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This is to certify that the
thesis entitled

THE EFFECTS OF AIR CELL INJECTION OF
PERFLUOROOCTANE SULFONATE PRIOR TO INCUBATION ON
THE DEVELOPMENT OF THE WHITE LEGHORN CHICKEN
(GALLUS DOMESTICUS) EMBRYO

presented by

Elizabeth Denisse Molina

has been accepted towards fulfillment
of the requirements for the

Master of Science degree in Animal Science

 

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THE EFFECTS OF AIR CELL INJECTION OF PERFLUOROOCTANE
SULFONATE PRIOR TO INCUBATION ON THE DEVELOPMENT OF THE
WHITE LEGHORN CHICKEN (GALLUS DOMESTICUS) EMBRYO

By

Elizabeth Denisse Molina

A THESIS
Submitted to
Michigan State University
In partial fulfillment of the requirements for the degree of
MASTER OF SCIENCE

Department of Animal Science

2004

ABSTRACT

THE EFFECTS OF AIR CELL INJECTION OF PERFLUOROOCTANE
SULFONATE PRIOR TO INCUBATION ON THE DEVELOPMENT OF THE
WHITE LEGHORN CHICKEN (GALLUS DOMESTICUS) EMBRYO
By
Elizabeth Denisse Molina
Fifty White Leghorn chicken (Gal/us domesticus) eggs per group were injected
with 0.1, 1.0, 10.0 or 20.0 pg perfluorooctane sulfonate (PFOS)lg egg prior to
incubation to investigate the effects of PFOS on the developing embryo. At hatch,
chicks were weighed and examined for gross developmental abnormalities and
then transferred to a battery brooder where they were raised for seven days.
Twenty chicks per treatment were randomly chosen for necropsy, weighed and
killed by cervical dislocation. The brain, heart, kidneys and liver were removed
and weighed. Five livers were immediately frozen on dry ice and stored at -80°C
for subsequent determination of PFOS concentrations and five livers were
preserved in a 10% formalin solution for subsequent histological examination.
Hatchability was significantly reduced in all treatment groups in a dose-
dependent manner. The calculated LDso was 0.97 ug PFOS/g egg. PFOS did not
affect post-hatch body or organ weights. Exposure to PFOS caused pathologic
changes in the liver characterized by bile duct hyperplasia, periportal
inflammation and hepatic cell necrosis at doses as low as 1.0 pg PFOS/g, wet wt
egg. Hepatic PFOS concentrations increased in a dose-dependent manner.

Based on reduced hatchability, the lowest observed adverse effect level (LOAEL)

was 0.1 ug PFOS/g egg.

DEDICATION
To my family, Marni, Papi, Cesar y Fernando Molina and my husband Jesse

Sweeney who supported me throughout this entire venture.

TABLE OF CONTENTS

PAGE
LIST OF TABLES ........................................................................... vi
LIST OF FIGURES ......................................................................... vii
INTRODUCTION ............................................................................ 1
LITERATURE REVIEW .................................................................... 6
Fluorine ....... 6 ...................................................................... 6
F luorocarbons ....................................................................... 7
Sulfonyl-Based Fluorochemicals ............................................... 9
PFOS in the Environment ....................................................... 11
Toxic Effects ........................................................................ 14
Mechanisms of Action ............................................................ 16
MATERIALS AND METHODS .......................................................... 20
RESULTS .................................................................................... 25
DISCUSSION ............................................................................... 35
CONCLUSION .............................................................................. 45
REFERENCES ............................................................................ 46

TABLE

LIST OF TABLES
PAGE

Concentrations of perfluorooctane sulfonate in liver

(pg/g, wet wt),blood plasma (pg/ml), eggs (pg/ml) and

muscle (nglg, wet wt) of humans, aquatic mammals,
fish—eating water birds, waterfowl and fish throughout

the world ...................................................................... 4

Effect of perfluorooctane sulfonate injected into the
air cell of White Leghorn chicken embryos prior to
incubation on hatchability ............................................. 26

Effect of perfluorooctane sulfonate injected into the
air cell of White Leghorn chicken embryos on body
weights upon hatching .................................................. 27

Effect of perfluorooctane sulfonate injected into the air
cell of White Leghorn chicken embryos on body weights
at one week of age ...................................................... 28

Effect of perfluorooctane sulfonate injected into the air
cell of White Leghorn chicken embryos on absolute
organ weights at one week of age .................................. 29

Effect of peifluorooctane sulfonate injected into the air
cell of White Leghorn chicken embryos on relative organ
weights at one week of age ........................................... 30

Histopathology of livers from seven-day-old White
Leghorn chickens exposed to perfluorooctane sulfonate
in ovo via air cell injection prior to incubation ..................... 32

Hepatic perfluorooctane sulfonate concentrations of
seven-day-old White Leghorn chickens exposed to
perfluorooctane sulfonate in ovo via injection into the

air cell ...................................................................... 33

Concentrations of petfluorooctane sulfonate in
chicken eggs from domestic commercial
sources .................................................................... 34

FIGURE

LIST OF FIGURES

PAGE
The structure of perfluorooctanesulfonyl fluoride
(POSF) which is the basic building block of
perfluorooctyl sulfonates ................................................ 2
The structure of perfluorooctane sulfonate(PFOS)
which is the ultimate degradation product of
POSF-compounds ....................................................... 3

vi

INTRODUCTION

Sulfonated fluorochemicals were first manufactured in Decatur, AL by
Minnesota Mining and Manufacturing Company (3M) in 1961. They have been
widely used in a variety of products as surfactants, as carpet, textile and paper
protectants and as fire fighting foams (USEPA, 2000a). The basic building block
of sulfonated fluorochemicals is perfluorooctane sulfonyl fluoride (POSF) (Figure
1). POSF is used to create many different products, all of which may degrade to
a compound called perfluorooctane sulfonate (PFOS) (Figure 2), which cannot
be metabolized any further (USEPA, 2000a). Time frames for POSF-derived
product degradation are variable, in that some fluorochemical-containing
polymers are stable for long periods of time and some degrade very quickly.
Thus, the rate at which POSF—derived products degrade to PFOS remains
uncertain (USEPA, 2000b).

PFOS is widespread in the environment. Its presence has been reponed

in water, soil, fish, birds and mammals including mink (Mustela vison), river otters
(Lutra canadensis), polar bears (Ursus man'timus), seals (Phoca hispida) and

humans (Table 1). Its stability, persistence, ubiquitous presence and potential for
bioaccumulation makes PFOS a chemical of concern. As a result, the United
States Environmental Protection Agency (USEPA) mandated that PFOS and all
related perfluorinated compounds be phased out of the market within a three
year period beginning in 2000 and the mayor manufacturer of PFOS (3M) agreed

(USEPA, 2000a).

F F F F F F F o
I l I I I I I II
F-C-C-C—C—C-C-‘C-S-O-F
I I I I I I I II
F F F F F F F 0

Figure 1.The structure of perfluorooctanesulfonyl fluoride (POSF) which is the
basic building block of perfluorooctyl sulfonates.

