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Chemical fingerprinting of naphthenic acids and oil

sands process waters—A review of analytical methods
for environmental samples
a a a b c
J. V. Headley , K. M. Peru , M. H. Mohamed , R. A. Frank , J. W. Martin , R. R.O.
d e f b b
Hazewinkel , D. Humphries , N. P. Gurprasad , L. M. Hewitt , D. C.G. Muir , D.
a g h h i h
Lindeman , R. Strub , R. F. Young , D. M. Grewer , R. M. Whittal , P. M. Fedorak ,
j k l m m n
D. A. Birkholz , R. Hindle , R. Reisdorph , X. Wang , K. L. Kasperski , C. Hamilton
n n o p p c
, M. Woudneh , G. Wang , B. Loescher , A. Farwell , D. G. Dixon , M. Ross , A. Dos
c c q r a s
Santos Pereira , E. King , M. P. Barrow , B. Fahlman , J. Bailey , D. W. Mcmartin ,
t u u u v b
C. H. Borchers , C. H. Ryan , N. S. Toor , H. M. Gillis , L. Zuin , G. Bickerton , M.
b b g w x
Mcmaster , E. Sverko , D. Shang , L. D. Wilson & F. J. Wrona
a
Water Science & Technology Directorate , Environment Canada , Saskatoon ,
Saskatchewan , Canada
b
Water Science & Technology Directorate , Environment Canada , Burlington , Ontario ,
Canada
c
Department of Laboratory Medicine and Pathology, Division of Analytical and
Environmental Chemistry , University of Alberta , Edmonton , Alberta , Canada
d
Alberta Environment , Edmonton , Alberta , Canada
e
Alberta Innovates–Technology Futures , Vegreville , Alberta , Canada
f
Water Science & Technology Directorate, Environment Canada , Edmonton , Alberta ,
Canada
g
Water Science & Technology Directorate , Environment Canada , Vancouver , British
Columbia , Canada
h
Department of Biological Sciences , University of Alberta , Edmonton , Alberta , Canada
i
Department of Chemistry , University of Alberta , Edmonton , Alberta , Canada
j
ALS Laboratory Group Environmental Division , Edmonton , Alberta , Canada
k
Vogon Laboratory Services Ltd. , Calgary , Alberta , Canada
l
Departments of Immunology and Pediatrics , National Jewish Health , Denver , Colorado ,
USA
m
Natural Resources Canada , IETS CanmetENERY-Devon , Devon , Alberta , Canada
n
AXYS Analytical Services Ltd. , Sidney , British Columbia , Canada
o
Maxxim Analytics , Burnaby , British Columbia , Canada
p
Department of Biology , University of Waterloo , Canada
q
Department of Chemistry , University of Warwick , Coventry , United Kingdom
r
Canadian Forest Service–Great Lakes Forestry Centre , Natural Resources Canada , Sault
Ste. Marie , Ontario , Canada
s
Environmental Systems Engineering , University of Regina , Regina , Saskatchewan , Canada
t
University of Victoria-Genome BC Proteomics Centre, Department of Biochemistry &
Microbiology , University of Victoria , Victoria , British Columbia , Canada
 u
Level Science Inc. , Saskatoon , Saskatchewan , Canada
v
Canadian Light Source Inc. , Saskatoon , Saskatchewan , Canada
w
Chemistry Department , University of Saskatchewan , Saskatoon , Saskatchewan , Canada
x
Water Science & Technology , Environment Canada , Victoria , British Columbia , Canada
Published online: 06 May 2013.

To cite this article: J. V. Headley , K. M. Peru , M. H. Mohamed , R. A. Frank , J. W. Martin , R. R.O. Hazewinkel , D.
Humphries , N. P. Gurprasad , L. M. Hewitt , D. C.G. Muir , D. Lindeman , R. Strub , R. F. Young , D. M. Grewer , R. M.
Whittal , P. M. Fedorak , D. A. Birkholz , R. Hindle , R. Reisdorph , X. Wang , K. L. Kasperski , C. Hamilton , M. Woudneh ,
G. Wang , B. Loescher , A. Farwell , D. G. Dixon , M. Ross , A. Dos Santos Pereira , E. King , M. P. Barrow , B. Fahlman , J.
Bailey , D. W. Mcmartin , C. H. Borchers , C. H. Ryan , N. S. Toor , H. M. Gillis , L. Zuin , G. Bickerton , M. Mcmaster , E.
Sverko , D. Shang , L. D. Wilson & F. J. Wrona (2013): Chemical fingerprinting of naphthenic acids and oil sands process
waters—A review of analytical methods for environmental samples, Journal of Environmental Science and Health, Part A:
Toxic/Hazardous Substances and Environmental Engineering, 48:10, 1145-1163

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 Journal of Environmental Science and Health, Part A (2013) 48, 1145–1163
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ISSN: 1093-4529 (Print); 1532-4117 (Online)

DOI: 10.1080/10934529.2013.776332

Chemical fingerprinting of naphthenic acids and oil sands

process waters—A review of analytical methods for
environmental samples

J.V. HEADLEY1, K.M. PERU1, M.H. MOHAMED1, R.A. FRANK2, J.W. MARTIN3, R.R.O. HAZEWINKEL4,
D. HUMPHRIES5, N.P. GURPRASAD6, L.M. HEWITT2, D.C.G. MUIR2, D. LINDEMAN1, R. STRUB7,
R.F. YOUNG8, D.M. GREWER8, R.M. WHITTAL9, P.M. FEDORAK8, D.A. BIRKHOLZ10, R. HINDLE11,
R. REISDORPH12, X. WANG13, K.L. KASPERSKI13, C. HAMILTON14, M. WOUDNEH14, G. WANG14,
B. LOESCHER15, A. FARWELL16, D.G. DIXON16, M. ROSS3, A. DOS SANTOS PEREIRA3, E. KING3,
Downloaded by [University of Saskatchewan Library] at 14:59 10 June 2013

M.P. BARROW17, B. FAHLMAN18, J. BAILEY1, D.W. MCMARTIN19, C.H. BORCHERS20, C.H. RYAN21,
N.S. TOOR21, H.M. GILLIS21, L. ZUIN22, G. BICKERTON2, M. MCMASTER2, E. SVERKO2, D. SHANG7,
L.D. WILSON23 and F.J. WRONA24
1
Water Science & Technology Directorate, Environment Canada, Saskatoon, Saskatchewan, Canada
2
Water Science & Technology Directorate, Environment Canada, Burlington, Ontario, Canada
3
Department of Laboratory Medicine and Pathology, Division of Analytical and Environmental Chemistry, University of Alberta,
Edmonton, Alberta, Canada
4
Alberta Environment, Edmonton, Alberta, Canada
5
Alberta Innovates–Technology Futures, Vegreville, Alberta, Canada
6
Water Science & Technology Directorate, Environment Canada, Edmonton, Alberta, Canada
7
Water Science & Technology Directorate, Environment Canada, Vancouver, British Columbia, Canada
8
Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada
9
Department of Chemistry, University of Alberta, Edmonton, Alberta, Canada
10
ALS Laboratory Group Environmental Division, Edmonton, Alberta, Canada
11
Vogon Laboratory Services Ltd., Calgary, Alberta, Canada
12
Departments of Immunology and Pediatrics, National Jewish Health, Denver, Colorado, USA
13
Natural Resources Canada,, IETS CanmetENERY-Devon, Devon, Alberta, Canada
14
AXYS Analytical Services Ltd., Sidney, British Columbia, Canada
15
Maxxim Analytics,, Burnaby, British Columbia, Canada
16
Department of Biology, University of Waterloo, Canada
17
Department of Chemistry, University of Warwick, Coventry, United Kingdom
18
Canadian Forest Service–Great Lakes Forestry Centre, Natural Resources Canada, Sault Ste. Marie, Ontario, Canada
19
Environmental Systems Engineering, University of Regina, Regina, Saskatchewan, Canada
20
University of Victoria-Genome BC Proteomics Centre, Department of Biochemistry & Microbiology, University of Victoria,
Victoria, British Columbia, Canada
21
Level Science Inc., Saskatoon, Saskatchewan, Canada
22
Canadian Light Source Inc., Saskatoon, Saskatchewan, Canada
23
Chemistry Department, University of Saskatchewan, Saskatoon, Saskatchewan, Canada
24
Water Science & Technology, Environment Canada, Victoria, British Columbia, Canada

