Environmental Toxicology and Pharmacology 23 (2007) 1–9
Effects of perfluorooctane sulfonate on mallard and northern
bobwhite quail exposed chronically via the diet
John L. Newsted a,∗ , Katherine K. Coady a , Susan A. Beach b ,
John L. Butenhoff c , Sean Gallagher d , John P. Giesy e,f,g
a ENTRIX, Inc., 4295 Okemos Road, Okemos, MI 48864, USA
3M Company, Environmental Laboratory, Maplewood, MN 55144, USA
c 3M Company, Medical Department, St. Paul, MN 55144, USA
d Wildlife International, LTD, Easton, MD 21601, USA
e Department of Zoology, National Food Safety and Toxicology Center, Center for Integrative Toxicology,
Michigan State University, E. Lansing, MI 48824, USA
f Department of Veterinary Biomedical Sciences and Toxicology Centre, University of Saskatchewan,
Saskatoon, Saskatchewan, Canada
g Department of Biology and Chemistry, City University of Hong Kong, Hong Kong, SAR, China
b
Received 23 May 2005; accepted 13 April 2006
Available online 3 June 2006
Abstract
Adult mallard ducks and northern bobwhite quail were exposed to 0, 10, 50, or 150 mg perfluorooctane sulfonate (PFOS)/kg in the diet for
up to 21 weeks. Adult health, body and liver weight, feed consumption, gross morphology and histology of body organs, and reproduction were
examined. Due to mortality, birds exposed to 50 or 150 mg PFOS/kg feed were terminated by Week 7. In quail, the lowest observable adverse
effect level (LOAEL) was 10 mg PFOS/kg feed based on decreased survivorship of 14-day-old quail offspring. For adult female quail fed 10 mg/kg
feed, there was a slight but statistically significantly PFOS-related increase in liver weight when compared to controls. When liver weight was
normalized to body weight, the statistically significant differences were still observed indicating that PFOS affected liver size. However, no other
pathological effects were observed livers of quail from this treatment group which suggests that this enlargement may have been an adaptive
response. For adult mallards, no treatment-related effects on feed consumption, body or liver weight, growth, or reproductive performance were
observed. There was a slightly greater incidence of small testes (length) in adult male mallards and quail exposed to 10 mg PFOS/kg, feed when
compared to controls. However, spermatogenesis was not affected and there was no effect on the rates of egg fertilization. Due to transfer to eggs,
concentrations of PFOS measured in the liver and blood at study termination were greater in male birds than female birds.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Bird; Toxicity; PFOS; Reproduction
1. Introduction
Perfluorooctane sulfonate (PFOS) is a fluorine-saturated,
eight-carbon acid with a terminal sulfonate functional group.
PFOS or materials that can degrade to PFOS have been
used in many commercial products such as surfactants, polymers, wetting agents, lubricants, adhesives, pesticides, corrosion
inhibitors, as well as stain resistant treatments for leather, paper
∗
Corresponding author at: 4295 Okemos Road, Okemos, MI 48864, USA.
Tel.: +1 517 381 1434; fax: +1 517 381 1435.
E-mail address: jnewsted@entrix.com (J.L. Newsted).
1382-6689/$ – see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.etap.2006.04.008
and clothing (Key et al., 1997; Giesy and Kannan, 2002). Due
to the rigidity associated with the carbon–fluorine bonds and
the saturation of carbon atoms with fluorine, PFOS is resistant
to chemical and biological changes and does not degrade or
volatilize to a significant extent in the environment (Key et al.,
1998; Beach et al., 2006). PFOS has also been shown to accumulate in wildlife by binding to proteins in blood and liver tissues
(Giesy and Kannan, 2001; Jones et al., 2003). PFOS has been
measured globally in air, surface waters, sediments, and the tissues of aquatic invertebrates, fish, fish-eating water birds, mink,
otter, and other wildlife (Kannan et al., 2001a,b, 2002a,b).
Experimental evidence has demonstrated that PFOS is
biologically active and can cause peroxisomal proliferation,
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J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
increased activity of lipid and xenobiotic metabolizing enzymes,
and alterations in other biochemical processes in exposed organisms (Sohlenius et al., 1993; Berthiaume and Wallace, 2002;
Luebker et al., 2002). In acute studies with northern bobwhite
quail and mallards there were no overt signs of toxicity or mortality observed at dietary PFOS concentrations less than or equal
to 70.3 mg/kg feed, equivalent to 24 mg/kg bw/day in quail, the
more sensitive species (Newsted et al., 2005). However, because
data on chronic effects of PFOS on avian species were unavailable at the time of this study, studies with the quail and mallard
were undertaken to measure the effects of PFOS on reproductive
and developmental endpoints.
2. Material and methods
2.1. Test materials
Potassium perfluorooctane sulfonate was obtained as a white powder from
3M (Lot #217) (St. Paul, MN, USA). Purity was determined to be 86.9% by liquid
chromatography/mass spectrometry and other elemental analysis techniques. All
PFOS concentrations in the feed and standards were adjusted to reflect purity.