F F F F F F F o

I I I l I I I II
F-C-C-C—C-C—C-C-S—O‘

I I I I I I I II

F F F F F F F 0

Figure 2. The structure of perfluorooctane sulfonate (PFOS) which is the ultimate
degradation product of POSF-based compounds

Table 1. Concentrations of perfluorooctane sulfonate in liver (nglg, wet wt), blood
plasma (pg/ml), eggs (pg/ml) and muscle (nglg, wet wt) of humans, aquatic
mammals, fish-eatirqwater birds, waterfowl and fish throughout the world.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

. . . PFOS b
SpeCIes LocatIon Tissue Concentration. Reference
Humans Liver <4.5-570 nglg 2

Plasma <6.1-58.3 nglg 2
Aquatic mammals
Ringed seal Baltic Sea Liver 0.13-1.10 (0.490) 4
Polar bear Alaska Liver 0.18-0.68 (0.35) 6
Mink Illinois Liver 0093-514 (1.61) 5
. . 0.097-0994
River otter Oregon LIver (0 579) 5
Ringed seal Canadian Arctic Plasma <0003-0012 6
Ringed seal Baltic Sea Plasma 0016-023 (0.110) 6
. . 0.011-0049
Gray seal CanadIan Arctic Plasma (0.028) 6
Fish-eating water
birds
Brandt’s . .
c o rm orant San DIego, CA LIver 0046-178 (0.907) 7
Sea gull Tokyo Bay, Japan Liver 0.230 1
Common .
connorant Tokyo Bay, Japan LIver 0.17-0.65 (0.39) 1
Bald eagle Midwestern US Plasma 0001-257 (0.36) 6
Laysan albatross North Pacific Ocean Plasma 0006-0034 (0014) 6 I
Double crested Lake Winnipeg,
corrnorant Canada Egg 013-032 (0.21) 6 I
Double crested
cormorant Great Lakes Egg 0.57-1.800 (1.3) 8 I
Caspian tern Great Lakes Egg 1.9-3.4 (2.6) !
Waterfowl
Mallard Tokyo Bay, Japan Liver 0.493
. . . 0.239-0497
PintaIl duck Tokyo Bay, Japan LIver (0. 368)
Fish
Chinook salmon Michigan waters Liver $031369 1 70
Yellow fin tuna North Pacific Ocean Liver >0007
. . . 0254-0310
Blue gill Lake BIwa, Japan LIver (0.282)
Ornate jobfish 33:31“ (Km 3“” Liver 0593-79 (3.25)
Lake whitefish Micfigan waters Egg 0.15-0.38 (0.26) 6
Carp Saginaw Bay, Ml Muscle 006-03 (0.12) 6
Data are expressed as the range with the mean in parentheses.
I’1 = Kannan et al., (2003), 2 = Olsen et al. (2003c), , 3 = Taniyasu et al., (2003), 4 = Kannan
et al., (20023), 5 = Kannan et al., (2002b), 6 = Giesy and Kannan, (2001), 7 = Kannan et al.,
(2001a)I 8 = Kannan, (Ersonal communication) L

PFOS has not been studied as thoroughly as other persistent organic pollutants
Such as organobromines and organochlorines. Epidemiological studies have
indicated the presence of PFOS in blood plasma and livers of occupationally
exposed humans as well as members of the general population (Olsen et al.,
1999; USEPA, 1999; Hansen et al., 2001). No adverse effects have yet been
associated with reported concentrations of PFOS in humans.

In mammals, PFOS caused hepatic changes including induction of hepatic
enzymes, hepatic vacuolization, as well as hypertrophy and disruption of gap
junction intercellular communication. Additional changes included gastrointestinal
effects, weight loss, convulsions and death (Goldenthal et al., 1978b; Seacat et
al. 2003). PFOS may also cause reproductive toxicity, characterized by neonatal
mortality and developmental delays (Lau et al., 2003).

Only a few studies assessing the effects of PFOS in avian species have
been conducted (Gallagher et al., 2000a; 2000b). Two dietary studies conducted
in the mallard (Anas platyrhynchos) and the northern bobwhite (Colinus
virginianus) reported L050 values of 628 and 293 pg PFOS/g feed, respectively.
Mortality of 90% of the mallards and 100% of the northern bobwhites occurred at
the highest dietary concentration (1171 pg PFOS lg feed) before day seven of
the trial.

During preliminary trials conducted in our laboratory, PFOS was detected
in non-injected eggs collected at the Michigan State University’s Poultry Science
Research and Teaching Center (0.33 ug/g egg) and in those purchased from a

local supermarket (0.34 to 0.70 ug/g egg). This finding suggests maternal

the effects of PFOS on the development of the avian embryo using an egg
injection technique. Specific objectives of this study were to determine the dose
of PFOS that resulted in 50% mortality (LDso) and to evaluate the chicken as a

suitable model to further study the effects of PFOS in wild avian species.

LITERATURE REVIEW

Fluorine

Fluorine (F) is responsible for the distinctive properties of all
fluorochemicals. Fluorine is a yellow, poisonous, highly corrosive gas that does
not exist in its elemental form due to its high reactivity. Fluorine reacts with most
organic and inorganic compounds and all of the elements in the periodic table
except for nitrogen, chlorine, oxygen and the inert gases. Because it is the most
electronegative of all elements in the periodic table, F forms very strong bonds
with electropositive elements (Banks and Tatlow, 1994).

One of the first uses of F can be traced to the 15th century. A naturally
occurring F-containing mineral called fluorspar (Can), also known as fluorite,
was used to facilitate the smelting of ores. Fluorspar was later used to obtain
hydrofluoric acid (HF). It is believed that HF may have been prepared in the
1700s by an unknown English glassworker who discovered that, by adding
sulfuric acid to fluorite, he could obtain a corrosive vapor that could be used to
etch glass. Due to F toxicity and high reactivity, many researchers were severely
injured or died during their ill-fated attempts to isolate this element. Not until 1886

was elemental F isolated in France by Henri Moissan, who used electrolysis of

anhydrous HF and potassium fluoride (Weeks, 1945).

Today, fluorine continues to have many uses. Sodium fluoride is added to
city water supplies and is used in toothpaste, in combination with stannous
fluoride and sodium monofluorophosphate, to help prevent tooth decay. HF is
used in etching semiconductor devices and in the cleaning and etching of glass,
including most of the glass used in light bulbs. Hydrofluoric acid is also used in
the synthesis of uranium tetrafluoride and uranium hexafluoride, both of which
are used in the nuclear industry to separate isotopes of uranium (Liteplo, 2002).
Fluorocarbons

One of the most significant uses of F is in the production of fluorocarbons.
When F, the most electronegative of the elements in the periodic table, is joined
to carbon (C), the C-F bond that results is one of the strongest bonds in nature
(~110 kcal). This bond requires a large amount of energy to form or break. As a
result, biological synthesis of the C-F bond is rare, and those fluorocarbons that
are synthesized chemically are very stable, extremely difficult to break down and,
in most cases, are persistent in the environment (USEPA, 1999).

Fluorocarbons can be synthesized by electrochemical fluorination (ECF)
through a process developed by Dr. Joseph Simmons at Pennsylvania State
University in 1944. The ECF process entails substituting hydrogen atoms in
hydrocarbon molecules dispersed in anhydrous hydrogen fluoride and passing
an electric current through the solution causing the replacement of hydrogen
atoms by fluorine (Alsmeyer et al., 1994).

One type of fluorocarbon is the chlorofluorocarbon (CFC).

One type of fluorocarbon is the chlorofluorocarbon (CFC).
Chlorofluorocarbons are organic compounds that contain chlorine, fluorine and
carbon. Frédéric Swarts first synthesized chlorofluoromethane in 1890. In 1928,
dichlorodifluoromethane was identified as a safe refrigerant for domestic
refrigerators built by Frigidaire, a General Motors (GM) subsidiary. General
Motors approached DuPont to jointly manufacture and market this product, which
was subsequently introduced under the trade name Freon” (DuPont, 2003).
DuPont introduced Freon 12 in 1931 followed by other FreonTM mixtures. The
most common CFCs were dichlorodifluoromethane (CFC-12) and
trichlorofluoromethane (CFC-11). CFCs were widely used in air conditioning
refrigeration systems and in aerosol spray cans until 1994 when they were
phased out by DuPont, because of damage caused to the ozone layer (Powell
and Stevens, 1994).

