M Oliveira
email: migueloliveira@ua.pt

I Ahmad

V L Maria

A Serafim

M J Bebianno

M Pacheco

M A Santos

Hepatic metallothionein concentrations in the golden grey mullet (Liza aurata) -relationship with environmental metal concentrations in a metal-contaminated coastal system in Portugal

Keywords: Liza aurata, Metallothioneins, Cadmium, Copper, Zinc, Mercury, Aquatic pollution

This field survey was designed to assess the environmental metal contamination status of Ria de Aveiro (Portugal). To achieve that goal, the concentrations of Cd, Hg, Cu and Zn in the sediments and water were assessed and Liza aurata hepatic metallothionein (MT) determined. The relationships between MT and environmental metal concentrations and hydrological factors were examined. Results revealed a wide distribution of metals both in water and sediments throughout the lagoon, mainly at Rio Novo do Principe (RIO) and Laranjo (LAR), at concentrations that may affect biota. MT concentrations were higher at the sites with high metal content (RIO and LAR). A significant positive correlation was found between MT and Cd in the sediments as well as with MT and Hg and Cu in the water. Moreover, a negative correlation between MT and salinity was found. Thus, the current data support MT use as a biomarker of metal exposure emphasizing the importance of hydrological parameters in its concentrations. Results suggest the continued monitoring of this lagoon system.

Introduction

Metal contamination in aquatic systems is a critical environmental issue due to metal uptake and toxicity in many aquatic organisms, as well as the possibility of their trophic transfer along food chains, eventually reaching humans. Most of these compounds have high affinity for particulate matter and are readily adsorbed onto suspended particles eventually becoming part of sediments where they will remain even after input cessation. Nevertheless, metals may return to the water column or be transferred to biota through physical, chemical and/or biological processes [START_REF] Schneider | Sediment contaminants off the coast of Sydney, Australia: A model for their distribution[END_REF][START_REF] Bervoets | Use of transplanted zebra mussels (Dreissena polymorpha) to assess the bioavailability of microcontaminants in Flemish surface waters[END_REF].

Normal fish cellular metabolism requires essential metals (such as Cu and Zn) which may be taken up from water, food and/or sediment. However, non-essential ones (such as Cd and Hg) may also be absorbed and accumulated in tissues [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF]. Thus, environmental pollution monitoring frequently includes assessment of metals in sediment, suspended particulate matter or biota. However, the quantification of environmental or tissue concentrations of a variety of metals does not take into account the biological response or significance of the accumulated metals [START_REF] Chan | Metallothionein: Potential biomarker for monitoring heavy metal pollution in fish around Hong Kong[END_REF] since metals can accumulate to high levels in organisms and yet be metabolically unavailable, thus unable to induce toxicological effects [START_REF] Vijver | Internal metal sequestration and its ecotoxicological relevance: a review[END_REF]Banni et al., 2007). Metal exposure generally increases total tissue concentrations of metals. However, the increase of essential metals in some tissues may not be significant due to their regulated accumulation (Atli and Canli, 2008).

Metallothioneins (MT) are low molecular weight cytosolic proteins rich in SH groups, known to be over-expressed in organisms from environments with high metal concentrations [START_REF] Sarkar | Molecular Biomarkers: Their significance and application in marine pollution monitoring[END_REF][START_REF] Viarengo | The use of biomarkers in biomonitoring: A 2-tier approach assessing the level of pollutantinduced stress syndrome in sentinel organisms[END_REF]. In fish, the large amount of MT induction occurs in liver [START_REF] Canli | The relationships between heavy metal (Cd, Cr, Cu, Fe, Pb, Zn) levels and the size of six Mediterranean fish species[END_REF], an organ playing a key role in the storage and inactivation of metals. Considering that MT induction is one of the first detectable signs of the presence of metals [START_REF] Filipovic | Metallothionein and metal levels in cytosol of liver, kidney and brain in relation to growth parameters of Mullus surmuletus and Liza aurata from the Eastern Adriatic Sea[END_REF][START_REF] Chesman | Hepatic metallothionein and total oxyradical scavenging capacity in Atlantic cod Gadus morhua caged in open sea contamination gradients[END_REF][START_REF] Van Campenhout | A field survey of metal binding to metallothionein and other cytosolic ligands in liver of eels using an on-line isotope dilution method in combination with size exclusion (SE) high pressure liquid chromatography (HPLC) coupled to Inductively Coupled Plasma time-of-flight Mass Spectrometry (ICP-TOFMS)[END_REF] and the relationship found between metal concentrations in the environment and MT concentrations in fish tissues [START_REF] Hylland | Hepatic metallothionein and heavy-metals in dab Limanda limanda from the German Bight[END_REF], they have been used to characterize metal contamination in the environment [START_REF] Chan | Metallothionein: Potential biomarker for monitoring heavy metal pollution in fish around Hong Kong[END_REF][START_REF] Linde | Metallothionein as bioindicator of freshwater metal pollution: European eel and brown trout[END_REF]. Ria de Aveiro (Portugal), a coastal lagoon, provides an important habitat for a number of fish species, including the golden grey mullet (Liza aurata). The feeding habits of this species include benthic organisms and detritus, making it particularly adapted to accumulate sediment-associated contaminants and thus an important bio-indicator species. Previous studies concerning this species, in this complex aquatic system, revealed the existence of critical sites within Ria de Aveiro, capable of inducing oxidative and genetic damage (Oliveira et al., 2009a,b,c) as well as endocrine disruption [START_REF] Oliveira | Fish thyroidal and stress responses in contamination monitoring -An integrated biomarker approach[END_REF]. High concentrations of metals have been detected in this aquatic system [START_REF] Coelho | Macroalgae response to a mercury contamination gradient in a temperate coastal lagoon (Ria de Aveiro, Portugal)[END_REF][START_REF] Monterroso | Metal-contaminated sediments in a semi-closed basin: Implications for recovery[END_REF] and MT inductions as well as inhibitions were observed in L. aurata gill, at some of those critical sites (Oliveira et al., 2009a).

Thus, the hepatic MT assessment is highly relevant due to liver central role in metal storage and inactivation. In this perspective, this study assessed the relation between metal concentrations found in water and sediment compartments of Ria de Aveiro and L. aurata hepatic MT. The influence of hydrological factors on MT concentrations was also studied.

Material and Methods

Study Area

Ria de Aveiro is a lagoon permanently connected to the ocean (Figure 1), about 45 km long and 8.5 km wide. Sampling sites were selected on a geographic distribution basis taking into account the known and supposed sources of metal contamination as well as the selection of a (theoretically) unpolluted reference point. Sampling sites were: Torreira (TOR), an intermediate region of the longest channel (S. Jacinto-Ovar channel), far from the main polluting sources and thus assumed as reference site; Barra (BAR), the initial part of the Mira channel close to the lagoon entrance and subject to considerable naval traffic; Gafanha (GAF) situated in the vicinity of a deep-sea fishing port and dry-docks, also connected with the main channel coming from Aveiro city carrying domestic discharges; Rio Novo do Principe (RIO), located at the terminal area of the Vouga River, 6.5 km distant from a pulp/paper mill effluent outlet, that discharged to this water course during nearly five decades (until the year 2000); Laranjo (LAR), close to a chlor-alkali plant (6 km), an important source of metal contamination (mainly mercury); Vagos (VAG), located at the terminal part of the Ílhavo channel, receiving municipal and domestic effluents with high concentrations of polycyclic aromatic hydrocarbons (PAHs).

Sampling:

Liza aurata was caught in October 2005, during low tide using a traditional beach-seine net named "chincha". Juvenile specimens were selected on the basis of their size, having an average length of 14.5±2.5 cm and weighing 21.6±3.7 g. Immediately after catching, fish was sacrificed, liver removed and frozen in liquid nitrogen. In the laboratory the samples were stored at -80º C until further treatments.

At each sampling site hydrological parameters were assessed (Table I) according APHA (1998) guidelines. Sediments were collected from the surface (15 cm deep) with a grab and stored at -20ºC until analysis. Sub-surface water samples were collected by hand, using polyethylene gloves and glass bottles of 1 L.

Metal determination in the water and sediments

Total metal concentrations in the water samples were determined after liquid-liquid extraction with ammonium pyrrolidine dithiocarbamate (APDC) and methyl isobutyl ketone (MIBK) followed by graphite furnace atomic absorption spectrophotometry (PerkinElmer). This procedure was adopted to preconcentrate the metals present in the water. Certified standard freshwater reference material (SRM 1643d from NIST) was analyzed to check the accuracy of metal determinations in the water samples.

