Gagné
email: francois.gagne@ec.gc.ca.

Neuroendocrine disruption in Mya arenaria clams during gametogenesis at sites under pollution stress

Keywords: Gametogenesis, vitellogenin-like proteins, serotonin/dopamine ratio, oxidative stress, inflammation, Mya arenaria

This study examined the neuroendocrine status of clams on intertidal mud flats in the St.

Lawrence Estuary and Saguenay Fjord areas during late gametogenesis. The impact of pollution was determined by a test battery of early stress markers (metallothioneins, heme levels, glutathione S-transferase activity), tissue damage (lipid peroxidation and DNA damage) and morphologic characteristics (age, soft-tissue weight ratio and growth index).

Neuroendocrinal status was examined by tracking serotonin and dopamine metabolism, monoamine adenylate cyclase activity in synaptosomes, monoamine oxidase and arachidonate cyclooxygenase activities in relation to gametogenetic activity: pyrimidine synthesis, (aspartate transcarbamoylase activity or ATC), vitellogenin-like proteins and gonado-somatic index. The results show that clam soft tissue weights were reduced at sites close to harbours and higher at sites near domestic wastewater outfalls. The age-tolength ratio of clams was generally higher at impacted sites, suggesting reduced growth.

The biomarkers of stress or damage confirmed that oxidative stress, DNA damage, metallothioneins and glutathione S-transferase activity were significantly increased at varying degrees, at the polluted sites. Vitellogenin-like proteins and gametogenetic activity were significantly increased at sites influenced by domestic wastewaters.

Furthermore, the clams were still in active gametogenesis and not ready for spawning, as indicated by the concordance of the serotonin/dopamine ratio with vitellogenin-like proteins and pyrimidine synthesis. However, gonadal cyclooxygenase activity was increased at polluted sites and significantly correlated with most of the stress biomarkers, suggesting that the clams were in a state of inflammation rather than at the spawning stage. Finally, a multivariate analysis revealed that the sites were readily discriminated with high efficiency (> 71%) and that both neuroendocrine physiological markers and stress responses were identified as the major components, thus explaining the global physiological response of the clams. We conclude that the effects of pollution compromise the clams' health status and that the initiation of gametogenesis in environments contaminated by municipal wastewaters or harbours contributes to the toxic effects of pollution.

Introduction

The St. Lawrence Estuary, along with its unique fjord (Saguenay River), is subject to all the different types of contaminants resulting from urban, industrial and agricultural activities. Indeed, intensive boating activities release chemicals such as polycyclic aromatic hydrocarbons (PAHs) and metals, including organotins, near harbours and marinas. In a previous study, gonadal tissues of Mya arenaria clams in the same area of Quebec, Canada, were contaminated with tributyltin (TBT) at concentrations as high as 400 ng/g dry weight (dw) [START_REF] Pellerin | Evidence of endocrine disruption in Mya arenaria located near the mouth of the Saguenay Fjord linked to TBT sediment contamination[END_REF]. The proportion of male clams was also higher at a harbour site contaminated by TBT [START_REF] Gagné | Sex alteration in soft-shell clams (Mya arenaria) in an intertidal zone of the Saint-Lawrence River (Québec, Canada)[END_REF]. In marine environments, domestic wastewaters are often released into the sea following minimal treatment, if treated at all. These wastewaters are known to contain various contaminants, with clear evidence of endocrine-disrupting effects. Indeed, municipal effluents are recognized to contain both estrogenic and serotonergic compounds, as suggested by increased vitellogenin-like proteins, cyclooxygenase activity and spawning in exposed mussels (Blaise et al., 2003a;[START_REF] Tilton | Evaluation of estrogenic activity from a municipal wastewater treatment plant with predominantly domestic input[END_REF][START_REF] Gagné | Serotonergic effects of municipal effluents: induced spawning activity in freshwater mussels[END_REF]. Clam populations at polluted sites adjacent to townships displayed various signs of pollution-related stress, including altered vitellogenin production.

To further investigate the effects of pollution on gametogenesis, specimens of feral clam populations living in intertidal mud flats in the Saguenay Fjord and St. Lawrence Estuary were collected from sites influenced by domestic effluents and harbours (Figure 1). In an attempt to relate reproductive endocrine disruption with anthropogenic pollution, we used a comprehensive test battery of biomarkers to evaluate exposure to pollutants, defense mechanisms, tissue damage, general health condition and reproductive (gametogenetic) function. Biomarkers selected for monitoring early biological effects and defense mechanisms were metallothioneins (MT) and total heme levels, for heavy metal effects [START_REF] Viarengo | A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs[END_REF][START_REF] Simionatto | Fluorometric measurement of tin-protoporphyrin in biological samples[END_REF], and glutathione S-transferase, a major enzyme responsible for the conjugation of electrophilic organic molecules such as (hydroxylated) PAHs [START_REF] Gowland | Implications from a field study regarding the relationship between polycyclic aromatic hydrocarbons and glutathione S-transferase activity in mussels[END_REF]. Next, tissue damage was determined by measuring lipid peroxidation and DNA damage [START_REF] Aldridge | Stages in the Induction of Toxicity[END_REF][START_REF] Doyotte | Antioxidant enzymes, glutathione and lipid peroxidation as relevant biomarkers of experimental or field exposure, in the gills and the digestive gland of the freshwater bivalve Unio tumidus[END_REF].

Total heme levels were also measured to evaluate hemoprotein breakdown by toxicants such as organotins (Siminiatto et al., 1984). Increased hemoprotein breakdown could result in delayed steroidogenesis owing to decreased cytochrome P450 activity.

Neuroendocrine parameters of gametogenesis and spawning were examined by following gonadal serotonin and dopamine metabolism, mono-amine oxidase, cyclooxygenase (COX) activities, vitellogenin-like proteins, aspartate transcarbamoylase (ATC) and the gonado-somatic index. During gametogenesis, dopamine and estradiol-17 levels are increased to support vitellogenesis (oogenesis) and spermatogenesis [START_REF] Osada | Estrogen effect on the seasonal levels of catecholamines in the scallop Patinopecten yessoensis[END_REF][START_REF] Li | Changes in vitellin during oogenesis and effect of estradiol on vitellogenesis in the Pacific oyster Crassostrea gigas[END_REF][START_REF] Gauthier-Clerc | Estradiol-17ß and testosterone concentrations in male and female Mya arenaria (Mollusca bivalvia) during the reproductive cycle[END_REF]. ATC is a step-limiting enzyme complex for pyrimidine biosynthesis to support nucleic acid production in the gametes [START_REF] Mathieu | Utilization of aspartate transcarbamylase activity in the study of neuroendocrinal control of gametogenesis in Mytilus edulis[END_REF]. Its activity is also enhanced and closely follows the gonado-somatic index in bivalves. In female gonads, oocytes accumulate vitellogenin-like proteins as the main energy source for the developing embryo [START_REF] Osada | Vitellogenin synthesis in the ovary of the scallop, Paatinopecten yessoensis: control by estradiol-17 and the central nervous system[END_REF][START_REF] Li | Changes in vitellin during oogenesis and effect of estradiol on vitellogenesis in the Pacific oyster Crassostrea gigas[END_REF].

Levels of serotonin and prostaglandin synthase or COX are gradually increased during the final maturation of eggs and spermatocytes, reaching maximum values to assist the spawning process [START_REF] Osada | The levels of prostaglandins associated with the reproductive cycle of the scallop, Patinopecten yessoensis[END_REF][START_REF] Osada | Implication of catecholamines during spawning in marine bivalve molluscs[END_REF][START_REF] Fong | Dopamine-reduced intensity of serotonin-induced spawning in the zebra mussel Dreissena polymorpha (Pallas)[END_REF].

The objective of this study was to characterize the neuron-endocrine parameters in feral Mya arenaria clams at the ripe (late) stage of gamete formation just before spawning.

Moreover, the impacts of pollution on the neuron-endocrine status of gametogenesis were also examined in clam populations under the influence of harbors and domestic wastewaters. To this end, biomarkers of health status and tissue damage in clams were examined in relation to those of gametogenetic activity and neuro-endocrine parameters (monoamine metabolism and cyclooxygenase). An attempt was made to examine the interaction of gametogenesis and pollution stress in respect to the health status in feral clam populations.

Methods

Sampling sites and clam handling

Soft-shell clams (Mya arenaria) were collected from six sites in the Saguenay Fjord and St. Lawrence Estuary during a field survey conducted in June 2004 (Figure 1): Baie Sainte-Catherine (BSC) where the clam population was located between the mouth of a river draining a small town and a wharf with heavy boat traffic (< 100 m); Anse St-Jean (ASJ), within (< 100 m downstream the source) the municipal effluent dispersal plume of a population of 2000 residents; Baie-Éternité (BE), close (<10 m) to a wharf for a seasonal commercial sight-seeing boat operation and a marina for small pleasure craft; Anse aux Érables (AE) bay, the farthest upstream site in the Saguenay Fjord (70 km) and influenced by several aluminum and paper mills located in the cities of Alma, Jonquière, Chicoutimi and La Baie, which discharge their effluents into the Saguenay River and Fjord. The wastewaters of 15 seasonal cottages and the upstream waters of Alma also affect site AE. Anse Saint-Étienne (ASE) and Baie du Moulin à Baude (BAU) are not affected by any direct source of anthropogenic activity and therefore considered as the "pristine" sites for the Saguenay Fjord and St. Lawrence Estuary, respectively, in this study (Figure 1). Thirty animals with a shell length greater than 4 cm were collected at each site for biochemical measurements. The clams were immediately placed in coolers chilled with ice (T = 0-4 o C) for transport to our field laboratory. Twelve animals were randomly selected for each site, frozen in dry ice and brought back to the St. Lawrence Centre laboratories, where they were placed in a -85 o C freezer until analysis. The remaining clams were dissected for gill and gonadal tissue weights and sex determinations [START_REF] Gagné | Sex alteration in soft-shell clams (Mya arenaria) in an intertidal zone of the Saint-Lawrence River (Québec, Canada)[END_REF]. For the chemical analyses, soft tissues of Mya arenaria clams were collected (n=12 individuals per site) and homogenized with a Polytron tissue grinder apparatus. The homogenized sample was then analyzed for heavy metals by ion-coupled plasma mass spectrometry (NLET 1994). Blank values consisted of distilled water that was processed with the Polytron mixer.

Individual morphometrics and tissue processing

Twelve clams of similar size from each sample site were thawed on ice. Each animal was measured (shell length in mm) and weighed (g wet weight of soft tissue) to determine the soft tissue weight ratio (g wet weight of soft tissue/g wet weight of clam). Age was estimated from the number of major grooves on shells. Each specimen's digestive gland (hepato-pancreas), gonadal and gill tissues were removed and homogenized with a Teflon pestle tissue grinder in ice-cold 25 mM Hepes-NaOH buffer, pH 7.4, containing 125 mM NaCl, 1 mM EDTA and 1 mM dithiothreitol at a 1:5 weight/volume ratio. Aliquots of each homogenate were taken for total protein determinations [START_REF] Bradford | A sensitive method for the total protein determination using the principle of protein-dye binding[END_REF], monoamines, heme levels, DNA strand breaks and lipid peroxidation (LPO).

Concentrations of these biomarkers were normalized with the total protein concentrations of each homogenate. A portion of the homogenate was centrifuged at 1500 x g for 10 min for the preparation of synaptosomes and mitochondria. The remaining homogenate was centrifuged at 15 000 x g for 30 min at 2 o C. The supernatant was then carefully separated from the pellet for cyclooxygenase (COX), metallothioneins (MT) and glutathione Stransferase (GST) activity determinations. These biomarkers were also normalized with total protein concentrations determined in the supernatant.

