Sterols of a contemporary lacustrine sediment

Geochimjca

et Cosmochimica

Acta,

1976. Vol. 40, pp. 1221 to 1228. Pergamon Press. Printed 1” Great Britain

Sterols of a contemporary lacustrine sediment SIMON J. GASKELL* and GEOFFREYEGLINTON Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol BS8 lTS, U.K. (Receioed 1 November

1975; accepted in revisedform ! April 1976)

C2,, CzB and CZ9 sterols, including A’-, A*‘- and As***-sterols and both 5c(- and 5fi-stanols, have been identified in the contemporary lacustrine sediment of Rostherne mere (Cheshire, England). Amounts of total sterols decrease from ca. 400 ppm extracted sediment dry weight for the

Abstract-&,,

(r7 cm sediment section to cu. 40 ppm for the corresponding 18-30 cm section. Sa-stanols are of far greater abundance than the S/&tan01 isomers. The carbon number distributions of unsaturated and saturated sterols and the increase in stanol: A5-sterol ratio with sediment depth provide indirect

evidence for the operation of a sterol hydrogenation process. CZ9 sterols preponderate at lower sediment depths, suggesting a predominantly higher plant input, whereas C2, sterols are more abundant in the surface

sediment.

INTRODUCTlON

DESPITE wide acceptance of the hypothesis that the steranes identified in ancient sediments (eg. BURLINGAMEet al., 1965; HENDERSONet al., 1968; KIMBLE et al., 1974) and petroleum (eg. HILLS et al., 1970) are derived from sterols present in living organisms, the origin and fate of sterols in Recent sediments remains poorly understood. The presence of sterols in Recent aquatic sediments was demonstrated by SCHWENDINGER and ERDMAN (1964) and several unsaturated sterols were identified in marine sediments by ATTAWAY and PARKER (1970). In a survey of the sterol composition of a sedimentary sequence, extending to the Pleistocene, HENDERSONet al. (1972) identified A5-sterols and both SC+ and 5/?-stanols. A sterol hydrogenation process within the sediment was inferred but no regular trend to increased saturation with age of sediment was observed. An increase in stanol:A’-sterol ratio with sediment depth was, however, found by OGURA and HANYA (1973) for the sediments of Lake Biwa, Japan. Recently, A7- and A5-sterols and Scr-stanols have been identified by IKAN et al. (1975) in two contemporary marine sediments. Direct evidence, based on radiolabelling techniques, for the rapid (within weeks) hydrogenation of A5-sterols in a contemporary lacustrine sediment has been reported (GASKELL and EGLINTON, 1975). In the present paper we report detailed sterol analyses of several depths (corresponding to O-W. 150yr in age) of the same sediment and of possible contributors to the sedimentary organic material. Early stages of the work have been reported previously (GASKELL and EGLINTON, 1974). The sediment chosen for study is that of Rostherne mere, a freshwater lake of post*Present address: Department of Chemistry, University of Glasgow, Glasgow, G12 SQQ, U.K. t By Dr. R. S. Cambray, A.E.R.E., Harwell. $ By Dr. R. Thompson, versity of Edinburgh.

Department

of Geophysics,

Uni-

glacial origin in the Cheshire-Shropshire basin (England). Rostherne is an eutrophic lake with a high primary productivity (BELCHER and STOREY, 1968). A high input of organic material and poor water circulation contribute to a permanent oxygen depletion of the bottom waters and underlying sediment in the deepest part (30 m) of the lake. Eh data for a 30 cm sediment core indicated a minimum, of 0 mV, at approximately 4 cm depth. Estimates, based on 13’Cs determinations? suggest a current sedimentation rate of 4-5 mm/yr. Measurement of the natural remanent magnetization profilet (MACKERETH, 1971; THOMPSON, 1973) indicates that &30 cm depth of sediment covers the time period cl50 yr B.P. A full description of the sediment together with further details of the lipid chemistry will be provided elsewhere (GASKELL and EGLINTON, in preparation).