Total metal concentrations in the sediments were determined in the < 63 µm fraction, to minimize the effect of grain size on metal distribution. Samples were manually wet sieved using 63 µm nylon sieves and Milli-Q water. After that, this sediment fraction was dried at 40 ºC. For analysis, approximately 2 g of the sediment fraction were accurately weighed and placed into 50 ml digestion vessels. After the addition of 5 ml 69% nitric acid (HNO 3 ), the samples were digested for 2 hours at 80ºC. The digestion mixture was cooled to room temperature and 2 ml 30% hydrogen peroxide (H 2 O 2 ) and 3 ml Milli-Q water carefully added. The digestion vessels were heated for another hour. The residue was allowed to cool and diluted with Milli-Q water to a final volume of 50 ml. Then samples were allowed to settle down for 24 hours and quantitatively transferred to 50 ml vessels for analysis.

The concentrations of Cd, Cu, Hg and Zn were determined in the supernatant using a Perkin-Elmer atomic absorption spectrophotometer (AAS) model A Analyst 800. All metals except Cd and Hg were analyzed by flame atomic absorption spectrophotometry. Cadmium was analysed by an electrothermal atomic absorption spectrometry and mercury by cold vapour atomic spectrophotometry with an automatic hydride generator system.

Metallothionein (MT) content

After liver homogenization in 20 mM Tris buffer (pH 8.6) containing 150 mM of NaCl, the homogenates were centrifuged 45 minutes at 30000 g (4ºC). Supernatant aliquots were heat-treated at 80ºC for 10 minutes and recentrifuged at 30000 g for 45 minutes (4ºC). MT quantification was made by differential pulse polarography (DPP) according to [START_REF] Bebianno | Quantification of metallothioneins in marine invertebrates using differential pulse polarography[END_REF] using a 646VA Processor autolab type II and an ECO Chemie IME663 mercury drop electrode. Comparisons of peak heights with those of standard additions of purified rabbit MT enabled MT quantification. Accuracy of the DPP analytical method was obtained by internal calibration using commercial rabbit liver MT-I. Recovery studies of our cytosols spiked with the MT-I rabbit liver standards revealed recovery rate from 97 to 103%. Results were expressed as µg MT/g wet weight.

Statistical analysis:

Results were expressed as means ± SE (standard error) corresponding to experimental groups of six fish (n=6).

Statistical data analysis was done using Statistica software (StatSoft, Inc., Tulsa, OK, USA). Assumptions of normality and homogeneity of data were verified. One way ANOVA was performed in order to assess significant effects, followed by post-hoc Tukey test to signal significant differences between groups [START_REF] Zar | Biostatistical Analysis[END_REF]. The significance of results was ascertained at = 0.05. Describing factors were grouped using principal component analysis (PCA), reducing the number of axis without loosing relevant information. Principal components (PCs) having eigenvalues of less than one were discarded, and varimax rotation maximising the loading of a variable on one component was then applied on the retained PCs. The resulting components were then used as independent factors in multiple regression analyses taking MT as the dependent variable.

Results

Hydrological Parameters

The hydrological parameters, expressed in Table I, show differences in terms of turbidity and salinity. Thus, waters were more turbid at LAR and VAG (0.3 m) and less turbid at BAR (1.2 m). Salinity at RIO was 21 whereas the other sites displayed values between 29 (LAR) and 34 (GAF and BAR).

Metals in Water and Sediment

The water analyses revealed that RIO displayed significantly higher concentrations of Hg, Cu and Zn than TOR and the other studied sites (with the exception of Zn concentrations which were not different from VAG) (Table I).

Concerning Cd, the highest concentrations in water were found at LAR (significantly higher than in all other sites). BAR displayed lower concentrations of Cd, Hg, Cu and Zn when compared to TOR (though not significantly in the case of Cd). The metal concentrations in water at GAF were only significantly different (higher) from TOR in the case of Hg. At LAR, the concentrations of all the analysed metals were significantly higher than TOR whereas VAG displayed lower Hg and Cu and higher Zn concentrations in water.

Concerning the concentrations of metals in the sediments (Table I), the highest concentrations of Hg, Cu and Zn were found at LAR (significantly higher than all other studied sites). Concerning Cd, the highest concentrations in the sediments were found at RIO (significantly higher than all other studied sites) and LAR (significantly higher than TOR, BAR, GAF and VAG). Hg concentrations in sediments at BAR and RIO were also higher than in TOR, GAF and VAG. The concentrations of Cu in sediments at RIO were also significantly higher than TOR whereas Zn concentrations at BAR, GAF and VAG were significantly lower than TOR.

Metallothionein

RIO displayed significantly higher MT concentration when compared to all studied sites (Figure 2). Thus, compared to TOR, MT concentration was 256% higher at RIO. LAR also displayed significantly higher MT concentration than TOR (107%), BAR, GAF, and VAG. At VAG, MT concentration was significantly lower than TOR (74%).

Principal Component Analysis and Linear Multiple Regression

Three principal components were defined as explaining the major amount of total variance (89.97%) of the assessed parameters (except MT) (Table II). Thus, the first component which includes salinity, temperature, Cd in the sediments, Hg, Cu and Zn in the water explains 36.19% of total variance.

The second component including Hg, Zn and Cu in the sediments and Cd in the water, explains 28.84%. The third components including turbidity, pH and dissolved oxygen explain 24.93% of variance. According to the multiple regression analyses the first two factors account for 98.2 % of MT variability and the first factor alone is responsible for 77%. Cd in the sediments, Hg and Cu in the water and salinity displayed the strongest (significant) correlations with MT (Figure 3).

Discussion

Biological factors such as age, sex, reproductive status as well as hormone levels may influence MT induction, increasing response variability [START_REF] Hamza-Chaffai | Physicochemical forms of storage of metals (Cd, Cu and Zn) and metallothionein-like proteins in gills and liver of marine fish from the Tunisian coast: ecotoxicological consequences[END_REF]. Moreover, it was demonstrated in L. aurata that total length, body weight and age are biometric parameters correlated with MT [START_REF] Filipovic | Metallothionein and metal levels in cytosol of liver, kidney and brain in relation to growth parameters of Mullus surmuletus and Liza aurata from the Eastern Adriatic Sea[END_REF]. In this perspective, L. aurata specimens selected for the current study were juvenile with the same age and reproductively quiescent and the stress of capture and handling was reduced as much as possible.

Metal concentrations

Metals mobility and reactivity in estuaries are highly influenced by the organic matter content, salinity, redox conditions and pH [START_REF] Sauve | Solid-solution partitioning of metals in contaminated soils: Dependence on pH, total metal burden, and organic matter[END_REF][START_REF] Liang | Spatial and temporal organic and heavy metal pollution at Mai Po Marshes Nature Reserve, Hong Kong[END_REF]. Sediment metal concentrations usually exceed those in the water column. However, metals bound to the sediment may enrich interstitial and overlying waters with metals [START_REF] Bryan | Bioavailability, accumulation and effects of heavy metals in sediments with special reference to United Kingdom estuaries -a review[END_REF] upon changes in the environment. In the current study, the concentrations of two of the most dangerous metals to aquatic organisms, Cd and Hg, listed in the priority substances of EU Directive 2008/105/EC, as well as two essential metals, Cu and Zn, that in high concentrations may also induce toxic effects in aquatic organisms [START_REF] Oliveira | Glutathione protects heavy metalinduced inhibition of hepatic microsomal ethoxyresorufin O-deethylase activity in Dicentrarchus labrax L[END_REF] were quantified in the water and sediments of Ria de Aveiro. The studied metals displayed a distribution throughout the lagoon. Zn displayed the highest concentrations in sediments, followed by Cu, Cd and Hg. The highest concentrations of metals in the sediments were found in LAR (for Zn, Cu and Hg) and RIO (for Cd). At RIO, LAR and BAR Hg was higher than the defined Water Framework Directive environmental quality standard for sediments (0.36 µg.g -1 ). Most of the available studies on metals concentrations in Ria de Aveiro focused on Hg contamination in LAR, due to the emission history of a chlor-alkali plant [START_REF] Ramalhosa | Distribution of mercury in the upper sediments from a polluted area (Ria de aveiro, Portugal)[END_REF]. Thus, at LAR area, the concentration of Hg detected in the sediments reached values as high as 51.7 µg.g -1 [START_REF] Coelho | Macroalgae response to a mercury contamination gradient in a temperate coastal lagoon (Ria de Aveiro, Portugal)[END_REF]. In the current study, the highest concentration of Hg in the sediments was found in LAR (2.33 µg.g -1 )

and was within the same order of magnitude of other studies [START_REF] Pereira | Seasonal fluctuations of tissue mercury contents in the European shore crab Carcinus maenas from low and high contamination areas (Ria de Aveiro, Portugal)[END_REF][START_REF] Ramalhosa | Accumulation versus remobilization of mercury in sediments of a contaminated lagoon[END_REF]. The present data also revealed considerable Hg in the sediments of BAR and RIO (0.88 and 0.86 µg.g -1 respectively), probably due to tidal export from Laranjo area [START_REF] Pereira | An estimation of industrial mercury stored in sediments of a confined area of the Lagoon of Aveiro (Portugal)[END_REF] and/or from nonpoint sources. Despite most studies on metal concentrations at LAR focused on Hg, elevated concentrations of other metals were also found there by [START_REF] Monterroso | Metal-contaminated sediments in a semi-closed basin: Implications for recovery[END_REF]. The concentrations of Cd, Cu and Zn found in the current study at LAR sediments were similar to those found by [START_REF] Monterroso | Metal-contaminated sediments in a semi-closed basin: Implications for recovery[END_REF].

