terrigenous dissolved organic matter along an estuarine

Terrigenous dissolved organic matter along an estuarinegradient 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 organicmatter (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±0.2 mm (VHDOM) nominal molecular weightfractions. 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 terrigenousmaterial in the river and turbid region and a predominantly algal/planktonic signal in the lower estuary and coastalocean. 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 andturbid 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 highmolecular weight DOM comprised 4% of total DOC. The annual ¯ux of TOM from the Delaware Estuary to thecoastal ocean was estimated at 2.0�1010 g OC yearÿ1 and 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 etal., 1995), but much less is known about the dynamics of

dissolved organic matter (DOM). Rivers transport0.25�1015 g of dissolved organic carbon (DOC) per yearto the ocean (Meybeck, 1982), yet the terrestrial con-

tribution to this overall ¯ux is not well known (Hedgeset 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 etal., 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 bethe 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 estuarineenvironments (Moran et al., 1991; Argyrou et al., 1997).The presence of dissolved lignin oxidation productsalong 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 etal., 1991; Moran and Hodson, 1994). Observations of

0146-6380/00/$ - see front matter # 2000 Elsevier Science Ltd. All rights reserved.

PI I : S0146-6380(00 )00099-1

Organic Geochemistry 31 (2000) 1611±1625

www.elsevier.nl/locate/orggeochem

* Corresponding author. Tel.: +1-410-326-7206; fax: +1-

410-326-7341.

E-mail address: [email protected] (H.R. Harvey).

dissolved lignin phenols in the Arctic Ocean (Kattner etal., 1999; Opsahl et al., 1999), Gulf of Mexico (Bianchiet al., 1997) and equatorial Paci®c (Meyers-Schulte andHedges, 1986) also illustrate contributions of TOM to

oceanic environments. Recent evidence of lignin phenolsin UDOM (>1 kDa DOM) from several sites anddepths within the tropical Paci®c Ocean and Atlantic

Ocean demonstrates widespread distribution of TOMwithin oceanic DOM (Opsahl and Benner, 1997). Sinceterrestrial ecosystems are highly productive, their con-

tribution to oceanic DOM could be substantial due tothe refractory nature of TOM, particularly lignin, andmay in¯uence global carbon cycling.

To determine the contribution of TOM to highmolecular weight DOM within a temperate estuary andits export the coastal ocean, we measured the abundanceand distribution of lignin phenols along the salinity

gradient of the Delaware Estuary, USA. The relativelynew procedure, thermochemolysis with tetra-methylammonium hydroxide (TMAH), was applied to

release lignin phenols and other potential biomarkersfrom the macromolecular matrix. Previous studies ofd13C and d15N POM in Delaware Bay indicate a dom-

inance of planktonic material, although the presence oflignin phenols throughout the year within suspendedparticles also suggest watershed inputs of terrestrial

material (Cifuentes et al., 1988; Cifuentes, 1991). Thecontribution of TOM to the DOM pool and its exportto the coastal ocean through the dissolved pool has notbeen previously examined. Constraining the inputs of

TOM to the ocean is critical to understanding theocean's carbon cycle.

2. Methods

2.1. Sampling and bulk analyses

Seven stations were sampled along a transect of theDelaware Estuary from riverine waters to the coastal

ocean (Fig. 1). Sample collection and ®ltration weredescribed 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 0.2 mm pore size. The ®ltrate was

then separated into three nominal size fractions: 30kDa±0.2 mm [very high molecular weight (VHDOM)],1±30 kDa [high molecular weight (HDOM)] and <1

kDa low molecular weight (LDOM)] using an AmiconDC-10L tangential ¯ow ultra®ltration unit with theS10Y30 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. Immediatelyfollowing initial fractionation and concentration, the

two high molecular weight fractions were desalted usingthe Amicon unit with 6±9 l of low organic deionizedwater. Samples for DOC analysis were collected fromthe <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 ShimadzuTOC 5000 (Benner and Strom, 1993). Remaining

sample retentates were stored frozen, concentratedfurther by rotary evaporation and lyophilized to drypowders. 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 450�C) What-

man GF/F ®lters by vacuum ®ltration. Organic carbonand total nitrogen content were measured using anExeter Analytical CHN analyzer for POM and DOMsamples. Stable carbon isotopes were quanti®ed on