EXPERIMENTAL Sample collection Sediment core samples [30 x 6 cm (dia.)] were taken from the deepest part of Rostherne mere using a Gilson mud sampler (Freshwater Biological Association, Ambleside, U.K.) and sealed with rubber bungs. After transport to the laboratory, cores for lipid analyses were stored at -20°C. Sphagnum moss and the fern Dryopteris dilatata were wrapped in aluminium foil following collection. Peat samples were recovered from a depth (of peat) of 75 cm (age, by 14C dating: ca. 700 yr) in Gale Bog, Rostherne mere. Samples were wrapped in aluminium foil and stored in the laboratory at -20°C. The blooming of Microcystis aeruginosa (identified by D. Rogers, Nature Conservancy) in Rostherne mere facilitated its collection. Samples were collected in clean glass bottles and extracted immediately after return to the laboratory. Lipid extraction Three 30 cm sediment cores were cut at 7 and 18 cm depth and the corresponding sections combined. Total lipids obtained by heptane-isopropanol extraction, as previously described (BROOKSet al., in press), are here termed the ‘pre-hydrolysis extract’. After acidification of the sediment residues to pH 1, heating under reflux for 6 hr and subsequent extraction with heptane-isopropanol gave the

1221

S. J. GASKELLand G. EC;LINTON Table I. Mass spectrometric data for TMS ethers of sterols from Rostherne sediment’

Computerized GC-MS analysis of the trimethylsilyl ethers (see Experimental). For structures see Appendix b. Loss of trimethylsilanol and a ring A fragment comprising Cl-C3. c. S.C.= side chain. d. < 1% relative intensity. e. The stereochemistry at the AZ2 bond was not determined. f. C,, Sa-stanol also present in small amounts. Mass spectrum analogous to those of other Sa-stanols. g. Cz, AZ2-sterol also tentatively identified. a.

‘post-hydrolysis extract’. Lipid constituents of peat, moss and fern samples were obtained by 6 hr Soxhlet extraction with chloroform-methanol (3/l, v/v). The algal sample was similarly extracted after filtration through a pre-extracted Soxhlet thimble. Sepuration of sterol fractions

Pre-hydrolysis sediment extracts were separated by silica column chromatography. The toluene-ethyl acetate (95/5, v/v) eluates were separated by silica thin-layer chromatography (TLC) with hexane-diethyl ether (SO/SO, v/v) as developing solvent. Total sterol fractions so obtained were further separated by TLC on silver nitrate-impregnated silica (l/3, w/w:Ag+ TLC) with chloroform as developing solvent (CHATTOPADHYAY and MOSBACH,1965) to give ‘A5 sterol’, ‘5a-stanol’ and ‘5fi-stanol’ fractions. Post-hydrolysis sediment extracts and lipid extracts from the peat, fern, moss and algal samples were separated into acids and neutrals by shaking with aqueous KOH (7% w/v). Separation of neutral lipids was carried out by silica TLC (hexanediethyl ether, 50/50, v/v) and sterol fractions further separated by TLC on silver nitrate-impregnated alumina (l/3, w/w) with chloroform-acetone (95/5, v/v) developing solvent (KAMMERECKet al., 1967). Improved separations between A5-sterols and Sa-stanols were achieved using alumina Ag+ TLC rather than the silica Ag+ TLC system. Gas-liquid chromatography (GLC) and combined matography-mass spectrometry (GC-MS)

gas chro-

GLC analyses of sterol fractions were performed, following derivatization to the trimethylsilyl (TMS) ethers (by treatment, at room temperature, with N,O-bis-trimethylsilylacetamide), on Varian 1200 or Perkin-Elmer F17 instruments, each equipped with flame ionization detection. Separations were achieved on 3 m x 0.16 cm o.d. silanized stainless steel columns packed with l&120 mesh Gas Chrom Q, coated with 3% by weight of OV-17 or Dexsil 300 liquid phases (Phase Separations Ltd., Flintshire, U.K.). High resolution GLC of a peat sterol TMS ether

