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Origin and transport of n-alkane-2-ones in a subtropical estuary: potential biomarkers for seagrass-derived organic matter
Organic Geochemistry 32 (2001) 21±32
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Origin and transport of n-alkane-2-ones in a subtropical estuary: potential biomarkers for seagrass-derived organic matter Maria E. Hernandez, Ralph Mead, Maria C. Peralba, Rudolf Jae * Environmental Chemistry and Geochemistry Laboratory, Southeast Environmental Research Center and Department of Chemistry, Florida International University, Miami, Florida 33199, USA Received 6 June 2000; accepted 19 October 2000 (returned to author for revision 24 August 2000)
Abstract n-Alkane-2-ones are lipids commonly found in sediments and soils. This group of compounds, frequently reported in the literature, usually occurs in the form of a homologous series ranging from about C19 to C33 characterized by a strong odd over even carbon number predominance. In this paper we report a dierent molecular distribution, centered about the C25 homologue as the dominant ketone. The relative abundance of the C25 compared to the C27 homologue in a sediment transect increased from the upper to the lower end of a South Florida estuary, and was found to correlate with surface water salinity in extracts from suspended solids. Analyses of dierent varieties of seagrasses showed these to be the most likely source of the C25 n-alkane-2-ones, while the C27+ homologues were mainly derived from mangroves and freshwater marsh vegetation. Compound-speci®c stable isotope measurements and statistical analyses support this ®nding, suggesting that molecular distributions of n-alkane-2-ones can be used to identify seagrass-derived organic matter in coastal environments. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biomarkers; Ketones; Lipids; Seagrass; Zostera; Thalassia; Halodule; Syringodium
1. Introduction Organic matter from both autochthonous and allochthonous sources accumulates in estuarine systems, and may be derived from coastal wetland and/or salt marsh vegetation, fringe forests (such as mangroves), benthic vegetation (such as seagrasses), riverine transport of eroded soils, and freshwater and marine plankton. It is essential to determine the relative contribution of different sources of organic carbon to the biogeochemical cycles in estuarine and coastal environments to better understand their ecological importance. In order to trace the origin, transport and fate of organic matter from such diverse sources, isotopic and/or molecular
* Corresponding author. Tel.: +1-305-348.24.56; fax: +1305-348.40.96. E-mail address: jaer@servms.®u.edu (R. JaeÂ).
marker (biomarker) approaches have been applied (e.g. Meyers and Ishiwatari, 1993; Prahl et al., 1994; Chmura and Aharon, 1995; Jae et al., 1995, 1996a, 2000; Wakeham, 1995; Canuel et al., 1997; Bull et al., 1999; Mannino and Harvey, 1999). Although each method, or the combination of both, has been quite successful, there are limitations. For example, in some aquatic environments, commonly used biomarkers such as fatty acids, n-alkanes, sterols and fatty alcohols have both autochthonous and allochthonous origins (e.g. Jae et al., 1995, 2000). Only a few biomarkers are truly taxonspeci®c (Cranwell, 1982; Volkman et al., 1999), so that most studies employ multiple tracers (e.g. Canuel et al., 1997; Jae et al., 1995, 1996a, 2000). Seagrasses are submerged vascular plants that grow in extensive beds in many coastal and estuarine areas of the world (e.g. Fourqurean et al., 1999). They serve several important functions by providing habitat for a wide variety of plant and animal species, and by physically
0146-6380/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00157-1
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
stabilizing coastal areas, reducing erosion. As such, seagrasses have been found to be important contributors to the organic matter pool in coastal environments (Thayer et al., 1978; de Leeuw et al., 1995a; Canuel et al., 1997; Bianchi et al., 1999). In these studies, dierent approaches were followed to trace seagrass-derived organic matter, such as compound-speci®c stable isotope measurements of sterols, fatty acids and n-alkanes (Canuel et al., 1997), analyses of lignin phenols (Bianchi et al., 1999) and a,bdihydroxy fatty acid distributions (de Leeuw et al., 1995a,b). Similarly, Volkman et al. (1980) demonstrated that the seagrass Zostera muelleri was a major source of speci®c a-hydroxy, o-hydroxy and a,o-dicarboxylic acids in an intertidal sediment. Although the lipid composition of seagrasses has been studied in quite some detail (Volkman et al., 1980; Nichols et al., 1982; ; Nichols and Johns, 1985; de Leeuw et al., 1995a; Canuel et al., 1997), no unambiguous seagrass-speci®c biomarkers have been identi®ed so far. This paper presents data to show that molecular distributions of n-alkane-2-ones (also referred to as methyl ketones or n-alkan-2-ones) in sediments and suspended particulate matter have the potential to serve as an indicator of seagrass-derived organic matter. Homologous series of n-alkane-2-ones have been isolated from a wide variety of depositional environments including marine and lacustrine sediments, soils and peats (Cranwell, 1981; Volkman et al., 1983; AlbaigeÂs et al., 1984; Cranwell et al., 1987; Jae et al., 1993, 1996b). The molecular distributions found show a high predominance of odd numbered carbon chain-lengths maximizing at C27 or C29. Their close resemblance to the terrigenous n-alkane distributions has led several authors to propose microbial oxidation of n-alkanes as the source of n-alkane-2-ones with b-oxidation of fatty acids followed by decarboxylation as an alternate pathway (Allen et al., 1971; AlbaigeÂs et al., 1984; Cranwell et al., 1987; Lehtonen and Ketola, 1990; Jae et al, 1993). Although some studies have suggested a correspondence of the n-alkane-2-ones distribution with that of the nalkanes or the fatty acids, it is not always close enough to substantiate precursor-product relationships between these classes of compounds (Volkman et al., 1980, 1983). Jae et al. (1993) suggested that dierent diagenetic processes such as binding to sediments and biodegradation could be the cause for such lack of correlation. It has also been suggested that when the n-alkanes derive from two dierent sources, i.e. when an algal signal is superimposed on a higher plant distribution, microbial oxidation of the latter prior to incorporation in the sediment could generate the type of n-alkane-2-one distribution usually observed (Volkman et al., 1980). More recently, n-alkane-2-ones have been reported in higher plant and phytoplankton biomass (Rieley et al., 1991; Qu et al., 1999) suggesting direct biological inputs to sediments. However, independent of their origin, n-alkane-2-ones
have been found to be ubiquitous in aquatic environments, and similar molecular distributions have been reported for sediments characterized by higher plant or microbial organic matter inputs. This study presents the ®rst report on a C25 n-alkane-2-one dominated molecular distribution in sediments from a South Florida estuary and discusses possible sources. 2. Experimental methods A transect of 10 sediment samples (Fig. 1) was collected starting at the freshwater peats of the Shark River Slough (Everglades National Park), through the Harney River estuary into the Florida Shelf. Samples were collected from a boat using an Eckman Dredge (Wildco, Michigan) for the Harney River samples and from the R-V Bellows with a box corer for the Florida Shelf samples. Mangrove leaves (Rhizophora mangle), sawgrass (Caladium sp.), periphyton, and four seagrasses (Thalassia testudinum, Halodule wrightii, Syringodium ®liforme, and Zostera marina) were also analyzed following a similar procedure to that described for the sediments. Seagrass samples were collected by divers, both in Florida Bay, Florida (T. testudinum, H. wrightii, S. ®liforme) and in the San Francisco Bay area, California (Z. marina), placed in zip-lock bags and kept frozen until analysis. Seagrass samples were rinsed with distilled water and major epiphytic growth was physically removed prior to freezing. Samples of T. testudinum and of H. wrightii with signi®cant epiphytic cover were also analyzed without the cleaning step. Mangrove leaves, sawgrass and periphyton samples were collected by hand from the Shark River Slough area, and treated similarly to the seagrasses. Surface sediment samples were transferred immediately to clean glass jars with Te¯on lined caps, placed on ice and stored frozen at ÿ8 C until analysis. Bulk sediment characteristics are shown in Table 1. Surface water samples were collected at sites 2, 3 and 4 (see Fig. 1) on a monthly basis from December 1997 to August 1998, placed in 50 l polyethylene bottles using a portable pump, and ®ltered through GF/C glass ®ber ®lters within a 24 h period after collection. The ®ltered particulate matter was kept frozen until analysis. Water quality parameters were determined using standard analytical procedures as described elsewhere (Boyer et al., 1999). The ®lters and sediment samples were freezedried and Soxhlet extracted for 24 h with high purity methylene chloride (Optima, Fisher, USA). Extracts were then saponi®ed and separated into neutral and acid fractions. The neutral fraction was further separated into 8 fractions by adsorption chromatography over silica gel as previously described (Jae et al., 1995). All fractions were analyzed by GC/MS (Hewlett Packard 5973 model) using a DB5-MS capillary column (25 m, 0.25 mm i.d., 0.25 mm from J&W, Flossom, California).