POM and DOM fractions as CO2 on a MicromassOptima instrument interfaced with a CHN elementalanalyzer (Fry et al., 1992; Macko et al., 1997). Pre-

cision of the method is typically �0.1%. In thelaboratory samples are commonly measured against atank of carbon dioxide which has been calibrated

against NBS 22 which is referenced to the PeeDeeBelemnite standard.

Fig. 1. Map of the Delaware Estuary with station locations.

1612 A. Mannino, H.R. Harvey /Organic Geochemistry 31 (2000) 1611±1625

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 acidand 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 55�C overnight. Internal standardswere then placed onto each ®lter, and ®lters were sub-

sequently dried under vacuum, sliced into small stripsand placed inside ampules. All ampules were evacuatedfor 2 h or longer, ¯ame sealed and placed within an

oven to react at 250�C for 30 min (McKinney et al.,1995). Ampules were subsequently cooled to room tem-perature, cracked open, and extracted three times withadditions of 1 ml of CH2Cl2. Combined extracts were

placed in 4 ml amber vials, dried under a gentle streamof N2 and 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 (0.32mm I.D., 0.25 mm ®lm thickness). Hydrogen served as

the carrier gas (2 ml minÿ1), and a temperature programof 10�C minÿ1 from 50�C to 120�C followed by 3�Cminÿ1 to 200�C and thereafter 4�C minÿ1 to 300�C was

used. Reagent blanks processed simultaneously witheach sample group indicated no contamination fromreagents or handling. Samples were also analyzed byGC±MS (HP-5890II GC coupled to a HP-5970B MSD),

and individual spectra were compared with referencemass spectra for compound identi®cation (Hatcher,pers. comm.; NIST, 1998). Helium served as the carrier

gas for GC±MS, and the temperature program abovewas used. The MSD was operated in electron impactmode at 70 eV with acquisition over 50-600 a.m.u.

Molecular weights of select compounds were con®rmedby GC±MS using positive chemical ionization with CH4

(1.7 torr) as the ionizing gas (HP-5890II GC coupled toa 5989A MS).

The thermochemolysis reaction is a thermally assistedbase-catalyzed reaction which cleaves ester and etherbonds including the b-O-4 bonds within lignin (see Fil-

ley et al., 1999, for details) with subsequent methylationof carboxylic and acidic hydroxy groups, including bothphenolic and non-acidic side chain hydroxyls (Cli�ord

et al., 1995; del Rio and Hatcher, 1996). The proposedmechanism for b-O-4 bond cleavage involves formationof an intramolecular epoxide following deprotonation

of the side chain a or g alcohols (with tetra-methylammonium alkoxy salts as intermediates) whichact as nucleophiles to displace the phenoxide (Filley etal., 1999). Functional groups of non-lignin compounds

such as fatty acids and sterols are also methylated byTMAH. To compare the relative response of o-coumaricacid with phenolic and non-phenolic standards, ferulic

acid, vanillic acid and diphenylamine (Sigma) wereanalyzed individually in the presence of o-coumaric acidand nonadecanoic acid. These analyses demonstratedthat o-coumaric acid is quantitatively recovered, with

coe�cients of variation ranging from 3.5 to 13%. On amass basis, however, the o-coumaric acid yielded lowerresponses than the nonadecanoic acid. Relative respon-

ses of the nonadecanoic acid and 5a-cholestane wereequivalent, indicating e�cient esteri®cation of fattyacids. 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+S components (sum of guaiacyl and syringyl lignin

phenol yields in mg/100 mg OC) and <26% for totalfatty acids. Proteins including RuBPcase, Trypsinogenand Lysozyme as well as three amino acid mixtures (allprotein amino acids, aromatics plus proline and histi-

dine, and aliphatic amino acids) were also reacted withTMAH to examine the contribution of proteinaceousmaterial 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) whichindicates a mass balance comparable to other publishedresults (81±128%; Guo and Santschi, 1996; Benner etal., 1997). The molecular weight distribution of ultra-