The metal concentrations in water reported in the current study can only be compared with other studies in Ria de Aveiro in terms of Hg in LAR, being in the same order of magnitude of previously reported concentrations [START_REF] Ramalhosa | Accumulation versus remobilization of mercury in sediments of a contaminated lagoon[END_REF][START_REF] Guilherme | Erythrocytic nuclear abnormalities in wild and caged fish (Liza aurata) along an environmental mercury contamination gradient[END_REF]. Unlike sediments which displayed higher Hg, Cu and Zn concentrations at LAR, in water, RIO displayed the highest concentrations of Hg, Cu and Zn. Another clear difference to sediments was found in BAR that displayed very low Hg concentrations in water (below detection limit). Moreover, Cd concentrations at all studied sites were above the defined environmental quality standard for water of Water Framework Directive (0.21 µg.g -1 ).

One of the outstanding difficulties encountered in environmental impact assessments is to determine the biological significance of contaminants [START_REF] Langston | Metallothionein in liver of eels Anguilla anguilla from the Thames Estuary: an indicator of environmental quality?[END_REF]. In this perspective numerical quality guidelines have been developed, identifying concentrations of chemicals of potential concern that may cause or contribute to adverse effects [START_REF] Macdonald | Development and evaluation of consensus-based sediment quality guidelines for freshwater ecosystems[END_REF].

However, its use should be carefully interpreted since factors like organic matter, grain size and percentage of clay may be confounding elements for direct comparison. Nevertheless, the comparison of the present metal data with some quality guidelines [START_REF] Long | Incidence of adverse biological effects within ranges of chemical concentrations in marine and estuarine sediments[END_REF][START_REF] Macdonald | Development and evaluation of sediment quality guidelines for Florida coastal waters[END_REF] reveals that the concentrations detected in the sediments mainly at LAR and RIO and BAR may lead to frequent adverse effects in aquatic organisms. The concentrations of metals in the water are highly dynamic, being influenced by abiotic factors among which the water flow. Nevertheless, the water concentrations of Hg at RIO as well as Cu at RIO, LAR and GAF, can lead to damage in aquatic species considering they were higher than the US EPA (2006) estimated highest concentration to which an aquatic community can be exposed indefinitely without resulting in an unacceptable effect.

The bioavailability of a metal does not necessarily lead to harmful effects in organisms since they evolved defence mechanisms which allow them to decrease metal reactivity in cells. Among those defences, MT are considered as the most suitable biomarker for Cd, Cu, Zn and Hg exposure [START_REF] Hylland | Natural modulation of hepatic metallothionein and cytochrome P4501A in flounder, Platichthys flesus[END_REF], reflecting bioavailability and tissue pharmacokinetics of metal uptake [START_REF] Handy | A proposal for the use of biomarkers for the assessment of chronic pollution and in regulatory toxicology[END_REF]. In this perspective, the study of L. aurata liver MT becomes highly relevant, taking into account that liver is the main metal accumulating organ in this species [START_REF] Usero | Heavy metals in fish (Solea vulgaris, Anguilla anguilla and Liza aurata) from salt marshes on the southern Atlantic coast of Spain[END_REF]. However, despite the known presence of metals in Ria de Aveiro and the suitability of fish MT as biomarkers of exposure to metals, the studies focusing on environmental metal concentrations and fish MT concentrations are very scarce.

Measurable (constitutive) concentrations of MT are always expected even in uncontaminated sites, due to MT homeostatic role in transferring essential elements to and from metalloproteins. The observed MT concentrations ranged from 45.6 to 627.7 (µg.g -1 wet weight), being in the same order of magnitude of the MT concentrations found in other species. For example, [START_REF] Zorita | Assessment of biological effects of environmental pollution along the NW Mediterranean Sea using red mullets as sentinel organisms[END_REF] found MT concentrations in red mullets (Mullus barbatus) between 100 and 710 (µg.g -1 wet weight). However, the current hepatic MT concentrations were much lower than in L. aurata from Eastern Adriatic coastal zone (2320 µg.g -1 wet weight) [START_REF] Filipovic | Metallothionein and metal levels in cytosol of liver, kidney and brain in relation to growth parameters of Mullus surmuletus and Liza aurata from the Eastern Adriatic Sea[END_REF].

Differences may be linked with different age, weight and size of the surveyed specimens. Supporting this hypothesis, a positive and statistically significant correlation between L. aurata total length and body weight with MT and metals in liver cytosol was found by the authors [START_REF] Filipovic | Metallothionein and metal levels in cytosol of liver, kidney and brain in relation to growth parameters of Mullus surmuletus and Liza aurata from the Eastern Adriatic Sea[END_REF].

In the current study, MT concentrations were higher at the sites displaying the highest metal concentrations, supporting its usefulness in environmental monitoring even in complex environments where interference of other xenobiotics can be found. MT use as biomarker of response to metals exposure is more sensitive to metals such Cd and Hg and to a much lower extent to essential metals [START_REF] Olsvik | Brown trout (Salmo trutta) metallothioneins as biomarkers for metal exposure in two Norwegian rivers[END_REF]Banni et al., 2007;Atli and Canli, 2008;) which have their cytosolic concentrations closely regulated through homeostatic mechanisms often binding to structures other than MT [START_REF] Laflamme | Interrenal metallothionein and cortisol secretion in relation to Cd, Cu, and Zn exposure in yellow perch, Perca flavescens, from Abitibi lakes[END_REF][START_REF] Langston | Metallothionein in liver of eels Anguilla anguilla from the Thames Estuary: an indicator of environmental quality?[END_REF][START_REF] Linde | Phenomenon of hepatic overload of copper in Mugil cephalus: Role of metallothionein and patterns of copper cellular distribution[END_REF]. In this perspective, other sulfhydryl group containing substances such as GSH may also play an important role in cellular protection against metal cytotoxicity [START_REF] Viarengo | Mechanisms of heavy metal cation homeostasis in marine invertebrates[END_REF][START_REF] Oliveira | Glutathione protects heavy metalinduced inhibition of hepatic microsomal ethoxyresorufin O-deethylase activity in Dicentrarchus labrax L[END_REF]. Nevertheless, a high positive correlation of MT and Cu cytosolic concentrations was found in liver of L. aurata [START_REF] Filipovic | Metallothionein and metal levels in cytosol of liver, kidney and brain in relation to growth parameters of Mullus surmuletus and Liza aurata from the Eastern Adriatic Sea[END_REF]. Moreover, considering the complex environment that is an estuary, the combined effects of other xenobiotics should also be considered as demonstrated by [START_REF] Romeo | Metallothionein determination in the liver of the sea bass Dicentrarchus labrax treated with copper and B(a)P[END_REF] and [START_REF] Van Den Hurk | Interactive effects of cadmium and benzo[a]pyrene on metallothionein induction in mummichog (Fundulus heteroclitus)[END_REF]. Despite the demonstration that simultaneous exposure to metals such as Cd and PAHs may yield higher MT concentrations than metal exposure alone, our study clearly shows MT induction in environments with high metal concentrations (RIO and LAR) and low concentrations in sites with low metal concentrations (TOR, BAR and VAG). The previous (and only available study) on MT concentrations of L. aurata caught at Ria de Aveiro (Oliveira et al., 2009a), showed higher gill MT concentrations at LAR and suggested that this site was the most contaminated with metals. Moreover, no significant differences to TOR were found in that study in MT concentrations from RIO. These results seem to confirm that the hepatic MT concentrations are more reliable to the environmental metal contamination assessment than gill MT.

Relation between MTs and abiotic factors

The multiple regression analyses showed that the assessed metals and hydrological parameters grouped in components 1 and 2 could explain 98.2 % of the variability of MTs. Thus, salinity, temperature, Cd in the sediments Hg, Cu and Zn in water were responsible for 77% of MT variability.

