Marine Chemistry, 36 (1991) 233-248Elsevier Science Publishers B.Y., Amsterdam

233

Evolutionary trends in the lipid biomarkerapproach for investigating the biogeochemistry of

organic matter in the marine environment

A. Saliot, J. Laureillard, P. Scribeand M.A. SicreLaboratoire de Physique et ChimieMarinesde l'Universite Pierre et MarieCurie, Unite Associee auCentreNational de la Recherche Scientifique no. 353, 4 Place Jussieu, 75252Paris Cedex05, France

(Received 23 November 1990; revision accepted 23 July 1991 )

ABSTRACT

Saliot, A., Laureillard, J., Scribe, P. and Sicre, M.A., 1991. Evolutionary trends in the lipid biomarkerapproach for investigating the biogeochemistry of organic matter in the marine environment. Mar.Chern., 36: 233-248.

The detailed investigation of organic carbon cycling in estuarine and marine environments hasstimulated the development of multidisciplinary concepts, research and sampling strategies as well asanalytical tools in the last 10 years. Among other approaches, the molecular biomarker one has alsoundergone significant improvements. Some examples dealing with specific markers includingsterols,alkenes and fatty acids are discussed in this paper in terms of source identification and transformationprocesses.

Although some limitations exist for the elucidation of their stereochemistry, sterols have been usedextensively to study biochemical processes affecting the organic matter in the water column and atthe ocean/sediment interface. Sterols also appear as promising tracers of terrestrial vs. marine inputsin complex estuarine systems. The specificity of individual fatty acids or groups ofacids has also beenused to assess the origins and transformation processes of organic matter in marine samples. Newapproaches are presented including the investigation of intact lipid classes and the elucidation of theposition of double bonds of unsaturated fatty acids and alkenes. Finally, we discuss the use of che­mometric techniques due to increasing chemical information and the interest of combining molecu­lar-level and stable isotope approaches.

INTRODUCTION

The organic matter in the ocean is one of the larger reactive reservoirs ofcarbon on the Earth's surface. Our knowledge of the chemical nature andquantity of organic components and their interactions with other chemicaland biological systems is important to the understanding and modeling of thecarbon cycle. Figure 1 shows the respective reservoirs of both inorganic andorganic carbon in the atmosphere, on the continents and in the oceans. Thisfigure underlines the huge stock of organic carbon present as dissolved or col-

0304-4203/91/$03.50 © 1991 Elsevier Science Publishers B.V. All rights reserved.

234

CONTINENTAtmosphere 692

Interface ontment IOcean

Organic Carbon

A. SAUOT ET AL.

OCEAN

InorganicCarbon

...

Soils Sediments

Fig. I. Main carbon reservoirs on the Earth with key interfaces. The amount of carbon of eachreservoir is estimated (unit 109 metric tons C) from Bolin and Cook (1983). The seawaterDOC reservoir is evaluated at 600 X 109 mTC by Williams and Druffel (1987).

loidal species. Interactions exist between the marine organic and inorganicreservoirs through photosynthesis, heterotrophy and mineralization. In orderto determine the processes and fluxes at the interfaces shown in Fig. l , werequire detailed information on the autochthonous production of organic car­bon, inputs of continentally derived material, food web dynamics includingmicrobial loop, fate of organic compounds in the atmosphere, in the watercolumn and in the surficial sediment. It is also essential to know how anthro­pogenic inputs may impact oceanic processes. Environmental systems can beobserved at different spatial and temporal scales depending on the process

LIPID BIOMARKERSIN MARINE BIOGEOCHEMISTRY 235

under study taking into account the limit of detection and reproducibility ofthe analytical tool used. Similarly different levels of investigation of the or­ganic carbon must be distinguished from bulk parameters (such as dissolvedorganic carbon (DOC) and particulate organic carbon (POC)) to molecularcomponents (such as amino acids, sugars, pigments, lipid compounds) viacompound classes (such as proteins, carbohydrates, lipids and humic sub­stances). To know better how to work out the 'giant puzzle' of organic carbonin the ocean, not only as a whole but also at the level of detail of every pieceindividually, is a challenge.