Reproductive function

Gametogenesis was estimated by measuring the gonado-somatic index (GSI; weight of gonad/soft tissue weight), relative levels of vitellogenin-like proteins (Vtg) and ATC activity. Sex was determined by microscopic examination of gonad smears at 400X enlargement. The presence of Vtg in gonad was determined by the organic alkali-labile phosphate (ALP) technique using the acetone fractionation procedure [START_REF] Gagné | Sex alteration in soft-shell clams (Mya arenaria) in an intertidal zone of the Saint-Lawrence River (Québec, Canada)[END_REF]. Vtg-like protein levels were expressed as µg of alkali-labile phosphate (ALP)/mg of gonad protein. Gamete activity was determined by measuring the activity of aspartate transcarbamoylase (ATC) in the S15 fraction of gonad homogenate (Mathieu et al., 1982) using the spectrophotometric detection of liberated phosphate in the presence of carbamoylphosphate [START_REF] Herries | The simultaneous estimation of orthophosphate and carbamoylphosphate and application to the aspartate transcarbamoylase reaction[END_REF].

General health status and stress biomarkers

Metallothionein (MT) levels were determined in the digestive glands of the clam specimens. Relative levels of MT proteins were determined by the thiol spectrophotometric assay [START_REF] Viarengo | A simple spectrophotometric method for metallothionein evaluation in marine organisms: an application to Mediterranean and Antarctic molluscs[END_REF]. The homogenate was centrifuged at 15 000 x g for 30 min at 2 o C and the supernatant carefully removed from the upper lipid layer. The MT fractionation and assay procedure was performed on the 15 000 x g supernatant. Standards of reduced glutathione (GSH) were used for calibration. The data are expressed as µmoles of GSH equivalents per mg protein. DNA damage (double and single DNA strand breaks) was assessed in gills, digestive glands and gonad by means of an alkaline precipitation assay [START_REF] Olive | DNA precipitation assay: a rapid and simple method for detecting DNA damage in mammalian cells[END_REF] with fluorescence quantification of DNA strands [START_REF] Bester | Cholate and pH reduce interference by SDS in the determination of DNA with Hoescht[END_REF]. The assay principle is based on K-assisted SDS precipitation of genomic DNA (associated with proteins), which leaves protein-free DNA breaks (double and single strands) in the supernatant. Salmon sperm DNA standards were added for DNA calibration. The results were expressed as µg of supernatant DNA/mg of proteins. LPO was determined by the thiobarbituric acid method, which measures the production of malonaldehyde [START_REF] Wills | Evaluation of Lipid Peroxidation in Lipids and Biological Membranes[END_REF]. Briefly, 20 µL of the homogenate was diluted to 80 µL with distilled water and mixed with 50 µL of 20% trichloroacetic acid solution containing 1 mM FeSO 4 and 100 µL of 0.67% thiobarbituric acid. The mixture then stood in a hot water bath (80 o C) for 10 min. Blanks and standards of tetramethoxypropane were prepared in the presence of the homogenization buffer. All assay tubes then stood at room temperature for 15 min to allow denatured proteins to settle. Fluorescence was measured at 520 nm for excitation and 590 nm for emission using a fluorescence microplate reader (Dynatech, Fluorolite-1000). Because the reagent could react with other aldehydes, results were expressed as µg of thiobarbituric acid reactants (TBARS)/mg of homogenate protein. Glutathione S-transferase (GST) was determined by the spectrophotometric method of [START_REF] Boryslawskyj | Elevation of glutathione-Stransferase activity as a stress response to organochlorine compounds, in the freshwater mussel, Sphaerium corneum[END_REF] using 2, 4dichloronitrobenzene as the substrate. Data are expressed as increase in absorbance/min/mg proteins. Total heme was determined by fluorescence according to the method of [START_REF] Simionatto | Fluorometric measurement of tin-protoporphyrin in biological samples[END_REF]. Briefly, 20 µL of digestive gland homogenate was mixed with 500 µL of 1 M NaOH in methanol. The mixture was allowed to stand for 5 min at room temperature and centrifuged at 10 000 x g for 5 min. Total heme was determined in the supernatant at 400 nm excitation and 685 nm emission. Standard solutions of hemin were used for calibration. Total heme levels were expressed as µg hemin/mg proteins. Cyclooxygenase (COX) activity was measured by the oxidation of 2, 7-diclhorofluorescin in the presence of arachidonate [START_REF] Fujimoto | The endocrine disruptor nonylphenol preferentially blocks cyclooxygenase-1[END_REF]. Briefly, 50 µL of gonad or gill 15 000 x g supernatant was mixed in 200 L of 50 µM arachidonate, 2 µM dichlorofluorescin and 0.1 µg/mL of horseradish peroxidase in 50 mM Tris-HCl buffer, pH 8, containing 0.05% Tween 20. The reaction mixture was incubated for 0, 10, 20 and 30 min at 30 o C, and fluorescence was measured at 485 for excitation and 520 for emission (Dynatech, Fluorolite 1000). The data were expressed as the increase in relative fluorescence units/(min x mg proteins).

Neuroendocrine assessment of gametogenesis

Gonad (containing nerve fibers) homogenates were centrifuged at 1500 x g for 15 min at 4 o C. The supernatant was overlaid on 0.8 M sucrose containing 10 mM Hepes-NaOH, pH 7.4, and 1 mM EDTA, and centrifuged at 10 000 x g for 20 min at 4 o C to separate the synaptosomes (in the 0.8 M sucrose phase) from mitochondria (pellet). The synaptosomes were mixed with two volumes of distilled water to prevent osmotic shearing of synaptosomes. Monoamine (serotonin and dopamine) levels were measured in the gonad homogenates using fluorescence-based methodologies [START_REF] Orsinger | A reliable and sensitive method for the simultaneous determination of dopamine, noradrenaline, 5hydroxytryptamine and 5-hydroxy-indol acetic acid in small brain samples[END_REF][START_REF] Szabo | A modified screening method for rapid simultaneous determination of dopamine, noradrenaline and serotonin in the same brain region[END_REF]. For serotonin, 5-hydroxyindolacetate (5-HIAA) and dopamine determinations, a sub-sample of homogenates was mixed with one volume of butanol containing 0.1 N HCl and 0.2 mM reduced glutathione (GSH). The mixture was centrifuged at 2000 x g for 15 min to remove precipitated material and the supernatant was extracted once with one volume of hexane. For serotonin, the aqueous phase was mixed with one volume of ophthalaldehyde in 2 N HCl and heated to 70 o C for 15 min. Fluorescence was measured at 360 nm excitation and 480 nm emission against standards of serotonin. For 5-HIAA, the deaminated metabolite of serotonin, the organic phase was mixed with one volume of 100 mM sodium bicarbonate pH 9.0, and levels were determined by the o-phthalaldehyde method as described above. For dopamine, the aqueous phase was mixed with a 0.1 volume of alkaline iodide solution (I 2 /KI, Lugol, Sigma Chemical Co.), heated at 75 o C for 60 min and fluorescence was measured at 325 nm excitation and 385 nm emission.

Standards of serotonin, 5-HIAA and dopamine were used for calibration. Data were expressed as ng of monoamine/mg proteins. Serotonin and dopamine transport in the presence of ATP was determined in synaptosomes using the liberation of inorganic phosphate from ATP methodology [START_REF] Kakko | The synaptosomal membrane bound ATPase as target for the neurotoxic effects of pyrethroids, permethrin and cypermethrin[END_REF]. Synaptosomes (10 µg/mL) were incubated with either 100 µM serotonin or dopamine in the presence of 100 µM ATP in the following assay buffer: 140 mM NaCl, 1 mM KH 2 PO 4 , 1 mM CaCl 2 , 10 µM GTP and 10 mM Hepes-NaOH, pH 7.4. The mixture (200 µL) was allowed to stand at 30 o C for 0, 10, 20, 30 and 40 min and the reaction was stopped with 5 µL of 100% trichloroacetic acid. The samples were centrifuged at 10 000 x g for 3 min to reduce turbidity. The levels of inorganic phosphates liberated from ATP hydrolysis were determined using the phosphomolybdate assay [START_REF] Stanton | Colorimetric determination of inorganic phosphate in the presence of biological material and adenosine triphosphate[END_REF]. MAO activity was determined using the serotonin analogue tryptamine as the substrate. Mitochrondria (0.1 to 0.5 mg/mL proteins) were incubated in the presence of 10 µM dichlorofluorescin, 100 µM tryptamine in 10 mM Hepes-NaOH, pH 7.4, containing 125 mM NaCl, 0.1 M aminotriazole (a catalase inhibitor) and 1 g/mL of horseradish peroxidase (Sigma-Aldrich Co., USA). The reaction was allowed to proceed at 30 o C for 0, 15, 30 and 60 min. Fluorescence was measured at 485 nm excitation and 520 nm emission. Enzyme activity was expressed as nmole of fluorescein formed/(min x mg proteins). Standard solutions of fluorescein were used for calibration.

Data analysis

Twelve clams (n = 12) from each site were held back for biomarker measurements. Data distribution was analysed using Levene's test for homogeneity of variances. When the data proved to be homogeneous, a two-way factorial analysis of variance was performed, with site and gender as the main factors; critical differences between groups were appraised using Dunnett's t test. In the case of nonhomogeneous data distributions, data were log 10 -transformed before performing a two-way factorial ANOVA. Correlation analyses between biomarkers were determined using the Pearson-moment correlation and factorial analysis (principal components method). Site classification according to biomarker response was determined by discriminant analysis. Biomarker data (biomarkers of stress or damage) from the spatial survey were also integrated using a ranked-sum approach to yield a biomarker index (Chèvre et al., 2003). Biomarker responses were ranked from 1 to 6 (because there were six sites), where the lowest rank value corresponds to the mean value of the reference site (i.e. 1). The biomarker responses were corrected to yield values > 1 to rank their relative intensity (e.g. a 0.2-fold decrease was corrected to produce 1.2-fold). Inter-site differences were verified using the Mann-Whitney U test where sites were assigned the same rank value when no difference was obtained between them. The rank values were then summed for each site to yield an index value. All statistical tests were performed using the Statistica software package (version 7.1).

Results

Clams were collected at various sites subject to differing types of contamination, as shown in Figure 1. To reiterate, site BE is contaminated by boating activities; site ASJ is contaminated by domestic wastewaters; and sites AE and BSC are contaminated by both these types of contamination. To confirm this, heavy metal loadings were determined in the soft tissues of clams (Table 1). Indeed, these sites were generally more contaminated in cobalt, chromium, copper, iron, mercury, manganese and nickel. The strongest increases were found for iron (14 fold increase), cobalt (2.8 fold increase), chromium (2.7 fold increase) and iron (1.9 fold increase) thus corroborating contamination of Mya arenaria clams at these sites. A two-way analysis of variance revealed that site location, but not gender, had a significant effect on soft-tissue weight ratios (Figure 2A). The softtissue weight ratio was significantly lower at BSC, but very high at the site contaminated by domestic wastewaters (ASJ; Figure 2A). Age also varied significantly across sites and gender (Figure 2B). Females were somewhat older (mean age of 6.3 y) than males (mean age value 6 y) with no apparent interaction of gender with site. Clams at BSC were significantly younger than those at BAU (the reference site for the St. Lawrence Estuary) and clams from sites ASJ and BE were significantly younger compared to those at site ASE (the reference site for the Saguenay Fjord). Clams at the most upstream site (AE) were significantly older than those from the reference site (ASE). The age-to-shell-length ratio, a measure of growth, varied with gender and site (Figure 2C). Again, females had somewhat higher growth index than males. The age-to-shell-length ratio was significantly higher at sites BSC, ASJ and AE, suggesting reduced shell growth in clams in the 5-to 8year-old age groups. A correlation analysis revealed that age was negatively correlated with the soft-tissue weight ratio (R = -0.3; p < 0.01).

