volkman, 1986 (ols) (geoq)

Org. Geochem. Vol. 9, No. 2, pp. 83 99, 1986 0146-6380/86 $3.00 + 0.00 Printed in Great Britain. All rights reserved Copyright ;C, 1986 Pergamon Press Ltd

A review of sterol markers for marine and terrigenous organic matter

JOHN K. VOLKMAN CSIRO Division of Oceanography, G.P.O. Box 1538, Hobart, Tasmania, Australia 7001

(Received 23 May 1985; accepted 18 October 1985)

Abstract--A review of literature on the occurrence of 4-desmethyl sterols in unicellular algae indicates that 'few sterols are sufficiently restricted in distribution to be considered unambiguous markers for specific algal groups. Almost all of the 4-desmethyl sterols found in higher plants occur in marine algae, sometimes as major constituents, so sterol distributions do not always allow one to distinguish between marine or terrigenous organic matter. Some of the problems associated with the use of sterols as markers for specific sources are highlighted by comparison of the sterol distributions in selected marine sediments and seawater samples. Although each sediment represents a very different depositional environment the sterol distributions are surprisingly similar. The sterol distributions in a saline Antarctic lake and samples of particulate matter from oligotrophic waters off the east Australian coast show that marine phytoplankton biosynthesize a wide range of sterols, including large amounts of 24-ethylcholest-5-en-3fl-ol which is often used as a marker for terrigenous organic matter. Similar distributions occur in marine sediments from the upwelling area off Peru and in temperate intertidal sediments despite large differences in algal productivity between the two areas. In deeper sediments, most of the sterols are not derived from phytoplankton but from higher plants. These data indicate that inferences drawn from sterol distributions regarding sources of organic matter must be made with caution and should be supported using other lipid data. It further follows that in ancient sediments and crude oils a high proportion of C29 steranes need not indicate that most of the organic matter was derived from vascular plants.

Key words: sterols, phytoplankton, biological markers, Recent sediments, seawater particles, early diagenesis.

INTRODUCTION

Many studies have highlighted the advantages of sterols as biological markers and much is now known about the distributions of sterols in sediments from many different types of environments (e.g. Brassell and Eglinton, 1983a,b, Mackenzie et al., 1982). Sterols are comparatively stable and hence they have a long geological record. They also possess structural features, such as positions of double bonds and patterns of side-chain alkylation, which are restricted to a few groups of organisms. However, some sterols are widely distributed in biological systems which makes them of limited value for assigning sources of organic matter in sediments. Sterols are also excellent biomarkers for tracing diagenetic transformations in both Recent and immature ancient sediments (Mackenzie et al., 1982).

In mature ancient sediments and crude oils, distributions of 5~(H)-steranes are found which can be related to precursor sterols by reasonably well docu- mented diagenetic pathways. Sterane mixtures are often complex, as are the distributions of sterols in Recent sediments, but there has been little progress in using these distributions as source indicators. In most cases, the only parameter determined is the ratio of C27 to C2~ steranes which is thought to reflect the

83 O G 9 2 l )

relative amounts of autochthonous and allochthon- ous (higher plant) inputs (e.g. Huang and Mein- schein, 1976; 1979). A high proport ion of C29 steranes is thought to indicate a large input of terrigenous organic matter, since precursor C29 sterols such as 24-ethylcholest-5-en-3fl-ol are abundant in vascular plants.

The results presented in this paper were chosen to highlight some of the strengths and ambiguities in the use of sterols as biomarkers for elucidating sources of organic matter. Marine organisms were expected to be major lipid sources in each of the samples studied, and yet C29 sterols predominate in each case. A detailed examination of the sterol distributions, combined with data for other lipid classes, indicates that marine organisms can be major sources of 24-ethylcholest-5-en-3fl-ol and other sterols presently used as biomarkers for terrigenous organic matter. The literature on sterols in unicellular algae is also reviewed and this suggests that a reassessment of many sterol biomarkers is needed.

EXPERIMENTAl,

Data for 3 sediments and 2 seawater samples are presented in this paper. Details of the sampling locations are given in Table 1.

84 JOHN K. VOLKMAN

Table 1. Sediment and seawater samples

Sample code Type ~ Depth Water column Location Sterols

C.I./41-50 S 41-50 cm 0 m 38"49'S 146"20'E 1.6 ,ug/g h Ace 31/50-55 S 50 -55cm 2 3 m 6828 .4 'S 78"11,1'E 1170~g/g PBC7/16-19 S 16-19 cm 8 5 m 15"02.2'S 75'~30.9'W 9.4 # g / g L.C./0-10 c S 0-10 cm 32 m - - ~ 80/~g/g SPI 1/83/23/0 SW 0 m > 2000 m 32 '00'S 155 0 0 ' W 0.3 ,ug/I SPI 1/83/23/60 SW 60 m > 2000 m 32 '00'S 155' 00"W 0.2/1 g/I

uS: sediment; SW: seawater. hConcentration of free sterols per g of dry sediment. ' D a t a for Loch Clair ( 0 -10cm) sediments f rom Cranwell and Volkman (1981).

. i d~

Samples

(a) Seawater. Seawater samples were obtained using a 81 Niskin bottles from depths of 0m (SP11/83/23/0) and 60m (SP11/83/23/60) during cruise SP11/83 of R / V Sprightly in June, 1983 from a station about 220 km off the East Australian coast north-east of Sydney. Each sample was filtered on- board through precleaned G F / F filters to obtain the particulate matter. Material greater than 200 #m in size was excluded using a mesh prefilter. The filters were frozen for later analysis on-shore. Total solvent extractable (TSE) lipids were obtained by extracting the filters 3 times with CHCI3:CH3OH (2:1, 10ml) using ultrasonication. The combined extracts were washed with saline solution and a portion was then analyzed for total lipid classes using Iatroscan thin layer chromatography-flame ionisation detection (TLC-FID). A second portion was saponified in KOH solution (10 ml, H20:CH3OH 2:1, pH 12) for 6hr. Neutral lipids were extracted using hexane- diethyl ether mixtures.

(b) Intertidal sediment. A 2 m x 5 cm o.d. sediment core was obtained in August, 1976 from the intertidal area of Old Yankie Beach, Corner Inlet, Victoria, Australia (38°49'S, 146°20'E). Details of the distributions of sterols and other lipids in surface sediments from this site are given by Volkman (1980c, 1981a). Data from a core section 41-50 cm deep (C.I./41-50) are reported in this paper.

(c) Antarctic lake sediments. Several sediment cores were obtained in June 1978 by H. Burton from the middle of Ace Lake. This small (1.3 x l0 s m2), saline, meromictic lake is situated in the Vestfold Hills of Princess Elizabeth Land, Eastern Antarctica. The lake is ice-covered from March to November which prevents wind-driven mixing of the lake waters giving rise to permanent stratification. Information on the phytoplankton and bacteria in the lake and their lipid and pigment compositions is given by Volkman et al., (1986).

Core 31 from the centre of the lake at a water depth of 23 m was sectioned into 5 cm sections at the collection site and immediately frozen (ambient air temperature <0°C). Portions of each core section were thawed in December 1983 and extracted with propan-2-ol (20 ml) and then CHCI3:CH3OH (2: 1, 20ml, 6 times) using ultrasonication. The initial extraction with propan-2-ol was included to inhibit

any enzyme activity which might promote trans- esterification of lipids containing fatty acids. Previous work with chloroform-methanol extraction of Peru sediments had shown that high concentrations of fatty acid methyl esters can be formed unless such precautions are taken. The solvent extracts were then combined, washed with saline solution and evapor- ated to dryness. Data from a core section 50-55 cm deep (Ace 31/50-55) are given here.

(d) Peru sediment. This sample was collected by J. W. Farrington and R. B. Gagosian using a MkIIl box corer deployed at a site (BC7) about 7 km from the Peru coast. Full details of the collection and extraction of this sample are given by Volkman et al. (1983) and Gagosian et al. (1983a, b).

