A Preliminary Investigation of the Polar Lipids in Recent Tropical Sediments from Aquatic Environments at Campos dos Goytacazes , Brazil

s of the 7 Latin American Congress on Organic Geochemistry, Foz do Iguaçu, Brazil, 2000; Santos, C.Y.M.; Azevedo, D.A.; Aquino Neto, F.R.; Atmos. Environ. 2002, 36,


Introduction
Organic matter (OM) in lacustrine sediments is derived from production within the aquatic system, inputs of terrestrial material from the surroundings and bacterial production within the sediments themselves.The relative importance of these two sources will be determined by local factors such as climate, nutrient supply, hydrodynamic conditions, and the biogeochemistry of lake sediments.Thus, changes in any of these aspects may be reflected in the organic components of the sediments.
2][3] Autochthonous and allochthonous OM inputs are easily discernible, but the former tend to be more labile than terrestrially derived counterparts. 4lthough lipids usually represent a small fraction of the total organic carbon (TOC), their diversity and specificity makes them useful compounds to study the sources, and the transformation of OM.Alkanoic acids are among the most abundant biomarkers, but are considered to be relatively nonspecific source indicators.Conversely, they are one of the few indicators of bacterial contributions and may also be used to distinguish marine from Azevedo J. Braz.Chem.Soc.
terrigenous and algal from zooplanktonic inputs. 5Alkanoic acids in surface sediments have been used to identify sources of the organic matter 6,7 as was observed in Ria Formosa Lake, Portugal. 8][11][12][13] The "biomarkers approach" has several limitations, including the wide distribution of some compounds, their different reactivity, and the multiplicity of processes (biological, chemical, physical and geochemical), which modify the original source signature.
In this study, we have analyzed the polar lipids present in the extractable organic matter of sediments, which have not yet been analyzed for biolipid content, in order to elucidate their possible sources.The tropical rivers and lake, under study, are important to the city of Campos dos Goytacazes (RJ, Brazil) because they are situated in a still relatively unpolluted and scantily inhabited region that, despite the presence of many sugar-cane plantations and other farming activities, is still free from most of the problems inherent to highly populated areas.We thus set out: i) to determine the origin of the organic matter (allochthonous vs. autochthonous) present in the sediments; ii) to determine if their polar lipids have a non-biogenic source; iii) to determine if there have been contributions from biomass burning; iv) to verify if there is any impact of anthropogenic activities on this environment; v) to verify if any similarities exist between the organic matter contributed to the four sites, since they are all connected.
The organic geochemistry of the northern part of the State of Rio de Janeiro remains practically unknown.On the other hand, such cannot be said about its hydrogeochemistry, 14 its hydrobiology 15 and its inorganic geochemistry. 16The lack of organic geochemical information led our group to study this area.As a result, we have published data regarding the aliphatic and aromatic hydrocarbon contents of these sediments. 17,18

Description of the sampling site
The city of Campos dos Goytacazes is an important city in the northern part of the State of Rio de Janeiro, in which sugar cane plantation is the main economic activity (Figure 1).Sugar cane plantations surround the Urubu, Imbé, Ururaí rivers and Lake de Cima.To facilitate manual harvesting and to increase production, the sugar cane crops are always burned before being harvested.This technique generates a great cloud of smoke, which hovers over the city and the surrounding areas for many days.
Located at latitude 21 o 45'15"S and longitude 41 o 19'28"W, roughly 28 kilometers from Campos dos Goytacazes city, Lake de Cima has a circumference of 18 kilometers and a maximum depth of 4 meters.Farms, sugar cane plantations and natural vegetation surround it and there is little traffic around.Besides, the Imbé and Urubu rivers originate in the Imbé Mountains.We may, thus, assume that these sites have suffered little or no impact at all from anthropogenic activities.Their geographic location is also shown in Figure 1.

