Eutrophication is considered to be one of the mainthreats to marine coastal ecosystems. Among otherdeleterious effects, this process involves changes in thefunctioning of ecosystems, leading to negative socio-economic impacts (Nixon 1995, Cloern 2001). The natu-rally nutrient-limited Mediterranean Sea is thought tobe highly vulnerable even to low anthropogenic nutri-ent inputs (UNEP/FAO/WHO 1996). Although there isa wealth of evidence of drastic shifts in macrophytecommunities following long-term exposure to high nu-trient loads in the Adriatic Sea (Munda 1993) and alongSpanish and French coastlines (Soltan et al. 2001,Pinedo et al. 2007), changes due to low anthropogenicdischarges are less conspicuous because they are usu-ally confounded with the effects of natural disturbances
(Cloern 2001). Early detection of nutrient enrichment isrequired in order to prevent further degradation ofaquatic habitats. However, initial effects are likely to bemissed if measurements are restricted to physicochem-ical water properties (e.g. nutrient concentrations,salinity, plume dispersion) and community composi-tions, as these are known to vary greatly in time andspace (e.g. Carballo et al. 1996). Moreover, these analy-ses do not provide evidence of an uptake of sewagematter by biomasses (Costanzo et al. 2001). Such diffi-culties may be overcome by using indicator organismswhich integrate pollution signals in their tissues and re-flect the quality of the habitat in which they live(Oehlmann & Schulte-Oehlmann 2003).
One method that can detect these signals is stableisotope analysis. This application is based on the factthat nitrogen originating from human effluents (e.g.
Potential early indicators of anthropogenicallyderived nutrients: a multiscale stable isotope analysis
S. Vermeulen*, N. Sturaro, S. Gobert, J. M. Bouquegneau, G. Lepoint
MARE Centre, Laboratory of Oceanology, University of Liège, Sart Tilman B6c, 4000 Liège, Belgium
ABSTRACT: Increasing human pressure along Mediterranean coastlines raises the need to definesensitive bioindicators that provide an early response to nutrient enrichment. We performed multi-scale carbon and nitrogen stable isotope analyses on the limpet Patella caerulea, the snail Monodontaturbinata, epilithic biofilms, and the macroalga Rissoella verruculosa inhabiting the rocky midlittoralzone. Samples were seasonally collected in 2006 from 5 sites exposed to a range of anthropogenic dis-charges in the Revellata Bay area and in Marseille harbour (France). All bioindicators exhibitedstrongly elevated δ15N values at impacted sites compared to pristine ones, which revealed the biolog-ical availability of anthropogenically derived nutrients. Only epilithic biofilms tended to show boththe occurrence of nutrient pulses during the tourist season and a δ13C response at impacted sites. Incontrast to macroalgae, which exhibited a somewhat equivocal signal, gastropods and especiallyM. turbinata provided the best time-integrated picture of the graduated exposure of the 5 sites toanthropogenic impact. Results also showed first evidence of large isotopic variability at a scale of tensof metres, close to that found at the kilometre scale. The intra- and interspecific isotopic variability ingastropods may be explained by the patchiness of resources and specific morphological and behav-ioural features, but these factors do not greatly hamper their potential as early bioindicators of waste-water disturbances.
Resale or republication not permitted without written consent of the publisher
Mar Ecol Prog Ser 422: 9–22, 2011
high trophic level faecal material) has a typically15N-enriched isotopic composition compared to natu-rally occurring dissolved inorganic nitrogen (DIN) andthat both sources differ isotopically and are availableat the base of food webs. These allochtonous nutrientsalso undergo a fractionation process towards 15N-enriched values through both the preferential loss of14N isotope during ammonia volatilization and the bac-terial enzymatic affinity for 14N during degradation(McClelland et al. 1997, Costanzo et al. 2001). Stablecarbon isotope ratios are a useful means to identifyconsumers’ food sources (DeNiro & Epstein 1978) andto determine the terrestrial or marine origin of organicmatter (Fry & Sherr 1984). Moreover, as nutrientenrichment exerts bottom-up forces at the base of foodwebs, the carbon isotopic values of microalgal assem-blages can reveal changes in physiological states(Brutemark et al. 2009), biodiversity (Vuorio et al.2006) and thus in the food sources available to con-sumers (Vadeboncoeur et al. 2003, Gil et al. 2006).
Numerous studies have successfully detected, quan-tified and traced the biological availability of waste-water-derived nutrients in marine food webs (Tewfiket al. 2005, Armitage & Fourqurean 2009), even underrelatively low nutrient discharges (McClelland et al.1997). However, some of these took samples from asingle point and at one spatial scale and/or did notspecify the spacing of samples collected from pristineand impacted sites (Riera et al. 2000, Waldron et al.2001). This could have some confounding effects indetecting pollution impact (i.e. over- or underestima-tion of the spatial extent of the impact), especiallywhen using low-mobility invertebrate grazers aspotential indicators, because of the natural spatialheterogeneity of their habitat and of the uneven influ-ence of contaminants in the environment (Bishop et al.2002). Another potential source of confusion is theintra- and inter-specific variability of the signals deliv-ered by bioindicators (e.g. morphological and behav-ioural features). Such analysis is required in order tovalidate and facilitate their routine use in the context offuture monitoring schemes (Segar et al. 1987).
Due to negligible tidal amplitude, the Mediterra-nean midlittoral zone is an easily accessible areawhere biota is directly exposed to human activities(Soltan et al. 2001). The endemic prosobranch gas-tropods, the limpet Patella caerulea (Linnaeus, 1758)and the snail Monodonta turbinata (Born 1780), areamong the most frequently encountered consumers ofthis narrow rocky fringe. These long-lived species areaccessible all-year round and often present wheremussels, a frequently used bioindicator, are not. Lim-pets and snails are coexisting microphagous grazersknown to feed on epilithic and epiphytic biofilmswhich are ubiquitous structures composed of bacteria,
microalgae, macroalgal sporelings, protozoans, anddetritus (Steneck & Watling 1982, Burns & Ryder 2001,Staal et al. 2007). Although it is known that biofilmscan play a key role in fuelling freshwater food webs(Vadeboncoeur et al. 2003), their marine counterpartsare still poorly investigated, probably because of sam-pling difficulties and a lack of knowledge regardingtheir taxonomic compositions (Hill & Hawkins 1991).Nevertheless, biofilms could be a major pathway ofanthropogenically derived material towards uppertrophic levels (Burns & Ryder 2001). The endemic redfoliose macroalga Rissoella verruculosa (Agardh 1849)is another emblematic species of the Mediterraneanmidlittoral zone, where it forms well-developed belts(Pérès & Picard 1964). These organisms and assem-blages exhibit features of good potential bioindicators,being of limited mobility or sedentary, abundant, andeasy to identify (Oehlmann & Schulte-Oehlmann2003). They belong to different trophic levels andexhibit 2 nutrient incorporation paths, with biofilmsand macroalgae taking nutrients from the surroundingwater and gastropods relying essentially on microflora.Furthermore, they presumably have different turnoverrates and thus could provide differentiated temporalinformation about anthropogenic impacts.
