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Estuarine, Coastal and Shelf Science 78 (2008) 267e279

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A preliminary investigation of the fish food web in the Gironde estuary,

France, using dietary and stable isotope analyses
S. Pasquaud a,*, P. Elie a, C. Jeantet a, I. Billy b, P. Martinez b, M. Girardin a
a
Cemagref, Groupement de Bordeaux, Estuarine Ecosystems and Diadromous Fish Research Unit, 50 avenue de Verdun, 33612 Cestas Cedex, France
b
Université Bordeaux 1, UMR CNRS 5805 EPOC, Paleoclimates Research Unit, Avenue des Facultés, 33405 Talence Cedex, France
Received 23 August 2007; accepted 17 December 2007
Available online 18 January 2008

Abstract

Carbon and Nitrogen stable isotopes and stomach contents analyses were used to investigate an estuarine fish food web and identify the
contribution of these two methods to the knowledge and understanding of the food web’s structure and its functioning. The nine most abundant
fish species during the warm period in the Gironde estuary (southwest France, Europe) are examined. Observation of the stomach contents re-
flects a variety of feeding modes between fish species that consume a diverse assortment of prey, with limited dietary overlap. Nevertheless,
when regarding the whole fish community, few prey species dominate the stomach contents. Nitrogen isotope ratios indicate a high intraspecific
variability inducing an interspecific covering of the signatures. However, a tendency to d15N enrichment according to the trophic position of the
species studied was observed. Fish assemblages show a trend towards enrichment of their carbon isotopic signatures from the upper estuary
(20.8  1.8&) towards the lower estuary (18.3  1.6&). But whatever the capture zone considered, most of the individual d13C values
for each fish analysed are comprised between 22 and 16&. Only few specimens, belonging to migratory amphihaline species, have signif-
icantly lighter values.
The stomach contents method of analysis has the advantage of giving an initial view of the ichthyological trophic structure of the system by
describing the food relations between a fish species and its prey. From these results, hypotheses can be drawn about the network’s functioning,
suggesting a sharing of resources between species and a ‘‘wasp-waist’’ control of this estuarine food web. The stable isotope analysis method
enables us to improve our structural knowledge by positioning the different species in a food web, with their position being determined by the
number of energy transfers (analysis of d15N). Conversely, in environments as complex and changing as estuaries, it appears difficult to precisely
identify and quantify the sources of the organic matter at the base of the fish estuarine food webs using analysis of d13C isotopic signatures.
Nevertheless, the results obtained by using these two methods in parallel suggest that more detailed functional ecological studies could be
carried out in future.
Ó 2008 Elsevier Ltd. All rights reserved.

Keywords: d13C; d15N; stomach contents; fish; food web; estuarine ecosystem

Regional index terms: Europe; France; Gironde estuary; Lat. 45 200 N; Long. 0 450 W

1. Introduction fluctuations in physicochemical factors and the biotic commu-

Estuaries are particular ecosystems in which the abiotic en-
vironment is especially characterized by rapid and intense
* Corresponding author.
E-mail address: stephanie.pasquaud@bordeaux.cemagref.fr (S. Pasquaud).
nities are characterized by a strong spatial and seasonal hetero-
geneity and variability (McLusky and Elliott, 2004).
Nevertheless, they are amongst the most productive systems
at various levels (Day et al., 1981; Costanza et al., 1997) and
they play a crucial role in maintaining biodiversity in aquatic
systems, particularly for fish. However, these ecosystems are
often subjected to considerable anthropogenic pressures such

