Terrigenous dissolved organic matter along an estuarine
gradient and its ¯ux to the coastal ocean
Antonio Mannino, H. Rodger Harvey *
Chesapeake Biological Laboratory, University of Maryland Center for Environmental Science, PO Box 38, Solomons, MD 20688, USA
Abstract
The contribution of terrigenous organic matter (TOM) to high molecular weight dissolved and particulate organic matter (POM) was examined along the salinity gradient of the Delaware Estuary. Dissolved organic matter (DOM) was fractionated by ultra®ltration into 1±30 kDa (HDOM) and 30 kDa±0.2mm (VHDOM) nominal molecular weight fractions. Thermochemolysis with tetramethylammonium hydroxide (TMAH) was used to release and quantify lipids and lignin phenols. Stable carbon isotopes, fatty acids and lignin content indicated shifts in sources with terrigenous material in the river and turbid region and a predominantly algal/planktonic signal in the lower estuary and coastal ocean. Thermochemolysis with TMAH released signi®cant amounts of short chain fatty acids (C9±C13), not seen by
traditional alkaline hydrolysis, which appear to be associated with the macromolecular matrix. Lignin phenol dis-tributions in HDOM, VHDOM and particles followed predicted sources with higher concentrations in the river and turbid region of the estuary and lower concentrations in the coastal ocean. TOM comprised 12% of HDOM within the coastal ocean and up to 73% of HDOM within the turbid region of the estuary. In the coastal ocean, TOM from high molecular weight DOM comprised 4% of total DOC. The annual ¯ux of TOM from the Delaware Estuary to the coastal ocean was estimated at 2.01010g OC yearÿ1and suggests that temperate estuaries such as Delaware Bay can
be signi®cant sources of TOM on a regional scale.#2000 Elsevier Science Ltd. All rights reserved.
Keywords:Lignin; DOM; TMAH; Protein; Fatty acids; Estuary; Terrestrial; Turbidity maximum
1. Introduction
Organic matter transport in coastal systems as parti-culate organic matter (POM) has been studied exten-sively (e.g. Prahl et al., 1994; Sicre et al., 1994; Yunker et al., 1995), but much less is known about the dynamics of dissolved organic matter (DOM). Rivers transport 0.251015g of dissolved organic carbon (DOC) per year
to the ocean (Meybeck, 1982), yet the terrestrial con-tribution to this overall ¯ux is not well known (Hedges et al., 1997). The presence of terrigenous organic matter (TOM) in oceanic settings is well documented, and sig-ni®cant amounts of TOM have been measured in pela-gic waters and sediments using isotopic and various
lipid and lignin biomarkers (e.g. Westerhausen et al., 1993; Opsahl and Benner, 1997; see review by Hedges et al., 1997). While TOM entering coastal systems in par-ticles is predominantly deposited in the coastal zone (Hedges, 1992; Prahl et al., 1994), DOM is thought to be the major conduit for transporting TOM beyond the coastal zone.
Lignin phenols have been applied as the principal molecular organic tracers of TOM within DOM pri-marily in large rivers such as the Amazon (Ertel et al., 1986) and other freshwater ecosystems (e.g. Ertel et al., 1984; Standley and Kaplan, 1998). Only a few studies have examined dissolved lignin phenols in estuarine environments (Moran et al., 1991; Argyrou et al., 1997). The presence of dissolved lignin oxidation products along the shelf of the southeastern US indicated an export of TOM to the ocean, with lignin phenols con-tributing 11 to 75% of the dissolved humics (Moran et al., 1991; Moran and Hodson, 1994). Observations of
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* Corresponding author. Tel.: 410-326-7206; fax: +1-410-326-7341.
dissolved lignin phenols in the Arctic Ocean (Kattner et al., 1999; Opsahl et al., 1999), Gulf of Mexico (Bianchi et al., 1997) and equatorial Paci®c (Meyers-Schulte and Hedges, 1986) also illustrate contributions of TOM to oceanic environments. Recent evidence of lignin phenols in UDOM (>1 kDa DOM) from several sites and depths within the tropical Paci®c Ocean and Atlantic Ocean demonstrates widespread distribution of TOM within oceanic DOM (Opsahl and Benner, 1997). Since terrestrial ecosystems are highly productive, their con-tribution to oceanic DOM could be substantial due to the refractory nature of TOM, particularly lignin, and may in¯uence global carbon cycling.
To determine the contribution of TOM to high molecular weight DOM within a temperate estuary and its export the coastal ocean, we measured the abundance and distribution of lignin phenols along the salinity gradient of the Delaware Estuary, USA. The relatively new procedure, thermochemolysis with tetra-methylammonium hydroxide (TMAH), was applied to release lignin phenols and other potential biomarkers from the macromolecular matrix. Previous studies of
d13C andd15N POM in Delaware Bay indicate a
dom-inance of planktonic material, although the presence of lignin phenols throughout the year within suspended particles also suggest watershed inputs of terrestrial material (Cifuentes et al., 1988; Cifuentes, 1991). The contribution of TOM to the DOM pool and its export to the coastal ocean through the dissolved pool has not been previously examined. Constraining the inputs of TOM to the ocean is critical to understanding the ocean's carbon cycle.