Gametogenesis was appraised by measuring the gonado-somatic index (GSI), vitellogenin-like proteins (Vtg) and gamete activity by aspartate transcarbamoylase (ATC) activity (Figures 3A-C). A two-way analysis of variance revealed that only site had a significant effect on GSI (Figure 3A). GSI was significantly reduced at sites BSC, ASJ and BE, but not at site AE. The levels of Vtg proteins were significantly different across the study sites and gender had a significant effect at p = 0.0.05 (Figure 3B). As expected, VTG was significantly higher in females than in males but no significant interaction between site and gender were observed. Vtg was significantly increased at sites ASJ and AE, but not at site BE, for both female and male clams. Gonad ATC activity varied with site where gender had no significant effects (Figure 3C). ATC activity was readily induced at site ASJ alone, suggesting enhanced pyrimidine biosynthesis. A correlation analysis of these reproductive endpoints revealed that GSI was positively correlated with age (R = 0.41; p < 0.01) and negatively correlated with soft-tissue weight ratio (R = -0.3; p < 0.05). Levels of Vtg were positively correlated with soft-tissue weight ratio (R = 0.34; p < 0.05), age (R = 0.27; p < 0.05) and growth index (R = 0.39; p < 0.01), suggesting that growth is directly associated with the production of egg-yolk proteins. ACT activity was positively correlated with soft-tissue weight index (R = 0.63; p < 0.001) and slightly so with Vtg (R = 0.25; p = 0.05).

Several biomarkers of defence and tissue injury were examined to assess the health status of clams (Table 2). A two-way ANOVA indicated that MT levels in digestive gland varied with site but no with gender. MT levels were significantly higher at all polluted sites in respect to their corresponding reference sites. Glutathione S-transferase (GST) activity in the digestive gland varied significantly across all sites, but not with gender.

GST activity was readily induced at BSC site alone. Total heme levels varied across all sites, but not with gender. Heme levels were readily increased in gonadal tissues at the ASJ site, while they were unchanged at the other sites relative to the reference sites. A two-way ANOVA for digestive gland DNA strand breaks revealed that only site was significant. DNA strand breaks were readily increased at all impacted sites. LPO in gills and digestive gland varied significantly with site, but not with sex. LPO in digestive gland was significantly induced at all impacted sites with no effect with gender, with the exception of site BE where its activity was not significantly different than the reference site. Gill LPO revealed significant differences within sites, gender and site-gender interactions (2 way ANOVA p<0.01 for site, gender and site-gender interaction). Female had more LPO in gills than in males. LPO in gills was reduced in females at impacted sites in the Saguenay fjord sector while a significant gill LPO decrease at sites BSC in males only. The biomarker responses were incorporated into an index by which sites were ranked according to toxic stress: BSC > AE = ASJ > BE > BAU > ASE. A correlation analysis revealed that MT was significantly correlated with growth index (R = 0.4; p < 0.01), GST in digestive gland (R = 0.76; p < 0.01), LPO (R = 0.35; p < 0.01), and DNA strand breaks (R = 0.26; p < 0.05) in digestive gland (Table 2). Digestive gland GST was significantly correlated with GSI (R = -0.39; p < 0.05), LPO (R = 0.26; p < 0.05) and DNA strand breaks in digestive gland (R = 0.39; p < 0.01). Total heme levels in gonad were significantly correlated with soft-tissue weight ratio (R = 0.45; p < 0.01), Vtg (R = 0.38; p < 0.05) and ACT (R = 0.49; p < 0.01). DNA damage in digestive gland was significantly correlated with GSI (R = -0.31; p < 0.05), Vtg (R = 0.37; p < 0.01) and LPO in digestive gland (R = 0.51; p < 0.01). Digestive gland LPO was significantly correlated with GSI (R = -0.3; p < 0.05) and growth index (R = 0.4; p < 0.01). LPO in gills was significantly correlated with GSI (R = 0.41; p < 0.01).

The influence of site quality was examined in various neuroendocrine mediators such as serotonin, 5-HIAA, dopamine, COX and MAO activities in gonadal (and visceral ganglion) tissues (Table 4). Serotonin levels were not influenced by gender, but changed significantly within sites. Serotonin levels were readily increased at contaminated sites in both areas, with the exception of site BE. Two-way ANOVA revealed that both site and gender were significant factors for 5-HIAA levels. The metabolite of serotonin, 5-HIAA , was somewhat higher in males than in females while its levels were significantly reduced at the BSC and AE sites and elevated at site ASJ. Meanwhile, serotonin transport activity (serotonin activation of adenylate cyclase activity) remained unchanged across all sites and with gender. The serotonin-to-5H1AA ratio varied marginally with gender (p=0.08, females having increased serotonin/5HIAA ratio), but did change within sites. The ratio was significantly higher at sites AE and BE, suggesting an accumulation of serotonin in tissues. Dopamine levels were also significantly different within sites, but not with gender. Dopamine levels were significantly elevated at all polluted sites. Moreover, dopamine transport activity (dopamine activation of adenylate cyclase activity in prepared synaptosomes) varied within sites, but gender had no effect. Dopamine transport activity was significantly induced at the most stressed sites (i.e. BSC and ASJ sites) relative to the corresponding control sites (i.e. BAU and ASE, respectively). The serotonin/dopamine ratio varied significantly within sites and with gender. The serotonin/dopamine ratio was higher in females than in males (1.36 fold higher). The serotonin/dopamine ratio was significantly induced at the polluted sites AE and ASJ. A two-way ANOVA for gonad MAO activity showed that both site and sex produced significant changes. MAO activity was 1.15 fold higher in females than in males. MAO activity was significantly elevated at sites in the Saguenay Fjord area (i.e. ASJ, BE and AE). A two-way ANOVA for gonad COX activity showed that only site was significant.

COX activity was significantly elevated at sites BSC and AE. A correlation analysis revealed that serotonin was significantly correlated with growth index (R = 0.49; p < 0.01), gonad ACT (R = 0.34; p < 0.05) and gonad COX (R = 0.32; p < 0.05) activity, and digestive gland MT (R = 0.49; p < 0.01) and GST (R = 0.31; p < 0.05) activity. Serotonin transport activity, while remaining unchanged with site, was significantly correlated with growth index (R = 0.34; p < 0.05), serotonin/5H1AA ratio (R = 0.42; p = 0.01) and gonad COX activity (R = 0.37; p < 0.05). The serotonin metabolite 5-H1AA was significantly correlated with total gonad heme level (R = 0.43; p < 0.05) only. The serotonin/5HIAA ratio was significantly correlated with MAO activity (R = 0.49; p < 0.01). Dopamine levels were correlated with growth index (R = 0.48; p < 0.01), gonad ACT levels (R = 0.34; p = 0.01), Vtg (R = 0.72; p < 0.01), digestive gland GST (R = 0.31; p < 0.05) and MT levels (R = 0.49; p < 0.01) and gonad COX activity (R = 0.32; p < 0.05). Dopamine transport activity was significantly correlated with gonad ACT level (R = 0.41; p < 0.01), Vtg (R = 0.4; p < 0.05), digestive gland GST (R = 0.54; p < 0.01), DNA strand breaks (R = 0.32; p < 0.05) and MT levels (R = 0.35; p < 0.05), gonad heme levels (R = 0.88; p < 0.05) and dopamine levels (R = 0.43; p < 0.05). The serotonin/dopamine ratio was significantly correlated with soft-tissue weight (R = 0.4; p = 0.01), gonad ACT activity (R = 0.37; p < 0.05) and Vtg (R = 0.57; p < 0.01), DNA strand breaks in digestive gland (R = 0.38; p = 0.01) and dopamine transport activity (R = 0.35; p = 0.05). Gonad MAO activity was significantly correlated with Vtg (R = 0.55; p < 0.01), digestive gland LPO (R = 0.48; p < 0.01) and DNA strand breaks (R = 0.4; p < 0.01), dopamine levels (R = 0.37; p < 0.05) and serotonin/5-HIAA ratio (R = 0.49; p < 0.01). Finally, COX activity was significantly correlated with the following biomarkers: GSI (R = -0.27; p < 0.05), soft-tissue weights (R = -0.38; p < 0.01), growth index (R = 0.42; p < 0.01), digestive gland MT (R = 0.68; p < 0.01), LPO (R = 0.69; p < 0.01), GST (R = 0.54; p < 0.01) and DNA strand breaks (R = 0.37; p < 0.01).

A multivariate data analysis was performed using discriminant analysis (Figure 4) followed by a principal component (factorial) analysis to highlight the most discriminative physiological responses (Table 4). The discriminant analysis revealed that most sites were correctly classified with more than 85% efficiency with the exception of the AE site (71%). For site AE, some clams were misclassified to sites BAU (3) and BSC

(3) indicating that some clams were not responding and some displayed a response pattern close to BSC (intense traffic harbor and municipal effluent). In the Saguenay Fjord area, ASJ was the site that differ the most from the pristine site (ASE). A factorial analysis revealed that 63% of the total variance was explained by four factors (Table 5).

In descending order of factorial weights (0.79 to 0.61), the biomarkers associated to the factors were: gonad dopamine, soft-tissue weights, heme levels, GST, serotonin, COX, DNA strand breaks and Vtg. These biomarkers were associated with general health status, defense biomarkers and neuroendocrinal aspects of gametogenesis.

Discussion

Clams from the Saguenay Fjord area (sites AE, BE and ASJ) and those from the St.

