Production of perfluorinated carboxylic acids (PFCAs) from the biotransformation of polyfluoroalkyl phosphate surfactants (PAPS): Exploring routes of human contamination

Article abstract of DOI:10.1021/es070126x

Perfluorinated acids are detected in human blood worldwide, with increased levels observed in industrialized areas. The origin of this contamination is not well understood. A possible route of exposure, which has received little attention experimentally, is indirect exposure to perfluorinated acids through ingestion of chemicals applied to food contact paper packaging. The current investigation quantified the load of perfluorinated acids to Sprague-Dawley rats upon exposure to polyfluoroalkyl phosphate surfactants (PAPS), nonpolymeric fluorinated surfactants approved for application to food contact paper products. The animals were administered a single dose at 200 mg/kg by oral gavage of 8:2 fluorotelomer alcohol (8:2 FTOH) mono-phosphate (8:2 monoPAPS), or the corresponding di-phosphate (8:2 diPAPS), with blood taken over 15 days post-dosing to monitor uptake, biotransformation, and elimination. Upon completion of the time-course study the animals were redosed using an identical dosing procedure, with sacrifice and necropsy 24 h after the second dosing. Increased levels of perfluorooctanoic acid (PFOA), along with both 8:2 PAPS congeners, were observed in the blood of the dosed animals. In the 8:2 monoPAPS-dosed animals, 8:2 monoPAPS and PFOA blood concentrations peaked at 7900 ± 1200 ng/g and 34 ± 4 ng/g respectively. In the 8:2 diPAPS-dosed animals, 8:2 diPAPS peaked in concentration at 32 ± 6 ng/g, and 8:2 monoPAPS and PFOA peaked at 900 ± 200 ng/g and 3.8 ± 0.3 ng/g, respectively. Several established polyfluorinated metabolites previously identified in 8:2 FTOH metabolism studies were also observed in the dosed animals. Consistent with other fluorinated contaminants, the tissue distributions showed increased levels of both PFOA and the 8:2 PAPS congeners in the liver relative to the other tissues measured. Previous investigations have found that PAPS can migrate into food from paper packaging. Here we link ingestion of PAPS with in vivo production of perfluorinated acids.

Full text of DOI:10.1021/es070126x

Environ. Sci. Technol. 2007, 41, 4799-4805
been observed in the blood of humans (2) and wildlife (3)
Production of Perfluorinated
Carboxylic Acids (PFCAs) from the
Biotransformation of Polyfluoroalkyl
Phosphate Surfactants (PAPS):
Exploring Routes of Human
Contamination
worldwide. Commonly discussed fluorinated contaminants
are the perfluorinated acids, which consist of perfluorinated
carboxylic acids (PFCAs) and perfluorinated sulfonic acids
(PFSAs). Despite their low pKa values (4), which render them
relatively involatile, perfluorinated acids are widespread in
the environment (5, 6). This ubiquity is not intuitive, and as
such has incited interest into possible modes of dissemina-
tion. Two fields of thought have emerged to explain current
levels found in the environment. One theory relies on direct
input of PFOA from production facilities (7, 8), and the other
relies on volatile fluorinated alcohol precursor emissions from
manufactured materials as an indirect source of contamina-
tion (9-11). The hub of major debate has been Arctic
contamination (6-11). The issue of human exposure is
increasingly complicated as several sources are likely in-
volved, with relative contributions varying with lifestyle and
location (2, 12). The reported concentrations of PFOA in
human serum vary from nondetect to >30 ng/mL worldwide
(2). Human contamination is of concern as perfluorinated
acids have no known degradation pathway, are slowly
excreted by humans (13), and an advisory board to the U.S.
EPA has published a draft document proposing PFOA be
deemed a rodent carcinogen with relevance to humans (14).
To properly estimate human exposure, relative contributions
of different exposure pathways need to be deciphered.
Discussed here is an indirect source of human exposure to
PFCAs via ingestion and metabolism of FTOH-based PAPS,
which are approved for application to food contact materials.
Fluorinated chemicals have primarily been produced via
two manufacturing processes: telomerization and electro-
chemical fluorination (ECF). ECF chemistry generates a
characteristic distribution of 20-30% structural isomers (4),
whereas telomerization produces only the straight chain
isomer (4). 3M, a major manufacturer of ECF-based fluo-
rochemicals, phased-out their perfluorooctyl-products in
2001 due to environmental concerns (15). The void left by
3M was largely replaced by telomer-based products, resulting
in a dramatic shift in the market toward telomer, and hence
linear, compounds (16, 17). The PFCA isomer distribution in
pooled human blood samples from the Midwestern United
States between 2004 and 2005 was >98% linear (18). With a
half-life for PFOA of 4.4 years in human serum (13), in the
absence of significant isomer discrimination, the predomi-
nance of the linear isomer suggests exposure to current-use
fluorinated materials and not the historical load present in
the environment.
PFCAs may be present in consumer articles treated with
fluorinated polymers. Extraction tests and product informa-
tion suggest that direct exposure to PFOA from the ap-
propriate use of products treated with fluorochemicals is
not a significant source of contamination to the general
population (19). FTOHs and perfluorinated sulfonamides
have been observed in both indoor (20) and outdoor (20-
22) air. FTOHs are metabolized to PFCAs (23), while per-
fluorinated sulfonamides are metabolized to PFSAs (24). As
such, inhalation of these neutral precursors may contribute
to the load of perfluorinated acids observed in human blood.
Indirect exposure to perfluorinated acids may also occur by
ingestion of PAPS, which are used to impart oil and water
repellency to certain food contact paper products (25). Begley
et al. (26) have shown that PAPS will migrate into food
simulants under appropriate test conditions. Cleavage of the
phosphate ester linkage of PAPS within a biological system
would likely result in production of perfluorinated acids via
metabolism of the released fluorinated alcohol. Although
J E S S I C A C . D ’ E O N A N D
S C O T T A . M A B U R Y *
Department of Chemistry, University of Toronto, 80 St. George
Street, Toronto, Ontario, Canada M5S 3H6
Perfluorinated acids are detected in human blood world-
wide, with increased levels observed in industrialized
areas. The origin of this contamination is not well understood.
