)
[71 [6]
Females
Steel 488.5“ 0.7“ ND
(326.98 - 729.68) (0.34 - 1.44)
[71
Lead 206.0“B 13.4B ND
(123.98 - 342.20) (9.72 - 18.54)
[11 [1]
Tungsten-iron 354.1“B ND 8.4
(237.06 - 529.01) (3.69 - 19.14)
[21
Tungsten-polymer 21 1 .8B 0.6A 8.0
(141.80 - 316.43) (0.36 - 1.00) (1.06 - 60.09)
[6] [7]

 

 

‘ Data presented as means (95% confidence intervals). Sample size is 8 for all groups

except for lead, which is 6. Means with different superscripts are significantly
different within the column (p < 0.5). Numbers in brackets refer to the number of
males or pooled samples of females having a tissue concentration below detection
limits. ND refers to not detected. Lead and tungsten detection limits are 0.5 and
3.0 mg/kg dry weight, respectively.

 

 

 

Table 28. The effect of treatment shot on concentrations (mg/kg dry weight) of lead
and tungsten in the kidneys of mallards on a ISO-day dosing test“.

 

 

 

 

 

 

 

Treatment Lead Tungsten

Steel NE 4.0
(1.94 - 8.24)

[12] [14]

Lead 621 .0B 5.9
(488.82 - 789.03) (3.18 - 11.10)

[9]

Tungsten-iron NB 9.5
(7.15 - 12.61)

[13] [3]

Tungsten-polymer 0.8A 6.8
(0.55 - 1.17) (4.46 - 10.48)

[11] [9]

 

 

' Data presented as means (95% confidence intervals). Sample size is 16 for all
groups except lead, which is 12. Means with different superscripts are significantly
different within the column (p < 0.5). Numbers in brackets refer to the number
of pooled samples having a tissue concentration below detection limits. NE
refers to non-estimable because most values in the data set were 0.0, which is
not log transformable.

 

82

 

 

 

Table 29. The effect of treatment shot on concentrations (mg/kg dry weight) of iron
in the kidneys of male and female mallards on a ISO-day dosing test“.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Iron
Males

Steel 678.8“
(591.40 - 778.99)

Lead 484.9B
(413.56 - 568.50)

Tungsten-iron 563.1A
(490.63 - 646.26)

Tungsten-polymer 509.013
(443.46 - 584.12)

Females

Steel 825.0“
(712.44 - 955.37)

Lead 406.7B
(343.30 - 481.74)

Tungsten-iron 623.6C
(538.51 - 722.13)

Tungsten-polymer 398.9B

 

(344.50 - 461.97)

 

" Data presented as means (95% confidence intervals). Sample size is 8 for
all groups except for lead, which is 6. Means with different superscripts are

Significantly different within the column (p < 0.5).

 

83

 

males. In the females, lead-dosed ducks had a significantly lower concentration of iron
when compared to steel- and tungsten-iron-dosed ducks. Tungsten-iron- and tungsten-
polymer-dosed females had significantly lower concentrations of iron when compared to
the steel-dosed females, while ttmgsten-iron-dosed females had a significantly higher
concentration of iron than tungsten-polymer—dosed females.

Iron concentrations in the liver from tungsten-polymer-dosed mallards were
significantly lower compared to the other 3 groups. Lead-dosed mallards had a
significantly higher concentration of lead in the liver compared to steel-, tungsten-iron-,
and tungsten-polymer-dosed ducks, while tungsten-polymer—dosed ducks had a
significantly lower concentration of lead than the steel-dosed ducks (Table 30). Lead was
detected in 12 of 16 ducks in the steel-dosed group, 9 of 16 ducks in the tungsten-iron-
dosed group, and 4 of 16 mallards in the tungsten-polymer-dosed group. Tungsten was
detected in the liver of all tungsten-iron-dosed mallards and in 2 of 16 tungsten-polymer-
dosed mallards.

Shot Recovery and Percent Shot Erosion

Approximately 90% of the lead pellets administered were recovered when the
lead-dosed ducks were necropsied between day 9 and day 25 of the trial (Table 31). Over
half of the steel pellets were recovered at day 150 as opposed to less than 3% of the
tungsten-polymer shot. Approximately 40% of the tungsten-iron shot was recovered at
day 150. Of the 4 Shot types, lead Shot eroded the least in both males and females,
followed by steel, tungsten-iron, and tungsten-polymer shot, respectively. Based on the
weight of the pellets recovered, there was nearly complete erosion of the tungsten-

polymer shot and 64% and 80% erosion of tungsten-iron shot in males and females,

84

 

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

 

 

 

 

 

 

 

 

Treatment Iron Lead Tungsten
Steel 10128.4“ 1.5“ ND
(7599.97 - 13496.69) (1.25 - 1.90)
[4]
Lead 9897.9“ 218.3?“ ND
(7109.50 - 13799.67) (176.92 - 269.37)
Tungsten-iron 6890.5A l .2“ 70.4
(5170.89 - 9182.92) (0.94 - 1.53) (50.69 - 97.71)
[7]
Tungsten-polymer 1 157.2B 0.7C NE
(868.35 - 1542.25) (0.47 - 0.97) [14]
[12]

 

 

" Data presented as means (95% confidence intervals). Sample size is 16 for

all groups except lead, which is 12. Means with different superscripts are
significantly different within the column (p < 0.5). Numbers in brackets

refer to the number of mallards having a tissue concentration below detection

limits. ND refers to not detected. Tungsten detection limit is 3.0 mg/kg dry weight.
NE refers to non-estimable because most values in the data set were 0.0, which is

not log transformable.

