Diagenesis of oleic acid in an estuarine sediment

Chemical Geology, 17 (1976) 319--324 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands

319

Short Communication DIAGENESIS OF OLEIC ACID IN AN ESTUARINE SEDIMENT

S.J. GASKELL*', M.M. RHEAD .2, P.W. BROOKS .3 and G. EGLINTON Organic Geochemistry Unit, School of Chemistry, University of Bristol, Bristol (Great Britain) (Received October 21, 1975; accepted for publication January 6, 1976)

ABSTRACT Gaskell, S.J., Rhead, M.M., Brooks, P.W. and Eglinton, G., 1976. Diagenesis of oleic acid in an estuarine sediment. Chem. Geol., 17: 319--324. Radiolabelled (14C, 3H) oleic acid, when incubated in estuarine sediment for 3--14 days in the laboratory, was rapidly degraded to 14CO2. A small portion of the labels was also incorporated into C~:--C~ 8 n-alkanoic acids in relative proportions approximating to the carbon-number distributions observed for the sediment itself. The implication is that these alkanoic acids in aquatic sediments result predominantly from in situ microbial synthesis. INTRODUCTION D e s p i t e t h e i r s i m p l i c i t y o f s t r u c t u r e , straight-chain c a r b o x y l i c acids continue to be v a l u a b l e subjects o f s t u d y in organic g e o c h e m i s t r y . In m a n y cases, c a r b o n - n u m b e r d i s t r i b u t i o n s in c o n t e m p o r a r y s e d i m e n t s m a y be c o r r e l a t e d w i t h t h o s e o f the biological i n p u t ( F a r r i n g t o n a n d Quinn, 1973; Cranwell, 1 9 7 4 ; E g l i n t o n e t al., 1 9 7 4 ; B r o o k s et al., 1976). E x a m i n a t i o n o f the variation o f f a t t y - a c i d c o m p o s i t i o n w i t h s e d i m e n t d e p t h p r o v i d e s i n d i r e c t e v i d e n c e f o r t h e h y d r o g e n a t i o n o f C12--C,s n - a l k e n o i c acids in s e d i m e n t s ( P a r k e r a n d Leo, 1 9 6 5 ; F a r r i n g t o n a n d Quinn, 1 9 7 1 ; Ishiwatari, 1974). T h e w o r k of R h e a d et al. ( 1 9 7 1 , 1 9 7 2 ) w i t h r a d i o l a b e l l e d oleic acid p r o v i d e d direct evid e n c e f o r t h e c o n v e r s i o n t o C~2-C~8 s a t u r a t e d acids, a l b e i t in m o d e s t yield (a m a x i m u m o f ca. 2% in eleven days), in an e s t u a r i n e s e d i m e n t . T w o bioc h e m i c a l p a t h w a y s w e r e p o s t u l a t e d ( R h e a d et al., 1971). T h e first c o n s i s t e d of s i m p l e h y d r o g e n a t i o n a n d d e c a r b o x y l a t i o n , w h e r e a s t h e s e c o n d i n v o l v e d d e g r a d a t i o n o f t h e c a r b o n chain to C2 units a n d s u b s e q u e n t resynthesis. As p a r t o f a c o n t i n u i n g p r o g r a m m e investigating, b y radiolabelling t e c h n i q u e s , Present addresses: *~ Department of Chemistry, University of Glasgow, Glasgow G12 8QQ, Great Britain. *: School of Environmental Sciences, Plymouth Polytechnic, Plymouth PL4 8AA, Great Britain. .3 Masspec Analytical Specialty Services, Ltd., Woodchester, Stroud, Gloucestershire, Great Britain.

