Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol31.Issue6.2000:
Organic Geochemistry 31 (2000) 509±521
www.elsevier.nl/locate/orggeochem
A molecular and stable carbon isotopic study of lipids in
late Quaternary sediments from the Arabian Sea
Stefan Schouten a,*, Marcel J.L. Hoefs b, Jaap S. Sinninghe Damste a,b
a
Netherlands Institute for Sea Research, Department of Marine Biogeochemistry and Toxicology, PO Box 59,
1790 AB Den Burg, Texel, The Netherlands
b
Utrecht University, Institute of Earth Sciences, PO Box 80021, 3508 TA Utrecht, The Netherlands
Received 20 July 1999; accepted 9 March 2000
(returned to author for revision 2 December 1999)
Abstract
The distribution of apolar and polar lipids and their stable carbon isotopic compositions were determined for a
number of sediment samples from dierent sites in the Arabian Sea. Lipids are mainly derived from planktonic
Archaea and a range of algae, including Haptophytes, Eustigmatophytes and diatoms. The stable carbon isotopic
compositions of compounds from diatoms, highly branched isoprenoids, fall into distinct groups suggesting other
sources beside the diatom species Rhizosolenia setigera for these compounds. High amounts of sterol ethers, which may
be derived from diatoms, were also detected. A recently identi®ed triterpenoid, malabaricatriene, was also present in
high abundance in these samples. The terrestrial input of lipids consists of n-alkanes and their carbon isotopic compositions show that they are derived from aeolian dust input from the Arabian Peninsula. The isotopic compositions of
C37 alkenones from two cores during the last 15 ka is relatively constant and suggests that growth conditions for
Haptophyte algae (averaged over 500 years) did not change signi®cantly over this period. # 2000 Elsevier Science Ltd.
All rights reserved.
Keywords: Algae; Diatoms; 13C of lipids; Alkenones; Malabaricatriene; Highly branched isoprenoids
1. Introduction
Sediments from the Arabian Sea have become a subject of great interest since the area is sensitive to changes
in climate, especially with respect to glacial cycles, which
eect monsoon intensity and, in turn, upwelling and
primary productivity. The Arabian Sea is uniquely situated since it is very sensitive to these atmospheric forces,
which leads to great seasonal variability in this part of
the ocean (Smith et al., 1998). In addition, the combination of high productivity and moderate ventilation of
the thermocline leads to an intense oxygen minimum
zone (OMZ) between 150 and 1250 m, in turn leading to
* Corresponding author. Fax: +31-222-369569.
E-mail address: [email protected] (S. Schouten).
deposition of relatively organic matter-rich sediments
enabling organic geochemical analyses.
A number of organic geochemical studies have reported
on sediments from the Arabian Sea. Ten Haven et al.
(1992) investigated a number of Pleistocene sediments of
upwelling regions, including those taken at oshore
Oman. A wide variety of extractable lipids were found,
including alkanediols, long-chain alkenones and sterols
in dierent relative concentrations. Eglinton et al.
(1997) analysed the 13C and 14C-content of a number of
biomarkers in a sediment from the Arabian Sea at oshore Oman. They found a widespread variability in
their 13C- and 14C-contents indicating multiple sources,
even for compounds with the same carbon skeleton, and
indicating dierent positions of organisms in the water
column. Schubert et al. (1998) investigated the distribution of several algal biomarkers in a core taken at oshore Pakistan, which spanned the last 200 ka. Using
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00031-0
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S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
standard biomarkers and TOC data, they inferred from
the data that the phytoplanktonic community was stable
during deposition of the sediments.
Although a number of organic geochemical studies
have been done in this area, studies on apolar hydrocarbons or stable carbon isotopic compositions of lipids
to determine their sources in recent sediments have been
limited. Thus, a detailed molecular and isotopic organic
geochemical study was undertaken of several sediment
cores from the Arabian Sea taken during the Netherlands Indian Ocean Program 1992±1993 (Van Weering
et al., 1997; Table 1, Fig. 1). Three sites (921, 451 and
453) were examined in detail for the distribution and
stable carbon isotopic composition of apolar hydrocarbons. Since these components are present in very low
amounts, large composite samples were taken from trip
cores. Three cores from dierent sites (455, 464 and 921)
were subsampled in a time range covering c. 15 ka and
total lipid fractions were analysed. Finally, long-chain
ketones were isolated from all samples and analysed for
their stable carbon isotopic compositions.
2. Methods
2.1. Samples
The samples investigated in this study are listed in
Table 1. Sites 451, 453, 455 and 464 were sampled in the
Arabian Sea during the 1992±1993 expedition of the
R.V. Tyro in the Indian Ocean (Netherlands Indian
Ocean Program; Fig. 1). From site 451 and 453 only a
trip core sample was studied. Sediment samples from
NIOP 455 and 464 were obtained by subsampling piston
cores from this site at speci®c intervals (Table 1). The
samples represent an average of approximately 500 years.
Site 921 is oshore Oman and was sampled during a different leg of the same Tyro-expedition. One large sample
(depth of 5±8 cm) from the core and several smaller subsamples were analysed from this site (Table 1).
2.2. Extraction and fractionation of soluble organic matter
Sediment samples were freeze-dried and ground in a
rotary disc mill and subsequently Soxhlet extracted with
dichloromethane:methanol (7.5:1, v/v) for 24 h. The
extracts were concentrated with a rotary evaporator at
30 C. Part of the extracts (total lipid fraction) were
methylated by diazomethane and silylated using BSTFA/
pyridine and analysed by gas chromatography (GC), and
GC±mass spectrometry (GC±MS). A selected number of
extracts from composite samples of sites 451, 453 and
921 were separated using a column (252 cm; column
volume 35 ml) packed with alumina (activated for 2.5 h at
120 C). The apolar and polar fractions were eluted with
hexane:dichloromethane (9:1, v/v; 150 ml) and methanol:dichloromethane (1:1, v/v, 150 ml), respectively. The
apolar fractions were further separated by argentatious
thin layer chromatography (TLC) using hexane as
developer. The AgNO3-impregnated silica plates (2020
cm; thickness 0.25 mm) used for this purpose were prepared by dipping them in a solution of 1% AgNO3 in
methanol/ bidistillated water (4:1, v/v) for 45 s and
subsequent activation at 120 C for 1.5 h. Four fractions
(A1, Rf=0.9±1.00; A2, Rf=0.3±0.9; A3, Rf=0.1±0.3;
A4, Rf=0.00±0.1) were scraped o the TLC plate and
Table 1
Samples analysed in this study
Site
Location
Water depth (m)
Sediment interval (cm)
Approximate age (ka)a
451
23 400 5300 N
66 020 9700 E
23 150 3000 N
65 440 5000 E
23 330 4000 N
65 570 3000 E
542
0±100
±
1556
0±100
±
995
464
22 150 4000 N
63 350 1000 E
1470
921
16 040 2300 N
52 360 2800 E
455
34±36
74±75
89±91
139±141
25±28
48±50
64±67
84±88
115±118
5±8
0±0.9
0.9±1.8
1.8±2.7
2.7±3.6
3.6±4.5
7.7
10
12
13
1.4
5.1
7.6
10.8
14.4
±
±
±
±
±
±
453
455
a
Reichart et al. (1997) and personal communication.
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
511
Fig. 1. Map showing the location of the samples investigated in this study.
ultrasonically extracted using ethyl acetate (3).
Separation of the fractions from silica/Ag+ proceeded
using a small pipette (volume 2 ml) half ®lled with alumina (not activated), by elution with hexane:dichloromethane (9:1, v/v).
Ketone fractions were isolated from polar fractions or
total extracts by separation using a small column/pipette
(volume 2 ml) half ®lled with alumina (not activated) and
elution with hexane:dichloromethane (1:1, v/v).
All total lipid and TLC fractions were analysed by
GC, GC±MS and isotope-ratio-monitoring gas chromatography±mass spectrometry (irm-GC±MS). Since the fractions sometimes contained polyunsaturated compounds
they were hydrogenated in ethyl acetate containing a few
drops of acetic acid with H2/PtO2 for 1 h at room temperature. The hydrogenated fractions were analysed by
GC, GC±MS and irm-GC±MS.
Some of the total lipid extracts or polar fractions were
treated with HI/LiAlH4 to release ether bound compounds as described previously (Hoefs et al., 1997).
Brie¯y, the fractions were re¯uxed in a solution of 56 wt%
HI in water for 1 h and the released alkyl iodides were
treated with LiAlH4 in 1,4-dioxane for 1h to convert them
to hydrocarbons.