fraction was performed on a 50 m x 0.25 mm (i.d.) stainless steel column coated with Dexsil 300 liquid phase (Perkin-Elmer Ltd., Beaconsfield U.K.). All GLC analyses were performed at a temperature of 270”. GC-MS analyses were performed using a Varian 1200 gas chromatograph coupled via an all-glass, single stage Watson-Biemann helium separator to a Varian CH7 mass spectrometer. Chromatographic separation was achieved with the packed Dexsil column used in the GLC analysis. All spectra were recorded at an electron energy of 70 eV. The instrument was operated in cyclic scanning mode, with a cycle time of ca. 6 s, with a pseudo-linear scan function. Time and intensity data were output to a DEC PDPS/e computer, cia a Carrick interface (Instem Ltd., Stone, U.K.). The mass spectra were counted and normalized and automatic methods of library searching (GRBNNEBERG et al., 1975) spectrum classification (SMITH, 1972) and spectrum interpretation (GRAYand GR~NNEBERG, 1975; GRAY et al., 1975) then applied.

RESULTS Sterol constituents

of’ Rostherne

sediment

Sterol constituents of each sediment section were identified on the basis of Rr values (Ag+ TLC) and GLC retention times and mass spectra of the TMS ethers. Mass spectrometric data for the TMS ethers of sedimentary sterol constituents are given in Table 1. Diagnostic features of the mass spectra of TMS ethers of sterols have been fully discussed by other authors (BROOKS et al., 1968, 1973). The important distinction between A5-sterols and the saturated analogues may be based on the characteristic occurrence of a pair of ions, m/e 129 and [M-129]+, in the spectra of A5-sterol TMS ethers. The major sterol con-

Sterols

of a contemporary

of Rostherne sediment were characterized by mass spectrometry as 4-desmethyl sterols. Several minor sterol components present at low concentrations were tentatively identified. In most of the Sa-stanol TMS ether fractions examined, a minor component (~5% of the total fraction) eluted during GLC analysis prior to 5a-cholestan-3/?-ol TMS. GC-MS analysis of the TMS ethers of Scl-stanol constituents of the 18-30 cm sediment section gave a mass spectrum for this component consistent with the TMS ether of a CZ6, 4-desmethyl stanol with a seven carbon side chain (Table 1). * In addition to the series of Sa-stanols observed in each level of Rostherne sediment, an unsaturated component was found, in the nominal ‘5a-stanol’ fractions, eluting during GLC analysis immediately after, and incompletely resolved from 24-methyl-5a-cholestan-3fi-ol TMS. The tentative assignment, of 24-ethyl-5a-cholest-22-en-3b-ol TMS, was made on the basis of GLC data and comparison of the mass spectrum (Table 1) with published spectra of the related compounds, 24-ethyl-5/%cholest-22-en-3P-o1 TMS (ENEROTH et al., 1965) and (24S)-24-ethyl-5a-cholest-22en-3/3-yl acetate (LENFANTet al., 1967). Several of the ‘5a-stanol’ fractions extracted from levels of Rostherne sediment contained a component whose retention time suggested the possible structure, 24-methyl-5a-cholest-22-en-3fi-ol TMS. Where mass spectra stituents

