Organic

Geochemistry
Organic Geochemistry 37 (2006) 787–797
www.elsevier.com/locate/orggeochem

25-Norhopanes: Formation during biodegradation of

petroleum in the subsurface
a,*
Barry Bennett , Milovan Fustic a, Paul Farrimond b, Haiping Huang a,
Stephen R. Larter a
a
Petroleum Reservoir Group (PRG), Department of Geology and Geophysics, University of Calgary, 2500 University Drive NW,
Calgary, Alberta, Canada T2N 1N4
b
Integrated Geochemical Interpretation Limited, Hallsannery, Bideford, Devon EX39 5HE, UK

Received 3 January 2006; received in revised form 1 March 2006; accepted 13 March 2006
Available online 6 June 2006

Abstract

Quantitative data from petroleum systems in China (Liaohe basin) and Canada (Athabasca tar sands) support the the-
ory that 25-norhopanes are produced during biodegradation of petroleum in the subsurface. Within a single oil column,
both case histories showed increasing severity of degradation, indicated by destruction of hopanes and production of 25-
norhopanes downward to the oil–water contact. In the Athabasca samples between the [Peters, K.E., Moldowan, J.M.,
1993. The Biomarker Guide: Interpreting Molecular Fossils in Petroleum and Ancient Sediments. Prentice Hall, Engle-
wood Cliffs, New Jersey, p. 363] scale of biodegradation levels 5–9, concentrations of C28 20S triaromatic steroids and
other biodegradation-resistant compounds increased by 35%, reflecting a concentration effect as a consequence of removal
of more degradable compounds. Over the same interval, the concentrations of C28 17a 25-norhopane and C29 17a 25-
norhopane increased by an order of magnitude, thus requiring that the balance be met by their net production during
degradation.
A detailed molecular investigation of the Athabasca bitumen revealed that C30 17a hopane degrades faster than C29 17a
hopane, whilst the rate of formation of both C29 17a 25-norhopane and C28 17a 25-norhopane are similar, complicating a
straightforward interpretation of demethylation of hopanes to form 25-norhopanes. Hopane degradation in the Athabasca
tar sand may also occur without the production of 25-norhopanes. The results show that even within a single petroleum accu-
mulation, a number of mechanisms control changes in the abundance and composition of hopanes and 25-norhopanes.
 2006 Elsevier Ltd. All rights reserved.

1. Introduction degradation. However, despite this useful character-

istic, the origin of 25-norhopanes remains a
The presence of 25-norhopanes in crude oils is controversial subject. Three hypotheses prevail for
commonly recognised as an indicator of severe bio- the origin of 25-norhopanes in biodegraded oils:
(i) microbial demethylation of hopanes to 25-norho-
* panes through the removal of the methyl group at
Corresponding author. Tel.: +1 403 210 3916; fax: +1 403
2840074. C-10 in the hopane nucleus (Seifert and Moldowan,
E-mail address: bennettb@ucalgary.ca (B. Bennett). 1979; Peters and Moldowan, 1991; Moldowan and