2.2. Dietary exposure
Each dose was prepared independently by mixing PFOS directly into the
game bird diet in a Hobart mixer with concentrations in the diet adjusted to 100%
purity. The target nominal concentrations for mallards and quail were 10, 50,
and 150 mg PFOS/kg feed. To evaluate homogeneity, stability, and verification
of nominal concentrations, dose preparations were extracted with methanol and
analyzed by high-pressure liquid chromatography with triple quadrupole mass
spectrometry. Separations were achieved using a Keystone Betasil C18 analytical
column and PFOS was detected at 499 m/z while the main product ion was
monitored at 99 m/z (Gallagher et al., 2003a,b). Internal standards were not used
to quantify PFOS. Instead matrix spikes were used to evaluate recoveries. All
concentrations were reported on a wet weight basis (ww).
2.3. Exposure
Adult mallard ducks and northern bobwhite quail were purchased from
Whistling Wings, Inc. (Hanover, IL) and Morris Quail Farm (Goulds, FL),
respectively. The birds were phenotypically indistinguishable from wild mallards and quail. Both species were acclimated to laboratory conditions for 2
weeks prior to the study and observed for general health and physical injuries.
To ensure the health of the birds, all housing and husbandry practices were
conducted as established by the National Research Council (NRC, 1996). All
birds were 24 weeks of age at study initiation. At study initiation, body weights
for mallards ranged from 781 to 1444 g and 177 to 250 g for quail. After
the acclimation phase, bird species were exposed to PFOS in the diet. Food
and water were provided ad libitum throughout the study to both adults and
offspring.
Each treatment group contained 16 replicate pens with each pen containing a pair of birds, one male and one female. Quail were housed in
25 cm × 51 cm × 26 cm pens constructed of wire mesh, and mallards were
housed in 75 cm × 90 cm × 45 cm pens constructed of vinyl-coated wire. Birds
were held at ambient laboratory temperature (15–30 ◦ C). A light cycle of 8 h
light:16 h dark was administered for the first several weeks prior to photostimulation. In addition, four additional bird pairs per treatment were maintained for
blood collection that occurred at 5-week intervals throughout the duration of the
study. These pairs were not used to assess reproduction.
Due to overt signs of toxicity noted in the 150 mg PFOS/kg feed treatment by
Week 3, the treatment level was reduced to 20 mg PFOS/kg feed. All birds in each
treatment group were observed daily for signs of toxicity or abnormal behavior. Body weights were measured at Weeks 2, 4, 6, 8 and at study termination
(Week 21).
Feed consumption was measured weekly as the difference in weighed
amount of feed given to each breeding pair at start (Day 0) and that was remaining at the end of the feeding period (Day 7). Due to the experimental design,
feed consumption could not be calculated on a bird basis nor could feed consumption be calculated by sex. The amount of the feed wasted by the birds was
not quantified, therefore the measured feed consumption values are presented
as an estimate of total feed consumption on a pen basis.
Adult quail and mallards were brought into their reproductive phase by
photostimulation at Weeks 7 and 11, respectively. At this time the light cycles
were altered from 8 h of light per day to 17 h of light per day. Once egg production
was initiated, eggs were set weekly for incubation. Eggs were incubated at
temperatures ranging from 37.2 to 37.8 ◦ C and humidity of 50–60%. Eggs were
candled so that the developmental stage and any abnormalities could be recorded
for each egg set. Reproductive endpoints evaluated during this study included:
egg production, embryo viability, hatchability, and hatchling health and survival.
After hatching, quail and mallard chicks were housed in 72 cm × 90 cm × 23 cm
and 62 cm × 92 cm × 25.5 cm brooding pens, respectively. The light cycle for
hatchlings was maintained at 16 h light:8 h dark and temperatures in the brooding
pens were within the range of 36.9–37.5 ◦ C. Hatchlings were fed an untreated
diet for 14 days. After 14 days post-hatch, chick body weights were determined
and blood and tissue samples were collected from 10 chicks from each treatment
group.
At the time of study termination (Week 21), blood samples were collected
from surviving adult birds and resulting serum was analyzed for PFOS. Following blood collection, the adult birds and chicks were euthanized and subjected to
gross necropsy. Liver, brain, kidney, gonad, proventriculus, gall bladder, adipose,
and Bursa of Fabricius tissues were removed for histopathological evaluation.
In addition, liver weights were determined and samples collected for PFOS
analysis.
PFOS concentrations were determined in pooled samples of egg yolks collected during Weeks 19, 20, and 21 of the study from each treatment group.
In addition, the yolk composites were separated into three fractions, very low
density lipoprotein (VLDL), phosvitin, and lipovitellin fractions by the method
of Stifani et al. (1990). Briefly, yolk homogenates were diluted with a solution
containing 0.67 M MgS04 and centrifuged to separate the yolk into a VLDL and
a high-density fraction (HDF). The VLDL fraction was diluted in Tris-NaCl
solution and re-centrifuged. The supernatant was discarded and the VLDL was
precipitated and dissolved in methanol for analysis. The HDF fraction was separated by centrifugation into supernatant (lipovitellin) and precipitate (phosvitin).
Both fractions were precipitated and the fractions dissolved in methanol for
chemical analysis.
2.4. Quantification of PFOS
Analysis of PFOS in red blood cells (RBCs) and serum collected from both
species as part of the time course experiment were analyzed by HPLC tandem mass spectrometry according to published methods (Hansen et al., 2001).