Of these parameters, linear regression analyses signalled as more correlated Cd in the sediments, Cu and Hg in water and salinity (inversely). It has been generally accepted that the toxicity of certain metals to aquatic animals is influenced by ambient salinity. Thus, metals are usually more toxic at low salinities probably associated with increased uptake rates, as a result of an increase in free metal ion concentrations. In this perspective, a highly significant negative correlation between MT and salinity was also found in the Brazilian flounder (Paralichthys orbignyanus) collected from an estuary (Amado et al., 2006). Thus, a determinant role of salinity in the metal bioavailability can be on the basis of the negative correlation currently found between that abiotic parameter and hepatic MT. However, a definitive linkage between these variables is complicated by the occurrence of different patterns of contamination and salinity levels in the sites under comparison.

Thus, the usefulness of L. aurata hepatic MT as biomarkers of metal exposure was demonstrated in fish caught at Ria de Aveiro despite the known ability of other xenobiotics to interfere in MT concentrations and the known existence of different type of xenobiotics in Ria de Aveiro.

Conclusions

High concentrations of metals can be found in the sediments and water of Ria de Aveiro which may result in pernicious effects to aquatic organisms.

The highest concentrations of metals in the sediments and water were found in LAR and RIO. Thus, in sediments, LAR for Zn, Cu and Hg and RIO for Cd The results of the current study suggest continued monitoring of Ria de Aveiro.

Acknowledgments

Amado, L.L., Robaldo, R.B., Geracitano, L., Monserrat, J.M., Bianchini, A., 2006. Biomarkers of exposure and effect in the Brazilian flounder Paralichthys orbignyanus (Teleostei: Paralichthyidae) from the Patos Lagoon estuary (Southern Brazil). Marine Pollution Bulletin 52, 207-213. Atli, G., Canli, M., 2008. Responses of metallothionein and reduced glutathione in a freshwater fish Oreochromis niloticus following metal exposures.

Environmental Toxicology and Pharmacology 25, 33-38. Banni, M., Dondero, F., Jebali, J., Guerbej, H., Boussetta, H., Viarengo, A., 2007. Assessment of heavy metal contamination using real-time PCR analysis of mussel metallothionein mt10 and mt20 expression: a validation along the Tunisian coast. Biomarkers 12, 369 -383. Table II

  displayed the highest concentrations. In water, RIO displayed the highest concentrations of Hg, Cu and Zn and LAR the highest concentrations of Cd.L. aurata hepatic MT concentrations can be ranked as:RIO>LAR>GAF>TOR>BAR>VAG. The highest hepatic MT concentrations displayed at the most contaminated sites (RIO and LAR) reveal a strong L. aurata defence capacity against metal toxicity.The correlation between environmental concentrations of metals and L. aurata hepatic MT, supports MT applicability as a biomarker of metal pollution in contaminated aquatic systems, though the influence of abiotic factors namely salinity should be considered.

Financial

  support from Fundação para a Ciência e Tecnologia -FCT (Government of Portugal) provided through POCI/MAR/61195/2004, SFRH/BD/27584/2006, SFRH/BPD/26970/2006, SFRH/BPD/34326/2006 and by CESAM -Aveiro University Research Institute are gratefully acknowledged.

Figure 1 -

 1 Figure 1 -Map of the sampling sites ( ) in the Ria de Aveiro. The respective coordinates (latitude, longitude) are: reference site -Torreira (TOR)

Figure 2 -Figure 3 -

 23 Figure 2 -Hepatic metallothionein (MT) of L. aurata collected at different sites in Ria de Aveiro. Values represent mean ± standard error (n=6). Distinct

  Figure 1

Table I -

 I Environmental characterization: hydrological parameters and metals concentrations in water and sediments. Distinct letters indicate significant differences between sites (p<0.05).


Stéphane Caut
email: stephanecaut@gmail.com

Elodie Guirlet

Marc Girondot

Effect of tidal overwash on the embryonic development of leatherback turtles in French Guiana

Keywords: tidal inundation, leatherback turtle, embryonic mortality, French Guiana, development, site selection

In marine turtles, the physical conditions experienced by eggs during incubation affect embryonic development. In the leatherback, hatching success is known to be low in relation to other marine turtles as a result of high embryonic mortality. Moreover, the hatching success on Yalimapo in French Guiana, one major nesting beach for this species, is lower compared to other nesting sites. We assessed the rate of leatherback turtle embryonic mortality in order to investigate the tolerance of leatherback turtle clutches laid on Yalimapo beach to tidal overwash, and we highlight causes of poor hatching success. Of the 89 nests studied, 27 were overlapped by tide at least once during the incubation period (of which 5 nests were lost by erosion). The hatching success was on average significantly lower in overwashed nests than in non-overwashed, highlighting the existence of embryonic developmental arrest linked to tidal inundation. The stages of developmental arrest and their proportion are linked with time, frequency and level of overwash events. In the context of global warming and associated sea level rise, understanding the detrimental effect of tidal inundation on the development of marine turtle nests is of interest in nesting sites where turtles are likely to be forced to nest closer to the tide line, thus exposing their nests to greater risk of nest overlap with sea and tidal inundation.

Introduction

For animals that lay their eggs in a nest, the selection of a nest site may strongly influence embryo development and offspring survival and may therefore have important consequences for the reproductive fitness of the adult. Indeed, for most oviparous species, the most vulnerable part of the life cycle is often the embryo stage, since eggs are typically left in fixed locations throughout their development without parental care [START_REF] Wood | Relation of temperature, moisture, salinity, and slope to nest site selection in Loggerhead sea turtles[END_REF]. Hatching success for organisms that lay eggs in a nest, such as sea turtles, is believed to be influenced by numerous biotic and abiotic factors such as predation, type of substrate, porosity, temperature, moisture content, salinity, slope of the beach, nest elevation, rainfall and tidal inundation [START_REF] Mortimer | The influence of beach sand characteristics on the nesting behavior and clutch survival of green turtles[END_REF][START_REF] Horrocks | Nest site location and nest success in the hawksbill turtle Eretmochelys-Imbricata In Barbados, West-Indies[END_REF][START_REF] Ackerman | The nest environment and the embryonic development of sea turtles[END_REF][START_REF] Wood | Relation of temperature, moisture, salinity, and slope to nest site selection in Loggerhead sea turtles[END_REF][START_REF] Allen | Effects of fire ants (Hymenoptera : Formicidae) on hatching turtles and prevalence of fire ants on sea turtle nesting beaches in Florida[END_REF][START_REF] Bilinski | The effects of nest environment on calcium mobilization by leatherback turtle embryos (Dermochelys coriacea) during development[END_REF][START_REF] Donlan | Predation of Caretta caretta (Testudines : Cheloniidae) eggs by larvae of Lanelater sallei (Coleoptera : Elateridae) on Key Biscayne, Florida[END_REF][START_REF] Foley | Effects of sand characteristics and inundation on the hatching success of loggerhead sea turtle (Caretta caretta) clutches on lowrelief mangrove islands in southwest Florida[END_REF]. In many oviparous reptiles, however, environmental factors not only influence embryo survivorship [START_REF] Horrocks | Nest site location and nest success in the hawksbill turtle Eretmochelys-Imbricata In Barbados, West-Indies[END_REF][START_REF] Burger | Effects of incubation temperature on behavior of hatchling pine snakes -Implications for reptilian distribution[END_REF][START_REF] Resetarits | Oviposition site choice and life history evolution[END_REF], but also hatchling size [START_REF] Packard | The physiological ecology of reptilian eggs and embryos[END_REF], performance [START_REF] Janzen | An experimental-analysis of natural-selection on body size of hatchling turtles[END_REF], growth [START_REF] Bobyn | Interclutch and interpopulation variation in the effects of incubation conditions on sex, survival and growth of hatchling turtles (Chelydraserpentina)[END_REF], behaviour [START_REF] Burger | Effects of incubation temperature on behavior of hatchling pine snakes -Implications for reptilian distribution[END_REF], and sex determination [START_REF] Spotila | Effects of incubation conditions on sex determination, hatching success, and growth of hatchling desert tortoises, Gopherus agassizii[END_REF]. After the emergence of hatchling turtles, their survival may be strongly related to the distance at which the nest is laid from the sea and from supra-littoral vegetation behind the beach [START_REF] Mrosovsky | Ecology and nest-site selection of leatherback turtles, Dermochelys coriacea[END_REF].

Placement of nests close to the sea increases the likelihood of inundation and egg loss to erosion, whereas placement of nests farther inland increases the likelihood of desiccation, hatchling misorientation, and predation on nesting females, eggs, and hatchlings [START_REF] Bustard | Physical and chemical factors affecting hatching in the green turtle, Chelonia mydas (L.)[END_REF][START_REF] Fowler | Hatching success and nest predation in the green sea turtle, Chelonia mydas, at Tortuguero, Costa Rica[END_REF][START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF][START_REF] Spencer | Experimentally testing nest site selection: Fitness trade-offs and predation risk in turtles[END_REF].