Address correspondence to J. V. Headley, Water Science & Technology Directorate, Environment Canada, 11 Innovation Blvd.,
Saskatoon, SK, Canada, S7N 3H5; E-mail: John.Headley@ec.gc.ca
Received November 12, 2012.
 1146 Headley et al.
This article provides a review of the routine methods currently utilized for total naphthenic acid analyses. There is a growing need
to develop chemical methods that can selectively distinguish compounds found within industrially derived oil sands process affected
waters (OSPW) from those derived from the natural weathering of oil sands deposits. Attention is thus given to the characterization
of other OSPW components such as oil sands polar organic compounds, PAHs, and heavy metals along with characterization of
chemical additives such as polyacrylamide polymers and trace levels of boron species. Environmental samples discussed cover the
following matrices: OSPW containments, on-lease interceptor well systems, on- and off-lease groundwater, and river and lake surface
waters. There are diverse ranges of methods available for analyses of total naphthenic acids. However, there is a need for inter-
laboratory studies to compare their accuracy and precision for routine analyses. Recent advances in high- and medium-resolution
mass spectrometry, concomitant with comprehensive mass spectrometry techniques following multi-dimensional chromatography
or ion-mobility separations, have allowed for the speciation of monocarboxylic naphthenic acids along with a wide range of other
species including humics. The distributions of oil sands polar organic compounds, particularly the sulphur containing species (i.e.,
Ox S and Ox S2 ) may allow for distinguishing sources of OSPW. The ratios of oxygen- (i.e., Ox ) and nitrogen-containing species (i.e.,
NOx , and N2 Ox ) are useful for differentiating organic components derived from OSPW from natural components found within
receiving waters. Synchronous fluorescence spectroscopy also provides a powerful screening technique capable of quickly detecting
the presence of aromatic organic acids contained within oil sands naphthenic acid mixtures. Synchronous fluorescence spectroscopy
provides diagnostic profiles for OSPW and potentially impacted groundwater that can be compared against reference groundwater
Downloaded by [University of Saskatchewan Library] at 14:59 10 June 2013

and surface water samples. Novel applications of X-ray absorption near edge spectroscopy (XANES) are emerging for speciation of
sulphur-containing species (both organic and inorganic components) as well as industrially derived boron-containing species. There
is strong potential for an environmental forensics application of XANES for chemical fingerprinting of weathered sulphur-containing
species and industrial additives in OSPW.
Keywords: Bitumen, chemical profiles, mass spectrometry, heavy metals, PAHs, Athabasca River, polar organics.

