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Forensic Fingerprinting of Biomarkers for Oil Spill
Characterization and Source Identification
Zhendi Wang
a
, Scott A. Stout
b
& Merv Fingas
a
a
Emergencies Science and Technology Division, Environmental Technology Centre,
Environment Canada , Ottawa, Ontario, Canada
b
NewFields , Rockland, MA
Published online: 23 Feb 2007.
To cite this article: Zhendi Wang , Scott A. Stout & Merv Fingas (2006) Forensic Fingerprinting of Biomarkers for Oil Spill
Characterization and Source Identification, Environmental Forensics, 7:2, 105-146, DOI: 10.1080/15275920600667104
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Environmental Forensics, 7:105146, 2006
Copyright
C
Taylor & Francis Group, LLC
ISSN: 15275922 print / 15275930 online
DOI: 10.1080/15275920600667104
Forensic Fingerprinting of Biomarkers for Oil Spill
Characterization and Source Identication
Zhendi Wang,
1
Scott A. Stout,
2
and Merv Fingas
1
1
Emergencies Science and Technology Division, Environmental Technology Centre, Environment Canada,
Ottawa, Ontario, Canada
2
NewFields, Rockland, MA
Biomarkers are one of the most important hydrocarbon groups in petroleum. Biomarkers can be detected in low quantities (ppm and
sub-ppm level) in the presence of a wide variety of other types of petroleum hydrocarbons by the use of the gas chromatography/mass
spectrometry (GC/MS). Relative to other hydrocarbon groups in oil such as alkanes and most aromatic compounds, biomarkers
are more degradation-resistant in the environment. Furthermore, biomarkers formed under different geological conditions and ages
may exhibit different biomarker ngerprints. Therefore, chemical analysis of biomarkers generates information of great importance
to environmental forensic investigations in terms of determining the source of spilled oil, differentiating and correlating oils, and
monitoring the degradation process and weathering state of oils under a wide variety of conditions. This article briey reviews biomarker
chemistry, biomarker characterizationandquantication, biomarker distributions, weatheringeffects onbiomarker composition, bicyclic
biomarker sesquiterpanes anddiamondoids, diagnostic ratios andcross-plots of biomarkers, unique biomarkers, applicationof biomarker
ngerprinting techniques for spill source identication, and application of multivariate statistical analysis for biomarker ngerprinting.
Keywords: oil spill identication, biomarker ngerprinting, terpanes, steranes, sesquiterpanes, aromatic steranes
Introduction
Liquid petroleum (crude oil and the products rened from it)
plays a pervasive role in modern society. As the population of
the world increases and developing countries become more in-
dustrialized, the demand for energy grows worldwide (Figure 1).
Oil is currently the dominant energy source and it is expected to
remain so over the next several decades. The worldwide extrac-
tion, transportation, and use of petroleuminevitably results in its
release to the environment. Oil spills have become a global prob-
lem. Based on analysis of data from a wide variety of sources,
about 260,000 metric tons of petroleum each year on average
are released to the waters off North America. Annual worldwide
estimates of petroleum input to the sea exceed 1,300,000 metric
tons (NRC, 2002). In Canada, about 12 spills of more than 4000
Lare reported each day, of which about one spill is into navigable
waters. Most spills take place on land, including oil spills from
pipelines, underground storage tanks, and aboveground storage
containers. In the United States, about 25 such spills occur each
day into navigable waters and about 75 occur on land (Fingas,
2001). Although large oil spills from tankers (such as the 1989
Exxon Valdez, 1999 Erika, and 2002 Prestige spills) occur in the
marine environment, these spills only make up about 5% of oil
Received 8 August 2005; accepted 25 January 2006.
Address correspondence to Zhendi Wang, Emergencies Science
and Technology Division, Environmental Technology Centre, Envi-
ronment Canada, 335 River Rd., Ontario, K1A 0H3, Canada. E-mail:
zhendi.wang@ec.gc.ca
entering the sea. Most oil pollution in the oceans comes from
the run-off of oil and fuel from land-based sources rather than
from accidental spills. Oil poses a range of environmental risks
and causes wide public concerns when released into the envi-
ronment, whether as catastrophic spills or chronic discharges.
Therefore, to unambiguously characterize, identify, categorize,
and quantify all sources of hydrocarbons entering the environ-
ment is very important for environmental damage assessment,
evaluation of the relative risks to the ecosystem posed by each
spill, and selecting appropriate spill response and taking effec-
tive cleanup measures.
Biomarkers play a very important role in characteriza-
tion, correlation, differentiation, and source identication in
environmental forensic investigations of oil spills. Biological
markers or biomarkers are one of the most important hydro-
carbon groups in petroleum for chemical ngerprinting. They
are complex molecules derived from formerly living organisms.
The biomarkers found in crude oils, rocks, and sediments are
stable and show little or no changes in structures from their par-
ent organic molecules, or so-called biogenic precursors (e.g.,
terpenoids, sterols, and steroids), in living organisms, and thus
carry information about the nature, source, type, geological con-
ditions, and thermal history of these organisms. Biomarkers
have for many years been widely used in petroleum exploration
and reservoir geochemistry to obtain information valuable to
geochemists such as the thermal maturity of the oil, the type
of source material, the depositional environment of the source
rock, the approximate geological age of the source rock, and
105
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Figure 1. Worldwide demand and supply for liquid petroleum from 1970 to 2004 (data from the US Department of Energy ref. DOE, 2004).
the degree of biodegradation of the oil. An excellent book, The
Biomarker Guide: Interpreting Molecular Fossils in Petroleum
and Ancient Sediments, on fundamentals of biomarker charac-
terization, their application in geochemistry, and interpretation
of biomarker data for oil exploration, was published in 1993
(Peters and Moldowan, 1993) and the second edition of the book
recently has been fully updated and expanded (Peters et al.,
2005). This new two-volume book provides a comprehensive
account of the role that biomarker technology plays both in
petroleum exploration and in understanding earth history and
process.
Biomarkers can be detected in low quantities (ppm and sub-
ppm level) in the presence of a wide variety of other types of
petroleum hydrocarbons by the use of the gas chromatogra-
phy/mass spectrometry (GC/MS). Relative to other hydrocar-
bon groups such as alkanes and most aromatic compounds,
biomarkers, in particular hopenoids and steroids (including
aromatic steroids), are more degradation-resistant in the envi-
ronment. Furthermore, biomarkers formed under different ge-
ological conditions and ages may exhibit different biomarker
ngerprints. Therefore, chemical analysis of biomarkers gener-
ates information of great importance to environmental forensic
investigations in terms of determining the source of spilled oil,
differentiating and correlating oils, and monitoring the degrada-
tion process and weathering state of oils under a wide variety of
conditions. Biomarkers have also proven useful in identication
of petroleum-derived contaminants in the marine and aquatic en-
vironments (Bence et al., 1996; Boehm et al., 1997; Hostettler
et al., 1999a; Kvenvolden et al., 1995, 2002; Stout et al., 2002;
Volkman et al., 1997; Wang et al., 1994a, 1994b, 1999a; Zakaria
et al., 2000) and in indicating chronic industrial and urban re-
leases (Kaplan et al., 1997; Stout et al., 1998; Volkman et al.,
1992a).
This review focuses on discussion of biomarker chem-
istry, overview of biomarker separation and analysis, biomarker
distributions and weathering effects on biomarker distribu-
tions, sesquiterpanes and diamondoids, diagnostic ratios and
cross-plots of biomarkers, unique biomarkers, application of
biomarker ngerprinting techniques for spill oil source iden-
tication, and application of multivariate statistical analysis for
biomarker ngerprinting and oil identication.
Biomarker Chemistry
Chemical Composition of Oil
Crude oils consist of complex mixtures of hydrocarbons and
non-hydrocarbons that range from small, volatile compounds
to large, non-volatile ones. Hundreds to thousands of com-
pounds have been identied in crude oils. Ultrahigh-resolution
Fourier transform ion cyclotron resonance mass spectrometry
(Marshall and Podgers, 2004) has recently revealed that crude
oil contains heteroatom-containing (N, O, S) organic compo-
nents having more than 20,000 distinct elemental compositions
(C
c
H
h
N
n
O
o
S
s
). In general, petroleumcomponents are classied
in bulk groups of saturates, olens, aromatics, resin (wide vari-
ety of compounds containing sulfur, oxygen, and nitrogen), and
asphaltenes (also called SARA analysis).
Saturates are a group of hydrocarbons composed of only
carbon and hydrogen with no carboncarbon double bonds.
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Forensic Fingerprinting of Biomarkers 107
Saturates are the predominant class of hydrocarbons in most
crude oil. Saturates include straight chain and branched
chain (also called parafns) and cycloalkanes (also called
naphthenes). Biomarker terpanes and steranes are branched
cycloalkanes consisting of multiple condensed ve- or six-
carbon rings. Sesquiterpanes and diamondoids are smaller cyclic
biomarkers, which can be particularly valuable for source iden-
tication of lighter petroleum products. Alkenes, commonly
referred to as olens, are partially unsaturated hydrocarbons
characterized by one or multiple double carboncarbon bonds.
These compounds are rare in crude oil but may be present in
some petroleum products, having been formed during the re-
ning process. Aromatic hydrocarbons are cyclic, planar com-
pounds that resemble benzene in electronic conguration and
chemical behavior. Aromatics in petroleum include the mono-
aromatic hydrocarbons such as benzene, toluene, and ethylben-
zene, and o, m, and p-xylene isomers (BTEX) and other alkyl-
substituted benzene compounds (C
n
-benzenes), and polycyclic
aromatic hydrocarbons (PAHs) (which include oil-characteristic
alkylated C
0
- to C
4
-naphthalene, phenanthrene, dibenzothio-
phene, uorene, and chrysene homologous series and other
United States Environmental Protection Agency (US EPA) pri-
ority PAHs). Polar compounds are those with distinct regions
of positive and negative charge, as a result of bonding with
atoms such as nitrogen, oxygen, or sulfur. Heavy oils generally
contain greater proportions of higher-boiling, more aromatic,
and heteroatom-containing (N-, O-, S-, and metal-containing)
constituents. In the petroleum industry, the smaller polar com-
pounds are called resins. Asphaltenes are a class of very large
heteroatom-containing compounds (Berkowitz, 1997; Speight,
1999). They are not dissolved in petroleum but are dispersed as
colloids. Asphaltenes are generally dened, based on the solu-
tion properties of petroleum residues in various solvents, as the
oil constituents precipitated from oils and bitumen by natural
processes or in laboratory by addition of excess n-pentane or
n-hexane. Table 1 summarizes the typical bulk composition of
some oils and petroleum products.
Table 1. Typical bulk composition of crude oils and petroleum products
Compound Light Heavy
Group class Gasoline Diesel crude crude IFO Bunker C
Saturates 5060 6595 5590 2580 2545 2040
alkanes 4555 3545
cyclo-alkanes 5 3050
waxes 01 020 010 210 515
Olens 510 010
Aromatics 2540 525 1035 1540 4060 3050
BTEX 1535 0.52 0.12.5 0.012 0.051 01
PAHs 0.55 0.53 14 110 110
Polar compounds 02 115 540 1525 1030
resins 02 010 225 1015 1020
asphaltenes 010 020 510 520
Sulphur <0.05 0.050.5 02 05 0.52 24
Metals (ppm) 3050 100500 1001000 1002000
BTEX = benzene, toluene, ethylbenzene, and xylenes; PAHs = polycyclic aromatic hydrocarbons.
The Isoprene Rule and Biomarker Families
In 1887, German chemist Otto Wallach determined the struc-
tures of several terpenes and discovered that all of them are
composed of two or more ve-carbon units of isoprene [2-
methyl-1,3-butadiene, CH
2
= C(CH
3
) CH = CH
2
]. The iso-
prene unit maintains its isopentyl structure in a terpene, usually
with modication of the isoprene double bonds. Compounds
composed of isoprene subunits are called terpenoids or iso-
prenoids. Terpenoids can be classied according to the number
of isoprene units from which they are biogenetically derived,
even though some carbons may have been added or lost (Con-
nolly and Hill, 1991). As for the oil saturated terpenoids, they
are generally categorized into families based on the approxi-
mate number of isoprene subunits they contain. The various oil
terpane families are composed of a wide variety of acyclic and
cyclic structures (Peters et al., 2005):
Hemiterpane (C
5
): containing one isoprene subunit;
Monoterpanes (C
10
): containing two isoprene subunits;
Sesquiterpanes (C
15
): containing three isoprene subunits;
Diterpanes (C
20
): containing four isoprene subunits;
Sesterterpanes (C
25
): containing ve isoprene subunits;
Triterpanes & containing six isoprene subunits;
steranes (C
30
):
Tetraterpanes (C
40
): containing eight isoprene subunits;
Polyterpanes (C
5n (n>8)
): containing nine or more isoprene
subunits.
As an example, Figures 2 and 3 show molecular structures of
some representative acyclic and cyclic terpenoid compounds
in oil, respectively. The most common cyclic terpenoids in
oil are terpanes, steranes and aromatic steranes. As a sum-
mary, Table 2 lists important biomarker terpane, sterane, and
aromatic sterane compounds, used frequently for oil spill
identication.
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108 Z. Wang et al.
Table 2. Source specic biomarker terpane and sterane compounds for oil spill studies
Empirical
Peak Compound Code formula Target ions
Sesquiterpanes (BicyclicTerpanes)
Drimanes and drimane-structure terpanes C
14
H
26
, C
15
H
28
, C
16
H
30
123
Diamondoids
Adamantanes C
10
H
16
, alkyl-C
10
H
15
136, 135, 149, 163, 177
Diamantanes C
14
H
20
, alkyl-C
14
H
19
188, 187, 201, 215, 229
Terpanes
1 C
19
tricyclic terpane TR19 C
19
H
34
191
2 C
20
20
H
36
191
3 C
21
21
H
38
191
4 C
22
22
H
40
191
5 C
23
23
H
42
191
6 C
24
24
H
44
191
7 C
25
tricyclic terpane (a) TR25A C
25
H
46
191
8 C
25
tricyclic terpane (b) triplet: C
24
tetracyclic terpane + TR25B C
25
H
46
191
9 C
26
(S + R) tricyclic terpanes TET24+TR26A+TR26B C
24
H
42
+C
26
H
48
191
10 C
28
28
H
52
191
11 C
28
tricyclic terpane (b) TR28B C
28
H
52
191
12 C
29
29
H
54
191
13 C
29
tricyclic terpane (b) TR29B C
29
H
54
191
14 Ts: 18(H),21(H)-22,29,30-trisnorhopane Ts C
27
H
46
191
15 17(H),18(H),21(H)-25,28,30-trisnorhopane TH27 C
27
H
46
191, 177
16 Tm: 17(H),21(H)-22,29,30-trisnorhopane Tm C
27
H
46
191
17 C
30
tricyclic terpane 1 TR30A C
30
H
52
191
18 C
30
tricyclic terpane 2 TR30B C
30
H
52
191
19 17(H),18(H),21(H)-28,30-bisnorhopane H28 C
28
H
48
191
20 17(H),21(H)-25-norhopane NOR25H C
29
H
50
191, 177
21 17(H),21(H)-30-norhopane H29 C
29
H
50
191
22 18(H),21(H)-30-norneohopane (C
29
Ts) C29Ts C
29
H
50
191
23 17(H)-diahopane DH30 C
30
H
52
191
24 17(H),21(H)-30-norhopane (normoretane) M29 C
29
H
50
191
25 18(H) and 18(H)-oleanane OL C
30
H
52
191, 412
26 17(H),21(H)-hopane H30 C
30
H
52
191