Within less than 15 years, marine organic chemistry has developed consid­erably due to the evolution of concepts, strategies in a multidisciplinary con­text, analytical tools, and data treatment possibilities (Andersen, 1977; Leeand Wakeham, 1989). Nevertheless, many questions addressed remain un­solved owing to the extreme complexity of the organic matter cycle.

Among important questions the reassessment of the non-volatile dissolvedorganic carbon in the ocean is presently of the highest priority (Sugimura andSuzuki, 1988; Cauwet et al., 1990; Suzuki and Tanoue, 1991). Since the firstdetermination of the age of the DOC in the deep Pacific Ocean by Williamset al. (1969) it is now possible to use recent radiogenic and stable isotopetechniques (Popp et al., 1989, and references therein). The investigation ofthe polymerized material, which is the predominant component of the DOC,is another major issue for which pyrolysis methods are promising (Van deMeent et al., 1983; Whelan et al., 1983; Saliot et al., 1984, 1991). We alsoneed to investigate the nature of organic matter at the molecular level. This isnow possible for amino acids and carbohydrates with newly developed highperformance liquid chromatography techniques (Lindroth and Mopper,1979), and for most lipid compounds.

This paper is a presentation of the lipid biomarker approach through sev­eral applications in various environments. This synthesis is, for the most part,based on data acquired over the past 10 years by our group, essentially in themarine (both open-sea and estuarine) environment. Potential improvementslinked to the sophistication of analytical methods for each individual com­pound series are also discussed.

THE LIPID BIOMARKER APPROACH

The global view of carbon cycling considers the ocean as an entity interact­ing with the atmosphere, the continent, the sediment and all forms of biota atdifferent time and space scales through complex interfaces (Fig. 1). Thiscomplexity has led marine chemists to address several questions that mightbe solved using the molecular markers. Although limitations exist, the devel­opment of new research strategies and analytical techniques allows moreproblems to be solved.

236 A. SALIOT ET AL.

Unambiguous markers are necessary to identify accurately material sources,but unfortunately most chemical structures are common to several types ofliving organisms, both terrestrial and marine. Moreover, many inventoriesare incomplete, often limited to selected species and often non-extendable tonatural environments, particularly when they have been obtained from in vi­tro experiments or from cultures performed far from natural conditions. Thesame difficulty is encountered with anthropogenic inputs (human, agricul­tural, petrogenic, and pyrolytic), to relate precisely sources to moleculartracers.

We need to know the transport pathways of organic compounds from theirassociation with aerosols, living organisms or dead cells, aggregates, floes, col­loidal or 'truly dissolved' states in the water column, till their arrival at theocean-sediment interface and during the first steps of their incorporation intothe surficial sediment. We also need to know their rates of production, kinet­ics of degradation, and their residence times in the different marine reser­voirs, taking into account physical, chemical and biological processes. Herethe coupling between the physical-chemical approach and modeling, and fieldmeasurements could be very helpful (Readman et al., 1987; Wu andGschwend, 1988).

The use ojsterols

Sterols are a very popular series of biogeochemical markers. They areubiquitous constituents of the living kingdom. The first inventories providedpowerful information from a limited number of identified sterols in marinewaters by Kanazawa and Teshima (1971), Saliot and Barbier (1973) andGagosian (1975, 1976), and in recent marine sediments (Lee et al., 1977,1979, among others). As sterols display a wide biosynthetic diversity leadingto a wide structural variety and sometimes characteristic distributions, theyhave been successfully used as tracers of inputs from various species of ma­rine and terrestrial plants and animals, and of transformation processes suchas oxidation and reduction.