In the present study, multi-spatial scales and sea-sonal dual C and N stable isotope analyses were per-formed on gastropods, epilithic biofilms, and macroal-gae collected from rocky midlittoral areas exposed to arange of anthropogenically derived nutrients in orderto (1) assess their sensitivity and verify their use asearly indicators of eutrophication; (2) validate the useof gastropods as bioindicators by analysis of their intra-and inter-specific isotopic variability.
MATERIALS AND METHODS
Study area. This study was conducted in the mid-littoral zone of the Revellata Bay area (northwest Cor-sica, France) and of Marseille harbour (southernFrance) (Fig. 1). Due to negligible tidal range (about40 cm), the midlittoral zone is restricted to a narrowfringe located at the air–sea interface and experienceshigh variability of environmental factors (Pérès &Picard 1964). The waters of Revellata Bay are oligo-trophic, and primary production mainly relies uponNH4
+ recycling (Velimirov & Walenta-Simon 1992).Rainfall is typical of the Mediterranean climate (i.e.sporadic and seasonal) and results in intermittent riverflows and runoff which supply very limited amounts ofnutrients. As neither agricultural nor industrial activityis present, the main sources of allochthonous nutrientsare (1) the occasional entrance of deep nutrient-richwaters driven by NNE winds, favouring early spring
10
Vermeulen et al.: Early indicators of anthropogenically derived nutrients
phytoplankton blooms (Skliris et al. 2001) and, (2)domestic wastewaters that undergo a physicochemicaltreatment before being discharged at 36 m depth(Fig. 1). The amount of nutrients discharged into thebay (mainly NH4
+ and PO43–) is very low most of the
year but reaches a maximum in summer (i.e. whenwaters are ultra-oligotrophic) due to tourism, whichresults in a 10-fold increase in the local population ofabout 5000 people. Average amounts of dischargedKjeldahl nitrogen (organic and ammoniacal forms) andtotal phosphorus (organic and mineral forms) rangedrespectively from 53 to 536 kg d–1 and from 2 to 5 kgd–1 in December 2005 and July 2006 (CompagnieMéditerranéenne d’Exploitation des Services desEaux, pers. comm. 2006). The Marseille area is denselyurbanized and is inhabited by about 1.5 million people.The fishing and leisure harbour (1450 moorings) con-tinuously receives organic and wastewater loads fromdiffuse sources (Ifremer 2007).
Sample collection and processing. Samples wereseasonally and randomly handpicked on granite sub-strata over a distance of 10 m in February (winter),June (summer) and November (autumn) 2006. A large-scale (kilometre) sampling was carried out accordingto the ‘control vs. impacted’ status of 4 sites located inRevellata Bay and its vicinity (Fig. 1). We chose 2 con-
trol sites (Alga, Bianca), a moderately impacted one(STAtion de REcherche Sous marine et Océano-graphique [STARESO], which is locally influenced byseptic tank discharges) and an impacted one (Calvisewer). Additionally, samples from Marseille harbourwere collected in winter and summer, allowing forcomparison with a heavily and continuously impactedsite. Small-scale sampling was conducted in autumn atpoints 30 metres apart along horizontal transects atBianca and Calvi sewer (Fig. 1). Points were numberedfrom 1 to 4 so that cumulative distances betweenextreme points were 90 m. Only the most frequentlysampled points over seasons were included in theoverall spatial and temporal analyses of data (i.e. point1 for Bianca, 4 for Calvi sewer).
Samples of epilithic biofilms were collected with atoothbrush over 100 cm2 areas. Biofilms were appar-ently growing in the same set of environmental condi-tions, e.g. light and wave exposure. The red macroalgaRissoella verruculosa, which forms seasonal belts atthe sea surface level, was collected in summer. Sixreplicates of Patella caerulea and Monodonta turbinatawere collected, where and when available, togetherwith biofilms from the same location. Due to logisticalconstraints, we were not able to perform sampling atMarseille harbour in autumn. Shell metrics were
11
Fig. 1. Location of study areas. Inset shows Calvi (Corsica) and Marseille harbour; the main map shows the sites in Revellata Bay(Alga, STARESO, Calvi sewer), its vicinity (Bianca) and midlittoral small-scale sampling points (1 to 4) along near sea-surfacelevel horizontal transects. Q: sewage outfall; 30 m: distance between points on transects; dark grey shading: built-up area of Calvi
Mar Ecol Prog Ser 422: 9–22, 2011
recorded using a calliper (~0.1 mm) and we were care-ful to select individuals within a narrow size range foranalysis: shell length 25–30 mm for P. caerulea andshell height 10– 15 mm for M. turbinata. Additionally,we investigated the size-related variability of dataamong 26 individuals of P. caerulea ranging from 11 to37 mm in shell length at STARESO in autumn.
Pedal muscles of gastropods were isolated under abinocular microscope and the sex of Patella caeruleaindividuals was determined (Orton et al. 1956). In thesize range defined for snails Monodonta turbinata,individuals did not reach sexual maturity, like theirAtlantic counterparts M. lineata (Crothers 1994). Sam-ples of epilithic biofilms were centrifuged and rinsedwith MilliQ water (3 times at 1880 × g for 10 min) andexposed to fuming HCl 37% in order to remove saltsand inorganic carbonates, respectively. The use of thisgentle acidification procedure avoids loss of acid-solu-ble organic matter (Hedges & Stern 1984). Epiphyteswere scraped from Rissoella verruculosa with a razorblade. Samples were then oven-dried at 50°C for 48 hand ground into a homogeneous fine powder usingmortar and pestle.
Stable isotope and elemental content analyses.Measurements were performed with a mass spectrom-eter (Optima, Micromass) coupled to a CNS elementalanalyzer (Carlo Erba). Isotopic ratios were computed inδ values (‰). Data were expressed relative to theVienna Pee Dee Belemnite standard (vPDB) and toatmospheric N2 for carbon and nitrogen respectively.Reference materials from the International AtomicEnergy Agency (IAEA) were N1 (ammonium sulphate,δ15N = 0.4 ± 0.2 ‰) and CH6 (sucrose, δ13C = –10.4 ±0.2 ‰). The analytical precision based on the standarddeviation of a gastropod muscle replicate was 0.3 ‰ forcarbon and 0.2 ‰ for nitrogen.
Statistics. Spatial and interspecific differences in iso-topic compositions as well as an interaction term (Site ×Species) were tested using the Scheirer-Ray-Hare non-parametric 2-way ANOVA following the procedurerecommended by Zar (1984) and Dytham (2003). Asthis technique was restricted to balanced sampledesigns, tests were performed on gastropod isotopicdata from the Revellata Bay area over the 3 seasons.Further spatial and temporal multivariate data analy-ses were conducted using matrices of normalizedEuclidean distances calculated from δ15N and δ13C val-ues. Similarity matrices were analyzed using the non-parametric analysis of similarity (ANOSIM). Outputs ofthis randomization procedure are the R statistics,which provide an absolute measure of the degree ofseparation between groups, with values close to zeroand unity showing complete similarity and discrimina-tion respectively. Two-way crossed ANOSIM was per-formed to test for spatial (among sites) and temporal
(among seasons) differences in δ15N and δ13C valuesamong gastropods, and to compare the interspecificisotopic patterns across seasons. Runs were first per-formed on δ13C and δ15N values together (Global R)and then on each variable separately in order to deter-mine their respective contribution to the between-group differences. The R statistics were also used forpairwise comparisons between sites. The small-scaledata were analyzed using 1-way ANOSIM, consider-ing each 30 m-apart point as independent. This proce-dure was also employed to test for spatial differencesamong data from Rissoella verruculosa. In addition,nMDS (non-metric multidimensional scaling) ordina-tion analyses graphically supported the results fromANOSIM. The stress values provided by nMDS indi-cate the goodness-of-fit of the representation of dis-tances between samples in the 2-dimensional space ofthe ordination plot. A stress value below 0.05 is consid-ered to provide an excellent picture of a matrix config-uration. Multivariate analyses were completed usingPRIMER v.6 software (Clarke & Gorley 2006). The co-efficients of variation, calculated by dividing the stan-dard deviation by the mean (CV in %), were providedand express a dispersion measure of the isotopicdata.The potential size-related effect on isotopic val-ues (Patella caerulea) and the intrapopulation isotopicvariance were assessed using linear regressions (Sta-tistica v.8, StatSoft 2007).