Another fluorochemical of particular interest is polytetrafluoroethylene
(PTFE), also known as Teflon”, a fluoropolymer originally discovered by Roy
Plunkett at DuPont Laboratories in 1938. Plunkett was working with Freon-
related gases when he inadvertently froze and compressed a sample of
tetrafluoroethylene, which caused polymerization of the chemical resulting in
PTFE. Large scale production of PTFE began in 1946 by DuPont for commercial
use as a lining for frying pans and other utensils and as a soil and stain repellant
for fabrics and textile products. Inert to virtually all chemicals, PTFE is
considered the most slippery material in existence (Dupont, 2003; Banks and

Tatlow, 1994). Fluoropolymers are unique among plastics because of their

be developed. However, perfluorooctanoic acid (PFOA), a component of
Teflon”, is currently being reviewed due to a preliminary assessment by the
USEPA that indicated potentially nationwide human exposure to low
concentrations of PFOA. Based on animal studies, there is a potential risk of
developmental and other adverse effects associated with PFOA in humans
(USEPA, 2003).

Sulfonyl-based Fluorochemicals

In 1944, 3M acquired the rights for the ECF process patent. Sulfonyl-
based fluorochemicals were then synthesized by ECF, utilizing 1-octanesulfonyl
fluoride, HF, and an electric current to yield a mixture of perfluorinated
fluorocarbons. The majority of the resulting products in this mixture have the
same chain length and skeletal arrangement as the organic molecules used as
feedstock. However, some fragmentation and rearrangement of the molecules
occurs resulting in products of various chain lengths, branched molecules and
cyclic compounds that are later used in a number of different products (USEPA,
1999)

Once fluorochemicals were manufactured, the challenge to create new
products from sulfonyl-based fluorochemicals ensued. In 1953, Dr. Patsy
Sherman, a 3M researcher, discovered the surfactant properties of these
fluorochemicals by accident. A laboratory assistant in Shennan’s lab spilled a
small amount of this experimental chemical on ShemIan’s tennis shoes. Their
inability to clean up the spill, both on the floor and on her shoe, with water,

alcohol, or any other solvent, led to the realization that this compound could

protect fabrics from water and soiling. Three years later, Scotchguardm Protector
was launched in the marketplace followed by many other Scotchguard'" brand
products (3M, 2003).

The characteristics of sulfonated fluorochemicals are a direct result of their
chemical structure. The general structure of a sulfonated fluorochemical consists
of a suffonated moiety with an attached fluorinated tail. The sulfonated portion of
the molecule makes the molecule highly soluble (hydrophilic) and the fluorinated,
non-polar, insoluble portion repels both oil and water. The combination of a
hydrophilic moiety with a hydrophobic and oleophobic portion gives fluorinated
chemicals their desired surfactant properties. Moreover, their surfactant
properties make them more effective than their non-fluorinated surfactants
predecessors (hydrocarbons and silicone surfactants). This is due to the
molecule’s stability and the resistance of the C-F bond to breakdown, even in the
most extreme chemical and thermal conditions (USEPA, 2000a).

SulfonyI-based fluorochemicals have been used in various products that
can be divided by function into three groups: surface treatments, paper
protectors, and performance chemicals. In 2000, SM produced approximately
6.6 million pounds of sulfonyl-based fluorochemicals for all uses in the US
(USEPA, 2000a).

Surface treatment provides protection against oil, water and soil for
carpets, upholstery, furniture and apparel fabric. This protectant may be applied
by carpet manufacturers, by the consumer (Scotchguardm) or through

aftermarket carpet treatments. Production volume for surface treatments was

10

approximately 2.4 million pounds, 36% of the total amount of sulfonyl-based
fluorochemicals produced (USEPA, 2000a).

Paper protectors provide oil, grease and water resistance. They are widely
used in food packaging applications including food wraps, bags, containers and
plates. They are also utilized in non-food contact applications such as folding
cartons and carbonless forms. Production volume for paper protectors was
approximately 2.7 million pounds, 41% of the total production (USEPA, 2000a).

Performance chemicals are used in many specialized industrial
applications including fire fighting foams, mining and oil well surfactants,
photographic film, shampoos, dental cleaners, carpet spot cleaners, floor
polishes and insecticides. Production volume for performance chemicals was
approximately 1.5 million pounds, 23% of the total amount of sulfonyl-based
fluorochemicals produced (USEPA, 2000a).

Surface treatments and paper protectants comprised the largest volume of
sulfonyl-based chemical production and may present the greatest potential for
human exposure. This is due to the great volume in which they were produced
and their proximity to the human body through food, clothing and personal
hygiene products (USEPA, 2000a).

Sulfonyl-based fluorochemicals derived from POSF degrade to
perfluorooctane sulfonate (PFOS) (Figure 2), which cannot be metabolized any
further. PFOS is an eight-carbon fluorinated molecule that has a sulfonate group
on the terminal carbon and is part of the sulfonyl-based fluorochemical family

(USEPA, 20003).

11

PFOS in the Environment

Fully fluorinated (perfluorinated) molecules like PFOS are very stable and
extremely difficult to break down and thus are persistent in the environment
(USEPA, 1999). The degree of fluorination has an effect on both the bond length
and strength. When fluorination is increased, the bond length is decreased
making the bond tighter and stronger. As a result, perfluorinated compounds tend
to be resistant to hydrolysis, photolysis and microbial and metabolical
degradation by vertebrates (Giesy and Kannan, 2001).

The occurrence of organofluorines in the environment was reported as
early as 1960. However, it was not until the 19905 that the technology became
available to identify specific perfluorinated compounds in biological matrices and
to quantify their presence with accuracy.

Epidemiological studies on occupationally exposed humans reported the
presence of PFOS in blood plasma at concentrations below 6ppm; however, no
adverse effects were associated with these concentrations of PFOS in humans
(Olsen et al., 1999). PFOS was reported in the serum of the general population in
concentrations lower than those found in occupationally exposed workers.
Analyses of commercially available serum samples and blood bank pooled
serum from the US revealed mean PFOS concentrations of approximately 28.4
ng/ml and 29.7 ng/ml, respectively (USEPA, 1999; Hansen et al., 2001).

The presence of PFOS in human livers was recently reported in a human
donor study. The mean hepatic PFOS concentration was 18.8 ng/g (Olsen et al.,

2003c). The PFOS liver to serum ratio was 1.3:1, indicating that the blood mainly

12

distributes PFOS as it concentrates in the liver (Hansen et al., 2001). This ratio
can be useful in human risk assessment estimates as PFOS blood serum
concentrations can estimate PFOS liver concentrations (Olsen et al., 2003c).

PFOS is easily absorbed through the digestive tract and there Is potential
for dermal absorption. Unlike organochlorines and organobromines such as
polychlorinated byphenyls (PCBs) and polybrominated byphenyls (PBBs), PFOS
does not accumulate in fatty tissue; conversely, it has high affinity for proteins. It
is more likely to be found in blood plasma and muscle and tends to concentrate
in the liver.

In February of 2000, researchers at Michigan State University reported the
presence of PFOS in wildlife. This study revealed the extent of PFOS distribution.
Various animal species throughout the world were sampled including polar bears,
marine mammals, fish-eating water birds, fish and frogs. Tissue samples
including bird and fish eggs, liver, blood plasma, kidneys and muscle were
analyzed for the presence of PFOS. PFOS was found in most of the samples
including those collected from remote locations such as the Artic (Giesy and
Kannan, 2001).

F ish-eating water birds, including bald eagles from the Great Lakes region,
had blood plasma concentrations of up to 2750 ng PFOS/ml. An average PFOS
concentration of 330 nglml was reported for blood samples of nestling bald
eagles less than 70 days old. The presence of relatively high concentrations in
young birds may indicate maternal transfer of PFOS through egg proteins

(Kannan et al., 2001a). In fact, eggs from Caspian terns and double crested

13

cormorants had concentrations ranging from 1900 to 3400 nglml and 570 to 1800
nglml, respectively (Kannan, K., personal communication).

PFOS was also found in fish-eating predators such as mink and river
otters from the midwestem and east coast regions of the United States. Greater
concentrations were found in mink collected from urbanized areas, with liver
concentrations as high as 5140 ng/g. Ranch mink fed diets containing 40%
Saginaw River fish, which contained an average of 160 ng PFOS/g, had average
liver PFOS concentrations of 3250 ng/g. This finding suggested that PFOS may
bioaccumulate, since PFOS concentrations detected in mink were considerably
greater than that those found in fish (Kannan et al., 2002a).

