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 Estuarine, Coastal and Shelf Science 73 (2007) 299e315
www.elsevier.com/locate/ecss

A seasonal survey of the food web in the Lapalme Lagoon

(northwestern Mediterranean) assessed by carbon and nitrogen
stable isotope analysis
Antoine Carlier a,*, Pascal Riera b, Jean-Michel Amouroux a, Jean-Yves Bodiou a,
Karine Escoubeyrou a, Martin Desmalades a, Jocelyne Caparros a, Antoine Grémare a
a
Université Pierre et Marie Curie-Paris 6 and CNRS, UMR 7621, F-66650 Banyuls-sur-Mer, France
b
UMR 7144 CNRS, Adaptation et Diversité en Milieu Marin, Station Biologique de Roscoff, Université Pierre et Marie Curie-Paris 6,
CNRS-INSUE, Place Georges Teissier, BP 74, 29682 Roscoff Cedex, France
Received 11 October 2006; accepted 22 January 2007
Available online 23 March 2007

Abstract

We used carbon and nitrogen stable isotope analysis to describe the food web of the Lapalme Lagoon, one of the best preserved coastal lagoons
along the French Mediterranean coast. Three surveys, corresponding to contrasting situations both in terms of continental inputs and of connection
between the lagoon and the open sea, were conducted in June and September 2004 and in February 2005. There were significant spatio-
temporal changes in the isotopic ratios of both primary producers and consumers. Temporal changes were mostly linked to important
13
C-depleted continental inputs caused by the long period of heavy rainfall before the June survey. Conversely, isotopic ratios were rather similar
in September and February despite the opening of the connection of the lagoon with the sea between these two surveys. The interpretation of the
results in terms of the structure of the trophic network differed between the June period and the two other ones. In September 2004 and February
2005, the food web was mostly based on SOM and lagoon POM pools with only a few consumers departing from this general trend. In June 2004,
a significant proportion of consumers were conversely 13C-depleted probably due to the assimilation of significant amounts of continental inputs by
grazers and filter-feeders. This stresses the necessity of carrying out seasonal surveys to get a sound idea of the structure of the food web in highly
variable ecosystems such as coastal lagoons. Spatial changes in isotopic ratios were likely linked to: (1) the salinity/confinement gradient with a trend
toward lower d13C and d15N values (i.e., higher continental influence) in the inner part of the main lagoon; and (2) the high d15N values of primary
producers and discretely motile consumers in a semi-confined site located downstream a vineyard receiving large amounts of fertilizers. We
hypothesised that these inputs are quickly 15N-enriched through denitrification and ammonia volatilisation processes and then contribute to the
15
N-enrichment of salt marsh plants and seagrass at this site. This interpretation is supported by the fact that such a 15N-enrichment of primary
producers only occurred in February (i.e., after the dispersion of the fertilizers). Conversely, discretely motile consumers were 15N-enriched all
year round, which suggests that they were mostly exploiting the detritic pool derived from 15N-enriched salt marsh plants.
Ó 2007 Elsevier Ltd. All rights reserved.

Keywords: food webs; d13C; d15N; coastal lagoons; spatio-temporal changes; food sources; northwestern Mediterranean

1. Introduction and/or feeding grounds because they constitute sheltered and

In spite of their small surface coastal lagoons are key zones in
the life history of many organisms (Boutière, 1980). These eco-
systems are used by marine fishes and migratory birds as nursery
* Corresponding author.
E-mail address: antoine.carlier@obs-banyuls.fr (A. Carlier).
highly productive areas (Nixon, 1981). Due to continental inputs
of nutrients and organic matter, primary production in coastal la-
goons is typically between 200 and 400 gC m2 yr1 (Nixon,
1981). In Mediterranean lagoons, phytoplanktonic production
can reach up to 300 gC m2 yr1 (Vaulot and Frisoni, 1986).
Consequently, fish landings represent up to 150 kg ha1 year1
(Amanieu and Lasserre, 1981), which is 20 times higher than in