Introduction considered to be economically recoverable.[5] Surface mine-

The purpose of this communication is to report the key find-
ings of a Naphthenic Acids Strategies Workshop, Saska-
toon, Canada, November 24 and 25, 2011. The workshop
was limited to invited practitioners to review routine meth-
ods for analyses of total naphthenic acid (NA) mixtures
and the growing need to develop chemical methods capa-
ble of distinguishing compounds found within industrial oil
sands process-affected waters (OSPW) from those released
during natural weathering of the McMurray oil sands for-
mation. Particular emphasis of this review is placed on the
complex organic acid mixtures found within oil sands im-
pacted waters, hereafter referred to as oil sands naphthenic
acids and as oil sands naphthenic acid fraction components
(NAFC), in view of recent advances using high resolution
mass spectrometry for analyses of these mixtures.[1–4]
However, an evaluation of chemical methods used for
other components such as PAHs, heavy metals, tracer
chemicals, and additional organic or inorganic compounds
will also be addressed. Contained within this review is a
summary and evaluation of currently utilized analytical
approaches, those under development, and existing knowl-
edge gaps concerning the structural and analytical char-
acterization of such components. Some of the material is
not yet published in the peer-reviewed scientific literature,
and thus, due care has been taken to protect the intellectual
property of researchers, while ensuring that this review re-
flects current approaches in use by practitioners. Although
assessment of the fingerprinting methods required for risk
assessment, along with detailed discussion of applications
for environmental effects monitoring, are important, they
are outside the scope of this communication.
The reserves of oil sands bitumen in northern Alberta,
Canada are estimated at 1.7 trillion barrels, with 173 billion
able bitumen is extracted from the oil sands mixture using
a modification of the Clark caustic hot water method,[6] a
procedure that requires large volumes of water.[7] Follow-
ing the extraction, the resulting OSPW is retained on-site
in vast tailings ponds and settling basins.[8]
Environmentally sustainable oil sands production is thus
a topic of increasing importance. As oil sands mining activ-
ities increase, there is a growing need to develop methods
for the analyses of oil sands NAs and other related com-
pounds to determine the impacts on water quality and to
measure the potential effects on the surrounding ecosys-
tem (Canadian Fisheries Act 1985).[9] OSPW contains sub-
stances known to be acutely toxic in aquatic environments;
namely organic components including polycyclic aromatic
hydrocarbons (PAHs)[10–15] and oil sands NAs.[1,12,16–20]
Recent research has drawn attention to PAHs and
heavy metals released into the aquatic environment in the
Athabasca oil sands regions originating from industrial
development processes.[11,21] Likewise, the characterization
of oil sands NAs in OSPW and environmental samples
is a topic of much interest.[16,22–24] The individual princi-
pal toxic components have not yet been identified for the
OSPW NAFCs, primarily due to the complexity of the
organic acid mixtures, which necessitates further develop-
ment of analytical methods. However, recently some indi-
vidual components in OSPW from two different operators
have been identified,[25–27] others tentatively identified [28]
and the toxicity of specific components reported.[29]
NAs are defined traditionally as mono-carboxylic acids
which include chain compounds and compounds with one
or more alicyclic ring structures. The NAFCs (Fig. 1), as
defined by Headley et al.[2] can be used more liberally
to describe the wide range of polar organic compounds
found within oil sands bitumen and OSPW.[2,3,7,17,18,30] In
 Chemical fingerprinting of naphthenic acids
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Fig. 1. Representative structures of naphthenic acid fraction com-
ponents (NAFC) in OSPW (adapted with permission from ref.[2]
Copyright 2011 Taylor & Francis).
comparison to classical NAs with the general formula
Cn H2n+Z O2 , the broader definition of NAFC, or oil sands
NAs, includes oil sands acid extractable organics with aro-
matic ring functional groups, nitrogen and sulphur atoms,
along with unsaturated groups.[1–3,31,32] OSPW contains
thousands of polar organic compounds, many of which
can now be resolved using ultrahigh resolution mass spec-
trometry. Although the differences that may be diagnostic
for environmental forensics can be masked by interferences
in the sample matrix,[33–35] the advantages of ultrahigh res-
olution for the characterization of oil sands acids in the
presence of high matrix interferences are well described in
the literature.[1,17,18,36,37]
The unequivocal determination of whether oil sands
NAFC present in surface waters originate from industrial
processes or from natural weathering of deposits is diffi-
cult to fully address. The problem is that leakage of past
tailings pond containments will likely be in the form of
mixtures of NAFC, metals, salts, chemical additives, and
1147
other unknown components. The chemical fingerprints of
such mixtures are subject to further erosion and weather-
ing, also affecting analogous mixtures leached from natural
oil sands deposits. Consequently, for chemical fingerprint-
ing oil sands bitumen and NAFC from OSPW in envi-
ronmental samples, a detailed understanding is needed of
many co-factors that can affect the fate and distribution
of such components. These include: (i) the relative adsorp-
tion of the mixture of compounds to tailings pond con-
tainment materials, sediments, and soils beneath tailings
ponds; (ii) groundwater interactions (oxygen-limited envi-
ronments and potential sorption to aquifer minerals); and
(iii) a general understanding of the geology and weathering
processes that may occur at sample sites.
This review will first discuss the analytical techniques
employed for the chemical characterization of bitumen as
source material; followed by methods used for the charac-
terization of water soluble components in OSPW, intercep-
tor well systems, groundwater, river water, sediments, and
biological tissues. Attention will be given to techniques that
are best suited to analyses of the complex mixtures in real-
world environmental samples. The extent to which a given
method is able to resolve interferences by chromatogra-
phy, mass resolution, or both, will be critical to assessing
the potential of the technique for routine analyses of total
oil sands NAs and application for environmental forensics.
Likewise, the goodness of a given approach which can dis-
tinguish features that are unique for industrially derived
versus naturally derived oil sands components will be of
key importance for fingerprinting analyses of oil sands ma-
terials in environmental samples.
Oil sands and oil sands process-affected water – What is
the composition?
Characterization of Athabasca oil sands – implications
for chemical fingerprinting
Defined in the simplest terms, “Oil sand is basically regu-
lar sand like the kind you would find on a beach that has
some oil trapped in it. It is black, sticky and smells like
oil.”[38] Oil sands are made up of approximately 83% sand,
4% water, 3% clay, and 10% bitumen. In addition to hy-
drocarbons, bitumen contains large amounts of chemical
impurities including S, N, Ni and V, mainly incorporated in
complex asphaltene aggregates. The extraction process for
obtaining oil from Athabasca oil sands is energy and water
intensive. Full details are given elsewhere,[39] describing the
processes for: (i) the separation of the bitumen from the
oil sand using hot alkaline water; (ii) the removal of wa-
ter and solids in a froth treatment step employing solvents
to clean up the hydrocarbon; (iii) the upgrading of the bi-
tumen where high temperatures, high pressure, hydrogen
and catalysts are used to convert the isolated bitumen to
 1148
synthetic crude oil; and finally (iv) the refinery process to
produce crude oil, gasoline and diesel. Hydrotreating is a
critical step in the upgrading process for the removal of
sulphur and nitrogen contained in the oil.
For surface mined oil sands, between 10 and 11 barrels
of water are required to produce one barrel of bitumen.[39]
Because much of this OSPW is recycled, the resulting net
balance is approximately three to four barrels of new wa-
ter being consumed for every one barrel of bitumen that is
produced. The residual tailings, consisting of water, sand
and clay, are stored in on-site containments, commonly re-
ferred to as tailings ponds.[39] Treatment technologies are
under development to help accelerate the dewatering of
the tailings to form solids for eventual reclamation of the
affected boreal forest and muskeg wetlands. For example,
Downloaded by [University of Saskatchewan Library] at 14:59 10 June 2013
under certain conditions, fluid fine tailings can be dewa-
tered rapidly with appropriate polymer addition followed
by beach deposition.[38]
Typically, high molecular weight, medium charge den-
sity polyacrylamides are used to modify the clay behavior
and enhance dewatering.[38] To date, little is known about
the fate and transport of such additives in aquatic environ-
ments. Thus, there may be potential to use the detection
of such additives in OSPW, or their transformation prod-
ucts resulting from weathering, degradation and oxidation,
as indicators to distinguish industrially-derived oil sands
components from those resulting from the natural erosion
and seepage of oil sands outcrops. Research to assess the
utility of chemical additives for fingerprinting purposes is
recognized for its potential importance and preliminary in-
vestigations are underway.
Research has shown that bitumen from surface mined
oil sands has a propensity for emulsion formation due to
elevated levels of surfactants.[38] These surfactants can con-
tribute to a phase layer formation in various froth treatment
operations as well as in other water and solids removal pro-
cesses. The high surfactant concentration in mined bitumen
may thus be a potential marker, or chemical fingerprint,
for differentiating industrial sources of oil sands compo-
nents from those derived from natural leaching of oil sand
into aquatic environments. To this end, Headley et al.[4]
described preliminary results for high resolution MS char-
acterization of Athabasca oil sands samples by employing
diagnostic Ox Ss species (which includes surfactants); details
of which are described in “Atmospheric Pressure Photoion-
ization (APPI) and ESI FTICRMS of oil sands acids in
OSPW.”
Researchers at Natural Resources Canada (NRCan) in
Devon, AB, Canada are at the forefront of research in the
development of new technologies for oil sands processing
as well as treatment and reclamation of the fine tailings.
New initiatives are underway in collaboration with En-
vironment Canada to pioneer treatment technologies in-
cluding wetland plants to help dewater mature fine tails
(MFT) and sequester contaminants for possible phytore-
mediation.[18,33,40] Specific details of this initiative and the
Headley et al.
implications for fingerprinting analyses are discussed later
in subsequent sections on “Overview and current state of
the art – Do we know what we are measuring for OSPW
acid extractable organics?” and “Orbitrap MS of oil sand
organic acids in plant tissue.” The underlying objective is to
improve the oil sands extraction and upgrading processes;
making them more energy efficient and environmentally
sustainable.
Fourier Transform Ion Cyclotron Resonance Mass
Spectrometry (FTICRMS) Characterization
of Athabasca bitumen
FTICRMS has played a key role in the rebirth of
petroleomics. Ultrahigh resolution (450,000 – 650,000 at
m/z 500) enables identification of isobaric species that dif-
fer in mass by 3 mDa or less, and high mass accuracy
(better than 300 ppb mass error) allows for unambiguous
molecular formula assignment. Rodgers et al.[41] have per-
formed detailed characterization of heavy oils by use of
(+/−) electrospray and atmospheric pressure photoion-
ization FTICRMS. The characterizations include a sum-
mary of the class, type and molecular weight trends for
petroleum species and a comparison of the results to pro-
posed asphaltene structural models. Athabasca oil sands
bitumen contains a complex mixture of hydrocarbons typ-
ical of heavy oils. According to the Boduszynski model,
“most of petroleum components do not exceed a molecu-
lar weight of about 2000.”[41]
FTICRMS fingerprinting studies are contributing to a
better understanding of the intermolecular interactions
among asphaltene molecules.[41] For example, the interac-
tion preferences among bitumen molecules, as represented
by heterocyclic and metalloporphyrin fragments,[42] as well
as between bitumen molecules and zeolite nanoparticles,[43]
are described in the literature. Filling the knowledge gaps
for improved understanding of the intermolecular interac-
tions in bitumen is critical to the development of advanced
processing technologies that require less energy and opti-
mizing water use. Research and development of this type is
encouraged as there is high potential for the new technolo-
gies to result in a reduction in the environmental footprint
of the oil sands industry.
Kim et al.[44] have developed statistical tools for high res-
olution FTICR mass spectra of crude oils based on Prin-
cipal Component and Correlation Analysis (PCA). Corre-
lational relationships between chemical composition in the
mass spectra and properties of crude oil were identified.
For example, sulphur-containing classes such as S1 , S2 , and
NS correlated well with sulphur content. The O2 and O4
