Organic Geochemistry 34 (2003) 165–183
www.elsevier.com/locate/orggeochem
Analysis of d13C, d15N, and d34S in organic matter from the
biominerals of modern and fossil Mercenaria spp.
Thomas H. O’Donnella,*, Stephen A. Mackob, Janel Choub,
Kathy L. Davis-Harttenc, John F. Wehmillerc
a
Academy of Natural Sciences, Philadelphia, PA 19103, USA
Department of Environmental Sciences, University of Virginia, Charlottesville, VA 22902, USA
c
Department of Geology, University of Delaware, Newark, DE 19176, USA
b
Abstract
The response of primary producers to changes in environmental and sea-level conditions is an important topic in the
study of estuarine ecosystems. Currently, sea levels are rising, and North American estuaries are in declining ecological
health (USEPA, 2002 National Coastal Condition Report. USEPA, Washington, DC). This investigation examines the
base of the estuarine food web by evaluating the diets of modern and fossil Mercenaria spp. These clams are infaunal,
primary consumers that feed on suspended organic matter. Proteinaceous materials secreted within the biominerals of
shell-building organisms are derived from the same diet sources as soft tissue and, therefore, are also capable of
recording diet information. We have developed an empirical relationship between the isotopic composition of shell
organic matter and soft tissue, which allows food web analyses in fossils to proceed in the same manner as modern
food web studies. The tissue-shell for d13C, d15N, and d34S is 0.1, 0.7, and 1.8%. Isotopic analyses of modern Mercenaria spp. shells from coastal Virginia (d13C: 12.8 to 15.9%, d15N: 11.3 to 13.7%), and the Gulf of Mexico coast of
Florida (d13C: 19.4 to 21.0%, d15N: 7.2–9.9%) demonstrate that diets are derived from phytoplankton and local
salt marsh plants. Shell organic-matter d13C varies from 13.1 to 27.5% and d15N varies from 2.4 to 9.8% in Quaternary Mercenaria spp. from coastal Virginia, North and South Carolina, and Georgia. These data demonstrate that
past diet sources have changed in space and time. The fact that past sea-level changes have been accompanied by shifts
in local primary production is evidence that significant changes might also occur in association with presently rising sea
levels.
# 2002 Elsevier Science Ltd. All rights reserved.
1. Introduction
Stable isotope investigations have made significant
contributions to the understanding of food web
dynamics in aquatic systems. This is largely due to the
discovery that different types of primary production
have different isotopic compositions, and that some
isotopes in the diets of animals are fractionated to a
predictable degree by metabolic processes (Michener
and Schell, 1994). Carbon, nitrogen, and sulfur isotopes
when used together provide methods to identify important primary producers in estuarine systems, to deter-
* Corresponding author. Fax: +1-215-299-1028.
E-mail address: [email protected] (T.H. O’Donnell).
mine relative proportions of different sources of food
for different animal groups, and to place different animal groups into a trophic structure. The average d13C
composition of C3 terrestrial vegetation is 13–14%
lower than C4 salt marsh plants. Most varieties of seagrass are enriched in 13C relative to terrestrial plants by
8–24% (McMillan et al., 1980). Both live and detrital
components of terrestrial, marsh, and seagrass plants
are important primary producers in estuarine systems.
Marine phytoplankton, which are another important
source of primary production, have d13C values that are
intermediate between C3 terrestrial vegetation and C4
saltmarsh plants. Marine phytoplankton can also be
distinguished from terrestrial and estuary plants on the
basis of d15N and d34S compositions. The d15N of phytoplankton is enriched by 8–10% relative to terrestrial
0146-6380/03/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0146-6380(02)00160-2
166
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
plants and d34S is enriched by 14% or more. Nitrogen
isotopes are also particularly useful for placing animals
into a trophic hierarchy because whole organism d15N is
enriched by an average of 3% over d15N in diets. The
multiple stable isotope approach has helped to identify
different functional roles of organisms in aquatic communities by clarifying producer–consumer relationships.
Understanding the inter-relationships of various organisms improves the ability to predict how ecosystems
might respond to changes in fundamental parameters
such as diet sources.
Actual isotopic compositions and fractionation factors of individual plants and animals do vary in nature.
The common procedure that is used in isotope studies of
aquatic systems compares the average isotopic compositions of primary producers to consumers. Identification of trophic relationships employs average isotopic
fractionation factors. Isotopic data from particular
organisms that do not follow the norm could lead to
misinterpretation of trophic relationships or diet
sources. Currin et al. (1995) measured a d34S range of
about 22% in live, senescent, and dead Spartina alterniflora in a North Carolina marsh. A d34S range of this
magnitude could lead to an inaccurate estimate of the
contribution of S. alterniflora to a local food web. Fantle et al. (1999) demonstrated that blue crabs fractionate
dietary nitrogen according to the nutritional quality of
their diet. The d15N of crabs fed on plant detritus were
enriched by 2.5% but there was no fractionation in
crabs that were fed a diet of zooplankton. The
contribution of zooplankton to the crab diet would
be missed using a general trophic level fractionation
of +3%. In spite of exceptions to average diet
source compositions and fractionation factors such as
these, the commonly used procedure for interpreting
stable isotope data clarifies many aspects of ecosystem
functioning.
The ability to predict changes in ecosystem functioning and population structure within aquatic systems is
important. North American estuaries are currently only
in fair to poor ecological condition as defined by the
USEPA (2002). Future impacts to estuarine organisms
from the cumulative effects of environmental changes
are a fundamental concern. Part of this concern can be
addressed by evaluating the response of primary productivity to changes in environmental conditions.
Knowledge of food dynamics provides insight needed to
predict population responses to changes in diet resources. One method to anticipate potential changes in primary productivity is to look for changes that have
occurred in the past. For example, isotopic analysis of
organic matter preserved in estuarine fossils might provide information about the types of primary productivity that existed in the geologic past and changes in
primary productivity that have occurred over time. In
recent geologic history, extending through the Pleisto-
cene period (1.8 Ma), sea levels have risen and fallen
numerous times (Imbrie et al., 1993). Climate and sea
level conditions during the Pleistocene constitute major
environmental cycles that could be used to evaluate
changes in estuarine ecosystems.
Reported here is a comparison of the isotopic
composition of bulk organic matter preserved in the
shells of modern and fossil Mercenaria mercenaria
and Mercenaria campechiensis. This investigation
demonstrates an isotopic approach for evaluating the
diets of individuals from one trophic level of recent
and ancient aquatic ecosystems. Mercenaria spp. are
infaunal suspension- feeding clams. These clams suction
suspended food particles through an inhalant siphon,
and thereby survive from ingested particulate, organic
matter that is generally smaller than about 15 m (Chesapeake Research Consortium, 1991). A record of the
isotopic composition of this diet is preserved as organic
matter in the calcified shell tissue of Mercenaria spp.
The d13C, d15N, and d34S compositions of organic matter from the soft tissue and from the calcified tissue of
modern clams have been measured to provide a framework for relating soft tissue to shell organic matter.
Once this relationship is known, then the methods for
interpreting isotopic data from modern food web studies can be used on fossils where the soft tissue has long
since disappeared. This report is the first to document
the relationship between the isotopic compositions of
organic matter from the soft tissue and calcified tissue of
individual aquatic invertebrates. Other isotopic studies
of fossils have provided environmental information by
analyzing the mineral component of the shells, as for
example, in d13C and d18O profiles of Mercenaria spp.
shell (Jones and Allmon, 1995).
The present study builds on prior investigations of
Mercenaria spp. by using fossil shells that have been
analyzed for regional amino-acid studies (Wehmiller et
al., 1988; Mirecki et al., 1995). All of the fossil specimens are Quaternary in age. Each fossil has also been
placed into a relative geochronological sequence using
the principles of aminostratigraphy (Miller and Hare,
1980). Some of the fossils come from a single location
and are different ages. Other specimens of similar age
come from different locations along the Atlantic and
Gulf of Mexico Coastal Plain.
Deegan and Garritt (1997) demonstrated that diet
resources for estuarine communities are locally derived.
Organisms that live in the oligohaline parts of estuaries
have diets that are dependent on terrestrial detritus, if it
is available, and on fresh to brackish-water primary
production (Incze et al., 1982). Organisms from the
same estuary but living in higher salinity waters derive
most of their diet from estuarine and marine sources of
primary production. The fossil shells used in this study
are morphologically the same as modern clam shells and
are assumed to have depended on local primary pro-
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
duction for food. The fossils, however, can not be
assumed to have lived at exactly the same time even if
they came from the same deposits. The purpose of this
investigation was to evaluate an isotopic technique for
studying past primary production in estuarine systems.