Analyses o f sterols

Aliquots of the TSE lipids from each of the sediment samples were separated into major lipid classes by column chromatography on silicic acid (7g) deactivated with 5% distilled water (Gagosian et al., 1983b). After elution of fractions containing less polar lipids, a fraction containing 4-desmethyl sterols was obtained using 20% ethyl acetate in hexane. These sterols were then converted to trimethylsilyl ether (TMS-ether) derivatives using bis(trimethylsilyl)trifluoroacetamide (BSTFA), for GC and G C - MS analysis.

Initial gas chromatographic analysis of the Peru sample was carried out using a Carlo Erba FTV 2150 gas chromatograph equipped with a 20 m × 0.32 mm i.d. pyrex WCOT capillary column as described by Volkman et al. (1983). Sterols from the Antarctic lake sediments and seawater samples were analyzed using a Shimadzu GC-9A gas chromatograph fitted with an SGE OCI-3 cool on-column injector and Hewlett Packard high performance grade bonded-phase fused silica column (25 m x 0.2 mm i.d.) coated with methyl silicone gum (0.11 #m fii~ thickness). Hydrogen was used as the carrier gas at a linear velocity of 34cm/sec. A dual ramp temperature program was employed with the oven heated from 40 to 150C at 30°C/min and then from 150 to 32OC at 4 C/rain. FID detection was used with a detector temperature of 350C. The Peru and Antarctic sediment samples were reanalyzed using a Hewlett Packard HP5890A capillary gas chromatograph with comparable results.

Sterol m a r k e r s

Table 2. Percentage composition of 4-desmethyl sterols in sediments and seawater samples

85

Peak" number

Sample"

C.I. Ace 31 PBC7 Structure ̂ Identification 41 50 5(~55 16-19

t .C. (~10

SPI 1/83 23/0

SP11/83 23,/60

1

2 3 4 5 6 7

8

9

10

11 12

13

14 15 16

17

18

19 20

la 24-nor-cholesta-5,22E-dien-3fl-ol TR 0.6 0.1 l la 24-nor- 5~t-cholest-22E-en-3fl-ol TR 0.7 TR lb cholesta-5,22E-alien-3fl-ol 2.0 2. I 1.5 l ib 5ct-cholest-22E-dien-3fl -ol 1.3 0.5 0.2 Ic cholest-5-en-3fl-ol 5.5 8.4 12.9 IIc 5ct-cholestan-3fl-ol 7.0 2.9 3.5 Id 24-methylcholesta-5,22E-dien-3B-ol 9.2 11. I 3.6 l id 24-methyl-5ct -cholest-22E-en-3fl-ol 5.4 1.7 1.7 lllc 5ct -cholest-7-en-3fl -ol TR a TR a 0.4 l l ld 24-methyl-5e-cholesta-7,22-dien-3fl -ol TR 2.4 ND le 24-methylcholesta-5,24(28)-dien-3fl -ol 2. I TR 1.3 lie 24-methyl- 5ct-cholest-24(28)-en-3fl-ol TR TR 0.2 If 24-methylcholest-5-en-3fl-ol 7.7 2.0 7.0 Ilf 24-methyl-5ct -cholestan-3fl -ol 3.9 1.2 2. I Ig 23,24-dimethylcholesta-5,22E-dien-31~-ol 3.7 1.5 1.3 l lg 23,24-dimethyl-5~-cholest-22E-en-3fl-ol 1.0 TR 3.4 lh 24-ethylcholesta-5,22E-dien-3fl -ol 6.4 10.9 9.7 l lh 24-ethyl-5ct-cholest-22E-en-3B-ol 3.1 2.8 3.0 IIlf 24-met hyl-50t-cholest-7-en-3fl -ol ND 6.6 TR Ii 23,24-dimethylcholest-5-en-3fl-ol 1.1 ND 0.6

f lli 23,24-dimethyl-5~t-cholestan-3fl-ol TR ND 3.2 Ij 24-ethylcholest-5-en-3fl-ol 29.3 35.0 28.0 Ik 24-ethylcholesta- 5,24(28)E-dien-3fl -ol ND TR TR

f llj 24-ethyl-5ct-cholestan-3fl-ol 11.3 4.3 11.8 Ilk 24-ethyl-5ct-cholest-24(28)E-en-3fl-ol ND ND ND 11 24-ethylcholesta-5,24(28)Z-dien-3fl -ol ND TR 0.7 1II 24-ethyl-5ct-cholest-24(28)Z-en-3fl-ol ND 0.4 TR Illj 24-ethyl- 5ct-cholest-7-en-3fl-ol TR 4.9 0.9

TR ND - - TR ND

5.2 4.3 - - 0.8 TR 5.4 27.5 29.5 5.8 4.0 1.6 1.6 12.8 12.0 1.2 4.3 1.8 - - ND ND

ND ND - - 9.3 8.0 - - TR TR 5.1 2.6 1.6 4.1 1.0 1.6

- - TR TR - - TR ND 8.1 7.8 6.4 3.3 1.2 1.2

ND ND - - ND ND - - ND ND

33.6 17.6 27.7 - - TR TR

31.8 5.9 4.3 - - ND ND - - TR ND

ND ND ND ND

°Peak numbers refer to Fig. 1. bStructures are given in the Appendix. CSample designations are given in Table 1. L.C./0-10: Loch Clair (~10 cm aND: not detected ( < 0.1%); TR: trace ( < 0.5%); - - : not reported.

free sterols--data from Cranwell and Volkman (1981).

Each sterol fraction was analyzed by capillary gas chromatography mass-spectrometry (GC-MS) using a VG70/70 magnetic sector mass spectrometer coupled to a Pye 204 gas chromatograph fitted with an SGE OCI-1 on-column injector. An SGE 25 m × 0.2 mm i.d. bonded phase fused silica BP-I (methyl silicone) column was used with chromatographic conditions similar to the above. The column was inserted directly into the ion source of the MS through an interface heated to 280°C. The MS was scanned from 600 to 20 amu with a recycle time of 2.2 sec. Sterols were identified from their electron impact mass spectra, retention index data and coinjection with standards (Table 2), as described in earlier studies (Volkman et al., 1980a; 1981a, b; 1984; De Leeuw et al., 1983; Gagosian et al., 1983a, b).

RESULTS

The percentage compositions of 4-desmethyl sterols in the three sediments and two seawater samples are presented in Table 2. Data for trace constituents and some unidentified sterols have not been included but in each case 90-95% of the total 4-desmethyl sterols were identified. Selected com- positional data expressed in terms of structural type and carbon number are shown in Table 3. Capillary gas chromatograms of the sterols in two of the sediment samples are shown in Fig. 1. Sterol structures are given in the Appendix.

The concentrations of total sterols are very different in the three environments (Table 1), reflecting very dissimilar amounts of organic matter contributed to each sediment. However, differences in

Table 3. Compositions of 4-desmethyl sterol fractions according to carbon number and structure

C.I. Ace 31 PBC7 L.C. SPI 1/83 SPI I,/83 41-50 50-55 1619 0-10 23/0 23/60

% C26 sterols TR 1.3 TR - - TR TR % C27 sterols 15.8 13.9 18.5 11.2 37.5 35.4 % C28 sterols 28.3 25.0 15.9 12.0 30.0 25.0 % Cz9 sterols 55.9 59.8 62.6 76.8 32.5 39.6 % 24*ethylcholesterol 40.6 39.3 39.8 65.4 23.5 32.3

plus 24-ethyl-5ct-cholestanol Total 5~t (H)-stanols/Atstenols 0.49 0.20 0.44 0.86 0.21 0.12 5ct-cholestanol/cholesterol 1.3 0.35 0.27 1.07 0.15 0.05 24-et hyl- 5~t-cholestanol/ 0.38 0.12 0.42 0.95 0.34 0.16

24-ethylcholesterol

86 JOHN K. VOLKMAN

Ace Loke

50 - 55 cm

5

3

17

13

114

'°11 11'511 20

12 18

~L

Peru Box Core 7

16 - 19 cm

3

~ ' - - - ' - " - - T i 36 38 40 4 2

13

I 4 4

17

18

24

I I I 46 48 50

Time ( rain )

Fig. I. Partial capillary gas chromatograms of free sterols (as TMS-ethers) in (a) Ace Lake 50 55 cm sediment and (b) Peru coastal sediment 16-19 cm. GC conditions are given in the experimental section.