 

85

 

 

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respectively. Fluoroscopy of the mallards during the trial substantiated the relatively
rapid erosion of the 2 types of tungsten shot.
Date First Egg was Laid and Number of Days Required to Lay 21 Eggs

Tungsten-polymer—dosed females began laying eggs approximately 7 days earlier
than females in the steel and tungsten-iron groups, which began laying around day 92 of
the study (Table 32). Females in all 3 groups required 24 to 25 days to lay 21 eggs.
There were 2 steel-dosed females and 3 tungsten-polymer—dosed females that did not lay
any eggs. Of those ducks that laid eggs, there was 1 steel-dosed female, 2 tungsten-iron-
dosed females and l tungsten-polymer-dosed female that did not lay at least 21 eggs.
Percent Egg Production, Fertility, and Hatchability

Percent egg production was similar among groups and ranged from 36% to 46%
(Table 33). Tungsten-polymer-dosed females had significantly lower percent fertility
when compared to tungsten-iron-dosed females, but percent hatchability was not different
between the steel, tungsten-iron, and tungsten-polymer groups.
Egg Weight and Shell Thickness

Eggs laid by tungsten-iron-dosed females were significantly heavier than eggs laid
by steel- or tungsten-polymer-dosed females and the shells of these eggs were

significantly thicker compared to shells of eggs laid by steel-dosed females (Table 34).

87

 

Table 32. The day the first egg was laid and the number of days required
for mallards to lay 21 eggs'.

 

 

 

 

Treatment Day first egg was laic?’ Days required to lay 21 eggsc
Steel 92.0 i 1.31 25.4 i 0.31
(l4) (l3)
Tungsten-iron 91.7 i 0.96 24.1 i 0.33
(16) (14)
Tungsten-polymer 84.5 i 1.02 25.6 i 0.45
(13) (12)

 

 

 

 

‘ Data presented as mean + standard error of the mean.

b Numbers in parentheses refer to the number of egg-laying females.

° Numbers in parentheses refer to the number of females that laid 21 eggs.

 

88

 

 

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9O

Metal Residues in Egg Shell and Contents

Iron was detected in 2 of 14, 5 of 16, and 2 of 13 shells of eggs laid by stee1-,
tungsten-iron-, and tungsten-polymer-dosed females, respectively, and in the contents of
all eggs analyzed (Table 35). There were no significant differences in iron concentration
of the eggshell or contents between the 3 groups. Lead was not detected in either the
shell or contents of eggs from the 3 groups. Tungsten was detected in the shell of 9 of 16
and 3 of 13 eggs laid by tungsten-iron- and tungsten-polymer-dosed females and in the
contents of 6 of 16 eggs laid by tungsten-iron-dosed females. There were no significant
differences in tungsten concentration in the eggshell or contents.
Survivability, Body Weight and Hematocrit of Ducklings

Survivability of ducklings through day 14 of age was equivalent for all 3 groups
(Table 36). Body weight over the 14-day period was also similar for ducklings in the 3
groups. Hematocrit of ducklings in the tungsten-iron group was significantly lower when
compared to ducklings in the steel group.
Duckling Organ Weights

Absolute and relative kidney weights of ducklings in the tungsten-polymer group
were significantly higher when compared to ducklings in the steel and tungsten-iron
groups (Tables 37 and 38). Absolute and relative weights of the liver, spleen, bursa,

heart, and brain were equivalent across the 3 groups (Tables 37 — 39).

91

 

 

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Table 36. The effect of treatment shot on duckling survivability, body weight (gm)

from day 0 through day 14, and hematocrit on day 14'.

 

 

 

 

Treatment % Survivability Body weight Hematocrit
Steel 99.1 165.4 1», 2.08 39.7 3; 0.247‘
(96.31 - 100.00) (156) (156)
Tungsten-iron 98.0 165.3 : 1.82 38.6 : 0.21B
(94.52 - 99.80) (202) (202)
Tungsten-polymer 95.7 167.0 : 2.24 39.1 : 0.26AB
(90.31 - 98.97) (135) (135)

 

 

 

 

 

' Data for % survivability presented as means (95% confidence intervals). Data for

body weight and hematocrit presented as mean : standard error of the mean.
Numbers in parentheses refer to sample size except % survivability. Means with
different superscripts are significantly different within the column (p < 0.5).

 

93

 

 

 

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96

Histopathology of Duckling Liver and Kidneys

Liver samples from ducklings in the steel, tungsten-iron, and tungsten-polymer
groups had mild to moderate diffuse hepatocellular vacuolation with the exception of
samples fi'om 1 female in the steel group and 1 female in the tungsten—polymer group. No
kidney lesions were observed (Tables 40 and 41).
Metal Residues in Tissues of Ducklings

Concentrations of iron in the femur, kidneys, and liver of ducklings were similar
across the 3 treatment groups (Table 42). Lead was detected in 2 of 16, 3 of 16, and 4 of
16 femur samples in the steel, tungsten-iron, and tungsten-polymer groups, respectively.
Lead was also detected in 1 of 16, 4 of 16, and 3 of 16 kidney samples in the steel,
tungsten-iron, and tungsten-polymer groups, respectively. Furthermore, lead was
detected in 1 of 16, 1 of 16, and 2 of 16 liver samples in the steel, tungsten-iron, and
tungsten-polymer groups, respectively. Tungsten was detected in the femur of 4 of 16
samples from tungsten-iron and 4 of 16 samples from tungsten-polymer ducklings.
Tungsten was also detected in 2 of 16 kidney samples from tungsten-iron ducklings and l
of 16 kidney samples from tungsten-polymer ducklings. Two of 16 liver samples from
tungsten-iron and tungsten-polymer ducklings contained tungsten. There were no
significant differences in lead and tungsten concentrations in the femur, liver, and kidney

samples between the 3 groups.

97

 

 

 

Table 40. The histopathological effects of treatment shot on the liver and kidneys of
male ducklings.