Larger scale studies using fossilized estuarine organisms
would require careful selection of multiple samples that
would be representative of a single stratigraphic
sequence.
2. Previous investigations
The basis for relating isotopes in animal tissue to diet
comes from previous studies of animal metabolism and
field studies where feeding relationships were clear.
Michener and Schell (1994) have summarized the results
of many of these studies. In general, each step-wise
increase in trophic level is accompanied by approximately 1 per mil (%) whole body enrichment in d13C
relative to dietary carbon. An average trophic level
enrichment of 3% occurs in whole body d15N during
metabolism although field studies have shown variations
of 1–2% in d15N fractionation. Little or no fractionation occurs in d34S. Deegan and Garritt (1997) verified a
3% enrichment in d15N among various primary consumers from Plum Island Sound that lived along a salinity gradient. These average fractionation factors apply
to the bulk organic matter at each consumer level under
natural conditions. Fantle et al. (1999) conducted an
experimental study of blue crabs and discovered that the
fractionation of both nitrogen and carbon isotopes varied by 2–3% depending on the protein quality of the
diet. Although diet quality does influence isotope compositions, the general consistency of fractionation factors for carbon, nitrogen, and sulfur has formed the
basis for interpreting producer–consumer interactions in
aquatic systems.
Isotopic fractionation factors associated with diet and
metabolism differ between the major biochemical components and between different tissue types (DeNiro and
Epstein, 1978, 1981; Koch et al., 1994). DeNiro and
Epstein (1977) found that lipids are characteristically
depleted in d13C by 3% relative to diet. The protein
fraction is generally enriched relative to dietary carbon
by 0.7–1.4% and closely matches fractionations found
in whole-body d13C samples (Rau et al., 1983). Among
various tissue fractions, those that are accompanied by a
high rate of metabolism most closely reflect the isotopic
composition of recent diet (Tieszen et al., 1983). It is
often impractical to conduct whole body analysis;
therefore, analysis of protein-rich tissue such as muscle
is often a preferred source of bulk isotopic data. Muscle
tissue, which was tested in the research reported here, is
readily available for sampling from most aquatic animals.
167
The first extensive isotopic study of an aquatic community was a survey-based investigation, which measured the d13C in estuarine organisms from a small area
of Redfish Bay, Texas (Parker, 1964). d13C values varied
from 5 to 26% (PDB) among different primary producers and higher trophic level consumers. The utilization of multiple stable isotopes for the study of food
webs became possible when nitrogen isotopes could be
routinely analyzed in conjunction with carbon (Miyake
and Wada, 1967; Wada, 1980). A multiple isotope
approach using d15N and d13C compositions in marine
organisms from the Antarctic Ocean documented a
trophic-level fraction of 3.3% in d15N (Wada et al.,
1987). This factor spanned four trophic levels and a
d15N range of 10%. Peterson et al. (1985) and Peterson
and Howarth (1987) introduced d34S along with nitrogen and carbon isotopes into an estuarine system-wide
study of food web dynamics. Their data indicated that
d34S in marine plankton is 18.8 0.6% which is about
14–17% heavier than terrestrial C3 plants, and 17–21%
heavier than S. alterniflora. The addition of d34S measurements to food web analyses has significantly
improved the ability to distinguish contributions from
marine-derived, organic matter. The isotopes of these
three elements can now be more routinely used to provide insight necessary to deconvolute complex dietary
dynamics and processes. Currin et al. (1995) applied
these isotope techniques to North Carolina marsh systems and documented ranges in d13C, d15N, and d34S of
21 9, and 26%, respectively. Deegan and Garritt (1997)
documented similar ranges in these isotopes during food
web studies of Plum Island Sound, Massachusetts. It is
now possible to identify and estimate the significance of
diet contributions to estuarine systems from upland,
local, and marine sources.
Previous studies have also demonstrated that food
resources are a dynamic feature of natural systems and
subject to change throughout the year. Organisms at
specific trophic levels, for example, may have variable
diet-derived isotopic signals during the year or within
different parts of an aquatic system that is a function of
local food sources, watershed characteristics, or tidal
flow hydrodynamics (Fogel et al., 1992; Deegan and
Garritt, 1997). Peterson et al. (1985) discovered that
mussels living on substrates above low tide had d34S
values of 12.1% while Mercenaria mercenaria living in
the bottom sediments had d34S values of 2.6%. Mussels
derived more of their diet from suspended, marine
organic matter during high-tide; however, M. mercenaria derived their food from marine and terrestrial
organic matter carried through their benthic habitat
during a complete tidal cycle. A number of other studies
have demonstrated spatial variation in primary production and food dynamics using stable isotope compositions of individual aquatic taxon, for example, mussels
(Incz et al., 1982), diatoms (Fry and Wainwright, 1991),
168
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
and blue crabs (Fantle et al., 1999). Diet sources for
estuarine organisms are related to local productivity and
local environmental and hydrodynamic conditions.
Shell building organisms sequester organic matter,
primarily protein amino acids, in their shell both as a
matrix component and within their mineral phase
(Crenshaw, 1980). These organic components are incorporated into the shell as it grows. Shells like those of
Mercenaria spp. develop annual growth bands that can
be easily identified in shell cross section, particularly
when annual growth is significant (Jones et al., 1990).
Essential and nonessential protein amino acids in mollusk shells originate from the animal’s diet (Neff, 1972),
the same as soft tissue proteins. Molecules are synthesized by the animal and then secreted through a thin
tissue layer, the epithelium, onto the shell growth surface. Weiner and Hood (1975) demonstrated that these
proteins could influence the nucleation and habit of
shell crystals. Some of the organic matter in Mercenaria
spp. becomes encased in growing carbonate crystals
while the remainder constitutes the intercrystalline,
matrix component of these biominerals. The proteins
that are encased by crystalline material survive for long
periods of time. Hare and Abelson (1968) measured low
concentrations of protein amino acids from shells that
were middle Miocene in age (10–15 Ma).
The use of carbon isotopes from the organic matrix of
invertebrate shells to investigate past environmental
conditions is limited. This is largely due to the lag in
developing techniques for isotopic analysis of micromolar concentrations of organic matter. Goodfriend
(1988) examined carbon isotopes in the organic matrix
of land snails to study Holocene environmental changes
in the Negev Desert. In this study, modern snails that
partly fed on C4 plants in arid areas had shell organic
matter with d13C values more enriched than 22.7%.
This isotopic value was used to reconstruct C3 and C4
vegetation zones in the region. CoBabe and Ptak (1995)
evaluated lipids from the shell of various bivalves as
potential habitat and diet biomarkers. This research
included analysis of the d13C value of individual fatty
acids. The isotopic composition of shell lipids followed
the pattern of variations in the isotopic composition of
tissue lipids. O’Donnell (2002) used the d13C of individual amino acids extracted from shells to infer the presence of original amino acids in fossils and the primary
diet sources for a suite of modern and fossil Mercenaria
spp. In this study, the isotopic compositions of amino
acids in modern and fossil shells varied over a range of
20–25%. The carbon isotopic compositions of amino
acid enantiomers from fossil shells were also used as an
indicator of organic matter preservation in land snails
(Engel et al., 1994).
The racemization of amino acids in fossil invertebrate
shells has been studied for a long time (Wehmiller and
Miller, 2000). In fact, the fossil shells used for the cur-
rent investigation were partially selected because existing racemization and stratigraphic data provided a
chronostratigraphic framework for the samples. Racemization dating uses the ratio of the d- and l-enantiomers of chiral amino acids. The d-enantiomer is formed
from the l-enantiomers upon death of an organism. The
rate of this transformation is dependent on a rate constant, time, and temperature. Time since death of an
organism can be calculated after an estimate of effective
long-term temperatures is made for a fossil locality and
the rate constant is known for a particular amino acid
(Wehmiller and Miller, 2000). Amino acid dates can also
be calibrated by other geochronological methods (Goodfriend and Rollins, 1998). Often the amino acid data is
presented as d/l ratios and shells are organized into
relative aminochronological ages (Wehmiller et al., 1998).
The isotopic composition of carbon and oxygen in the
carbonate phase of invertebrate shells has been successfully used to reconstruct environmental conditions in
many different situations (Wefer and Berger, 1991).
More recently, McConnaughey et al. (1997) and Smith
et al. (2000) have described procedures to identify the
influence of respired or metabolic carbon on the d13C
values of carbonates from aquatic and terrestrial mollusks. The combined information from isotopic studies
of both the mineral and organic components of fossil
shells can serve to expand the knowledge of past
estuarine ecosystems.