0272-7714/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ecss.2007.12.014
268 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279
as fishing, harbour activities, dredging or industrial and urban
discharges, threatening their integrity. Describing and attempt-
ing to understand their structure and their functioning, through
the study of natural variations and anthropogenic effects, con-
stitutes an essential step in maintaining and restoring the qual-
ity of estuarine areas and providing sustainable management
for these systems (Pasquaud et al., 2007).
Assuming, like Platt and Denman (1978), that ‘‘the struc-
ture of a community of species resulted from trophic interac-
tions’’, several recent scientific works emphasize food web
ecology as central to an understanding of how aquatic systems
function (Ulanowicz, 1986; De Jonge, 1990; Baird et al., 1991;
Baird and Ulanowicz, 1993; Livingston, 2002). These authors
put interactions between the different living parts of the
system at the heart of ecosystem considerations. The above
considerations dictate the need to describe the estuarine food
web by characterising trophic relationships, sources of organic
matter and energy flows between the components of the sys-
tem (West et al., 2003).
Through the necessity to synthesize and to improve our
knowledge about the trophic structure of estuarine ecosystems,
new techniques to describe and quantify organic matter flows
have appeared with the development of data-processing and in-
terpretative tools, such as trophic models (e.g., Wolff et al.,
2000).
Whipple et al. (2000) put forward that ‘‘Construction,
validation and application of models is an useful method to
evaluate predation and fisheries mortality and the impacts of
these processes on population dynamics’’. Most of the models
tackled by this author, especially process-based models, re-
quire qualitative or quantitative data about species interactions
in the system, as well as the analysis of dietary compositions.
So, local and relevant studies are often necessary, in particular
to describe the trophic regimes of species.
An Ecopath model (Polovina, 1984), one of the main static
approaches in modelling trophic flows, has been recently real-
ized in the Gironde estuary from available data and knowledge
about this system (see Lobry et al., in press for details). This
work highlights the need of diet biomass data, particularly
for the main fish taxa present in the system, and similarly
the need to study the trophic seasonal dynamics.
That’s why a recent work has been undertaken on the deter-
mination of the seasonal trophic relationships concerning the
main Gironde fish species (from summer 2003 to spring 2004;
unpublished data). Stomach contents analysis has appeared to
be the most reliable method in order to obtain this kind of infor-
mation (Pasquaud et al., 2007). This technique is considered to
be a standard practice for diagnosing fish trophic relationships
(Hyslop, 1980; Cortès, 1997). It consists of the direct observa-
tion of stomach contents, providing an instantaneous record of
what an animal has consumed and in what quantity (Costa
et al., 1992; Elliott and Hemingway, 2002).
In recent years, increased interest and utilisation of stable
isotopes of organic matter has been used to study the architec-
ture of the aquatic food webs (Deegan and Garritt, 1997). The
stable isotope method is based on the fact that the atoms that
make up living beings are derived from the atoms of their food
(DeNiro and Epstein, 1978; Minagawa and Wada, 1984;
Hesslein et al., 1993). Stable isotopes therefore represent an
integrative record of the food that has really been assimilated
by the organism during a period prior to the sampling. The
isotopes that are most commonly used in aquatic ecology
are carbon, 13C and 12C, and nitrogen, 15N and 14N (Peterson
and Fry, 1987; Peterson, 1999). The analysis involves studying
the isotope ratio, 13C/12C (or d13C), and/or 15N/14N (or d15N).
For a living being, the carbon isotope ratio provides an esti-
mate of the origin (fluvial, marine, etc.) of the assimilated
organic matter (DeNiro and Epstein, 1978; Fry and Sherr,
1984; Post, 2002) and the nitrogen to carbon ratio gives an
indication as to its trophic level (DeNiro and Epstein, 1981;
Minagawa and Wada, 1984; Wada et al., 1991).
The application of these stable isotopes has become a pow-
erful tool for identifying food web linkages (Wada et al., 1991)
in rivers (Finlay, 2001), flood plains (Lewis et al., 2001), salt
marshes (Currin et al., 1995), lakes (Kling et al., 1992; Vander
Zanden and Rasmussen, 1999) and marine systems (Michener
and Schell, 1994). But, few studies use this method on the
scale of entire fish communities in an estuarine environment
(e.g. Kwak and Zedler, 1997; Garcia et al., 2007).
The overall aim of this study is to evaluate to what extent
the stomach contents and stable isotope analytical methods
can provide us with knowledge of the structure and function-
ing of estuarine fish food webs. Our first objective is to present
the results of these two methods on the main species of fish in
the Gironde estuary and the second is to analyse their respec-
tive contributions.
2. Materials and methods
2.1. Study area
The Gironde is a macrotidal estuary, located in South West
France (45 200 N, 0 450 W; Fig. 1) opening into the Bay of Bis-
cay. It is 76 km long, up to 12 km in width and has a surface
area of 635 km2 at high tide; it is thus one of the most exten-
sive estuary systems in western Europe (Salomon, 2002). It is
fed by two rivers, the Garonne and the Dordogne, which drain
a watershed of 81 000 km2. Their confluence forms the Bec
d’Ambès, the approximate upstream salinity limit. In this
vast area where continental freshwater and ocean seawater
are mixed together, many hydro-sedimentary processes occur,
making it one of the most turbid estuaries in Europe (Castaing
and Allen, 1981; Sautour and Castel, 1995). This high turbid-
ity restricts light penetration considerably and thus limits the
production of phytoplankton. The base of the food webs in
this zone therefore consist, for the most part, of a varied nutri-
tional pool containing a high proportion of detritus (Irigoien
and Castel, 1995).
The climate of the region is temperate and under oceanic
influence. There is moderate variability in water temperature
(between 2  C in January and 26  C in August) and monthly
rainfall fluctuates between 50 mm in summer and 100 mm in
winter (Tank et al., 2002). The river drainage has a seasonal
precipitation pattern; with high discharge periods in the
 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279
Fig. 1. Map of the marine part of the Gironde estuary, black stars showing the location of the sampling sites.
autumn (rain) and in the spring (snowmelt), and lower dis-
charges in the late summer. Thus, during a typical year, tem-
perature, salinity and river flow in the estuary exhibit a clear
seasonality with lower water flow and higher temperatures
and salinities over the summer months (July, August,
September).
As part of established fish monitoring and surveillance
schemes, the waters of the Gironde have been sampled regu-
larly since the early 1980s by two different and complementary
methods (see Lobry et al., 2003 for details). These sampling
surveys have initiated some recent works (Lobry et al., 2003,
2006), improving the structural and dynamic knowledge of
Gironde fish assemblages.
As a whole, the Gironde’s fish assemblages seem to be
dominated by marine species. The relative proportion of resi-
dent species is very small. Moreover, the Gironde would
appear to be the European estuary with the largest migratory
amphihaline assemblage (Lobry et al., 2003). Annual commu-
nity variability exists, but seasonal variations are undoubtedly
more important when determining the species composition of
the Gironde’s estuarine assemblages. Three main ecological
seasons corresponding to three distinctive fish assemblages
that have been described (Lobry et al., 2006). Among them,
269
the cold (approximately from November to March) and
warm (approximately from July to October) seasons show
the most distinct structures (Lobry et al., in press). In that pre-
liminary investigation, the warm period was chosen for further
study, preferred to the cold season due to higher species diver-
sity over the summer months.
2.2. Fish sampling
Sampling for stable isotope analysis was carried out in
September 2004. The study of the fish stomach contents, aim-
ing at characterizing their seasonal trophic relationships, was
undertaken from summer 2003 to spring 2004 (unpublished
data). In that study, data collected in July and in September
have been selected to consider the same period of the year
for the two studied methods, taking into account the integra-
tion time of the isotopic signal (Perga and Gerdeaux, 2005).
Date were collected once monthly across seven sampling
stations (Fig. 1), located in the marine part of the Gironde es-
tuary. Fish sampling events were restricted to daylight hours at
high tide by relative small coefficients (comprised between 48
and 75) in order to standardize them. Benthic and demersal
fish were sampled using an otter trawl with a 4 m vertical
270 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279

