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VIROTspogyL pubs.acs.org/est iencee nno
Identification of Novel Perfluoroalkyl Ether Carboxylic Acids (PFECAs)
and Sulfonic Acids (PFESAs) in Natural Waters Using Accurate Mass
Time -of -Flight Mass Spectrometry (TOFMS)
Mark Strynar,*'t Sonia Dagnino,t'$ Rebecca McMahen,t'$ Shuang Liang,t'$ Andrew Lindstrom,t
Erik Andersen,t Larry McMillan,§ Michael Thurman," Imma Ferrer,11and Carol Ball1
National Exposure Research Laboratory, U.S. Environmental Protection Agency, Research Triangle Park, North Carolina 27711,
United States
*Oak Ridge Institute for Science and Education, Oak Ridge, Tennessee 37831 United States
§National Caucus and Center on Black Aged, Inc., Durham, North Carolina 27713, United States
"Center for Environmental Mass Spectrometry, University of Colorado Boulder, Boulder, Colorado 80309, United States
J-Agilent Technologies Inc., Wilmington, Delaware 19808, United States
© Supporting Information
ABSTRACT: Recent scientific scrutiny and concerns over
exposure, toxicity, and risk have led to international regulatory
efforts resulting in the reduction or elimination of certain
perfluorinated compounds from various products and waste
streams. Some manufacturers have started producing shorter-9
y
chain per- and polyfluorinated compounds to try to reduce the
potential for bioaccumulation in humans and wildlife. Some of
these new compounds contain central ether oxygens or other
minor modifications of traditional perfluorinated structures. At Sequential
resent there has been very limited information published on Samplings
present, ry p Non -Targeted _ - 1 Time -of -flight Mau
these "replacement chemistries" in the peer -reviewed Screening Spec JTocMS►
literature. In this study we used a time -of -flight mass Discovery
spectrometry detector (LC-ESI-TOFMS) to identify fluori-
nated compounds in natural waters collected from locations Mass Wad all features (upst(eam" Do Sueam)
with historical perfluorinated compound contamination. Our
workflow for discovery of chemicals included sequential .,, ° • ,
sampling of surface water for identification of potential .. ' • " ;
sources, nontargeted TOFMS analysis, molecular feature
extraction (MFE) of samples, and evaluation of features <�}• a : e s •:':
unique to the sample with source inputs. Specifically, t € • ; „ {
compounds were tentatively identified by (1) accurate mass
determination of parent and/or related adducts and fragments
from in -source collision -induced dissociation (CID), (2) in-
depth evaluation of in -source adducts formed during analysis, d „
and (3) confirmation with authentic standards when available.
We observed groups of compounds in homologous series that
differed by multiples of CF2 (m/z 49.9968) or CF20 (m/z 65.9917). Compounds in each series were chromatographically
separated and had comparable fragments and adducts produced during analysis. We detected 12 novel perfluoroalkyl ether
carboxylic and sulfonic acids in surface water in North Carolina, USA using this approach. A key piece of evidence was the
discovery of accurate mass in -source n-mer formation (H+ and Na*) differing by m/z 21.9819, corresponding to the mass
difference between the protonated and sodiated dimers.
■ INTRODUCTION
PerIluoroalkyl and polyfluoroalkyl substances (PFASs) have
Received: March 10, 2015
unique physical and structural properties that make them Revised: August 7, 2015
extremely resistant to chemical and thermal degradation. As a Accepted: August 30, 2015
result, PFASs have been used in a wide range of consumer
® ACS Publications m XXXX American Chemical Society A DM 10.1021/acs.est.5b01215
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+Sample 1 _ Sample 1(control)
Sample 2 (experiment)
:S�ample
ial Source input 1
ce Sample 2 Non -targeted
TOFMS
Sam le re _ Sample 2 (control)
P P P• screening Sample 3(experiment)
3... ,�
Source input 2...
Potential
PSource
Source
Proposed, N`ov�el Compounds
Acquisition of authentic standard for
confirmation If possible
Chemical database searching
Step 6
firmatlon &
supjppoorr"ting data
Figure 1. Sample workflow for TOWS discovery.
Investigation of co -eluting peaks (adducts -to
acetate and dlmers - Na-, K.)
Diagnostic fragment ions
Isotope peak matching (distribution, spacing)
Exact mass caliper tool
Exact mass off sets (CF,, CF,O..) homologous series
Kendrick Mass Defect Plots
Step 5
products and industrial applications! Between 1950 and 2000,
the eight -carbon PFASs, including perfluorooctanoic acid
(PFOA) and perfluorooctanesulfonate (PFOS), were among
the most commonly produced and used perfluorinated
compounds. PFASs have been detected in many environmental
and biological matrices across the globe for a number of years,2
and this ubiquity has led to mounting concerns about exposure
and potential toxicity. As a result, many international regulatory
efforts have been enacted to reduce, substitute, or eliminate
long -chain (8 consecutive perfluorinated carbons or longer)
PFASs from products and waste streams. In the United States,
eight major fluorochemical manufacturers entered into a
voluntary stewardship agreement with the U.S. Environmental
Protection Agency (EPA) to phase out the use and production
of long -chain perfluorinated chemistries by 2015 3 To take their
place, manufacturers have started using alternative chemistries
that may include both shorter -chain perfluorinated (<C8) and/
or polyfluorinated materials. For example, some of these
replacement compounds still have the traditional perfluorinated
carbon regions, but they are broken into shorter units by
insertion of ether oxygens at regular intervals. These ether
linked compounds are still technically classified as perfluori-
nated if all carbons are substituted with fluorine and
polyfluorinated if any carbon is occupied by a hydrogen rather
than a fluorine. The rationale is that the addition of oxygens
would likely make the replacement chemistry more labile to
degradation, and thus more favorable for usage. However, to
date the chemical manufacturers have disclosed little
information publicly concerning the chemical structures and
potential toxicities of the replacement PFASs now being
produced. This makes it extremely difficult for independent
[molecular feature Visualization of features unique totlon(MFE) Compound Exchange File (CEF)exportation
Reduction of data by extracted ion chromato-
gram (EIC) of traditional PFCs
Investigation of large, negative mass defect
eat t
spaatnl
Wm...
Advanced -��--- - .._�---''_—•
techniques
Background subtracted for clean spectra
Formulae generation (C, H, 0, N, S, F, Cl, P, Na...)
Step 4
research groups to evaluate their environmental distributions
and potential toxicities.
A rare exception is a peer -reviewed publication on 4,8-dioxa-
3H-perfluorononanoate (ADONA) (Supporting Information
Figure S1), a polyfluorinated alternative to PFOA which is said
to have a "more favorable toxicological profile".5 This single
journal article was published by its manufacturer, but standards
for this new material have not been made available (to our
knowledge) for comprehensive ecological or toxicological
evaluation. Manufacturers typically submit more detailed
information concerning compound structure, potential toxicity,
environmental fate, and projected production volumes to
regulatory authorities prior to large-scale production (e.g., the
U.S. EPA's premanufacture notice process), but this informa-
tion is held as confidential business information and cannot be
disclosed to the general research community.
One example of industrial producers manufacturing replace-
ment compounds is outlined in an executed draft consent order
from the state of West Virginia to DuPont Corporation
allowing the discharge of a new fluorinated compound into the
Ohio river, at the Washington Works facility (West Virginia) 6
This is the same facility that has been the source of historical
PFOA contamination of local surface and groundwater due to
industrial manufacture and discharge and is the center of a
court -ordered investigation by the C8 science panel to
determine if PFOA exposure in the local human population
has led to adverse health effects. A number of recent
epidemiological studies have indicated that exposure to
PFOA at this location has been associated with adverse
human health outcomes 8,9
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Typical environmental occurrence investigations have for
years focused on targeted analysis for specific analytes of
interest. Highly precise and sensitive LC and GC MS/MS
methods have been developed to quantify contaminants of
concern. A more recent approach for environmental sample
analysis has been the use of high -resolution mass spectrometry
(HRMS) for these efforts. Some researchers have used
nontargeted or suspect screening effectively in demonstrating
many chemical classes in wastewater effluent without the use of
reference standards."—" These approaches have begun to rely
more heavily on the use of databases, accurate mass searches,
and HRMS molecular formula prediction capabilities for
discovery. Confirmation with authentic standards still remains
the "gold standard". Schymanski et al.14 reported on five levels
of confidence in identifying small molecules using HRMS
ranging from exact mass matches, through molecular formula
prediction, tentative candidate structures, library match
searches, and finally confirmation with a reference standard.
This is the type of approach that we use in this work to identify
previously undescribed PFASs in surface water.
As it is mostly unknown what is being produced to replace
traditional perfluorinated compounds, a series of sampling
events was conducted to collect water samples with
determination of new per- and polyfluorinated substances as
a goal. In Nakayama et a1.,10 a sampling of surface water in the
Cape Fear River Basin in North Carolina indicated sources of
perfluorinated compounds intermittently spread throughout
the drainage basin. A follow-up investigation with more focused
sampling to determine sources was not conducted. However,
based on this early work, there was some indication that there
were PFASs sources in the Fayetteville, NC area. More recent
surface water sampling trips in this region indicated continued
elevated concentrations of traditional Ferfluorinated com-
pounds, supporting the Nakayama et al.' findings. One such
source was known to be downstream of the industrial effluent
discharge of a fluorochemicai production facility. Further
investigation of these samples for identification and occurrence
of potential replacement chemistries was undertaken.
■ MATERIALS AND METHODS
Sample Workflow for Discovery. A diagram of the
workflow we used for novel compound discovery in surface
water samples is shown in Figure 1. It is a six -step process that
starts with sequential sampling of water, followed by non -
targeted screening, visualization of detected peaks, further
investigation of suspect features, advanced TOFMS techniques,
and, last, compound confirmation with authentic standards
when available. Using this approach, it becomes possible to
detect chemicals that enter a waterway from a source (point or
nonpoint) between sampling locations. This can then be used
for such purposes as source elucidation, compound discovery,
or both. In the following sections we will be referring to Figure
1 and the specific step in the process to which we are referring.
Sample Collection. In the summer of 2012, water samples
were collected at locations on the Cape Fear River and its
tributaries, with some locations having previously been found
to contain measurable PFASs.'-' Grab samples were taken from
the bank and bridge crossings with a lab -made dip sampler or a
stainless steel Kemmerer sampler, and stored in a 1-L HDPE
bottle following the procedures described in Nakayama et al.16
Samples (n = 9) were acquired from the main flow of the Cape
Fear River including from just south of the city of Fayetteville,
NC to the bridge crossing in the town of Tar Heel, NC.
Duplicate samples, trip spikes, and trip blanks were included in
this sampling. This sampling also included tributaries to the
Cape Fear River along the same stretch of river where
reclaimed wastewater treatment plant (VVWTP) and industrial
effluent streams occurred (Figure 1, step 1).
Sample Analysis for Traditional PFASs. Samples were
prepared and analyzed according to the methods of Nakayama
et al.16 In brief, water samples were stabilized with addition of
nitric acid and stored at ambient temperature prior to analysis
(<7 days). Each water sample was measured in a graduated
cylinder, and the sampling bottle was washed with 10 mL of
methanol to solubilize analytes that may have become sorbed to
the container wall during shipping and storage. The water was
then placed back in the methanol -washed bottle with the
methanol rinsate, spiked with a mixture of internal standards,
and filtered through a glass fiber filter. A volume, typically 500
mL, of water was concentrated on a Waters WAX SPE cartridge
using a positive displacement pump. The cartridge was eluted
with basic methanol (0.3% N1440H) and the eluent was
concentrated via evaporation under Nz gas to 1 mL and
prepared for analysis. A procedural blank was prepared as
described above using 500 mL of in-house DI water.
Traditional PFASs (C4—C10 perfluorocarboxylic acids
(PFCAs); perfluorobutanesulfonate (PFBS), perfluorohexane-
sulfonate (PFHxS), and PFOS) analysis was performed using a
Waters Acquity ultra performance liquid chromatograph
interfaced with a Waters Quattro Premier XE triple quadrupole
mass spectrometer (UPLC-MS/MS) (Waters, Milford, MA,
USA).
TOFMS Investigation of Novel Fluorinated Com-
pounds. The extraction method used for traditional PFASs
analysis was also used for elucidation of novel compounds.
Elevated concentrations of traditional PFASs in samples were
used as an indication of samples that were likely closer to
sources (Figure S2). Sample analysis was performed using an
Agilent 1100 series HPLC interfaced with a 6210 series
Accurate -Mass LC-TOF system (Agilent Technologies, Palo
Alto, CA). The mass spectrometer was operated in electrospray
ionization (ESI) negative mode and any drift in the mass
accuracy of the TOF was continuously corrected by infusion of
two reference compounds (purine (m/z 119.03632) and the
acetate adduct of hexakis (1H,1H,3H-tetrafluoropropoxy)
phosphazine (m/z 980.016375)) via dual-ESI sprayer.
Chromatographic separation was accomplished using an Eclipse
Plus C8 column (2.1 X 50 nun, 3.5 µm; Agilent). The method
consisted of the following conditions: 0.2 mL/min flow rate;
column at 30 °C; mobile phases: A. ammonium formate buffer
(0.4 mM) and DI water/methanol (95:5 v/v), and B:
ammonium formate (0.4 mM) and methanol/DI water (95:5
v/v); gradient: 0-15 min a linear gradient from 75:25 A/B to
15:85 A/B; with a 4 min post time for equilibration.
Identification of Suspect Features. The total ion
chromatogram (TIC) of each water sample was subjected to
the software molecular feature extraction algorithm (MFE) and
was restricted to the 100 most intense features m/z 50-1700
based on peak height. A molecular feature is defined as a single
accurate mass with a specific retention time and an integrated
area count. Compounds identified in the solvent and
procedural blank samples were used to develop a mass
exclusion list to subtract from subsequent unknown samples.
After molecular features were identified, they were exported as