3. Methods
The source of specimens used for this investigation is
listed in Table 1. All of the fossil clams for this investigation are also part of ongoing amino acid racemization
studies of coastal sedimentary deposits along the Atlantic Coastal Plain (Wehmiller and Miller, 1998). The
shells came from exposures that have assigned ages
based on regional stratigraphic mapping (Toscano and
York, 1992), paleontological correlation (Hulbert and
Pratt, 1998), amino acid chronologies (Mirecki et al.,
1995), or radiogenic age dates (Szabo, 1985). The sample ages shown in Table 1 are the best current estimates
based on the aminostratigraphic data. The shells have
yielded d/l amino acid ratios consistent with regional
stratigraphic correlation and with the exception of the
JW 95-43 and JW 95-46 series samples, are physically
well preserved. Sub-samples of many of the specimens
have also been used to investigate diagenetic alteration
of the shells using geochemical and thin section techniques (Davis-Hartten, 2001). The data reported in this
study builds on a strong base of previous geological
work. All of the modern and fossil specimens except JW
96-132 were collected from sedimentary deposits typical
of estuarine sands, silts, or clays. JW 96-132 was
collected from Holocene age, open-shelf, sand deposits
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
169
Table 1
Locality and age information for shell fragments of Mercenaria spp used in this study (Davis-Hartten, 2001).
Collection location
Sample name
Local and state
Species
Estimated age
Assateague, Chowder C and smaller clams
JW 99 026-1
JW 96 132
GP 85-181
JW 95 043-1
JW 95 043-8-2
JW 95 046-20
JW 95 046-17
JW 95 038-1
JW 95 038-2
JW 94 220
JW 95 059-2
Assateague Channel Chincoteague, VA
Cedar Key, FL
Bass Site, NC shelf.
Gomez Pit, Virginia Beach, VA
Gomez Pit, Virginia Beach, VA
Gomez Pit, Virginia Beach, VA
Gomez Pit, Virginia Beach, VA
Gomez Pit, Virginia Beach, VA
Gomez Pit, Virginia Beach, VA
Gomez Pit, Virginia Beach, VA
Mark Clark Pit, Charleston, SC
Jones Site, Skidaway, Is. GA
m
c
m
m
m
m
m
m
c
c
m
m
Modern
Modern
Holocene
Late Pleist., IIaa
Late Pleist., IIa
Late Pleist., IIa
Mid-Pleist., IIc
Mid-Pleist., IIc
Early-Pleist., IIe
Early-Pleist., IIe
Late Pleistocen
Late Pleistocene
m=mercenaria, c=campechiensis.
a
Gomez Pit superposed aminozones of Mirecki et al. (1995), IIa is youngest, IIe is oldest.
off the coast of North Carolina. This is the only specimen that lived in an open marine environment rather
than estuarine environments represented by the other
shells. The specimens are spatially distributed along
Pleistocene estuarine environments extending from Virginia to Georgia, although the number of specimens
from any one location does not permit a comprehensive
analysis of any single sedimentary environment.
Soft tissue samples of adductor muscle, foot, and
mantle were dissected from modern Assateague Channel, Virginia, specimens, dried at temperatures of 40–
50 C, hand ground and homogenized. Soft tissue isotopic analyses were performed to establish a correlation
between the isotopic composition of soft tissue and shell
organic matter in the same organism. The soft tissue
data were also useful to compare with the isotopic
composition of primary producers common to the area
where the modern shells were collected. The dual-isotope plots used for comparing isotopic data are frequently used in food web studies such as those
referenced earlier. Isotopic analyses of soft tissue were
performed with a Carlo Erba CNS Elemental Analyzer
(EA) coupled to a VG-Micromass Optima isotope ratio
mass spectrometer (IRMS). Elemental concentrations
were determined by comparison with standards using
the TCD detector on the EA or by comparing ion mass
signal intensities detected by the Optima on standards
and samples.
Shell fragments were cut with a diamond wafer sawblade from the outer 2–3 mm of the ventral margins of
modern bivalves or from discrete parts of the inner or
middle layer of the shell (Fig. 1). Analyses of 4–7 fragments taken from individual modern valves and 2–14
fragments taken from single fossil valves are reported.
Samples were also cut from the ventral margin anterior
to the pallial line of juvenile and young adult clams.
Sample weights varied but in modern specimens about
100 mg of shell was found to be optimal. In fossil shells
300 mg provided sufficient organic matter for analysis.
These fragments were taken to provide data and insight
into the temporal variability of shell organic matter in
individual clams. The variability of organic matrix
material is directly related to diet changes. Mercenaria
spp. are relatively large, thick-shelled clams and samples
can be taken from portions of the shell that represent
annual growth increments or from parts of the shell that
represent intra-annual time increments (Jones and
Quitmyer, 1996). Shell fragments were acid hydrolyzed,
desalted with hydrofluoric acid, transferred in water,
dried, and then analyzed in the same manner as soft
tissue samples.
Lipids were separated from protein in soft tissue using
dichloromethane (DCM). Both the lipid fraction and
residual proteins were dried for analysis. Ground tissue
was placed in teflon-lined glass vials that had been previously ashed at 550 C. Sample vials with DCM were
then heated and refluxed at about 40 C for 24 h. Splits of
untreated tissue, extracted tissue and solvent were dried
for bulk isotopic analysis using the Micromass Optima
IRMS. Lipid analyses were performed to establish that
Mercenaria spp. muscle and mantle tissues are low in fats
that might complicate correlation with shell organic
matter. The mantle was analyzed for lipids because it was
assumed to contain a higher fat content then the adductor or foot muscles. The shells of modern M. mercenaria
contain 1500–2000 mg of protein per gram of shell (Wilbur and Simkiss, 1968) and about 50 mg of lipid material
per gram of shell (Hare and Hoering, 1977).
Analytical results of isotope analyses were calculated
by comparing sample and standard mass ratio signals
170
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Fig. 1. Sketch of Mercenaria spp. showing shell features mentioned in the text: (a) vertical view illustrating saw-cut orientation for
taking shell slabs; (b) cross section view showing nomenclature for shell growth layers and shell mineral texture layers. The translucent
layers and opaque layers are the result of periods of slow and fast growth, respectively (from Jones and Quitmeyer, 1996).
and reporting results in units of per mil (%) according
to the following convention:
dsample ¼
Rsample Rstandard =Rstandard 1000
atmospheric air standard and sulfur is reported relative to
the Canyon Diablo Troilite (CDT) standard. Elemental
concentrations of standards were reproducible within 10%.
where the isotopic composition of the sample, dsample, is
calculated from the ratio (R) of heavy to light isotope in
a standard and sample.
The ratios of samples and standards were compared
and corrections were made for cross-mass overlap, background noise, and blanks. The reproducibility of isotopic
analyses of standards was: d13C—0.10%, d15N—0.15%,
and d34S—0.30%. Carbon isotope compositions are
reported relative to the PeeDee Formation Belemite
Standard (PDB). Nitrogen is reported relative to the
4. Results and discussion
4.1. Soft tissue
The overall, average isotopic composition of soft tissue measured in adult M. mercenaria from Assateague
Channel (n=6–12) is d13C 17.1, d15N +11.7, and d34S
+16.0% (Table 2). The standard deviation for triplicate
analyses of single tissue samples was generally less than
0.4 for d13C, d15 N, and d34 S.
171
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Table 2
Average d13C, d15N, and d34S compositons of M. mercenaria
soft tissue collected from a two square meter area in Assateague Channel, Virginia, in August, 1998
Sample height (mm) Foot
Mantle
topic compositions shown in Table 3, and the following
equation relating isotopic compositions to end-member
concentrations, indicate that lipids constitute 9 2 wt.%
of the mantle tissue of these bivalves.
Muscle
34 (n=1)
47–131 (n=10)
S.D. d13C
S.D. d13C
S.D.
d13C
17.5 0.09 17.6 0.09 17.2 0.17
17.2 0.13 17.1 0.13 16.8 0.11
34 (n=1)
47–131 (n=10)
d15N S.D. d15N S.D. d15N S.D.
11.0 0.10
10.5 0.11
10.9 0.20
11.6 0.29
11.1 0.21
12.3 0.24
57–131 (n=9)
d34S S.D. d34S S.D. d34S S.D.
16.9 0.4
16.0 0.3
15.1 0.3
Isotope compositions are reported in standard per mil units.
na=not analyzed; S.D.=standard deviation; n=number of
specimens that were analyzed.