Table

2. Major

lacustrine

1223

were obtained they were of low intensity but consistent with this structure. Table 2 records the amounts of individual sterol components identified in extracts from three levels of Rostherne sediment. The sterol fractions with lowest mobility on Ag+ TLC may include other unsaturated sterols but were not further examined. Sterolsfrom

other sources in the Rostherne environment

Contributors to Rostherne sediment were also analysed for sterol content. A sample from the decaying peat bog, located at the northern end of the lake, was examined as a possible contributor of non-contemporary geological material and found to contain approximately 150 ppm (dry weight) total sterols of which cu. 90% consisted of two components separable by high resolution GLC. GC-MS analysis and Fourier transform ‘H NMR analyses (after fractionation by Ag+ TLC) indicated the identities 24ethylcholest-5-en-3p-ol and 24-ethyl-5a-cholestan-3b-01, present in the ratio 7.2: 1. Minor sterol components were tentatively identified, on the basis of GLC retention times and mass spectra of the TMS ethers, as 24-methylcholest-5-en-3/?-01, 24-ethycholesta-5,22dien-3fi-01, and 24-methyl-Sa-cholestan-3/?-01. Analysis of Sphagnum moss associated with the peat bog indicated the presence of a suite of unsaturated sterols (predominantly C2s and CZs components).

sterol constituents

r

sediment

of Rostherne STEROL AND-

sediment SEDIMENT SLZTIONS

WOK

(PPd" STSuCTuSALASSIGmNTS'

0 - 7om

7 -1Scm

18 - 3OOcm

7

(2)

?

(11)

0.6(2.5)

A'-sterols cAoL!wm.-5,22-DIm-3p-oL cHoLEsT+-EN-3p-oL~ 2d-ME~CHOLESTA-5,ZZ-lr~N-jS-OL 24-MEWfLCHOLEST-5-EN-3~-OL* 24-EW,'LCHOLEST.k-5,22-DIXN-3P-OL* 24-CI1MCHOLPST-5-EN-i-)P_OLX

(IV) (I)

3? (i?) 132

(27)

11

(-1

(d

7

(12)

4

(6)

l.O(l.2)

(II)

28

(8)

7

(2)

0.2(O.S)

7

(1.5)

O.j(O.5)

(9)

2.3(3.8)

(va

10

(3)

(III)

53

(22)

(VII)

a3

(VIII) (IX)

52

~-stands C26 STANOL 5a-CHOLESTAN-3P-OF 24-MEZhn-5a-CHOLESThN-3P-OL" 24-ETKL-%-CHOLEST~3~-OL*

-

(0.4?)

-

(0.06?) 1

(0.1)

(15)

7

(1.n)

3

(C.6)

S

(:I

4

(0.9)

2

(0.7)

17

(9)

27

(1.8)

16

(I.51

5P-stands3 5~-CROlESTitN-3~-OLx 2d-EI1M5@-CHOLESTAN-)s-OL

(~1

0.6

0.01

0.01

(XI)

0.1

0.03

0.02

1. Assignments based on GLC retention times and mass spectra (recorded by computerized GC-MS: see Experimental). Asterisk (*) indicates availability of authentic standard; other sterols identified by comparison with literature data. As-sterols listed in order of increasing GLC retention time. ‘?’ indicates probable presence but no positive identification. ‘-’ indicates absence. For structures see Appendix. 2. Amounts expressed as ppm sediment dry weight (after extraction). Quantitation by measurement of GLC peak areas and comparison with internal standard (cholestane) with allowance for differing GLC response for cholestane, sterol TMS and stanol TMS. 3. 5B-stanol TMS constituents of post-hydrolysis fractions obscured during GLC analysis by the presence of other, unidentified constituents.

1224 Table

S. J. GASK~LLand G. EGLIWON 3. Sterol

constituents of Sphuynum

Rostherne

peat

and

1. Assignments based on GLC retention times and mass spectra of TMS ethers (recorded by computerized GC-MS: see Experimental). Authentic parison with all components. 2. Quantitation by GLC. ment.

standards available for comFor structures see Appendix. “?’ indicates tentative assign-