Sterol identifications are listed in Table 2.

stanol/stenol ratios (Table 3) are surprisingly small when one considers the large variation in depositional conditions and redox potentials.

Corner Inlet in ter t idal sed iment

Sterols are present in very low concentrations in this sediment compared with other, more organic- rich, environments. The total concentration of 4-desmethyl sterols is only 1.6/~g/g which is only slightly less than in surface sediments from this area (1.8 /~g/g; Volkman et al., 1981a). Total concentrations show little change with depth, but the proportions of individual sterols vary considerably (unpublished data). At the surface, the major 4-desmethyl sterol is 24-methylcholesta-5,22E-dien-3fl-ol (24.9%; Volk- man et al., 1981a) but in C.I./41 50 it is less abundant than 24-ethylcholest-5-en-3fl-ol (9.2% cf. 29.3%; Table 2). Other major sterols include 24-ethyl-5~- cholestan-3fl-ol (11.3%), 24-methylcholest-5-en-3fl- ol (7.7%), and 24-ethylcholesta-5,22E-dien-3fl-ol (6.4%). C29 sterols collectively comprise ~ 56% of the total sterols whereas C27 sterols represent only 15.8%.

Major sources of lipids in the intertidal zone

include diatoms, bacteria, seagrass detritus and vascular plants with minor contributions from other algae. In surface sediments diatoms are major contributors (Volkman et al., 1981a), but in deeper sediments vascular plants and seagrass detritus are clearly more important contributors. In the 41 50 cm section, the n-alkane and fatty acid distributions are dominated by C20-C30 components typical of higher plants (Johns et al., 1978).

Antarc t i c lake sediments

The concentration of4-desmethyl sterols in the Ace Lake sediment is very high ( l l70~g/g ; Table 2) reflecting high algal productivity, good preservation and the absence of benthic fauna. A previous study of Antarctic lake sediments from the dry valleys of Victoria Land found much lower concentrations of sterols (range: 0.13-16.7/~g/g; Matsumoto et al.,

1982), and even in organic rich sediments deposited in highly productive upwelling areas off Namibia and Peru and sterol concentrations fall typically in the range 80-1201~g/g (Wardroper et al., 1978; Smith et al., 1982; Gagosian et al., 1983a), with somewhat

Sterol markers 87

higher concentrations occurring at the sediment- water interface (Smith et al., 1983b).

The 4-desmethyl sterol distributions in the Ace Lake sediments are very complex, consisting of over 25 identified constituents and several sterols of unknown structure (Table 2). This diversity of structures, ~sJ typical of marine ecosystems, although complex sterol distributions are found in a few freshwater lacustrine environments (e.g. Robinson et al., 1984). In the Antarctic lake sediments studied by Matsumoto et al. (1982) only 9 sterols were reported, although the use of packed GC columns in that study would have made it difficult to recognize any minor sterols which might have been present.

The major sterol is 24-ethylcholest-5-en-3fl-ol (35.0%) despite the fact that vascular plants are not present in the Vestfold Hills. Another common plant sterol, 24-ethylcholesta-5,22E-dien-3fl-ol is also abundant (10.9%). 24-Methylcholesta-5,22E-dien- 3fl-ol represents 11.1% of the 4-desmethyl sterols and yet there are few diatom cells in the lake. Scales of the green alga P y r a m i m o n a s gel idicola are abundant throughout the core (H. R. Burton, personal communication) so one would expect this alga to be a major source of lipids. There are also smaller populations of a dinoflagellate, a Cryptophyte and a small biflagellate alga which may be Prymnesiophyte (Volkman et al., 1986). These 4 algae must contribute most of the sterols in the sediment. The high concentrations of cholest-5-en-3fl-ol in the sediments are probably mainly derived from the only animal in the lake, the copepod Paralab idocera antarc t ica , since in common with most copepods it contains cholest- 5-en-3fl-ol as the major sterol (unpublished data). Cyanobacterial mats fringe the lake but since cells have not been found in the water column it seems unlikely that they are contributors of lipids in sediments deposited in the centre of the lake.

An unusual feature of the sterol distribution is the presence of sterols with AT-unsaturation (Table 2). The identifications of 24-methyl-5~-cholesta-7,22E- dien-3fl-ol (2.4%), 24-methyl-5~-cholest-7-en-3fl-ol 6.6%) and 24-ethyl-5~-cholest-7-en-3fl-ol (4.9%) were confirmed by coinjection with authentic standards; other minor A7-sterols may be present but standards were not available for confirmation. Sterols with A s- and AS~14)-unsaturation have similar mass spectra but these are readily identified from retention index data (Itoh et al., 1982), and these were not detected in the sediment.

There are very few reports of A7-sterols in sediments. Small amounts of 5c~-cholest-7-en-3fl-ol and other A7-sterols, have been identified in a few marine sediments (lkan et al., 1975; Smith et al., 1982, 1983b) including surface sediments from Corner Inlet (Volkman et al., 1981a). Marine invertebrates such as sponges and asteroids are common sources of A7-sterols in the marine environment (Faulkner and Anderson, 1974; Ballantine et al., 1979), but these organisms are not present in Ace Lake so the A 7-

sterols must be derived from algal sources. A~-Sterols have been reported in several species of diatoms (Orcutt and Patterson, 1975), but more recent studies of diatoms have not identified these sterols as significant components (Ballantine et al., 1979; Volkman et al., 1980a; Gillan et al., 1981; Lin et al., 1982), nor are they abundant in diatomaceous oozes from Walvis Bay and Peru (Wardroper et al., 1978; Gagosian et al., 1983a, b; Smith et al., 1982, 1983). AT-Sterols are found in several species of green algae (Table 4) and thus these sterols have been proposed as markers for lipids from this algal class (Volkman et al., 1981a). AT-Sterols were not detected in P. gel idicola (unpublished data) so this green alga is probably not the source of AT-sterols in Ace Lake. Cyanobacteria are a potential source of A7-sterols in some environment (Table 4), but the source of these sterols in Ace Lake is uncertain at this stage.

Peru sed iment

The concentration of sterols in the Peru sediment is 125 times less than in the Antarctic core section (Table 1), but the distribution of sterols is similar in many respects (Table 3). The major constituents are the Cz9 sterols 24-ethylcholest-5-en-3/~-ol, 24-ethyl- 5~-cholestan-3/~-oi and 24-ethylcholesta-5,22E-dien- 3/~-ol, which comprise 49.5% of the total sterols. Cholest-5-en-3/~-ol is the only other sterol to exceed 10% in relative abundance (Table 2). Surface sediments from the same general area contain much higher concentrations of sterols and the distributions are considerably more complex with C28 sterols such as 24-methylcholesta-5,22E-dien-3/3-ol and 24- methyicholesta-5,24(28)-dien-3/~-ol as major components (Gagosian et al., 1983a, b; Smith et al., 1983).

The distribution of sterols in this sediment section resembles that found in lacustrine sediments such as those from Loch Clair (Cranwell and Volkman, 1981 ) where much of the organic matter is derived from vascular plants (Table 2). This is surprising since the two environments are very different; many studies have established that the organic matter deposited in the Peru coastal area results mainly from high phytoplankton productivity (Gagosian et al., 1983a, Smith et al., 1983a, b). On the basis of inferences drawn from the Antarctic sediment data, one might argue that most of the Cz9 sterols in the Peru sediments are also derived from marine algae but this is highly unlikely. Previously studies have suggested that phytoplankton could contribute some of the 24- ethylcholest-5-en-3/~-ol in surface sediments from this area (Gagosian et al., 1983a; Smith et al., 1983b), but data from sediment trap studies clearly show that 24-ethylcholest-5-en-3/~-ol and other C29 sterols are minor constituents of phytoplankton in this region (Gagosian et al., 1983a). Also, this sediment section contains high concentrations of n-alkanes and fatty acids whose distributions clearly indicate substantial inputs of terrigenous organic matter (Volkman et al.,

88 JOHN K. VOLKMAN

Table 4. ,~,5-Unsaturated 4-desmethyl sterols in unicellular green algae (Chlorophyceae and Prasino- phyceae) and cyanobacteria

Sterol"