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
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Fig. 1. Location of sediment sampling sites in Everglades National Park and the Florida Shelf.
Table 1 Bulk sediment characteristics Site
1
2
3
4
5
6
7
8
9
%C C/N d13Ca kb
41.4 21.4 ÿ28.9 4.7
31.3 16.9 ÿ27.6 3.6
18.7 16.0 ÿ26.7 1.1
12.5 13.9 ÿ25.7 0.97
2.3 9.4 ÿ22.5 0.30
6.9 7.0 ÿ20.0 0.31
16.0 7.1 ÿ19.4 0.11
11.5 7.9 ÿ19.6 0.01
10.8 7.5 ÿ19.5 0.02
a b
d13C reported as %. k=total n-alkane-2-ones in mg/g.
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Quantitation was based on an internal standard (perdeuterated phenanthrene). n-Alkane-2-ones (fraction 5) were identi®ed based on chromatographic retention and mass spectra characteristics (m/z=59). Note that although the m/z=59 ion is present in the mass spectra of all the n-alkane-2-ones, its abundance relative to the m/z 58 ion varies with chain-length. Therefore, the data presented here are semiquantitative, and the relative abundance of the lower molecular weight homologues may be somewhat underestimated. Detailed biomarker data from these samples have been reported elsewhere (Jae et al., 2000). Stable isotope analyses (d13C) were performed on a Finnigan Delta Plus (for bulk sediments) and on a
Hewlett Packard 5890 gas chromatograph coupled to a Finnigan Delta C (for compound speci®c d13C analysis; irm-GC/MS). The irm-GC/MS analyses were performed based on the technique described by Hayes et al. (1990). A standard mixture of aromatic hydrocarbons and alkanes was injected to test the reproducibility and analytical errors of the instrument. Typically 1 ml of the standard was injected for each run. The reproducibility and the accuracy of the measurements were satisfactory (within 0.1 and 0.3% respectively). Pulses of standardized CO2 were introduced into the ion source during each run. Although this method of external isotopic calibration fails to compensate for the physical conditions to which analytes were being subjected during
Fig. 2. Molecular distributions of n-alkane-2-ones (m/z=59 ion chromatograms) in sediment and peat samples throughout the Harney River transect (sites 1±5 and P1) and sediment samples of the Florida Shelf (sites 6±9).
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
their passage though the gas chromatograph and the combustion interface, it eliminated possible interferences between the analytes and co-injected standards. Moreover, since the square pro®le delivers, on average, more CO2 per unit of peak width than a gaussian peak with equal height and width (Merritt et al., 1994), the precision of related isotopic analysis was improved. 3. Results and discussion 3.1. Molecular distribution of n-alkane-2-ones in sediments and plant biomass All of the ketone fractions contained a series of nalkane-2-ones, ranging from C21 to C33 with a strong predominance of odd chain-lengths. The molecular distribution showed a gradual change from the freshwater to the marine end-members of the Harney River estuary, by shifting from a C27, C29 and C31, C33 dominated pro®le for peat and mangrove organic matter in¯uenced sediments (sites 1 and 2 respectively) to a C25 dominated signal for the marine in¯uenced sediments (sites 4 and 5; Fig. 2).
25
Florida Shelf samples 6 and 7 were also characterized by such a C25 predominance, while samples 8 and 9 exhibited the typical higher plant distribution (Fig. 2). Although n-alkane-2-ones have been detected in a wide variety of depositional environments, their distribution has been found to be remarkably similar, typically showing a maximum at C27 or C29 and in some cases at C25 (e.g. Rieley et al., 1991; Ying and Fan, 1993; Qu et al., 1999). Although dierent Cmax values have been reported for n-alkane-2-one distributions in sediments and plant biomass, their molecular distribution is usually characterized by a series of higher molecular weight odd carbon number homologues, and is not strongly dominated by any particular homologue. The strong predominance of the C25 homologue observed here for the marine-in¯uenced samples is, to the best of our knowledge, the ®rst such report in the literature suggesting a dierent origin for this compound. While the analyses of mangrove, sawgrass and periphyton samples resulted in the typical n-alkane-2-one distribution centered around C27±C31, the three local seagrass samples (seagrass blades) showed the C25 ketone being by far the most dominant homologue (Fig. 3). The
Fig. 3. Ion chromatograms (m/z=59 ion chromatograms) of the n-alkane-2-ones in three seagrasses (Thalassia testudinum, Halodule wrightii and Syringodium ®liforme). Presence of signi®cant epiphytic surface cover of seagrass blades is indicated.
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Z. marina sample, however, showed no preference for this particular homologue (data not shown). Root samples from T. testudinum were also analyzed and showed similar C25-dominated n-alkane-2-one distributions. These results are interesting in several ways. Although n-alkane-2-ones are often believed to arise from the microbially mediated b-oxidation of the alkanes (see above) the ®nding of signi®cant amounts of n-alkane-2ones present in plant biomass seems to indicate that a direct biological origin for these compounds is signi®cant in this environment. In agreement with this observation, a lack of correspondence between the nalkane and n-alkane-2-one distributions was observed (e.g. Fig. 4) in these plants, as previously reported by Ying and Fan (1993). The unusually high abundance of even-carbon n-alkenes in the aliphatic hydrocarbon fraction of the mangrove leaf extract (Fig. 4), has been discussed in more detail elsewhere (Jae et al., 2000). A similar distribution has been reported for a coastal macrophyte (Juncus roemericanus) by Canuel et al. (1997). Total n-alkane-2-one concentrations found in the seagrass samples were in the range of 2±21 mg/g. For all seagrass samples which were relatively free of epiphytes,
the C25 n-alkane-2-one represented between 82 and 88% of the ketone fraction. For samples with signi®cant epiphytic cover, this relative abundance was reduced by about 50%. In contrast, the C25 homologue in periphyton, sawgrass and mangrove samples was only 8±9% of total ketones. This predominance of the C25 homologue (and to some extent that of the C23 homologue) in the seagrasses, compared to the predominance of higher molecular weight homologues (C27±C33) for terrestrial higher plants, clearly suggests the applicability of the relative abundance of the C25 homologue as a potential indicator of seagrass-derived organic matter in coastal and estuarine sediments. For example, the C25/C27 ratio could be applied for such a purpose. Note that seagrass with abundant epiphytes showed a signi®cantly reduced C25/C27 ratio compared to `clean' seagrass, indicative of a C27+ n-alkane-2-one contribution from the epiphytic organisms. Cyanobacteria have recently been reported as a source for n-alkane-2-ones (Qu et al., 1999), suggesting that epiphytic prokaryotes and perhaps microalgae could also be responsible for a reduced C25/C27 ratio. Surprisingly, the C25 dominated molecular distribution of the n-alkane-2-ones was not observed for the Z. marina samples. Both of the eelgrass samples analyzed showed
Fig. 4. Ion chromatograms of the n-alkane-2-ones (m/z=59) and n-alkanes/alkenes (m/z=57) of a Thalassia testudinum and a Rhizophora mangle sample.
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
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a typical higher plant molecular distribution for the nalkane-2-ones, maximizing at the C27 homologue. Although the reasons for the dierent molecular composition between the local tropical/sub-tropical seagrasses and the eelgrass (abundant in temperate climates) is dicult to assess, phytogenetic studies of seagrasses have shown that Z. marina, T. testudinum and the pair S. ®liformis and H. wrightii fall into three distinct groups (Les et al., 1997). The lack of an enhanced C25 homologue for Z. marina suggests that the applicability of this feature as a seagrass-derived organic matter indicator may be limited to tropical and sub-tropical environments where Thalassia, Syringodium and Halodule are abundant.
observed between the ketone ratio and salinity (Fig. 6), no correlation was found with the chlorophyll concentration or turbidity (r2 <0.20). The positive salinitydependence of the particle-associated C25/C27 n-alkane2-one ratio clearly suggests a signi®cant marine origin for the C25 homologue, and that tidal in¯uence has a strong eect on the distribution of marine-derived organic matter in the Harney River estuary (see also Jae et al., 2000). The lack of correlation with chlorophyll concentration, a measure of phytoplankton abundance in water, indicates that marine phytoplankton is not the main source of the C25 n-alkane-2-ones in the suspended particulate matter, leaving seagrasses as the most likely source.