RESULTS
Epilithic biofilms and macroalgae
Results from epilithic biofilms showed high kilome-tre-scale and temporal variability, with large differ-ences in carbon (Δ13C) and nitrogen (Δ15N) isotopic val-ues occurring between sites in winter (Δ13C = 10.6 ‰,Δ15N = 6.7 ‰) and across seasons (Δ13C = 10.3 ‰, Δ15N =3.2 ‰) (Table 1). The data from Marseille harbour andin summer for Calvi sewer showed some 15N and13C-enriched values, contributing to their separationfrom other sites in the nMDS plot (Fig. 2a). This trendalso appeared along the horizontal transect at Calvisewer (Table 2) where the results showed some largevariations of isotopic ratios over short distances. TheC:N contents of biofilms also exhibited an overall sea-sonal trend towards increasing values (i.e. lowerN contents) from winter to autumn (Table 3).
The isotopic values of Rissoella verruculosa showedlarge spatial differences (Global R = 0.66) (Fig. 3a,b,Table 4). The most 15N-enriched values occurred atCalvi sewer and provided, when compared to the datafrom STARESO and Bianca, a maximum Δ15N range of3.0 ‰ allowing a good discrimination between sites
12
Vermeulen et al.: Early indicators of anthropogenically derived nutrients
(Fig. 2b). The values from Alga were 15N-enrichedrelative to those from Bianca and STARESO.Moreover, the lowest N contents (0.9%, Table 3)along with the most 13C-depleted compositionsclearly set R. verruculosa individuals from Algaapart from others (e.g. Alga vs. Bianca, R δ13C = 1,Table 5).
Gastropods
The kilometre-scale variability of isotopic valueswas particularly high for both species (Global R =0.72–0.78) compared to the seasonal variability(Global R = 0.26–0.30, Tables 1 & 4). The δ15N val-ues accounted for most of this spatial variability (Rδ15N = 0.72–0.76) so that the nMDS discriminatedvery well between sites (Fig. 2c,d) and allowed 3clusters to be identified, from the most enriched tothe most 15N-depleted values: (1) Marseille har-bour, Calvi sewer, (2) STARESO, Bianca (3) Alga(Fig. 3a). The results from ANOVA corroboratedthe occurrence of these high spatial differences(p < 0.0001). The largest differences of mean δ15Nvalues (Δ15N) were consistently observed betweenAlga and Calvi sewer, with values ranging from4.6 to 6.7 ‰ for Patella caerulea and from 2.0 ‰ to3.9 ‰ for Monodonta turbinata in summer andautumn respectively. The data from Marseille har-bour provided some close to maximum Δ15N val-ues for P. caerulea (Δ15N = 6.0 and 6.4 ‰) and afurther extended range for M. turbinata (Δ15N =4.5 ‰). As suggested by the nMDS plots(Fig. 2c,d), the Δ15N values between Calvi sewerand Marseille harbour were more pronounced forM. turbinata (R δ15N = 1) than for P. caerulea(R δ15N = 0.46, Table 5). Moreover, only snailsshowed some 15N-enriched values at STARESOcompared to Bianca (R δ15N = 0.37). The kilome-tre-scale variability of δ13C values was lower thanthe δ15N variability (R δ13C = 0.41–0.48, Fig. 3b,Table 4). Spatial differences in δ13C values werealso detected by the ANOVA (p < 0.0001). For bothspecies, the 13C-enriched compositions from Algaweighted in the observed spatial pattern andclearly set this site apart from others (Fig. 2c,d,Table 1). The maximum Δ13C values usually oc-curred between STARESO and Alga and ranged,according to season, from 4.8 to 6.8 ‰ forP. caerulea and from 2.3 to 3.8 ‰ for M. turbinata.There were no, or only slight, differences in δ13Cvalues between Bianca, STARESO, Calvi sewerand Marseille harbour (Table 5).
The small-scale differences in isotopic values ac-cording to sampling points were particularly high,
13
Tab
le 1
. S
easo
nal
an
d k
ilom
etre
-sca
le v
aria
bil
ity
of i
soto
pic
val
ues
fro
m P
atel
la c
aeru
lea,
Mon
odon
ta t
urb
inat
a(n
= 6
, m
ean
±S
D i
n ‰
, co
effi
cien
t of
var
iati
on C
V i
n %
) an
d e
pil
ith
ic b
iofi
lms
(n =
1, i
n ‰
) b
etw
een
. For
Mar
seil
le h
arb
our,
gap
s in
dic
ate
no
dat
a av
aila
ble
Win
ter
Su
mm
erA
utu
mn
δ13C
δ15N
δ13C
δ15N
δ13C
δ15N
Mea
nS
DC
VM
ean
SD
CV
Mea
nS
DC
VM
ean
SD
CV
Mea
nS
DC
VM
ean
SD
CV
Pat
ella
cae
rule
aA
lga
–10.
61.
211
.22.
00.
733
.1–
9.3
0.8
8.9
2.4
0.8
33.5
–8.
10.
67.
01.
70.
421
.8B
ian
ca–1
2.5
3.8
30.2
3.1
0.7
23.7
–13.
81.
06.
93.
60.
615
.8–1
4.1
2.0
14.5
4.5
0.6
13.9
Sta
reso
–15.
40.
85.
03.
50.
14.
2–1
4.2
1.7
12.1
3.5
0.4
11.6
–14.
91.
17.
14.
80.
510
.5C
alvi
sew
er–1
3.4
0.7
5.3
7.6
0.7
8.9
–14.
01.
611
.47.
00.
45.
7–1
3.0
3.5
27.1
8.4
1.5
17.3
Mar
seil
le h
arb
our
–14.
11.
39.
08.
40.
34.
2–1
3.4
1.6
12.2
8.4
0.7
8.7
Mo
no
do
nta
tu
rbin
ata
Alg
a–1
3.6
1.0
7.4
3.3
0.4
12.1
–13.
70.
43.
23.
40.
27.
5–1
3.0
0.8
6.0
2.5
1.2
47.6
Bia
nca
–16.
50.
42.
23.
80.
26.
4–1
6.3
0.8
4.9
4.0
0.3
6.5
–15.
21.
17.
23.
00.
414
.2S
tare
so–1
7.2
1.1
6.2
4.3
0.2
4.3
–17.
