Organic Geochemistry 42 (2011) 1363–1374

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Organic Geochemistry

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Geochemical appraisal of palaeovegetation and climate oscillation in theLate Miocene of Western Bulgaria

Maya Stefanova a,⇑, Dimiter A. Ivanov b, Torsten Utescher c

a Bulgarian Academy of Sciences, Institute of Organic Chemistry, Acad. G. Bonchev Bl. 9, 1113 Sofia, Bulgariab Bulgarian Academy of Sciences, Institute of Biodiversity and Ecosystem Research, Acad. G. Bonchev Bl. 23, 1113 Sofia, Bulgariac University of Bonn, Geological Institute, Nussallee 8, 53115 Bonn, Germany

a r t i c l e i n f o

Article history:Received 12 May 2011Received in revised form 25 July 2011Accepted 22 August 2011Available online 3 September 2011

0146-6380/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.orggeochem.2011.08.015

⇑ Corresponding author. Tel.: +359 29606162; fax:E-mail address: maia@orgchm.bas.bg (M. Stefanov

a b s t r a c t

Palaeobotanical methods and geochemical techniques were used to assess plant contribution and pala-eoenvironment for the Staniantsi Basin, Bulgaria. The aim was to connect palaeovegetation change andclimate oscillation based on pollen and statistical analysis with organic geochemical proxies for a LateMiocene lacustrine to paludial sedimentary succession.

Three samples from lignite/marl cycles were studied. The biomarker assemblage and bulk chemicaldata indicated that gymnosperms were not important in the palaeomire. The presence of des-A-triterp-enoids, 17,21-seco-triterpenoids, hopanes, a high content of a D-ring monoaromatic hopane, and aroma-tized triterpenoids suggested that photochemical and microbial processes significantly contributed to thealteration of the organic matter (OM). A prolonged period of high water table and severe mechanicaldestruction promoted microbial activity prior to burial and enhanced decay. A geochemical appraisalof short term climate oscillation (ca. 21.7 kyr) was attempted within the limitations of the small numberof samples studied. The cycles are expressed as lignite/marl–clay layers combined with cyclic changes inswamp vegetation related to cyclic changes in groundwater level and inundation of the basin. In periodsof low water level (swamp phase) lignite accumulation took place. Preliminary results for selected sam-ples suggest that the oscillation may be reflected in the content of friedelin vs. possible degradation prod-ucts. The ratio of a chromatographic peak tentatively assigned as A-norfriedel-8-en-10-one to friedelin isproposed as a means of detecting short term environmental cycles, where values <1 represent the swampphase and those >1 reflect periods of inundation. However, time-series analysis using densely sampledlignite–clay layer oscillations are needed to confirm the value of this biomarker ratio for environmentalreconstruction.

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1. Introduction

The Neogene basins in Bulgaria, part of a system of sedimentarycomplexes on the Balkan Peninsula, contain important informationon the development of the flora during the Miocene. Because of itsgeographical location between the Tethyan and Paratethyan ba-sins, the Balkans territory played a crucial role in the evolution ofMediterranean flora and appears to be one of the major migrationroutes in the floral exchange between Asia Minor and Europe.Changes in vegetation, the emergence of sclerophyllous woodyplants and the dissemination of herbaceous communities reflectingregional palaeogeography, as well as the strong dependence ofplant composition on climate conditions, offers a unique opportu-nity for assessing climate in ancient times. Reconstructing palaeo-

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+359 28700225.a).

climate is essential for understanding recent and future changes inthe climate system under the influence of internal/external forcingfactors.

Recently, various quantitative palaeobotanical techniques forreconstructing palaeoclimate have been developed [e.g. ClimateLeaf Analysis Multivariate Programme, CLAMP (Wolfe, 1993),Coexistence Approach (Mosbrugger and Utescher, 1997), ClimateAmplitude Method (Fauquette et al., 1998), European Leaf Physiog-nomic Approach, ELPA (Traiser et al., 2005, 2007)] and refined (e.g.Utescher et al., 2009a). Besides traditional palaeobotanical meth-ods, geochemical techniques can supply new insights into plantcomposition and inferred palaeoenvironment. The application ofboth approaches significantly improves knowledge about the evo-lution of the climate system and vegetation dynamics.

Bulgarian Neogene coals are characterized by low grade techno-logical parameters but are interesting from a fundamental point ofview as they still retain precursor – product relationships.Oxygen-containing derivatives of terpenes, i.e. phenols, ketones,

1364 M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374

quinones, keto phenols, etc., occur in Miocene lignites. Hopanesprovide evidence for microbial activity, while oxygenated triterpe-noids, i.e. lupenone/lupenol, amyrin/amyrone, etc., are biomarkersfor deciduous plants (Angiospermae). Sesquiterpenoids and diter-penoids, and a high concentration of phyllocladane and other diter-penoids are typical of Cenozoic swamp taxa. Various studies havecorrelated biomarkers with modern taxa and tried to draw system-atic and phylogenetic relationships (e.g. Otto and Wilde, 2001;Otto et al., 2002, 2003, 2005, 2007; Auras et al., 2006; Hautevelleet al., 2006; Nguyen-Tu et al., 2007; Zanetti et al., 2007). Ratiosfor evaluating gymnosperm/angiosperm contributions to organicmatter (OM) have been used by Bechtel et al. (2003, 2008), Habereret al. (2006), Nakamura et al. (2010) and others. Once sufficientand unequivocal information for source specific markers is avail-able, palaeoclimatic reconstruction should be possible.

In previous studies, source specific biomarkers in the BulgarianNeogene coals were described and their diagenetic transforma-tions assumed (Stefanova et al., 1995, 2005, 2008, 2010). Laterinvestigations focussed on the total biomarker assemblage of mac-roscopically identified macrofossils (Stefanova, 2004; Stefanovaand Simoneit, 2008). The purpose was to determine the main bio-markers in the basins and relate them to the peat-formingvegetation.

The aim of the present study was to relate palaeobotanicalobservations to chemical data for Late Miocene sediments, somefrom lignite–clay cycles. For the late Miocene of western Bulgaria,we use geochemical proxies to test assumptions of palaeovegeta-tion change and climate oscillation based on quantitative analysisof palynological data.