0146-6380/$ - see front matter  2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.orggeochem.2006.03.003
788 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797
McCaffrey, 1995), (ii) a relative enrichment of 25-
norhopanes derived from source rocks and already
present in the nonbiodegraded oil as a result of their
greater resistance to degradation (Chosson et al.,
1992) and (iii) the microbes responsible for severe
petroleum degradation may produce 25-norhopanes
without any direct relationship to degradation of
hopane (Peters et al., 2005).
The formation of 25-norhopanes through the
microbial removal of the methyl group at C-10 in
the hopane nucleus during the biodegradation of
petroleum is well documented in the literature (Seif-
ert and Moldowan, 1979; Rullkötter and Wendisch,
1982; Volkman et al., 1983; Requejo and Halpern,
1989; Peters and Moldowan, 1991; Moldowan and
McCaffrey, 1995; Peters et al., 1996; Nascimento
et al., 1999; Alberdi et al., 2001; Tocco and Alberdi,
2002), and evidence supporting microbially induced
demethylation of hopanes to the 25-norhopanes is
clear. In a continuous oil column in a diatomite res-
ervoir in California, Moldowan and McCaffrey
(1995) were able to show a concomitant and inverse
change with depth in concentrations of C35 hopane
and C29 hopane and their C34 25-norhopane and
C28 25-norhopane counterparts. A mass balance
approach using nondegraded diasteranes as ‘‘inter-
nal standards’’ also demonstrated that the concen-
trations of 25-norhopanes in heavily degraded oils
were similar to the concentrations of hopanes in
related nonbiodegraded oils (Volkman et al., 1983;
Peters and Moldowan, 1991).
Evidence supporting the existence of 25-norho-
panes in source rocks is also in no doubt, although
the C28 and C29 demethylated hopanes are minor in
comparison to C29 17a hopane (Chosson et al.,
1992) and their distributions are usually only repre-
sented by the C29 17a 25-norhopane with a few
other members of the series, rather than the full
C29-C35 pseudo-homologous series typically found
in degraded oils. They have been reported as free
hydrocarbons in source rocks from Western Austra-
lia, although their origin was attributed to a
reworked component of organic matter deriving
from biodegraded seeps (Noble et al., 1985). 25-
Norhopanes have also been found in supposedly
nondegraded oils (Blanc and Connan, 1992), sup-
porting the potential explanation that their high rel-
ative abundance in some degraded oils is due to
removal of more labile components (Chosson
et al., 1992).
Despite conflicting opinions on the origin of 25-
norhopanes, it is widely accepted that crude oil con-
taining n-alkanes, isoprenoid alkanes, and 25-
norhopanes is a mixture of a heavily degraded oil
(palaeobiodegraded oil containing 25-norhopanes)
and ‘‘fresh oil’’ containing n-alkanes (Volkman
et al., 1983; Philp, 1983; Howell et al., 1984; Blanc
and Connan, 1992; Peters and Moldowan, 1993;
Mason et al., 1995; Campos et al., 1996; Rooney
et al., 1998; López et al., 1998; George et al.,
1998, 2002; Scotchman et al., 1998; Dzou et al.,
1999; Nascimento et al., 1999; Grimalt et al.,
2002; Pan et al., 2003). Considering the number of
studies that use 25-norhopanes as indicators of pal-
aeodegraded oil, it is important to define the true
significance and limitations of 25-norhopanes when
identified in petroleum accumulations. In this paper,
we investigate the inter-relationship of 25-norho-
panes and hopanes in two petroleum systems using
a quantitative mass balance approach. We show
that although hopanes decrease while 25-norho-
panes increase during severe biodegradation, mass
balance arguments show that the relationship of
hopanes to 25-norhopanes does not appear to be a
straightforward conversion.
2. Methods
2.1. Samples
The sample suite consisted of oil samples from
the Liaohe petroleum system of China and two
sets of previously frozen core samples from the
Athabasca tar sand deposit of Alberta, Canada.
A description of the Liaohe petroleum system
and effects of biodegradation on the petroleum
accumulation is in Huang et al. (2003). Briefly,
the Liaohe suite consists of eight oil samples from
1490.8 to 1645.5 m within a 160 m oil column in
the heavily biodegraded petroleum accumulation
in the youngest member of the Eocene Shahejie
Formation (ES1). For the Athabasca study, core
material was obtained from the Lower Cretaceous
McMurray Formation sandstone reservoir in the
Athabasca tar sands from two different locations
in a north–south direction. The main investigation
was based on seven bitumen-rich core samples
(Athabasca-1 well) representing the depth interval
from 4.4 m to a water saturated sand below
90.4 m. A second suite of samples (Athabasca-2
well) consisted of three bitumen-rich cores col-