50.
42.
44.
10.
37.
6–1
5.2
0.4
2.5
3.9
0.4
10.3
Cal
vi s
ewer
–16.
51.
27.
35.
70.
34.
6–1
5.3
0.5
3.5
5.4
0.5
9.5
–15.
30.
95.
76.
40.
914
.1M
arse
ille
har
bou
r–1
5.8
0.7
4.7
7.8
0.3
3.6
Ep
ilit
hic
bio
film
sA
lga
–21.
70.
8–2
2.8
0.9
–18.
01.
5B
ian
ca–2
3.1
0.3
–21.
01.
4–2
6.0
2.6
Sta
reso
–24.
9–
0.2
–21.
61.
0–2
2.6
1.8
Cal
vi s
ewer
–25.
90.
3–1
8.3
3.5
–15.
63.
4M
arse
ille
har
bou
r–1
5.3
6.5
–15.
44.
0
Mar Ecol Prog Ser 422: 9–22, 201114
Fig. 2. nMDS ordinations of δ15N and δ13C individual values from (a) epilithic biofilms, (b) Rissoella verruculosa, (c) Monodontaturbinata and (d) Patella caerulea over study time and at kilometre-scale. Graded shading shows the exposure of sites to nutrientenrichment: Alga (white), Bianca (light grey), STARESO (medium grey), Calvi sewer (dark grey), Marseille harbour (black).
For epilithic biofilms, letters indicate the seasonal variability of values: (A) autumn, (W) winter and (S) summer
Table 2. Small-scale variability of δ15N and δ13C values from Patella caerulea, Monodonta turbinata (n = 6, mean ± SD in ‰, coef-ficient of variation CV in %) and epilithic biofilms collected along horizontal transects (1 to 4; see Fig. 1) (n = 1, in ‰ except for
‘biofilms overall (1 to 4)’ mean ± SD, CV%) at Bianca and Calvi sewer in autumn. Gaps indicate no data available
Vermeulen et al.: Early indicators of anthropogenically derived nutrients
especially for Monodonta turbinata atCalvi sewer (Global R = 0.65, Tables 2 &4). Snails showed Δ15N (3.6 ‰) and Δ13C(2.4 ‰) ranges, which were comparableto the kilometre-scale ranges but oc-curred within distances of 30 to 60 mand lead to large between-points sepa-ration in the nMDS diagram (R δ15N =0.58, Fig. 4a). The overall δ15N coeffi-cients of variation were 37.2% at Calvisewer as opposed to 13.9% at Bianca forM. turbinata. The δ15N values for Patellacaerulea did not show such discrepancybetween points along the horizontaltransect (Fig. 4b). For both species atBianca, the main causes of small-scaleisotopic variability were the δ13C valueswith Δ13C ranges of 2.0 ‰ for M.turbinata and 2.6 ‰ for P. caerulea over30 to 60 m (Table 2). No particular trendappeared when comparing the coeffi-cients of variation between kilometer-scale and small-scale results.
Intra- and interspecific variability inisotopic ratios
A slight negative correlation betweenthe shell length and the δ15N valuesof Patella caerulea was found atSTARESO (r2 = 0.18, p = 0.03), while nosignificant difference was observed forthe δ13C values (r2 = 0.07). The mainvariability in δ15N values was attributedto only few individuals belonging to the15 to 20 mm class (Fig. 5a). The plot ofδ15N and δ13C individual values showeda strong negative relationship at a sin-gle sampling point for P. caerulea(STARESO, r2 = 0.55, p = 0.0001) andfor both limpets (r2 = 0.48, p = 0.0002)and snails (r2 = 0.31, p = 0.004) overmultiple sampling points at Bianca(Fig. 5b). Along this transect, the use ofindividual instead of mean δ15N valuesrevealed a large variability of resultsranging from about 2.0 to 3.7 ‰ forsnails and to 5.7 ‰ for limpets. Theoccurrence of elevated δ15N values wasnevertheless lower than at Calvi seweror Marseille harbour. We did not de-tect any differences in isotopic valuesthat could be ascribed to the sex of indi-viduals.
15
Table 3. C:N ratios (w/w) and nitrogen contents (% dry wt) of epilithic biofilms be-tween winter, summer and autumn and for Rissoella verruculosa in summer
Fig. 3. Kilometre-scale (a) δ15N and (b) δ13C values from Patella caerulea (darkgrey), Monodonta turbinata (light grey) and Rissoella verruculosa (white;except for Marseille harbour) over study period. Thick horizontal lines indi-cate medians, boxes the interquartile ranges and whiskers mark the range of
data excluding outliers (open circles, >1.5× interquartile range)
Mar Ecol Prog Ser 422: 9–22, 2011
Although they provided the same overall isotopicpatterns, differences occurred among gastropods, withMonodonta turbinata consistently showing more re-stricted ranges of isotopic values and an overall deple-tion in 13C isotope compared to Patella caerulea. The
use of ANOVA allowed detection of large differences incarbon isotopic compositions between snails andlimpets (p < 0.0001) but did not find significant differ-ences in δ15N (p = 0.62) or in Sites × Species interactioneffects for either δ15N (p = 0.13) or δ13C values (p = 0.90).
The largest and more consistent inter-specific differences in mean δ13C val-ues were seen at Alga (R δ13C = 0.90,Table 6). Differences were marked forboth isotopic ratios at other sites of theRevellata Bay area (0.58 > Global R >0.73) and also emerged from small-scale data at Bianca (Global R = 0.62).The isotopic values of limpets consis-tently exhibited higher variability thanthose of snails, leading to some be-tween- and within-sites overlap ofvalues (Fig. 3). The coefficients of vari-ation from M. turbinata were generallylower for the δ15N values and con-sistently showed values for δ13C thatwere below 10% while those fromP. caerulea frequently exceeded thisvalue.
16
Fig. 4. nMDS ordinations of small-scale δ15N and δ13C values from (a) Monodonta turbinata and (b) Patella caerulea at Bianca(squares) and Calvi sewer (circles) in autumn. Graded shading shows increasing distances between the points 30 m apart along
Table 4. Summary of ANOSIM R statistics computed for Patella caerulea, Mono-donta turbinata and Rissoella verruculosa over the study period, and in autumnfor small-scale data from the samples collected along the horizontal transects(Fig. 1). Global R: combined analysis of δ13C and δ15N. Significant differences are
Rissoella verruculosaGlobal R 0.66R δ13C 0.42R δ15N 0.51
Table 5. Output of ANOSIM main pairwise comparisons of δ13C and δ15N values for Patella caerulea, Monodonta turbinata (allseasons considered), and Rissoella verruculosa. Global R: combined analysis of δ13C and δ15N. Significant differences are p < 0.01
except *p < 0.05, ns: not significant. Gaps indicate no data available
Patella caerulea Monodonta turbinata Rissoella verruculosaGlobal R R δ13C R δ15N Global R R δ3C R δ15N Global R R δ13C R δ15N
Vermeulen et al.: Early indicators of anthropogenically derived nutrients
DISCUSSION
The isotopic results from gastropods,macroalgae and biofilms exhibited largespatial variability at kilometre-scale.They consistently showed 15N enrichmentat impacted sites compared to pristineones, thus corroborating previous find-ings that stable nitrogen isotopes can beused as an environmental tracer in orderto detect the influence of anthropogeni-cally derived nutrients in coastal ecosys-tems (e.g. McClelland et al. 1997).Although biofilms showed a 13C enrich-ment trend at impacted sites, the δ13Cspatial patterns did not discriminatebetween the terrestrial (–23 to –30 ‰ forC3 plants) or marine (–18 to –24 ‰ forphytoplankton) origin of materials (Fry &Sherr 1984).
