Pierre-Yves Pascal
email: pypascal@univ-ag.fr

John W Fleeger

DIVERSE DIETARY RESPONSES BY SALTMARSH CONSUMERS TO CHRONIC NUTRIENT ENRICHMENT

Abstract

We examined the effect of whole-ecosystem nutrient enrichment on herbivory in saltmarsh creek-wall habitats in Plum Island Estuary (Massachusetts-USA). Located between the macrophyte-dominated high marsh and adjoining mudflats, creek walls are steep vertical habitats vegetated with productive filamentous algae and associated epiphytes. Annual nitrate and phosphate loading rates were increased approximately 10-15x in creeks mimicking shortterm (2-month) and chronic (6-year) eutrophication. We assessed the diets of epifaunal invertebrates (3 gastropods and 1 amphipod species) that potentially graze on benthic algae using natural isotope abundance data and per capita grazing rate measurements derived from 13 C pre-labeled algae. Substantial dietary contributions from benthic algae were observed in all consumers even though previous research has indicated most rely on Spartina detritus as the principal food resource. The amphipod Orchestia grillus and the snail Melampus bidentatus grazed benthic algae in excess of 500 µg algal C g C -1 h -1 whereas the snail Nassarius obsoletus and hydrobiid snails grazed at lower rates. Few dietary changes were detected with short-term enrichment. Algal grazing rates of N. obsoletus and M. bidentatus increased with chronic enrichment probably as a functional response to increased algal productivity. O. grillus grazed at a high rate and parasitic infection did not affect its consumption of benthic algae. The abundance and frequency of occurrence of O. grillus on creek-wall habitats increased with chronic nutrient enrichment suggesting amphipods contribute to top-down control on benthic algae and slow algal growth as nutrient enrichment occurs.

Introduction

Estuaries and associated salt marshes provide important ecological and economic services including those that contribute to nutrient regulation and that support fisheries [START_REF] Costanza R, D'arge | The value of the world's ecosystem services and natural capital[END_REF][START_REF] Valiela | Comparative evidence that salt marshes and mangrove may protect seagrass meadows from land-derived nitrogen loads[END_REF]. However, these environments are under increasing anthropogenic pressure, for example, as the amount of nitrogen entering watersheds increases globally [START_REF] Galloway | Transformation of the nitrogen cycle: recent trends, questions and potential solutions[END_REF][START_REF] Gruber | An earth-system perspective of the global nitrogen cycle[END_REF][START_REF] Canfield | The evolution of Earth's nitrogen cycle[END_REF][START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss[END_REF]. Nutrient loading and subsequent cultural eutrophication of the coastal environment has been documented worldwide in coastal environments [START_REF] Diaz | Spreading dead zones and consequences for marine ecosystems[END_REF], becoming major issues for both scientists and managers.

Nutrient enrichment can potentially relieve nitrogen or phosphorus limitations affecting biomass and/or production of primary producers in estuaries [START_REF] Bertness | Anthropogenic modification of New England salt marsh landscapes[END_REF][START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF][START_REF] Darby | Consequences of eutrophication to salt marsh roots, rhizomes, and soils[END_REF]. Fast-growing macroalgae, epiphytes and phytoplankton can outcompete macrophytes in nutrient-enriched estuaries [START_REF] Taylor | Responses of coastal lagoon plant comminuties to different forms of nutrient enrichment-A mesocosm experiment[END_REF][START_REF] Wear | Effects of water-column enrichment on the production dynamics of three seagrass species and their epiphytic algae[END_REF].

Surface-dwelling invertebrates (epifauna) exerting top-down control on algae [START_REF] Duffy | Strong impacts of grazing amphipods on the organization of a benthic community[END_REF] may consequently be affected by resource changes [START_REF] Rosemond | A test of top-down and bottom-up control in a detritus-based food web[END_REF][START_REF] Johnson | High-marsh invertebrates are susceptible to eutrophication[END_REF]. Because services provided by salt marshes depend on these higher trophic levels, it is critical to understand how they will be affected by nutrient addition.

The present study takes advantage of a whole-ecosystem manipulation designed to evaluate the effects of nutrient enrichment on salt marshes in the Plum Island Estuary, Massachusetts, USA (The 'TIDE' project ; [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF]. TIDE mimics the nutrient loadings and the mechanism of nutrient delivery typically associated with anthropogenic eutrophication in marsh creeks. Dissolved N and P have been added to creeks with the flooding tide throughout whole growing seasons since 2004. The large spatial (60,000 m 2 ) and temporal (6 years of seasonal nutrient enrichment when our study was conducted) scales make this experimental perturbation more similar to human-induced eutrophication than the typical application of dry fertilizer to small plots [START_REF] Sardá | Decadal shifts in a salt marsh macroinfaunal community in response to sustained long-term experimental nutrient enrichment[END_REF]. These features should contribute to a better understanding of the rate and mechanisms of change associated with chronic nutrient enrichment.

TIDE experiments previously revealed that nutrient enrichment modifies grazing pressure on benthic algae by i) causing dietary shifts in benthic grazers [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF][START_REF] Galván | The diet of saltmarsh consumers[END_REF], Johnson et al. 2009[START_REF] Lockfield | Chronic nutrient enrichment effects on mummichog, Fundulus heteroclitus in a northeastern Massachusetts salt marsh[END_REF], ii) increasing per capita algal consumption [START_REF] Pascal | Response of the benthic food web to short-and long-term nutrient enrichment in saltmarsh mudflats[END_REF]) and iii) stimulating increases in the density of some grazers [START_REF] Johnson | High-marsh invertebrates are susceptible to eutrophication[END_REF][START_REF] Lockfield | Chronic nutrient enrichment effects on mummichog, Fundulus heteroclitus in a northeastern Massachusetts salt marsh[END_REF][START_REF] Johnson | Chronic nutrient enrichment increase the density and biomass of the mudsnail, Nassarius obsoletus[END_REF]. A detailed understanding of the response of benthic grazers is needed to better understand the process by which nutrient enrichment brings about eutrophication and increased algal biomass in salt marshes.

Creek walls are steep vertical habitats located between the macrophyte-dominated marsh platform and adjoining creek mudflats. Although creek-wall habitats cover a relatively small fraction of the total marsh surface area, this understudied habitat may play a disproportionate role in the structure and function of salt marshes because they harbor productive filamentous algal and associated epiphyte communities and the highest infaunal abundance across the marsh landscape [START_REF] Johnson | Worm holes and their space-time continuum: spatial and temporal variability of macroinfaunal annelids in a northern New England salt marsh[END_REF], Fleeger et al. 2008). Moreover, the largest response to nutrient addition among benthic infauna was observed in creek-wall habitat [START_REF] Fleeger | Top-down and bottom-up control of infauna varies across the saltmarsh landscape[END_REF].

The aim of the present study was to evaluate changes in herbivory induced by nutrient enrichment in the creek-wall habitat. We focused here on resident and transient epifaunal benthic grazers of this habitat, and we used two approaches: variation in natural abundance of isotopes ( 13 C and  15 N) and grazing rate measurements with 13 C pre-labeled algae. Chronic and short-term nutrient enrichment were both studied. Our results i) give a picture of creek-wall habitat herbivory function under natural conditions and ii) facilitate an evaluation of changes induced by nutrient enrichment over time.

Materials and methods

TIDE project

TIDE, stands for "Trophic Cascades and Interacting Control Processes in a Detritus-Based Aquatic Ecosystem". This multi-institutional, multi-disciplinary, multi-year project is a manipulation, at the ecosystem-scale, of the extensive salt marshes of Plum Island Estuary (PIE), Massachusetts, (42°44'N, 70°52'W), USA (Figure 1). PIE is a 25-km long mesotidal estuary with a mean tidal range of 3 m. Spartina alterniflora and S. patens are the dominant macrophytes on the marsh platform in the seawater-dominated intertidal zone where the salinity ranges from 8 to 28‰. Here, we focus on the creek-wall habitat found adjacent to the marsh edge at a lower tidal elevation. It is a steep, almost vertical wall about 1.5 m in height constituted by cohesive sediments (Figure 1).

Creek wall is a distinct habitat with a ~30-cm wide band of dense macroalgae (Enteromorpha spp.), filamentous algae (Rhizoclonium spp.) and associated epiphytic diatoms that form a distinct and physically complex algal turf extending ~1 cm above the sediment surface.

Mudflats of poorly consolidated sediments delimit the lower border of this habitat. ). Along the Rowley River, nutrient enrichment experiments were conducted in three intertidal marsh creeks (West, Clubhead, Sweeney) having similar physical dimensions, water chemistries, plant and infaunal communities (details in [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF], Johnson et al. 2007). Sweeney (hereafter referred to as the chronically fertilized creek or CFC) was fertilized for 6 field seasons (2004)(2005)(2006)(2007)(2008)(2009), Clubhead (hereafter referred to as the short-term fertilized creek or SFC) was fertilized for about 2 months (in 2009) and West (hereafter referred to as the reference creek or RC) was unfertilized (Figure 1). In CFC, for approximately 150 d during the growing season, nitrate and phosphate levels were increased to approximately 70 µmol L -1 NO 3 -and 4 µmol L -1 PO 4 3on each incoming tide. These concentrations were chosen as they designated an estuary to be "moderately to severely eutrophied" (US EPA, 2002). In order to maintain a relatively constant enrichment through each tidal cycle, a fertilizer solution was pumped into CFC near its mouth at a flow rate based on a hydrologic model of volume of flooding water. For SFC, fertilization methods were identical to CFC but began about 2 months before our experiments were conducted. Detailed background information of experimental design, biogeochemistry of the tidal creeks, and the effects of fertilization have been previously described [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF][START_REF] Drake | Salt marsh ecosystem biogeochemical responses to nutrient enrichment: a paired 15 N tracer study[END_REF][START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss[END_REF].