Leatherback turtles (Dermochelys coriacea), classified as critically endangered by the Species Survival Commission (IUCN, 2006)), often place their nests in the open sand near the water, but rarely in vegetation [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF][START_REF] Kamel | Nest site selection in leatherbacks, Dermochelys coriacea: Individual patterns and their consequences[END_REF]Caut et al., 2006a). For this reason, researchers have investigated possible strategies and cues that might influence nest placement. [START_REF] Mrosovsky | Ecology and nest-site selection of leatherback turtles, Dermochelys coriacea[END_REF], at the population level, and [START_REF] Eckert | Environmental unpredictability and leatherback sea turtle (Dermochelys coriacea) nest loss[END_REF], at the individual level, suggested that leatherbacks have developed a scatter nesting strategy whereby nests are randomly distributed along a beach to maximize clutch survival in an unpredictable nesting area. However, leatherback turtles often nest in places where their eggs are destroyed by high tides. Poor nest site selection, where nests have been shown to completely fail, ranges from <2.5% in Malaysia to around 40% in the Guianas and appears to be related to beach topography [START_REF] Mrosovsky | Ecology and nest-site selection of leatherback turtles, Dermochelys coriacea[END_REF]. Leatherbacks generally have low hatching success (~50%) relative to other marine turtle species (~85% or more; see [START_REF] Bell | Low reproductive success of leatherback turtles, Dermochelys coriacea, is due to high embryonic mortality[END_REF].

Predation could partly explain the low hatching rate, with the longer incubation period for leatherbacks increasing exposure to predators [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF]. But this low hatching success of leatherbacks also seems to be mainly the result of nest site selection and embryonic mortality [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF][START_REF] Bell | Low reproductive success of leatherback turtles, Dermochelys coriacea, is due to high embryonic mortality[END_REF]. Indeed, leatherbacks generally nest nearer to the sea than other marine turtles and nests are therefore subjected to erosion and inundation, sometimes leading to 50% nest loss on certain beaches in South America [START_REF] Patino-Martinez | Globally significant nesting of the leatherback turtle (Dermochelys coriacea) on the Caribbean coast of Colombia and Panama[END_REF]. These overwashes of nests by sea water may lead to egg mortality from suffocation (lack of respiratory oxygen; [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF] and/or disruption of metabolism as a result of exposure to higher salinities and therefore chloride toxicity [START_REF] Bustard | Physical and chemical factors affecting hatching in the green turtle, Chelonia mydas (L.)[END_REF]) and/or critical cooling below minimum temperature for embryonic development [START_REF] Hewavisenthi | Incubation environment and nest success of the flatback turtle (Natator depressus) from a natural nesting beach[END_REF]. Moreover, leatherback nests are deeper than those of others marine turtles, and water content and salinity are known to increase with depth, leading to a decrease in hatching success [START_REF] Foley | Effects of sand characteristics and inundation on the hatching success of loggerhead sea turtle (Caretta caretta) clutches on lowrelief mangrove islands in southwest Florida[END_REF]. The greater embryonic mortality in leatherback nests is therefore mainly due to the nest-site selection resulting in more nests being overwashed.

However, the effect of tidal overwash on hatching success and embryonic development remains poorly understood and represents an important question for the future. Indeed, threats to coastal areas are increasing [START_REF] Huang | Climate change and integrated coastal management: a challenge for small island nations[END_REF] with the projected rise in sea level resulting from anthropogenic global warming; the increase in the number and concentration of greenhouse gases in the atmosphere has the potential to cause an elevation in the global mean air temperature and mean sea level of 1-4.5 °C and 31-150 cm, respectively, by the year 2100 (IPCC, 2007). Therefore, many nesting beaches are particularly susceptible to coastal hazards such as storm surges and coastal erosion. However, although much effort has been expended over the last two decades to understand and mitigate the threats to marine turtles [START_REF] Watson | Fishing methods to reduce sea turtle mortality associated with pelagic longlines[END_REF], the threat of climate change on this taxon has, until recently, been given little attention (for more information, see [START_REF] Hawkes | Climate change and marine turtles[END_REF]. Studies of the effects of global warming on sea turtle populations have focused essentially on the loss of nesting beach habitat as a result of an increase in sea level and on the changes in sex ratio [START_REF] Daniels | Sea-level rise -Destruction of threatened and endangered species habitat in South-Carolina[END_REF][START_REF] Davenport | Temperature and the life-history strategies of sea turtles[END_REF][START_REF] Nicholls | Coastal vulnerability assessment for sealevel rise: evaluation and selection of methodologies for implementation[END_REF][START_REF] Nicholls | Increasing flood risk and wetland losses due to global sea-level rise: regional and global analyses[END_REF][START_REF] Fish | Predicting the impact of sea-level rise on Caribbean sea turtle nesting habitat[END_REF][START_REF] Baker | Potential effects of sea level rise on the terrestrial habitats of endangered and endemic megafauna in the Northwestern Hawaiian Islands[END_REF][START_REF] Jones | Australian sandy beach ecosystems and climate change: ecology and management[END_REF][START_REF] Fish | Construction setback regulations and sea-level rise: Mitigating sea turtle nesting beach loss[END_REF][START_REF] Mazaris | Evaluating the impacts of coastal squeeze on sea turtle nesting[END_REF]. Most of these studies examined the potential effect of sea-level rise on marine turtle nesting beaches with different models (e.g. elevation model, inundation flooding model), but they did not take into account how life history and ecological parameters that determine extinction risks will be affected by climate change [START_REF] Isaac | Effects of climate change on life history: implications for extinction risk in mammals[END_REF] . The recent review on climate change and marine turtles [START_REF] Hawkes | Climate change and marine turtles[END_REF]) therefore indicated the importance for future research on climate change effects on key habitats upon which turtles depend and factors that influence nest site selection and hatchling success. The effect of inundation and, indirectly, the sea-level rise on leatherback hatching success have been poorly studied [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF] to date, despite the tendency of this species to lay its clutches closer to the high-tide line than other sea turtle species. Results of studies concerning global warming have highlighted threats for coastal zones with accelerated beach erosion and greater frequency of flooding events [START_REF] Kumar | Potential vulnerability implications of sea level rise for the coastal zones of Cochin, southwest coast of India[END_REF].

The beach of Yalimapo has one of the highest nesting densities of leatherback females, with about 30% of the world's population of nesting females and approximately 50% of all leatherbacks nesting in the region of French Guiana and Suriname [START_REF] Girondot | Leatherback turtles, Dermochelys coriacea, nesting in French Guiana, 1978-1995[END_REF]; it should be noted that the exact proportion varies according to the year. However, hatching success on this beach is lower than at other leatherback nesting sites [START_REF] Boulon | Leatherback turtles (Dermochelys coriacea) on St. Croix, U.S. Virgin Islands: fifteen years of conservation[END_REF]. Depressed hatching success compounds the problem of population decline that results from adult mortality caused by incidental fisheries capture [START_REF] Kaplan | A risk assessment for Pacific leatherback turtles (Dermochelys coriacea)[END_REF][START_REF] Martinez | Conservation and biology of the leatherback turtle in the Mexican Pacific[END_REF], and understanding the causes of low hatching success would therefore be an important conservation step towards preventing extinction in a species [START_REF] Ralph | Effect of egg location and respiratory gas concentrations on developmental success in nests of the leatherback turtle, Dermochelys coriacea[END_REF]. In this study, we determined the level and frequency of tidal overwash and hatching success of leatherback nests in order to investigate the tolerance of nests to tidal inundation in the context of global warming and sea-level rise. We assessed the rate and stages of leatherback turtle embryonic mortality in order to investigate tolerance of leatherback turtle clutches laid on Yalimapo beach to tidal overwash and we highlight causes of poor hatching success.

Materials and methods

Study site and data collection

Research was carried out on the Awala Yalimapo beach in French Guiana (53°57W-5°45'N). The beach is located within the Amana Nature Reserve, on the inshore plain of coastline between the Mana and Maroni Rivers. Guyana has a semi-diurnal tide (two daily high tides) with a period of approximately 12-and-a half hours. The beach is 4 km in length and the width varies from a few metres to 20 m, depending on the tide line. For this study, we chose a 300-m long section of beach with the same slope (approximately 10%), which was sufficiently frequented by turtles but also distant from sites frequented by tourists. The width of the sand beach depends on the strength of the tide (~ 5 to 30 metres).

The nests analysed in the study were laid from 20 May to 4 June 2002. At the time of oviposition or later, during nest covering, we measured the minimum straight carapace length of the nesting female (SCLmin) [START_REF] Bolten | Research and Management Techniques for the Conservation of Sea Turtles[END_REF] and the female identity was recorded (Passive Integrated Transponder (PIT) tags). For each freshly laid nest encountered, we measured the minimum distance from the nest to the last high-tide line and to the vegetation line (sparse creeping vegetation (Ipomoea pes-caprae), sand still visible) with a plastic measuring tape.