classes of compounds, presumably with COOH functional
groups, had a strong correlation with the total acid number
(TAN). Although the research of Kim et al.[44] pertains to
crude oils, the question arises as whether the same approach
would work equally well for oil sands bitumen; namely pre-
dicting the environmental fate and properties of oil sands
 Chemical fingerprinting of naphthenic acids
bitumen from the molecular information obtained by the
use of FTICRMS or other ultrahigh resolution MS. Fur-
ther development of the approach to predict the environ-
mental effects of oil sands water soluble components and
their fate would address this knowledge gap and be invalu-
able for fingerprinting and monitoring programs.
On this note, recent application of high resolution FTI-
CRMS[4] and Orbitrap Fourier Transform Mass Spectrom-
etry (FTMS)[3] instrumental techniques are proving to be
valuable tools for fingerprinting environmental samples in
the Athabasca oil sands region. The FTMS technique has
the potential to differentiate sources of industrial oil sands
components and to distinguish such components from nat-
ural leaching of oil sands to aquatic environments. Salient
points on recent developments of FTMS fingerprinting of
Downloaded by [University of Saskatchewan Library] at 14:59 10 June 2013
environmental samples are discussed in the section “At-
mospheric Pressure Photoionization (APPI) and ESI FTI-
CRMS of oil sands acids in OSPW.”
PAHs and biomarkers for fingerprinting
of petroleum sources
Although the fingerprinting of petroleum hydrocarbons
is well developed for heavy crude oils,[41] there are few
published reports in the open literature for applications
to fingerprinting Athabasca oil sands in environmental
samples. Yang et al.[45] have recently described an inte-
grated quantitative chemical characterization of Alberta
oil sands bitumen and other related Alberta oils using
gas chromatography-flame ionization detection (GC/FID)
and gas chromatography-mass spectrometry (GC/MS).
In general, the fingerprinting techniques are based
on the characterization of target hydrocarbons, includ-
ing n-alkanes, unsubstituted polycyclic aromatic hydrocar-
bons (PAHs) and their alkylated homologues (APAHs),
biomarker terpanes and steranes, bicyclic sesquiterpanes,
and diamondoids. Yang et al.[46] have shown that the chem-
ical features of bitumen in oil sands are distinguishable from
most conventional crude oils. Yang et al.[46] reported that
n-alkanes are nearly absent in bitumen extracted from oil
sands. Oil sands bitumen is characterized by a chromato-
graphic hump of unresolved complex mixtures (UCMs)
eluting between n-C10 to n-C40 , and by almost identical
abundance of biomarker terpanes and cage-like adaman-
tanes, while the levels of biomarker steranes and bicyclic
sesquiterpanes were indicative of physical weathering or
biodegradation.[45]
A key attraction of these hydrocarbons is the relative
ease of analyses and the wide availability of the required
instrumentation in most laboratories. Furthermore, load-
ing of PAHs in the Athabasca oil sands region is a hot
topic of environmental concern as the distribution of levels
in various environmental compartments may be indicative
of industrial releases from the oil sands industry. A sum-
mary of the key findings of Kelly et al.,[11] Timoney and
1149
Lee,[47] and others in the context of chemical fingerprinting
is discussed in the following section.
Athabasca River Basin: levels of PAHs, metals
and NAs
Loadings of PAHs and metals to the Athabasca watershed
by oil sands mining and processing
Kelly et al.[11,21] concluded that the industrial development
of Athabasca oil sands releases greater levels of PAHs and
heavy metals to the environment than previously realized.
Kelly et al.[11] state, “In 2008, within 50 km of oil sands
upgrading facilities, the loading to the snowpack of air-
borne particulates was 11,400 T over 4 months and in-
cluded 391 kg of polycyclic aromatic compounds (PAC),
equivalent to 600 T of bitumen, while 168 kg of dissolved
PAC was also deposited.”
These authors also concluded that the elevated concen-
trations of dissolved PAC in the Athabasca River and its
tributaries were related to oil sands developments within
the past two years. Kirk, Muir and Wang, Environment
Canada (Personal communication) have recently confirmed
the findings of Kelly et al.[11,21] using multi-element data (45
elements + methyl-mercury) for 28 snow samples. Further-
more, the analysis of snowmelt samples for elements not
previously examined (Ba, Bi, Ce, Cs, Ga, Ge, In, La, Li,
Nb, Pd, Pt, Rb, Sc, Te, W, Y, and Zr) suggest that snowpack
deposition of many of these elements, especially in the par-
ticulate phase, is higher near oil sands development than at
background sites (Kirk, Muir and Wang, personal commu-
nication). These studies highlight the value of relatively sim-
ple measurements of multi-elements, PAHs, and alkylated
homologues for monitoring and fingerprinting purposes.
The value of PAH data for fingerprinting of oil sands
activity in the Athabasca region is further illustrated
by Timoney and Lee,[47] who reviewed the temporal
trends and levels in Athabasca River Delta sediment. The
historically deposited levels of PAHs in sediments were
reported to correlate with the annual bitumen production,
mined sand volume, extent of landscape disturbance, and
particulate emissions. However, Timoney and Lee [47]
concluded that carefully designed studies are required to
further elucidate which factors best explain the observed
variability in sediment PAH concentrations.
The levels of PAHs in snow packs or snow melt have
important potential for fingerprinting oil sands industrial
activity in the Athabasca region. The recent research of
Parrott et al.[48] also illustrates this point where they used
embryo-larval fathead minnows in a preliminary assess-
ment of the toxicity of snowmelt samples. Samples were
collected in the vicinity of oil sands process facilities along
the Athabasca River and upstream of the development
area. Samples were collected in early March 2011, prior to
the spring melt and then shipped frozen to the laboratory.
Snow samples from upstream and far downstream of the oil
 1150
sands were not toxic to larval fathead minnows while sam-
ples from the near vicinity of oil sands mining and refining
areas were toxic. Whereas the snow melt waters had negli-
gible concentrations of naphthenic acids, several contained
elevated levels of PAHs that likely include particulates re-
suspended from the open pit mines and airborne refinery
emissions.[48]
Metals also have potential for fingerprinting. The levels
of trace metals in bitumen can range from less than one
part per billion (ppb) to more than several thousand parts
per million (ppm). The most abundant metals are nickel
and vanadium with values ranging up to 200 ppm Ni and
2000 ppm V. Abundances of other trace metals are gener-
ally less than 100 ppm. As reported by Kelly et al.[21] the
oil sands industry releases certain metals via air and water,
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to the Athabasca River and its watershed. “Canada’s or
Alberta’s guidelines for the protection of aquatic life were
exceeded for seven priority pollutants (Cd, Cu, Pb, Hg,
Ni, Ag, Zn) in melted snow and/or water collected near or
downstream of development.” As a result, the development
of fingerprinting approaches should include measurement
of the levels of metals in environmental samples, further
discussion of which is expanded upon in Section “Induc-
tively Couple Plasma (ICP)/MS of heavy metals in OSPW
and oil sands environmental samples.”
Overview and current state of the art – Do we know what we
are measuring for OSPW acid extractable organics?
Grewer et al.[1] and Headley et al.[2–4,30] have recently drawn
attention to the need for establishing a clear definition of
what is being measured in OSPW acid extractable organ-
ics. The use of the term naphthenic acids can be mislead-
ing because the NA fraction contains much more than
classical NAs. For example, the acid extractable fraction
of OSPW contains between 1 and 7% sulphur.[1] Grewer
et al.[1] showed that the mass spectra contained between
300 and 1880 peaks, with > 99% of the peaks having m/z
between 145 and 600. In most cases, < 20% of the peaks
were assigned as classical NAs and oxy-NAs. The classical
NAs from the OSPW were predominantly Z = −4 and −6
(Z refers to the hydrogen deficiency incurred by ring for-
mation), whereas those from natural waters were mainly Z
= 0, with palmitic and stearic acids being the major com-
ponents.
What is measured in the OSPW fraction is discussed be-
low in detail with reference to the works of Barrow et al.,[12]
Rowland et al.[25,28,49] and others[2,3] who used either (i) ad-
vances in FTMS for speciation of components according to
classes of compounds or (ii) GCxGC/MS for identification
and confirmation of specific compounds, many of which are
not classical NAs. Barrow et al.[12] demonstrated that the
combination of ionization techniques employing electro-
spray ionization (ESI) and atmospheric pressure photoion-
ization (APPI) provide complementary information on the
types of classes present in OSPW.
Headley et al.
Headley et al.[2,3,30,36,50] further demonstrated that the
range of compounds measured depends on co-factors such
as salinity, pH, and solvent effects observed by electro-
spray ionization methods. Consequently, the measured
components are not entirely attributable to the components
present in the original acid extractable fraction of OSPW,
but rather, additional components originating from many
field and laboratory procedural variables. Therefore, anal-
yses of NA fraction compounds must be mindful of such
variables to avoid misleading or possibly erroneous conclu-
sions.
Toxicity – Interactions of salts and OSPW NAs in aquatic
environments
The interactions of salts with oil sands NAFC are not well
understood for field environments. Furthermore, the impli-
cations of such interactions for analyses of NAFCs are just
now emerging. Leung et al.[51] demonstrated that despite the
simultaneous and uncontrolled variation of environmen-
tal factors in the field, a significant change in taxonomic
composition of 40% was linked to the levels of naphthen-
ate and major ion concentrations (as indexed by conduc-
tivity). Kavanagh et al.[52] suggested that aquatic habitats
with high NA concentrations (>25 mg/L) and conductiv-
ities (>2000 µS/cm) are not conducive for successful fish
reproduction. Jack pine growth is affected by saline tailings
water.[53] Likewise, it was reported that simulated runoff or
seepage waters from chemically amended and dried ma-
ture fine tailings were phytotoxic, due to combined effects
caused by salts, NAFC and pH.[40]
OSPW is known to have elevated levels of chloride ions
above the background in the Athabasca River. Headley
et al.[30] demonstrated that the solubility of oil sands or-
ganic acids can be affected by the salt content of the water,
which in turn affects the concentration of total NAs as well
as the distribution of organics in aqueous environments.
Salt interactions were shown to affect the distribution
of polar organics observed by negative-ion FTICRMS
analysis of environmental samples (OSPW, river water and
groundwater). Acidic oxygen containing species (Ox in
which x = 2–10, were prevalent in most samples, the rela-
tive distribution of which depended on the sample location
and chloride levels of the water. Likewise, the distribution
of heteroatomic sulphur species (Ox Ss species in which x =
2–8 and s = 1–2) also depended on the sample location and
chloride ion concentration of the waters investigated.[30]
Analyses of oil sands polar organics and NAs in envi-
ronmental samples should therefore be conducted concur-
rently with measurement of chloride ions and other salts.[30]
Furthermore, as suggested by Nero et al.[54] there is poten-
tial to use the interactive effects of ionic content, NAs, and
PAHs on measured histopathological responses as strong
indicators of exposure to OSPW.
 Chemical fingerprinting of naphthenic acids 1151
Analytical methods of oil sands NAs example, there was evidence for selective removal of lower
molecular weight NAs with carbon numbers less than or
GC/MS, with ion-trap or linear quadropole detection, equal to 21, and this corresponded with lower toxicity in
fingerprinting of oil sands components the waters investigated. Due to the sheer complexity of the
mixture, it is now understood that the GC/MS method of
Holowenko et al.[55] and Scott et al.[56] demonstrated the
Holowenko et al.[55] is prone to misclassification of NAs
utility of gas chromatography-electron impact mass spec-
in OSPW, by carbon number and Z-series (see following
trometry for the characterization of NAs in water samples
section “High Performance Liquid Chromatography/Time-
derived from oil sands extraction processes. The GC/MS
of-Flight – Mass Spectrometry (HPLC/TOF-MS) of naph-
analysis required a derivatization step using N-methyl-N-
thenic acids”), but these early analytical advancements
(tert-butyldimethylsilyl)trifluoroacetamide. However, this
enforced the need to better understand how the structure
was a significant advancement compared to the Fourier
of NAs could influence environmental fate and toxicity.
transform infra-red (FTIR) method, as the analysis was
The key advantage of the GC/MS approach is the ease of
more sensitive, and the m/z information could potentially
use and wide availability in most laboratories. The method
be used to profile the NAs by the relative abundance of
specificity may be adequate for routine analyses of the total
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each carbon number and Z series (0 to −12).