A B C D E F G H Others Reference

Chlorophyceae Dunaliella minuta ~ 36 2 8 Coccomyxa elongata 48 33 Trebouxia decolorans + 31 3 Chlorella ellipsoidea 28 16 Chlorella saccharophila 30 7 Chlorella pringsheimii 23 1 Trebouxia sp. 23 13 Dunaliella primolecta +

Prasinophyceae Tetraselmis tetrathele 5 34 Tetraselmis chui 99 Tetraselmis sueica c 1 48 Pyramimonas gelidicola

Cyanobacteria Anacystis nidulans a 29 48 Fremyella diplosiphon d 57 36 Cvanidium caldarium + 6 39 Microcystis aeruginosa 55 6 20 Anabaena cylindrica 8 2 Spirulina platensis 6 1 51 Spirulina sp. 15 4 33 Calothrix sp. 10 7 48 Nostoc commune 10 3 4

26 7 21" 1 19 2

1 64 3 56 4 60 3 4

4 72 4 4 64 5

1 20 80" 6

2 58 1 t 7

51 8 95 5 9

6 13 90

3 7 22 2 8 8

20 10 1 1 4 3

23 10 7 10

55 II 12 13 14 14 14 14

Notes: ~A---cholest-5-en-3/%ol; B--24-methylcholest-5-en-3~-ol; C--24-ethylcholest-5-en-3//-ol; D--cholesta-5,22E-dien-3/~-ol; E--24-methylcholesta-5,22E-dien-3/~-ol; F--24-ethylcholesta- 5,22E-dien-3/~-ol; G--24-methylcholesta-5,24(28)-dien-3/~-ol; H--24-ethylcholesta-5,24(28)E or Z-dien-3/~-ol; hstationary phase, composition differs in exponential phase; Cas Platymonas sueica; aapproximate values calculated from Fig. 3 in Ref. (10); emainly AT-sterols.

References cited: (1) Ballantine et al. (1979); (2) Patterson (1974); (3) Lenton et al. (1973); (4) Patterson (1974); (5) Goad et al. (1972); (6) Prahl et al. (1984); (7) Volkman, unpublished data (1984); (8) L ine t aL (1982); (9) Volkman, unpublished data (1985) (10) Reitz and Hamilton (1968); (11) Seckbach and lkan (1972); (12) Nishimura and Koyama (1977); (13) Teshima and Kanazawa (1972); (14) Paoletti et al., (1976).

For reviews of sterols in green algae see Patterson (1971) and Holden and Patterson (1982); for cyanobacteria see also De Souza and Nes (1968) and Martiniz Nadal (1971).

1983). There is no reason to suspect that this core section received significantly less marine organic matter than other sections in the core, so the en- hanced abundance of lipids from terrigenous sources probably results from selective diagenetic removal of marine lipids (Volkman et al., 1983; Meyers et al., 1984). The factors determining the relative rates of degradation of marine vs. terrigenous organic matter are still poorly understood but the association of plant lipids with resistant biopolymers may enhance their survival compared with more readily decom- posed algal cells.

Seawater samples

Sterols were present in low concentrations in seawater particles from the East Australian coast (Table 1), which prevented positive identification of some minor sterols by GC-MS. Quantitative data for major constituents are shown in Table 2. These concentration values (0.2 and 0.3/~g/l) are comparable to values obtained for other seawater samples, such as western North Atlantic (0.1-1.3/~g/1; Gagosian and Nigrelli, 1979), Black Sea (0.45- 0.5/~g/l; Gagosian and Heinzer, 1979), Sargasso Sea (0.5-2.1/~g/1; Gagosian, 1976) and Arabian Sea (0.04#g/l; Saliot et al., 1982). In surface waters,

most of the sterols are associated with particulate organic matter (e.g. Gagosian, 1976), and their concentrations would be expected to vary in response to fluctuations in phytoplankton abundances and species distributions.

Major sterols include cholest-5-en-3/3-ol, 24-ethylcholest-5-en-3/3-ol, 24-methylcholesta-5,24(28)-dien- 3/3-ol and 24-methylcholesta-5,22E-dien-3/3-ol (Table 2). The two C28 sterols are common in seawater where they are derived mainly from diatoms (e.g. Volkman et al., 1983 and refs therein). Cholest-5-en- 3/3-ol is usually the major sterol in seawater, and it is thought to originate mainly from zooplankton lipids, with minor contributions from phytoplankton (e.g. Gagosian and Nigrelli, 1979; Gagosian et al., 1983b). 24-Ethylcholest-5-en-3/3-ol is often present in significant concentrations in seawater, but there has been uncertainty as to its origin. Some authors have used it as a marker for terrigenous organic matter (e.g. Saliot et al., 1982; Gagosian et al., 1983b), but others have suggested the possibility of a phytoplankton origin (e.g. Saliot and Barbier, 1974). The relative importance of these two sources will depend on the type of environment, the proximity of the site to sources of land-derived organic matter and the abundance and type of phytoplankton present.

Sterol markers 89

S P 1 1 / 8 3 S t a t i o n 2 3

s u r f a c e p a r t i c u l a t e m a t t e r

t o t a l f a t t y acids

( m e t h y l es ters )

16:0

18:0

14: (

i 15:0 16:1

o 15:0

I I I '12 16 20 40

18:1 _ --/,9

I I I

25 30 35

T i m e ( ra in )

Fig. 2. Partial capillary gas chromatogram of free fatty acids (methyl esters) in particulate matter from surface waters at 32°S, 155°W off the east coast of Australia. GC conditions: 20 m × 0.2 mm i.d. fused silica methyl silicone capillary column; 100 to 320°C at 4°C/min. H 2 carrier gas. The nomenclature used

is number of carbon atoms: number of double bonds.

In the two samples studied here most of the sterols, including 24-ethylcholest-5-en-3fl-ol, are probably derived from phytoplankton since the sampling site is far from land. Other lipid classes such as fatty acids, alcohols or hydrocarbons did not contain significant quantities of biomarkers indicative of higher plant lipids (unpublished data) precluding inputs from airborne terrigenous sources (cf. Simoneit, 1977). The phytoplankton in these oligotrophic waters are mainly small flagellates (Prasinophyceae and Prymnesiophyceae) and cyanobacteria (Hallegraeff, 1981, 1983, 1984), whereas in areas of upwelling and high productivity diatoms are usually abundant.

The distribution of free fatty acids in the surface waters (Fig. 2) gives an indication as to likely sources of the organic matter and hence sterols. One compli- cation in any comparison with the fatty acid compositions of the different phytoplankton groups is that some of the saturated fatty acids in the particulate matter, perhaps as much as 50% in this case, are associated with detritus and not living cells. With this exception, the distribution is most similar to those found in Prymnesiophycean algae (Volkman et al.,

1981b), particularly with respect to the presence of 18:4~3, 18:5~o3, 20:5o~3 and 22:6~3. Similar

distributions are also found in some dinoflagellates (Joseph, 1975), but 4-methyl sterols typical of these algae such as dinosterol are very minor constituents of the sterols in these seawater samples. It should be noted that in phytoplankton the fatty acid 18:5~3 has only been found in Prymnesiophycean algae and dinoflagellates.

Palmitoleic acid (16:leJ7) is a relatively minor component of the fatty acid distribution (Fig. 2), but it is usually the major fatty acid in diatoms indicating that these algae cannot be the major source of the fatty acids. Unicellular green algae typically syn- thesize a high proportion of 18:1e~9, 18:2(o6 and 18:3co3 fatty acids but they usually also contain significant amounts of 16:4co3 which is barely detectable in the seawater samples. Cyanobacteria produce different types of fatty acids: some species contain 16:0 and 16:1o~7 as major components, whereas others contain a high proportion of 18:1~ 9 (Piorreck e t al., 1984 and refs therein). The fatty acid data do not exclude a significant contribution from these algae. It is of interest that fatty acids typical of bacteria such as vaccenic acid (18:1~ 7) and branched fatty acids comprise less than 3% of the total fatty acids.

90 JOHN K. VOLKMAN

D I S C U S S I O N

In any sedimentary environment there are many potential sources of organic matter. These include for example, phytoplankton, macroalgae, vascular plants, yeasts, fungi, protozoa, lower plants, zooplankton, benthic fauna and bacteria. This material can be produced in situ or brought in from distant areas by currents or from aeolian inputs. The composition of the organic matter is further affected by chemical and biological modifications, to the extent that the distribution of lipids may bear little resemblance to that produced in the overlying water column.