2. Geological setting

The Staniantsi Basin is located within an NW–SE trending intra-mountain depression in western Bulgaria (Fig. 1) and continuesinto eastern Serbia. The maximum width is ca. 3–4 km and the ba-sin is ca. 10 km long (22 km including the Serbian part; Vatsev,1999). The basement is formed by middle Triassic and Jurassicage limestones (Haydoutov et al., 1995). The Neogene fill of the

Fig. 1. Sketch map of Staniantsi Coal Basin [redrawn from Yovchev (1960) withcorrections]. 1, Quaternary conglomerates, sands and sandy clays; 2, grey to greenclays; 3, grey fine clays and sandy clays; 4, brown coal; 5, dark grey clays; 6, Triassicbasement; 7, assumed limit of coalbearing layer; 8, erosional valleys of temporarystreams.

basin can be subdivided into four units. The basal unit consists oflacustrine clay up to 40 m thick. This is followed by a brown coalseam with variable thickness of up to 25 m in the central part.The next unit is composed of lacustrine clays and marls, ca. 25 mthick, with single limestone beds in the upper part, possibly repre-senting palaeosoil (Utescher et al., 2009b). The top of the sequence(the fourth unit, ca. 20 m thick) consists of comparatively homog-enous light green calcareous clays containing carbonate nodules.The basin fill terminates in red Pleistocene sands and conglomer-ates discordantly resting on the Neogene strata. As a small grabenstructure in Triassic carbonate rocks, the Staniantsi Basin had noconnection with neighbouring Cenozoic depositional areas (Vatsev,1999). The terrestrial ecosystems around the lake and in the mirewere therefore potentially sensitive to environmental changes,such as water level and climate.

According to mammal findings, the lower part of the brown coalbelongs to the mammal zone MN13, most probably to its earlier,latest Maeotian part (Nikolov, 1985; Utescher et al., 2009b). Theclastic sediments above the brown coal seam (between 30 and45 m) contain large and small mammal fauna, indicating late (Pon-tian) MN13 age (see Utescher et al., 2009b). Palaeomagnetic datafrom the Staniantsi Basin (C3 Chron: Utescher et al., 2009b) pro-vide evidence that lignite accumulation took place during the lat-est Maeotian to early Pontian (Late Messinian) and the magneticreversal observed at a depth of ca. 11 m in the section can be cor-related with the C3A/C3 Chron boundary (6.033 Ma).

3. Material and methods

3.1. Samples

The position of the samples is shown in the section of the openpit brown coal mine where they were collected (Fig. 2). The sectionincludes 27.5 m of brown coal, displaying ca. 18 cyclic alternatinglayers of brown coal and marl/clay (Utescher et al., 2009b). Thethickness corresponds to cycles ranging between 1.8 m and 2.0 min most cases. The samples originate from two intervals of the ex-posed section: Lignite and Clay A (7–10 m) – one sample from lig-nite and one from marl/clay, and Clay B (23–26 m) – one samplefrom clayey sediments. The three samples therefore originate fromthe lignite/marl cycles described by Utescher et al. (2009b) fromthe main coal seam. Their characteristics are shown in Table 1.

Fig. 2. View from open cast Staniantsi mine showing position of studied profile andapproximate depths.

M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374 1365

3.2. Pollen analysis

Pollen analysis was based on 63 samples taken from the mainsection. Each sample contained homogenized bulk material froma single layer or from 50 cm sediment at the maximum in the caseof constant lithology. For interpretation of vegetation/climatechange we focussed on samples from the two sections mentioned

Table 1Rock Eval characteristics, bitumen yield and fractionation balance.

Characteristic Sample

Lignite Clay A Clay B

Rock Eval data:TOC, wt.% 35.39 38.38 38.91Tmax (�C) 411 419 406Hydrogen index (HI), mg HC/g TOC 237 146 127Oxygen index (OI), mg CO, CO2/g TOC 116 120 136

Content of ‘‘free’’ bitumen (mg/g Corg) 65.9 21.5 24.4

Bitumen fractionation (%)Neutrals 5.0 5.7 4.6Aromatics/polars 19.2 11.9 14.8NSO polar 38.8 54.0 59.6Asphaltenes 37.0 28.4 21.0

Fig. 3. Lithological column of Staniantsi section with stratigraphic data [redrawnfrom Utescher et al. (2009a,b) with changes]. Bars next to profile indicate positionof samples.

above (Lignite and Clay A – 7–10 m; and Clay B – 23–26 m) de-picted in Fig. 3.

The samples were initially processed according to the standardtechnique for Neogene sediments, which includes successive treat-ment with HCl, HF, KOH, and heavy liquid separation (ZnCl2) priorto storage in glycerine.

3.3. Solvent extraction and separation

For bitumen analysis, samples (ca. 30 g) were Soxhlet extracted(30 h) using CHCl3. The data in Table 1 represent mean values fortwo experiments. Asphaltenes were precipitated in hexane (1:50,v/v) and soluble aliquots after concentration were separated inthree fractions (Stefanova et al., 2005). Silica gel mini-columnswere used and separation into neutrals (n-hexane eluent), aromat-ics/polars (toluene eluent) and NSO compounds (acetone eluent)carried out.

3.4. Instrumental analysis

3.4.1. Rock–EvalRock–Eval analysis was carried out with a ‘‘Turbo’’ model RE6

pyrolyzer (Vinci Technologies). The basic operating principles havebeen described by Lafargue et al. (1998). The instrument allowsdetermination of total organic carbon (TOC), free hydrocarbons(S1 peak), Tmax, S2 peak profile, hydrogen index (HI) and oxygen in-dex (OI).

3.4.2. Gas chromatography–mass spectrometry (GC–MS)Neutrals and aromatic/polar fractions were analyzed using a

Hewlett–Packard 5972 MS instrument equipped with a DB-5 col-umn (0.22 mm � 30 m; 0.25 lm film thickness), a flame ionizationdetector (300 �C) and a split/splitless capillary injector (300 �C)used in the splitless mode (valve reopened 1 min after injection).After 0.5 min hold at 85 �C, the oven temperature was increasedto 200 �C at 20�/min and then to 320 �C at 5�/min. Componentassignments were made by comparison of mass spectra with theNIST library or literature data (Philp, 1985). Concentrations werecalculated using comparison of peak areas with those from internalstandards (deuteriated n-C24 alkane and binaphthyl). Concentra-tions were normalized to TOC and are expressed as lg/g Corg.