concentration of NAs in natural waters, or for trend analy-
Nonetheless, detection was based on low resolution MS
ses and general monitoring purposes. For example, despite
and thus subject to possible interferences from components
the known limitations of the method, Humphries[57] have
with the same nominal mass as classical NAs under inves-
applied the early GC/MS method reported by Holowenko
tigation. Based on GC-MS profiles of NAs, Holowenko
et al.[55] for preliminary fingerprinting analyses of oil sands
et al.[55] provided the first insight on the degradation and
components in environmental samples (Fig. 2).
toxicity of oil sands NA for fingerprinting analyses. For

3 3

Full Data Set Athabasca River

2 2

1 1

0 0
-3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3

-1 -1

-2 -2

-3 -3

-4 -4

3 3
Muskeg Creek Small Creeks
2 2

1 1

0 0
-3 -2 -1 0 1 2 3 -3 -2 -1 0 1 2 3
-1 -1

-2 -2

-3 -3

-4 -4

Fig. 2. PCA of GC/MS “fingerprints” of environmental samples collected in the Athabasca oil sands region (color figure available
online).
 1152
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Fig. 3. Comparison of GC/MS and HPLC/QTOF-MS ESI results for OSPW showing misidentification of Z = 0 revealed by
high resolution TOF-MS. (Reprinted [adapted] with permission from[58]. Copyright 2006 American Chemical Society) (color figure
available online).
The PCA of a preliminary investigation[57] of environ-
mental samples suggests that the GC/MS approach, de-
spite its limitations to specificity, may be helpful for source
determination of NAFC. However, complementary meth-
ods are recommended for more detailed confirmation and
isolation of the individual components at the molecular
level. For definitive source fingerprinting, there may be a
need for follow-up studies with more advanced techniques,
employing further separations (e.g., LCxLC, GCxGC, ion-
mobility and high resolution mass spectrometry). Salient
points of the latter for fingerprinting analyses of oil sands
NAFC are discussed in the following sections.
Martin et al.[35] described the advantages of HPLC/
TOF-MS in comparison to direct infusion ESI low res-
olution MS for the analysis of NAs in pure commercial
mixtures and environmental samples. The low resolution
MS method resulted in false positives and misclassification
of Z = 0 NAs in oil sands NAFC; whereas the interferences
were not observed in the HPLC/TOF-MS method (Fig. 3).
The latter was also less prone to matrix effects as salts were
separated on-line by the use of HPLC.
A three-fold lower response factor for total OSPW NAs
by HPLC/TOF-MS was largely attributable to contribu-
tions in low resolution MS from other organic compounds
in the OSPW NAFC, including hydroxylated NAs, O4
containing species, S- and N-containing heteroatomics,
along with other components.[12,17] The need for the de-
velopment of target analyses using authentic standards
is evident as quantification of NAFC expressed as total
NAs is currently at best, semi-quantitative. The individ-
ual authentic standards prepared by Rowland et al.[25,28,49]
should help expedite the development of better quanti-
tative methods for specific NAFC. In the meantime, the
semi-quantitative methods have substantial value, partic-
ularly where the environmental fate or relative concentra-
Headley et al.
tion changes are the desired endpoints rather than absolute
concentrations.[58]
Further development of HPLC/TOF-MS was reported
by Noestheden [59] in which improved chromatographic sep-
aration of OSPW NAFC was observed. Noestheden [59]
reported a 30–80% reduction in ion suppression by opti-
mizing sample dilution, injection volume and chromato-
graphic separation of oil sands NAFC. Chromatography
in particular gives an extra degree of resolving power by re-
ducing analyte co-elution. The combined chromatographic
and high resolution separation of components has high po-
tential for fingerprinting analyses of oil sands components
in environmental samples. The latter is part of an ongoing
collaborative initiative with Environment Canada.
GCxGC/MS of OSPW
There has been a rebirth in the development and application
of comprehensive GC×GC/MS for the characterization of
OSPW and NA in petroleum samples.[25,28,49] The technique
is particularly well suited for the identification of individ-
ual components with confirmation by use of authentic stan-
dards (Figs. 4 and 5). Rowland et al.[25,28,49] have synthesized
newly identified diamondoid and other individual compo-
nents in OSPW based on GC x GC/MS characterization of
OSPW. Specifically, identification of individual tricyclic oil
sands NAs in OSPW was performed by use of comprehen-
sive two-dimensional gas chromatography/time-of-flight
mass spectrometry (GC × GC/TOF-MS) of the methyl
esters. The acids were diamondoid adamantane acids, re-
sulting from a proposed biotransformation of the corre-
sponding alkyladamantane hydrocarbons.
The GC × GC/MS approach has great potential for
fingerprinting sources and following the fate of specific
 Chemical fingerprinting of naphthenic acids 1153
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Fig. 4. GC × GC/TOF-MS total ion current chromatogram of oil sands NAs extracted from OSPW as methyl esters, illustrating
high chromatographic separation compared to GC/MS (white line on black background). (Reprinted [adapted] with permission from
ref.[25] Copyright 2012 Wiley-Blackwell) (color figure available online).