There are small variations in the isotopic composition
of different tissue types that may reflect fine scale
aspects of bivalve metabolism, biochemical synthesis,
bulk chemical composition, or may result from some
other resource allocation process. In adult specimens,
the average muscle tissue for example was enriched in
15
N by 0.5% and enriched in 34S by 1.0–1.8% relative to
other tissue types. However, the apparent distinctions
are only statistically significant for sulfur.
The d13C composition of soft tissue from M. mercenaria mantle was not substantially influenced by the
presence of lipids (Table 3). d13C values for untreated
and DCM extracted tissue samples were within 0.6%.
Mass balance estimates using the average carbon iso-
d13 Ctissue ¼ d13 Ctreated
x ¼ percent
tissue
þ ð1 xÞd13 Clipid
Wilbur and Simkiss (1968) reported that shell organic
matter from M. mercenaria contains only trace amounts
of lipids. CoBabe and Ptak (1999) analyzed shell
organic matter from five genera of mollusks and reported lipid concentrations averaging 1146 ng/g of shell.
Lipids influence the d13C values of M. mercenaria soft
tissue by less than 0.5%, an amount that minimizes a
potential complication associated with comparing soft
tissue and acid hydrolyzed organic matter.
The diet sources for these clams were evaluated by
comparing the carbon, nitrogen, and sulfur isotope
compositions of soft tissue to average values of major
primary producers (Fig. 2). The distribution of data
implies that marine phytoplankton and S. alterniflora
are the only significant sources of food for the clams and
that these primary producers alone are adequate to
generate the isotope signals. A mass balance analysis,
using the same form of equation described above, and
the following end member d34 S values: plankton
+20%, M. mercenaria +16%, and S. alterniflora
+6%, indicates that the clams derive > 70% of their
sulfur from marine plankton. Carbon and nitrogen
requirements are obtained in the same general diet proportions. This interpretation of diet sources is compatible with field observations of site conditions. The shells
were collected from sub-tidal deposits of sand or clay
Table 3
d13C and d15N in samples of untreated Mercenaria spp. mantle tissue, solvent treated tissue, and solvent extract following refluxing
with dichloromethane
Assateague Channel, Virginia Specimens
Specimen height (mm)
d13C
d15N
Untreated tissue
Treated tissue
Diff.
Untreated tissue
Treated tissue
Diff.
70
63
74
64
79
70
17.0
17.2
17.2
17.4
18.0
17.7
16.6
17.3
16.7
17.1
17.6
17.4
0.4
0.1
0.5
0.3
0.4
0.3
11.9
12.0
12.8
12.4
12.2
12.0
12.4
11.9
12.6
12.3
11.6
11.5
0.5
0.1
0.2
0.2
0.6
0.5
Average
S.D.
17.4
0.3
17.1
0.4
0.3
0.3
12.2
0.4
12.0
0.4
0.2
0.4
Lipid fraction of mantle tissue
21.7
3.6
Isotopic compositions are reported in per mil units relative to PDB and air standards.
172
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Fig. 2. Comparison of the average isotopic compositions of Mercenaria mercenaria soft tissue to typical primary producers. Bivalve
samples were collected from Assateague Channel, Virginia, in August, 1998. Isotopic values for primary producers are from the literature (e.g. Michener and Schell, 1994). Soft tissue isotope measurements are corrected for trophic level shifts of +1.0 for d13C and
+3.0 for d15N.
adjacent to a S. alterniflora salt marsh within about 1 km
of the Assateague Inlet, which opens to the Atlantic Ocean.
4.2. Shell organic matter from juvenile and young adult
M. mercenaria
Acid hydrolyzable organic matter from the ventral
margin of different sizes of M. mercenaria has d13C,
d15N, and d34S values that vary over a relatively small
range (Table 4; Fig. 3). Nitrogen isotopic compositions
vary from 10.0 to 12.4% with values greater than 11.0%
associated with mostly smaller, juvenile clams. d13C
values range over 4% with the more enriched values of
about 14 to 16% occurring in the smaller clams. d34S
values vary from 12 to 14% with more enriched values
occurring in the larger clams. These trends are opposite
of those measured in soft tissue samples where the
juvenile clams have d13C values that are more depleted
173
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Table 4
d13C, d15N, d34S, organic carbon, organic nitrogen, and organic sulfur in bulk organic matter from the ventral margin of Mercenaria
mercenaria collected live from Assateague Channel, Virginia, in August 1998
Shell height (mm)
Carbon
13
Nitrogen
15
C/N
d C
at. %
d N
at. %
31
34
47
57
58
59
60
75
14.8
15.2
16.0
18.3
16.9
18.4
17.6
17.3
0.17
0.19
na
0.22
0.30
0.26
0.22
0.13
11.6
12.4
10.8
10.4
0.5
10.1
10.3
11.6
0.057
0.062
na
0.060
0.10
0.084
0.060
0.041
Average of young adults with height >45 mm
17.4 1.4
10.60.5
3.0
3.1
na
3.7
3.0
3.1
3.7
3.2
Sulfer
d34S
at. m%
12.4
12.8
13.4
na
na
13.7
na
na
0.003
0.002
0.001
na
na
0.002
na
na
13.6
Isotope compositions are reported in per mil units relative to PDB and air standards. na=not analyzed.
than adult clams by 0.4%, and d15N values that are
more depleted by 0.9%. These two organic matter
reservoirs are likely to be linked in those metabolic and
resource allocation processes that influence isotopic signatures. More enriched soft tissue values are associated
with more depleted shell organic matter compositions.
Young adults (> 45 mm height) have shell d13C values
that average17.4 1.2%, compared to an average soft
tissue d13C value of17.3 0.1% in the same clams. The
shell d15 N of young adults averages 10.6 0.5%, compared with 11.3 0.1% in soft tissue compositions of the
same clams. The d34S values in the young adults are
more distinct than the soft tissue, 13.6 0.2% versus
15.4 0.8% in soft tissue.
Variation in the isotopic composition of Mercenaria
spp. juveniles and adults has not been systematically
studied but may result from more than one process.
Smaller clams may have access to different food sources
simply because of the smaller size of their inhalant
syphons (Rau et al., 1990). The smaller clams may also
have organic-matrix shell proteins with amino acid
compositions that are slightly different than larger
clams. Larval and juvenile abalone, for example, contain protein moities that contain three times as much
glycine as the adult forms (Cariolou and Morse, 1988),
and glycine is typically enriched in 13 C relative to other
amino acids (Abelson and Hoering, 1961). There may
also be slightly different isotope fractionations associated with amino acid synthesis at the cellular level
based on different growth strategies and resource allocation needs as the clams mature. The influence of
gametogenesis in sexually mature clams, for example,
has been evoked to explain differences in the isotopic
composition of d13C and d18O in the shells of marine
bivalves (Krantz et al., 1987).
Alternatively, O’Donnell (2002) measured a kineticdisequilibrium isotope trend in d13C and d18O values in
the shell component of Mercenaria spp. that generates
isotope depletions of 3–7%. Regardless of the cause for
isotopic differences with age, the young adult clams
have soft tissues and shell-matrix organic matter with
d13C values that are the same within analytical uncertainty (0.1%) and an organic matrix that is depleted in
d15N by about 0.7%. Organic matrix materials are also
depleted in d34S by about 1.8%. Trophic-level interpretations based on diet are made with the general
assumption that carbon isotopes are enriched in consumers by an average of 1.0% (Koch et al., 1994).
Likewise, nitrogen isotopes are enriched in consumers
by an average of 3.0% at each trophic level and sulfur
isotopes remain unchanged (Michener and Schell, 1994).
These trophic-level fractionations must be balanced
against fractionations that occur between soft tissue and
shell organic matter before shell organic data can be
used for diet reconstruction. The following carbon,
nitrogen and sulfur adjustments to shell organic matter
isotopic compositions were calculated from the isotopic
data collected in this study:
13
Ctissue-shell ¼ 17:3 ð17:4Þ ¼ þ0:1 per mil
15
N
¼ 11:3 ð10:6Þ ¼ þ0:7 per mil
34 tissue-shell
Stissue-shell ¼ 15:4 ð13:6Þ ¼ þ1:8 per mil
4.3. Shell organic matter from modern and fossil
Mercenaria spp.
The isotopic compositions of bulk organic carbon
and nitrogen preserved in the calcified tissue of individual, modern and fossil Mercenaria spp. varies in samples
taken from different parts of the same shell (Table 5).