GLC analysis of a fraction corresponding in Rf value to stanols indicated the presence of minor constituents, none of which, however, corresponded to more than 1”; of the total sterols. Sterol constituents of Rostherne peat and Sphagnum are summarized in Table 3. Analysis of a species of fern, Dryopteris dilatatu, abundant in the Rostherne environment, indicated a preponderance of 24-ethylcholest-5-en-3b-ol (ca. 70 ppm dry weight) with minor amounts of components tentatively identified as 24-methylcholest-5-en-3b-ol and 24-ethylcholesta-5,22-dien-3fl-01. A sample of the major algal species of Rostherne mere. the blue-green Micwcy,sti.s cteruginosa. was also analysed for possible sterol constituents. Though GLC analysis indicated their possible presence, the quantities were extremely small&three orders of magnitude less, for example, than the quantities of free saturated acids obtained from the same sample. No characterization was attempted.

DISCUSSION GCMS. following preliminary separation of total sterols by Ag+TLC, provides a useful general procedure for the analysis of sterol mixtures of geological origin, particularly where techniques of computerized acquisition and interpretation of mass spectral data may be applied (GR~NNEBERGrt al.. 1975: GRAY and GR~NNEBERG,1975; GRAY ef al., 1975). The analytical methods employed here in the analysis of sterol constituents of Rostherne sediment have led to the identification of stanols. corresponding to the major unsaturated sterols, and to the tentative identification of minor components whose structures are of particular interest. The occurrence of a CZb stanol in Rostherne sediment represents, to our knowledge, the first report of such a sterol skeleton from a non-marine source. C26 unsaturated sterols, containing a side chain with a terminal isopropyl group, have been

reported in a number of marine organisms (e.g. IDLER et al., 1970). FEREZOU et (I/. (1974) identified 24-norcholesta-5,22-dienol in a species of red alga but were unable to detect radioactivity in the CZ6 sterol in feeding experiments with radiolabelled precursors. The hypothesis of bacterial synthesis, proposed by the authors, may suggest a possible origin for the CZh stanol in Rostherne sediment. The identification of A**-sterols in Rostherne sediment is also of interest, particularly in view of their possible origin from A’.** -sterols ciu an hydrogenation process within the sediment. Monounsaturated sterols with a A** double bond are not widespread in organisms. though they have been reported from such diverse sources as a marine sponge (BERGMANN et a/., 1945), a slime mould (HEFTMANNet al., 1960) and the roots of a plant (TAKEDA and RAPER, 1958). 24-Ethyl-5fi-cholest-22-en-3fi-ol has been identified as a metabolite of stigmasterol in cultures of a rat faecal bacterium (EY%XN et cd., 1973) and in human faeces (ENEROTH et ul., 1965). It is noteworthy that Rostherne mere, in common with Recent sediments examined by other workers, does not contain detectable quantities of 4-methyl sterols. In contrast, the abundances of 4-methyl sterols and their sterane analogues in the Eocene Messel shale were found to exceed those of the corresponding 4-desmethyl compounds (MATTERN et d., 1970: KIMBL~ et (II., 1974). Unusual features of the Messel flora or the bacterial population of the sediment provide the most plausible explanations. Saturclted

srerols

in Rosthcrrw

sediment

Examination of the data of Table 2 indicates a parallel in carbon number distributions between the A’-sterol, Sa-stanol and 5(&stanol fractions obtained from each sediment level by solvent extraction alone (GASKELL.and EC;LINTON, 1974). The abundances of the See-stanols far exceed those of S/&stanols in the sediment levels examined (Table 2). Thus the 5a-:58stanol ratio for each level is high (rn. 70:1, 900: 1 and 700: 1 for the O-~7,7 I8 and I8 30 cm levels, respectively) and contrasts with the low ratios observed for estuarine sediments grossly polluted with sewage (Goodfellow and Eglinton, unpublished data). The somewhat lower Sr-:S/&stanol ratio for the c-7 cm level. compared with lower sediment depths may reflect the small measure of sewage pollution of the lake (GASKELL. 1974) but production of 5P-stanols in the sediment certainly also occurs in siru (see below). The comparison between A”-sterol and Srx-stanol distributions is illustrated in Fig. 1, where carbon number distributions are also shown for the equivalent fractions obtained (in lesser amounts; Table 2) by extraction after acid hydrolysis of the solventextracted sediment. The close parallel in the pre-hydrolysis carbon number distributions provides strong. though circumstantial, evidence for a sterol hydrogenation process in the sediment rather than a sedimentary input of both saturated and unsaturated