C O2H
C O2H
C11

C O2H
C O2H C O2H C O2H

C12

C O2H
C O2H C O2H

C13

C O2H
C O2H

C14

Fig. 5. Structures of recently identified tricyclic C11-14 NAs (Z = −6) in OSPW reported by Rowland et al.[25,28,49] All structures were
confirmed by use of authentic standards (many more isomers were present).
 1154
components in aquatic and other environments. To date,
the main advantage of the GC×GC/MS approach is the
identification and confirmation of specific components as
opposed to simply characterizing broad classes of compo-
nents in OSPW. Such selectivity should be well suited for
quantification of target components in the environment.
Development of this aspect is encouraged for environmen-
tal samples in the Athabasca oil sands region. A limitation
of the method is that the MS applied to date have not been
of ultra high resolution.
Despite the significant chromatographic separation at-
tained, some components with similar nominal mass may
not be fully resolved. Future developments of the approach
may therefore entail interfacing the GC×GC to FTMS
and/or tandem approaches employing ion-mobility for
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structural separation of isomers. The importance of struc-
tural identification of isomers is discussed further below
(Section “LC × LC/MS of OSPW”). The future develop-
ments will make the already powerful technique even more
directly applicable for study of the toxicity of OSPW com-
ponents.
LC × LC/MS of OSPW
Martin et al.[60] presented preliminary separations using a
comprehensive LC × LC system with TOF-MS, or infor-
mation dependent acquisition (IDA) MS/MS, to minimize
interferences and to investigate the possible separation of
isomeric species. This approach may be ideally suited for
studies of the persistence and natural fate of the toxic con-
stituents of OSPW in the environment. Martin et al.[60] have
previously demonstrated that oil sands NA constituents are
recalcitrant due to their cyclic and alkyl branched struc-
tures.[58,61] The LC × LC/MS method may also prove to
be a powerful technique for fingerprinting the source and
fate of OSPW constituents in the environment. Future de-
velopments should explore the coupling of ion-mobility
separations with LC × LC/MS to further enhance the
identification of components according to their molecular
structure.
Molecular structure is a key factor controlling the toxi-
city of OSPW components; therefore, the development of
such methods is encouraged. For example, Frank et al.[62]
demonstrated in an investigation of NAs of equal molecu-
lar weight, that the United States Environmental Protection
Agency (USEPA) ECOSAR quantitative structure activity
relationship (QSAR) model predicted an increased toxic
potency for NAs containing longer alkyl chains and fewer
carbon rings. Frank et al.[62] also showed that NA struc-
tures with a linear grouping of carbon rings had a greater
predicted toxic potency than structures containing carbon
rings in a clustered grouping. Likewise, Frank et al.[62] used
a series of model NA surrogate compounds to show the
importance of structure and number of carboxylic groups
on the measured toxicity. The addition of a second car-
boxylic acid moiety resulted in a significant toxicity reduc-
tion (Daphnia magna LC50 values of 10 ± 1.3 mM and
Headley et al.
27 ± 2.2 mM for monocarboxyl and dicarboxyl NA-like
surrogates of similar MW, respectively).
LC/MS/MS of naphthenic acids in aqueous samples
LC/MS/MS has been used with success for the characteri-
zation of crude oil for about a decade.[24,63–65] For example,
Rudzinski et al.[65] employed tandem MS along with 13C
NMR for study of commercial standards of petroleum-
derived NAs. MS/MS was used to determine the acid-
ity, ring type, and carbon number distribution. The early
MS/MS experiments confirmed that a batch of Fluka NA
mixtures consisted primarily of carboxylic acids with dom-
inant hydrogen deficiency values of −6 and 0, relative to an
alkanoic acid reference standard.
In contrast to the early applications of LC/MS/MS for
the analysis of NAs originating from crude oil, approaches
for analyses of oil sands NAFC in environmental sam-
ples have received little attention. This lack of develop-
ment is due in part to the difficulty encountered for tandem
MS experiments with some instrumentation. The extent
of product ions observed for the parent NA negative-ions
is strongly dependent on the type of instrumentation em-
ployed. Up till now, MS/MS of NA negative-ions appears
to work best for high energy collision induced dissociation
(CID) of the carboxylate anion in ion-traps. Negligibly low
abundant product-ions are observed under the CID condi-
tions employed for most commonly available quadrupole
and sector instruments.
Recently, Wang and Kasperski[63] utilized an HPLC-
MS/MS method for chromatographic separation and tan-
dem MS detection of model NAs. The advantage of this
method is that inorganic salts are separated on-line from
the acids without sample extraction or pre-treatment. Dif-
ferent NAs give characteristic retention times due to their
structures, thus enabling measurements of the individual
concentrations. Wang and Kasperski[63] suggested that the
tandem MS procedure could be employed to determine
the fate and stability of individual NAs in the environ-
ment and thus provide insight to potential environmental
impacts of oil sands processing. However, as the appli-
cation of the method to complex mixtures of NAFC in
environmental samples has yet to be established, further
development of the procedure for fingerprinting purposes
is encouraged.
On this note, Woudneh et al.[64] have reported a
HPLC/MS/MS method for quantification of NAs in sur-
face waters. This approach is complementary to the method
used by Wang and Kasperski [63] and utilizes positive-
ion ESI instead of negative-ion ESI. Woudneh et al.[64]
utilized 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide
(EDC) derivatives which under positive-ion detection give
both strong parent and product ions of the derivatized
NAs. Quantification is based on the tandem MS transi-
tion [parent] > [parent-129] over a linear range of 0.25
and 250 ng/mL for model carboxylic acids. Woudneh
et al.[64] have applied the tandem MS procedure with use of
 Chemical fingerprinting of naphthenic acids
deuterium labeled internal standards for quantification of
NAs (as pyrenebutyric acid equivalent concentrations) in
northern Alberta river samples. Hence, there is potential for
the procedure to be used for analyses of the levels of NAs
in environmental samples. A full assessment of the tech-
niques for delineating oil sand sources will likely hinge on a
detailed comparison with other methods, probably includ-
ing FTMS, GC × GC/MS, and LC × LC/MS. Therefore,
studies of this type are encouraged for future investigations.
Atmospheric Pressure Photoionization (APPI) and ESI
FTICRMS of oil sands acids in OSPW
Barrow et al.[12] compared the characterization of OSPW by
Downloaded by [University of Saskatchewan Library] at 14:59 10 June 2013
use of ESI and APPI. The FTICR mass spectrometer was
operated in both the positive-ion and negative-ion modes,
with detection of ions in the range of m/z 147.4–3000.
Sodium adduct ions were observed in the positive-ion mode
using ESI. Although positive-ion mode ESI was well-suited
for the observation of Ox , Ox S, Ox S2 and NOx classes,
negative-ion mode ESI mass spectra were dominated by Ox
classes (and, to a lesser degree, Ox S), making it particularly
useful for the study of NAs (Fig. 6). Naphthene-based [66]
species were more amenable to analysis by APPI than ESI,
and the positive-ion mode APPI mass spectra were notable
for the greater intensity of the NOx and N classes. A key ad-
vantage of APPI is the detection of aromatic hydrocarbons
which do not contain a heteroatom. Because the latter are
not detected using ESI, application of APPI may be better
suited for comparison with synchronous fluorescence spec-
troscopy (SFS) data for fingerprinting analyses of aromatic
acids in aquatic environments.[12]
FTICRMS negative-ion ESI analyses revealed that oil
sands NA-fortified samples treated with UV and microwave
radiation have disparate relative abundances of classes.[33]
Species with relatively high Ox content, namely, the O3 , O5 ,
and O6 classes, were present only in UV- and microwave-
treated samples. The relatively high Ox content is consis-
tent with oxidation of the parent acids in treated samples.
Headley et al.[33] postulated that these differences in abun-
dances of classes have potential implications for environ-
mental forensics. For example, the monitoring of the ratio
of species containing SO2 :O2 , or tracking the relative abun-
dances of species containing O2 , O3 , O4 , O5 , and O6 classes,
may provide insights for distinguishing naturally derived oil
sands components from those that are of industrial origin
in aquatic environments.
Follow-up studies were reported for preliminary finger-
printing analyses of Athabasca oil sands polar organics in
environmental samples using FTICRMS.[4] Headley et al.[4]
evaluated the potential of negative ion ESI high-resolution
FTICRMS for comparing oil sands polar organics from
OSPW, interceptor well systems, groundwater, as well as
river and lake surface waters (Fig. 7).
1155
PCA was performed for a wide range of species, in-
cluding: (i) O2 class (often assumed to be monocarboxylic
naphthenic acids), (ii) humic substances in the river and
lake samples: Ox where x = 1–16; NOx and N2 Ox where
x = 1–13; and (iii) Ox S and Ox S2 surfactant-like species
where x = 1–10 and 1–8, respectively. The distributions of
the oil sands NAFC, particularly the sulphur-containing
species, Ox S and Ox S2 , were demonstrated to have poten-
tial for distinguishing sources of OSPW. Likewise, the ratios
of species containing Ox along with nitrogen-containing
species (i.e., NOx , and N2 Ox ), were useful for differentiat-
ing organic components derived from OSPW from those
found in natural waters following weathering of oil sands
outcrops. Headley et al.[4] concluded that further applica-
tion of the FTICRMS technique for fingerprinting should
be extended to a diverse range of OSPW of various ages
and composition, as well as the surrounding groundwater
wells.
Orbitrap MS of oil sand organic acids in plant tissue
Headley et al.[3] described a new procedure employing high
resolution Orbitrap MS for direct analysis of oil sands NAs
in plant tissue. Previous to this development, methods for
the uptake of NAFC in plant tissue were based on in-
direct analyses of water samples, before and after expo-
sure to plant systems. The direct Orbitrap MS analyses
is considered to be a first step toward the full quantifica-
tion of oil sands acids by demonstrating the identification
of NAFC in Alder species tissue.[3] The advantage of the
Orbitrap MS method for the analyses of plant tissue com-
pared with low resolution MS approaches is the accurate
determination (<2 ppm error) of diagnostic ions (Fig. 8).
Such types of ions were present in spiked plant tissue but
absent in the blank plant matrix. For example, diagnostic
ions at m/z 195.139, 247.170, and 277.181 correspond to
the deprotonated ion with molecular formulae C12 H19 O2 ,
C16 H23 O2 , and C17 H25 O3 , respectively. Whereas the first
two ions correspond to classic NAs, with C12 H19 O2 indicat-
ing a Z-number of –4 and C16 H23 O2 indicating a Z-number
of –8, the third diagnostic ion contains three oxygen atoms
and is likely a hydroxyl substituted carboxylic acid.[3]
Quantitative analyses of complex oil sands mixtures in
biota are best based on congener-specific analysis. The re-
cent availability of synthesized individual compounds can
now serve as authentic standards for selected components
in OSPW.[25,49] For a full quantitative method, reproducible
recoveries should be demonstrated using authentic stan-
dards of specific components of oil sands components ex-
tracted from the plant matrix. The same is true for other
biota, including fish tissue, where background interferences
can mask the detection of oil sands NAFC. However, de-
spite the high background interferences, Young et al.[67]
utilized a careful choice of target ions and GC/MS with
low resolution MS to measure the distribution of total
 1156 Headley et al.
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Fig. 6. Comparison of plots of compound classes observed by use of FTICRMS ESI and APPI in both positive-ion and negative-ion
mode for Athabasca OSPW. (Reprinted [adapted] with permission from ref.[12] Copyright 2010 American Chemical Society) (color
figure available online).

