Complex patterns of steroidal biomarkers in Tertiary
lacustrine sediments of the Biyang Basin, China
Junhong Chen
1, Roger E. Summons *
Australian Geological Survey Organisation, GPO Box 378, Canberra ACT 2601, Australia
Received 21 February 2000; accepted 20 September 2000 (returned to author for revision 25 May 2000)
Abstract
Gas chromatography (GC), gas chromatography±mass spectrometry (GC±MS) and gas chromatography±mass spectrometry±mass spectrometry (GC±MS±MS) with co-injected synthetic standards were used to analyse the bio-marker patterns of some Tertiary lacustrine clayey dolomites from the Biyang Basin, China. The lithology, low Pr/Ph ratio and high gammacerane content of these sediments indicated that high salinity prevailed during their deposition. The distributions of steroidal hydrocarbons were particularly unusual and several pseudohomologous series, including regular steranes (C27±C29), 4-methyl steranes (C28±C30including dinosteranes), 3b-ethyl steranes (C29±C31), lanostanes
(C30±C32), and a variety of other 3b-alkylated steranes were identi®ed. 3b-n-Propylcholestane, 3b-n-propylstigmastane
and 4,4-dimethylcholestane were identi®ed using authentic standards and this is the ®rst time these compounds have been unambiguously characterised in sediments. Crown Copyright # 2001 Published by Elsevier Science Ltd. All rights reserved.
Keywords:Biomarkers; Steroids; Hydrocarbons; Dinosterane; 3b-Alkylated steranes; Lanostanes; 4,4-Dimethyl steranes; Lacustrine
sediments; Tertiary; Hypersaline; Biyang Basin; China
1. Introduction
Lipid biomarker compounds have been widely used to assess depositional environments, types of organic input, thermal maturity of organic matter and to demonstrate the relationship between oils and their sources (e.g. Mackenzie et al., 1980, 1981; Brassell et al., 1986, 1987; ten Haven et al., 1987; Volkman, 1988; Peters and Moldowan, 1993; Ritts et al., 1999). Steroids are an important class of biomarker and unambiguous determination of their chemical structures is funda-mental to understanding their sources and application in paleoenvironmental reconstruction. With continuing improvements in analytical techniques numerous
ster-oids, many with only subtly dierent molecular struc-tures, have been reported (e.g. Maxwell et al., 1980; Brassell and Eglinton, 1981; Robinson et al., 1984; Summons and Capon, 1988, 1991; Chen et al., 1989; Moldowan et al., 1990; Nichols et al., 1990; Volkman et al, 1990; Dahl et al., 1992, 1995).
Lacustrine sediments often show complex and dis-tinctive biomarker compositions, since these settings can, through time, receive a wide spectrum of organic inputs. Lakes also show a wide variety of water column chemistries with consequent variability in diagenetic conditions. This provides organic geochemists with opportunities for detailed study of the controls on bio-marker distribution. The Eocene Biyang Basin located in central China has previously been investigated for its petroleum geology and geochemical characteristics of the oils and sediments (Zhu et al., 1981; Jiang and Jia, 1986; Chen et al., 1988; Philp et al., 1992). A pseudo-homologous series of C30±C32lanostanes was identi®ed
there for the ®rst time (Chen et al., 1989). In this paper,
0146-6380/01/$ - see front matter Crown Copyright#2001 Published by Elsevier Science Ltd. All rights reserved. P I I : S 0 1 4 6 - 6 3 8 0 ( 0 0 ) 0 0 1 4 5 - 5
www.elsevier.nl/locate/orggeochem
* Corresponding author. Fax: +61-6-249-9956.
E-mail address:rsummons@agso.gov.au (R.E. Summons).
we report further aspects of the Biyang biomarker dis-tributions and the use of synthetic standards to aid characterisation of conventionally and unconventionally alkylated steroids.
2. Geological setting and samples
2.1. Geological setting
The Biyang Basin is an Eocene faulted lake basin located in the southern part of Henan Province in central China. Although it has an area of only 1000 km2, it has
been a prospective target for oil exploration (Zhu et al., 1981; Chen et al., 1988). The basin developed on the background of the Qinling mountains and gradually evolved from a freshwater environment to a much smaller and increasingly saline lake. The development of the basin was controlled by the presence of two large faults on the southern border, trending NNE and NWW. The sediments were deepest in the south and the burial depth decreased from south to north in the basin. In the early stages of its evolution 3000±4000 m of red clastic sediments were deposited relatively rapidly dur-ing periods of river ¯ooddur-ing to form the Yuhuangddur-ing and Dachangfang formations which are not oil prone. The latter stage was one of steady sediment deposition to produce the Oligocene Hetaoyuan formation, which is divided into three sections: Eh1, Eh2and Eh3. Eh3is
the oldest and was deposited as a suite of dark grey or grey claystones interbedded with oil shales and sand-stones. This unit is the major source of the oils in the Biyang Basin. The second section of the Hetaoyuan formation (Eh2) represented a generally more saline
environment, rich in organic matter and with a lower thermal maturity than Eh3. The youngest section is Eh1,
which is immature and has little contribution to oil accumulation. In the ®nal stage of lake evolution (Hetaoyuan to Niaozhuang formation), uplift occurred, the water volume of the lake decreased and the salinity increased. Clastic sediments with little petroleum potential were deposited from rivers onto the ¯ood-plains.