2.3. Gas chromatography
GC was performed using a Hewlett Packard 5890
series II chromatograph equipped with an on-column
injector and ®tted with a 250.32 mm fused silica
capillary column coated with CP-Sil 5 (®lm thickness
0.12 mm). Helium was used as the carrier gas and the
oven was programmed from 70 to 130 C at 20 C/min,
followed by an increase of 4 C/min to 320 C (10 min
hold time). Detection was performed using a ¯ame
ionization (FID).
2.4. Gas chromatography±mass spectrometry
GC±MS analyses were performed using the same
chromatograph and conditions as described for GC.
The column was directly inserted into the electron
impact ion source of a VG-Autospec Ultima mass spectrometer, operated with a mass range of m/z 40±800, a
cycle time of 1.8 s and ionization energy of 70 eV.
2.5. Isotope-ratio-monitoring gas chromatography±mass
spectrometry
Isotope-ratio monitoring was performed using a
DELTA-C irm-GC±MS system (Schouten et al., 1998a),
equipped with an on-column injector and ®tted with a
250.32 mm fused silica capillary column coated with
CP-Sil 5 (®lm thickness 0.12 mm). Helium was used as
carrier gas and the oven was programmed from 70 to
130 C at 20 C/min, followed by an increase of 4 C/min
to 320 C (20 min hold time). Isotopic values were calculated by integrating the mass 44, 45 and 46 ion currents
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S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
of the peaks produced by combustion of the chromatographically separated compounds and those of CO2-spikes
produced by admitting CO2 with a known 13C-content at
regular intervals into the mass spectrometer. Two analyses
were carried out for each sample and the results were
averaged to obtain a mean value and to calculate the
standard deviation.
3. Results and discussion
Five sediment cores from dierent sites of the Arabian Sea were sampled and analysed for total lipids.
From three cores (NIOP 451, 453 and 921) samples were
taken and analyzed for the distribution and stable carbon isotopic compositions of apolar hydrocarbons as
well. The fractions of samples 451 and 453 were hydrogenated prior to irm-GC±MS analysis to simplify the
mixture of compounds. Two other cores (NIOP 455 and
464) were subsampled at several intervals and analyzed
for total lipids. In addition, long-chain ketones were
isolated from the total lipid extracts or polar fractions
and analyzed for their 13C-contents.
It is interesting to note that no sulfur compounds
were detected either in the apolar and polar fractions of
the sediments. Indeed, ¯ash pyrolyses of some of the
kerogens also yielded very low amounts of sulfur compounds (Hoefs et al., 1995a and unpublished results).
This suggests that no sulfur was incorporated into the
organic matter, consistent with the observation that no
sulfate reduction was observed in the pore waters at
these sites (Passier et al., 1997). Hence, no free hydrogen
sul®de or polysul®des were available to react with the
organic matter (Sinninghe Damste et al., 1989).
3.1. C37 alkenones
All sediments contain relatively high amounts of C37
and C38 diunsaturated methyl and ethylketones (I, see
Appendix; e.g. Fig. 2). These lipids are biosynthesized
by Prymnesiophyte algae (e.g. Volkman et al., 1980,
1995; Marlowe et al., 1984) and have been reported in
numerous sediments, including in several parts of the
Indian Ocean (e.g. Sonzogni et al., 1997). Both Gephyrocapsa oceanica and Emiliania huxleyi occur abundantly
in the northern part of the temporary Indian Ocean (e.g.
Kleijne et al., 1988) and both could be the source for the
alkenones. Based on the dominance of the C37 ketones
over the C38 ketones and the Uk'
37 correlation with temperature, Sonzogni et al. (1997) suggested that E. huxleyi is the main source of alkenones in sediments from
the Indian Ocean. The distribution of the di- vs the triunsaturated C37 ketone is used, via the Uk'
37 ratio (Brassell, 1986), as an indication for paleotemperatures. As
noted by Sonzogni et al. (1997), the diunsaturated
ketone is dominating, yielding Uk'37 always higher than
0.9. This is due to the high water temperatures (26±
28 C) of the Indian Ocean.
Since the total lipid fraction consisted of a complex
mixture of coeluting compounds, analysis of their stable
carbon isotopic compositions was impossible. The analysis of the C37 diunsaturated methyl ketone was, however,
feasible after isolation using column chromatography (see
experimental). Bidigare et al. (1997) and Popp et al. (1998)
have shown with cultures of E. huxleyi that the stable
carbon isotopic composition of this compound depends
on the isotopic composition and concentration of
[CO2]aq and the growth rate of its parent organism.
Since the isotopic composition of [CO2]aq remained
fairly constant in the time frame that our sediments
were deposited (the d13C values of the inorganic carbonate in the cores are relatively constant, i.e for 464
varying between +0.75 to +1.15%; Reichart et al.,
1997), pCO2 remained constant within a factor of 1.2 in
the last 15 ka (IndermuÈhle et al., 1999) and the temperatures were relatively constant between 25 and 27 C
based on the Uk'37 values, the 13C-value of this compound should provide information on the average
growth rate of E. huxleyi during times of deposition.
Since our samples are an average of c. 0.5 Ka short term
patterns will be averaged out and only long term trends
will be visible. Fig. 3 shows the 13C-contents of the C37:2
methylketone in the Arabian Sea sediments which shows
that at both at sites 464 and 455 no signi®cant variations
are observed and in fact remains rather constant at
ÿ23.50.3%. This suggests that growth rates did not
signi®cantly vary at the sampled resolution during the
last 15 Ka. The average value in itself is relatively high
compared to data reported from the Equatorial Paci®c,
Santa Monica Basin and Bermuda Atlantic but lower
than that for the Peru Upwelling area (Bidigare et al.,
1997), suggesting excellent growth conditions for haptophyte algae due to a high input of nutrients in this
upwelling area.
3.2. Highly branched isoprenoids
All samples contained C25 and C30 highly branched
isoprenoid (HBI) polyenes. The A3 and A4-fractions
contained C25 (II) and C30 HBI's (III) with 2±4 and 4±6
double bonds, respectively (Fig. 4). In addition, a C35
HBI (IV) with 7 double bonds was present as well, as
reported previously (Hoefs et al., 1995b). These compounds are encountered in numerous sediments (Rowland and Robson, 1990) and the C25 and C30 carbon
skeletons are known to be biosynthesised by two diatom
species Haslea ostrearia and Rhizosolenia setigera
(Volkman et al., 1994; Wraige et al., 1997; Sinninghe
Damste et al., 1999) and are used as diatom biomarkers.
Indeed, diatoms are the main phytoplankton living in
the temporary Arabian Sea and comprise on average
86% of the total phytoplanktonic population (Sawant
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
513
Fig. 2. GC-traces of total lipid fractions of samples (a) site 455, sample 139±141 cm and (b) site 464, sample 84±88 cm.
and Madhupratap, 1996). One of the more common
species occurring in the diatom community are dierent
strains of Rhizosolenia spp. among which is R. setigera.
Thus R. setigera could be the main source for the C25
and C30 HBI's though it is likely that there are more
diatom sources as well (see below).
Most 13C-contents of the diatom-derived biomarkers
are generally between ÿ22 to ÿ24% (Fig. 4; Table 2).
However, two speci®c C30 HBI isomers are very depleted
in 13C with values of ÿ37%. A similar phenomenon was
noted in an Arabian Sea sediment by Eglinton et al. (1997).
They found HBI-isomers with values between ÿ19.9 and
ÿ23.2% and one C30 HBI isomer with a d13C value of
ÿ37.1%. Kohnen et al. (1992) and Schouten et al. (1997)
also detected dierent HBI-isomers (albeit in sulphurized
form) with widely dierent isotopic compositions in sediments from the Vena del Gesso basin and the Monterey
Formation, respectively. The isotopic dierence between
514
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
Fig. 3. Stable carbon isotopic composition of the C37:2 alkenone in samples 464 (circles) and 455 (squares) through time.
the C25 and C30 HBI isomers in the Arabian Sea sediments indicate that R. setigera cannot be the only source
for the HBI's and that alternative (diatom) sources have
to be considered.
The depleted values of the C30 HBI-isomers indicate
maximum fractionation of the algae. Assuming that the
HBI is 5% depleted compared to the diatom biomass
(Schouten et al., 1998a) and the isotopic composition of
CO2[aq] is approximately ÿ8%, this would mean an estimated maximum Rubisco fractionation of approximately ÿ24%. This extreme fractionation may be
explained if they exclusively derive from diatom species
which have extremely low nitrogen-limited growth rates
and a relatively high cell area-to-volume ratio (Popp et
al., 1998). The fact that the lipids are so depleted and
require maximum Rubisco fractionation implicitly
implies that they did not derive their inorganic carbon by
actively using bicarbonate as suggested before from data
of d13C values of diatom sterols in the Peru upwelling
region (Pancost et al., 1997). However, this may be a
distinct possibility for the diatoms which biosynthesized
the HBI isomers with carbon isotope values between
ÿ22 and ÿ24%.