320 the early diagenesis of various biolipids (e.g. phytol and cholesterol) in contemporary sediments (Brooks and Maxwell, 1974; Gaskell and Eglinton, 1975), we have re-examined the fate of radiolabelled oleic acid in an estuarine sediment with a view to determining the major labelled products of incubation and to correlating the labelled fatty-acid products with the fatty acids normally present in the sediment. METHODS 1-~4C-, U-~4C- and 9,10-3H-oleic acid were obtained from the Radiochemical Centre (Amersham, Great Britain) with specific activities of 210, 1,170 and 6,800 p Ci/mg, respectively. Following methylation (BF3--MeOH), the labelled material was purified on thin layers of silica impregnated with silver nitrate (10% by weight) with development in hexane--diethyl ether (90/10, v/v). The free acid was recovered by hydrolysis with methanolic KOH (7%, w/v), addition of water and extraction with hexane--diethyl ether (50/50, v/v). Determination of radioactivity (14C, 3H) of the oleic acid and of fractions following sediment incubation was by liquid-scintillation counting (ICN Tracerlab Spectromatic). Sediment samples were taken from tidal mud flats in the Severn estuary (National Grid reference ST 642983) approximately 22 km up-river from the site used by Rhead et al. (1971, 1972) and 24 km from the Avonmouth industrial complex. Radiolabelled oleic acid was injected, as the sodium salt in aqueous solution, into sediment samples (taken from 5--10 cm sediment depth) in the form of a slurry (~ 50 ml, in a sealed container) or sealed intact core (4 × 20 cm). Incubations were for periods of days in the laboratory at ambient temperature and in the dark. For experiments with sediment slurries, incubations were terminated by the injection of aqueous KOH (8%, 5 ml) and thorough mixing. Aliquots of the slurry were removed for acidification and trapping, with 2-phenylethylamine, of the released CO2 and subsequent 14C determination. Following acid hydrolysis (HCI: pH 1, eight hours reflux) of sediment samples, lipid extractions and separations were performed as previously described (Rhead et al., 1972). Polar acid products of incubation were separated as their methyl esters by thin-layer chromatography (TLC; hexane--diethyl ether--methanol, 40/10/1, v/v). Further evidence for the presence of m o n o h y d r o x y acids was obtained by acetylation (acetic anhydride--pyridine (1/1, v/v)) and TLC (hexane--diethy ether, 90/10) with radio-scanning (~4C). RESULTS AND DISCUSSION Table I records the amounts of radioactivity recovered following incubation. In experiment A, 75% of the '4C activity was recovered as '4CO2, while a similar proportion of the 3H activity was recovered in the aqueous extracts,

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322 presumably mainly as 3H20. Small amounts of ~4C activity in the aqueous e x t r act may be associated with short-chain acids and other water-soluble metabolites. A shorter incubation (three days, e x p e r i m e n t C) of U-~4C-oleic acid yielded ca. 20% of the activity as 14CO> Since a single d e c a r b o x y l a t i o n of the precursor U-14C-oleic acid would yield only ca. 5% of the activity as ~4CO2, the higher observed yield clearly demonstrates that degradation occurs by sequential oxidation of the carbon chain. Of the lipid extracts (experiments A and B) the majority of the radiolabel was associated with unchanged 3H,~ac-oleic acid. The next m ost highly labelled fraction o f the total lipids was m or e polar and probabl y consisted mainly of m o n o h y d r o x y acids (on the basis of T L C mobility of the m e t h y l esters and acetyl derivatives of the m e t h y l esters). F u r t h e r characterisation was n o t attemp ted . The recoveries of radiolabel corresponding to C,2--C~8 n-alkanoic acids (determined by preparative gas c h r o m a t o g r a p h y of the m e t h y l esters) are shown in Fig.1 (for e x p e r i m e n t B). The conversion of 3H,14C-oleic acid to 80

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Fig.1. Distribution of radiolabel in even-numbered n-alkanoic acids in Severn estuary sediment following incubation with 3H,~4C-labelledoleic acid (experiment B; Table I). Alkanoic acids (as methyl esters) obtained by TLC on AgNO3-impregnated plates (Brooks et al., 1976), of the total lipid extract (of the acid-hydrolysed sediment) after methylation. Radiolabel associated with even-numbered n-alkanoic acid methyl esters determined by liquid-scintillation counting of individual components trapped from preparative gas chromatography (Rhead et al., 1972). Components eluting between even-numbered n-alkanoic acid methyl esters contained low levels of activity. Negligible activity was associated with compounds of retention time greater than the C~8 acid methyl ester. A control experiment (zero-time incubation) revealed insignificant levels of activity in total alkanoic acids and in individually trapped fractions. total-saturated acids was n o t greater than 0.01%, much less than the conversions o f up to 2% observed in Rhead's earlier experiments with sediment from, and at, a different site (Rhead et al., 1971, 1972). Using the m e t h o d of Rhead et al. (1971, 1972), based on observed 3H:~4C ratios, the proportion of each n-alkanoic acid (C12, C,4 and Ct6) derived by complete degradation (to acetyl-CoA) of the oleic-acid precursor and resynthesis of the carbon chain (resynthesis pathway) was estimated ( e x p e r i m e n t B) to be ca. 37, 54

323 and 41%, respectively. A similar calculation for stearic acid is complicated by the possibility of cycling between stearic and palmitic acids (Rhead et al., 1971). Hence, the overall conversion to labelled n-alkanoic acids is much lower than, and the proportions of each acid formed by the resynthesis pathway somewhat different from, those observed in Rhead's earlier work. The situation is u n d o u b t e d l y complex and dependent on the precise biological environment. It is significant, however, that the distributions of both aH and 14C label (Fig. 1) resemble the carbon-number distributions observed, in the C,2--CIs region, for the natural n-alkanoic acids of the sediment (Fig.2).