2.2. Samples
The core samples employed in this study were col-lected from a clayey dolomite formation belonging to the second section of the Hetaoyuan formation (Eh2) in
well Y2 located in the central area of the Biyang Basin. Previous studies have shown that sample nos. 1 (1992 m) and 3 (2036 m) contained C30±C32lanostanes (Chen
et al., 1989). In our present study, these two samples and another two core samples (nos. 2 and 4), also from well Y2 (1994 and 2085.5 m, respectively), were analysed by GC, GC±MS and GC±MS±MS.
3. Experimental
3.1. Bitumen isolation
Sediment samples were ground to a ®ne powder and extracted by Soxhlet using dichloromethane:methanol (87:13). After removal of elemental sulfur, the extracts were further separated into saturated, aromatic and polar-asphaltene fractions using column chromato-graphy on silica gel.
3.2. GC analysis
GC analyses of saturated hydrocarbons were carried out to examine the distributions of n-alkanes, iso-prenoids and the relative contents of steranes, hopanes and other compounds. These experiments were con-ducted with a HP 6890 GC using a 25 m0.25 mm i.d. DB-1 capillary column, coupled to an auto sampler with on-column injection and hydrogen carrier gas. The oven temperature was programmed from 60C (held 2 min)
to 310C at 4C/min, followed by an isothermal period
of 15 min.
3.3. GC±MS and GC±MS±MS analysis
The full-scan and selected ion monitoring (SIM) of GC±MS and metastable reaction monitoring (MRM) of GC±MS±MS were carried out with a VG Autospec Ultima-Q coupled to a CarloErba GC (8000 series), to further evaluate the distribution of sterane biomarkers and to compare with unambiguously identi®ed synthetic standards. Chromatography was conducted using a 60 m0.25 i.d. mm DB-5 capillary column with H2carrier
gas. For the identi®cation of 4,4-dimethylcholestanes, a polar column (SGE, type: BP-10; length: 50 m; i.d. 0.22 mm; ®lm: 0.25mm) was also used. Samples were injected using a vaporising injector at 300C in the splitless
mode. The oven temperature was programmed from 70 to 210C at 10C/min and then to 310C at 2C/min,
then held at the ®nal temperature for 20 min. The mass spectrometer was operated at 70 eV with a source tem-perature of 240C. During the fullscan acquisition mode
the mass spectrometer was scanning from m/z600±50, with a scan time of 1.0 s. For GC±MS±MS metastable analysis, we analysed 17 dierent parent ion to daughter ion transitions, each with 30 ms scanning acquisition and 50 ms delay periods.
3.4. Synthesis of standards
iso-mers was carried out as previously described (Abbott et al., 1984; Summons and Capon, 1988; Bisseret and Rohmer, 1990). Lanostane and 18a(H)-oleanane were obtained from Chiron Laboratory (Norway).
4. Results and discussion
4.1. General geochemical characteristics
Lithological and organic parameters for the Biyang Basin sediment samples are summarised in Table 1. Saturated hydrocarbon fractions isolated from the bitu-mens had low Pr/Ph ratios (0.19±0.73) and relatively high gammacerane/C30 hopane ratios (0.15±0.73) and
both features are associated with sedimentation from saline waters. This conclusion is supported by the pre-sence of relatively high contents ofb-carotane, a noted component of oils and bitumens from saline lacustrine environments (Fu et al., 1985; Volkman, 1988; Peters and Moldowan, 1993). The saturated hydrocarbon fractions of sample nos. 1±3 were dominated byn-C17or
phytane, hydrocarbons considered to signal input from cyanobacteria and/or algae. Sample no. 4 was unusual in that C30hopane was the most abundant hydrocarbon
(Fig. 1). Waxy n-alkanes had low odd over even pref-erence (OEP=1.16±1.39), which indicates that there may have been a minor input from vascular plant waxes, an observation supported by the presence of oleanane in sample nos. 1 and 3 (Fig. 3). Stigmastane was the predominant desmethyl sterane in all samples except no. 1 and 24-n-propylcholestane was notable for its absence from all samples.
All samples are immature as shown by the pre-dominance of steranes with biological stereochemistry (i.e. 20R>>20S and aaa>>abb) (Fig. 2) and high contents of C30moretane (Fig. 3). Values of the ratio of
C29sterane-20S/(S+R) range from 0.22 to 0.25 and the
ratio of C30hopane/moretane from 1.59 to 4.27 (Table
1). 17a(H)-22, 29, 30-Trisnorhopane (C27Tm) was the
most abundant C27 hopane with more
thermo-dynamically stable 18a(H)-22, 29, 30-trisnorneohopane (C27Ts) almost absent, which was consistent with the
low maturity of these samples. The C31methyl hopanes
detected in the Biyang Basin samples included a mixture of isomers of 2a-, 2b- and 3b-methylhopanes, with the less thermodynamically stable 2b- and 3b-methyl isomers in approximately equal abundance. These A-ring methy-lated hopanes have been detected in a variety of other sediments and oils (Summons and Walter, 1990; Collis-ter et al., 1992; Summons and Jahnke, 1992). Possible precursors for C31±C36methyl hopanes include a range
of organisms including acetic bacteria, methylotrophs, methanotrophs and cyanobacteria (e.g. Rohmer et al., 1984; Bisseret et al., 1985; Zundel and Rohmer, 1985a,b;
Summons et al., 1999). Isotopic evidence supports the Ta
idea that sedimentary 3-methylhopanes are derived from methanotrophs (Freeman et al., 1990; Collister et al., 1992) while a recent survey of cultures and microbial mats indicates that their 2-methyl counterparts prob-ably re¯ect inputs from cyanobacteria, especially in lacustrine sedimentary environments (Summons et al., 1999).