opening and a cod-end with a mesh size of 8 mm. Pelagic fish
were sampled using a surface grid 4 m  1 m, with a net with
a mesh size of 18 mm in the main part of the device and
2.8 mm in the end part. To optimise stomach content analysis,
sampling hauls were limited to 15 min in order to minimise
regurgitation or abnormal feeding. All the sampled fish were
identified, enumerated, measured (total length), weighed and
then stored on dry ice on board the sampling vessel, before
freezing in the laboratory at 20  C while awaiting further
analysis. At the laboratory, the most abundant fish species
and the same age classes were chosen for the both analyses
(Table 1).
2.3. Stomach content analyses
The stomach contents of 494 individuals from the nine
most abundant species during the sampling period in the
Gironde estuary were analysed (Table 1). All items present
in the stomachs were examined under a binocular microscope,
identified to the lowest possible taxonomic level and enumer-
ated. The volume of each prey was assessed by multiplying the
number of individuals by a prey-dependant volumetric coeffi-
cient (Hyslop, 1980).
Dietary analysis is traditionally assessed by occurrence, nu-
merical and volumetric/gravimetric methods (Hynes, 1950;
Hyslop, 1980). Baldó and Drake (2002) based their analysis
on these traditional indices and came to the conclusion that,
because of the degree of correlation, all were suitable for
describing trophic relationships. Nevertheless, each of these
measures provides different insight into feeding habits of
predators (Cortès, 1997). The volume or gravimetric measures
reflect dietary nutritional value (Macdonald and Green, 1983).
It is for this reason that in the current study, results are ex-
pressed by the volumetric percentage, calculated for each
item consumed by a fish species. The occurrence, numerical
and volumetric percentages of each preys for all analysed
stomachs were also calculated, indicating the relative contri-
bution of these different preys for the entire fish assemblages
(Baldó and Drake, 2002). Fish samples with empty stomachs
were excluded from this analysis.
2.4. Stable isotope analyses
Analyses of isotopic abundances require different pre-treat-
ments of the biological samples. To limit, as much as possible,
the variability of the lipid body compartment of the fish having
any influence on the analysis, dissection of the dorsal white
muscle was preferred (Hesslein et al., 1993; Dufour and
Gerdeaux, 2001; Pinnegar et al., 2003; Bardonnet and Riera,
2005). For small fish, all the muscle tissue was recovered by
removing the digestive tube, the skin, the head, the fins and
the main bones, thus eliminating all sources of nitrogen and
carbon which might distort the analyses (Bardonnet and Riera,
2005). All samples were then rinsed in Milli-Q water, freeze-
dried for 48h and ground into a fine, uniform powder using
a pestle and mortar. To remove any traces of carbonates, the
small fish that had been recovered whole were treated with
hydrochloric acid 1 M (Bardonnet and Riera, 2005) before
once again being freeze-dried and ground.
Measurements were carried out using an Isoprime isotope
mass spectrometer (Micromass, UK), coupled to a Carlo
Erba elemental analyser. Values are reported relative to atmo-
spheric nitrogen (N2) for nitrogen and the Pee Dee Belemnite
standard, a marine limestone fossil, for carbon, in the delta
notation (d) expressed per mil (&) using the formula:


d ¼ Rsample =Rstandard  1  1000;
R ¼ heavy isotope=light isotope
Precision of the isotopic analyses was 0.2& for carbon and
0.3& for nitrogen.
2.5. Statistical analyses
In order to test possible spatial, intraspecific and interspe-
cific variability in isotopic results, a two-factor variance anal-
ysis (ANOVA) was carried out on the signals obtained for d13C
and d15N. The alpha threshold, the significance threshold for
the test, was fixed at 1%. Since the ANOVA analysis requires
data to be standardised, this was verified beforehand using the
KolmogoroveSmirnov test.