0272-7714/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.ecss.2007.01.012
300 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
open Mediterranean (Margalef, 1985). Coastal lagoons are sub-
mitted to strong seasonal changes in main environmental param-
eters. The increase of temperature, the lack of wind and the
degradation of organic matter typically reduce oxygen availabil-
ity during summertime eventually leading to dystrophic crises
(Boutière et al., 1982; Bachelet et al., 2000). Coastal lagoons
thus constitute extreme environments, where only a restricted
number of species is usually present. The diversity of benthic
macrofauna is typically low whereas both its abundance and
biomass are high (Guelorget and Perthuisot, 1983), which
may facilitate the assessment of the architecture of benthic
food webs. However, the diversity of potential food sources
(e.g., continental inputs, salt marsh plants, seagrasses, macro-
algae and phytoplankton) makes coastal lagoons complex eco-
systems and the description of their trophic relationships
challenging. This explains why, there is currently no paradigm
stating, which main source of organic matter fuels the food
web of coastal lagoons, and how relative contributions may
shift spatially and/or temporally (Fry, 1984; Sprung, 1994;
Kwak and Zedler, 1997).
Analysis of the natural abundance of stable isotopes of
carbon (d13C) and nitrogen (d15N) had proved to be a useful
tool for the study of coastal marine food webs (Peterson and
Fry, 1987; Kwak and Zedler, 1997). Many studies have dealt
with the assessment of trophic relationships in ecosystems fea-
turing strong salinity gradients. However, coastal lagoons have
been much less studied than estuaries (Peterson et al., 1985;
Deegan and Garritt, 1997; Bardonnet and Riera, 2005), and
salt marshes (Peterson and Howarth, 1987; Riera et al.,
1999) in this particular context. Stable isotope data on food
webs of Mediterranean coastal lagoons are still scarce. The
Stagnone di Marsala (Western Sicily) was the subject of fre-
quent investigations on: (1) suspended particulate organic mat-
ter (Sarà et al., 1998); (2) seagrasses as a food source for
higher trophic levels (Vizzini et al., 2002); and (3) the diets
of Atherina boyeri (Vizzini and Mazzola, 2005) and Syngna-
thus spp. (Vizzini and Mazzola, 2004). The food web of the
Lake of Sabaudia (central Italy), was investigated seasonally
but without any assessment of spatial changes (Vizzini and
Mazzola, 2003). To our knowledge, the Vaccarès and the Mau-
guio Lagoons are the only French Mediterranean lagoons,
whose food web have been studied through the use of stable
isotopes (Persic et al., 2004; Vizzini et al., 2005). However, al-
though these large lagoons are both characterised by a salinity
gradient, their investigations were based on one single and two
sampling sites only, respectively.
Besides stable isotope analysis, the assessment of the amino
acid composition of potential food sources may also help in
assessing their relative contributions to a food web. Bioavail-
able amino acids can be quantified by mimicking digestion
through an enzymatic hydrolysis (Mayer et al., 1995). The rel-
ative contribution of enzymatically hydrolysable amino acids
(EHAA) to the total hydrolysable amino acids (THAA) pool
can then be used as an index of nutritive value (Grémare
et al., 1997; Dauwe et al., 1999).
The Lapalme Lagoon is a small coastal lagoon located on
the French Mediterranean coast. Its small catchment area is
only slightly disturbed by anthropogenic pressure. Conse-
quently, its water is of good quality and dystrophic crises
are nearly absent (Wilke and Boutière, 2000; Ifremer, 2006).
Moreover, its only connection with the open sea has not
been modified by human activities and is thus still functioning
naturally with a succession between periods of opening and
closure depending on meteorological conditions. This charac-
teristic is important since the free circulation of water plays an
essential role in the functioning of Mediterranean lagoons
(Petit, 1953). Overall, the Lapalme Lagoon is one of the
most genuine along the French Mediterranean coast and can
therefore be considered as a comprehensive example of undis-
turbed Mediterranean coastal lagoons.
The aims of the present study were to assess: (1) spatio-
temporal changes in the d13C and the d15N of primary
producers and consumers in the Lapalme Lagoon; and (2)
how these changes are linked to the relative importance of
continental and marine inputs, and to anthropogenic effects.
2. Materials and methods
2.1. Study area
The study was conducted in the Lapalme Lagoon (Languedoc-
Roussillon), which is located 60 km north of the French-Spanish
Mediterranean border. Its surface is about 600 ha, and its
mean water depth 60 cm. The lagoon is divided in 3 basins, which
are connected by narrow channels (Fig. 1). It has a unique
temporary connection with the open sea, which is usually
closed during summer, opens in fall and then remains open until
spring.
The Lapalme Lagoon shows marked seasonal changes in
salinity (i.e., between 3.6 and 76.8 with a mean annual salinity
of 28), because of seasonal rainfall (500 mm per year), and in-
tense sunshine during summertime (Wilke and Boutière,
2000). It is submitted to strong (>16 m s1) winds (150 days
per year) with maximum speed up to 56 m s1. Northwesterly
winds dominate and southeast winds are less frequent. They
both play a major role in the hydrodynamism of the lagoon
(especially in the opening of the connection with the open
sea) and generate strong resuspension events.
The Lapalme Lagoon receives freshwater from a small
catchment area (about 65 km2), which is almost not affected
by human activities. Several karstic springs, which flow almost
constantly all year round (about 0.6 m3 s1), constitute the
main freshwater inputs. The peripheric channel bordering
the salt pans north of the lagoon is a second significant input
of freshwater with an annual mean flow of 0.24 m3 s1. A few
small streams (the most important being the Rieu River) con-
tribute for sporadic (annual mean of 0.03 m3 s1) inputs of
freshwater. They are almost dry in summer, but their flow
can reach up to 3 m3 s1 in fall and winter after strong rainfall.
There are only 2 potential sources of pollution around the
lagoon: (1) the waste waters from the La Palme village (about
1000 inhabitants) which are processed in a sewage treatment
plant and then injected in the main karstic springs in the north
of the lagoon; and (2) inputs of fertilizers in association with
 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
Fig. 1. Map showing the locations of the 12 stations, and the 4 sites sampled during the present study.
the culture of the vine in the inner west part of the lagoon
(Wilke and Boutière, 1999).
About half of the bottom of the lagoon is covered by mac-
rophytes. Mean vegetal standing crop is approximately
1.7 kg WW m2 (Wilke and Boutière, 2000). Seagrasses
(i.e., Ruppia cirrhosa and Zostera noltii) dominate, followed
by macroalgae (i.e., Chaetomorpha sp., Cladophora sp.,
Ulva sp., Enteromorpha sp., Acetabularia acetabulum and
Characeae), which bloom during late spring and summer.
The Lapalme Lagoon is considered as oligotrophic and phyto-
planktonic production is limited by phosphate availability
(Wilke and Boutière, 2000).
2.2. Field observations and sample collection
The opening of the connection of the lagoon with the sea
was checked periodically between July 2003 and June 2005
by M. Wilke (CEH Aquaexpert) and L. Fonbonne (Leucate
301
Civic Center). Monthly rainfall were measured at the Météo-
France station of Perpignan (i.e., about 30 km north of the
lagoon). In the lagoon, samples were collected in June 2004,
September 2004 and February 2005 at 12 stations located
along a salinity/confinement gradient (Fig. 1). These stations
were later pooled into 4 sampling sites. Site 1 corresponded
to the northwest part of the main basin. It is the deepest and
the most brackish part of the lagoon. Site 2 corresponded to
the remaining of the main basin. This area is shallower and
sandier than the first one. Site 3 corresponded to the two little
basins located between the railway and the connection with
the open sea. Site 4 corresponded to a little pond located
west of the main basin near the mouth of the Rieu River.
This site was surrounded by dense salt marsh vegetation and
was isolated from the main lagoon in September 2004 and
February 2005. Salinity and temperature were assessed at all
stations and during all surveys (except during June at site 2)
using a WTW LF196 thermosalinometer.
302 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
Leaves of the major upland plant species in the catchment
area (i.e., Vitis vignifera, Olea europea, Populus nigra, Prunus
dulcis, Pinus halepensis, Cupressus oemphuineus, Elaeagnus
angustifolia and Ficus carica) were collected in June 2004.
The salt marsh plants bordering the lagoon (i.e., Arthrocne-
mum macrostachyum, Sarcocornia fruticosa, Halimione portu-
lacoides, Limonium vulgare, Juncus spp., Phragmites
communis and Elytrigia juncea), seagrasses and dominant
macroalgae were collected during all 3 surveys as their isoto-
pic composition could vary seasonally (Machas et al., 2003).
The salt marsh plants A. macrostachyum and H. portulacoides
and the seagrass Ruppia cirrhosa were sampled at sites 1, 3
and 4 during all 3 surveys, except for R. cirrhosa, which
was sampled at site 4 only in February. Seagrasses and algae
were carefully scraped with a scalpel and/or a teeth brush to
remove epiphytes, which were analysed separately.
Suspended particulate organic matter (POM) was sampled
both in the tributaries of the lagoon and at 12 stations within
the lagoon (except at site 2 in June) (Fig. 1). The Rieu River
was sampled in June and February. The peripheric channel
was sampled in September when salty water was entering
the lagoon. The main karstic spring was sampled during all
3 surveys and water was collected at the exit of the La Palme
sewage treatment plant in February. In all cases, 5 l of water
were collected about 10 cm below the surface. Sediment cores
were hand-collected at 5 stations (i.e., the 6 stations indicated
on Fig. 1, except site 4) during all 3 surveys and their first cen-
timeter was sliced and kept for the analysis of sediment or-
ganic matter (SOM).
The benthic fauna of the lagoon was sampled at all
12 stations and during all 3 surveys. Benthic invertebrates
were sampled by sieving hand-shoveled sediment on a 1 mm
mesh. Small invertebrates associated with the vegetation
(Gammarus aequicauda and Idotea sp.) were collected with
a hand-towed net (mesh size: 1.5 mm). Fishes were caught
with a hand-towed trawl net (mesh size: 1 cm). Overall, 15
species of benthic invertebrates and 14 fish species were col-
lected. Care was taken to get comparable samples from the dif-
ferent locations in particular relative to size.
2.3. Sample processing
Water samples were pre-filtered through a 200-mm-mesh to
remove zooplankton and large detritus. 1 to 5 l of water were
filtered on pre-combusted (500  C; 4 h) Whatman GF/F filters
under moderate vacuum (10 mbar), until clogging. Filters
were acidified with HCl (10%), rinsed with distilled water,
dried at 60  C and kept frozen (20  C) until analysis. Epi-
phytes were resuspended in filtered (0.45 mm) lagoon water,
collected on GF/F filters and then processed as described
above for POM. SOM samples were freeze-dried and ground
with a mortar and pestle. A subsample (w500 mg DW) was
acidified with HCl (10%) and dried (50  C) under a fume ex-
tractor to evaporate acid. SOM samples were not rinsed to pre-
vent any loss of dissolved organic matter (Riera et al., 1996).
They were instead mixed with distilled water and dried. This
procedure was repeated twice. SOM samples were then ground
and kept frozen (20  C) until analysis. Invertebrates were
starved overnight in filtered (0.45 mm) lagoon water and then
killed. The flesh of molluscs and crustaceans was separated
from their calcareous shells or external skeletons. Each spec-
imen of invertebrates was then analysed as a whole. Con-
versely, only the muscles of the dorsal region of fishes were
analyzed as their isotopic composition is less variable than
that of other body parts (Pinnegar and Polunin, 1999). All an-
imal and plant samples were briefly acidified with 10% HCl,
rinsed with distilled water, dried at 60  C, ground in fine pow-
der and then kept at 20  C until analysis. Acidification of
samples is a key step as it eliminates carbonates, which inter-
fere with d13C measurements. Its effect on d15N values is still
unclear. Some authors reported that it significantly affects
d15N values to a degree that may lead to confounding interpre-
tations (Bunn et al., 1995; Pinnegar and Polunin, 1999),
whereas other found only weak (Jacob et al., 2005) or even
no significant effect (Bosley and Wainright, 1999). Overall,
significant effects of acidification on d15N values appear to
be caused by a prolonged (i.e., from 1 to 3 h) incubation in
acid (Carabel et al., 2006), which may induce a loss of dis-
solved organic matter during rinsing. Our own incubation
times were much shorter (i.e., typically less than 10 min),
and we are therefore confident in stating that our acidification
procedure was appropriate for measuring d15N. Powdered
samples were weighed according to sample types
(15 mg DW for SOM, 1.0 mg DW for animals; 1.5 mg DW
for plants; and 2.0 mg DW for plant detritus) and put into
clean tin cups before stable isotope analysis.
2.4. Stable isotope analysis
Isotope analysis was carried out using a Europa Scientific
ANCA-NT 20-20 Stable Isotope Analyser with an ANCA-NT
Solid/Liquid Preparation Module (Europa Scientific, Crewe,
UK). A 1:4 leucine/citric acid mixture was used as standard.
The numbers of samples analysed for each component, each
location and each sampling survey are provided in Table 1.
The isotopic composition (d13C or d15N) was expressed as the
relative difference between isotopic ratios in the sample and
in conventional standards (Vienna Pee Dee Belemnite for car-
bon and atmospheric N2 for nitrogen):
d13 C or d15 N ð&Þ ¼ ½ðRsample =Rstandard Þ  1  1000
where: R ¼ 13C/12C or 15N/14N.
The precision for d13C was w0.1&. The precision for d15N
was w0.4& (for samples with N content >5% DW).
2.5. Biochemical analysis of potential food sources
Organic carbon and nitrogen contents were measured after
acidification (1 N HCl) using a CHN Perkin Elmer 2400 ana-
lyzer. Total hydrolysable amino acids (THAA) were extracted
as described in Medernach et al. (2001). 10 to 30 mg DW of
samples were submitted to a strong acid hydrolysis (500 ml
of 6 N HCl, 100  C, 24 h, under vacuum). 0.4 ml subsamples
 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315 303