4. Results and discussion

4.1. Vegetation (characteristic) based on pollen data

A comparatively rich spore and pollen flora has been found inthe sediments of the Staniantsi Basin, comprising a total of 88 fossiltaxa (Ivanov et al., 2008; Utescher et al., 2009b). It consists ofplants from different taxonomic groups, e.g. algae, pteridophytes,gymnosperms and angiosperms. The latter provide the greatesttaxonomic diversity, viz. 70% of the taxonomic composition ofthe flora. Based on principal component analysis performed withproportions of the most common components, Utescher et al.(2009b) described two deciduous upland forest communitiesreaching up to 25% of non-bisacctes. A wetland forest association,with frequent Betulaceae in the arboreal vegetation fraction, at-tains up to 40% and is interpreted as probably being present at wet-ter stands, marginal to the mire. As peat bog vegetation, twoassociations were described, comprising mainly Pteridophytes (cf.Thelypteridaceae, Cyperaceae, Taxodiaceae/Osmundaceae). Thefrequencies of both associations show pointed anticyclic variationsand may attain >50% of the non-bisaccates. In addition, severalallochthonous palynomorph groups were described, as well as

Fig. 4. Simplified pollen diagram showing distribution of main pollen types. Bars indicate position of samples.

1366 M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374

lacustrine plankton. Rarer elements, including several thermophi-lous, ‘‘palaeotropical’’ elements were not grouped in the study.

Based on the pollen diagram for the Staniantsi section (Fig. 4),the taxonomic composition of the fossil flora could be derived fromthe main features of the spore/pollen complex as follows:

(i) In terms of biodiversity, the comparatively low proportion ofthermophilous taxa and palaeotropical elements is notewor-thy. Comparatively scanty is the content of palaeotropical ele-ments, e.g. Engelhardia, Platycarya, Reevesia, Symplocos,Sapotaceae, Arecaceae, Araliaceae, etc. These are only sporad-ically (or in low quantity) represented in the fossil pollen flora.

(ii) The most abundant angiosperm pollen is from species of thegenera Betula, Quercus, Fagus, Carya, Ulmus, and the Oleaceaefamily, attesting to the dominance of deciduous mixedmesophytic in forests surrounding the basin. Some ever-green plants (Ilex, Buxus) and lianas (Vitaceae, Humulus) con-stitute the understory of these communities. Theproportions of the other components of the mesophytic for-est vary within small ranges, mainly between 1% and 5%, e.g.pollen grains of Corylus, Carpinus, Ostrya, Eucommia, Zelkova,Fraxinus, Castanea, Tilia, Acer, Engelhardia, Pterocarya andJuglans.

(iii) Hygrophytic forest communities (swamp forests), the mainsource of the lignite, are well developed. Taxodiaceae pollenwas present in low quantities, leading to the conclusion thatthese plants were not important in the palaeovegetation.The same is true for other late Miocene basins in Bulgaria,e.g. Karlovo Basin (Ivanov and Slavomirova, 2004; Ivanovet al., 2010), Beli-Breg Basin (Ivanov et al., 2007) andGotse-Delchev Basin (Ivanov et al., in press). The deciduousalder (Alnus) played major role in the structure of swampforests, evidenced by its greater quantitative representationin the pollen spectra.

(iv) There is no clear evidence for the existence of a well devel-oped fluviatile system, or of riparian vegetation and compo-nents of such forests, respectively (Platanus, Planera,Liquidambar, Pterocarya and Salix). All of these occur in thepollen spectra in low quantity.

(v) Pinus pollen occurs in high proportions and dominates allthe pollen spectra. Pinus diploxylon type prevails over thePinus haploxylon type and Cathaya. High values of Pinus canbe attributed to long distance transportation from neigh-bouring mountains. Members of the Pinaceae have ane-mophilous pollen characterized by air filled sacs (sacci),that assist in atmospheric dispersal (Niklas, 1985). Asobserved from biomarkers (see below), gymnosperms wereobviously not important in the peat-forming vegetation,confirming long distance transportation of Pinus pollen.

(vi) Herbaceous plants are well represented with respect to theirtaxonomic diversity, but are in low abundance in the pollenspectra. The amount of non-arboreal pollen (NAP) usuallyranges between 5% and 15%, except at the top of the sectionwhere values up to 80% are reached.

(vii) Two pteridophyte species played an important role in thevegetation (and apparently in the peat-forming vegetation)– Osmunda and Laevigatosporites (Polypodiaceae/Thelipterida-ceae) – both growing in wet or swampy areas. Apparentlythey also played an important role in the peat formingprocess.

(viii) Spirogyra and other zygospores (Zygnemataceae), as repre-sentatives of the lacustrine periphyton, are abundant at sin-gle levels, reaching up to 14%. They usually indicate clean,eutrophic freshwater and a shallow environment (Berryand Lembi, 2000). Thus, high proportions of Spirogyrazygospores at certain levels may correspond to a higherwater level during mire formation (Chmura et al., 2006;Stefanova et al., 2008).

M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374 1367

4.2. Bulk characteristics

Rock–Eval pyrolysis data assign a terrestrial character to thesamples (Table 1). HI and OI values correspond to Type II–III ker-ogen (Espitalié et al., 1985; Disnar et al., 2003) and the Tmax val-ues correspond to immature OM typical for the thermalbreakdown of biological constituents such as cellulose and/or lig-nin, two major constituents of woody and herbaceous tissues.The Rock Eval characteristics are in agreement with results re-ported by Zdravkov et al. (2011) for samples from the StaniantsiBasin and by Papanicolaou et al. (2000) for coal samples fromthe Balkan region.

Fig. 5. TIC of Clay B second fraction (PAHs, polycyclic aromatic hyd

Table 2Homologous series and species in neutral fractions (lg/g Corg).

