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 C19to C33characterized by a
strong odd over even carbon number predominance. In this paper we report a dierent molecular distribution, centered about the C25homologue as the dominant ketone. The relative abundance of the C25compared to the C27homologue
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 C25n-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 dif-ferent 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
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 taxon-speci®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 sev-eral important functions by providing habitat for a wide variety of plant and animal species, and by physically
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* Corresponding author. Tel.: 305-348.24.56; fax: +1-305-348.40.96.
stabilizing coastal areas, reducing erosion. As such, sea-grasses 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 andn-alkanes (Canuel et al., 1997), analyses of lignin phenols (Bianchi et al., 1999) anda,b -dihydroxy fatty acid distributions (de Leeuw et al., 1995a,b). Similarly, Volkman et al. (1980) demonstrated that the seagrassZostera muelleriwas a major source of speci®c a-hydroxy, o-hydroxy and a,o-dicarboxylic acids in an intertidal sediment. Although the lipid com-position 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 orn-alkan-2-ones) in sediments and suspended particulate matter have the potential to serve as an indicator of seagrass-derived organic matter.
Homologous series ofn-alkane-2-ones have been iso-lated 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 pre-dominance of odd numbered carbon chain-lengths maximizing at C27or C29. Their close resemblance to the
terrigenous n-alkane distributions has led several authors to propose microbial oxidation ofn-alkanes as the source ofn-alkane-2-ones withb-oxidation of fatty acids followed by decarboxylation as an alternate path-way (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 n -alkanes 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 pro-cesses such as binding to sediments and biodegradation could be the cause for such lack of correlation. It has also been suggested that when then-alkanes derive from two dierent sources, i.e. when an algal signal is super-imposed on a higher plant distribution, microbial oxi-dation of the latter prior to incorporation in the sediment could generate the type ofn-alkane-2-one dis-tribution 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 environ-ments, 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
mole-cular 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 epi-phytic growth was physically removed prior to freezing. Samples of T. testudinum and of H. wrightiiwith sig-ni®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 immedi-ately to clean glass jars with Te¯on lined caps, placed on ice and stored frozen atÿ8C until analysis. Bulk sediment
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 41.4 31.3 18.7 12.5 2.3 6.9 16.0 11.5 10.8
C/N 21.4 16.9 16.0 13.9 9.4 7.0 7.1 7.9 7.5
d13Ca
ÿ28.9 ÿ27.6 ÿ26.7 ÿ25.7 ÿ22.5 ÿ20.0 ÿ19.4 ÿ19.6 ÿ19.5
kb 4.7 3.6 1.1 0.97 0.30 0.31 0.11 0.01 0.02 a d13C reported as%.
b
Quantitation was based on an internal standard (per-deuterated phenanthrene). n-Alkane-2-ones (fraction 5) were identi®ed based on chromatographic retention and mass spectra characteristics (m/z=59). Note that although them/z=59 ion is present in the mass spectra of all then-alkane-2-ones, its abundance relative to the
m/z58 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®cd13C 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 1ml 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
standar-dized CO2were introduced into the ion source during
each run. Although this method of external isotopic calibration fails to compensate for the physical condi-tions to which analytes were being subjected during
their passage though the gas chromatograph and the combustion interface, it eliminated possible inter-ferences between the analytes and co-injected standards. Moreover, since the square pro®le delivers, on average, more CO2per 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 ofn-alkane-2-ones in sediments and plant biomass
All of the ketone fractions contained a series of n -alkane-2-ones, ranging from C21 to C33 with a strong
predominance of odd chain-lengths. The molecular dis-tribution showed a gradual change from the freshwater to the marine end-members of the Harney River estuary, by shifting from a C27, C29and C31, C33dominated pro®le for
peat and mangrove organic matter in¯uenced sediments (sites 1 and 2 respectively) to a C25dominated signal for
the marine in¯uenced sediments (sites 4 and 5; Fig. 2).
Florida Shelf samples 6 and 7 were also characterized by such a C25predominance, while samples 8 and 9
exhib-ited the typical higher plant distribution (Fig. 2). Althoughn-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 C27or C29and 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 typicaln-alkane-2-one distribu-tion centered around C27±C31, the three local seagrass
samples (seagrass blades) showed the C25ketone being
by far the most dominant homologue (Fig. 3). The
Z. marina sample, however, showed no preference for this particular homologue (data not shown). Root sam-ples fromT. testudinumwere also analyzed and showed similar C25-dominatedn-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 ofn -alkane-2-ones present in plant biomass seems to indicate that a direct biological origin for these compounds is sig-ni®cant in this environment. In agreement with this observation, a lack of correspondence between the n -alkane 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±21mg/g. For all seagrass samples which were relatively free of epiphytes,
the C25n-alkane-2-one represented between 82 and 88%
of the ketone fraction. For samples with signi®cant epi-phytic cover, this relative abundance was reduced by about 50%. In contrast, the C25homologue in periphyton,
sawgrass and mangrove samples was only 8±9% of total ketones. This predominance of the C25homologue (and
to some extent that of the C23 homologue) in the
sea-grasses, compared to the predominance of higher mole-cular weight homologues (C27±C33) for terrestrial higher
plants, clearly suggests the applicability of the relative abundance of the C25 homologue as a potential
indi-cator 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/C27ratio 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 forn-alkane-2-ones (Qu et al., 1999), suggesting that epiphytic prokaryotes and perhaps microalgae could also be responsible for a reduced C25/C27ratio.