Middle layer shell fragments were sampled from different ontogenetic ages of the animal (Jones et al., 1990),
therefore, variations in the isotopic compositions of the
174
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Fig. 3. Comparison of d13C, d15N, and d34S in soft tissue and the shell organic matter of modern juvenile and young adult M. mercenaria (height of 45–70 mm). Samples were collected from Assateague Channel, Virginia, in August, 1998. Error bar represents
standard deviations for young adult samples.
organic matter are also related to time. The middle layer
fragments listed in Table 5 represent different years of
shell growth (inter-annual), or in the case of samples
identified with a number followed by a letter, different
months of shell growth (intra-annual). For example, ‘4a’
indicates approximate growth during the later part of
the growing year and ‘4b’ represents growth during the
early part of the growth period. The numeral in this case
is added to distinguish different interannual fragments.
Inner layer samples incorporate multiple but unquantified years of growth. Ventral margin fragments contain
the most recent growth layers of the shell.
The isotopic compositions of shell organic matter
measured in modern M. mercenaria from Assateague
Channel are plotted on Fig. 4 after adjusting for trophic
level shifts and the shell organic-matrix fractionation
described earlier. Assateague sample fragments have
d13C values that range from 12.8 to 15.9%. The d13C
value of 15.9 % from the inner layer fragment
approaches an average value for the entire clam. The
d13C value for this fragment was close to the 18.1%
measured in the soft tissue of the clam and the 18.3%
measured in the soft tissue of young adult clams (n=5)
collected from the same area. The remaining fragments
had more enriched d13C values that reached 12.8%,
which is typical for S. alterniflora. The compositions of
d15N adjusted for trophic level varied from 8.6 to
11.4%. The more enriched value is 2.1% heavier than
the soft tissue. Currin et al. (1995) summarized literature
values for d15N values in estuarine systems and reported
175
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Table 5
d13C, d15N, organic carbon, and organic nitrogen in bulk organic matter from the shells of modern and fossil Mercenaria spp.
Sample
Fragment
Carbon
d13C
Modern
Virginia
at. %
d15N
C/N
at. %
12.8
13.3
14.6
11.6
14.1
15.9
0.081
0.14
0.106
0.118
0.094
0.044
13.4
13.7
11.9
11.6
11.0
11.3
0.027
0.046
0.034
0.038
0.032
0.014
3.0
3.0
3.1
3.1
2.9
3.1
layer
layer
layer
layer
19.4
21.0
20.3
20.5
0.084
0.12
0.10
0.13
9.9
7.4
7.2
7.7
0.029
0.036
0.026
0.043
2.9
3.3
3.8
3.0
middle layer
middle layer
ventral margin
19.7
18.5
15.8
0.054
0.061
0.025
5.2
6.9
7.1
0.007
0.010
0.006
7.7
6.1
4.2
JW95 043-1
middle layer
middle layer
middle layer
23.4
20.7
25.5
0.073
0.049
0.076
11.3
na
4.5
0.009
0.003
0.009
8.1
16
8.4
JW95 043-8-2
middle layer
ventral layer
inner layer
21.1
21.4
20.7
0.043
0.044
0.027
8.5
7.9
13.2
0.006
0.007
0.004
7.2
6.3
6.8
JW95 046-20
middle layer
middle layer
ventral margin
21.2
22.9
21.5
0.047
0.035
0.045
7.2
7.5
7.9
0.006
0.004
0.007
7.8
8.8
6.4
JW95 046-17
middle layer
ventral margin
inner layer
inner layer
na
21.6
na
27.5
0.059
0.056
na
0.036
7.7
9.3
6.5
8.9
0.006
0.008
0.006
0.004
9.8
7.0
7.8
9.0
JW95 038-1
middle layer
middle layer
16.1
14.4
0.020
0.024
0.004
na
5.0
–
JW95 038-2
middle layer
ventral margin
inner layer
inner layer
na
21.6
na
27.5
0.059
0.056
na
0.036
7.7
9.3
6.5
8.9
0.006
0.008
0.006
0.004
9.8
7.0
7.8
9.0
16.5
23.0
20.4
21.4
19.8
22.4
22.1
17.3
17.5
18.0
0.040
0.046
0.023
0.025
0.020
0.023
0.026
0.009
0.027
0.029
3.7
3.8
na
na
na
na
9.8
na
7.6
8.3
0.008
0.010
0.005
na
na
0.003
0.004
na
0.007
0.005
5.0
4.6
4.6
–
–
7.7
6.5
–
3.9
5.8
Assateague
Chowder C
middle layer 4b
middle layer 4a
middle layer 3b
middle layer 3a
ventral margin
Inner layer
Nitrogen
Florida
JW99 026-1
Fossil
Virginia
GP 181
middle
middle
middle
middle
na
na
N. Carolina
JW96 132
middle
middle
middle
middle
middle
middle
middle
middle
middle
middle
layer
layer
layer
layer
layer
layer
layer
layer
layer
layer
176
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Table 5 (continued)
Sample
Fragment
Carbon
d13C
Nitrogen
at. %
C/N
d15N
at. %
middle layer
ventral margin
inner layer
18.4
16.2
16.6
0.034
0.029
0.034
na
4.2
6.4
na
0.006
0.011
–
4.8
3.1
middle layer
middle layer
20.3
21.5
0.023
0.026
4.5
3.8
0.006
0.006
4.3
3.8
middle layer
middle layer
24.1
21.8
0.035
0.044
7.2
na
0.004
0.008
8.8
5.5
S. Carolina
JW94 220
Georgia
JW95 059-2
Fragments from the middle layer are arranged from oldest specimens at the top to youngest at the bottom. Pleistocene fossils from
Viginia are also arranged from oldest at the top to youngest. Isotopic compositions are reported in per mil relative to PDB and Air
standards. Concentrations are reported in atom percent of shell. Na=not analyzed
Fig. 4. Comparison of the isotopic compositions of bulk organic matter extracted from the shell of one adult Mercenaria mercenariafrom Assateague Channel, VA. and one adult Mercenaria campechiensis from Cedar Key, Florida. Isotopic values have been
adjusted for average trophic level fractionations of +1 for d13 C and +3 for d13 N and also for the shell isotope fractionation of
0.7% for d13N. Open circles connected with lines are intra-annual samples. The other samples represent inter-annual shell increments.
Values for primary production are from the literature (e.g. Michener and Schell, 1994). Error bar represents the spread of isotope values
measured in young adult samples from Virginia and, hence, are an indication of potential variability within a local population.
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
only two locations where primary production had
values of d15N > 10%. In both cases the analyses were
from S. alterniflora. Fogel et al. (1989) predicted d15N
values reaching 10–15% in S. alterniflora particulate
matter during early stages of microbial degradation.
These researchers also predicted a 1–2% depletion of
d13C values during early stages of degradation. The diet
represented by fragments 3a and 3b may have originated from partially degraded S. alterniflora particulate
matter. Macko and Estep (1984) determined experimentally that amino acid substrates could be enriched in
d15N by up to 22% as a result of microbial processes.
The magnitude of the d15N fractionation was dependent
on the amino acid composition. Qian et al. (1992)
demonstrated that condensation reactions of amino
acids and carbohydrates during diagenesis could also
lead to at least a 2–3% enrichment in d15N in the residual substrate. The enriched shell d15N values in the M.
mercenaria could also represent a diet contribution from
an undocumented and d15N enriched microzooplankton
source. The additional trophic level enrichment from a
primary consumer group would introduce an enriched
d15N to the particulate organic matter pool. The enriched shell values could also represent a component of
dissolved or suspended organic matter that has not been
analyzed, such as nanno or picoplankton. Regardless of
the factors that are controlling the isotopic composition
of the shell organic matter, these data indicate that a
significant diet-related, ontogenetic variation in isotopic
compositions can be measured in the shells of modern
Mercenaria spp.
The dual isotope plot (Fig. 4) of d13C versus d15N for
modern M. campechiensis collected from the Gulf Coast
of Florida at Cedar Key provides further evidence of the
ability of isotopes to record primary diet sources. The
sample fragments suggest a mixed diet of marine
phytoplankton and some combination of C3-terrestrial
plant detritus, Juncus, or benthic microalgae. Cedar Key
does not receive direct river runoff and the marsh areas
are dominated by Juncus; therefore, the primary food
sources in addition to phytoplankton are most likely to
be Juncus detritus and benthic microalgae. The shell
fragment with an enriched d15N suggests that the diet
contribution of phytoplankton may be more significant
at certain times of the year.
The fossil samples collected at Gomez Pit, Virginia
are from three different stratigraphic units that are different in age (Table 1). Each superposed unit has been
assigned to a unique aminozone by Mirecki et al. (1995).