Toxic Effects

PFOS is moderately toxic to rats, having an oral LD50 of 251 pg/g (Dean et
al., 1978). Rusch and Reinhart (1979) reported that rats exposed to PFOS by
inhalation for one hour to doses ranging from 0.0 to 24 mg PFOS/L exhibited
labored breathing, decreased activity, lung and liver discoloration and death of all
rats in the highest dose group.

PFOS was mildly irritating to the eyes and non-irritating to the skin of
rabbits (O’Malley and Ebbens, 1980). PFOS was negative for mutagenicity when
tested in various strains of Salmonella (strains TA-1535, TA-1537, TA-1538, TA-
98 and TA-100), and Sacharomyses cereviciae (strain D4) (Jagannath and
Brusic, 1978). PFOS did not cause chromosomal aberrations in mice when
administered orally at doses as high as of 950 pg PFOS/g (Murli, 1996).

Various studies on sub-chronic effects of PFOS when administered orally

14

Various studies on sub-chronic effects of PFOS when administered orally
have been conducted in rats and primates. Clinical signs observed in rats fed
PFOS at concentrations from 0.0 to 3000 pg/g feed included emaciation,
convulsions, reduced body and organ weights, hepatomegaly, gastrointestinal
and immune system effects, hypoactivity and death. Serum chemistry changes
included increases in creatinine phosphokinase and alkaline phosphatase
activities and decreases in cholesterol and triglyceride concentrations. Plasma
aspartate aminotransferase and alanine aminotransferase activities were
increased. Histological changes in the liver included hepatocellular hypertrophy,
vacuolation and necrosis (Goldenthal et al., 1978a).

Adverse effects seen in a 90-day, oral gavage, rhesus monkey study
included weight loss, hypoactivity, emesis, diarrhea, prostration, hemorrhage,
tremors and death at doses as low as 10 pg PFOS/glday and death of all
monkeys by day 20 of the trial (Goldenthal et al., 1978b). Serum chemistry
analyses in a repeated-dose, cynomolgus monkey study (Seacat et al., 2002)
indicated a reduction in serum cholesterol and high-density lipoprotein
concentrations.

Two dietary studies have been conducted to assess the toxic effects of
PFOS in avian species. Mallards and northern bobwhite quail were fed diets
containing 0, 9.1, 18.3, 36.6, 73.2, 146, 293, 586 or 1171 pg PFOS/g feed for a
period of five days. No treatment related mortality occurred in mallards in the
lowest dose groups. However, there was 20% mortality in the 293 pg PFOS/g

feed group, 30% mortality in the 586 pg lg feed group and 90% mortality in the

15

1171 pg lg feed group. Signs of toxicity included ruffled appearance, lethargy,
lower limb weakness, decreased muscle mass, reduced weight gain, depression,
convulsions and ultimately death. Toxicity signs were first observed on the
second day of the five-day exposure period and mortality occurred as early as
day four in the 1171 pglg feed group. Gross examination findings in the 293 pglg
feed treatment group and higher included empty crops, loss of muscle mass,
small and pale spleen, emaciation, and a generally thin body condition
(Gallagher et al., 2000a).

Treatment-related mortality (11%) first occurred in the northern bobwhite
at 146 pglg feed. Mortality was 80% in the 233 pglg feed group and 100% in the
586 pglg feed and 1171 pglg feed groups. Signs of toxicity included ruffled
appearance, wing droop, reduced reaction to stimuli (sound and motion), loss of
coordination, lethargy, depression, lower limb weakness, rigidity, prostration,
convulsions and ultimately death. Toxicity signs were first observed on the
second day of the five-day exposure period. There was 100% mortality as early
as day three in the 1171 pglg feed group and day seven in the 586 pglg feed
group. Body weight gain was reduced in the 146, 293 and 569 pg PFOS/g feed
treatment groups and feed consumption decreased at 293 pg PFOS/g feed and
higher. Goss examination findings for the northern bobwhite quail included loss
of muscle mass, autolysis of tissues in the abdominal cavity, pale organs,
prominent keel bone, emaciation and generally thin body condition (Gallagher et
aL,2000b)

Mechanisms of Action

16

Related perfluorinated compounds have been found to interfere with gap junction
intercellular communication (GJIC), mitochondrial biogenesis and induction of
liver enzymes involved in fatty acid metabolism. These compounds are also
known to be peroxisome proliferators. Recent studies using PFOS have
confirmed these findings (Hu et al., 2002; Berthiaume and Wallace, 2002).

GJIC is described as the process in which plaque-like structures in the cell
plasma membrane form tunnel-like structures that serve as a channel to aid
synchronization of cell functions. These channel structures allow the exchange
of cytosolic molecules like ions, second messengers and metabolites between
adjacent cells (Yamasaki, 1996). GJIC plays a vital role in maintaining the
homeostasis of tissues; therefore, disruption of GJIC results in abnormal cell
growth, which is associated with tumor formation and promotion (Hu et al., 2002).
Disruption of GJIC can also lead to neurological, cardiovascular, reproductive
and endocrinological dysfunction (Trosko et al., 1998).

Perfluorinated compounds such as perfluorooctanoic acid, perfluorooctane
sulfonamide, perfluorohexane sulfonic acid and perfluorodecanoic acid can
inhibit GJIC in a dose-related manner (Upham et al., 1998). However, potency of
inhibition is dependent on the length of the fluorinated carbon chain; compounds
with less than five carbons or more than 16 have not shown inhibition of GJIC
(Upham et al., 1998). PFOS, an eight-carbon compound, caused inhibition of
GJIC in rat liver and dolphin kidney cell lines, and in the Sprague-dawley rat.
Inhibition of GJIC was rapid, yet reversible, after removal of the compound.

Inhibition was neither gender related nor tissue specific as reported by Hu et al.

17

(2002).

Many perfluorinated compounds like PFOA, N-ethyl perfluorooctane
sulfonamide ethanol (N-EtFOSE) and PFOS are peroxisome proliferators.
Although many perfluorinated compounds affect both mitochondrial biogenesis
and peroxisomal proliferation, it was discovered that not all peroxisome
proliferators have an effect on mitochondrial biogenesis. PFOS did not affect
mitochondrial biogenesis in male rats when injected via single intraperitoneal
injection (Berthiaume and Wallace, 2002).

Peroxisomes are organelles found mainly in hepatocytes and the kidney
cell cytoplasm and are involved in the metabolism of long chain fatty acids
(peroxisomal 8 -oxidation) (Baudhuin et al., 1965). Peroxisomes are rich in the
enzymes peroxidase, catalase and d-aminoacid oxidase, which are involved in
peroxide metabolism (Dorland, 1995).

When peroxisome proliferators, such as PFOA, clofibrate or various
phthalate esters, are given to rodents, they cause an increase in the number and
size of peroxisomes in the cell as well as an increase in liver size due to
hyperplasia (Berthiaume and Wallace, 2002; Timbrell, 2000). This results in an
increase in the rate of fatty acid metabolism, which may ovenNhelm the
mechanisms necessary to metabolize hydrogen peroxide into water. This
situation causes the formation of hydroxyl radicals and reactive oxygen species
leading to oxidative stress in the cell. Reactive oxygen species will also cause
DNA, protein, and cell damage. Cell proliferation due to hyperplasia along with

cell damage can cause cells to progress into tumors. However, tumor occurrence

18

as a result of exposure to peroxisome proliferators does not appear to have the
same effect in all species. Humans seem to be non-responsive to this

phenomenon (T imbrell, 2000).