4.2. C30A-ring methylated steranes with base peak at
m/z231
The A-ring methylated C30 steranes detected in the
Biyang sediment samples comprise dinosteranes, 4a -methylstigmastane, 2a-methylstigmastane and 3b -methylstigmastane (Fig. 4). Dinosteranes were predom-inant only in Biyang Basin sample no. 1 but were present as minor components in the other three samples that were dominated by 3b-methylstigmastane and 4a -methylstigmastane (Fig. 4). Lower homologues,
that is methylated cholestanes and ergostanes, also comprise mixtures of 2-, 3- and 4-methyl analogues in the Biyang samples.
Mixtures of C30 4-methyl steranes, including
dinos-teranes (i.e. 4,23,24-trimethylcholestanes) and 4-methyl-stigmastane, have long been regarded as indicating dino¯agellate input to geological samples (e.g. Boon et al., 1979; de Leeuw et al., 1983). However, Edmunds and Eglinton (1984) suggested that dinosterol and rela-ted sterols were not exclusive markers for dino-¯agellates, since they may be produced by other organisms. Nichols and co-workers (1990) detected dinosterol as a minor component (0.1±3.2% of total sterols), with two other novel 4-methyl-C30 sterols, in
sea-ice diatom communities. The predominance of dinosteranes over 4-methyl stigmastanes in lacustrine sediments is quite unusual (Goodwin et al., 1988; Sum-mons et al., 1992) and, in the present case, may be a re¯ection of water column chemical conditions. Dia-genesis in sediments subject to high pH, low Eh and/or sulfate reduction, as compared to those deposited under
Fig. 1. GC traces for saturated hydrocarbon fractions of the Tertiary lacustrine sediment samples from Well Y2, Biyang Basin. The peak marked with an asterisk is 3-methylhenicosane (a-C22), the internal standard.
freshwater, may lead to enhanced preservation of dinosterol, a side-chain unsaturated steroid, through formation of sul®de adducts. The main precursor of 4-methylstigmastane, on the other hand, is likely to be the 4-methyl-24-ethylcholestanol and diagenetic conditions (Eh or pH) should not preferentially in¯uence its pres-ervation. In other words, the high abundance of 23,24-dimethyl isomers of the 4-methyl steranes compared to the 24-ethyl analogues, generally observed in marine settings, is possibly due to protection of the unsaturated dinosterol side-chain feature from oxidation. Such a mechanism has been proposed to explain the relative abundance of oleanane in marine sediments through preferential preservation of oleanoid triterpene pre-cursors. In contrast, the same precursors in non-marine settings appear to follow diagenetic pathways toward partial or complete aromatisation (Murray et al., 1997).
4.3. C29-C31steranes with a base peak atm/z245
A pseudohomologous series of C29±C31steranes with
base peak m/z 245 is prominent in the Biyang Basin
samples. They could have either one C2 or two C1 sub-stituents in ring-A+B+C. Compounds identi®ed by co-injection with the 20Sand 20Risomers of C29and C31
synthetic standards comprised the 3b-ethylcholestanes and 3b-ethylstigmastanes with 3b-ethylergostanes iden-ti®ed, by analogy as the C30components. As observed
with the desmethyl steranes, all compound series were dominated by 5a, 14a, 17a(H)-20Risomers (Fig. 5) and indicated a low maturity for the samples.
Full scan mass spectral analyses of synthetic 3b-ethyl steranes and 4,4-dimethyl analogues showed that their mass spectra are virtually identical. Chromatographic behaviour, therefore, oers the best means to distin-guish them in complex mixtures and we observed that the 4,4-dimethyl steraneaaa-20Risomer eluted earlier than its 3b-ethyl counterpart on both DB-5 and Ultra-1 columns. Furthermore, under the GC condition dis-cussed above, the 3b-ethylcholestanes could be com-pletely separated from their 4,4-dimethylcholestane counterparts (Fig. 5). While the 20S isomer of 4,4-dimethylstigmastane was totally separated from the 20S
Fig. 3. Hopanes, gammacerane and oleanane distribution pat-terns evident in GC±MSm/z191 selected ion chromatograms for four samples from the Biyang Basin.Tm=17a (H)-22,29,30-trisnorhopane.
isomer of 3b-ethylstigmastane, the 20R epimers could not be fully separated (Fig. 6) under routine conditions. For unambiguous identi®cation we conducted GC±MS (MRM) analyses on a polar (SGE BP-10) column and con®rmed the target 20Risomer of 4,4-dimethylcholes-tane also eluted with the standard. We also observed that GC±MS analyses using a delayed GC program enabled a complete separation of all 3b-ethyl and 4,4-dimethyl isomers to be achieved (Chen et al., 1993).