Table 1
Numbers of samples analysed for each component, each survey and each location. * indicates samples collected at the immediate vicinity of the sewage treatment
plant
Species Surveys
June, 2004 September, 2004 February, 2005
Upland plants
Cupressus oemphuineus 3
Elaeagnus angustifolia 3
Ficus carica 3
Olea europea 3
Pinus halepensis 3
Prunus dulcis 3
Populus nigra 3
Vitis vignifera 2
Tamarix gallica 3
Sampling sites
1 2 3 4 1 2 3 4 1 2 3 4
Salt marsh plants
Halimione portulacoides 2 2 1 5 2 2 6 3 3
Arthrocnemum macrostachyum 2 1 1 6 3 3 6 3 3
Sarcocornia fruticosa 2 2 1
Suaeda sp. 2
Juncus acutus 1 1 3 1 4
Limonium vulgare 1 1 1 4
Phragmites communis 1 1 1 2
Phragmites communis* 1 1 1
Elytrigia juncea 1
Seagrasses
Ruppia cirrhosa 2 3 6 3 3 6 3 6 3
Zostera noltii 2 3
Decaying leaf litter 2 3 3
Macroalgae
Characeae 1 2 1
Acetabularia acetabulum 6
Enteromorpha sp. 2 1 1 1 3
Ulva sp. 2 5 2 1
Chaetomorpha sp. 3 3 2 3
Cladophora sp. 2 3 2 1 3
Pheophyta 2 3
Rhodophyta 2 1 1 1
Epiphytes
Seagrass epiphytes 1 1
POM
Lagoon 3 3 1 5 1 4 5 2 6 2
Karstic spring 3 3 2
Rieu River 1 3
Peripheric channel 1 1
Sewage treatment plant 1
SOM
Lagoon 1 1 2 1 4 1 5
Invertebrates
Nephtys sp. 1
Nereis diversicolor 12 3 15 3 9 3 6 3 6 3 6 3
Gammarus aequicauda 4 2 3 2 1 2 1 2
Crangon crangon 6 11 3 6 6 1 1 6 3
Palaemon serratus 2 2 2
Carcinus maenas 6 6 3 6 6 3 3 1 2
Idotea sp. 2 1 4 1 1 1 1
Sphaeroma serratum 1 2
Balanus amphitrite 1
Cerastoderma glaucum 9 1 17 3 9 3 6 3 12 3 12
Loripes lacteus 1 1 5 3
Crassostrea gigas 1
Mytilus galloprovincialis 4 3 1
(continued on next page)
304 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315

Table 1 (continued )
Species Surveys
June, 2004 September, 2004 February, 2005
Abra ovata 8 2 18 9 3 5 3 12 3 12 3
Scrobicularia plana 1
Fishes
Anguilla anguilla 1 2 3 6
Belone belone 1 1 3
Gasterosteus aculeatus 2 3
Atherina boyeri 6 7 3 5 6 3 3 2
Atherina boyeri (juveniles) 6
Salaria pavo 1 3 1 1
Gobius niger 1
Gobius sp. 1
Pomatoschistus spp. 6 18 3 6 6 1 6 11 3
Dicentrarchus labrax 3 5
Chelon labrosus 2
Liza aurata 5 9 3 6 6 1 4 2 1
Mugil cephalus 4 4 7 1
Solea solea 7 3 3 2
Syngnathus abaster 5 3 6 3 6