Indicators of anthropogenically derivednutrients
Despite the scarcity of stable isotopemeasurements from natural epilithicbiofilms, our overall low δ13C and δ15Nvalues were in accordance with experi-mental results obtained from artificialsubstrates in seawater (Staal et al. 2007)and freshwater (MacLeod & Barton 1998,Hill & Middleton 2006). Most of our δ15Nvalues were typical of primary producersfrom the Mediterranean Sea. This can beexplained by the occurrence of the lowerδ15N values of DIN in this part of theworld compared to more eutrophic areas(e.g. Krom et al. 2004). Epilithic biofilms
tended to concomitantly exhibit some 13C- and 15N-enriched values at impacted sites, suggesting that boththe nature and the isotopic compositions of dissolvedinorganic carbon (DIC) and nitrogen (DIN) sourcesavailable to microalgae were variable at the kilometre-scale and, notably, according to the availability ofanthropogenically derived nutrients (Costanzo et al.2001, Vizzini et al. 2005). Rather than assigning a par-ticular δ13C value to DIC originating from wastewater,we hypothesized that a supply of anthropogenic nutri-ents may have stimulated the growth of microalgae,leading to a low discrimination against both the 13Cisotope and the enriched 15NH4
+ pool (Brutemark et al.2009). Furthermore, as nutrient requirements and iso-topic fractionation processes are taxa-specific, a prolif-eration of opportunistic nitrophilous taxa was possible
17
Fig. 5. Relationships between δ15N and δ13C values for size classes (in mm)of Patella caerulea individuals from (a) STARESO and (b) for small-scalevalues from P. caerulea and Monodonta turbinata at Bianca (n = 6) inautumn. Shading in (b) refers to increasing distances between the points30 m apart along the horizontal transects (Fig. 1): 1 (white), 2 (light grey),
3 (medium grey), 4 (black)
Table 6. Per-site output of ANOSIM showing interspecific dif-ferences in δ13C and δ15N values between Patella caeruleaand Monodonta turbinata. The small-scale results (points30 m apart along the horizontal transects, Fig. 1) from Biancaand Calvi sewer are also shown. Global R: combined analysisof δ13C and δ15N. Significant differences are p < 0.01 except
*p < 0.05, ns: not significant
Patella caerulea vs. Monodonta turbinataGlobal R R δ13C R δ15N
at impacted sites (Wilmotte & Demoulin 1988, Vuorioet al. 2006). For example, isotopic values from theheavily and steadily impacted Marseille harbourremained constant over time, as opposed to those fromCalvi sewer where summer pulses of nutrients mayhave stimulated a shift in the composition ofmicroflora. This seemed to show that, probably due totheir high turnover rates (Burns & Ryder 2001,Vadeboncoeur et al. 2003), biofilms respond rapidly inrelation to fluctuations in δ15N of nitrogen sources (i.e.caused by the influx of tourists in summer) and thatthey could be good indicators of seasonal changes innutrient loads. The emergence of compound-specificstable isotope analyses will likely refine our knowl-edge of the taxonomic composition and metabolism ofbiofilms by targeting the isotopic composition of spe-cific fractions (autotrophs, heterotrophs, detritus),which we were not able to dissociate in this study (e.g.Oakes et al. 2005).
In contrast to biofilms, the isotopic values of gas-tropods exhibited low seasonal variability, suggesting aslow turnover of muscle tissue over a period of severalmonths (Lorrain et al. 2002). This is in agreement withfeeding experiments conducted on the marsh periwin-kle Littorina irrorata which showed half-life of a 15Nlabel ranging from 99 to 231 d (turnover model fromMcIntyre & Flecker 2006 applied to data from Kemp etal. 1990). The δ15N values of gastropods reflected theexposure of sites to anthropogenic impact, showingthat they were likely feeding from a food web forwhich the δ15N baseline was set higher at impactedsites compared to pristine ones. The δ15N baseline con-cept refers to the time-integrated signal provided byprimary consumers which allows the characterizationof the base of food webs (Cabana & Rasmussen 1996,Vander Zanden & Rasmussen 1999). The use of micro-phagous gastropods exhibiting plasticity in their feed-ing behaviour (Hawkins & Hartnoll 1983) may haveincreased the probability of assimilating material thatincorporated 15N from human effluents. Moreover, asan indirect effect of nutrient enrichment, the abun-dance of small animals (ostracods, copepods, smallmolluscs) was likely high in relation to that of microal-gal biomass (Gil et al. 2006, Armitage & Fourqurean2009). Small animals are frequently observed in thegut of limpets and snails (Hill & Hawkins 1991, Ver-meulen 2006, Camus et al. 2008), and and a recentstudy provides an insight into the assimilation of suchfood items by chitons and key-hole limpets (Camus etal. 2009).
Conversely, the lower δ15N values from control sitesand especially at Alga were more typical of Mediter-ranean first consumers (Lepoint et al. 2000). The con-comitantly 13C-enriched and 15N-depleted values fromAlga undoubtedly show that both the DIC and DIN
pools and the composition of food sources available tograzers differed in comparison with other sites. Fur-thermore, Alga receives large amounts of litterdeposits from the adjacent Posidonia oceanica sea-grass meadow. Although sampling was performedaway from the deposition area, the degradation of litterwas likely to influence the isotopic values of inorganicpools used by primary producers. The assimilation bygastropods of some pieces of decaying leaves with δ13Cand δ15N values of about –13.0 and 1.3 ‰, respectively,as well as their fouling organisms (bacteria, fungi,blue-green algae, diatoms) was considered possible(Kurata et al. 2001, Sturaro et al. 2010).
The fact that N contents and isotopic values of Ris-soella verruculosa at Alga were set apart from those ofother locations also supports the idea that Alga is a dis-tinct area as regards DIC and DIN fluxes. However, theisotopic pattern of R. verruculosa was inconsistent withthat of gastropods since values for this species at Algawere 13C-depleted and 15N-enriched compared tothose from STARESO and Bianca. The turnover rate ofR. verruculosa, likley being intermediate between thatof biofilms and gastropods, may be responsible for pro-viding a different isotopic signal. Nevertheless, macro-algae exhibited maximum Δ15N ranges similar toMonodonta turbinata between Calvi sewer and Bian-ca, showing that, despite its seasonal occurrence, R.verruculosa provides a reliable snapshot for detectingthe influence of sewage.