4,4-Dimethylsterols such as 4,4-dimethyl-5a -cholesta-8(14),24-dien-3b-ol and 4,4-dimethyl-5a -cholesta-8(14)-en-3b-ol may be regarded as precursors of 4,4-dime-thylcholestane. They have been reported in living organisms, being intermediates in the biosynthetic pathway from lanosterol to desmethyl sterols through the loss of the C-14 methyl group (e.g. Bouvier et al., 1976; Seher, 1976; Nes and McKean, 1977; Weete, 1980, and references therein). 4,4-Dimethyl steranes, to our knowledge, have not been unambiguously identi®ed in geological samples. A report of the identi®cation of 4,4-dimethylpregnanes and 4,4-dimethylhomopregnanes has been made (ten Haven et al., 1985), but these results
were based on the appearance of mass spectra, without appreciating the possible interference of 3b-ethyl ster-anes that were not known at the time.
In our present study, a further attempt to identify 4,4-dimethyl steranes from the geological record was made by GC±MS±MS analysis. Only C29
4,4-dimethylcholes-tane with a con®guration of 20Rwas found to occur in Biyang sediments, and it was in very low concentration (Fig. 5). Furthermore, 4,4-dimethylergostane (C30) and
4,4-dimethylstigmastane (C31), compounds having
alkyl-ation at C-24, were not detected in these samples (Fig. 6). This is consistent with the observed order of biosyn-thetic reactions whereby alkylation at C-24 follows loss of the methyl at C-14 and at least one of those at C-4 (Nes and McKean, 1977). In contrast to the 4,4-dime-thylsteroids, 3b-ethyl steroids (and other 3b-alkylated steroids) have not been reported in any natural living system while occurring ubiquitously in geological sam-ples (Summons and Capon, 1988, 1991; Dahl et al., 1992, 1995).
4.4. C30±C32steranes with a base peak atm/z259
A series of C30±C32lanostanes with base peakm/z259
had been detected in previous studies of the Biyang Basin samples using GC±MS analysis. These com-pounds were characterised by comparisons of mass spectra and co-injection with a synthetic standard for the C30analogue (Chen et al., 1989,1993). In the present
study, a second series of C30±C32compounds with base
peak m/z 259 was observed and characterised. They eluted after the lanostane series and the major series members were hypothesised to be the 5a, 14a, 17a (H)-20R isomers of 3b-n-propylcholestane, 3b-n -propy-lergostane and 3b-n-propylstigmastane, respectively. This was con®rmed (Fig. 7) by co-injection with authentic standards. 3b-Propyl steranes have been reported to occur in crude oils and rock extracts (Dahl et al., 1992, 1995) with the assignment being made on the basis of their being part of homologous series of 3b -alkyl sterane isomers. Given the complexity of the M+
!259 reaction chromatograms of some petroleum and
bitumen samples, our characterisation here provides necessary rigour to the compound class assignments.
Both lanostanes and 3b-n-propyl steranes show m/z 259 (rings-A+B+C) as the base peak in their main beam mass spectra. The 3b-n-propyl steranes also have a strong ion atm/z191, the latter being analogous to the rings-A+B fragment at m/z 149 in regular desmethyl steranes. On the other hand, lanostanes have a very strong ion at m/z 190 instead of m/z 191 and conse-quently highm/z190 vs 191 ratios in lanostanes serve to distinguish the two carbon skeletons. These two series of compounds can also be readily dierentiated based on their relative elution times from a DB-5 capillary col-umn. Recently, a third compound class has been
®ed usingm/z 414!259 reaction chromatograms. The
so-called TPP or `C30tetracyclic polyprenoids'
(Schaef-fer et al. 1994; Holba et al., 2000) have been identi®ed in lacustrine sediments and in their derived oils and attributed to a freshwater algal source. Comparison of the m/z 414!259 reaction chromatograms for the
Biyang Basin sediment sample no. 1 with those of freshwater lacustrine oils, from Indonesia (LAC oils of Murray et al., 1994) enabled the TPP doublet to be assigned (Fig. 7) on the basis of relative retention times. The m/z 259 selected ion chromatograms and m/z 414!259 reaction chromatograms of the latter oils
presented an intense doublet of peaks, with higher homologues apparently absent, and we presumed these to be the C30TPP of Holba et al. (2000).
In their recent study, Peng et al. (1998) identi®ed C30
and C31lanostane sul®des occurring in an immature oil
from Paleocene evaporite source rocks in Jianghan Basin, China. No saturate counterparts were found in this sample, indicating that the lanosterol precursors were completely converted to sul®des under an envi-ronment with ready availability of H2S or polysul®de.
Accordingly, occurrence of lanostanes in sedimentary bitumens and oils might be controlled by a combination of source and diagenetic factors such as sul®de avail-ability.