A possible route of exposure, which has received little
attention experimentally, is indirect exposure to perfluorinated
acids through ingestion of chemicals applied to food
contact paper packaging. The current investigation quantified
the load of perfluorinated acids to Sprague-Dawley rats
upon exposure to polyfluoroalkyl phosphate surfactants
(PAPS), nonpolymeric fluorinated surfactants approved for
application to food contact paper products. The animals
were administered a single dose at 200 mg/kg by oral gavage
of 8:2 fluorotelomer alcohol (8:2 FTOH) mono-phosphate
(8:2 monoPAPS), or the corresponding di-phosphate (8:2
diPAPS), with blood taken over 15 days post-dosing to monitor
uptake, biotransformation, and elimination. Upon completion
of the time-course study the animals were redosed
using an identical dosing procedure, with sacrifice and
necropsy 24 h after the second dosing. Increased levels of
perfluorooctanoic acid (PFOA), along with both 8:2 PAPS
congeners, were observed in the blood of the dosed animals.
In the 8:2 monoPAPS-dosed animals, 8:2 monoPAPS and
PFOA blood concentrations peaked at 7900 ( 1200 ng/g and
34 ( 4 ng/g respectively. In the 8:2 diPAPS-dosed
animals, 8:2 diPAPS peaked in concentration at 32 ( 6
ng/g, and 8:2 monoPAPS and PFOA peaked at 900 ( 200
ng/g and 3.8 ( 0.3 ng/g, respectively. Several established
polyfluorinated metabolites previously identified in 8:2
FTOH metabolism studies were also observed in the dosed
animals. Consistent with other fluorinated contaminants,
the tissue distributions showed increased levels of both
PFOA and the 8:2 PAPS congeners in the liver relative
to the other tissues measured. Previous investigations have
found that PAPS can migrate into food from paper
packaging. Here we link ingestion of PAPS with in vivo
production of perfluorinated acids.
Introduction
Since 1968 when Taves (1) discovered organic fluorine in
human blood samples, fluorinated organic compounds have
* Corresponding author phone: (416) 978-1780; fax: (416) 978-
3596; e-mail: smabury@chem.utoronto.ca.
9
10.1021/es070126x CCC: $37.00
Published on Web 05/23/2007
2007 American Chemical Society
VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4799

dephosphorylation processes are common in biological
systems (27), the significance of the fluorinated chains present
in PAPS-based materials on this process is not known.
N-methyl perfluorooctane sulfonamido ethanol (NMe-
FOSE) and N-ethyl perfluorooctane sulfonamido ethanol
(NEtFOSE) were the functional units used by 3M in their
fluorinated coatings (28). NMeFOSE was primarily incor-
porated into polymeric surface treatment products for fabrics
and carpets, a technology that was commercialized in the
1950s (28, 29), whereas the primary application of NEtFOSE
was in PAPS-based nonpolymeric surfactant materials used
in food contact paper products, for which human food contact
applications were introduced in 1974 (28, 29). In a study of
the historical load of fluorochemicals in human blood, Olsen
et al. (29) observed a 4-fold increase in the acetate adduct
of perfluorooctane sulfonamide (PFOSA) between 1974 and
1989. The authors attribute this increase to the incorporation
of NEtFOSE-PAPS materials into human food contact paper
products (29). A study published in 2006 by Calafat et al. (12)
found the acetate adducts of both NMeFOSE and NEtFOSE
in pooled blood samples from the United States from 2001
and 2002. The presence of these fluorinated metabolites,
which are relatively quickly eliminated or metabolized to
perfluorooctane sulfonate (PFOS) (24, 29), suggests recent
exposure to indirect fluorochemical sources. The presence
of the NEtFOSE acetate adduct suggests this indirect source
may be a PAPS-based material.
TABLE 1. Common Names, Acronyms, and Structures for the
Analytes of Interest
The relevance of PAPS to human fluorochemical exposure
extends beyond their use in food packaging applications, as
PAPS are approved by the U.S. EPA as an inert defoaming
additive to pesticide formulations (30). Although the load of
PAPS used in this capacity is not publicly known, nonpoly-
meric fluorinated surfactants akin to PAPS account for 20%
of the 12 million kg of fluorinated materials produced
annually (16).
To interrogate whether PAPS can contribute to the load
of PFCAs observed in the human population, we dosed male
Sprague-Dawley rats with in-house synthesized and purified
8:2 FTOH mono- or di-substituted PAPS (8:2 monoPAPS, 8:2
diPAPS). We were particularly interested in the biological
availability of FTOH-based PAPS as they are currently
commercially available for food contact paper applications
(31-33), and specific restrictions, which take effect in 2008,
have been placed on their use as inert additives within
pesticide formulations (34).
The animals were administered a single dose of either 8:2
PAPS congener by oral gavage with subsequent blood
sampling for 15 days post-dosing. As 8:2 FTOH is the
fluorinated moiety in both 8:2 PAPS congeners, PFOA is the
major PFCA biotransformation product expected (23, 35). In
addition to PFOA, several established 8:2 FTOH intermediate
metabolites were monitored to link PFOA production with
8:2 FTOH exposure via 8:2 PAPS biotransformation (23, 35).
Perfluorononanoic acid (PFNA), perfluoroheptanoic acid
(PFHpA), and perfluorohexanoic acid (PFHxA) were moni-
tored to quantify contributions from R-oxidation, and any
potential degradation of the perfluorinated chain (23, 35).
Direct absorption of the 8:2 PAPS congeners was also
monitored. All analytes are shown in Table 1. To investigate
the distribution of 8:2 PAPS among select tissues, the animals
were redosed upon completion of the time-course, with
sacrifice and necropsy 24 h after the second dosing. Hy-
drolysis experiments were performed in concert with the
biological experiments to characterize the abiotic degradation
potential of the 8:2 PAPS congeners.
a 8:2 FTOH was not monitored in this investigation. b No analytical
standards were available for the 7:3 uAcid.
lowing equilibrium reaction, which is a bench-scale variation
of a 1963 patented process (36). To establish anhydrous
conditions, triethylamine (TEA) was dried by distillation
under nitrogen, and tetrahydrofuran (THF) was dried by reflux
over sodium with benzophenone as an indicator.