Like other lipids, sterols are better preserved in sedimentary environmentsthan most other biological products such as carbohydrates and amino acids.Thus sterol fingerprints can record plant and animal organic matter inputs.Generally the sterol distributions found in lake sediments tend to be domi­nated by C29 sterols, which are major constituents of higher plants; they differsignificantly from the distributions observed in plankton and marine sedi­ments, where C27 and C28 sterols are more abundant (Huang and Meinschein,1976; Nishimura and Koyama, 1977). Calculations based on the ratio of C27

to C29 sterols were proposed by Huang and Meinschein (1976) to determinethe contributions of terrigenous and autochthonous organic material in ma­rine and lacustrine sediments. Triangular diagrams plotting C27, C28 and C29

LIPID BIOMARKERS IN MARINE BIOGEOCHEMISTRY 237

sterol contents of reference materials such as marine plankton, higher terres­trial plants, soils, lacustrine and marine sediments have been proposed andused extensively up to the 1980s to discriminate between input sources andcharacterize ecological systems (Huang and Meinschein, 1979). However,caution has to be taken (see the review by Volkman, 1986) since analyticalpossibilities to study complex mixtures of sterols, often present in lowamountsin marine samples, did not allow for differentiation of isomers. A typical ex­ample of this is the elucidation of the C29 P-sitosterol (24a), mainly ofterres­trial origin and c1ionasterol (24P)' mainly of marine origin. More recentlyinvestigations ofbiogeochemical processes affecting the organic matter in thewater column were carried out using sterol imprints by several groups (Saliotet al., 1982; Lee and Wakeham, 1989, and references therein). Extensivestudies were conducted in surficial sediments (Smith et al., 1982; De Leeuwet al., 1983; Volkman, 1986; Volkman et al., 1987; Lajat et al., 1990) to in­vestigate the ultimate fate of deposited material. Very few studies have fo­cused on interstitial waters (Saliot and Tusseau, 1984; Saliot et al., 1988a) orestuarine environments (Bayona et al., 1989; Lajat and Saliot, 1990).

Application to suspended particles in the open oceanMost studies of sterols in seawater deal with settling and sinking particles

obtained by filtration or collection in sediment traps, and in a few cases withthe dissolved phase. Although sterols represent a minor component of totalorganic carbon, they have been found to be model constituents for mixed layerstudies. Using budget calculations of free sterols from the Sargasso Sea, Ga­gosian and Nigrelli (1979) showed that 0.05-3% of the sterols produced inthe euphotic zone would reach the sea floor. Total sterol concentrations werecorrelated with particulate organic carbon (PaC), particulate organic nitro­gen (PaN) and chlorophyll a. The average residence time of sterols in sea­water was estimated to be 1-4 months in the euphotic zone, therefore, allow­ing the recording of seasonal processes, and between 20-150 years in the deepwater column.

Tracingcontinentaland marine inputsin estuariesEstuaries are subject to intense production, mixing and deposition of detri­

tal, autochthonous riverine and marine organic matter. Sterols may be usedto characterize the origins and the processes governing the transfer of the or­ganic matter from rivers to coastal zones. The large Changjiang estuary inChina is an example of the extreme complexity resulting from various mixingprocesses and inputs of organic matter from different origins. The detailedanalysis of sterols associated with the suspended particles allowed the evolu­tion of terrigenous imprints from the river mouth to the eastern marine zoneto be described (Lajat and Saliot, 1990). Sterol distributions allowed the

238 A.SAI.IOT ET AL.

characterization of several phytoplanktonic population signatures through­out the river mouth and adjacent East China Sea.

Campesterol, stigmasterol and p-sitosterol have been shown to be reliableterrestrial markers off the Rhone river. Using ratios of these three sterols vs.total free sterols, a clear distinction was observed for the particulate materialin the water column between a set ofstations with decreasing influence on theRhone river from deltaic to open-sea stations of the northwestern basin of theMediterranean sea (Scribe et al., 1989, 1991b).

Sources and seasonal variability of sterols have been investigated in coastalwaters of the Western Mediterranean. Both concentrations and compositionsof sterols found in suspended particles of the Ebro delta were seasonally de­pendent, reflecting changes in the phytoplanktonic populations. Diatom-pro­duced LJ5_, LJ5.22_ and LJ5.24(28)_stenols and LJ4-stenones were the major compo­nents during marine productive periods (spring and fall), whereas the 400­methylstanols, considered as dinoflagellate markers, predominated during thelowest productivity periods (summer and winter) (Bayona et al., 1989).