27 17(H)-30-nor-29-homohopane NOR30H C
30
H
52
191
28 17(H),21(H)-hopane (moretane) M30 C
30
H
52
191
29 22S-17(H),21(H)-30-homohopane H31S C
31
H
54
191
30 22R-17(H),21(H)-30-homohopane H31R C
31
H
54
191
31 Gammacerane GAM C
30
H
52
191, 412
32 17(H),21(H)-hopane Internal standard Internal standard 191
33 22S-17(H),21(H)-30,31-bishomohopane H32S C
32
H
56
191
34 22R-17(H),21(H)-30,31-bishomohopane H32R C
32
H
56
191
35 22S-17(H),21(H)-30,31,32-trishomohopane H33S C
33
H
58
191
36 22R-17(H),21(H)-30,31,32-trishomohopane H33R C
33
H
58
191
37 22S-17(H),21(H)-30,31,32,33-tetrakishomohopane H314S C
34
H
60
191
38 22R-17(H),21(H)-30,31,32,33-tetrakishomohopane H34R C
34
H
60
191
39 22S-17(H),21(H)-30,31,32,33,34-pentakishomohopane H35S C
35
H
62
191
40 22R-17(H),21(H)-30,31,32,33,34-pentakishomohopane H35R C
35
H
62
191
Steranes
41 C
20
5(H),14(H),17(H)-sterane S20 C
20
H
34
217, 218
42 C
21
21
H
36
217, 218
43 C
22
22
H
38
217, 218
44 C
27
20S 13(H),17(H)-diasterane DIA27S C
27
H
48
217, 218, 259
45 C
27
20R 13(H),17(H)-diasterane DIA27R C
27
H
48
217, 218, 259
46 C
27
20S 13(H),17(H)-diasterane DIA27S2 C
27
H
48
217, 218, 259
47 C
27
20R 13(H),17(H)-diasterane DIA27R2 C
27
H
48
217, 218, 259
48 C
28
28
H
50
217, 218, 259
49 C
28
28
H
50
217, 218, 259
50 C
29
29
H
52
217, 218, 259
51 C
29
29
H
52
217, 218, 259
52 C
27
20S 5(H),14(H),17(H)-cholestane C27S C
27
H
48
217, 218
53 C
27
20R 5(H),14(H),17(H)-cholestane C27R C
27
H
48
217, 218
54 C
27
20S 5(H),14(H),17(H)-cholestane C27S C
27
H
48
217, 218
55 C
27
20R 5(H),14(H),17(H)-cholestane C27R C
27
H
48
217, 218
56 C
28
20S 5(H),14(H),17(H)-ergostane C28S C
28
H
50
217, 218
57 C
28
20R 5(H),14(H),17(H)-ergostane C28R C
28
H
50
217, 218
58 C
28
20S 5(H),14(H),17(H)-ergostane C28S C
28
H
50
217, 218
59 C
28
20R 5(H),14(H),17(H)-ergostane C28R C
28
H
50
217, 218
60 C
29
20S 5(H),14(H),17(H)-stigmastane C29S C
29
H
52
217, 218
61 C
29
20R 5(H),14(H),17(H)-stigmastane C29R C
29
H
52
217, 218
62 C
29
20S 5(H),14(H),17(H)-stigmastane C29S C
29
H
52
217, 218
63 C
29
20R 5(H),14(H),17(H)-stigmastane C29R C
29
H
52
217, 218
64 C30 steranes C30S C
30
H
54
217, 218
Monoaromatic Steranes 253
Triaromatic Steranes 231
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Figure 2. Molecular structures of representative acyclic terpenoid compounds in oil.
Labeling System and Nomenclature of Biomarkers
The chemical structures of terpenoids are more complicated than
that of normal alkanes and isoalkanes, in particular the three-
dimensional nature of the terpenoids structure. For many ter-
penoid classes, several names has been proposed for the carbon
skeleton, but the basic rules of the International Union of Pure
and Applied Chemistry (IUPAC) system are used for nomencla-
ture of biomarkers. For example, acyclic phytane (C
20
) is named
as 2,6,10,14-tetramethylhexadecane.
The labeling system for cyclic penta-cyclic triterpanes and
regular steranes is shown in Figure 4 and described as follows:
1) Each carbon atomand the rings in biomarker molecules are la-
beled systematically. Rings are specied in succession from left
toright as the A-ring, B-ring, C-ring, D-ring, etc. 2) Acapital C
followed immediately by a subscript number refers to the num-
ber of carbon atoms in a particular compound (e.g., C
30
hopane
and C
27
sterane mean that they contain 30 and 27 carbon atoms,
respectively). 3) A capital C followed by a dash and numbers
refers to a particular position within the compound (e.g., C-17
and C-21 in the 17, 21-hopane is the carbon atoms at posi-
tions 17 and 21). 4) Prexes are used to indicate the changes
to the normal biomarker carbon skeleton, which include the
prex nor-, seco-, and neo-, and others. Table 3 summarizes
the nomenclature used to modify the structural specication of
cyclic biomarkers. The prex nor- is used to indicate loss of
carbons from a carbon skeleton. For example, 25-norhopane is
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110 Z. Wang et al.
Figure 3. Molecular structures of representative cyclic terpenoid compounds in oil.
identical to hopane except that a methyl group at the C-25 posi-
tion has been lost from its point of attachment at the C-10 posi-
tion (Figure 4). If two or three carbons are lost, the prex bisnor-
or trisnor- is usedrespectively. Similarly, the prexhomo- is used
to indicate addition of carbons to a carbon skeleton. The prex
Figure 4. Labelling system for carbon atoms in C
30
hopane (left) and C
27
sterane (right).
seco- is used to indicate cleavage of a bond, with the locants for
both ends of the broken bond given, e.g., 3,4-secoeudesmane
and 17,21-secohopane. For more information, see the Chemical
Abstract Index Guide: Appendix IV (CAS, 2002), which uses
these prexes extensively for some classes of terpenoids.
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Table 3. Common modiers and other nomenclature related to cyclic biomarkers
Modier Description Example Biomarker
homo- One additional carbon on the parent molecular
structure
C
31
17(H),21(H)-30-homohopane
bis-, tris-, tetrakis-, pentakis-
(also di-, tri-, tetra-, and
penta-)
Two to ve additional carbons on the parent
molecular structure
C
32
17, 21-30,31-homohopane
C
33
17, 21-30,31,32-homohopane
C
34
17, 21-30,31,32,33-homohopane
seco- Cleaved C-C bond C
24
17,21-secohopane (tetracyclic)
nor- One less carbon on the parent molecular
structure
25-norhopane
bisnor- Two less carbons on the parent molecular
structure
28,30-bisnorhopane
trisnor- Three less carbons on the parent molecular
structure
25,28,30-trisnorhopane
neo- Methyl group shifted from C-18 to C-17
position on hopanes
C
29
Ts: 30-norneohopane
Asymmetric carbon in ring with H down 17(H),21(H)-hopane
Asymmetric carbon in ring with H up 17(H),21(H)-hopane
R Asymmetric carbon in acyclic moiety of
biomarkers obeying convention in a
clockwise direction
C
27
20R cholestane
S Asymmetric carbon in acyclic moiety of
biomarkers obeying convention in a
clockwise direction
C
27
20S cholestane
and Stereoisomers
Stereoisomers are isomers whose atoms are bonded together
in the same sequence but differ from each other in the orienta-
tion of the atoms in space. Hydrogen atoms that are below the
plane of the molecule are called hydrogens, and the bond is
drawn with a dashed line. Conversely, hydrogen atoms located
above the plane of the molecule are called hydrogens, and
the bond is drawn with a wedge bond. In many common ring
systems the hydrogen atoms found at ring junctions are gener-
ally omitted from drawings for clarity. Hopanes are previously
considered to exist as three stereoisomers: 17(H), 21(H)-
hopane, 17(H), 21(H)-hopane, and 17(H), 21(H)-hopane
(Peters and Moldowan, 1993; Waples and Machihara, 1991).
Hopanes in the series are also called moretanes. Hopanes
with the 17 -conguration in the range of C
27
to C
35
are
characteristic of petroleum because of their greater thermody-
namic stability compared with other epimeric series ( and
). The series is generally not found in petroleum because
it is thermally unstable. The series was previously considered
not present in oil because of low stability in comparison with
the and series. However, mechanics calculations have
shown that the -hopanes should be less stable than - and
-hopanes, but more stable than -hopanes. Recently, Nytoft
and Bojesen-Koefoed (2001) showed that moderate quantities of
17(H), 21(H)-hopanes are present in several sediments and
oils. The ratios of C
30
17(H), 21(H)-hopane to C
30
17(H),
21(H)-hopane are typically 0.020.04 in crude oils and mature
sediments, but ratios up to 0.10 have been found in immature
sediments.
R and S Stereoisomers
For biomarkers, the use of R and S nomenclature is gener-
ally restricted to carbon atoms which are not part of a ring. The
steranes including cholestanes (C
27
H
48
), ergostanes (C
28
H
50
),
and stigmastanes (C
29
H
52
) can have R- and S-conguration at
the acyclic (chain position) carbon atom C-20 (Figure 4), re-
sulting in two homologue series with 20R (20R and 20R
) and 20S (20S and 20S ) congurations. Hopanes
with 30 carbons or less show asymmetric centers at C-21 and
all ring-juncture carbons including C-5, C-8, C-9 C-10, C-13,
C-14, C-17, and C-18. Common homohopanes (C
31
to C
35
) have
an extended side chain with an additional asymmetric center at
C-22, resulting in two homologues with 22R and 22S cong-
urations. These two homologous homohopanes (22R and 22S)
can be well separated by GC/MS as well-resolved doublet peaks,
prominent in gas chromatograms of oils.
Biomarker Analysis Methodologies
Oil Sample Preparation, Cleanup, and Fractionation
There are a variety of common sample extraction and prepara-
tion techniques including sonication, Soxhlet, supercritical uid,
and accelerated solvent extraction (Fakness et al., 2002a; Kaplan
et al., 1996; Page et al., 1995; Sauer and Boehm, 1991; Short
et al., 1996; Stout et al., 2002; Wang and Fingas, 2003) and some
of them have been promulgated by the US EPA and American
Society for Testing and Material (ASTM) as oil-contaminated
soil extraction and analysis methods such as ASTM Methods
3328 (ASTM, 1997a) and 5739 (ASTM, 1997b), and some
EPA 600 (EPA, 1983 and 1986) and 8000 series Methods (EPA,
1997). Solvent extractions and separations of oil hydrocarbons
are based on the like-dissolves-like principle. Surrogates are
often added to monitor extraction efciency. Glass columns
are often used for sample cleanup and fractionation prior to the
GC/MS analysis of biomarkers. The advances of oil ngerprint-
ing methodologies, overview of selected US EPA and ASTM
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112 Z. Wang et al.
methods and their limitations, and qualitative and quantitative
aspects and comparison of major oil ngerprinting methods have
been described and discussed by Faksness et al. (2002a) Stout
et al. (2002), and Wang et al. (1999a).
As an example, the following briey describes oil-related
sample preparation and biomarker characterization procedures
by the Environment Canada Oil Spill Research Laboratory.
Oil Samples: Oil samples are directly dissolved in hexane at a
concentration of 50100 mg/mL. For heavy oils, small amounts
(<5% of the total solvent volume) of dichloromethane (DCM)
may be added to help the oil dissolution.
Oil-contaminated water samples: Oil-contaminated water
samples are weighed, spiked with appropriate amounts of sur-
rogate standards, and extracted according to the EPA Method
3510: separatory funnel liquid-liquid extraction (EPA, 1996).
Oil-contaminated sediment samples: Oil-contaminated sedi-
ment or soil samples are weighed, dried with anhydrous sodium
sulphate then spiked with appropriate amounts of surrogate stan-
dards. The samples are extracted ultrasonically with 1:1 (v/v)
hexane/ DCM, and then twice with DCMalone (if there is visible
color in the third extraction, additional extraction should be per-
formed); or Soxhlet-extracted with DCMfor 24 hours/overnight.
Extracts are combined and concentrated by rotary evaporation,
solvent-exchanged to hexane, and evaporated under a gentle
stream of dry nitrogen to a nal volume of 2 to 10 mL (the nal
volume depends on the concentration of hydrocarbons in the -
nal extract). An aliquot of the concentrated extract (0.51.0 mL)
is taken and blown down with N
2
to dryness and the residue
is weighed on a microbalance to obtain a total solvent ex-
tractable material (TSEM)/weight expressed as mg/g of sam-
ple. TSEM provides an equal basis for quantitative comparison
of petroleum hydrocarbon groups between samples. Based on
the TSEM value, appropriate aliquot of the concentrated ex-
tract (containing approximately 20 mg oil) is transferred to a
3-g silica gel chromatographic column for sample cleanup and
fractionation.
Silica gel is frequently used for cleanup and fractionation of
oil extracts (Wang et al., 1994a). Saturated hydrocarbons are
eluted with hexane (Fraction 1, labeled F1). Aromatic hydrocar-
bons are eluted with mixture of hexane:dichloromethane (v/v,
1:1, Fraction 2, labeled F2). Saturated biomarkers are eluted
with other saturates in F1. Aromatic steranes are eluted in the
aromatic fraction, F2.
Internal Standards and Quantication of Biomarkers
For each sample, half of F1 is used for analysis of the total GC-
detectable saturates, nalkanes and isoprenoids, and biomarker
compounds; and half of F2 is used for analysis of alkylated
PAH homologues and other EPA priority parent PAHs, and aro-
matic steranes. The remaining halves of the F1 and F2 are
combined into one fraction (Fraction 3, labeled F3) and used
for the determination of Total Petroleum Hydrocarbons (TPH)
and Unresolved complex mixture of hydrocarbons (UCM). The
three fractions are concentrated under a stream of nitrogen
to appropriate volumes, spiked with appropriate internal stan-
dards, and then adjusted to an accurate pre-injection volume
(0.501.00 mL) for gas chromatography/ame ionization detec-
tion (GC-FID) and GC/MS analyses. The C
30
17(H), 21(H)-
hopane is used as internal standard for quantication of C
30
17(H), 21(H)-hopane and other terpanes (in the range of C
19
to C
35
) and steranes. The deuterated d
3
monoaromatic steranes
[5(H)/5(H), C
21
H
27
D
3
] are usedas internal standardfor quan-
tication of monoaromatic and triaromatic steranes. The deuter-
ated d
18
-decahydronaphthalene (cis) and d
16
-adamantane are
usedas internal standards for quanticationof bicyclic sesquiter-
panes and diamondoid compounds, respectively.
Capillary Gas Chromatography/Mass Spectrometry (GC/MS)
The mass spectrometer has long been recognized as the most
powerful detector for a gas chromatograph due to its high sen-
sitivity and specicity and its capability to elucidate compound
structures. Today, GC/MS (e.g., benchtop quadrupole GC/MS,
GC/MS-MS, high-resolution GC/MS, and GC-ion trap/MS)
have become the principal and routine techniques used in most
oil and environmental forensics laboratories to analyze a wide
range of petroleum hydrocarbons including biomarkers. The
new-generation GC/MS, which combines chemical separation
by capillary column GC, spectral resolution by MS and comput-
erized data preprocessing, enables separation, identication, and
quantication of trace amounts of biomarker compounds in oil.
Early use of mass chromatograms in organic geochemistry was
pioneered at Chevron and led to a stereochemical understanding
of steroids and the rst practical method of oil ngerprinting
based on terpanes and steranes (Seifeit, 1977).
Benchtop Quadrupole GC-MS
The quadrupole is the most common mass separator in use to-
day. The benchtop quadrupole GC/MS systems, although lack-
ing the high-resolution capabilities of larger and more expen-
sive magnetic-sector instruments, have sufcient sensitivity and
selectivity for most purposes of biomarker analysis. Benchtop
quadrupole GC/MS can be operated in various modes, includ-
ing the most widely used selected ion monitoring (SIM) mode
and scan mode. For quantication of individual biomarker com-
pounds, the SIMmode is mostly selected. In the SIMmode, only
a limited number of characteristic ions (Table 2) are selected and
monitored.
Example benchtop GC/MS conditions (Environment Canada
Oil Spill Research Laboratory): Analyses of biomarkers are
performed on an Agilent 6890 GC coupled with an Agilent
5973 mass selective detector (MSD). System control and data
acquisitions are achieved with the Agilent G1701 BA MSD
ChemStation. A 30 m 0.25 mm i.d. (0.25 m lm thick-
ness) HP-5MS fused-silica capillary column is used. The chro-
matographic conditions are as follows: carrier gas, helium (1.0
mL/min); injection mode, splitless; injector and detector tem-
perature, 280 and 300
C, respectively. The temperature program