19

MATERIALS AND METHODS

Perfluorooctane sulfonate (heptadecafluorooctane sulfonic acid,
potassium salt, MW== 538.22, 96% purity) was purchased from TCI, America
(Portland, OR). PFOS was dissolved in dimethyl sulfoxide (DMSO). The choice
of vehicle was based on the results of a preliminary study in our laboratory that
evaluated DMSO, methanol, methylene chloride, acetone and saline in terms of
PFOS solubility and hatchability of chicken eggs.

Doses and Design

Fifty White Leghorn chicken (Gal/us domesticus) eggs were randomly
assigned to each of six treatment groups; non-injected control, vehicle (DMSO)
control and 0.1, 1.0, 10.0, and 20 pg PFOS/g egg. The injection volume was 0.1
pllg egg. PFOS was injected into the air cell of the egg following the procedure of
Powell et al (1996). Preliminary studies in our laboratory using methylene blue
dye injected into the egg showed that PFOS is better distributed in the egg
albumin. Doses of PFOS were chosen to bracket concentrations detected in the
blood plasma and eggs of birds collected from the Great Lakes region.

Dosing solutions were prepared from stock solutions of 1,000, 10,000,
100,000 and 200,000 pg PFOS/ml by cold filtering (0.22 micron filter, Millipore
Products Division, Bedford, MA) appropriate volumes of the PFOS stock
solutions and DMSO into sterilized glass injection vials to give final
concentrations of 1.0, 10.0, 100 and 200 pg PFOS/ml. All PFOS solutions were
stored at room temperature.

Egg preparation

20

White Leghorn chicken eggs were obtained from the Michigan State
University Poultry Science Teaching and Research Center fertile egg flock. Eggs
were lightly sanded to remove excess dirt and stored in a cooler at 14 °C for 24
hr prior to injection. Eggs were candled to detect cracks in the eggshell and
weighed to determine the injection volume. The air cell was outlined with a pencil
and eggs were labeled with an individual identification number. Eggs were then
placed in egg trays with the blunt end up.

Injection Procedure

Eggs were injected via the air cell on day 0 of incubation using a 10 ul
syringe (Hamilton Company, Reno, Nevada). Immediately prior to injection, the
egg surface was wiped with 70% ethanol and a small hole was made above the
air cell with a pin wiped with 70% ethanol to insert the syringe into the air cell.
The pin and the syringe needle were wiped with 70% ethanol between injections.
After injection, the hole was sealed with melted paraffin and eggs were placed in
the incubator (Petersime, Gettysburg, Ohio) with their blunt end up.
lncubafion

The temperature of the incubator was maintained at 37.5-37.7‘C (99.5-
99.75‘F), while the relative humidity was maintained at 60% (29.4-30.6‘C, wet
bulb temperature). Eggs were automatically rotated every two hours. Eggs were
candled weekly to assess embryo viability and to estimate the stage of
development at which the embryo died. Eggs that did not develop were
considered infertile and were excluded from the analysis.

On day 18 of incubation, eggs were placed in pedigree baskets and were

21

transferred to a hatcher (Natureforrn, Jacksonville, FL). Temperature and
humidity of the hatcher were set at 37.5-37.7'C and 65% (31.1-37.7°F, wet bulb
temperature), respectively, and were monitored daily.
Post-hatch

Hatching began on day-21 of incubation and was allowed to continue until
day-24. Hatching dates of individual chicks were recorded. Hatchlings were
identified with a Swiftak identification tag (Heartland Animal Health, Fairplay, MO),
weighed and examined for gross deformations within 24hr of hatching and
transported to a Petersime battery brooder in a containment room at the MSU
Poultry Science Research and Teaching Center. Eggs that did not hatch by day
24 were opened to assess the stage of embryo death and the incidence of gross
developmental abnormalities. Chicks were provided Purina Chick Starter (Purina
Mills, St. Louis, MO) and water ad libitum for seven days. Chicks were introduced
to their surroundings by dipping their beaks in feed and water before placing
them in the breeder.
Necropsy

At seven days of age (post-hatch), 20 chicks per treatment were randomly
chosen, weighed and killed by cervical dislocation. The brain, heart, kidneys and
livers were removed and weighed. Five livers per treatment were frozen on dry
ice before placement in an ultra-cold freezer (-78°C) for subsequent
' determination of PFOS concentration and five livers per treatment were placed in
a 10% formalin solution for subsequent histopathological examination.

Contaminant Analysis

22

Hepatic concentrations of PFOS were determined as described by
Kannan et al. (2001a), using high performance liquid chromatography (HPLC)
coupled with mass spectrometry (MS). For extraction of liver samples, a liver
homogenate of 1 9 liver to 5 ml of Milli-Q water was prepared. One ml of the
homogenate was transferred to a polypropylene tube and 1 ml of 0.5 M tetra
butyl ammonium hydrogen sulfate solution (pH adjusted to 10) and 2 ml of 0.25
M sodium carbonate buffer were added. After thorough mixing, 5 ml of methyl-
tert-butyl ether (MTBE) were added, and the mixture was vigorously shaken for
20 min. The organic and aqueous layers were separated by extraction and 4 ml
of MTBE were removed from the solution and transferred to a fresh 15 ml
polypropylene tube. The solvent was evaporated under nitrogen before adding 1
ml of methanol. The sample was mixed for 30 sec and 1 ml was transferred into
a 1.5 ml auto vial. Analyte separation was performed using a Waters 2695
Alliance high performance liquid chromatograph (HPLC) (Milford, MA). A total of
10 pl of extract was injected onto a 50 x 2 mm (5 pm) Keystone Betasil®
(Runcom, PA) C18 column with a 2 mM ammonium acetatel methanol mobile
phase starting at 10% methanol. The gradient was increased to 100% methanol
at seven min at a flow rate of 200 pllmin before reverting to original conditions at
15 min. Column temperature was maintained at 25 °C. Although PFOS eluted at
9.8 min, a longer chromatographic run (20 min) was necessary to completely
elute all extractables from the column. For quantitative determination, the HPLC
system was interfaced to a Micromass ZQ atmospheric pressure ionization mass

spectrometer (Beverly, MA) operated in the electrospray negative mode.

23

Instrumental parameters were optimized to transmit the [M-Kj' ion. For all
analyses, the capillary was held at 3 kV. Desolvation temperature and gas flow
were held at 300°C and 300 llhr respectively.
Statistical Analyses

All comparisons were made with respect to the vehicle control. PFOS
effects on hatchability were evaluated using logistic regression. Body and organ
weights (brain, heart, kidney and liver) were analyzed by one way analysis of
varianw (ANOVA) followed by Dunnett’s test for comparisons with the vehicle
control using SAS Software (SAS; Statistical Analysis Systems, Cary, NC). LDso
values were obtained by probit analysis (Version 1.5, USEPA). The level of

statistical significance was p< 0.05 unless otherwise specified.

24

RESULTS

Hatchability was significantly (p<0.0001) reduced in all dose groups in a
dose-dependent manner (Table 2). The LDso was 0.97 pg PFOS/g egg (95% CL
0014-5584 ug PFOS l 9 egg). Post-hatch mortality through seven days of age
was not affected by treatment.

There was a slight incidence of gross abnormalities that included the
vehicle control group. One embryo in the control group had a deformed leg. One
embryo in the 10 ug PFOS / 9 egg dose group had no skull or eyes and a cross
bill, and at 20 ug PFOS / 9 egg, one case of celosomia or exposed viscera.

There were no significant changes in chick body weights at hatch and at
seven days of age that were associated with PFOS exposure (Tables 3 and 4).
Similarly, absolute and relative organ weights (liver, brain, kidney and heart)
were not affected by injection of PFOS (Tables 5 and 6).

Morphological changes in the liver were assessed by gross examination
and light microscopy. Livers in all groups were pale, which was attributed to
absorption of the yolk through thefirst week of life. Lesions observed during
histological examination included hepatic vacuolation, bile duct hyperplasia,
periportal inflammation, multifocal hepatocellular necrosis and multiple
subcapsular hepatocellular degeneration and one case of fatty cysts in the 1.0
pglg dose group. Hepatic vacuolation was observed in all dose groups, but
vacuolation tended to be more prevalent in the 1.0, 10.0 and 20.0 pglg egg dose
groups compared to the vehicle control and the 0.1 pglg egg groups (T able 7).