Algal biomass

To assess the effect of nutrient enrichment on surface algae on the creek-wall habitat, ten 2.2-cm inner diameter creek-wall sediment cores from each creek were sampled at the time of grazing experiments and frozen. Benthic algae sampled in each core included filamentous algae and its epiphytes. Algal biomass in the surficial centimeter of sediment of each creek was assessed using chlorophyll a as a proxy and measured after extraction with acetone using a Turner fluorometer (APHA 1992). Carbon algal biomass was estimated using a carbon:chlorophyll ratio of 40 [START_REF] Jonge | Fluctuations in the organic carbon to chlorophyll-a ratios in estuarine benthic diatom populations[END_REF].

Grazer collections

Gastropods and amphipods were collected by hand in each of the 3 creeks in areas with similar tidal elevations ~100 m upstream from the location where fertilizer was added to CFC and SFC and in areas physically similar in RC. Sampling was conducted during the first week of July 2009 for natural isotope analysis and for grazing experiments. Most invertebrates were collected directly on creek walls although some were collected less than 1 m from creek walls in adjoining habitats.

Stable isotope composition

Gastropod taxa selected for natural abundance stable isotope analysis were hydrobiid snails (mean shell height  sd = 2.6 mm  0.6), Melampus bidendatus (mean shell height  sd = 10.9 mm  1.2) and Nassarius obsoletus (mean shell height  sd = 17.7 mm  2.3). The tissue of five specimens of each was manually removed from their shells and pooled together.

In addition, five brown color morphs (mean length  sd = 13.2 mm  3.5) and five orange specimens (mean length  sd = 14.0 mm  2.2) of the amphipod Orchestia grillus were pooled after being sorted by color. Samples were homogenized using a tissue grinder, freeze dried and a fraction of each sample was used for stable isotope analyses. Samples were analyzed at the Marine Biological Laboratory's Stable Isotope Laboratory using a continuous flow isotope ratio mass spectrometer. Samples are reported relative to the standards, atmospheric N 2 and Vienna PeeDee Belemnite (VPDB) carbon. Stable isotope values are reported in  notation:

 13 C or  15 N = [ (R sample /R standard ) -1] X 10 3
where R is respectively 13 C/ 12 C or 15 N/ 14 N.

Grazing experiments

In each of the 3 creeks, 10 plots of 314 cm 2 were haphazardly located 1 m apart from each other and marked on creek-wall habitat. NaH 13 CO 3 (> 99% 13 C-enriched) was dissolved in seawater and this solution was applied daily over 6 days during the daytime low tide with a sprayer directly onto the sediment surface. Each plot was sprayed at a concentration of 1 g m -2 [START_REF] Middelburg | The fate of intertidal microphytobenthos carbon. An in situ 13 C labelling study[END_REF]. After 6 days, sprayed sediment was gently cored (internal diameter = 20 cm) and removed from creek-wall habitat at low tide without disturbing its structure.

Each cohesive disk of sediment with approximate thickness of 5 cm was placed in an independent mesocosm (inner diameter = 20 cm; 314 cm 2 ). These cores were collected less than 5 h before the start of grazing experiments.

Five specimens of each consumer of the previously described sizes were placed in each mesocosm. Invertebrates were collected less than 24 h before grazing experiments and provided with food (sediment and filamentous algae from creek-wall habitat) in order to limit starvation. From each creek, killed controls (n = 3) were performed in which all specimens were first frozen (-20°C) for 24 h. All incubations lasted 5 h and were conducted at the same time, in similar conditions of light and temperature close to natural ones. Sediments were not water covered at any time during the experimental period. Incubations were stopped by freezing whole mesocosms (-20°C). Samples were thawed and all isotope samples were prepared and analyzed as previously described.

In each mesocosm, filamentous algae (approximately 1 g wet weight) and their epiphytes were collected at the end of the experiment, cleaned of foreign debris, pooled, homogenized using a tissue grinder, and freeze dried. A fraction of each sample was prepared for stable isotope analyses.

Carbon and nitrogen content of each taxon were obtained along with isotopic ratios during isotopic analysis. Incorporation of 13 C was defined as excess above background 13 C (from control mesocosms) and was expressed in term of specific uptake (I). I was calculated as the product of excess 13 C (E) and biomass of C per grazer. E was the difference between the background (F background ) and the sample (F sample ) 13 C fraction: E=F sample -F backgroun d, with F = 13 C / ( 13 C + 12 C) = R / (R + 1) and Uptake = I / (F enriched algae X incubation time). F enriched algae was determined using  13 C of algae from each microcosm.

Data analyses

One-way analysis of variance (ANOVA) and Tukey a posteriori tests, were used to assess differences in individual grazing rates on benthic algae among creeks. Kruskall-Wallis tests were used to compare natural isotopic composition in consumers from different creeks.

A Bayesian isotopic mixing model was used to determine possible contribution of primary producers (epiphytes, filamentous algae, S. alterniflora (Table 1)) to the diet of epifauna from RC. SIAR (Stable Isotope Analysis in R; [START_REF] Parnell | Source partitioning using stable isotopes: coping with too much variation[END_REF])) incorporates the variability of consumers and trophic enrichment factor (TEFs) to produce a mean and a 95% confidence interval of the percent contribution of each source to a consumer. A TEF of 0.5% was used for  13 C [START_REF] Fry | Stable isotope ecology[END_REF]) and 3.4% for  15 N for all consumers [START_REF] Minagawa | Stepwise enrichment of 15 N along food chain: further evidence and the δ 15 N and animal age[END_REF].

Results

Algal biomass and primary producer isotope composition

Benthic algal biomasses were not significantly different among creeks (ANOVA, p = 0.128) at the time of the grazing experiment, and means were respectively 6.17 ± 1.05 in RC, 4.47 ± 1.16 in SFC and 5.96 ± 2.03 gC m 2 in CFC (n = 10, ± sd). Natural abundance isotope composition of creek-wall habitat primary producers from RC (Table1) previously sampled at the same time of the year [START_REF] Galván | The diet of saltmarsh consumers[END_REF]) was used in dietary comparisons (Figure 2) and as source values in the SIAR mixing model. Primary producer isotope composition data from [START_REF] Galván | The diet of saltmarsh consumers[END_REF] from CFC after two years of nutrient enrichment were also used to help interpret consumer diet.

Natural isotopic composition

The SIAR model revealed large ranges in the possible dietary contributions of Spartina detritus, filamentous algae and epiphytes in the reference creek (Table 2). The highest basal contribution came from filamentous or epiphytic algae in all consumers. Further, the natural isotope composition of carbon (Kruskall-Wallis test, p < 0.005) and nitrogen (Kruskall-Wallis test, p < 0.05) significantly differed among epifaunal species in the reference creek. N.

obsoletus was distinct from other epifauna with a higher  15 N of 8.4‰ (Figure 2), and the SIAR mixing model revealed a higher possible mean contribution of S. alterniflora in the diet of N. obsoletus than other epifauna (Table 2). All other epifauna presented a similar 13 C composition (Kruskall-Wallis test, p = 0.68), indicative of a diet based principally on epiphytes and filamentous algae.

Some differences in isotopic composition were noted in consumers between the nutrient enriched and reference creeks suggesting dietary changes occurred with nutrient enrichment.

O. grillus (both orange and brown color morphs) presented significantly higher  13 C values in CFC compared to RC (Kruskall-Wallis test, p < 0.05). M. bidentatus presented a significantly higher  13 C in SFC compared to RF or CFC (Kruskall-Wallis test, p < 0.05) (Figure 3).

All epifauna from the reference creek were less depleted in  15 N than epifauna from creeks that were nutrient enriched either recently (2 months) or chronically (six years) (Figure 3), and this difference was significant for hydrobiid snails and M. bidentatus (Kruskall-Wallis test, p < 0.05). This difference is probably not related to change in diet but instead to 15 N changes induced by the fertilizer used to enrich creek water. The most common method of fertilizer production is industrial fixation of atmospheric nitrogen, resulting in products with

 15 N values close to zero.

Grazing rates

Benthic algae became substantially enriched in 13 C in the sprayed plots.

Discussion

Our natural isotopic composition studies were conducted concurrently with grazing rate measurements, and both suggest that chronic nutrient enrichment increased the contribution of benthic algae in the diet of some benthic consumers. Below, we discuss methodological considerations, trophic relationships in the reference creek, and the time course of changes induced by nutrient enrichment.

Methodological considerations

Ecosystem-scale experiments are useful in that they incorporate a broad range of phenomena [START_REF] Carpenter | Replication and treatment strenght in whole-lake experiments[END_REF]). However, replication is challenging because similar ecosystems may be rare [START_REF] Carpenter | Ecosystem experiments[END_REF], and the maintenance of large-scale treatments is expensive and labor intensive. Our whole-creek nutrient enrichment technique facilitated quantification of trophic dynamics over large spatial (60 000 m 2 creek per treatment) and long temporal (6 years) scales, and studied creeks were similar physically and biologically before nutrient addition began, limiting bias due to inter-creek variability [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF], Johnson et al. 2007). Treatments (creeks with differing histories of nutrient enrichment) were not replicated in our study and statistical comparisons utilized pseudo-replicates. Previous research in PIE suggests, however, that geochemical, faunal and floral responses to nutrient enrichment are very similar in replicate creeks [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF], Johnson et al. 2007[START_REF] Drake | Salt marsh ecosystem biogeochemical responses to nutrient enrichment: a paired 15 N tracer study[END_REF]).

Using similar methods, [START_REF] Galván | Natural abundance stable isotopes and dual isotope tracer additions help to resolve resources supporting saltmarsh food web[END_REF] found that epiphytes and filamentous algae became equally enriched in 13 C in the PIE creek-wall habitat, and we consequently made the assumption that enrichment was equal in our study. Differential enrichment between filamentous algae and their epiphytes could lead to incorrect estimation of grazing rates if epifauna feed selectively.