Surprisingly, the C25dominated molecular distribution
of then-alkane-2-ones was not observed for theZ. 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
a typical higher plant molecular distribution for the
n-alkane-2-ones, maximizing at the C27 homologue.
Although the reasons for the dierent molecular compo-sition between the local tropical/sub-tropical seagrasses and the eelgrass (abundant in temperate climates) is dicult to assess, phytogenetic studies of seagrasses have shown thatZ. marina, T. testudinumand the pair
S. ®liformis and H. wrightii fall into three distinct groups (Les et al., 1997). The lack of an enhanced C25
homologue forZ. marinasuggests that the applicability of this feature as a seagrass-derived organic matter indicator may be limited to tropical and sub-tropical environments whereThalassia, SyringodiumandHalodule
are abundant.
3.2. Hydrodynamics
Since hydrodynamic sorting of sediments during deposition can control their chemical characteristics, the molecular distribution of then-alkane-2-ones of a size-fractionated 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 <75mm, 44% was in the range 75±150 mm, and 17% in the range 150±250 mm. The remaining 3% comprised a coarse fraction (>250mm) 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 dis-tribution is not discussed here. Dierent grain-size frac-tions had markedly similar molecular composifrac-tions 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 C25ketone homologue, the
n-alkane-2-one fraction showed a mixed seagrass and higher plant signal in the coarse fractions and a strong seagrass signal in the ®ner (<75mm) sediment fraction (Fig. 5). Although the bulk amount of the seagrass-derived 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 indi-cation that resuspended ®ne sediments and associated seagrass-derived organic matter can be transported over long distances in coastal environments and into estu-aries 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/C27n-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
observed between the ketone ratio and salinity (Fig. 6), no correlation was found with the chlorophyll con-centration or turbidity (r2<0.20). The positive
salinity-dependence of the particle-associated C25/C27n
-alkane-2-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 C25n-alkane-2-ones in the suspended
particulate matter, leaving seagrasses as the most likely source.
3.3. Corroboration of seagrass origin for the C25
n-alkane-2-one
In order to further con®rm the origin and applic-ability of the C25n-alkane-2-one relative abundance as
an indicator of seagrass-derived organic matter, corre-spondence 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 respec-tively.
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
Station 1, located in the lower left quadrant, consists almost exclusively of mangrove detritus as indicated by the predominance of the C31n-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 ann-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
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, high-density 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
enrich-ment 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 toÿ27.8%) 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 (C-3 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 parti-culates from temperate regions haved13C values ranging
from aboutÿ24 toÿ16%(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).
Thed13C values for individualn-alkane-2-one
homo-logues were in agreement with the bulk sedimentd13C
data. Note that lipids usually are about 5±8% lighter
than bulk carbon measurements. Carbon isotopic com-positions of individual n-alkane-2-ones isolated from sediments and plants are summarized in Table 2. At station 2, the three measuredn-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 C25ketone becomes enriched
in13C, 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 C25n-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 toÿ15%; Thayer et al. 1978; Canuel et al., 1997). Thus, an increase in the input of seagrass-derived
Fig. 6. Correlation of then-alkane-2-one C25/C27ratio in surface water suspended particles collected from sites 2, 3 and 4 vs. surface
organic matter (and therefore seagrass derivedn- alkane-2-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
homo-logues 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 C25n-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-dominatedn-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 environ-ments, 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 ofn-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 domi-nated by the C25 homologue. Such molecular
distribu-tions were con®rmed to be derived from seagrasses though statistical analyses and compound-speci®c stable isotope analyses. The application of the C25/C27ratio 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
Fig. 7. Correspondence factorial analysis (CFA) plot of then-alkane-2-one molecular abundance in sediments. AC (Absolute Contribu-tion) and RC (Relative ContribuContribu-tion) values expressed as percent for factors I and II, are shown on the margin, and provide the con-tribution 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) ÿ31.30.1 ÿ30.20.1 ÿ30.80.2 Site 3 (sediment) ÿ29.80.1 ÿ24.80.2 ÿ30.00.1 Site 4 (sediment) ÿ27.60.3 ÿ17.20.1 ÿ29.60.2
Periphyton na ÿ32.80.2 ÿ33.40.3
Thalassia testudinum na ÿ15.00.2 ÿ14.90.1
Halodule wrightii na ÿ14.70.1 na
shows the application of the relative abundance of the C25n-alkane-2-one as a seagrass indicator in estuarine
and coastal environments. The potential use of this molecular tool in paleoenvironmental studies, particu-larly in tropical and sub-tropical environments, where species such as Thalassia testudinum, Halodule wrightii
andSyringodium ®liformisare 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. Four-qurean for supplying the seagrass samples and for help-ful discussions, Dr. M. A. Sicre for helphelp-ful comments on the use of the CFA, and the crew of theR-V Bellowsfor sampling assistance. Special thanks go to Dr. J. Volk-man 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 irm-GC/MS and the irm-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
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