All nest locations were localized to within 1 m by triangulation using numbered stakes placed every 10 m along the vegetation of the beach. To locate specific nests after incubation, we placed a numbered ring of plastic-insulated copper in the sand above the clutch during the covering of the nest by the female. We used a metal detector that captured signals from probes in the copper at the end of incubation.

Embryonic development

Nest status was checked each day for signs of emergence, predation by dogs or erosion. We waited sufficient time for any live embryos to hatch and leave the nest before analysing the remaining embryos. All nests were excavated 48 hours after the first signs of emergence; in the absence of evidence, we expected a period of 70 days of incubation, so no live embryos were excavated or sacrificed (authorization no. 1516 1D/1B/ENV of 27 August 2001, DIREN Guyane). We recorded the total number of yolked eggs (noted YE, including hatched and unhatched yolked eggs) and shelled albumin globs (noted 'yolkless' eggs). We dissected unhatched yolked eggs in situ and staged them in category using these criteria:

-Category 1: Egg with no visible signs of development or dead embryo < 10 mm in length.

-Category 2: Egg with dead embryo 10 mm and < 30 mm in length.

-Category 3: Egg with dead embryo 30 mm and < 60 mm in length.

-Category 4: Egg with dead embryo 60 mm (including dead hatchling in a pipped egg).

Embryonic size and time of incubation

To analyse the effect of tidal overwash on embryonic development, we determined for each category of embryonic size a corresponding time of incubation. [START_REF] Renous | Caractéristiques de développement embryonnaire de la tortue luth, Dermochelys coriacea (Vandelli, 1761)[END_REF] presented a stage-by-stage description of leatherback development and reported that many measurements of embryonic growth could be related to developmental time (incubation period). We used Renous' stage description to estimate rates of growth. These growth curves were performed by [START_REF] Renous | Caractéristiques de développement embryonnaire de la tortue luth, Dermochelys coriacea (Vandelli, 1761)[END_REF] on the same beach (Awala Yalimapo, French Guiana) with a random sampling of nests in natural environment and between the end of May and the beginning of August. We fitted these data using an exponential function: y = 2.85 exp (0.05x) R 2 = 0.99, with the embryonic size on the y axis (mm, with maximum 110 mm) and the time of incubation on the x axis. We determined the incubation period for each size class of each category:

Category 1 corresponded to the period 1 0 to 23 days; Category 2 to the period 2 24 to 43 days; Category 3 to the period 3 44 to 56 days; Category 4 to the period 4 57 to 67 days.

Percentage of embryos and hatchling success

We estimated the percentage of embryos for each category (p(E)) as:

p(E n ) = E n / YE.
Where: n = the category 1 to 4; E= Number of embryos in the category n; YE = Total number of Yolked Eggs

We also estimated the percentage of embryos for categories 2 to 4 of the total yolked eggs that can continue their development at each corresponding period (pc(E)) as:

pc(E 2 ) = E 2 / (YE -E 1 ) pc(E 3 ) = E 3 / (YE -E 1 -E 2 ) pc(E 4 ) = E 4 / (YE -E 1 -E 2 -E 3 )
Hatching success was estimated by the number of large shell fragments divided by the total number of yolked eggs. When eggshells were fragmented, pieces were grouped together to represent one egg. The use of shell fragments to estimate hatching success had been previously validated in an open-air hatchery study (Caut et al., 2006b)

Measurements of overwash

During each day of incubation time, we measured the minimum distance from each nest to the daily high-tide line (represented by the high-water mark of the two daily highs, noted DDHTL (Daily Distance to the High Tide Line; "+" positive for distance non overwash and "-" negative for distance overwash), see Figure 1). To define the characteristics of overwash for each of the four specific periods (period 1 : 0 to 23 days, period 2 : 24 to 43 days, period 3 : 44 to 56 days, and period 4 : 57 to 67 days), we define four different parameters (see -The First Overwashed Day (FOD): the first day of the incubation for which an overwash was recorded for each specific period.

Statistical analysis

As all females laid only one clutch during the study period, they could be treated independently in the analysis. We decided to eliminate nests laid in vegetation and nests depredated prior to a completed period (about 67 days). First, we performed one-way ANOVAs using either SCLmin, the number of yolked eggs or the number of yolkless eggs as the dependent variable, and the state of nest (overwashed or non-overwashed) was treated as the independent variable. Generalized linear models (GLM) were also performed to assess the effect of overwash on (1) hatching success and (2) percentage of dead embryos (corrected or not) in the different categories 1 to 4. Nests were considered as overwashed when they were covered by tide at least once during the incubation time. Second, GLM were performed to assess the effect of TD of each different period on the corrected percentage of corresponding dead embryos for overwashed and non-overwashed nests. Finally, GLM were performed among overwashed nests to investigate variables responsible for developmental arrest through incubation periods (OD, FOD, NOD). Preliminary analysis was carried out to estimate the correlation between OD and NOD; if this correlation was significant, we selected only OD for the GLM. One model with first-order interactions was fitted for each period and corresponding embryonic category n:

G n (pcE n ) = OD n + FOD n + NOD n + error
where G is a link function and n corresponds to one period as defined above. OD, FOD and NOD were treated as continuous independent variables. As dependent variables were percentages (pcE n ), we used binomial error distribution and logit link function. Computations were performed with the SAS package (procedure MIXED, v. 9.1.3, SAS Institute Inc., 2004).

Results

Over a period of 15 days, we marked and followed 97 nests along a 300 m stretch of beach. At the end of the study, 5 nests were lost by erosion, 6 nests by dog predation and 2 nests by human poaching. Of the remaining 84 nests, 22 had been overwashed at least once, 55 nests were non-overwashed and 7 were nested in vegetation. Therefore, the remaining 77 nests were analysed for hatching success and the effect of overwash on embryonic mortality assessed.

Hatching success and embryonic mortality

First, we tested whether there were significant differences between the characteristics of females (SCLmin) and clutch size for overwashed and non-overwashed nests. We found no difference between the general characteristics in these two categories; the SCLmin and the number of yolked and yolkless eggs were very similar (Table 1A).

However, the hatching success and the percentage of total dead embryos were significantly different between nests that were overwashed or not (Table 1A). Hatching success was significantly smaller for nests that were overwashed. The percentage of total dead embryos was significantly higher in overwashed nests (Table 1A).

Embryonic arrest vs. time of overwash

We calculated the percentage of nests with dead embryos for each size of embryo for overwashed and non-overwashed nests and we compared this with the percentages of overwashed nests for each day of incubation (Fig. 2). We observed different patterns of embryo distribution:

-Period 1 between 0 and 23 days (corresponding to an embryo size of <10 mm) in which there was the same frequency of occurrence for the overwashed and non-overwashed nests, approximately 100%, meaning that all nests had embryos of <10 mm. However, the percentage of dead embryos of <10 mm was significantly different between nests that were overwashed or not (50.32% and 29.73%, respectively, Table 1B). During this period, the overwashed nests were frequently overwashed during the 16 first days.

-Period 2 between 24 and 43 days (corresponding to an embryo size of 10 to 30 mm) in which there was only one embryo. This was probably due to the fact that we had no nests overwashed between the times of incubation.

-Period 3 between 44 days and 56 days (corresponding to an embryo size of 30 to 60 mm), in which there was a majority of embryos from overwashed nests. During this period 15/22 nests were inundated. As tidal overwash began to occur in nests (day 44), the percentage of nests with dead embryos began to increase (Fig. 2). The percentage of dead embryos 30-60 mm (corrected or not) was significantly different between nests that were overwashed or not (Table 1B).

-Period 4 between 57 to 67 days (corresponding to an embryo size >60 mm), in which we observed an important percentage of nests with dead embryos from overwashed and nonoverwashed nests. However, the percentage of dead embryos of >60 mm was significantly different between overwashed and non-overwashed nests (pcE 4 : 25.87% and 4.21%, respectively, Table 1B). During this period 15/22 nests were overwashed.

Sensitivity of embryos vs. parameters of overwash

To study more precisely the effect of overwash on embryonic mortality, we selected three different periods corresponding to the presence of embryo size: period 1 (1 to 24 days), period 3 (44 to 56 days) and period 4 (57 to 67 days). First, we performed GLM to assess the general effect of TD (sum of daily distance to the high-tide line) during two periods (period 1 and period 4 ) on the percentage of corresponding dead embryos (pcE n ) for overwashed and non-overwashed nests (Table 2A). We did not perform GLM for period 2 (no overwash) or for period 3 , because the TD and the number of dead embryos were very low (Fig. 3). For the two periods (1 and 4), TD was found to be significant for overwashed nests and not significant for non-overwashed nest (Table 2A). The percentage of dead embryos increased with TD.