Lawrence Estuary (BSC) were recognized to be suffering anthropogenic stress. Previous studies have shown that clams from BSC site were contaminated by various heavy metals, including organotin products, with clear evidence of reproductive perturbations: decreased GSI, Vtg and ATC and increased male/female ratio and oxidative stress in gonadal tissues [START_REF] Gagné | Sex alteration in soft-shell clams (Mya arenaria) in an intertidal zone of the Saint-Lawrence River (Québec, Canada)[END_REF][START_REF] Gagné | Health status of Mya arenaria bivalves collected from contaminated sites in Canada (Saguenay Fjord) and Denmark (Odense Fjord) during their reproductive period[END_REF]. The male-to-female sex ratio, Vtg and ATC were not statistically different in the present study, but GSI, age and soft-tissue weight ratio still remained significantly reduced. This suggests that pollution inputs are not constant over the years and clams can recover. Nonetheless, clams from site BSC had increased MT, GST, LPO and DNA strand breaks in the digestive gland. Based on the correlative analysis, MT was associated with neither Vtg-like proteins nor GSI but was related with serotonin and COX levels. This suggests that MT could be influenced during the spawning process and the occurrence of inflammation perhaps through infection [START_REF] Shi | Vitellogenin is a novel player in defence reactions[END_REF] in addition to heavy metal exposure. Although GST is considered a conjugating enzyme of reduced glutathione to less-polar electrophilic organic compounds, its activity was readily increased by organotin contamination in digestive gland acini of the scallop Pecten maximus (LePennec and LePennec, 2003). In another study, the population status of Mya arenaria clams in the same sector revealed that oxidative stress, in addition to reduced vitellogenesis and GSI, was associated with low clam density and mean age values in the local population (Blaise et al., 2003a). These results support the observation that this population is under constant anthropogenic stress in this area, but the long-term trends within each biomarker response could vary over the years. Despite these observations, the biomarker index revealed that this site (BSC) was the most impacted one in the area, followed by those affected by domestic wastewaters and industrial inputs (BSC > ASJ = AE > BE). The neuroendocrine biomarker data suggest these clams were experiencing delayed gametogenesis or that they were perhaps in a state related to inflammation. This latter condition was shown by the increased gonadal COX activity accompanied by increased dopamine and dopamine adenylate cyclase activity (dopamine transport in synaptosomes) and decreased serotonin levels. In the scallop Argopecten purpuratus, dopamine levels rose during the first phases of gamete development while serotonin was more closely associated with the later steps of gamete maturation and spawning [START_REF] Martinez | Role of monoamines in the reproductive process of Argopecten purpuratus[END_REF]. In the scallop, no difference was observed between the sexes (as with the clams in this study) and gonad dopamine was lowest during spawning [START_REF] Osada | Estrogen effect on the seasonal levels of catecholamines in the scallop Patinopecten yessoensis[END_REF]. Interestingly, COX activity was elevated but poorly associated with serotonin level and serotonin transport activity. During the spawning process, serotonin and COX are induced for gamete release in the scallop [START_REF] Osada | The levels of prostaglandins associated with the reproductive cycle of the scallop, Patinopecten yessoensis[END_REF], and thus significantly correlated with each other. However, a dampening of gametogenesis, as suggested by reduced GSI and increased ATC activity, points to other factors as being responsible for the increased COX activity. The association of COX activity with the condition of inflammation rather than the later stage of gametogenesis was further supported by an analysis of covariance of COX activity using serotonin and serotonin transport activity as covariates, which showed that COX activity remained significantly elevated at impacted sites.

Inflammation might be the results of infections from pathogenic micro-organisms such as viruses, bacteria and macroparasites [START_REF] Canesi | Bacteria-hemocyte interactions and phagocytosis in marine bivalves[END_REF]. However, cockles' vulnerability to pollution was enhanced by parasites through the deficit of MT synthesis [START_REF] Baudrimont | Impact of digenean parasite infection on metallothionein synthesis by the cockle (Cerastoderma edule): A multivariate field monitoring[END_REF]. Although MT synthesis was increased during gonad maturation, MT levels from parasitized animals were lower. In the present study, although decreased GSI were observed at impacted sites, we did not observe either the occurrence of major infections of macroparasites in gonad or reduced MT levels at polluted sites. In the Arctic bivalve, the influence of salinity and sexual status was practically negligible in comparison with tissue weight and the copper concentration in tissues [START_REF] Amiard-Triquet | Metallothioneins in Arctic bivalves[END_REF]. In a 2 year study with Mytilus galloprovincialis, contamination by PAHs were highest during winter but with less tissue lesions in respect to warmer months [START_REF] Bodin | Seasonal variations of a battery of biomarkers and physiological indices for the mussel Mytilus galloprovincialis transplanted into the northwest Mediterranean Sea[END_REF] suggesting that toxicity follows temperature increase and seasons. Changes in salinity were recognized to have moderate effects of some biomarkers such as heat shock proteins and GST activity (Lyon et al., 2003).

However, the interaction of temperature and salinity had a significant effect on oxidative stress [START_REF] Damiens | Evaluation of biomarkers in oyster larvae in natural and polluted conditions[END_REF]. Indeed, TBARS levels increased as a function of salinity when the temperature was maintained at 20-21 o C, whereas at 25.8 o C no effect of salinity was obtained in addition to lack of change in GST and catalase activities at 20-21 o C.

Hence, the effects of salinity, temperature and spawning were considered minimal since clams were collected during the same week before the spawning period at moderate temperatures (in early June corresponding to 12-20 o C).

Municipal effluents were recently recognized to produce an inflammatory syndrome in freshwater mussels, with increased response in oxidative stress and COX activity and reduced phagocytotic activity in hemocytes [START_REF] Gagné | Inflammatory properties of municipal effluents to Elliptio complanata mussels: lack of effects from antiinflammatory drugs[END_REF]2006). Moreover, COX activity was significantly correlated with MT, GST and LPO in the digestive gland, suggesting the implication of pollutant-related effects with bacterial infection on the state of inflammation in Mya arenaria clams. In a recent study, it has been shown that infection by Vibrio tapetis lead to modifications in several defence-related factors such as increased hemolymph protein and lysozyme activity [START_REF] Allam | Effects of the pathogenic Vibrio tapetis on defence factors of susceptible and non-susceptible bivalve species: II. Cellular and biochemical changes following in vivo challenge[END_REF]. In addition, COX activity was negatively correlated with soft-tissue weight ratio in clams, perhaps pointing to a harmful effect of this syndrome. Inflammation is a physiological state that precedes various pathologic manifestations where oxidative stress, nitric oxide, prostaglandin production or COX are increased. In general, induced catecholamines (i.e. dopamine and noradrenaline) were shown to stimulate the immune response via the induction of interleukin-1, tumor necrosis factor and interleukin-8 production usually implicated in pro-inflammation conditions [START_REF] Esch | Proinflammation: a common denominator or initiator of different pathophysiological disease processes[END_REF]; cytokines that are also found in bivalves, but the interaction between increased dopaminergic activity and inflammation is not well understood at present in invertebrates [START_REF] Hughes | Evidence for the conservation of an immunoreactive monokine network in invertebrates[END_REF]. MT proteins were induced during infection and inflammation in the striped bass Morone saxitilis (Regala and Rice, 2004), which suggests that the inflammatory state (COX and oxidative stress) is likely to contribute to the MT response. A study on caged freshwater mussels exposed to a municipal effluent dispersion plume found that even though metals were less bioavailable, MT was still induced in the digestive gland [START_REF] Gagnon | Exposure of caged mussels to metals in a primary-treated municipal wastewater plume[END_REF]. This finding suggests that the inflammatory properties of pollution, and not the presence of metals in municipal effluents, contributed to increased MT synthesis. This hypothesis is supported by an analysis of covariance that revealed that COX activity (covariate) had the greatest effect on inter-site differences in MT levels.

Besides the contamination from commercial and pleasure boating activities, the contribution of domestic or municipal effluents could also have an impact on the health status of clam populations. Although clams from sites ASJ and AE had decreased GSI, their levels of Vtg and ATC activity were significantly increased. Moreover, the growth index was higher in clams from sites ASJ and AE, but the mean age value was significantly lower and higher, respectively, than the pristine site ASE. The increase in Vtg-like proteins at site ASJ and the slight increase at site AE were previously reported elsewhere [START_REF] Blaise | Molluscan shellfish biomarker study of the Quebec, Canada, Saguenay Fjord with the soft-shell clam Mya arenaria[END_REF], showing a sustained estrogenic effect of domestic wastewaters on feral clam populations. Although serotonin levels were elevated at polluted sites and weakly correlated with COX activity, the serotonergic signal might have been produced by exposure to domestic wastewaters. Indeed, serotonergic properties were recently identified in freshwater mussels exposed to municipal effluents [START_REF] Gagné | Serotonergic effects of municipal effluents: induced spawning activity in freshwater mussels[END_REF]. It was observed that exposing freshwater mussels to municipal wastewaters increased COX activity in gonadal tissues, with a concomitant increase in monoamine metabolism that favoured serotonergic over dopaminergic effects. Because clams were collected during gametogenesis, the serotonin/dopamine ratio should have been lower. The contrary was true, however, with the serotonin/dopamine ratio being much higher at polluted sites, especially those under the influence of municipal wastewaters (sites ASJ and AE). Interestingly, gonad dopamine levels were more highly correlated with Vtg-like protein and gonad ATC activity, which usually occurs at the beginning of gametogenesis in females, than with COX activity. Thus, exposure to urban wastewaters at the beginning of gametogenesis represents a sensitive period in the manifestation of toxic effects in feral clam populations. Indeed, dopamine levels were also closely associated with oxidative stress (LPO) and DNA strand breaks, suggesting that the increase in catecholamine synthesis during the initiation of gametogenesis might have some DNA-damaging effects, perhaps through oxidative stress. Catecholamines are well known for their sensitivity to oxidation and recognized for enhancing DNA damage by oxidative stress such as nitric oxide production during inflammation conditions (Yoshie and Oshima, 1997). Based on a correlation analysis, DNA damage seems equally linked to the processes of inflammation (COX activity) and gametogenesis, as determined by dopamine metabolism biomarkers (i.e. no significant difference between correlation coefficients was observed). Therefore, the induction of COX activity by urban pollutants at the time of gametogenesis, where dopamine production is higher, appears to increase oxidative stress and lead to lipid peroxidation and DNA strand breaks in clams.

In conclusion, the neuroendocrine status shows clear signs of disruption in clams influenced by different types of contaminating pollution. The biomarker data revealed delayed gametogenesis and an inflammatory syndrome in these clams, as evidenced by their increased serotonin/dopamine ratio, COX activity and oxidative stress. Furthermore, this inflammatory condition appears more closely related to urban pollution, especially domestic effluents, as suggested by the close relationships with MT, GST and LPO in the digestive gland and the absence of trends with GSI or Vtg. We conclude that pollution not only delays but indeed disrupts the neuroendocrine signals of gametogenesis.

Gametogenesis, therefore, appears to increase the susceptibility of organisms to pollution. Clams were collected at sites (identified by stars) in June 2004. The sites Anse de Saint-Étienne (ASE) and Baie-du-Moulin à Baude (BAU) were considered as the reference (pristine) sites, while Anse aux Érables (AE), Baie-Éternité (BE), Anse Saint-Jean (ASJ) and Baie Sainte-Catherine (BSC) were those affected by pollution. Clams were collected at low tide and brought back to the laboratory for analysis of soft tissue weights (A), age (B) and growth index, as determined by the age-to-shell-length relationship (C). Significance was set at p < 0.05.

Figure 1 .

 1 Figure 1. Site location in the Saguenay Fjord area.

Figure 2 .

 2 Figure 2. Morphological characteristics of clam populations in the St. Lawrence Estuary and Saguenay Fjord.

Figure 4 .

 4 Figure 4. Multivariate analysis of biomarker data.Biomarker data were analysed by discriminant analysis for site classification. The classificant performance for each sites are expressed as percentages in the legend.

Gametogenetic activity in clams collected from impacted sites.

					A	0,52 0,54 0,56	A	0,18 0,16	Mean	Mean Stand. error Stand error
					Soft tissue weight	(g tissues/g whole weight)	0,36 0,38 0,40 0,42 0,44 0,46 0,48 0,50	Gonado-somatic index	(g gonad/g soft tissues)	0,04 0,06 0,08 0,10 0,12 0,14
							0,34			0,02	BSC	BAU	ASE	ASJ	AE	BE
							0,32				Sites
	B			350			0,30				BSC *	BAU	ASE Sites	ASJ	AE	BE	Mean
	Vitellogenin-like proteins	(ug alkali-labile phosphates/g gonad)	0 50 100 150 250 300 200	Age B (years)	5,4 5,6 5,8 6,0 6,2 6,4 6,6 6,8 7,0 7,2 7,4 7,6 7,8 8,0 BSC BAU ASE ASJ AE BE Mean Stand. error *	BSC BAU ASE ASJ AE BE Stand. error Stand. dev iation * *
							5,2
							5,0			BSC Female	BAU	ASE Sites Sites	ASJ	AE	BE	Male
			0,24			0,13
	Aspartate transcarbamoylase activity	(ug Pi/min/g gonad)	0,04 0,06 0,08 0,10 0,12 0,14 0,16 0,18 0,20 0,22	Development index C (age/shell length)	0,08 0,09 0,10 0,11 0,12		Mean Stand. error Mean Stand, error
			0,02
			0,00			0,07	BSC	BSC BAU	BAU ASE	ASE	ASJ ASJ	AE AE	BE BE
											Sites Sites
	Clams were collected and the gonad analyzed for the gonado-somatic index (A), for
	levels of vitellogenin-like proteins (B) and aspartate transcarbamoylase activity (C).
	Significant at p<0.05.