Homologous series and species

n-Alkanes (nC15–nC35)nC29 max

Des-A-triterpenoids [i.e. des-A-lupane, M+ 330, m/z 123 (100%) C24H42]17,21-Seco-hopanes [i.e. C24, M+ 330, m/z 191 (100%), C24H42]Products of triterpenoid degradation

Trimethyl-1,2,3,4-tetrahydrochrysene [M+ 274, m/z 218 (100%), C21H22]3,3,7,12a-Tetramethyl-1,2,3,4,4a,11,12,12a-octahydrochrysene [M+ 292, C22H28]C17H14 M+ 218 [m/z 218 (100%)]

Products of triterpenoid aromatization24,25-Dinoroleana-1,3,5(10),12-tetraene [M+ 376, m/z 158 (100%), C28H40]24,25-Dinoroleana-1,3,5(10)-triene [M+ 378, m/z 145 (100%), C28H42]Diaromatic triterpenoid [M+ 374, m/z 195 (100%), C28H38]

HopanesH27b, 22,29,30-trisnorhopane [M+ 370, m/z 149 (100%), C27H46]H29a,b, 17a(H),21b(H)-30-norhopane [M+ 398, m/z 191 (100%), C29H50]H29b,b, 17bH, 21bH-30-morhopane [M+ 398, m/z 177 (100%), C29H50]H30bb, 17bH, 21bH-Hopane [M+ 412, m/z 191 (100%), C30H52]

Aromatized hopanesD-ring monoaromatic hopane [M+ 364, m/z 211 (100%), C27H40]

Aromatized steranes [i.e. M+ 394, m/z 211 (100%), C29H46]Diterpenoids

18-Norabieta-triene [M+ 256, m/z 241 (100%), C19H28]Dehydroabietane [M+ 270, m/z 255 (100%), C20H30]16a(H)-Phyllocladane [M+ 274, m/z 123 (100%), C20H34]Simonellite [M+ 252, m/z 237 (100%), C19H24]Retene [M+ 234, m/z 219 (100%), C18H18]

4.3. Molecular composition of bitumen

The extract yields are relatively low at 2–7 wt.%. Extractswere separated into aliphatics (ca. 5%), aromatics/polars (10–20%) and NSO fractions, the latter in highest abundance (Table 1).Normalized to TOC content, differences are most apparent fornon-polar components, where Clay A (7–10 m) had twice asmuch (155 lg/g TOC) than Clay B (23–26 m; 75 lg/g TOC), whilethe quantities of polar compounds were similar, in the range109–116 lg/g TOC.

GC–MS revealed the presence of n-alkanes and terpenoids (Fig. 5;Tables 2 and 3). The n-alkanes and a C27 D-ring monoaromatic

rocarbons); d – n-alkan-2-one; peak identifications in Table 3.

Sample

Lignite Clay A Clay B

81.77 95.01 53.9120.2 20.28 8.91

4.17 3.54 0.370 7.61 3.76.37 2.92 4.02

3.22 1.8 0.871.21 1.12 3.10 0 0.05

2.05 4.77 1.450 1.35 0.821.45 0.92 0.130.6 2.5 0.5

3.09 9.58 4.031.3 3.64 1.330.87 1.94 1.240.92 4 0.480 0 0.98

13.64 3.09 30.37 3.113.64 27.7 3.1

0.79 1.45 0.950 0 3.21

0 0 0.90 0 0.520 0 1.20 0 0.530 0 0.06

Table 3Homologous series and species in aromatic/polar fraction (lg/gCorg).

Homologous series and species Peak in Fig. 5 Sample

Lignite Clay A Clay B

Alkan-2-ones (nC25–nC35) 31.92 7.67 3.65n-C29 max 12.72 2.02 1.23

Mid-chain ketones (i.e. C29, C31), mid-chain alcohol (C29) 0.4 1.3 a

Nonacosan-10-one, M+ 422, m/z 155, 295, C29H58O 9 0.4 1.3 a

Nonacosan-10-ol, M+ 424, m/z 157, 297, C29H60O 10

Iso ketone, i.e.trimethylpentadecan-2-one [M+ 268, m/z 58 (100%), C18H36O] a a a

PAHs, 3–4 aromatic rings, alkylated 3.08 4.47 3.31

Polar diterpenoids 0 0 2.63Ferruginol [M+ 286, m/z 271 (100%), C20H30O] 3 0 0 1.687-Oxo-dehydroabietane [M+ 284, m/z 269 (100%), C20H28O] 1 0 0 0.45Dehydroferruginol [M+ 284, m/z 202, C20H28O] 2 0 0 0.5

Keto triterpenoids 38.5 29.62 28.67ba – Friedelin [M+ 426, m/z 95 (100%), C30H50O] 18,19 26.34 24.8 21.3ba – Amyrone [M+ 424, m/z 218 (100%), C30H48O] 14.15 2.88 3.06 4.29Lupan-3-one [M+ 426, m/z 205 (100%), C30H50O] 16 6.00 0.76 0.78Taraxerone [M+ 424, m/z 204 (100%), C30H48O] 2.58 tr. tr.Olean-9(11),12-dien-3-one [M+ 422, m/z 255 (100%), C30H46O] 13 0.7 1.0 2.34

Keto hopanes 5.23 5.34 4.6517,21-Seco-pentakisnor-hopan-17-one [M+ 358, m/z191 (100%), C25H42O] 8 3.81 4.00 3.0017a(H)-Trisnorhopane-21-one [M+ 384, m/z 191, C27H48O] 12 1.42 1.34 1.65

Keto sterane, i.e. stigmastan-3,5-dien-7-one [M+ 410, m/z 174, C29H46O] 17 1 1.1 1.46

Degradated keto triterpenoids 29.93 59.74 70.42A-norfriedel-8-en-10-one [M+ 342, m/z 342 (100%), m/z 123 (50%), C24H38O] 6 27.3 44.27 45.45A-norfriedel-dien-10-one [M+ 340, m/z 325 (100%), C24H36O] 5 2.63 12.37 22.47A-norfriedelan-10-one [M+ 358, m/z 358, C25H42O2] 7 0 3.1 2.5

Othersa-Tocopherol [M+ 430, m/z 165 (100%), C29H50O2] 11 2.4 0.65 0.844,8,12,16-Tetramethylheptadecan-4-olide [M+ 324, m/z 99 (100%), C21H40O2] 4 a a a

a Present in low content and not integrated.

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hopane were present in the first fraction, while alkan-2-ones andpolar triterpenoids were in the second. Extremely high quantitiesof keto triterpenoids and products of their possible degradationwere found, i.e. tentatively assigned A-norfriedel-8-en-10-one,reaching 50–60% in the clays and 30% in the lignite sample (Table 4).Homologoues series are discussed separately.