Experimental
Surficial sediment samples were collected (05- 13-1996) from the Urubu, Imbé and Ururaí rivers and Lake de Cima.Only one sample was collected from each site for this preliminary investigation.Sampling was carried out using a mechanical shovel.The samples were stored in a glass jar, and then frozen.Samples were lyophilized, and extracted ultrasonically for four 20min periods with fresh 50 mL of methylene chloride: methanol (9:1-Omnisolv, Merck).The extracts were first concentrated on a rotary evaporator followed by a stream of nitrogen and separated into aliphatic and aromatic hydrocarbons, and polar compounds, by thin layer chromatography (TLC) on silica gel (developer hexane).The bands corresponding to these fractions, visualized using iodine vapor were scraped from the plates, eluted with dichloromethane, concentrated by rotary evaporation and then in a stream of nitrogen.The polar fractions were derivatized using diazomethane (to esterify alkanoic acids), followed by reaction with N,Obis-(trimethylsilyl)-trifluoroacetamide (BSTFA, 30 min at 60 o C) to convert alcohols into trimethylsilyl ethers.
The fractions were analyzed by gas chromatography (GC) and gas chromatography-mass spectrometry (CG/MS) Where possible, structural assignments were achieved by comparison of mass spectra with those of authentic standards, or with the help of the Wiley 138 standard library.Whenever mass spectra were unavailable, interpretation of fragmentation patterns was used together with GC relative retention times.
Quantification was performed from GC profiles using n-dodecanol (Merck -as trimethylsilyl ether) as the internal standard.
The elemental composition (C,H,N) of the samples was determined using a Perkin-Elmer 2400 CHN Analyzer and a AD-4 Autobalance.

Results and Discussion
Total ion chromatograms (TIC) of the polar fractions are shown in Figure 2. The identifications of labeled peaks are given in Table 1.
As seen in Figure 3, phytosterol concentration in the Imbé, Urubu and Lake de Cima sediments, increased in the order C 29 >C 28 >C 27 .On the other hand, the mass fragmentograms m/z 215 and 129 (characteristic ions for stanols and sterols, respectively) show, in Figure 4, that cholesterol is dominant in Ururaí sediments with minor contributions of other sterols In the sediment samples, stenols were almost as abundant as the corresponding stanols, indicating that the early diagenetic transformations of these compounds had already occurred.
C 27 and C 28 sterols reflect an algal contribution to the OM. 194] The relative importance of these two sources in the formation of OM will depend on the type of environment surrounding the site, on the proximity to sources of land-derived organic matter, and on the abundance and type of phytoplankton present. 11he sampled sites in the present study were situated close to land and places like or such that, so the origin of 14ethylcholest-5-en-3β-ol is pointed as terrigenous.
The lipid material is composed of plant wax, and of components deriving from microbial detritus and algae.This interpretation is also supported by the enhanced      concentration of cholesterol, abundant in many freshwater algae and zooplankton.Hence, its occurrence in the Ururaí sediment probably arose mainly from an autochthonous planktonic input.The presence of substantial concentrations of 24-ethylcholesta-5,22-dienol (C 29 ∆ 5,22 ) and 24-ethylcholest-5-enol (C 29 ∆ 5 ), and of less amount of their 24-methyl counterparts in the Urubu, Imbé, and Lake de Cima sediments indicate the abundance of a vascular plant input.Note that these compounds have also been detected in high concentrations in soils adjacent to other tropical riverine systems. 25ydrogenation of the ∆ 5 double bond yields stanols with 5α(H) and 5β(H) configurations.Hydrogenation in oxygenated sediments favors the 5α form, whereas the 5β form is favored in strongly anoxic sediments. 26In the sediments under study, only 5α(H)-stanols (Table 1) were detected, indicating an oxygenated sedimentary environment.
Olean-12-en-3β-ol (β-amyrin) and friedelan-3-one were prevalent among the triterpenoids in the Imbé and Urubu samples.α-Amyrin was not detected in these samples although it is usually found in conjunction with β-amyrin in many recent sediments.Other triterpenoids are also present, but they could not be identified (see Table 1) in this and on the other two sediments.It is important to note that pentacyclic 3-oxytriterpenoids were very abundant in the soil samples from the Orinoco Basin studied by Jaffé and coworkers (1996). 27It has been suggested that the bulk of the dissolved and particulate organic carbon in tropical rivers derives from highly degraded soil materials, and that, the lipid fraction of such organic phases show a relatively high contribution of autochthonous organic matter. 25,27The same patterns seem to occur in the tropical sediments now under study.
n-Alkanols are present in all four samples.They range from C 14 to C 32 with a maximum at C 16 and a strong even carbon number predominance (see Table 2 and Figure 5).A better visualization of the alkanols was obtained from fragmentograms of n-alkanol trimethylsilyl ethers using the [M-15] + ion.The Imbé and Urubu samples display a bimodal distribution with maxima at C 16 and C 28 .This indicates both algal and /or bacterial (C 16 -C 18 ) and higher plant (>C 22 ) contributions, as illustrated in Figure 6(A-C) and Table 2. Thus, in Figure 6C indicates that Lake de Cima receives a large input of n-alkanols from algae/zooplankton but minor contributions of higher (C 24 -C 30 ) n-alkanols.A very similar trend was observed for the Ururaí samples.
The distribution, and the relative concentration, of nalkanols in the Imbé and Urubu samples seem to indicate a predominant higher plant, allochthonous source.Contrastingly, the presence of lower molecular weight compounds in appreciable concentrations in Lake de Cima and Ururaí sediments suggests the existence of an additional algal/bacterial source.