Surprisingly, in Revellata Bay, the Δ15N valuesbetween impacted and control sites were far higherand also more consistent compared to those previouslymeasured in other Mediterranean coastal open waterand non-estuarine areas (Vizzini & Mazzola 2006, Las-sauque et al. 2010). In fact, our Δ15N ranges for gas-tropods at Revellata Bay fell within those reportedfrom areas receiving high nitrogen loads in Mediter-ranean lagoons (Carlier et al. 2008) and worldwide, inboth freshwater and marine systems (Cabana & Ras-mussen 1996, Riera et al. 2000, McKinney et al. 2001,2002, Tewfik et al. 2005) (Table 7). With respect to therelatively low nutrient loads in the Revellata Bay, thisunexpectedly strong signal seemed to show a greaterthan previously thought influence of sewage and/orhighlighted the importance of even occasional suppliesof anthropogenic nitrogen in a naturally nutrient-lim-ited system.
The small-scale data provided qualifed support forthese conclusions and the first evidence of large δ13Cand δ15N variability at a scale of tens of metres within asingle, apparently homogeneous habitat (see Guest etal. 2004 for small-scale inter-habitat δ13C variability).At Calvi sewer, samples of biofilms and Monodontaturbinata taken from points 30 m apart exhibitedranges of δ15N values that were respectively higher
18
Vermeulen et al.: Early indicators of anthropogenically derived nutrients
than and close to those found between impacted andcontrol sites (i.e. at kilometre-scale). This could beinterpreted as showing an uneven influence of sewagealong the shore due to the effects of diffuse sources ofwastewater and the mixing between naturally occur-ring DIN and the 15N enriched pools coming from themain sewage outfall. Thus, it appears that establishinga δ15N baseline from a single sampling point is contro-versial because it does not integrate the whole habitatsignal. For biofilms and gastropods, such samplingmay lead to either a failure in detecting wastewaterinfluence or an over-estimation of its spatial extent(Bishop et al. 2002).
Potential confounding effects in gastropods
Gastropods can be considered as reliable indicators,providing a time-integrated but spatially uneven sig-nal of anthropogenically derived nitrogen. The inter-and intra-specific variability in isotopic values couldlead to some additional confounding effects due to spe-cies particular morphological and behavioural fea-tures. For example, the occurrence of large isotopicvariances like those observed for Patella caerulea maylimit the reliability of this species as a bioindicator ofthe exposure of sites to urban effluents. The responseof snails was closer to the pattern of graduated expo-sure of sites to anthropogenic impact, exhibiting thegreatest difference between the heavily impactedMarseille harbour and Calvi sewer and a 15N-enrich-ment signal at the moderately impacted STARESO site.Monodonta turbinata individuals also consistentlyshowed more depleted 13C values compared toP. caerulea, suggesting differences in diet compositionbetween these coexisting species. Although gas-tropods likely experience specific isotopic fractionation
while processing food into their tissues (Bearhop et al.2004), we assumed that the existence of distinct radu-lar apparatus was a good explanation for interspecificdifferences in isotopic composition. Whereas the rhipi-doglossan radula of M. turbinata brushes substrate, theminerally hardened teeth of the docoglossan radula ofP. caerulea act as a rasp (Hawkins & Hartnoll 1983).Therefore, as supported by their high isotopic vari-ances, P. caerulea individuals could have relied on abroader range of food items (endolithic and endobioticmaterials) than M. turbinata (Della Santina et al. 1993).The δ13C mismatch between biofilms and gastropodsseemed to indicate that these generalist grazers didnot, as previously thought, rely exclusively on biofilmsas a food source (Hawkins & Hartnoll 1983). The eluci-dation of these gastropods’ diet would require detailedgut content analyses and further isotopic analyses fromseveral other potential food sources (e.g. Kurata et al.2001), which is beyond the scope of our study.
Commonly reported reasons for intra-population iso-topic variance refer to physiological processes thatvary according to size (or age), sex, developmentalstages, and nutritional states of consumers (Adams &Sterner 2000, Bearhop et al. 2004). Our results forlimpets did not show strong evidence of size-relatedisotopic variability, even though the time required forisotopic equilibration (i.e. tissue replacement followinga shift in diet composition) in small individuals isthought to be shorter than for larger ones (Sweeting etal. 2005).
The strong negative relationship found between theδ13C and δ15N values from gastropods provided evi-dence for alternative explanations, other than size, forthe isotopic variance found in this study. A similar iso-topic pattern was previously described for some mis-cellaneous taxonomic groups of primary consumersand was attributed to the typical depth-related iso-
19
Table 7. Reported maximum ranges of Δ15N values (impacted – control mean values in ‰) among bivalves and gastropods. Pollution sources refer to wastewater discharge (w) and agricultural fertilizers (af). Functional feeding groups are: bivalve
suspension (Bv-Sf) and deposit feeders (Bv-Df), gastropod deposit feeders (G-Df) and grazers (G-g)
Elliptio spp. Bv-Sf 7.7 w, af Lakes, USA McKinney et al. (2002)Geukensia demissa Bv-Sf 2.7 w, af Semi-enclosed estuaries, USA McKinney et al. (2001)Abra ovata Bv-Df 10.6 w, af Coastal lagoons, NW Mediterranean Carlier et al. (2008)Strombus gigas G-Df 4.2 w Coastal, Dominican Republic Tewfik et al. (2005)Strombus costatus G-Df 5.5 w Coastal, Dominican Republic Tewfik et al. (2005)Pinna carnea Bv-Sf 2.9 w Coastal, Dominican Republic Tewfik et al. (2005)Crassostrea gigas Bv-Sf 9.5 w, af Estuaries, NW Europe Riera et al. (2000)Littorina littorea G-g 7.5 w, af Estuaries, NW Europe Riera et al. (2000)Littorina saxatilis G-g 5.1 w, af Estuaries, NW Europe Riera et al. (2000)Mussels Bv-Sf 7.8 w Lakes, Quebec Cabana & Rasmussen (1996)Patella caerulea G-g 6.7 w Coastal, Mediterranean Present studyMonodonta turbinata G-g 4.5 w Coastal, Mediterranean Present study
Mar Ecol Prog Ser 422: 9–22, 2011
topic values of primary producers (i.e. particulargrowth conditions) reflected in consumers’ tissues(Vander Zanden & Rasmussen 1999). Here, we re-ported such relationships within a single habitat andfor single species of first consumers. We suggest thatsome patch-specific conditions, where available foodvaried in taxonomic composition, physiological stateand nutritional value, led to inter- and intra-specificvariability in isotopic composition (Lancaster & Wal-dron 2001). Indeed, due to their low mobility and theirspecific foraging behaviour (limpets’ homing vs. snailsroaming, with no fixed resting site), the selected gas-tropods presumably relied on local resources pro-duced in distinct microhabitats (Hawkins & Hartnoll1983, Hutchinson et al. 2007). A kind of gardening iseven thought to occur among these species as themucus they produce was recognised as a suitablemedium for bacterial activity enhancing the growth ofmicroalgae (Peduzzi & Herndl 1991, Johnson et al.2008).