employed for biomarkers and alkylated PAHs is: 50
C hold for
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2 minutes, then ramp at 6
C/min to 300
C and hold for 20 min-

utes. Prior to sample analysis, the GC/MS is tuned with peru-
orotributylamine (PFTBA). The total run time is 60 minutes.
Example benchtop GC/MS conditions (Petrobras Geochem-
istry Laboratory): A 60 m 0.25 mm i.d. (0.25 m lm thick-
ness) HP-5MS or equivalent 60-m capillary column is used
to achieve improved resolution for biomarkers. The temper-
ature program is as follows: 55
C hold for 2 minutes, ramp

at 20
C/min. to 150
C and then 1.5
C to 310
C and hold for

15 minutes. The total run time is 128 minutes.
The 30-m capillary column is used in many environmental
forensic labs for most oil spill works. However, the 60-m capil-
lary column with a slow temperature rate and longer running
time can offer further improved resolution for some paired
biomarker isomers, which may be not well-resolved by the use
of a 30-m column. An example of this is given in the next
section.
Figure 5. Mass spectrometric fragmentation of common biomarkers.
Characteristic Fragment Ions of Biomarkers
Characterization of major biomarker groups is largely achieved
using the following MS fragment ions. Figure 5 shows mass
spectrometric fragmentation of common biomarkers.
alkyl-cyclohexanes: m/z 83
methyl-alkyl-cyclohexanes: m/z 97
isoalkanes and isoprenoids: m/z 113, 127, 183
sesquiterpanes: m/z 123
adamantanes: m/z 135, 136, 149, 163, 177,
and 191
diamantanes: m/z 187, 188, 201, 215 and
229
tri-, tetra-, penta-cyclic terpanes: m/z 191
25-norhopanes: m/z 177
28,30-bisnorhopanes: m/z 163, 191
steranes: m/z 217, 218
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114 Z. Wang et al.
5(H)-steranes: m/z 149, 217, 218
5(H)-steranes: m/z 151, 217, 218
X diasteranes: m/z 217, 218, 259
methyl-steranes: m/z 217, 218, 231, 232
monoaromatic steranes: m/z 253
triaromatic steranes: m/z 231
The m/z 191 fragment is often the base peak of mass spec-
tra of cyclic terpanes. It is derived from rings (A + B) of the
molecule (Figure 5), but rings (D + E) may also be the source.
The m/z 177 fragment is most likely derived from rings (A+B)
of triterpane molecules that have lost a methyl group from posi-
tion 10, that is, 25-norhopanes (Grahl-Nielsen and Lygre, 1990;
Volkman et al., 1983a, 1983b). The notable feature of mass spec-
tra for 25-demethylated hopanes is that the m/z 177 fragment has
higher intensity than the m/z 191 fragment. Demethylated triter-
panes contain different information than the triterpanes and have
been suggested as markers for biodegradation (Volkman et al.,
1983b). The other triterpanes also give the m/z 177 fragment
on electron impact in the mass spectrometer but in lower abun-
dance than the m/z 191 fragment. The fragment is formed by
the loss of CH
2
from the m/z 191 fragment, and can be seen in
all mass spectra of triterpanes. C
29
18, 21-30-norneohopane
(C
29
Ts), which elutes immediately after C
29
-hopane, has a
greater abundance of the m/z 177 ion than of the m/z 191 ion.
The proposed mechanism is that m/z 177 ion is derived from
the (D + E) ring fragment and stabilized by methyl group at
position 14 (Gallegos, 1971; Killops and Howell, 1991). By a
similar mechanism, C
29
17, 21-30-norhopane (normoretane)
also shows signicantly greater abundance of the m/z 177 ion
than of the m/z 191 ion. The m/z 217 and 218 fragment ions are
derived from rings (A + B + C) of most 14(H)- and 14(H)-
steranes. The and steranes have a base peak at m/z
217, whereas the base peak of steranes is m/z 218. The
relative intensities of m/z 149 to m/z 151 fragment ions in the
mass spectra of steranes have been used to determine and dis-
tinguish between 5- and 5-stereoisomers. If the ratio of the
m/z 149 ion to the m/z 151 ion is greater than 1, it is 5-sterane;
otherwise, it is 5--sterane.
The SIM chromatogram of one ion at given m/z with the
GC retention time is often diagnostic of a class of homolo-
gous compounds with similar structures but different carbon
numbers and isomerism, and can be used for identication. As
an example, Figure 6 shows the SIM chromatograms for com-
mon biomarker classes in a Kuwait crude oil obtained by using
a 60-m column (Barbanti, personal communication, 2004). In
total, 38 terpanes fromC
19
tricyclic terpane to C
35
homohopanes
(m/z 191) and 19 steranes fromC
21
to C
29
steranes (m/z 217 and
218) have been unambiguously identied and characterized in
this Kuwait oil. Less abundant C
30
steranes can be clearly rec-
ognized as well although conrmation of their identity normally
requires MS-MS. Paired biomarker isomers (H29 and C29Ts,
H30 and NOR30H) and triplet (TET24 +TR26A+TR26B) are
well-resolved.
Quality Assurance and Quality Control
The reliabilityof methods for biomarker analysis is dependent on
the quality control (QC) procedures followed. In order to support
oil spill forensic investigations, quality management (including
quality assurance [QA] and QC system, updated standard op-
erational procedures, personnel training program and record,
up-to-date methodology, equipment management, sample man-
agement, and data management) is a fundamental element of
the analytical QA program. The chemical measurements must
be conducted within the framework of highly stringent, defensi-
ble andreliable QAandQCprograms (ASTM, 1997a and1997b;
Douglas et al., 1996 and 2004; EPA, 1997, 1998a, 1998b, 2001;
ETC, 2005; Faksness et al., 2002b; Page et al., 1995; Stout et al.,
2002; Wang et al., 1999a, 2003a). The QA/QCprograms used by
different laboratories may differ, but the principle and purposes
are similar.
Biomarker Distributions
Distribution of Biomarkers in Crude Oils
Crude oil compositions vary widely, depending on the oil
sources, the thermal regime during oil generation, and the geo-
logical migration and reservoir conditions. Crude oils can have
1) large differences in distribution patterns of the n-alkane and
cyclic-alkanes as well as UCM proles; 2) signicantly differ-
ent relative ratios of isoprenoids to normal alkanes; and 3) large
differences in distribution patterns and concentrations of alkyl-
ated PAH homologues and biomarkers. Various biomarkers can
occur in different carbon ranges of crude oils (Figure 7). In
general, GC/MS chromatograms of terpanes (m/z 191) are char-
acterized by the terpane distribution in a wide range from C
19
to
C
35
with C
29
- and C
30
-pentacyclic hopanes and C
23
and
C
24
tricyclic terpanes being often the most abundant. As for ster-
anes (at m/z 217 and 218), the dominance of C
27
, C
28
, and C
29
20S/20R homologues, particularly the epimers of -steranes,
among the C
20
to C
30
steranes is often apparent.
Figures 8and9showGC-MSchromatograms at m/z 191, 217,
and 218 for light (API > 35), medium (API: 2535), and heavy
(API < 25) crude oils including Alaska North Slope (ANS),
Cook Inlet, Arabian Heavy, Orinoco Bitumen (Venezuela), and
California API-11. For comparison, the GC-FIDchromatograms
for these oils are also shown in Figure 8. Different from most
crude oils, the very light Scotia Light (API = 59) only con-
tains trace amount of C
19
C
35
biomarkers (only 29 g/g oil
in total, not shown here). The Alaska North Slope oil contains
a wide range of terpanes with the C
30
and C
29
hopane
as the most abundant. The triplet C
24
tetracyclic +C
26
(S +
R) tricyclic terpanes is highly abundant as well. In contrast,
the terpanes in Arabian Heavy and Cook Inlet are largely lo-
cated in the C
27
to C
35
pentacyclic hopane range containing only
small amounts of tricylic terpanes. In addition, the abundance of
C
29
hopane is higher than that of C
30
hopane in Arabian
Heavyoila feature commonincrude oils sourcedfromcarbon-
ate source rock sequences. Notably, the biodegraded California
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115
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116
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e
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a
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118 Z. Wang et al.
Figure 7. Carbon number range distribution of common cyclic biomarker classes in crude oil and petroleum products.
and Orinoco oils exhibit strong biomarker distribution, which
demonstrates the resistance of biomarkers to biodegradation.
The steranes are present in all ve light to heavy crude
oils but with different distribution patterns. The characteris-
tic V-shaped C
27
-C
28
-C
29
regular sterane (m/z 218) dis-
tribution is clearly demonstrated, which indicates high thermal
maturity. The relative abundances of C
27
-C
28
-C
29
steranes in
oils reect the carbon number distribution of the sterols in
the organic matter in the source rocks for these oils (Peters
and Moldowan, 1993; G urgey, 2002). Compared with other
oils, the Alaska North Slope oil contains higher amounts of
diasteranes as well as C
21
and C
22
regular steranes. The Ara-
bian Heavy displays markedly higher concentration of C
29

steranes than C
27
cholestane series and C
28
ergostane
series.
The dominance of C
28
17(H), 18(H), 21(H)-28,30-
bisnorhopane (BHN28) is particularly prominent in California
API-11. Ahigh concentration of C
28
-28,30-bisnorhopane is typ-
ical of petroleum from highly reducing to anoxic depositional
environments (Melloet al., 1990). The California API-11oil also
demonstrates signicantly higher concentration of C
35
homo-
hopanes (22S+22R) than C
34
homohopanes (22S+22R), further
indicating a highly reducing marine environment of deposition
with no available free oxygen (Peters and Moldowan, 1993). For
Orinoco Bitumen, C
23
terpane is the most abundant, followed
by the C
30
and C
29
-hopane. The presence of triplets with
different relative distributions is apparent for most heavy oils.
Different from many other heavy oils which have somewhat the
V-shaped C
27
-C
28
-C
29
regular sterane (m/z 218) distribu-
tion, California API-11 contains far higher levels of steranes,
with a more abundant C
28
ergostane than C
27
and C
29
sterane
series. This is also the case for several other heavy California
oils, including California API-15, Platform Irene, and Sockeye
oil (data not shown here).
The distribution of petroleumbiomarkers has been applied to
investigations of oil spill accidents (Barakat et al., 1999; Bence
et al., 1996; Kvenvolden et al., 1993; Page et al. 1988; Wang
et al., 1994b, 1995a, 1998a; Zakaria et al., 2000, 2001) and
to trace the record of hydrocarbon input to San Francisco Bay
(Hostettler et al., 1999a). Dahlmann (2003) demonstrated that
Nigerian crude oils from Niger Delta are characterized by the
presence of oleanane in relatively high concentrations. The same
author (Dahlmann, 2003) also found that gammacerane is in
high concentrations (up to 50% in relation to C
30
-hopane)
in the African Angola Cabinda and Gabon crude oils. Recently,
biomarkers, together with PAHs and other hydrocarbon charac-
terization results, have been extensively applied to the controver-
sial issue over the origin of petrogenic hydrocarbon background
in Prince WilliamSound of Alaska, site of the 1989 Exxon Valdez
oil spill (i.e., whether the petrogenic hydrocarbon background
is mainly from eroding Tertiary shales and residues of natural
oil seepage, or mainly from Berling River coals and oil from the
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Figure 8. GC-MS chromatograms at m/z 191 for light to heavy crude oils. For comparison, the GC-FID chromatograms for these oils are also shown in
Figure 8.
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120 Z. Wang et al.
Figure 9. GC-MS chromatograms at m/z 217 and 218 for light to heavy crude oils.
Katalla area) by two groups of scientists (Boehm et al., 2001,
2002; Gerald et al., 2002; Hostettler et al., 1999b; Page et al.,
1995, 1996, 2002; Short and Heintz, 1997; Short et al., 1999,
2004).
Distribution of Biomarkers in Petroleum Fuels
Rened petroleum fuels are obtained from crude oil through
a variety of rening processes (Olah and Monnar, 1995;
Simanzhenkov and Idem, 2003; Speight, 2002; Stout et al.,
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2002). Depending on the chemical composition of their parent
crude oil feed stocks, varying rening approach and conditions,
wide range of applications, regulatory requirements, and eco-
nomic requirements, rened products can have wide variety in
chemical compositions.
Light distillates are typically mostly volatile products in the
C
4
to C
13
carbon range. They include aviation gas, naphtha (a
liquid petroleum product that boils from about 30
C to approxi-
mately 200
C, the term petroleum solvent is often used synony-