ID# Hen# Treatment Observation(s)a

Y13 2014 Steel Mild, diffuse hepatocellular vacuolation
Y15 2002 Steel Mild, diffuse hepatocellular vacuolation
Y48 2022 Steel Moderate, diffuse hepatocellular vacuolation
Y129 2010 Steel Moderate, diffuse hepatocellular vacuolation
Y172 2018 Steel Moderate, diffuse hepatocellular vacuolation
DSX3869 2008 Steel Moderate, diffuse hepatocellular vacuolation
Y93 2030 Steel Moderate, diffuse hepatocellular vacuolation
Y187 2032 Steel Mild, diffuse hepatocellular vacuolation
DSX3 807 3018 Tungsten-iron Moderate, diffuse hepatocellular vacuolation
DSX3651 3018 Tungsten-iron Moderate, diffuse hepatocellular vacuolation
B203 3018 Tungsten-iron Moderate, diffuse hepatocellular vacuolation
B78 3018 Tungsten-iron Moderate, diffuse hepatocellular vacuolation
B59 3018 Tungsten-iron Moderate, dififuse hepatocellular vacuolation
B258 3018 Tungsten-iron Mild, diffuse hepatocellular vacuolation
3171 3018 Tungsten-iron Mild, diffuse hepatocellular vacuolation

B75 3018 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

 

 

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

board-certified veterinary pathologist

 

98

 

 

Table 40 continued. The histopathological effects of treatment shot on the liver and

 

 

kidneys of male ducklings.
ID# Hen# Treatment Observation(s)a
P87 4018 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

DSX3 849 4020 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
DSX3 845 4016 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
P164 4008 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

P144 4030 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
DSX3 693 4014 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation

P79 4004 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation

 

P171 4026 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

 

 

Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald,
board-certified veterinary pathologist

 

 

99

 

 

 

Table 41. The histopathological effects of treatment shot on the liver and kidneys of
female ducklings.

ID# Hen# Treatment Observation(s)"

Y20 2030 Steel Moderate, diffuse hepatocellular vacuolation

DSX3 803 2014 Steel Moderate, diffuse hepatocellular vacuolation

Yl4l 2032 Steel Mild, diffuse hepatocellular vacuolation

Y84 2010 Steel Moderate, diffuse hepatocellular vacuolation

DSX3539 2026 Steel Mild, diffuse hepatocellular vacuolation

Y82 2012 Steel Normal

Y217 2006 Steel Moderate, diffuse hepatocellular vacuolation

Y222 2022 Steel Moderate, diffuse hepatocellular vacuolation

B82 3026 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

B57 3012 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

DSX3635 3032 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

DSX3 877 3010 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

8276 3014 Tungsten-iron Moderate, diffuse hepatocellular vacuolation

B295 3002 Tungsten-iron Mild, diffuse hepatocellular vacuolation

B168 3022 Tungsten-iron Mild, diffuse hepatocellular vacuolation

DSX3625 3018 Tungsten-iron Mild, diffuse hepatocellular vacuolation

 

 

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

board-certified veterinary pathologist

 

100

 

 

Table 41 continued.

The histopathological effects of treatment shot on the liver and

 

 

kidneys of female ducklth
ID# Hen# Treatment Observation(s)'
DSX3813 4018 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
DSX3545 4020 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
P129 4016 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
P122 4008 Tungsten-polymer Normal
P35 4030 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
DSX3 893 4014 Tungsten-polymer Moderate, diffuse hepatocellular vacuolation
P13 4004 Tungsten-polymer Mild, diffuse hepatocellular vacuolation
P173 4026 Tungsten-polymer Mild, diffuse hepatocellular vacuolation

 

 

Histopathological assessment of tissues was performed by Dr. Scott Fitzgerald,
board-certified veterinary pathologist

 

 

101

 

‘m 'a.

 

Table 42. The effect of treatment shot on concentrations (mg/kg dry weight) of iron,

lead, and tungsten in tissues of ducklings‘.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Treatment Iron Lead Tungsten
Femur
Steel 118.8 3: 3.23 0.2 3; 0.14 ND
(14)
Tungsten-iron 112.1 1 3.23 0.2 i 0.14 1.1 :1; 0.59
(13) (12)
Tungsten-polymer 110.1 : 3.23 0.3 i 0.14 1.7 :|_- 0.59
(12) (12)
Kidneys
Steel 242.5 j; 6.62 0.1 i 0.16 ND
(15)
Tungsten-iron 251.3 1; 6.62 0.4 i 0.16 1.2: 0.56
(12) (14)
Tungsten-polymer 247.5 : 6.62 0.2 i 0.16 0.4 i 0.56
(13) (15)
Liver
Steel 463.8 : 46.06 0.03 i 0.063 ND
(15)
Tungsten-iron 521.3 : 46.06 0.03 i 0.063 0.6 i 0.34
(15) (14)
Tungsten-polymer 399.4 : 46.06 0.14 i 0.063 0.6 i 0.34
(14) (14)

 

 

 

 

“ Data presented as mean 1 standard error of the mean. Sample size is 16 for

all groups. Numbers in parentheses refer to the number of pooled samples having a

tissue concentration below detection limits. ND refer to non-detect. Tungsten

detection limit is 3 mg/kg dry weight.

 

 

102

Dias-2122
Adult Mortality

Only the lead-dosed ducks died during the ISO-day trial and mortality was 100%
by day 25 (Table 1). These results suggest that waterfowl fed a nutritionally inadequate
diet consisting of corn are more susceptible to the toxic effects of lead than ducks fed a
diet high in protein. Jordan and Bellrose (1950) reported that 86% of Pekin ducks (Anas
platyrhynchos) dosed with 25 #4 lead shot and maintained on a corn diet died within 17
days. In a subsequent study, Jordan and Bellrose (1951) reported that only 1 or 2 #6 lead
shot pellets were sufficient to cause lead poisoning in 50% of game-farm mallards fed a
whole-com diet. Grandy et al. (1968) and Longcore et al. (1974) reported 100% mortality
within 7 to 28 days in pen-raised mallards that were dosed with 8 #6 lead shot and
maintained on corn. Sanderson et a1. (1992) dosed mallards with 2, 4, or 8 #2 lead shot
or 4 #2 lead shot plus 4 #2 bismuth shot and maintained the ducks for up to 30 days on a
diet of shelled corn. Mortality was 95% with only 2 ducks (dosed with 2 #2 lead shot)
surviving. In contrast, Rattner et al. (1989) reported no mortality after 14 days in pen-
raised and wild black ducks (Anas rubripes) and game-farm and wild mallards
maintained on duck pellets that were closed with a single #4 lead shot. The same ducks
were then dosed with either 2 or 4 #4 lead shot and maintained on a pellet diet for another
49 days. Mortality of wild black ducks was 40% and that of wild mallards was 45%.
Jordan and Bellrose (1950) reported that only 33% of Pekin ducks dosed with 25 #4 lead
shot died within 17 days when fed duck pellets. Kelly et al. (1998) reported 50%
mortality after 30 days in game-farm mallards dosed with 8 #4 lead shot and maintained

on a commercial duck pelleted diet.