3.3. C30±C32 1,15-alkanediols
The total lipid fractions contain relatively high
amounts of C30±C32 1,15- and 1,14-alkanediols (e.g. V;
Fig. 2) and a C32 alka-15-keto-ol. C30±C32 1,15-alkanediols occur in a range of marine and freshwater sediments
(for a review see Versteegh et al., 1997) and are well known
products from marine and freshwater Eustigmatophytes
(Volkman et al., 1992, 1999). Ten Haven et al. (1992)
already reported the presence of C30 1,15-alkanediols in
sediments at oshore Oman. The occurrences of these
compounds in all sediments investigated in this study
suggest that Eustigmatophytes occurred throughout the
last 15 ka in the Arabian Sea. Unfortunately, due to the
complex mixture in which the alkanediols elute it was
not possible to determine the stable carbon isotopic
composition of these compounds directly. However, a
number of extracts (921 and 451) were treated with HI
to release ether-bound acyclic and cylic biphytanes (see
below). During this treatment the C30 alkanediols,
together with any ester-bound counterparts, are transformed into a C30 n-alkane. Although other ester- or
ether-bound C30 moieties may contribute as well, the
relatively high abundance of the C30 alkanediols before
HI-treatment and the C30 n-alkane after HI-treatment
suggest that the latter is predominantly derived of the
former. The d13C value of this n-alkane varied between
ÿ28.6% (site 455) and ÿ30.9% (site 921). These values
are relatively depleted compared to other algal biomarkers
in these sediments. Indeed the same phenomenon was
observed for samples in the Black Sea (Eglinton et al.,
unpublished results). This ®ts well with the fact that the
Eustigmatophyte algae are relatively small in size (e.g. 2±4
mm for Nannochloropsis; van den Hoek et al., 1995) and
therefore, may be less limited in their uptake of inorganic
carbon than other algae (Popp et al., 1998).
3.4. Sterenes
The major compounds resolved in the gas chromatograph in A2-fractions of samples of sites 451 and 921 were
C27±C30 sterenes with the 2 sterenes (VI) dominating
(Fig. 4). A complex mixture of C27±C30 sterenes, steradienes and steratrienes dominated the steroids present in
the A3-fraction, with double bonds at positions 2, 5, 22
and/or 24(28). These components are the result of early
diagenetic dehydration of the alcohol groups of C27±C30
sterols, compounds present in relatively low abundance
in the total lipid fraction (Fig. 2). The sterenes are
derived from numerous algal sources (Volkman, 1986).
The stable carbon isotopic composition of the cholestenes range from ÿ23.5% to ÿ24.8% and average
around ÿ24.70.9% (Table 2), identical to the value of
ÿ25.0% reported by Eglinton et al. (1997) for cholest-2ene in an Arabian Sea sediment sample. The 13C-contents of higher homologs were dicult to obtain due to
the complex mixture of sterenes, even after hydrogenation. The C27 sterenes are likely diagenetic products of
C27 sterols, especially cholesterol. These compounds are
either derived from de novo biosynthesis of algae
(Volkman, 1986) or are derived from higher carbon
number sterols through modi®cation by zooplankton
herbivory (e.g. Goad, 1981). Since the latter process has
no signi®cant eect on the stable carbon isotopic composition of the precursor sterol (Grice et al., 1998), the
13
C-content of the C27 sterenes are thought to re¯ect an
average value of 13C-content of sterols.
Schouten et al. (1998a) showed that the isotopic
composition of the C27 sterol of R. setigera, grown in
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
515
Fig. 4. GC-traces of TLC fractions of sediment sample at site 921. (a) A2-fraction, (b) A3-fraction and (c) A4-fraction. i.s.=internal
standard. Numbers in italic indicate stable carbon isotopic compositions of compounds.
batch culture, is identical to that of the C25:5 HBI it
biosynthesizes. If this holds for all HBI-synthesizing
diatoms, then the isotopic composition of the C27 sterenes should be similar to that of the HBI's in case the
diatoms are the main source of sterols in the Arabian
Sea. Indeed, the weighted-average isotopic composition of
the HBI's in the dierent sediments is around ÿ25%,
similar to the d13C value of the C27 sterenes. However,
haptophyte algae, which also produce a range of sterols
(Volkman, 1986) may also bear an imprint on the isotopic
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S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
Table 2
Stable carbon isotopic compositions of selected biomarkers in
sediments from the Arabian Sea
Site 451a
Compound
Site 921
n-Alkanes
C29
C31
ÿ28.30.1 ÿ28.50.2
ÿ27.90.3 ÿ27.80.3
Highly branched isoprenoids
C25:0
C25:3
C25:30
C25:4
C30:0
C30:5
C30:6
C30:60
C35:7
Steroids
C27:0
C27:15
C27:25,22
C28:25,22
Decyl C275-sterol ether
Decyl C275,22-sterol ether
ÿ23.30.3 ÿ21.7
ÿ19.90.1
ÿ19.40.2
ÿ21.70.8
ÿ27.70.2 ÿ29.60.7
ÿ20.90.3
ÿ37.20.1
ÿ37.30.5
ÿ22.31.6
ÿ24.80.2 ÿ23.50.1
ÿ24.60.7
ÿ24.20.9
ÿ25.20.7
ÿ25.80.1
ÿ24.80.1
Hopanoids
Hop-(22,29)-ene
17b,21b-hopane
ÿ22.70.7
Triterpenoids
13a -malabaricatriene
13b-malabaricatriene
13-malabaricane
ÿ21.70.4
ÿ21.90.2
Miscellaneous
C30 alkanediolsb
C37 alkenones
C40 tricyclic biphytaneb
a
b
Site 453a
ÿ23.10.2 ÿ24.00.1
ÿ22.30.3
ÿ30.90.3
ÿ21.90.1 ÿ22.20.3
ÿ19.70.1
Hydrogenated compounds.
After HI-treatment of extract of subsample 0±0.9.
patterns of the C27 sterenes. Bidigare et al. (1997) and
Schouten et al. (1998a) have shown that haptophyte
algal sterols are approximately 3% depleted compared
to the long-chain alkenones in E. huxleyi and Isochrysis
galbana, respectively. Since long-chain alkenones in
these sediments are on average ÿ22.10.2%, sterols
biosynthesized by alkenone-producing algae should
have isotopic compositions of c.ÿ25%, similar to the
value observed for the sedimentary sterenes. Hence, in
the sediments studied here it is not possible to make a
distinction between diatoms or haptophyte algae as the
main source of the C27 sterenes, since their isotopic
values are similar to those expected for sterols of both
species.
3.5. C27±C29 sterol ethers
Some A4-fractions contain relatively high amounts of
tentatively identi®ed C27±C29 5-sterol ethers with a
decyl moiety ether bound to the C-3 position (VII) (Fig.
4). Their mass spectra have similar features as those of
silylated sterols with respect to the steroid fragmentation pattern (Fig. 5), with the fragment of m/z 141
belonging to the fragmentation of the decyl side-chain
(Boon and de Leeuw, 1979). It may be suggested that
these compounds are derived from modi®cations by
zooplankton of dietary algal sterols. However, the distribution of the sterol ethers is dierent from that of the
sterols, i.e. the sterols have a relatively high abundance
of the C29 5-sterol whilst the sterol ethers are dominated by the C27 5-sterol ether. Furthermore, these
compounds are rarely encountered in sediments and the
alkyl-chain is restricted to the C10 homolog. This suggests that sterol ethers represent a direct biological input
rather than a diagenetic product. Sterol ethers have been
reported before in Walvis Bay diatomaceous ooze by
Boon and de Leeuw (1979) and in Miocene sediments
from the Monterey Formation (Schouten et al., 2000).
Both settings are known to have had high input of lipids
of diatoms, as have the sediments of this study judging
from the relatively high amounts of HBI's and the
known abundance of diatoms in the Arabian Sea. This
tentatively suggests that diatoms may be the source for
these sterol ethers.
The isotopic composition of the decyl cholesterol ether
(ÿ25.8%) is composed of two biosynthetically distinct
parts: a C10 n-alkyl moiety and a C27 sterol. Presuming
that the n-alkyl part is ultimately derived from a linear
fatty acid and the sterol is built from isoprenoid units, it
can be expected that they will be isotopically dierent.