Two moderate lesions were observed in the 1.0 pglg egg group, one lesion was

25

 

Table 2. Effect of perfluorooctane sulfonate injected into the air cell of
White Leghorn chicken embryos prior to incubation on

 

 

 

 

 

 

 

 

hatchability ‘ .
Dose Number of hatchlings/ . . o
(um Number of fertile eggs “Wham” W
Vehicle 42/49 85.7‘3
0.1 27/44 61 .4D
1.0 23l48 47.9r
10.0 20/50 40.0c
20.0 17/46 370°

 

 

 

1Non-injected controls were excluded from the statistical analysis. Values with
different superscripts are significantly different at p<0.05.Vehicle used was

dimethyl sulfoxide.

 

26

 

 

 

Table 3. Effect of perfluorooctane sulfonate injected into the air cell of
White Leghorn chicken embryos on body weights upon

 

 

 

 

 

 

 

hatching .
Dose (595) n Wei ht
Vehicle 42 - 39.93 1: 052"
0.1 27 40.33 :I: 064‘I
1.0 23 39.65 t 070"
10.0 20 41.35 :l: 0.753
20.0 17 42.15 :t 0.812'

 

 

 

 

1Data presented as mean 1: standard error of the mean. Values with different
superscripts are significantly different at p<0.05 Vehicle used was dimethyl
sulfoxide.

 

27

 

 

Table 4. Effect of perfluorooctane sulfonate injected into the air cell of
White Leghorn chicken embryos on body weights at one week

 

 

 

 

 

 

 

of age 1.
Dose (ugLrL) n Wei ht
Vehicle 37 59.59 :I: 1.453
0.1 27 56.72 :I: 1.70al
1.0 21 55.38 :l: 1.93a
10.0 19 58.50 i 2.083
20.0 16 57.09 t 2.213

 

 

 

 

1Data presented as mean :I: standard error of the mean. Values with different
superscripts are significantly different at p<0.05 Vehicle used was dimethyl
sulfoxide.

 

28

 

 

Table 5. Effect of perfluorooctane sulfonate injected into the air cell of

White Leghorn chiCken embryos on absolute organ weights (9)
at one week of age 1.

 

Dose

Liver
(uglg)

Brain

Kidney

Hean

 

Vehicle 2.24 t 0.1 (18)

1.16 :l: 0.03 (11)

0.84 i 0.04 (13)

0.66 t 0.03 (13)

 

01 205:01um

1.10 :l: 0.03 (10)

0.75 :1: 0.04 (11)

0.57 e 0.03 (11)

 

10 200:01um

110100300

0.74 t 0.04 (10)

0.61 :l: 0.03 (11)

 

10.0 2.19 e 0.1(15)

 

1.15 i 0.03 L9)

0.84 1 0.04 (10)

0.59 e 0.03 (10)

 

20.0 2.28 e 0.1 (15)

 

1.10:0.03(10)

 

0.73 1 0.04 (10)

 

0.61 a: 0.04 (9)

 

 

1Data presented as mean :I: standard error of the mean. Number in parentheses
is sample size. Vehicle used was dimethyl sulfoxide.

 

29

 

 

Table 6. Effect of perfluorooctane sulfonate injected into the air cell of
White Leghorn chicken embryos on relative organ weights (9) at
one week of age ‘.

 

Dose

(ug/g)

Liver Brain Kidney Heart

 

Vehicle 3.78 :l: 013(18) 1.89 t 0.1 (11) 1.34 :l: 0.05 (13) 1.05 t 004(13L

 

0.1 3.79:0.1206) 1.99:0.0400) 1.99:0.0401) 104200501)

 

1.0 3.57:0.1405) 199100400) 132500400) 109100401)

 

10.0 3.34 :l: 0.13 (15) 1.96 1 004(9) 1.37 x 0.0500) 1.05 :I: 00400)

 

 

 

 

 

20.0 3.96 t 0.1 (15) 1.85 :l: 0.04 (10) 1.30 :l: 0.05 (10) 1.03 :l: 004(9)

 

1Data presented as mean :I: standard error of the mean. Number in parentheses
is sample size. Vehicle used was dimethyl sulfoxide.

 

 

 

30

observed in the 10.0 pglg egg group and two cases were observed in the 20.0
pglg egg group. Bile duct hyperplasia was observed at PFOS concentrations
greater than 1.0 pglg egg. Multifocal hepatocellular degeneration and necrosis
were observed in five liver samples, three in the 1.0 pglg egg dose group and
one each in the 10 and 20 pg PFOS/g egg groups (Table 7).

PFOS concentrations in livers significantly increased proportional to dose
(Table 8). Concentrations in the 10.0 and 20.0 ug PFOS/g egg groups were
significantly greater compared to concentrations in the vehicle control, 0.1 ug

PFOS/g egg and 1.0 ug PFOS/g egg groups (Table 8).

31

  

Table 7. Histopathology of livers from seven-day-old White Leghorn
chickens exposed to perfluorooctane sulfonate in ovo via air
cell injection prior to incubation 1.

    
 
 

  

 

 

  

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

   
  

 

 

 

 

 

 

 

Treatment ChiCK Hepatocgflbfaerwat|cBrIlse duct
0
(09/9 egg) ID Vacuolation hyperplasia
Vehicle B854 1 0
Vehicle 8857 1 0 I
Vehicle B861 1 0
Vehicle 8882 1 O
0.1 P831 1 0
0.1 P840 1 O
0.1 P845 1 0
0.1 P864 1 0
0.1 P869 1 0
0 1+ Periportal
0‘1 P871 1 inflammation
0 1+ Periportal
1 '0 PC399 1 inflammation
Multiple subcapsular
1 .0 P0872 2 1 hepatocellular
egeneration
Multifocal hepatocellular
1 '0 PC873 2 1 W‘egenerationlnecrosis
ultifocal hepatocellular
1'0 PC877 1 1 necrosis; Fatty cyst
1.0 P0888 1 0
10.0 Y397 1 0 I
10.0 Y403 2 1
10.0 Y404 1 2 Hepatocellular necrosis
10.0 Y406 1 1 I
20.0 YC41 1 1 1
20.0 YC412 1 1 |
20.0 YC415 2 1 I
Multifocal hepatocellular
20'0 YC416 2 1 degeneration/necrosis
20.0 YC421 1 1
Vehicle used was dimethyl sulfoxide. Severity of lesion was graded using the
. following scale: 0 = normal; 1 = mild; 2 = moderate; 3 = severe.

 

32

 

Table 8.Hepatic perfluorooctane sulfonate concentrations of seven-day-
old White Leghorn chickens exposed to perfluorooctane
sulfonate in ovo via injection into the air cell ‘.

 

 

 

 

 

 

 

 

Dose (ug/g) Concentration (pglg, wet wt)
Vehicle 1.101 a: 0.53678
0.1 1.411 :1: 0.5367“
1.0 1.762 :t 0.60003
10.0 3.165 1: 0.53677
20.0 4.752 :t 05367”

 

 

 

1Data presented as mean :1: standard error of the mean. Values with different

superscripts are significantly different at p<0.05. Vehicle used was dimethyl
sulfoxidej

 

 

 

 

33

 

 

 

  
 
 

Table 9. Concentrations of perfluorooctane sulfonate in chicken eggs
from domestic commercial sources‘.

 

 

 

 

Source n PFOS Concentrations
(ug/g wet wt)
“Organic” eggs from local supermarket 4 0.703 :t 001‘
Eggs from local supermarket 4 0.344 :I: 0.05b

 

Eggs from MSU Poultry Science and b
Research Center 3 0'329 i 0'07
1Data are presented as the mean 1: standard error of the mean. Values with

different sugerscripts are significantly different at g<005.