Each of the sediment samples studied in this paper represents a different depositional environment and yet the sterol distributions are remarkably similar. Although marine organisms would be expected to be the most important sources of organic carbon, the major sterol is 24-ethylcholest-5-en-3fl-ol which is usually thought of as a marker for terrigenous organic matter. Vascular plants do not contribute to the lipids in the Ace Lake sediments or the seawater samples, so clearly there are important algal sources for this sterol. In the Corner Inlet and Peru sediments the major source of 24-ethylcholest-5-en-3fl-ol is vascular plants despite the fact that most of the organic matter in surface sediments from these areas is derived from marine sources, principally diatoms.

These results highlight a major difficulty which may occur when attempts are made to reconstruct palaeoenvironments from lipid data (Didyk et al., 1978). It is axiomatic that the lipids found in ancient sediments are those which best resist chemical and biological degradation. If these lipids do not reflect the bulk of the organic matter deposited in the sediment, as seems to be the case in the deeper Peru and Corner Inlet sediments, then any reconstruction of the palaeoenvironment is unlikely to be accurate. Indeed, it is highly unlikely that any geochemist given

the fatty acid, hydrocarbon and sterol distributions in the Peru sediment would have predicted that this sample came from an area of upwelling and high productivity. These examples highlight the need to integrate data from a variety of lipid classes with other chemical and biological information such as physical characteristics of the sediment, presence of specific phytoplankton species and carbon isotope data.

The similarity of the sterol distributions is even more surprising when one considers the great differences in species compositions in the different environments. Although there is only one major phytoplankter in Ace Lake, and 3 minor ones, there is still a surprising diversity of sterol structures found in the sediments of the lake. Even in this comparatively simple ecosystem, it is not possible to assign unequivocal origins for many of these sterols, and some undoubtedly have several sources. In most oceanic and lacustrine environments the number of potential sources of sterols are likely to be orders of magnitude greater, which could make it difficult to assign origins to even some of the major sterols.

An assessment of sterol biomarkers for unicellular algae

A major difficulty facing organic geochemists has been the small proportion of algal species which have been analysed for sterols using modern analytical techniques. Furthermore, the taxonomy of many algal groups is in a state of flux and it is not uncommon to see some species transferred to com- pletely different classes and species names changed several times. As new data are obtained it is apparent that many sterols are more widely distributed than had been thought and some earlier sterol identifications are now known to be in error.

In Tables 4-8, I have summarized much of the recent literature concerning the distribution of sterols in unicellular algae. For reasons of space, the tables

Table 5. AS-Unsaturated 4-desmethyI sterols in Prymnesiophycean algae

Sterol"

B C- D E F G H Others Reference

Chrysochromulina po(vlepis 74 26 1 Prvmnesium patellifi, ra 94 6 2 Ochrosphaera neapolitana 59 1 12 12 141 I Ochrosphaera verrucosa 36 18 8 35 3 t I Pavlova lutheri h 27 18 23 1 31 3 Pavlova lutheri + 16 73 10 2 Emiliania huxleyi 2 98 + 4 Isochrvsis galbana 4 96 4 lsocho'sis galbana 1 + 2 97 2 lsochrysis sp.' 2 98 2 lsocho'sis sp. I 97 5 Cho'sotila lamellosa 4 + ~ 86 I 0 1 Cho'sotila lamellosa 6 49 44 6 Cho'sotila stipitata 3 + 50 47 1 l-lymenomomas carterae '~ 54 33 13 t 4 Coccolithus pelagicus" 88 12 4

Notes: "sterols are given in Table 4; ~ as Monochrysis lutheri: ~as Pseudoisocho'sis paradoxa; d _ Cricos- phaera carterae: " = Co'stallolithus hyalinus; I _ mainly 23,24-dimethylcholesta-5,22E-dien-3[~-ol.

References cited: (I) Marlowe et al. (1984); (2) Ein et al. (1982); (3) Baitantine et al. (1979); (4) Volkman et al. (1981b); (5) Berenberg and Patterson (1981): (6) Raederslorff and Rohmer (1984).

Sterol markers 91

Table 6. ALUnsatura ted 4-desmethyl sterols in diatoms (Bacillariophyceae)

Sterol"

A B C D E F G H Others Re~rence

89 5 6 I 67 21 5 6 I 2 60 3 2 23 4 2 + ' 6 3 42 4 54 1 40 4 I 12 43 4 25 17 17 40 I 3 25 6 II 58 5

I 39 1 59 d 1 95 5 6

2 1 82 7 3 1 4 7 100 8

2 98 1 + 98 2 I

1 4 1 91 3 3 91 9 a 1

21 79 9 I1 33 54 2 I 2 18 21 18" 41 d I 4 38 40 4 12 2 5 2 36 6 57 J I

+ 9 4 + 83 4 10 17 80 3 1 + + 99 + 10

100 I1 33 I 2 9 4 + 26 13 12 12

Nitzschia hmgissima Melosira granulata Thalassionema nitzschoides Nitzschia .fi'ustulum Chaetoceros simph'x Skeletonema costatum Thalassiosira [luviutilis Fragilaria sp. Asterionella glacialis Biddulphia sinensis Cvclotella nana h Novicula pelliculosa Nitzschia closterium Phaeodactylum tricornutum Phaeodactylum tricornutum Stauroneis amphioxys Nitzschia ovalis Biddulphia aurita Chaetoceros sp. Thalassiosira pseudonana Thalassiosira pseudonana Amphora exigua Amphora sp. Nitzschia alba Rhizoselenium spp.

Notes: %terols are given in Table 4; bCyclotella nana - Thalossiosira pseudonana; ' + = < I%; '~mainly A 7 or A ~191 unsaturated sterols; 'Tucosterol.

References cited: (1) Orcutt and Patterson (1975); (2) Nishimura and K o y a m a (1977); (3) Ballantine et al. (1979); (4) Boutry and Barbier (1974); (5) Lin et aL (1982); (6) Volkman and Gagosian (1982)--unpublished results; (7) Volkman et al. (1980a); (8) Kanazawa et al. (1971); (9) Gillan et al. (1981); (10) Berenberg and Patterson (1981); (1 I) Kates et al. (1978); (12) Morris and Carre (1984).

are restricted to the 8 most commonly encountered AS-unsaturated sterols found in sediments. Data from some early studies have not been included because the identifications are suspect or have proved to be erroneous. Species which do not contain significant amounts of AS-sterols such as many green algae (Holden and Patterson, 1982), some dinoflagellates (Kokke e t al . , 1981a), and a cyanobacterium (De Souza and Nez, 1968) have also been excluded. In a few cases the name of a species or its taxonomic classification has been amended according to lists published by Parke and Dixon (1976).

There are still too few published sterol analyses to state that a particular sterol is characteristic of a

given algal class, or even to define a typical sterol distribution for each group of algae. A possible exception is dinosterol which appears to be a reliable biomarker for dinoflagellates, although some dinoflagellates do not contain this sterol (Teshima e t al . ,

1980; Kokke e t al. , 1981a; Goad and Withers, 1982). Nonetheless, among the better studied algal classes some trends can be discerned:

(i) many species of green algae (Chlorophyta) contain A 7, A 5'7 and A 7'22 sterols (Holden and Patterson, 1982), but a few contain mainly A 5- unsaturated sterols (Table 4). In this group, 24-methylcholest-5-en-3fl-ol and 24-ethylcholesta-

Table 7. ALUnsatura ted 4-desmethyl sterots in dinoflagellates (Dinophyceae)

Sterol °


Gonyaulax spp. 100 I Peridinium foliaceum h 1 O0 2 Peridinium .[oliaeeum 80 20 3 Gonyaulax diegensis' 39 29" 32 4 Pyroeystis lunula 76 6 2 I 15 t 5 Gonyaulax polygramma 36 1 9 7 47 t 6 Gymnodinium wih'zeki 26 39 35 7 Glenodinium hallii 8 50 42 8 Noetiluca milaris a 1 I 5 73 6 14 9 Gymnodinium simplex 53 47 10 Proroeentrum cordatum 7 5 63 25t I 1

Notes: %terols are given in Table 4; has Gh, nodinium[bliaceum; 'as Gonyaulax diagenesis; das Noctiluca milialis; "isofucosterol; tmainly 23,24-dimethyl substituted sterols.