Culture experiments of Schouten et al. (1998a) showed
that the C27 sterol is enriched by approximately 1%
compared to the n-C16 fatty acid in two diatom strains.
If this is also the case for the diatoms producing the
sterol ethers then the stable carbon isotopic composition
of the sterol ethers should be only approximately 0.3%
depleted compared to sterols produced by the same
algae. Indeed, the isotopic compositions of the decyl
cholesterol ether is slightly depleted compared to that of
the sterenes (Table 2), although this dierence is within
analytical uncertainty.
3.6. Hopenes
Small amounts of C30±C31 hopenes (e.g. diploptene;
VIII) and small amounts of C27 and C30 neohopenes
(Sinninghe Damste et al., unpublished results) were
detected in A2-fractions of samples 451, 453 and 921
(e.g. Fig. 4). Similar to the sterenes, the hopenes are
likely early diagenetic products of hopanols such as
diplopterol, although neohopenes may represent a direct
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
517
Fig. 5. Mass spectrum of decyl cholesterol ether present in the A4-fraction of sediment sample at site 921.
biological input (Sinninghe Damste et al., unpublished
results).
Hopenes may be derived from numerous bacteria,
although it should be noted that major components of
the picoplankton in the Indian Ocean are cyanobacteria
of the genus Synechococcus (Burkill et al., 1993) and
Prochlorococcus (e.g. Liu et al., 1998). These species may
possibly be major sources of the sedimentary hopanoids
especially since several strains of Synechococcus are
known to produce abundant hopanoid derivatives (Llopiz et al., 1996; Summons et al., 1999). However, the
isotopic compositions of the hopanoids suggest otherwise. The isotopic compositions of the normal hopenes
vary are ÿ23 to ÿ24% (Table 2), again very similar to
the data of Eglinton et al. (1997), and are thus slightly
isotopically heavier than the sterenes. Popp et al. (1998)
showed that the stable carbon isotopic composition of
Synechococcus is relatively independent of the CO2
concentration and growth rate. This was attributed to
the relatively small cell size, which give rise to a high cell
area to carbon content ratio, which in turn enhances
passive diusion of dissolved CO2. Recently, however,
Keller and Morel (1999) suggested that this was due to
the high amount of active bicarbonate uptake. Whatever the cause, based on the empirical observations by
Popp et al. (1998) a similar degree of fractionation of
13
C can be expected for the picoplanktonic Prochlorococcus and thus a consistent depletion of
approximately 17±18% of the biomass compared to the
13
C-content of [CO2]aq. Combined with the observation
of Sakata et al. (1997) that hopanoids of the cyanobacterium Synechocystis are 6±8% depleted compared to
biomass it may be tentatively expected that hopanoids
derived from Synechococcus or Prochlorococcus have
13
C-values between ÿ31 to ÿ34%. Since the hopanoids
in the sediments studied here are considerably more
enriched it is tempting to suggest that they are not
derived from picoplanktonic cyanobacteria but other
bacteria.
3.7. Acyclic and cylic biphytanes
In a previous communication we showed that, after
ether-bond cleavage of the polar fractions of these sediments, high amounts of C40 acyclic and cyclic biphytanes (IXa) are released and that they are by far the
most dominant lipids in these sediments (Hoefs et al.,
1997). In addition, the sediments contain relatively high
amounts of C40 acyclic and cyclic biphytanediols (IXb;
Fig. 2) (Schouten et al., 1998b). The diols have not yet
been identi®ed in organisms, but based on their structural similarities with ether-bound C40 acyclic and cyclic
biphytanes and their occurrence in sediments, Schouten
et al. (1998b) suggested that they are biosynthesized by
planktonic Archaea, similar to their ether-bound counterparts (Hoefs et al., 1997; DeLong et al., 1998). Thus a
large part of the lipids in the sediments studied are
derived from these organisms. Their isotopic compositions are enriched compared to algal sterols (Hoefs et
al., 1997; Table 2). However, since it is unknown what
the carbon acquisition mechanism of these Archaea is, it
is dicult to interpret these values.
3.8. Malabaricatrienes
Two tricyclic triterpenoids were present in equal, relatively high abundance in samples 451 and 921 and in lower
abundance in 453. They were identi®ed as 17(E)-13a(H)malabarica-14(27),17,21-triene and 17(E)-13b(H)-malabarica-14(27),17,21-triene (X) based on their identical
mass spectra and retention times of the compounds
reported by Behrens et al. (1999) and Werne et al.
518
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
(2000). Furthermore, after hydrogenation four isomers
were generated with mass spectra and retention times in
accordance with the expected hydrogenated derivatives
of the malabaricatrienes, i.e. 17R/S-13a/b (H)-malabaricane (XI). The 13a isomer has been previously identi®ed
in a lake sediment (Lake Cadagno; Behrens et al., 1999),
whilst the 13b isomer was detected in a marine sediment
(Cariaco Basin; Werne et al., 2000). The isotopic compositions of the malabaricatriene isomers are the same at
site 921 (ÿ21.7% and ÿ21.9%; Table 2), suggesting one
source, and are relatively enriched compared to algal
sterenes. Their origin remains unknown though their
occurrence in these and other sediments (Behrens et al.,
1999; Werne et al., 2000) suggest that they may be derived
from organisms living in low oxygen environments.
3.9. n-Alkanes
The A1-fractions of the samples of sites 451, 453 and 921
contained mainly saturated hydrocarbons of which C18±
C33 n-alkanes are the most dominant compounds. The
C25±C33 n-alkanes have an odd-over-even carbon-number
predominance suggesting that they are derived from a
terrestrial source (Eglinton and Hamilton, 1963).
At sites 921 and 453 the stable carbon isotopic compositions of the terrestrially derived C29 and C31 nalkanes could be determined. Their d13C-values are very
similar compared to each other and in each sample with
an average value of ÿ28.10.3%. These values are
comparable to those reported by Eglinton et al. (1997)
for C27 and C29 alkanes. The values indicate that vegetation using the C3-metabolism pathway may not be the
only source for these compounds since they have typically n-alkane values between ÿ30 and ÿ37% (Collister
et al., 1994; Lockheart et al., 1997). It seems likely that
C4- and/or CAM plants were also a source these nalkanes. The dominant terrestrial input into these sediments is likely to be transported via aeolian dust moved
by hot, north-westerly winds originating from dry, arid
areas in the Arabian peninsula and south-west Asia
(Reichart et al., 1997). This ®ts well with our hypothesis
that the terrestrial lipid input into the sediments are
partly of C4/CAM origin since these type of plants
especially thrive in arid climates (Mauseth, 1998). Goni
et al. (1997) also reported a signi®cant input of C4
plants in surface sediments in the Gulf of Mexico.
4. Conclusions
Lipids in Late Quaternary sediments from the Arabian Sea are mainly derived from planktonic Archaea
and a range of algae, including Prymnesiophytes,
Eustigmatophytes and diatoms. HBI's derived from
diatoms have widely dispersed isotopic compositions
suggesting additional sources beside the diatom species
Rhizosolenia setigera. The isotopic composition of the
unsaturated C37 alkenones from two cores during the
last 15 ka is remarkably constant and suggests that 500
year-averaged growth conditions for Prymnesiophyte
algae did not change signi®cantly over this period.
Long-chain alkyldiols are indicative of input of Eustigmatophyte algae and their relatively depleted stable
carbon isotopic compositions suggest that these algae
are less limited in their inorganic carbon uptake than
other algae. The terrestrial input of lipids is restricted to
n-alkanes and their carbon isotopic compositions show
that they are partly derived from plants using C4/CAMpathways.
Acknowledgements
We thank the Netherlands Indian Ocean Program
and Professor Dr. C.H. van der Weijden (University of
Utrecht) and Dr. W. Helder (NIOZ) for making available the samples used in this study. Drs. G. Reichart
and H.-J. Visser are thanked for subsampling. Ms. M.
Baas, W.I.C. Rijpstra and M. Dekker are thanked for
analytical assistance. Drs. P. Schaeer and X. de las
Heras are thanked for their constructive reviews. This
work was supported by a PIONIER-grant to J.S.S.D.
by the Netherlands Organization for Scienti®c Research
(NWO). Shell Internationale Petroleum Maatschappij
BV is thanked for ®nancial support for the irm-GC±MS
facility. This is NIOZ contribution No. 3398.