®ltered DOC was non-conservative in the DelawareEstuary (Fig. 2). HDOM-C concentration peaked at theturbidity maximum (133 mM C; 61% of DOC) and was

lowest in the coastal ocean (42 mM C; 25% of DOC).VHDOM-C concentration was low (<5 mM C) andvariable throughout the estuary, comprising <3% of

total DOC. POC was highest at the turbidity maximum(318 mM) and lowest in the coastal ocean (53 mM).The carbon to nitrogen atom ratio (C:Na) varied

along the estuarine gradient for POM and both high

molecular weight DOM fractions. Carbon rich (nitrogenpoor) material was found in the upper and turbidregions of the estuary, whereas nitrogen rich material

was observed within the lower estuary and coastal ocean(Table 2). POM was enriched in nitrogen compared toDOM 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:Na for HDOM was24 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

followed similar patterns along the Delaware Estuary

A. Mannino, H.R. Harvey /Organic Geochemistry 31 (2000) 1611±1625 1613

(Table 2). The most enriched d13C 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 heaviest d13C values,

ranging from ÿ23.2% at station 6 to ÿ19.6% down-stream of the chlorophyll maximum. The variation inthe d13C signature along the estuary was smaller forHDOM, which ranged from ÿ25.5% at the turbidity

maximum to ÿ22.2% 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 oflignin phenols was observed at the riverine site forVHDOM and HDOM. The turbid sites (stations 2 and3) contained equivalent amounts of particulate lignin,

even though total suspended particle (TSP) load wasmore 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 inTSP and an increase in chlorophyll a. Lignin phenols inVHDOM followed the salinity based conservative mix-

ing line very closely, except for a slightly higher value atstation 7. Downstream of station 3, HDOM lignin phe-nol concentrations declined in tandem with conservativemixing of river and coastal ocean waters. A list of lignin

derived thermochemolysis products is shown in Table 3(Cli�ord et al., 1995; Hatcher et al., 1995; McKinney etal., 1995). In addition to lignin phenols, thermo-

chemolysis with TMAH released a complex suite ofother molecules from Delaware Bay POM and DOM(Figs. 4 and 5).

On a carbon basis, VHDOM contained similar orhigher amounts of lignin than POM or HDOM. Guaia-cyl structures (3,4-dimethoxyphenyls) dominated the

lignin composition of macromolecular DOM, especiallyfor VHDOM (Table 4). In contrast, POM contained amixture of p-hydroxy, guaiacyl and syringyl phenolswith the syringyl forms as least abundant. Ratios of p-

hydroxy phenols to guaiacyl phenols (P/G) did not varyfor 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 of p-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.

Fig. 2. Distributions of organic carbon in particles and high

molecular weight dissolved fractions in Delaware Bay and the

coastal ocean. POM, particulate organic matter; HDOM, 1±30

kDa dissolved organic matter; VHDOM, 30 kDa±0.2 mm dis-

solved organic matter.


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 upstreamsites. S/G declined with increasing salinity for HDOMbut 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 apredominance 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 trimethoxybenzenes (del Rio et al., 1998).Several other aromatic compounds which appear to

be of non-lignin origin were found in Delaware BayPOM 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 23 mgmg OCÿ1, respectively). Other aromatic compounds,excluding lignin phenols, proteinaceous-derived com-

pounds and methyl acetophenone, showed variable dis-tributions throughout the estuary (Table 4). Onepotential thermochemolysis product of hydroquinone,

1,4-dimethoxybenzene (Hatcher et al., 1995), declined inconcentration between the upper and turbid regions ofthe estuary and the coastal ocean. 1,2,4-Trimethoxy-

benzene, believed to originate from polysaccharidessuch 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 terrestrialorigin. In particles, however, the greatest amounts of1,2,4-trimethoxybenzene were observed in the coastalocean. Small amounts of 1,3,5-trimethoxybenzene were

detected in HDOM and POM and could originate fromtannin (Pulchan et al., 1997) and cutan (McKinney etal., 1996) of vascular plants.