AnalyticaldevelopmentThe development of analytical techniques such as high resolution gas chro­

matography (HRGC) and computerized HRGC-mass spectrometry(HRGC-MS ), has allowed the refinement ofmethods to recognize numerousnovel sterols with modified side chains and new epimers in both marine or­ganisms and sediments (Maxwell et al., 1980; Brassell and Eglinton, 1983).Investigation by HRGC-MS of extractable sterols in marine sediments re­vealed complex distributions. Up to 69 different sterols were characterized inthe Neogene deep-sea sediments from the Japan Trench by Brassell and Eglin­ton (1983). The chromatographic separation of the C24isomers was resolvedby Maxwell et al. (1980); however, the GC run time necessary for this sepa­ration is prohibitive for routine analysis.

Even though the development of analytical techniques has allowed variousinputs of organic matter to be distinguished through the determination of alarge number of structures, there is a need for a multimarker approach. Thisapproach would combine different compound series such as sterols, hydro­carbons, fatty acids, alcohols and pigments, resulting from different biosyn­thetic pathways, and having different residence times in marine waters andsediments.

The use ofalkenes

Most marine and freshwater phytoplanktonic species contain biogenicmonoenes and polyenes. Extensive studies have been published over the lasttwo decades on the alkane and alkene contents of representative phytoplank-

LIPID BIOMARKERS IN MARINE BIOGEOCHEMISTRY 239

tonic species: (Blumer et al., 1971; Weete, 1976; Saliot, 1981). But in mostcases the location of the double bonds was not elucidated. Olefins have alsobeen characterized in the mixing zone of marine and fresh waters (Albaigeset al., 1984; Saliot et al., 1988b; Scribe et al., 1991a). The distributions ofalkenes associated with particles in a riverine / estuarine system tend to reflectthe spatial and temporal heterogeneities of algal growth due to steep chemicalgradients and the changing environmental factors occurring in such environ­ments. Recently, derivatization techniques using dimethyl disulfide (DMDS)such as developed by Francis and Ve1and (1981) allowed the identificationof five heptadecenes with a double bond located at the 1, 3, 5, 7 and 8 posi­tions (Scribe et al., 1990). These findings are promising for the use of a1kenesas freshwater phytoplankton indicators in river and coastal waters (Scribe etal., 1989, 1991a).

The use offatty acids

Occurrence and source determinationAnother complementary class of lipid tracers is provided by the fatty acids.

This series represents the major lipid constituents of marine organisms and,therefore, the predominant lipids in particles and sediments. Nevertheless,they can be ambiguous source indicators since many of them are present inmany organisms. However, the specificity of some individuals or groups ofacids has been used to assess the origins of organic matter in marine samples.Their use as taxonomic indicators for algae has been described in several pa­pers (see Volkman et al., 1989, and references therein). For example, plank­tonic inputs lead to a mixture of 14: 0, 16: 0, 16: 1w7, 18: lw9, 18: 0,20: 5w3,22:6w3. High values of the l:C I6/EC I8 (E = sum of saturated and unsatu­rated fatty acids) and C16: 1/C I6: 0 ratios are characteristic of diatoms (e.g,Claustre et al., 1988-1989). The presence oflong-chain, from 24: 0 up to 40: 0,monocarboxylic acids with a pronounced predominance of even over oddcarbon chains usually indicates terrestrial inputs from higher plants (Kolat­tukudy and Walton, 1973; Matsumoto et al., 1981). Nevertheless, diatomshave been found to be responsible for the accumulation of high molecularweight fatty acids (up to 26:0-28:0) in recent sediments (Volkman et a1.,1980; Nichols et al., 1986b). Branched 15: 0 and 17: 0 iso and anteiso acids,as well as 18: 1w7are commonly used as bacterial biomarkers because of theirstrong predominance in microorganisms (Perry et al., 1979; Volkman et al.,1980) .