4.3.1. n-AlkanesThe n-alkanes strongly dominated in all three samples (60–

70%). The distribution is characterized by a strong dominance of

Table 4Homologoues series in two fractions (from MS; rel.%).

Homologous series Lignite Clay A Clay B

Neutrals:n-Alkanes 73.1 61.2 71.2Des-A-triterpenoids 3.7 2.3 0.5Seco-Hopanes 0 4.9 6.3Degradared triterpenoids 5.7 1.9 5.4Hopanes 2.7 6.2 5.2Aromatic hopanoids 12.2 19.7 4Aromatic steroids 0.8 0.8 1.3Atomatic triterpenoids 1.8 3 1.8Diterpenoids 0 0 4.3

Aromatic/polars:Polycyclic aromatic hydrocarbons (PAHs) 2.7 4.1 2.8Alkanones 28.7 8.2 4.2Ketohopanes 4.7 4.9 4.0Ketosteranes 0.9 1.0 1.3Ketotriterpanes 34.2 27.0 24.5Degraded ketotriterpanes 26.6 54.4 60.2Polar diterpenoids 0.0 0.0 2.3a-Tocopherol 2.1 0.6 0.7

n-C29. Lockheart et al. (2000) determined a similar pattern for Fag-aceae leaves from the Miocene Clarkia lake deposit (Idaho, USA).The content of <C21 n-alkanes is lower (6–16% of all n-alkanes) thanfor >C25 (63–80%). The carbon preference index (CPI) was calcu-lated as:

CPI ¼ 1=2XðC25 � C33Þ=

XðC26 � C34Þ

h

þXðC25 � C33Þ=

XðC24 � C32Þ

i

Values range from 2.8 to 4.5 and confirm the dominance of oddhigher molecular weight homologues typical for low rank coalswith an epicuticular wax contribution from higher plants, sporesand pollen (Chaffee et al., 1986). The distribution coincided withthat described by Zdravkov et al. (2011).

4.3.2. IsoprenoidsTraces of the regular isoprenoids pristane (Pr, isoC19) and phy-

tane (Ph, isoC20) were found. Pr is commonly found in coal extractsand is thought to be derived from oxidation and decarboxylation ofphytol, tentatively recorded in the second fraction of the samples.Isoprenoids are widespread in plants and are often found in the ex-tracts and pyrolysates of Bulgarian Neogene coals (Stefanova et al.,2008). One potential precursor could be the a-tocopherol presentin the samples (Table 3). In brief, it is supposed that, as a resultof the immaturity of the samples, regular isoprenoids are stillincorporated into the macromolecular matrix. The origin andtransformations of regular isoprenoids in Staniantsi lignites wasdiscussed recently by Zdravkov et al. (2011). Since Pr can beformed from a-tocopherol (present in the samples) and Ph frombis phytanyl ether (Philp, 1994), Pr/Ph may not be diagnositic ofeither source or depositional conditions. Moreover, coupled

M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374 1369

pristanyl and phytanyl units in head-to-head linked isoprenoidsoccur in bitumens from another Bulgarian low rank coal, MaritzaEast lignite (Stefanova, 2000).

Homophytanic acid c-lactone (4,8,12,16-tetramethyl heptadec-an-4-olide; M+� 324, m/z 99 (100%), C21H40O2, is also present and isinterpreted as an oxidation product of a-tocopherol. The isopren-oid ketone, phytone (6,10,14-trimethylpentadecan-2-one) appearsin the m/z 58 chromatograms of the second fraction. It is quitecommon in nature and occurs widely in sediments and coals. Sed-imentary phytone and homophytanic acid c-lactone are indicatorsof oxic diagenesis (Jaffé et al., 2006).

4.3.3. n-AlkanonesTwo homologoues series of n-alkanones were detected via m/z

58 chromatograms. The first comprises long chain alkan-2-onespresent in all the samples (Fig. 5, Table 3) but in especially highabundance in the lignite sample (ca. 30%; Table 4). An odd prefer-ence is obvious for all samples and the distributions of long chainn-alkanes and n-alkan-2-ones (i.e. C24–C33) are similar, maximizingat C29. The similarity suggests a product-precursor relationship.Saturated n-alkan-2-ones with minor n-alkan-3-ones and n-al-kan-4-ones are common in geological samples, as described byTuo and Li (2005), who discuss possible sources. According to Rie-ley et al. (1991), alkan-2-ones are not constituents of leaf wax butare formed in very early diagenesis.

Mid-chain ketones, n-alkan-10-ones (C29, C31) were present inall the samples; n-nonacosan-10-ol and the corresponding ketoneoccur in the waxes of various higher plants (Tulloch, 1976). Itcan be therefore assumed that the aliphatic lipids in the samplesoriginated from epicuticular leaf waxes of higher plants. Ketonescannot be considered as source specific tracers because they arecommon in the plant kingdom.

4.3.4. n-AlkanolsLong chain n-alkanols from C26 to C30 with a strong even pre-

dominance were observed as such without derivatization. Themid-chain alcohol, n-nonacosan-10-ol also was detected. It occursin the waxes of various higher plants (Tulloch, 1976) and we as-sume that the aliphatic lipids in the extract originated from theepicuticular leaf waxes of higher plants (Lockheart et al., 2000).

4.3.5. SteroidsThe aliphatic fractions reveal an extremely low content of ste-

roids. A monoaromatic sterane, (M+� 394, m/z 211 (100%), C29H46)and one sterone [stigmastan-3,5-dien-7-one (M+� 410, m/z 174100%, C29H46O)] were the only steroid structures found. C29 ste-roids are abundant in the plant kingdom and reflect an input ofdetritus from higher plants (Simoneit, 2004, 2005) but are non-specific markers here because sitosterol, the biological precursor,is ubiquitous in nature.

4.3.6. DiterpenoidsStudies of Neogene lignites have demonstrated that the preser-

vation potential of certain terpenoids as phenols or ketones is sig-nificant (Stefanova et al., 2002; Stefanova and Simoneit, 2008).These compounds provide valuable information about precursorOM and its transformation during coal burial and coalification.Diterpenoids can therefore be used as (palaeo)chemosystematicindicators for the taxonomical systematics and phylogeny of coni-fers (Otto et al., 2002, 2003).