Thermochemolysis revealed a suite of aromatic andnon-aromatic proteinaceous-derived products in all

three size fractions within Delaware Bay organic matter.Most of the aromatic compounds were identi®ed (Table1) and appeared to have a proteinaceous origin based on

protein and amino acid standards (Fig. 6). Severalnitrogen containing aliphatic products have yet to beidenti®ed. Although thermochemolysis appears to liber-

ate many proteinaceous-derived products, yields mustbe interpreted with caution as only 38 to 64% of theprotein or amino acid standards were recovered basedon the o-coumaric acid internal standard (38% for

lysozyme, 55% for trypsinogen, 44% for aliphaticamino acids and 64% for aromatic+Pro and His aminoacids). 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-dimethylthymine were also observed in Delaware Bay particlesand macromolecular DOM. Distributions of thermo-chemolysis TMAH products from Delaware Bay

organic matter demonstrated compositional di�erencesamong size fractions with POM containing higher rela-tive abundances of proteins and lipids (fatty acids and

sterols) and lower hydrocarbon content than VHDOMor HDOM (Figs. 4 and 5).Yields of C14±C26 fatty acids released by thermo-

chemolysis were equivalent to fatty acids measured bythe traditional solvent extraction±KOH saponi®cationprocedure (Fig. 7). However, thermochemolysis released

higher amounts of short chain fatty acids (C9±C13) thanthe traditional procedure at stations 1, 2 and 4 forVHDOM, stations 1, 3, 4 and 6 for HDOM and stations1, 3, 4, 5 and 6 for POM (Fig. 8). In particles, the con-

centration of the 26:0 acid, a biomarker of terrestrialplants, also di�ered between the two procedures in theupper river and turbid sites with much higher amounts

Table 2

Carbon to nitrogen ratios and d13C 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 ÿ26.82 Turbidity maximum 100 0.67 30.0b b 10.4 ÿ25.5b b ÿ26.23 Turbid 66.4 9.07 17.3 19.1 9.3 ÿ24.4 ÿ22.6 ÿ24.24 Chl a maximum 45 13.2 17.9 13.5 6.5 ÿ23.8 ÿ19.8 ÿ20.55 High Chl a 28.4 22.6 18.7 13.0 7.0 ÿ22.7 ÿ19.6 ÿ18.96 Coastal ocean ÿ16.3 29.4 16.9 23.9 7.1 ÿ24.9 ÿ23.2 ÿ24.57 Coastal ocean ÿ51.5 29.5 19.7 14.0 7.4 ÿ22.2 ÿ21.1 ÿ23.5

a Distance, distance upstream from the bay mouth; C:Na, atom ratio of organic carbon to total nitrogen; HDOM, 1±30 kDa DOM;

VHDOM, 30 kDa to 0.2 mm DOM; chl a, chlorophyll.b >1 kDa fraction only; high particle density precluded ®ltration of the large volume of water required for isolation of >30 kDa

fraction.


released by thermochemolysis (Fig. 9). In addition,thermochemolysis released higher amounts of the 24:0acid 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 forPOM, VHDOM and HDOM indicated a predominanceof terrigenous material (mostly C3 plants) in the river

and turbid regions of the Delaware Estuary and greaterplanktonic material in the lower estuary and coastalocean (Table 2). In nearby Chesapeake Bay which also

receives multiple sources of organic matter, the d13C of>1 kDa DOM ranged from ÿ23 to ÿ31% (Guo andSantschi, 1997). Peterson et al. (1994) observed d13C of

bulk DOM to range between ÿ22 and ÿ29% at themarine and freshwater termini of several estuaries. Atthe riverine station of Delaware Bay, stable carbon iso-

topes for POM (d13C=ÿ26.8%) and HDOM (d13C=ÿ24.7%) indicate terrestrially derived plant material orfreshwater algae (Fry and Sherr, 1984). In addition,

lignin phenol concentrations for HDOM and VHDOMwere greatest at the riverine site (Fig. 3). At stations 1and 3, VHDOM d13C values suggest a mixed signal ofterrestrial and autochthonous organic matter, but the

presence of coprostanol, a biomarker of mammalianfeces, in these samples is consistent with some fraction ofthis DOM originating from treated sewage (Mannino

and Harvey, 1999). The C:Na of POM at station 1 (8.8) islow compared to terrestrial plants (20±500; Hedges et al.,1997), indicating that plankton contributed to the low