During an investigation of the biogeochemistry of organic matter in a kars­tic river estuary, the Krka (Yugoslavia), fatty acids were analyzed and com­pared with pigments. Significant correlations were observed between the con­centrations ofrepresentative fatty acids and pigments for Cryptophyceae andChlorophyceae. Another correlation between the ratio of EC I6/EC I8 and the

240 A.SALlOTETAL.

percentage of fucoxanthin for predominating diatom populations was alsoobtained. These correlations, observed for a large salinity range (0-30%0),suggest the potential value of such biomarkers in estuaries (Scribe et al.,199Ic). However, they should be used with care as the amounts of biosyn­thesized pigments and fatty acids are known to be influenced by light inten­sity (Holdsworth, 1985), ageing, or nitrogen and phosphorus limitations incultures (Piorreck et al., 1984; Siron et al., 1989). These correlations ob­served in the Krka estuary suggest quite stable conditions. Extrapolations toother natural environments must be made with care.

Transformation processesThe fatty acid analysis also enables the alteration of organic matter both

within the water column and in the atmosphere to be followed (Sicre et al.,1990). Recently, a series of mid-chain (6: 0-18: 0) isomeric ketocarboxylicacids, with an odd-carbon numbered predominance, was found in remotemarine aerosols (Kawamura and Gagosian, 1990). Their presence was attrib­uted to the photochemical oxidation of semivolatile monocarboxylic acids.These latter compounds could result, together with diacids and oxoacids fromthe oxidative degradation of unsaturated fatty acids emitted from the sea sur­face (Kawamura and Gagosian, 1987).

Most of the particulate organic matter (PaM) biosynthesized by phyto­plankton is recycled in the upper ocean through ingestion by zooplankton andfish. Fatty acids, and especiallypolyunsaturated fatty acids (PUFAs), are im­portant in marine food webs. The material defecated by the animals containsextensively modified algal lipids. Fecal pellets have been shown to be de­pleted in co3 PUFA, present in the animal's diet, as they are nutritionally es­sential for marine species. Fatty acids have been shown to be more assimi­lated than carbohydrates in Euphausia superba feeding (Tanoue et a1., 1982),and directly transferred to the storage lipids (Bourdier and Amblard, 1989;Fraser and Sargent, 1989). Pelagic crustacean feeding ultimately appears tocontrol fatty acid and sterol distributions in surface sediments and thus de­serves special attention.

Analytical developmentNew approaches are being developed to assess more precise biological fin­

gerprints through fatty acid analyses. These new approaches include the in­vestigation of intact lipid classes instead of the commonly analyzed total sa­ponified acids, and the elucidation of the position of double bonds ofunsaturated fatty acids.

Lipid classes can be identified and quantified in complex mixtures at thelow concentrations found in marine environmental samples using a IATROS­CAN thin layer chromatography/flame ionization detection (TLC/FID)system. Marine lipid classes are useful tracers for the study of variability in

LIPID BIOMARKERS IN MARINE BIOGEOCHEMISTRY 241

dissolved and particulate matter (Parrish and Ackman, 1983), sediments,and animals (Volkman et al., 1986). The TLCjFID technique is a conve­nient tool for quickly accumulating synoptic data on lipid extracts from largesample sets to estimate phytoplanktonic or bacterial sources (Goutx et al.,1990). Quantitation of biogenic lipids using the IATROSCAN technique inaddition to the analyses of their fatty acid moities allows us to complete ourunderstanding offood web processes (Bourdier and Amblard, 1989) and or­ganic matter sources in marine samples. As a rule, the percentage of neutrallipids, waxes and triglycerides, reflects the physiological status of the orga­nisms. In contrast, the structural phospholipids have a relatively constant fattyacid composition that can be used for complementary specific information(Laureillard et al., 1990).