2. Methods
2.1. Sampling and bulk analyses
Seven stations were sampled along a transect of the Delaware Estuary from riverine waters to the coastal ocean (Fig. 1). Sample collection and ®ltration were described previously (Mannino and Harvey, 1999). Brie¯y, large volume water samples (13 to 104 l) for analysis of DOM were collected at 1 m depth, and par-ticles were removed by sequential passage through car-tridge ®lters of 3 and 0.2mm pore size. The ®ltrate was then separated into three nominal size fractions: 30 kDa±0.2 mm [very high molecular weight (VHDOM)], 1±30 kDa [high molecular weight (HDOM)] and <1 kDa low molecular weight (LDOM)] using an Amicon DC-10L tangential ¯ow ultra®ltration unit with the S10Y30 and the S10N1 ultra®lters following the meth-ods of Benner (1991). Because of the high particle load at the turbidity maximum (station 2), only 13 l were ®l-tered and the >1 kDa fraction retained. Immediately following initial fractionation and concentration, the
two high molecular weight fractions were desalted using the Amicon unit with 6±9 l of low organic deionized water. Samples for DOC analysis were collected from the <0.2 mm ®ltrate and each DOM size fraction (stored frozen) and analyzed by high temperature com-bustion in triplicate (S.D. 45%) using a Shimadzu TOC 5000 (Benner and Strom, 1993). Remaining sample retentates were stored frozen, concentrated further by rotary evaporation and lyophilized to dry powders. LDOM carbon was analyzed for mass balance purposes, but no further characterization was made.
For analysis of particles, additional whole water was ®ltered through pre-combusted (4±6 h at 450C)
What-man GF/F ®lters by vacuum ®ltration. Organic carbon and total nitrogen content were measured using an Exeter Analytical CHN analyzer for POM and DOM samples. Stable carbon isotopes were quanti®ed on POM and DOM fractions as CO2 on a Micromass
Optima instrument interfaced with a CHN elemental analyzer (Fry et al., 1992; Macko et al., 1997). Pre-cision of the method is typically 0.1%. In the laboratory samples are commonly measured against a tank of carbon dioxide which has been calibrated against NBS 22 which is referenced to the PeeDee Belemnite standard.
2.2. Thermochemolysis with TMAH
For powdered DOM, a sub-sample (0.5±1.8 mg OC) was placed in a 2 ml glass ampule along with three internal standards:o-coumaric acid, nonadecanoic acid and 5a-cholestane and mixed with 100 ml of TMAH (25% in methanol; Sigma). Whatman 47 mm GF/F ®l-ters containing suspended particles (0.58±1.5 mg OC) were ®rst dried at 55C overnight. Internal standards
were then placed onto each ®lter, and ®lters were sub-sequently dried under vacuum, sliced into small strips and placed inside ampules. All ampules were evacuated for 2 h or longer, ¯ame sealed and placed within an oven to react at 250C for 30 min (McKinney et al.,
1995). Ampules were subsequently cooled to room tem-perature, cracked open, and extracted three times with additions of 1 ml of CH2Cl2. Combined extracts were
placed in 4 ml amber vials, dried under a gentle stream of N2and re-dissolved in CH2Cl2.
The TMAH derivatives were quanti®ed by capillary gas chromatography with ¯ame ionization detection (HP-5890II) using a 30 or 60 m DB-5MS column (0.32 mm I.D., 0.25mm ®lm thickness). Hydrogen served as the carrier gas (2 ml minÿ1), and a temperature program
of 10C minÿ1 from 50C to 120C followed by 3C
minÿ1to 200C and thereafter 4C minÿ1to 300C was
used. Reagent blanks processed simultaneously with each sample group indicated no contamination from reagents or handling. Samples were also analyzed by GC±MS (HP-5890II GC coupled to a HP-5970B MSD), and individual spectra were compared with reference mass spectra for compound identi®cation (Hatcher, pers. comm.; NIST, 1998). Helium served as the carrier gas for GC±MS, and the temperature program above was used. The MSD was operated in electron impact mode at 70 eV with acquisition over 50-600 a.m.u. Molecular weights of select compounds were con®rmed by GC±MS using positive chemical ionization with CH4
(1.7 torr) as the ionizing gas (HP-5890II GC coupled to a 5989A MS).
The thermochemolysis reaction is a thermally assisted base-catalyzed reaction which cleaves ester and ether bonds including theb-O-4 bonds within lignin (see Fil-ley et al., 1999, for details) with subsequent methylation of carboxylic and acidic hydroxy groups, including both phenolic and non-acidic side chain hydroxyls (Cliord et al., 1995; del Rio and Hatcher, 1996). The proposed mechanism forb-O-4 bond cleavage involves formation of an intramolecular epoxide following deprotonation of the side chain a or g alcohols (with tetra-methylammonium alkoxy salts as intermediates) which act as nucleophiles to displace the phenoxide (Filley et al., 1999). Functional groups of non-lignin compounds such as fatty acids and sterols are also methylated by TMAH. To compare the relative response ofo-coumaric acid with phenolic and non-phenolic standards, ferulic
acid, vanillic acid and diphenylamine (Sigma) were analyzed individually in the presence ofo-coumaric acid and nonadecanoic acid. These analyses demonstrated that o-coumaric acid is quantitatively recovered, with coecients of variation ranging from 3.5 to 13%. On a mass basis, however, theo-coumaric acid yielded lower responses than the nonadecanoic acid. Relative respon-ses of the nonadecanoic acid and 5a-cholestane were equivalent, indicating ecient esteri®cation of fatty acids. Variability from duplicate analyses of two sam-ples (one POM and one HDOM sample) showed coe-cients of variation of 24% for lignin phenols, 30% for
G+Scomponents (sum of guaiacyl and syringyl lignin
phenol yields inmg/100 mg OC) and <26% for total fatty acids. Proteins including RuBPcase, Trypsinogen and Lysozyme as well as three amino acid mixtures (all protein amino acids, aromatics plus proline and histi-dine, and aliphatic amino acids) were also reacted with TMAH to examine the contribution of proteinaceous material in thermochemolysis products from Delaware Bay samples (Table 1).