lected from 486 to 488 m just above an oil–water
contact and above the regional unconformity with
Devonian carbonates.
 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797
2.2. Recovery of aliphatic hydrocarbons from oils and
core extracts
Saturated hydrocarbons were isolated from oil
and core extracts following the procedure described
by Bennett and Larter (2000). The Athabasca cores
were tar sands with the bitumen readily extracted by
repeatedly washing the core with solvent (dichloro-
methane; CH2Cl2) until the extracts were colourless.
The core extracts were de-asphaltened to obtain a
hexane-soluble fraction that was amenable to the
following solid-phase extraction (SPE) procedure.
Typically, 100 mg of oil or 50 mg de-asphaltened
core extract was absorbed onto a pre-weighed C18
nonendcapped (NEC) SPE cartridge. The hydrocar-
bon-containing fraction was collected in n-hexane
(5 ml). The solvent volume was reduced to 1/2 ml
under a gentle stream of nitrogen gas. The saturated
hydrocarbons were isolated from the hydrocarbon
fraction using a silver nitrate impregnated silica
(Ag + silica) SPE cartridge (Bennett and Larter,
2000). An aliquot (50 ll from 1/2 ml) of the n-hex-
ane (hydrocarbon-containing) eluent recovered dur-
ing C18 NEC SPE was added dropwise onto the
Ag + silica, and the saturated hydrocarbons were
eluted in 2 ml of n-hexane. Aromatic hydrocar-
bons were subsequently recovered in 4 ml of
CH2Cl2. The aliphatic and aromatic hydrocarbons
were analysed by GC-MS in selected ion monitoring
(SIM) mode.
2.3. Gas chromatography-mass spectrometry
Mass spectral characterisation of compounds in
the hydrocarbon fractions was carried out using
gas chromatography-mass spectrometry (GC-MS)
on a Hewlett-Packard 5890 GC (using splitless
injection) interfaced to a HP 5970B quadrupole
mass selective detector (electron input energy
70 eV, source temperature 250 C).
The saturated hydrocarbons were analysed using
a DB-5 fused silica capillary column (30 m ·
0.32 mm i.d. · 0.25 lm film thickness; J&W Scien-
tific). The oven temperature program was: 40 C
(initial time, 2 min) to 175 C at 10 C/min to
225 C at 6 C/min to 300 C at 4 C/min and held
at 300 C for 20 min.
The internal standards 5b(H)-cholane and squa-
lane were added to the core sample or oil prior to
C18 NEC SPE. Peak area integration during GC-
MS analysis was by MASS LAB. The relative
response factor (RRF) for individual saturated
789
hydrocarbons versus the internal standard 5b(H)-
cholane was assumed to be 1. Corrections for mass
spectral responses were introduced during the quan-
tification of C28 17a 25-norhopane (m/z 177) and
C30 17a hopane (m/z 191) to account for the fact
that both the AB and DE ring fragments contribute
to the peak areas in their respective mass chromato-
grams. The ratios of the AB/DE ring fragments
were determined from mass spectra of neighbouring
carbon number homologues. The relative abun-
dance of the AB and DE ring fragments from the
C29 17a 25-norhopane (m/z 177/m/z 191), C30 17a
22S and 22R 25-norhopanes (m/z 177/m/z 205)
are similar, giving a ratio of 0.47. For the correction
of C30 17a hopane the AB/DE ratio of 0.7 was
obtained from the relative response in the C29 17a
norhopane (m/z 191/m/z 177) and C31 17a 22S
and 22R homohopanes (m/z 191/m/z 205). These
response factors were determined in oil samples rel-
atively enriched in the 25-norhopanes and hopanes,
respectively, to avoid complications arising from co-
elution (see Fig. 1b, c).
3. Results and discussion
3.1. Determining the levels of petroleum
biodegradation and identification of 25-norhopanes
We focus our discussion on case studies from two
petroleum systems from China (Liaohe) and Wes-
tern Canada (Athabasca) where a range of biodeg-
radation levels was been encountered within each
petroleum accumulation (e.g., Huang et al., 2004).
The levels of petroleum degradation are usually
ranked according to the 10 point scale of Peters
and Moldowan (1993), with Peters and Moldowan
level 1 (abbreviated to PM 1) representing slightly
biodegraded oil and Peters and Moldowan level 10
(abbreviated to PM 10) severely degraded oil
(hopanes and diasteranes absent, C26–C29 aromatic
steroids attacked). The presence of 25-norhopanes
in biodegraded oils is usually an indication that an
accumulation has been heavily biodegraded (PM 6
or greater). In one of the most severely biodegraded
oils we analysed, the hopanes were degraded so
extensively (equivalent to PM 8) that the dominant
compound in the mass chromatogram typically
employed to identify hopanes (m/z 191) is C29 17a
25-norhopane (Fig. 1a). The distribution of 25-
norhopanes in the same oil consists of a fully devel-
oped regular series of C26–C34 25-norhopanes
including the demethylated counterparts of Ts and
790 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797