103

1'] “mi-fluff n..' '_'"‘v ~ .

 

{‘3’

In the present study, none of the birds dosed with tungsten-iron or tungsten-
polymer shot died. In a similar toxicity study in which game-farm mallards where dosed
with 8 BBs of tungsten-iron or tungsten-polymer shot, no mortalities were recorded
during the 30-day trial (Kelly et al., 1998). In addition, Ringelman et al. (1993) dosed
mallards with 12 to 17 pellets composed of 39% tlmgsten, 44% bismuth, and 16 % tin,
and reported no mortalities during the 32-day trial.

Tungsten has been reported to cause mortality in birds. Nell et al. (1980) dosed
broiler cockerels with sodium tungstate by intramuscular injection at 5 mg tungsten from
day l to day 11, 10 mg from day 12 to day 21, and 20 mg from day 22 to day 35. They
reported that 4 of 10 birds died on day 29 of the trial. However, the tungsten was in a
soluble form injected in animals that were relatively small, resulting in a higher exposure
rate based on mg/kg body weight, which might enhance toxicity.

Adult Clinical Signs

Lead-dosed mallards were the only ducks that had obvious clinical signs. The
classic signs of lead poisoning, seen in more chronic cases, usually develop in the
following sequence: anorexia and lethargy; greenish diarrhea that stains the feathers
surrounding the vent; muscular weakness first evident as an inability to fly and then as an
inability to walk or move; coma; and death. There is a progressive weight loss and
atrophy of the breast muscle resulting in a “hatchet-breast” appearance (Wobeser, 1981;
Friend, 1987; Locke and Thomas, 1996). Mallards dosed with tungsten-iron and
tungsten-polymer shot appeared normal throughout the ISO-day trial. These results agree
with those reported by Kelly et al. (1998) who dosed mallards with 8 BBs of tungsten-

iron or tungsten-polymer shot and Ringelman et al. (1993) who dosed mallards with

104

 

tungsten-bismuth-tin shot. Nell et al. (1980) reported that clinical signs in chickens
administered tungsten were anorexia, reduced weight gain, diarrhea, and labored
breathing before death.

Adult Body Weights

Lead-dosed mallards lost a significant amount of body weight (54 — 73%) after the
first 25 days of the trial (Table 1). Generally, waterfowl that die of chronic lead
poisoning lose from 40-60% of their body weight before death. Sanderson and Irwin
(1976) reported that 8 of 20 male game-farm mallards on a diet of corn and dosed with 5
#4 lead pellets died of acute lead poisoning an average of 7.6 days post-dosing afier
losing 20.5% of their body weight. The 12 remaining ducks died of chronic lead
poisoning an average of 20.7 days post-dosing and lost 47.6% of their body weight.
Sanderson et al. (1992) reported the average weight loss of game-farm mallards on a diet
of corn and dosed with 2, 4 or 8 #2 lead shot was 42.2 %, with a range of 16% to 56% for
individual ducks.

In the present study, there were statistically significant differences in body weights
at specific time points between the tungsten-iron- or tungsten-polymer-dosed ducks and
steel-dosed ducks (Tables 3 - 4). However, over the ISO-day period body weights
changed little. In males, there was a 3% drop in body weight in the steel- and tungsten-
iron-dosed ducks, while tungsten-polymer-dosed ducks had no change in body weight.
Steel-dosed females gained 9%, tungsten-iron-dosed females gained 8% and tungsten-
polymer-dosed females gained 14% of their original weight over the 150-day period. The
weight gain of the females was probably associated with an increase in food consumption

during the reproductive phase of the trial. Sanderson et al. (1997) reported that body

105

 

 

pan

weights of mallards dosed with 8 #4 bismuth alloy shot on days 0, 30, 60, 90 over a 150-
day period were similar compared to controls. The females, which were reproductively
active, were heavier than males at day 120. Ringelman et al. (1993) reported that
mallards dosed with 12 to 17 pellets of tungsten-bismuth-tin shot gained a similar amount
of weight as controls over 32 days. Mallards dosed with 8 BBs of tungsten-iron or
tungsten-polymer shot gained a slight amount of weight (0.9 to 5.8%) during a 30-day
period (Kelly et al., 1998).
Adult HCT, Hb Concentration, ALAD Activity

The low hematocrit, hemoglobin concentration and delta aminolevulinic acid
deyhdratase (ALAD) activity in lead-dosed mallards at day 7 are all indicators of lead
toxicity (Table 5). Lead poisoning is associated with two basic hematologic defects:
shortened erythrocyte lifespan and impairment of heme synthesis. Shortened lifespan of
the red blood cell may be due to increased mechanical fragility of the cell membrane. The
impairment of heme synthesis is due to the inhibition of ALAD. ALAD is a key enzyme
in the synthesis of heme, which is an integral component of hemoglobin (Goyer, 1996).
Pain and Rattner (1988) reported that hematocrit and hemoglobin concentrations were
significantly depressed in black ducks administered 1 #4 shot within 6 days of dosing but
recovery was apparent by 30 days post-dosing. ALAD activity was inhibited by 100% at
1 day postodosing, increased slightly between 3-9 days post-dosing (approximately 70%
inhibition) and then declined again until the end of the 30-day study. Finley et al. (1976)
reported that mallard drakes fed 25 ppm lead exhibited a 40% decrease in blood ALAD
activity 3 weeks after post-dosing and enzyme activity remained at this level through the

12-week treatment period. In the same study, ducks fed 5 ppm of lead in the diet for 12

106

 

weeks had a 36% decrease in blood ALAD activity. Since the inhibition of ALAD
activity has been shown to be a sensitive indicator of lead poisoning, the elevated ALAD
activity in tungsten-polymer-dosed ducks at day 7 was considered not to be biologically
significant