Diterpenoids were found only in the Clay B sample (23–26 m),amounting up to 4.3% of the aromatic hydrocarbons and 2.3% ofthe polar diterpenoids (Table 4). The components could be as-signed to the abietane and phyllocladane groups (Tables 2 and3); 16a(H)-phyllocladane was present in the neutral fraction, whileabietanes occurred as aromatic hydrocarbons (dehydroabietane,

simonellite, retene) and polar derivatives, such as ferruginol, dehy-droferruginol and 7-oxo-dehydroabietane, in the aromatic/polarfraction (Fig. 5). Abietic acid methyl ester has little chemotaxo-nomical value as it is highly abundant and is considered an artifact.Nevertheless, it was found only in Clay B. It is essential to note that,in this part of the profile, pollen of the Abies genus (Fig. 4) was re-corded in higher abundance. In the major part of the profile, fir isrepresented in the Staniantsi section in low quantity (1–2%), butin the part from 23 to 26 m (cf. Fig. 4) it reaches >5–7%. This couldbe explained as a result of cooling and expansion of high mountainvegetation.

Abietanes could be divided into ‘‘regular’’ and ‘‘phenolic’’ abie-tanes (Otto and Wilde, 2001). The latter, e.g. ferruginol and deriv-atives, are widely distributed in conifer families, especially theCupressaceae, Taxodiaceae and Podocarpaceae, but seem to be lar-gely absent from the Pinaceae. Polar diterpenoids are highly abun-dant in some Bulgarian Miocene brown coals and are the majorbiomarkers in the Taxodium dubium progenitor macrofossil (Stefa-nova et al., 2002; Stefanova and Simoneit, 2008).

The biomarker assemblage of the Clay B sample from 23–26 mgave grounds to assume a presence of gymnosperm vegetation(Taxodium, Glyptostrobus) in the palaeomire but in low abundanceas evident from pollen data. The relative content of diterpenoidsand their polar counterparts was low but still sufficient for quanti-tative determination. It should be emphasized that they were pres-ent only in the Clay B sample, which is characterized by anincreased proportion of some conifers (Abies, Picea, Tsuga) anddeciduous trees (Fagus, Ulmus) in this part of the section (Fig. 4:23–26 m).

4.3.7. HopanoidsA series of tetracyclic 17,21-seco-hopanes was detected in Clay

A from 7–10 m, 4.9% and Clay B from 23–26 m, 6.3% (Table 4). Thedistributions were similar, maximizing at C24 [M+�m/z 330, m/z 191(100%); Table 2]. Other tetracyclanes, i.e. C23 and C25, were seen inthe m/z 191 chromatograms but their total ion current (TIC) inten-sities were low and difficult to integrate.

Intensities for hopanes and 17,21-seco-hopanes in m/z 191chromatograms were comparable. The significance of the hopa-noids in Bulgarian Neogene coals has been discussed previously(Stefanova et al., 1995, 2005; Bechtel et al., 2005). The m/z 191chromatograms also revealed one hopene, hop-17(21)-ene (M+�

410) and series of H27-H31 bb hopanes (H28 absent and H27 bb max-imizing). The high abundance of bb hopanes vs. ab hopanes indi-cates the immaturity of the samples. The hopanoid distributionpattern is very similar with that described by Zdravkov et al.(2011) for Staniantsi lignites.

Particularly abundant in the hopanoid distribution was a D-ringmonoaromatic hopane [M+�m/z 364, m/z 211 (100%)] in the neutralfraction of the Clay A sample (7–10 m) at ca. 20%, and highly abun-dant in the other samples (Tables 2 and 4). Greiner et al. (1976)first described pentacyclic compounds with various degrees of aro-matization (1–4 rings) from the Eocene Messel oil shale(Germany). They explained that the aromatization started in ringD and progressively spread during diagenesis to ring A. A highabundance of the M+� 364 hopane has been observed in Lomlignites (Stefanova et al., 2008).

Two keto hopanes occurred in the second fraction, i.e. a C27

hopanone [17a(H)-trisnorhopan-21-one, M+� 384, C25H42O and17,21-seco-pentakisnor-hopan-17-one, M+� m/z 358 with compara-ble abundance in the samples at 4–5% (Tables 3 and 4)]. Theirpresence provides evidence of microbial activity.

4.3.8. Polycyclic aromatic hydrocarbons (PAHs)PAHs occurred in similar abundance (2–3%; Fig. 5 and Table 4)

in all the aromatic fractions. Phenanthrene/anthracene (M+� 178)

1370 M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374

and their 1–4 alkylated homologues were present. Four ring PAHswere represented by pyrene/fluoranthene (M+� 202). Phenylnaph-thalene (M+� 204) was also observed. Perylene (M+� 252), a commonPAH in recent sediments, was not found.

4.3.9. Other triterpenoidsTriterpenoid derivatives with lupane, ursane, oleanane and re-

lated skeletons are biomarkers for angiosperms (Simoneit, 2004,2005). The most abundant higher plant triterpenoids are oleananederivatives such as b-amyrone. It should be noted that the pres-ence of C-3 functionalized triterpanes, i.e. alcohols and ketones,makes them more susceptible than hopanoids to microbial or pho-tochemical degradation, leading to des-A-triterpenoids. The onlydes-A-triterpenoid found in the samples, des-A-lupane (M+� 330,

Cla

Cla

Lign

Fig. 6. Content of homologo

m/z 123 100%) occurs in all the samples (Table 2). It is assumedthat lupane derivatives were present in greater abundance in thepalaeomire than b-amyrone (oleanane-type) bioterpenoids. Espe-cially high is the content of lupan-3-one [M+� 426, m/z 205(100%)] in the lignite sample. Lupane structures generated fromdicotyledonous angiosperms have chemotaxonomic value as theyreveal the presence of representatives of the Betulaceae family.Maritza East lignites are the other Bulgarian Neogene coals charac-terized by a high abundance of lupane structures (Stefanova et al.,1995; Bechtel et al., 2005). The conclusion of a significant contribu-tion from the birch family corroborates the palaeobotanical dataand provides evidence for a significant role of Alnus in the swampvegetation (Fig. 3). Typically, the pollen of this deciduous treereaches 20–30% in the pollen spectra and significantly dominates

y B

y A

ite

ues series (lg/g TOC).