We used the isotopic composition of primary producers previously sampled in RF and CFC at the same time of the year by Galván (2008, Table 1) to infer the diet of consumers.

The level of variation detected by Galván was relatively modest; however, this variation adds additional uncertainty regarding our dietary estimates of consumers. We also found significant variation in the isotope composition of the amphipod O. grillus between RC and CFC (amphipods were less depleted in 13 C in the creek with nutrient enrichment, and differences between creeks  13 C were 2.1 ‰ for brown O. grillus and 2.0 ‰ for orange O.

grillus). This variation exceeded that found in each potential basal resource [START_REF] Galván | The diet of saltmarsh consumers[END_REF]).

The maximum  13 C expressed by epiphytes resulted in a difference of 1.4 ‰ for  13 C between creeks and 1.5 ‰ among years (Table 1), and the variation associated with Spartina and filamentous algae was even less. Furthermore, [START_REF] Galván | The diet of saltmarsh consumers[END_REF] did not find a significant effect of nutrient enrichment on the  13 C of primary producers in PIE after two years of enrichment (Table 1). Relatively few studies have examined the effect of nutrient enrichment on  13 C of primary producers although [START_REF] Vermeulen | Potential early indicators of anthropogenically derived nutrients: a mutiscale stable isotope analysis[END_REF] found that the  13 C of macroalgae was not affected by nutrient enrichment. We infer from these observations that the changes we detected in the isotope composition of O. grillus in CFC were due to changes in diet induced by nutrient enrichment rather than a change in isotope composition of primary producers.

Grazing rates on benthic algae were measured over a five-hour period. Because feeding behavior can potentially vary diurnally and daily, our grazing rates may not be representative of longer time periods. All grazing experiments were performed simultaneously with epifauna collected in the field at the same time in order to limit bias in feeding behavior. Any bias associated with short-term measures should be similar in all creeks, suggesting comparisons among creeks are informative of nutrient-enrichment effects.

Epifaunal diet in the reference creek SIAR indicated that benthic algae were the principal basal resource for all consumers studied and the estimated combined contributions of filamentous algae and epiphytes ranged from 76-95%. N. obsoletus fed at a higher trophic level as evidenced by a higher  15 N value [START_REF] Deniro | Influence of the diet on the distribution of nitrogen isotopes in animals[END_REF]. N. obsoletus is an omnivore and scavenger requiring a high protein diet for reproduction [START_REF] Hurd | On the importance of carrion to reproduction in an omnivorous estuarine neogasteropode, Ilyanassa obsoleta (Say)[END_REF]. The observed higher  15 N value of this snail is likely due to a mixed diet of animal and plant resources [START_REF] Curtis | On the broad nutritional requirements of the mudsnail, Ilyanassa (Nassarius) obsoleta (Say) and its polytrophic role in the food web[END_REF]. Per capita grazing rates in N. obsoletus expressed as g algal C g C -1 h -1 were more than 10 times lower than in M.

bidentatus and O. grillus and 1.8 times lower than hydrobiid snails in the same habitat.

Experiments run in the adjacent mudflat environment in PIE lead to a similar range of algal ingestion rates for N. obsoletus: 0.7-2.7 µg ind -1 h -1 in creek-wall habitat versus 1.1 -2.3 µg ind -1 h -1 in mudflat [START_REF] Pascal | Response of the benthic food web to short-and long-term nutrient enrichment in saltmarsh mudflats[END_REF]. Although the principal habitat of N. obsoletus is saltmarsh mudflats, some individuals migrate with the rising tide onto the creek-wall habitat (Johnson, personal communication). However, the low grazing rates, limited foraging time on the creek-wall habitat and the low density suggest N. obsoletus exerts little grazing pressure on benthic algae on creek walls. [START_REF] Pascal | Response of the benthic food web to short-and long-term nutrient enrichment in saltmarsh mudflats[END_REF] found that hydrobiids were responsible for ~50% of the total grazing pressure exerted by benthic consumers on mudflats adjacent to the creek-wall habitat in PIE.

However, per capita grazing rates were much lower (by 28X) on creek walls, presumably due to differences in algal species composition between each habitat. Hydrobiid snails are highly transitory in salt marshes and do not appear to aggregate on creek-wall habitat in reference or nutrient-enriched creeks. The potential for hydrobiid snails to graze significant amounts of benthic algae on creek walls therefore also appears to be low.

M. bidentatus is an herbivore/detritivore that uses a radula to scrape food. Although ingestion of Spartina detritus occurs, this species prefers fungi growing on plant material [START_REF] Lee | Competitive displacement of a detritivorous salt marsh snail[END_REF]. In addition, benthic algae (i.e., diatoms) occasionally dominate its diet [START_REF] Thompson | Comparison of the diets of the tidal marsh snail, Melampus bidentatus and the amphipod, Orchestia grillus[END_REF]. The high per capita algal grazing rates measured here show that M.

bidentatus can efficiently feed on benthic algae. Natural abundance isotope data on specimens collected from the high marsh platform also indicate that benthic algae provide an important basal resource [START_REF] Galván | The diet of saltmarsh consumers[END_REF]. M. bidentatus is prone to large fluctuations in density in PIE and densities were very high in 2009 in all creeks (Fleeger unpublished). At least during years of high density, individuals are found on the creek-wall habitat in PIE but in much lower abundance than the high marsh platform.

O. grillus is commonly considered to be a leaf-shredding detritivore [START_REF] Lopez | The effect of grazing by the detritivore Orchestia grillus on Spartina litter and its associated microbial community[END_REF].

In PIE, stable isotope and gut content analyses revealed that the diet of this species was dominated by filamentous algae and epiphytes, at least in our summer studies when benthic algae are most productive in PIE (This study; [START_REF] Galván | The diet of saltmarsh consumers[END_REF]. The high grazing rates and results of the SIAR mixing model indicate that O. grillus consumes mostly filamentous algae and epiphytes, although Spartina detritus may contribute up to 25% of the basal resource (Table 2).

Hydrobiid snails are the initial host of the microphallid trematode L. byrdii, and talitrid amphipods are the intermediate host. Infected amphipods develop a bright orange color, lose their strong negative phototaxis [START_REF] Bousfield | Systematics distributional ecology and some host-parasite relationships of Uhlorchestia uhleri (Shoemaker) and U spartinophila new species (Crustacea: Amphipoda) endemic to salt marshes of the Atlantic Coast of North America[END_REF], and migrate from vegetated habitats to exposed creek-walls habitats making them much more frequent and abundant on nutrient-enriched creek walls. Theses traits also increase amphipod susceptibility to predation by shorebirds, the parasite's definitive host (Johnson et al. 2009). Our experiments showed that infected orange O. grillus color morphs fed at rates statistically indistinguishable from brown O. grillus. Because it takes up to 30 days for behavioral and morphological changes to manifest after infection (D. S. Johnson pers. com.), some brown amphipods may have also been parasitized without exhibiting symptoms of infection. Feeding rate in parasitized amphipods has been studied in several species and has been shown to be higher [START_REF] Crompton | An ecological approach to acanthocephalan physiology[END_REF], unchanged [START_REF] Fielding | Effects of the acanthocephlan parasite Echinorhynchus truttae on the feeding ecology of Gammarus pulex (Crustacea: Amphipoda)[END_REF]) and lower (McCahon et al. 1988[START_REF] Pascoe | Gammarus pulex (L.) feeding bioassay -effects of parasitism[END_REF] compared to non-parasitized individuals, indicating that the effects of parasites are complex and/or variable among host species.

Effect of nutrient enrichment

Previous research in salt marshes [START_REF] Rosemond | A test of top-down and bottom-up control in a detritus-based food web[END_REF][START_REF] Keats | The effect of nitrogen loading on a brackish estuarine faunal community: a stable isotope approach[END_REF][START_REF] Fox | Restructuring of benthic communities in eutrophic estuaries: lower abundance of preys leads to trophic shifts from omnivory to grazing[END_REF][START_REF] Johnson | High-marsh invertebrates are susceptible to eutrophication[END_REF]) and seagrass beds (Jaschinski and Sommer 2010) suggests that nutrient enrichment increases dietary contributions from epiphytic or benthic algae to benthic consumers. We examined variation in the diet of consumers based on i) isotope composition (as a long-term dietary indicator) and ii) grazing rate measurements (as a short-term indicator). The natural isotopic composition of gastropods did not vary significantly with nutrient enrichment. However significantly higher per capita grazing rates were observed with chronic enrichment in M. bidentatus and N. obsoletus. Higher algal grazing rates with nutrient enrichment are usually thought to be associated with a functional response to increasing benthic algal production (Jaschinski and Sommer 2010). In addition to higher per capita consumption rates, both species were significantly increased in abundance with chronic enrichment in PIE [START_REF] Johnson | High-marsh invertebrates are susceptible to eutrophication[END_REF][START_REF] Johnson | Chronic nutrient enrichment increase the density and biomass of the mudsnail, Nassarius obsoletus[END_REF], suggesting a greater total consumption of benthic algae with nutrient enrichment by these species.

The diet of the amphipod O. grillus was affected by nutrient enrichment in a contrasting fashion. Per capita grazing rates on benthic algae were high (550 µg algal g C -1 h -1 ) and not significantly different between reference and nutrient-enriched creeks (Figure 4). However higher  13 C values of O. grillus were observed in fertilized creeks suggesting an increase in the use of Spartina detritus as a basal resource. Increased dietary diversity was previously observed in seagrass [START_REF] Armitage | Stable isotope reveal complex changes in trophic relationships following nutrient addition in a coastal marine ecosystem[END_REF] and estuarine [START_REF] Keats | The effect of nitrogen loading on a brackish estuarine faunal community: a stable isotope approach[END_REF]) food webs in response to changes in the availability of resources associated with fertilization. In PIE, chronic nutrient addition also enhanced macrophyte root and leaf decomposition rates [START_REF] Deegan | Coastal eutrophication as a driver of saltmarsh loss[END_REF]) potentially making Spartina detritus and/or associated microbes more attractive or accessible to O. grillus. However, the high grazing rates of O. grillus on benthic algae and its increased abundance and utilization of the creek-wall habitat with nutrient enrichment (due to increased frequency of parasitic infections) suggest this species has the potential to impact benthic algal standing crop in areas undergoing chronic enrichment.