Liver and serum samples collected from adult and offspring at study termination
were extracted by a solid phase (SPE) method. Briefly, liver and serum samples
were homogenized with water, extracted with acetonitrile, and centrifuged. The
supernatant was passed through a conditioned C18 (SPE) column and the analyte was analyzed by HPLC electrospray tandem mass spectrometry (Gallagher
et al., 2003a). To determine PFOS in egg yolk and yolk fractions, samples were
extracted with methanol. Activated charcoal was added to the extract and this
solution was then filtered and analyzed by electrospray LC/MS/MS. All tissue
PFOS concentrations are reported on a wet weight basis.
2.5. Statistical methods
Analysis of variance (ANOVA) and Dunnett’s multiple comparison procedure were used to evaluate differences between treatment and control groups.
Student’s t-tests were used for statistical comparisons in those instances where
only the control and a treatment group were compared. The criterion used for
significance in all statistical tests was p < 0.05. Feed consumption and reproduction parameters were evaluated on a pen basis while adult body weight was
evaluated on an individual bird basis. Statistical analyses of body weights were
conducted separately for males and females. Percentage data (reproduction data)
J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
were examined using Dunnett’s method following arcsine square root transformation. Statistical analysis of the data was performed using Avian Reproduction
Data System (ARDS) Software, a validated software package developed by
Wildlife International, Ltd. (Easton, MD). Average daily intake (ADI) of PFOS
for each treatment group was estimated on a pen basis where food consumption
and adult body weights were averaged over the duration of the exposures at each
exposure treatment Eq. (1):
ADI =
Average feed consumption
× Feed concentration
Average body weight
(1)
where ADI is in units of mg PFOS/kg body weight/day, feed consumption is in
g feed/bird/day, average body weight is in g/bird, and feed concentration is in
mg PFOS/kg feed.
3. Results
3.1. Verification of PFOS treatment concentrations
Actual concentrations of PFOS in the diets containing nominal concentrations of 10, 50, and 150 mg PFOS/kg feed determined during Week 1, Day 0 were within 2% of nominal with
an average coefficient of variation (CV) of 2.7%. During the
study, sampling to verify feed concentrations showed measured
mean concentrations of 10.2 ± 0.55, 20.8 ± 2.14, 50.9 ± 2.04
and 161 ± 15.6 mg PFOS/kg feed for the nominal dietary concentrations of 10, 20, 50, and 150 mg PFOS/kg feed, respectively. These measured values were approximately 102–108%
of the nominal concentrations. Stability of PFOS in the diet held
at ambient conditions for 7 days averaged 108, 100 and 103% of
Day 0 values for the 10, 50 and 150 mg PFOS/kg feed treatment
groups, respectively. This result indicates that PFOS was stable
during the study.
The limit of quantitation (LOQ) was set to 2.0 mg PFOS/kg
feed for both the mallard and quail diets. Chemical analyses of
the dietary treatments showed that in the control diets, the presence of PFOS or other co-eluting compounds were not observed
(Gallagher et al., 2003a,b).
3
3.2. Mortality and clinical observations
No PFOS-related deaths were observed for quail or mallards
fed 10 mg PFOS/kg in the diet. However, starting at Week 5,
clinical signs that included reduced reaction to external stimuli,
ruffled appearance, and lethargy were observed in quail from
the 10 mg PFOS/kg feed treatment. No treatment-related clinical
effects were observed in mallards in the 10 mg PFOS/kg feed
treatment.
In the 50 mg PFOS/kg feed treatment groups, 5 treatmentrelated deaths were observed in quail while 3 incidental
(non-treatment-related) and 10 treatment-related deaths were
observed in mallards within the first 5–7 weeks of the study.
In the 150/20 mg PFOS/kg feed treatments, three quail mortalities and four mallard mortalities occurred within the first
5 weeks of the study. Due to these effects, all surviving birds
in the 150/20 mg PFOS/kg feed treatments were euthanized at
Week 5, while all surviving birds in the 50 mg PFOS/kg feed
groups were euthanized at either Week 7 or Week 8. Signs of
toxicity in the 50 or 150/mg PFOS/kg feed treatments for commonly seen in moribund birds of both species included reduced
reaction stimuli, wing droop, loss of coordination, thin appearance, lacrimation, loss of righting reflex, lower limb rigidity,
convulsions, shallow and rapid respiration, ruffled appearance,
lower limb weakness, lethargy, gaping, prostrate posture and
spasms.