Sterols of a contemporary lacustrine sediment

21

23

29

27

28

29

Fig. 1. Carbon number distributions of sterol constituents of three sections of Rostherne sediment. For amounts expressed as ppm extracted sediment dry weight, see Table 2. Abundances of individual sterols here given relative to most abundant component of fraction.

Solid lines refer to sterol constituents of pre-hydrolysis extract, dashed lines to post-hydrolysis extract. sterols. The input, from contemporary organisms, of the observed quantities (Table 2) of saturated sterols is unlikely in view of their minor abundance in living organisms so far studied. There is no doubt, however, that organisms in the Rostherne environment, such as those examined in the present work, contribute unsaturated sterols to the sediment, so that a postulation of older, eroded sedimentary material as the source of stanols is not consistent with the observed parallel in unsaturated sterol and stanol distribution. The observation of an increase in the Sot-stanol to A$-sterol ratio with sediment depth (Table 4) furnishes further indirect evidence for a sterol hydrogenation process. Similar observations have been made for a 200 m core from Lake Biwa, Japan (OGURA and HANYA, 1973). Definitive evidence for the conversion of cholesterol to both Sa- and 5/&cholestanol has been obtained by radiolabelling experiments (GASKELLand EGLINMN, 1975). The sterol analyses (of a t&10 cm sediment section) performed in conjunction with radiolabelling experiments are not directly comparable with the analyses of sediment sections described here, but comparison of the two sets of data suggests no major discrepancy in terms

1225

of carbon number distributions. The amounts of both 5a- and 5/J-stanols observed in the analysis associated with the radiolabelling experiment (GASKELL and EGLINTON,1975), however, were much higher than those reported here. Jnhomogeneity of the sediment in terms of the operation of the sterol hydrogenation process would seem the most likely explanation. Acid hydrolysis and subsequent extraction of the solvent-extracted sediment yielded further quantities of sterols, though in lesser amounts than those obtained from the initial solvent extraction. The carbon number distributions observed in the post-hydrolysis sterol fractions differ from those observed in prehydrolysis extracts (Table 2; Fig. 1) and no close parallel is observed between corresponding A5-sterol and 5a-stanol distributions. Moreover, the Sol-stanol to A5-sterol ratio shows no consistent trend with sediment depth (Table 4). A similar observation was made in carboxylic acid analyses of Rostherne sediment, where a trend to increased saturation with depth was observed in the pre-hydrolysis, but not post-hydrolysis, extracts (GASKELL,1974). The effects of the acid hydrolysis procedure are probably complex, but may include the release of bound material and the destruction of microbial cell walls, with the consequent release of intra-cellular lipids reflected in the composition of the post-hydrolysis extract (BROOKSet al., in press). The observation of relatively high abundances of Cl5 and C,, branched acids in the posthydrolysis extracts of Rostherne sediment (GASKELL, 1974) is consistent with the release of microbial lipids, since these acids are frequently cited as characteristic of microbial origin LEO and PARKER,1966; EGLINTON et al., 1968; ECLINTDN,1973; CRANWELL,1973). The evidence of sterol and fatty acid analyses suggests that bound unsaturated lipids may be afforded some degree of preservation in the sediment. Variation of sterol composition with sediment depth In general, variations in lipid composition with sediment depth may be attributed to changes in sediment input and/or diagenetic effects. With many lipid Table 4. Variation of 5a-stanol:A’-sterol ratio with Rostherne sediment depth’

1. Amounts of C,,, Czs and Ca9 Sa-stanols, and AS-sterols, respectively summed for determination of 5a-stanol: A’-sterol ratio. For amounts of individual sterols, expressed as ppm sediment dry weight, see Table 2. Amounts of sterols extracted pre-hydrolysis exceed those of sterols extracted post-hydrolysis (Table 2). Sa-stanol: 5/?stanol ratios for the pre-hydrolysis extracts are ca. 70, 900 and 700 for the O-7,7-1 8 and 18-30 cm levels, respectively.