Per capita grazing rates of two gastropods were higher in CFC than in SFC suggesting that dietary changes were slow to develop. A similar trend was observed in local mudflat grazers of PIE [START_REF] Pascal | Response of the benthic food web to short-and long-term nutrient enrichment in saltmarsh mudflats[END_REF]) suggesting that eutrophication generally may have cumulative effects that are not apparent within a year for algal grazers. Similarly, food webchanges in a tundra river fertilization experiment were different between first and following years of fertilization [START_REF] Peterson | Biological response of a tundra river to fertilization[END_REF]). Thus, some effects of nutrient enrichment on food webs may take years to appear.

The intensity and timing of benthic algal responses to long-term nutrient enrichment has been found to differ among subhabitats types within PIE (Mitwally & Fleeger In press).

Benthic algal biomass in tall form S. alterniflora (at the marsh-creek edge) increased by the 4th year of fertilization, and responses in mudflat and creek-wall algal communities were smaller, slower and variable over time (Mitwally & Fleeger In press). Benthic algal biomass did not differ between the reference and nutrient enriched creeks at the start of our grazing experiments. One explanation for the slow pace of change in benthic algal biomass, even if algal production is increased by nutrient enrichment, is that increased grazing exerts top-down control slowing increases in algal standing crop with nutrient enrichment [START_REF] Hillebrand | Marine microbenthic community structure regulated by nitrogen loading and grazing pressure[END_REF][START_REF] Hillebrand | Control of microbenthic communities by grazing and nutrient supply[END_REF]. Increased algal biomass is often used as an indicator of eutrophication [START_REF] Juanes | Macroalgae, a suitable indicator of the ecological status of coastal rocky communities in the NE Atlantic[END_REF][START_REF] Giordani | Simple tools for assessing water quality and trophic status in transitional water ecosystems[END_REF][START_REF] Ferreira | Overview of eutrophication indicators to assess environmental status within the European Marine Framework Strategy Framework Directive[END_REF]. Top-down control of grazers can potentially mask bottom-up effects of nutrient enrichment in marsh creeks [START_REF] Posey | Top down vs. bottom up control of benthic community composition on an intertdal tideflat[END_REF] and mudflats [START_REF] Pascal | Response of the benthic food web to short-and long-term nutrient enrichment in saltmarsh mudflats[END_REF]. Grazer control could similarly mask the increase of algal biomass in the creek-wall habitat.

The TIDE project has a unique potential to gauge responses to nutrient enrichment at large spatial scales because nutrient-enriched creek water spreads across the landscape with the rising tide. Benthic grazers may control the rate of change in nutrient-enriched marshes because they can limit or slow the rate of increase of algal biomass. Several taxa (N.

obsoletus, O. grillus, M. bidentatus, Hydrobia spp., nematodes, ostracods and mummichogs)

have been shown to increase in abundance and/or to increase per capita grazing rates on benthic algae with nutrient enrichment, and TIDE research to-date suggests that the species most responsible for increased grazing pressure with nutrient enrichment vary across the landscape. In the creek-wall habitat, O. grillus is the largest and most active epifaunal taxon at low tide, and utilization of the creek-wall habitat increases with nutrient enrichment (Johnson & Fleeger 2009). This increased abundance coupled with relatively high per capita algal grazing rates indicate that O. grillus contributes most among benthic species to grazing pressure on filamentous and epiphytic algae of nutrient enriched creek walls. In the mudflat habitat, large increases in the population density of N. obsoletus and increases in per capita grazing rates by epifaunal and meiofaunal grazers with chronic nutrient enrichment lead to an increase in grazing pressure of 235% on the epipelic algal community [START_REF] Pascal | Response of the benthic food web to short-and long-term nutrient enrichment in saltmarsh mudflats[END_REF].

Fundulus heteroclitus (the mummichog) also increased in abundance in nutrient enriched creeks and a surrogate for algal grazing rate (gut content photosynthetic pigment concentration) increased by 115% with nutrient enrichment [START_REF] Lockfield | Chronic nutrient enrichment effects on mummichog, Fundulus heteroclitus in a northeastern Massachusetts salt marsh[END_REF]). As a result, this abundant nektonic species likely contributed to increased grazing pressure on filamentous algae in creek-wall habitat, marsh edge and high marsh habitats, and manipulative reductions in abundance led to increases in algal standing crop in PIE [START_REF] Deegan | Susceptiblity of salt-marshes to nutrient enrichment and predator removal[END_REF]). On the marsh platform, the abundance of M. bidentatus approximately doubled with chronic nutrient enrichment [START_REF] Johnson | High-marsh invertebrates are susceptible to eutrophication[END_REF]. Although usually not considered a grazer of benthic algae, we found high grazing rates and a large increase in per capita grazing rates, suggesting this species increases grazing pressure on benthic algae in areas experiencing chronic nutrient enrichment. Our research suggests that many species have the potential to contribute to increased grazing pressure with nutrient enrichment in salt marshes. 2. Mean and 95% confidence intervals of the % contributions of epiphyte, filamentous algae, and S. alterniflora to the diets of various taxa from RC based on a Bayesian mixing model (SIAR) for isotopes of carbon and nitrogen. (18,5" (18" (17,5" (17" (16,5" (16" (15,5" (15" (14,5" (14" (13,5" (13" Ilyanassa"

Tables

Figures

Hydrobia"

Orches<a"B"

Orches<a"O" Melampus"

Epiphy<c"algae"

Filamentous"algae"

Sapr<na"alterniflora"

424Figure 1 .FigureFigure 2 .

 12 Figure 1. Map of the Rowley River region salt marshes of the Plum Island Estuary, MA.

Figure 3 .

 3 Figure 3. Natural isotopic composition of nitrogen ( 15 N(‰)) plotted against carbon ( 13 C(‰)) of snails (N. obsoletus, M. bidentatus, hydrobiid

Figure 4 .

 4 Figure 4. Individual grazing rates (algal carbon, per individual grazer, per hour) of snails (N. obsoletus, M. bidentatus, hydrobiid snail) and

Table 1 .

 1 Mean isotopic composition of carbon and nitrogen (‰) of each primary producer previously measured in RC in 2004 and in RC and CFC in 2005 the second year of nutrient enrichment)[START_REF] Galván | Stable isotope addition reveals dietary importance of phytoplankton and microphytobenthos to saltmarsh infauna[END_REF]). In the present study, values from RC in 2004 were used for all SIAR modeling, as inter-annual variations of sources isotopic compositions were smaller than inter-grazers variations in 2013.

	Creek	Year	Epiphytes	Filamentous algae	S. alterniflora
			 13 C	 15 N	 13 C	 15 N	 13 C	 15 N
	RC	2004	-20.4	5.8	-19.1	5.5	-13.8	6.7
	RC	2005	-18.9	5.8	-19.8	6.1	-13.5	5.9
	CFC	2005	-20.3	2.9	-19.8	2.0	-13.7	6.3

Acknowledgments

Pierre-Yves Pascal conducted this research while being supported by a postdoctoral fellowship funded by Department of Energy Office of Biological and Environmental Research Award DE-FG02-05ER64070 and the Louisiana State University College of Science.

We thank the Marshview field crew for maintaining nutrient enrichment experiment and for valuable help in field collecting invertebrates. We thank Marshall Otter for stable isotopic analyses. The National Science Foundation funded this work under grant Nos. 9726921, 0213767, 0816963, 1058747, and 0423565.

Key Words

Nutrient enrichment, food web, epiphytes, filamentous algae, estuary, creek-wall habitat


Pierre-Yves Pascal
email: ppascal@lsu.edu

John W Fleeger

Fernando Galvez

Kevin R Carman

THE TOXICOLOGICAL INTERACTION BETWEEN OCEAN ACIDITY AND METALS IN COASTAL MEIOBENTHIC COPEPODS

Keywords: Ocean acidification, metal, cadmium, copper, harpacticoid copepod

Increased atmospheric CO 2 concentrations are causing greater dissolution of CO 2 into seawater, and are ultimately responsible for today's ongoing ocean acidification. We manipulated seawater acidity by addition of HCl and by increasing CO 2 concentration and observed that two coastal harpacticoid copepods, Amphiascoides atopus and Schizopera knabeni were both more sensitive to increased acidity when generated by CO 2 . The present study indicates that copepods living in environments more prone to hypercapnia, such as mudflats where S. knabeni lives, may be less sensitive to future acidification. Ocean acidification is also expected to alter the toxicity of waterborne metals by influencing their speciation in seawater. CO 2 enrichment did not affect the free-ion concentration of Cd but did increase the free-ion concentration of Cu. Antagonistic toxicities were observed between CO 2 with Cd, Cu and Cu free-ion in A. atopus. This interaction could be due to a competition for H + and metals for binding sites.

Introduction

Over the last century, the atmospheric concentration of CO 2 has risen at a rate 100 times faster than any change observed during the past 650,000 years [START_REF] Siegenthaler | Stable carbon cycleclimate relationship during the last Pleistocene[END_REF].