3.2. Hydrodynamics
3.3. Corroboration of seagrass origin for the C25 n-alkane-2-one
Since hydrodynamic sorting of sediments during deposition can control their chemical characteristics, the molecular distribution of the n-alkane-2-ones of a sizefractionated sediment sample from a Harney River (site 4, Fig. 1) was examined. For this purpose, the sample was sequentially passed through 250, 150 and 75 mm brass sieves. Approximately 36% of the sediment was composed of material <75 mm, 44% was in the range 75±150 mm, and 17% in the range 150±250 mm. The remaining 3% comprised a coarse fraction (>250 mm) mainly made up of shells and coarse-grained minerals. The amount of extracted organic material obtained from it was insigni®cant and thus, its molecular distribution is not discussed here. Dierent grain-size fractions had markedly similar molecular compositions indicating that similar processes aected the organic material. For example, the odd/even preference (carbon preference index of ca. 5.2) of the n-alkanes and the maximum at C29, was approximately the same from the coarser to the ®ner fractions. However, based on the relative abundance of the C25 ketone homologue, the nalkane-2-one fraction showed a mixed seagrass and higher plant signal in the coarse fractions and a strong seagrass signal in the ®ner (<75 mm) sediment fraction (Fig. 5). Although the bulk amount of the seagrassderived organic matter in this sample seems to be present as coarse detrital material (>75 mm), only the C25 homologue was detected in the ®ne sediments, an indication that resuspended ®ne sediments and associated seagrass-derived organic matter can be transported over long distances in coastal environments and into estuaries during tidal exchange. To further assess the transport of seagrass-derived organic matter in this estuary, suspended solids were sampled monthly at three stations during a 9 month period. The C25/C27 n-alkane-2-one ratio in these samples was correlated with water quality parameters such as salinity, chlorophyll concentration and turbidity. While a reasonably good linear correlation (r2=0.77) was
In order to further con®rm the origin and applicability of the C25 n-alkane-2-one relative abundance as an indicator of seagrass-derived organic matter, correspondence factorial analysis (CFA) of the data was implemented using a commercially available statistical package (Statistica). CFA, which has successfully been applied to biomarker interpretations in estuarine systems (Sicre et al., 1988, 1993), is an exploratory multivariate technique that converts a matrix of nonnegative data into a particular type of graphical display in which the rows and columns of the matrix are depicted as points. It is a generalization of a simple concept: the scatterplot (Fig. 7). The ®rst two factors calculated from the data matrix accounted for 87% of the total inertia. The values of the absolute contributions (AC) and relative contributions (RC) were used to interpret the axis. AC values show that the C31 and the C25 n-alkane-2-ones are the main contributors to the construction of the ®rst factor accounting for 44 and 47% of its inertia respectively. From this observation, we can assume that the ®rst axis discriminates between seagrass and higher plant sources. AC values for the second axis indicate that this axis distinguishes between the latter group and another one dominated by the C27 n-alkane-2-one presumably due to the in¯uence of sawgrass and periphyton inputs. For comparison, data obtained from the analysis of mangroves leaves, sawgrass and seagrass blades and periphyton were plotted in a supplementary way (i.e. not included in the statistical analysis). The relative proximity of the two seagrass samples to sediment from station 7 supports the idea that the station is mainly in¯uenced by seagrass inputs. The lower right quadrant of the graph consists of stations with marine in¯uence and some degree of seagrass input. The upper part of the quadrant shows those stations with little seagrass in¯uence, while the lower part of the quadrant hosts stations 5 and 7, which have a very strong seagrass signal.
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Fig. 5. Molecular distribution of n-alkane-2-ones (m/z=59 ion chromatograms) in a grain size-fractionated sediment sample (site 4). Size fractions correspond to 250±150, 150±75 and <75 mm respectively. Overall n-alkane-2-one grain-size based distribution is indicated at the bottom of the ®gure.
Station 1, located in the lower left quadrant, consists almost exclusively of mangrove detritus as indicated by the predominance of the C31 n-alkane-2-one. The peat sample and the sediments at station 2 plotted very close to each other in the upper left quadrant halfway
between sawgrass and periphyton. Station 8 appears to have an n-alkane-2-one distribution very similar to that of higher plants. The scatter of the shelf samples re¯ects the patchiness of the organic matter in this area, thus agreeing with the irregular distribution of drift particulate
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
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Fig. 6. Correlation of the n-alkane-2-one C25/C27 ratio in surface water suspended particles collected from sites 2, 3 and 4 vs. surface water salinity.
material throughout the Florida Shelf (Zieman et al. 1989). In agreement with the CFA results, stations 8 and 9 fall into areas characterized by low seagrass cover, while sites 6 and 7 are in the vicinity of extensive, highdensity seagrass beds (Fourqurean et al., 1999). Stable isotope (d13C) data of the bulk organic matter in the sediments supports the seagrass in¯uence towards the marine end-member of the transect. The bulk d13C values throughout the Harney River transect ranged from ÿ28.9% at station 1, to about ÿ19% for the Florida Shelf samples (Table 1). Chmura and Aharon (1995) showed that such trends of 13C isotopic enrichment paralleling the salinity gradient between the upper and lower estuaries are quite typical and are mainly controlled by the gradual transition from freshwater marsh-dominated C-3 plants (ÿ23.5 to ÿ27.8%) to C-4 plant community dominated salt marshes (ÿ11.6 to ÿ15.5%). However, the vegetation of the lower estuaries of South Florida is usually dominated by mangroves (C3 plants), and the isotopic pattern presented here is most likely caused by the in¯uence of a combination of marine plankton and seagrass-derived organic matter in the sediments. While marine plankton and suspended particulates from temperate regions have d13C values ranging from about ÿ24 to ÿ16% (Michener and Schell, 1994; Fry, 1996; Johnston and Kennedy, 1998), seagrasses
(although being C-3 plants) re¯ect an isotopic signature similar to that of C-4 plants (Canuel et al., 1997). Therefore, the gradual enrichment in 13C between the upper and lower estuary suggests a transition from higher plant-derived organic matter to that increasingly in¯uenced by marine-derived organic matter, including seagrasses (see also Jae et al., 2000). The d13C values for individual n-alkane-2-one homologues were in agreement with the bulk sediment d13C data. Note that lipids usually are about 5±8% lighter than bulk carbon measurements. Carbon isotopic compositions of individual n-alkane-2-ones isolated from sediments and plants are summarized in Table 2. At station 2, the three measured n-alkane-2-ones (C23, C25 and C27) have similar isotopic compositions in the range of ÿ31 to ÿ30%. However, starting at station 3, the isotopic composition of the C25 ketone becomes enriched in 13C, reaching a value of ÿ17.2% at station 4. From the previous analysis of the molecular distributions of the samples, it was suggested (see above) that stations 3 and 4 have a greater seagrass in¯uence as evidenced by high relative C25 n-alkane-2-one abundances. Seagrasses are aquatic C-3 plants that fractionate carbon similarly to C-4 plants, resulting in a heavier isotopic signature (ÿ10 to ÿ15%; Thayer et al. 1978; Canuel et al., 1997). Thus, an increase in the input of seagrass-derived
30
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Fig. 7. Correspondence factorial analysis (CFA) plot of the n-alkane-2-one molecular abundance in sediments. AC (Absolute Contribution) and RC (Relative Contribution) values expressed as percent for factors I and II, are shown on the margin, and provide the contribution of a parameter to the variance of a factor and the extent to which a factor explains the variance of a parameter, respectively. Table 2 Compound-speci®c d13C values for n-alkane-2-ones in plant biomass and sediment samples n-Alkane-2-one