The slight, but statistically significant, decrease in hematocrit of tungsten-
polymcr-dosed females from day 90 through day 150 (Table 7) was not thought to be
treatment related but rather reflected to the reproductive status of all females in each
group. Hematocrits measured during this time were lower than hematocrits measured
during the first 60 days of the trial when birds were not reproductively active (Table 6).
Bell et al. (1965) and Sturkie (1976) reported that lowered hematocrit was associated with
egg production in birds. Similar results were reported by Sanderson et al (1997), in that
female mallards repeatedly dosed with 8 #4 bismuth alloy shot had a decline in average
hematocrit during reproduction. Tungsten has been shown to have no effect on
hematocrit in short-tenn studies (< 32 days) using game-farm mallards (Ringelman et al,
1993, Kelly et al 1998).
Adult Plasma Chemistries

The administration of lead shot caused a number of changes in day 7 plasma
chemistry values. The decrease in sodium concentration in lead-dosed mallards (Table 8)
may have been indicative of early renal disease associated with renal tubular damage
(Campbell and Coles, 1986). Plasma sodium concentration in the tungsten-polymer-
dosed group was statistically lower compared to the steel-dosed group, but within the

normal range reported for mallards (Lewandowski et al., 1986; Kelly, 1997).

107

 

The elevated concentrations of blood urea nitrogen and creatinine in leadodosed
mallards (Table 8) could indicate one of the following possibilities: pre-renal azotemia
(dehydration), renal azotemia (primary renal damage), and post-renal azotemia
(obstruction of the ureters) (Campbell and Coles, 1986). Histopathological examination
of the kidneys from the lead-dosed ducks suggests that pre-renal and renal azotemia are
the causes of the elevation in blood urea nitrogen and creatinine. However, blood urea
nitrogen, creatinine, and blood urea nitrogen/creatinine ratio are not considered useful
diagnostic tests for renal function in birds (Campbell and Coles, 1986). The depressed
plasma protein and albumin concentrations in the lead-dosed mallards (Table 8) was
probably associated with early signs of chronic renal disease and malnutrition (Campbell
and Coles, 1986). The elevation in albumin/globulin ratio in lead-dosed mallards (Table
8) reflected the depressed total protein and albumin concentrations.

Birds lack the enzyme biliverdin reductase needed to reduce biliverdin to
bilirubin, thus bilirubin accounts for only a small percentage of the total bile pigment
(Campbell and Coles, 1986). Since histopathological examination of lead-dosed mallards
indicated biliary dysfunction rather than biliary obstruction, the elevated concentration of
bilirubin in the lead-dosed mallards (Table 8) was associated with biliary dysfunction.
Elevated phosphorus concentration may be associated with renal disease, in which
concentrations can be 9.5 mg/dL or greater (Campbell and Coles, 1986). In the present
study, the significantly elevated phosphorus concentration in lead-dosed mallards (Table
8) was only slightly above concentrations considered to be normal (5.38 vs 2 - 4.5 mg/dL;
Campbell and Coles, 1986). The elevated concentration of uric acid in the lead-dosed

ducks (Table 8) may be an indication of starvation or renal disease. The increase in uric

108

I“!

 

acid concentration is thought to be a result of a decreased rate of tubular excretion plus
poor nutritional status, which can cause an increase in uric acid production as body
proteins are degraded (Campbell and Coles, 1986; March et al., 1976). However, while
the lead-dosed mallards had a marked increase in blood uric acid concentration, the value
was within the normal range (2 to 15 mg/dL) as reported by Campbell and Coles (1986).

The hepatic enzymes alkaline phosphatase, alanine aminotransferase, aspartate
aminotransferase, and lactate dehydrogenase can be useful diagnostic tests to determine
lead-poisoning in mallards. The depressed alkaline phosphatase activity observed in the
lead-dosed ducks (Table 8) is due to direct inhibition of the enzyme by lead (Rozman et
al., 1974). Although increases in the plasma activities of alanine aminotransferase,
aspartate aminotransferase, and lactate dehydrogenase are not specific to liver disease in
birds, the increased activities in the lead-dosed ducks (Table 8) were probably associated
with hepatocellular damage (Campbell and Coles, 1986). Rozrnan et al. (1974) and Kelly
et al. (1998) reported significant increases in plasma alanine aminotransferase activity in
mallards dosed with lead shot.

Triglyceride concentration tends to be low in fasted birds and then increases when
birds are refed (Herrnier et al., 1984). The decrease in triglyceride concentration in lead-
dosed ducks (Table 8) was probably due to inappetence associated with lead poisoning.

Since changes in chloride generally follow those of sodium, the decrease in
chloride concentration in male and female lead-dosed mallards (Table 9) was assumed to
be associated with the decrease in sodium concentration (Table 8). The chloride
concentration for tungsten-polymer-dosed females was within normal range (108 - 112

mmol/l) reported for mallards (Kelly, 1997). Calcium concentrations in male and female

109

 

lead-dosed ducks and tungsten-iron-dosed males were significantly decreased at day 7
(Table 9) and while hypocalcemia is associated with renal failure, the lead-dosed mallards
and tungsten-iron-dosed males had concentrations within the normal range (8 - 18
mg/dL) rcported by Campbell and Coles (1986). Lead-dosed male and female ducks had
marked elevated activities of creatinine phosphokinase (Table 9), which has been
reported to be associated with lead toxicity (Campbell and Coles, 1986, Kelly et al.,
1998). Since a diagnostic use for serum amylase has not been well investigated in birds
(Campbell and Coles, 1986), it is not clear what the biological relevance is of the
decrease in plasma amylase activity in the lead—dosed males and females (Table 9).

The plasma values reported for steel-, tungsten-iron-, and tungsten-polymer-dosed
mallards from day 30 through day 60 (Tables 10 - 12) and from day 90 through day 150
(Tables 13 — 14) are within the range of normal values reported in other studies
(Campbell and Coles, 1986; Lewandowski et al., 1986; Fairbrother et al., 1990;
Ringelman et al., 1993; Kelly, 1997; Kelly et a1. 1998). Thus, the occasional significant
difference in values between the tungsten-dosed ducks and the steel-dosed ducks were not
considered to be biologically relevant.

Adult Gross Pathology

The linings of the gizzards of 6 of 12 ducks in the lead-dosed group were
discolored (Tables 15 and 16). This effect has been described in 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 3 groups had gross lesions
within their gizzards. Other gross observations noted in the leadodosed ducks included

urate crystals surrounding the heart in one bird, which is consistent with visceral gout,

110

If. U l-‘La. .u

’IAI'JA h. P".