Associate EditorÐJ.O. Grimalt
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
Appendix
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S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
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www.elsevier.nl/locate/orggeochem
A molecular and stable carbon isotopic study of lipids in
late Quaternary sediments from the Arabian Sea
Stefan Schouten a,*, Marcel J.L. Hoefs b, Jaap S. Sinninghe Damste a,b
a
Netherlands Institute for Sea Research, Department of Marine Biogeochemistry and Toxicology, PO Box 59,
1790 AB Den Burg, Texel, The Netherlands
b
Utrecht University, Institute of Earth Sciences, PO Box 80021, 3508 TA Utrecht, The Netherlands
Received 20 July 1999; accepted 9 March 2000
(returned to author for revision 2 December 1999)
Abstract
The distribution of apolar and polar lipids and their stable carbon isotopic compositions were determined for a
number of sediment samples from dierent sites in the Arabian Sea. Lipids are mainly derived from planktonic
Archaea and a range of algae, including Haptophytes, Eustigmatophytes and diatoms. The stable carbon isotopic
compositions of compounds from diatoms, highly branched isoprenoids, fall into distinct groups suggesting other
sources beside the diatom species Rhizosolenia setigera for these compounds. High amounts of sterol ethers, which may
be derived from diatoms, were also detected. A recently identi®ed triterpenoid, malabaricatriene, was also present in
high abundance in these samples. The terrestrial input of lipids consists of n-alkanes and their carbon isotopic compositions show that they are derived from aeolian dust input from the Arabian Peninsula. The isotopic compositions of
C37 alkenones from two cores during the last 15 ka is relatively constant and suggests that growth conditions for
Haptophyte algae (averaged over 500 years) did not change signi®cantly over this period. # 2000 Elsevier Science Ltd.
All rights reserved.
Keywords: Algae; Diatoms; 13C of lipids; Alkenones; Malabaricatriene; Highly branched isoprenoids
1. Introduction
Sediments from the Arabian Sea have become a subject of great interest since the area is sensitive to changes
in climate, especially with respect to glacial cycles, which
eect monsoon intensity and, in turn, upwelling and
primary productivity. The Arabian Sea is uniquely situated since it is very sensitive to these atmospheric forces,
which leads to great seasonal variability in this part of
the ocean (Smith et al., 1998). In addition, the combination of high productivity and moderate ventilation of
the thermocline leads to an intense oxygen minimum
zone (OMZ) between 150 and 1250 m, in turn leading to
* Corresponding author. Fax: +31-222-369569.
E-mail address: [email protected] (S. Schouten).
deposition of relatively organic matter-rich sediments
enabling organic geochemical analyses.
A number of organic geochemical studies have reported
on sediments from the Arabian Sea. Ten Haven et al.
(1992) investigated a number of Pleistocene sediments of
upwelling regions, including those taken at oshore
Oman. A wide variety of extractable lipids were found,
including alkanediols, long-chain alkenones and sterols
in dierent relative concentrations. Eglinton et al.
(1997) analysed the 13C and 14C-content of a number of
biomarkers in a sediment from the Arabian Sea at oshore Oman. They found a widespread variability in
their 13C- and 14C-contents indicating multiple sources,
even for compounds with the same carbon skeleton, and
indicating dierent positions of organisms in the water
column. Schubert et al. (1998) investigated the distribution of several algal biomarkers in a core taken at oshore Pakistan, which spanned the last 200 ka. Using
0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(00)00031-0
510
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
standard biomarkers and TOC data, they inferred from
the data that the phytoplanktonic community was stable
during deposition of the sediments.
Although a number of organic geochemical studies
have been done in this area, studies on apolar hydrocarbons or stable carbon isotopic compositions of lipids
to determine their sources in recent sediments have been
limited. Thus, a detailed molecular and isotopic organic
geochemical study was undertaken of several sediment
cores from the Arabian Sea taken during the Netherlands Indian Ocean Program 1992±1993 (Van Weering
et al., 1997; Table 1, Fig. 1). Three sites (921, 451 and
453) were examined in detail for the distribution and
stable carbon isotopic composition of apolar hydrocarbons. Since these components are present in very low
amounts, large composite samples were taken from trip
cores. Three cores from dierent sites (455, 464 and 921)
were subsampled in a time range covering c. 15 ka and
total lipid fractions were analysed. Finally, long-chain
ketones were isolated from all samples and analysed for
their stable carbon isotopic compositions.
2. Methods
2.1. Samples
The samples investigated in this study are listed in
Table 1. Sites 451, 453, 455 and 464 were sampled in the
Arabian Sea during the 1992±1993 expedition of the
R.V. Tyro in the Indian Ocean (Netherlands Indian
Ocean Program; Fig. 1). From site 451 and 453 only a
trip core sample was studied. Sediment samples from
NIOP 455 and 464 were obtained by subsampling piston
cores from this site at speci®c intervals (Table 1). The
samples represent an average of approximately 500 years.
Site 921 is oshore Oman and was sampled during a different leg of the same Tyro-expedition. One large sample
(depth of 5±8 cm) from the core and several smaller subsamples were analysed from this site (Table 1).
2.2. Extraction and fractionation of soluble organic matter
Sediment samples were freeze-dried and ground in a
rotary disc mill and subsequently Soxhlet extracted with
dichloromethane:methanol (7.5:1, v/v) for 24 h. The
extracts were concentrated with a rotary evaporator at
30 C. Part of the extracts (total lipid fraction) were
methylated by diazomethane and silylated using BSTFA/
pyridine and analysed by gas chromatography (GC), and
GC±mass spectrometry (GC±MS). A selected number of
extracts from composite samples of sites 451, 453 and
921 were separated using a column (252 cm; column
volume 35 ml) packed with alumina (activated for 2.5 h at
120 C). The apolar and polar fractions were eluted with
hexane:dichloromethane (9:1, v/v; 150 ml) and methanol:dichloromethane (1:1, v/v, 150 ml), respectively. The
apolar fractions were further separated by argentatious
thin layer chromatography (TLC) using hexane as
developer. The AgNO3-impregnated silica plates (2020
cm; thickness 0.25 mm) used for this purpose were prepared by dipping them in a solution of 1% AgNO3 in
methanol/ bidistillated water (4:1, v/v) for 45 s and
subsequent activation at 120 C for 1.5 h. Four fractions
(A1, Rf=0.9±1.00; A2, Rf=0.3±0.9; A3, Rf=0.1±0.3;
A4, Rf=0.00±0.1) were scraped o the TLC plate and
Table 1
Samples analysed in this study
Site
Location
Water depth (m)
Sediment interval (cm)
Approximate age (ka)a
451
23 400 5300 N
66 020 9700 E
23 150 3000 N
65 440 5000 E
23 330 4000 N
65 570 3000 E
542
0±100
±
1556
0±100
±
995
464
22 150 4000 N
63 350 1000 E
1470
921
16 040 2300 N
52 360 2800 E
455
34±36
74±75
89±91
139±141
25±28
48±50
64±67
84±88
115±118
5±8
0±0.9
0.9±1.8
1.8±2.7
2.7±3.6
3.6±4.5
7.7
10
12
13
1.4
5.1
7.6
10.8
14.4
±
±
±
±
±
±
453
455
a
Reichart et al. (1997) and personal communication.
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
511
Fig. 1. Map showing the location of the samples investigated in this study.
ultrasonically extracted using ethyl acetate (3).
Separation of the fractions from silica/Ag+ proceeded
using a small pipette (volume 2 ml) half ®lled with alumina (not activated), by elution with hexane:dichloromethane (9:1, v/v).
Ketone fractions were isolated from polar fractions or
total extracts by separation using a small column/pipette
(volume 2 ml) half ®lled with alumina (not activated) and
elution with hexane:dichloromethane (1:1, v/v).
All total lipid and TLC fractions were analysed by
GC, GC±MS and isotope-ratio-monitoring gas chromatography±mass spectrometry (irm-GC±MS). Since the fractions sometimes contained polyunsaturated compounds
they were hydrogenated in ethyl acetate containing a few
drops of acetic acid with H2/PtO2 for 1 h at room temperature. The hydrogenated fractions were analysed by
GC, GC±MS and irm-GC±MS.
Some of the total lipid extracts or polar fractions were
treated with HI/LiAlH4 to release ether bound compounds as described previously (Hoefs et al., 1997).
Brie¯y, the fractions were re¯uxed in a solution of 56 wt%
HI in water for 1 h and the released alkyl iodides were
treated with LiAlH4 in 1,4-dioxane for 1h to convert them
to hydrocarbons.