Under nitrogen atmosphere at -78 °C, 3 mol equiv of dry
TEA in 10 mL of dry THF was added over 10 min to 1 mol
equiv of phosphorus oxychloride in 10 mL of dry THF. For
the di-substituted surfactant, 2 mol equiv of either 8:2 FTOH
or 9:1 FTOH was added to the reaction mixture in 30 mL of
dry THF over 1 h. For the monosubstituted surfactant 0.3
mol equiv (to minimize production of di- and tri-substituted
8:2 PAPS congeners) of 8:2 FTOH was added to the reaction
mixture in 30 mL of dry THF over 1 h. After addition of the
alcohol the reaction mixture was allowed to warm to room
temperature (1-2 h), then 50 mL of distilled deionized water
was added over 1 h. Product isolation and purity analysis are
described in the Supporting Information. Both 8:2 PAPS
congeners were >97% pure with respect to fluorinated
materials of interest. 8:2 monoPAPS contained <0.01% PFOA
and 0.6% 8:2 FTOH. 8:2 diPAPS contained <0.02% PFOA,
1.5% 8:2 FTOH, and <1% 8:2 monoPAPS. Unless otherwise
stated all percentages are expressed on a per mole basis.
Abiotic Hydrolysis Procedure. The hydrolysis procedure
involved detection of 8:2 FTOH, produced by cleavage of the
phosphate ester linkage from either 8:2 PAPS congener at
50 °C and pH 9, via a purge-and-trap system described
elsewhere (9), with analysis using a Hewlett-Packard 6890
gas chromatograph coupled to a 5973 inert mass spectrometer
operating in single ion monitoring mode (GC-MS). Experi-
mental and chromatographic details are provided in the
Supporting Information.
Experimental Section
Synthesis of the Polyfluoroalkyl Phosphate Surfactants
(PAPS). 8:2 monoPAPS, 8:2 diPAPS, and 9:1 diPAPS (using
9:1 FTOH, F(CF2)9CH2OH) were synthesized using the fol-
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In Vivo Model. This research was conducted under an
animal use protocol approved by the University Animal Care
Committee, and was supervised by a licensed veterinarian.
Twelve 7-week-old male Sprague-Dawley rats were obtained
from Charles River Laboratories Inc. (Wilmington, MA). The
animals were doubly housed and randomly assigned into
three groups corresponding to 8:2 monoPAPS exposure, 8:2
diPAPS exposure, and control. Food and water were available
ad libitum throughout the experiment. After one week
acclimatization, the animals in the exposure groups were
administered a single bolus dose of either 8:2 monoPAPS or
8:2 diPAPS at 200 mg/kg in 0.5% aqueous methylcellulose at
5 mL/kg by oral gavage without prior fasting. Shaking by
hand and sonication were used to dissolve the 8:2 PAPS
congeners in 0.5% aqueous methylcellulose. Although the
administered doses were cloudy, they were uniform and void
of visible lumps. Control animals received 5 mL/kg of undosed
0.5% aqueous methylcellulose. In accordance with the
approved animal use protocol, the dose concentration
corresponded to a no observable adverse effect level (NOAEL)
for 8:2 FTOH from the literature (37). Whole blood was
harvested from the animals via heparinized syringes using
the lateral tail vein 24 h prior to dosing and at 0.2 (4 h), 1,
2, 3, 5, 9, and 15 days post-dosing. The total volume of blood
collected per animal over the course of the experiment was
limited to 10% of their total blood volume (estimated using
50 mL of blood per kg of animal). Individual blood samples
ranged from 50 to 500 mg, with a mean mass of 189 mg.
Whole blood samples were stored at -20 °C until extraction
and analysis. Urine was not collected, however feces samples
were obtained, if available, from animals during blood
sampling. Five days after the last time-course sample was
taken, the animals were redosed using the same procedure
as above, with sacrifice by carbon dioxide asphyxiation 24-h
post-dosing. Blood, liver, kidney, muscle, fat, spleen, and
brain were harvested from the animals and stored at -20 °C
until extraction and analysis. The animals did not show any
clinical signs of toxicity throughout the course of the
experiment, and the liver somatic index was not statistically
different among the three groups of animals (8:2 monoPAPS-
dosed, 8:2 diPAPS-dosed, control), as determined using a
Kruskal-Wallis test (p ) 0.397), suggesting the administered
dose had little toxicological significance.
quantified using internal calibration with the following
internal standards: 13C2-PFHxA (PFHxA), 13C2-PFOA (PFHpA,
PFOA), 13C5-PFNA (PFNA), 13C2-8:2 FTUCA (8:2 FTCA, 8:2
FTUCA, 7:3 Acid, 7:3 uAcid), 13C2-perfluorodecanoic acid (8:2
monoPAPS), and 9:1 diPAPS (8:2 diPAPS). Internal calibration
of 8:2 monoPAPS with 13C2-perfluorodecanoic acid was
validated using standard addition, where three samples
analyzed by both methods were statistically similar using a
paired t-test (p ) 0.411). A spike and recovery experiment
(n ) 4) was performed using blood harvested upon necropsy
from a control animal. Whole rat blood (300 µL) was stored
for 24 h at -20 °C with 2.5 ng of all analytes of interest (Table
1). Extraction and analysis was performed as described above.
The spike and recovery results were as follows: 107 ( 4%
(PFHxA), 64 ( 5% (PFHpA), 111 ( 6% (PFOA), 108 ( 7%
(PFNA), 101 ( 8% (8:2 FTCA), 63 ( 5% (8:2 FTUCA), 174 (
19% (7:3 Acid), 70 ( 16% (8:2 monoPAPS), and 66 ( 8% (8:2
diPAPS). Reported values were not corrected for recovery.