There is broad consensus that this ongoing change is a direct result of human activity, principally by fossil fuel burning, cement production and changing land use [START_REF] Hansen | Dangerous human-made interference with climate: a GISS modelE study[END_REF]. Atmospheric levels of CO 2 have consequently increased from pre-industrial levels of 280 ppm to a concentration of approximately 380 ppm [START_REF] Feely | Impact of anthropogenic CO 2 on the CaCO 3 system in the oceans[END_REF]. Almost 50% of all anthropogenic CO 2 emitted to the atmosphere has diffused passively into the ocean, significantly decreasing the rate of global warming [START_REF] Sabine | The oceanic sink for anthropogenic CO 2[END_REF]. Concentrations of atmospheric CO 2 are rising at a rate of 3.3% per year and will likely continue to rise [START_REF] Canadell | Contributions to accelaerating atmospheric CO 2 growth from economic activity, carbon intensity, and efficiency of natural sinks[END_REF].

Political, social and environmental pressures to reduce atmospheric CO 2 have led governments to seek new options for CO 2 mitigation. A potential approach is the injection of CO 2 in underground porous reservoir rocks. Sub-seabed sequestration of CO 2 is considered to be a practical tool [START_REF] Gibbins | Scope for future CO 2 emission reductions from electricity generation through the deployment of capture and storage technologies[END_REF], and is already in use in the Norwegian sector of the North Sea [START_REF] Holloway | Underground sequestration of carbon dioxide-a viable greenhouse gas mitigation option[END_REF]. Subsurface leakage is possible over time and has the potential to considerably increase local CO 2 concentration [START_REF] Hawkins | No exit: thinking about leakage from geologic carbon storages sites[END_REF].

The net effect of CO 2 enrichment to seawater is to increase the concentration of carbonic acid (H 2 CO 3 ), bicarbonate ion (HCO 3 -), and hydrogen ion (H + ), and to decrease the concentration of carbonate (CO 3

2-

). The production of H + lowers the pH and causes the phenomenon called "ocean acidification". Carbonate ions can react with excess H + to form HCO 3 -, and it was thought that the carbonate buffering system would allow oceans to retain a stable pH despite rising emissions. However, as the partial pressure of CO 2 increases, the buffering capacity of seawater decreases and the ocean has been reduced by 0.1 pH unit since start of the industrial revolution, representing a 30% increase in the concentration of H + ions [START_REF] Caldeira | Anthropogenic carbon and ocean pH[END_REF][START_REF] Key | A global ocean carbon climatology: result from GLOPAD[END_REF]. Recently, an increase in the area exposed to corrosive seawaters during seasonal upwelling was observed along the North American Pacific continental shelf [START_REF] Feely | Evidence for upwelling of corrosive "acidified" water onto the continental shelf[END_REF]. Hydrological models predict that, based on proposed future emissions of CO 2 , the average oceanic pH will decline by 0.3-0.5 units by the year 2100 and by 0.7 units within the next 300 years [START_REF] Caldeira | Anthropogenic carbon and ocean pH[END_REF]. Leakage from CO 2 seabed storage would create locally faster and stronger acidification than that induced by atmospheric CO 2 [START_REF] Hawkins | No exit: thinking about leakage from geologic carbon storages sites[END_REF].

Reduced availability of carbonate ions, induced by increased CO 2 concentrations, decreases calcification rates of organisms producing CaCO 3 shells or skeletons [START_REF] Gattuso | Effect of calcium carbonate saturation of seawater on coral cacification[END_REF][START_REF] Kleypas | Impact of ocean acidification on coral reefs and other marine calcifiers: a guide for future research[END_REF][START_REF] Riebesell | Reduced calcification of marine plankton in response to increase atmospheric CO 2[END_REF]. In addition to calcification, a number of other physiological processes, including growth, development, metabolism, ionoregulation and acid-base balance, can be affected directly by increases in CO 2 (hypercapnia) or resulting acidosis [START_REF] Fabry | Impacts of ocean acidification on marine fauna and ecosystem processes[END_REF][START_REF] Pörtner | Ecosystems effects of ocean acidification in times of ocean warming: a physiologist's view[END_REF][START_REF] Pörtner | Biological impact of elevated ocean CO 2 concentrations: lessons from animal physiology and earth history[END_REF][START_REF] Widdicombe | Predicting the impact of ocean acidification on benthic biodiversity: what can animal physiology tell us?[END_REF]).

It appears inevitable that marine biota will be faced with ocean acidification for decades or centuries to come. Further, effects of acidification may exacerbate the potential effects of other anthropogenic stressors such as heavy-metal enrichment, especially in coastal sediments. Specifically, ocean acidification will change the organic and inorganic speciation of metals and will modify interactions of metals with marine organisms [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF].

In the general context of ocean acidification, as well as in the context of CO 2 leakage from seabed storage, data are urgently needed to quantify the potential effects of CO 2 on organisms from shallow coastal areas [START_REF] Proyle | Seacarb, an R package to calculate parameters of the seawater carbonate system[END_REF]. Meiofauna are the most abundant metazoans living in soft sediment, and this environment constitutes the majority of seabed habitats. The first aim of the present study was to compare toxic impacts of acidity induced by HCl and CO 2 on two species of harpacticoid copepods: Schizopera knabeni and Amphiascoides atopus in aqueous exposures. A wide range of acidity conditions was tested (including conditions that caused 100% mortality) to examine the entire H + concentrationresponse curve. A second aim of the present study was to evaluate the interactive toxicity of CO 2 with cadmium (Cd) and copper (Cu) for the copepod A. atopus. Those heavy metals are produced by human activity and both are widespread in the marine environment. Cd and Cu were chosen for study because they have different behaviors in CO 2 acidified water, which may create differences in toxicity.

Material and method

Test organism and laboratory culture

A culture of Schizopera knabeni was initiated in 1993 using animals from a salt marsh near Port Fourchon, Louisiana [START_REF] Lotufo | Toxicity of sediment-associated PAHs to an estuarine copepod: effect on survival, feeding, reproduction and behavior[END_REF], and a culture of A. atopus was established in 1992 [START_REF] Lotufo | Description of Amphiascoides atopus, new species (Copepoda: Harpacticoida) from a mass culture system[END_REF]. Both cultures were kept at 25°C in 1 L sediment-free Erlenmeyer flasks containing 600 mL of 30 psu artificial seawater (ASW), which was renewed every 14 days. Copepods were fed weekly with T-Isochrisis paste (Brine Shrimp Direct, Odgen, UT, USA). Continuous reproduction in both species was apparent. Copepod were harvested by rinsing culture medium through a 125 µm aperture screen and retained copepods were sorted under a stereo dissection microscope via Pasteur pipette.

96-h toxicity tests

All materials used to conduct bioassays were acid-cleaned prior to use. Fifteen nonovigerous adult female copepods (A. atopus or S. knabeni) were placed in 600 mL ASW plastic flasks, covered with a lid to avoid exchange with CO 2 from the air, and three replicates were used for each treatment. ASW was previously oxygenated by bubbling air for 24 h. Incubations at each condition were performed for 96 h at 25°C and 16:8 light:dark photoperiod in an environmental chamber.

Acidity was manipulated by adding mineral acid (HCl) to ASW in flasks. Rangefinding tests were used to determine acidities that resulted in 100% mortality to both copepod species. A. atopus and S. knabeni were exposed to 5 (ranging from 5.62 to 6.22) and 8 (ranging from 4.75 to 5.67) different pH conditions, respectively (N = 3  15 animals for each pH level). The pH (NBS scale) was measured, using a pH meter (Oakton) at the beginning and at the end of the incubation. pH varied by an average of 0.059 ± 0.015 units over the course of the exposures, representing an average H + concentration variation of 0.250 ± 0.145 µmol.L -1 . Carbon dioxide effects were studied by bubbling pure CO 2 in ASW. This CO 2 -rich water (pH = 4.5) was mixed with air-bubbled ASW in order to achieve desired pH levels. CO 2 concentrations were tested at increasing levels until 100% mortality was achieved. A. atopus and S. knabeni were exposed to 10 (ranging from 5.47 to 6.67) and 9 (ranging from 5.29 to 6.44) different CO 2 -induced pH conditions, respectively (N = 3  15 specimens). Mean variations of pH and concentration of H + measured between the end and the beginning of incubation were 0.089 ± 0.053 and 0.132 ± 0.093 µmol.L -1 respectively. Total alkalinity in the ASW used under the conditions of our experiments was determined potentiometrically using a Gran function [START_REF] Dickson | Guide to best practices for ocean CO 2 measurements[END_REF]. The pCO2 was calculated from pH and total alkalinity using the R package seacarb [START_REF] Proyle | Seacarb, an R package to calculate parameters of the seawater carbonate system[END_REF].

In the present study, the pH values measured at the end of the incubations were used for the calculation of LC 50 s.

Only A. atopus was studied for metal exposure. A small volume (20 µL) of acidified solution with the calculated amount of Cd and Cu to achieve a target concentration was spiked into each 600 mL incubation flask. At pH of 8.20, 6.30, 6.23 and 6.07, respectively 9, 6, 5 and 5 different Cd concentrations were tested (N = 3  15 specimens). At pH of 8. 20, 6.36, 6.23, 6.15 and 6.08, respectively 6, 3, 3, 4 and 3 different Cu concentrations were tested (N = 3  15 specimens).

After 96 h, water was sampled for pH and metal concentrations. Contents of each microcosm were rinsed through a 125 µm mesh sieve and copepods retained were enumerated as live or dead. Missing copepods were presumed dead and percent mortality was calculated.

For A. atopus and S. knabeni, mean fractions of missing copepods represented 7.27 ± 14.22 % and 6.42 ± 14.18 % of the total animals, respectively.