C23
C25
C27
Site 2 (sediment) Site 3 (sediment) Site 4 (sediment) Periphyton Thalassia testudinum Halodule wrightii Rhizophora mangle
ÿ31.30.1 ÿ29.80.1 ÿ27.60.3 na na na ÿ32.30.3
ÿ30.20.1 ÿ24.80.2 ÿ17.20.1 ÿ32.80.2 ÿ15.00.2 ÿ14.70.1 ÿ32.60.1
ÿ30.80.2 ÿ30.00.1 ÿ29.60.2 ÿ33.40.3 ÿ14.90.1 na ÿ32.70.2
organic matter (and therefore seagrass derived n-alkane2-ones) should shift the isotopic signature of the C25 ketone to a heavier signal as is seen in station 4 (ÿ17.2%). The isotopic enrichment of the C27 homologues was signi®cantly less pronounced at station 3 and 4 due to the low relative contribution of the C27+ homologues from seagrasses, resulting in less dilution of the original terrestrial signal. When the carbon isotopic compositions of T. testudinum, H. wrightii, R. mangle and a periphyton sample were measured, it was found that the C25 n-alkane-2-ones isolated from the mangroves and the periphyton were ca. ÿ32%, while that for the seagrasses was ca. ÿ15%. Similar carbon isotopic values have been reported for other seagrass-derived lipids
(Canuel et al., 1997; Hammer et al., 1998). These ®ndings seem to con®rm that seagrasses are an important source of the C25-dominated n-alkane-2-one distributions. 4. Conclusions n-Alkane-2-ones have been found to be present in mangroves, sawgrass, periphyton mats and seagrasses from South Florida aquatic and terrestrial environments, suggesting that the presence of these lipids in sediments is not exclusively derived from the oxidation of other linear lipids such as alkanes, alkenes and fatty acids. In addition, the molecular distribution of the predominantly odd carbon numbered homologous series of n-alkane-2-ones for the dierent biomass components is not identical. Although both terrestrial higher plants and planktonic organisms (periphyton) showed similar molecular distributions, seagrasses had a distinctively dierent distribution, with a homologous series dominated by the C25 homologue. Such molecular distributions were con®rmed to be derived from seagrasses though statistical analyses and compound-speci®c stable isotope analyses. The application of the C25/C27 ratio as a means to distinguish marine, seagrass-derived organic matter from terrestrial/planktonic organic matter was tested through the analysis of suspended matter and correlation with water quality parameters. This study
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
shows the application of the relative abundance of the C25 n-alkane-2-one as a seagrass indicator in estuarine and coastal environments. The potential use of this molecular tool in paleoenvironmental studies, particularly in tropical and sub-tropical environments, where species such as Thalassia testudinum, Halodule wrightii and Syringodium ®liformis are abundant, is suggested. Acknowledgements The authors thank the National Science Foundation (NSF) for partial support for this project through grant No. 9450394, Drs. R. Jones and J. Boyer for providing logistical support and water quality data, Dr. J. Fourqurean for supplying the seagrass samples and for helpful discussions, Dr. M. A. Sicre for helpful comments on the use of the CFA, and the crew of the R-V Bellows for sampling assistance. Special thanks go to Dr. J. Volkman whose helpful comments signi®cantly improved this manuscript. This research was made possible thanks to instrumentation grants from NSF (No. 9512385) and the FIU Division of Sponsored Research, for the irmGC/MS and the GC/MS systems respectively. M.H. and R.M. thank NSF for a student fellowship (No. 9450394) and FIU for a teaching assistantship, respectively. SERC Contribution No. 136. Associate EditorÐJ. Volkman References AlbaigeÂs, J., Algaba, J., Grimalt, J., 1984. Extractable and bound neutral lipids in some lacustrine sediments. Organic Geochemistry 6, 502±507. Allen, J.E., Forney, F.W., Markovetz, A.J., 1971. Microbial subterminal oxidation of alkanes and alk-1-enes. Lipids 6, 448±452. Bianchi, T.S., Argyrou, M., Chippett, H.F., 1999. Contribution of vascular-plant carbon to surface sediments across the coastal margin of Cyprus (eastern Mediterranean). Organic Geochemistry 30, 287±297. Boyer, J.N., Fourqurean, J.W., Jones, R.D., 1999. Seasonal and long term trends in the water quality of Florida Bay (1989±1997). Estuaries 22, 417±430. Bull, I.D., van Bergen, P.F., Bol, R., Brown, S., Gledhill, A.R., Gray, A.J., Harkness, D.D., Woodbury, S.E., Evershed, R.P., 1999. Estimating the contribution of Spartina anglica biomass to salt-marsh sediments using stable isotope measurements. Organic Geochemistry 30, 477±484. Canuel, E.A., Freeman, K.H., Wakeham, S.G., 1997. Isotopic composition of lipid biomarker compounds in estuarine plants and surface sediments. Limnology and Oceanography 42, 1570±1583. Chmura, G.L., Aharon, P., 1995. Stable carbon isotope signatures of sedimentary carbon in coastal wetlands as indicators of salinity regime. Journal of Coastal Research 11, 124±135.
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Cranwell, P.A., 1981. Diagenesis of free and bound lipids in terrestrial detritus deposited in a lacustrine sediment. Organic Geochemistry 3, 79±89. Cranwell, P.A., 1982. Lipids of aquatic sediments and sedimenting particulate matter. Progress in Lipid Research 21, 271±308. Cranwell, P.A., Eglinton, G., Robinson, N., 1987. Lipids of aquatic organisms as potential contributors to lacustrine sediments-II. Organic Geochemistry 6, 513±527. Fourqurean, J.W., Durako, M.J., Hall, M.O., Hefty, L.N., 1999. Seagrass distribution in south Florida: A multi-agency coordinated monitoring program. In: Porter, J.W., Porter, K.G. (Eds.), Linkages Between Ecosystems in the South Florida Hydroscape: The River of Grass Continues. CRC Press, Boca Raton, Florida, pp. 48. Fry, B., 1996. 13C/12C fractionation by marine diatoms. Marine Ecology Progress Series 134, 283±294. Hammer, B.T., Fogel, M.L., Hoering, T.C., 1998. Stable carbon isotope ratios of fatty acids in seagrass and redheaded ducks. Chemical Geology 152, 29±41. Hayes, J.M., Freeman, K.H., Popp, B.N., Hoham, C.H., 1990. Compound-speci®c isotopic analysis: a novel tool for reconstruction of ancient biogeochemical processes. Organic Geochemistry 16, 1115±1128. JaeÂ, R., Cabrera, A., Hajje, N., Carvajal-Chitty, H., 1996a. Organic biogeochemistry of a hypereutrophic tropical, freshwater lake Ð Part 1: particle associated and dissolved lipids. Organic Geochemistry 25, 227±240. JaeÂ, R., Elisme, T., Cabrera, A.C., 1996b. Organic geochemistry of seasonally ¯ooded rain forest soils: molecular composition and early diagenesis of lipid components. Organic Geochemistry 25, 9±17. JaeÂ, R., Cabrera, A., Hausmann, K., Carvajal-Chitty, H., 1993. On the origin and fate of n-alkane-2-ones in freshwater environments. In: Manning, D. (Ed.), Organic Geochemistry: Applications in Energy and the Natural Environment. Manchester University Press, Manchester, UK, pp. 356±359. JaeÂ, R., Mead, R.N., Hernandez, M.E., Peralba, M.C., DiGuida, O.A., 2000. Origin and transport of sedimentary organic matter in two subtropical estuaries: a comparative, biomarker-based study. Organic Geochemistry, in press. JaeÂ, R., Wol, G.A., Cabrera, A.C., Carvajal-Chitty, H., 1995. The biogeochemistry of lipids in rivers from the Orinoco Basin. Geochimica et Cosmochimica Acta 59, 4507±4522. Johnston, A.M., Kennedy, H., 1998. Carbon stable isotope fractionation in marine systems: Open ocean studies and laboratory studies. In: Griths, H. (Ed.), Stable Isotopes: Integration of Biological, Ecological and Geochemical Processes. BIOS Scienti®c, Oxford, pp. 239±256. de Leeuw, J.W., Frewin, N.L., van Bergen, P.F., Sinninghe DamsteÂ, J.S., Collinson, M.E., 1995b. Organic carbon as a palaeoenvironmental indicator in the marine realm. In: Boscence, D.W.J., Allison, P.A. (Eds), Marine Palaeoenvironmental Analysis from Fossils. Geological Society Special Publication No. 83, pp. 43±71. de Leeuw, J.W., Rijpstra, W.I.C., Nienhuis, P.H., 1995a. Free and bound fatty acids and hydroxy fatty acids in the living and decomposing eelgrass Zostera marina L. Organic Geochemistry 23, 721±728. Lehtonen, K., Ketola, M., 1990. Occurence of long-chain acyclic methyl ketones in Sphagnum and Carex peats of various degrees of humi®cation. Organic Geochemistry 15, 275±280.