C:Na of suspended particles at the riverine site (C:Na ofphytoplankton=6.6; C:Na of bacteria�4.3; Lee andFuhrman, 1987).

The much higher C:Na in DOM size fractions despitethe similar lignin concentrations in POM and HDOMrevealed di�erences in composition among size frac-tions. Thermochemolysis products of Delaware Bay

organic matter demonstrated compositional di�erencesamong POM and DOM size fractions comparable toconventional 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#,

syringyl.


and Harvey, 1999, 2000). On a carbon basis, Delaware

Bay particles contained greater amounts of proteins andlipids (fatty acids and sterols) and lower hydrocarboncontent than VHDOM or HDOM (Figs. 4 and 5; Man-

nino and Harvey, 1999, 2000). Thus, thermochemolysiswith TMAH can be applied to various organic samples todistinguish relative compositional di�erences 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 materialwithin the turbid region (Fig. 3). Higher particulate lig-nin concentrations within the turbid region coincidedwith higher C:Na and TSP. Biggs et al. (1983) suggested

that the turbidity maximum in the Delaware Estuary isderived from a combination of ¯occulation induced bygravitational circulation and tidal resuspension of bot-

tom sediments. Dilution or ¯occulation of dissolved lig-

nin phenols between the riverine site and the turbiditymaximum 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 ofthe bay mouth) and 2±5-fold higher lignin content insurface sediments than in suspended particles. In addi-

tion, Spartina alterni¯ora dominated marshes within themiddle and lower regions of the Delaware Estuary(Roman and Daiber, 1984) could release dissolved lignininto the bay. However, Cifuentes (1991) concluded that

marsh vegetation did not contribute signi®cant amountsof lignin to suspended particles in Delaware Bay. Ourresults also indicate minor inputs of dissolved lignin

Fig. 4. Gas chromatogram of thermochemolysis TMAH products from station 1 POM (¯ame ionization detection). Peaks are labeled

according to IDs in Tables 1 and 3. A1, 2-methyl acetophenone?; A2, 1,4-dimethoxybenzene; A3, 2,5-dimethoxytoluene; A4, 1,2,4-

trimethoxybenzene; A5, 1,3,5-trimethoxybenzene. Fatty acid methyl esters are labeled as carbon number # double bonds; i, iso; a,

anteiso; n, normal; br, branched. IS, internal standard: methylated forms of o-coumaric acid (cis and trans isomers at 20.8 and 25.2

min, respectively) and nonadecanoic acid (42.6 min) and 5a-cholestane (54.9 min).


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, lipidbiomarkers 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 suspendedparticles revealed predominantly diatoms in the lowerestuary (stations 4 and 5) and a mixed plankton assem-

blage of diatoms, nano¯agellates and dino¯agellates inthe coastal ocean. Nevertheless, lignin phenols werefound throughout the estuary, albeit at much lower

concentrations in the coastal ocean.Thermochemolysis with TMAH appears an e�ective

technique for quantifying fatty acids. Substantially

higher yields of short chain fatty acids in VHDOM andHDOM were released by thermochemolysis withTMAH versus the traditional procedure (Fig. 8).Although double-bond scission within long chain unsa-

turated fatty acids could contribute to the short chainfatty acids observed, thermochemolysis with TMAH ofa monounsaturated acid standard (18:1�9) did not yield

any short chain fatty acid methyl esters. A more likelyexplanation 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 fattyacids have previously been found within humin andhumic acids of soils using thermochemolysis withTMAH (Hatcher and Cli�ord, 1994; Grasset and

Ambles, 1998). Formation of geopolymers within soilsand natural waters provides a mechanism for preservingotherwise 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 extractionand base hydrolysis (Fig. 9). Although the sources of theshort chain fatty acids cannot be unequivocally resolved

at present, our results imply a terrestrial origin,although not necessarily a vascular plant origin. Soilmicrobes are a potential source of short chain fattyacids in Delaware Bay organic matter. In coastal marine

sediments dominated by autochthonous organic matter,Goni and Hedges (1995) speculated that the short chainfatty acids they observed originated from bacteria.