3. Results
The three DOC size fractions accounted for 85-110% of the total DOC (Mannino and Harvey, 1999) which indicates a mass balance comparable to other published results (81±128%; Guo and Santschi, 1996; Benner et al., 1997). The molecular weight distribution of ultra-®ltered DOC was non-conservative in the Delaware Estuary (Fig. 2). HDOM-C concentration peaked at the turbidity maximum (133mM C; 61% of DOC) and was lowest in the coastal ocean (42mM C; 25% of DOC). VHDOM-C concentration was low (<5 mM C) and variable throughout the estuary, comprising <3% of total DOC. POC was highest at the turbidity maximum (318mM) and lowest in the coastal ocean (53mM).
The carbon to nitrogen atom ratio (C:Na) varied
along the estuarine gradient for POM and both high molecular weight DOM fractions. Carbon rich (nitrogen poor) material was found in the upper and turbid regions of the estuary, whereas nitrogen rich material was observed within the lower estuary and coastal ocean (Table 2). POM was enriched in nitrogen compared to DOM with C:Na ranging from 6.5 at the chlorophyll
maximum (station 4) to 10.4 at the turbidity maximum. For VHDOM, C:Na was highest at coastal ocean
sta-tion 6 and downstream of the turbidity maximum, 24 and 19, respectively, and lowest within the high chlor-ophyll region (stations 4 and 5). C:Nafor HDOM was
24 and 30 at the riverine and turbidity maximum sites, respectively, and ranged from 17 to 20 at the remaining stations.
The d13C of POM and high molecular weight DOM
(Table 2). The most enrichedd13C value for POM was
found downstream of the chlorophyll maximum,
ÿ18.9%, and the lightest value at the riverine station,
ÿ26.8%. VHDOM contained the heaviestd13C values,
ranging from ÿ23.2% at station 6 to ÿ19.6% down-stream of the chlorophyll maximum. The variation in the d13C signature along the estuary was smaller for
HDOM, which ranged from ÿ25.5% at the turbidity maximum toÿ22.2%at coastal ocean station 7.
Concentrations of lignin phenols derived from ther-mochemolysis with TMAH were higher in the river and
turbid region of the estuary with lower concentrations in the coastal ocean (Fig. 3). The highest concentration of lignin phenols was observed at the riverine site for VHDOM and HDOM. The turbid sites (stations 2 and 3) contained equivalent amounts of particulate lignin, even though total suspended particle (TSP) load was more than double at the turbidity maximum (Fig. 3A). Steep declines in lignin phenols between stations 3 and 4 in all three size fractions coincided with a decrease in TSP and an increase in chlorophylla. Lignin phenols in VHDOM followed the salinity based conservative mix-ing line very closely, except for a slightly higher value at station 7. Downstream of station 3, HDOM lignin phe-nol concentrations declined in tandem with conservative mixing of river and coastal ocean waters. A list of lignin derived thermochemolysis products is shown in Table 3 (Cliord et al., 1995; Hatcher et al., 1995; McKinney et al., 1995). In addition to lignin phenols, thermo-chemolysis with TMAH released a complex suite of other molecules from Delaware Bay POM and DOM (Figs. 4 and 5).
On a carbon basis, VHDOM contained similar or higher amounts of lignin than POM or HDOM. Guaia-cyl structures (3,4-dimethoxyphenyls) dominated the lignin composition of macromolecular DOM, especially for VHDOM (Table 4). In contrast, POM contained a mixture of p-hydroxy, guaiacyl and syringyl phenols with the syringyl forms as least abundant. Ratios ofp -hydroxy phenols to guaiacyl phenols (P/G) did not vary for HDOM or VHDOM along the estuarine gradient. For POM, P/G values and syringyl to guaiacyl phenol
Table 1
Protein and potential nucleic acid derived thermochemolysis products in Delaware Bay POM, VHDOM and HDOMa
Compound ID Precursor Molecular Ion (BP)
Aromatic
Benzene acetic acid ME (POM only) 1 Tyr/Phe 150 (91) 1-Methyl-1H-indole 2 Trp 131 (131) Benzenepropanoic acid ME 3 Phe 164 (104) 3-Phenyl-2-propenoic acid ME 4 Tyr/Phe 162 (131) 4-Methoxybenzene-propanoic acid ME 5 Tyr 194 (121) trans-4-Methoxybenzene-propenoic acid ME
(product ofp-coumaric acid)
6 Tyr 192 (161)
Non-aromatic
Leucine dimethyl ester (POM only) 7 Leu 145 (86) Aspartic acid dimethyl ester 8 Asp 159 (100) Butanedioic acid dimethyl ester 9 Pr? 146 (115)
N-Methyl-proline ME 10 Pro 143 (84)
Methyl-butanedioic acid dimethyl ester 11 Pr? 160 (59) Glutamic acid dimethyl ester (POM only) 12 Glu 173 (114) 1-Methyl-2,5-pyrrolidinedione 13 Al/Pr 113 (113) 1,3-Dimethyl uracil (POM only) 14 RNA? 140 (140) 1,3-Dimethyl thymine 15 DNA? 154 (68)
a BP, base peak from mass spectral trace; ME, methyl ester; Tyr, tyrosine; Phe, phenylalanine; Trp, tryptophan; Leu, leucine; Asp,
aspartic acid; Pr, protein; Pro, proline; Glu, glutamic acid; Al, aliphatic amino acids.