29N
m/z 191 (a)

29H
30H

28N
m/z 177 29N (b)

30N
26N 26N 31N
(Ts) (Tm) 32N
33N 34N
Intensity

30H
m/z 191 (c)

29H

31H
Tm 29N 32H
Ts 33H
34H 35H

Retention time (min)

Fig. 1. Mass chromatograms (a) m/z 191, displaying degraded hopanes and (b) m/z 177, demethylated hopanes in a severely (PM 8)
biodegraded oil, and (c) m/z 191, from a moderately biodegraded oil (PM 4). Homologous series of 25-norhopanes and hopanes having
17a stereochemistry are denoted by the carbon number followed by the letter N and H, respectively. The two commonly occurring
trisnorhopanes are designated Ts and Tm, while the demethylated compounds are labelled as 26N.

Tm (Fig. 1b). Overall, the distribution of the
25-norhopanes bears a strong resemblance to the
regular hopane series in a ‘‘less altered’’ oil sample
(compare Fig. 1b, c; c.f., Volkman et al., 1983), if
a correction is applied for the enhanced responses
in the m/z 177 and m/z 191, respectively, for C28
17a 25-norhopane and C30 17a hopane (Fig. 2). It
has been reported in some cases that demethylated
Ts and Tm are not observed and that the extended
C35-hopanes also show resistance during petroleum
degradation under surface weathering conditions
(Requejo and Halpern, 1989). These observations
have also been reported in biodegraded oils from
California, USA (Peters and Moldowan, 1991;
Moldowan and McCaffrey, 1995) and during petro-
leum degradation under laboratory aerobic condi-
tions (Bost et al., 2001). In contrast, the extended
hopanes may be degraded prior to the regular
hopanes during laboratory experiments (Goodwin
et al., 1983; Chosson et al., 1992) and in some bio-
degraded crude oils (Seifert and Moldowan, 1979;
Rullkötter and Wendisch, 1982; Seifert et al.,
1984). These differences in hopane and 25-norho-
pane characteristics imply that biodegradation
alteration of hopanes can proceed in numerous
ways.
 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797 791

2500
(a)
2000

1500
µg g-1 oil
1000

500

0
Tm

C35 22R
C29 17α

C33 22R
C31 22R

C32 22R

C34 22R

C35 22S
C31 22S

C32 22S

C33 22S

C34 22S
C30 17 α
Ts

2000
(b)

1500
µg g-1 oil

1000

500

C34 22R
C30 22R

C32 22R

C33 22R
Tm - C26

C30 22S

C32 22S

C33 22S

C34 22S
Ts - C26

C28 17α

C29 17α

C3122R
C3122S

Fig. 2. Histogram plot of the concentrations of (a) C27– C35 17a hopanes in a moderately degraded oil (PM 4, Fig. 1c) and (b) C26–C34 17a
25-norhopanes in a severely degraded oil (PM 8, Fig. 1b), following correction for the enhanced mass spectral contribution (see Section
2.3) from C28 17a 25-norhopane and C30 17a hopane.