 

and enlarged gallbladders. Both of these lesions have been previously associated with
lead toxicosis (Slauson and Cooper, 1990; Alden and Frith, 1991; Popp and Cattley,
1991).

During the 90-day reproductive trial, 4 of 5 female ducks that did not lay eggs had
abnormalities that probably were responsible for their failure to lay eggs. The lack of
gross changes in mallards dosed with tungsten-iron and tungsten-polymer shot agrees
with findings reported by Ringelman et al. (1993) and Kelly et al. (1998), although
exposure periods in these studies were considerably shorter than in the present study.
Adult Organ Weights

The higher relative kidney, heart, brain, and gizzard weights of lead-dosed ducks
(Table 19) are associated with the significant weight loss (-61%) due to chronic lead
poisoning. Additionally, lower relative spleen weight in lead-dosed ducks (Table 19) can
be attributed to lead-induced atrophy of this organ (Rocke and Samuel, 1991). These
results are similar to those in the study by Kelly et al. (1998) who reported that relative
kidney and heart weights were significantly higher in mallards dosed with 8 #4 lead shot
as compared to control ducks. Sanderson et al. (1997) reported mallards dosed with 8 #4
lead shot had greater gizzard and kidney weights than controls.

The difference in relative gonad weights of lead-dosed male and female mallards
compared to the other 3 groups (Table 20) was due to the fact that lead-dosed ducks died
before becoming reproductively active. The increase in relative liver weight in lead-
dosed males (Table 20) was associated with the marked decrease in body weight. There
were no differences in relative liver weights of females. Although the depressed body

weight in lead-dosed females caused an increase in their relative organ weights, the liver

11]

"N"?

 

weights of the females in the other 3 groups were high because these ducks were
reproductively active.
Histopathology of the Adult Gonads, Liver and Kidneys

Microscopic renal lesions (acute tubular necrosis or nephrosis) were found only in
lead-dosed ducks (Tables 21 — 24). Acute tubular nephrosis is associated with lead
toxicosis in many animal species (Alden and Frith, 1991). The absence of renal lesions in
the steel-, tungsten-iron-, and tungsten-polymer-dosed ducks suggested that these metals
were non-toxic to the renal tubular epithelium, or that they were not absorbed in sufficient
quantities to produce renal tubular toxicity.

The primary hepatic lesions observed (Tables 21-24) were categorized as
substantial biliary stasis or liver hemosiderosis. The accumulation of bile within
hepatocytes or within canaliculi is somewhat nonspecific, as it may occur because of
obstruction of bile ducts, or primary hepatocellular dysfunction (Popp and Cattley, 1991).
In the present study, no evidence of cholelithiasis or other obstructive biliary disease was
detected, thus biliary stasis was considered evidence of hepatocellular dysfunction. As
previously mentioned, the increase found in plasma total bilirubin concentration in lead-
dosed mallards at day 7 suggested hepatocellular dysfunction, rather than biliary
obstruction. The degree of biliary stasis was graded, and only the lead-dosed group had
detectable biliary stasis. Hemosiderosis was only found in the steel and tungsten-iron
groups with the exception of one male from the tungsten-polymer group. Hemosiderosis
(deposition of iron in the form of hemosiderin) commonly occurs when ducks are fed
iron-containing shot (Locke et al., 1967). Additionally, intrahepatocellular fatty

vacuolation was present in at least half of the ducks in each of the 4 experimental groups

112

.‘O‘ . this ‘I'nk'. .110. t .' ~ "1" -. u.’ r» ;. ~

 

with the exception of males in the steel, tungsten-iron, and tungsten-polymer groups.
Fatty accumulation can be due to a variety of causes and was judged as an incidental
finding in this study.

The gonads from the lead-dosed mallards were inactive and no histologic lesions
were found. The testes and ovary from steel, tungsten-iron, tungsten-polymer groups
were all normal.

Metal Residues in Tissues of Adults

Iron was detected in femur, gonads, kidneys, and liver samples in all treatment
groups (Tables 26, 27, 29, 30). In general, the concentration of iron was highest in the
tissue samples from the tungsten-iron- and steel-dosed ducks. Moreover, the iron
concentrations in samples of the femur, kidneys, and gonads from tungsten-iron- and
steel-dosed females were generally higher than in the males. The sex-related difference
in iron concentration was related to physiological changes in the female in preparation for
the egg-laying season. Underwood (1971) reported a 5-fold increase in iron in the serum
of ducks during the egg-laying season. In contrast, the physiological changes due to egg-
laying obviously do not apply to lead-dosed mallards because none of these ducks were
reproductively active. The elevated concentrations of iron in the lead-dosed females
could be attributed to lead-induced interference of heme synthesis, which caused an
accumulation of iron in the liver. The high concentration of iron in the liver of lead-
dosed ducks agrees with results reported by Sanderson et al. (1992) and Kelly et al.
(1998)

The high concentration of iron in liver samples from steel- and tungsten-iron-

dosed ducks were associated with the histological findings of hemosiderosis. Locke et

113

Jfi’
‘.

V

:

 

 

 

al. (1967) dosed mallards with 8 pellets of iron shot, which resulted in hemosiderosis of
the liver and hepatic iron concentrations ranging fi'om 3,185 to 6,131 ppm. Because liver
hemosiderosis commonly occurs when ducks are fed iron-containing shot, Rozrnan et a1.
(1974) investigated the effects of hemosiderosis on the hepatic enzymes alkaline
phosphatase, aspartate arninotransferease, and alanine aminotransferase and found no
significant changes of these enzymes in groups of ducks receiving up to 64 #4 steel shot
when compared to control ducks. In the present study, the activity of plasma enzymes
alkaline phosphatase, aspartate aminotransferease, and alanine aminotransferase had no
significant changes in the steel- and tungsten-iron-dosed ducks when values were
compared to those of control mallards fi'om F airbrother et al. (1990).