Alkanoic acid
The distribution of n-alkanoic acids in our four samples is given in Figure 6(D-G), which shows that hexadecanoic acid (nC 16 ; Figure 2) predominates.In sediments from the Imbé, Urubu and Lake de Cima, alkanoic acids range from C 12 to C 30 , with a bimodal distribution and a second maximum at C 24 /C 26 .Although long chain alkanoic acids (C 22 -C 30 ) derive primarily from higher plants, 24 short-chain acids are ubiquitous, being found in algae, 20,28 higher plants, 24,29 bacteria and fungi. 4ome unsaturated alkanoic acids, e.g., hexadecenoic acid (C 16:1 ), octadecenoic acid (C 18:1 ) and octadecadienoic acid (C 18:2 ) were also observed in the samples.The presence of unsaturated alkanoic acids, mainly the C 18:1 with some C 16:1 and C 20:1 , are indicators of recent biogenesis, and are   present in all recent sediment samples. 30C 18:1 was also reported in aerosol vegetation wax and river sediments in the Amazonian region. 31Iso-and anteiso-pentadecanoic, heptadecanoic, iso-tetradecanoic and hexadecanoic acids were also present in the four samples.The presence of isoand anteiso-acids, especially in surficial sediments, is characteristic of a bacterial input. 32Anteiso-and isoalkanoic acids (C 13 -C 17 ) are commonly found in microbial lipids, lending further support to a possible recent biological origin. 25,33lkenoic acids (nC 14:0 , nC 16:0 , nC 16:1 ) are abundant in phytoplankton, while nC 16:0 , nC 18:1 and nC 18:0 often predominate in zooplankton.Diatoms are rich in nC 14:0 , nC 16:0 and nC 16:1 alkenoic acids, and low in C 18 homologues.In contrast, nC 18:1 , nC 18:2 , nC 18:3 and nC 18:4 predominate in flagellates and freshwater algae.However, higher plants also contain the more specific saturated C 22 -C 32 series.C 14 -C 16 alkanoic acids are found in higher plants, green algae or bacteria.
Because alkenoic acids are known to be more susceptible to degradation than their saturated counterparts, their abundant presence in the sediments being analyzed, indicate the relative freshness of the collected material.
The distribution of alkanoic acids in these tropical sediments suggests a dominant phytoplankton, zooplankton and microbial input (short-chain and unsaturated acids; < C 20 ) with a minor contribution from higher plants (long-chain; >C 21 ).Branched alkanoic acids, such as the iso-and anteiso-C 15 compounds reflect bacterial contributions to the OM.