3. Results
Table 1
Total length range (mm) of the nine fish species studied and number of sam- 3.1. Feeding ecology
ples used for the two methods of analysis
Method Stomach content Stable isotope The feeding ecology of each fish species was described
analysis analysis from the stomach contents analyses. Of the 494 fish stomachs
Species TL range Sample TL range Sample analysed, 421 contained prey (85%) and were considered for
(mm) number (mm) number this analysis. Forty-two taxa, from a range of locations in
Sprattus sprattus 46e133 63 63e110 6 the water column (zooplankton, hyperbenthos, nekton, epiben-
Engraulis encrasicolus 38e80 51 49e59 7 thos, benthos) were identified (Table 2). Upon first examina-
Potamotoschistus minutus 23e77 110 29e65 7
tion, these fish species showed a wide variety of feeding
Dicentrarchus labrax 53e220 14 79e195 6
Dicentrarchus punctatus 90e151 9 75e189 5 modes, evident from a diverse assortment of prey. The dietary
Argyrosomus regius 30e235 142 54e300 20 overlap among species appears limited.
Anguilla anguilla 289e740 26 267e760 21
Platichthys flesus 73e272 37 121e333 11 e Sprattus sprattus and Engraulis encrasicolus, (marine pe-
Solea solea 77e265 42 90e198 12
lagic fish), present a similar zooplanktivore trophic
Table 2
Importance of prey taxa (O, occurrence, N, numerical and V, Volumetric percentages) in all analysed stomachs and relative volumetric diet composition (%V) of nine fish species collected during the warm period
in the Gironde estuary
%O %N %V %V
All stomachs Sprattus Engraulis Pomatoschistus Dicentrarchus Dicentrarchus Argyrosomus Anguilla Platichthys Solea
sprattus encrasicolus minutus labrax punctatus regius anguilla flesus solea
Size range (TL; mm) 46e133 38e80 23e73 53e220 90e151 30e235 286e740 73e272 77e265
Number of full stomach 421 53 41 86 14 7 132 21 30 37
Zooplankton
Nauplii larvae 14.7 72.7 15.5 87.2 31.9 1 0.3
Mysis larvae 4.1 0.3 0.6 0.4 4.5 1.8
Cypris larvae 15.2 3.0 2.1 2.4 5.8 6.6
Eggs 2.9 3.6 0.8 0.1 20.5 1.2 0.8

S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279

Mollusc larvae 7.2 1.0 0.2 1.1 1
Copepod
Acartia spp. 13.8 8.5 0.7 7.9 16.9 0.1 0.2
Eurytemora affinis 0.5 0.5 1.3 0.5 0.1 0.3
Copepods spp. 0.5 0.0 0.0 0.1
Hyperbenthos
Mysidacea
Neomysis integer 8.7 0.3 0.7 7.2 5.2 1.6
Mesopodopsis slabberi 33.6 4.1 9.5 9.3 34.3 0.7 33.5 0.1 0.1
Schystomysis spp. 7 0.4 1.0 2.4 6.0 3.3
Gastrosaccus spinifer 1.0 0.0 0.1 0.2
Mysids spp. 2.4 0.1 0.1 0.3 6.7 0.4
Isopoda
Idotea pelagica 2.9 0.1 0.3 11.9 0.6 0.1
Synidotea laticauda 9.4 1.3 12.1 1.5 32.0 56.7 4.5 73.9 5.2
Isopods spp. 0.2 0.0 0.0 0.1
Amphipoda
Gammarus spp. 1.4 0.0 0.2 4.5 0.5 0.2
Decapoda natantia
Palaemon spp. 11.4 0.5 6.1 23.6 9.3 13.3 11.8 1.1 0.2
Crangon crangon 20.8 0.8 8.8 11.2 16.5 11.2 24.5 1.5 1.5 4.5
Shrimps spp. 0.2 0.0 0.1 0.2
Nekton
Teleost fishes
Sprattus sprattus 0.2 0.0 0.2 0.8
Engraulis encrasicolus 0.2 0.0 0.2 4.7
Pomatoschistus spp. 3.6 0.1 3.3 11.7 2.1
Argyrosomus regius 0.2 0.0 0.2 4.7
Solea spp. 0.5 0.0 0.6 14.1
Fishes spp. 1.4 0.0 1.2 22.4 2.3
Epibenthos
Isopoda
Cyathura carinata 3.6 0.2 1.4 10 0.4
Amphipoda
Corophium volutator 1.7 0.1 0.5 0.6 0.9 1.4
Decapoda brachyura
Carcinus maenas 0.5 0.0 0.3 2.4 2.1

271
(continued on next page)
272 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279

behaviour, feeding essentially on larvae, mainly repre-

Solea
solea

76.4
0.4
3.4
7.9
0.2
4.2

0.5
sented by nauplius stages of crustacean (respectively
%V ¼ 87.2% and 31.9%) and on copepods of the genus
Acartia (%V ¼ 7.9% and 16.9%). Nevertheless, the diet
Platichthys

of E. encrasicolus is distinguished from that of S. sprattus

by a consumption of mysids (Mesopodopsis slabberi,
flesus

11.6
4.3
1.5

1.2
62 Neomysis integer and Schistomysis spp.).
e Pomatoschistus minutus, estuarine resident species, shows
a highly diversified diet containing zooplanktonic, hyper-
Anguilla
anguilla

benthic and benthic prey. However, the mysid Mesopodop-

5.1

0.3
1
1
1

sis slabberi (%V ¼ 34.3%), the isopod Idotea pelagica

(%V ¼ 11.9%) and the shrimp Crangon crangon
Argyrosomus

(%V ¼ 11.2%), hyperbenthic species, represent the great-

est volumes ingested by P. minutus.
regius

e The marine demersal fish Dicentrarchus labrax, Dicen-

trarchus punctatus and Argyrosomus regius base their
feeding mainly on hyperbenthic and nekton compartments
Dicentrarchus

with a high amount of Synidotea laticauda, shrimps (Pa-

punctatus

laemon spp. and Crangon crangon) and teleost fish (e.g.

Sprattus sprattus, Engraulis encrasicolus, Pomatoschistus
0.1

spp., Solea spp.) found in their stomachs. Argyrosomus.

regius is distinguished from the others by a high consump-
Dicentrarchus

tion of mysids (%V Mesopodopsis slabberi ¼ 33.5%).

e The feeding of Anguilla anguilla, a diadromous bentho-
labrax

demersal fish, consists almost exclusively of hyperbenthic

0.9

and epibenthic species with the highest volumetric

percentage of the isopod Synidotea laticauda (%V ¼
73.9%), the shrimps Palaemon spp. (%V ¼ 11.8%), and
Pomatoschistus

crabs, mainly Pachygrapsus marmoratus (%V ¼ 5.1%).