1226

S. J.

GASKELL

and G. EGLINTON

classes, the two effects may not be readily distinguishable. The sterols may be of unique value as geochemical markers in providing clear indications of both the history of organic input to the sediment and the operation of short-term diagenetic processes. As discussed above, the change in the ratio of saturated to unsaturated sterols with Rostherne sediment depth is indicative of an early diagenetic process. Although de-alkylation and degradation of the sterol side-chain are acknowledged bacterial processes under specific conditions (eg. MALLORY and CONNOR, 1971; MARSHECK, 1971), the carbon number distributions of sterols are likely to be largely dictated by input to the sediment. Sterol analyses indicate major variations in input to Rostherne sediment with time. The pre-hydrolysis extracts from the lower sediment levels show a predominance of the CZ9 sterol skeleton, consistent with a mainly higher plant input, while CZ7 sterols are most abundant in the (r7 cm sediment section. Hydrocarbon analyses of the surface sediment sample (GASKELL, 1974) show a high relative abundance of n-heptadecane in the &7 cm level, suggesting a recently increased algal contribution. Such a conclusion is substantiated by the observed preponderance in Rostherne mere in recent years of the blue-green alga Microc+s aeruginosa, whose life cycle leads to an exceptionally high input to the sediment (REYNOLDSand ROGERS, 1976). The sterol content of the alga, however, is low so that other sources must be responsible for the abundance of the CZ7 sterols in the surface sediment. Changes in the biology of the water column associated with the changes in the algal population are a possible explanation. A general similarity is apparent between the lipid components of the 7-18 and 18-30 cm levels of Rostherne sediment (GASKELL, 1974). An exception is the proportion of A5v22-sterols, which are (relative to A5-sterols) more abundant in the 18-30 cm section (Table 2). This difference between the 7--18 and 18-30 cm levels may be associated with changes in the Rostherne environment during planting of woodland in the last century (D. A. Rogers, Nature Conservancy, personal communication). Differences in sterol composition between the posthydrolysis extracts of the sediment sections are less pronounced than in the corresponding pre-hydrolysis extracts though there remains a trend to increased relative abundance of CZ9 sterols with sediment depth (Fig. 1). Possible explanations for this observation include some selectivity in the binding of sterols to the sediment and a contribution from microbial constituents, released by the acid hydrolysis procedure. Mechanism

of sterol hydrogenation

The temperature of Rostherne sediment is low (6-10’); indeed, the failure to detect a conversion of “C-cholestero1 to “C-cholestanol in experiments conducted in the laboratory at ambient temperature (GASKELL, 1974) suggests that a raised temperature may inhibit the hydrogenation process in the sedi-