Recently, by selective extractions, Goossens et al. (1989) isolated specificlipid classes, such as amide-bound products in acidic extracts. Generally, thepresence of P-hydroxy fatty acids is considered to reflect a contribution frombacteria. In fact eukaryotes cannot be precluded, but amide-boundP-OH acidsare only found in bacteria as lipopolysaccharides. Therefore, p-hydroxy fattyacid distributions in the acidic extract are fingerprints ofbacterial populations.

Gas chromatography-mass spectrometry (GC-MS) analysis of the DMDSderivatives of monounsaturated fatty acid methyl esters showed major ionsattributable to fragmentation between the two CH 3S groups located at theoriginal site of unsaturation. Discrimination between cis and trans isomershas been described by Dunkelblum et al. (1985), Nichols et al. (1986a) andScribe et al. (1988): the threo isomer (originally the trans fatty acid) elutesprior to the erythro isomer (originally the cis fatty acid) under capillary GCconditions. The occurrence of both the cis and the, often present, trans iso­mers in the marine environment indicates that care needs to be taken whenidentifying these compounds (Nichols et al., 1989). As an example, from thelimited data available, it appears that both bacteria and marine invertebratesmay be sources of 16: 1co 1Oc, whilst 16: I co 1Ot has been found in a large va­riety of marine animals.

The use ofchemometric techniques

As previously emphasized, the sophistication of the analytical techniquesdeveloped for the investigation of environmental samples in the past 15yearshas led to a profusion of chemical information. Not only has the number ofcompound classes investigated for every sample analyzed increased, but alsothe number of individual components identified within an homologous serieshas increased, as structural elucidation is getting more and more precise. Sub­sequent handling and interpretation of larger databases often require ad­vanced methods such as multivariate data analysis or pattern recognitiontechniques.

The efficiency of a method for extracting information and optimizing inter-

242 A. SAUOT ET AL.

pretation depends on the quality and relevance of the data. These techniquesare generally based on the concept of relationship or similarity existing be­tween individuals or groups of objects in a set of observations or results, andthe hypothesis that similar compounds showing similar statistical behaviorare likely to have the same or related sources. However, very few organic geo­chemists have attempted to apply the multivariate methods to determinesources of organic material, which is a main goalwhen using biomarkers. Thesemathematical methods have been developed for investigating sample pointconfiguration in a two-dimensional space with minor loss of information, asthe representation of the sample is usually multidimensional and, therefore,impossible to inspect visually. However, some difficulties must be borne inmind when interpreting geochemical data, mostly because our understandingof the chemical composition of sources is far from complete, but also becausethe specificity of some biomarkers is still questionable.

To illustrate the potential use of chemometric methods for geochemicalstudies, we will discuss one application of these methods, recently publishedby our group (Sicre et al., 1988), to characterize seawater samples usingbiomarker fatty acids. A multivariate method, correspondence factorial anal­ysis (CFA) and a non-parametric procedure, hierarchical clustering classifi­cation (HCC) were used in conjunction to investigate fatty acid sources inorder to further characterize the composition of seawater samples. This studywas performed on dissolved and particulate fatty acids collected in the micro­layer and surface Mediterranean waters. Hierarchical clustering classificationwas first applied to these data to assess the similarities between the differentfatty acid methyl esters (FAMEs) and identify their sources for this particu­lar data set. Since FAMEs were obtained upon saponification of the tota11ipidextract they thus include free fatty acids as well as esterified forms. From theirsimilarity dendrogram it was possible to differentiate between phytoplank­tonic, zooplanktonic and microbial FAMEs. The information gained from thisfirst statistical treatment was exploited to ascertain the relative importanceof the different sources in the dissolved and particulate material of the sea­water samples using CFA. This method demonstrated that esters in the par­ticulate fraction were of phytoplanktonic origin except in one sample wherezooplanktonic FAMEs, dominated. On the other hand, the dissolved com­partment indicated a strong microbial fingerprint. The statistical behavior ofthe 16: 1OJ 10, previously unreported in seawater suggested a bacterial originof this FAME.

