Occurrence of 2,6,10-trimethyl-7-(3-methylbutyl)-dodecane and related hydrocarbons in the green alga Enteromorpha prolifera and sediments

Org. Geochem. Vol. 8, No. 3, pp. 207-213, 1985 Printed in Great Britain. All rights reserved

0146-6380/85$3.00+ 0.00 Copyright © 1985Pergamon Press Ltd

Occurrence of 2,6,10-trimethyl-7-(3-methylbutyl)dodecane and related hydrocarbons in the green alga Enteromorpha prolifera and sediments S. J. ROWLAND*, D. A. YON?, C. A. LEWIS and J. R. MAXWELL Organic Geochemistry Unit, School of Chemistry, University of Bristol, Cantock's Close, Bristol BS8 ITS, U.K. (Received 15 August 1984; accepted 13 November 1984)

Abstract--The isoprenoid alkane 2,6,10-trimethyl-7(3-methylbutyl)-dodecane, a related monoene and a pseudohomologous C25 diene have been identified in field specimens of the green alga Enteromorpha prolifera. The identifications were based on gas chromatographic retention data, microscale hydrogenation, mass spectral evidence and, for the C20 alkane, comparison with the synthesised hydrocarbon. The same compounds, and related hydrocarbons with the same two carbon skeletons, appear to occur widely in freshwater and marine sediments and sedimenting particles from different parts of the globe. Some of these highly branched sedimentary compounds may be algal in origin. Key words: green alga, Enteromorpha, acyclic isoprenoids, 2,6,10-trimethyl-7-(3-methylbutyl)dodecane,

branched hydrocarbons, sedimentary hydrocarbons

INTRODUCTION There are several reports of a group of C20 and C25 highly branched hydrocarbons in freshwater and marine sediments and in sedimenting material. The ! C20 compounds reported to date comprise an alkane and two isomeric monoenes (shown by hydro"rr genation to have the same carbon skeleton) whilst the C25 hydrocarbons occur as an alkane and alkenes 2,6,10,14-tetramethyl-7-(3-methylpentyl)pentadecane with one to four double bonds (e.g Gearing et al., (II; Yon, 1982; Bayona et al., 1983). 1976; Prahl et al., 1980; Barrick et al., 1980; Barrick Although they were abundant (up to 17~g g-~ and Hedges, 1981; Cranwell, 1982; Volkman et al., organic carbon) in sediments from Puget Sound 1983; Yon et al., 1982; Requejo and Quinn, 1983). (Washington State, U.S.A.) neither I, II or the related Hydrogenation of sedimentary hydrocarbon frac- alkenes were present in local atmospheric dust or tions containing the various alkenes produces a C20 river particulate samples (Barrick et al., 1980). and a C25 alkane which have been shown to be Volkman et al. (1983) and Smith et al. (1983) pseudohomologous by the use of gas chro- identified a number of C~5 alkenes of type II in coastal matographic retention data and mass spectrometry sediments from Peru and concluded that they origi(Barrick et al., 1980). Electron impact spectra of the nated from an autochthonous source in the water C20 and the C25 alkane are characterised by the column. They argued that, since a large proportion of presence of small molecular ions and doublets at m / z the lipids of these sediments are derived from phyto168, 169 and 238, 239 respectively, resulting from plankton, the C2s alkenes might also derive from this fragmentation at branch points (Barrick et al., 1980; source (e.g. diatoms which predominate in the area). Requejo and Quinn, 1983; Bayona et al., 1983). It has It remains to be seen whether diatoms contain the C20 been shown recently (Yon et al., 1982), by synthesis and C25 alkenes related to I and II. More recently, on of the C20 alkane, that it is 2,6,10-trimethyl- the basis of sedimentary concentration profiles, it has 7-(3-methylbutyl)dodecane (I). On the basis of mass been proposed that the C2s alkenes (and associated spectral comparison with the C20 alkane it has been C~o alkenes) in an anoxic river basin originated from suggested that the C25alkane is the pseudohomologous in situ bacterial synthesis (Requejo et al., 1984). So far, to our knowledge, no biological source of these compounds has been reported. *Present address: Department of Environmental Sciences, In this paper we report the identification, in field Faculty of Science, Plymouth Polytechnic, Drake specimens of the green alga Enteromorpha prolifera, Circus, Plymouth, Devon PIA 8AA, U.K. tPresent address: Dow Chemicals, Agricultural Research of I, a related monoene and a C25 diene which on hydrogenation gives the alkane proposed to be II. and Development, Kings Lynn, Norfolk, U.K. 207

S.J. ROWLANDet al.