mously with naphtha), and automotive gasoline.
Mid-range distillates are typically products that can span a
relatively broader carbon range (C
6
to C
28
) and include kerosene,
aviation jet fuels, and lighter diesel products. Jet fuels (includ-
ing Jet-A, Jet-B, JP-4, JP-5, and JP-8) are similar in gross com-
position, and compositional differences are often attributable
to additives designed to control some fuel parameters. Diesels
consist of hydrocarbons in a carbon range of C
8
to C
28
and
contain high levels of n-alkanes, alkyl-cyclohexane, and PAHs.
The properties of a given diesel are largely a function of the
crude oil feedstock. The GC chromatogram of diesel fuel No. 2
is dominated by a nearly normal distribution of n-alkanes with
maxima around n-C
11
to n-C
14
. Also, a central UCM hump is
obvious.
Classic heavy fuel types include fuel No. 5 and No. 6 (also
known as Bunker C) fuel. The heavy residual fuels are largely
used in marine diesel and industrial power generation. Currently,
many Bunker type fuels are produced by blending residual oils
with diesel fuels or other lighter fuels in various ratios to produce
fuel of acceptable viscosity for marine or power plant use.
GC/MS chromatograms for biomarker ngerprinting (at m/z
191 and 218) of common light petroleum products and heavy
fuel oils are shown in Figures 10 and 11, respectively. No tar-
get terpane and sterane compounds are detected in Jet A fuel.
Only traces of smaller terpanes are detected in Diesel No. 2
sampled from an Ottawa gas station in 2002 and the diesel used
for 1994 Mobile Burn study (16.3% weathered). For the 2002
Quebec spill diesel sample, both terpanes and steranes with a
wider carbon range (C
20
to C
30
) are detected. Relative to other
light petroleum products, biomarkers with a much wider car-
bon range are quite abundant in Korea Diesel No. 2. Clearly,
diesels obtained from different crude oil feed stocks and re-
ning process have correspondingly varying biomarker nger-
prints. Venezuela Fuel No. 6 is characterized by a distribution in
a wide range from C
19
to C
35
, with C
23
tricyclic terpanes being
the most prominent. As for steranes, the dominance of C
27
, C
28
,
and C
29
20S/20R homologues is apparent. The relative propor-
tion of C
27
-C
28
-C
29
steranes shows a consistent decrease
with increasing carbon number (C
27
>C
28
>C
29
). The suspected
spill source candidate of Bunker C from Quebec has different
biomarker distribution pattern with the C
29
and C
30
hopane and
C
27
-C
28
-C
29
sterane homologous series being prominent. C
23
and C
24
tricyclic terpanes are the most abundant terpanes in
HFO 6303, whereas the level of homohopanes (i.e., from C
31
to
C
35
) is close to the detection limit. Furthermore, contrary to most
Bunker C type oils, the concentrations of terpanes and steranes
are quite lowin HFO6303 (the total target biomarker concentra-
tion is only 255 g/g oil). As for Fuel No. 5, the dominance of
C
29
sterane peaks, in particular the 20S C
29
sterane, in SIM
chromatogram (m/z 217, not shown here) is very pronounced.
Distribution of Biomarkers in Lubricating Oils
Small-scale spills of lubricatingoil are commondue totheir wide
application. Lubricating oil is distinguished fromother fractions
of crude oil by its high (>340
C) boiling point region. Lubricat-