C Figure 3.

Table 1 .

 1 Heavy-metal analysis in whole soft tissues of Mya arenaria clams.Data were expressed as the mean with the standard error in µg/g dry weight from N=4 groups of 10 clams per site. Bold value indicates significance relative to the reference site (either BAU or ASE) at p<0.05 level.

	SITE	BSC	BAU	ASE	ASJ	BE	AE
	Metal
	As	0.91±0.05	0.74±0.07	0.84±0.04	0.48±0.03	0.47±0.025	0.42±0.02
	Cd	0.04±0.002	0.06±0.004	0.1±0.01	0.12±0.01	0.11±0.008	0.12±0.007
	Co	3.6±0.2	3.8±0.4	2.7±0.2	7.6±0.3	3.3±0.3	6.7±0.5
	Cr	11.2±1	7.6±0.5	6.4±0.8	16.3±2	6.4±0.1	15.8±0.9
	Cu	1.6±0.15	1.7±0.15	1.3±0.1	1.7±0.2	1.4±0.1	1.45±0.1
	Fe	753±60	450±40	580±40	1130±75	744±60	575±45
	Hg	0.03±0.005 0.008±0.004	Nd	0.032±0.003	0.046±0.005	0.044±0.005
	Mn	21.6±3	9.2±0.5	11.7±1	162±10	50±5	54±4
	Ni	6±0.5	1±0.1	1.5±0.1	2.6±0.2	2.2±0.2	1.8±0.15

Table 2 . Biomarkers of defence and tissue injury in Mya arenaria clams.

 2

								Biomarker index
				Total heme	DNA damage	LPO (digestive
	Site	MT (digestive gland)	GST (digestive gland)	(gonad)	(digestive gland)	gland)	LPO (gill)	Sum Rank **
	BSC	175 ±10*	0.81 ±0.03*	0.29 ± 0.1	63 ± 3*	3.9±0.26*	1.4±0.23*	16	5
	BAU	104 ±7	0.36 ±0.02	0.36 ± 0.1	41 ± 4	1.95±0.25	2.5±0.46	8	2
	ASE	49 ±5	0.38 ±0.03	0.41 ± 0.07	44 ± 2	2.49±0.26	2.4±0.36	6	1
	ASJ	88 ±6*	0.43 ±0.026	0.80 ± 0.03*	63 ± 5*	3.37±0.26*	1.8±0.39	13	4
	AE	111 ±8*	0.34 ±0.026	0.34 ± 0 .06	60 ± 3*	4.73±0.38*	1.5±0.17*	13	4
	BE	71 ±6*	0.32 ±0.027*	0.39 ± 0.06	58 ± 6*	3.0±0.27	1±0.17*	10	3
	* Significance was set at p < 0.05 relative to the reference sites (BAU and ASE).

** Sites are ranked in ascending order of toxicological stress.

Table 3 . Trend analysis of biomarker data.

 3

			Correlation
	Variable 1	Variable 2	coefficient	P value
	Gonado-somatic index	Age	0.34	< 0.01
		GST	0.39	< 0.01
		LPO in digestive gland	-0.28	0.02
		LPO in gill	0.41	< 0.01
		DNA damage in gill	-0.34	0.02
		DNA damage in digestive gland	-0.31	0.018
		COX	-0.27	0.038
	Tissue weight ratio (soft-tissue	Age	-0.32	< 0.01
	wet weight/total weight)	Development index	-0.21	0.04
		ACT	0.58	< 0.01
		DNA damage in gill	0.61	< 0.01
		Heme	0.45	< 0.01
		Serotonin/dopamine ratio	0.4	0.01
		COX	-0.38	< 0.01
	Age	Development index	0.75	< 0.01
		DNA damage in gill	-0.33	0.024
		Serotonin/5H1AA ratio	0.33	0.013
	Developmental index	MT in digestive gland	0.4	< 0.01
	(shell length/age)	LPO in digestive gland	0.41	< 0.01
		Dopamine	0.34	0.029
		Serotonin	0.48	< 0.01
		Serotonin/5H1AA ratio	0.49	< 0.01
		COX	0.42	< 0.01
	ACT	DNA damage in gill	0.47	< 0.01
		Heme	0.49	< 0.01
		Dopamine	0.32	0.039
		Serotonin	0.34	0.012
		Serotonin/dopamine ratio	0.37	0.018

Table 4 . Neuroendocrine effects of site quality.

 4

									COX
		Serotonin	Serotonin	5-HIAA	Seratonin/	Dopamine	Dopamine	Serotonin/	activity	MAO act
	Site	(ng/ g ww)	transport activity	(ng / g ww)	5-HIAA	(ng/g ww)	transport	dopamine	(x 10 3 )	(x 10 3
							activity
	BSC	15 ± 2	28 ± 7	7 ± 3.4* 1	2.5 ± 0.7	16.4 ± 3.9*	143 ± 47*	2.5 ±0.7	1.7± 0.08*	1.8 ± 0
	BAU	21.7 ± 2*	26 ± 8	16.5 ± 1.*	1.5 ± 0.3	5.04 ±1	55 ±12	2 ± 0.5	0.9± 0.05	1.6 ± 0.
	ASE	16 ± 2	18 ± 5	7.4 ± 1.9	0.6 ± 0.1	3.97 ± 1	42 ± 13	1 ± 0.27	0.9±0.09	1.5 ± 0
	ASJ	21 ± 1.8*	27 ± 7	11 ± 1.5*	1.9 ± 0.3	20.5 ± 7*	130 ± 33*	1.9 ± 0.3*	0.74±0.04	2.8 ± 0.
	AE	19 ± 1.7*	51 ± 9	3.4 ± 0.2*	5.8 ± 0.4*	14.6 ± 1*	21 ± 5	5 ± 0.4*	1.3± 0.07*	2.9 ± 0.
	BE	13 ± 1.3*	19 ± 3	4.7 ± 1.5	3.1 ± 1.2*	12.4 ± 3.6*	41 ± 5	1.6 ± 0.6	0.9±0.1	2.6 ± 0.
	N.B.: Asterisks (*) indicate significance at p < 0.05

Table 5 . Factorial analysis of biomarker data. Biomarkers

 5

	Factor 1	Factor 2	Factor 3	Factor 4

Regala,R.P., & Rice, C.D., (2004). Mycobacteria, but not mercury, induces metallothionein (MT) protein in striped bass, Morone saxitilis, phagocytes, while both

Acknowledgments. The authors are grateful for the technical assistance of Sophie Trépanier, Brian Walker and Chantal André of the St. Lawrence Centre of Environment Canada and to Pascal Rioux and Gilles Lemay of ISMER (UQAR). The manuscript was edited by Patricia Potvin. This work was funded by the St. Lawrence Action Plan and NSERC through the Discovery grant attributed to Jocelyne Pellerin.


Maia Aricelso

Angela De L Rebello Limaverde
email: angela@rdc.puc-rio.br

Marcos A Wagener

Arthur De L Fernandez

Ricardo Scofield

Coutinho

Aricelso Maia Limaverde

Angela De L Rebello Wagener

Marcos A Fernandez

Arthur De L Scofield

Ricardo Coutinho

Stramonita haemastoma as a bioindicator for organotin contamination in coastal environments

Keywords: organotins, Stramonita (Thais) haemastoma, bioassay, Guanabara Bay

Stramonita haemastoma was investigated as a suitable bioindicator of TBT and TPhT contamination in the tropical Atlantic Ocean by: 1. Imposex induction in healthy females after inoculation with TBT and TphT in the laboratory; and 2. Determining incidence of imposex in S. haemastoma collected from areas with various levels of tributyltin (TBT) and triphenyl tin (TphT) and determining the concentrations of these chemical in its tissues and that of its prey , the mussell Perna pernas. Imposex intensities and organotin concentrations in tissues showed good correlation, indicating S. haemastoma as a reliable bioindicator of TBT and TPhT contamination in coastal waters. Body burden threshold of TBT and TPhT for imposex induction was estimated to be 10-20 ng.g -1 .

Introduction

Organotins (OT) are man-made organometallic compounds widely used as biocides, mainly as antifouling paints on ship hulls. TBT and TPhT (triphenyltin) liberated from the hulls in contact with seawater have been shown to be very toxic to the marine biota [START_REF] Hoch | Organotin compounds in the environment -an overview[END_REF][START_REF] Champ | A review of organotin regulatory strategies, pending actions, related costs and benefits[END_REF]. The most characteristic sub-lethal effect of organotins is hormonal disruption in gastropods, leading to the imposition of male sexual attributes in the females, i.e., imposex [START_REF] Gibbs | Reproductive failure in populations of the dog-whelk Nucella lapillus, ceased by imposex induced by tributyltin from antifouling paints[END_REF][START_REF] Horiguchi | Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera[END_REF][START_REF] Depledge | Ecological significance of endocrine disruption in marine invertebrates[END_REF]. Imposex, has been observed in several areas of the globe where OT contamination is present [START_REF] Mathiessen | Critical appraisal of the evidence for tributyltin-mediated endocrine disruption in molluscs[END_REF]. The neogastropod Stramonita haemastoma is a muricid, closely related anatomically to other species of the genus Thais. This genus is widespread in tropical and temperate waters, including the Mediterranean [START_REF] Rilov | Unregulated use of TBT-based antifouling paints in Israel (Eastern Mediterranean) : High contamination and imposex levels in two species of marine gastropods[END_REF], North America, the Caribbean and South America [START_REF] Rios | Seashells of Brazil[END_REF], Australia and Asia (Stewart, De Mora, Jones & Miller, 1992;Tan, 1997;Bech, 2002a), living even in cool waters in Korea and Japan [START_REF] Horiguchi | Imposex and organotin compounds in Thais clavigera e T. bronni in Japan[END_REF]Shin , Kahng, Hong, Kim, Kim & Shim, 2000). The incidence and intensity of imposex in S. haemastoma varies inversely with the distance from OT sources, as for instance, harbors, marinas and shipyards [START_REF] Spence | The mollusk Thais haemastoma -an exhibitor of imposex and potential biological indicator of tributyltin pollution[END_REF][START_REF] Castro | Imposex in Thais heamastoma (Linnaeus, 1767)(Mollusca:Gastropoda) an indication of organotin contamination in the coast of Fortaleza-Ceara-Brazil[END_REF][START_REF] Rilov | Unregulated use of TBT-based antifouling paints in Israel (Eastern Mediterranean) : High contamination and imposex levels in two species of marine gastropods[END_REF][START_REF] Fernandez | Occurrence of imposex in Thais haemastoma: possible evidences of environmental contamination derived from organotin compounds in Rio de Janeiro and Fortaleza, Brazil[END_REF][START_REF] Fernandez | Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil[END_REF]. Among species of the Thais genus, imposex was shown to occur in T. orbita (Stewart et al, 1992), T. clavigera [START_REF] Horiguchi | Imposex and organotin compounds in Thais clavigera e T. bronni in Japan[END_REF]Tan, 1997;[START_REF] Blackmore | Imposex in Thais clavigera (neogastropoda) as na indicator of TBT (tributyltin) bioavailability in coastal waters of Hong Kong[END_REF]Shin et al, 2000;[START_REF] Hung | Organotins and imposex in the rock shell, Thais clavigera, from oyster mariculture areas in Taiwan[END_REF], T. bronni [START_REF] Horiguchi | Imposex and organotin compounds in Thais clavigera e T. bronni in Japan[END_REF], T. bitubercularis (Tan, 1997;Bech, 2002a), T. distinguenda (Tan, 1997;Bech, 2002a), T. jubilacea (Tan, 1997), and T. rufotincta (Bech, 2002a). A few authors used organotins in bioassays to induce imposex in T. clavigera [START_REF] Horiguchi | Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera[END_REF] used injection of the toxicants, while [START_REF] Liu | Organotins promoting the development of imposex in the oyster drill Thais clavigera[END_REF] tested direct exposure to the toxicant in water. [START_REF] Bech | Development of imposex and accumulation of butyltin in the tropical muricid Thais distinguenda transplanted to a TBT contamination site[END_REF] tested transplantation of T. distinguenda from non-contaminated to contaminated sites coupled to body burden chemical evaluation. All studies gave positive results for imposex induction by organotins.