Second, GLM were performed among overwashed nests to investigate more precisely variables responsible for embryonic arrest (OD, FOD, NOD). Preliminary analysis found a significant correlation between OD and NOD (R=0.87). We selected therefore only OD for the GLM (G 1 (pcE 1 ) = OD 1 + FOD 1 + error and G 4 (pcE 4 ) = OD 4 + FOD 4 + error for period 1 and period 4 , respectively). The GLM model for period 1 : OD 1 and FOD 1 was significant (Table 2B). The percentage of embryos E< 10 increased with OD 1 and when the FOD 1 decreased (the earlier the nest was overwashed, the more abundant were the dead embryos).

The GLM model for period 4 : Only OD 4 was found to be significant. The percentage of embryos E >60 increased with OD 4 . The non-significance of FOD 4 was probably due to the short duration of this period (Table 2B).

Discussion

In marine turtles, the physical conditions experienced by the egg during incubation in the nest have been shown to strongly affect embryonic development and hatching rate [START_REF] Mortimer | The influence of beach sand characteristics on the nesting behavior and clutch survival of green turtles[END_REF][START_REF] Ackerman | The nest environment and the embryonic development of sea turtles[END_REF][START_REF] Marco | Soil acidification negatively affects embryonic development of flexible-shelled lizard eggs[END_REF]. As female marine turtles do not provide parental care to eggs or offspring, female nesting behaviour and nest site selection are important factors that can largely affect the development of embryos and the survival of juveniles [START_REF] Kamel | Nest site selection in leatherbacks, Dermochelys coriacea: Individual patterns and their consequences[END_REF]. Our results showed first that many nests were inundated at least once during the incubation periods and that these nests were not automatically associated with null hatching success, but presented a higher proportion of dead embryos than in non-overwashed nests. This result highlights that overwash may affect important variables for embryonic development leading to embryo mortality. Mortality in our study is probably caused by fluctuations of those important physical or chemical variables for embryo development, caused by overwash, rather than overwash alone. Embryonic mortality (stage and proportion) also seems to be linked with the date of the overwash event during the incubation, the frequency and the level of overwash.

Effects of overwash

The effects of the hydric environment and immersion of the nest during incubation have been studied in reptiles [START_REF] Losos | Effect of immersion in seawater on egg survival in the lizard Anolis sagrei[END_REF] but relatively little attention has been given to those effects in marine turtle. Indeed, [START_REF] Kennett | Underwater nesting by the Australian freshwater turtle Chelodina rugosa: effect of prolonged immersion and eggshell thickness on incubation period, egg survivorship, and hatchling size[END_REF] studied the northern longnecked turtle, Chelodina rugosa, the only reptile known to deposit its eggs underwater (Kennett et al., 1993a). They showed that prolonged immersion of eggs in water results in smaller hatchlings and shorter incubation times, but does not increase egg mortality during incubation. However, other studies have shown that even short-term immersion is fatal for reptilian embryos (Tryonix muticus muticus, [START_REF] Plummer | Some aspects of nesting success in the turtle, Tryonix muticus[END_REF]Crocodylus porosus, Webb et al., 1977; Alligator mississipiensis, [START_REF] Kushlan | Environmental variability and reproductive success of Everglades alligators[END_REF]Pseudemys nelsoni, Kam, 1994). Embryo death normally results from the influx of water into the egg, with consequent egg swelling and rupture of shell and membranes, but these changes do not occur in C. rugosa eggs (Kennett et al., 1993b). Moreover, for marine species the effect is more important, especially due to the variation in water salinity that may be related to rainfall and tidal inundation. Studies generally concern Caretta caretta or Chelonia mydas in sea turtles [START_REF] Foley | Effects of sand characteristics and inundation on the hatching success of loggerhead sea turtle (Caretta caretta) clutches on lowrelief mangrove islands in southwest Florida[END_REF][START_REF] Mortimer | The influence of beach sand characteristics on the nesting behavior and clutch survival of green turtles[END_REF]. The effect of moisture and/or immersion on eggs of leatherback turtles has, for example, been little studied, while their nests are the most likely to be inundated [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF]. In our study, immersion of eggs is not necessarily fatal to the whole clutch, thus highlighting that the eggs can withstand flooding episodes The failure of development during tidal overwash may actually be explained by the fluctuations of the three most important physical variables affecting the survival of reptilian embryos: temperature, water content and respiratory gases [START_REF] Packard | The physiological ecology of reptilian eggs and embryos[END_REF]. Because leatherbacks nest closer to the tide line than other marine turtles, their nest are more exposed to fluctuations of these important variables. There is an optimal range of temperatures above and below which hatchability decreases. Hatchability is generally known to decrease significantly at lower temperature. The effects of large daily temperature fluctuations on embryonic development are unknown, but prolonged exposure of reptilian eggs below optimum temperature can result in developmental abnormality and embryonic mortality [START_REF] Packard | The physiological ecology of reptilian eggs and embryos[END_REF]. Moreover, local conditions such as rainfall and tidal overwash may induce pronounced cooling and decrease hatchability if temperature is below minimum temperature for embryonic development [START_REF] Hewavisenthi | Incubation environment and nest success of the flatback turtle (Natator depressus) from a natural nesting beach[END_REF]. During overwash, eggs can also be confronted with increased water content and lower gas content, leading to higher moisture and limited or blocked gas exchange responsible for asphyxiation and death of the embryo [START_REF] Ackerman | Growth and gas exchange of embryonic sea turtles (Chelonia, Caretta)[END_REF][START_REF] Özdemir | Embryonic mortality in loggerhead turtle (Caretta caretta) nests: A comparative study on Fethiye and Göksu delta beaches[END_REF]. Indeed, the hatching success of loggerhead turtle nests has been shown to decrease when water content increases in this way [START_REF] Foley | Effects of sand characteristics and inundation on the hatching success of loggerhead sea turtle (Caretta caretta) clutches on lowrelief mangrove islands in southwest Florida[END_REF]. Finally, eggs can be confronted with increased salinity, which is known to reduce the ability of eggs to absorb water [START_REF] Bustard | Physical and chemical factors affecting hatching in the green turtle, Chelonia mydas (L.)[END_REF]) and to disrupt cellular metabolism as a result of exposure to higher salinities and therefore chloride toxicity [START_REF] Bustard | Physical and chemical factors affecting hatching in the green turtle, Chelonia mydas (L.)[END_REF]).

Mortality during inundation may be related to the stage of development at which inundation occurs. Our results showed a relationship between time of incubation and embryonic arrest. Early and late embryonic stages were identified as the critical periods in the embryonic development of turtles [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF][START_REF] Girondot | Hatchling success for Dermochelys coriacea in a French Guiana hatchery[END_REF]Girondot et al., , Özdemir et al., 2008)). The present study is consistent with these results, since embryonic mortality was higher in both of these stages than during the middle stage. However, there were few inundation events during the middle stages of incubation in our study nests. This study is the first to analyse the daily situation of the nest, and we found a relationship between overwash event and mortality of embryo, with a clear match between days of overwash and embryonic development arrest, judging by the size of dead embryos (see Fig. 2). More research is needed to determine precisely whether the middle stages of incubation are more resilient to the negative impacts of overwash. [START_REF] Bell | Low reproductive success of leatherback turtles, Dermochelys coriacea, is due to high embryonic mortality[END_REF] studied the development of leatherback embryos in the laboratory and showed that the early stages appeared to be critical.

The late stage seems to be a sensitive period for the development for all nests in general. We found this stage of arrested development in each nest (overwashed or not), although the rate was higher for overwashed nests. This sensitivity appears to result from an increase in gas exchange between the embryo and the nest environment at the end of development (higher metabolism) and a period of immersion at this stage is likely to be more damaging because of higher sensitivity to hypoxia [START_REF] Thompson | Oxygen-consumption and energetics of development in eggs of the leatherback turtle, Dermochelys coriacea[END_REF]. In addition, we found that mortality during overwash could also be largely dependent on the frequency and the length of overwash.