a Compound Exchange File (.CEF) and imported into Mass
Profiler to compare upstream with downstream samples.
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.10,
1.06 F F`
• t66.09t1 E--'-� �
1
OA6 F
0.9 F F F
F
O.t16
[2M-H]-
os
0.75 F F
0.7 F F [2M-2H+Na]-
F F
0.66 r f F
F F F
f F F` \
/ O F F
6.65 F F ¢ \[ O�f F G
0.5 X.ONOy� F F
/ f f
0.45
o
O
OA FA-
0.3O GP
6.� 284.9793 Ns
GF O O F F
016
Q2 666.0432
0.16 9" 0000
Q1 119.0369 660.9249
0.05 112. 56 160, 540
0
6G 160 120 110 IiO 160 2V0 2i0 210 2L0 2i0 360 SiO 310 3!0 390 400 420 440 4C0�6�) 662,0 NO 0W. M. 7G0 720 710 760 7p SW' M. t10 80 Mi 9C0 MI 910 OW oeo
x10 t -681 Scan (6.334.43 min, 7 Sant) Fng-W,OV WoO MINW A (�
7M 457 D
/ D
1
\F OF/ o--'\F o Fb" f F
[2M-2H+Na]-
6.9 F F F\/' M• P F F F
'Quo
6.9 F F F F F F i F F F
6 /\y�/- `, �
&S c
[2M-H]-
o '
4
3S E F \
F
F 1 /
9 \
ME
11 i 1 1 293.1757
360 360 400 420 40 44W 4WC—ft (W MNeaabCharye (60 60
J 620 60 6
Figure 2. ESI negative spectra for two example perfluorinated ether carboxylic acids found in water: (A) C6HF1103 (retention time 5.9-6.0 min)
and (B) C61-IF1106 (retention time 6.5-6.6 min). Note: in spectrum (A and B) m/z 112.9856, 119.0363, and 966.0007 are reference masses for
continuous mass calibration. Peaks not pointed out are not associated with the compound of interest.
Pairwise comparisons of sequential samples in river flow
(Figure 1 step 2) were used to identify those requiring further
investigation. For instance, features found in the downstream
sample that were not found in the upstream sample indicate a
source (point or nonpoint) exists between the two sampling
locations (Figure 1 step 3). Further investigation of peaks with
larger area counts using the "identify compounds" feature in the
software and additional analyses of samples were used for
confirmation. Perfluorinated compounds already identified with
traditional UPLC-MS/MS analysis were eliminated from this
remaining list further narrowing the unknown compounds
requiring follow-up scrutiny.
Poly- and perfluorinated compounds tend to have a negative
mass defect due to the presence of multiple fluorine and oxygen
atoms. Negative mass defect means that the exact mass of a
compound is less than the nominal mass.17 For instance, the
exact mass of perfluorooctanoate (C8171502) is 412.9664,
whereas the nominal mass is 413 Da. In this instance negative
mass defect compounds only found in the downstream sample
were further explored. An additional characteristic of samples
that are contaminated with per- and polyfluorinated com-
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Table 1. Accurate Mass of Polyfluorinated Compounds and In -Source Artifacts Found in Extracted Water Samples
number formula
Monoether PFECAs
1 C3HF503
2 C4HF703
3 CSHF903
4 C,HF1103
5 C,HF1303
6 CsHF1503
Polyethelr+ PFECAs
7 `.'7HF1307
8 C6HF1106
9 CSHF905
10 C4HF704
PFESAs
11 C7HF1305S
12 C7H2F1405S
Other
Na'
H'
CF'0
CF2
[M _ H1- 12M - H1-
CAS no. name [M]' m/x 12M - 2H + Nal- m/z m/z
863090-89-5
13252-13-6 undecafluoro-2-methyl-3-oxahexanoic acid
39492-91-6 perfluoro-3,5,7,9,11-pentaoxadodecanoic
acid
39492-90-5
perfluoro-3,5,7,9-butaoxadecanoic acid
39492-89-2
perfluoro-3,5,7-propaoxaoctanoic acid
39492-88-1
perfluoro-3,5-dioxahexanoic acid
66796-30-3b
179.9846 178.9773
229.9813 228.9740
279.9782 278.9709
329.9750 328.9677
379.9718 378.9645
429.9686 428.9613
443.9515 442.9442
377.9598
376.9525
311.9681
310.9608
245.9764
244.9691
443.9337
442.9264
463.9399
462.9326
22.9892
1.0073
65.9917
49.9968
"Indicates the monoisotopic mass of the neutral species. bCAS number for Nafion copolymer.
pounds is the presence of multiple peaks that differ by exactly ±
m/z 49.9968 and/or 65.9917, corresponding to a difference of
CF2 and CF20, respectively. This happens because PFAS
synthesis is an industrial process that yields a distribution of
products that differ primarily in chain length and/or degree of
isomerization. Compounds identified as having a negative mass
defect and differing from another peak by +_ m/z 49.9968 and
65.9917 are highly likely to be polyfluorinated compounds.
Selected samples were also analyzed by QTOF to aid with
confirmation of proposed structures (generously provided by
colleagues mentioned in the Acknowledgments).
TOFMS Experimental Workflow. Once an unknown
compound with negative mass defect was noted, it was isolated
from other mass spectral data in the following way: An
extracted ion chromatogram (EIC) was generated from the
total ion chromatogram (TIC) using the m/z identified. The
EIC was then used to do a background subtraction of the
spectral region both immediately before and after the detected
peak (typically ±0.1 min) (Figure 1 step 4). The center of the
detected peak was extracted for the mass spectral information,
and the background spectrum was subtracted for a clean
spectrum. This was necessary to eliminate, to the best of our
ability, competing spectral peaks that were coeluting and not
associated with the spectrum being investigated. This would
include, but is not limited to, spectral signals originating from
the reference compounds (constantly infused to maintain a lock
on mass assignments) and their fragments. Once suspect
spectral signals were isolated, a series of experiments were
conducted to induce in -source fragmentation ions to aid with
compound identification based on the work of Ferrer and
Thurman.18 Specifically, a series of methods with differing
fragmentor voltages (80-190 V) were run in sequence to look
for diagnostic ions that emerged as fragmentor voltage
increased. In addition, common adducts such as Na, NH,,
acetate, etc. were used to aid with compound identification.
Detected spectral features were subjected to molecular formula
generation using the elements (C, H, 0, N, S, P, Cl and F).19
380.9438
358.9619
480.9372
458.9553
580.9310
558.9491
680.9247
658.9427
780.9182
758.9363
880.9118
858.9299
908.8776
886.8957
776.8942
754.9123
644.9108
622.92,89
512.9274
490.9455
Formulas generated were scored based on accurate mass,
isotope abundance (compound mass distributions attributable
to elemental mass distributions e.g., 1.196 13C, 0.2% 180, etc.)
and isotope distribution (for example, slight differences in
isotope mass such as when an m+2 peak is from two 13 C or a
single 180) . Only scores >75% were considered for further
exploration. Figure 1 steps 4 and 5 outline the advanced
TOFMS techniques used to discover novel chemical species.
■ RESULTS
With the approach described above, sites were identified for
further investigation when a large increase in number and
magnitude (area counts) of unknown compounds was found in
downstream samples. In addition, a very large increase (greater
than 2 orders of magnitude) in concentration of historically
measured PFAAs also persisted for many river miles down-
stream of a certain point (Figure S2) consistent with
observations in Nakayama et al.10 Interestingly, the profile of
the historically measured PFASs contributing to the total
PFASs found at each location was seen to dramatically change
near this location (Figure S3). The major compounds
contributing to this increase were perfluoropentanoic acid
(PFPeA), followed by perfluoroheptanoic acid (PFHpA) and
perfluorobutanoic acid (PFBA). It was evident that there was a
significant source (total historically measured PFASs concen-
tration increased by >100 times) of per- and polyfluorinated
compounds near this location. Plotting the samples (CFR004
upstream vs CFRO02 downstream) in MassProfiler for
visualization indicated that there were 77 features that were
unique to the downstream sample (Figure S4) with many (n =
69) having negative mass defects. In this figure (S4) the size of
the symbol is proportional to the area counts of the molecular
feature. Traditional PFASs with known concentrations (in
Figure S2) are included for comparison purposes. Peaks with
larger areas were investigated first under the assumption that
these concentrations would be among the highest. Additional
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pairwise comparisons were done on all sequential samples,
however results indicated that samples downstream of CFR003
were simply dilutions of a source input. Samples were chosen
for additional scrutiny where there was sufficient analytical
signal without saturation of the detector which compromises
mass accuracy.
Dimers and Fragments. It became evident that in multiple
instances several of the larger peaks had a number of common
characteristics: (1) negative mass defect, (2) multiple related
chromatographic peaks differing by ± m/z 49.9968 and/or
65.9917 (± a CF2 group and/or a CF2O group), and (3)
apparent noncovalent homodimers linked by either a proton or
sodium ion. For example, at a retention time of 5.9-6.0 min,
two large peaks emerged from the spectra with m/z of
658.9462 and 680.9256 (Figure 2A). After careful evaluation, it
became apparent that these were the proton -bound and
sodium -bound in -source homodimers of undecafluoro-2-
methyl-3-oxahexanoic acid (C6HF11O3) (Table 1). These
differed by m/z 21.9819, the difference between the Ne and
H+ versions of the dimer. It is important to note that the [M -
H]- peak (m/z 328.9677), which one might expect to be the
most prominent, was barely distinguishable from the back-
ground signal at this point because the in -source ionization
conditions so heavily favored the formation of these dimers.
Given the prominence of the C6HF11O3 compound in this
sample, we postulated that a homologous series of related
perfluorinated ether carboxylic acids (PFECAs), differing from
the first compound by either the addition or deletion of CF2
units (m/z 49.9968) might also be present. An EIC for each of
the hypothetical masses, based on sequential addition or
deletion of CF2 units (m/z 49.9968) was extracted from the
TIC, with the resulting series of related PFECAs presented in
Figure S5. Although not specifically shown in this Figure, we
also observed the presence of the analogous sodium and proton
bound dimers for each compound identified in this
homologous series of PFECAs (C(n_3_8)HF(i_5_15)O3 (Table
1). To obtain additional evidence supporting the proposed
structure of the compounds eluting at 5.9 min, additional
experiments were conducted by altering the fragmentor voltage
in order to try to form diagnostic ions from the m/z of
658.9427 and 680.9247 dimers eluting at this time. Figure S6
shows diagnostic dimer and fragment ions resulting from in -
source CID that are consistent with the structure proposed for
the C6HF11O3 compound. The major ions found are also
shown in Figure 510. Extracted spectra comparable to those
shown in Figure S6, differing only by the loss of CF2 groups,
were investigated as well (Figure S7) lending credence to the
postulated structure(s) and discussed in more detail in the
following section.
Confirmation of Structure. An online search of the
formula and proposed structure of the C6HF11O3 compound
eluting at 5.9 min led to a tentative match in a "grey -literature"
citation that mentions a new -generation processing aid for the
production of high-performance fluoropolymers identified only
as C3 dimer acid/salt (CAS 13252-13-6) (Figure S6).e With a
CAS number, it was possible to purchase an authentic standard
(Synquest Laboratories, Alachua, FL) to compare for retention
times and mass spectral ionization patterns. The authentic
standard compared very well with the tentatively identified
compound found in water samples (±0.051 min RT; ± m/z
0.0002 (0.51 ppm) (Figure 1 step 6). Additional authentic
standards for other compounds in this homologous series were
not commercially available. However, common spectral
patterns and adducts were observed differing only by repeating
CF2 units, as supporting information for compound identi-
fication of a homologous series of PFECAs (Figure S7).
Additional PerFluorinated Ether Acids. Figure S4 shows
that there are a number of features that are present in the water
sample downstream of a potential input location that are not in
the upstream sample. Evident in Figure S4 are a pronounced
series of peaks that appear as vertical lines, likely from coeluting
and related chemicals (see later discussion on in -source n-mer
formation). A number of these peaks exhibited negative mass
defects. Further investigation of the TOFMS spectra indicated
two peaks coeluting at 6.5 min (m/z 776.8942 and 754.9123)
with a mass difference of m/z 21.9819, again suggesting the
presence of in -source proton- and sodium -bound homodimers
(Figure 2B). Knowing this information, we postulated an exact
mass of m/z 376.9525 for the [M - H]- ion (Table 1). We
further postulated a homologous series based on addition and
deletion of CF2 units, but exact masses corresponding to ± m/z
49.9968 were not detected. Given reports of industrial
producers making greater use of ether oxygens to limit the
size of perfluorinated regions within a given molecule, we
postulated that a homologous series could also be based on the
repeating units of CF2O, with an exact mass offset of ± (m/z
65.9917).1 " Searching the sample for m/z 376.9525 ± this
hypothetical CF2O offset did yield another homologous series
(Figure SS). In addition, as was previously the case, each of
these new compounds was also found to form sodium -bound
and proton -bound homodimers, providing further support for
the proposed structures (Table 1). Confirmation of these
compounds with authentic standards was not possible. It
should be noted that in the homologous series shown, the
Chemical C3HF5O3 (m/z 178.9773) is a common feature
(Table 1) to the previously identified homologous series.
Taken together, the exact mass of the proposed structures, the
offset by a CF2O, and the exact mass of the in -source dimers
formed, all support the occurrence of this additional
homologous series. One compound in this homologous series
(C4HF7O4 [M - H] 244.9691 Table 1) was subjected to
QTOF analysis leading to a series of accurate mass fragments
consistent with the proposed structure (Figure S9). The
accurate mass fragments show the sequential breakage of the
ether oxygen bonds and the resulting fragment ions.
In -Source n-mer Formation. Multiple peaks originally
thought to be of polymeric origin (m/z 1176.8353 and
1576.7769) were determined to be sodium -bound n-mers of
m/z 376.9525 already identified (Figure S12). The retention
time of these peaks (6.5 min) indicates this is an in -source
phenomenon, as they coelute (Figure S4). In -source artifacts
occur in the ES1 source and can include such things as the
formation of adducts (formate, Na*, H*, NH4'), fragments,
dimers, trimers, and combinations of the above. Peaks that do
not coelute, or are chromatographically separated, are generally
considered not to be in -source artifacts. The observed spectra
(Figure S12) include the formation of n-mers17 including 2 and
3 bound sodium cations in addition to the compounds shown
in Figure S 11. The formation of other n-mers with > 1 bound
sodium was seen as well for additional PFECAs found in this
work (data not shown). Recent work by Trier et al.17 indicates
that the formation of these polyfluorinated surfactant clusters
occurs in the MS source in the gas phase and are pH and
concentration dependent.17 However, this phenomenon may
also be instrument -specific as sample dilution in this study did
not lead to loss of these in -source dimers being formed. Similar
DOI:10.1021 /acs.est5 b01215
Fnviron. Sci. Technol. XXXX, XXX, XXX-XXX
DEQ-CFW 00000262