naphthenic acids in tissues of laboratory-exposed fish and
in wild fishes from near the Athabasca oil sands in Alberta,
Canada.
Reconstructed ion chromatograms (m/z = 267) selec-
tively detected NAs in fish that were exposed to naphthenic
acids.[68] However, for analyses of NAFC in biota, it is rec-
ommended that steps be taken to resolve interferences from
components of interest either (i) chromatographically, by
use of (ii) high resolution MS, or by (iii) methods employing
a combination of both separation methods ((i) and (ii)).
Emerging technologies for analyses of oil sands
components in aquatic environments
SFS of OSPW and environmental samples
Kavanagh et al.[31] demonstrated the use of SFS for the
detection of OSPW components in water systems (Fig. 9).
The potential for screening and analyses of environmental
samples was evident from the early studies of Kavanagh
et al.[31] in which water samples were characterized from
 Chemical fingerprinting of naphthenic acids
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Fig. 7. PCA of FTICRMS ESI negative-ion results of compound classes observed for environmental samples from the Athabasca oil
sands region. (Reprinted [adapted] with permission from ref.[4] Copyright 2011 Wiley-Blackwell) (color figure available online).
ponds containing OSPW and selected sites in the Alberta
oil sands region.
OSPW ponds consistently displayed a minor peak at
282.5 nm and a broad major peak ranging between 320
and 340 nm (Fig. 9). In contrast, water from various refer-
ence sites within the oil sands region had little fluorescence
at 282.5 nm but greater fluorescence beyond 345 nm. In-
terestingly, the SFS spectra for a commercial NA mixture
and a NA extract prepared from OSPW had similar spec-
tral features.[31] However, these different NA mixtures are
known to have significantly different aquatic toxicity and
chemical composition.[16,17]
Clearly, a greater understanding of what components
are responsible for the SFS peaks is needed for further
development of the technique as a fingerprinting method.
The presence of aromatic acids closely associated with the
naphthenic acids may account for unique fluorescence at
320–340 nm.[28,32] The primary advantage of the SFS ap-
proach will likely be its application as a rapid and in-
expensive pre-screening technique for other approaches
employing, for example, GC × GC/MS or FTMS meth-
ods to obtain detailed fingerprint signatures of the oil sands
1157
NAFC. As such, SFS could serve as an important tool for
initial screening of the release of OSPW into ground and
surface waters in the oil sands region.[28]
Ion-mobility MS structural analysis of OSPW
Gabryelski and Froese[69] demonstrated the use of ESI
high-field asymmetric waveform ion mobility spectrome-
try (FAIMS) combined with quadrupole, TOF-MS/MS
for the characterization of isomer distributions in commer-
cial and naturally occurring NA mixtures. ESI-FAIMS-
MS is especially useful for separation of isomers with the
same exact mass that cannot be resolved readily by other
methods. Recently, there have been considerable advances
in ion-mobility instrumentation that facilitate further de-
velopment of the early work of Gabryelski and Froese.[69]
Commercially available systems such as the Synapt G2 offer
complementary approaches to FAIMS.
Russell and Becker[70] demonstrated the utility of
ion-mobility spectrometry (IMS) for applications in
petroleomics. Separations were based on the shape and
size of ions for resolving crude oil sample components at
 1158 Headley et al.
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Fig. 8. Orbitrap MS profiles of spiked plant tissue. (A) denotes Athabasca oil sands NAFC; (B) denotes Alder stems spiked with oil
sands NAFC; and (C) denotes blank Alder stems. (Reprinted [adapted] with permission from ref.[3] Copyright 2011 Wiley-Blackwell).