 

 

 

 

 

 

DISCUSSION

This study was conducted to assess PFOS toxicity in the developing
chicken embryo following introduction of the chemical into the air cell prior to
incubation. The objectives of this study included determining an LDso value as
well as the suitability of the chicken embryo as an animal model for studying the
adverse effects of PFOS in wild avian species.

Injection doses for this study (0.1-20.0 pglg egg) were selected based on
PFOS concentrations detected in eggs of wild avian species collected from the
Great Lakes basin. The Great Lakes is a highly contaminated area because of
the high volume of industries in the states surrounding the lakes. PFOS, like
many other persistent organic pollutants, is present at higher concentrations in
urbanized and industrialized areas like the Great Lakes basin. Fish and fish-
eating water birds in these areas are now known to have relatively high
concentrations of PFOS (Kannan et al., 2001a). Tissues of fish from the Great
Lakes, including lake Whitefish (Coregonus clupeaformis), brown trout (Salmo
trutta), chinook salmon (Oncomynchus tshawytscha) and carp (Cyprinus carpio),
contained detectable PFOS concentrations in eggs, whole blood, blood plasma,
liver and muscle (Giesy and Kannan,2002). Carp from the midwestem US had
muscle PFOS concentrations up to 0.3 pglg (Giesy and Kannan, 2001). The
presence of PFOS in fish and its occurrence in fish-eating water birds suggests
that fish could be a source of PFOS. Bald eagles (Haliaeetus Ieucocephalus)
from the Great Lakes region had blood plasma concentrations as great as 2.75

pg PFOS/ml. (Giesy and Kannan, 2001). Other birds, such as double crested

35

cormorants (Phalacmcorax aun'tus), herring gulls (Lams argentatus) and ring
billed gulls (Larus delawarensis) contained concentrations of PFOS in various
tissues including blood plasma and liver. Moreover, nestling bald eagles had
blood PFOS concentrations as great as 0.33 pg/ml. The presence of relatively
high concentrations in young birds may indicate maternal transfer of PFOS
through egg proteins (Kannan et al., 2001a). In fact, eggs from Caspian terns
(Stema caspia) and double crested cormorants were found to have PFOS
concentrations ranging from 1.9 to 3.4 pg/ml and 0.57 to 1.8 pg/ml, respectively
(Kannan, unpublished data).

PFOS was also found in fish-eating mammals such as mink (Muste/a

vison) and river otters (Lutra canadensis) from the midwestem and east coast

regions of the United States. The greatest concentrations were found in mink
collected from urbanized areas, with liver concentrations as high as 5.14 pglg.
Ranch mink fed diets containing 40% Saginaw River fish, which contained an
average of 0.16 pg PFOS/g, had an average liver PFOS concentration of 3.25
pglg. This finding suggested that PFOS may bioaccumulate, since PFOS
concentrations detected in mink were considerably greater than those found in
fish (Kannan et al., 2002b).
Hatchability and LDso

In the present study, PFOS significantly lowered hatchability of chicken
eggs in a dose-related manner when compared to the vehicle control group
(Table 2). The LDso for chicken eggs injected with PFOS via the air cell prior to

incubation (Day 0) was 0.97 pg PFOS/g egg.

36

 

PFOS concentrations causing embryo mortality in this study were
comparable to PFOS concentrations found in eggs collected from wild species.
Egg samples from urbanized areas had higher concentrations of PFOS when
compared to eggs from isolated regions. Double crested cormorants from the
Great Lakes area contained PFOS concentrations from 0.57 to 1.8 pglg and
Caspian tern eggs from the same region contained PFOS at concentrations from
1.9 to 3.4 pglg (Kannan, unpublished data). Double crested cormorant eggs from
Canada had slightly lower PFOS concentrations, ranging from 0.02 to 0.32 pglg
(Giesy and Kannan, 2001). It is possible that hatching success of eggs in the wild
may be compromised by PFOS if the sensitivity for these species in terms of
mortality is similar to the chicken and air cell injection accurately represents
whole egg concentrations achieved by dietary exposure of adults.

LD50 values have been derived for other species including the rat, the
northern bobwhite quail (Colinus virginianus) and the mallard (Anas
platyrhynchos), all under different treatment regimes and conditions (age of
animal, route of exposure and length of exposure). Pregnant Sprague-Dawley
rats were given 1 mm pg PFOS/g body weight daily by gavage beginning on
gestation day (GD) 2 and continuing until GD 21. The LDso for rat pups exposed
in utero was 3 pglg. Pregnant mice were dosed under similar conditions with 1 to
20 pg PFOS lg from the first day of gestation through GD 18. The LDso for mice
pups was 10 pglg (Lau et al., 2003). In two avian dietary studies, young northern
bobwhites and mallards were fed diets containing 0, 9.1, 18.3, 36.6, 73.2, 146,

293, 586 or 1171 pg PFOS/g feed for a period of five days. The LCso derived for

37

the northern bobwhite quail was 293 pglg feed and 628 pglg feed for the
mallards (Gallagher et al., 2000a; 2000b), which is equivalent to a total PFOS
dose of 9 pglg body weight and 748 pglg body weight respectively. Even though
there are various LDso values reported, comparisons are irrelevant because of
the unique conditions under which each one was determined. However, the
results of the present study suggest that PFOS is very toxic to the chicken
embryo when administered prior to embryo development.

There are two additional factors that may affect the viability and hatching
success of the chicken embryo in addition to the toxicant concentration: the site
of injection (yolk sac compared to air cell) and the time of injection (prior to
incubation compared to day four of incubation). Injection of toxicants into the yolk
sac causes considerably greater mortality to the chicken embryo when compared
to air cell injection. Chicken embryos injected with 2, 3, 7, 8-tetrachlorodibenzo-
p-dioxin (TCDD) into the yolk sac had greater mortality than embryos injected
with equal concentrations of TCDD via the air cell (LDso values of 122 pg
TCDD/g egg and 297 pg TCDDIg egg, respectively) (Henshel et al. 1997). In a
preliminary study conducted in our laboratory, it was also observed that there
was higher mortality when PFOS was injected into the yolk compared to the air
cell. Air cell injection was selected based on the relatively high water solubility of
PFOS. The egg albumen is composed primarily of water (88%) with the reminder
being proteins and glycoproteins. The yolk, on the other hand, has greater lipid
content and lesser water content than the albumin. It was anticipated that PFOS,

and the vehicle DMSO, would be more evenly distributed in the water-rich

38

environment of the albumin.

The avian egg is exposed to toxins from the time it starts growing in the
ovary until the yolk is completely absorbed four days post-hatch. In this study, the
injection was conducted just prior to incubation to mimic this natural process of
exposure via deposition of the compound into the egg by the female. The time of
injection or state of development at which the substance is in contact with the
embryo may adversely affect hatchability. TCDD was more toxic to embryos
exposed to the compound on day four of incubation compared to days eight and
14 of incubation (Bruggemann et al. 2003).

Post-hatch survival was not affected by PFOS in the seven-day period the
chicks remained in the battery brooder. In contrast, PFOS induced mortality in
rat pups exposed in utem to 1-10 pg PFOS/g body weight and in mice pups
exposed in utero to 1 - 20 pg PFOS/g body weight within the first 24 hr after birth
(Lau et al., 2003). Although all animals were born alive, the neonates in the
highest dosage groups (10 pglg body weight for rat and 20 pglg body weight for
mouse) became pale, inactive, and moribund within 30 to 60 min, and all died
soon afterward. In the 5 pglg (rat) and 15 pglg (mouse) groups, the neonates
also became moribund, but survived for a longer period of time (8 to12 hr).
Teratogenicity

PFOS did not induce significant abnormalities in chicken embryos. The
few abnormalities observed in this study fell within the normal range (6 to 7%) of
spontaneous malformations for chicken embryos (Romanoff, 1972). Occasionally,

malformed embryos have more than one malformation as was observed in this

39

trial. Three embryos had multiple abnormalities including deformed extremities
(0.6%), anophthalmia or absence of one or both eyes (0.3%), exencephalia or
exposed brain (0.3%), celosomia or exposed viscera (0.3%) and
mandibular/maxillary malformation (0.3%). None of the embryos with
abnormalities hatched with the exception of two chicks. One had curled toes and
died before the end of trial and the other had a protruding eye and survived to the
end of the trial.