Environmental Science & Technology
A B
aoF F F
s
Gas. F F
ws O F F F
suspected oxidation species
0
CAS 76090-14-5
F F
O F
o.q
d1 W
o.,
i u u xe i, e- u i( as 4e 64YP..%4:'{.�7�]omm Iw 164 iae li nx u4 u.z i 16 ii 1zz 1dA
a.b''. 20.0003 -... HF D1111—nm
6 1.41 ppm mass accuracy
e6
e
)5..: ME 92N
1621327
66
8
4
3!
2.b'
2
Is
1 I
05'
0
N0 442 441 446 446 4,1 4 66t3{U 4tSt444:�4Ym�:tmn}2 4G *6 460 470 472 474
.tax
1 F F F F A
3+ adz 6M4 F 0 F
F
os �I O F
F
0a HOtF
F Molecular Formula: C,HF,30,.S
07 Ogg Monoisotopic Mass: 443.9337 Da
of F ]M-H]-: 442.9264 m/z
0s IMeasured [M-H] 442.9264 (0.19 ppm mass accuracy)
04
I
0.3
I
02 j
4436266 .46270
0.1
i
442.E 443 —6 0. M66 ^w M 5
.16 I
7 4626328 B
65. r F F F F H
6
Bs F O F
5 O F FF F
F
as F Molecular Formula: C,H,F
4 HO1405S
\
Monolsotoplc Mass: 463.9399 Da
as O Of F [M-MI.:462.9326 mh
0
25i Measured [M-H) 462.9327 (0.11 ppm mass accuracy)
2
1,6
1 IN 62N
461.0306
06 IQ 7662 <6]Im 464.5676 466.1972 MOM 1659291
0
462.6 463 4N2 MA 4636 4634 4N 465.2 4664 46i6 4066 466
Figure 3. Suspected perfluoro ether sulfonic acids identified in water. Oxidation species are likely products of CAS 16090-14-5, a perfluorinated ether
sulfonyl fluoride. Chromatogram shown of two resolved peaks (A,B) and isotope cluster matching of measured versus exact mass. Red boxes indicate
the exact mass for the suspected formulas, compared to the accurate mass profile spectrum. Mass difference between two species is an HF (m/z
20.0063-1.41 ppm mass accuracy).
sodium- and proton -bound, as well as mobile phase, modifier
(i.e., formate) adducts have been shown by Trier et al.17 for
perfluorocarboxylic acids and mono-PAPs (perfluorinated alkyl
phosphates).
Perfluoroalkyl Ether Sulfonic Acid (PFESA) Identifica-
tion. Records of groundwater monitoring at a fluorochemical
manufacturing plant with wells in close proximity to our
sampling site indicate PFOA in µg/L concentrations. 21 This
document also indicated a sulfonated tetrafluoroethylene based
fluoropolymer-copolymer with the trade name Nafion was also
produced at this facility. Our data provide evidence that
materials related to this compound are also emitted into the
river at this point. Figure 3 shows the chemical structures of the
suspected oxidation species and resulting transformation
products tentatively identified in our samples. An EIC of m/z
442.9264, which is the exact mass of the [M — H]- moiety of
the copolymer containing the sulfonate group (IUPAC name
1,1,2,2-tetrafluoro-2-({ 1,1,1,2,3,3-liexafluvi u-3- [ ( trifluorovi iyl)-
oxy]-2-propanyl}oxy)ethanesulfonic acid), suggests this com-
pound is present in the water. In addition, spectral evidence
supports the detection of a related compound which is
consistent with the addition of an HF ([M — H]- m/z
462.9327) (IUPAC name 1,1,2,2-tetrafluoro-2-{[1,1,1,2,3,3-
hexafluoro-3-(1,1,2,2-tetrafluoroethoxy) propan-2-yl]oxy}-
ethanesulfonic acid). The two suspected compounds differ by
the mass of HF (m/z 20.0062-1.41 ppm mass accuracy)
(Figure 3). These compounds elute as two discrete chromato-
graphic peaks, likely explained by the position occupied by the
H and F additions. It is possible that these chemicals originate
from the oxidation of ethanesulfonyl fluoride, 2-[1-[difluoro-
[ (t (fluoroethenyl) oxy] methyl]-1,2,2,2-tetrafluoroethoxy] -
1,1,2,2-tetrafluoro- (CAS 16090-14-5) which may have been in
use at this industrial location.22 These compounds would thus
be classified as perfluoroalkyl ether sulfonic acids (PFESA) 4
However, unlike previous perfluorinated ether carboxylic acid
compounds found in this study, in -source proton- and sodium -
bound dimers were not found for these perfluorinated ether
sulfonates. In addition, a homologous series based on CF2 or
CF20 additions was not found. We have not been able to
obtain an authentic standard for confirmation of this proposed
structure.
■ DISCUSSION
It has been well-known for a number of years that, due to the
toxicity and persistence of perfluorinated compounds, industrial
producers are moving toward shorter chain per- and
polyfluorinated compound homologues. However, it is not
well-known which compounds are being produced as replace-
ments for historic perfluorinated compounds (e.g., PFOS and
PFOA). Peer -reviewed literature showing the chemical
structure of manufactured per- and polyfluorinated replacement
compounds is sparse. An example of one such publication from
3M Corporation indicated that the compound ADONA (4,8-
dioxa-3H-perfluorononanoate) is being made to replace
ammonium perfluor000nanoate (APFO) as an emulsifier in
the manufacture of fluoropolymers.5 The data presented in this
study and Figure S1 indicate that the manufacture and release
of per- and polyfluorinated compounds with CF2 units
interspersed with ether oxygen linkages likely is ongoing. A
more recent study suggests that polyfluoroalkyl ether
G DOI: 10.1021/acs.est.5b01215
Environ. Sci. TechnoL XXXX, XXX, XXX—XXX
DEQ-CFW 00000263