References cited: ( I) Alam et al. (1979a); (2) Alam et al. (1979b); (3) Withers et al. (1979); (4) Alam et al. (1978); (5) Kokke et al. (1982); (6) Volkman et al. (1984); (7) Nichols et al. (1984); (8) Alam et al. (1981); (9) Tesbima et al. (1980); (10) Goad and Withers (1982); (I I) Robinson et al. (1984).

For other analyses of sterols see Swenson et al. (1980); Kokke et al. (1979, 1981a,b); Jones et al. (1983); Wengrovitz et al. (1981); Withers et al. (1978, 1982); Withers (1983).

92 JOHN K. VOLKMAN

Table 8. ALUnsaturated 4-desmethyl sterols in other unicellular algae

Sterol"


Chrysophyceae Ochromonas danica 5 9 13 58 15 a I Ochromonas malhamensis I 98 1 1 Ochromonas sociabilis 90 2 Unidentified sp. 2 17 22 20 10 1 28" 3a

1 10 13 10 4 I 6 5Y 3b

Xanthophyceae Botrydium granulatum 14 86 4 Monodus subterraneus 33 67 4 Tribonema aequale 31 69 4

Euglenophyceae Euglena gracilis 12 16 3 69 t 5 Astasia Ionga 27 3 45 6 5 14 6

Bangiophyceae Goniotrichum elegans 24 44 5 27 7 Porphyridium cruentum 3 + 63 + + 34 d 8,9 Porphyridium aeurigeurn h 45 4 13 38 a 9

Cryptophyceae Crvptomonas sp. 95 5 10 Nematochrvsopsis roscoffensis 12 + 6 2 25 + 2 53: I 1

Raphidophyceae Fibrocapsa japonica c + 2 62 38 12

Notes: °sterols are given in Table 4; ~see also Cargile et al. (1975); Cas FCRG51--chemotaxonomic data suggest a biochemical link to dinoflagellates; amainly ergosterol; emainly C~0 sterols; /mainly A 7- sterols.

References cited: (1) Gershengorn et al. (1968); (2) Avivi et aL (1967); (3a) Rohmer et al. (1980); (3b) Kokke et al. (1984); (4) Mercer et al. (1974); (5) Brandt et al. (1970); (6) Rohmer and Brandt (19733; (7) Brothers and Dickson (1980); (8) Beastall et al. (1971); (9) Beastall et al. (1974); (10) Goad et al. (1983); (11) Raederstorff and Rohmer (1984); (12) Nichols et aL (1983).

5,22E-dien-3fl-ol usually predominate with moderate amounts of 24-ethylcholest-5-en-3fl-ol (Table 4). Most research has concentrated on the Chloro- phyceae and not other classes which is unfortunate since the Prasinophyceae can be important constituents of the phytoplankton in oceanic waters. Three species of Tetraselmis contain 24-methylcholest-5- en-fl-ol and 24-methylcholesta-5,24(28)-dien-3fl-ol as major sterols (Table 4).

(ii) Cyanobacteria (blue-green algae) have not been regarded as a major source of sterols in seawater or sediments but this view may need to be revised. Although earlier research had suggested that sterols are not present in cyanobacteria, more recent studies have confirmed their presence, albeit in low concentration compared with many unicellular algae. Most species seem to contain simple mixtures in which cholesterol and 24-ethylcholest-5-en-3fl-ol predominate (Table 4), but A 7 and AS'7-sterols including ergosterol are also found (De Souza and Nez, 1968; Seckbach and Ikan, 1972).

(iii) Prymnesiophytes usually contain from 1 to 5 major sterols, and commonly cholesterol or 24-methylcholesta-5,22E-dien-3fl-ol predominates (Table 5). Moderate amounts of 24-ethylcholest-5- en-3fl-ol and 24-ethylcholesta-5,22E-dien-3fl-ol are found in several species. Diatoms (Bacillariophyceae) contain a wider variety of sterols (Table 6), although in more than half of those analyzed a single sterol represents over 80% of the total. In most species the major sterol is either 24-methylcholesta-5,22E-dien-

3fl-ol, cholest-5-en-3fl-ol or 24-methylcholesta-5,24- (28)-dien-3fl-ol but in Amphora species 24-ethylcholesta-5,22E-dien-3fl-ol predominates (Table 6). Some species have been reported to contain large amounts of A7-unsaturated sterols (Orcutt and Patterson, 1975), but this has not been substantiated by more recent studies. 4-Methyl sterols have not been detected in either Prymnesiophycean algae or diatoms.

(iv) Dinoflagellates (Dinophyceae) are generally considered to be the major source of 4-methyl sterols in sediments (e.g. De Leeuw et al., 1983 and refs therein), but they are also potential contributors of 4-desmethyl sterols. In most species, 4-methyl sterols predominate but exceptions are known (e.g. Teshima et al., 1980), Generally, one would not expect dinoflagellates to be a major source of 4-desmethyl sterols in a sediment where 4-methyl sterols are not abundant. Complex mixtures of sterols are usually found (Table 7), and many species contain unusual sterols having A 7 or A 8 double bonds and unusual patterns of side-chain alkylation such as 23,24- dimethyl substitution. Some species of Gonyaulax contain large amounts of cholest-5-en-3fl-ol but there does not appear to be one distribution of 4-desmethyl sterols which characterizes these algae.

From the preceding discussion it is apparent that many sterols are widely distributed and few can be considered as characteristic of a particular algal class. Despite this, sterol distributions in sediments

Sterol markers 93

and seawater can still be useful indicators of possible sources of organic matter provided that the distribution as a whole is studied. It is not adequate to simply check for the presence or absence of a particular sterol; rather one should determine the most reason- able source of each sterol and examine whether this is consistenl with all information known about the sample and other lipid data. Probable sources for selected AS-unsaturated 4-desmethyl sterols arc discussed below.

In marine environments, high concentrations of cholest-5-en-3/~-ol are generally attributed to zooplankton or other marine fauna since this is the major sterol of most marine animals. However, a wide diversity of phytoplankton also contain cholest-5-en- 3[:~-ol (Tables 4 8), and in many species it is the major sterol present. Very few dinoflagellates do not contain cholest-5-en-3/~-ol and in many species it is the major 4-desmethyl sterol present (Table 7). High concentrations are found in Gonyaulax species which occasionally form massive blooms known as red tides (Alam et al., 1979a, b). It is also abundant in cultured zooxanthellae (dinoflagellates) from marine invertebrates (Withers et al., 1982). Thus, it samples containing high concentrations of dinoflagellate sterols it is possible that much of the cholest-5-en-3/~-ol is also derived from these algae.

Cholest-5-en-3/~-ol is also present in many diatoms but most do not contain large amounts (Table 6). Nonetheless, in some environments they could be significant sources of cholest-5-en-3//-ol if those species rich in cholest-5-en-3//-ol happen to predominate. A few species from the Bangiophyceae, Xanthophyceae and Euglenophyceae also contain large amounts (Table 8) and it appears to be a major sterol in most cyanobacteria although reliable quantitative data are lacking. Cholest-5-en-3/~-ol is not common in the Chrysophyceae or Chlorophyceae.

A source of cholest-5-en-3/3-ol which has largely been ignored is Prymnesiophycean algae. Although high concentrations of cholest-5-en-3/~-ol are not typical of this group it is nonetheless the major sterol in several important marine species. In oligotrophic waters a significant amount of cholest-5-en-3/~-ol may be derived from these algae. In the seawater samples examined here most zooplankton were excluded using a prefilter although some juvenile stages may have been included. The cholest-5-en-3/~-ol found must therefore be associated with the smaller size fractions which are mainly phytoplankton and cyanobacteria. The dominant flagellates off the East Australian coast are Prymnesiophytes and many of these are species of Chrysochromulina (Hallegraeff, 1983). In C. polylepis, cholest-5-en-3/~-ol represents 74% of the total sterols (Table 5), and it would be valuable to establish whether high concentrations of cholest-5-en-3/~-ol are typical of this genus.