nominal m/z conditions. Furthermore, Russell and Isotope ratio MS

Becker [70] applied ion-mobility to differentiate monomers
There are few reports of isotope ratio MS for the study of
from aggregate asphaltene species in heavy oil. Previ-
oil sands NAFC in environmental samples.[14,15,72–74] The
ous analysis of asphaltenes demonstrated that monomer
studies range from research of specific isotopes of model
sample components span approximately 2000 mass units
compounds [72] microbial degradation of oil sands NAs
with a distribution maximum at ∼800 m/z.[41,71] Sample
using stable isotope tracers [73] to investigations of trends
components above m/z 2500 were identified as aggregate
in stable isotopic signatures observed for experimental oil
species.[41,71]
sands reclamation ponds [14] and rivers in the Athabasca oil
To date, detailed ion-mobility MS studies have not been
sands region.[20] Although studies of this type are useful for
reported for fingerprinting of OSPW or oil sands NAFC.
gaining insight on the fundamentals of compound-specific
Development of such applications are encouraged for fin-
degradation, general applicability to the complex mixtures
gerprinting oil sands components in environmental sam-
in OSPW is not known. Other studies have examined stable
ples as structural analysis will be critical to understanding
isotopes of bacteria grown in oil sands NA media to better
and predicting the toxicity of specific isomers. The unique
understand the potential application of isotopic tracers in
feature of ion-mobility to separate ions of the same exact
the environment.[73]
mass but different structures has great potential for struc-
Farwell et al.[14] reported the successful use of isotope
ture elucidation of specific isomers in oil sands NAFC.
ratio MS for study of a series of experimental reclama-
This potential will be further enhanced for future appli-
tion ponds of similar age and size. Trends of 13C depletion
cations that combine the power of ion-mobility with hy-
and 15N enrichment were observed for benthic invertebrates
phenated techniques such as tandem MS, LC × LC, or
along a gradient of increased levels of industrially derived
GC × GC.
 Chemical fingerprinting of naphthenic acids
SFS Spectra of River Water
600
282 320 333
500
400
Emission Intensity
300
200
100
0 were reacted with iron powder to convert the active acids
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260 280 300 320 340
Excitation wavelength (nm), λ em= λ ex + 18 nm
Fig. 9. Synchronous fluorescence spectra of river samples col-
lected in the Athabasca oil sands region. NAE is an oil sands
NAFC extracted from OSPW (color figure available online).
oil sands components. The latter was based on levels of
mature fine tailings and consolidated tailings in a survey
of 16 sites. Farwell et al.[14] suggested that the extraction
process by-product, NH+
4 , has high potential for use in sta-
ble nitrogen isotope fingerprinting to define exposure of
biota to OSPW affected natural waters in the Athabasca
oil sands region. Carbon and nitrogen isotope signatures
of fish species provided insight on site fidelity and base-
line data for sites along the Athabasca River and its tribu-
taries.[15] Therefore, the further development of 15N isotope
MS fingerprinting of environmental samples is encouraged.
There is a need to develop other types of isotope ra-
tio mass spectrometry for fingerprinting oil sands sources.
Ahad et al.[74] described the potential of the technique to
monitor biodegradation and possibly discriminate sources
of organic contaminants in the Athabasca oil sands re-
gion. The future development of the fingerprinting analy-
ses would rely on the inherent age differences between 14C-
depleted organic compounds derived from oil sands and
more modern 14C-enriched organic matter. The assump-
tion holds that diagnostic differences will occur for a given
sample site based on differences in isotope geochemistry.
Research is ongoing in collaboration with Environment
Canada to assess the utility of compound-specific stable
carbon (δ 13C) and hydrogen (δ 2H) isotopes to differentiate
oil sands sources of PAHs and possibly NAFC. The tech-
nique will likely improve the understanding of the natural
variability in PAH inputs across recent geological record
and provide valuable insights for remediation strategies.
Furthermore, if for example, specific oil sands PAHs or
NAFC are derived from several different sources for a given
site, it is anticipated that the isotope ratio MS fingerprints
will provide great potential for environmental forensics.[74]
1159
Inductively Couple Plasma (ICP)/MS of heavy metals
in OSPW and oil sands environmental samples
NAE
Athabasca R. As discussed in the “Oil Sands and Oil Sands Process-
Steepbank R.
Ells R.
Affected Water – What is the Composition?” section, bi-
Firebag R. tumen is known to contain heavy metals including several
that have been detected in environmental samples exceed-
ing provincial Alberta and Canadian Federal guidelines.[21]
ICP/MS is the method of choice for the broad spectrum
n=3
analyses of trace metals, for which applications are well
developed for the characterization of crude oil. Rakoton-
n=3
n=3
dradany et al.[75] described an interesting application of
n=5 ICP/MS for the characterization of iron naphthenates in
heavy oils. Labile NAs in bitumen-derived heavy gas oils
360 380 400
into the corresponding iron naphthenates, leaving the less
reactive species as free acids in solution. The reacted mix-
ture of iron powder, iron naphthenate, and free NA was
subsequently separated by filtration. An ion-exchange resin
was used to remove the free NAs and the naphthenates were
recovered by use of toluene as an extraction solvent.
Although the speciation of the NA described by Rako-
tondradany et al.[75] was geared to understanding the con-
tribution of the reactive NAs for process related reasons, the
question arises whether similar strategies to chemically, or
microbially, isolate labile and refractory NAFC can provide
insights for environmental endpoints. The fact that NAs
are present as their naphthenate metal salts under the weak
alkaline conditions found in environmental samples tend
to be overlooked for fingerprinting potential as well as by
studies of their aquatic toxicity. As noted by Oiffer et al.[76]
despite subsurface residence times exceeding 20 years, there
is minimal mobilization of trace metals in groundwater.
Application of XANES for speciation of sulphur species
in OSPW
Ryan et al.[77] reported the first application of X-ray
absorption near-edge spectroscopy (XANES) for the mea-
surement of oil sands NAFC. The XANES technique was
sufficiently sensitive for speciation of sulphur in oil sands
NAFC for which total NA concentration, as measured by
low resolution ESI/MS, was 65 mg/L and total sulphur
concentration was 5.3 mg/L, as measured by ICP/MS.
The speciation of sulphur at such levels is considered low
enough to challenge the detection capabilities of the beam
lines. Early results are thus promising for the identification
and quantification of sulphur components at environmen-
tally relevant concentrations. The occurrence of sulphur
species may serve as potential markers for delineating
anthropogenic sources of oil sands components from
those that are naturally occurring. For example, different
chemical states of sulphur were found for OSPW NAFC
in which there are predominant contributions of sul-
phates, and additional contributions from organosulphur
 1160
compounds similar to thioethers, benzothiophenes, and
sulphoxides.[78]
Ryan et al.[77] also described an application of XANES
for the speciation of boron species in a relatively small num-
ber of environmental samples. Preliminary results indicate
that at least two classes of boron are present in environ-
mental samples in the Athabasca region. The predominant
form found in all samples is an electron-deficient boron
species such as borates and borosilicates. The second form
of boron species was only observed in samples containing
oil sands NAFC collected from locations with a known
history of industrial activity. The latter boron species are
believed to be impurities or additives from the industrial
processing and include boron carbide and boron nitride
features.
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Further development and application of XANES for fin-
gerprinting oil sands components in environmental sam-
ples is currently under evaluation as a collaborative effort
between the Ryan group at Level Science Inc. and Environ-
ment Canada. The technique is anticipated to be comple-
mentary to other fingerprinting methods as it is amenable to
the simultaneous detection of metal species along with both
organic and inorganic sulphur-containing components in
oil sands NAFC.
Conclusions and recommendations
Recently, there have been major advancements in the de-
velopment of chemical analyses of oil sands components
in environmental samples, employing a diverse range of
instrumental techniques. Each technique has specific ad-
vantages and limitations as outlined in this review. Thus,
there is no single universal technique that meets all require-
ments and provides the entire synopsis of highly complex
oil sands NAFC mixtures; the number and complexity of
components in oil sands bitumen and OSPW is simply
too vast for a single technique. Rather, a battery of such
methods provides complementary characterization. Meth-
ods adopted for environmental forensics applications will
depend largely on the availability of instrumentation, lab-
oratory expertise, and intended end-use of the results.
In view of the many knowledge gaps identified in the re-
spective sections above (for brevity, a listing of the knowl-
edge gaps are not reiterated here) there is an urgent need
to form a working group to formulate and agree upon pro-
tocols for standardized analyses of environmental samples
containing oil sands materials. The working group should
be comprised of technical practitioners from all stakehold-
ers (government, academia, and industry) who are actively
working on chemical analyses of oil sands mixture compo-
nents. At a minimum, inter-laboratory studies are necessary
to allow for comparison and validation of results from the
various approaches.
Headley et al.
As more laboratories perform analyses of OSPW and
environmental samples, there is a pressing need for read-
ily available authentic standards of oil sands NAFC and
individual NA compounds. Potential suppliers, such as
Environment Canada’s Information Quality Management
group or commercial companies, are encouraged to provide
such certified materials.
Although the high potential for bio-monitoring ap-
proaches was only mentioned in passing, attempts should
be made to further integrate chemical and biological (ge-
nomic) fingerprinting methods. For example, significant
insight can be gained by coupling chemical fingerprint-
ing efforts with a battery of aquatic bioassays, including
assessments of toxicity to invertebrates, mussels, fish, and
wetland plants.
Acknowledgments
The participants who contributed formal presentations at
the Naphthenic Acids Strategies Workshop, November 24,
25, 2011, are listed as co-authors of this communication.
Other participants are thanked for their helpful discussions.
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