Other successful applications of chemometric methods have been pub­lished by Van Graas et al. (1981), Boon et al. (1983), Van de Meent et al.(1983), Shaw and Johns (1986), and Gomez-Belinchon et al. (1988). Im­proved procedures for correlation studies and graphical methods for investi­gating the spatial variation trends of large data sets will be necessary in the

LIPID BIOMARKERS IN MARINE BIOGEOCHEMISTRY 243

future to overcome the difficulties of extracting information at different lev­els of sophistication in a more effective and objective way.

Combined lipidbiomarker-stable isotope approaches

The combination of the biomarker and stable isotope approaches that isbeing investigated now in several laboratories will constitute a step forwardin assessing sources in terms of marine vs. terrestrial origins of organic mat­ter. Preliminary data are encouraging. One example of this is the work re­cently published by Saliot et al. (1988b) for a temperate macrotidal region.They observed a correlation between J I3C values ( -27%0, -29%0 for terres­trially derived material; - 20%0, - 21%0 for the marine material) and carbonpreference index (CPI) values calculated for n-alkanes ranging from n-C23 ton-C35 in suspended particles collected in the Loire estuary. The CPI value haslong been considered for differentiating between terrestrial matter (CPI range:5-10) and degraded or autochthonous material (CPI: low values 1-2). Al­though the CPI constitutes a qualitative criterion, it has been considered asan efficient index to ascertain different types of organic matter. Another ex­ample is provided by a recent detailed study of aromatic compounds in aMediterranean deltaic environment, the Rhone delta. Compounds of natural,petrogenic, pyrolytic and diagenetic origins were detected in particulate mat­ter, in waters and in surficial sediments (Bouloubassi and Saliot, 1991; Lip­iatou and Saliot, 1991 ). One interesting feature is the relationship found forsurficial sediments between J 13C and tetrahydrochrysenes, which are aro­matic compounds derived from the amyrin series, originating from terrestrialhigher plants (Lipiatou et al., 1991).

CONCLUSIONS

Among a large variety of lipid compounds, sterols, alkenes and fatty acidsappear very promising as source indicators. Although many sterols are widelydistributed in higher plants, marine algae and animals, their fingerprints havebeen successfully used to record the seasonal variability of organic matter in­puts to Mediterranean sediments, which reflect the succession of predomi­nant phytoplanktonic populations: diatoms and dinoflagellates.

The spatial and temporal heterogeneities of algal growth can be followed incomplex estuarine systems by analyzing n-alkenes or n-fatty acids.

The fatty acid analysis also enables the transformation of organic matter inthe water column and in the atmosphere to be followed.

Within the last 20 years the development of the biomarker approach hasundoubtedly been driven by an increased sophistication of analytical tech­niques. Knowledge of the detailed structure of an organic tracer has increasedits potential use in the investigation of sources, transformation and transport

244 A. SALIOT ET AL.

pathways. The development of analytical techniques such as the determina­tion ofthe C-24 isomery for C29 sterols would allow the distinction to be madebetween different source inputs, i.e. terrigenous vs. marine.

Even though the development of analytical techniques has allowed the de­termination of an increasing number of structures, there is a need to developa multimarker approach, combining different compound series such as ster­ols, ketones, fatty acids, alcohols, pigments, and hydrocarbons, which belongto different biosynthetic pathways. This multimarker approach merits beingcombined with other tools such as pyrolysis methods and stable isotopes.

We also need to know better the residence times of these compounds in thevarious carbon reservoirs to assess transformation processes. Finally, we haveto promote powerful tools for the determination of sources of organic mate­rial such as chemometric methods.

ACKNOWLEDGMENTS

We thank J. Dagaut, C. Pepe, J. Fillaux and A. Lorre for their participationin recent cruises and for their laboratory work. We also thank all the youngscientists involved in our programs, whose data have been used throughoutthe preparation of this manuscript: Ch. Barreau, 1.Bouloubassi, A. Conde, V.Denant, Ch. Fuche, M. Lajat, E. Lipiatou, L. Mejanelle, Y.J. Qiu, and E. Bro­zek for preparation of the manuscript.

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