An eel predation on Argyrosomus regius has also been
minutus

0.1

0.4

highlighted in that study.

3

e The diets of the two flatfish sampled in this study, Solea

solea and Platichthys flesus, consist of hyperbenthic (e.g.
encrasicolus

Synidotea laticauda, Crangon crangon) and benthic prey

Engraulis

(polychaetes). The polychaete, Nereis sucinea, is the

0.1
0.2

most represented prey in ingested volume term. Plati-

chthys flesus has a more diversified trophic range, consum-
ing a relatively large amount of epibenthic prey, in
Sprattus
sprattus

particular the isopod Cyathura carinata (%V ¼ 10%).

0.4
%V

When regarding the whole fish community overall, the

24.7
0.5
0.1
0.1
0.1

0.1
1.3
1.9
0.0
2.5
0.0

0.3
0.1
%V

crustacean larvae, the copepod Acartia, the mysid Mesopodop-

sis slabberi, the isopod Synidotea laticauda, the shrimps
Palaemon spp. and Crangon crangon, and the polychaete
0.0
0.0
0.0
0.0

1.3
0.0
0.1
0.2
0.0
0.1
0.0

0.2
0.6
%N
All stomachs

Nereis succinea were the most important consumed prey items

over this period of the year.
10.9
0.7
0.2
0.2
0.2

0.2
1.7
1.9
0.2
3.1
0.7

8.7
1.7
%O

3.2. Trophic levels

Pachygrapsus marmoratus

The trophic levels of the nine fish species are characterized

Rhithropanopeus harrisii

by d15N values. The ANOVA analysis carried out on their

Streblospio shrubsolii
Liocarcinus holsatus

Plant/algal material
Nereis diversicolor

nitrogen content showed no spatial variability ( p ¼ 0.49).

Annelida polychaeta
Table 2 (continued )

Polychaetes spp.
Nereis succinea

Consequently, the capture zone was not taken into consider-

Polydora spp.

ation for this approach. The average d15N content of the estu-
Nereis spp.
Crabs spp.

Molluscs

arine ichthyofauna is presented in ascending order (Fig. 2).

Pollen
Benthos

Comparison of d15N contents according to size class for the

Other

different fish species showed no significant differences, except

 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279 273

D. labrax*
A. anguilla
P. flesus
A. regius'
D. punctatus
D. Labrax**
S. solea
S. sprattus
P. minutus
A. regius''
E. encrasicolus

8,0 8,5 9,0 9,5 10,0 10,5 11,0 11,5 12,0 12,5 13,0 13,5 14,0
Lighter δ15N Heavier

Fig. 2. Mean d15N signatures and standard deviations (&) of the ichthyofauna in the Gironde estuary. *: total length range ¼ 186e195 mm; **: TL range ¼ 85e
95 mm; 0 : TL range ¼ 150e300 mm; 00 TL range ¼ 54e125 mm.

in Dicentrarchus labrax (R2 ¼ 0.72) and Argyrosomus regius
(R2 ¼ 0.60). These two species were therefore separated by
size class (Fig. 2).
The d15N signals obtained for each species that made up the
ichthyofauna sampled were around 9.1&  0.6& for Engrau-
lis encrasicolus and 13.6&  0.1& for large Dicentrarchus
labrax. Overall, in the values obtained for each species, there
were considerable standard deviations, in excess of 1& in S.
solea and the group of small Argyrosomus regius. This may
therefore lead to variable positioning across different trophic
levels, depending on the individual. The average d15N content
of the fish sampled in the Gironde estuary thus shows a high
level of intraspecific variability.
However, a trend of higher d15N enrichment levels with
a higher trophic position can nevertheless be seen. The small
pelagic fish (Engraulis encrasicolus, Sprattus sprattus) have
lower levels of d15N, with average values of between 9.1
This trend is also observed when considering the mean d13C
signatures of each fish captured in upper, middle and lower
zones of the studied area (Fig. 4). But whatever the capture
zone considered, most of the individual d13C values for each
fish analysed fell between 22 and 16&. Only few speci-
mens have significantly lighter values. Some Anguilla an-
guilla, sampling in the upper and in the middle zones and
two Platichthys flesus, one catching in the middle zone and
the other in the lower zone, have the lowest values.
4. Discussion
Knowledge of the dietary pattern of fish is extremely im-
portant for an understanding of the biological interactions
-24
‰
-23
Lighter

and 112& whereas the fish considered as top-predators

(Dicentrarchus labrax, Argyrosomus regius, Dicentrarchus -22
punctatus) and benthic/epibenthic fish (Platichthys flesus,
Solea solea, Anguilla anguilla) have the highest levels, be- -21

tween 11.9 and 13.6&. -20

-19

3.3. Organic matter sources -18

Because of a low d13C enrichment level during trophic tran- -17

δ13C

sition (about 1&), the levels of this isotope ratio in consumers

-16
provide information on the main sources of organic matter at
the bottom of the food webs. As consumers tend to assimilate -15
organic matter in the area where they were caught, the average
-14
d13C levels in ichthyofauna were separated according to the
three sampling zones where they were caught (Fig. 3). The -13
high standard deviations produced an overlap of d13C levels
-12
in fish caught in the different zones studied. However, the
Heavier

overall trend observed showed an enrichment of the isotope -11

signal from the upper estuary towards the lower estuary,
with average values of 20.8  1.8& for the upper estuary
(n fish analysed ¼ 13), 20.6  2.3& for the middle estuary
(n ¼ 44) and 18.3  1.6& for the lower estuary (n ¼ 38).
-10
Fig. 3. Mean d13C signatures and standard deviations (&) of the ichthyofauna
in the upper zone , middle zone and lower zone of the Gironde estuary.
274 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279