ing oils can be generally divided into two categories: petroleum
derived or synthetic (Speight, 2002). Synthetic lubricating oils
are created by the chemical reaction of several ingredients. Typi-
cal carbon-range of lubricating oils is C
20
to C
45+
and n-alkanes
are usually removed by solvent extraction. Figure 12 shows SIM
chromatograms of biomarker distributions in lubricating oils.
Signicant features of the biomarker distribution in petroleum-
derived lubricating oils include:
r
Biomarkers are predominantly located in the high carbon
number end.
r
Lubricating oils, in general, contain high levels of target
biomarkers in comparison with most crude oils and petroleum
products.
r
The dominance of characteristic pentacyclic C
31
to C
35
homohopanes is particularly prominent. For example, the
concentrations of 22S/22R C
31
to C
33
homohopanes in the
Extreme Pressure Gear Oil 80W-90 are only slightly lower
than the most abundant C
30
hopane. The dominance of
C
27
, C
28
, and C
29
20S/20R homologues is apparent too.
r
The synthetic lubricating oil has a different biomarker distri-
bution pattern from petroleum-derived lube oils. The target
biomarkers are low in abundance, and the unresolved humps
are particularly pronounced.
Lubricating oil contamination through engine exhaust and
through leakage and spillage occurs everywhere (Kaplan et al.,
2001). Bieger et al. (1996) have reported the use of terpane
ngerprints of rened oils and motor exhausts to indicate the
presence of and trace the origin of diffuse lubricating oil con-
tamination in plankton and sediments around St. Johns of
Newfoundland, eastern Canada. Different types of lubricating
oils and motor exhausts were found to consistently feature dis-
tinct terpane distributions. Variable inputs of automotive and
outboard motor oils were clearly recognized through character-
ization and recognition of the terpane distributions.
Distribution of Aromatic Steranes in Oils
and Petroleum Products
Aromatic steranes are another group of biomarker compounds
that are highly resistant to biodegradation and can be used
for oil-to-oil correlation and oil source tracking (Peter and
Moldowan, 1993). Figure 13 shows the mass chromatograms
of the C-ring monoaromatic (MA, m/z 253, left panel) and
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122 Z. Wang et al.
Figure 10. GC-MS chromatograms (at m/z 191 and 218) for lighter petroleum products.
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Figure 11. GC-MS chromatograms (at m/z 191 and 218) for heavier fuels.
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124 Z. Wang et al.
Figure 12. GC-MS chromatograms (at m/z 191 and 218) for biomarker distributions in lubricating oils.
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Figure 13. Left panel: mass chromatograms of the triaromatic steranes (TA, m/z 231). Right panel: mass chromatograms of the monoaromatic steranes
(MA, m/z 253) in the aromatic hydrocarbon fractions of crude oils (NIST SRM 1582 oil and California Platform Irene) and rened products (a lubricating
oil, and a Diesel No. 2 from Korea).
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126 Z. Wang et al.
Table 4. Peak identication of the triaromatic steranes (TA-steranes, m/z 231) and
monoaromatic (MA-steranes, m/z 253) in the NIST SRM 1582 oil
Peak No. Compounds Molecular formula
Triaromatic steranes (TA-steranes, m/z 231)
1 C
20
TA-sterane (X=ethyl) C
20
H
20
2 C
21
TA-sterane (X=2-propyl) C
21
H
22
3 C
22
TA-sterane (X=2-butyl) C
22
H
24
(a and b are epimers at C-19)
4 C
26
TA-chloestane (20S) C
26
H
32
5 C
26
TA-chloestane (20R) C
26
H
32
+C
27
TA-ergostane (20S) C
27
H
34
6 C
28
TA-stigmastane (20S) C
28
H
36
7 C
27
TA-ergostane (20R) C
27
H
34
8 C
28
TA-stigmastane (20R) C
28
H
36
Monoaromatic steranes (MA-steranes, m/z 253)
1 C
21
MA-sterane (X=ethyl) C
21
H
30
2 C
22
MA-sterane (X=2-propyl) C
22
H
32
3 C
23
MA-sterane (X=2-butyl) C
23
H
34
(a and b are epimers at C-20)
4 C
27
5(H) MA-cholestane (20S) C
27
H
42
5 C
27
MA-diacholestane (20S) C
27
H
42
6 C
27
5(H) MA-cholestane (20R) C
27
H
42
+ C
27
MA-diacholestane (20R)
7 C
27
5(H) MA-cholestane (20S) C
27
H
42
8 C
28
5(H) MA-ergostane (20S) C
28
H
44
+ C
28
MA-diaergostane (20S)
9 C
27
5(H) MA-cholestane (20R) C
27
H
42
10 C
28
5(H) MA-ergostane (20S) C
28
H
44
11 C
28
5(H) MA-ergostane (20R) C
28
H
44
+ C
28
MA-diaergostane (20R)
12 C
29
5(H) MA-stigmastane (20S) C
29
H
46
+ C
29
MA-diastigmastane (20S)
13 C
29
5(H) MA-stigmastane (20S) C
29
H
46
14 C
28
5(H) MA-ergostane (20R) C
28
H
44
15 C
29
5(H) MA-stigmastane (20R) C
29
H
46
16 C
29
5(H) MA-stigmastane (20R) C
29
H
46
17 C
30
5(H) MA-sterane (20S) C
30
H
48
ABC-ring triaromatic steranes (TA, m/z 231, right panel) in the
aromatic hydrocarbon fractions of crude oils (NIST 1582 oil
and California Platform Irene) and rened products (IFO-180,
a lubricating industrial oil, and a Diesel No. 2 from Korea), re-
spectively. Peak identication is summarized in Table 4. The
m/z 231 mass chromatograms are characterized by series of
20R and 20S C
26
-C
27
-C
28
triaromatic steranes plus C
20
to C
22
TA-steranes. All target TA-steranes are well resolved under the
present GC conditions except that the C
26
20R isomer coelutes
with the C
27
20S isomer. The m/z 253 mass chromatograms
are featured by series of 20R and 20S C
27
-C
28
-C
29
5(H) and
5(H) MA-steranes, as well as rearranged ring-C 20S and
20R MA-diasteranes (Peters and Moldowan, 1993). The struc-
ture of rearranged MA-steranes has been established as 10-
desmethyl 5- and 5-methyl (20S and 20R) MA-diasterane
isomers (Moldowan and Fago, 1986; Riolo and Albrecht, 1985).
The triaromatic sterane mass fragmentograms at m/z 231 clearly
show fewer and better resolved peaks and are therefore eas-
ier to use than the more complicated m/z 253 fragmentograms
of monoaromatic steranes, which typically contain several co-
eluting peaks.
Figure 13 shows apparent differences in the relative distri-
butions and absolute concentrations of TA- and MA-steranes
between oils and rened products. Lubricating oils demonstrate
signicantly lower concentrations of TA-steranes than crude oils
because most aromatic hydrocarbons have been removed during
the rening process. The Korea Diesel No. 2 also contains a
relatively large quantity of high carbon number TA-steranes.
Furthermore, triaromatic steranes are much more abundant than
monoaromatic steranes for all oils studied. In many lighter oils
such as Cook Inlet, Federated, West Texas, and Scotia Light, only
trace MA-steranes are detected. This implies that TA-steranes
can be more valuable marker compounds than MA-steranes in
forensic investigations. Similarly, lubricating oils do not or only
contain trace levels of MA-steranes. Synthetic lubricants (such
as Marinus Turbine oil and Valve oil), as expected, should not
contain any TA- or MA-sterane compound. However, GC/MS
analyses showthat apparent TA-steranes are present in the syn-
thetic 10W-30 lubricating oil (chromatogram not shown), in-
dicating that this lube oil may not be 100% synthesized as
claimed and may be composed of portions of petroleum-derived
hydrocarbons.
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Sesquiterpanes in Oils and Lighter Petroleum Products
The bicyclic biomarkers comprise one of the terpenoid classes.
Sesquiterpanes with the drimane skeleton are ubiquitous com-
ponents of crude oils and ancient sediments. Most bicyclic
sesquiterpanes probably originate not only from higher plants,
but also from algae and bacteria (Alexander et al., 1984; Fan
et al., 1991; Philp, 1985). Philp et al. (1981) have suggested tri-
cyclic diterpanes from higher plants as a source of bicyclic ter-
panes through opening of C-ring during maturation. The relative
concentration of C
14
sesquiterpanes decreases with increasing
maturation of the parent organic matter. The concentrations of
C
14
bicyclic sesquiterpanes are higher at the immature stage,
whereas those of C
15
drimane and C
16
homodrimane are rel-
atively lower. As their higher molecular weight precursors de-
hydroxylate, the concentrations of drimane (C
15
) and homod-
rimanes (C
16
) gradually increase and the concentrations of C
14
sesquiterpanes reduce (Cheng et al., 1991).
For lighter petroleum products, the high boiling-point penta-
cyclic triterpanes and steranes are generally absent or in low
abundance (Figure 10), while the smaller sesquiterpanes are
more concentrated in these distillates. The sesquiterpanes are
monitored using m/z 123, a base fragment ion common to all
sesquiterpanes (Figure 5). Conrmation ions for the sesquiter-
panes include m/z 179 (the ion after sesquiterpane C
14
H
26
loses
CH
3
), 193 (the ion after C
15
H
28
loses CH
3
and after C
16
H
30
loses C
2
H
5
, and 207 (the ion after C
16
H
30
loses CH
3
). Exami-
nation of GC/MS chromatograms of these characteristic ions of
sesquiterpanes provide highly diagnostic means of correlation,
differentiation, and source identication for lighter petroleum
products, in comparison to the use of other hydrocarbon groups.
The sesquiterpanes ranging fromC
14
to C
16
elute between n-C
13
and n-C
16
in the SIM chromatogram of saturated hydrocar-
bon fraction. Peaks 1 and 2, peaks 3 to 6, and peaks 7 to 10
are identied as C
14
, C
15
, and C
16
sesquiterpanes, respectively.
Among these ten compounds, Peaks 5 and 10 are identied to
be 8(H)-drimane and 8(H)-homodrimane, respectively. The
distribution patterns of sesquiterpanes generally vary in crude
oils and in rened petroleum products from different sources.
Figure 14 shows GC/MS chromatograms of sesquiterpanes at
m/z 123 for example crude oils and petroleum products. ANS,
Arabian Light, and Scotia Light have high concentrations of
sesquiterpanes, with peak 10 (C
16
) being the most abundant for
the Alaska North Slope and Arabian Light, and peak 3 (C
15
)
the most abundant for Scotia Light, respectively. The Arabian
Light has the lowest concentration of C
14
sesquiterpanes (peaks
1 and 2), indicating this oil is highly mature. By contrast, the
heavy California API 11 oil has the highest concentration of
C
14
sesquiterpane (peak 1), indicating that this oil may be rela-
tivelyimmature. Sesquiterpanes are absent inverylight kerosene
and in heavy lubricating oils. However, the IFO-180 and HFO-
6303 (a Bunker C fuel) have relatively high concentrations of
sesquiterpanes. Jet A is characterized with peaks 3, 5, and 1 be-
ing the most abundant, while the diesel sample (2002, Ottawa)
is characterized with peaks 5 and 10 being equally abundant
and followed by peak 3. Fingerprinting a large number of mid-
dle distillate fuels like diesels demonstrate that sesquiterpanes
are quite abundant in diesels. The differences in distribution
patterns and concentrations of sesquiterpanes are often quite
apparent between diesels (Stout et al., 2005; Wang et al., 2005).
Furthermore, diagnostic ratios of selected paired sesquiter-
panes for a large number of oils and petroleum products have
also developed. In general, oils from different regions have ra-
tios that cover quite a wide range. For example, Figure 15 plots
the ratio of peak 4/peak 5 (C
15
) versus the ratio of peak 3/peak 5
(C
15
) for more than 50 crude oils and rened products (both iso-
mers 3 and 4 are normalized relative to isomer 5). It is apparent
there is a large scatter in this set of oils in the cross-plot data.
The values of peak 4/peak 5 and peak 3/peak 5 fall in ranges
of 0.2 to 1.2 and 0.1 to 2.1, respectively. Another feature of
Figure 15 is that related oils produce tight clusters on the plot
(such as the circle for Orimulsion samples fromdifferent batches
and of the original Orinoco Bitumen). This observation implies
that the ratios of sesquiterpane isomers, in combination with
other ngerprinting data, may be used to discriminate different
oils and to identify the source of spill samples. Oil spills were
reported and sampled on March 17 and 23, 1998, at a sewer
outlet owing into the Lachine Canal in Quebec. Following the
accident, a diesel fuel, which was suspected to be the source
of the spill, was collected from a reservoir at a pumping sta-
tion located in Lachine, Quebec. Biomarker ngerprinting of
the samples revealed that only trace amounts (<10 g/g oil)
of C
19
-C
24
tricyclic-terpanes, regular C
20
-C
22
steranes, and di-
asteranes were detected. However, the spill samples contained
signicant amount of sesquiterpanes. The GC/MS/SIM chro-
matogram (at m/z 123) and diagnostic ratios of target sesquiter-
panes of the spill samples were foundtobe nearlyidentical tothat
of the suspected-source diesel. The only noticeable difference
is that, compared with the suspected-source diesel, the spilled
sample had slightly higher abundances due to weathering. These
similarities, in combination with other hydrocarbon quantica-
tion results such as bulk hydrocarbon groups, n-alkane distri-
bution, and ngerprints of alkylated PAHs and diagnostic ratios
of source-specic PAH compounds (Wang et al., 2000), argued
strongly that the suspected diesel collected from the pumping
station close to the spill site was the source of the spilled diesel.
Diamondoid Compounds in Oils and Lighter
Petroleum Products
The group of diamondoids (collective term for adamantane
(C
10
), diamantane (C
14
), and their alkyl homologous series) is
another group of low-boiling cyclic biomarkers of interest to
environmental forensic scientists. Admantane was rst discov-
ered in crude oils in the early 1930s. Diamondoids are rigid,
three-dimensionally fused cyclohexane-ring alkanes that have
a diamond-like (cage-like) structure (Chen et al., 1996; Dahl
et al., 1999; Grice et al., 2000) and have been identied in crude
oils (Petrov, 1987; Williams et al., 1986; Wingert, 1992). The
diamond structure endows these molecules with a high ther-
mal stability and resistance to biodegradation. The laboratory
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128 Z. Wang et al.
Figure 14. Left panel: GC-MS chromatograms of sesquiterpanes at m/z 123 for light, medium and heavy crude oils including Alaska North Slop (ANS),
Arabian Light, Scotia Light oil (Nova Scotia), West Texas, and California API 11. Right panel: Comparison of SIM chromatograms of sesquiterpanes at
m/z 123 for petroleum products from light kerosene to heavy fuel oil.
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Figure 15. Cross plots of double ratios of Peak 4/Peak 5 versus Peak 3/Peak 5 of sesquiterpanes for over 50 different oils and rened products.
thermal cracking experiments (Dahl et al., 1999) showed that
diamondoids have a higher thermal stability than most other
hydrocarbons during thermal cracking of oil; therefore, dia-
mondoids become increasingly enriched in the residual oil or
condensate. The increase in methyldiamantane (C
15
) concentra-
tion is directly proportional to the extent of cracking, indicat-
ing that, under the conditions of the experiments, diamondoids
are neither destroyed nor created. Instead, they are conserved
and concentrated and hence can be considered a naturally oc-
curring internal standard by which the extent of oil crack-
ing can be determined. Adamantanes and diamantanes elute
in the ranges of n-C
10
and n-C
13
and n-C
15
and n-C
17,
re-
spectively, in the GC/MS chromatogram of saturated hydro-
carbon fraction. As an example, Figure 16 shows the total ion
chromatograms of the diamonoid compounds in Prudhoe Bay
oil. At least 26 diamondoid compounds can be identied and
among these 17 are adamantanes and nine diamantanes. Peak
assignments are presented in Table 5. Identication of diamon-
doid hydrocarbons are based on mass spectral data, compar-
ison with standards, and assignments in the literature (Chen
et al., 1996; Wingert, 1992). Adamantane and its alkyl sub-
stituted homologues are analyzed at m/z 136 (for adamantane),
135 (for methyl-adamantanes), 149 (for dimethyl-adamantanes),
163 (for trimethyl-adamantanes), and 177 (for tetramethyl-
adamantanes), whereas diamantane and its alkyl substituted ho-
mologues are measured at m/z 188 (for diamantane), 187 (for
methyl-diamantanes), 201 (for dimethyl-diamantanes), and 215
(for trimethyl-diamantanes). Studies on application of diamon-
doid hydrocarbons in the chemical ngerprinting of natural gas
condensate and gasoline (Stout and Douglas, 2004) and of crude
oils and rened products (Wang et al., 2005) have been recently
reported.
Comparison of Biomarker Distribution for Oil Correlation
and Differentiation
Oils and petroleum products from different sources may have
different distribution patterns of biomarkers. Various biomark-
ers can occur in different carbon ranges (Figure 7). Also,
the abundances or concentrations of individual biomarkers
could be markedly different. Therefore, qualitative and quan-
titative comparisons of biomarker distribution are impor-
tant for spill/source identication and correlation. In general,
comparison of biomarker distribution for oil correlation and
differentiation involves the following steps:
r
Whether target biomarkers detected in spill samples can be
found in the same dened carbon range of suspected source
candidates;
r
Whether the distribution patterns and proles of biomarkers
are matching;
r
Whether the abundances of target biomarkers are matching;
r
Whether there are any unique or unknown biomarker com-
pounds; and
r
Whether the diagnostic ratios of the major biomarkers are
matching.