3.3. Adult body weight and feed consumption
Compared to controls, there were no apparent treatmentrelated effects on body weight for quail or mallards exposed
to 10 mg PFOS/kg feed (Table 1). However, starting at Week 2,
there was a marked and statistically significant reduction in body
weights among both mallard and quail of both sexes in the 50
and 150/20 mg PFOS/kg feed treatment groups. No treatmentrelated effects on feed consumption rates were observed in
Table 1
Mean food consumption and body and liver weights (±one standard deviation) of northern bobwhite quail and mallards exposed to various concentrations of PFOS
in the definitive chronic study
Nominal PFOS
(mg/kg)
Average daily intake
(mg PFOS/kg body weight/day)
Feed consumption
(g feed/bird/day)a
Quail
Mallard
Quail
Mallard
Control
<LOQb
<LOQ
19 ± 2
205 ± 53
10
0.77 ± 0.06
1.48 ± 0.19
19 ± 3
50
2.64 ± 0.24
6.36 ± 1.05
150
7.32 ± 1.26
20.9 ± 7.24
Sex
Body weight (g)a
Liver weight (g)a
Quail
Mallard
Quail
Mallard
M
F
215 ± 16 (16)
247 ± 20 (13)
1164 ± 60.0 (15)
1155 ± 134 (16)
4.43 ± 0.50
8.66 ± 1.11
29.4 ± 3.86
41.7 ± 10.51
193 ± 38
M
F
208 ± 11 (16)
249 ± 22 (14)
1127 ± 105 (16)
1144 ± 128 (16)
4.48 ± 0.55
10.9 ± 1.60*
30.6 ± 4.63
42.2 ± 10.27
9 ± 2*
112 ± 45
M
F
179 ± 20* (15)
185 ± 22* (16)
NA
NA
8 ± 2*
194 ± 73*
M
F
187 ± 14* (13) 1016 ± 85* (14)
172 ± 29* (15) 880 ± 76* (14)
NA
NA
916 ± 109* (15)
781 ± 74.0* (16)
NA is not applicable due to no sample being recorded.
a For the control and 10 mg PFOS/kg feed treatments, body and liver weights and feed consumption were from Week 20 data while for the 50 and 150 mg PFOS/kg
feed treatments data were from Weeks 6 and 4, respectively. Sample size in parentheses.
b LOQ, limit of quantitation. LOQ for dietary PFOS was 2.0 mg/kg feed.
* Indicates a statistical difference from controls at p < 0.05. Comparisons between controls and 10 mg PFOS/kg feed groups based on Week 20 data. Comparisons
50 and 150 mg PFOS/kg treatments and controls (data not shown) were based on Weeks 6 and 4 data, respectively.
4
J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
mallards or quail from the 10 mg PFOS/kg feed treatments when
compared to controls (Table 1). However, starting at Week 1, a
treatment-related reduction in feed consumption was observed
for both species in the 50 and 150/20 mg PFOS/kg feed treatment groups. While feed consumption rates improved in quail
from the 150/20 mg PFOS/kg feed treatment group by Week 4,
they remained less than that observed in the controls through
dose group termination. In mallards, greater feed consumption
was also observed and by Week 3 there was a statistically significant increase in feed consumption from control levels observed
for this treatment group. This apparent recovery was most likely
due to the reduction of PFOS concentrations from 150 to 20 mg
PFOS/kg feed after Week 3.
3.4. Liver weight
When compared to controls, there were no treatment-related
effects on liver weight of male quail in the 10 mg PFOS/kg feed
treatment group (Table 1). There was a statistically significant
(p < 0.01) treatment-related increase in liver weight of female
quail from the 10 mg PFOS/kg feed treatment group. When
female liver weights were normalized to body weight, differences between the control and 10 mg PFOS/kg feed treatment
group were still statistically significant (p = 0.001).
For mallards, no treatment-related effects on liver weights or
liver weights normalized to body weight were noted for either
males or females from the 10 mg PFOS/kg feed treatment group
when compared to controls (Table 1). Livers were not collected
from mallards or quail in the 50 or 150/20 mg PFOS/kg feed
treatment groups.
3.5. Gross pathology and histopathology
No lesions were observed at either the gross or histological
levels of examination were observed in female quail or mallards fed 10 mg PFOS/kg feed. This indicates that there were
no PFOS-related effects in exposed birds when compared to the
controls. However, in male mallards and quail from the 10 mg
PFOS/kg feed treatment group there was a greater incidence of
smaller (length) testis when compared to the controls. In male
quail, 6 of 16 birds fed 10 mg PFOS/kg feed exhibited testes that
were smaller than those of the untreated controls. In the control
group 1 bird out of 16 males had testes that were smaller than
expected. Likewise, 7 out of 16 mallards in the 10 mg PFOS/kg
feed treatment had reduced testes size as compared to 2 out of
16 males from the control. All other pathology findings in males
were considered incidental and not related to treatment.
No treatment-related effects were observed in the histopathological evaluation of liver, kidney, brain, proventriculus, adipose tissue, or Bursa Fabricius from mallards or quail of the
10 mg PFOS/kg feed treatment. In quail, the greater incidence
of small testis size in the 10 mg PFOS/kg feed treatment was
not accompanied by any morphological change in spermatogenesis. However, in mallards with small testes, one of the control
(one of two) and four treated (four of seven) birds had altered
spermatogenesis that was characterized by fewer or no maturing/mature spermatozoa in the seminiferous tubules. In the other
birds with smaller testis, spermatogenesis appeared to be normal
when compared to control.
3.6. Reproduction
For quail, no treatment-related effects on egg production or
embryo viability were noted in the 10 mg PFOS/kg feed treatment (Table 2). However, there were slight, but not statistically
significant reductions in egg fertility and hatchability in the
10 mg PFOS/kg feed treatment. There was a slight but statistically significant reduction in the number of 14-day-old survivors
as a percentage of the number of eggs set in the 10 mg PFOS/kg
feed treatment.