4.5. Other A-ring alkylated steranes
Additional A-ring alkylated steranes were present in the Biyang samples and identi®ed as A-ring-C4steranes
(C31±C33) (Fig. 8), and 3b-n-pentyl steranes and 3b-i
-pentyl steranes (C32±C34) (Fig. 9). The 3b-n-pentyl
ster-anes and 3b-i-pentyl steranes (C32±C34) were con®rmed
Fig. 6. m/z428!245 GC±MS±MS chromatograms showing the elution order and co-injections of standards of isomerised 3b-ethylstigmastane (20S+20R) and isomerised 4,4-dimethyl-stigmastane (20S+20R). Under the analytical conditions there is only partial separation of 20Risomers while 20Sisomers are completely separated. No 4,4-dimethylstigmastane is evident in contrast to the equivalent experiment for cholestanes shown in Fig. 5.
Fig. 7. M+
with co-injected isomerised 3b-n-pentylcholestane and 3b-i-pentylcholestane. The relative content of 3b-n -pen-tyl steranes is much higher than that of 3b-i-pentyl ster-anes and supports the hypothesis of Dahl et al. (1995) that the ratio of 3b-n-pentyl steranes to 3b-i-pentyl steranes is related to source environments. In their ear-lier study, Dahl et al. (1995) found that the Green River Formation and Rozel Point oil (both lacustrine samples) showed a preference for 3b-n-pentyl steranes while most other samples (e.g. marine Monterey oil) were domi-nated by 3b-i-pentyl steranes.
Although no standards were employed in the identi®-cation of the A-ring-C4compound series, they appear to
be dominated by 20Risomers of the 3b-n-butyl analo-gues based on the normal structure and 20R stereo-chemistry of the co-occurring series and the low maturity of the sediments.
4.6. Signi®cance and implications
Except for 4,4-dimethylcholestane (20R), the steroids discussed above were found occurring in all four Biyang
Basin samples. Based on the biomarker ratios and par-ticularly relative abundances of gammacerane, Pr, Ph and b-carotane (Table 1), these lacustrine sediments were immature and deposited from highly saline waters carrying a biota dominated by algae and bacteria with only minor inputs from vascular plants. Samples nos. 2 and 3 had very similar geochemical characteristics and biomarker distributions despite a large dierence in TOC values. Sample no. 1 was dierentiated by higher relative abundance of phytane and C27 steranes (37%)
and predominance of dinosteranes over 4-methyl-stigmastanes, while no. 4 was characterised by very high concentration of C30hopane and the dominance of C29
steranes (50%). This is probably a re¯ection of changes of depositional conditions, since sample no. 4 has a much higher content of clay minerals than others (Table 1), which may indicate a depositional environment with in¯ux of fresher water and more terrigenous debris. This is also consistent with the high Pr/Ph ratio and low gammacerane content for sample no. 4.
The biological origins of lanostanes and 3b-alkyl steranes (C1±C5) remain to be determined. Lanosterol,
Fig. 8. M+
!273 GC±MS±MS chromatograms of C31±C33 A-ring-C4sterane compounds in sample no. 1.
Fig. 9. M+
the most likely precursor of lanostane, is normally associated with sterol biosynthesis in animals and fungi and has been identi®ed as a precursor to the methyl and dimethyl sterols of the methylotrophic bacterium
Methylococcus capsulatus (Bouvier et al., 1976).
Tri-terpenes with a lanostane carbon skeleton have been identi®ed in the fern Adiantum venustum(Alam et al., 2000) but these compounds did not have alkylation at C-24. Although there has been a report of 24-methylla-nosterol (C31) occurring in fungi (e.g.Aspergillus
fumi-gatus and Penicillium expansum etc., Weete, 1980 and
references therein), 24-ethyllanostane has no known natural precursor identi®ed in any organisms. Although 3b-alkylated steroids have not yet been found to occur as natural products in living organisms, 3b-carboxyl steroids have been found in sediments (Dany et al., 1990) and kerogen hydrolysates (Barakat and Rull-koÈtter, 1994). The evidence from their carbon number and isomer distributions suggests they are possibly diagenetic products of sedimentary 2-sterenes
(Sum-mons and Capon, 1991; Dahl et al., 1992, 1995). 4,4-Dimethylcholestane (20R) was unambiguously characterised in Biyang Tertiary lacustrine sediments. The very low concentration of 4,4-dimethylcholestane is most probably a re¯ection of the low concentration of its precursors in algae or bacteria (e.g. 4,4-dimethyl-5a -cholesta-8(14),24-dien-3b-ol and 4,4-dimethyl-5a -cho-lesta-8(14)-en-3b-ol), which are intermediates in the bio-synthetic pathway from lanosterol to desmethyl sterols. Our failure to detect 4,4-dimethylergostane (C30) and
4,4-dimethylstigmastane (C31) is possibly a consequence
of the ordering of reactions in sterol biosynthesis, whereby alkylation at C-24 generally takes place after removal of the C-14 methyl group and at least one C-4 methyl group (Nes and Mckean, 1977; Weete, 1980).