In Brazil, very little is known about the presence and threats imposed by organotins in coastal environments. [START_REF] Fernandez | Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil[END_REF] and [START_REF] Almeida | Speciation of organotin compounds in sediment cores from Guanabara Bay, Rio de Janeiro (Brazil) by gas chromatography-pulsed flame photometric detection[END_REF] found high concentrations of TBT in sediments of Guanabara Bay, in Rio de Janeiro, and reported that TBT may be persistent in the anoxic sediments of the bay. [START_REF] Godoi | Determination of tributyltin compounds in surface sediments from the São Paulo state coast (Brazil) by gas chromatographypulsed flame photometric detection[END_REF] also reported high concentrations in sediments from the Santos Bay, where boat and ship traffic is very intense. As for biological studies, [START_REF] Fernandez | Imposex em Thais haemastoma no litoral do estado do Rio de Janeiro, Brasil. Um levantamento preliminar[END_REF], [START_REF] Magalhães | Evidences of imposex in Stramonita haemastoma (Gastropoda : prosobranchia) population from harbor areas of the state of São Paulo[END_REF] and [START_REF] Castro | Imposex in Thais heamastoma (Linnaeus, 1767)(Mollusca:Gastropoda) an indication of organotin contamination in the coast of Fortaleza-Ceara-Brazil[END_REF] reported the occurrence of imposex in Stramonita haemastoma in Rio de Janeiro, Santos and Recife.

Imposex was also found in Stramonita Rustica in the Northeastern coast of Brazil [START_REF] Camillo | Imposex in Thais (Stramonita) rustica (Mollusca:Neogastropoda)(Lamark, 1822) as an indicator of organotin pollution in Maceio coast (Northeastern Brazil)[END_REF][START_REF] Castro | Thais rustica (Lamark, 1822)(Mollusca:Gastropoda:Thaididae): bioindicator of contamination by organotin compounds in the coast of Natal city, Rio Grande do Norte state, Northeastern Brazil[END_REF], however, in most cited studies the cause of such an alteration has not been proven to be TBT by correlating, for instance, the anomaly intensity with chemical body burden.

Brazil, as most Latin American countries, does not regulate the use of organotins on vessels.

An International Convention under IMO authority is under way to globally ban the use of organotins as antifouling agents. However, these substances will be present in the marine environment long after banning, either released from ship hulls still carrying these substances, or from anoxic sediments where TBT may show a half-live of several years [START_REF] Dowson | Persistence and degradation pathways of tributyltin in freshwater and estuarine sediments[END_REF]Stewart & Thompson, 1997;[START_REF] Alzieu | Tributyltin: case study of a chronic contaminant in the coastal environment[END_REF]. Enforcing such a convention will imply monitoring environmental conditions, a process that may be tedious and expensive, especially for developing countries such as Brazil, with more than 7,000 km of coast.

The use of a reliable bioindicator as a tool for screening hotspots is a viable option to the expensive chemical analysis of samples from every site suspected of contamination (Axiak Vella, Micallef, Chircop & Mintoff, 2003[START_REF] Fernandez | Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil[END_REF]. Having this in mind the present work tests for a cause-and-effect relationship between TBT and TPhT exposure and the development of imposex in Stramonita haemastoma. Two approaches were taken: (1) a laboratory bioassay, or dosing experimenet; and (2) a field investigation of imposex coupled to organotin determination in the gastropod as well as in its natural prey (Perna perna) in Guanabara Bay.

Our goal was to validate the use of this widespread gastropod species, and of the mussel Perna perna, as sentinels for organotin contamination in coastal environments in Brazil, and in other areas where they occur.

Methods

Sampling

For the bioassay, gastropods, and mussels used as feed, were collected in January 2001, from the Pontal do Atalaia in Arraial do Cabo, Rio de Janeiro. This is a pristine area where Stramonita populations were healthy and showed no evidences of imposex [START_REF] Ribeiro | Evaluation of Stramonita haemastoma (Kool, 1987) as a biological indicator of environmental contamination due to organotin compounds[END_REF].

For the survey in Guanabara Bay samples of Stramonita haemastoma and Perna perna were collected in September, 2000 and in October-December, 2001, from rocky substrates in sites at different distances from known sources (see Figure 1) of organotins, so as to obtain an imposex gradient in the field. From each site a minimum of 20 individuals were collected [START_REF] Ellis | Widespread neogastropod imposex: a biological indicator of global TBT contamination?[END_REF]. Sexual maturity of the gastropods was inferred from shell size that was, in general, close to 40 mm, and the presence of marked ribs at the shell edge, near the operculum [START_REF] Minchin | Effect of freezing on the length of the penis in Nucella lapilus[END_REF][START_REF] Ellis | Widespread neogastropod imposex: a biological indicator of global TBT contamination?[END_REF]. During transportation to the laboratory all organisms were kept in aquaria equipped with an air pump.

Bioassay

From the total of 400 collected individuals measuring 40 ± 5 mm of shell length (measures were taken with vernier calipers), 25 were randomly picked and examined for confirmation that the sampled population was imposex free. The assay was performed in 8 aquaria containing 42 gastropods and 10 mussels each. The aquaria were filled with 35 L of seawater, collected from the sampling site given above, and equipped with air pumps, and activated charcoal filters. Five days were given for adaptation of the organisms to the new conditions prior to starting the inoculations. Two of the 8 aquaria were used as controls. Animals from one of the control batches were inoculated with ethanol (Merck, P.A.), the solvent used for diluting TBT and TPhT standards. The remaining aquaria were divided in two groups of three aquaria; the first group was used for inoculation of the organisms with increasing concentrations of TBT (aquaria TBT-1, TBT-2, TBT-3), and the second group was used for inoculation of TPhT aquaria TPhT-1, TPhT-2, TPhT-30). To ensure better control of the desired dose, the relation between shell length (SL) and fresh weight of soft parts (FW) was tested in a separate batch of organisms ( SL (mm) = 3,46 x FW (g) + 29,97; r = 0,725). This relation was thereafter used to estimate the animal mass from the shell length (for example, an organism of 40 mm shell length has an average weight of 2.9g). Based on the work of [START_REF] Horiguchi | Imposex and organotin compounds in Thais clavigera e T. bronni in Japan[END_REF] the applied dose was of the order of 10-20 ng.g -1 . Solutions of tributyltin chloride 96% and triphenyltin chloride 95% (Aldrich Chem. Co.) were then prepared by diluting the standards with ethanol (Merck, P.A.). Inoculation was performed by injecting 1 µL (containing the following amounts of organotin as Sn: TBT-1 = 14.9 ng; TBT-2 = 29.9 ng; TBT-3 = 59.6 ng; TPhT-1 = 12.9 ng; TPhT-2 = 25.9 ng; and TPhT-3 = 51.8 ng) of the diluted solution in the animal foot with the aid of a 5 µL chromatographic syringe. An air-solution-air sandwich was used to ensure that the desired volume was fully injected. Elapsed 14 days (Group1) from inoculation, 15 organisms were removed from each aquarium for determination of the imposex frequency and intensity. The same evaluation was applied to the remaining organisms 28 days after the inoculation (Group 2).

A previous transplantation test had shown that this time was sufficient for imposex development in this species [START_REF] Ribeiro | Evaluation of Stramonita haemastoma (Kool, 1987) as a biological indicator of environmental contamination due to organotin compounds[END_REF].

During the 4 weeks of the bioassay duration, water in the aquaria was exchanged weekly.

Temperature and salinity were always kept at the initial conditions (20 o C and 34.5 g kg -1 , respectively). Living mussels were also weekly replaced by freshly harvested animals, and those by chance dying during the experiment were immediately removed from the aquaria.

Field Gradient Study

Animals were sampled at five sites in Guanabara Bay (Figure 1) previously studied for imposex intensity by [START_REF] Fernandez | Organotin compounds in Guanabara Bay (Rio de Janeiro): distribution and possible impacts[END_REF]. Sampling stations were selected as to possibly provide animals with a broad range of imposex intensity (from an expected maximum in the near of the Rio de Janeiro Yacht Club, in Urca wave breaker, to a minimum in the less impacted area of the Cagarras Islands, located outside the bay). From each site at least 20 individuals were collected [START_REF] Ellis | Widespread neogastropod imposex: a biological indicator of global TBT contamination?[END_REF]. The sexual maturity of the gastropods was inferred from the shell size (40.0 ± 5.0 mm), and the presence of marked ribs at the shell edge, near the operculum [START_REF] Ellis | Widespread neogastropod imposex: a biological indicator of global TBT contamination?[END_REF][START_REF] Minchin | Effect of freezing on the length of the penis in Nucella lapilus[END_REF]. During transportation to the laboratory all organisms were kept in air-pump-equipped aquaria.

Imposex intensity was determined in the gastropods from each station. Thereafter, these animals, along with a sample of 20 mussels Perna perna per station, were separated for chemical analysis.

Imposex intensity evaluation

Sex of the animals was determined by the presence of the prostate gland in males, and the albumen, sperm-ingesting and capsule gland in females (Gibbs & Bryan, 1987[START_REF] Rios | Seashells of Brazil[END_REF][START_REF] Horiguchi | Imposex and organotin compounds in Thais clavigera e T. bronni in Japan[END_REF]. Penis length in males and in imposexed females was also measured with calipers [START_REF] Axiak | Imposex in Hexaplex trunculus (Gastropoda: Muricidae): first results from biomonitoring of tributyltin contamination in the Mediterranean[END_REF], and vas deferens development was observed under a binocular microscope.