208

R iclmslon Bridge

Sampie collection site

N I

\ Metres o 1oo 200 30o 400 500 I

I

I

I

I

Herbraf~Iston o

I

Seno~ Haven o

•

\']

s

L •

EmllimCttam~

o

so

~eomj

Extent of algal- ¢lornic~tad ~ n d

Fig. 1. Location of sampling site for Enteromorpha and Sandyhaven sediments. EXPERIMENTAL

Samples Enteromorpha prolifera was collected by hand at low tide from sand banks at Sandyhaven, Dyfed, Wales at a site described previously, (Fig. 1; Rowland et al., 1981). Samples were collected in an aluminium screw-top tube. Sub-samples were sent to the Marine Algae Section, British Museum, London for identification. A further sample also considered to be Enteromorpha was collected from the shore at Mumbles Head, Swansea, West Glamorgan, Wales. No assessment of the viability of the organism was made in either case.

Extraction and fractionation Enteromorpha (ca 250 g wet weigh 0, washed with water to remove adhering sand particles as far as possible, was cut into fine pieces, extracted with

propan-2-ol/hexane (ultrasonicat/on, 100 ml, 4: 1) and partitioned between hexane (72 ml) and water (48 ml). Evaporation of the hexane afforded an extract which was fractionated by column chromatography (AI20~, grade I, hexane eluant). The resulting hydrocarbon fraction was adducted with urea ( x 2) and the combined non-adducts examined by GC and GC-MS before and after microscale hydrogenation (PtO2, ethyl acetate, 30min, room temperature).

Gas chromatography (GC) and combined gas chromatography-mass spectrometry (GC-M S ) GC was performed on an Erba Science FTV 2151 chromatograph fitted with wall-coated open tubular columns coated with OV-I ( 2 0 m x 0 . 3 m m ) or DEGS/PEGS (3/1, 100 m x 0.2 ram). The temperature was programmed from 60-265°C at 6°C/min

Hydrocarbons in E. prolifera and sediments

209

5

Time

Fig. 2. Capillary gas chromatogram of urea non-adduct of hydrocarbon fraction of Enteromorpha. (For conditions see text.) Key: Peak 1 =n-heptadecadiene; 2=n-heptadecen¢; 3 =C20 highly branched isoprenoid monoene; 4 ~-2,6,10-trimethyl-7-(3-methylbutyl)dodecaneand 2,6,10,14-tetramethylpentadecane (pdstane); 5 = 2,6,10,14-tetramethylhexadecane (phytone); 6,7 = 2,6,10,14-tetramethylhexadecanes; 8--Czs highly branched isoprenoid dien¢.

(OV-1) or 40-120°C at 4°C/min (DEGS/PEGS). For resolution of diastereomers of I the temperature was programmed from 40-80°C at 4°C/min (DEGS/PEGS). G C - M S was performed on a Finnigan 4000 mass spectrometer coupled to a Finnigan 9610 gas chromatograph with on-line INCOS 2000 data system. Chromatography under these conditions was performed on a 25 m x 0.33 mm fused silica column coated with methylsilicone fluid (Hewlett-Packard). All injections were made with a Grob-type split/splitless injector in the splitless mode. The temperature was programmed from 50-270°C at 6°C/min. The spectrometer scanned masses 50-600 every I sec. Filament current was 0.35 mA, electron voltage 40 eV. RESULTS