Environmental Science & Technology
compounds are being produced and that the number and
spacing of ether oxygen linkages and size of the resulting per -
and polyfluorinated compound may be manufacturer -specific
including other major fluorochemical producers (DuPont, 3M,
Solvay, and Asahi). ° However, there are little to no data in the
peer -reviewed literature on most of these compounds. The use
of HRMS.appears to be an ideal technique to use for discovery
and occurrence of new PFASs being released into the
environment.
As illustrated in this research, identification of previously
undescribed compounds in environmental matrices can be
made difficult by the formation of a multitude of in -source
artifacts such as in -source gas phase adducts, n-mers, and the
presence of a homologous series of per- and polyfluorinated
compounds. D'Agostino and Mabury identified novel fluori-
nated surfactants in aqueous film -forming foams (AFFF) and
commercial surfactant concentrates using high -resolution mass
spectrometry.23 Their results imply that there are a number of
additional fluorinated compounds being produced and emitted
into the environment that are unknown to most analysts. The
use of TOFMS or other high -resolution mass spectrometers
and the workflow we demonstrated (Figure 1) appears to be a
useful way to determine occurrence and emissions of new and
emerging fluorinated compounds for environmental occurrence
efforts.
Schymanski et A. report on identifying small molecules via
HRMS, with five levels of confidence.' As has been noted
earlier, one of the pitfalls of discovery work is the lack of
authentic standards at times. As such the highest level of
confidence for confirmed structure (Level 1) can be made to
one compound in this effort, with the remaining compounds
falling in the categories of tentative candidate(s) (Level 3) or
probable structure (Level 2).14 An additional line of evidence
we present that Schymanski et at. appear not to is the presence
of a homologous series (CF2, CF2O) as evidence for compound
identification.
The manufacture of shorter -chain perfluorinated and
polyfluorinated compounds may have an advantageous effect
on biological persistence and bioaccumulation. Two recent
publications deemed "The Helsingor Statements24 and the
"Madrid Statement"25 explore the state of the science related to
poly- and perfluorinated alkyl substances. Shorter straight -chain
homologues of perfluorocarboxylic acids (<C6) and sulfonic
acids (<PFBS) have been shown to be cleared quickly from
mammalian species tested and thus are not expected to
bioaccumulate as readily as PFOS and PFOA.26 However, the
environmental persistence of these shorter chain perfluorinated
compounds is likely to be no different from that of the related
longer -chain acids. The conventional wisdom is the perfluori-
nated ether compounds will be more labile than the n-alkyl
perfluorinated homologues with ether oxygen linkages being
more susceptible to chemical or microbial attack. However, as
the CF2 bond in perfluorinated compounds is not at all open to
microbial attack due to its bond strength, even the slightest
degree of degradation of a per- or and polyfluorinated ether
compound would be considered "more" labile. Environmental
degradation or toxicology studies associated with the
compounds identified in this study were not found by the
authors in the peer -reviewed literature at the time of writing
this Article. Although data are scarce, the few perfluoropo-
lyethers (PFPEs) to have undergone any kind of degtradation
studies show little to no biodegradation or hydrolysis. ° These
data mainly come from the European Chemical Agency
(ECHA) database of registered substances and associated
information.27 Past studies have demonstrated poor removal
efficiency for historical PFASs from source water to drinking
water in conventional systems (i.e., treatments that do not
include granular activated charcoal (GAC)). It is unknown if
these PFECAs and PFESAs are removed by conventional
WWTP processes.
Environmental Implications. We demonstrate the pres-
ence of a series of novel perfluorinated ether carboxylic and
sulfonic acid(s) found to be in natural waters using a
nontargeted workflow (Figure 1). The compounds consist of
a homologous series of per- and polyfluorinated compounds
with repeating units of CF2 or CF2O subunits. LC-TOFMS
investigations of accurate mass dimers and n-mers (proton- or
sodium -bound) in addition to diagnostic fragment ions support
these findings. Further unidentified compounds with negative
mass defects are likely also of per- and polyfluorinated origin,
but have not yet been identified and are undergoing further
analytical scrutiny. Nontargeted screening and discovery of
novel species in samples such as reported here is an ongoing
process. Additional environmental samples from other locations
may be needed to identify additional chemistries.
Once a xenobiotic compound is identified in the environ-
ment, it falls upon the scientific community to begin
monitoring efforts to find the extent of contamination of
newly discovered species. In addition, toxicological and
degradation investigations of identified compounds can
commence once a compound has been identified. The
procurement of authentic compounds for use in these types
of investigations may be a major limiting factor in conducting
such investigations, as most of these compounds appear not to
be commercially available. More research will need to be
conducted on these perfluorinated ether carboxylic and sulfonic
acids concerning environmental occurrence, toxicology, and
degradation potential.
■ ASSOCIATED CONTENT
© Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.est.5b01215.
Additional information including tables, figures, chem-
icals structures, and spectra (PDF)
■ AUTHOR INFORMATION
Corresponding Author
*Phone: [phone]; a -mail: strynar.mark()epa.gov.
Notes
The authors declare no competing financial interest.
■ ACKNOWLEDGMENTS
We thank Agilent Technologies for their support of this effort
through a TOFMS and travel/training CRADA with the U.S.
EPA (CRADA 437-A-12) and in particular Joe Weitzel for his
support of this work In addition, we thank Mike Hays
(USEPA), and Chris Higgins and Simon Roberts (Colorado
School of Mines) for the use of their QTOFs in confirmation of
select compounds identified. We also thank John Offenberg,
Michelle Angrish, and Mike Hays for their review of this
manuscript. The United States Environmental Protection
Agency through its Office of Research and Development
funded and managed the research described here. It has been
subjected to Agency review and approved for publication.
DOI:10.1021 /acs.est.Wl 215
Envlron. Sci. Technol. MOM XXX, XXX—XXX
DEQ-CFW 00000264