Fig. 5. Gas chromatogram of thermochemolysis TMAH products from station 2 HDOM. See Fig. 4 for details.


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) andwas not observed as a CuO oxidation product of pro-teins or tyrosine (Goni and Hedges, 1995). Its high

concentration in TMAH treated proteins and aromaticamino acid mixtures as well as its distribution in Dela-ware Bay particles supports a proteinaceous origin inthermochemolysis TMAH derived products (Fig. 6). It

seems likely that p-coumaric acid originates from pro-teins and possibly other plant tissues, which wouldexplain its stable carbon isotopic range (ÿ19 to ÿ28%)

previously observed in thermochemolysis products fromcoastal marine sediments (Pulchan et al., 1997). Studies

using CuO oxidation have shown that other p-hydroxyphenols may form from non-lignin tissues (Hedges andParker, 1976; Goni and Hedges, 1995; Opsahl and Ben-

ner, 1995). Proteins and amino acid standards analyzedby thermochemolysis contained only trace amounts of4-methoxybenzaldehyde and 4-methoxybenzene acetic

acid methyl ester, although their respective CuO oxida-tion products were attributed to tyrosine (and also p-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 similaritiesare 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.e coel, Coelution of peaks.


Thermochemolysis with TMAH yields a greaterdiversity of lignin products than the traditional CuO

oxidation procedure, yet total lignin content appears tobe equivalent (Hatcher et al., 1995). Overall yields oflignin phenols from CuO oxidation for guaiacyl andsyringyl monomers vary from 30 to 90% (Sarkanin and

Ludwig, 1971; Opsahl and Benner, 1995), indicatinglignin measurements are conservative estimates of thetotal lignin present. Because lignin monomers are linked

to each other by several types of bonds in addition tothe b-O-4 bonds (Crawford, 1981), thermochemolysis

with TMAH is also likely to yield comparatively con-servative estimates of total lignin. In order to comparethe amount of terrigenous organic matter in DOMfractions 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

Fig. 6. Gas chromatograms of thermochemolysis TMAH products from protein and amino acid standards: (A) lysozyme, (B) ali-

phatic amino acids (Ala, Gly, Ser, Thr, Val, Leu, Ile, Cys, Asp, Asn, Met, Glu, Gln, Lys and Arg) and (C) aromatic+other amino

acids (Tyr, Phe, Trp, Pro and His). Peaks are labeled according to IDs listed in Table 1. 6c, cis-4-methoxybenzene-propenoic acid ME.


and Benner, 1997), �G+S values (sum of guaiacyl andsyringyl phenols listed in Table 3) were calculated andcompared to the �G+S content in the Delaware River

(1180 and 948 mg/100 mg OC for HDOM andVHDOM, respectively; [%TOM=(�G+S sample/�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). The �6

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 theturbid region of the Delaware Estuary due to contribu-

tions from resuspended sedimentary material within thisregion (Fig. 10). Although this is an obvious over-estimation of TOM, the low DOC content in VHDOMmay have resulted in greater sensitivity of VHDOM to

additional inputs than HDOM. Nevertheless, the strongrelation of VHDOM lignin concentrations (G+S andtotal lignin) with salinity (adjusted R2=0.94;