M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374 1371

the pollen of Taxodiaceae, which were the dominant trees in manyMiocene peat bogs (Schobert, 1987). Ivanov (1995) and Ivanovet al. (2002) pointed out that the transition from the Middle Mio-cene to Late Miocene swamp vegetation in Bulgaria was character-ized by a decrease in Taxodiaceae and an invasion of deciduousalder in wet habitats.

It should be mentioned that alcohol derivatives of triterpenoidsalso were observed, i.e. lupan-3-ol [M+� 428, m/z 189, m/z 207(100%), C30H52O], D:A-friedooleanan-3-ol [M+� 428, m/z 231, m/z165 (100%), C30H52O], ursa-9(11),12-dien-3-ol, [M+� 424, m/z 255(100%), C30H48O] and others. Being in lower quantity than the ketotriterpenoids they were not quantified and are not included in thetables.

The problem of the application of oleananes as source markersin terrestrial systems is that the main fate of the precursor olean-enes is not reduction but rather partial or complete aromatization.It has been demonstrated that progressive aromatization startsvery soon after deposition, via microbial mediation. Wakehamet al. (1980), Stout (1992) and Murray et al. (1997) proposed sev-eral pathways for the transformation of C-3 functionalized precur-sors. Some are applicable to explain our set of components: (i)progressive aromatization and demethylation with preservationof the pentacyclic skeleton [24,25-dinoroleana-1,3,5(10),12-tetrae-ne is a prominent peak in the first eluting fractions] and (ii) loss ofring A and formation of des-A-triterpenoids, i.e. des-A-lupane.

5. Friedelin and degradation products

The oxygenated pentacyclic triterpenoid, ab friedelin [M+� 426,C30H50O, m/z 69 (100%)] occurs in comparable abundance (ca.25 lg/g Corg) in all the samples (Table 3, Fig. 6). It is a common epi-cuticular wax component (Logan et al., 1995). Lower amounts of3a/3b� friedelanol (M+� 428) were also observed. Taken on itsown, a high abundance of friedelin is not unusual for Miocene sed-iments. High contents were found in Neogene lignites from a Sofiacoal bearing province (Bulgaria; Stefanova and Simoneit, 2008). Itis also highly abundant in clay sediments and fossil plants fromthe Miocene Clarkia Formation, Idaho, USA (Logan et al., 1995; Ottoet al., 2005) and in oak covered soil (Trendel et al., 2010). The novelfinding in this study is the peak tentatively assigned as A-norfri-edel-8-en-10-one on the basis of a recent study of Bakar et al.

O

Friedeline

O

M+ 358

C25 H42O

A-Norfriedelan-10-one

M+ 426

C30H50O

O

C24H38O

M+ 342

A-Norfriedel-8-en-10-one

O

A-Norfriedel-5(6),8-dien-10-one

C24H36O

M+ 340

Fig. 7. Formulae cited in text.

(2011), and which is at extremely high abundance in the clay sam-ples (ca. 45 lg/g Corg) and lower in the lignite sample, (27.3 lg/gCorg; Fig. 6). The ratio A-norfriedel-8-en-10-one/friedelin, is <1.0for the lignite sample and twice that for the clay sediments.

A-norfriedel-8-en-10-one can be regarded as a product of loss ofring A in friedelin (Fig. 7). Corbet et al. (1980) described 3,4-seco-triterpenoids as major products of photochemical/photomimeticalteration of 3-oxygenated triterpenoids. Later, Yanes et al.(2006) and Simoneit et al. (2009 and references therein) investi-gated the reactivity of higher plant 3-oxy-triterpenoids to sunlightand assumed an analogy with the behaviour in sediments duringdiagenetic reworking of the parent triterpenoids. Bakar et al.(2011) described the A-norfriedelan-10-one (M+� 358) and A-nor-fridel-8-en-10-one (M+� 342) in sediments from Bera, Malaysia asbeing products of the photochemical degradation of friedelin.

An additional compound of the friedelin series was present, A-norfriedel-dien-10-one [M+� m/z 340, m/z 325 (100%), C24H36O]with a distribution pattern similar to A-norfriedel-8-en-10-one(Fig. 7). Its retention time, as well as eluting just before A-norfri-del-8-en-10-one [M+� m/z 342, m/z 123 (100%)] suggests the‘‘diene’’ structure (Fig. 7). The molecular mass also could be ful-filled by A-norfriedel-quinone (C23H32O2). Although this is consid-ered less likely, the compound cannot be assigned to a specificstructure (see Fig. 8).

High quantities of tentatively assigned A-norfridel-8-en-10-oneare present in the Staniantsi Basin (this study) and Balsha coal(Stefanova, unpublished data). Products of friedelin degradationwere not observed in other samples characterized by high friedelincontent. Their formation may therefore be related to specific con-ditions of deposition in the palaeomire. For example, carbonaterocks in the Staniatsi Basin might have played a catalytic role infriedelin transformation. The lack of connection of the StaniantsiBasin to neighbouring Cenozoic depositional areas made terrestrialecosystems potentially sensitive to ground water level and climate.Additionally, products of friedelin degradation were more pro-nounced where not diluted with allochthonous OM.

Triterpenoids, friedelin in particular, are located both internallyand in the epicuticular leaf waxes that are exposed and thus aremore readily degraded by microorganisms than resinous matter.Mechanical degradation, resulting in extensive disintegration to af-ford microscopic particles promotes microbial activity. The sedi-mentary environment at Staniantsi was characterized byprolonged periods of high water table, additionally facilitating deg-radation. Leaf tissue was not observed in petrographic analysis andwas explained by way of an unfavourable setting for the palaeoen-vironment, characterized by severe mechanical degradation priorto burial (Zdravkov and Kortenski, 2008). In a pioneering study,Corbet et al. (1980) proposed a hypothesis of ‘‘sun-drenched’’ sed-iments, whereby ketones would undergo photolysis. Perhaps longperiods of a high water table in the Staniantsi Basin and severereworking of the progenitors were responsible for the degradationof friedelin and formation of the tentatively identified A-norfridel-8-en-10-one. These processes were more important for the claythan the lignite, as reflected in the higher A-norfriedel-8-en-10-one/friedelin ratio values.