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Les, D.H., Cleland, M.A., Waycott, M., 1997. Phylogenetic studies in Alismatidae, II: evolution of marine angiosperms (seagrasses) and hydrophily. Systematic Botany 22, 443±463. Mannino, A., Harvey, H.R., 1999. Lipid composition in particulate and dissolved organic matter in the Delaware Estuary: Sources and diagenetic patterns. Geochimica et Cosmochim. Acta 63, 2219±2235. Merritt, D.A., Brand, W.A., Hayes, J.M., 1994. Isotope-ratiomonitoring gas chromatography±mass spectrometry: methods for isotopic calibration. Organic Geochemistry 21, 573± 583. Meyers, P.A., Ishiwatari, R., 1993. Lacustrine organic geochemistry: an overview of indicators of organic matter sources and diagenesis in lake sediments. Organic Geochemistry 20, 867±900. Michener, R.H., Schell, D.M., 1994. Stable isotope ratios as tracers in marine aquatic food webs. In: Lajtha, K., Michener, R. (Eds.), Stable Isotopes in Ecology and Environmental Science. Blackwell, Oxford, pp. 138±157. Nichols, P.D., Johns, R.B., 1985. Lipids of the tropical seagrass Thalassia hemprichii. Phytochemistry 24, 81±84. Nichols, P.D., Klumpp, D.W., Johns, R.B., 1982. Lipid components of the seagrasses Posidonia australis and Heterozostera tasmanica as indicators of carbon source. Phytochemistry 21, 1613±1621. Prahl, F.G., Ertel, J.R., GonÄi, M.A., Sparrow, M.A., Eversmeyer, B., 1994. Terrestrial organic carbon contributions to sediments on the Washington margin. Geochimica et Cosmochimica Acta 58, 3035±3048. Qu, W., Dickman, M., Sumin, W., Ruijin, W., Pingzhong, Z., Jianfa, C., 1999. Evidence for an aquatic origin of ketones found in Taihu Lake sediments. Hydrobiologia 397, 149± 154. Rieley, G., Collier, R.J., Jones, D.M., Eglinton, G., 1991. The biogeochemistry of Ellesmere Lake, U.K.-I: source correla-
tion of leaf wax inputs to the sedimentary lipid record. Organic Geochemistry 17, 901±912. Sicre, M.A., Paillasseur, J.L., Marty, J.C., Saliot, A., 1988. Characterization of seawater samples using chemometric methods applied to biomarker fatty acids. Organic Geochemistry 12, 281±288. Sicre, M.A., Tian, R.C., Broyelle, I., Saliot, A., 1993. Aquatic distribution of 4-desmethyl sterols in the Chang Jiang Estuary, China. Marine Chemistry 42, 11±24. Thayer, B.P., Parker, P.L., LaCroix, M.W., Fry, B., 1978. The stable carbon isotope ratio of some components of an eelgrass, Zostera marina. Oecologia 35, 1±12. Volkman, J.K., Barrett, S.M., Blackburn, S.I., 1999. Eustigmatophyte microalgae are potential sources of C29 sterols, C22-C28 n-alcohols and C28-C32 n-alkyl diols in freshwater environments. Organic Geochemistry 30, 307±318. Volkman, J.K., Farrington, J.W., Gagosian, R., Wakeham, S.G., 1983. Lipid composition of coastal sediments from the Peru up welling region. In: Bjorùy, M. et al. (Eds.), Advances in Organic Geochemistry 1981. John Wiley & Sons, New York, pp. 228±240. Volkman, J.K., Johns, R.B., Gillan, F.T., Perry, G.J., Bavor Jr., H.J., 1980. Microbial lipids of an intertidal sediment Ð I: fatty acids and hydrocarbons. Geochimica et Cosmochimica Acta 44, 1133±1143. Wakeham, S.G., 1995. Lipid biomarkers for heterotrophic alteration of suspended particulate organic matter in oxygenated and anoxic water columns of the ocean. Deep Sea Research 42, 1749±1771. Ying, G.G., Fan, P., 1993. Origin of ketones in sediments of Qinghai Lake. Science in China Series B 36, 237±241. Zieman, J.C., Fourqurean, J.W., Iverson, R.L., 1989. Distribution, abundance and productivity of seagrasses and macroalgae in Florida Bay. Bulletin of Marine Science 44, 292±311.
www.elsevier.nl/locate/orggeochem
Origin and transport of n-alkane-2-ones in a subtropical estuary: potential biomarkers for seagrass-derived organic matter Maria E. Hernandez, Ralph Mead, Maria C. Peralba, Rudolf Jae * Environmental Chemistry and Geochemistry Laboratory, Southeast Environmental Research Center and Department of Chemistry, Florida International University, Miami, Florida 33199, USA Received 6 June 2000; accepted 19 October 2000 (returned to author for revision 24 August 2000)
Abstract n-Alkane-2-ones are lipids commonly found in sediments and soils. This group of compounds, frequently reported in the literature, usually occurs in the form of a homologous series ranging from about C19 to C33 characterized by a strong odd over even carbon number predominance. In this paper we report a dierent molecular distribution, centered about the C25 homologue as the dominant ketone. The relative abundance of the C25 compared to the C27 homologue in a sediment transect increased from the upper to the lower end of a South Florida estuary, and was found to correlate with surface water salinity in extracts from suspended solids. Analyses of dierent varieties of seagrasses showed these to be the most likely source of the C25 n-alkane-2-ones, while the C27+ homologues were mainly derived from mangroves and freshwater marsh vegetation. Compound-speci®c stable isotope measurements and statistical analyses support this ®nding, suggesting that molecular distributions of n-alkane-2-ones can be used to identify seagrass-derived organic matter in coastal environments. # 2001 Elsevier Science Ltd. All rights reserved. Keywords: Biomarkers; Ketones; Lipids; Seagrass; Zostera; Thalassia; Halodule; Syringodium
1. Introduction Organic matter from both autochthonous and allochthonous sources accumulates in estuarine systems, and may be derived from coastal wetland and/or salt marsh vegetation, fringe forests (such as mangroves), benthic vegetation (such as seagrasses), riverine transport of eroded soils, and freshwater and marine plankton. It is essential to determine the relative contribution of different sources of organic carbon to the biogeochemical cycles in estuarine and coastal environments to better understand their ecological importance. In order to trace the origin, transport and fate of organic matter from such diverse sources, isotopic and/or molecular
* Corresponding author. Tel.: +1-305-348.24.56; fax: +1305-348.40.96. E-mail address: jaer@servms.®u.edu (R. JaeÂ).