of the hydrolysates were neutralised with 0.4 ml of 6 N NaOH
and buffered with 0.8 ml of H3BO3 (0.4 M, pH 8). Fluorescent
derivatives were obtained by adding 6 ml of an orthophtaldial-
dehyde solution (125 mg in 2.5 ml of methanol and 0.125 ml
of mercaptoethanol) and 400 ml of H3BO3 to 100 ml of those
samples. THAA identification was based on retention times
within an HPLC column (Lindroth and Mopper, 1979) and
achieved through comparison with a standard containing 19
amino-acids. THAA quantification was based on fluorescence
measurements (excitation wavelength: 335 nm, emission
wavelength: 450 nm). Enzymatically hydrolysable amino
acids (EHAA) were extracted following the biomimetic ap-
proach proposed by Mayer et al. (1995). 10 to 200 mg DW
of samples were poisoned with 1 ml of a solution containing
2 inhibitors of bacterial active transport systems (0.1 M so-
dium arsenate and 0.1 mM pentachlorophenol within a pH 8
sodium phosphate buffer). This mixture was let to incubate
for 1 h. 100 ml of proteinase K solution (1 mg ml1) were
then added and the samples incubated for 6 h at 37  C. They
were then centrifuged to discard remaining particulate mate-
rial. 75 ml of pure trichloracetic acid were added to 750 ml
of supernatant to precipitate macromolecules, which are con-
sidered to be non-suitable for absorption. 750 ml of the super-
natant were then hydrolysed and processed as described for
THAA. In addition, a blank accounting for possible degrada-
tion of the enzyme was carried out. EHAA/THAA ratios rep-
resent the contribution of EHAA to the THAA pool and were
expressed in percent. These ratios were used as indices of nu-
tritional value (Grémare et al., 1997; Medernach et al., 2001).
2.6. Statistical analysis
The effects of surveys and sampling sites on d13C and d15N
values of main potential food sources and consumers were
assessed using two-way ANOVAs. These ANOVAs were first
carried out for all potential food sources and consumers, which
were sampled at sites 1, 3 and 4 during all surveys. Site 2 was
not included because Nereis diversicolor was the only species
which had been sampled at this site during all 3 surveys. The
results of these ANOVAs showed the specificity of site 4,
which was isolated from the main basin during both Septem-
ber 2004 and February 2005. Spatio-temporal changes in the
isotopic ratios of potential food sources and consumers within
the main basin were thus also assessed using two-way
ANOVAs carried out for all the organisms, which were present
at sites 1 and 3 during all surveys.
3. Results
3.1. Rainfall, salinity, temperature
The connection of the lagoon with the sea was closed be-
tween July 2003 and the beginning of June 2004 (Fig. 2A).
An artificial connection was then established during one
week to eliminate the flood caused by heavy rainfall (see
below). The connection then closed again until the beginning
of September 2004 before re-opening until March 2005. The
June and September 2004 surveys were thus carried when
the lagoon had been isolated from the sea for a long period
of time, whereas the February one was achieved when the la-
goon had been connected to the sea for about 6 months. Rain-
fall was high before the June 2004 survey, low before the
September 2004 one and intermediate before the February
2005 survey (Fig. 2A). The lowest salinities were recorded
in June, and the highest ones in February (Fig. 2B). In Septem-
ber and February, there were clear salinity gradients (from
12.4 to 23.3, and 19.9 to 31.0, respectively). Conversely, there
was almost no salinity gradient in June. Salinity recorded at
site 4 did not match with salinity gradients in the main basin
both in September and February. Salinity at site 4 was ex-
tremely high in September (46.7) and conversely extremely
low in February (9.3). There were marked temporal changes
in water temperature, which was highest in June (mean of
22.7  C) and lowest in February (mean of 6.5  C) (Fig. 2C).
 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
Fig. 2. Temporal changes in monthly rainfall at Perpignan (A) and spatio-
temporal changes in water temperature (B) and salinity (C). The black hori-
zontal bar at the top indicates the periods during which the connection of
the lagoon with the open sea was closed. Vertical dash lines represent the three
sampling times. * indicates periods during which site 4 was isolated from the
main part of the lagoon.
There was no clear gradient in the main basin, and temperature
at site 4 never departed from those recorded in the main
lagoon.
3.2. Biochemical characteristics of potential food
sources
The main biochemical characteristics of potential food
sources are listed in Table 2. Mean C/N ratios were high
(>14.8) for all macrophytes and low both for POM (<6.6)
305
and SOM (7.6). Low EHAA/THAA ratios were recorded for
upland plants (<18.4%) and to a lesser extent seagrasses
(<33.4%). Conversely mean EHAA/THAA ratios were high
for salt marsh plants (>50%) and macroalgae (>72.3%).
The mean EHAA/THAA ratio of SOM was 36.7%.
3.3. Temporal changes in d13C and d15N of potential
food sources and consumers
The relationships between the d13C and d15N values of all
sampled potential food sources and consumers are shown in
Fig. 3 for each survey. The main categories of potential food
sources mostly differed by their overall mean d13C values
(Table 3). Freshwater POM (karstic springs and Rieu River),
upland plants and salt marsh plants were the most 13C-
depleted (overall mean d13C values of 28.8, 27.7 and
26.2&, respectively). Conversely, seagrasses were the
most 13C-enriched (overall mean d13C value of 13.8&).
The d13C values of macroalgae were highly variable (overall
mean values between 28.0& and 16.6&) with both Aceta-
bularia acetabulum (overall mean value of 16.6&) and
Characeae (overall mean value of 17.2&) being more
13
C-enriched than all other macroalgae (overall mean values
between 28.0 and 23.1&). d13C values of seagrass epi-
phytes (overall mean of 15.7&) were close to those of Char-
aceae and seagrasses. d13C values of SOM and lagoon POM
tended to be similar (overall mean values of 21.0 and
22.2&, respectively) and were intermediate between those
of 13C-depleted and 13C-enriched pure food sources (see
above). Potential food sources also featured a wide range of
d15N values (Table 3). The overall mean d15N ranged from
0.2& for the upland plant Cupressus oemphuineus to
12.0& and 15.6& for Phragmites communis and POM col-
lected at the output of the sewage treatment plant. Conversely
to d13C, d15N did not allow for a clear discrimination between
the main categories of potential food sources. Lagoon POM,
freshwater POM and SOM were however more 15N-depleted
than aquatic macrophytes and salt marsh plants. The d15N
values of particulate nitrogen in karstic springs were highly
variable (i.e., between 1.9 and 8.4&).
During all surveys, the d13C range of consumers was nar-
rower than those of potential food sources. Indeed, if we do
not consider the bivalve Loripes lacteus, which bears endo-
symbiotic bacteria and is then strongly 13C-depleted, d13C
range of consumers was [26.2&; 16.7&] in June 2004,
[23.7&; 15.2&] in September 2004, and [21.6&;
14.8&] in February 2005. During all 3 surveys, the d13C
of consumers were intermediate between the isotopic ratios
of 13C-depleted potential food sources (salt marsh plant, mac-
roalgae) and 13C-enriched seagrasses. During both September
and February, the isotopic ratios of almost all consumers were
grouped in a single cloud (Fig. 3B,C). Conversely, in June,
these isotopic ratios grouped into 2 distinct clouds (Fig. 3A).
This was due to the fact that some primary consumers
(i.e., the benthic invertebrates Gammarus aequicauda, Idotea
sp., Sphaeroma serratum, Cerastoderma glaucum and Mytilus
galloprovincialis) and the fishes Belone belone, Gasterosteus
306 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315

Table 2
Main biochemical characteristics (mean  SD) of potential food sources. OC: organic carbon; N: nitrogen; EHAA: enzymatically hydrolysable amino acids;
THAA: totally hydrolysable amino acids
Potential food sources OC N C/N EHAA THAA EHAA/THAA
(%DW) (%DW) (nmol mg DW1) (nmol mg DW1) (%)
Upland plants
Vitis vignifera 45.6  1.4 2.9  0.6 16.2  2.6 111.6 616.1 18.1
Pinus halepensis 49.3  1.3 1.3  0.2 38.1  6.9 59.9 577.7 10.4
Tamarix gallica 45.7  0.2 2.5  0.0 18.6  0.3 103.3 560.7 18.4
Salt marsh plants
Halimione portulacoides 34.1  2.0 1.7  0.1 21.3  0.8 239.5 397.6 60.2
Arthrocnemum macrostachyum 37.9  1.8 2.3  0.4 16.7  2.1 252.7 421.4 59.7
Phragmites communis 43.4  0.3 3.0  0.4 14.9  1.5 333.9 667.6 50.0
Seagrasses
Ruppia cirrhosa 41.2  1.1 2.0  0.4 22.1  4.0 160.2 483.2 33.4
Zostera noltii 42.6  1.7 2.0  0.7 22.8  7.4 46.3 380.4 12.2
Seagrass leaf litter 41.9  0.9 2.0  0.4 21.6  4.2 e e e
Macroalgae
Acetabularia acetabulum 37.5  2.2 1.0  0.1 39.5  3.7 134.4 151.6 88.7
Ulva sp. 36.4  0.6 2.7  1.0 14.8  5.2 342.1 473.3 72.3
Chaetomorpha sp. 39.9  0.5 2.4  0.0 16.9  0.5 402.6 519.0 77.6
Cladophora sp. 32.4  0.8 2.2  0.2 15.0  1.8 376.4 445.0 84.6
Epiphytes
Seagrass epiphytes e e 7.7  0.5 e e e
POM
Lagoon e e 5.7  0.4 e e e
Karstic springs e e 6.2  1.7 e e e
Rieu River e e 6.6  1.5 e e e
Peripheric channel e e 5.7  0.6 e e e
Sewage treatment plant e e 4.2 e e e
SOM
Lagoon 1.3  1.2 0.2  0.1 7.6  1.7 15.2 46.8 36.7