Metal measurements

Total Cd concentrations were determined by flame r atomic absorption spectroscopy (Varian AA240FS), and Cu concentrations were analyzed using ICP-OEC (Inductively Coupled Plasma, Optical Emission Spectrometer). All modeling for Cd and Cu was performed using the geochemical equilibrium modeling software program MINEQL+, version 4.6. Modeling simulations were run using components from the existing internal MINEQL+ database including, Cl -, Na + , SO 4 2-, Mg 2+ , Ca 2+ , K + , HCO 3-, Br -, BO 3 , Sr, and F -, at the nominal concentrations typical of 30 psu ASW (Instant Ocean). Two successive iterations were performed; the first at a fixed pH of 4.5 with an open atmosphere in order to obtain the dissolved inorganic carbon (DIC) concentration, and the second using this DIC in a closed system, and using the components at the concentrations as described above, plus the different measured total Cd and Cu concentrations added to the components. These conditions mimicked the experimental protocols used in this study, which included bubbling CO 2 into ASW to a pH of 4.5 (under open atmosphere conditions), then mixing this water with airbubbled ASW to achieve a desired pH (as described above) before sealing the container to container with copepods inside.

Concentrations of H + , metals and free-ion metal concentrations that caused 10% (LC 10 ) and 50% (LC 50 ) lethality were estimated, as well as their 95% confidence intervals using log-probit analysis (SPSS Version 17.0.1, Chicago, IL).

The sum toxic unit approach [START_REF] Sprague | Measurement of polluant toxicity to fish: II. Utilizing and applying bioassay results[END_REF] was used to assess the joint effects of binary mixtures of (i) CO 2 /Cd, (ii) CO 2 /Cu and (iii) CO 2 /Cu free-ion. Toxic units (TU) are a means of expressing the toxicity of a mixture of compounds as a portion of its threshold effect concentration [START_REF] Sprague | Measurement of polluant toxicity to fish: II. Utilizing and applying bioassay results[END_REF]. The toxicity of a mixture of compounds is expressed as a sum of the ratios of the exposure concentration and threshold-effect concentrations (LC 50 expressed as concentration that causes 50% mortality [LR 50 ]) of the individual compounds in the mixture, where: sum toxic units = (concentration x / LR 50 x) + (concentration y / LR 50 y).

If compounds in a mixture are acting additively, the sum TU required to cause a 50% mortality of the exposed organisms will equal 1. A TU including 1 within the 95% confident interval was considered additive, a TU that did not include 1 in its high end 95% confidence interval (<1) was considered greater than additive (synergistic), while a TU that did not include 1 in its low end 95% confidence interval was considered less than additive (antagonistic).

Statistical analysis

Differences between (i) acidity tolerances of A. atopus vs. S. knabeni and (ii) acidity induced by HCl vs. CO 2 were tested with analysis of covariance (ANCOVA) [START_REF] Green | Life-stage-specific toxicity of sedimentassociated chlorpyrifos to a marine, infaunal copepod[END_REF] using SAS software (SAS-Institute, 1985). The observed survival proportions were transformed via angular transformation [START_REF] Anscombe | The transformation of Poisson, binomial and negative binomial data[END_REF].

Results

Acid toxicity

Over the 96-h exposure period, mortality of A. atopus and S. knabeni ranged, respectively, from 0 to 2.2 % and from 0 to 4.4% in control experiments.

A. atopus had an LC 10 of 5.99 (5.86 -6.19) pH units and the LC 50 was 5.75 (5.63 -5.84) pH units when acidity was manipulated with HCl. When water was acidified with CO 2 , LC 10 was 6.41 (6.29 -6.82) pH units and LC 50 was 6.11 (6.04 -6.17) pH units, equivalent to LC 10 of 23.6 (9.1 -31.1) and LC 50 of 47.2 (41.0 -55.4) matm CO 2 (Fig. 1).

S. knabeni, had an LC 10 of 5.51 (5.38 -5.90) pH units and the LC 50 was 5.00 (4.90 -5.06) pH units when acidity was manipulated with HCl. When water was acidified with CO 2 , LC 10 was 6.06 (5.92 -6.39) pH units and LC 50 was 5.54 (5.49 -5.59) pH units, equivalent to LC 10 of 52.9 (24.7 -73.1) and LC 50 of 175.1 (156.6 -197.1) matm CO 2 (Fig. 2).

ANCOVA of the angular-transformed survival proportions showed that both species were significantly more sensitive to acidification from CO 2 relative to acidification from HCl (p < 0.001). Furthermore, regardless of the method used to acidify the ASW, A. atopus was significantly more sensitive to acidity than S. knabeni (p < 0.001).

Metal toxicity

The 96-h LC 50 of A. atopus for Cd was 1.38 mg.L -1 (1.26 -1.52) at pH 8.20. When acidity was changed by adding CO 2 , Cd LC 50 was 1.41 mg.L -1 (1.22 -1.60) at pH 6.30, 0.98 mg.L -1 (0.72 -1.18) at pH =6.23 and 0.05 mg.L -1 (0 -0.307) at pH 6.07 (Fig. 3). The Toxic Unit required to create 50% mortality was 1.47 mg.L -1 (1.28-1.62) (Fig. 4). As TU did not include 1 in its low end 95% confidence interval, CO 2 and Cd can be considered to have antagonistic toxicity. MINEQL+ modeling revealed that the Cd free-ion concentration was not affected by CO 2 under the conditions of the experiment.

The 96-h LC 50 of A. atopus for Cu was 0.65 mg.L -1 (0.40 -0.99) at pH 8.20. When acidity was changed by adding CO 2 this Cu LC 50 was 0.32 mg.L -1 (0.25 -0.55) at pH 6.37 and 0.26 mg.L -1 (0.19 -0.48) at pH = 6.23 (Fig. 5). The Toxic Unit required to create 50% mortality was 1.17 (1.01 -1.25). TU was slightly > 1 in its low end 95% confidence interval (Fig. 6), which indicated that the toxicity of CO 2 and Cu were antagonistic. MINEQL+ modeling revealed that the free-ion concentration of Cu 2+ was affected by CO 2 . The 96h LC 50 free-ion Cu 2+ concentration was 2.73 µM (2.08-3.32) at pH 8.2. Free-ion concentrations of Cu 2+ were evaluated for each incubation condition and TU was calculated according to the individual toxicities of CO 2 and free-ion Cu. The TU that created 50% mortality was 1.53

(1.24 -1.92) (Fig. 7). Consequently, when calculated with free-ion concentrations, toxicological interactions between CO 2 and Cu appeared to be more strongly antagonist than when calculated with total Cu concentrations (TU = 1.17 (1.01 -1.25)). Interactions between CO 2 and Cd as well as CO 2 and free-ion Cu displayed a similar range of antagonist effects with TU LC 50 s of 1.47 (1.28-1.62) and 1.53 (1.24 -1.918) respectively.

Discussion

Methodological consideration

To our knowledge, this is the first study of the influence of CO 2 on shallow marine harpacticoid copepods. However, the present results should be interpreted with caution as several methodological aspects may underestimate the toxicity of elevated CO 2 concentrations in seawater. Sublethal effects may produce CO 2 sensitivity affecting many physiological parameters. Mechanisms that mitigate acidity by acid-base balance comes at a metabolic cost and, while not life threatening, could decrease growth and reproductive capacity [START_REF] Langenbuch | High sensitivity to chronically elevated CO 2 levels in a eurybathic marine sipunculid[END_REF]. For instance, egg production in the pelagic copepod Acartia steueri was found to decrease at pH of 6.8 [START_REF] Kurihara | Effect of raised CO 2 concentration on the egg production rate and early development of two marine copepods (Acartia steueri and Acartia erythraea)[END_REF]. The fact that only the response of adults was studied, may also lead to an underestimation of CO 2 toxicity. It is widely accepted that early life history stages may be more sensitive to high pCO 2 [START_REF] Pörtner | Physiology and climate change[END_REF], especially in invertebrates (Dupont and Thorndyke, 2009a;[START_REF] Kurihara | Effects of CO 2 -driven ocean acidification on the early developmental stages of invertebrates[END_REF][START_REF] Mayor | CO 2 -induced acidification affect hatching success in Calanus finmarchicus[END_REF]. Early developmental stages of copepods are especially sensitive to contaminants [START_REF] Green | Life-stage-specific toxicity of sedimentassociated chlorpyrifos to a marine, infaunal copepod[END_REF][START_REF] Lotufo | Toxicity of sediment-associated PAHs to an estuarine copepod: effect on survival, feeding, reproduction and behavior[END_REF].

However, other aspects of the present study may lead to an overestimate of the impact of ocean acidification. Transfers of animals to each condition were abrupt and consequently not representative of the gradual changes that animals would experience in natural environments. Specifically, our acute exposures did not allow for either physiological acclimation or genetic modification in response to altered environmental conditions. A multigenerational experiment on the pelagic copepod Acartia tonsa showed that only one generation is needed to cope with impacts of ocean acidification on life cycle dynamics (Dupont and Thorndyke, 2009b).

Such potential biases must be kept in mind when interpreting results dealing with the toxicity of acidity. However, the impact of those biases would not apply to a (i) comparison of toxicity between CO 2 and HCl, (ii) comparison of CO 2 tolerance between species or (iii) comparison of metal and CO 2 interactive toxicity.

CO 2 vs. HCl toxicity

The present study suggests that acidification using CO 2 produced an LC 10 of pH 6.41 for A. atopus (Fig. 1) and pH 6.06 for S. knabeni (Fig. 2). A model of the Southern North Sea suggests an average decrease of 0.1 pH unit over the next 50 years [START_REF] Blackford | pH variability and CO 2 induced acidification in the North Sea[END_REF], while a global model predicts ocean pH values as low 7.5 within the next 300 years [START_REF] Caldeira | Anthropogenic carbon and ocean pH[END_REF]. Both predictions are, however, well above observed toxicities.