Procedural contamination was quantified by including two
extraction blanks with each set of samples. Samples were
blank corrected where appropriate using the mean blank
value obtained from all analyses. For analytes present in the
procedural blanks (PFHxA, PFHpA, PFOA (time-course),
PFNA) the limit of detection (LOD) was defined as three
standard deviations (3σ) from the mean blank level, and the
limit of quantitation (LOQ) was defined as 10σ from the mean
blank level (40). For analytes absent in the procedural blanks
(PFOA (tissue distribution), 8:2 FTCA, 8:2 FTUCA, 7:3 Acid,
8:2 monoPAPS, 8:2 diPAPS) the LOD was empirically deter-
mined as the concentration producing a signal-to-noise ratio
of 3, and the LOQ was defined as the concentration producing
a signal-to-noise ratio of 10 (40). LOD and LOQ values were
transformed from extract concentrations to blood or tissue
concentrations using the mean mass of blood harvested over
the time-course (189 mg) and the mean mass of tissue
extracted (0.715 g). LOD and LOQ values for all analyses are
provided in the Supporting Information. Values less than
LOD are reported as nondetect (nd) and given a value of
zero, values less than LOQ are reported unaltered but are
indicated using brackets in tables and an asterisk (*) in figures.
Background concentrations in the control animals were either
nondetect or below LOQ for all analytes, and are reported
in the Supporting Information.
Extraction Procedure. Whole blood samples were ex-
tracted using a modified version of the ion-pairing method
developed by Hansen et al. (38). Tissue and feces samples,
except fat, were extracted using the abovementioned ion-
pairing method with the addition of a fluorosolvent protein
precipitation step described in detail in Furdui et al. (39).
Due to the high lipid content, fat samples were extracted
using a methanol extraction technique. Extractions are
described in detail in the Supporting Information.
Instrumental Analysis. All samples were analyzed by
liquid chromatography coupled to negative electrospray
ionization tandem mass spectrometry (LC-MS/MS). Blood
samples from the time-course were analyzed for PFHxA,
PFHpA, PFOA, PFNA, 8:2 FTCA, 8:2 FTUCA, 7:3 Acid, 7:3
uAcid, 8:2 monoPAPS, and 8:2 diPAPS, using an API 4000 Q
Trap (Applied Biosystems/MDS Sciex) coupled to an Agilent
1100 autosampler. All tissue samples, including blood
obtained at necropsy, were analyzed for PFOA, 8:2 mono-
PAPS, and 8:2 diPAPS, using a Micromass Ultima (Micromass,
Manchester, United Kingdom) coupled to a Waters 717 plus
autosampler (Waters, Milford, United Kingdom). Chromato-
graphic details and mass transitions are provided in the
Supporting Information.
Results and Discussion
Abiotic Study. PFCA production from 8:2 PAPS will likely
proceed via cleavage of the phosphate ester linkage, releasing
free 8:2 FTOH, with subsequent biotransformation to PFOA.
Although phosphate triesters are relatively labile toward
hydrolysis (41), phosphate monoesters and diesters are stable,
with lifetimes on the order of several years with respect to
hydrolysis at environmental conditions (42). To confirm these
findings, the hydrolytic stability of both 8:2 monoPAPS and
8:2 diPAPS was investigated under aggressive conditions of
pH 9 and 50 °C. We observed <0.1% degradation over a
2-week period for both 8:2 PAPS congeners, corresponding
to a minimum lifetime of 26 years with respect to hydrolysis.
As this lifetime is consistent with previous investigations (42),
we expect both 8:2 monoPAPS and 8:2 diPAPS to be stable
toward abiotic hydrolysis throughout the entire gastrointes-
tinal tract. In biological systems phosphates are subject to
dephosphorylation by phosphatase enzymes, which are
prevalent in the body as the phosphate anion is involved in
energy transfer reactions and as a major component of
hydroxyapatite (27). Phosphatase enzymes are also present
in the intestinal tract and are involved in the dephospho-
rylation of certain nutrients, such as the phosphate ester of
thiamin (27). As both 8:2 PAPS congeners are insensitive to
abiotic hydrolysis, any dephosphorylation presumably in-
Quality Control Procedure. Concentration and spike and
recovery values are reported using the mean concentration
and standard error. Units are reported as mass of analyte
(ng) per mass of blood or tissue extracted (g). Analytes were
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VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4801

FIGURE 1. Mean concentrations in whole blood. Error bars indicate standard error. (a) 15-Day time-course for 8:2 monoPAPS and PFOA
in the 8:2 monoPAPS-dosed animals. (b) 3-Day time-course for PFOA, PFHpA, 8:2 FTCA, 8:2 FTUCA, and 7:3 Acid in the 8:2 monoPAPS-dosed
animals. (c) 15-Day time-course for 8:2 diPAPS, 8:2 monoPAPS, and PFOA in the 8:2 diPAPS-dosed animals. (d) 3-Day time-course for PFOA,
8:2 FTCA, 8:2 FTUCA, and 7:3 Acid in the 8:2 diPAPS-dosed animals. Values less than LOD are reported as zero, and values less than LOQ
are indicated with an asterisk (*).
volves phosphatase enzymes either in the gut or within the
body.
animals. The time-course from 0 to 3 days post-dosing for
the 7:3 Acid, 8:2 FTCA, and 8:2 FTUCA is shown in Figure 1b.
The 7:3 uAcid was not observed, however this analyte was
monitored using a mass transition obtained from Martin et
al. (23) without further optimization as no analytical standards
were available. The transient nature of the observed me-
tabolites in blood is evident from their concentration peak
at 4 h post-dosing, with subsequent rapid decrease or absence
in the remainder of the time-points. The biological mech-
anism involved in the transformation from 8:2 FTOH to PFOA
has been discussed in detail in previous studies (23, 35), and
is not the focus of this investigation, however these transient
species provide considerable evidence that the observed
increase in PFOA resulted from 8:2 FTOH exposure. As well
as the transient metabolites, Figure 1b also includes the initial
3-day time-course for PFOA and PFHpA. Aside from PFOA,
PFHpA was the only PFCA observed above background levels
in the control animals. PFHpA attained a maximum con-
centration of 6.1 ( 1.1 ng/g 24 h post-dosing. PFHpA has
previously been observed in rats dosed with 8:2 FTOH (35).