aculeatus, Liza aurata and Mugil cephalus featured lower d13C
in June than in the two other surveys.
3.4. Spatio-temporal changes in d13C and d15N of
selected potential food sources and consumers
The significance of spatio-temporal changes in isotopic
values was tested for potential food sources and consumers in
two sets of two-way ANOVAs. The first one included species
sampled at sites 1, 3 and 4 during all 3 sampling surveys
(Table 4), and the second one included those which were sam-
pled at sites 1 and 3 during all 3 sampling surveys (Table 5).
Spatio-temporal changes in isotopic values are shown for: (1)
the three main species of macrophytes (Arthrocnemum macro-
stachyum, Halimione portulacoides and Ruppia cirrhosa)
(Fig. 4); and (2) the three main categories of primary consumers
(i.e., Cerastoderma glaucum for suspension-feeders, Abra
ovata for deposit-feeders and Gammarus aequicauda for
grazers) and a carnivorous fish (Pomatoschistus spp.) (Fig. 5).
When considering d13C measured of food sources and ani-
mals collected at sites 1, 3 and 4 (Figs. 4 and 5, Table 4), there
were significant interactions between sites and surveys for
Halimione portulacoides, Carcinus maenas, Crangon crangon
and Liza aurata. These interactions were due to different sea-
sonal changes at site 4 compared to the 2 other sites with low
values in September and February for H. portulacoides and
C. crangon, and low values in June and February for L. aurata.
There were no significant interaction between sites and sur-
veys for Arthrocnemum macrostachyum, Nereis diversicolor
and Pomatoschistus spp. d13C were lower in June and Septem-
ber than in February for A. macrostachyum with no significant
difference between sites. For N. diversicolor, there was signif-
icant difference in d13C values neither between sites nor be-
tween surveys. For Pomatoschistus spp., d13C values did not
significantly differ between surveys but were significantly
lower at site 4 than at sites 1 and 3. When considering d15N
measured of food sources and animals collected at sites 1, 3
and 4 (Figs. 4 and 5, Table 4), there were significant interac-
tions between sites and surveys for A. macrostachyum, H. por-
tulacoides, C. crangon and Pomatoschistus spp. For both salt
marsh plants, interactions were due to the occurrence of
very high d15N values at site 4 during February, whereas inter-
actions for C. crangon and Pomatoschistus spp. reflected the
occurrence of very high d15N values both in September and
February but not in June. There was no significant interaction
between sites and surveys for N. diversicolor, C. maenas and
L. aurata. For all species, d15N were significantly higher at
site 4 than at sites 1 and 3. There was also significant differ-
ence in surveys for N. diversicolor and C. maenas (with lower
d15N values in June than in February), but not for L. aurata.
When only considering d13C measured at sites 1 and 3 (Figs.
4 and 5, Table 5), we found significant interactions between
sites and surveys for Ruppia cirrhosa, Cerastoderma glaucum,
Carcinus maenas and Liza aurata. Concerning R. cirrhosa, this
 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315 307

Fig. 3. Plot of d13C and d15N values of potential food sources and consumers in (A) June 2004, (B) September 2004 and (C) February 2005. Isotopic ratios are
pooled for all sites during each survey. Bars are standard deviations. * indicates samples collected at the immediate vicinity of the sewage treatment plant.

reflected the occurrence of low d13C values at site 1 only in June, and
high d13C values at site 3 only in February. Concerning C. glaucum,
this reflected a lower difference between sites in February than in
the two other seasons. For C. maenas this was conversely due to
higher difference between sites in February than in the two
other seasons. There were significant seasonal changes in
d13C for POM, Arthrocnemum macrostachyum and Halimione
portulacoides (with lower values in June than in February)
and for Abra ovata (with lower d13C values in June than in Sep-
tember). There were significant spatial changes for A. ovata,
308 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315

Table 3 during all surveys for discretely motile consumers but only in
Carbon and nitrogen isotopic ratios (overall mean  SD) of potential food September and February for mobile consumers; (3) low d13C
sources. * indicates samples collected at the immediate vicinity of the sewage
treatment plant
values at site 4 for consumers; and (4) lower d13C values at
site 1 than at site 3 for Ruppia cirrhosa and most of consumers.
Potential food sources d13C (&) d15N (&)
Upland plants 4. Discussion
Cupressus oemphuineus 26.2  0.6 0.2  0.3
Elaeagnus angustifolia 28.0  1.6 0.9  0.4
Ficus carica 28.5  1.3 5.8  0.9 4.1. Isotopic characterisation of potential food sources:
Olea europea 27.2  0.7 1.6  2.9 importance of continental inputs
Prunus dulcis 29.8  1.7 4.7  1.5
Populus nigra 30.4  0.7 4.7  1.6 Our overall mean d13C and d15N values of salt marsh plants
Vitis vignifera 25.5  0.0 6.2  0.9
Pinus halepensis 27.0  0.8 0.7  1.2
were similar to previous data reported for other coastal lagoons
Tamarix gallica 27.0  0.7 3.7  1.7 (Kwak and Zedler, 1997; Machas et al., 2003). High d15N
Salt marsh plants values were however observed for Phragmites communis col-
Halimione portulacoides 26.3  1.3 7.6  1.3 lected at the immediate vicinity of the sewage treatment plant.
Arthrocnemum macrostachyum 26.7  1.2 8.5  1.8 This may reflect the incorporation of 15N-enriched NHþ 4 and/or
Sarcocornia fruticosa 28.6 7.0
Suaeda sp. 29.9 7.2
NO 3 of wastewater origin as previously reported (Lapointe,
Juncus acutus 25.1  0.7 4.9  0.8 1997; McClelland and Valiela, 1998; Riera et al., 2000). The
Limonium vulgare 26.8  1.1 8.6  1.9 overall mean d13C value of seagrasses was slightly lower than
Elytrigia juncea 27.6 3.5 previous measurements carried out in the Mediterranean
Phragmites communis 26.6  1.0 6.8  2.2 (Dauby, 1989; Vizzini and Mazzola, 2003) as well as in other
Phragmites communis* 25.8  1.1 12.0  1.1
Seagrasses
areas worldwide (Hemminga and Mateo, 1996). Similarly,
Ruppia cirrhosa 13.1  1.2 7.0  1.0 the macroalgae Chaetomorpha sp. and Cladophora sp. were
Zostera noltii 14.4  1.6 6.9  0.1 overall more 13C-depleted than samples from the Stagnone di
Seagrass leaf litter 16.9  1.3 5.4  2.0 Marsala (Vizzini et al., 2002; La Loggia et al., 2004), the
Macroalgae Lake of Sabaudia (Vizzini and Mazzola, 2003) and the Bay
Characeae 17.2  0.3 4.1  1.1
Acetabularia acetabulum 16.6 4.4
of Calvi (Dauby, 1989; Lepoint et al., 2000; Pinnegar and
Ulva sp. 28.0  3.6 7.4  0.9 Polunin, 2000). The low d13C values of these submerged
Enteromorpha sp. 27.5  1.5 8.5  1.3 macrophytes in the Lapalme Lagoon may reflect a significant
Chaetomorpha sp. 24.5  1.9 7.9  1.5 influence of continental inputs, which would result in a
Cladophora sp. 25.7  2.8 7.8  1.9 13
C-depletion of dissolved inorganic carbon available for pho-
Pheophyta 24.8 7.3
Rhodophyta 23.1  0.3 6.3  0.2
tosynthesis (Lin et al., 1991; Hemminga and Mateo, 1996).
Epiphytes Our overall mean d13C value for lagoon POM (22.1&)
Seagrass epiphytes 15.7  2.4 4.2  0.8 was: (1) close to values reported for POM in northwestern
POM Mediterranean open Sea (Lepoint et al., 2000; Darnaude
Lagoon 22.1  1.3 2.2  2.2 et al., 2004); and (2) lower than those obtained in the Stagnone
Karstic springs 29.5  1.5 3.7  5.2
Rieu River 28.1  0.9 3.8  2.9
di Marsala (16.2&), where seagrass detritus strongly con-
Peripheric channel 25.1  1.7 2.6 tribute to the bulk suspended POM (Vizzini and Mazzola,
Sewage treatment plant 25.0 15.6 2004). The low C/N value of POM in the Lapalme Lagoon
SOM suggests that this pool mainly originates from phytoplanktonic
Lagoon 21.0  0.7 4.3  0.5 production rather than from a detritic mixture between sea-
grass and 13C-depleted macrophytes. As for lagoon POM,
d13C of SOM was in the middle of the range of potential sour-
Pomatoschistus spp. and Atherina boyeri, with lower d13C ces. In most of lagoon ecosystems, the SOM pool results from
values at site 1 than at site 3. When only considering d15N mea- a mixture of several sources of organic matter (Nichols and
sured at sites 1 and 3 (Fig. 4 and 5, Table 5), there was not any Allen, 1981). Our SOM d13C values were in the range
significant interactions between sites and surveys, except for (i.e., 27 to 20&) of those reported for several estuarine
POM. There were significant differences between surveys for and near-shore systems (Fry and Sherr, 1984). Most of d13C
SOM, A. macrostachyum (with no consistent seasonal pattern) values reported for SOM of coastal lagoons are however higher
and for all consumers but Pomatoschistus spp. (with lower (i.e. from 20 to 10&) because of the presence of large sea-
values in June and higher ones in February, except for L. aur- grass meadows (Fry et al., 1977; Vizzini et al., 2002; Jones
ata). In addition, significant spatial differences in d15N were et al., 2003), algal mats (Peters et al., 1978) or Spartina sp.
found for A. macrostachyum and H. portulacoides (with lower (Botto et al., 2005). The d13C of SOM collected in the Lapalme
values at site 3) and for C. glaucum and Nereis diversicolor Lagoon was thus rather low, which suggests an overall higher
(with higher values at site 3). contribution of 13C-depleted sources (i.e., salt marsh plants,
Overall these results show: (1) higher d15N at site 4 only in macroalgae). The C/N ratio of SOM (7.6) was lower than
February for primary producers; (2) high d15N values at site 4 those of salt marsh plants, seagrasses and macroalgae, which
 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315 309