In mallards from the 10 mg PFOS/kg feed treatment, no statistically significant differences were noted for any reproductive
parameters measured in the study when compared to controls
(Table 2).
3.7. PFOS concentrations in adults and juvenile birds
Chemical analysis of serum and liver samples showed that
adult birds in the 10 mg PFOS/kg feed treatment accumulated
PFOS during the study (Table 3). At study termination, postreproductive liver and serum samples from male mallards and
quail had greater PFOS concentrations than female birds from
the same treatment groups. In quail, the average liver and serum
PFOS concentrations in males were approximately 18- and 16times greater than that found in adult females from the same
treatment groups (Table 3). In mallards, sex-dependent differences in liver and serum PFOS concentrations were also
observed where the concentrations in males were approximately
5–6 times greater that that observed in females from the same
treatment group, respectively (Table 3). Despite the sex-related
differences in serum and liver PFOS concentrations, the ratio of
PFOS concentrations in serum to liver were similar for males and
females of the same species sampled from the 10 mg PFOS/kg
feed treatment. For example, the serum to liver PFOS ratio was
1.6 and 1.8 for male and female quail, respectively. For mallards, the serum to liver ratio for both males and females was
approximately 1.5.
PFOS concentrations were measured in serum and liver samples collected from juvenile birds sampled 14-days post-hatch
(Table 4). While PFOS concentrations in liver and serum of
juvenile birds from parents of the 10 mg PFOS/kg feed treatment were greater than those measured in juveniles from control
parents, they were less than the concentrations measured in the
adult birds (Table 3). In addition, no sex-specific differences
were observed in juvenile liver or serum PFOS concentrations
unlike that observed for post-reproductive adults.
PFOS concentrations in yolks of eggs collected from both
quail and mallards fed 10 mg PFOS/kg feed were greater than
control levels (Table 5). The ratio of PFOS concentrations in egg
yolks to that of serum in adult females sampled at study termination was 7.1 for quail and 3.2 for mallards. However, since
samples were not available for additional exposure concentrations or sampling times, the utility of these ratios to predict egg
concentrations from adult female serum PFOS concentrations is
J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
5
Table 2
Summary of reproductive performance from a northern bobwhite quail and a mallard reproduction study with PFOS
Reproductive parameter
Northern bobwhite
No. replicates
Total eggs laida
Eggs set
Viable embryos
Live 3-week embryos
Hatchlings
Eggs laid/hen/day
14-Day old survivors/hen
Normalized percent datab
Viable embryos/eggs set (%)
Live 3-week embryos/viable embryos (%)
Hatchlings/live 3-week embryos (%)
14-Day old survivors/hatchlings (%)
Hatchlings/eggs set (%)
14-Day old survivors/eggs set (%)
Hatchlings/max. set (%)
14-Day old survivor/max. set (%)
Mallard
Control
10 mg/kg
Control
10 mg/kg
13
605
521
494
490
473
0.50
35
13
592
526
470
466
414
0.49
30
15
719
644
567
564
460
0.67
30
16
877
781
664
653
522
0.76
32
94 ± 13
99 ± 1
97 ± 3
97 ± 4
90 ± 12
87 ± 12
64 ± 20
62 ± 19
89 ± 15
99 ± 2
89 ± 13
93 ± 8
78 ± 18
72 ± 17*
56 ± 20
52 ± 19
88 ± 26
100 ± 1
81 ± 18
99 ± 2
71 ± 28
70 ± 27
48 ± 28
47 ± 28
87 ± 24
98 ± 6
80 ± 21
97 ± 2
69 ± 28
67 ± 27
51 ± 22
50 ± 21
a For bobwhite and mallards, values are based on 93 and 72 days of egg production, respectively. This value represents the total number of eggs layed in each
group.
b Values represent pen means for each experimental group.
* Differences between control and each treatment were statistically significant (p < 0.05).
Table 3
Mean concentrations (±one standard deviation) of PFOS in liver and serum of adult quail and mallards at study terminationa
Treatment (mg/kg PFOS)
Sex
Liver concentration (␮g/g)
Serum concentration (␮g/mL)
Quail
Mallard
Quail
Mallard
<LOQb
Control
M
F
(20)
<LOQ (20)
<LOQ (20)
<LOQ (20)
<LOQ (19)
<LOQ (17)
0.19 ± 0.39 (18)
0.06 ± 0.04 (20)
10
M
F
88.5 ± 28.5 (20)
4.9 ± 1.0 (20)
60.9 ± 19.5 (20)
10.8 ± 8.45 (20)
141 ± 30 (20)
8.7 ± 2.6 (18)
87.3 ± 12.3 (20)
16.6 ± 12.0 (20)
a
b
Sample size given in parentheses. Liver PFOS concentrations given on wet weight basis.
LOQ, limit of quantitation; for liver it was 0.02–0.050 ␮g/g, while for serum it was 0.04–0.10 ␮g/mL.
not great and may only reflect the relative distribution as defined
by the experimental design of these studies. In both mallards and
quail, the greatest concentration of PFOS was associated with
the VLDL fraction in yolk while the concentrations in phosvitin
and lipovitellin were at least 10-fold less than VLDL levels. In
mallards the ranking of PFOS concentrations in yolk fractions
from greatest to least was VLDL > phosvitin > lipovitellin while
in quail it was VLDL > lipovitellin > phosvitin.