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Figure 16. GC-MS chromatograms of diamondoid compounds in Prudhoe Bay oil for peak identication.
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Table 5. Peak identication of diamondoid compounds in Prudhoe Bay oil
Peak No. Compounds Abbreviations Base peak M
+
(m/z) Formula
Adamantanes
1 Adamantane A 136 136 C
10
H
16
2 1-Methyladamantane 1-MA 135 150 C
11
H
18
3 1,3-Dimethyladamantane 1,3-DMA 149 164 C
12
H
20
4 1,3,5-Trimethyladamantane 1,3,5-TMA 163 178 C
13
H
22
5 1,3,5,7-Tetramethyladamantane 1,3,5,7-TeMA 177 192 C
14
H
24
6 2-Methyladamantane 2-MA 135 150 C
11
H
18
7 1,4-Dimethyladamantane, cis 1,4-DMA, cis 149 164 C
12
H
20
8 1,4-Dimethyladamantane, trans 1,4-DMA, trans 149 164 C
12
H
20
9 1,3,6-Trimethyladamantane 1,3,6-TMA 163 178 C
13
H
22
10 1,2-Dimethyladamantane 1,2-DMA 149 164 C
12
H
20
11 1,3,4-Trimethyladamantane, cis 1,3,4-TMA, cis 163 178 C
13
H
22
12 1,3,4-Trimethyladamantane, trans 1,3,4-TMA, trans 163 178 C
13
H
22
13 1,2,5,7-Tetramethyladamantane 1,2,5,7-TeMA 177 192 C
14
H
24
14 1-Ethyladamantane 1-EA 135 164 C
12
H
20
15 1-Ethyl-3-methyladamantane 1-E-3-MA 149 178 C
13
H
22
16 1-Ethyl-3,5-dimethyladamantane 1-E-3,5-DMA 163 192 C
14
H
24
17 2-Ethyladamantane 2-EA 135 164 C
12
H
20
Diamantanes
18 Diamantane D 188 188 C
14
H
20
19 4-Methyldiamantane 4-MD 187 202 C
15
H
22
20 4,9-Dimethyldiamantane 4,9-DMD 201 216 C
16
H
24
21 1-Methyldiamantane 1-MD 187 202 C
15
H
22
22 1,4 & 2,4-dimethyldiamantane 1,4 & 2,4-DMD 201 216 C
16
H
24
16
H
24
24 Trimethyldiamantane TMD 215 230 C
17
H
26
25 3-Mthyldiamantane 3-MD 187 202 C
15
H
22
16
H
24
In most cases, disparity (i.e., no matching) among biomarker
distributions is strong evidence for lack of correlation be-
tween spill sample(s) and suspected source(s). Matching may
be an indication of correlation of spill sample(s) and suspected
source(s), but it is not necessarily a proof that samples are
from the same source. Based on analysis of triterpane distribu-
tion patterns and determination of two pentacyclic C
27
triter-
panes, Shen (1984) distinguished four Arabian crudes, which in
their weathered forms were extremely similar. Volkman et al.
(1992b) determined the distribution of various biomarker com-
pounds in a range of aquatic sediment samples to conrm the
presence of oil contamination and identify possible oil sources.
Among a number of pollution sources, lubricating oils were
identied as a major source of hydrocarbon pollution in many
estuaries and coastal areas around Australia. Currie et al. (1992)
proposed utilization of triterpanes to distinguish tarballs origi-
nated from Southeast Asia from those of Australian petroleum
sources. Barakat et al. (1997) studied the biomarker distribution
within ve crude oils from the Gulf of Suez, Egypt. The results
revealed signicant difference in biomarker distribution within
the oils, and the oils can be categorized into three groups. Type 1
oils show a high relative abundance of gammacerane, indicat-
ing a marine saline-source depositional environment. Further-
more, these oils have a predominance of C
35
to C
34
17(H)-
homohopanes. Type 2 oils have an oleanane content of more
than 20% of the concentration of C
30
hopane, indicating
they originated from an angiosperm-rich, tertiary source rock.
Type 3 oil has geochemical characteristics intermediate between
type 1 and 2 oils. Wang et al. (1999b) studied oil spilled after
a re broke out at a carpet factory in Acton Vale, Quebec, on
June 29, 1998. The GC/FID chromatograms of the spill samples
were markedly different fromthe suspected-source sample. Spill
samples were highlyweathered. However, the GC/MSbiomarker
analysis demonstrated that the distribution pattern of biomarker
terpanes and steranes were nearly identical for the highly weath-
ered spill and the relatively fresh suspected-source samples. In
addition of the presence of the regular biomarkers from C
21
to
C
35
, C
30
- hopane of high abundances were also observed. It
was concluded that the spilled oil was a Bunker C type fuel,
and it matched with the oil in the heat exchange equipment near
the boiler, suggesting the oil spilled in the river came from the
burned factory.
In some cases, unknown oil samples may have very similar
bulk chemical composition but markedly different biomarker
distribution. On March 28, 2001, three unknown oil sam-
ples were received from Montreal for product characterization,
correlation, and differentiation. The GC/FID screening results
show that three samples are hydraulic uid type products. The
samples have very similar GC proles. However, biomarker
characterization results demonstrated that samples 1 and 2 are
nearly identical in biomarker distribution patterns and concen-
trations, but sample 3 shows signicantly different biomarker
distribution from samples 1 and 2 (Wang et al., 2002). Con-
centrations of C
29
and C
30
hopanes in sample 3 match those
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132 Z. Wang et al.
in samples 1 and 2. Conversely, concentrations of C
23
and C
24
and the sum of C
31
to C
35
homohopanes are markedly lower and
higher, respectively, than those of the corresponding compounds
in samples 1 and 2. Consequently, the diagnostic ratios of target
biomarkers are very similar for samples 1 and 2, but apparently
different from sample 3. All these observations point toward the
conclusion that samples 1 and 2 are identical, whereas sample 3
comes from another source.
Diagnostic Ratios (Indices) and Cross-Plots
of Biomarkers
Biomarker diagnostic parameters have been long established and
are widely used by geochemists for oil correlation; determina-
tion of organic input and precursors; depositional environment;
assessment of thermal maturity; and evaluation of in-reservoir
oil biodegradation (Peters et al., 2005). Many diagnostic ratios
currently used in oil spill studies and environmental forensics
originate from geochemistry.
Diagnostic Ratios of Biomarkers
Genetic oiloil correlations are based on the concept that the
composition of biomarkers in spill samples does not differ from
those of the candidate source oils. Most biomarkers in spill sam-
ples and source oils, in particular those homologous series of
biomarkers with similar structure, show little or no changes in
their diagnostic ratios. An important benet of comparing diag-
nostic ratios of spilled oil and suspected source oils is that con-
centration effects are minimized. In addition, the use of ratios
tends to induce a self-normalizing effect on the data because
variations due to the uctuation of instrument operating con-
ditions day-to-day, operator, and matrix effects are minimized.
Therefore, comparison of diagnostic ratios reects more directly
differences of the target biomarker distributionbetweensamples.
Diagnostic ratios (DRs) can either be calculated fromquanti-
tative (i.e., compound concentrations) or semi-quantitative data
(i.e., peak areas or heights). Ratios of compound concentrations
or double normalized DRs as those proposed by Christensen
et al. (2004) describe the relative chemical composition. Three
types of DRs are listed in the following.
DR = a
S
n
/a
S
n
(1)
D = a
S
n
/
a
S
n
+a
S
n
(2)
DR =
DR
S
DR
S
+ DR
R
(3)
DR
S
= a
S
n
/a
S
n
or DR
S
= a
S
n
/
a
S
n
+a
S
n
DR
R
= a
R
n
/a
R
n
or DR
R
= a
R
n
/
a
R
n
+a
R
n
where a
S
n
is the peak area, peak height, or concentration of com-
pound n or the sumof several compounds in an oil sample and a
R
n
is the peak area, peak height, or concentration of the correspond-
ing compound(s) in the reference oil. Diagnostic biomarker ra-
tios frequently used as defensible indices by the environmental
chemists for identication, correlation, and differentiation of
spilled oils are summarized in Table 6. These ratios consist of
alkanes, terpanes, steranes, sesquiterpanes, and diamondoids.
Ratios are dened from Eq. (1) for simplicity, but can readily be
redened using Eqs. (2) and (3). Selection of diagnostic ratios
employed in oil spill studies is mainly based on source-specic
variables (e.g., specicity, diversity, and analytical precision). It
is important to realize that the suit of DRs as listed in Table 6
is neither inclusive nor appropriate for all oil spill identication
cases. In some spill cases, it may be prudent to include some
particularly characteristic ratios. In other situations, the abun-
dance of some biomarkers may be too low to obtain reliable
DRs. Thus, maintaining exibility in the selection of DRs to be
used in specic cases is important.
The triplet ratio, if present, generally varies in oils from dif-
ferent sources and is dependent upon sources, depositional envi-
ronment, and maturity. The ratio was rst used by Kvenvolden
et al. (1985) to study a North Slope crude, in which the ratio
is 2. The spilled Exxon Valdez oil (an Alaska North Slope
crude) and its residues also have triplet ratios of 2. Con-
versely, many tarballs and residues collected from the shore-
lines of the Prince William Sound were similar to each other
but chemically distinct from the spilled Exxon Valdez oil with
triplet ratios of 5. The triplet ratio, combined with other di-
agnostic biomarker ratios and isotopic compositions, revealed
that these non-Valdez tarballs originated from California with a
likely source being the Monterey Formation (Kvenvolden et al.,
1995). During the Arrow oil spill work, the ratio of the most
abundant C
29
to C
30
hopane as well as C
23
/C
24
, Ts/Tm, and
/( +) of C
27
, C
28
and C
29
steranes were dened
and used by Wang et al. (1994b) as reliable source indicators.
Similar approaches, combined with determinations of a number
of other source-specic marker ratios, were applied to charac-
terize oil samples fromthe Arctic Bafn Island spill (Wang et al.
1995a), oil on birds (Wang et al., 1997), the 25-year old wetland
Nipisi spills (Wang et al., 1998a), a mystery spill in Quebec
(Wang et al., 2001a), and the Detroit River oil spill (Wang et al.,
2004). Barakat et al. (2002) have proved the molecular ratios of
triaromatic steranes including C
28
TA 20R/C
28
TA 20S, C
27
TA
20R/C
28
TA 20R, and C
28
TA 20S/(C
26
TA 20R + C
27
TA 20S)
were useful source indicators for correlating naturally weath-
ered oil residues in the Egyptian Western Desert to a fresh crude
oil sample of the Western Desert-sourced oil. In January and
February 1996, a signicant number of tarball/patty incidents
occurred along the coasts of Vancouver Island, Washington,
Oregon, and California. Samples of the tarballs were collected
from the affected beaches and characterized by GC/FID and
GC/MS, and further analyzed using a carbon isotopic tech-
nique (Wang et al., 1998b). Biomarker characterization revealed
that the British Columbia and California samples have similar
diagnostic ratios of most biomarkers, but the California sam-
ples show lower ratios of C
23
/C
30
and C
24
/C
30
than the British
Columbia samples. Only after in combination with characteriza-
tion results of PAHs and PAHDRs, it was defensively concluded
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Table 6. Diagnostic ratios of biomarkers frequently used for the environmental forensic studies
Biomarker
classes Diagnostic ratios Code
Acyclic pristane/phytane pri/phy
isoprenoids pristane/n-C
17
pri/C
17
phytane/n-C
18
phy/C
18
Terpanes C
21
/C
23
tricyclic terpane
C
23
/C
24
tricyclic terpane,
C
23
tricyclic terpane/C
30
hopane
C
24
tricyclic terpane/C
30
hopane
C
24
tertracyclic/C
26
tricyclic (S)/C
26
tricyclic (R) terpane
C
27
18, 21-trisnorhopane/C
27
17, 21-trisnorhopane
C
28
bisnorhopane/C
30
hopane
C
29
-25-norhopane/C
30
hopane
C
29
-30-norhopane/C
30
hopane
oleanane/C
30
hopane
moretane(C
30
hopane)/C
30
hopane
Gammacerane/C
30
hopane
tricyclic terpanes (C
19
-C
26
)/C
30
hopane
C
31
homohopane (22S)/C
31
homohopane (22R)
C
32
bishomohopane (22S)/C
32
bishomohopane (22R)
C
33
trishomohopane (22S)/C
33
trishomohopane (22R)
Relative homohopane distribution
(C
31
-C
35
)/C
30
hopane
homohopane index
TR21/TR23
TR23/TR24
TR23/H30
TR24/H30
triplet ratio
Ts/Tm
H28/H30
NOR25H/H30
H29/H30
OL/H30
M30/H30
GAM/H30
(TR19-TR26)/H30
H31S/H31R
H32S/H32R
H33S/H33R
H31:H32:H33:H34:H35
(H31-H35)/H30
H31/(H31-H35) to
H35/(H31-H35)
Steranes Relative distribution of regular C
27
-C
28
-C
29
steranes
C
27
/C
29
steranes (at m/z 218)
C
28
/C
29
steranes (at m/z 218)
C
27
/(C
27
+C
28
+C
29
) (at m/z 218,
C
28
/(C
27
+C
28
+C
29
) (at m/z 218)
C
29
/(C
27
+C
28
+C
29
) (at m/z 218)
C
27
, C
28
, and C
29
/ epimers (at m/z 217)
C27:C28:C29 steranes
C27(S+R)/C29(S+R)
C28(S+R)/C29(S+R)
C27/(C27+C28+C29)
C28/(C27+C28+C29)
C29/(C27+C28+C29)
C27/C27
C28/C28
C29/C29
C
27
, C
28
, and C
29
20S/(20S + 20R) steranes C27 (20S)/C27 (20R)
C28 (20S)/C28 (20R)
C29 (20S)/C29 (20R)
C
30
sterane index: C
30
/(C
27
to C
30
) steranes selected C30/(C27 to C30) steranes
diasteranes/regular steranes
Regular C
27
-C
28
-C
29
steranes/C
30
-hopanes C27-C28-C29 steranes/H30
Sesquiterpanes Relative distribution of sesquiterpanes
Peak 5/Peak 3
Peak 10/Peak 3
Peak 1/Peak 3
Diamondoids Methyl adamantane index: 1-MA/(1- + 2-MA)
1,4-DMA, cis/1,4-DMA, trans
MAI
Dimethyl admantane index: 1,3-DMA/(1,3- + 1,4- +
1,2-DMA)
1,3,4-DMA, cis/1,3,4-DMA, trans
DMAI
Trimethyl admantane index: TMAI
1,3,4-DMA, cis/(1,3,4-DMA, cis + 1,3,4-DMA, trans)
Ethyl admantane index: 1-EA/(1- + 2-EA) EAI
Methyl-diamantane index: 4-MD/(1- + 3- + 4-MD) MDI
Relative distribution of diamantanes:
C
0
-D:C
1
-D:C
2
-D:C
3
-D
Triaromatic steranes C
20
TA/(C
20
TA + C
21
TA)
C
26
TA (20S)/sum of C
26
TA (20S) through C
28
TA (20R)
C
27
TA (20R)/C
28
TA (20R)
C
28
TA (20R)/C
28
TA (20S)
C
26
TA (20S)/[C
26
TA (20S) +C
28
TA (20S)]
C
28
TA (20S)/[C
26
TA (20S) +C
28
TA (20S)]
Monoaromatic steranes C
27
-C
28
-C
29
monoaromatic steranes (MA)
distribution.
The diagnostic ratios in Table 6 consist of alkanes, terpanes, steranes, sesquiterpanes, and diamondoids. Ratios are dened from Eq. (1)
for simplicity, but can be readily redened using Eqs. (2) and (3). For example, in accordance with Eq. (2), the ratio of C
29
-30-
norhopane/C
30
hopane (H29/H30) can be readily redened as H29/(H29 + H30).
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134 Z. Wang et al.
that: 1) California/Oregon samples were chemically similar and
consistent with the same source of a bunker type fuel; 2) British
Columbia tarball samples were chemicallysimilar andconsistent
with the same source (also bunker type fuel). They were sim-
ilar to the California/Oregon samples but may have a different
source; 3) the spilled samples had been highly weathered since
release, and the California samples were more heavily weathered
than the BCsamples; and 4) the source of the tarball samples was
neither Alaska North Slope nor California Monterey Miocene
oil.
In application of DRs of biomarkers for spill studies, it is im-
portant to acknowledge that regardless of diagnostic parameters
used, a basic rule applied to all correlations and differentiations
should be:
r
No matching of biomarker distribution and/or DRs is strong
evidence for lack of a correlation between spill sample(s) and
suspected source(s); and
r
Matching may be an indication of correlation of spill sam-
ple(s) and suspected source(s), but is not necessarily a proof
for identity.
Hence, in order to make more reliable and defensible corre-
lations, the use of a multi-criteria approach is often a prereq-
uisite. In a multi-criteria approach the nal conclusion is based
on analysis and evaluation of the distribution of more than one
suite of petroleum compounds (Christensen et al., 2004; Daling
et al., 2002; Peters et al., 2005; Stout et al., 2002; Wang et al.,
1999a).
Cross-Plots of Biomarkers
Cross-plots (i.e., plot of one diagnostic biomarker ratio vs. an-
other ratio) are frequently used in oil geochemistry for oiloil
correlation and determination of oil source and depositional
environment. Cross-plots have, for example, been used exten-
sively (Jiang and Li, 2002; Jiang et al., 2001; Li and Jian,
2001) in the project of Bakken/Madison petroleum systems
in the Canadian Williston Basin for oil-source rock correla-
tions and for distinguishing two source rocks with distinct dis-
tribution patterns of biomarkers. G urgey (2002) analyzed 56
rock and 28 crude oil samples from the sub-salt and supra-
salt section of the southern Precaspian Basin. Source-specic
biomarker ratios reveal two populations: Population 1 and Pop-
ulation 2. Seifeit and Moldowan (1986) applied cross-plots of
C
29
/( +) sterane versus C
29
20S/(20S + 20R)
steranes as a particularly effective measure in describing the
thermal maturity of source rocks or oils. Based on cross-plots of
relative abundance (at m/z 123) of various isomeric sesquiter-
panes versus relative abundances of bicadinanes to C
30
hopane
on the m/z 412 mass chromatogram(bicadinane-T/C
30
-hopane),
crude oils from the eastern Pearl River Mouth Basin were clas-
sied into two groups (Zhang et al., 2003).
Malaysiancoasts are subjectedtovarious threats of petroleum
pollution including deliberate and accidental oil spills fromvari-
ous sources. The identication of detailed sources of the oil pol-
lution, therefore, is essential to reduce the oil pollution through
effective regulation. Based on chemical evidence that Middle
East crude oils were characterized by C
29
17, 21-norhopane
and C
31
-C
35
homohopanes, whereas these compounds were
depleted in Southeast Asian crude oils, Zakaria et al. (2000
and 2001) proposed utility of the cross-plots of C
29
/C
30
hopane ratio versus the homohopane index (C