3.2. A quantitative investigation into the relationship noids have been completely removed, and traces
of hopanes and 25-norhopanes of 25-norhopanes are present, indicating that the
oil is moderately biodegraded (PM 5), whereas at
3.2.1. Liaohe case study the base of the oil column, hopanes are being
We first describe the quantitative changes in the destroyed, indicating a petroleum degradation
hopanes and 25-norhopanes in a biodegraded level of PM 8. The behaviour of C30 17a hopane
petroleum accumulation that originated from and the presumed product C29 17a 25-norhopane
lacustrine source rocks in China. The sample suite are shown in Fig. 3a. Toward the base of the oil
was acquired from the Liaohe Basin and was part column C29 17a 25-norhopane gradually increases
of a detailed investigation of the changes in the in abundance, coinciding with the loss of C30
hydrocarbons and nitrogen compounds resulting 17a hopane; this is also depicted by the ratio
from varying degrees of biodegradation (Huang C29 17a 25-norhopane/C30 17a hopane versus
et al., 2003, 2004). The petroleum was generated depth (Fig. 3b). Cases where 25-norhopanes show
from deeply buried Eocene ES3 Formation source an inverse relationship with hopanes have been
rocks and accumulated in freshwater lacustrine recognised in other heavily degraded oilfields,
sediments of the Eocene ES3 and ES1 formations. including the Wabasca Field in Alberta, Canada
The bulk petroleum composition and molecular (Brooks et al., 1988), the Tia Juana Field in the
data show systematic biodegradation gradients Maracaibo Basin, Venezuela (Tocco and Alberdi,
within the oil column (160 m). At the top of the 2002) and the Cymric Field in California (Moldo-
ES1 oil column, the n-alkanes and acyclic isopre- wan and McCaffrey, 1995).
792 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797
(a)
Concentrations - µg/g oil
0 5000 10000
1450
C30 17α Hopane
Depth (m) 1500
1550
1600
1650
Fig. 3. (a) Concentrations of C30 17a hopane and C29 17a 25-norhopane and (b) ratio of C29 17a 25-norhopane/C30 17a hopane versus
depth in the ES1 oil column. The oil–water contact is located at 1650 m.
A number of lines of evidence from the present
case history support the formation of 25-norho-
panes in the reservoir. In terms of mass balance,
the concentrations of C29 17a 25-norhopane range
from 30 lg/g oil within the top of the oil column
to 1400 lg/g oil toward the base. A concentration
increase from 30 to 1400 lg C29 17a 25-norhopane
per gram oil without formation of the compound
would require that more than 90% of the oil was
consumed between biodegradation levels PM 5–8,
which seems unreasonable. Furthermore, in geneti-
cally related petroleum from the deeper but less bio-
degraded (PM 1–4; Huang et al., 2003) ES3
reservoir, no 25-norhopanes were detected, implying
that their presence in the more degraded ES1 reser-
voir is most likely linked to their neoformation dur-
ing higher levels of degradation.
The decrease in hopane and corresponding
increase in 25-norhopane abundance down hole
(Fig. 3a) is not a quantitative conversion (c.f.,
Moldowan and McCaffrey, 1995); C30 17a hopane
loss represents 8,700 lg/g oil, whilst C29 17a 25-
norhopane increases by only 1060 lg/g oil over the
depth interval 1561 to 1645 m. Thus, it appears that
demethylation of hopane may represent only one of
a number of degradation reactions contributing to
hopane loss.
3.2.2. Athabasca case study
Requejo and Halpern (1989) showed that in some
cases during biodegradation the extended (C31+)
hopanes may be preserved compared to the C29
and C30 hopanes. The converse also applies in some
cases (Seifert and Moldowan, 1979). If 25-norho-
panes are formed directly from hopanes, then one
(b)
C29 17α 25-Norhopane
C30 17α Hopane
0 500 1000 0 0.10 0.20 0.30
C29 17α 25-Norhopane
would expect to find the inverse response of 25-
norhopane and hopane abundance; i.e., where
extended hopanes are preserved, a lack of extended
25-norhopanes would be expected. The hopane and
25-norhopane composition data from Athabasca
affords such a comparative investigation.
Here we present quantitative data for hopanes
and 25-norhopanes in two wells from the Athabasca
tar sands deposit to further investigate the inter-
relationship of hopanes and 25-norhopanes.
Bitumens extracted from the Athabasca-1 well
samples show a range of biodegradation from PM
5 in the main oil column increasing downward to
PM 9 at the oil–water contact, where even diaster-
anes are attacked. Compositional changes have also
affected the hopane distributions, where C30 17a
hopane decreases more than C29 17a hopane
(Fig. 4). This feature has also been observed in oil
degradation experiments conducted under aerobic
conditions in the laboratory (Watson et al., 2002)
and in severely biodegraded oils from the South Bel-
ridge Field, California (Walters, 1993). Based on the
changing hopane distributions observed in the m/z
191 mass chromatogram, one would expect to find
higher amounts of C29 17a 25-norhopane compared
to C28 17a 25-norhopane in the m/z 177 mass chro-
matogram. For comparative purposes, the distribu-
tions of the 25-norhopanes as a function of depth
are shown in Fig. 4. At 27.7 m, trace contributions
from 25-norhopanes may be discernable, but with
increasing depth toward the oil–water contact, the
25-norhopanes become the major components in
the m/z 177 mass chromatogram (Fig. 4). Over the
depth interval 79–90.4 m, the relative abundances
of C28 and C29 17a 25-norhopanes are similar, with
 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797 793