Environmental Science & Technology
Mention of trade names or commercial products does not
constitute endorsement or recommendation for use.
■ REFERENCES
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(2) Houde, M.; De Silva, A. 0.; Muir, D. C. G.; Letcher, R. J.
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(5) Gordon, S. C. Toxicological evaluation of ammonium 4,8-dioxa-
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(6) West Virginia Department of Environmental Protection. Executed
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(12) Schymanski, E. L.; Singer, H. P.; Longr6e, P.; Loos, M.; Ruff,
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(14) Schymanski, E. L.; Jeon, J.; Gulde, R; Fenner, K; Ruff, M.;
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(15) Nakayama, S.; Strynar, M. J.; Helfant, L.; Egeghy, P.; Ye, X.;
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(16) Nakayama, S. F.; Strynar, M. J.; Reiner, J. L.; Delinsky, A. D.;
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(21) NCDENR. Onsite PFOA Groundwater Monitoring Report 12-9-
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(22) USEPA. Envirofacts. http://iaspub.epa.gov/enviro/tsca.get_
chem_info?v_registry_id=110000559609. 2015.
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T.; Wang, Z.; Webster, T. F. Helsingor Statement on poly- and
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Cousins, I.; Diamond, M.; Fletcher, T.; Higgins, C.; Lindeman, A A.;
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(26) Lau, C.; Anitole, K; Hodes, C.; Lai, D.; Pfables-Hutchens, A.;
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DOI:10.1021 /acs.est.5b01215
Environ. Sci. Technol VM XXX, XXX—XXX
DEQ-CFW 00000265