CONCLUSIONS
The nitrogen stable isotope analyses applied to gas-tropods, macroalgae and epilithic biofilms highlightedtheir good sensitivity as indicators of the biologicalavailability of anthropogenically derived nitrogen. Bio-indicators exhibited different time-dependent res-ponses to nutrient enrichment. Epilithic biofilms pro-vided a short-term signal suggesting the influence ofnutrient pulses during the tourist season. Their use inthe early detection of wastewater disturbance is pro-mising but requires further investigation, especially asregards their δ13C response at impacted sites, theircomposition and the mechanisms involved in thepatchiness process. Although satisfactory, the signaldelivered by the macroalga Rissoella verruculosa wasless reliable compared to that of gastropods. Limpetsand snails exhibited a time-integrated signal for sev-eral months, suggesting that 1 annual sampling cam-paign should be sufficient to determine the anthro-pogenic impact. We consider that Monodontaturbinata delivered the most suitable signal becausemean values were associated with low variance andthe discrimination between sites best mirrored thegraduated exposure to anthropogenic impact. Thelarge variability of our δ15N baseline along small-scaletransects (tens of metres) warned against drawing gen-eral conclusions from a pollution signal on only onesampling scale, especially when low-mobility inverte-brate grazers are selected as potential bioindicators.This must be taken into account in future studies deal-ing with the assessment of anthropogenic impact andthe understanding of food web functioning.
Acknowledgements. We thank R. Biondo and STARESOresearch station staff for field and laboratory assistance, andProfessors V. Demoulin and P. Dauby, Drs. M. Poulicek,G. Quinn (CAMbio, Ireland) and anonymous referees for theircritical comments on this work. We are grateful to J. Richirand L. Michel for statistical advice. This study was funded bythe ARC-Rapid Assessment for the marine Coastal Environ-ment 05/10-333, by the FRS-FNRS (Belgian National Fund forScientific Research) FRFC 2.4.502.08.F and the French Bel-gian community. S.V. is funded by a PhD grant from the ARC-RACE and G.L. is a Research Associate FRS-FNRS. This pub-lication is MARE number 199.
LITERATURE CITED
Adams TS, Sterner RW (2000) The effect of dietary nitrogencontent on trophic level 15N enrichment. Limnol Oceanogr45:601–607
Armitage AR, Fourqurean JW (2009) Stable isotopes revealcomplex changes in trophic relationships following nutri-ent addition in a coastal marine ecosystem. Estuar Coasts32:1152–1164
Bishop MJ, Underwood AJ, Archambault P (2002) Sewageand environmental impacts on rocky shores: necessity ofidentifying relevant spatial scales. Mar Ecol Prog Ser 236:121–128
Brutemark A, Lindehoff E, Granéli E, Granéli W (2009) Car-bon isotope signature variability among cultured micro-algae: influence of species, nutrients and growth. J ExpMar Biol Ecol 372:98–105
Burns A, Ryder DS (2001) Potential for biofilms as biologicalindicators in Australian riverine systems. Ecol ManagRestor 2:53–64
Cabana G, Rasmussen JB (1996) Comparison of aquatic foodchains using nitrogen isotopes. Proc Natl Acad Sci USA93:10844–10847
Camus PA, Daroch K, Opazo LF (2008) Potential for omnivoryand apparent intraguild predation in rocky intertidal her-bivore assemblages from northern Chile. Mar Ecol ProgSer 361:35–45
Camus PA, Cid YY, Cisterna L, Cáceres CW (2009) Consump-tion and digestion of animal food by rocky intertidal herbi-vores: an evaluation of digestive flexibility and omnivoryin three grazing species. Lat Am J Aquat Res 37:191–197
Carballo JL, Naranjo SA, García-Gómez JC (1996) Use ofsponges as stress indicators in marine ecosystems at Alge-ciras Bay (southern Iberian Peninsula). Mar Ecol Prog Ser135:109–122
Carlier A, Riera P, Amouroux JM, Bodiou JY, Desmalades M,Grémare A (2008) Food web structure of two Mediter-ranean lagoons under varying degree of eutrophication.J Sea Res 60:264–275
Clarke KR, Gorley RN (2006) PRIMER v6: user manual/tutor-ial. PRIMER-E, Plymouth
Cloern JE (2001) Our evolving conceptual model of thecoastal eutrophication problem. Mar Ecol Prog Ser 210:223–253
Costanzo SD, O’Donohue MJ, Dennison WC, Loneragan NR,Thomas M (2001) A new approach for detecting and map-ping sewage impacts. Mar Pollut Bull 42:149–156
Crothers JH (1994) Student investigations on the populationstructure of the common topshell, Monodonta lineata, onThe Gore, Somerset. Field Stud 8:337–355
20
Vermeulen et al.: Early indicators of anthropogenically derived nutrients
Della-Santina P, Sonni C, Sartoni G, Chelazzi G (1993) Foodavailability and diet composition of three coexistingMediterranean limpets (Patella spp.). Mar Biol 116:87–95
DeNiro MJ, Epstein S (1978) Influence of diet on the distribu-tion of carbon isotopes in animals. Geochim CosmochimActa 42:495–506
Dytham C (2003) Choosing and using statistics: a biologist’sguide. Blackwell, Berlin
Fry B, Sherr E (1984) δ13C measurements as indicators of car-bon flow in marine and freshwater ecosystems. ContribMar Sci 27:13–47
Gil M, Armitage AR, Fourqurean JW (2006) Nutrient impactson epifaunal density and species composition in a subtrop-ical seagrass bed. Hydrobiologia 569:437–447
Guest MA, Connolly RM, Loneragan NR (2004) Carbonmovement and assimilation by invertebrates in estuarinehabitats at a scale of metres. Mar Ecol Prog Ser 278:27–34
Hawkins SJ, Hartnoll RG (1983) Grazing of intertidal algae bymarine invertebrates. Oceanogr Mar Biol Annu Rev 21:195–282
Hedges JI, Stern JH (1984) Carbon and nitrogen determina-tions of carbonate-containing solids. Limnol Oceanogr 29:657–663
Hill AS, Hawkins S (1991) Seasonal and spatial variation ofepilithic microalgal distribution and abundance and itsingestion by Patella vulgata on a moderately exposedrocky shore. J Mar Biol Assoc UK 71:403–423
Hill WR, Middleton RG (2006) Changes in carbon stable iso-tope ratios during periphyton development. LimnolOceanogr 51:2360–2369
Hutchinson N, Davies MS, Ng JSS, Williams GA (2007) Trailfollowing behaviour in relation to pedal mucus productionin the intertidal gastropod Monodonta labio (Linnaeus).J Exp Mar Biol Ecol 349:313–322
Ifremer (Institut français de recherche pour l’exploitation dela mer) (2007) Synthèse de l’état de la contaminationchimique du golfe de Marseille. Report No. R.INT.DOP/LER-PAC/07-05. Ifremer, Paris
Johnson MP, Hanley ME, Frost NJ, Mosley MWJ, Hawkins SJ(2008) The persistent spatial patchiness of limpet grazing.J Exp Mar Biol Ecol 365:136–141
Krom MD, Herut B, Mantoura RFC (2004) Nutrient budget forthe eastern Mediterranean: implications for phosphoruslimitation. Limnol Oceanogr 49:1582–1592