3.8. Serum PFOS time course
Both red blood cell and serum samples from whole blood
collected over the duration of the study were analyzed for PFOS
(Fig. 1). Given the relatively great variability in the RBC measurements (mean CV = 59% for mallards, mean CV = 94% for
quail), the serum measurements were deemed to be a more
reliable and representative measure of the PFOS body burden.
Table 4
Mean concentrations (±one standard deviation) of PFOS in liver and serum of 14-day-old juvenile quail and mallards at study terminationa
Treatment (mg/kg PFOS)
Sex
Liver concentration (␮g/g)
Serum concentration (␮g/mL)
Quail
Mallard
Quail
Mallard
<LOQb
Control
M
F
(6)
<LOQ (4)
< LOQ (3)
< LOQ (7)
< LOQ (6)
< LOQ (4)
0.068c ± 0.037 (3)
<LOQ (7)
10
M
F
5.76 ± 0.67 (5)
5.49 ± 1.14 (5)
3.17 ± 1.30 (3)
3.61 ± 1.26 (7)
12.6 ± 3.37 (5)
12.4 ± 3.46 (5)
4.41 ± 0.931 (3)
4.97 ± 1.12 (7)
a
b
c
Sample size given in parentheses. Liver concentrations are on a wet weight basis.
LOQ, limit of quantitation; LOQ for liver = 0.02–0.05 ␮g/g; LOQ for serum = 0.10–0.40 ␮g/ml.
The serum average calculated using half the LOQ when samples were below the LOQ.
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J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
Table 5
Mean concentrations of PFOS (±one standard deviation) in mallard and quail
egg componentsa
Treatment
(mg/kg PFOS)
Fraction
Control
10
PFOS concentration (␮g/g)b
Quail
Mallard
Yolk-whole
<LOQ
<LOQ
Yolk-whole
Yolk-VLDL
Yolk-phosvitin
Yolk-lipovitellin
62 ± 15
39.8
0.83
1.32
52.8 ± 0.58
42.2
8.87
3.59
a Two composite samples from collected from control and PFOS groups for
analysis. Each composite consisted of 12 egg each. All concentrations given on
a wet weight basis.
b LOQ, limit of quantitation, for quail LOQ = 0.01 ␮g/g; for mallard
LOQ = 10 ␮g/g.
The serum measurements showed that there were sex-related
differences in PFOS concentrations. In adult females of both
species, there was a decrease in serum PFOS concentrations that
coincided with the onset of the egg-laying phase. These results
indicate that female birds reduced their body burden by transferring PFOS into eggs during reproduction. In contrast, male
serum PFOS concentrations reached an apparent steady state by
Week 10 and remained relatively constant over the remainder of
the study.
In both male quail and mallards there was a positive relationship between exposure duration and serum PFOS concentrations
that was not greatly affected during the egg-laying phase of the
studies as was observed in adult females. To study the relationship between dietary exposure and the uptake of PFOS by birds,
the serum PFOS data was fitted to a kinetic model and pharmacokinetic parameters were determined Eq. (2):
Ct = Css (1 − e−k2 t )
(2)
where Ct is the concentration of PFOS in the blood serum
(␮g/ml) at time (t); Css the estimated steady state concentration of PFOS in serum (␮g/ml); k2 the elimination rate constant
(day−1 ); and t is the time (day).
The above model assumes that PFOS is accumulated into
and excreted from a single compartment in the bird and that
the kinetics are first order (Wagner, 1979). That is, elimination of PFOS from male birds can be accounted for by a single
rate constant rather than that of females where loss mechanisms include excretion and losses to eggs. The kinetic parameters were determined by fitting the nonlinear model Eq. (2) to
serum PFOS concentration data collected at several time periods
using Marquardt iterative methods (PROC NLIN; SAS Institute,
1999). The uptake rate constant (k1 ) was estimated using the
concentration at steady state and the elimination rate constant
Eq. (3).
k1 =
Css k2
Cfeed
(3)
Fig. 1. Time course of PFOS concentrations in serum and red blood cells from adult mallards and bobwhite quail. Photostimulation indicates the time at which
female birds were stimulated for egg production. Sample size at each time period and for each matrix was four. (a) Concentration of PFOS in serum of adult quail;
(b) concentration of PFOS in red blood cells of adult quail; (c) concentration of PFOS from adult mallards; (d) concentration of PFOS in red blood cells of adult
mallards.
J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
7
Table 6
Estimated kinetic parameters for serum PFOS concentrations in male quail and mallards exposed to 10 mg PFOS/kg feeda
Species
Css (␮g/ml)
k1 (g/ml day)
k2 (day−1 )
Half life (day)
Quail
Mallard
161 ±
118 ± 7.80
0.539
0.602
0.0335 ± 0.0168
0.0510 ± 0.0202
20.7
13.6
a
b
18.5b
Css is serum PFOS concentration at steady state, k1 is uptake rate constant from diet, k2 is elimination rate constant from serum.