1 Supporting Information for:
2 identification of novel perfluoroalkyl ether carboxylic acids (PFECAs) and sulfonic acids (PFESAs)
3 in natural waters using accurate mass time -of -flight mass spectrometry (TOFMS)
4 Mark Strynar', Sonia Dagnin02, Rebecca McMahen 2, Shuang Liang2, Andrew Lindstrom', Erik
5 Andersen', Larry McMillan3, Michael Thurmano, Imma Ferrero, Carol Balls
6 1. National Exposure Research Laboratory, U.S. Environmental Protection Agency, Research
7 Triangle Park, NC;
8 2. ORISE fellow at the United States Environmental Protection Agency, National Exposure
9 Research Laboratory, Research Triangle Park, North Carolina;
10 3. National Caucus and Center on Black Aged, Inc, Durham, NC;
11 4. Center for Environmental Mass Spectrometry, University of Colorado in Boulder CO;
12 5. Agilent Technologies Inc., Wilmington, DE
13
14
15
16 This document includes one table, and 12 SI figures, on 13 pages.
17
18
19
20
21
22
23
24
25
0
S1
DEQ-CFW 00000266

27 Table S1. Water samples description and GPS coordinates.
28
Sample ID
Description
Latitude
Longitude
CFR 001
Cape Fear river Tar Heel, NC
34.74525
-78.78574
CFR 002
Cape Fear river below Huske lock and dam #3
34.83026
-78.82246
CFR 003
unnamed tributary
34.83179
-78.82375
CFR 004
Cape Fear river above Huske lock and dam #3
34.83544
-78.82347
CFR 005
Cape Fear river below Rockfish creek
34.96820
-78.81579
CFR 006
Rockfish Creek
34.95610
-78.84424
CFR 007
Rockfish Creek WWTP effluent
34.96834
-78.82765
CFR 008
Cape Fear River at Fayetteville boat ramp access
34.99669
-78.85076
CFR 009
Regional drinking water sample
1 34.94199
-78.92422
29
30
31
32
F F F F F
F�O)
F F
33
H F 0
O O� NH4
F F
34 Figure S1. The structure of ADONA (4,8-dioxa-3H-perfluorononanoate) a polyfluorinated
35 compounds used to replace classic perfluorinated compounds such as APFO.
36
37
38
S2
DEQ-CFW 00000267