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Fig.2. Carbon-number distribution of total n-alkanoic acids (C12--C3o) from Severn estuary sediment. Alkanoic acids isolated as described in Fig.1. Quantitation by gas chromatography of the methyl esters; amountsare expressed as percentages of most abundant component (C~6). Branched acid components (C~s, C~7 mainly) amount to less than 10% of the straight-chain components. Examination of the earlier data (Rhead et al., 1971, 1972) indicates similar distributions of radiolabel. This close correspondence between distributions of radiolabel (Fig. 1) and the natural pattern of straight-chain fatty-acid composition (Fig.2) suggests that a major portion of the even-numbered n-C~2--n-C~8 acids in this, and probably other, aquatic sediments, may result from the operation of biochemical degradative and synthetic processes on many possible precursors. The origin of the odd-numbered n-alkanoic acids, of low relative abundance and n o t specifically studied in this work, is less clear. However, the conversion of labelled oleic acid to labelled C~s and C~7 branched acids observed by Rhead et al. (1972) reflects the contribution of microorganisms to the alkanoic-acid composition of the sediment, n-Alkanoic acids of greater chain length (> C2o) may be attributed to a higher-plant input to the sediment (Brooks et al., 1976). In summary~ the short-term (days) fate of oleic acid in a predominantly anoxic estuarine sediment is almost complete biological degradation to CO2, and conversion to minor amounts of polar acids and, to a lesser extent, nalkanoic acids. From the results, it would appear that the majority of the C~2--Cls n-alkanoic acids in some aquatic sediments is derived by biological, probably microbiological, degradation and synthesis within the sediment.

324

ACKNOWLEDGEMENTS T h e t e c h n i c a l a s s i s t a n c e o f Mrs. J. P i l l i n g e r a n d Mr. I. M a n n i n g is g r a t e f u l l y acknowledged. This work was supported by the Natural Environment Research Council (Grants GR/3/655 and GR/3/1695), the National Aeronautics and Space Administration (NGL 05-003-003; sub-contract from the University of California, Berkeley), and the Science Research Council (Studentship to PWB). The liquid-scintillation counter was kindly made a v a i l a b l e b y D r . J. M a c M i l l a n .

REFERENCES Brooks, P.W. and Maxwell, J.R., 1974. Early stage fate of phytol in a recently deposited lacustrine sediment. In: B. Tissot and F. Bienner (Editors), Advances in Organic Geochemistry, 1973, Technip, Paris, pp. 977--991. Brooks, P.W., Eglinton, G., Gaskell, S.J., Maxwell, J.R. and Philp, R.P., 1976. Lipid constituents of Recent sediments, Part I: Straight-chain hydrocarbons and carboxylic acids of some temperate lacustrine and sub-tropical lagoonal--tidal sediments. Chem. Geol. 18 (in press). Cranwell, P.A., 1974. Monocarboxylic acids in lake sediments: indicators, derived from terrestrial and aquatic biota, of palaeoenvironmental trophic levels. Chem. Geol., 14: 1--14. Eglinton, G., Maxwell, J.R. and Philp, R.P., 1974. Organic geochemistry of sediments from contemporary aquatic environments. In: B. Tissot and F. Bienner (Editors), Advances in Organic Geochemistry, 1973, Technip, Paris, pp. 941--961. Farrington, J.W. and Quinn, J.G., 1971. F a t t y acid diagenesis in Recent sediments from Narragansett Bay, Rhode Island. Nature (London), Phys. Sci., 230: 67--69. Farrington, J.W. and Quinn, J.G., 1973. Biogeochemistry of fatty acids in Recent sediments from Narragansett Bay, Rhode Islar~d. Geochim. Cosmochim. Acta, 37 : 259--268. Gaskell, S.J. and Eglinton, G., 1975. Rapid hydrogenation of sterols in a contemporary lacustrine sediment. Nature (London), 254: 209--211. Ishiwatari, R., 1974. F a t t y acids in a 200-meter sediment core from Lake Biwa. In: S. Horie (Editor), Paleolimnology of Lake Biwa and the Japanese Pleistocene. Kyoto University, Kyoto, pp. 202--217. Parker, P.L. and Leo, R.F., 1965. F a t t y acids in blue-green algal mat communities. Science, 148: 373--374. Rhead, M.M., Eglinton, G., Draffan, G.H. and England, P.J., 1971. Conversion of oleic acid to saturated fatty acids in Severn Estuary sediment. Nature (London), 232: 327--330. Rhead, M.M., Eglinton, G. and England, P.J., 1972. Products of the short-term diagenesis of oleic acid in an estuarine sediment. In: H.R. von Gaertner and H. Wehner (Editors), Advances in Organic Geochemistry, 1971, Pergamon, Oxford, pp. 305--315.