Kurata K, Minami H, Kikuchi E (2001) Stable isotope analysisof food sources for salt marsh snails. Mar Ecol Prog Ser223:167–177
Lancaster J, Waldron S (2001) Stable isotope values of lotic in-vertebrates: sources of variation, experimental design, andstatistical interpretation. Limnol Oceanogr 46:723–730
Lassauque J, Lepoint G, Thibaut T, Francour P, Meinesz A(2010) Tracing sewage and natural freshwater input in anorthwest Mediterranean bay: evidence obtained fromisotopic ratios in marine organisms. Mar Pollut Bull 60:843–851
Lepoint G, Nyssen F, Gobert S, Dauby P, Bouquegneau JM(2000) Relative impact of a seagrass bed and its adjacentepilithic algal community in consumer diets. Mar Biol136:513–518
Lorrain A, Paulet YM, Chauvaud L, Savoye N, Donval A,Saout C (2002) Differential δ13C and δ15N signaturesamong scallop tissues: implications for ecology and phy-siology. J Exp Mar Biol Ecol 275:47–61
MacLeod NA, Barton DR (1998) Effects of light intensity,water velocity, and species composition on carbon andnitrogen stable isotope ratios in periphyton. Can J FishAquat Sci 55:1919–1925
McClelland JW, Valiela I, Michener RH (1997) Nitrogen-stable isotope signatures in estuarine food webs: a recordof increasing urbanization in coastal watersheds. LimnolOceanogr 42:930–937
McIntyre PB, Flecker AS (2006) Rapid turnover of tissue nitro-gen of primary consumers in tropical freshwaters. Oecolo-gia 148:12–21
McKinney RA, Nelson WG, Charpentier MA, Wigand C(2001) Ribbed mussel nitrogen isotope signatures reflectnitrogen sources in coastal salt marshes. Ecol Appl 11:203–214
McKinney RA, Lake JL, Charpentier MA, Ryba S (2002) Usingmussel isotope ratios to assess anthropogenic nitrogeninputs to freshwater ecosystems. Environ Monit Assess74:167–192
Munda IM (1993) Changes and degradation of seaweedstands in the northern Adriatic. Hydrobiologia 260–261:239–253
Nixon SW (1995) Coastal marine eutrophication: a definition,social causes, and future concerns. Ophelia 41:199–219
Oakes JM, Revill AT, Connolly RM, Blackburn SI (2005) Mea-suring carbon isotope ratios of microphytobenthos usingcompound-specific stable isotope analysis of phytol.Limnol Oceanogr Methods 3:511–519
Oehlmann J, Schulte-Oehlmann U (2003) Molluscs as bioindi-cators. In: Market BA, Breure AM, Zechmeister G (eds)Bioindicators and biomonitors. Elsevier Science Publish-ers, New York, NY, p 577–635
Orton JH, Southward AJ, Dodd JM (1956) Studies on the biol-ogy of limpets, II. the breeding of Patella vulgata L. inBritain. J Mar Biol Assoc UK 35:149–176
Peduzzi P, Herndl GJ (1991) Mucus trails in the rocky inter-tidal: a highly active microenvironment. Mar Ecol Prog Ser75:267–274
Pérès JM, Picard J (1964) Nouveau manuel de bionomie ben-thique de la mer Méditerranée. Recl Trav Stn MarEndoume 31:5–137
Pinedo S, García M, Satta MP, De Torres M, Ballesteros E(2007) Rocky-shore communities as indicators of waterquality: a case study in the northwestern Mediterranean.Mar Pollut Bull 55:126–135
Riera P, Stal LJ, Nieuwenhuize J (2000) Heavy δ15N in inter-tidal benthic algae and invertebrates in the Scheldt estu-ary (the Netherlands): effect of river nitrogen inputs.Estuar Coast Shelf Sci 51:365–372
Segar DA, Phillips DJH, Stamnan E (1987) Strategies for long-term pollution monitoring of the coastal oceans. In: BoyleTP (ed) New approaches to monitoring aquatic ecosys-tems. American Society for Testing and Materials.Philadelphia, PA, p 12_27
Skliris N, Elkalay K, Goffart A, Frangoulis C, Hecq JH (2001)One-dimensional modelling of the plankton ecosystem ofthe north-western Corsican coastal area in relation tometeorological constraints. J Mar Syst 27:337–362
Soltan D, Verlaque M, Boudouresque CF, Francour P (2001)Changes in macroalgal communities in the vicinity of aMediterranean sewage outfall after the setting up of atreatment plant. Mar Pollut Bull 42:59–70
Staal M, Thar R, Kühl M, Van Loosdrecht MCM, Wolf G, DeBrouwer JFC, Rijstenbil JW (2007) Different carbon iso-tope fractionation patterns during the development ofphototrophic freshwater and marine biofilms. Biogeo-sciences 4:613–626
Steneck RS, Watling L (1982) Feeding capabilities and limita-tion of herbivorous molluscs: a functional group approach.Mar Biol 68:299–319
Sturaro N, Caut S, Gobert S, Bouquegneau JM, Lepoint G
21
Mar Ecol Prog Ser 422: 9–22, 2011
(2010) Trophic diversity of idoteids (Crustacea, Isopoda) in-habiting the Posidonia oceanica litter. Mar Biol 157:237–247
Sweeting CJ, Jennings S, Polunin NVC (2005) Variance inisotopic signatures as a descriptor of tissue turnover anddegree of omnivory. Funct Ecol 19:777–784
Tewfik A, Rasmussen JB, McCann KS (2005) Anthropogenicenrichment alters a marine benthic food web. Ecology86:2726–2736
UNEP/FAO/WHO (1996) Assessment of the state of eutrophi-cation in the Mediterranean sea. MAP Technical SeriesNo. 10. United Nations Environment Programme (UNEP),Athens
Vadeboncoeur Y, Jeppesen E, Vander Zanden MJ, SchierupHH, Christoffersen K, Lodge DM (2003) From Greenland togreen lakes: cultural eutrophication and the loss of benthicenergy pathways in lakes. Limnol Oceanogr 48: 1408–1418
Vander Zanden MJ, Rasmussen JB (1999) Primary consumerδ13C and δ15N and the trophic position of aquatic con-sumers. Ecology 80:1395–1404
Velimirov B, Walenta-Simon M (1992) Seasonal changes inspecific growth rates, production and biomass of a bacte-rial community in the water column above a Mediterra-nean seagrass system. Mar Ecol Prog Ser 80:237–248
Vermeulen S (2006) Utilisation des isotopes stables du C et du
N dans l’évaluation du potentiel de bioindicateur d’eu-trophisation de deux gastéropodes herbivores de la zonemédio-littorale en Baie de Calvi (Corse). MSc dissertation,University of Liège
Vizzini S, Mazzola A (2006) The effects of anthropogenicorganic matter inputs on stable carbon and nitrogen iso-topes in organisms from different trophic levels in a south-ern Mediterranean coastal area. Sci Total Environ 368:723–731
Vizzini S, Savona B, Chi TD, Mazzola A (2005) Spatial vari-ability of stable carbon and nitrogen isotope ratios in aMediterranean coastal lagoon. Hydrobiologia 550:73–82
Vuorio K, Meili M, Sarvala J (2006) Taxon-specific variationin the stable isotopic signatures (δ13C and δ15N) of lakephytoplankton. Freshw Biol 51:807–822
Waldron S, Tatner P, Jack I, Arnott C (2001) The impact ofsewage discharge in a marine embayment: a stable iso-tope reconnaissance. Estuar Coast Shelf Sci 52:111–115
Wilmotte A, Demoulin V (1988) Studies of marine epiphyticalgae, Calvi, Corsica. II. Seasonal variations in the popula-tions of epiphytic blue-green algae in three harbours withdifferent pollution loads. Br Phycol J 23:259–266