m/z 191 30H m/z 177

27.7 m 27.7 m 29H (a)
29H
23T

Tm

35H 28N

m/z 191 m/z 177 29H

79 m 23T 29H 79 m (b)

29N

30N
28N s
* R+30H
Intensity

35H

m/z 191 m/z 177

90.4 m 90.4 m
23T 29H (c)

29N

28N

30N
s
R+30H
* 35H

Time

Fig. 4. Representative m/z 191 and m/z 177 mass fragmentograms of the saturated hydrocarbon fractions isolated from Athabasca-1 tar
sand samples (a) 27.7 m, (b) 79 m and (c) 90.4 m depth. Key to 191 mass fragmentograms: 23T, C23 tricyclic terpane; Tm, C27 17a-
22,29,30-trisnorhopane; 29H, C29 17a hopane; 30H, C30 17a hopane; 35H, C35 17 a (H) 22S and 22R homohopane, * = contribution due
to C29 17a (H) 25-norhopane. Key to m/z 177 mass fragmentograms: 28N, C28 17a 25-norhopane; 29N, C29 17a 25-norhopane; 30N, C30
17a 25-norhopane.

no apparent enhancement of the C29 17a 25-norho-
pane to match the more rapid decrease in the C30
17a hopane; thus a direct relationship between
hopane and 25-norhopane appears ambiguous.
The quantitative data in Figs. 5 and 6 confirm that
the concentrations of 25-norhopanes increase while
the hopanes decrease. Conversion of hopanes to 25-
norhopanes would be an appropriate explanation of
these down-hole concentration trends. However,
considering the data in more detail, the more rapid
removal of C30 17a hopane compared to C29 17a
hopane is not matched by greater production of
C29 17a 25-norhopane compared with C28 17a 25-
norhopane.
In a second Athabasca case study, three core
samples from the Athabasca-2 well were sampled
from just above the oil–water contact, lying above
the regional unconformity. The degradation of
hopane suggested a biodegradation level of PM 8,
although unlike the Athabasca-1 well samples, the
steranes and diasteranes were intact. However, like
the Athabasca-1 well samples, greater removal of
C30 17a hopane compared to C29 17a hopane was
observed toward the oil–water contact. The concen-
tration of C30 17a hopane fell from 2390 to 1040 lg/
g extract compared to a slight increase from 2600 to
2690 lg/g extract for C29 17a hopane. However,
there were no detectable 25-norhopanes in Athaba-
sca-2 well. The subsurface environment represented
by the Athabasca-2 well does not appear to support
25-norhopane formation, whereas in the Athabasca-
1 well 25-norhopanes are produced. The oil column
in the Athabasca-1 well overlies a water leg that is
within the McMurray Formation sandstones,
794 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797