Concentrations observed in the blood of the 8:2 diPAPS-
dosed animals are displayed in Figure 1c and d. 8:2 diPAPS
peaked at 4 h post-dosing with a concentration of 32 ( 6
ng/g. 8:2 monoPAPS was observed at a maximum concen-
tration of 900 ( 200 ng/g 24 h post-dosing. The 15-day time-
course for PFOA shows a clear uptake profile, which plateaus
In Vivo Study. Elevated levels of PFOA were observed in
the blood from both dose groups as compared to background
levels in the control animals; where 80% of the control
samples were below the LOD of 1.8 ng/g, with a maximum
observed concentration of 2.0 ng/g. In the 8:2 monoPAPS-
dosed animals, 8:2 monoPAPS was the most prevalent
fluorinated contaminant observed (Figure 1a), with a peak
concentration of 7900 ( 1200 ng/g 24 h post-dosing. Overlaid
with 8:2 monoPAPS in Figure 1a is the time-course for PFOA.
PFOA peaked in concentration between 24 and 48 h post-
dosing, with a mean value of 34 ( 4 ng/g at both time-points.
This plateau suggests the true concentration peak was likely
not captured by the sampling routine. Using 24 h as the peak
uptake value, we calculated an elimination half-life of PFOA
from blood of 20 days. This half-life is longer than the value
of 4.1-9.0 days reported by Fasano et al. (35) for the
elimination half-life of PFOA from the blood of adult male
rats administered a single bolus dose of 8:2 FTOH at 125
mg/kg by oral gavage. The additional dephosphorylation step
involved in PAPS metabolism may be responsible for the
longer half-life observed here.
In addition to 8:2 monoPAPS and PFOA, several poly-
fluorinated metabolites, consistent with 8:2 FTOH exposure,
were observed in the blood of the 8:2 monoPAPS-dosed
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TABLE 2. Mean Concentrations in Tissues Harvested at Necropsy (Errors are Reported Using the Standard Error; Values Less than
LOD are Reported as Nondetect (nd) and Values Less than LOQ are Reported in Brackets)
concentration (ng/g)a,b
8:2 monoPAPS-dosed
8:2 monoPAPS
8:2 diPAPS-dosed
8:2 monoPAPS
tissuec
PFOA
8:2 diPAPS
PFOA
8:2 diPAPS
blood
liver
kidneys
130 ( 15
320 ( 50
100 ( 20
{130 ( 21}
1700 ( 300
{440 ( 70}
-
-
-
480 ( 100
1500 ( 200
230 ( 30
820 ( 90
9200 ( 600
3700 ( 200
{85 ( 11}
600 ( 200
nd
a Analysis was performed using a Micromass Ultima mass spectrometer. b Tissues harvested from the control animals were nondetect for the
analytes of interest. c Muscle, fat, and brain samples from the dosed animals were nondetect for the analytes of interest. Spleen and testes from
the dosed animals were nondetect for all analytes except PFOA observed in the testes of the 8:2 diPAPS-dosed animals at 57 ( 7 ng/g and PFOA
observed in the spleen of the 8:2 diPAPS-dosed animals at 5.5 ( 0.7 ng/g.
around 48 h at 3.8 ( 0.3 ng/g, however the excretion profile
is not obvious, with a PFOA concentration of 4.0 ( 0.7 ng/g
present in the blood of the animals 15 days post-dosing. It
must be noted that the LOQ for PFOA in the whole blood
samples analyzed here is 6.1 ng/g (see Supporting Informa-
tion), driven by minor contamination present in the pro-
cedural blanks. Values are provided as illustration of the
observed trends, without further interpretation. The 3-day
time-course for PFOA and the intermediate metabolites for
the 8:2 diPAPS-dosed animals is displayed in Figure 1d.
Despite the low levels of PFOA observed, both the 7:3 Acid
and 8:2 FTCA were detected 4 h post-dosing. Aside from
PFOA, no PFCAs were observed above background levels
present in the control animals for the 8:2 diPAPS-dosed
animals.
Upon completion of the time-course the animals were
FIGURE 2. Schematic outlining two potential pathways for PFOA
dosed a second time, with sacrifice and necropsy 24 h post-
dosing. In contrast to the results for the time-course, the
levels observed in the 8:2 diPAPS-dosed animals were greater
than those in the 8:2 monoPAPS-dosed animals. Reasons for
this discrepancy are not apparent, and with only one time
point subsequent to the second dosing the two situations
are difficult to compare. Concentrations observed in the
analyzed tissues are displayed in Table 2. Within the dose
groups, concentrations for each analyte were greatest in the
liver, which is consistent with previous rat studies involving
PFOA (43).
The quality of the reported results depends on the purity
of the administered dose. There is considerable evidence
that the compounds observed in the dosed animals are a
result of PAPS metabolism and not due to contamination
within the dosed materials. Byproducts of the 8:2 PAPS
synthetic routine include congeners of different substitutions
and residual starting materials. After purification, both 8:2
monoPAPS and 8:2 diPAPS were >97% pure with respect to
relevant fluorinated materials. Of particular concern within
this purity range is residual 8:2 FTOH. The percent by mass
of 8:2 FTOH in the synthesized 8:2 monoPAPS and 8:2 diPAPS
was 0.6% and 0.7%, respectively. The 8:2 monoPAPS-dosed
animals had almost 1 order of magnitude more PFOA in
their blood 24 h post-dosing compared to the 8:2 diPAPS-
dosed animals (Figure 1). As 8:2 FTOH contamination is
consistent by mass between the doses, the difference in PFOA
exposure between the dose groups must be due to increased
biological processing of 8:2 monoPAPS as compared to 8:2
diPAPS, and not from residual 8:2 FTOH present in the dosed
materials.
production from the biotransformation of the 8:2 PAPS congeners.
Dosed compounds are indicated by boxes. The solid arrows
represent intestinal dephosphorylation followed by absorption of
the free 8:2 FTOH. The dotted arrows indicate absorption of 8:2
PAPS followed by in vivo dephosphorylation.
diPAPS. Changes of this magnitude in the chemical com-
position of the feces cannot be explained by uptake of 8:2
diPAPS or excretion of 8:2 monoPAPS via enterohepatic
circulation, as the sum of these compounds observed in the
blood of the animals was <0.05% of the administered dose.