Table 4
Results of two-way-ANOVA tests and LSD tests performed on food sources and consumers. The sites 1, 3 and 4 are included in the ‘‘Site’’ factor. J: June 2004;
S: September 2004; F: February 2005
Two-way ANOVA LSD tests
Survey Site Interaction Survey Site
13
d C
Food sources
Lagoon POM e e e e e
SOM e e e e e
A. macrostachyum <0.001 0.183 0.064 J¼S<F ¼
H. portulacoides <0.001 0.062 0.033 J¼S<F ¼
R. cirrhosa e e e e e
Invertebrates
C. glaucum e e e e e
A. ovata e e e e e
N. diversicolor 0.196 0.095 0.438 ¼ ¼
C. maenas 0.221 <0.001 <0.001 ¼ 4<1<3
C. crangon 0.661 <0.001 0.015 ¼ 4<3
Fish
Pomatoschistus spp. 0.637 <0.001 0.225 ¼ 4<1<3
A. boyeri e e e e e
L. aurata 0.001 0.020 0.020 J<S 4<1¼3
d15N
Food sources
Lagoon POM e e e e e
SOM e e e e e
A. macrostachyum <0.001 <0.001 <0.001 S<J<F 3<1<4
H. portulacoides <0.001 0.007 <0.001 J¼S<F 3<1¼4
R. cirrhosa e e
Invertebrates
C. glaucum e e e e e
A. ovata e e e e e
N. diversicolor <0.001 <0.001 0.525 J¼S<F 1<3<4
C. maenas <0.001 <0.001 0.666 J<S<F 1¼3<4
C. crangon <0.001 <0.001 <0.001 J<S¼F 1¼3<4
Fish
Pomatoschistus spp. <0.001 <0.001 <0.001 J<S¼F 1¼3<4
A. boyeri e e e e e
L. aurata 0.809 0.040 0.273 ¼ 1¼3<4

suggests that sedimented phytoplankton could also contribute
substantially to the SOM pool. Unfortunately, it was not pos-
sible to better assess the relative contributions of each pure
sources to the SOM pool through the use of mixing models
(Phillips and Gregg, 2003) with both C and N tracers. d15N
of SOM was for example lower than those of all pure sources
in February probably indicating a 15N-depletion during plant
decomposition as already reported (Zieman et al., 1984; Cur-
rin et al., 1995). Changes in d15N of plant during their decom-
position can be highly variable and the d15N of decaying
tissues may be either higher or lower than the d15N of living
plant, depending on species and season (Cloern et al., 2002).
This uncertainty prevents the use of the N tracer in isotopic
mixing model to more precisely assess the contributions of
each pure source to SOM.
4.2. Temporal changes in isotopic ratios: description of
the trophic network
Coastal lagoons are located at the frontier between land and
sea, and are therefore submitted to both continental and
marine influences. Rainfall and the opening of the connection
with the sea are key factors in controlling the relative impor-
tance of these two influences in Mediterranean lagoons (Petit,
1953; Boutière, 1980). Our three surveys were rather different
in this particular context. The June survey took place after
a long period of heavy rainfall and closed connection with
the sea. It can thus be considered as representative of maximal
continental influence. Conversely, the February survey took
place after a long period of low rainfall and open connection
with the sea. It can thus be considered as representative of
maximal marine influence. The September survey was inter-
mediate because preceded by a period of low rainfall but
with no open connection with the sea.
The d13C values of consumers were not similar in all three
surveys. They formed a single cloud during September 2004
and February 2005 but two distinct ones during June 2004,
due to low d13C values of many consumers. The results of
both the September and the February survey suggest the exis-
tence of a main pathway of organic matter originating from
a main basis, which is quite similar to what we recently observed
in a northwestern marine open bay (Carlier et al., 2007). None of
310 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315