2.3. Gas chromatography
GC was performed using a Hewlett Packard 5890
series II chromatograph equipped with an on-column
injector and ®tted with a 250.32 mm fused silica
capillary column coated with CP-Sil 5 (®lm thickness
0.12 mm). Helium was used as the carrier gas and the
oven was programmed from 70 to 130 C at 20 C/min,
followed by an increase of 4 C/min to 320 C (10 min
hold time). Detection was performed using a ¯ame
ionization (FID).
2.4. Gas chromatography±mass spectrometry
GC±MS analyses were performed using the same
chromatograph and conditions as described for GC.
The column was directly inserted into the electron
impact ion source of a VG-Autospec Ultima mass spectrometer, operated with a mass range of m/z 40±800, a
cycle time of 1.8 s and ionization energy of 70 eV.
2.5. Isotope-ratio-monitoring gas chromatography±mass
spectrometry
Isotope-ratio monitoring was performed using a
DELTA-C irm-GC±MS system (Schouten et al., 1998a),
equipped with an on-column injector and ®tted with a
250.32 mm fused silica capillary column coated with
CP-Sil 5 (®lm thickness 0.12 mm). Helium was used as
carrier gas and the oven was programmed from 70 to
130 C at 20 C/min, followed by an increase of 4 C/min
to 320 C (20 min hold time). Isotopic values were calculated by integrating the mass 44, 45 and 46 ion currents
512
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
of the peaks produced by combustion of the chromatographically separated compounds and those of CO2-spikes
produced by admitting CO2 with a known 13C-content at
regular intervals into the mass spectrometer. Two analyses
were carried out for each sample and the results were
averaged to obtain a mean value and to calculate the
standard deviation.
3. Results and discussion
Five sediment cores from dierent sites of the Arabian Sea were sampled and analysed for total lipids.
From three cores (NIOP 451, 453 and 921) samples were
taken and analyzed for the distribution and stable carbon isotopic compositions of apolar hydrocarbons as
well. The fractions of samples 451 and 453 were hydrogenated prior to irm-GC±MS analysis to simplify the
mixture of compounds. Two other cores (NIOP 455 and
464) were subsampled at several intervals and analyzed
for total lipids. In addition, long-chain ketones were
isolated from the total lipid extracts or polar fractions
and analyzed for their 13C-contents.
It is interesting to note that no sulfur compounds
were detected either in the apolar and polar fractions of
the sediments. Indeed, ¯ash pyrolyses of some of the
kerogens also yielded very low amounts of sulfur compounds (Hoefs et al., 1995a and unpublished results).
This suggests that no sulfur was incorporated into the
organic matter, consistent with the observation that no
sulfate reduction was observed in the pore waters at
these sites (Passier et al., 1997). Hence, no free hydrogen
sul®de or polysul®des were available to react with the
organic matter (Sinninghe Damste et al., 1989).
3.1. C37 alkenones
All sediments contain relatively high amounts of C37
and C38 diunsaturated methyl and ethylketones (I, see
Appendix; e.g. Fig. 2). These lipids are biosynthesized
by Prymnesiophyte algae (e.g. Volkman et al., 1980,
1995; Marlowe et al., 1984) and have been reported in
numerous sediments, including in several parts of the
Indian Ocean (e.g. Sonzogni et al., 1997). Both Gephyrocapsa oceanica and Emiliania huxleyi occur abundantly
in the northern part of the temporary Indian Ocean (e.g.
Kleijne et al., 1988) and both could be the source for the
alkenones. Based on the dominance of the C37 ketones
over the C38 ketones and the Uk'
37 correlation with temperature, Sonzogni et al. (1997) suggested that E. huxleyi is the main source of alkenones in sediments from
the Indian Ocean. The distribution of the di- vs the triunsaturated C37 ketone is used, via the Uk'
37 ratio (Brassell, 1986), as an indication for paleotemperatures. As
noted by Sonzogni et al. (1997), the diunsaturated
ketone is dominating, yielding Uk'37 always higher than
0.9. This is due to the high water temperatures (26±
28 C) of the Indian Ocean.
Since the total lipid fraction consisted of a complex
mixture of coeluting compounds, analysis of their stable
carbon isotopic compositions was impossible. The analysis of the C37 diunsaturated methyl ketone was, however,
feasible after isolation using column chromatography (see
experimental). Bidigare et al. (1997) and Popp et al. (1998)
have shown with cultures of E. huxleyi that the stable
carbon isotopic composition of this compound depends
on the isotopic composition and concentration of
[CO2]aq and the growth rate of its parent organism.
Since the isotopic composition of [CO2]aq remained
fairly constant in the time frame that our sediments
were deposited (the d13C values of the inorganic carbonate in the cores are relatively constant, i.e for 464
varying between +0.75 to +1.15%; Reichart et al.,
1997), pCO2 remained constant within a factor of 1.2 in
the last 15 ka (IndermuÈhle et al., 1999) and the temperatures were relatively constant between 25 and 27 C
based on the Uk'37 values, the 13C-value of this compound should provide information on the average
growth rate of E. huxleyi during times of deposition.
Since our samples are an average of c. 0.5 Ka short term
patterns will be averaged out and only long term trends
will be visible. Fig. 3 shows the 13C-contents of the C37:2
methylketone in the Arabian Sea sediments which shows
that at both at sites 464 and 455 no signi®cant variations
are observed and in fact remains rather constant at
ÿ23.50.3%. This suggests that growth rates did not
signi®cantly vary at the sampled resolution during the
last 15 Ka. The average value in itself is relatively high
compared to data reported from the Equatorial Paci®c,
Santa Monica Basin and Bermuda Atlantic but lower
than that for the Peru Upwelling area (Bidigare et al.,
1997), suggesting excellent growth conditions for haptophyte algae due to a high input of nutrients in this
upwelling area.
3.2. Highly branched isoprenoids
All samples contained C25 and C30 highly branched
isoprenoid (HBI) polyenes. The A3 and A4-fractions
contained C25 (II) and C30 HBI's (III) with 2±4 and 4±6
double bonds, respectively (Fig. 4). In addition, a C35
HBI (IV) with 7 double bonds was present as well, as
reported previously (Hoefs et al., 1995b). These compounds are encountered in numerous sediments (Rowland and Robson, 1990) and the C25 and C30 carbon
skeletons are known to be biosynthesised by two diatom
species Haslea ostrearia and Rhizosolenia setigera
(Volkman et al., 1994; Wraige et al., 1997; Sinninghe
Damste et al., 1999) and are used as diatom biomarkers.
Indeed, diatoms are the main phytoplankton living in
the temporary Arabian Sea and comprise on average
86% of the total phytoplanktonic population (Sawant
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
513
Fig. 2. GC-traces of total lipid fractions of samples (a) site 455, sample 139±141 cm and (b) site 464, sample 84±88 cm.
and Madhupratap, 1996). One of the more common
species occurring in the diatom community are dierent
strains of Rhizosolenia spp. among which is R. setigera.
Thus R. setigera could be the main source for the C25
and C30 HBI's though it is likely that there are more
diatom sources as well (see below).
Most 13C-contents of the diatom-derived biomarkers
are generally between ÿ22 to ÿ24% (Fig. 4; Table 2).
However, two speci®c C30 HBI isomers are very depleted
in 13C with values of ÿ37%. A similar phenomenon was
noted in an Arabian Sea sediment by Eglinton et al. (1997).
They found HBI-isomers with values between ÿ19.9 and
ÿ23.2% and one C30 HBI isomer with a d13C value of
ÿ37.1%. Kohnen et al. (1992) and Schouten et al. (1997)
also detected dierent HBI-isomers (albeit in sulphurized
form) with widely dierent isotopic compositions in sediments from the Vena del Gesso basin and the Monterey
Formation, respectively. The isotopic dierence between
514
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
Fig. 3. Stable carbon isotopic composition of the C37:2 alkenone in samples 464 (circles) and 455 (squares) through time.
the C25 and C30 HBI isomers in the Arabian Sea sediments indicate that R. setigera cannot be the only source
for the HBI's and that alternative (diatom) sources have
to be considered.
The depleted values of the C30 HBI-isomers indicate
maximum fractionation of the algae. Assuming that the
HBI is 5% depleted compared to the diatom biomass
(Schouten et al., 1998a) and the isotopic composition of
CO2[aq] is approximately ÿ8%, this would mean an estimated maximum Rubisco fractionation of approximately ÿ24%. This extreme fractionation may be
explained if they exclusively derive from diatom species
which have extremely low nitrogen-limited growth rates
and a relatively high cell area-to-volume ratio (Popp et
al., 1998). The fact that the lipids are so depleted and
require maximum Rubisco fractionation implicitly
implies that they did not derive their inorganic carbon by
actively using bicarbonate as suggested before from data
of d13C values of diatom sterols in the Peru upwelling
region (Pancost et al., 1997). However, this may be a
distinct possibility for the diatoms which biosynthesized
the HBI isomers with carbon isotope values between
ÿ22 and ÿ24%.