(‰) -26.0
A.a
-25.5
A.a
Lighter

-25.0
-24.5 P.f A.a
A.a
-24.0 A.a A.a
-23.5 A.a A.a P.f

-23.0 A.a
A.a A.a
-22.5
-22.0 P.f
D.l A.a
-21.5 D.l A.a
P.f
P.m P.f P.m A.a
-21.0 A.a S.s
D.l D.p S.s A.a
E.e P.mA.r P.f
-20.5 P.f
D.l P.m A.r
S.s P.f
C

-20.0 E.e A.r E.e A.a

E.e
13

A.r Sp.s P.m

A.a
-19.5 A.r Sp.s
A.r A.r
A.r S.s E.e Sp.s
-19.0 A.a P.m
E.e P.f Sp.s
-18.5 E.e D.p Sp.s
A.r A.r A.r
-18.0
A.a P.f A.r A.r
Sp.s P.m A.r
D.p A.r A.r S.s A.r
-17.5 S.s S.s
A.r D.p A.r A.r
-17.0 D.l S.s
S.s A.a
P.f
-16.5 S.s
D.p
-16.0 S.s
S.s
Heavier

-15.5
D.l
-15.0

Upper Middle Lower

Fig. 4. Mean d13C signatures and standard deviations (&) of fish in the upper, middle and lower zones of the Gironde estuary. A.a: A. anguilla; A.r: A. regius; D.l:
D. labrax; D.p: D. punctatus; E.e: E. encrasicolus; P.f: P. flesus; P.m: P. minutus; S.s: S. solea; Sp.s: S. sprattus.