Imposex response in the populations was evaluated by using the RPLI (relative penis length index), RPSI (relative penis size index) and VDSI (vas deferens sequence index), according to the procedures given under Gibbs & Brian, 1986;[START_REF] Gibbs | The use of dog-whelk Nucella lapilus, as an indicator of tributyltin (TBT) contamination[END_REF][START_REF] Fioroni | The pseudohermaphroditism of prosobanchs; morfological aspects[END_REF]Stroben, Schulte-Oehlmann, Fioroni & Oehlmann, 1995. This last index was determined using a six-stage scale similar to the classic Gibbs scale for Nucella lapillus [START_REF] Gibbs | Biomonitoring of tributyltin pollution using the imposex response of neogastropod molluscs[END_REF] adapted for Stramonita haemastoma [START_REF] Fernandez | Occurrence of imposex in Thais haemastoma: possible evidences of environmental contamination derived from organotin compounds in Rio de Janeiro and Fortaleza, Brazil[END_REF]. In this scale, grade 0 is attributed to normal females. Grade I includes females presenting a small hump or pre-penis behind the right tentacle. In Grade II, a small penis is visible. In grade III, the penis is bigger, and an incomplete vas deferens could be observed in the palial cavity floor. In grade IV, the vas deferens is complete and female penis reach a size greater than half of the male's mean size. Grade V is characterized by vulva blockage and Grade VI by aborted egg mass in the capsule gland. After narcotization with a 7% MgCl 2 in a 1:1 solution of distilled water:local seawater [START_REF] Huet | Comparison of imposex response in three prosobranch species[END_REF] shells were crushed using a small vice. Narcotization was required to obtain precise measurements of penis size, since the unarcotized organism retracts after shell removal rendering manipulation and anatomic examination very difficult.

Sample pre-treatment for chemical analysis

Separate composite samples of female and male gastropods from each station were prepared for chemical analysis. Soft tissues selected for chemical analysis were homogenized in an Ultra Turrax and duplicate sub-samples of each homogenate were stored at -20ºC in clean glass vessels until analysis. Mussel's soft parts were removed from the shells with the aid of a stainless steel blade. A pooled sample (20 individuals) from each station was homogenized in an Ultra Turrax. Duplicate sub-samples were stored at -20 o C in clean glass containers until analysis.

Similarly, pooled samples of males and females from each aquarium of the bioassay were prepared and analyzed in duplicate. Gastropods were fed during the bioassays with mussels collected weekly; therefore, organotins were analyzed in sub samples of each batch of harvested mussels.

Analytical Procedure

Analytical protocol was based on [START_REF] Bayona | Partitioning of urban wastewater organic microcontaminants among coastal compartments[END_REF] and adapted as described in [START_REF] Fernandez | Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil[END_REF] to the determination by gas chromatography with pulsed flame photometric detection GC/PFPD. The analytical procedure including extraction, clean-up, derivatization and GC/MS determination is described in length in [START_REF] Limaverde | Compostos orgânicos de estanho (COE) em neogatrópodos afetados por imposex na Baía de Guanabaraanálise preliminar[END_REF]. In brief, after spiking 2g of tissue homogenate with tripropyltinchloride (TPrTCl 98%, Merck) and tricyclohexyltinchloride (TcyTCl 97%, Aldrich), samples were treated with methanol in HCl and extracted with tropolone solution in 3:2 ethyl ether/hexane and sonication. Derivatization proceeded with PeMgBr in diethyl ether under N 2 atmosphere. Cleanup was done in glass columns filled with activated silica and anhydrous Na 2 SO 4 . After elution with ethyl ether in hexane and volume reduction under N 2 stream, tetrabutyltin was added as internal standard.

A Varian 3600CX gas chromatograph fitted with an 8200 auto sampler, a pulsed photometric detector (Varian) and a DB-17 (50%-phenyl-methylpolysiloxane) fused silica column (J&W Scientific) were used. The detailed chromatographic conditions are given under [START_REF] Fernandez | Imposex and surface sediment speciation: A combined approach to evaluate organotin contamination in Guanabara Bay, Rio de Janeiro, Brazil[END_REF]. The determinations were made in triplicate. Quantification was performed by internal standard procedure, using TeBT added after derivatization. Procedural blanks were carried out for every batch of samples.

The analytical procedure was validated by analyzing the reference material NIES-11 (National Institute for Environmental Studies-Environmental Agency of Japan) certified for TBT. The values found for TBT (512 ± 58 ng.g -1 as Sn; n=6) and TPHT (1384 ± 260 ng.g -1 as Sn; n=6) are in good agreement with the certified value for TBT (475 ± 36 ng.g -1 dw as Sn) and the suggested value for TPHT (1942 ng.g -1 ), respectively. The calibration curves gave correlation coefficients (n=6) of: 0,999 (TBT, DBT, MBT, TCyT), 0,986 (TPhT and DPhT), 0,979 (MPhT) and 0,995 (TeBT). The concentration of butyltins and phenyltins were corrected for recovery of TPrT (73 ± 5 ng.g -1 ). The losses involved in the overall process were estimated by analyzing a sample low in organotins, spiked with a mixture of the standards (TPrT, TBT, DBT, MBT, TPhT, DPhT, MPhT and TCyT) of known concentrations. Equilibration was allowed for 2 hours before extraction. The average value found was 75 ± 12 % of the initial concentration (TBT gave 91 ±18%, and the lowest value, not included in the average, was of 60 ± 2% for MPhT). Mean relative standard deviations for n=5 were: 12.2% (TPrT), 3.5% (TBT), 15.0% (DBT), 14.8% (MBT), 27.4% (TPhT), and 36% (DPhT). Detection limits given as [Sn] for the various organotins are: TBT = 0.39 pg; DBT = 0.66 pg; MBT = 0,96 pg; TPhT = 0.90 pg; DPhT = 0.72pg.

Results and Discussion

Table 1 shows the results for the bioassay. Because of the small penis development in imposex females, RPLI and RPSI were not calculated, except for females harvested 28 days after TBT inoculation, which displayed penis of more than 1 mm (RPLI = 17,1; total number of females was equal to 7 from which 5 (71%) showed imposex). The average penis length found in females 28 days after inoculation of 20 ng.g -1 TBT (TBT-3) was 1.5 ± 0.9 mm. [START_REF] Horiguchi | Imposex and organotin compounds in Thais clavigera e T. bronni in Japan[END_REF] and [START_REF] Bech | Development of imposex and accumulation of butyltin in the tropical muricid Thais distinguenda transplanted to a TBT contamination site[END_REF] also found for gastropods of the same genus as S. haemastoma that TBT concentrations of 10-20 ng.g -1 of fresh tissue induced development of imposex.

Sampling 14 days after inoculation of TPhT was not carried out because of the high mortality observed. In this case, all survivals were harvested 28 days after inoculation, and among those only one female presented a measurable penis size (2 mm).

As for the VDSI, no significant differences were observed among organisms 14 days after inoculation with different TBT doses; all altered organisms were classified as in level 1 of the applied VDSI scale. Two animals in the control group, inoculated with ethanol, developed imposex. A good correlation was found between frequency of imposex incidence and inoculated doses (see Figure 2).

The groups of organisms sampled 28 days after inoculation with 59.6 ng of TBT (as Sn)

and 51.8 ng of TPhT (as Sn), respectively, had significant VDSI levels: 2.3 (n=6) for TBT inoculation, and 1.4 ( n= 5) for TPhT while the control group was imposex free. The 2 test and contingency tables were used to check for possible independence of F (healthy females) and I (imposexed females) factors from organotins inoculation dose. For TBT the test was applied first separately to the results for 14 (Group 1) and 28 (Group 2) days of experimentation, and then to the entire data set. The dependency relation found between F/I and the injected dose for Group 1 (x o = 12.72; x = 9.23, d.o.f. = 5, p < 0.05) is more evident for Group 2 data since the independence hypothesis was rejected with a value well above the rejection threshold (x o = 35.81; x = 9.23, d.o.f. = 5, p < 0.001). Similar relations were obtained for TPhT data in Group 2.

Chemical analysis revealed DBT (7.38 ± 2.53 ng g -1 ww as Sn for females and 13.70 ± 2.06 ng g -1 ww as Sn for males) contamination in the gastropods freshly harvested from the sampling site, and the presence of DBT (5.20 to 6.94 ng.g -1 ww as Sn) and MBT (0.54 to 1.05 ng g -1 ww as Sn) in the mussels. In the control batches, DBT and MBT concentrations increased over the 28 days of experiment, resulting in a DBT background value (as Sn) of 20.4 ± 0.6 ng.g -1 in the gastropods. This is an indication that seawater from the area, taken as pristine, contained traces of organotins, possibly derived from eventual navigation activities (pleasure and fishing boats). Although the contamination source for DBT was not clearly identified here, based on the observations of [START_REF] Horiguchi | Effects of triphenyltin chloride and five other organotin compounds on the development of imposex in the rock shell, Thais clavigera[END_REF] and in the present observations it can be assumed that the background DBT concentration in the gastropods played no role in the development of imposex.

The presence of MBT and MPT concentration in excess of the background in females after 28 days of inoculation is evidence of parent compound metabolism.

Table 2 shows organotin concentrations in the animals of the bioassay, and Figure 3 displays the mass balance obtained by using the inoculated dose, the background concentration, and the measured body burden after 14 and 28 days of inoculation. This balance provides an estimate of the average organotin fraction accumulated in the organism. TPhT concentrations in females was 30-40% higher than for TBT, and evidently the accumulation in males is remarkably smaller than in females, especially for TBT, suggesting a more effective biotransformation mechanism in males. The generally higher TPhT persistence in tissues, also observed by [START_REF] Horiguchi | Imposex in japonese gastropods (Neogastropoda and Mesogastropoda) : Effects of tributyltin and triphenyltin from antifouling paints[END_REF], suggests a slower metabolization of this compound in this species.

The field studies showed that organotin concentrations in gastropods (see Table 3) from Leme, Cagarras Islands, Vermelha Beach, Cotumduba and Urca are of the same order of those reported for gastropod of the same genus from other contaminated areas [START_REF] Horiguchi | Imposex in japonese gastropods (Neogastropoda and Mesogastropoda) : Effects of tributyltin and triphenyltin from antifouling paints[END_REF].

A single study by of [START_REF] Rilov | Unregulated use of TBT-based antifouling paints in Israel (Eastern Mediterranean) : High contamination and imposex levels in two species of marine gastropods[END_REF] along the coast of Israel reports imposex development as well as organotin concentrations in S. haemastoma. TBT, DBT and MBT concentrations of 447.7 ng.g -1 , 913.6 ng.g -1 and 111.3 ng.g -1 , respectively, were found in animals from Herzelia marina, heavily polluted by organotins. Mean VDSI was 4 and some sterile females where identified. At Ha Bonin, a natural reserve and reference area, concentrations were 18.9 ng.g -1 TBT, 51.2 ng.g -1 DBT and 12.2 ng.g -1 MBT, while 25% of females displayed imposex and RPSI was of 0.1. At Vermelha beach, some 4-5 km away from the main organotin sources in Guanabara Bay, TBT, DBT, MBT and TPhT average concentrations were 20.3 ng.g -1 , 36.9 ng.g -1 , 4,6 ng.g -1 and 7.45 ng.g -1 , respectively. Mean VDSI of 3 and RPLI of 30.7 for S.haemastoma in Vermelha Beach indicated a more severe response at comparable body burdens. The presence of TPhT, which also induces imposex in this species, may account for the apparent higher sensitivity of animals in Vermelha Beach.