As a result, the increasing amount of 8:2 monoPAPS relative
to 8:2 diPAPS in the feces of the 8:2 diPAPS-dosed animals
suggests that 8:2 diPAPS was dephosphorylated in the gut of
the animals. Intestinal dephosphorylation of 8:2 diPAPS to
8:2 monoPAPS releases a unit of 8:2 FTOH into the gut of the
animal, which may continue through the metabolic pathway
to PFOA (Figure 2). Although relative contributions from
reaction byproducts and from biotransformation of 8:2
diPAPS cannot be fully delineated, the concentration profiles
in the blood and feces suggest there is a measurable
contribution from biotransformation of 8:2 diPAPS to the
load of 8:2 monoPAPS and PFOA observed in the 8:2 diPAPS-
dosed animals.
Implications to Human Exposure. The overall conclu-
sions from this study are twofold. First, the phosphate ester
bonds in both 8:2 PAPS congeners are biologically labile.
Animals from both exposure groups had increased levels of
PFOA and intermediate 8:2 FTOH metabolites from 8:2 PAPS
dephosphorylation and subsequent biotransformation of 8:2
FTOH. Second, both 8:2 PAPS congeners are bioavailable,
with both 8:2 monoPAPS and 8:2 diPAPS observed in the
blood of their respective dose groups.
The concentration profiles for 8:2 diPAPS, 8:2 monoPAPS,
and PFOA in the 8:2 diPAPS-dosed animals, shown in Figure
1c, have staggered maxima. This absorption pattern is
consistent with the sequential dephosphorylation of 8:2
diPAPS shown schematically in Figure 2. The administered
8:2 diPAPS dose contained <1% by mass 8:2 monoPAPS,
however feces collected with blood at 4 and 24 h post-dosing
contained 3% and 9% by mass 8:2 monoPAPS relative to 8:2
Possible exposure pathways leading to the production of
PFOA from either 8:2 monoPAPS or 8:2 diPAPS are shown
schematically in Figure 2. The administered compounds are
indicated with boxes. Two distinct mechanisms are shown
in Figure 2. Following the solid arrows, the 8:2 PAPS congeners
are intestinally dephosphorylated with absorption of free
9
VOL. 41, NO. 13, 2007 / ENVIRONMENTAL SCIENCE & TECHNOLOGY 4803

8:2 FTOH, whereas the dotted arrows depict direct absorption
of 8:2 PAPS followed by dephosphorylation within the animal.
There is evidence to support the participation of both
mechanisms. The presence of both 8:2 PAPS congeners in
the blood of the animals establishes that dephosphorylation
could occur after absorption, and the concentration profile
of 8:2 monoPAPS in the feces of the 8:2 diPAPS-dosed animals
supports the hypothesis that intestinal dephosphorylation
is contributing to the observed transformation. As the kinetics
of the individual transformations are not known, contribu-
tions from each mechanism cannot be inferred from the
pharmacokinetics observed here.
Determining sources of human exposure to PFCAs may
have implications beyond controlling the final concentration
observed in human blood. Although direct PFCA exposure
and exposure to precursor compounds with subsequent
metabolism to PFCAs both manifest as elevated PFCA serum
levels, their toxicological significance may not be equivalent.
The metabolic pathway from FTOHs to PFCAs proceeds via
several reactive intermediates including the polyfluorinated
carboxylic acids monitored here. The toxicology of the 10:2
FTCA has been investigated using Daphnia magna, and was
found to be 4 orders of magnitude more toxic than the
corresponding PFCA (44). In addition to the studied toxi-
cological endpoints for FTCAs, several polyfluorinated al-
dehydes have been observed in the transformation from 8:2
FTOH to PFOA (23, 35). Saturated and unsaturated aldehydes
have well-documented toxicity and carcinogenic activity (45).
Due to their inherent reactivity, exposure to these transient
metabolites is likely of greater toxicological concern than
exposure to PFCAs alone. Unlike bioaccumulation potential,
which decreases with chain length (6), these toxicological
concerns regarding FTOH exposure may not be mitigated by
decreasing the length of the fluorinated chain.
procedure; complete extraction procedures for blood, tissues,
and feces; details regarding LC-MS/MS analysis including
MRM transitions and LC separation method; LOD and LOQ
values for all analyses; concentrations observed in control
animals; animal weights at dosing and necropsy; liver weights
and the liver somatic index. This material is available free of
charge via the Internet at http://pubs.acs.org.
Literature Cited
(1) Taves, D. R. Evidence that there are two forms of fluoride in
human serum. Nature 1968, 217, 1050-1051.
(2) Kannan, K.; Corsolini, S.; Falandysz, J.; Fillmann, G.; Kumar, K.
S.; Loganathan, B. G.; Ali Mohd, M.; Olivero, J.; Van Wouwe, N.;
Ho Yang, J.; Aldous, K. M. Perfluorooctanesulfonate and related
fluorochemicals in human blood from several countries. En-
viron. Sci. Technol. 2004, 38, 4489-4495.
(3) Houde, M.; Martin, J. W.; Letcher, R. J.; Solomon, K. R.; Muir,
D. C. G. Biological monitoring of polyfluoroalkyl substances:
A review. Environ. Sci. Technol. 2006, 40, 3463-3473.
(4) Kissa, E. Fluorinated Surfactants, 2nd ed.; Marcel Dekker: New
York, 2002.
(5) Yamashita, N.; Kannan, K.; Taniyasu, S.; Horii, Y.; Petrick, G.;
Gamo, T. A global survey of perfluorinated acids in oceans.
Mar. Pollut. Bull. 2005, 51, 658-668.
(6) Martin, J. W.; Smithwick, M. M.; Braune, B. M.; Hoekstra, P. F.;
Muir, D. C. G.; Mabury, S. A. Identification of long-chain
perfluorinated acids in biota from the Canadian Arctic. Environ.
Sci. Technol. 2004, 38, 373-380.
(7) Prevedouros, K.; Cousins, I. T.; Buck, R. C.; Korzeniowski, S. H.