3.3. C30±C32 1,15-alkanediols
The total lipid fractions contain relatively high
amounts of C30±C32 1,15- and 1,14-alkanediols (e.g. V;
Fig. 2) and a C32 alka-15-keto-ol. C30±C32 1,15-alkanediols occur in a range of marine and freshwater sediments
(for a review see Versteegh et al., 1997) and are well known
products from marine and freshwater Eustigmatophytes
(Volkman et al., 1992, 1999). Ten Haven et al. (1992)
already reported the presence of C30 1,15-alkanediols in
sediments at oshore Oman. The occurrences of these
compounds in all sediments investigated in this study
suggest that Eustigmatophytes occurred throughout the
last 15 ka in the Arabian Sea. Unfortunately, due to the
complex mixture in which the alkanediols elute it was
not possible to determine the stable carbon isotopic
composition of these compounds directly. However, a
number of extracts (921 and 451) were treated with HI
to release ether-bound acyclic and cylic biphytanes (see
below). During this treatment the C30 alkanediols,
together with any ester-bound counterparts, are transformed into a C30 n-alkane. Although other ester- or
ether-bound C30 moieties may contribute as well, the
relatively high abundance of the C30 alkanediols before
HI-treatment and the C30 n-alkane after HI-treatment
suggest that the latter is predominantly derived of the
former. The d13C value of this n-alkane varied between
ÿ28.6% (site 455) and ÿ30.9% (site 921). These values
are relatively depleted compared to other algal biomarkers
in these sediments. Indeed the same phenomenon was
observed for samples in the Black Sea (Eglinton et al.,
unpublished results). This ®ts well with the fact that the
Eustigmatophyte algae are relatively small in size (e.g. 2±4
mm for Nannochloropsis; van den Hoek et al., 1995) and
therefore, may be less limited in their uptake of inorganic
carbon than other algae (Popp et al., 1998).
3.4. Sterenes
The major compounds resolved in the gas chromatograph in A2-fractions of samples of sites 451 and 921 were
C27±C30 sterenes with the 2 sterenes (VI) dominating
(Fig. 4). A complex mixture of C27±C30 sterenes, steradienes and steratrienes dominated the steroids present in
the A3-fraction, with double bonds at positions 2, 5, 22
and/or 24(28). These components are the result of early
diagenetic dehydration of the alcohol groups of C27±C30
sterols, compounds present in relatively low abundance
in the total lipid fraction (Fig. 2). The sterenes are
derived from numerous algal sources (Volkman, 1986).
The stable carbon isotopic composition of the cholestenes range from ÿ23.5% to ÿ24.8% and average
around ÿ24.70.9% (Table 2), identical to the value of
ÿ25.0% reported by Eglinton et al. (1997) for cholest-2ene in an Arabian Sea sediment sample. The 13C-contents of higher homologs were dicult to obtain due to
the complex mixture of sterenes, even after hydrogenation. The C27 sterenes are likely diagenetic products of
C27 sterols, especially cholesterol. These compounds are
either derived from de novo biosynthesis of algae
(Volkman, 1986) or are derived from higher carbon
number sterols through modi®cation by zooplankton
herbivory (e.g. Goad, 1981). Since the latter process has
no signi®cant eect on the stable carbon isotopic composition of the precursor sterol (Grice et al., 1998), the
13
C-content of the C27 sterenes are thought to re¯ect an
average value of 13C-content of sterols.
Schouten et al. (1998a) showed that the isotopic
composition of the C27 sterol of R. setigera, grown in
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
515
Fig. 4. GC-traces of TLC fractions of sediment sample at site 921. (a) A2-fraction, (b) A3-fraction and (c) A4-fraction. i.s.=internal
standard. Numbers in italic indicate stable carbon isotopic compositions of compounds.
batch culture, is identical to that of the C25:5 HBI it
biosynthesizes. If this holds for all HBI-synthesizing
diatoms, then the isotopic composition of the C27 sterenes should be similar to that of the HBI's in case the
diatoms are the main source of sterols in the Arabian
Sea. Indeed, the weighted-average isotopic composition of
the HBI's in the dierent sediments is around ÿ25%,
similar to the d13C value of the C27 sterenes. However,
haptophyte algae, which also produce a range of sterols
(Volkman, 1986) may also bear an imprint on the isotopic
516
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
Table 2
Stable carbon isotopic compositions of selected biomarkers in
sediments from the Arabian Sea
Site 451a
Compound
Site 921
n-Alkanes
C29
C31
ÿ28.30.1 ÿ28.50.2
ÿ27.90.3 ÿ27.80.3
Highly branched isoprenoids
C25:0
C25:3
C25:30
C25:4
C30:0
C30:5
C30:6
C30:60
C35:7
Steroids
C27:0
C27:15
C27:25,22
C28:25,22
Decyl C275-sterol ether
Decyl C275,22-sterol ether
ÿ23.30.3 ÿ21.7
ÿ19.90.1
ÿ19.40.2
ÿ21.70.8
ÿ27.70.2 ÿ29.60.7
ÿ20.90.3
ÿ37.20.1
ÿ37.30.5
ÿ22.31.6
ÿ24.80.2 ÿ23.50.1
ÿ24.60.7
ÿ24.20.9
ÿ25.20.7
ÿ25.80.1
ÿ24.80.1
Hopanoids
Hop-(22,29)-ene
17b,21b-hopane
ÿ22.70.7
Triterpenoids
13a -malabaricatriene
13b-malabaricatriene
13-malabaricane
ÿ21.70.4
ÿ21.90.2
Miscellaneous
C30 alkanediolsb
C37 alkenones
C40 tricyclic biphytaneb
a
b
Site 453a
ÿ23.10.2 ÿ24.00.1
ÿ22.30.3
ÿ30.90.3
ÿ21.90.1 ÿ22.20.3
ÿ19.70.1
Hydrogenated compounds.
After HI-treatment of extract of subsample 0±0.9.
patterns of the C27 sterenes. Bidigare et al. (1997) and
Schouten et al. (1998a) have shown that haptophyte
algal sterols are approximately 3% depleted compared
to the long-chain alkenones in E. huxleyi and Isochrysis
galbana, respectively. Since long-chain alkenones in
these sediments are on average ÿ22.10.2%, sterols
biosynthesized by alkenone-producing algae should
have isotopic compositions of c.ÿ25%, similar to the
value observed for the sedimentary sterenes. Hence, in
the sediments studied here it is not possible to make a
distinction between diatoms or haptophyte algae as the
main source of the C27 sterenes, since their isotopic
values are similar to those expected for sterols of both
species.
3.5. C27±C29 sterol ethers
Some A4-fractions contain relatively high amounts of
tentatively identi®ed C27±C29 5-sterol ethers with a
decyl moiety ether bound to the C-3 position (VII) (Fig.
4). Their mass spectra have similar features as those of
silylated sterols with respect to the steroid fragmentation pattern (Fig. 5), with the fragment of m/z 141
belonging to the fragmentation of the decyl side-chain
(Boon and de Leeuw, 1979). It may be suggested that
these compounds are derived from modi®cations by
zooplankton of dietary algal sterols. However, the distribution of the sterol ethers is dierent from that of the
sterols, i.e. the sterols have a relatively high abundance
of the C29 5-sterol whilst the sterol ethers are dominated by the C27 5-sterol ether. Furthermore, these
compounds are rarely encountered in sediments and the
alkyl-chain is restricted to the C10 homolog. This suggests that sterol ethers represent a direct biological input
rather than a diagenetic product. Sterol ethers have been
reported before in Walvis Bay diatomaceous ooze by
Boon and de Leeuw (1979) and in Miocene sediments
from the Monterey Formation (Schouten et al., 2000).
Both settings are known to have had high input of lipids
of diatoms, as have the sediments of this study judging
from the relatively high amounts of HBI's and the
known abundance of diatoms in the Arabian Sea. This
tentatively suggests that diatoms may be the source for
these sterol ethers.
The isotopic composition of the decyl cholesterol ether
(ÿ25.8%) is composed of two biosynthetically distinct
parts: a C10 n-alkyl moiety and a C27 sterol. Presuming
that the n-alkyl part is ultimately derived from a linear
fatty acid and the sterol is built from isoprenoid units, it
can be expected that they will be isotopically dierent.