As for the mussels, butyltins were at concentrations comparable with those reported for mussels of the same genus (Perna viridis, L.) from contaminated areas of Thailand (Kan-Atireklap, Tanabe, Sanguansin, Tabucanon & Hungspreugs 1997), for Mytilus trossulus from Vancouver Harbour [START_REF] Horiguchi | Contamination of organotin compounds and imposex im molluscs from Vancouver, Canada[END_REF], and for mussels from Danish coastal waters (NERI 2000apud Strand & Jacobsen, 2005). Concentrations in the gastropod were, in general, in the following order: DBT>TBT>MBT. The same order was observed in the mussel tissues, except for animals from Urca where TBT>DBT>MBT. TPhT was the most important phenyl species in both cases. [START_REF] Berg | Sorption and desorption behavior of organotin compounds in sediment-pore water system[END_REF] and [START_REF] Langston | Determinants of TBT adsorption and desorption in estuarine sediments[END_REF] showed that TBT sorption on particulate organic matter is fast and reversible; therefore besides TBT in the dissolved phase, particulate organotins are also potentially available to filter feeders [START_REF] De Mora | The tributyltin debate: ocean transportation versus seafood harvesting[END_REF]. From all observed sites, Urca, located in a highly eutrophic embayment and in the near of an important leisure boat anchoring area, is the most likely to contain a significant fraction of organotins adsorbed on organic particles. These set of conditions may explain the high absolute and relative TBT concentrations in the organisms from this site. [START_REF] Bryan | Uptake and transformation of 14 C-labelled tributyltin chloride by dog-whelk, Nucella lapillus: importance of absorption from diet[END_REF] estimated that in carnivorous gastropod organotins body burden seem to result from equal contributions from diet and the surrounding environment. The bioconcentration (BC; uptake from water or particulate phase via body surface) in mussels Mytilus edulis reported by [START_REF] Laughlin | Accumulation of bis(tributyltin) oxide by the marine mussel Mytilus edulis[END_REF] is of the order of 5000 while the biomagnification factor (BM; through algae ingestion) was equal to 2. By using such a BC it is possible to roughly estimate that in the sampled sites butyltin concentration in the water column (assuming that biomagnification is of lesser importance) ranged from 1.5 ng L -1 in Arraial do

Cabo to 33 ng L -1 in Urca. Such conceentrations are of special concern since detrimental effects of TBT on plankton and oysters have been reported at the concentration range from 0.5 to 50 ng.L -1 [START_REF] Alzieu | Biological effects of tributyltin on marine organisms[END_REF]. In the study by Stroben, Schulte-Oehlmann, [START_REF] Huet | Comparison of imposex response in three prosobranch species[END_REF], relating imposex level in gastropods to TBT concentrations in water, sterility in Nucella lapillus, the only muricid tested that showed this phenomenon, commenced at 4 -6 ng.L -1 TBT and was widespread in populations exposed to 20 -30 ng L -1 TBT levels. These levels are similar to the estimate in Urca, where females of S.haemastoma could not be found. Because imposex development in this gastropod species can cause female sterility, which leads to profound alteration in populations, the absence of females in Urca seems to concur with the estimated level of 33 ng.L -1 TBT in water.

Some authors have studied the biomagnification of organotins in food webs [START_REF] Laughlin | Accumulation of bis(tributyltin) oxide by the marine mussel Mytilus edulis[END_REF]Strand & Jacobsen, 2005). Mensink, Boon, ten Hallers-Tjabbes, van

Hattun & Koeman, (1997) calculated for a North Sea whelk a BM of 8 in relation to Mytilus edulis, and TBT concentrations in snails lower than in mussel by a factor of 7. [START_REF] Stäb | Determination of organotin compounds in the foodweb of a[END_REF] also report no biomagnification in the food web of fresh water systems in Netherlands. In the present work, BM (BM =

[OT] gastr. /[OT] mussel , OT = organotins) values for TBT ranged from 0.5 to 2.7 (n = 6 observed sites); for DBT the range was from 0.9 to 1.6; for MBT 1.2-3.5; and for TPhT 1.9 to 7.6 (n = 4 sampling sites). Data were not normalized to lipid content because of the higher affinity of the studied compounds to proteins. For the butyltins the linear plot ([BT] gastropod = 13.91 + 0.74[BT] mussels ; r = 0.953; p = 0.0009, BT = butyltins) of concentrations in the gastropods versus concentrations in the mussels show that, in the low concentration range ([BT] gastropod 50 ng g -1 ww as Sn, BT = butyltins), the calculated BM > 1, decreasing smoothly above this limiting concentration. If it is considered that biomagnification does not occur, this maybe the threshold value above which bioelimination mechanisms assume higher weight than bioaccumulation processes. The relatively high biomagnification for TPhT may imply that biotransformation mechanisms are less efficient for this compound.

TBT contribution to the BT body burden was of 37 to 64% in gastropods and of 37 to 67 % in mussels. There was no relation between the fraction of TBT in gastropods and in the mussels. Only mussels from Urca showed TBT at higher levels than DBT, indicating that recent inputs of antifouling overwhelm on going degradation processes.

The following pairs of data were tested for correlation: (1) VDSI levels and TBT concentration;

(2) VDSI level and total butyltins; (3) VDSI level and the sum of all organotins.

Because no females were found in Urca and there were no significant differences in organotin concentration between females and males, a tentative VDSI level for females, when existing in Urca, was obtained from the organotin concentration in males from this station using the linear equations (y= 0.004x + 0.145; r 2 = 0.885 for OTs versus VDSI, and y = 0.046x + 0.148; r 2 = 0.903 for butyltins versus VDSI) obtained for data from all other sites. The obtained results of 6.2 ( OTs) and 6.4 ( butyltins), included in the above correlation tests, may explain the absence of females there, since at these imposex levels possible lethal alterations are expected, as for instance blockage of the vulva and presence of dark decaying tissue in the capsule gland. Some populations in heavily impacted areas are composed mostly of males, because of selective female mortality [START_REF] Gibbs | Biomonitoring of tributyltin pollution using the imposex response of neogastropod molluscs[END_REF]. Figure 4 shows the plot of VDSI versus butyltins and the good fit of the bioassay values (VDSI = 2.3 and butyltins = 28.2 ng.g -1 , 28 days after TBT inoculation) to the curve.

There was excellent correlation between the data obtained for mussels collected in the same sites and dates as the gastropods, and imposex levels in the later (r = 0.934; p < 0.05).

Because concentrations in mussels are closely related to that of contaminants in the water column, the above correlation suggests organotin induction of imposex in Stramonita haemastoma under local conditions in the observed sites.

Conclusions

The bioassay demonstrated that TBT and TPhT are capable of inducing imposex in S.

haemastoma. This evidence coupled to the field studies demonstrate that this species is an adequate bioindicator of organotins contamination, either by means of monitoring the body burden of OTs or/ and determining imposex incidence. The inclusion of mussels in the field studies was useful as an indicator of the presence of organotins in the water column and demonstrated that this species is also functional in monitoring organotin contamination. Mussels can be useful in evaluating whether anomalies in gastropod population distribution (as in the case of Urca where only male gastropods were found) and linked to higher levels of organotin contamination in the environment. The results from the field studies allied to the bioassay confirmed the reliability of the VDSI scale proposed by [START_REF] Fernandez | Organotin compounds in Guanabara Bay (Rio de Janeiro): distribution and possible impacts[END_REF] for S. haemastoma as demonstrated by the excellent correlation found between VDSI level and organotin concentrations in tissues.

The chemical analysis of animals used in the bioassay indicated metabolization and excretion of TBT and TPhT by females and males of S. haemastoma with indications of a faster elimination in male gastropods.

As for concentrations of organotins in snails measured in the field study, those found for TBT in Vermelha Beach and in Urca are above the inoculated concentrations in the bioassay that caused imposex development. These areas evidently show contamination levels that pose a threat to molluscs and other organisms at the lower trophic levels, which appear to be at higher risk from exposure to such substances.

Figure 1 :

 1 Figure 1: Sampling stations in Guanabara Bay.

Figure 2 :Figure 3 .

 23 Figure2: Imposex frequency and inoculated TBT concentrations in the different aquaria: 1control aquarium; 2 -control aquarium + ethanol; 3 -TBT 1 (41.5 ng.µL -1 ); 4 -TBT 2 (82.9 ng.µL -1 ); 5 -TBT 3 (165.9 ng.µL -1 )

Figure 4 .

 4 Figure 4. Correlation (linear fitting r= 0.941; p < 0.005; VDSI = 0.145 + 0.04 OTs) between

Table 1 -

 1 Biological results of the bioassay. A = results for sampling 14 days after inoculation; B = results for sampling 28 days after inoculation; C = final results of the bioassay after each

	treatment; F-healthy females; I -imposexed females; M -males; T-total number of animals;
	VDSI -level of VDSI in imposex females; % -imposex frequency; D* -average dose (ng Sn / g
	of fresh tissue); Mort. -mortality; F-T -total number of healthy females; I -T -total number of
	imposexed females; % T -total imposex frequency; nr -not retrieved.
				A					B				C
	Aquaria	F I	M T VDSI % D* F I	M T	VDSI % D* Mort F-T I-T %T
	Control	7 0 8 15 0	0	0	13 0	12 25 0	0 0	2	20	0	0
	Control + ethanol	6 2 7 15 1	25 0	15 0	10 25 0	0 0	2	21	2	8,7
	TBT 1	3 3 9 15 1	50 5	9	3	12 24 1	25 5	3	12	6	33,3
	TBT 2	3 5 7 15 1	63 10 6	5	14 25 1	45 12 2	9	10 52,6
	TBT 3	2 6 7 15 1	75 24 1	6	5 12 2,3	86 20 15	3	12 80,0
	TPT 1	nr nr nr -	-	-	-	3	1	5 9	1	25 5	33	3	1	25,0
	TPT 2	nr nr nr -	-	-	-	2	3	3 8	1	60 8	34	2	3	60,0
	TPT 3	2 5 8 15 1	71 23 8	5	10 23 1,4	38 28 4	10	10 50,0

Table 2 .

 2 Results of organotin determination (ng g -1 ww as Sn) in gastropods from aquaria TBT-3

	and TPhP-3 of the bioassay. Inoculated doses in ng g -1 ww as Sn: (a) 24.7; (b) 24.9; (c) 20.0 and
	(d) 20.0; (e) 22.8; (f) 23.5; (g) 28.3; (h) 25.3.
	Samplings	14 days	28 days
	Compound
		Females	Males	Females	Males
	TBT-3	(n = 8) a	(n = 8) b	(n = 7) c	(n = 8) d
	TBT	5.21± 0.13	<dl	< dl	< dl
	DBT	29.20±5.26	19.77±3.61	16.00±1.17	22.59±2.20
	MBT	< dl	2.21±0.68	12.35±0.49	3.06±0.03
	TPhT-3	(n = 7) e	(n = 8) f	(n= 13) g	(n = 10) h
	TPhT	16.08±0.75	4.31±0.74	14.14±2.03	8.23±1.85
	DPhT	1.09±0.09	< dl	0.71±0.21	< dl
	MPhT	< dl	1.14±0.67	< dl	4.10±1.77
	(DBT)	12.51±1.04	20.86±6.12	20.89±6.72	23.97±3.17

Table 3 .

 3 Imposex development expressed by VDSI (Vas Deferens Sequence Index*), organotin concentrations (ng g -1 ww as Sn) in S. haemastoma (S.h.), and organotin concentrations in the mussel P. perna (P.p.). AC stands for Arraial do Cabo, sampling site for the biassay; 1-5 refer to sampling stations in Guanabara Bay shown under Figure1.

	Sampling Sites

38 7.46 22.69 14.05 44.61 34.91 66.95 78.33 56.67 73.36 68.78 40.05 137.97 164.21 PhT 3.30 < dl 1.03 0.83 6.17 0.523 12.88 <dl 6.21 1.03 4.95 0.58 10.87 5.86 OTs 10.68 7.46 23.72 14.88 50.80 35.44 79.94 78.33 62.89 74.39 73.74 40.62 148.84 170.07

Acknowledgments

The authors are grateful for the financial support from the Rio de Janeiro Foundation for Science Funding (FAPERJ) and from the Federal Coordination for Tertiary Education (CAPES).

ACCEPTED MANUSCRIPT

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