ratios (S/G) were much higher in the lower estuary (stations 4 and 5) and station 7, because of lower amounts of guaiacyl phenols compared to upstream sites. S/G declined with increasing salinity for HDOM but did not change for VHDOM. In UDOM from coastal waters o the Gulf of Mexico, del Rio et al. (2000), using thermochemolysis with TMAH, found a predominance of G1 and 1,4-dimethoxybenzene, 2,5-dimethoxytoluene and 1,2,4-trimethoxybenzene with P6, G6 and S6 less abundant than Suwanee River DOM. They also observed lower amounts of aromatic acids (P6, G6 and S6) in ocean samples versus Suwanee River DOM but relatively higher amounts of dimethoxy-benzenes and trimethoxydimethoxy-benzenes (del Rio et al., 1998). Several other aromatic compounds which appear to be of non-lignin origin were found in Delaware Bay POM and DOM fractions (Table 4). A compound ten-tatively identi®ed as methyl acetophenone comprised substantial portions of VHDOM and HDOM through-out the estuary with maxima at station 3 (82.6 and 23mg mg OCÿ1, respectively). Other aromatic compounds,
excluding lignin phenols, proteinaceous-derived com-pounds and methyl acetophenone, showed variable dis-tributions throughout the estuary (Table 4). One potential thermochemolysis product of hydroquinone, 1,4-dimethoxybenzene (Hatcher et al., 1995), declined in concentration between the upper and turbid regions of the estuary and the coastal ocean. 1,2,4-Trimethoxy-benzene, believed to originate from polysaccharides such as cellulose (Pulchan et al., 1997; Fabbri and Hel-leur, 1999), also decreased in DOM between the upper estuary and coastal ocean which supports a terrestrial origin. In particles, however, the greatest amounts of 1,2,4-trimethoxybenzene were observed in the coastal ocean. Small amounts of 1,3,5-trimethoxybenzene were detected in HDOM and POM and could originate from tannin (Pulchan et al., 1997) and cutan (McKinney et al., 1996) of vascular plants.
Thermochemolysis revealed a suite of aromatic and non-aromatic proteinaceous-derived products in all three size fractions within Delaware Bay organic matter. Most of the aromatic compounds were identi®ed (Table 1) and appeared to have a proteinaceous origin based on protein and amino acid standards (Fig. 6). Several nitrogen containing aliphatic products have yet to be identi®ed. Although thermochemolysis appears to liber-ate many proteinaceous-derived products, yields must be interpreted with caution as only 38 to 64% of the protein or amino acid standards were recovered based on the o-coumaric acid internal standard (38% for lysozyme, 55% for trypsinogen, 44% for aliphatic amino acids and 64% for aromatic+Pro and His amino acids). For Delaware Bay samples, however, proteinac-eous products from thermochemolysis comprised <10% of total hydrolyzable amino acids for POM and <30% for DOM. Two compounds which may originate from nucleic acids, 1,3-dimethyl uracil and 1,3-dimethyl thymine were also observed in Delaware Bay particles and macromolecular DOM. Distributions of thermo-chemolysis TMAH products from Delaware Bay organic matter demonstrated compositional dierences among size fractions with POM containing higher rela-tive abundances of proteins and lipids (fatty acids and sterols) and lower hydrocarbon content than VHDOM or HDOM (Figs. 4 and 5).
Yields of C14±C26 fatty acids released by
thermo-chemolysis were equivalent to fatty acids measured by the traditional solvent extraction±KOH saponi®cation procedure (Fig. 7). However, thermochemolysis released higher amounts of short chain fatty acids (C9±C13) than
the traditional procedure at stations 1, 2 and 4 for VHDOM, stations 1, 3, 4 and 6 for HDOM and stations 1, 3, 4, 5 and 6 for POM (Fig. 8). In particles, the con-centration of the 26:0 acid, a biomarker of terrestrial plants, also diered between the two procedures in the upper river and turbid sites with much higher amounts
Table 2
Carbon to nitrogen ratios andd13C of dissolved and particulate fractions through the Delaware Estuarya
Station Site Distance Salinity C:Na d13C
Descriptor (km) (psu) HDOM VHDOM POM HDOM VHDOM POM
1 Riverine 197 0.11 24.0 15.2 8.8 ÿ24.7 ÿ23.0 ÿ26.8 2 Turbidity maximum 100 0.67 30.0b b 10.4
ÿ25.5b b
a Distance, distance upstream from the bay mouth; C:N
a, atom ratio of organic carbon to total nitrogen; HDOM, 1±30 kDa DOM;
VHDOM, 30 kDa to 0.2mm DOM; chla,chlorophyll.
b >1 kDa fraction only; high particle density precluded ®ltration of the large volume of water required for isolation of >30 kDa
released by thermochemolysis (Fig. 9). In addition, thermochemolysis released higher amounts of the 24:0 acid at stations 2 and 3 but similar amounts at other stations (Fig. 9). Algal production sustained the com-paratively high 24:0 acid content in the lower estuary (stations 4 and 5).