that occur within an ecosystem (Odum, 1953). Stomach con-
tents analysis appears to be the most appropriate method for
understanding these interactions in estuarine environments
(Pasquaud et al., 2007).
At a species level, even if the stomach content analysis pro-
vides only a snapshot of the diet, it has the advantage of giving
useful taxonomic data about prey items and an intake estima-
tion for each of them at a given moment. This knowledge is
particularly useful when attempting to improve upon the
construction and validation of trophic models (Lobry et al.,
in press). Many fish species demonstrate a degree of plasticity
in prey item choice and change their diet seasonally and/or in
response to prey availability (Costa et al., 1992; Cabral, 2000;
Laffaille et al., 2001; Elliott and Hemingway, 2002; Mollmann
et al., 2004; Salgado et al., 2004). Nevertheless, similarities
were found between qualitative dietary data of some fish
species (Sprattus sprattus, Pomatoschistus minutus, Dicen-
trarchus labrax, Argyrosomus regius, Anguilla Anguilla,
Platichthys flesus and Solea solea) from other estuarine envi-
ronments (Costa, 1988; Costa et al., 1992; Cabral, 2000;
Cabral and Ohmert, 2001; Maes and Ollevier, 2002; Salgado
et al., 2004; Vinagre et al., 2005), as well as in the Gironde,
as determined over the course of the present study. Note that
this work also provides the first results of S. sprattus and
Dicentrarchus punctatus feeding ecology in an estuarine eco-
system. Moreover, the dietary patterns of the fish studied seem
to be closely linked with the position of these predators in the
water column. For example, the flatfish species S. solea and P.
flesus, which dominate the substrate, feed most often on ben-
thic prey (e.g. the polychaete Nereis succinea), whereas small
pelagic fish (such as S. sprattus and Engraulis encrasicolus)
consume zooplanktonic and hyperbenthic prey exclusively.
So, the use of this method gives us useful information on
the dietary requirements and behaviours of species, allowing
a first description of fish trophic relationships.
On the scale of an entire fish community, analysis of stom-
ach contents provides an image of the trophic structure of the
system (Fig. 5) and represents the first stage in the understand-
ing of its overall functioning (e.g. West et al., 2003).
Indeed, in this study the dietary overlap of the fish species
considered appears fairly weak, due to different assemblages
of consumed prey. Even among species with dietary similari-
ties (Sprattus sprattus and Engraulis encrasicolus or Dicen-
trarchus labrax, Dicentrarchus punctatus and Argyrosomus
regius or Solea solea and Platichthys flesus), some have a diver-
sified diet (E. encrasicolus, A. regius, P. flesus). This suggests
a considerable sharing of resources which would seem to limit
interspecific competition for access to food resources. How-
ever, at this warm period of the year, the ‘‘fish’’ food web is
based essentially on only a small number of prey items (crus-
tacean larvae, Acartia spp., Mesopodopsis slabberi, Synidotea
laticauda, Palaemon spp., Crangon crangon and Nereis
 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279
Water column
position
Nauplii larvae
Eggs
Planktonic/
Pelagic
Acartia spp.
M. slabberi
I. pelgica
S. laticauda
Proximo-
benthic
Epibenthic
C. carinata
Polychaetes spp.
N. succinea
Benthic
Fig. 5. Structure of the fish food web during the warm period in the Gironde estuary from stomach content analyses. Arrows represent preferential relationships
(%V 10). Ellipses: preys taxa. Rectangles: fish species analysed.
succinea). According to the literature, these prey are extremely
abundant in the Gironde estuary at this time of year (Sorbe,
1981; Brosse et al., 2000; David et al., 2005; Girardin, pers.
com.). This observation would suggest a ‘‘wasp-waist’’ control
of the estuarine food web, i.e. control by the species that pre-
dominate numerically (see Cury et al., 2000).
However, using the stomach content analysis method does
not give us detailed knowledge of the different trophic levels,
nor can we identify the origin of the source of the organic mat-
ter (Pasquaud et al., 2007).
The concept of trophic level is very important in both
theoretical and applied ecology as it indicates the position of an
individual within a food web, as determined by the number of en-
ergy transfers (Pimm and Lawton, 1977, 1978; Levine, 1980;
Pace et al., 1999; Post, 2002; Williams and Martinez, 2004).
With this in mind, the use of the nitrogen isotope ratio (d15N)
has proved extremely useful in many studies (Cabana and Ras-
mussen, 1994; Vander Zanden et al., 1999; Post et al., 2000).
Analysis of d15N content in the fish caught in the Gironde
estuary showed a considerable intraspecific variability in sig-
nals. This can be explained by the size and dietary diversity
of the species considered.
275
S. sprattus
E. encrasicolus
A. regius
D. punctatus
P. minutus
D. labrax
Palaemon spp. A. anguilla
C. crangon
S. solea
P. flesus
Trophic
position
The relative size of a species plays an important role in the
structure of food webs. In any given web, predators are often
larger than their prey and they attain a higher trophic posi-
tions as their size increases (Cohen et al., 1993; Woodward
and Hildrew, 2002; Deudero et al., 2004). In fact, generally,
the bigger the predator, the better it is able to eat large
prey, in other words prey at a high trophic level. However,
this relationship between size and d15N (synonymous with
trophic level) was only demonstrated for two predatory fish
species, Argyrosomus regius and Dicentrarchus labrax, and
not for any of the other species due to either the number of
samples per species being too small or their size distributions
were too uniform. Note that for the same reasons, the relation-
ship between predator size and dietary variability was not
demonstrated by the stomach content analysis method. Estua-
rine fish populations are generally dominated by specimens
that are small in size (juveniles or sub-adults), and the pres-
ence of large predators of marine origin is rare and only occurs
sporadically (see Baldó and Drake, 2002; Hajisamae et al.,
2003; Maes et al., 2003), especially in the middle and upper
parts of the estuary. A similar observation in the Gironde es-
tuary reinforces this point and confirms the protective function
276 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279
attributed to estuarine habitats for juvenile fish (e.g. Elliott and
Taylor, 1989).
Fish in estuaries are often characterized as omnivorous,
sharing common resources, and flexible, being able to exploit
temporary peaks in prey populations (Ley et al., 1994). Thus
the ability of certain species to feed at more than one trophic
level can also increase the intraspecific variability of d15N
signals. In this study, for example, Solea solea consumes
both primary producers (plant/algal material) and various
consumers (e.g. Crangon crangon, Nereis succinea). While
the proportion of different items assimilated varies according
to the individuals, their isotopic signatures are also clearly
distinguished.
Despite this intraspecific variability, which leads to an
overlap of these signals between most the fish, an interspe-
cific variability in the average d15N level emerges, showing
trophic enrichment with lower values for the small pelagic
species (Engraulis encrasicolus, Sprattus sprattus) and higher
values for the top-predators (Dicentrarchus labrax, Dicen-
trarchus punctatus, Argyrosomus regius). Thus, using stable
isotope analysis has an advantage over analysis of stomach
contents in providing an integrated signal of what has really
been assimilated by the fish and positioning the species one
alongside the other. In this study, the benthic/epibenthic fish
(Platichthys flesus, Solea solea, Anguilla anguilla) have very
high trophic positions, which is sometimes surprising, given
their dietary ecology as determined from analysis of their
stomach contents. For example, P. flesus, a predator of poly-
chaete annelids and benthic isopods, has a higher average
d15N level than A. regius and D. punctatus, fish which tend