µg /g extract whereas the oil column in Athabasca-2 well directly

overlies the Devonian the carbonates. Therefore,
0 500 1000 1500 2000 2500 differences in the local water chemistry may affect
10
the development of the microbial environment. Fur-
ther work is required in the Athabasca region with
supporting aquifer data to understand what envi-
20 ronmental factors control 25-norhopane formation.
In Eocene Wilcox oils from South Texas, the onset
of hopane degradation was indicated in the C29 and
C30 hopanes, but no 25-norhopanes were identified
40
(Williams et al., 1986). In studies of seep oils from
Depth (m)

Greece (Seifert et al., 1984), the Wessex Basin (Bigge

and Farrimond, 1998), and heavily degraded seep
oils from the Central Adriatic Basin (Moldowan
60
et al., 1992), no 25-norhopanes were identified even
though hopanes were degraded. Active seeps from
offshore West Africa and North West Scotland
80 (Wenger and Isaksen, 2002) showed extensive petro-
leum degradation without the formation of 25-
norhopanes. A suite of tar balls collected from
along the California coast was also devoid of 25-
100
norhopanes even though hopanes were degraded
Fig. 5. Plot of concentrations of C28 17a 25-norhopane (h), C29 (Hostettler et al., 2004).
17a 25-norhopane (D), C29 17a norhopane (j), and C30 17a Although 25-norhopanes have been reported in
hopane (m) versus depth (m) in an Athabasca-1 tar sand core.
source rocks and nondegraded oils (Blanc and Con-
nan, 1992) we present mass balance evidence that
requires the formation of 25-norhopanes to explain
the geochemical trends observed in the Athabasca
µg /g extract cores. We have quantified a suite of compounds that
0 50 100 150 200 250
are known to be highly resistant to petroleum degra-
0 dation: monoaromatic C30 8,14-secohopanes, C28
20S tri-aromatic steroids and C21aaa pregnane.
The concentrations of these ‘‘resistant’’ compounds
20
increase towards the oil–water contact (Fig. 6) con-
sistent with their relative enrichment as other more
easily degradable components are selectively
removed. On average, the concentrations of these
Depth (m)