4. Discussion
Isotopic signatures and lignin phenol distributions for POM, VHDOM and HDOM indicated a predominance of terrigenous material (mostly C3 plants) in the river
and turbid regions of the Delaware Estuary and greater planktonic material in the lower estuary and coastal ocean (Table 2). In nearby Chesapeake Bay which also
receives multiple sources of organic matter, thed13C of
>1 kDa DOM ranged fromÿ23 toÿ31% (Guo and Santschi, 1997). Peterson et al. (1994) observedd13C of
bulk DOM to range between ÿ22 and ÿ29% at the marine and freshwater termini of several estuaries. At the riverine station of Delaware Bay, stable carbon iso-topes for POM (d13C=
ÿ26.8%) and HDOM (d13C=
ÿ24.7%) indicate terrestrially derived plant material or freshwater algae (Fry and Sherr, 1984). In addition, lignin phenol concentrations for HDOM and VHDOM were greatest at the riverine site (Fig. 3). At stations 1 and 3, VHDOM d13C values suggest a mixed signal of
terrestrial and autochthonous organic matter, but the presence of coprostanol, a biomarker of mammalian feces, in these samples is consistent with some fraction of this DOM originating from treated sewage (Mannino and Harvey, 1999). The C:Naof POM at station 1 (8.8) is
low compared to terrestrial plants (20±500; Hedges et al., 1997), indicating that plankton contributed to the low C:Naof suspended particles at the riverine site (C:Naof
phytoplankton=6.6; C:Na of bacteria4.3; Lee and
Fuhrman, 1987).
The much higher C:Nain DOM size fractions despite
the similar lignin concentrations in POM and HDOM revealed dierences in composition among size frac-tions. Thermochemolysis products of Delaware Bay organic matter demonstrated compositional dierences among POM and DOM size fractions comparable to conventional analytical methods (Figs. 4 and 5; Mannino
Fig. 3. Total lignin phenol concentrations in POM, VHDOM and HDOM within the Delaware Estuary. Conservative mixing lines based on salinity using stations 1 and 6 as estuarine end-members are shown for comparison. Chl a, cholorophyll a; TSP, total suspended particles.
Table 3
Lignin phenols in Delaware Bay organic matter released by thermochemolysis with TMAHa
Compounds Symbol Molecular ion (BP)
4-Methoxybenzene-ethylene P3 134 (134) 4-Methoxybenzaldehyde P4 136 (135) 1,2-Dimethoxybenzene G1 138 (138) 4-Methoxyacetophenone P5 150 (135) 3,4-Dimethoxytoluene G2 152 (152) 3,4-Dimethoxybenzene-ethylene G3 164 (164) 4-Methoxybenzoic acid methyl ester P6 166 (135) 1,2,3-Trimethoxybenzene S1 168 (168) 4-Methoxybenzene acetic acid
methyl ester
P24 180 (121)
3,4-Dimethoxyacetophenone G5 180 (165) 3,4,5 Trimethoxytoluene S2 182 (182) 3,4-Dimethoxyphenyl
1-methoxy-ethylene
G7/G8 194 (194)
3,4-Dimethoxybenzoic acid methyl ester
G6 196 (196)
3,4,5-Trimethoxyacetophenone S5 210 (195) 3,4,5-Trimethoxybenzoic acid
methyl ester
S6 226 (226)
a Lignin phenol symbols: P#,p-hydroxy; G#, guaiacyl; S#,
and Harvey, 1999, 2000). On a carbon basis, Delaware Bay particles contained greater amounts of proteins and lipids (fatty acids and sterols) and lower hydrocarbon content than VHDOM or HDOM (Figs. 4 and 5; Man-nino and Harvey, 1999, 2000). Thus, thermochemolysis with TMAH can be applied to various organic samples to distinguish relative compositional dierences of major biochemical components (i.e. lignin, lipids and proteins). The distributions of lignin phenols in DOM and POM along the estuary indicated sources from the Delaware River and release from sedimentary material within the turbid region (Fig. 3). Higher particulate lig-nin concentrations within the turbid region coincided with higher C:Naand TSP. Biggs et al. (1983) suggested
that the turbidity maximum in the Delaware Estuary is derived from a combination of ¯occulation induced by gravitational circulation and tidal resuspension of
bot-tom sediments. Dilution or ¯occulation of dissolved lig-nin phenols between the riverine site and the turbidity maximum and release of lignin from resuspended sedi-mentary OM within the turbid region could explain the high concentrations of dissolved lignin observed at sta-tion 3. Cifuentes (1991) found the highest lignin content, on a carbon basis, in suspended particles within this turbid region of Delaware Bay (65±127 km upstream of the bay mouth) and 2±5-fold higher lignin content in surface sediments than in suspended particles. In addi-tion,Spartina alterni¯oradominated marshes within the middle and lower regions of the Delaware Estuary (Roman and Daiber, 1984) could release dissolved lignin into the bay. However, Cifuentes (1991) concluded that marsh vegetation did not contribute signi®cant amounts of lignin to suspended particles in Delaware Bay. Our results also indicate minor inputs of dissolved lignin
from salt marshes within the main-stem of the estuary. Although stable carbon isotopes for POM at stations 2, 3 and 6, HDOM from stations 3 and 6, and VHDOM from station 6 suggest a mixed source composition, lipid biomarkers were consistent with algal, sewage and ter-restrial organics at the turbidity maximum and plank-tonic material at coastal ocean station 6 (Mannino and Harvey, 1999). Microscopic examination of suspended particles revealed predominantly diatoms in the lower estuary (stations 4 and 5) and a mixed plankton assem-blage of diatoms, nano¯agellates and dino¯agellates in the coastal ocean. Nevertheless, lignin phenols were found throughout the estuary, albeit at much lower concentrations in the coastal ocean.