Culture experiments of Schouten et al. (1998a) showed
that the C27 sterol is enriched by approximately 1%
compared to the n-C16 fatty acid in two diatom strains.
If this is also the case for the diatoms producing the
sterol ethers then the stable carbon isotopic composition
of the sterol ethers should be only approximately 0.3%
depleted compared to sterols produced by the same
algae. Indeed, the isotopic compositions of the decyl
cholesterol ether is slightly depleted compared to that of
the sterenes (Table 2), although this dierence is within
analytical uncertainty.
3.6. Hopenes
Small amounts of C30±C31 hopenes (e.g. diploptene;
VIII) and small amounts of C27 and C30 neohopenes
(Sinninghe Damste et al., unpublished results) were
detected in A2-fractions of samples 451, 453 and 921
(e.g. Fig. 4). Similar to the sterenes, the hopenes are
likely early diagenetic products of hopanols such as
diplopterol, although neohopenes may represent a direct
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
517
Fig. 5. Mass spectrum of decyl cholesterol ether present in the A4-fraction of sediment sample at site 921.
biological input (Sinninghe Damste et al., unpublished
results).
Hopenes may be derived from numerous bacteria,
although it should be noted that major components of
the picoplankton in the Indian Ocean are cyanobacteria
of the genus Synechococcus (Burkill et al., 1993) and
Prochlorococcus (e.g. Liu et al., 1998). These species may
possibly be major sources of the sedimentary hopanoids
especially since several strains of Synechococcus are
known to produce abundant hopanoid derivatives (Llopiz et al., 1996; Summons et al., 1999). However, the
isotopic compositions of the hopanoids suggest otherwise. The isotopic compositions of the normal hopenes
vary are ÿ23 to ÿ24% (Table 2), again very similar to
the data of Eglinton et al. (1997), and are thus slightly
isotopically heavier than the sterenes. Popp et al. (1998)
showed that the stable carbon isotopic composition of
Synechococcus is relatively independent of the CO2
concentration and growth rate. This was attributed to
the relatively small cell size, which give rise to a high cell
area to carbon content ratio, which in turn enhances
passive diusion of dissolved CO2. Recently, however,
Keller and Morel (1999) suggested that this was due to
the high amount of active bicarbonate uptake. Whatever the cause, based on the empirical observations by
Popp et al. (1998) a similar degree of fractionation of
13
C can be expected for the picoplanktonic Prochlorococcus and thus a consistent depletion of
approximately 17±18% of the biomass compared to the
13
C-content of [CO2]aq. Combined with the observation
of Sakata et al. (1997) that hopanoids of the cyanobacterium Synechocystis are 6±8% depleted compared to
biomass it may be tentatively expected that hopanoids
derived from Synechococcus or Prochlorococcus have
13
C-values between ÿ31 to ÿ34%. Since the hopanoids
in the sediments studied here are considerably more
enriched it is tempting to suggest that they are not
derived from picoplanktonic cyanobacteria but other
bacteria.
3.7. Acyclic and cylic biphytanes
In a previous communication we showed that, after
ether-bond cleavage of the polar fractions of these sediments, high amounts of C40 acyclic and cyclic biphytanes (IXa) are released and that they are by far the
most dominant lipids in these sediments (Hoefs et al.,
1997). In addition, the sediments contain relatively high
amounts of C40 acyclic and cyclic biphytanediols (IXb;
Fig. 2) (Schouten et al., 1998b). The diols have not yet
been identi®ed in organisms, but based on their structural similarities with ether-bound C40 acyclic and cyclic
biphytanes and their occurrence in sediments, Schouten
et al. (1998b) suggested that they are biosynthesized by
planktonic Archaea, similar to their ether-bound counterparts (Hoefs et al., 1997; DeLong et al., 1998). Thus a
large part of the lipids in the sediments studied are
derived from these organisms. Their isotopic compositions are enriched compared to algal sterols (Hoefs et
al., 1997; Table 2). However, since it is unknown what
the carbon acquisition mechanism of these Archaea is, it
is dicult to interpret these values.
3.8. Malabaricatrienes
Two tricyclic triterpenoids were present in equal, relatively high abundance in samples 451 and 921 and in lower
abundance in 453. They were identi®ed as 17(E)-13a(H)malabarica-14(27),17,21-triene and 17(E)-13b(H)-malabarica-14(27),17,21-triene (X) based on their identical
mass spectra and retention times of the compounds
reported by Behrens et al. (1999) and Werne et al.
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S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
(2000). Furthermore, after hydrogenation four isomers
were generated with mass spectra and retention times in
accordance with the expected hydrogenated derivatives
of the malabaricatrienes, i.e. 17R/S-13a/b (H)-malabaricane (XI). The 13a isomer has been previously identi®ed
in a lake sediment (Lake Cadagno; Behrens et al., 1999),
whilst the 13b isomer was detected in a marine sediment
(Cariaco Basin; Werne et al., 2000). The isotopic compositions of the malabaricatriene isomers are the same at
site 921 (ÿ21.7% and ÿ21.9%; Table 2), suggesting one
source, and are relatively enriched compared to algal
sterenes. Their origin remains unknown though their
occurrence in these and other sediments (Behrens et al.,
1999; Werne et al., 2000) suggest that they may be derived
from organisms living in low oxygen environments.
3.9. n-Alkanes
The A1-fractions of the samples of sites 451, 453 and 921
contained mainly saturated hydrocarbons of which C18±
C33 n-alkanes are the most dominant compounds. The
C25±C33 n-alkanes have an odd-over-even carbon-number
predominance suggesting that they are derived from a
terrestrial source (Eglinton and Hamilton, 1963).
At sites 921 and 453 the stable carbon isotopic compositions of the terrestrially derived C29 and C31 nalkanes could be determined. Their d13C-values are very
similar compared to each other and in each sample with
an average value of ÿ28.10.3%. These values are
comparable to those reported by Eglinton et al. (1997)
for C27 and C29 alkanes. The values indicate that vegetation using the C3-metabolism pathway may not be the
only source for these compounds since they have typically n-alkane values between ÿ30 and ÿ37% (Collister
et al., 1994; Lockheart et al., 1997). It seems likely that
C4- and/or CAM plants were also a source these nalkanes. The dominant terrestrial input into these sediments is likely to be transported via aeolian dust moved
by hot, north-westerly winds originating from dry, arid
areas in the Arabian peninsula and south-west Asia
(Reichart et al., 1997). This ®ts well with our hypothesis
that the terrestrial lipid input into the sediments are
partly of C4/CAM origin since these type of plants
especially thrive in arid climates (Mauseth, 1998). Goni
et al. (1997) also reported a signi®cant input of C4
plants in surface sediments in the Gulf of Mexico.
4. Conclusions
Lipids in Late Quaternary sediments from the Arabian Sea are mainly derived from planktonic Archaea
and a range of algae, including Prymnesiophytes,
Eustigmatophytes and diatoms. HBI's derived from
diatoms have widely dispersed isotopic compositions
suggesting additional sources beside the diatom species
Rhizosolenia setigera. The isotopic composition of the
unsaturated C37 alkenones from two cores during the
last 15 ka is remarkably constant and suggests that 500
year-averaged growth conditions for Prymnesiophyte
algae did not change signi®cantly over this period.
Long-chain alkyldiols are indicative of input of Eustigmatophyte algae and their relatively depleted stable
carbon isotopic compositions suggest that these algae
are less limited in their inorganic carbon uptake than
other algae. The terrestrial input of lipids is restricted to
n-alkanes and their carbon isotopic compositions show
that they are partly derived from plants using C4/CAMpathways.
Acknowledgements
We thank the Netherlands Indian Ocean Program
and Professor Dr. C.H. van der Weijden (University of
Utrecht) and Dr. W. Helder (NIOZ) for making available the samples used in this study. Drs. G. Reichart
and H.-J. Visser are thanked for subsampling. Ms. M.
Baas, W.I.C. Rijpstra and M. Dekker are thanked for
analytical assistance. Drs. P. Schaeer and X. de las
Heras are thanked for their constructive reviews. This
work was supported by a PIONIER-grant to J.S.S.D.
by the Netherlands Organization for Scienti®c Research
(NWO). Shell Internationale Petroleum Maatschappij
BV is thanked for ®nancial support for the irm-GC±MS
facility. This is NIOZ contribution No. 3398.
Associate EditorÐJ.O. Grimalt
S. Schouten et al. / Organic Geochemistry 31 (2000) 509±521
Appendix
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