Phytoplankton must be considered as potentially major sources of the cholest-5-en-3/~-ol present in the smaller size fractions (< 100/xm) of marine particu-

late matter, and the caution expressed by Cargile et al. (1975) regarding the use of cholest-5-en-3//-ol as a "zoogenous" marker seems to be well founded. Notwithstanding this, zooplankton are undoubtedly a major repository of cholest-5-en-3/~-ol in the marine environment and they convert much of the sterols produced by algae into cholest-5-en-3/~-ol. Also, the demonstration that copepods excrete significant amounts of cholest-5-en-3/t-ol (up to 4 ng/pellet; Volkman et al., 1980b; Prahl et al., 1984) indicates that zooplankton faecal pellets are a major source of cholest-5-en-3/t-ol in marine sediments.

24-Methylcholest-5-en-3/t-ol is much less widely distributed than cholesterol and only rarely is it the major sterol in algae. Significant amounts are found in a few diatoms and dinoflagellates and it is common in those green algae which do not biosynthesize pre- dominantly AT-unsaturated sterols. It is usually ab- sent from the Prymnesiophyceae and Xanthophyceae and a very minor constituent of a few cyanobacteria.

Small amounts of cholesta-5,22E-dien-3/~-ol are found in most marine sediments and in seawater, and yet this sterol is not common in unicellular alga. It is the major sterol (82%) in the common marine diatom Biddulphia sinens& (Volkman et al., 1980a), and it is present in at least 3 other diatoms (Table 6). It is also a minor constituent of a few dinoflagellates (Table 7). Cholesta-5,22E-dien-3//-ol is the major sterol in unicellular red algae of the genus Porphyridium (Beastall et al., 1974; Cargile et al., 1975), but these algae are not abundant in seawater, and they also contain significant amounts of ergosterol which has not been identified in marine sediments or seawater. Diatoms therefore appear to be the most likely primary source for this sterol in phytoplankton samples. In sediments, significant amounts could be derived from zooplankton faeces and moults (Gagosian et al., 1983b).

24-Methylcholesta-5,22E-dien-3//-ol has often been used as a biomarker for diatom lipids since it is present in most species and often represents over 90% of the total sterols (Table 6). This is probably valid in areas of high productivity where diatoms predominate but this sterol is also found in many other algal groups. Studies by Volkman et al., (1981b) and Marlowe et al. (1984) established that 24-methylcholesta-5,22E-dien-3/~-ol is also common in Prym- nesiophycean algae, and in almost half of the species examined to date it represents over 80% of the total sterols (Table 5). In oligotrophic waters such as those studied in this paper, Prymnesiophytes are probably a more likely source of 24-methylcholesta-5,22E- dien-3/3-ol than diatoms. Small amounts are also found in some Chrysophycean algae, unicellular red algae and dinoflagellates. It is the major sterol in 2 dinoflagellates Gymnodinium simplex and Noctiluca milialis, the latter associated with blooms known as red tides, and a cyanobacterium Anabaena eylindrica (Table 4). Limited data are available for the Crypto- phyceae but in those species examined, 24-methyl-

94 JOHN K. VOLKMAN

cholesta-5,22E-dien-3/3-ol is the major sterol (Goad et al., 1983).

24-Ethylcholesta-5,22E-dien-3/3-ol is also common in marine sediments although only rarely does it represent more than 10% of the 4-desmethyl sterols. It is a major sterol in a few diatoms, but it seldom occurs in dinoflagellates, its place being taken by sterols with 23,24-dimethyl side-chain substitution. Many Prymnesiophycean algae however do contain moderate amounts and this probably account for some of the 24-ethylcholesta-5,22E-dien-3/3-ol found in oligotrophic waters. This sterol also predominates in species of Ochromonas (Chrysophyceae; Table 8), and it is found in many green algae from the Chloro- phyceae but not from other genera of the Chloro- phyta. However, these species are rarely abundant in seawater. Significant amounts have also been found in a species of Cryptophyceae (Table 8), but too little is known about the sterols of these algae to generalize at this stage.

24-Methylcholesta-5,24(28)-dien-3/3-ol is abundant in some sediments, particularly those from the Peru upwelling area (Gagosian et al., 1983a, b). Diatoms are major components of the phytoplankton of this region and Thalassiosira eccentrica is often abundant. The major sterol of this species is 24-methylcholesta- 5,24(28)-dien-3/3-ol (~90%; J. K. Volkman, unpublished data), which accounts for the predominance of this sterol in seawater and sediment samples from this region. However 24-methytcholesta- 5,24(28)-dien-3/3-ol is the major sterol in only a few diatoms (Table 6) and it cannot be said to be a typical constituent of these algae. In such cases, inferences about sterol origins derived from studies of more typical algal sterol distributions are of limited value. Note that this sterol is only a minor constituent of diatomaceous oozes from another area of upwelling, Walvis Bay (Wardroper et al., 1978).

24-Methylcholesta-5,24(28)-dien-3fl-ol is not common in other phytoplankton groups and thus in most samples it is probably derived from diatoms. How- ever, high concentrations have been found in two dinoflagellates and 2 species of Tetraselmis (Prasino- phyceae). Green algae from the Prasinophyceae can be important constituents of the phytoplankton in oligotrophic waters (e.g. Hallegraeff, 1983), but more studies are required to establish whether this sterol is common in these species.

The C29 sterols 24-methylcholesta-5,24(28)-dien- 3/3-ol can exist as either the 24(28)E or 24(28)Z isomers but neither is common in unicellular algae. The 5,24(28)E isomer (fucosterol) is the major sterol of nearly all macroscopic brown algae (Phaeophy- ceae), whereas the 24(28)Z isomer (isofucosterol) is the major constituent of some macroscopic green algae (Charophyceae; Patterson, 1972). Small amounts of both isomers have been found in several diatoms and a moderate amount of isofucosterol occurs in the dinoflagellate Gonyaulax diegensis ( = diagenesis) (Alam et al., 1978).

An assessment o f sterol biomarkers .for plant inputs The ma jo r sterols found in higher plants (Goad

and Goodwin, 1972) are (24ct)-24-ethylcholest-5-en- 3fl-ol (sitosterol), (24cQ-24-ethylcholesta-5,22E-dien- 3/3-ol (stigmasterol) and (24~)-24-methylcholest-5- en-3/3-ol (campesterol). Small amounts of cholesterol are also found in many species, and a few plants contain (24/3)-24-methylcholesta- 5,22E-dien- 3/3-ol (brassicasterol). Sterols with A v-, A 5'24~28~- and A -~'25- unsaturation also occur as minor components in some plants (Goad and Goodwin, 1972). Most C> sterols have the 24ct configuration but recent studies have demonstrated that the C2s sterols can occur as both 24~ and 24/3 epimers.

With the possible exceptions of differences in C-24 stereochemistry, all of the common higher plant sterols are major constituents of the Ace Lake sediments and seawater samples and hence cannot be considered as unambiguous biomarkers for terrigenous organic matter. All of these sterols are found in unicellular algae, and the usefulness of 24-ethylcholest-5-en-3/3-ol as a biomarker for vascular plants is seriously open to question particularly in samples where most of the organic matter is derived from marine organisms. By extension, the presence of 24-ethylcholestane in ancient sediments and crude oils is not unambiguous evidence for organic matter derived from land plants.

Other researchers have speculated that unicellular algae could be significant sources of 24-ethylcholest- 5-en-3/3-ol in sediments (e.g. Lee et al., 1980; Volk- man et al., 1981a; Matsumoto et al., 1982; Gagosian et al., 1983; Smith et al., 1983a), usually on the basis that terrigenous sources seemed unlikely, but it has not been clear which phytoplankton species might be potential contributors. Lee et al. (1980) and Volkman et al. (1981a) suggested that diatoms might be important whereas in Antarctic lake sediments green algae and cyanobacteria are thought to be major sources (this paper and Matsumoto et al., 1982).