Data presented as mean and standard error of the mean.
where k1 is the uptake rate constant in blood (g/ml/day); Css the
estimated steady state concentration of PFOS in serum (␮g/ml);
Cfeed the PFOS concentration in diet (␮g/g); and k2 is the elimination rate constant (day−1 ). Based on the results of these
analyses, it was determined that serum PFOS concentrations
in males achieved steady state concentration during the study
and that PFOS had a relatively short residence time in serum
with half-lives of 20.7 and 13.6 days for quail and mallards,
respectively (Table 6).
4. Discussion
The mortality of quail and mallards caused by dietary exposure to 50 or 150 mg PFOS/kg feed in this study was similar to
that observed in other avian studies where the onset of PFOSrelated lethality occurred within a narrow concentration range
and was related to cumulative dose (Newsted et al., 2005). In the
current studies, the total cumulative dose associated with overt
signs of toxicity in mallards and quail fed 150 mg PFOS/kg feed
was 293 and 204 mg PFOS/kg bw, respectively. Furthermore, in
the current study, PFOS-related reductions in feed consumption
and body weight occurred at dietary concentrations equal to and
greater than 50 mg PFOS/kg feed while in two pilot reproduction
studies, significant reductions in these endpoints were observed
in quail and mallard fed 17.6 mg PFOS/kg feed (Gallagher et al.,
2003c,d). In both the current and pilot studies, no PFOS-related
effects were noted on these endpoints in birds exposed to 10
and 6.2 mg PFOS/kg feed, respectively. Some of the differences
observed in effects noted between the pilot and current studies
may have been due to experimental design. In the pilot studies,
both species were first brought into reproductive condition prior
to dietary exposure while in the current reproduction studies,
the birds were fed treated diet for up to 10 weeks prior to photostimulation to induce egg laying. Because of the differences
in exposure regimes, it is difficult to make direct comparisons
between the studies. In particular, the potential effects of accelerated exposure/reproduction phase in the pilot studies, relative
to alterations in feed consumption and bioenergetics of the birds
in comparison to the longer exposure phase of the current study
is difficult to evaluate. However, despite the differences in study
design between these sets of studies, taken together these data
indicate that the threshold for effects on body weight and feed
consumption occurs between 10 and 20 mg PFOS/kg feed.
The sensitivities of the two bird species evaluated in this study
were similar to thresholds for PFOS-related effects on body
weight, body weight gain and feed consumption observed in
mammals. Several sub-chronic exposures of rats, mice and monkeys to PFOS have resulted in effects on body weight, weight
gain and/or feed consumption at doses from 0.4 to 20 mg/kg
bw/day. In a 14-week study of the effects of PFOS on rats, the
rate of food consumption but not body weight gain was decreased
by exposure to 20 mg/kg feed, which was equivalent to 1.3 mg
PFOS/kg bw/day in females and 1.56 mg PFOS/kg bw/day in
males (Seacat et al., 2003). Likewise, in a two-generation study
of the effects of PFOS on rats, body weights and weight gains
of males of the parental generation (F0 ) exposed to 0.4 mg
PFOS/kg bw/day were significantly less than those measured
in the untreated controls (Luebker et al., 2005b). In a study of
pregnant rats dosed by gavage during gestation Days 2–20, significant reductions in maternal body weight gain were observed
at doses of 2–10 mg PFOS/kg bw/day (Thibodeaux et al., 2003).
Pregnant CD-1 mice dosed during gestation showed reduced
body weights at 20 mg PFOS/kg bw/day (Thibodeaux et al.,
2003). In monkeys exposed by dosing with encapsulated PFOS
for 6 months, body weight was reduced at 0.75 mg PFOS/kg
bw/day (Seacat et al., 2002). Overall, the results from the mammalian and avian studies indicate that mammals and birds have
similar thresholds for PFOS-related effects.
The reversible increase in liver weight and absence of tissue
damage in rodents exposed to PFOS was similar to the results
observed in this study. The statistically significant increase in
absolute and relative liver weight of quail, in the absence of any
other pathological findings, is indicative of an adaptive response
to PFOS exposure, but not necessarily a toxic effect (Keenan et
al., 1995; Cotran et al., 1999; USEPA, 2002; Bailey et al., 2004).
The minor differences between responses of rats and the two bird
species was most likely due to several factors, including differences in species sensitivity, differences in absorption of PFOS
via the gut, differences in metabolism and excretory mechanisms
between rats and birds.
Taken together with the fact that other treated mallards
with small testes did not exhibit any alteration in spermatogenesis, PFOS may have accelerated early post-reproductive
phase regression in exposed birds, a normal physiological phenomenon. In many seasonal breeders such as quail, males display
a cyclic reduction in fertility during non-breeding season. This
reduction is accompanied by regression of testicular germinal
epithelium leading to a decline in spermatozoa production and
testicular size (Rosenstrauch et al., 1994; Wilkelski et al., 2003).
Thus, because no effects were noted on egg fertilization, the
small testes in males did not appear to significantly affect reproductive performance in the males exposed to PFOS in the diet.