Table 5
Results of two-way-ANOVA tests and LSD tests performed on food sources and consumers. The sites 1 and 3 are included in the ‘‘Site’’ factor. J: June 2004; S:
September 2004; F: February 2005
Two-way ANOVA LSD tests
Survey Site Interaction Survey Site
13
d C
Food sources
Lagoon POM 0.010 0.388 0.440 F<S ¼
SOM 0.613 0.784 0.252 ¼ ¼
A. macrostachyum <0.001 0.116 0.055 J¼S<F ¼
H. portulacoides <0.001 0.574 0.681 J<S<F ¼
R. cirrhosa <0.001 <0.001 0.029 J¼S<F 1<3
Invertebrates
C. glaucum <0.001 <0.001 <0.001 J<F<S 1<3
A. ovata 0.009 0.011 0.654 J¼F<S 1<3
N. diversicolor 0.057 0.068 0.483 ¼ ¼
C. maenas 0.007 0.008 <0.001 J<S 1<3
C. crangon 0.207 0.084 0.163 ¼ ¼
Fish
Pomatoschistus spp. 0.078 <0.001 0.483 ¼ 1<3
A. boyeri 0.616 0.001 0.237 ¼ 1<3
L. aurata 0.002 0.508 0.009 J<F¼S ¼
d15N
Food sources
Lagoon POM <0.001 0.024 0.025 J¼S<F 3<1
SOM 0.044 0.209 0.339 F<S ¼
A. macrostachyum 0.007 0.002 0.857 S<F¼J 3<1
H. portulacoides 0.136 0.009 0.809 ¼ 3<1
R. cirrhosa 0.096 0.155 0.136 ¼ ¼
Invertebrates
C. glaucum <0.001 <0.001 0.387 J<S¼F 1<3
A. ovata 0.017 0.875 0.430 J<F ¼
N. diversicolor 0.003 0.027 0.743 J¼S<F 1<3
C. maenas <0.001 0.526 0.442 J¼S<F ¼
C. crangon <0.001 0.237 0.379 J<S¼F ¼
Fish
Pomatoschistus spp. 0.063 0.613 0.194 ¼ ¼
A. boyeri 0.019 0.078 0.298 J<S¼F ¼
L. aurata 0.031 0.834 0.749 S<J ¼

the dominant pure sources (i.e. living salt marsh plants, macro-
algae, living seagrass and seagrass epiphytes) could constitute
the unique basis of such a food web, since the d13C values of
most of consumers were always intermediate between those of
13
C-depleted salt marsh plants and most of macroalgae on one
side, and 13C-enriched seagrass and their epiphytes on the other
side. Besides, some of the potential food sources could be ruled
out. Firstly, karstic springs were not likely to represent a signif-
icant food source for consumers as: (1) their d13C were much
lower than those of consumers; and (2) even if they constituted
the main freshwater input in the lagoon (Wilke and Boutière,
2000), their POM loads always remained very low (Kiener
and Petit, 1968). Secondly, the output of the sewage treatment
plant were not likely to significantly fuel the food web as well,
because the d15N values of both primary producers and con-
sumers at site 1 never showed marked increases related to waste
water discharges. During both September and February, the iso-
topic ratios of most consumers were consistent with a food web
based on SOM and lagoon POM, taking into account the usual
enrichments in 13C and 15N at each trophic level (DeNiro and
Epstein, 1978, 1981; Minagawa and Wada, 1984; Post, 2002).
The rather high content of bioavailable amino acids in the
SOM pool is consistent with a major contribution of this compo-
nent to the basis of the food web. The EHAA/THAA ratio which
accounts for the proportion of amino acid pool that can be ab-
sorbed by benthic primary consumers, was higher in the
Lapalme Lagoon’s SOM (w40%) than in sediment of north-
western Mediterranean (between 15 and 34%) (Grémare et al.,
2003, 2005). The value obtained in Lapalme Lagoon is rather
close to those found in very productive area like upwelling
coastal water off Central Chile (Grémare et al., 2005). Neverthe-
less, the d13C values of a limited number of consumers collected
in September and February indicated that significant contribu-
tions of other sources than lagoon POM and SOM cannot be ex-
cluded. For example, the carnivorous fish Anguilla anguilla, the
detritivorous crustacean Sphaeroma serratum and the filter-
feeder Mytilus galloprovincialis all featured low d13C indicating
that they also depend on 13C-depleted sources (e.g., detritus of
macroalgae or salt marsh plants). At the opposite, the two
shrimps Crangon crangon and Palaemon serratus showed
high d13C compared with most of consumers, suggesting the as-
similation of 13C-enriched sources. These additional sources
A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315 311

may be seagrass epiphytes and/or 13C-enriched macroalgae, but

probably not seagrasses, as suggested by the high EHAA/THAA
ratio of Acetabularia acetabulum and the rather low EHAA/
THAA ratio of Ruppia cirrhosa. Overall the results of the Sep-
tember and the February surveys are thus indicative of the occur-
rence of a main food web based on SOM/lagoon POM as already
suggested by Vizzini and Mazzola (2005) for a semi-enclosed
coastal bay, the Stagnone di Marsala.
The outcome of the June survey is different because it sug-
gests the use of several food sources by different categories of
consumers. The low d13C values observed for an important
proportion of consumers during this survey were probably
linked to important continental inputs (as opposed to February
and September) associated with heavy rainfall (Fig. 2). More
specifically, in June all suspension-feeders and grazers pre-
sented lower d13C values than deposit-feeders and omnivores.
Continental inputs that preceded the June survey consisted in
POM and dissolved inorganic carbon (DIC) which are both
usually 13C-depleted relative to marine POM and DIC (Fry
and Sherr, 1984). This suggests that in June, both suspen-
sion-feeders and grazers had assimilated a greater proportion
of terrestrial organic matter than deposit-feeders as illustrated
by d13C values of Cerastoderma glaucum, Gammarus aequi-
cauda and Abra ovata in Fig. 5. The low d13C values of Rup-
pia cirrhosa obtained in June at site 1 (Fig. 4) also suggest that
major inputs of DIC had a significant impact on the d13C
values of this primary producer. The d13C value of lagoon
POM was however not affected in June. We do not believe
that this is indicative of the lack of effect of DIC input on
the isotopic ratios of phytoplankton since POM measurements
are only indicative of short-term environmental changes. In-
deed lagoon POM is immediately affected by terrestrial inputs,
whereas a time lag exists between terrestrial inputs and
decrease of d13C in suspension-feeders’ tissues (Riera and
Richard, 1997). The d13C of SOM and deposit-feeders were
respectively not and weakly affected by the sampling survey.
This may either indicate a low contribution of continental in-
puts in the diet of deposit-feeders and/or reflect the fact that
continental inputs were quickly diluted in the pool of resident
SOM. The isotopic ratios of omnivores were not affected by
terrestrial inputs in June as well, which may result from the
fact that: (1) omnivores were mostly feeding on SOM and/or
deposit-feeders; and (2) omnivores occupy a higher trophic
level than suspension-feeders and grazers and are thus
integrating temporal changes in the isotopic compositions of
primary producers over longer periods of time. In any case,
it should be underlined that the analysis of the results of the
September and the February surveys on one side and of those
of the June one on the other side, would lead to very different
conclusions relative to the structure of the trophic network in
the Lapalme Lagoon. Our results thus clearly stress the neces-
sity of carrying out seasonal isotopic surveys to get a sound