A gas chromatogram (OV-1) of the branched and cyclic hydrocarbons isolated from Enteromorpha prolifera (Sandyhaven) is shown in Fig. 2. No quantitative data were obtained. The major hydrocarbons (peaks 1 and 2) were identified from their mass spectra, GC retention positions and hydrogenation

behaviour as a n-heptadecadiene and a nheptadecene (cf. Youngblood et al., 1971; Youngblood and Blumer, 1973). These n-aikenes appear to have been incompletely adducted by urea. G C - M S showed peak 3 to correspond to a C20 hydrocarbon with one degree of unsaturation (M .+ 280). The spectrum (Fig. 3A) was very similar to that shown by Cranwell (1982) for a C20 monoene in freshwater sediment from Upton Broad (U.K.). The spectrum contains characteristic ions at m/z 69, 83, I I I , 196, and 210 as also found by Barrick et al. (1980) for two isomeric C20 monoenes in sediments from Puget Sound (U.S.A.). The compounds found by us and Cranwell 0982) and those found by Barrick et al. 0980) appear to have the same carbon skeleton judging from the similarities in their mass spectra. Hydrogenation of the urea non-adduct from Enteromorpha enhanced peak 4 which was shown to contain I by co-chromatography with the synthesised alkane. Coelution was confirmed on a second, more polar, GC stationary phase (DEGS/PEGS), suggesting that the C20 hydrocarbon is a 2,6,10-trimethyl-7-(3-methylbutyl)dodeccne. Mass chromatography of key ions (m/z 168/169,

210

S.J. ROWLAND et al. 57 100"

(A) 69

50 83

126 97 111 140

196

210 280

J| loo

m/Z

10o"

--

!

,,

I

300

200

55 69 (B)

95

83 50 109 123 I

137 151

I

I

100

207 l

2O0

235

266

320

348

I

3oo

m/z

Fig. 3. Mass spectra of highly branched isoprenoid hydrocarbons from Enteromorpha. (For conditions see text). (A) C20 monoene, (cf. Fig. 2) which affords 2,6,10-trimethyl-7-(3-methylbutyl)-dodecane on hydrogenation; (B) C2s diene (cf. Fig. 2).

Hydrocarbons in E. prolifera and sediments 183) and G C - M S coinjection with standards showed that peak 4 (Fig. 2) contained a mixture of 2,6,10,14-tetramethylpentadecane (pristane) and 2,6,10-trimethyl-7-(3-methylbutyl)dodecane (I). Peak 5 was shown to be due to 2,6,10,14-tetramethylhexadecane (phytane) by G C - M S , whilst mass spectra and retention positions of peaks 6 and 7 closely matched those of phytenes (Urbach and Stark, 1975). Enhancement of peak 5 upon hydrogenation confirmed the latter assignments. The spectrum of peak 8 (Fig. 2, retention index 2082, OV-1) was consistent with that of a highly branched C25 hydrocarbon with two degrees of unsaturation (M .+ 348; Fig. 3B). Notable ions occurred at m / z 320 (M.+-C2H4), 266 (M+-C6:2), 235 (M+.-C8:o), 207 M+.-C10:o). Requejo and Quinn (1983) recently observed the same compound in sediments from Rhode Island, U.S.A. Hydrogenation of an aliquot of the urea non-adduct from E n t e r o m o r p h a converted the hydrocarbon to an alkane (retention index 2112, OV-1) which had a retention index and mass spectrum virtually identical to that reported by Barrick et al. (1980), Bayona et al. (1983) and Requejo and Quinn (1983) for the hydrogenation product of the C25 alkenes identified in their studies (Table 2). The spectrum exhibited a notable ion pair doublet at m / z 238, 239; the latter ions probably result from fragmentation about the C6-C 7 bond in II which has been proposed as the most likely structure of the alkane (Yon, 1982; Bayona et al., 1983). Synthesis or isolation of the C25 alkane is needed in order to confirm the structure (cf. Yon et al., 1982). Atteml~LS to establish the positions of the double bonds by derivatisation were unsuccessful. The presence of a small unresolved complex mixture (UCM) in the chromatogram in Fig. 2 probably reflects minor contamination of the alga by fossil fuel hydrocarbons (e.g. Rowland et al., 1981).