marker (biomarker) approaches have been applied (e.g. Meyers and Ishiwatari, 1993; Prahl et al., 1994; Chmura and Aharon, 1995; Jae et al., 1995, 1996a, 2000; Wakeham, 1995; Canuel et al., 1997; Bull et al., 1999; Mannino and Harvey, 1999). Although each method, or the combination of both, has been quite successful, there are limitations. For example, in some aquatic environments, commonly used biomarkers such as fatty acids, n-alkanes, sterols and fatty alcohols have both autochthonous and allochthonous origins (e.g. Jae et al., 1995, 2000). Only a few biomarkers are truly taxonspeci®c (Cranwell, 1982; Volkman et al., 1999), so that most studies employ multiple tracers (e.g. Canuel et al., 1997; Jae et al., 1995, 1996a, 2000). Seagrasses are submerged vascular plants that grow in extensive beds in many coastal and estuarine areas of the world (e.g. Fourqurean et al., 1999). They serve several important functions by providing habitat for a wide variety of plant and animal species, and by physically
0146-6380/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved. PII: S0146-6380(00)00157-1
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
stabilizing coastal areas, reducing erosion. As such, seagrasses have been found to be important contributors to the organic matter pool in coastal environments (Thayer et al., 1978; de Leeuw et al., 1995a; Canuel et al., 1997; Bianchi et al., 1999). In these studies, dierent approaches were followed to trace seagrass-derived organic matter, such as compound-speci®c stable isotope measurements of sterols, fatty acids and n-alkanes (Canuel et al., 1997), analyses of lignin phenols (Bianchi et al., 1999) and a,bdihydroxy fatty acid distributions (de Leeuw et al., 1995a,b). Similarly, Volkman et al. (1980) demonstrated that the seagrass Zostera muelleri was a major source of speci®c a-hydroxy, o-hydroxy and a,o-dicarboxylic acids in an intertidal sediment. Although the lipid composition of seagrasses has been studied in quite some detail (Volkman et al., 1980; Nichols et al., 1982; ; Nichols and Johns, 1985; de Leeuw et al., 1995a; Canuel et al., 1997), no unambiguous seagrass-speci®c biomarkers have been identi®ed so far. This paper presents data to show that molecular distributions of n-alkane-2-ones (also referred to as methyl ketones or n-alkan-2-ones) in sediments and suspended particulate matter have the potential to serve as an indicator of seagrass-derived organic matter. Homologous series of n-alkane-2-ones have been isolated from a wide variety of depositional environments including marine and lacustrine sediments, soils and peats (Cranwell, 1981; Volkman et al., 1983; AlbaigeÂs et al., 1984; Cranwell et al., 1987; Jae et al., 1993, 1996b). The molecular distributions found show a high predominance of odd numbered carbon chain-lengths maximizing at C27 or C29. Their close resemblance to the terrigenous n-alkane distributions has led several authors to propose microbial oxidation of n-alkanes as the source of n-alkane-2-ones with b-oxidation of fatty acids followed by decarboxylation as an alternate pathway (Allen et al., 1971; AlbaigeÂs et al., 1984; Cranwell et al., 1987; Lehtonen and Ketola, 1990; Jae et al, 1993). Although some studies have suggested a correspondence of the n-alkane-2-ones distribution with that of the nalkanes or the fatty acids, it is not always close enough to substantiate precursor-product relationships between these classes of compounds (Volkman et al., 1980, 1983). Jae et al. (1993) suggested that dierent diagenetic processes such as binding to sediments and biodegradation could be the cause for such lack of correlation. It has also been suggested that when the n-alkanes derive from two dierent sources, i.e. when an algal signal is superimposed on a higher plant distribution, microbial oxidation of the latter prior to incorporation in the sediment could generate the type of n-alkane-2-one distribution usually observed (Volkman et al., 1980). More recently, n-alkane-2-ones have been reported in higher plant and phytoplankton biomass (Rieley et al., 1991; Qu et al., 1999) suggesting direct biological inputs to sediments. However, independent of their origin, n-alkane-2-ones
have been found to be ubiquitous in aquatic environments, and similar molecular distributions have been reported for sediments characterized by higher plant or microbial organic matter inputs. This study presents the ®rst report on a C25 n-alkane-2-one dominated molecular distribution in sediments from a South Florida estuary and discusses possible sources. 2. Experimental methods A transect of 10 sediment samples (Fig. 1) was collected starting at the freshwater peats of the Shark River Slough (Everglades National Park), through the Harney River estuary into the Florida Shelf. Samples were collected from a boat using an Eckman Dredge (Wildco, Michigan) for the Harney River samples and from the R-V Bellows with a box corer for the Florida Shelf samples. Mangrove leaves (Rhizophora mangle), sawgrass (Caladium sp.), periphyton, and four seagrasses (Thalassia testudinum, Halodule wrightii, Syringodium ®liforme, and Zostera marina) were also analyzed following a similar procedure to that described for the sediments. Seagrass samples were collected by divers, both in Florida Bay, Florida (T. testudinum, H. wrightii, S. ®liforme) and in the San Francisco Bay area, California (Z. marina), placed in zip-lock bags and kept frozen until analysis. Seagrass samples were rinsed with distilled water and major epiphytic growth was physically removed prior to freezing. Samples of T. testudinum and of H. wrightii with signi®cant epiphytic cover were also analyzed without the cleaning step. Mangrove leaves, sawgrass and periphyton samples were collected by hand from the Shark River Slough area, and treated similarly to the seagrasses. Surface sediment samples were transferred immediately to clean glass jars with Te¯on lined caps, placed on ice and stored frozen at ÿ8 C until analysis. Bulk sediment characteristics are shown in Table 1. Surface water samples were collected at sites 2, 3 and 4 (see Fig. 1) on a monthly basis from December 1997 to August 1998, placed in 50 l polyethylene bottles using a portable pump, and ®ltered through GF/C glass ®ber ®lters within a 24 h period after collection. The ®ltered particulate matter was kept frozen until analysis. Water quality parameters were determined using standard analytical procedures as described elsewhere (Boyer et al., 1999). The ®lters and sediment samples were freezedried and Soxhlet extracted for 24 h with high purity methylene chloride (Optima, Fisher, USA). Extracts were then saponi®ed and separated into neutral and acid fractions. The neutral fraction was further separated into 8 fractions by adsorption chromatography over silica gel as previously described (Jae et al., 1995). All fractions were analyzed by GC/MS (Hewlett Packard 5973 model) using a DB5-MS capillary column (25 m, 0.25 mm i.d., 0.25 mm from J&W, Flossom, California).
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
23
Fig. 1. Location of sediment sampling sites in Everglades National Park and the Florida Shelf.
Table 1 Bulk sediment characteristics Site
1
2
3
4
5
6
7
8
9
%C C/N d13Ca kb
41.4 21.4 ÿ28.9 4.7
31.3 16.9 ÿ27.6 3.6
18.7 16.0 ÿ26.7 1.1
12.5 13.9 ÿ25.7 0.97
2.3 9.4 ÿ22.5 0.30
6.9 7.0 ÿ20.0 0.31
16.0 7.1 ÿ19.4 0.11
11.5 7.9 ÿ19.6 0.01
10.8 7.5 ÿ19.5 0.02
a b
d13C reported as %. k=total n-alkane-2-ones in mg/g.
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Quantitation was based on an internal standard (perdeuterated phenanthrene). n-Alkane-2-ones (fraction 5) were identi®ed based on chromatographic retention and mass spectra characteristics (m/z=59). Note that although the m/z=59 ion is present in the mass spectra of all the n-alkane-2-ones, its abundance relative to the m/z 58 ion varies with chain-length. Therefore, the data presented here are semiquantitative, and the relative abundance of the lower molecular weight homologues may be somewhat underestimated. Detailed biomarker data from these samples have been reported elsewhere (Jae et al., 2000). Stable isotope analyses (d13C) were performed on a Finnigan Delta Plus (for bulk sediments) and on a
Hewlett Packard 5890 gas chromatograph coupled to a Finnigan Delta C (for compound speci®c d13C analysis; irm-GC/MS). The irm-GC/MS analyses were performed based on the technique described by Hayes et al. (1990). A standard mixture of aromatic hydrocarbons and alkanes was injected to test the reproducibility and analytical errors of the instrument. Typically 1 ml of the standard was injected for each run. The reproducibility and the accuracy of the measurements were satisfactory (within 0.1 and 0.3% respectively). Pulses of standardized CO2 were introduced into the ion source during each run. Although this method of external isotopic calibration fails to compensate for the physical conditions to which analytes were being subjected during
Fig. 2. Molecular distributions of n-alkane-2-ones (m/z=59 ion chromatograms) in sediment and peat samples throughout the Harney River transect (sites 1±5 and P1) and sediment samples of the Florida Shelf (sites 6±9).
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
their passage though the gas chromatograph and the combustion interface, it eliminated possible interferences between the analytes and co-injected standards. Moreover, since the square pro®le delivers, on average, more CO2 per unit of peak width than a gaussian peak with equal height and width (Merritt et al., 1994), the precision of related isotopic analysis was improved. 3. Results and discussion 3.1. Molecular distribution of n-alkane-2-ones in sediments and plant biomass All of the ketone fractions contained a series of nalkane-2-ones, ranging from C21 to C33 with a strong predominance of odd chain-lengths. The molecular distribution showed a gradual change from the freshwater to the marine end-members of the Harney River estuary, by shifting from a C27, C29 and C31, C33 dominated pro®le for peat and mangrove organic matter in¯uenced sediments (sites 1 and 2 respectively) to a C25 dominated signal for the marine in¯uenced sediments (sites 4 and 5; Fig. 2).
25
Florida Shelf samples 6 and 7 were also characterized by such a C25 predominance, while samples 8 and 9 exhibited the typical higher plant distribution (Fig. 2). Although n-alkane-2-ones have been detected in a wide variety of depositional environments, their distribution has been found to be remarkably similar, typically showing a maximum at C27 or C29 and in some cases at C25 (e.g. Rieley et al., 1991; Ying and Fan, 1993; Qu et al., 1999). Although dierent Cmax values have been reported for n-alkane-2-one distributions in sediments and plant biomass, their molecular distribution is usually characterized by a series of higher molecular weight odd carbon number homologues, and is not strongly dominated by any particular homologue. The strong predominance of the C25 homologue observed here for the marine-in¯uenced samples is, to the best of our knowledge, the ®rst such report in the literature suggesting a dierent origin for this compound. While the analyses of mangrove, sawgrass and periphyton samples resulted in the typical n-alkane-2-one distribution centered around C27±C31, the three local seagrass samples (seagrass blades) showed the C25 ketone being by far the most dominant homologue (Fig. 3). The
Fig. 3. Ion chromatograms (m/z=59 ion chromatograms) of the n-alkane-2-ones in three seagrasses (Thalassia testudinum, Halodule wrightii and Syringodium ®liforme). Presence of signi®cant epiphytic surface cover of seagrass blades is indicated.