5. Summary
In this study we employed GC as well as GC±MS in full scan, SIM and MRM modes to investigate the detail of steroidal hydrocarbons in lacustrine clayey dolomites from the Eocene of the Biyang Basin. Isomerised stan-dards of A-ring alkylated cholestanes and stigmastanes were critical to the unambiguous characterisation of
several groups of compounds. In our experience, these sediments are unusual for showing such a broad range of steroids in a single sample. Sample no. 1, for example, contained isomeric mixtures of lanostane, 24-methyllanostane, 24-ethyllanostane, 4,4-dimethylcholes-tane, 4-methylcholes4,4-dimethylcholes-tane, 4-methylergos4,4-dimethylcholes-tane, 4-methyl-stigmastane and 4,23,24-trimethylcholestane (dinosterane). All of these compounds, with the exception of 24-ethyl-lanostane can be traced to known sterol precursors which have been identi®ed in algae, fungi, methylo-trophic bacteria and plants. In addition, the samples contained series of 2a- and 3b-alkyl steroids without known precursors, the most prominent being the latter with chain lengths of C1±C5. Because of the complexity
of co-eluting and closely eluting peaks, metastable analysis with coinjections of 20S+20Rsterane standards was required to distinguish between 3b-ethylcholestane and 4,4-dimethylcholestane on the one hand, and the 3b-n-propylcholestane (C30) to 3b-n-propylstigmastane
(C32) and lanostane (C30±C32) series on the other.
GC analyses showing low Pr/Ph ratios and prominent peaks for gammacerane and b-carotane indicate sedi-mentation in highly saline waters. These conditions were evidently hospitable to an unusual microbial ¯ora and the probable source of the diverse array of steroids. Minor plant inputs were also evident through the pre-sence of waxy hydrocarbons with a low odd/even pref-erence and oleanane in some samples.
Acknowledgements
We would like to thank Zarko Roksandic, Graham Logan and Paul Greenwood for their assistance with GC±MS analysis and Peng Ping'an for collecting some reference material. Rob Capon provided the synthetic standards for 4,4-dimethylcholestane and 4,4-dimethyl-stigmastane while Janet Hope synthesised the 3b-alkyl steranes. Graham Logan and Jochen Brocks provided comments that improved this manuscript. We also thank Michael Moldowan and an anonymous reviewer for their constructive reviews. Roger Summons pub-lishes with the approval of the CEO of AGSO.
Associate EditorÐB.R.T. Simoneit
Appendix. Chemical structures of tetracyclic biomarkers discussed in text
References
Abbott, G.D., Lewis, C.A., Maxwell, J.R., 1984. Laboratory simulation studies of steroid aromatisation and alkane iso-merisation. Organic Geochemistry 6, 31±38.
Alam, M.S., Chopra, N., Ali, M., Niwa, M., 2000. Normethyl pentacyclic and lanostane-type triterpenes from Adiantum
venustum. Phytochemistry 54, 215±220.
Barakat, A.O., RullkoÈtter, J., 1994. Occurrence of bound 3b -carboxylsteroids in geological samples. Energy & Fuels 8, 481±486.
Bisseret, P., Rohmer, M., 1990. Bromine,n-bromosuccinimide and sulfur induced isomerisations in the hopane series. Tet-rahedron Letters 31, 7445±7448.
Bisseret, P., Zundel, M., Rohmer, M., 1985. Prokarytic tri-terpenoids. 2. 2b-Methylhopanoids fromMethylobacterium
organophilum and Nostoc muscorum, a new series of
pro-karytic triterpenoids. European Journal of Biochemistry 150, 29±34.
8(14)-steroids in the bacteriumMethylococcus capsulatus. Biochemistry Journal 159, 267±271.
Brassell, S.C., Eglinton, G., 1981. Biogeochemical signi®cance of a novel sedimentary C27stanol. Nature 290, 579±582. Brassell, S.C., Eglinton, G., Fu, J.M., 1986. Biological marker
compounds as indicators of the depositional history of the Maoming oil shale. In: Leythaeuser, D., RullkoÈtter, J. (Eds.), Advances in Organic Geochemistry 1985, Organic Geo-chemistry, Vol. 10. Pergamon, Oxford, pp. 927±941. Brassell, S.C., Eglinton, G., Howell, V.J., 1987.
Palaeoenvi-ronmental assessment of marine organic-rich sediments using molecular organic geochemistry. In: Brooks, J., Fleet, A.J. (Eds.) Lacustrine Petroleum Source Rocks Geological Society Special Publication No. 26, pp. 179-198.
Chen, J.Y., Wang, Q.J., Ma, W.Y., 1988. Geochemical features of source rocks in an alkaline lake basin. In: 2nd Conference on Petroleum Geochemistry and Exploration in the Afro-Asian Region (abstract), Beijing, p. 145.
Chen, J.H., Philp, R.P., Fu, J.M., Sheng, G.Y., 1989. The occurrence and identi®cation of C30±C32lanostanes: a novel series of tetracyclic triterpenoid hydrocarbons. Geochimica et Cosmochimica Acta 53, 2775±2779.