39
40
41
42
43
44
45
46
47
61000
51000
■ C10
41000
- —
--- n PFO5
31000
---
------- 9 C9
Streamflow
Direction
21000
-- _
-- - — C8
J
040411000
-
- ■ PFHS
c
"
u _
■ C7
0- 1000
--- -- -- --- -- —
— - - ----
W
--
900
— ------ --
■ C6
800
-- —
■ PFBS
700
600
---.----- ------
■ C5
500
...
400
_..___ __------.----------. ___ ._-_-��Y
_._ �� ■C4
300
_
-
200
100
__...
--
CFR 001 CFR 002 CFR 003
CFR 004
CFR 005 CFR 006
CFR 007 CFR 008 CFR 008 CFR 009
repl rep2
Figure S2. PFAAs found in water samples from the Cape Fear River. Note the y-axis is a split
scale.
S3
DEQ-CFW 00000268

CFR 001- downstream (- 10.7 km)
C8 C9 CIO C4
C7 6% 2% 2% 4%
6%
C6
3%
CFR 002 - downstream (-300 meters)
C8 C9 CIO
C7 1% 1% 0% C4
9% 7%
1%
1%
48
49
C8 C9 CIO CFR 003 -
C7 0% 0% 0%
8%
C6 C4
1% 7%
CFR 004 - upstream (-300 meters)
C4
5%
7%
7%
50 Figure S3. Proportion of PFAAs contribution to the total for select water samples from the Cape
51 Fear River.
S4
DEQ-CFW 00000269

1800
1600
1400
1200
p 1000
a 800
i
600
400
200
0
Mass vs. Retention Time
• Control - upstream sample; CFR 004
• Experiment — downstream sample CFR 002 (a)
0•
-V' _.• •
1 1 2 t 4 5 6 7 8 9 1c
Retention Time (min)
—•-—<lono vf•
77 Features present only in Experiment
1800
1600
(� )
1400
1200
S 1000
• .r
N
600
400
PFaA PFP:A
N �
200 -
i • • PFHp4
+• _ •
0
0 1 2 3 4 ~5 6 7 8 9 10
Retention Time (min)
52 mn w a en.. w..
53
54 Figure S4. Mass Profiler visualization of molecular features found in (a) control and
55 experimental sample and (b) 77 features unique only to experimental sample, with select peaks
56 previously identified (PFBA, PFPeA and PFHpA). Note: The size of the symbol in this plot is
57 proportional to the area of the peak. Pronounced co -eluting peaks that appear as vertical lines
58 are likely related (i.e. fragments, M-H-, n-mers).
59
60
61
62
63
64
S5
DEQ-CFW 00000270

65 X10^
3
2
66 `
o
,m"
67 ,
0.5
GQ »
z
69 `
v
7D mp
o
^
71 u
v
X103
7.5
72 ,
2.5
73 *v
'
74 ,
o
to F
F+ F
OH
ESI EIC(379.5645) ',-an Fraa-SO.OW)orklistDatald F+ F F
, [--�4F
F OH
F 30,�F OH
+�F V
0511.522.533.544.555.56 5.577.538.09U.D 1Um,,,11Z"1=13=^14=
����*�"n=*�
7S
76
77 FiguneS5. Extracted Ion Chromatogram (E|[)ofmsuspected homologous series of
78 perfluorinated ether carboxylic acids. Note: The chromatographic peak indicated by an * is the
79 substance associated with this homologous series based on the H+ and Na+ climer co -elution at
80 this retention time. The second later eluting peak inthis chromatogram iaasimilar nn/z,
81 possibly anisomer.
82
83
84
OS
86
S6
OEQ-CFVV_00000271

Target Compound CAS # 13262-13-6
2,3,3,3-ietratuom-24hepiatucewq*xy3proPanoate
F IY6-W2
O
F F
F F F
m/z 284.9779
Mdlemftffatm Ae: CBHFjjOS
Formula WeWW. 330.OSM
Compoaasow. C(21.83%) H(a.31 %) F(63.32%) 0(14 54%)
Mondsatapk Mew. 329.97M Da
32&9w Do
O
[2M-2H+Na]- _ a=
F F F
F F F
F F F F
F F
F F
mlz 680.9247
87
88
F
F \C
F
F1'! F
F
mlz 168.9894
F
F O
F
F F
mfz 184.9843
F [2M-H F
F- " -F
F •"'J•
F F
F Y
F O F
F X,
H$
Fxr U F IF
F
mfz 658.9427
89 Figure 56. Identified perfluorinated ether carboxylic acid and diagnostic fragment and dimer
90 ions.
91
S7
DEQ-CFW 00000272

92
93
x10 5 A
6.9
64 2.5 ppm error
6.2 5--958 "29
6
1.8
4.6
4.4
42 O
4
3.8 F O
3.6 178.9779
3.4 O
32
3
2.8 F F
2.6 F F
2.4
22
2
1.8
1.6
1.4
1.2
1
0.8
0.6
OA 292.9704 j '
02 146.9236 192.9290209.9193 246.9651263.9562 '. 314.9526 342.. 74
0
90 1" 110 120 130 140 150 160 170 180 190 260 210 CW a230 1 2 ( ) 250 290 300 310 320 330 3<0 350 360 370 380 390 400
x10 a
7.25 B
7
6.75 184.9851ts O
6.25 O
6 " F
VS
5.5 O F
5.25
5
4.75 F
4.5 F F F
4.25
4 F
3.75
3.5
3.25
3
2.75
2.5
2.22 0.46 ppm error
9.3
1•25 458.9555
1
0.75
0.5 480.9974
0.25 1Y8A925 134.9740 2n'91748 270.9027 313.9618 364.9487 408.9586 �, 500.8229 566.9Y81
0 1 � I
100 ,20 140 160 180 200 220 240 260 ' 280 300"31% �%3 380 �) 420 440 460 480 500 $20 540 500 590 600
S8
DEQ-CFW 00000273

94
234.9620
4
3-8
3.6
3.4
2.6
IA
1.6
1.4
1.2
1
0.8
0.6
OA Z/
0.2 118.9926 154.9740 211.0463 2781711 320.9592 363.9486
100 uo 140 160 160 zoo 220 2io 26o 280 3W320nt, 34) w. M 3
X10 6 l
6.4 a
6.6
6.4
s.2
5
4.8
4.6
4.4
4.2
2.8
2.6
2.4
2.2
6
4
2
1
8
6
° 134.9977
2 I
0 '
80 160 120 140 160
284.9793
a MOM
180 200 220 240 260 280 300 320 340 C380 380 41
Count, N w.
0
C
-1.23 ppm error
660A706
414.9464 606.MN
1140l 480.93#2 $24.9466 _ t 1 , 6164136
0 420 "0 460 490 500 $20 $40 660 680 600 620 640
❑C
-0.85 ppm error
nos=
65$.9422
t
464.9423
460 490 500 620 540 560 580 600 620 640 660 680 700 720 740
P (.)
96 Figure S7 Spectra for Figure S5 chromatographic plot EICs. The spectra shown are for the first
97 four eluting mono ether PFECAs shown in Figure S5. Plot A corresponds to m/z 178.9773; Plot B
98 corresponds to m/z 228.9741; Plot C corresponds to m/z 278.9709; Plot D corresponds to m/z
99 328.9677. Error values shown are for the corresponding [2M-H]- (Table 1)
100
S9
DEQ-CFW 00000274