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Z. marina sample, however, showed no preference for this particular homologue (data not shown). Root samples from T. testudinum were also analyzed and showed similar C25-dominated n-alkane-2-one distributions. These results are interesting in several ways. Although n-alkane-2-ones are often believed to arise from the microbially mediated b-oxidation of the alkanes (see above) the ®nding of signi®cant amounts of n-alkane-2ones present in plant biomass seems to indicate that a direct biological origin for these compounds is signi®cant in this environment. In agreement with this observation, a lack of correspondence between the nalkane and n-alkane-2-one distributions was observed (e.g. Fig. 4) in these plants, as previously reported by Ying and Fan (1993). The unusually high abundance of even-carbon n-alkenes in the aliphatic hydrocarbon fraction of the mangrove leaf extract (Fig. 4), has been discussed in more detail elsewhere (Jae et al., 2000). A similar distribution has been reported for a coastal macrophyte (Juncus roemericanus) by Canuel et al. (1997). Total n-alkane-2-one concentrations found in the seagrass samples were in the range of 2±21 mg/g. For all seagrass samples which were relatively free of epiphytes,
the C25 n-alkane-2-one represented between 82 and 88% of the ketone fraction. For samples with signi®cant epiphytic cover, this relative abundance was reduced by about 50%. In contrast, the C25 homologue in periphyton, sawgrass and mangrove samples was only 8±9% of total ketones. This predominance of the C25 homologue (and to some extent that of the C23 homologue) in the seagrasses, compared to the predominance of higher molecular weight homologues (C27±C33) for terrestrial higher plants, clearly suggests the applicability of the relative abundance of the C25 homologue as a potential indicator of seagrass-derived organic matter in coastal and estuarine sediments. For example, the C25/C27 ratio could be applied for such a purpose. Note that seagrass with abundant epiphytes showed a signi®cantly reduced C25/C27 ratio compared to `clean' seagrass, indicative of a C27+ n-alkane-2-one contribution from the epiphytic organisms. Cyanobacteria have recently been reported as a source for n-alkane-2-ones (Qu et al., 1999), suggesting that epiphytic prokaryotes and perhaps microalgae could also be responsible for a reduced C25/C27 ratio. Surprisingly, the C25 dominated molecular distribution of the n-alkane-2-ones was not observed for the Z. marina samples. Both of the eelgrass samples analyzed showed
Fig. 4. Ion chromatograms of the n-alkane-2-ones (m/z=59) and n-alkanes/alkenes (m/z=57) of a Thalassia testudinum and a Rhizophora mangle sample.
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
27
a typical higher plant molecular distribution for the nalkane-2-ones, maximizing at the C27 homologue. Although the reasons for the dierent molecular composition between the local tropical/sub-tropical seagrasses and the eelgrass (abundant in temperate climates) is dicult to assess, phytogenetic studies of seagrasses have shown that Z. marina, T. testudinum and the pair S. ®liformis and H. wrightii fall into three distinct groups (Les et al., 1997). The lack of an enhanced C25 homologue for Z. marina suggests that the applicability of this feature as a seagrass-derived organic matter indicator may be limited to tropical and sub-tropical environments where Thalassia, Syringodium and Halodule are abundant.
observed between the ketone ratio and salinity (Fig. 6), no correlation was found with the chlorophyll concentration or turbidity (r2 <0.20). The positive salinitydependence of the particle-associated C25/C27 n-alkane2-one ratio clearly suggests a signi®cant marine origin for the C25 homologue, and that tidal in¯uence has a strong eect on the distribution of marine-derived organic matter in the Harney River estuary (see also Jae et al., 2000). The lack of correlation with chlorophyll concentration, a measure of phytoplankton abundance in water, indicates that marine phytoplankton is not the main source of the C25 n-alkane-2-ones in the suspended particulate matter, leaving seagrasses as the most likely source.
3.2. Hydrodynamics
3.3. Corroboration of seagrass origin for the C25 n-alkane-2-one
Since hydrodynamic sorting of sediments during deposition can control their chemical characteristics, the molecular distribution of the n-alkane-2-ones of a sizefractionated sediment sample from a Harney River (site 4, Fig. 1) was examined. For this purpose, the sample was sequentially passed through 250, 150 and 75 mm brass sieves. Approximately 36% of the sediment was composed of material <75 mm, 44% was in the range 75±150 mm, and 17% in the range 150±250 mm. The remaining 3% comprised a coarse fraction (>250 mm) mainly made up of shells and coarse-grained minerals. The amount of extracted organic material obtained from it was insigni®cant and thus, its molecular distribution is not discussed here. Dierent grain-size fractions had markedly similar molecular compositions indicating that similar processes aected the organic material. For example, the odd/even preference (carbon preference index of ca. 5.2) of the n-alkanes and the maximum at C29, was approximately the same from the coarser to the ®ner fractions. However, based on the relative abundance of the C25 ketone homologue, the nalkane-2-one fraction showed a mixed seagrass and higher plant signal in the coarse fractions and a strong seagrass signal in the ®ner (<75 mm) sediment fraction (Fig. 5). Although the bulk amount of the seagrassderived organic matter in this sample seems to be present as coarse detrital material (>75 mm), only the C25 homologue was detected in the ®ne sediments, an indication that resuspended ®ne sediments and associated seagrass-derived organic matter can be transported over long distances in coastal environments and into estuaries during tidal exchange. To further assess the transport of seagrass-derived organic matter in this estuary, suspended solids were sampled monthly at three stations during a 9 month period. The C25/C27 n-alkane-2-one ratio in these samples was correlated with water quality parameters such as salinity, chlorophyll concentration and turbidity. While a reasonably good linear correlation (r2=0.77) was
In order to further con®rm the origin and applicability of the C25 n-alkane-2-one relative abundance as an indicator of seagrass-derived organic matter, correspondence factorial analysis (CFA) of the data was implemented using a commercially available statistical package (Statistica). CFA, which has successfully been applied to biomarker interpretations in estuarine systems (Sicre et al., 1988, 1993), is an exploratory multivariate technique that converts a matrix of nonnegative data into a particular type of graphical display in which the rows and columns of the matrix are depicted as points. It is a generalization of a simple concept: the scatterplot (Fig. 7). The ®rst two factors calculated from the data matrix accounted for 87% of the total inertia. The values of the absolute contributions (AC) and relative contributions (RC) were used to interpret the axis. AC values show that the C31 and the C25 n-alkane-2-ones are the main contributors to the construction of the ®rst factor accounting for 44 and 47% of its inertia respectively. From this observation, we can assume that the ®rst axis discriminates between seagrass and higher plant sources. AC values for the second axis indicate that this axis distinguishes between the latter group and another one dominated by the C27 n-alkane-2-one presumably due to the in¯uence of sawgrass and periphyton inputs. For comparison, data obtained from the analysis of mangroves leaves, sawgrass and seagrass blades and periphyton were plotted in a supplementary way (i.e. not included in the statistical analysis). The relative proximity of the two seagrass samples to sediment from station 7 supports the idea that the station is mainly in¯uenced by seagrass inputs. The lower right quadrant of the graph consists of stations with marine in¯uence and some degree of seagrass input. The upper part of the quadrant shows those stations with little seagrass in¯uence, while the lower part of the quadrant hosts stations 5 and 7, which have a very strong seagrass signal.
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M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Fig. 5. Molecular distribution of n-alkane-2-ones (m/z=59 ion chromatograms) in a grain size-fractionated sediment sample (site 4). Size fractions correspond to 250±150, 150±75 and <75 mm respectively. Overall n-alkane-2-one grain-size based distribution is indicated at the bottom of the ®gure.