ment. Such evidence is consistent with a process effected by psychrophilic bacteria. Bacterial hydrogenation of cholesterol has been widely studied (e.g. SCHUBERTand KAUFMANN,1965; BJ~~RKHEM and GusTAFSSON,1971; EYSSENet al., 1973) though at incubation temperatures (2540”) substantially higher than those involved in the present work. The stereochemistry of the product varies with the system concerned. Thus for example, intestinal bacteria convert cholesterol to 5P-cholestanol (BJ~RKHEM and GUSTAFSSON, 1971), whereas a soil bacterium has been shown to produce 5a-cholestanol (SCHUBERT and KAUFMANN, 1965). The tentative identification of cholest-4-en-3one, 5a- and 5b-cholestan-3-one as radiolabelled products of incubation of “C-cho1esterol in Rostherne sediment (GASKELL and EGLINTON, 1975) suggests that the hydrogenation mechanism may be analogous to that observed in other biological, including microbial, systems (e.g. EYSSENet al., 1973). The variation observed in relative amounts of 5~ and S/&stanols in samples of Rostherne sediment may be attributable to an inhomogeneity in the microbial population of the sediment. In the obviously different microbial environment of anaerobic sewage sludge, 5,&, rather than 5~, stanols predominated both as natural constituents and radiolabelled products of incubation of L4C-cho1estero1 (GASKELL and EGLINTON, 1975). The parallel carbon number distributions observed for the A*-sterol, 5~ and 5p-stanol constituents of each Rostherne sediment level suggest that the hydrogenation process is insensitive to the alkyl substitution. ROSENFELDand HELLMAN(1971) showed that 3H-cho1esterol and 14C-sitostero1 were converted to the corresponding 5fl-stanols at similar rates by human intestinal bacteria. CONCLUSIONS (i) A5-, A”- and A5.“-sterols, 5a- and 5fl-stanols have been identified in a contemporary (O-150 yr B.P.) lacustrine sediment. A CZ6 stanol with a C, sidechain has been identified, though not fully characterized. 4-methyl sterols were not observed. (ii) Carbon number distributions of 5~ and 5b-stanol sediment constituents, obtained by solvent extraction alone, parallel the corresponding A5-sterol distributions. Similar parallels were not observed for the less abundant sterol constituents released from the sediment by acid hydrolysis. (iii) Amounts of Sa-stanols exceed those of 5P-stanols in Rostherne sediment. Variations in 5a-: S/j’-stanol ratio between sediment samples from similar depths suggest an inhomogeneity of the sediment. (iv) The sterol composition of sediment cores was found to vary markedly with depth, reflecting both the effects of a sterol hydrogenation process (leading to an increased 5a-stanol:A5-sterol ratio with depth) and a changing input to the sediment (leading to a variation in carbon number distribution with depth).

Sterols of a contemporary Cz9 sterols, of probable higher plant origin, predominate at lower sediment depths (7-30 cm), whereas C2, sterols, possibly derived from autochthonous sources, are the more abundant in the surface sediment. (v) Sphagnum moss and a species of fern, Dryopteris dilatata, both growing in the Rostherne environment, contained A’- and A5pz2-sterols. A peat sample taken from a site adjacent to the lake contained mainly 24-ethylcholest-5-en-3b-ol and its Sstanol analogue.

lacustrine sediment

1227

EGLINTONG. (1973) Chemical fossils: a combined organic geochemical and environmental approach. Pure Appl. Chem. 34, 611-632.

EGLINTONG., HUNNEMAN D. H. and DOURAGHI-ZADEH K. (1968) Gas chromatographic-mass spectrometric studies of long-chain hydroxy acids II. The hydroxy acids and fatty acids of a 5000-year old lacustrine sediment. Tetrahedron 24, 5929-5941. ENEROTH P.,HELLSTROM K. and RYHAGER. (1965) Identification of two neutral metabolites of stigmasterol found in human faeces. Steroids 6, 707-720. EY~~ENH. J., PARMENTIER G. C., COMPERNOLLE F. C., DE PAUW G. and PIESSENS-DENEF M. (1973) BiohydrogenaAcknowledgements-We are grateful to the Nature Consertion of sterols by Eubacterium ATCC 21,408-Nova spevancy for permission to take samples and perform expercies. Eur. J. Biochem. 36, 411-421. iments in the Rostherne mere National Nature Reserve FEREZOUJ. P., DEVYSM., ALLAISJ. P. and BARBIERM. and, in particular, to Mr. D. ROGERS,warden at Rostherne, (1974) Sur le sterol a 26 atomes de carbone de l’algue for practical assistance and informative discussions. The rouee Rhodvmenia oalmata. 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