Sources, fate, and transport of perfluorocarboxylates. Environ.
Sci. Technol. 2006, 40, 32-44.
(8) Armitage, J.; Cousins, I. T.; Buck, R. C.; Prevedouros, K.; Russell,
M. H.; Macleod, M.; Korzeniowski, S. H. Modeling global-scale
fate and transport of perfluorooctanoate emitted from direct
sources. Environ. Sci. Technol. 2006, 40, 6969-6975.
(9) Dinglasan-Panlilio, M. J. A.; Mabury, S. A. Significant residual
fluorinated alcohols present in various fluorinated materials.
Environ. Sci. Technol. 2006, 40, 1447-1453.
PAPS may be a significant source of PFCA contamination
to the human population. We have shown here that oral
exposure of rats to either 8:2 monoPAPS or 8:2 diPAPS will
result in increased PFOA blood levels, and that both 8:2 PAPS
congeners themselves absorb from the gut into the blood-
stream. The significance of PAPS exposure to the overall load
of PFCAs observed in the general population is difficult to
ascertain. PAPS products are manufactured with a variety of
different molecular structures (26), and the relative contri-
bution of different formulations to the commercial market
is not known. The overall prevalence of PAPS incorporation
into applications such as paper food packaging (25) and as
pesticide defoaming agents (30) is expected to be significant,
though specific values are not available. For the specific
application of food-contact paper packaging, the extent of
migration into food is not well documented, with only one
study looking at only one PAPS formulation (26). Once an
appreciation for these variables is available, human exposure
to PFCAs via PAPS exposure can be properly assessed.
(10) Ellis, D. A.; Martin, J. W.; De Silva, A. O.; Mabury, S. A.; Hurley,
M. D.; Sulbaek Andersen, M. P.; Wallington, T. J. Degradation
of fluorotelomer alcohols: A likely source of perfluorinated
carboxylic acids. Environ. Sci. Technol. 2004, 38, 3316-3321.
(11) Wallington, T. J.; Hurley, M. D.; Xia, J.; Wuebbles, D. J.; Sillman,
S.; Ito, A.; Penner, J. E.; Ellis, D. A.; Martin, J.; Mabury, S. A.;
Nielsen, O. J.; Sulbaek Andersen, M. P. Formation of C7F15COOH
(PFOA) and other perfluorocarboxylic acids during the atmo-
spheric oxidation of 8:2 fluorotelomer alcohol. Environ. Sci.
Technol. 2006, 40, 924-930.
(12) Calafat, A. M.; Kuklenyik, Z.; Caudill, S. P.; Reidy, J. A.; Needham,
L. L. Perfluorochemicals in pooled serum samples from United
States residents in 2001 and 2002. Environ. Sci. Technol. 2006,
40, 2128-2134.
(13) Butenhoff, J. L.; Gaylor, D. W.; Moore, J. A.; Olsen, G. W.; Rodricks,
J.; Mandel, J. H.; Zobel, L. R. Characterization of risk for general
population exposure to perfluorooctanoate. Reg. Toxicol. Phar-
macol. 2004, 39, 363-380.
(14) U.S. EPA. Draft Risk Assessment of Potential Human Health Effects
Associated with PFOA and Its Salts; U.S. EPA SAB, May 30, 2006;
U.S. EPA public docket EPA-SAB-06-006; Washington, DC, 2006.
(15) 3M. Phase-Out Plan for POSF-Based Products; Specialty Materials
Markets Group, 3M, July 7, 2000; U.S. EPA public docket EPA-
HQ-OPPT-2002-0043; St. Paul, MN, 2000.
(16) Telomer Research Program. Telomer Research Program Update;
Presented to U.S. EPA OPPT, November 25, 2002; U.S. EPA Public
Docket AR226-1141; Washington, DC, 2002.
(17) DuPont Global PFOA Strategy - Comprehensive Source Reduction;
presented to the U.S. EPA OPPT, January 31, 2005; U.S. EPA
public docket AR226-1914; Washington, DC, 2005
(18) De Silva, A. O.; Mabury, S. A. Isomer distribution of perfluo-
rocarboxylates in human blood: Potential correlation to source.
Environ. Sci. Technol. 2006, 40, 2903-2909.
(19) Washburn, S. T.; Bingman, T. S.; Braithwaite, S. K.; Buck, R. C.;
Buxton, L. W.; Clewell, H. J.; Haroun, L. A.; Kester, J. E.; Rickard,
R. W.; Shipp, A. M. Exposure assessment and risk characteriza-
tion for perfluorooctanoate in selected consumer articles.
Environ. Sci. Technol. 2005, 39, 3904-3910.
Acknowledgments
We thank Vasile Furdui (University of Toronto, Toronto, ON)
for technical assistance with LC-MS/MS analysis, Scott
Browning (University of Toronto, Toronto, ON) for assistance
with the design and execution of the 8:2 PAPS synthesis,
Amila De Silva (University of Toronto, Toronto, ON) for help
with rat blood sampling, and Gilles Arsenault (Wellington
Laboratories Inc., Guelph, ON) for providing the labeled
internal standards. This research was funded by the Natural
Science and Engineering Research Council of Canada
(NSERC) through a strategic grant.
(20) Shoeib, M.; Harner, T.; Ikonomou, M.; Kannan, K. Indoor and
outdoor concentrations and phase partitioning of perfluoroalkyl
sulfonamides and polybrominated diphenyl ethers. Environ.
Sci. Technol. 2004, 38, 1313-1320.
Supporting Information Available
A comprehensive list of chemicals; isolation and purity
analysis for the 8:2 PAPS congeners; abiotic hydrolysis
9
4804 ENVIRONMENTAL SCIENCE & TECHNOLOGY / VOL. 41, NO. 13, 2007

(21) Martin, J. W.; Muir, D. C. G.; Moody, C. A.; Ellis, D. A.; Kwan,
W. C.; Solomon, K. R.; Mabury, S. A. Collection of airborne
fluorinated organics and analysis by gas chromatography/
chemical ionization mass spectrometry. Anal. Chem. 2002, 74,
584-590.