Our results suggest that adult benthic copepods from coastal environment may be relatively insensitive to CO 2 and may not be highly affected by an increase of ocean acidity. However, leakage from CO 2 seabed storage can create a faster and stronger local acidification [START_REF] Hawkins | No exit: thinking about leakage from geologic carbon storages sites[END_REF] than the one induced by atmospheric CO 2 . The present study suggests that some harpacticoid species may be sensitive to those strong changes expected for seepage scenario.

The 96-h LC 50 observed for A. atopus and S. knabeni were, respectively, pH 5.75 (Fig. 1) and 5.00 (Fig. 2) when acidity was manipulated with HCl. In a similar type of experiment, the sensitivity of pelagic copepods to acidity was shown to be species-specific: among 10 oceanic zooplankton species studied, only two had lower sensitivity to acidification than we observed: Eucalanus bungii bungii and Themisto japonica LC 50 's of pH 5.16 and 5.00 respectively [START_REF] Yamada | Acute toxicity of lowered pH to some oceanic zooplankton[END_REF]. Thus, adult benthic copepods may be slightly more tolerant to acidity than pelagic copepods.

Unmixed sediments are characterized by strong geochemical gradients. pH changes of 1 unit with depth [START_REF] Fisher | High resolution vertical profiles of pH in recent sediments[END_REF] and 1.5 units over short time scales [START_REF] Gnaiger | pH fluctuation in an intertidal beach in Bermuda[END_REF] have been measured. In such variable environments, it is unlikely that benthic animals would be affected by small changes of pH. However, some meiofauna appear to be specialists, living at preferred depth locations within the sediment [START_REF] Fleeger | Does interference copetition determine the vertical distribution of meiobenthic copepods?[END_REF][START_REF] Steyaert | The importance of fine-scale, vertical profiles in characterizing nematode community structure[END_REF]. Depth specialists could be adapted to highly specific conditions from that sediment stratum and consequently some species may be intolerant to small changes in pH.

CO 2 sensitivity of benthic fauna has been studied in the deep sea in order to evaluate the potential impact of large-scale sequestration of CO 2 . Deep-sea harpacticoid copepods suffered 70-100% mortality when exposed to a reduction in pH of 0.75 units [START_REF] Thistle | Deepocean, sediment-dwelling animals are sensitive to sequestered carbon dioxide[END_REF].

Copepods may not be able to burrow deeper to avoid CO 2 [START_REF] Thistle | Simulated sequestration of industrial carbon dioxide at a deep-sea site: effect on species of harpacticoid copepods[END_REF], but they may be able to swim into the water column in order to attempt escape from the advancing front of carbon dioxide-rich seawater CO 2 [START_REF] Thistle | Exposure to carbon dioxide-rich seawater is stressful for some deep-sea species: An in situ, behavioral study[END_REF]. Other studies have illustrated sensitivity of deep-sea nematodes, foraminifera and euglenoid flagellates [START_REF] Barry | Effect of direct ocean CO 2 injection on deep-sea meiofauna[END_REF][START_REF] Fleeger | Simulated sequestration of anthopogenic carbon dioxide at a deep-sea site: effect on nematode abundance and biovolume[END_REF]Fleeger et al., In press;[START_REF] Ishida | In situ enclosure experiment using a benthic chamber system to assess the effect of high concentration of CO 2 on deep-sea benthic communities[END_REF][START_REF] Ricketts | Effects of carbon dioxide sequestration on California margin deep-sea foraminiferal assemblages[END_REF] at pH decreases of only 0.1-0.2 units [START_REF] Barry | Utility of deep sea CO 2 release experiments the biology of a high-CO 2 ocean: effect of hypercapnia on deep sea meiofauna[END_REF]. The deep sea is characterised by highly stable physico-chemical parameters. In contrast, shallow-dwelling aquatic animals have evolved acid-base regulatory capabilities to combat seasonal, or even daily fluctuations in water pH. Deep-sea animals are unlikely to possess well-developed acid-base regulatory capacity [START_REF] Pane | Extracellular acid-base regulataion short-term hypercapnia is effective in a shallow-water crab, but ineffective in a deep-sea crab[END_REF]. Consequently, the high CO 2 sensitivity of deep-sea copepods may not be observed among species from shallow coastal environments.

To our knowledge, this is the first study to examine the impact of changing seawater pH on harpacticoid copepods from a shallow-water environment. Among other members of shallow meiofauna, nematodes have a higher tolerance to CO 2 than do macrofauna [START_REF] Dashfield | Impacts of ocean acidification and burrowing urchins on within-sediment pH profiles and subtidal nematode communities[END_REF][START_REF] Widdicombe | Effects of CO 2 induced seawater acidification on infaunal diversity and sediment nutrient fluxes[END_REF] and this apparently short-term tolerance could be due to their impermeable proteinaceous cuticle. Three species of nematodes from subtidal coastal environments were highly resistant to high concentrations of CO 2 and lethal effects were only observed after increasing acidity to pH 5.5-6 [START_REF] Takeuchi | Impacts of high concentration of CO 2 on marine organisms; a modification of CO 2 ocean sequestration[END_REF]. At pH 7.5, a subtidal nematode community was unaffected by high CO 2 after several days of exposure [START_REF] Dashfield | Impacts of ocean acidification and burrowing urchins on within-sediment pH profiles and subtidal nematode communities[END_REF], but a pH of 7.3 produced a change in nematode community composition [START_REF] Widdicombe | Effects of CO 2 induced seawater acidification on infaunal diversity and sediment nutrient fluxes[END_REF]. Nematodes and copepods share the same habitat and have consequently evolved in similar environmental conditions. The present study suggests that, as with nematodes, shallow-water harpacticoid copepods would be largely unaffected by future ocean acidification.

Carbonate chemistry of seawater can be manipulated in various ways, and the most commonly used is by bubbling seawater with gases at different pCO 2 values. In the present study, different pCO 2 seawaters were prepared by mixing different ratios of water bubbled with pure CO 2 and water bubbled with air. This technique increases dissolved inorganic carbon at constant total alkalinity, and provides realistic simulations of past and predicted changes of ocean chemistry [START_REF] Gattuso | Technical note: approaches and software tools to investigate the impact of ocean acidification[END_REF]. Adding a strong acid (HCl) to a system does not alter the concentration of dissolved inorganic carbon but does modify total alkalinity. This method of acidification is thus more artificial because alkalinity is not expected to change over this century. Consequently at similar pH values, water acidified with CO 2 will contain more dissolved CO 2 , HCO 3 -and CO 3 2-than water acidified with a strong acid. CO 2 diffuses easily into intracellular compartments and rapidly reacts with water to form H + and HCO 3 -. This intracellular acidosis can affect many physiological processes and may explain why the strongest toxic effects were observed when CO 2 was used to manipulate acidity in the present study. Stronger toxicity of early developmental stages of sea bream was also observed when acidity was manipulated by CO 2 [START_REF] Kikkawa | Comparison of the lethal effect of CO 2 and acidification on red sea brean (Pagrus major) during the early developmental stages[END_REF]. These results emphasize that the addition of strong acid does not fully mimic the changes in carbonate chemistry expected with ocean acidification and would likely lead to an underestimation of the toxic effect.

S. knabeni and A. atopus sensitivity

Even though ocean acidification is expected to reduce biodiversity [START_REF] Widdicombe | Predicting the impact of ocean acidification on benthic biodiversity: what can animal physiology tell us?[END_REF], some species may benefit from these new environmental conditions (Dupont and Thorndyke, 2009a). In several experiments, increased pCO 2 has modified the meiofaunal community composition of deep-sea [START_REF] Bernhard | Impact of intentionally injected carbon dioxide hydrate on deep-sea benthic foraminiferal survival[END_REF][START_REF] Thistle | Simulated sequestration of industrial carbon dioxide at a deep-sea site: effect on species of harpacticoid copepods[END_REF] and shallow [START_REF] Widdicombe | Effects of CO 2 induced seawater acidification on infaunal diversity and sediment nutrient fluxes[END_REF] sediments. Those experiments indicate that not all species within a community have the same sensitivity to CO 2 . Differences were also observed between species of deep-sea copepod in their ability to escape CO 2 [START_REF] Thistle | Exposure to carbon dioxide-rich seawater is stressful for some deep-sea species: An in situ, behavioral study[END_REF]. Such natural differences could explain the observed lower sensitivity of S. knabeni relative to A. atopus.

Different physiological mechanisms allow organisms to counteract acidification via: (i) passive buffering of intra-and extracellular fluids, (ii) transport of CO 2 in the blood of species that have respiratory pigments and (iii) metabolic suppression during periods of elevated CO 2 .

Species adapted to hypercapnic environments have evolved such mechanisms to counteract CO 2 more efficiently than other species (Seibel andWalsh, 2001, 2003).

The two copepod species studied are associated with different environments. The natural habitat and the distribution of A. atopus are unknown, but species in this genus are exclusively found on beaches comprised of large particles (large sand grains to small cobble) [START_REF] Lotufo | Description of Amphiascoides atopus, new species (Copepoda: Harpacticoida) from a mass culture system[END_REF]. The porosity of such sediment being high, the porewater pH is similar to the pH of overlying water with little change over space and time. Cultures of S.

knabeni were initiated with specimens collected from estuarine sediments in Louisiana [START_REF] Lotufo | Toxicity of sediment-associated PAHs to an estuarine copepod: effect on survival, feeding, reproduction and behavior[END_REF]. The pH of sediment porewater in this environment is approximately 7.55 [START_REF] Ho | Interstitial water composition in Barataria bay (Louisiana) sediment[END_REF]. This type of muddy sediment is poorly permeable and pH decreases strongly with depth at the millimetre scale (e.g. [START_REF] Fisher | High resolution vertical profiles of pH in recent sediments[END_REF]. Moreover, in muddy sediments, the majority of meiofauna lives in the uppermost surficial layer. Bioturbation increases water penetration to depth [START_REF] Aller | Meiofauna and solute transport in marine muds[END_REF], creating millimetre lateral heterogeneity in pH [START_REF] Zhu | Two-dimensional pH distributions and dynamics in bioturbated marine sediments[END_REF]. In addition to differences in spatial and temporal pH heterogeneity, muddy sediments have generally lower oxygen availability and higher CO 2 concentration than sandy sediment and cobble. Mud-dwelling meiofauna may consequently be better adapted to hypercapnic environments than the fauna of sand and cobble. Our observations are consistent with this hypothesis. [START_REF] Widdicombe | Effects of CO 2 induced seawater acidification on infaunal diversity and sediment nutrient fluxes[END_REF] similarly observed that a nematode community from a mud environment was less sensitive to CO 2 -rich water than was a nematode community coming from sandy sediment.