Table Location N.E. Gulf of Mexico Puget Sound (Washington State, U,S.A.) Dabob Bay (Washington State, U,S.A.) Narragansett Bay (Rhode Island, U.S.A.) Alfacs Bay (Ebro River, Spain) Cariaco Trench ( o f f Venezuela) E. Mediterranean Peru Continental S h e l f

211

S t e r e o c h e m i s t r y o f I in Enteromorpha

When examined by GC under conditions which separated the eight possible isomers of synthesised I into a symmetrical doublet, I from E n t e r o m o r p h a (before hydrogenation of the hydrocarbons) produced only a single peak which co-chromatographed with the second peak of the doublet. This infers the presence of only one or a limited number of isomers (1 ~
DISCUSSION

Previous studies o f Enteromorpha

The hydrocarbons of an E n t e r o m o r p h a sp. have been examined previously (Youngblood et al., 1971). Neither I or II or related alkenes were reported. A compound identified tentatively as a C~7 cyclopropane was assigned from GC retention data on packed columns (1678 Apiezon-L, 1792 FFAP), nonhydrogenation with Pt in isooctane and 90~o removal with gaseous HC! in chloroform. However, these analytical data are also consistent with a highly branched C20 alkene with the carbon skeleton I,

1. Occurrences of (1) and (II) and related hydrocarbonsin young sediments Age/environment Compounds reported Reference Recent/marine C20:0, C25:1,C25:2,C25:3,C25:4 Gearinget al. (1976) Recent/land-lockedmarine C20:0, C20:t, C20:l, Cz5:3,C2s:3, Barricket al.. (1980); C25:4, C25:4 Barrick and Hedges (1981) Recent/land-lockedmarine C25:3 Prahl et aL (1980) Recent/estuarine

cC25:H*, cC2~:l:t*,C25:2,

Requejo and Quinn (1983)

Recent/estuarine

C20:0, C25:0,C2~:3,C25:3

Bayona et al. (1983)

Pleistocene/marine

C20:t

Yon (1982)

Pleistocene, Pliocene/marine Recent/marine

C:0:0, C25:~,C:5:2 C25:3, C25:3,C2s:4,C25:4

Comet (1982) Volkman et al. (1983); Smith et aL (1983) Rowland (unpublished results) Yon et al. (1982) Rowland (1982)

cC2~:2:2"

Sandyhaven, U.K. Recent/intertidal C20:0, C20:l,C25:2 Grasmere (Cumbria, U . K . ) Recent/freshwaterlake C20:0, C20:t, C25:t Rostherne Mere (Cheshire, Recent/freshwaterlake C20:0, C20:l, C25:1 U.K.) Pettaquamscutt River Recent/anoxic river basin C25:2, C25:2,C25:2,C25:3 (Rhode Island, U,S.A.) *It is possible that compounds denoted c (cyclic)are actually acyclic,i.e. C25:2 , C25:2 , C25:4,

Requejo et al. (1984)

212

S.J. ROWLANDet al.

Table 2. GC retentiondata for highlybranchedisoprenoidhydrocarbons Retention indexon apolar phases and Compound refereneC CNo' :degrees unsaturation