31
-C
35
)/C
30
hopane as key biomarker indicators and successfully distin-
guished a large number of tarball samples that originated from
Southeast Asian crude oil sources from those of Middle East
sources.
As described previously, cross-plots of double ratios of
sesquiterpanes peak 4/peak 5 (C
15
) versus peak 3/peak 5 (C
15
)
for more than 50 crude oils and rened products are shown in
Figure 15. Different oils clearly demonstrate different values of
peak 4/peak 5 and peak 3/peak 5. Furthermore, related oils (such
as Orimulsion samples from different batches) produce a tight
cluster on the plot, indicating that they originate from the same
source oil: Orinoco Bitumen.
Unique Biomarker Compounds
Biomarker terpanes with a hopane skeleton and steranes are
common constituents of crude oils. However, some biomarker
compounds including several geologically rare acyclic alkanes
are found to exist only in certain oils and can, therefore, be
used as unique markers for oil spill identication. The ratios
of these unique biomarkers can furthermore provide additional
diagnostic information on the types of organic matter that give
rise to the crude oil. Some unique biomarkers (Figure 17), which
have been used to identify new class of oils by geochemists and
environmental chemists, are summarized below:
Botryococcane (C
34
H
70
, m/z 183): Botryococcane is an ir-
regular C
34
-isoprenoid compound, which is derived from botry-
ococcene known in a fresh to brackish water lacustrine green
algae. The occurrence of botryococcane in petroleum is limited
to a fewgeographic regions, including Australia (Mckirdy et al.,
1986) and Sumatra (Seifeit and Moldowan, 1981). Therefore,
characterization of this geologically rare botryococcane can be
highly specic. Mckirdy et al. (1986) identied botryococcane
in a new class of Australian non-marine crude oils, and de-
tected exceptionally high concentrations of botryococcane (C
34
botr/n-C
29
= 0.30.7) in coastal bitumens from southern Aus-
tralia (Mckirdyet al., 1994). Hwanget al. (2002) usedbotryococ-
cane as an effective key source indicator to genetically separate
the highly similar Sumatra oils into ve groups. On the con-
trary, commonly used source indicators including homologous
distribution of steranes, diasteranes, and hopanes are unable to
genetically group most of the studied Sumatra oils.
Methyl-hopane (CH
3
-C
30
H
51
, m/z 205): Ring A and B
methyl-hopane were rst detected in a series of Jurassic oils
from the Middle East (Seifeit and Moldowan, 1978). The ring
A/Bfragment of the hopane series, normally m/z 191 (Figure 5),
is increased to m/z 205 by the additional methyl group. The
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Figure 17. Molecular structures of a selection of unique biomarkers: Botryococcane (C
34
H
70
), bicardinanes, C
30
17(H)-Diahopane, 18(H),21(H)-
30-norneohopane (C
29
Ts), 18(H)-oleanane, 18(H)-oleanane, 4-methyl-24-ethyl-cholestane, C
30
dinosteranes (4,23,24-trimethyl-cholestanes), and
macrocyclic alkanes and methylated macrocyclic alkane.
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136 Z. Wang et al.
most prominent series of these compounds identied in Pre-
cambrian oils has been identied as 2-methyl-17(H),21(H)-
hopane (Summons and Jahnke, 1992). Based on the fact that
methyl-hopanes are major polycyclic biomarkers in some Mid-
dle East oils from carbonate source strata but are uncommon
in Australian oils, lubricating oils, which contaminated many
estuaries and coastal areas around Australia, were identied
to be originated from Middle East crudes (Volkman et al.,
1992a).
Extended hopanes beyond C
40
: Hopanes with 30 or fewer
carbon atoms are often interpreted as diagenetic products of
C
30
hopanoids, while the extended hopanes (C
31
-C
35
) have
been related to C
35
precursors, such as bacteriohopane polyols,
aminopolyols, and a number of composite hopanoids (Ourisson
and Albrecht, 1992; Rohmer et al., 1992). A series of side-
chain extended 17(H), 21(H)-hopanes and 17(H), 21(H)-
hopanes up to C
44
has been identied in crude oils and source
rock extracts in the Liaohe Basin, northeast China (Wang et al.,
1996). These compounds may be viewed as the representatives
of a new class of molecules whose natural distribution and bio-
logical signicance remains to be determined.
Bicadinanes (C
30
H
52
, m/z 191 and 217 ): Bicardinanes are
C
30
-pentacyclic biomarker compounds and have three con-
gurations, labeled as W (cis-cis-trans-bicadinane), T (trans-
trans-trans-bicadinane), and R (cardinane). The mass spectra
of bicadinane contain prominent m/z 191 and 217 fragments,
whereas peaks can appear in corresponding chromatograms of
both hopanes and steranes. All three bicadinanes (W, T, and R
form) elute prior to C
29
hopane in the m/z 191 chromatogram.
But it can be conveniently monitored with little interference
using the m/z 412 fragmentogram. They are highly specic
for resinous input from certain families of higher plants (van
Aarssen et al., 1990). Based on bicadinane composition, crude
oils fromthe north, central, and south Sumatra Basins, Indonesia
were classied into three types (Sosrowidjojo et al., 1994), and
Group II oils were further distinguished from Group 1 oils by
their high abundance of bicadinanes relative to C
30
hopane on
the m/z 412 mass chromatogram.
C
30
17(H)-di ahopane(C
30
H
52
): Moldowan et al. (1991)
identied two rearranged hopanes from Prudhoe Bay oil,
C
30
17(H)-diahopane (C
30
) and 18(H), 21(H)-30-
norneohopane (C
29
Ts) by x-ray crystallography and advanced
NMRmethods. C
30
* elutes right after C
29
norhopane and C
29
Ts in the m/z 191 mass chromatogram. C
30
17(H)-diahopane
has been regarded as a possible terrestrial marker. Recently, El-
Gayar et al. (2002) characterized seven oils representing the dif-
ferent petroleum-bearing basins in the Western Desert, Egypt.
The characterization indicated that Type 2 and Type 3 oils are
similar and show relative high pristane/phytane ratios, paucity
of C
30
steranes, and high relative abundance of C
30
*, suggesting
that they probably originated from source rocks containing sig-
nicant proportions of higher plant material.
18(H)-oleanane: Oleanane has two isomers: 18(H)-
oleanane and 18(H)-oleanane. The -type conguration has
the higher thermodynamic stability, thus, it is the predominant
conguration in mature crude oils and rocks (Riva et al., 1988).
18(H)-Oleanane is a biomarker found only in Tertiary and
Cretaceous (<130 million years) rocks and oils. No examples
of oleananes have been found in oils known to be older than
Cretaceous. 18(H)-Oleanane is highly specic for angiosper-
mous plant input. The presence of 18(H)-oleanane in benthic
sediments in Prince William Sound, combined with its absence
in Alaska North Slope crude and specically in Exxon-Valdez
oil and its residue, conrmed another petrogenic source (Bence
et al., 1996; Kvenvolden et al., 1993; Page et al., 1996). Charac-
terization of 18(H)-oleanane in oils from the Anaco area and
Maturin subbasin, Venezuela, has been used for organic type
and age indicator for assessment of Venezuelan petroleum sys-
tem (Alberdi and Lopez, 2000).
4-Methyl steranes: The 4-methyl steranes can be divided
into two major classes: 1) C
28
-C
30
analogues of the steranes at
positions 4 and 24 (e.g., the C
30
sterane is 4-methyl-24-ethyl-
cholestane), and 2) C
30
dinosteranes (e.g., 4,23,24-trimethyl-
cholestanes). 4-Methyl-24-ethyl-cholestanes often occur in
relatively high abundance in Tertiary source rocks and related
oils from China (Fu et al., 1992). For example, almost all of
the oils from the eastern Pearl River Mouth Basin contain sig-
nicant amounts of 4-methyl-steranes (Zhang et al., 2003). Hu
(1991) found that 4-methyl-steranes in the range of C
28
-C
30
are
unusually rich (2040%of the total steranes) in certain oils from
terrestrial facies within the South China Sea. C
30
4-methyl-
sterane (M
+
= 414) is particularly abundant among the 4-
methyl-steranes. Rogers et al. (1999) have investigated oil seeps
and stains from the East Coast Basin, New Zealand. They found
that oils sampled from Hawkes Bay and Wairarapa have higher
abundances of C
30
regular steranes and 28,30-bisnorhopane
than oil seeps from the Raukumara Peninsula and Marlborough.
These biomarker analysis results have been used to distinguish
at least two distinct sources of hydrocarbons in the basin.
Dinosterane has only been reported in petroleum younger
than Triassic age (Summons et al., 1992). The presence of di-
nosterane in relatively high concentrations in asphaltic bitumens
fromsouthernAustralia (Mckirdyet al., 1994) suggests that their
source is no older than mid-Triassic. In a study to re-evaluate the
petroleumprospective potential in southeast Australia, Volkman
et al. (1992b) examined 10 bitumen samples collected between
1880 and 1915. The high proportions of C
27
steranes and pres-
ence of C
30
steranes including dinosteranes suggested that the
bitumens were derived from a marine source rock containing
mainly marine organic matter.
Macrocyclic alkanes: Murrisepp et al. (1994) rst reported
the presence of non-isoprenoidal macrocyclic-alkanes in sedi-
mentary material and tentatively identied these cyclic hydro-
carbons of the cyclododecane and cyclohexadecane series in the
non-aromatic hydrocarbon fractions of the semi-coking oil from
an Estonian oil shale. Recently, Audino et al. (2001 and 2002)
have unambiguously identied for the rst time a new class of
cyclic hydrocarbon biomarker, macrocyclic alkanes and their
methylated analogues in a Botryococcus brauniirich sediment
(torbanite) of Late Carboniferous age (Audino et al., 2001) and
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in two Indonesian crude oils (Audino et al., 2002). The com-
pounds consist of a homologous series of macrocyclic alkanes
in a wide range from C
15
to C
34
and their methylated deriva-
tives (ranging from C
17
to C
26
). The distribution of macrocyclic
alkanes was measured at the characteristic ion m/z 111. The
macrocyclic alkanes appear to be novel markers of B. braunii
and add to the catalogue of the characteristic hydrocarbons de-
rived fromthis algae. More importantly, these compounds could
be original markers specic to highly resistant algaenan of B.
braunii in sediments and crude oils.
The search for unique geochemical biomarkers continues to
be a fertile area of research for ngerprinting similar sources
of petroleum. Note, however, that reliable biomarker interpre-
tation is usually based on a whole biomarker distribution chro-
matogram and a series of biomarker parameters. There is no
single parameter that can be exclusively used for unambigu-
ous source identication of unknown spills. Individual unique
biomarker parameters only become valuable and meaningful
when they are used together and agree with other biomarker
parameters.
Effects of Weathering on Chemical Composition of
Oil and Biomarkers
Weathering is dened as the combination of a wide variety of
physical, chemical, and biological processes which affect the
composition of spilled oil in the environment (NRC, 2002; Jor-
dan et al., 1980; Wang and Fingas, 1995). The main weathering
processes include 1) evaporation, 2) emulsication, 3) natural
dispersion, 4) dissolution, 5) microbial degradation, 6) photoox-
idation, and 7) other processes such as sedimentation, adhesion
onto surface of suspended particulate materials, and oil-ne in-
teraction. In the short term after a surcial oil spill, evaporation
is usually the single most important and dominant weathering
process, in particular for the light petroleum products such as
gasoline. Evaporation has the greatest effect on the amount of
oil remaining on water or land after a spill. In the rst few
days following a spill, the loss can be up to 70% of the vol-
ume of light crudes and petroleum products, and gasoline can
evaporate nearly completely above zero degrees under certain
circumstances. For heavy or residual oils, such as Bunker C oil,
the losses due to evaporation comprise only a small percentage
of the total volume. The rate at which oil evaporates depends
primarily on the oil composition. The more volatile components
an oil or fuel contains, the greater the extent and rate of its
evaporation (Fingas, 1995).
Chemical Composition Changes of Oils Due to Weathering
Weathering causes considerable changes in the physical prop-
erties and the chemical composition of spilled oil. The degree
(lightly, moderately, or severely weathered) and rate of weath-
ering is different for each spill and is controlled by a number
of spill conditions and natural processes, such as: 1) type of the
spilled oil and the chemical composition and concentrations of
oil components; 2) spill site and environmental conditions; and
3) natural population of indigenous bacteria. Weathered oil sam-
ples can generally be divided into three groups based on degree
of changes in their chemical composition:
1. Lightly weathered oils (<15% naturally weathered): the
abundances of low-end n-alkanes are signicantly reduced.
Furthermore, BTEX and C
3
-benzene compounds are lost.
2. Moderately weathered oils (1530% weathered): a signi-
cant loss of n-alkanes and low-molecular-weight isoprenoid
compounds has occurred. BTEX and C
3
-benzenes could be
completely lost (Wang et al., 1995b), and the loss of lighter
C
0
and C
1
- naphthalenes can be signicant. The ratio of
GC-resolved peaks to UCM is decreased considerable due
to the preferential loss of resolved hydrocarbons over the
unresolved complex hydrocarbons.
3. Severely weathered oils: not only n-alkanes but also branched
and cyclo-alkanes are heavily or completely lost, and the
UCM is pronounced. The BTEX and alkyl benzenes are
completely lost. PAHs and their alkylated homologous se-
ries could also be highly degraded, resulting in development
of a prole in each alkylated PAHfamily with the distribution
of C
0
< C
1
< C
2
< C
3
.
Hence it is a difcult and often impossible task to identify
severely weathered oil samples through recognition of n-alkane
and PAHdistribution patterns. However, the biomarker distribu-
tion patterns are often unaltered even in severely weathered oil
samples. Thus, ngerprinting terpane and sterane biomarkers
provides a powerful tool for tracking the source and correlation
and differentiation of weathered oils.
Effects of Weathering on Composition of Biomarkers
The chemical composition changes of numerous oils and
petroleum products with various weathering percentages by
evaporation have been thoroughly studied as part of the En-
vironment Canada Oil Spill Research Program. The studied oils
include the Alberta Sweet Mix Blend oil (0% to 45% weath-
ered) (Wang and Fingas, 1995); 19 EPAoils (EPAReport, 2003;
Wang et al., 2003a), and six US Mineral Management Service
oils (Wang et al., 2003b). For example, when the Alaska North
Slope oil was weathered by evaporation from 0% to 31%, the
loss of lower molecular weight n-alkanes and PAHs and alter-
ations in their relative distributions were apparent. Conversely,
no evaporative losses were observed for biomarkers (Figure 18).
Microbial degradation of petroleumhydrocarbons represents
one of the main mechanisms by which oil and oil-related hydro-
carbons are removed from contaminated sites. The degree and
rate of biodegradation are different for each spill and are con-
trolled by a greater number of variables than is physical weath-
ering. These variables could include spill conditions and natural
processes, such as type of the oil spilled, spill site, the ambient
and seasonal environmental conditions (such as temperature,
oxygen, nutrients, water activity, salinity, and pH), and compo-
sition and activities of the autochthonous microbial community.
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138 Z. Wang et al.
Figure 18. Distribution of terpanes (m/z 191) and steranes (m/z 218) for the ANS oil samples with evaporative weathering percentages at 0 and 31%.
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A number of laboratory biodegradation studies have been con-
ducted under controlled laboratory conditions (Atlas and Bartha,
1992; Blenkinsopp et al., 1996; Foght et al., 1998; Prince, 1993;
Swannel et al., 1996; Wang et al., 1998c) to better understand
the effects of biodegradationonpetroleumhydrocarbons. Ingen-
eral, biodegradation affects the oil composition in the following
patterns (Blenkinsopp et al., 1996; Leahy and Colwell, 1990;
Wang et al., 1998c):
r
Smaller hydrocarbons are degraded faster than larger hydro-
carbons;
r
Straight-chain n-alkanes degrades faster than branched alka-
nes;
r
GC-resolved compounds are degraded more than GC-
unresolved complex hydrocarbons;
r
Lowmolecular weight aromatics are degraded faster than high
molecular weight aromatics;
r
Increase in alkylation level within alkylated homologous fam-
ilies signicantly decreases susceptibility to microbial attack;
and
r
Certainisomers withinisomeric PAHgroups are preferentially
degraded by microbes.
For biomarkers, lab biodegradation studies (Foght et al.,
1998; Blenkinsopp et al., 1996; Wang et al., 1998c) clearly
show that no sign of alteration in the composition of biomarkers
was observed, regardless of the oil type (light, middle or heavy),
incubation times (7, 14 and 28 days), or incubation conditions
(incubated at 4, 10, 15, and 22
C), with and without the presence