Thermochemolysis with TMAH appears an eective technique for quantifying fatty acids. Substantially higher yields of short chain fatty acids in VHDOM and HDOM were released by thermochemolysis with TMAH versus the traditional procedure (Fig. 8). Although double-bond scission within long chain unsa-turated fatty acids could contribute to the short chain fatty acids observed, thermochemolysis with TMAH of a monounsaturated acid standard (18:19) did not yield
any short chain fatty acid methyl esters. A more likely explanation is that the short chain fatty acids are ter-restrial in origin and are bound with lignin and other compounds within the humic matrix. Longer chain fatty acids have previously been found within humin and humic acids of soils using thermochemolysis with TMAH (Hatcher and Cliord, 1994; Grasset and Ambles, 1998). Formation of geopolymers within soils and natural waters provides a mechanism for preserving otherwise labile organic matter such as fatty acids. The higher amounts of the 26:0 acid released by thermo-chemolysis in the upper and turbid regions of the estu-ary indicate a terrestrial origin associated with more refractory POM which is resistant to solvent extraction and base hydrolysis (Fig. 9). Although the sources of the short chain fatty acids cannot be unequivocally resolved at present, our results imply a terrestrial origin, although not necessarily a vascular plant origin. Soil microbes are a potential source of short chain fatty acids in Delaware Bay organic matter. In coastal marine sediments dominated by autochthonous organic matter, Goni and Hedges (1995) speculated that the short chain fatty acids they observed originated from bacteria.
The thermochemolysis precursor of 4-methoxy-benzene propenoic acid methyl ester, p-coumaric acid, originates from tyrosine. However, the cupric oxide (CuO) oxidation product of p-coumaric has been sug-gested as a terrestrial marker of non-lignin plant tissues (e.g. Opsahl and Benner, 1995; Goni et al., 1998) and was not observed as a CuO oxidation product of pro-teins or tyrosine (Goni and Hedges, 1995). Its high concentration in TMAH treated proteins and aromatic amino acid mixtures as well as its distribution in Dela-ware Bay particles supports a proteinaceous origin in thermochemolysis TMAH derived products (Fig. 6). It seems likely that p-coumaric acid originates from pro-teins and possibly other plant tissues, which would explain its stable carbon isotopic range (ÿ19 toÿ28%)
previously observed in thermochemolysis products from coastal marine sediments (Pulchan et al., 1997). Studies using CuO oxidation have shown that otherp-hydroxy phenols may form from non-lignin tissues (Hedges and Parker, 1976; Goni and Hedges, 1995; Opsahl and Ben-ner, 1995). Proteins and amino acid standards analyzed by thermochemolysis contained only trace amounts of 4-methoxybenzaldehyde and 4-methoxybenzene acetic acid methyl ester, although their respective CuO oxida-tion products were attributed to tyrosine (and alsop -hydroxy lignin for the aldehyde) by Goni and Hedges (1995). Products released by CuO oxidation and ther-mochemolysis with TMAH appear speci®c to the ana-lytical procedure employed, although some similarities are to be expected.