1

2

3

4

20:0 1708 1705 --20:1 1702 1700 --20:1 1698 ---25:0 2109 2112 2111 2100 25:1 . . . . 25:2 -2082 2084 -25:2 --2079 -25:2 --2140 -25:3 2044 --2044 25:3 2090 --2092 25:3 . . . . 25:4 2078 -2097 2082 25:4 2124 --2129 *References: (1) Barriek et aL (1980);(2) this study, Enteromorpha sp.;(3) Requejoand Quinn(1983);(4)Volkmanet al. (1983). assuming a double bond in a position resistant to hydrogenation under the mild conditions used. Occurrences o f 1, II, and related hydrocarbons in sediments There are many reports of compounds now established as I, II and/or related alkenes in sediments. Table 1 summarises the occurrences in recent sediments. Table 2 summarises available GC retention index data on apolar phases. From the latter, and a comparison of mass spectra given in the literature, it is clear that the compounds are all closely related to each other, as suggested by previous workers (e.g. Gearing et al., 1976; Barrick et al., 1980; Yon et aL, 1982; Requejo and Quinn, 1983; Volkman et al., 1983) and to those in our Enteromorpha samples (Table 2). The isomeric alkenes in Tables 1 and 2 probably all have the same carbon skeleton and differ only in the position or geometry of the double bonds. It is not clear whether the cyclic C2s alkenes (Table 1) reported by Requejo and Quinn (1983) are indeed cyclic or if they are acyclic components which were incompletely hydrogenated. There seems little doubt, however, that these alkenes are closely related to the other C25 alkenes in Table 1. For example, the monocyclic C25:t component (cC25:H) of Requejo and Quinn (1983) has a mass spectrum which is very similar to that of the C25:2component which, in turn, is very similar (and has a similar retention index on apolar phases) to the C2s diene in the present samples of Enteromorpha. The occurrence of some of these highly branched isoprenoid hydrocarbons in Enteromorpha, an alga which occurs widely in both freshwater and marine aquatic environments (e.g. Levring et al., 1969) suggests that it may be a source of these compounds in some sediments. However, whilst the major n-alkenes (Ct7:~, Ct7:2) of the alga co-occur with hydrocarbons of types I and II in some sediments (e.g. Sullom Voe, Shetland; Sandyhaven, Dyfed; unpublished results) in others this is not the case. For example, the sediments of a small lake, Rostherne Mere (Cheshire, U.K.)

contain a distribution of hydrocarbons of types I and II which is quite similar to Enteromorpha, but no n-Ct7 alkenes are present (Rowland, 1982). Chromatograms shown by Bayona et al. (1983) for aliphatic hydrocarbons isolated from sediments and water particulates of the Ebro river estuary (Spain) also show the presence of I and II, but the relative abundance of n-C17 alkenes is low and again, unlike that of the alga. In some other sediments, such as those examined by Volkman et aL (1983) and Barrick et al. (1980), the differences between the alga and sediments are even more pronounced with n-Ct7 alkenes not reported to be present at all. There are several possible explanations for these differences; for example, if Enteromorpha is a source, it may be able to biosynthesise variable proportions of n-alkenes and branched alkenes. Another possibility is that the n-alkenes may be more rapidly biodegraded than the highly branched isoprenoid alkenes. Unfortunately, few comparative studies of nand branched alkene biodegradation appear to have been made. CONCLUSIONS Field samples of Enteromorpha prolifera have been shown to contain 2,6,10-trimethyl-7(3-methylbutyl)dodecane (I), a related monoene and C2s diene of carbon skeleton II. To our knowledge, this report describes the first biological occurrence of highly branched C20 and C25 isoprenoid hydrocarbons of the types frequently found in marine, coastal and fresh-water sediments. The presence of certain of these hydrocarbons in an alga suggests that the sedimentary compounds could be algal in origin. It should be borne in mind, however, that the samples were not euxinic cultures and it remains to be proved that the hydrocarbons were biosynthesised by the alga itself. Studies of cultured samples of E. prolifera and other green algae are required to determine the limits of biological occurrences of the C20 and C25 hydrocarbons. Acknowledgements--We thank SERC (SJR and DAY) and the British Petroleum plc (CAL) for studentships, and NERC for GC-MS facilities (GR3/2951 and GR3/3758). We are grateful to K. M. Cumbers (Western Australian Institute of Technology), D. M. Jones (University of Newcastle-upon-Tyne) and Dr P. J: Tibbetts (M. Scan Ltd, Ascot) for helpful discussions, and to S. Howells (Oil Pollution Research Unit, Wales) for all his help, including collection of samples.

REFERENCES

Barrick R. C., Hedges J. I. and Peterson M. L. (1980) Hydrocarbon geochemistryof the Puget Sound region--I. Sedimentary acyclic hydrocarbons. Geochim. Cosmochim. Acta 44, 1349-1362. Barrick R. C. and Hedges J. I. (1981) Hydrocarbon geochemistry of the Puget Sound region--II. Sedimentary diterpenoid, steroid and triterpenoid hydrocarbons. Geochim. Cosmochim. Acta 45, 381-392.