(35) Fasano, W. J.; Carpenter, S. C.; Gannon, S. A; Snow, T. A.; Stadler,
J. C.; Kennedy, G. L.; Buck, R. C.; Korzeniowski, S. H.; Hinderliter,
P. M.; Kemper, R. A. Absorption, distribution, metabolism, and
elimination of 8-2 fluorotelomer alcohol in the rat. Toxicol. Sci.
2006, 91, 341-355.
(22) Stock, N. L.; Lau, F. K.; Ellis, D. A.; Martin, J. W.; Muir, D. C. G.;
Mabury, S. A. Polyfluorinated telomer alcohols and sulfonamides
in the North American troposphere. Environ. Sci. Technol. 2004,
38, 991-996.
(23) Martin, J. W.; Mabury, S. A.; O’Brien, P. J. Metabolic products
and pathways of fluorotelomer alcohols in isolated rat hepa-
tocytes. Chem.-Biol. Interact. 2005, 155, 165-180.
(24) Xu, L.; Krenitsky, D. M.; Seacat, A. M.; Butenhoff, J. L.; Anders,
M. W. Biotransformation of N-ethyl-N-(2-hydroxyethyl)per-
fluorooctanesulfonamide by rat liver microsomes, cytosol, and
slices and by expressed rat and human cytochromes P450.Chem.
Res. Toxicol. 2004, 17, 767-775.
(25) U.S. FDA. Indirect Food Additives: Paper and Paperboard
Components; Code of Federal Regulations, 21 CFR 176.170.
(26) Begley, T. H.; White, K.; Honigfort, P.; Twaroski, M. L.; Neches,
R.; Walker, R. A. Perfluorochemicals: Potential sources of and
migration from food packaging. Food Addit. Contam. 2005, 22,
1023-1031.
(36) Brace, N. O.; Mackenzie, A. K. (Du Pont) U.S. Patent 3,083,224
(1963); CA 59, 5023f.
(37) Mylchreest, E.; Munley, S. M.; Kennedy, G. L., Jr. Evaluation of
the developmental toxicity of 8-2 telomer B alcohol. Drug Chem.
Toxicol. 2005, 28, 315-328.
(38) Hansen, K. J.; Clemen, L. A.; Ellefson, M. E.; Johnson, H. O.
Compound-specific, quantitative characterization of organic
fluorochemicals in biological matrices. Environ. Sci. Technol.
2001, 35, 766-770.
(39) Furdui, V. I.; Stock, N. L.; Ellis, D. A.; Butt, C. M.; Whittle, D. M.;
Crozier, P. W.; Reiner, E. J.; Muir, D. C. G.; Mabury, S. A. Spatial
distribution of perfluoroalkyl contaminants in lake trout from
the great lakes. Environ. Sci. Technol. 2007, 41, 1554-1559.
(40) Keith, L. H.; Libby, R. A.; Crummett, W.; Taylor, J. K.; Deegan,
J., Jr.; Wentler, G. Principles of environmental analysis. Anal.
Chem. 1983, 55, 2210-2218.
(41) Schwarzenbach, R. P; Gschwend, P. M.; Imboden, D. M.
Environmental Organic Chemistry, 2nd ed.; John Wiley & Sons,
Inc.: Hoboken, NJ, 2003.
(27) Brody, T. Nutritional Biochemistry; Academic Press: San Diego,
CA, 1999.
(28) 3M. Fluorochemical Use, Distribution and Release Overview;
3M Company, May 26, 1999; U.S. EPA public docket AR226-
0550; St. Paul, MN, 1999.
(29) Olsen, G. W.; Huang, H.-Y.; Helzlsouer, K. J.; Hansen, K. J.;
Butenhoff, J. L.; Mandel, J. H. Historical comparison of per-
fluorooctanesulfonate, perfluorooctanoate, and other fluoro-
chemicals in human blood. Environ. Health Perspect. 2005, 113,
539-545.
(30) U.S. EPA. Meeting with Registrant on Perfluorinated Chemical
(Masurf-FS-780, Fluowet PL 80-B, and Flowet PL-80) Use as
Inerts; Letter from the Office of Pollution Prevention and Toxics
to the Office of Pesticide Programs; Washington, DC, March
24th, 2006.
(42) 42) Wolfenden, R.; Ridgway, C.; Young, G. Spontaneous hy-
drolysis of ionized phosphate monoesters and diesters and the
proficiencies of phosphatases and phosphodiesterases as
catalysts. J. Am. Chem. Soc. 1998, 120, 833-834.
(43) Kennedy, G. L., Jr.; Butenhoff, J. L.; Olsen, G. W.; O’Connor, J.
C.; Seacat, A. M.; Perkins, R. G.; Biegel, L. B.; Murphy, S. R.;
Farrar, D. G. The toxicology of perfluorooctanoate. Crit. Rev.
Toxicol. 2004, 34, 351-384.
(44) MacDonald, M. M.; Sibley, P. K.; Dinglasan, M. J. A.; Mabury,
S. A.; Solomon, K. R. Aquatic toxicity of fluorotelomer acids.
Poster presentation at Fluoros International Symposium on
Fluorinated Organics: Toronto, ON, August 19-21, 2005.
(45) O’Brien, P. J.; Siraki, A. G.; Shangari, N. Aldehyde sources,
metabolism, molecular toxicity mechanisms, and possible
effects on human health. Crit. Rev. Toxicol. 2005, 35, 609-
662.
(31) Dupont Zonyl RP Paper Fluoro-Protectant; technical informa-
tion.
(32) Material Safety Data Sheet for Zonyl RP paper Fluoridizer;
DuPont, Inc.: Wilmington, DE, 2003.
(33) Masurf FS-115; technical information.
(34) U.S. EPA. Inert Ingredient; Revocation of the Tolerance Exemption
for Mono- and Bis-(1H, 1H, 2H, 2H,-perfluoroalkyl) Phosphates
Where the Alkyl Group is Even Numbered and in the C6-C12
Range; U.S. EPA public Docket OPP-2006-0253; Washington,
DC, 2006.
Received for review January 17, 2007. Revised manuscript
received April 4, 2007. Accepted April 16, 2007.
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