Directory UMM :Data Elmu:jurnal:O:Organic Geochemistry:Vol32.Issue1.2001:
Organic Geochemistry 32 (2001) 21±32
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
22
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).
23
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
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.
24
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.
26
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
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
22
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).
23
M.E. Hernandez et al. / Organic Geochemistry 32 (2001) 21±32
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.
24
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.
26
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