Metal toxicity

Anthropogenic activities have led to significant metal pollution in many parts of the world, especially in inshore sediments [START_REF] Nixon | Coastal marine eutrophication: a definition, social causes, and future concerns[END_REF]. Physical adsorption and chemical binding contribute to heavy metal concentrations that are several orders of magnitude higher in sediment than in overlying water. As a result, sediments generally represent sinks for pollutants. Sediment dwellers like copepods can potentially be exposed by taking up dissolved pore-water metals across their thin exoskeletons and soft tissues and/or by ingesting sediment and associated metal.

Compared to other heavy metals, Cd is relatively soluble, tends to bioaccumulate and is consequently considered to be a significant environmental threat [START_REF] Goldberg | Cadmium and the oceans[END_REF]. Cu can enter the aquatic environment primarily from mining activities [START_REF] Lewis | Copper in water and aquatic environments[END_REF] but also from antifouling paints. Cu is an essential micronutrient for all organisms. However at high concentrations, it becomes toxic for plants and animals. In the present study, the Cu 96-h LC 50 of A. atopus was 650 µg.L -1 at a pH of 8.2, which is in the range of LC 50 s found for the intertidal copepod genus Tigriopus [START_REF] Barka | Influence of different essential and nonessential metals on MTLP levels in the copepod Tigriopus brevicornis[END_REF][START_REF] Kwok | Toxicity of antifouling biocides to the intertidal copepod Tigriopus japonicus (Crustacea, Copepoda): effects of temperature and salinity[END_REF][START_REF] O'brien | Copper tolerance of the life history stages of the splashpool copepod Tigriopus californicus (Copepoda, Harpaticoida)[END_REF].

Metal / CO 2 toxicity

In the future ocean, marine life will have to face acidification simultaneously with other anthropogenic stressors such as metal contamination. Prediction of the toxicology of multiple stressors is challenging when the combined effects of toxicants cannot be predicted from the individual effect of each. A review of studies of mixtures of heavy metals reveals that synergisms and antagonisms are more common than response-additive toxicity [START_REF] Norwood | Effects of metal mixtures on aquatic biota: a review of observations and methods[END_REF]. Such interactive toxicology was observed in copepods [START_REF] Fleeger | Mixture of metals and polynuclear aromatic hydrocarbons elicit complex, nonadditive toxicological interactions in meiobenthic copepods[END_REF][START_REF] Hagopian-Schlekat | Acute toxicity of five sedimentassociated metals, individually and in a mixture, to the estuarine meiobenthic harpaticoid copepod Amphiascus tenuiremis[END_REF]. However, the combined effect of CO 2 and metal toxicity for marine organisms have rarely been studied [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF].

A consequence of ocean acidification is a decreased concentration of OH -and CO 3 2-.

These anions form strong complexes in ocean water with divalent and trivalent metals [START_REF] Millero | Effect of ocean acidification on the speciation of metals in seawater[END_REF]. This reduction is expected to change the speciation of numerous metal ions in seawater [START_REF] Byrne | Inorganic speciation of dissolved elements in seawater: the influence of pH on concentration ratios[END_REF]. Metals such as Cd 2+ form strong complexes with chloride.

However, because Cl -is pH insensitive, speciation of Cd will be minimally affected by ocean acidification. Indeed, modeling conducted in the present study revealed that the free-ion concentration of Cd was not affected by acidity. Other metals such as Cu 2+ form strong complexes with carbonate. Such metals will be more strongly affected by acidification and the concentration of their free ionic form will increase. In the present study, free-ion modeling with MINEQL+ revealed that in contrast to Cd, the free-ion form of Cu was increased with increasing acidity. Free-ion forms of metals are generally more toxic than complex forms [START_REF] Allen | Metal speciation. Effects on aquatic toxicity[END_REF]. Comparison between LC 50 calculated using total Cu or free-ion Cu allows an evaluation of the influence of CO 2 on speciation. The approach using total Cu measured permits an evaluation of toxicity of combined (i) Cu, (ii) CO 2 and (iii) increased concentration of free-ion Cu according to CO 2 conditions. With the Cu approach, only Cu and CO 2 effects are evaluated. The strongest antagonism observed with the free-ion approach confirmed that CO 2 increased the free-ion form of Cu, thus making it more toxic.

Acidification of the ocean also leads to an increased concentration of H + . As a result, competition for binding sites increases between H + and metals. Surface sites become less available to adsorb metals in the presence of increasing H + , potentially making acidification and metal toxicity antagonistic. In the present study, an antagonism was observed between CO 2 and Cd and of CO 2 and the free-ion of Cu, suggesting a competition for binding sites with H + for both metals. CO 2 concentration decreased toxicity of both metals by the same order of magnitude, suggesting that the binding sites of both metals may be similar. However, other processes could lead to the observed antagonist effect. Indeed, one mechanism to counteract acidosis is to depress metabolism [START_REF] Guppy | Metabolic depression in animals: physiological perspectives and biochemical generalizations[END_REF], thus reducing the rate of metal transport in the animals. Cd mimics Ca, and a reduced calcification rate can, for instance, reduce the rate of Cd uptake. The extent to which such processes operate in harpacticoid copepods is not well understood. To our knowledge, only one other study dealing with CO 2 and metal toxicity has been performed in the marine environment. In this case, antagonism was also observed between CO 2 and Cd: when CO 2 concentration was increased, the uptake of Cd decreased in the hatchling tissue of cuttlefish Sepia officinalis [START_REF] Lacoue-Labarthe | Effects of increased pCO 2 and temperature on trace element (Ag, Cd and Zn) bioaccumulation in the eggs of the common cuttlefish, Sepia officinalis[END_REF]. Studies of freshwater organisms are more common and reveal competition for binding sites between H + and metals in aquatic biota (e.g. [START_REF] Hare | Predicting animal cadmium concentration in lakes[END_REF][START_REF] Yu | Kinetic uptake of bioavailable cadmium, selenium, and zinc by Daphnia magna[END_REF]. Similarly, uptake of metal by soil-dwelling animals was reduced when pH was changed by adding lime (e.g. [START_REF] Oste | Cadmium uptake by eartworms as related to the availability in the soil and the intestine[END_REF].

Extrapolation to natural environment

The present study was conducted in water and allows evaluation of pharmacological interactions between metals and CO 2 . However, exposure in sediments can produce other kinds of interactions that are difficult to predict. The effects of mixtures in sediments are especially complex for deposit-feeding animals that are exposed to both dissolved contaminants and contaminated food/sediment. For example, the narcotic effect of PAH has been shown to slow the uptake of Cd-contaminated sediment in a bulk deposit-feeding oligochaete by reducing his feeding rate [START_REF] Millward | Pyrene bioaccumulation, effects of pyrene exposure on particle-size selection, and fecal pyrene content in the oligochaete Limnodrilus hoffmeisteri (Tubifidae, Oligochaete)[END_REF]. CO 2 also has a narcotic effect but its toxic interactions with metals in sediment remains uncertain, as copepods are not considered as bulk deposit feeders [START_REF] Green | Aqueous-phase, porewater and sedimentphase cadmium: Toxicity relationships for a meiobenthic copepod[END_REF]. Other interactions may differ between aquatic and sediment environments. High amounts of metal can potentially be bound to sediment and those complex forms of metals are not necessarily bioavailable and toxic for sediment dwelling animals such as harpacticoid copepods. Acidification releases bound metals from sediments [START_REF] Ardelan | Effect of experimental CO 2 leakage on solubility and transport of seven trace metals in seawater and sediment[END_REF] rendering them more toxic in a free form. The amount of free metal released from sediment may be more important than amount available from pelagic environments. Such mechanisms are largely unknown and therefore studies are needed to better evaluate such potential indirect effects of ocean acidification.

Figure captions

Figure

Figure 1 .

 1 Figure 1. Mortality (%) of A. atopus (± s.d., N = 3  15 specimens) versus pCO 2 (matm) and

Figure 2 .

 2 Figure 2. Mortality (%) of S. knabeni (± s.d., N = 3  15 specimens) versus pCO 2 (matm) and

Figure 3 .

 3 Figure 3. Mortality (%) of A. atopus (± s.d., N = 3  15 specimens) versus total Cd

Figure 4 .

 4 Figure 4. Effect of Cd-CO 2 mixture on A. atopus mortality (± s.d., N = 3  15 specimens).

Figure 5 .

 5 Figure 5. Mortality (%) of A. atopus (± s.d., N = 3  15 specimens) versus Cu concentrations

Figure 6 .

 6 Figure 6. Effect of Cu-CO 2 mixture on A. atopus mortality (± s.d., N = 3  15 specimens).

Figure 7 .

 7 Figure 7. Effect of free Cu 2+ ion-CO 2 mixture on A. atopus mortality (± s.d., N = 3  15

  Figure 1.

  Figure 2.

  Figure 3.

  Figure 4.

  Figure 6.

Acknowledgments

We thank Robert Gambrell for his help in the Cu analyses. We are also grateful to Thomas Lacoue-Labarthe for really fruitful comments provided about this study.