Station 1, located in the lower left quadrant, consists almost exclusively of mangrove detritus as indicated by the predominance of the C31 n-alkane-2-one. The peat sample and the sediments at station 2 plotted very close to each other in the upper left quadrant halfway
between sawgrass and periphyton. Station 8 appears to have an n-alkane-2-one distribution very similar to that of higher plants. The scatter of the shelf samples re¯ects the patchiness of the organic matter in this area, thus agreeing with the irregular distribution of drift particulate
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
29
Fig. 6. Correlation of the n-alkane-2-one C25/C27 ratio in surface water suspended particles collected from sites 2, 3 and 4 vs. surface water salinity.
material throughout the Florida Shelf (Zieman et al. 1989). In agreement with the CFA results, stations 8 and 9 fall into areas characterized by low seagrass cover, while sites 6 and 7 are in the vicinity of extensive, highdensity seagrass beds (Fourqurean et al., 1999). Stable isotope (d13C) data of the bulk organic matter in the sediments supports the seagrass in¯uence towards the marine end-member of the transect. The bulk d13C values throughout the Harney River transect ranged from ÿ28.9% at station 1, to about ÿ19% for the Florida Shelf samples (Table 1). Chmura and Aharon (1995) showed that such trends of 13C isotopic enrichment paralleling the salinity gradient between the upper and lower estuaries are quite typical and are mainly controlled by the gradual transition from freshwater marsh-dominated C-3 plants (ÿ23.5 to ÿ27.8%) to C-4 plant community dominated salt marshes (ÿ11.6 to ÿ15.5%). However, the vegetation of the lower estuaries of South Florida is usually dominated by mangroves (C3 plants), and the isotopic pattern presented here is most likely caused by the in¯uence of a combination of marine plankton and seagrass-derived organic matter in the sediments. While marine plankton and suspended particulates from temperate regions have d13C values ranging from about ÿ24 to ÿ16% (Michener and Schell, 1994; Fry, 1996; Johnston and Kennedy, 1998), seagrasses
(although being C-3 plants) re¯ect an isotopic signature similar to that of C-4 plants (Canuel et al., 1997). Therefore, the gradual enrichment in 13C between the upper and lower estuary suggests a transition from higher plant-derived organic matter to that increasingly in¯uenced by marine-derived organic matter, including seagrasses (see also Jae et al., 2000). The d13C values for individual n-alkane-2-one homologues were in agreement with the bulk sediment d13C data. Note that lipids usually are about 5±8% lighter than bulk carbon measurements. Carbon isotopic compositions of individual n-alkane-2-ones isolated from sediments and plants are summarized in Table 2. At station 2, the three measured n-alkane-2-ones (C23, C25 and C27) have similar isotopic compositions in the range of ÿ31 to ÿ30%. However, starting at station 3, the isotopic composition of the C25 ketone becomes enriched in 13C, reaching a value of ÿ17.2% at station 4. From the previous analysis of the molecular distributions of the samples, it was suggested (see above) that stations 3 and 4 have a greater seagrass in¯uence as evidenced by high relative C25 n-alkane-2-one abundances. Seagrasses are aquatic C-3 plants that fractionate carbon similarly to C-4 plants, resulting in a heavier isotopic signature (ÿ10 to ÿ15%; Thayer et al. 1978; Canuel et al., 1997). Thus, an increase in the input of seagrass-derived
30
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
Fig. 7. Correspondence factorial analysis (CFA) plot of the n-alkane-2-one molecular abundance in sediments. AC (Absolute Contribution) and RC (Relative Contribution) values expressed as percent for factors I and II, are shown on the margin, and provide the contribution of a parameter to the variance of a factor and the extent to which a factor explains the variance of a parameter, respectively. Table 2 Compound-speci®c d13C values for n-alkane-2-ones in plant biomass and sediment samples n-Alkane-2-one
C23
C25
C27
Site 2 (sediment) Site 3 (sediment) Site 4 (sediment) Periphyton Thalassia testudinum Halodule wrightii Rhizophora mangle
ÿ31.30.1 ÿ29.80.1 ÿ27.60.3 na na na ÿ32.30.3
ÿ30.20.1 ÿ24.80.2 ÿ17.20.1 ÿ32.80.2 ÿ15.00.2 ÿ14.70.1 ÿ32.60.1
ÿ30.80.2 ÿ30.00.1 ÿ29.60.2 ÿ33.40.3 ÿ14.90.1 na ÿ32.70.2
organic matter (and therefore seagrass derived n-alkane2-ones) should shift the isotopic signature of the C25 ketone to a heavier signal as is seen in station 4 (ÿ17.2%). The isotopic enrichment of the C27 homologues was signi®cantly less pronounced at station 3 and 4 due to the low relative contribution of the C27+ homologues from seagrasses, resulting in less dilution of the original terrestrial signal. When the carbon isotopic compositions of T. testudinum, H. wrightii, R. mangle and a periphyton sample were measured, it was found that the C25 n-alkane-2-ones isolated from the mangroves and the periphyton were ca. ÿ32%, while that for the seagrasses was ca. ÿ15%. Similar carbon isotopic values have been reported for other seagrass-derived lipids
(Canuel et al., 1997; Hammer et al., 1998). These ®ndings seem to con®rm that seagrasses are an important source of the C25-dominated n-alkane-2-one distributions. 4. Conclusions n-Alkane-2-ones have been found to be present in mangroves, sawgrass, periphyton mats and seagrasses from South Florida aquatic and terrestrial environments, suggesting that the presence of these lipids in sediments is not exclusively derived from the oxidation of other linear lipids such as alkanes, alkenes and fatty acids. In addition, the molecular distribution of the predominantly odd carbon numbered homologous series of n-alkane-2-ones for the dierent biomass components is not identical. Although both terrestrial higher plants and planktonic organisms (periphyton) showed similar molecular distributions, seagrasses had a distinctively dierent distribution, with a homologous series dominated by the C25 homologue. Such molecular distributions were con®rmed to be derived from seagrasses though statistical analyses and compound-speci®c stable isotope analyses. The application of the C25/C27 ratio as a means to distinguish marine, seagrass-derived organic matter from terrestrial/planktonic organic matter was tested through the analysis of suspended matter and correlation with water quality parameters. This study
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
shows the application of the relative abundance of the C25 n-alkane-2-one as a seagrass indicator in estuarine and coastal environments. The potential use of this molecular tool in paleoenvironmental studies, particularly in tropical and sub-tropical environments, where species such as Thalassia testudinum, Halodule wrightii and Syringodium ®liformis are abundant, is suggested. Acknowledgements The authors thank the National Science Foundation (NSF) for partial support for this project through grant No. 9450394, Drs. R. Jones and J. Boyer for providing logistical support and water quality data, Dr. J. Fourqurean for supplying the seagrass samples and for helpful discussions, Dr. M. A. Sicre for helpful comments on the use of the CFA, and the crew of the R-V Bellows for sampling assistance. Special thanks go to Dr. J. Volkman whose helpful comments signi®cantly improved this manuscript. This research was made possible thanks to instrumentation grants from NSF (No. 9512385) and the FIU Division of Sponsored Research, for the irmGC/MS and the GC/MS systems respectively. M.H. and R.M. thank NSF for a student fellowship (No. 9450394) and FIU for a teaching assistantship, respectively. SERC Contribution No. 136. Associate EditorÐJ. Volkman References AlbaigeÂs, J., Algaba, J., Grimalt, J., 1984. Extractable and bound neutral lipids in some lacustrine sediments. Organic Geochemistry 6, 502±507. Allen, J.E., Forney, F.W., Markovetz, A.J., 1971. Microbial subterminal oxidation of alkanes and alk-1-enes. Lipids 6, 448±452. Bianchi, T.S., Argyrou, M., Chippett, H.F., 1999. Contribution of vascular-plant carbon to surface sediments across the coastal margin of Cyprus (eastern Mediterranean). Organic Geochemistry 30, 287±297. Boyer, J.N., Fourqurean, J.W., Jones, R.D., 1999. Seasonal and long term trends in the water quality of Florida Bay (1989±1997). Estuaries 22, 417±430. Bull, I.D., van Bergen, P.F., Bol, R., Brown, S., Gledhill, A.R., Gray, A.J., Harkness, D.D., Woodbury, S.E., Evershed, R.P., 1999. Estimating the contribution of Spartina anglica biomass to salt-marsh sediments using stable isotope measurements. Organic Geochemistry 30, 477±484. Canuel, E.A., Freeman, K.H., Wakeham, S.G., 1997. Isotopic composition of lipid biomarker compounds in estuarine plants and surface sediments. Limnology and Oceanography 42, 1570±1583. Chmura, G.L., Aharon, P., 1995. Stable carbon isotope signatures of sedimentary carbon in coastal wetlands as indicators of salinity regime. Journal of Coastal Research 11, 124±135.
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