Table 4
Thermochemolysis products in Delaware Bay particles and DOM (mg/mg OC)a
Station
Compound Fraction 1 2 3 4 5 6 7
p-Hydroxy lignin phenols HDOM 2.70 1.10 1.97 3.15 2.59 0.87 1.99
VHDOM 1.97 ndd 1.32 nd 1.09 nd
POM 2.75 1.32 5.03 4.45 1.76 nd 2.21
Guaiacyl lignin phenols HDOM 7.18 2.49 6.94 5.72 4.36 3.54 2.12 VHDOM 7.55 15.3 8.65 3.31 1.05 3.18 POM 3.48 3.78 5.95 0.94 0.49 nd 1.06
Syringyl lignin phenols HDOM 4.63 1.31 1.72 1.95 1.28 0.90 nd VHDOM 1.93 3.64 2.60 1.08 0.32 nd POM 1.16 0.51 1.31 1.00 0.19 1.48 0.95
2-Methyl-acetophenoneb HDOM 4.87 3.28 8.21 7.19 8.02 11.0 3.96
134 (91) VHDOM 3.57 82.6 11.6 7.76 1.51 13.7 POM 4.80 nd 2.30 coele 0.57 coel 2.21
1,4-Dimethoxybenzene HDOM 6.60 1.87 1.18 2.50 2.49 2.12 0.69 138 (123) VHDOM 10.9 18.3 3.20 1.69 0.74 1.50 POM nd nd nd 0.38 0.05 nd 0.46
2,5-Dimethoxytoluene HDOM 3.06 1.12 0.76 1.26 1.10 1.33 0.35 152 (137) VHDOM 5.77 4.85 2.32 1.68 0.91 2.30 POM 0.52 nd 0.25 0.80 nd nd 0.47
1,24-Trimethoxybenzene HDOM 2.18 0.58 0.47 0.84 0.76 0.69 0.42 168 (168) VHDOM 3.48 3.46 3.52 1.16 0.88 0.93 POM 1.98 1.21 2.46 2.10 1.04 6.25 3.99
1,3,5-Trimethoxybenzene HDOM 1.64 0.58 coel 0.57 nd nd nd 168 (168) VHDOM nd nd nd nd nd nd nd POM 1.42 0.63 1.39 coel nd coel 0.50
Other aromaticsc HDOM 24.8 7.88 7.79 11.1 12.0 9.34 5.75
VHDOM 25.8 67.9 25.9 7.07 2.54 4.72 POM 13.4 1.84 9.77 5.52 3.87 6.25 9.22
a Molecular weight and base peak are indicated for individual compounds as in the previous table.
b Tentative identi®cation.
c Excluding lignin phenols, proteinaceous compounds and 2-methyl acetophenone.
d nd, Not detected.
Thermochemolysis with TMAH yields a greater diversity of lignin products than the traditional CuO oxidation procedure, yet total lignin content appears to be equivalent (Hatcher et al., 1995). Overall yields of lignin phenols from CuO oxidation for guaiacyl and syringyl monomers vary from 30 to 90% (Sarkanin and Ludwig, 1971; Opsahl and Benner, 1995), indicating lignin measurements are conservative estimates of the total lignin present. Because lignin monomers are linked
to each other by several types of bonds in addition to the b-O-4 bonds (Crawford, 1981), thermochemolysis with TMAH is also likely to yield comparatively con-servative estimates of total lignin. In order to compare the amount of terrigenous organic matter in DOM fractions from this study with published values from CuO oxidation which are based on the 6 parameter
(sum of vanillin, vanillic acid, acetovanillone, syr-ingealdehyde, syringic acid and acetosyringone; Opsahl
and Benner, 1997),G+Svalues (sum of guaiacyl and
syringyl phenols listed in Table 3) were calculated and compared to the G+Scontent in the Delaware River
(1180 and 948 mg/100 mg OC for HDOM and VHDOM, respectively; [%TOM=(G+Ssample/G+S
river)*100]). Estimates of TOM are typically very sensi-tive to selected terrestrial endmember values and should be interpreted with caution (Prahl et al., 1994). The6
aldehydes were not found in POM or DOM samples, and only trace amounts of the methylated forms of syr-ingic acid and acetosyringone were measured. Terrige-nous material comprised >100% of VHDOM in the turbid region of the Delaware Estuary due to contribu-tions from resuspended sedimentary material within this region (Fig. 10). Although this is an obvious over-estimation of TOM, the low DOC content in VHDOM may have resulted in greater sensitivity of VHDOM to additional inputs than HDOM. Nevertheless, the strong relation of VHDOM lignin concentrations (G+S and total lignin) with salinity (adjusted R2=0.94; P=0.0008) demonstrated conservative mixing of lignin phenols with salinity which can be inferred to signify a decrease in TOM with increasing salinity. Terrigenous organics comprised 12% of HDOM at coastal ocean station 7 and up to 73% at station 3. The high propor-tion of TOM at stapropor-tion 6 (38% of HDOM) along with a lower d13C (
ÿ24.9%) relative to stations 5 and 7 may have resulted from selective degradation of planktonic-derived HDOM. In comparison, TOM comprised 0.7% of UDOM from the Paci®c Ocean and 2.4% of UDOM from the Atlantic Ocean (Opsahl and Benner, 1997).
Through the high molecular weight DOM pool, Delaware Bay exports approximately 2.3108g of
lig-nin yearÿ1 (7
108 g of total lignin yearÿ1) into the
coastal ocean, based on the estimated concentration of 0.85 mg lignin Lÿ1 (2.8mg total lignin lÿ1) at the bay
mouth (Table 5). Our measurements for total lignin phenols in POM (0.15 to 1.23 mg/100 mg OC) fall within the lower range of previous measurements of lig-nin oxidation products in Delaware Bay suspended POM (<0.1 to 4.7 mg/100 mg OC), but most similar to spring values (<0.1 to 1.4 mg/100 mg OC; Cifuentes, 1991). The highest concentration of lignin phenols in
Fig. 7. Methods comparison of Delaware Bay particulate long chain fatty acid content by thermochemolysis with tetra-methylammonium hydroxide (TMAH) and traditional solvent extraction±saponi®cation procedure (solvent; Mannino and Harvey, 1999). Error bars indicate1 S.D. for one duplicate analysis for the TMAH procedure.