The type of environment will have a major influence on the species of phytoplankton present, and hence will determine the distribution of sterols incorporated into the sediment. Diatoms often dominate the phytoplankton in upwelling areas and thus they are potential contributors of 24-ethylcholest-5-en-3fl-ol in such environments. However, C29 sterols are not common in these algae (Table 6), so we would expect to find proportionally more C27 and C2~ sterols. For example, in a diatomaceous ooze from Walvis Bay, 24-ethylcholest-5-en-3/3-ol was only 4% of the total 4-desmethyl sterols (Wardroper et al., 1978).

In oligotrophic environments, Prymnesiophycean algae, green algae (mainly Prasinophyceae) and cyanobacteria usually dominate the phytoplankton (e.g. Hallegraeff, 1981). A few Prymnesiophycean algae and some Chlorophycean algae contain moderate amounts of 24-ethylcholest-5-en-3fl-ol (Tables 4 and 5) and thus are potential sources of this sterol in

Sterol markers 95

seawater and sediments. 24-Ethylcholest-5-en-3fl-ol has not been identified in the Prasinophyceae although many species common in seawate~ have not yet been studied. Green algae are common in both freshwater and saline lakes and can be major sources of 24-ethylcholest-5-en-3/3-ol and other "higher plant" sterols.

With few exceptions, cyanobacteria have been shown to contain 24-ethylcholest-5-en-3fl-ol in high concentration relative to other sterols (Table 4). The predominance of C29 sterols in cyanobacterial mats (Boon et al., 1983), indicates that cyanobacteria can be a major source of this sterol in such environments and perhaps in lacustrine and marine environments as well. Clearly there are many environments where high concentrations of 24-ethylcholest-5-en-3fl-ol could be derived from sources other than vascular plants. Conversely, a predominance of 24-ethylcholest-5-en-3fl-ol does not necessarily indicate a lacustrine environment, as shown by the sterol data for the Peru and Corner Inlet coastal samples (Table 2).

Distinguishing between sterols derived from vascular plants or marine phytoplankton is a difficult task as illustrated by the samples reported in this paper. One possibility is to look at the ratios of the three sterols 24-methylcholest-5-en-3fl-ol, 24- ethytcholesta-5,22E-dien-3/3-ol and 24-ethylcholest- 5-en-3/3-ol. In the Peru and Loch Clair sediments the ratios are I: 1.4:4.0 and 1 : 1.6: 6.6, with the latter being fairly typical of sediments where most of the sterols are derived from higher plants. Similar values are found in some of the samples studied here, so the method is fallible. However, in sediments where 24-ethylcholest-5-en-3/3-ol was less abundant than the other two sterols one would be reasonably confident that algal (not necessarily marine) sources of organic matter are important.

Another possibility is to check whether sterols with unusual side-chains such as 23,24-dimethyl, 24-nor, 27-nor, propylidine or cyclopropyl are present. Such sterols are not unique to marine organisms but appear to be mainly found in the marine environment. Many are derived from marine dinoflagellates (e.g. Goad and Withers, 1982; Withers et al., 1979) and it remains to be shown whether they are also common in freshwater species. High concentrations of gorgosterol (22,23-methylene-23,24-dimethylcholest-5-en-3/3-ol) characterize some sediments from Walvis Bay (Wardroper et al., 1978), and coral reef sediments (unpublished data), but as yet this sterol has not been identified in lacustrine sediments. The presence of A-nor steroids and sterols with shortened side-chains in sponges (Bohlin et al., 1982 and refs therein) may provide biomarkers for oxic marine conditions since only a few sponges are found in freshwater environments. High concentration of C30 4-desmethyl sterols have also been found in two species of marine Cryptophyceae (Raederstorff and Rohmer, 1984).

A better approach is to compare inferences drawn from the sterol data with information derived from other lipid classes. The presence of organic matter from terrestrial plants can be confirmed from such biomarkers as long-chain alkanes showing a high proportion of odd chain-lengths, certain triterpan- oids based on friedelane, oleanane and ursane skele- tons (e.g. Brassell and Eglinton, 1983a), saturated even-chain C40-C60 wax esters (e.g. Cranwell and Volkman, 1981), hydroxy acids and dicarboxylic acids characteristic of cutin and suberin, mid-chain alkyl ketones, fl-di-ketones and estolides amongst others.

C - 2 4 s t e reochemis t ry

In discussions on the sources of sterols in sediments little attention is usually given to the stereochemistry of the alkyl group at C-24, since the two epimers are not separable on conventional (25 m or less) apolar capillary columns. However, it is important to recognize that different algal groups produce sterols with either 24~ or 24fl stereochemistry, but rarely both. For example, the C29 sterols of vascular plants generally have the 24~ configuration, whereas green algae and dinoflagellates biosynthesize sterols with the 24fl configuration (Goad et al., 1974; Bohlin et al., 1981; Goad and Withers, 1982). Both ~ and fl isomers have been identified in an unidentified alga believed to be a Chrysophyte (Kokke et al., 1984); (24~)-24-ethylcholest-5-en-3fl-ol comprised 10.2% of 4-desmethyl sterols compared with 3.0% for the 24/3 epimer. Several algal species, including a diatom, two Prymnesiophycean algae and two Cryptophycean algae, have been shown to produce (24~)-24-methylcholesta-5,22-dien-3fl-ol (epibrassicasterol; Rubin- stein and Goad, 1974; Maxwell et al., 1980; Goad et al., 1983; Raederstorff and Rohmer, 1984). It seems likely from biosynthetic considerations that any C29 sterols produced by these algae will also have the 24~ configuration although work by Raederstorff and Rohmer (1984) suggests that this may not always be true. The presence of (24fl)-24-ethylcholest- 5-en-3fl-ol in sediments would probably be good evidence for lipid contributions from green algae, but not conclusive since the C-24 stereochemistry of 24-ethylcholest-5-en-3fl-ol in cyanobacteria has not been determined.

Sterols differing in C-24 stereochemistry can be separated on very long, polar glass capillary columns (Maxwell et al., 1980; Thompson et al., 1981), but long analysis times are involved, and to date the method has not been exploited by organic geochemists. Further work on this aspect would clearly be a valuable aid to assigning the sources of sterols in sediments (Maxwell et al., 1980), but even with this development we may still be unable to assign unequivocal origins for many sterols.

CONCLUSIONS

Studies of the sterol composition of sediments from

96 JOHN K. VOLKMAN

a saline Antarct ic lake of marine origin, and seawater particles from the east coast o f Australia demon-

strate that unicellular algae can be sources of a wide

variety of sterols, including some used as biomarkers for terrigenous organic matter. It is suggested that much of the 24-ethylcholest-5-en-3/~-ol in particulate matter from oligotrophic waters may be derived from green algae, Prymnesiophycean algae and cyanobacteria and not vascular plants. In the small size fraction of particulate matter (<200/xm) , Prym- nesiophycean algae are probably major sources of cholest-5-en-3/3-ol and 24-methylcholesta-5,22-dien- 3~-ol and not zooplankton and diatoms respectively.

A review of the sterols in the major phytoplankton groups indicates that few 4-desmethyl sterols can be considered as characteristic of a given algal class and many sterols are widely distributed. A further compli- cation in the use of sterols as biomarkers is that

species with "atypical" sterol composi t ions may dominate the phytoplankton of particular marine environments. Sterols associated with terrigenous organic mat ter appear to be less readily degraded than sterols o f marine origin so that the distributions of sterols in subsurface sediments from coastal zones, even in areas of intense upwelling and high productivity such as off Peru, are often almost entirely due to terrestrial plants.

Acknowledgements--I am very grateful to H. R. Burton, R. B. Gagosian and J. W. Farrington for providing sediment samples and for many stimulating discussions. G. Hallegraeff provided considerable assistance with algal taxomony and discussions of phytoplankton ecology of Australian waters. D. Allen and D. Everitt are thanked for valuable help in the laboratory. N. Frew and N. Davies assisted with GC-MS analyses. Financial support from a WHOI post-doctoral scholarship for the Peru study, and gifts of sterol standards from Dr W. C. M. C. Kokke, Professor T. Matsumoto and the Steroid Reference Collec- tion of the Medical Research Council (U.K.) are gratefully acknowledged. Dr D. M. McKirdy and Dr F. T. Gillan are thanked for very helpful reviews of the manuscript.

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APPENDIX

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R "

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R R

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" " j " " k ~ I

volkman, 1986 (ols) (geoq)

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