to be ichtyophagous. Sherwood and Rose (2005) have shown
that d15N levels in benthic species are higher than those in
pelagic species for an equivalent trophic position, which
would explain our observations. Thus a precise estimate of
the trophic positions of fish based on d15N values requires
a good knowledge of the variations in d15N at the bottom
of the food chains, and of the fractionation values between
predators and their prey (Cabana and Rasmussen, 1996;
Vander Zanden and Rasmussen, 1999; Sherwood and Rose,
2005). Further work regarding these aspects in the Gironde
estuary is in progress.
In environments like estuarine ecosystems which are con-
stantly changing, it is difficult to accurately identify the
source, or sources of the organic matter at the base of the
food webs. Analysis of the carbon isotope ratio (d13C) would
seem to be an useful tool with which this can be achieved (e.g.
Fry and Sherr, 1984).
The average d13C level for the ichthyofauna in each catch-
ment zone reveals a slight spatial variation in the isotope
signal, with an increase in the signal from the upper estuary
downstream towards the lower estuary. The same spatial trend
of the mean d13C values concerning the particulate organic
carbon was found by Fontugne and Jouanneau (1987) in the
Gironde estuary. Moreover, this has also been verified by sim-
ilar studies on other hydrosystems (Riera and Richard, 1996;
Deegan and Garritt, 1997; Bardonnet and Riera, 2005) and
can be explained by the gradual mixing together of sources
of fluvial (light d13C) and marine (heavy d13C) organic matter
(Fontugne and Jouanneau, 1987). With mean values comprised
between 20.8& in the upper estuary and 18.3& in the
lower estuary, the d13C data recorded in that study are closer
to marine signals than terrestrial signals. Indeed, according to
Fontugne and Jouanneau (1987), the marine reference in the
Gironde system (corresponding to a mean annual value of
the isotopic composition of particulate carbon) is 20.5&
and the d13C terrestrial reference is 25.9&. As in other stud-
ies on estuarine food webs (Wada et al., 1991; Riera and
Richard, 1996; Deegan and Garritt, 1997) the results can
hint towards a marine predominance of the organic matter
in the studied areas. In the Gironde, this result can be ex-
plained by the hydrodynamism of the estuary at this warm pe-
riod of the year and the integration time for the isotope signal
of the fish in relation to the moment when our samples were
taken. The Gironde is a macrotidal estuary and hence the sam-
pling areas, which were located downstream from the salinity
limit, are very much affected by the tide. Moreover, we can
consider that the integration period covers at least the end
of spring and summer (Perga and Gerdeaux, 2005), that is
to say when large quantities of sea water enter the estuary be-
cause of the low water flow associated with the warm period.
But, even if the marine species dominated the fish communi-
ties during this period (link to this hydrodynamism), accord-
ing to Lobry et al (in press), then the Gironde estuary is
totally under river influence in terms of energy and feeding.
So, a mixture between estuarine enriched sources such as
benthic diatoms, and other more d13C depleted sources such
as terrestrial detritus could better explain these heavy values.
Irigoien and Castel (1997) have shown that in this turbid estu-
ary the chlorophyll levels in the water column were largely
due to benthic diatoms (that are generally very d13C enriched
on muddy intertidal flats at the ones lining estuary), and that
they greatly support most of preys species consumed by fish
(see Connolly et al., 2005).
During the cold period (approximately from November to
March), the Gironde estuary is under strongly fluvial influence
(flood) and its fish assemblages are dominated by resident
benthic and demersal species (Lobry et al., 2006). It would
be interesting to conduct a similar d13C investigation at this
period of the year to check this latter hypothesis.
Most of the individual d13C values for the fish analysed in
this study were across a range between 22 and 16&.
Only a few individuals belonging to migratory amphihaline
species (Anguilla anguilla and Platichthys flesus) had a signif-
icantly lighter d13C signature. Given the ecology of these fish
(Daverat et al., 2004; Masson, unpublished data), they could
all have spent a part of their period of summer growth up-
stream from the zone studied here and hence could have assim-
ilated organic matter from a fluvial food chain, for instance,
before coming back down to a more marine zone where they
were caught, and where they could have assimilated estuarine
and/or marine organic matter. This observation highlights, as
other studies have done (e.g. Limburg, 1998; Bardonnet and
Riera, 2005; Guelinckx et al., 2006), the usefulness of this
isotope tool for revealing fish movements.
 S. Pasquaud et al. / Estuarine, Coastal and Shelf Science 78 (2008) 267e279
5. Conclusions
This study shows that the combined use of stomach content
analysis and nitrogen stable isotope analysis can provide a de-
tailed picture of the structure of an estuarine fish food web in
the following ways: by describing trophic relationships be-
tween different biological compartments (stomach contents);
by defining trophic levels and the pelagic and/or benthic nature
of the food chains (nitrogen stable isotope). From this knowl-
edge of the trophic structure we can put forward hypotheses as
to its functioning (e.g. ‘‘wasp-waist’’ control of the web, nurs-
ery function, omnivorous nature of the majority of fish)
providing the first building blocks towards gaining a better
understanding of estuarine environments.
Conversely, in environments as complex and changing as
estuaries, it seems difficult to identify precisely from the anal-
ysis of the carbon isotope ratio what proportions of the organic
matter is assimilated by the fish. Indeed, the d13C isotopic
signatures of the various sources of organic matter (terrestrial,
estuarine and marine sources) fluctuate and are often superim-
posed in space and in time (e.g. Fontugne and Jouanneau,
1987) due to most of the species studied being highly mobile
and able to change habitats regularly. Nevertheless, the use of
carbon isotope analysis, coupled with a firm grasp of the ecol-
ogy of the fish, can be an useful tool for revealing fish
movements.
This study also underlines the importance of using two
methods simultaneously and of carrying out exhaustive sam-
pling of all fish size classes and all their potential food sources
for even better precision in the results and hence a better un-
derstanding of the functioning of the system at a given period.
The results obtained using these methods in parallel enable
us to reflect on more detailed studies of functional ecology
using multispecific and ecosystemic models based on prey-
predator relations (e.g. Christensen and Pauly, 1993) to explore
functioning hypotheses (e.g. Chassot et al., 2005). Moreover,
we recommend the use of these two methods for a better
understanding of estuarine systems and to make inter-site
comparisons with a view to improve upon sustainable manage-
ment, or even the initiation of restoration programmes for
these often extremely disturbed environments.
Acknowledgements
The authors would like to thank Pierre Richard (CNRS-
CRELA, l’Houmeau), François Le Loc’h (IRD Sète), Pascal
Riera (Biological Station of Roscoff), Benoı̂t Sautour (Marine
Station of Arcachon) and Valérie David (University of La
Rochelle) for all their valuable advice. We also thank Bernard
Ballion, Romaric Le Barh, Jean-François Bigot, Françoise
Daverat, Nathalie Tapie, Mario Lepage and everyone who
took part in sampling surveys. This investigation was sup-
ported by the French Institute of Agricultural and Environ-
mental Engineering Research (Cemagref), the Regional
Council of Aquitaine and the Ecology and Economics of the
Garonne basin programme. We would particularly thank
277
Henk van Rein (University of Ulster, Northern Ireland) for
checking the English.
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