of nutrients. The concentrations of terpanes and steranes in
the tested oils were consistent, and the DRs of paired terpanes
and steranes remained constant. For example, the average of
the sum of eight target diagnostic biomarker ratios (including
C
23
/C
24
, Tm/Ts, C
29
/C
30
, C
32
(S)/C
32
(R), C
33
(S)/C
33
(R),
C
23
/C
30
, C
24
/C
30
, and C
27
/C
29
steranes) from 70
biodegradation samples of the Alberta Sweet Mix Blend oil
inoculated under various inoculums conditions during 1994
was 8.2 0.2 with relative standard deviation less than 4%.
As opposed to the biomarker compounds, n-alkanes, pristane,
and phytane were greatly reduced in the positive controls, and
n-C
17
/pristane, n-C
18
/phytane, and pristane/phytane ratios were
signicantly altered, indicating degradation of pristane and
phytane had also occurred.
Compared with the laboratory-controlled biodegradation,
biodegradation under eld conditions is generally a long-term
weathering process. Alterations in chemical composition of nat-
urally weathered oil spill samples are caused by the combina-
tion of various complex weathering processes. Figure 19 shows
the GC/FID and GC/MS chromatograms for TPH and n-alkane
analysis (right panel) and biomarker terpane analysis (left panel)
for 25-year-old Nipisi oil spill samples collected from the same
location and site but with different sampling depths. The sur-
face oil (02 cm) has been heavily weathered. Conversely, the
subsurface residual oil (>3040 cm) is still almost unaffected
by weathering, with GC chromatographic proles similar to the
reference oil. The microbial degradation of subsurface oil has
been slow since the peat in this wetland habitat is acidic, and
water saturated, i.e., largely anaerobic (Wang et al., 1998a). In
contrast to alkane and PAH groups, the biomarker composition
is nearly unaffected. The accumulation of terpanes relative to the
reference oil during the 25-year period of weathering is apparent,
especially for the severely weathered surface sample N2-1A: the
concentration of C
30
- hopane was approximately 1.8 times
that found in the reference oil, and ve diagnostic biomarker ra-
tios (C
23
/C
24
, Ts/Tm, C
29
/C
30
, C
32
22S/22R, and C
33
22S/22R)
were found to be consistent between samples as well.
Biodegradation of Biomarkers
Although terpanes and steranes are highly resistant to biodegra-
dation, several studies have shown that they can be degraded to
certain degree under severe weathering conditions (i.e., exten-
sive microbial degradation) (Chosson et al., 1991; Munoz et al.,
1997; Prince et al., 2002; Seifert et al., 1984). The Arrow(Wang
et al., 1994b) andBIOSoil spill studies (Prince et al., 2002; Wang
et al., 1995a) have demonstrated degradation of C
23
and C
24
tri-
cyclic terpanes. In addition, Tmis degraded faster relative to Ts,
even though Ts chromatographically elutes earlier than Tm. In
March 1986, sections of peaty mangrove in a tropical ecosys-
tem were polluted by Arabian Light crude oil. Eight years later,
Munoz et al. (1997) found that isoprenoids were severely de-
graded and the biomarker distribution altered. Norhopanes were
found to be the most biodegradation-resistant among the stud-
ied terpane and sterane groups and the C
30
-hopane appeared
more sensitive to weathering than its higher homologues. Based
on several geochemical studies, Peters and Moldowan (1993)
have created a quasi-stepwise sequence for assessing the ex-
tent to which biomarkers are degraded in the reservoir.
Three coastal sites, heavily oiled from the 1974 Metula oil
spill in the Strait of Magellan, Chile, were examined in May
1998 to determine the long-term fate and persistence of Metula
oil in a marine marsh environment (Wang et al., 2001b). Among
the characterized samples, the asphalt pavement samples were
the most heavily weathered, evidenced by a complete loss of
n-alkanes fromn-C
8
to n-C
41
and by depletion of more than 98%
of the alkylated PAHs. Even the most refractory biomarker com-
pounds were affected to varying degrees. Biomarkers showed
degradation in the following sequences:
r
Biomarkers were altered in the declining order of importance
as: diasteranes > C
27
steranes > tricyclic terpanes > penta-
cyclic terpanes > norhopanes (C
29
Ts) C
29
steranes.
r
Steranes degraded in the order of C
27
> C
28
> C
29
with the
stereochemical degradation sequence 20R steranes >
20(R+S) steranes > 20S steranes.
r
Degradation of terpane C
35
> C
34
> C
33
> C
32
> C
31
was
apparent with a signicantly preferential degradation of the
22R epimers over the 22S epimers.
r
C
30
--hopane appeared more degradable than the 22S
epimers of C
31
and C
32
homohopanes, but had roughly the
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140 Z. Wang et al.
Figure 19. GC-FID (left panel) and GC-MS-SIM chromatograms for TPH and n-alkane analysis and biomarker terpane analysis of 25-year-old Nipisi
oil spill samples collected from the same location and site but at varying sampling depths.
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same biodegradation rate as the 22R epimers of C
31
and
C
32
homohopanes and was signicantly more resistant to
degradation than the 22S and 22R epimers of C
34
and C
35
homohopanes.
r
C
29
-18(H), 21(H)-30-norneohopane and C
29
- 20R
and 20S stigmastanes were found to be the most degradation-
resistant terpane and sterane, respectively, among the studied
target biomarkers.
Determination of Weathered Percentages Using Biomarkers
Highly degradation-resistant oil components such as C
30
hopane or C2
29
norhopane have been applied as conserved
internal standards for more precise estimation of the weath-
ering degree and extent of the spilled residual oil (Butler et al.,
1991; Douglas et al., 1994; Prince et al., 1994; Wang et al.,
1995a):
P(%) =
1
C
S
C
W
100%
in which P is the weathered percentages of the weathered sam-
ples, and C
S
and C
W
are the concentrations of C
30
-hopane in
the source oil and weathered samples, respectively. It should be
noted, however, that the weatheredpercentages canbe still under-
estimated by using C
30
--hopane as an internal oil reference
for extremely degraded oil samples because C
30
--hopane un-
der such circumstances is itself partially depleted, such as in the
case of the Metula oil spill (Wang et al., 2001b). However, in
most cases, C
30
--hopane is the preferred choice for estimat-
ing weathered percentages because C
30
--hopane is often the
most abundant among C
19
to C
35
biomarkers and can thus be
quantied more accurately. For lighter rened products, such as
diesel samples, which generally do not contain high molecular
weight terpane and sterane compounds, the bicyclic sesquiter-
panes as well as a selection of the more conservative PAHs with a
high degree of alkylation such as C
3
- or C
4
-phenanthrenes can be
used as an alternative internal standard for estimating the degree
of weathering. Wang et al. (2005) have quantitatively studied the
effects of weathering on sesquiterpane distributions and devel-
oped a number of diagnostic sesquiterpane indices for source
identication of spilled diesels and for estimation of weathering
percentages of the spilled diesel samples.
Application of Multivariate Statistical Methods for
Biomarker Fingerprinting
Data analysis is an important part of chemical ngerprinting and
a broadcollectionof statistical techniques has beenusedfor eval-
uation of data in real oil spill cases. Data evaluation techniques
within environmental forensics and specically for spill/source
identication, oil correlation, and differentiation have tradition-
ally centered on univariate methods and comparison of DRs and
complex proles for subjective pattern matching such as those
applied in the tiered approaches (Uhler et al., 19981999; Wang
et al., 1999a) and the modied Nordtest method (Faksness et al.,
2002a). However, in spill cases with a large number of candi-
date sources (such as spills in the harbor area), subjective pattern
matching and univariate analysis becomes increasingly difcult
to use for defensibly linking spilled oil with its generic source. In
particular, when the spilled oil(s) and the suspected spill source
candidates are closely related, the use of multivariate statistical
methods would be benecial.
Multivariate methods have been used for data analysis in or-
ganic geochemistry since the 1980s (ygard et al., 1984; Telnaes
and Dahl, 1986) and have recently been adopted for oil spill iden-
tication within environmental forensic science (Aboul-Kassim
and Simoneit, 1995a, 1995b; Burns et al., 1997; Christensen
et al., 2004, 2005; De Luca et al., 2004; Hostettler et al., 2004;
Lavine et al., 1995; Mudge, 2002; Stella et al., 2002; Stout et al.,
2001) These powerful multivariate statistical tools include prin-
cipal components analysis (PCA), discriminant analysis, and
cluster analysis. Among them, PCA is the most widely used and
powerful multivariate analysis technique. It is used to transform
original sample composition data into new, smaller, and uncor-
related variables called principal components. For data sets with
a large number of interrelated variables, PCA is a powerful tool
for analyzing the structure of the data and reducing the dimen-
sionality of the pattern vectors.
Lavine et al. (1995) employed pattern recognition and PCAto
study spilled jet fuels that had undergone weathering in subsur-
face environment and then classied these fuels into types: JP-4,
Jet-4, JP-5, JP-8, and JPTS. Aboul-Kassimand Simoneit (1995a,
1995b) have used a variety of statistical techniques for source
oil identication. In their analysis of the aliphatic and aromatic
compositions in particulate fallout samples (PFSs) in Alexan-
dria, multivariate statistical analyses, including extended Q-
mode factor analysis and linear programming, were performed
to reduce the hydrocarbon data set into a meaningful number of
end members (sources). Their analyses indicated that there were
two signicant end members explaining 90% of the total varia-
tion among samples and conrming petrochemical (79.6%) and
thermogenic/pyrolytic (10.4%) sources in the PFS model. In a
study of sediment samples in the eastern harbor of Alexandria,
a similar multivariate statistical approach was used to determine
the end member compositions and evaluate sediment partition-
ing and transport in the eastern harbor area. They found that
untreated sewage was the main source of petroleum hydrocar-
bons in the eastern harbor area rather than direct inputs from
boating activities or urban run-off. Burns et al. (1997) used
PCA and a least-squares iterative matching procedure to allo-
cate PAHs in intertidal and subtidal sediment samples from the
Prince William Sound of Alaska to 30 potential sources. They
used PCA to identify 18 possible sources, including diesel oil,
diesel soot, spilled crude oil in various weathering states, natu-
ral background, creosote, and combustion products from human
activities and forest res. Subsequently, the least-squares model
was used to estimate the source mix, with the best least-squares
t of 36 PAH analytes including the parent and alkylated ho-
mologues of naphthalene, phenanthrene, uorene, dibenzothio-
phene, and chrysene. Isomers were grouped by number of car-
bons in side groups and PAH family and treated as individual
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142 Z. Wang et al.
analytes. Stout et al. (2001) analyzed a suite of diagnostic PAH
and biomarker ratios with PCA. The ratios were selected on the
basis of high analytical precision and lowusceptibility to weath-
ering. The analysis helped to identify the prime suspects for a
heavy fuel oil spill of unknown origin from66 candidate sources.
In a recent attempt to resolve the origin of background hydro-
carbons in the sediments of Prince William Sound and the Gulf
of Alaska, Mudge (2002) used partial least-squares regression
to reassess the percentage distribution of ve possible sources
(coal, seep oil, shales, and input from two rivers) to the hydro-
carbon loading in the Gulf of Alaska. The data analysis results
suggest mixed sources whose contributions varied signicantly
across the sampling area. Christensen et al. (2004) presented an
integrated methodology for forensic oil spill identication based
on GC/MS analysis, chromatographic data processing, variable-
outlier detection, multivariate data analysis, estimation of uncer-
tainties, and statistical evaluation. The combination of PCA and
statistical evaluation of sample similarities enabled the simulta-
neous analysis of a large number of diagnostic ratios of PAHs
and biomarkers. Weathering was taken into account by consid-
ering the sampling uncertainties estimated from replicate spill
samples. Statistical evaluation ensured an objective matching of
oil spill samples with suspected source oils as well as classi-
cation into positive match, probable match, and non-match. The
methodology correctly identied the source of two spill samples
(i.e., crude oils fromOsebergEast andOsebergFieldCenter) and
distinguished them from closely related source oils. Tarballs are
common along the southern California coastline. Hostettler et al.
(2004) ngerprinted a total of 128 tarballs and tar residues col-
lected fromcoastal locations using biomarkers and stable carbon
isotope parameters. Then a chemometric multivariate statistical
approach (hierarchical cluster analyses [HCA] and PCA) was
applied to the data to sort out the differences in the biomarker
ratios, to group samples according to genetic similarities, to test
for correlation between and within the sample groups, and to
attempt to relate the tar residues to possible local sources. The
data showed that the tarballs are of natural and not anthropogenic
origin and that all had originated from source rocks within the
Miocene Monterey Formation via shallow seeps offshore.
Recently, Christensen et al. (2005) presented a methodology
for objective analysis of sections of GC/MS chromatograms of
biomarker steranes at m/z 217. The method consisted of GC/MS
analysis, preprocessing of chromatograms (including derivatisa-
tion, normalization, and chromatographic alignment), and PCA
of selected chromatographic regions. The resolution power of
the PCA model was enhanced by deselecting the most uncer-
tain variables or scaling them according to their uncertainty,
using a weighted least-square (WLS) criterion. Furthermore,
the four principal components, found to be the optimal number
of components, was interpreted as: boiling point range (PC1),
clay content (PC2), carbon number distribution of sterols in the
source rock (PC3), and thermal maturity of the oil (PC4) from
a priori knowledge on biomarkers. The method was applied to
chromatograms of m/z 217 of oil spill samples and source oils.
Oil spill samples were collected from the coastal environment
in the weeks after the Baltic Carrier oil spill and were clusted in
principal components 1 to 4 with the potential source oil sam-
ples. In this case study, 101 digitized chromatograms at m/z
217 were used for forensic oil spill identication. The data is
composed of 51 source oils, 16 weathered Baltic Carrier oil
spill samples, 18 replicate references, six spill samples and 10
replicate analyses of selected source oils (101 oil samples in
total). The chromatograms comprised 2510 data points, which,
after preprocessing, were reduced to 1231 by omitting the parts
without chemical information. Finally, the spill samples were
matched to the source oils.
Numerous commercial software packages for unsupervised
multivariate statistical analysis are available on the market,
which enables the forensic chemist to apply a range of mul-
tivariate statistical methods on a complex data set. However, it
must be realized that these methods can be used by the chemist as
a black-box without prior knowledge of data preprocessing,
interpretation of results, and how the latter depends on initial
preprocessing and variable selection. This may lead to far from
optimal models with low-resolution powers and more severely to
misinterpretation of the outputs (e.g., loading and score plots).
Hence, multivariate methods have to be used and interpreted
with care, but if some general considerations are made during
the modeling, these methods can considerably improve the anal-
ysis of complex ngerprinting data of petroleum biomarkers.
Conclusions
Forensic biomarker ngerprinting and data interpretation have
made great advances in the past two decades. This article pro-
vided a brief overview and discussion of biomarker chemistry,
biomarker characterization and quantication, biomarker dis-
tributions, weathering effects on biomarker composition, bi-
cyclic biomarker sesquiterpanes and diamondoids, DRs and
cross-plots of biomarkers, unique biomarkers, application of
biomarker ngerprinting techniques for spill source identi-
cation, and application of multivariate statistical analysis for
biomarker ngerprinting. It should be noted, however, that in
any oil spill investigation cases, particularly for complex hy-
drocarbon mixtures or extensively weathered and degraded oil
residues, there is no single ngerprinting technique that can fully
and readily meet the objectives of forensic investigation and
quantitatively allocate hydrocarbons to their respective sources.
Combined and integrated multiple tools are often necessary un-
der such situations.
Advancements in spilled oil ngerprinting techniques includ-
ing use of emerging instrumental techniques and further de-
velopment of multivariate statistical tools for data evaluation
will continue. It can be anticipated that these developments will
further enhance the utility and defensibility of oil spill charac-
terization and source identication.
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C, respectively. The temperature program

C and hold for 20 min-

C hold for 2 minutes, ramp

C and then 1.5

C and hold for

C, the term petroleum solvent is often used synony-

C) boiling point region. Lubricat-

hopane ratio versus the homohopane index (C

C), with and without the presence

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