POM were observed during the fall, indicating higher inputs of TOM during this period of low phytoplankton production and low river discharge (Cifuentes, 1991). Assuming that particulate and dissolved lignin
con-centrations are similar as observed in this study, then our estimated annual ¯ux of dissolved lignin to the ocean is likely to be a conservative estimate with an actual ¯ux up to several fold greater. For comparison, the ¯ux of dissolved lignin from the Amazon River (which accounts for 15% of total DOC riverine input to the ocean; Richey et al., 1986), has been estimated at 1.21011 g yearÿ1 (Ertel et al., 1986). Because XAD
resins used to isolate lignin may be less ecient than ultra®ltration, measurements from the Amazon River may underestimate the ¯ux of lignin to the Atlantic Ocean (Hedges et al., 1997). Opsahl and Benner (1998) found that 90% of lignin oxidation products occurred in the >1 kDa fraction, but photo-oxidation resulted in 80% of lignin oxidation products to photodegrade to the LDOM fraction. In terms of the Delaware Estuary, photo-oxidation is likely to be important in the coastal ocean, butlessimportant in the bay due to higher par-ticle load from suspended sediments and algal produc-tion. Although the Delaware Bay watershed is dominated by angiosperms (Cifuentes, 1991), lower yields of syringyl lignin phenols compared to guaiacyl phenols indicated that dissolved TOM in Delaware Estuary was highly degraded (Table 4). Attributing changes in lignin composition along the estuarine gra-dient to either biotic degradation or photo-oxidation is beyond the scope of this study. Nevertheless, we can speculate that physical processes, speci®cally river discharge and resuspension of bottom sediments, would have the greatest impact on lignin phenol concentra-tions in the Delaware Estuary with both biotic degra-dation and photo-oxidation in¯uencing lignin composition.
Fig. 9. Comparison of (A) 24:0 and (B) 26:0 fatty acids in Delaware Bay POM released by TMAH and the solvent extraction procedure. Error bars indicate 1 S.D. for one duplicate analysis for the TMAH procedure.
Table 5
Dissolved lignin and terrigenous organic matter (TOM) ¯ux from the Delaware Bay to the Atlantic Oceana
Lignin G+S
TOM TOM/ DOCb
Lignin ¯ux
TOM ¯ux
Fraction (mg/L) (%) (%) (g yÿ1) (g OC yearÿ1)
HDOM 0.85 14.0 4.0 2.1108 1.8
1010
VHDOM 0.077 20.9 0.3 1.9107 2.1
109
UDOM 0.92 14.3 4.3 2.3108 2.0
1010
a Values are based on the estimated concentration of lignin
phenols at the bay mouth using salinity derived conservative mixing curves and mean ¯ow of 8000 m3sÿ1at the bay mouth
(Garvine, 1991). TOM estimated from Delaware River end-member lignin content (G+S=mg G+S lignin phenols per 100
mg OC; 1180 for HDOM and 948 for VHDOM).
b TOM/DOC, percentage of high molecular weight dissolved
TOM relative to total DOC.
Fig. 10. Terrigenous organic matter (TOM) in the Delaware Estuary, calculated as theG+S parameter and the average
lignin content in the Delaware River (%TOM=[G+Ssample/
In contrast to rivers with high discharge such as the Amazon, Mississippi and Lena Rivers where TOM dominates the composition of DOM (Opsahl and Ben-ner, 1997; Kattner et al., 1999), only about 14% of UDOM at the mouth of the Delaware Bay appears to be terrestrially derived (Table 5). In pelagic waters of the Atlantic Ocean, TOM comprised 2.4% of UDOM (Opsahl and Benner, 1997), compared to 12±38% for the inner-shelf waters of the Delaware system (Fig. 10). Only 4% of total DOC entering the coastal ocean is terrestrially derived high molecular weight DOM (Table 5). The annual TOM ¯ux from Delaware Bay to the coastal ocean was estimated at 2.01010 g OC yearÿ1
(equivalent to 0.2±0.7% of the dissolved TOM ¯ux from the Arctic Ocean to the Atlantic Ocean through the East Greenland Current; Opsahl et al., 1999). Although additional measurements throughout the year are nee-ded to obtain more precise calculations, our observa-tions suggest that inputs of TOM from temperate watersheds such as the Delaware Estuary (0.01% of global DOC riverine ¯ux) can be signi®cant on a regio-nal and likely global scale.
Acknowledgements
We thank the captain and crew of the R/V Cape Henlopen, D.L. Kirchman for the invitation to partici-pate in work on the Delaware Estuary and for providing chlorophyll a data, P.G. Hatcher and H. Knicker for advice and help related to thermochemolysis with TMAH procedure, S.A. Macko for carbon and stable carbon isotope measurements, and R. Benner for advice on ultra®ltration. Two anonymous reviewers provided constructive comments which improved an earlier ver-sion of this manuscript. This work was supported by NSF (OCE-9617892) and the Donors of the Petroleum Research Fund of the American Chemical Society. Contribution No. 3351, University of Maryland Center for Environmental Science.
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