Lead was generally detected in femur, gonad, kidney, and liver samples from all
treatment groups with the exception of gonad samples from the tungsten-iron-dosed
group (Table 25, 27, 28, 30). Concentrations of lead in the lead-dosed ducks were
approximately 100 to 6000 fold higher when compared to the other 3 groups. Kelly et al.
(1998) reported concentrations of lead in the femur, liver, and kidneys of all mallards on
trial with the highest concentrations being in the lead-dosed ducks. In the present study,
lead concentrations were highest in the kidneys, intermediate in the femur and liver, and
lowest in the gonads. In contrast, Havera et a1. (1992) reported wild mallards redosed
with lead shot had lead concentrations highest in the wing bone, intermediate in the
kidney, and lowest in the liver.

In the tungsten-iron-dosed ducks, the number of femur, gonad, kidney, and liver
samples that tungsten was detected in and the concentration of tungsten in these tissue

samples were substantially greater when compared to the tungsten-polymer—dosed ducks

114

 

(Tables 26—28, 30). The bone, liver, and kidneys are principle sites of tungsten deposition
in a number of different species (Kinard and Aull, 1945; Wase, 1956; Kaye, 1968; Bell
and Sneed, 1970; Aamodt, 1975) and the primary site of tungsten deposition is species-
specific. In the present study, the concentration of tungsten was highest in the liver,
intermediate in the femur, and lowest in the kidneys and gonads. These results agree with
Kelly et al. (1998) who reported tungsten concentrations highest in the liver, intermediate
in the femur, and lowest in the kidneys from mallards dosed with tungsten-iron or
tungsten-polymer shot. Ringelman et al. (1993) did not detect tungsten in either the liver
or kidneys from mallards dosed with tungsten-bismuth-tin shot. However, the proportion
of tungsten in the tungsten-bismuth-tin shot was 39%, while in the present study, tungsten
concentrations were 55% and 95.5 % for tungsten-iron and tungsten-polymer shot,
respectively.

Tungsten was also detected in the kidneys of 2 steel-dosed and 3 lead-dosed ducks
(Table 28). It was thought this was due to the normal variance one can expect from
readings near the instrument’s detection limit that may have been accentuated by “noise”
induced by a complex matrix such as animal tissue (personal communication, CT&E
Environmental Services).
Shot Recovery and Percent Shot Erosion

Lead-dosed ducks had the highest percent of shot recovered (86%), followed by
steel (59%), tungsten-iron (39%), and tungsten-polymer (2%). Since all lead-dosed ducks
died by day 25 and the steel-, tungsten-iron-, and tungsten-polymer-dosed groups
survived until day 150, the high recovery shot rate seen in lead-dosed ducks was

expected.

115

 

Percent shot erosion in male ducks dosed with steel, lead, tungsten-iron, and
tungsten-polymer shot was 50%, 22%, 64%, and 99%, respectively (Table 31). Percent
shot erosion in female ducks dosed with steel, lead, tungsten-iron, and tungsten-polymer
shot was 60%, 15%, 80%, 99%, respectively. These results were substantiated during
fluoroscopy of ducks in that steel and lead pellets were readily visible while the tungsten-
iron and particularly the tungsten-polymer pellets were often difficult to see because of
disintegration. Kelly et al. (1998) reported similar findings from a 30-day test with
percent shot erosion highest in tungsten-polymer-dosed ducks (80%), intermediate in
tungsten-iron- and lead-dosed ducks (55% and 50%, respectively), and lowest in steel-
dosed ducks (33%). Furthermore, Kelly et al. (1998) compared the percent shot erosion
in the lead-dosed ducks that survived the 30-day trial (71%) to the lead~dosed ducks that
died during the 30-day trial (34%). These results for the lead-dosed ducks that died in the
Kelly et al. (1998) study are similar to those reported in the present study.

Date First Egg was Laid and Number of Days Required to Lay 21 Eggs

The administration of tungsten-iron or tungsten-polymer shot did not have an
effect on the commencement or duration of egg laying by female mallards (Table 32).
The fact that 4 egg-laying females (1 steel-dosed, 2 tungsten-iron-dosed, 1 tungsten-
polymer-dosed) did not lay 21 eggs may have been the result of individual variation. The
removal of eggs from incubating mallards will result in the continuation of egg laying
whereas retention of the clutch will terminate egg-laying. It is possible that these 4
females mimicked the behavior seen in wild mallards, which terminate egg laying after a
clutch of eggs has been laid. In the present study, egg laying began at day 92, 92, and 85,

and the days required to lay 21 eggs were 25, 24, and 26 for the steel-, tungsten-iron-, and

116

 

11...:

”V

 

 

tungsten-polymer-dosed females, respectively. These results agree with those of
Sanderson et al. (1997) who reported that egg-laying in control mallards began on day 84,
on day 94 for iron-dosed females, and on day 92 for bismuth-dosed females The mean
range to lay 21 eggs was 26 to 27 days for the 3 groups in the latter study.
Percent Egg Production, Fertility, and Hatchability

In our study, tungsten did not have an apparent effect on the rate of egg
production, fertility, or hatchability (Table 33). These findings are similar to those of
Teekell and Watts (1959) who reported that supplementation of the diet of breeder hens
with 250 or 500 ppm tungsten had no adverse effect on rate of egg production or
hatchability. The slight decrease in percent fertility of eggs laid by tungsten-polymer-
dosed females may be because tungsten-polymer-dosed females became reproductively
active earlier than tungsten-polymer-dosed males. Four of the 13 tungsten-polymer-dosed
females that laid eggs did not begin to lay fertile eggs until after the 17th egg was laid.
Similarly, there were 3 steel-dosed females that did not produce fertile eggs until after the
12“1 egg was laid.
Egg Weight and Shell Thickness

The weight and shell thickness of eggs from tungsten-iron-dosed ducks were
statistically greater compared to eggs from steel- and tungsten-polymer-dosed females
(Table 34), but the difference was not considered biologically relevant. In our study, the
egg weights were 61, 63, and 61 grams and shell thicknesses were 0.372, 0.412, and
0.385 mm for the eggs from the steel-, tungsten-iron-, and tungsten-polymer-dosed ducks,

respectively. Sanderson et al. (1997) reported similar findings with egg weights of 61.2,

117

 

61.2, and 61.3 grams and shell thickness of 0.335, 0.338, and 0.335 mm for control, iron-
dosed and bismuth-dosed mallards, respectively.
Metal Residues in Egg Shell and Contents

Iron concentration in egg contents was highest in the steel-dosed group,
intermediate in the tungsten-iron-dosed group, and lowest in the tungsten-polymer—dosed
group (Table 35). The presence of iron in the contents of eggs is associated with a 5-fold
increase of iron in the serum during the egg-laying season in ducks (Underwood, 1971).