40 resistant markers increase by 35% over the well

60
80
100
Fig. 6. Plot of concentrations of C28 17a 25-norhopane (h), C29
17a 25-norhopane (D), C28 triaromatic steroid (·), C21 aaa
pregnane (), and mono-aromatic C30 8,14-secohopane (d)
versus depth (m) in Athabasca-1 tar sand core.
interval and behave in a consistent manner, suggest-
ing that they are responding to the same process. In
contrast, the concentrations of 25-norhopanes
increase by an order of magnitude, indicating that
25-norhopanes are being produced during degrada-
tion in the Athabasca petroleum system.
3.2.3. Hopanes and 25-norhopanes: not a
straightforward relationship!
Brooks et al. (1988) suggested that at least two
mechanisms of biodegradation affect the biomarkers
of the Cretaceous oil sands/heavy oils of Alberta:
firstly in oils where regular steranes have been
removed, no 25-norhopanes were identified
 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797
(Athabasca tar sands); secondly, when regular ster-
anes were present, 25-norhopanes were identified
(Wabasca tar sands). Contrary to the findings of
Brooks et al. (1988) we have identified 25-norho-
panes in some Athabasca samples coinciding with
the degradation of steranes and diasteranes. And
in other Athabasca samples where the steranes
appear to be intact and C30 17a hopane was signif-
icantly removed; no 25-norhopanes were identified.
In summary, detailed quantitative molecular
investigations into the origin of 25-norhopanes
reveal more complexity in the apparent inter-rela-
tionship of hopanes and 25-norhopanes. In the lit-
erature, strong evidence for the conversion of
hopanes to 25-norhopanes is shown by the inverse
relationship between hopane and 25-norhopane
abundance (Moldowan and McCaffrey, 1995).
Our study of the Liaohe and Athabasca petroleum
deposits confirms this relationship, but shows that
conversion is not quantitative. Furthermore, data
from the Athabasca case study suggest that the
degree of conversion may be different for individual
carbon number species. The quantitative depth-cor-
related relationship between hopanes and 25-
norhopanes observed by Moldowan and McCaffrey
(1995) in the Cymric Field (California, USA) led
those authors to suggest that an oxidised intermedi-
ate was unlikely to accumulate. In the case histories
presented here, a 1:1 quantitative relationship was
not observed, suggesting that the balance in the
quantitative relationship may have to be met by
the formation of as yet unknown products that
may or may not be intermediates in 25-norhopane
formation. Candidate intermediates include hopa-
noid structures bearing functionalities at the C-25
carbon, but so far only a tentative identification
of 28,30-bisnorhopan-25-oic acid has been pro-
posed (de Lemos Scofield, 1990). The occurrence
of hopanoic acids and 25-norhopanoic acids in bio-
degraded oil may offer parallel information to that
obtained from hydrocarbon biomarkers (Nasci-
mento et al., 1999) and help shed light on the
behaviour of hopanes and 25-norhopanes. How-
ever, the hopanoic acids and 25-norhopanoic acids,
when present in biodegraded petroleums, occur in
concentrations orders of magnitude less than their
hydrocarbon counterparts (Bennett et al., unpub-
lished), suggesting they represent minor products
or short-lived intermediates.
Attempting to understand the inter-relationship
between hopanes and 25-norhopanes, remains chal-
lenging since there appears to be no consistent trend
795
amongst the hopane and 25-norhopane data sets
presented herein and in the literature. In the Ath-
abasca-1 well, C30 17a hopane degrades faster than
C29 17a hopane, while the rate of C28 and C29 17a
25-norhopane generation are similar, one would
expect faster production of C29 17a 25-norhopane
if they originated by hopane degradation. In the
Athabasca-2 well, hopane degradation occurs with-
out formation of 25-norhopanes. The observed
variations require that hopane degradation and
25-norhopane formation proceed along different
pathways that may operate in association with the
many different combinations of environmental con-
ditions and microbial processes. The geochemical
data presented here indicate that a number of mech-
anisms of hopane degradation are probably operat-
ing even within a single petroleum deposit.
4. Conclusions
The presence of 25-norhopanes is commonly
used to indicate that a petroleum accumulation
has experienced at least heavy (PM 6) biodegrada-
tion. Quantitative geochemical data from case stud-
ies for the Liaohe Basin and Athabasca tar sands
suggest the operation of more than one mechanism
for hopane degradation and 25-norhopane forma-
tion. The formation of 25-norhopanes in both of
these biodegraded petroleum systems has been
shown using quantitative biomarker data, but mass
balance arguments show that only a proportion of
the degraded hopanes give rise to 25-norhopanes.
In some Athabasca samples, C30 17a hopane
degrades to a greater extent than C29 17a hopane,
but there is no concomitant increase in the genera-
tion of C29 17a 25-norhopane compared to C28
17a 25-norhopane. An added complication is that
in some other Athabasca samples where hopanes
are degrading, no 25-norhopanes could be identi-
fied. Thus, even within a single oil deposit it appears
that a number of mechanisms control the biodegra-
dation of hopanes, with or without the formation of
25-norhopanes.
Acknowledgements
The authors thank the Bacchus I sponsors (BP,
Chevron, ConocoPhillips, ExxonMobil, ENI E&P
Division, Enterprise Oil, JNOC, Norsk Hydro,
Petrobras, Shell, TotalFinaElf) for providing
financial support. Encana are thanked for provid-
ing sample material from the Athabasca-2 well.
796 B. Bennett et al. / Organic Geochemistry 37 (2006) 787–797
The Alberta Ingenuity Centre for In-Situ Energy is
also thanked for support (Milovan Fustic, PhD
student). The authors acknowledge that much of
the work was carried out at the NRG (Newcastle
University) prior to re-locating to Calgary (Barry
Bennett, Steve Larter, Huang Haiping) and Bide-
ford (Paul Farrimond). We are also grateful to
Drs. Mike Moldowan and Ken Peters for con-
structive reviews.
Associate Editor—Kenneth E. Peters
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