6. Palaeoenvironmental implications

Palaeoclimate data reconstructed by applying the CoexistenceApproach (Mosbrugger and Utescher, 1997) attest to a warm-tem-perate climate, with values for all temperature parameters at least2–3 �C higher than today and with rainfall rates that exceed thepresent rate (Utescher et al., 2009b). Average annual temperaturevaried from 12 �C to 18 �C. Reconstruction of winter temperature(coldest month mean, CMM) displays a relatively wide range from

50 100 150 200 250 300m/z

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283326123 298

341173 269159

145 177 25555 91 10581 187 243157143 297229171 25621579 1079567 201 299257 270 3129783 327 34265 313285 328 343

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11910581 17713355 91 203

147 175 328107 2321899567 145 159 190 205 30121917383 202 217 25797 324 344309281 29923357 271 329311 345

Fig. 8. Mass spectra of A-norfriedel-8-en-10-one (M+� 342, C24H38O) and A-norfriedel-5(6,8)-dien-10-one (M+� 340, C24H36O).

1372 M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374

0 �C to 12 �C, the most common intervals being from 3.8 �C to9.6 �C. The results for the average summer temperature (warmestmonth mean, WMM) are between 20 �C and 28 �C, but most

common are intervals between 24.7 �C and 26.4 �C. Coexistingintervals for the annual amount of precipitation span a relativelywide range – from 370 to 1500 mm/m2 – with a mean around or

M. Stefanova et al. / Organic Geochemistry 42 (2011) 1363–1374 1373

slightly above 1000 mm. Thus, all the data attest to a warm and hu-mid climate in western Bulgaria for the studied time interval.

The results provide evidence of climate change cyclicity. Cyclesoccur with different levels of hierarchy in terms of length of peri-ods. They were the subject of a discussion by Utescher et al.(2009b), where a generalized model of environmental conditionsfor the region during the Late Miocene was presented. The modelreflects the hierarchical cyclicity of the climate: (i) longer term cy-cles (period ca. 100 kyr), expressed by way of oscillation of ther-mophilous elements and triggered by climate shifts fromwarmer/wetter to cooler/drier periods; (ii) short term cycles (ca.21.7 kyr) interpreted as inundation cycles reflecting changes ingroundwater level and (iii) millennial scale climate variability(ca. 4.5 kyr). The present geochemical analysis confirms the pres-ence of short term cycles (ca. 21.7 kyr). These cycles are expressedas lignite/marl–clay layer alternations combined with cyclicchange in swamp vegetation. This type of cyclicity is interpretedas cyclic changes in groundwater level and inundation of the basin.In the periods of low water level (swamp phase in the evolution ofthe basin) lignite accumulation took place. It is characterized bythe spreading of swamp vegetation in the basin, which was thesource of buried OM and a higher rate of lignite accumulation. Atthe same time, the low inflow of water from the land explainsthe low input of terrigenous clastic material. In contrast, duringthe stages with high water level (lacustrine phases), increased run-off due to higher precipitation rate resulted in a greater input ofsiliciclastic material to the basin, causing additional dilution ofthe OM. In addition, a high lake level enhanced biogenic produc-tion of carbonate (presence of molluscs, charophytes). The distri-bution of swamp vegetation was reduced and was spread mainlyalong the margins of the basin. As a result, production and burialof plant material in the basin was reduced.

The ‘‘lacustrine phase’’ with its high water level was suitable forfriedelin degradation as discussed above. It provided sufficientlylong periods (ca.12 kyr) for transformation and sedimentation ofthe precursor material, resulting in the inferred A-norfriedel-8-en-10-one/friedelin values. Respectively, brown coal/marl cyclesrepresenting changes from lacustrine to paludal conditions in thebasin were most probably related to groundwater table changes.Our data indicate that the concentration of friedelin and its proba-ble degradation products may reflect these oscillations in water ta-ble. The ratio of A-norfriedel-8-en-10-one/friedelin, with values <1could represent the swamp phase, with values >1 being character-istic for periods of inundation, could probably be used in time-series analyses by way of biomarker ratios. However, detailedfollow-up studies are needed to verify the potential of theparameter for environmental reconstruction.

7. Conclusions

The biomarker assemblages accompanied by bulk chemical dataindicate that, in the palaeomire of the Staniantsi Basin, gymno-sperms were not important, suggesting a predominantly angio-sperm-rich flora as a source of the OM. The observations coincidewith palaeobotanical data and recent results of Zdravkov et al.(2011) for the Angiospermae prevalence in the peat-forming vege-tation. This differs from the interpretation of Utescher et al.(2009b), who came to the conclusion that the peat-forming vegeta-tion of the Staniantsi brown coals consisted primarily of pterido-phytes, with Taxodiaceae as an accessory element. In that study,Alnus, Betula and other, partly shrubby angiosperms were inter-preted as a wetland association thriving in marginal areas of themire and along the coast in the lake phases, respectively.

The presence of des-A-triterpenoids, 17,21-seco-triterpenoids,hopanes, high content of the D-ring monoaromatic hopane

[M+� 364, m/z 211 (100%) C27H40] and aromatized triterpenoids,suggested that microbially mediated processes significantly con-tributed to the alteration of the OM. Mechanical degradation,resulting in extensive disintegration of microscopic particles, pro-moted microbial activity by providing better contact. A prolongedperiod with a high water table and severe mechanical degradationprior to burial additionally enhanced decay.

The data suggest that a geochemical appraisal of short term cy-cles (ca. 21.7 kyr) in climate may be possible. These cycles are ex-pressed as lignite/marl–clay layer alternation combined with cyclicchanges in swamp vegetation. This is interpreted as cyclic changesin groundwater level and inundation of the basin. In the periods oflow water level (swamp phase) lignite accumulation took place.Preliminary results obtained from selected samples suggest thatthe oscillations may be reflected in the content of friedelin vs. itsproducts of degradation. The ratio of A-norfriedel-8-en-10-one/friedelin might be used to detect short term environmental cycles,where values <1 represent the swamp phase, while those >1 reflectperiods of inundation. Time-series analysis using densely sampled,lignite–clay layer oscillations are needed to confirm if this bio-marker ratio can be used in environmental reconstruction.

Acknowledgements

M.S. expresses gratitude to B.R.T. Simoneit for help in MS datainterpretation and to A. Bechtel for valuable discussions on the Sta-niantsi biomarker composition. The authors express their sincerethanks to A. Bechtel and an anonymous reviewer for thoughtfulcomments.

Associate Editor—C.C. Walters

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