Samples JW 95 043-1 and JW 95 043-8-2 are from the
youngest stratigraphic unit (late Pleistocene, aminozone
IIa) and were collected laterally within 10 feet of one
another. GP 181 is from the same lithologic unit but
from a different part of the Pit. Shells from this unit
have also been assigned to aminozone IIa, although d/l
values for GP 181 are 10% lower (younger) than JW 95
177
043 series shells. JW 95 046 series shells are from a lower
aminostratigraphic unit in the Pit and are middle Pleistocene in age. JW 95 038-2 is from the oldest stratigraphic unit exposed at the Pit and is early middle
Pleistocene in age. The isotopic compositions of shell
organic matter from Pleistocene Mercenaria spp. are
significantly different (Fig. 5; and Table 5). The fossil
shells are recording different diet sources. This feature
implies that the primary production for infaunal filter
feeders in this part of the Atlantic Coastal Plain and the
ancestral Chesapeake Bay estuarine system has varied
over the past approximately 1 million years. The isotopic compositions from the early Pleistocene JW 95
038-2 are similar to modern day samples from Assateague Channel that are dominated by the influence of
marine phytoplankton and S. alterniflora. Alternatively,
if S. alterniflora was not present at this location in the
early Pleistocene, some variety of seagrass with a similar
isotopic composition may have been an important primary producer in the ecosystem. The middle Pleistocene
samples, JW 95 046-17 and 20, yielded clustered isotope
data that indicate a terrestrial or marsh-grass diet influence in addition to marine phytoplankton. The late
Pleistocene specimens, JW 95 043-1 and 043-8-2, have
wide ranging isotopic values including some samples
with compositions that are the same as the middle
Pleistocene JW 95 046 series shells. Trophic adjusted
d15N compositions in JW 95 043 series shells range from
2.2 to 10.9%, which spans the complete range of terrestrial to marine d15N values. Relatively depleted d13C
compositions (21.5 to 26.5%) in the shells suggest
diet carbon sources that are influenced by terrestrial
detritus. The possibility that two shells with the same
age and from the same unit can record such a wide
range of diet sources is low (Deegan and Garritt, 1997).
If the shells are the same age then it is more likely that
fragments from one or both of the shells have been
contaminated. These deposits formed during past high
sealevel stands. The shells have been exposed to
groundwater for much of their history because sea level
has been lower. Contamination with depleted d13C from
terrestrial organic matter sources is possible. The isotopes in the JW 95 043-1 shell fragment with depleted
d15N and d13C resemble values typical of terrestrial
organic matter and may have been contaminated. The
late Pleistocene shell, GP 85 181, yielded isotopic data
that suggest a diet of benthic algae and some combination of terrestrial detritus or S. alterniflora, and benthic
microalgae.
Isotopic compositions of fossil Mercenaria spp. from
locations other than Gomez pit, Virginia, are shown in
Fig. 6. Bulk isotopic compositions of the Holocene M.
mercenaria, JW 96-132, display a wide range of trophic
adjusted d15N values (1.4–7.5%) that trend along a mix
of benthic microalgae and marine phytoplankton. This
shell was collected from the inner Continental Shelf
178
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Fig. 5. Isotopic composition of bulk organic matter from the shells of fossil Mercenaria spp collected from Gomez Pit, Virginia.
Sample locations and ages are listed in Table 1. Isotopic values have been adjusted for average trophic level fractions of +1 for d13C
and +3 for d15N and shell fractionation of and 0.7% for d15N. Sample fragments represent sub-annual and annual growth increments. Error bar represents the spread of isotope values measured in juvenile and young adult samples from Assateague Channel and,
hence, are an indication of variability within a local population. Values for primary production (modern) are from the literature (e.g.
Michener and Schell, 1994).
adjacent to North Carolina in an environment where
phytoplankton and benthic algae would be expected to
be important primary producers. One of the shell fragments appears to be strongly influenced by terrestrial
organic matter while another by significant amounts of
marine phytoplankton and perhaps a marsh grass like
Juncus. Late Pleistocene M. mercenaria samples from
South Carolina (JW 94-220) and Georgia (JW 95 059-2)
were collected from sedimentary deposits characteristic
of estuaries. The Georgia sample came from an area
that is also well known for marine and terrestrial vertebrate and invertebrate fossils (Hulbert and Pratt, 1998).
The isotope compositions of both of these shells suggest
diets of mixed terrestrial detritus and either a salt marsh
grass detritus or benthic microalgae. In either case, the
indicated diets are compatible with geologic conditions
thought to exist at the time that these fossils were alive
(Toscano and York, 1992).
Additional biogeochemical information is available
from the carbon and nitrogen concentrations of the shell
organic matter (Fig. 7). The C/N ratio has often been
measured in association with organic matter degradation (Fogel and Tuross, 1999; Hedges et al., 1997).
There is a negative correlation (slope 1.2) between the
C/N ratio and d13C of shell organic matter that could be
interpreted as indicating increasing amounts of shell
contamination from terrestrially derived organic matter.
There is also a 50–75% decrease in the concentrations of
carbon and nitrogen in the biominerals (Table 5), which
would cause small amounts of contamination to have
proportionally greater impact on isotopic compositions.
Changes in both concentration and C/N ratio reflect
diagenetic alteration of organic matter over time. The
relationship between diagenetic alteration of carbon and
nitrogen concentrations and alteration of isotopic compositions is not as well understood. Two important
issues are the degree to which the isotopic compositions
of carbon and nitrogen change with diagenesis and,
whether isotopic changes reflect contamination or selective loss of isotopically distinct organic matter (Fantle et
al., 1999; Keil and Fogel, 2001). The tendency for isotope values below about21 % (Fig. 7) to be associated
with higher C/N ratios may indicate a contribution of
terrestrially derived C3-plant material or microbial
alteration during diagenesis. However, the relationship
of d15N to C/N does not suggest a contribution from a
source that is relatively depleted in d15N as is common
in terrestrial environments. The trend of increasing C/N
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
179
Fig. 6. Isotopic composition of bulk organic matter from the shells of fossil Mercenaria spp. collected from coastal North and South
Carolina and Georgia. Sample locations and ages are listed in Table 1. Isotopic values have been adjusted for average trophic level
fractionation of +1 for d13C and +3 for d15N and shell fractionation of 0.7% for d15N. Sample fragments represent sub-annual and
annual shell increments. Error bar represents standard deviation of isotope values measured in young adult samples from Assateague
Channel, Virginia, and hence are within a local population. Values for primary production (modern) are from the literature (e.g.
Michener and Schell, 1994).
and decreasing d13C may just be a feature of the sample
set. In the previous discussion of isotopic data in shells
and primary producers, low d15N containing fragments
were suggested as having impacts from terrestrial carbon sources. The fragments implicated were taken from
JW 94 220, JW 95 043-1, and JW 96 132 (Fig. 6). Fragments JW 94 220 and JW 96 132 have C/N ratios
between 4 and 5, which are considerably lower than the
highest C/N values of around 8–10 (Fig. 7; Table 5).
Fragment JW 95 043-1, on the other hand, does have a
relatively low d15N, the most depleted d13C that was
measured, a high C/N ratio, and an isotopic composition suggesting a diet that is not compatible with the
other shell fragment that were tested. All of the data for
JW 95 043-1 suggest a contribution of terrestrially
derived organic matter as a contaminant. The issue of
contamination is not resolvable through the use of C/N
ratios alone. In combination with bulk isotope data
however, the C/N ratios provide more convincing evidence for the presence of contamination.
Additional testing based on the use of compound
specific isotopic analysis can also provide independent
evidence for the presence of contamination as described in Engel et al. (1994). O’Donnell (2002) measured
the d13C composition of individual amino acid enan-
tiomers from some of the shell fragments used for
this investigation. The test for indegeneity of original
amino acids compares the d13C value of the enantiomers. Amino acids are considered to be unaltered when
the enantiomers are isotopically identical. The amino
acid testing verified that d and l leucine were isotopically identical in the following sample fragments:
the oldest middle layer sample from JW 96 132, all of
the GP 181 fragments, the middle layer fragment from
JW 95 043-8-2, the middle layer from JW 94 220, and
the oldest middle layer fragment from JW 95 059-2. The
fact that these samples have not been contaminated with
amino acids supports the presence of unaltered bulk
organic matter.