However, overwashed nests in this study also showed high hatching rates, which suggests that nests in marine turtles have a certain tolerance to immersion and can survive a substantial period of time in saltwater. However, if immersion is too long or too frequent, mortality increases drastically. Calcium is another factor linked to inundation which has already been shown to affect development. Calcium is a major component of the eggshell [START_REF] Ewert | Normal and multiple eggshells in batagurine turtles and their implications for dinosaurs and other reptiles[END_REF]Packard et al., 1984a, Packard et al., 1984b) and is leached from the shell during immersion [START_REF] Seymour | Osmotic balance in the eggs of the turtle Chelodina rugosa during developmental arrest under water[END_REF]. Eggshell is an important source of calcium for the developing embryo [START_REF] Packard | Patterns of mobilization and deposition of calcium in embryos of oviparous, amniotic vertebrates[END_REF], and reduced calcium availability may limit embryonic growth. The loss of calcium also potentially alters eggshell permeability and leads to higher rates of water exchange (water uptake by the yolk and increases in yolk mass and size, [START_REF] Kennett | Underwater nesting by the Australian freshwater turtle Chelodina rugosa: effect of prolonged immersion and eggshell thickness on incubation period, egg survivorship, and hatchling size[END_REF]. Finally, when nests were greatly overwashed at the very beginning of the incubation (as in the nest in Fig. 1), hatching success was found to be null with pE 1 =100%; this period of immersion is likely to have caused the death of all the eggs. However, it should be noted that yolked eggs with no visible sign of development may not have been fertilized, although results on the population of leatherback along the Guianas [START_REF] Whitmore | Infertility, embryonic mortality and nest-site selection in leatherback and green sea turtles in Suriname[END_REF] and Costa Rica coasts [START_REF] Bell | Low reproductive success of leatherback turtles, Dermochelys coriacea, is due to high embryonic mortality[END_REF] showed a very low rate of infertile yolked egg ~ 6%.

In our study, only nests that were covered once or more by tide were included in the analysis of the effects of overwash on embryonic development. However, nests that have not been covered by tide might also suffer from the adverse effects of inundation because of immersion due to the water table below the surface of the beach. The water table is the level at which the ground water pressure is equal to atmospheric pressure, and is above the mean sea level [START_REF] Li | Beach water table fluctuations due to wave run-up: Capillarity effects[END_REF]. We were unable to assess the height of the water table and it is therefore likely that we underestimated the number of nests affected by the deleterious effects of immersion.

Nesting beaches threatened

Nest placement has major consequences for offspring survival and it is likely that this behaviour is or has been under strong selection [START_REF] Mrosovsky | Distorting gene pools by conservation: Assessing the case of doomed turtle eggs[END_REF]. If nest site selection has a genetic basis, then individual females should be consistent in their particular choice of nesting sites; that is, their choices should be repeatable [START_REF] Boake | Repeatability: Its role in evolutionary studies of mating behavior[END_REF]. The significant repeatability of nest-site choice with respect to distance from the high-tide line suggests that this behaviour may show evolutionary potential [START_REF] Kamel | Nest site selection in leatherbacks, Dermochelys coriacea: Individual patterns and their consequences[END_REF]. In this case, it appears that leatherbacks may have the opportunity for further evolution of nest-site choice in response to selection. This is particularly important in the context of current environmental changes and habitat destruction and alteration. Many of these areas are particularly susceptible to coastal hazards such as storm surges and coastal erosion and, with the projected rise in sea level resulting from anthropogenic global warming, threats to coastal areas are increasing [START_REF] Huang | Climate change and integrated coastal management: a challenge for small island nations[END_REF]. Increases in sea-level rise are thought to lead to increases in high-tide levels and the frequency of storm events [START_REF] Kurle | Regional differences in foraging of young-of-the-year steller sea lions Eumetopias jubatus in Alaska: stable carbon and nitrogen isotope ratios in blood[END_REF]. Coastal environment and nesting beaches are therefore threatened with more frequent flooding events that are supposed to decrease the viability of the turtles' nests. This threat is especially true for narrow beaches, such as Yalimapo, on which nests are already located close to the high-tide line, thus exposing them to a greater risk of nest overlap and tidal inundation. Reduction of the size of nesting beaches has implications for reproduction success, especially in major nesting sites such as Yalimapo where density-dependent effects have already had a detrimental effect on nest development (Caut et al., 2006b).

However, susceptibility to storm-related threats may vary according to species [START_REF] Pike | Sea turtle species vary in their susceptibility to tropical cyclones[END_REF]; species with lower nest-site fidelity, such as leatherback turtles, would be less vulnerable than those with higher site fidelity [START_REF] Lahanas | Molecular evolution and population genetics of greater Caribbean green turtles (Chelonia mydas) as inferred from mitochondrial-DNA control region sequences[END_REF][START_REF] Dutton | Global phylogeography of the leatherback turtle (Dermochelys coriacea)[END_REF][START_REF] Hatase | Population structure of loggerhead turtles, Caretta caretta, nesting in Japan: bottlenecks on the Pacific population[END_REF]. Moreover, highly dynamic and interseasonally variable nesting beaches in the Guianas [START_REF] Plaziat | Evolution of progradation/ erosion along the French Guiana magrove coast: a comparison of mapped shorelines since the 18th century with Holocene data[END_REF], allow turtles to maintain successful nesting [START_REF] Kelle | Monitoring of nesting leatherback turtles (Dermochelys coriacea): contribution of remote-sensing for real time assessment of beach coverage in French Guiana[END_REF], despite the fact that some beaches disappear between nesting years. Such behavioural flexibility may offer opportunities to colonise new beaches, with nesting now occurring at recently formed sites at areas opened after glacial retreats.Whether turtles can colonise nesting areas made available, either thermally or geographically, by climate change remains to be seen [START_REF] Hawkes | Climate change and marine turtles[END_REF]. Although it is possible that other beaches in the area could be used, extensive coastal development is occurring regionally, and alternatives are likely to become increasingly scarce. Until a critical habitat can be more clearly identified, it is essential from a conservation standpoint that the choice of beach and new site is not limited because reproductive success could be compromised.

Fig

  Fig. 1):

Figure 1 :

 1 Figure 1: Example of overwashed pattern in one nest during the 67 days of the study. Daily Distances of the High Tide Line (DDHTL) to the nest are shown. Negative distance represents

Figure 2 :

 2 Figure 2: Percentages of nests (1) with dead embryos for each size of embryo (according to nests overwashed and non-overwashed), (2) overwashed for each day of incubation. The x-

Figure 3 :

 3 Figure 3: Relationships between percentage of embryos corrected (pcE n ) and the Total Distance (TD) of the nest to the tide line on the Yalimapo beach. Overwashed nests are

  Figure 1 686 687

Table 1 :

 1 (A) Differences between overwashed nests and non-overwashed nests for different parameters: female size (SCLmin), clutch size (Number of yolked and yolkless eggs), hatching success (%) and the percentage of total dead embryos (pE Total ). (B) Percentage of dead embryos corrected (pcE n ) or not (pE n ) for different size categories corresponding to the four different periods. A nest was considered overwashed when it was overwashed during the period corresponding to the embryonic size.

		Overwashed nests		Non-overwashed nests
		Mean	SD	n	Mean	SD	n	F/χ²	P
	A / SCLmin	161.8	8.3	22	161.7	8.0	55	<0.01	0.958
	Number of yolked eggs	90.3	21.0	22	86.9	21.7	55	0.39	0.534
	Number of yolkless eggs	21.9	8.1	22	25.0	10.2	55	1.64	0.204
	Hatching success	28.3	26.8	22	44.9	17.9	55	168.42	<0.001
	pE Total	54.5	32.1	22	33.2	17.1	55	263.75	<0.001
	B / pE 1	50.3	31.0	15	29.7	16.9	62	166.69	<0.001
	pE 3	2.0	3.5	14	0.4	2.1	63	29.57	<0.001
	pcE 3	3.8	7.3	13	1.0	4.6	62	42.30	<0.001
	pE 4	11.9	16.7	16	2.8	2.7	61	184.65	<0.001
	pcE 4	25.9	29.2	16	4.2	4.0	59	238.57	<0.001

Table 2 :

 2 Variations of the corrected percentage of dead embryos during period 1 (0-23 days) and period 4 (57 to 67 days). (A) Effect of TD (Total Distance) of each different period on the corrected percentage of corresponding dead embryos for overwashed and non-overwashed nests. (B) Effect of OD (Overwashed Distance) and FOD (First Overwashed Day) of each different period on the corrected percentage of corresponding dead embryos for overwashed nests.

			Overwashed nests	Non-overwashed nests
	Variables	Predictors	χ²	P	χ²	P
	A / pcE 1	TD 1	224.35	<0.001	1.77	0.184
	pcE 4	TD 4	167.47	<0.001	0.54	0.462
	B / pcE 1	OD 1	126.29	<0.001
		FOD 1	97.22	<0.001
	pcE 4	OD 1	134.70	<0.001
		FOD 1	0.59	0.4440

Acknowledgments

We would like to thank L. Antoni, J. Bernard and S. Quetre for their help in the field, E.

Stanek for the design of the probe, E. Angulo for statistical analysis and Matthew Godfrey for his helpful comments on this paper. We wish to thank Jacqueline Minett for improvement of the English. DIREN-Guyane provided financial and legal support (1516 1D/1B/ENV of 27 August 2001) and the use of the facilities of the Amana Nature Reserve (Réserve Naturelle de l'Amana).