Body and organ weights

PFOS did not affect body weights (Tables 3 and 4) or absolute and
relative organ weights of chickens exposed in ovo (Table 5). However, previous
studies in different species reported body weight loss or a decrease in body
weight gain after repeated exposure to PF OS.

Primates, including rhesus and cynomolgus monkeys, had decreased
body weights as a result of exposure to PFOS. Rhesus monkeys given 0-300 pg
PFOS/g body weight! day orally for 90 days had significant body weight loss, but
organ weights were normal (Goldenthal et al., 1978b). In a subsequent 90-day
rhesus monkey study, lower doses of PFOS (0.5 to 4.5 pglg body weight/day)
caused body weight loss, presumably as a result of anorexia, emesis and
dehydration (Goldenthal et al., 1978b). Mean body weight gain was significantly
reduced in cynomolgus monkeys administered PFOS at 0.75 pglg body
weight/day for six months (Seacat et al., 2002).

PFOS reduced body weights in a dose dependent manner in rats fed 0.5

to 20.0 pg PFOS/g feed/day for a period of four or 14 weeks. In addition, relative

40

liver weights were increased after 14 weeks of exposure in the group
administered 20.0 pglg feed/day (Seacat et al., 2003). In a two-generation
developmental study, rat and mice pups exposed to PFOS prenatally (GD 2 to
GD 21) had deficits in body weight gain at concentrations as low as 2 pg PFOS
lg body weight/day (Lau et al., 2003).

During necropsy, we observed that three chicks in the 0.1 pglg egg dose
group had prominent keel bones. This was a common finding in the bobwhite and
mallard dietary study and was associated with decreased food consumption in
the higher treatment groups (293 to 1171 pg PFOS/g feed). In mallards, there
was significant body weight loss in the 73.2 to 1171 pglg treatment groups during
the five-day exposure period. By day 22 of the post-exposure period, body
weights appeared comparable in all dose groups. In bobwhite quail, significant
body weight loss also occurred during the five-day exposure period at 146 to 586
pg PFOS/g feed (Gallagher et al., 2000a; 2000b). However, the emaciation and
lethargic behavior seen in these studies were not observed in the present study
at the doses and exposure period tested.

Histological analysis

PFOS caused morphological changes in the livers of chicken embryos at
concentrations as little as 1.0 pglg egg. Changes included hepatocellular
vacuolation, bile duct hyperplasia, periportal inflammation, hepatocellular
degeneration and necrosis.

Mild hepatocellular vacuolation was observed among all dose groups;

however, moderate vacuolation was observed more frequently at doses of 1.0

41

pglg or greater (Table 7). The appearance of hepatic vacuolation has been
reported in previous PFOS studies. Hepatocellular vacuolation was observed in
rats fed diets containing PFOS at 5 or 20 pglg feed for a period of four or 14 wk
and in cynomolgus monkeys administered PFOS at a dose of 0.75 pglg body
weight/day for six months (Seacat et al. 2002, 2003).

Vacuolation is the result of accumulation of lipids in hepatocytes, which
occurs because of the absorption of the lipid-rich yolk sac that continues after
hatching. Although it is not always injurious to the liver, it can be indicative of
impairment such as blockage of triglyceride secretion into plasma or reduced
synthesis of carrier lipoprotein (Osweiler, 1996). Previous studies have reported
that PFOS may interfere with fatty acid binding to lipoprotein, contributing to lipid
accumulation in the liver (Luebker et al., 2002).

Mild to moderate bile duct hyperplasia was observed in the highest dose
groups (1.0, 10.0 and 20.0 pglg egg). This lesion consists of an abnormal
number of bile ducts occurring in the portal area accompanied by periportal
inflammation. This lesion is considered to be a common lesion and it is unclear
whether or not this can progress into benign or malignant tumors (Newberne,
1998)

Multifocal hepatocellular degeneration and necrosis were observed in five
liver samples. Because three of the samples were from the 1.0 pglg dose group,
it was not clear If this was a treatment-related effect. Hepatocellular necrosis has
only been reported in rhesus monkeys exposed to PFOS at concentrations as

high as 300 to 3000 pglg body weight.

42

Interestingly, some of the lesions observed in the present study have been
observed in monkeys and rats exposed to PFOS for prolonged periods of time.
In this study, chickens exposed before incubation experienced similar liver
changes after a single exposure in a period of only 28 d. This may be due to the
fact that fertile eggs were injected at a very early stage of development.
PFOS Concentrations in Eggs

PFOS concentrations in vehicle control chicks and those exposed in ovo
to 0.1, 1.0, 10.0 or 20.0 pg PFOS lg egg ranged from 1.0 to 4.8 pglg (Table 8).
The highest concentration of PFOS measured in chick liver samples was 7.2 pg
PFOS lg from a bird in the highest dose group (20.0 pglg egg). The lowest
concentrations were detected in the control group, yet, some individual liver
samples in the vehicle control group had PFOS concentrations as high as 1.7 pg
PFOS/g. PFOS concentrations in livers of wild avian species, mostly from
urbanized areas (Table 1), had concentrations similar to those found in liver
samples of embryos from the vehicle control, 0.1 and 1.0 pg PFOS/g egg
treatment groups (Table 8). For example, Brandt’s cormorants collected from
San Diego, CA had hepatic PFOS concentrations ranging from 0.05 to 1.78 pglg.
Common cormorant liver samples collected from Tokyo Bay, Japan had PFOS
concentrations that ranged from 0.17 to 0.65 pglg and mallards and pintails had
hepatic PFOS concentrations of 0.49 and 0.50 pg PFOS/g respectively (Table 1).

It is not known why control eggs contained measurable concentrations of
PFOS. Samples of laying mash and chick starter used to feed the hens from

which the eggs were derived and the hatchlings, respectively, were analyzed for

43

the presence of PFOS, and neither had detectable concentrations. A possible
source of PFOS contamination is water, but this was not confirmed in this study.
Eggs from a local supermarket had high concentrations of PFOS, ranging from
0.34 to 0.70 pg PFOS/g (Table 9). These concentrations are greater than those

found in eggs from wild birds (Table 1).

44

CONCLUSION

In summary, the results of this study indicate that PFOS caused mortality
of the chicken embryo after a single exposure prior to incubation. The LD50 for
the chicken embryo was 0.97 pg PFOS/g egg. PFOS did not affect post-hatch
body weights or organ weights. A single exposure to PFOS caused hepatic
changes including bile duct hyperplasia, periportal inflammation and necrosis at
doses as little as 1.0 pglg egg. Hepatic PFOS concentrations increased in a
dose-dependent manner.

Concentrations of PFOS at which toxic effects were observed in chicken
embryos were similar to concentrations observed in wild bird eggs. PFOS caused
significant embryo mortality in the 0.1 pglg egg dose group, which had a mean
hepatic PFOS concentration of 1.4 pglg (range 0.7 to 2.2 pglg). Similar
concentrations have been found in the livers and eggs of wild fish-eating water
birds. Even though previous studies have shown chickens to be more sensitive to
some chemicals, such as polychlorinated biphenyls (PCBs), dibenzo-p-dioxins
(PCDDs) and dibenzofurans (PCDFs) (Powell el al, 1996, 1997). It is difficult at
this point to determine how the chicken responds to PFOS exposure compared
to wild birds. If the chicken is more sensitive to PFSO compared to wild birds,
then risk assessments based on Toxicity Reference Values (TRVs) derived from

studies of the chicken would be protective of sensitive species.

45

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