5. Summary and conclusions
Mercenaria spp. preserve a record of their growth
environment as an isotope signal in the organic portion
of their calcified tissue or biomineral. Isotopic analyses
of bulk organic matter from the shell matrix have
demonstrated that proxies of primary productivity are
preserved in the shells. A first attempt has been made to
relate the isotopic composition of soft tissue to shell
180
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Fig. 7. Comparison of C/N ratios to d13C and d15N in bulk organic matter from the shells of modern and fossil Mercenaria spp. All
samples are adult clams.
organic matter. By doing this, modern isotopic methods
for evaluating food webs can be extended to fossilized
organic matter. The data indicate that d13C is identical
between these two organic matter reservoirs, d15N is
depleted in shell organic matter by 0.7 %, and that
d34S is depleted in shell organic matter by 1.8%. The
source of this difference is not certain but may relate to
available diet, or to small differences in the amino acid
composition of proteins in the shell matrix, or to isotopic effects that accompany different resource allocation activities.
A modern M. mercenaria from Assateague Channel,
Virginia, has soft tissue isotopic compositions (d13C,
d15N, d34S) that indicates a diet composed primarily of
marine plankton and S. alterniflora detritus. This diet
mix matches features of the local habitat, which is
dominated by S. alterniflora marsh and inflow from the
Atlantic Ocean. Isotopes in the shell organic matter
(d13C, d15N, d34S) also indicate that the proportion of
each food has varied over the last 4–5 years. In addition,
relatively high d15N values that exceed 12% suggest that
partially degraded S. alterniflora detritus is likely to be
an important component of the local food web at times.
Analyses of modern specimens from the Gulf Coast of
Florida yielded isotopic data that pointed to a diet
composed of locally present primary production, marine
phytoplankton and Juncus. The isotopic variability in
different shell fragments demonstrates that a record of
short-term diet sources is preserved. In this investigation, millimeter-sized shell samples from the middle shell
layer represent estimated time periods as brief as one
month.
Mercenaria spp. are infaunal clams that feed by
drawing suspended, particulate organic matter into their
stomachs. Food particles are transported to them along
tidal currents and diet sources are shown to be locally
derived. Interpreting food web data from fossils is more
difficult because of the limited control on spatial coverage among contemporaneous organisms. There is a
unique opportunity to evaluate local changes over time
at locations like Gomez Pit where three superposed
aminozones exist. This is true even if the position that
each clam occupied within the estuary is not exactly
known. Large differences in diet sources between fossil
systems suggests that primary productivity responds
differently with changing environmental conditions.
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Changes in the type of primary productivity may have
implications for the type and abundance of higher
trophic animals. The isotopic data collected from Mercenaria spp. at Gomez Pit, Virginia, suggests that significant changes in the type of primary production have
occurred during the Pleistocene. Each of the three
deposits at Gomez Pit represent separate sea-level highstands that have been correlated to marine oxygen isotope stages 5, 7, and 11 (Coleman and Mixon, 1988;
Szabo, 1985). Primary production during the middle
and late Pleistocene was dominated by benthic microalgae, terrestrial detritus, and possibly C3 salt marsh
detritus similar to Juncus. Early-middle Pleistocene primary production was similar to the present.
The geochemical study of fossils is always concerned
with the issue of contamination or diagenetic alteration.
The data presented here suggest that the use of the C/N
ratio alone as an indicator of organic matter contamination is not reliable. Supporting information in the
form of elemental concentrations and bulk isotopic
compositions is helpful to identifying shells that might
be contaminated. Additional independent criteria such
as compound specific isotope analysis can be used to
identify original amino acids in fossils. With the use of
information from uncontaminated shells, the techniques
described in this investigation have the potential to
clarify changes in aquatic ecosystems that were associated with extinction events or anthropogenic changes
in a more historical context.
Acknowledgements
The authors thank Linda York for providing information about various Mercenaria spp. specimens from
the collections of the University of Delaware and for
assisting in sample preparation. This paper was greatly
improved by the comments of Richard Mitterer and
Hope Jahren. Support for this project was provided by
National Science Foundation Grant No. 9315052.
References
Abelson, P., Hoering, T., 1961. Carbon isotope fractionation in
formation of amino acids by photosynthetic organisms. Proceedings National Academy of Science 47, 623–632.
Cariolou, M., Morse, D., 1988. Purification and characterization of calcium-binding conchiolin shell peptides from the
mollusc, Haliotis rufescens, as a function of development.
Comparative Physiology 157, 717–729.
Chesapeake Research Consortium, 1991. Hard Clam Mercenaria mercenaria. In Funderburk, S., Mihursky, J.,Jordan,
S., Riley, D. (Eds.), Habitat Requirements for Chesapeake
Bay living Resources. Chesapeake Research Consortium,
Inc, pp. 5-1–5-15.
181
CoBabe, E., Ptak, L., 1995. Molecular and isotopic compositions of lipids in bivalve shells: a new prospect for molecular
paleontology. Geochimica et Cosmochimica Acta 59 (1), 87–
95.
CoBabe, E., Ptak, L., 1999. Comparison of in situ mineralassociated lipid compositions in modern invertebrate skeletons: preliminary evidence of dietary and environmental
influence. Paleobiology 25 (2), 201–211.
Coleman, S., Mixon, R., 1988. The record of major Quaternary
sea-level changes in a large coastal plain estuary, Chesapeake
Bay, eastern United States. Paleogeography, Paleoclimatology, Paleoecology 68, 99–116.
Crenshaw, M., 1980. Mechanisms of shell formation and dissolution. In: Rhoads, D., Lutz, R. (Eds.), Skeletal Growth
of Aquatic Organisms. Plenum Press, New York, pp. 115–
128.
Currin, C., Newell, S., Pearl, H., 1995. The role of standing
dead Spartina alterniflora and benthic microalgae in salt
marsh food webs: considerations based on multiple stable
isotope analysis. Marine Ecology Progress Series 121, 99–
116.
Davis-Hartten, K., 2001. Diagenetic alteration of Quaternary
Mercenaria, US Atlantic Coastal Plain. Dissertation. Dept.
Geol., University of Delaware, 240 pp.
Deegan, L., Garritt, R., 1997. Evidence for spatial variability in
estuarine food webs. Marine Ecology Progress Series 147,
31–47.
DeNiro, M.J., Epstein, S., 1977. Mechanism of carbon isotope
fractionation associated with lipid synthesis. Science 197,
261–263.
DeNiro, M., Epstein, S., 1978. Influence of diet on the distribution of carbon isotopes in animals. Geochimica et Cosmochimica Acta 42, 495–506.
DeNiro, M., Epstein, S., 1981. Influence of diet on the distribution of nitrogen isotopes in animals. Geochimica et
Cosmochimica Acta 45, 341–351.
Engel, M., Goodfriend, G., Qian, Y., Macko, S., 1994. Indigeneity of organic matter in fossils: a test using stable isotope
analysis of amino acid enantiomers in Quaternary mollusk
shells. Proceeedings National Academy of Science 91, 10475–
10478.
Fantle, M., Dittel, A., Schwalm, S., Epifanio, C., Fogel, M.,
1999. A food web analysis of the juvenile blue crab, Callinectes sapidus, using stable isotopes in whole animals and
individual amino acids. Oecologia 120, 416–426.
Fogel, M., Sprague, E., Gize, A., Frey, R., 1989. Diagenesis of
organic matter in Georgia salt marshes. Estuarine, Coastal,
and Shelf Science 28, 211–230.
Fogel, M., Cifuentes, L., Velinsky, D., Sharp, J., 1992. Relationship of carbon availability in estuarine phytoplankton to
isotopic composition. Marine Ecology Progress Series 82,
291–300.
Fogel, M., Tuross, N., 1999. Transformation of plant biochemicals to geological macromolecules during early diagenesis. Oecologia 120, 336–346.
Fry, B., Wainright, S., 1991. Diatom sources of 13C-rich carbon
in marine food webs. Marine Ecology Progress Series 76,
149–157.
Goodfriend, G., 1988. Mid Holocene rainfall in the Negev
Desert from 13C of land snail shell organic matter. Nature
333, 757–760.
182
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Goodfriend, G., Rollins, H., 1998. Recent barrier beach retreat
in Georgia: dating exhumed salt marshes by aspartic acid
racemization and post-bomb radiocarbon. Journal Coastal
Research 14 (3), 960–969.
Hare, P., Ableson, P., 1968. Racemization of Amino Acids in
Fossil Shells. Carnegie Inst, Wash, YB.
Hare, P., Hoering, T., 1977. Organic Constituents of Fossil
Mollusc Shells. Carnegie Inst, Wash. YB.
Hedges, J., Keil, R., Benner, R., 1997. What happens to terrestrial organic matter in the oceans? Organic Geochemistry
2 (5/6), 195–212.