Chen, J.H., Summons, R.E., Fu, J.M., Sheng, G.Y., 1993. The distribution and implications of biomarkers from some Chi-nese Tertiary lacustrine sediments, Biyang Basin, In: éygard K. (Ed.), Organic Geochemistry: Poster Sessions from the 16th International Meeting on Organic Geochemistry, Sta-vanger, Norway, 20±24 September 1993. Falch Hurtigtrykk, pp. 313±316.
Collister, J.W., Summons, R.E., Lichtfouse, E., Hayes, J.M., 1992. An isotopic biochemical study of the Green River Oil Shale. Organic Geochemistry 19, 265±276.
Dahl, J., Moldowan, J.M., McCarey, M.A., Lipton, P.A., 1992. A new class of natural products revealed by 3b-alkyl steranes in petroleum. Nature 355, 154±157.
Dahl, J., Moldowan, J.M., Summons, R.E., McCarey, M.A., Lipton, P.A., Watt, D.S., Hope, J.M., 1995. Extended 3b -alkyl steranes and 3b-alkyl triaromatic steroids in crude oils and rock extracts. Geochimica et Cosmochimica Acta 59, 3717±3729.
Dany, F., Riolo, J.-M., Albrecht, P., 1990. 3-Carboxylsteranes, a novel family of fossil steroids. Journal of the Chemical Society, Chemical Communications, 1228-1230.
Edmunds, K.L.H., Eglinton, G., 1984. Microbial lipids and carotenoids and their early diagenesis in the Solar Lake laminated microbial mat sequence. In: Cohen, Y., Castle-nholtz, R.W., Halvorson, H.O. (Eds.), Microbial Mats: Stromatolites. A.R. Liss, New York, pp. 343±389.
Freeman, K.H., Hayes, J.M., Trendel, J.-M., Albrecht, P., 1990. Evidence from carbon isotope measurements for diverse origins of sedimentary hydrocarbons. Nature 343, 254±256.
Fu, J., Sheng, G., Peng, P., Brassell, S.C., Eglinton, G., Jiang, J., 1985. Peculiarities of salt lake sediments as potential source rocks in China. In: Leythaeuser, D., RullkoÈtter, J. (Eds.), Advances in Organic Geochemistry 1985. Organic Geochemistry, Vol. 10. Pergamon, Oxford, pp. 119±126. Goodwin, N.S., Mann, A.L., Patience, R.L., 1988. Structure
and signi®cance of C304-methylsteranes in lacustrine shales and oils. Organic Geochemistry 12, 495±506.
ten Haven, H.L., Baas, M., Kroot, M., de Leeuw, J.W.,
Schenck, P.A., Ebbing, J., 1987. Late Quaternary Medi-terranean sapropels. III: Assessment of source of input and palaeotemperature as derived from biological markers. Geo-chimica et CosmoGeo-chimica Acta 51, 803±810.
ten Haven, H.L., de Leeuw, J.W., Schenck, P.A., 1985. Organic geochemical studies of a Messian evaporitic basin, northern Appenines (Italy) 1: hydrocarbon biological markers for a hypersaline environment. Geochimica et Cosmochimica Acta 49, 2181±2191.
Holba, A.G., Ellis, L., Tegelaar, E., Singletary, M.S., Albrecht, P., 2000. Tetracyclic polyprenoids: indicators of fresh water (lacustrine) algal input. Geology 28, 251±254.
Jiang, N.H., Jia, F.Y., 1986. Source and migration of crude oils of Biyang Depression. Oil and Gas Geology 7, 51±58 (in Chinese).
de Leeuw, J.W., Rijpstra, W.I., Schenck, P.A., Volkman, J.K., 1983. Free, esteri®ed and bound sterols in Black Sea Unit I sediments. Geochimica et Cosmochimica Acta 47, 455±465. Mackenzie, A.S., Homann, C.F., Maxwell, J.R., 1981.
Mole-cular parameters of maturation in the Toarcian shales, Paris Basin, France III. Changes in aromatic steroid hydrocarbons. Geochimica et Cosmochimica Acta 45, 1345±1355.
Mackenzie, A.S., Patience, R.L., Maxwell, J.R., Vanden-broucke, M., Durand, B., 1980. Molecular parameters of maturation in the Toarcian shales, Paris Basin, France I. Changes in the con®guration of the acyclic isoprenoid alkanes, steranes and triterpanes. Geochimica et Cosmochi-mica Acta 44, 1709±1721.
Maxwell, J.R., Mackenzie, A.S., Volkman, J.K., 1980. Con®g-uration at C-24 in steranes and sterols. Nature 286, 694±697. Moldowan, J.M., Fago, F.J., Lee, C.Y., Jacobson, S.R., Watt, D.S., Slougui, N.-E., Jeganathan, A., Young, D.C., 1990. Sedimentary 24-n-propylcholestanes, molecular fossils diag-nostic of marine algae. Science 247, 309±312.
Murray, A.P., Summons, R.E., Boreham, C.J., Dowling, L.M., 1994. Biomarker andn-alkane isotope pro®les for Tertiary oils: relationship to source rock depositional setting. Organic Geochemistry 22, 521±542.