P=0.0008) demonstrated conservative mixing of ligninphenols with salinity which can be inferred to signify adecrease in TOM with increasing salinity. Terrigenous

organics comprised 12% of HDOM at coastal oceanstation 7 and up to 73% at station 3. The high propor-tion of TOM at station 6 (38% of HDOM) along with a

lower d13C (ÿ24.9%) relative to stations 5 and 7 mayhave 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 2.3�108 g of lig-

nin yearÿ1 (7�108 g of total lignin yearÿ1) into thecoastal ocean, based on the estimated concentration of0.85 mg lignin Lÿ1 (2.8 mg total lignin lÿ1) at the bay

mouth (Table 5). Our measurements for total ligninphenols in POM (0.15 to 1.23 mg/100 mg OC) fallwithin 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 tospring 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 indicate �1 S.D. for one duplicate

analysis for the TMAH procedure.

Fig. 8. Comparison of short chain fatty acids released by

TMAH and the solvent extraction procedures from Delaware

Bay (A); POM, (B) VHDOM and (C) HDOM. Error bars

indicate �1 S.D. for one POM and one HDOM duplicate

analyses for the TMAH procedure.


POM were observed during the fall, indicating higherinputs of TOM during this period of low phytoplanktonproduction and low river discharge (Cifuentes, 1991).Assuming that particulate and dissolved lignin con-

centrations are similar as observed in this study, thenour estimated annual ¯ux of dissolved lignin to theocean is likely to be a conservative estimate with anactual ¯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 tothe ocean; Richey et al., 1986), has been estimated at

1.2�1011 g yearÿ1 (Ertel et al., 1986). Because XADresins used to isolate lignin may be less e�cient thanultra®ltration, measurements from the Amazon River

may underestimate the ¯ux of lignin to the AtlanticOcean (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 in80% of lignin oxidation products to photodegrade tothe LDOM fraction. In terms of the Delaware Estuary,photo-oxidation is likely to be important in the coastal

ocean, but less important 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), loweryields of syringyl lignin phenols compared to guaiacylphenols indicated that dissolved TOM in Delaware

Estuary was highly degraded (Table 4). Attributingchanges in lignin composition along the estuarine gra-dient to either biotic degradation or photo-oxidation

is beyond the scope of this study. Nevertheless, wecan speculate that physical processes, speci®cally riverdischarge and resuspension of bottom sediments, wouldhave the greatest impact on lignin phenol concentra-

tions in the Delaware Estuary with both biotic degra-dation and photo-oxidation in¯uencing lignincomposition.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 2.1�108 1.8�1010VHDOM 0.077 20.9 0.3 1.9�107 2.1�109UDOM 0.92 14.3 4.3 2.3�108 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 m3 sÿ1 at 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 the �G+S parameter and the average

lignin content in the Delaware River (%TOM=[�G+S sample/

�G+S Delaware River] *100).


In contrast to rivers with high discharge such as theAmazon, Mississippi and Lena Rivers where TOMdominates 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 tobe terrestrially derived (Table 5). In pelagic waters ofthe Atlantic Ocean, TOM comprised 2.4% of UDOM

(Opsahl and Benner, 1997), compared to 12±38% forthe 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 (Table5). The annual TOM ¯ux from Delaware Bay to thecoastal ocean was estimated at 2.0�1010 g OC yearÿ1

(equivalent to 0.2±0.7% of the dissolved TOM ¯ux fromthe Arctic Ocean to the Atlantic Ocean through the EastGreenland Current; Opsahl et al., 1999). Althoughadditional measurements throughout the year are nee-

ded to obtain more precise calculations, our observa-tions suggest that inputs of TOM from temperatewatersheds 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 CapeHenlopen, D.L. Kirchman for the invitation to partici-pate in work on the Delaware Estuary and for providingchlorophyll a data, P.G. Hatcher and H. Knicker for

advice and help related to thermochemolysis withTMAH procedure, S.A. Macko for carbon and stablecarbon isotope measurements, and R. Benner for advice

on ultra®ltration. Two anonymous reviewers providedconstructive comments which improved an earlier ver-sion of this manuscript. This work was supported by

NSF (OCE-9617892) and the Donors of the PetroleumResearch Fund of the American Chemical Society.Contribution No. 3351, University of Maryland Centerfor Environmental Science.

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