While no PFOS-related effects were observed for any reproductive parameter monitored during the mallard reproduction
study, there were slight but significant effects on egg hatchability and survival of offspring of adult quail fed 10 mg PFOS/kg
8
J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
Table 7
NOAEL and LOAEL values in various matrices in adult and offspring mallards and quail in a chronic dietary study with PFOS
Measures of PFOS exposurea
Bobwhite quail
Mallard
NOAEL
LOAELb
NOAEL
LOAEL
Adult males
Dose (ppm)
ADI (mg PFOS/kg body weight/day)
Serum (␮g PFOS/mL)
Liver (␮g PFOS/g)
10
0.772
141
88.5
ND
ND
ND
ND
10
1.49
87.3
60.9
ND
ND
ND
ND
Adult females
Dose (ppm)
ADI (mg PFOS/kg body weight/day)
Serum (␮g PFOS/mL), pre-reproduction (5-weeks)
Serum (␮g PFOS/mL)
Liver (␮g PFOS/g)
ND
ND
ND
ND
ND
10
0.77
84
8.7
4.9
10
1.49
76.9
16.6
10.8
ND
ND
ND
ND
ND
Offspring
Yolk (␮g PFOS/mL)
Liver (␮g PFOS/g)c
Serum (␮g PFOS/mL)c
ND
ND
ND
62
5.5
12.5
52.7
3.39
4.69
ND
ND
ND
a
b
c
All concentrations are reported on a wet weight basis. All exposure measures made at 21 weeks (study termination) unless otherwise specified.
LOAEL was based on a decrease in the 14-day-old survivability of offspring, and a statistically significant increase in adult female liver weight.
Offspring liver and serum LOAEL values are averages of female and male concentrations.
feed. This is similar to the results of studies in which maternal
rats and mice were exposed to prior to mating and/or through
mating and gestation. In the rat study, post-natal survival was
less for pups born to dams exposed to 1.6 mg PFOS/kg bw/day
for approximately 6 weeks prior to mating as well as through
gestation (Luebker et al., 2005a). The effects on pups were also
similar to that observed for mice exposed to 10 mg PFOS/kg
bw/day through most of gestation (Lau et al., 2003). The absence
of obvious malformations observed in offspring of mallards or
quail treated with PFOS in the current chronic reproductive studies is similar to the results of studies with mammals where effects
noted on offspring of mammals exposed to PFOS during prenatal development were generally unremarkable when compared
to maternal effects from the same treatment groups (Case et al.,
2001; Lau et al., 2004).
The concentrations of PFOS in tissues associated with the
lowest observable adverse effect level (LOAEL) are summarized
(Table 7). In general, the PFOS concentrations associated with
LOAELs in rats and mice are greater than measured concentrations of PFOS in avian serum and liver that are associated with
PFOS-related adverse effects. In part, these differences may be
due to different experimental design and dosing, but may also
be related to pharmacokinetics. For instance, the estimated halflife of PFOS in the serum of quail and mallards were 20.7 and
13.6 days, respectively. While these results are preliminary estimates of elimination kinetics in birds, the analyses indicate a
significant departure from that observed in several mammals. In
male rats, the whole body elimination half-time for elimination
after a single IV dose was estimated to be >89 days (Johnson
et al., 1979) while in monkeys, the elimination half-life after
a single IV dose ranged between 88 and 149 days (Noker and
Gorman, 2003). In cynomologus monkeys exposed to PFOS
via intragastric intubation of a capsule for at least 28 days, the
elimination half-life ranged between 100 and 200 days (Seacat
et al., 2002). In mammals, PFOS is readily absorbed and distributed in serum and liver but poorly eliminated, partially due
to enterohepatic circulation (Johnson et al., 1984). However, the
role of this mechanism in maintaining body burdens of PFOS
in birds has not been studied. In addition, urinary excretion in
birds differs from that observed in mammals and could also be
a contributing factor in the more rapid loss of PFOS observed in
birds. Finally, the association of PFOS with serum lipids and in
particular lipoproteins, is an important elimination mechanism
in female birds via the eggs that does not exist in mammals that
could contribute to differences in the retention of PFOS in the
serum of birds from that observed in mammals. The latter point is
especially important when evaluating adult female serum PFOS
concentrations in that reproductive condition can have a significant influence on measured PFOS concentrations and could lead
to erroneous conclusions relative to the risk posed by PFOS to
avian populations.
A comparison of the results from the two avian reproduction studies indicates that quail were slightly more sensitive to
dietary PFOS than were mallards exposed to equivalent concentrations. Based on a reduction in the number of 14-day-old
offspring survivors as a percent of eggs set by quail fed PFOS
in the diet, a LOAEL of 10 mg PFOS/kg feed was determined.
While a NOAEL from the chronic quail study could not be determined, a NOAEL from a pilot reproduction study with quail
was determined to be 6.2 mg PFOS/kg feed (Gallagher et al.,
2003c). Based on the slight effects observed in birds fed 10 mg
PFOS/kg feed in the definitive study, in conjunction with the
results of the pilot study, birds populations exposed to dietary
concentrations of PFOS equal to or less than 6.2 mg PFOS/kg
feed would not be expected to experience adverse effects
on survival of adults of offspring, reproductive performance
of the adults or survival, growth, or development offspring
development.
J.L. Newsted et al. / Environmental Toxicology and Pharmacology 23 (2007) 1–9
Acknowledgements
The authors would like to thank 3M for financial support
for this project. We also thank Paul Jones and others for their
insightful comments and helpful advice on this manuscript.
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