Fig. 4. Spatio-temporal changes in d13C and d15N values of Arthrocnemum

macrostachyum, Halimione portulacoides and Ruppia cirrhosa. Plots are
mean values for each site and each survey. Bars are standard deviations.
312 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
Fig. 5. Spatio-temporal changes in d13C and d15N values of Cerastoderma glaucum, Gammarus aequicauda, Abra ovata and Pomatoschistus spp. Plots are mean
values for each site and each survey. Bars are standard deviations.
and/or more complete idea of the structure of the trophic net-
work in such a variable environment.
Until now, few investigations on seasonal variability in the
isotopic composition of marine primary producers and con-
sumers have been undertaken in coastal lagoons (Buskey
et al., 1999; Machas et al., 2003; Vizzini and Mazzola,
2003, 2005) and different conclusions have been drawn. In
the Lake of Sabaudia as well as in the Stagnone di Marsala
(Italia), a general 13C-enrichment of the whole food web
was observed from cold to warm seasons (Vizzini and
Mazzola, 2003, 2005). This pattern was explained by high
irradiance levels during summer, which results in a lower iso-
topic discrimination against 13C by primary producers. Con-
versely, Machas et al. (2003) found that filter-feeders
exhibited significantly lower d13C in summer than in winter
in the Ria Formosa Lagoon (Portugal). These authors proposed
that during summer, high respiratory activity may lead to high
concentration of 13C-depleted CO2, which in turn result in
13
C-depleted food sources (phytoplankton and benthic micro-
algae). Moreover, other factors such as temperature, salinity
and pH have been proposed to explain seasonal change in
d13C in coastal food webs (Simenstad and Wissmar, 1985).
As a result, it is difficult to draw general patterns on food
web structure in costal lagoons without considering their eco-
logical context and their temporal variations.
At last it is interesting to discuss, the specificity of temporal
changes in d13C of the symbiotic bacteria harboring bivalve
Loripes lacteus. Its overall d13C values were low and similar
to those of a closely related bivalve (Loripes lucinalis) which
also harbors endosymbiotic bacteria (Johnson et al., 1994).
They were much lower than those of bivalves without symbi-
onts (e.g., Cerastoderma glaucum, Mytilus galloprovincialis
 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
and Abra ovata). This discrepancy indicates a substantial che-
moautotrophic contribution to the carbon nutrition of L. lac-
teus. The percentage of host carbon provided by the
symbionts can fluctuate throughout the year (Johnson et al.,
1994), and that seems to be also the case in our study since
we recorded higher d13C values for L. lacteus in June than
in February. Such changes may be linked to a more heterotro-
phic feeding behavior in summer, when food sources are more
abundant (Sprung, 1994).
4.3. Spatial changes in the isotopic ratios: the impacts of
confinement and anthropogenic inputs
The most important spatial difference in isotopic composi-
tion of both primary producers and consumers originated from
values obtained at site 4. High d15N affected salt marsh plants
and Ruppia cirrhosa in February (Fig. 4), and high d15N and
low d13C affected discretely motile consumers in all seasons,
and mobile consumers only when site 4 was isolated from
the main lagoon (i.e., during September and February) as
shown in Fig. 5. Site 4 is located far away from the sewage
treatment plant; moreover d15N values of primary producers
and consumers were low at site 1. Therefore sewage outputs
cannot account for significant 15N-enrichment at site 4. Con-
versely, site 4 is located downstream of an important vineyard.
Wilke and Boutière (1998, 1999) already considered fertilizers
as a potential threat for the lagoon, and measured temporary
high NO 1
3 concentrations (around 2 mg l ) close to site 4.
15
Synthetic fertilizers are N-depleted due to the conversion
of atmospheric N2 (d15N ¼ 0&) during their manufacturing
(Fryer and Aly, 1974, in Lapointe, 1997, Riera, pers. obs.).
They are thus probably not contributing directly to a 15N-enriched
dissolved inorganic nitrogen (DIN) pool at site 4. However, bio-
geochemical processes can account for such a 15N-enrichment
of the DIN pool. Denitrification for example results in an in-
crease in the d15N of the remaining nitrate pool, which can
be 15 to 30& higher than the d15N value of fertilizer nitrate
(Cline and Kaplan, 1975; Kendall, 1998). Volatile loss of am-
monia also results in the isotopic fractionation of fertilizer ni-
trogen when it is applied in reduced forms (i.e. NHþ 4 and
organic nitrogen), and the resulting organic matter may be
15
N-enriched by up to 20& (Flipse and Bonner, 1985; Ken-
dall, 1998). Denitrification can be high in salt marsh anoxic
sediment (Kaplan et al., 1979) and especially at site 4, a small
and confined pond surrounded by dense vegetation that can
rapidly become anoxic because of the abundance of salt marsh
plant detritus (Carlier, pers. obs.). The strong 15N-enrichment
of primary producers and consumers observed at site 4 could
thus indirectly result from fertilizers inputs, which may un-
dergo isotopic fractionation through denitrification and ammo-
nia volatilisation. An interesting point is the lack of
synchronicity between the enrichment of primary producers
and consumers. Primary producers were only 15N-enriched
in February, whereas discretely motile consumers (i.e., Nereis
diversicolor, Cerastoderma glaucum and Abra ovata) were
15
N-enriched during all 3 surveys. Fertilizers are mostly dis-
persed during wintertime as granules, which then require
313
heavy rainfall to be dissolved (Amouroux, pers. obs.). Such
a schedule in the use of fertilizers may explain the high
d15N values observed for primary producers in February but
not in June and September. Conversely, the high d15N values
of discretely motile consumers observed all year round suggest
that they preferentially use a pool of organic matter, which is
constantly enriched in 15N. This is consistent with a significant
utilisation of a detritic organic matter pool mainly originating
from salt marsh plants, which are clearly dominant at site 4.
The ability of sedentary consumers to use this organic matter
pool is supported by the high EHAA/THAA ratios (i.e., the
high nutritive value) of these plants, which were recorded dur-
ing the present study. This hypothesis should now be further
tested by the assessment of temporal changes in d15N values
of SOM at site 4.
A classical feature in coastal lagoon is the occurrence of
a salinity/confinement gradient, which reflects the relative im-
portance of continental and marine influences (Guelorget and
Perthuisot, 1983). During the present study, there was a clear
salinity gradient in September and February but not in June,
when the lagoon had been isolated from the sea for a long pe-
riod of time. Overall, the salinity/confinement gradient was
also apparent in the isotopic ratios of several primary pro-
ducers and discretely motile consumers. The d13C values of
Ruppia cirrhosa were significantly lower at site 1 than at
site 3, whatever the season (Fig. 4). Along the same line, the
d13C values of Cerastoderma glaucum, Abra ovata and Carci-
nus maenas, together with the d15N values of both C. glaucum
and Nereis diversicolor were significantly lower at site 1 than
at site 3. This indicates that both dissolved inorganic and
organic matter pools are more influenced by 13C- and
15
N-depleted continental inputs in the inner part of the lagoon.
Consequently, seagrasses are influenced in the same way as al-
ready observed in several other similar environments such as
the Tomales Bay (Fourqurean et al., 1997) and the Ria For-
mosa Lagoon (Machas et al., 2003). Interestingly, d13C values
of mobile consumers such as Pomatoschistus spp. (Fig. 5) and
Atherina boyeri were also significantly lower at site 1 than at
site 3, whatever the season. This result confirms the existence
of small-scale spatial variability in the isotopic composition of
animals already pointed out by Vizzini and Mazzola (2005)
and Dubois et al. (in press).
As far as the isotopic ratios of consumers are concerned,
the most important differences between the 3 surveys were be-
tween the June and the other two surveys (see above). Changes
were more related to the importance of continental inputs
(high in June and low in September and February), than to
the opening of the connection of the lagoon with the sea
(closed in June and September and open in February). In
this sense, our results suggest that the continental inputs are
more important than the opening of the connection with the
sea in controlling trophic pathways in the Lapalme Lagoon.
However, it should be stressed that: (1) this conclusion is
partly due to the particular context (prolonged period of heavy
rainfall during Spring 2004 and restricted periods of opening
of the connection with the sea) of the present study; and (2)
both factors may be usually more tightly correlated since the
314 A. Carlier et al. / Estuarine, Coastal and Shelf Science 73 (2007) 299e315
connection with the sea often opens during strong winter
storms, which also generates heavy rainfall.
Acknowledgements
This work is in partial fulfillment of the PhD of Antoine
Carlier who was supported by a fellowship of the French Min-
istry of Education Research and Technology. Thanks are due
to L. Fonbonne for her observations regarding the opening
of the connection of the lagoon with the open sea. This
work was partly funded by the EEC network of Excellence
MARBEF through its responsive mode project FOODWEBIO.
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