Hydrocarbons in E. prolifera and sediments Bayona J. M., Grimalt J., Albaiges J., Walker W. II, de Lappe B. W. and Risebrough R. W. (1983) Recent contributions of high resolution gas chromatography to the analysis of environmental hydrocarbons. J. H.R.C and C.C. 6, 605-611. Comet P. A. (1982) The use of lipids as facies indicators. Ph.D. Thesis, University of Bristol, U.K. Cranwell P. A. (1982) Lipids of Aquatic sediments and sedimentary particulates. Prog. Lipid Res. 21, 271-308. Gearing P., Gearing J. N., Lytle T. F. and Lytle J. S. (1976) Hydrocarbons in 60 north east Gulf of Mexico Shelf sediments: a preliminary survey. Geochim. Cosmochim. Acta 40, 1005-1017. Levring T., Hoppe H. A. and Schmid O. J. (1969) Marine Algae. A Survey of Research and Utilization, pp. 135-136. Cram, de Gruyler, Hamburg. Prahl F. G., Bennett J. T. and Carpenter R. (1980) The early diagenesis of aliphatic hydrocarbons and organic matter in sedimentary particulates from Dabob Bay, Washington. Geochim. Cosmochim. Acta 44, 1967-1976. Requejo A. G. and Quinn J. G. (1983) Geochemistry of C25 and C30 biogenic alkenes in sediments of the Naragansett Bay estuary. Geochim. Cosmochim. Acta 47, 1075-1090. Requejo A. G., Quinn J. G., Gearing J. N. and Gearing P. J. (1984) C25 and C30 biogenic alkenes in a sediment core from the upper anoxic basin of the Pettaquamscutt River (Rhode Island, U.S.A.). Org. Geochem. 7, 1-10. Rowland S. J., Tibbetts P. J. C., Little D. I., Baker J. M. and Abbiss T. P. (1981) The fate and effects of dispersanttreated compared with untreated crude oil with particular reference to sheltered intertidal sediments, Part I. In Proc.

0(3 8 / ~ C

213

lOth Worm Oil Spill Conference, pp. 283-292. API/EPA/USCG. Rowland S. J. (1982) Origins and fate of sedimentary acyclic isoprenoids. Ph.D. Thesis, University of Bristol, U.K. Smith D. J., Eglinton G. and Morris R. J. (1983) The lipid chemistry of an interfacial sediment from the Peru Continental Shelf: Fatty acids, alcohols, aliphatic ketones and hydrocarbons. Geochim. Cosmochim. Acta 47, 2225-2232. Thompson S. and Eglinton G. (1976) The presence of pollutant hydrocarbons in estuarine epipelic diatom populations. Est. Coastal Mar. Sci. 4, 417-425. Urbach G. and Stark W. (1975) The C20 hydrocarbons of butterfat. J. Agric. Food Chem. 23, 20-24. Volkman J. K., Farrington J. W., Gagosian R. B. and Wakeham S. G. (1983) Lipid composition of coastal marine sediments from the Peru upwelling region. In Advances in Organic Geochemistry 1981 (Edited by Bjgroy M. et al.), pp. 228-240. Wiley. Yon D. A. (1982) Structural, synthetic and stereochemical studies of some sedimentary isoprenoid compounds. Ph.D. Thesis, University of Bristol, U.K. Yon D. A., Ryback G. and Maxwell R. J. (1982) 2,6,10-trimethyl-7-(3-methylbutyl)dodecane, a novel sedimentary biological marker compound. Tetrahedron Lett. 23, 2143-2146. Youngblood W. W., Blumer M., Guillard R. L. and Fiore F. (1971) Saturated and unsaturated hydrocarbons in marine benthic algae. Mar. Biol. 8, 190-201. Youngblood W. W. and Blumer M. (1973) Alkanes and alkenes in marine benthic algae. Mar. Biol. 21, 163-172.