Tungsten was detected in the shell and contents of eggs from tungsten-iron-dosed
ducks and in the shell of eggs from tungsten-polymer-dosed ducks (T able 35). The
concentration of tungsten in the eggs followed the same trend as in the adult tissue
samples. Tungsten was detected in 9 shells of eggs from tungsten-iron-dosed females at a
concentration that was higher compared to the concentration of tungsten detected in 3
shells of eggs from tungsten-polymer-dosed females. The presence of tungsten in shells
can be attributed to the fact that calcium-containing tissues are among the principle sites
of tungsten deposition (Kinard and Aull, 1945; Wase, 1956; Kaye, 1968; Bell and Sneed,
1970; Aamodt, 1975).
Survivability, Body Weight, and Hematocrit of Ducklings

The administration of tungsten-iron and tungsten-polymer shot had no adverse
effects on the survivability, body weight. or hematocrit of ducklings (Table 36). The
slight but significant decrease in hematocrit of tungsten-iron ducklings was not
considered biologically relevant. Sanderson et al. (1997) reported bismuth alloy shot
caused no adverse effects on duckling survivability, body weight (day 7), or hematocrit.

Duckling Organ Weights

118

 

Ducklings in the tungsten-polymer group had slightly, but significantly greater
absolute and relative kidney weights compared to ducklings in the other 2 groups (Tables
37, 38). This difference was considered not to be biologically relevant.

Histopathology of Duckling Liver and Kidneys

The most common finding in the liver of ducklings in all treatment groups was
mild to moderate hepatocellular vacuolation (Tables 40 and 41). Based on the ducklings’
young age, this condition was considered normal and was due primarily to hepatic
glycogen accumulation. Sanderson et al. (1997) reported a similar condition in the liver
of ducklings from a reproduction study that assessed the effects of bismuth alloy shot.
There were no histologic lesions present in the kidneys of the ducklings.

Metal Residues in Tissues of Ducklings

Iron concentration was highest in the liver, intermediate in the kidneys, and lowest
in the femur samples from ducklings (Table 42). Sanderson et al. (1997) reported similar
findings in that iron concentration was highest in the liver and lowest in the kidney from
ducklings of mallards dosed with bismuth alloy shot. Lead was detected in trace amounts
in the femur, kidneys, and liver samples from the ducklings. Sanderson et al. (1997) also
reported the presence of lead in the liver and kidneys of the ducklings. Tungsten was
detected in relatively few samples of the femur, kidneys, and liver from ducklings of

tungsten-iron- and tungsten-polymer-dosed females.
Conclusions

Male and female mallards administered 40 #4 tungsten-iron or tungsten-polymer
shot and maintained for 150 days were not adversely affected based on the variables

measured. All ducks, with the exception of lead-dosed mallards, survived the ISO-day

119

 

trial. No significant differences were observed in HCT, Hb concentration, and ALAD
activity at day 7 in the 2 tungsten shot groups when compared to the steel-dosed group.
The differences in hematocrit and plasma chemistry variables that occurred from day 30
through day 150 were within the normal range for mallards and thus were not considered
biologically relevant. The ducks appeared normal at the time of necropsy on day 150 of
the trial, and no deleterious changes were detected in weights of organs. Three of 8
tungsten-iron-dosed females, 8 of 8 tungsten-iron-dosed males, and 1 of 8 tungsten-
polymer-dosed males manifested mild to moderate liver hemosiderosis, which was not
considered deleterious. Similarly, liver hemosiderosis was present in 5 of 8 steel-dosed
females and 8 of 8 steel-dosed males. No other histopathological lesions were noted.
Tungsten residues were generally detected in the femur, gonads, kidneys, and liver of
tungsten-iron- and tungsten-polymer-dosed ducks. Concentrations of tungsten were
generally higher and occurred in more tissue samples from the tungsten-iron-dosed ducks
compared to tungsten-polymer-dosed mallards. The erosion rate of tungsten-polymer
shot was 28% greater than the erosion rate of tungsten-iron shot, which was 35% and
17% greater than the erosion rates of lead and steel shot, respectively. There were no
significant differences in percent egg production, fertility and hatchability in the 2
tungsten-dosed groups when compared to the steel-dosed group. Similarly, there were no
relevant differences in egg weight and shell thickness of eggs from tungsten-iron and
tungsten-polymer-dosed ducks. Tungsten-residues were detected in the shell of 9 of 16
eggs and in the contents of 6 of 16 eggs from tungsten-iron-dosed females. The
concentration of tungsten was slightly above the detection limit in the shell of 3 of 13

eggs from tungsten-polymer-dosed ducks. No relevant differences were observed in

120

 

 

 

duckling survivability, body weight, or hematocrit when compared to ducklings from
steel-dosed ducks. Absolute and relative kidney weights of ducklings from tungsten-
polymer ducks were slightly greater when compared to ducklings from steel-dosed ducks.
No other significant differences in duckling organ weights were observed. All ducklings
had mild to moderate hepatocellular vacuolation, which was considered normal. No other
histopathological lesions were noted. Tungsten residues were detected in the femur of 4 1
of 16 tungsten-iron and tungsten-polymer ducklings, in the kidney of 2 of 16 and l of 16

tungsten-iron and tungsten-polymer ducklings, and in the liver of 2 of 16 tungsten-iron

 

and tungsten-polymer ducklings. Because the two formulations of tungsten-shot were '1]
non-toxic to mallards after 150 days exposure, they have potential for permanent use for .
waterfowl hunting. If one were to compare steel, tungsten-iron, and ttmgsten-polymer

shot, tungsten-polymer shot seems preferable because of its high erosion rate and its low

concentrations in selected tissues, when compared to steel and tungsten-iron shot.

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