101
102
103
104
x10I I'ESI EIC(754.9123) Scan Frog-80.0V\lwkIWD-6.d
1
x102 I;ESI EIC(622.9289) Scan Fra9-80.OVWV klistData6.d
1 11 5 6o.
021
0.1
00
x10 1 I-ESI EIC(490.9865) Scan
31,
1
0
F F F F F
`�./,/` /�,� Molecular Formula: 0yHF710y
-0 ! -o 0H Monoisotopic Maas: 377,9698 Do
F F F F F (M•H]•: 376,962608
0
F F F F Molecular Formula: CBHF905
F\ / \ /o\ / Monoiaotopic Maw. 311.9608 Da
/X\ /X\ /�\ off Monoi: 311.9880 Do
0 0
F F F F
5 0
F F F
0
OH
F' O
F
x101 -ESI EIC(358.9621) Scan Fra9-SO.OV \YwkliatData6.d
1 8 I 1,262 0 I
0 F /
12 OH
1
0.8 F 0 F
0,6
O.a
02
0
Molecular Formula: C�HF70.
Monoisotopic Mass: 245.9763 Da
[M-H]-: 244.9690 Do
Molecular Formula: CSHFyO,
Monoisotopic Mass: 179.9646 Da
(M•H]•: 178.9773 Da
105 ` ' ' C-1. ('4) va. A-uiaition Tine
106 Figure S8. Extracted ion chromatogram of additional perfluorinated ether carboxylic acids
107 homologous series. Proton bound dimer EIC shown for chromatogram. Monomer structure
108 and exact mass shown
109
S10
DEQ-CFW 00000275

110
111
112
0
F F Molecular Formula: C,HFrO.
,/^i Monolsotopic Mass: 245.9763
OH [M.H)-: 244.9690
O F
�O FOF\ Molecular plcForMass:
20D.9
F'
VO�O Molecular Formula: CrFsO�
'�\ Monolsotopic Mass: 7509ffi4
F F
VO�C F Molecular Formula: C,Fs01
F -/ \ Monolsotoplc Mau; 134.9875
F F
F\ O
V Molecular formula: CFrO,
F'/<\ Monoisotopic Mau: 94.9907
F
Figure S9. QTOF fragment ions for C4HF704 (m/z 244.9691) pefluoroether carboxylic acid.
S11
DEQ-CFW 00000276

-ESISae65 S96I-2E )Nw O'A—,tD dS 1
R
E51 S:an139t2.4 atlmn.ES:Fra}FrFp.B°WYM4°sNFMEd SNFan
8 az [2M-2H+Na]-
s Clt FT7 Na Oe 680.4255 680.9247 -1.2 98,11
[M-H]-
(2M-H)- C32 H F2206' 658.9433 658.9427 -2.36 92.96
,7
(M-H]- Ca F1103' 328.9683 328.9677 -1.8 97.85
°e
06 Fragment 1 CS F110• 284.9789 284.9779 -3.49 96.35
°a
o: 2ss.7 J?90' YHJ] 17�529, 33105P7 Fragment2 C3F7- 168.9902 168.9894 •4.75 84.41
l2)5; 06 3%
0
113 Cwnb (•r,) y. MpfdaChagelm4)
114
115 Figure S10. Spectrum, isotope pattern matching and identified adducts and fragment of
116 molecular feature (m/z 328.9683) found in water. The table shows the formula, measured m/z,
117 the exact m/z, the error associated with the measurement and the software scoring of the
118 isotope cluster. The red boxes around each spectrum peak indicate the agreement between
119 the measured and theoretical isotope cluster. The scoring is a bundled measurement of the
120 accurate mass of the monoisotopic peak versus theoretical, the isotope abundance and the
121 isotope spacing.
122
S12
DEQ-CFW 00000277

x10t SuWKI
•ESI Ssanl6.%i5613 min. 6Sam)Frp.80,W4krMiftlJw6.d
5.5 -1139
d5 [2M-H]-
M S157
G�42ID
Scan (65*6.596 n•1n.2 $on) Ftp.800vt b.MHdbb6.d SL4*w
Fragment 1
02 198.6886 1",3723 I 205 7�M SSI6 2N 7780 2�061,06
0 t 105 �2005 1 20S � 2015 3 205 2 iCa5 .5 206
Cart 00-M—ta o'Wm )
•IO^ IEV LMt66MS ]Henna&wli••p.90Wt:bls•rowia SAett•
[2M-2H+Na]- H2O
[2M-2H+Na]- C32 F22 Na 012' 776.8952 776.8942 -1.31 95.92
[2M H] C32 H F22012' 754.9139 754.9122 -2.22 92.66
[M Hj C6 F3106 Ob eot 376.9525 --- --
Fragment 1 C3 F702. 200.9785 200.9792 3.71 95.18
[2M-2H+Na]- C32 H3 F22 Na 013 794,9016 794.9047 3.95 84.15
H
123 2O
124 Figure S11. Spectrum, isotope pattern matching and identified adducts and fragment of
125 molecular feature (m/z 376.9525) found in water. The table shows the formula, measured m/z,
126 the exact m/z, the error associated with the measurement and the software scoring of the
127 isotope cluster. The red boxes around each spectrum peak indicate the agreement between
128 the measured and theoretical isotope cluster. The scoring is a bundled measurement of the
129 accurate mass of the monoisotopic peak versus theoretical, the isotope abundance and the
130 isotope spacing.
131
132
133
S13
DEQ-CFW 00000278

.10 a -ESI Scan (6.547-6.646 min. 7 Sans) Frag=80.OV WwIdistDaia6,d Subtract
1-
0.95-
0.9 754.9138
F
0.85-
0.8 I2M-Hl-
f 0
0.75- F F
0.7 F F [2M-2H+Na]-
0.65 0 776.8953
0.6-
/y\ 0.55 F F
0.5-
0.15 4xoas
1
0.4- [M-H). F
0.35 376.9625
0.3
0.25
0.2 1176,8363
i8�7769
0.15- [3M-3H+2Na)-
g 1 [4M-4H+3Na]-
0.05-L200.9795 12W.9532
0
31 285.2076 418.9414 573.3179 688.9220 862.8761 952.1049 1110.8436 i 1391.6219 151017836 1662.7671
100 150 200 250 �300 390 400 450 500 550 600 850 700 750 t800 850 900 950 1000 1050 ltlq 1150 /200 1250 1300 1350 1400 1450 1500 1560 1800 1650 1700
134 Caunts — Mass+o-cna<ga (m7=)
135 Figure S12. Spectrum of novel perfluorinated ether carboxylic acids showing proton bound and
136 sodium bound n-mers found. The structure shown is the parent compound. The various in-
137 source n-mers formed are shown with shorthand nomenclature for dimers, trimers and
138 tetramers.
139
S14
DEQ-CFW 00000279