Organic matter
Sedimentary organic C/N ratios are useful in distinguishing between an algal and vascular land-plant origin of organic matter.The presence or absence of cellulose in the plants sourcing organic matter influences the C/N ratios in sediments.Nonvascular aquatic plants have low C/ N ratios, typically between 4 and 10, whereas vascular land plants, which contain cellulose, have C/N ratios of 20 or greater.Lakes for which the contribution of organic matter from vascular land plants is small, relative to that produced in the water-column, exemplified by Walker Lake (C/N=8) and Lake Michigan (C/N=9), show lower C/N ratios in their sediments than do lakes receiving substantial amounts of vascular plant debris, such as Lake Mangrove (C/N=13) and Lake Bosumtwi (C/N=14).Ratios of 13-14 for the surface sediments of lakes suggest a mixture of non-vascular and vascular contributions, a situation expected for most lakes. 26,34,35he organic matters in our samples appear to originate from both autochthonous and allochthonous sources.For example, the C/N ratios for sediments from Lake de Cima, and the Imbé and Urubu rivers (10, 15 and 17, respectively; see Table 2) are intermediate to those characteristic of landderived OM (20-100) and of aquatic algae and bacteria derived OM (5-8). 2,36For the Ururaí sample, the value (3; see Table 2) is closer to aquatic algae, bacteria and nonvascular aquatic plants.This low value might be due to plankton and/or microbial action on organic matter: microbial degradation generally decreases the C/N ratio (microbial immobilization of nitrogen and mineralization of carbon).Because the C content is low, the presence of ammonia in clay minerals could also cause the lower C/N ratio.

General observations
Hydrocarbon fractions from these sediments had already been analyzed by our group. 17,18In the sample from Lake de Cima, fluoranthene, pyrene and chrysene had been detected, and quantified, but solely at trace levels.No hopanes, steranes, nor an unresolved complex mixture (UCM) was observed in any samples.Moreover, most of the aromatic hydrocarbons were of biogenic origin; for example tetramethyloctahydropicene, 3,3,7-trimethyl-1,2,3,4-tetramethylhydrochrysene and cadalene.The same behavior has been verified now in this study of the polar organic compounds.
In addition to the local sources of organic matter, winds commonly transport material such as pollen and particulate matter from sources outside the local watershed.The eolian component is typically a small fraction of the total organic mixture, yet it can contain distinctive components potentially useful to paleolimnetic and paleoclimatic studies. 26In contrast to what was observed in surface sediments from the Amazon shelf, 37 the specific biomass burning biomarker, levoglucosan, though detected in the atmospheric particulate matter over Campos dos Goytacazes, 38 was not detected in the four samples discussed in this article.This fact precludes the contribution of significant inputs from airborne sources to these sediments, but a smaller anthropogenic contribution should not be excluded, as they are also common biomarkes transported by aerosols. 30,33,39,40n other words, although a great cloud of smoke, deriving from the burning of sugar cane hovers continually over the studied area, no parameter corroborating a significant input from anthropogenic origins could be identified or detected in theses samples.
Previous studies have suggested that the OM present in most tropical rivers derives from allochthonous OM.However, substantial contributions of autochthonous algal and bacterially-derived compounds to the total lipids is also evident in tropical river samples. 25In the present study, the same pattern observed by Jaffé and coworkers for the tropical rivers of the Orinoco Basin (Venezuela) was also observed.The rivers Imbé, Urubu and Lake de Cima received contributions from both allochthonous and autochthonous sources, but their samples show a marked predominance of an allochthonous input.Contrastingly, the Ururaí sediment displayed a significant contribution from autochthonous sources (greater abundance of C 27 sterol and a large input of C 16 n-alkanoic acid).

Figure 5 .
Figure 5. Mass fragmentogram (m/z 75) characteristic ion for nalkanols (as trimethylsilyl ethers) for the polar compounds fractions of the Urubu (numbers 14-32 refer to carbon chain length of homologous series).

Table 1 .
). * Phthalate contaminants.Compounds identified and quantified (ng g -1 ) in the samples nd = not detected; + = detected but not quantified; tr = identified in trace quantities; a two distinct isomers given as the methyl esters for the carboxylic acids and trimethylsilyl ether for alcohols.

Table 2 .
Sediment samples from the rivers Imbé, Urubu, Ururaí and Lake de Cima analytical results a CPI = Carbon Preference Index.b Carbon chain maxima in homologous series (dominant homolog is underlined)