Hulbert Jr, R., Pratt, A., 1998. New Pleistocene (rancholabrean) vertebrate faunas from coastal Georgia. Journal
of Vertebrate Paleontology 18 (2), 412–429.
Imbrie, J., Berger, A., Boyle, E., Clemens, S., Duffy, A.,
Howard, W., Kukla, G., Kutzbach, J., Martinson, D.,
McIntyre, A., Mix, A., Molfino, B., Morley, J., Peterson, L.,
Pisias, N., Prell, W., Raymo, M., Shackleton, N., Toggweiler, J., 1993. On the structure and origin of major glaciation
cycles 2: the 100,000 cycle. Paleoceanography 8 (6), 699–735.
Incz, L., Mayer, L., Sherr, E., Macko, S., 1982. Carbon inputs
to bivalve mollusks: a comparison of two estuaries. Canadian Journal Aquatic Science 39, 1348–1352.
Jones, D., Quitmyer, I., Arnold, W., Marelli, D., 1990. Annual
shell banding, age, and growth rate of hard clams (Mercenaria spp.) from Florida. Journal Shellfish Research 9 (1),
215–225.
Jones, D., Allmon, W., 1995. Records of upwelling, seasonality
and growth in stable-isotope profiles of Pliocene mollusk
shells from Florida. Lethaia 28, 61–74.
Jones, D., Quitmyer, I., 1996. Marking time with bivalve shells:
oxygen isotopes and season of annual increment formation.
Palaios 11, 340–346.
Keil, R., Fogel, M., 2001. Reworking of amino acids in marine
sediments: stable carbon isotopic compositions of amino
acids in sediments along the Washington coast. Limnology
and Oceanography 46 (1), 14–23.
Koch, P., Fogel, M., Tuross, N., 1994. Tracing the diets of
fossil animals using stable isotopes. In: Lajtha, K.M., Michener, R.H. (Eds.), Stable Isotopes in Ecology and Environmental Science. Blackwell Scientific Publications, Boston, pp.
63–93.
Krantz, D., Williams, D., Jones, D., 1987. Ecological and
paleoenvironmental information using stable isotope profiles
from living and fossil molluscs. Paleogeography, Paleoclimatology, Paleoecology 58, 249–266.
Macko, S., Estep, M., 1984. Microbial alteration of stable
nitrogen and carbon isotopic compositions of organic matter. Organic Geochemistry 6, 878–890.
McConnaughey, T., Burdett, J., Whelan, J., Paull, C., 1997.
Carbon isotopes in biological carbonates: respiration and
photosynthesis. Geochimica et Cosmochimica Acta 61 (3),
611–622.
McMillan, C., Parker, P., Fry, B., 1980. 13C/12 ratios in seagrasses. Aquatic Botany 9, 237–249.
Michener, R., Schell, D., 1994. Stable isotope ratios as tracers in
marine aquatic food webs. In: Lajtha, K.M., Michener, R.
(Eds.), Stable Isotopes in Ecology and Environmental Science.
Blackwell Scientific Publications, Boston, pp. 138–157.
Miller, G., Hare, P.E., 1980. Amino acid geochronology:
integrity of the carbonate matrix and potential of mulluscan
fossils. In: Hare, P.E., Hoering, T.C., King Jr, K. (Eds.),
Biogeochemistry of Amino Acids. John Wiley & Sons, New
York, pp. 415–443.
Mirecki, J., Wehmiller, J., Skinner, A., 1995. Geochronology of
Quaternary coastal plain deposits, southeastern Virginia.
USA Journal Coastal Research 11, 1135–1144.
Miyake, Y., Wada, E., 1967. The abundance ratio of 15N/14N
in marine environments. Records of Oceanographic Works 9
(1), 37–53.
Neff, J., 1972. Ultrastructure of the outer epithelium of the
mantle in the clam Mercenaria mercenaria in relation to calcification of the shell. Tissue & Shell 4 (4), 591–600.
O’Donnell, T., 2002. Stable Isotope Compositions and Amino
Acid Preservation in Mercenaria Shells. Dissertation submitted to University of Virginia.
Parker, P., 1964. The biogeochemistry of the stable isotopes of
carbon in a marine bay. Geochimica et Cosmochimica Acta
28, 1155–1164.
Peterson, B., Howarth, R., Garritt, R., 1985. Multiple stable
isotopes used to trace the flow of organic matter in estuarine
food webs. Science 227, 1361–1363.
Peterson, B., Howarth, R., 1987. Sulfur, carbon, and nitrogen
isotopes used to trace organic matter flow in the salt-marsh
estuaries of Sapelo Island, Georgia. Limnology and Oceanography 32 (6), 1195–1213.
Qian, Y., Engel, M., Macko, S., 1992. Stable isotope fractionation of biomonomers during protokerogen formation.
Chemical Geology 101, 201–210.
Rau, G., Mearns, A., Young, D., Olson, R., Schafer, H.,
Kaplan, I., 1983. Animal 13C/12 correlates with trophic level
in pelagic food webs. Ecology 64 (5), 1314–1318.
Rau, G., Teyssie, J.L., Rassoulzadegan, F., Fowler, S., 1990.
13
C/12 and 15N/14N variations among size-fractionated marine particles: implications for their origin and trophic relationships. Marine Ecology Progress Series 59, 33–38.
Smith, J., Schwarcz, H., Risk, M., 2000. Paleotemperatures
from deep-sea corals: overcoming ‘vital effects’. Palaios 15,
25–32.
Szabo, B., 1985. Uranium-series dating of fossil corals from
marine sediments of southeastern United States Atlantic
Coastal Plain. Geological Society of America Bulletin 96,
398–406.
Tieszen, L., Boutton, T., Tesdahl, K., Slade, N., 1983. Fractionation and turnover of stable carbon isotopes in animal
tissues: implications for d13C analysis of diet. Ocelogia 57,
32–37.
Toscano, M., York, L., 1992. Quaternary stratigraphy and sealevel history of the U.S. Middle Atlantic Coastal Plain.
Quaternary Science Reviews 11, 301–328.
USEPA, 2002. National Coastal Condition Report. United
States Environmental Protection Agency, Washington,
DC.
Wada, E., 1980. Nitrogen isotope fractionation and its significance in biogeochemical processes occurring in marine
environments. In: Goldberg, E., Horibe, Y., Saruhashi, K.
(Eds.), Isotope Marine Chemistry. Uchida, Tokyo, pp. 375–
398.
Wada, E., Terazake, M., Kabaya, Y., Nemoto, T., 1987. 15N
and 13C abundances in the Antarctic Ocean with emphasis
on the biogeochemical structure of the food web. Deep-Sea
Research 34 (5/6), 829–841.
T.H. O’Donnell et al. / Organic Geochemistry 34 (2003) 165–183
Wefer, G., Berger, W., 1991. Isotope paleontology: growth and
composition of extent calcareous species. Marine Geology
100, 207–248.
Wehmiller, J., Belknap, B., Boutin, B., Mirecki, J., Rahaim,
S., York, L., 1988. A review of the aminostratigraphy of
Quaternary mollusks from United States Atlantic Coastal
Plain. Geological Society of America, Special Paper 227 p.
69–110.
Wehmiller, J., Miller, G., 1998. Amino acid racemization. In:
Sowers, J., Noller, J., Lettis, W. (Eds.), Dating and Earthquakes: Review of Quaternary Geochronology and Its
Application to Paleoseisomology. US Nuclear Regulatory
Commission-5562, Washington, DC, pp. 2307–2359.
Wehmiller, J., Lamothe, M., Noller, J., 1998. Comparison of
approaches to dating Atlantic Coastal Plain sediments, Vir-
183
ginia Beach, Virginia. In: Sowers, J., Noller, J., Lettis, W.
(Eds.), Dating and Earthquakes: Review of Quaternary
Geochronology and Its Application to Paleoseisomology. US
Nuclear Regulatory Commission-5562, Washington, DC. pp.
3-4–4-31.
Wehmiller, J., Miller, G., 2000. Aminostratigraphic dating
methods in Quaternary Geology. In: Quaternary Geochronology: Methods and Applications. American Geophysical
Union Reference Shelf 4, Washington, DC, pp. 187–222.
Weiner, S., Hood, L., 1975. Soluble protein of the organic
matrix of mollusk shells: a potential template for shell formation. Science 190, 987–988.
Wilbur, K., Simkiss, K., 1968. Calcified shells. In: Florkin, M.,
Stotz, E. (Eds.), Comprehensive Biochemistry. Elsevier, New
York, pp. 229–295.