Murray, A.P., Sosrowidjojo, I.B., Alexander, R., Kagi, R.I., Norgate, C.M., Summons, R.E., 1997. Oleananes in oils and sediments: evidence of marine in¯uence during early diagen-esis? Geochimica et Cosmochimica Acta 61, 1261±1276. Nes, W.R., McKean, M.L., 1977. Biochemistry of Steroids and
Other Isopentenoids. University Park Press, Baltimore. Nichols, P.D., Palmisano, A.C., Rayner, M.S., Smith, G.A.,
White, D.C., 1990. Occurrence of novel C30sterols in Ant-arctic sea-ice diatom communities during a spring bloom. Organic Geochemistry 15, 503±508.
Peng, P., Morales-Izquierdo, A., Fu, J., Sheng, G., Jiang, J., Hogg, A., Strausz, O.P., 1998. Lanostane sul®des in an immature crude oil. Organic Geochemistry 28, 125±134. Peters, K., Moldowan, J.M., 1993. The Biomarker Guide:
Interpreting Molecular Fossils in Petroleum and Ancient Sediments. Pretince Hall, Englewood Clis, NJ.
Philp, R.P., Chen, J.H., Fu, J.M., Sheng, G.Y., 1992. A geo-chemical investigation of crude oils and source rocks from Biyang Basin, China. Organic Geochemistry 18, 933±945. Ritts, B.D., Hanson, A.D., Zinniker, D., Moldowan, J.M.,
China. American Association of Petroleum Geologists Bul-letin 83, 1980±2005.
Robinson, N., Eglinton, G., Brassell, S.C., Cranwell, P.A., 1984. Dino¯agellate origin for sedimentary 4a -methylster-oids and 5a(H)-stanols. Nature 308, 439±442.
Rohmer, M., Bouvier-Nave, P., Ourisson, G., 1984. Distribu-tion of hopanoid triterpenes in prokaryotes. Journal of General Microbiology 130, 1137±1150.
Schaeer, P., Poinsot, J., Hauke, V., Adam, P., Wehrung, P., Trendel, J.-M., Albrecht, P., Dessort, D., Connan, J., 1994. Novel optically active hydrocarbons in sediments: evidence for an extensive biological cyclisation of higher regular polyprenols. Angewandte Chemie International Edition 33, 1166±1169. Seher, A., 1976. Sterols in the chemistry and technology of
edible fats and oils. In: Paoletti, R., Jacini, G., Porcellati, G. (Eds.), Lipids, Vol. 2. Technology Raven Press, New York, pp. 293±313.
Summons, R.E., Boreham, C.J., Thomas, J., Maxwell, J.R., 1992. Secular and environment constraint on the distribution of dinosterane in sediments. Geochimica et Cosmochimica Acta 56, 2437±2444.
Summons, R.E., Capon, R.J., 1988. Fossil steranes with unprecedented methylation in ring-A. Geochimica et Cos-mochimica Acta 52, 2733±2736.
Summons, R.E., Capon, R.J., 1991. Identi®cation of sig-ni®cance of 3b-ethylsteranes in sediments and petroleum. Geochimica et Cosmochimica Acta 55, 2391±2395.
Summons, R.E., Jahnke, L.L., 1992. Hopenes and hopanes methylated in ring A: correlation of the hopanoids from extant methylotrophic bacteria with their fossil analogues. In: Moldowan, J.K., Albrecht, P., Philp, R.P. (Eds.), Biological
Markers in Sediments and Petroleum. Prentice Hall, Engle-wood Clis, NJ, pp. 182±194.
Summons, R.E., Walter, M.R., 1990. Molecular fossils and microfossils of prokaryotes and protists from Proterozoic sediments. American Journal of Science 51, 557±566. Summons, R.E., Jahnke, L.L., Logan, G.A., Hope, J.M., 1999.
2-Methylhopanoids as biomarkers for cyanobacterial oxy-genic photosynthesis. Nature 400, 554±557.
Volkman, J.K., 1988. Biological marker compounds as indica-tors of the depositional environments of petroleum source rocks. In: Fleet, A.J., Kelts, K., Talbot, M.R. (Eds.), Lacus-trine Petroleum Source Rocks. Geological Society Special Publication No. 40, pp. 103-122.
Volkman, J.K., Kearney, P., Jerey, S.W., 1990. A new source of 4-methyl sterols and 5a(H)-stanols in sediments: prymne-siophyte microalgae of the genus Pavlova. Organic Geo-chemistry 15, 489±497.
Weete, J.D., 1980. Lipid Biochemistry of Fungi and Other Organisms. Plenum Press, New York.
Zhu, S.A., Xu, S.R., Zhu, S.B., Wang, Y.X., 1981. Petroleum geological characterisics of the Biyang depression, Henan. Acta Petroleum Sinica 2 (2), 21±218 (in Chinese).
Zundel, M., Rohmer, M., 1985a. Prokaryotic triterpenoids. 1. 3b-Methylhopanoids from Acetobacter sp. and
Methylo-coccus capsulatus. European Journal of Biochemistry 150,
23±27.
Zundel, M., Rohmer, M., 1985b. Prokaryotic triterpenoids. 3. The biosythesis of 2b-methylhopanoids and 3b -methylhopa-noids of Methylobacterium organophilum and Acetobacter
pasteurianus spp. pasteurianus. European Journal of
