Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: Qualitative and quantitative aspects

Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: Qualitative and quantitative aspects

Marine Pollution Bulletin 61 (2010) 568–575 Contents lists available at ScienceDirect Marine Pollution Bulletin journal homepage: www.elsevier.com/l...

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Marine Pollution Bulletin 61 (2010) 568–575

Contents lists available at ScienceDirect

Marine Pollution Bulletin journal homepage: www.elsevier.com/locate/marpolbul

Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: Qualitative and quantitative aspects Laetitia Hédouin a,b,c, Marc Metian a,b, Thomas Lacoue-Labarthe b, Renaud Fichez c,1, Jean-Louis Teyssié a, Paco Bustamante b, Michel Warnau a,b,* a b c

International Atomic Energy Agency – Marine Environment Laboratories (IAEA-MEL), 4 Quai Antoine Ier, MC-98000 Principality of Monaco, Monaco Littoral, Environnement et Sociétés (LIENSs), UMR 6250 CNRS-Université de La Rochelle, 2 rue Olympe de Gouges, F-17042 La Rochelle, France Institut de Recherche pour le Développement (IRD-Nouméa Center), BP A5, 98848 Nouméa cedex, New Caledonia

a r t i c l e

i n f o

Keywords: Mollusks Isognomon isognomon Gafrarium tumidum New Caledonia Radiotracer Feeding

a b s t r a c t The present study aimed at examining the influence of food quality and quantity on the assimilation efficiency (AE) of metals in two abundant bivalves in the New Caledonia lagoon, the oyster Isognomon isognomon and the clam Gafrarium tumidum. Bivalves were exposed via their food to the radiotracers of three metals of concern in New Caledonia (54Mn, 57Co and 65Zn) under different feeding conditions (phytoplankton species, cell density, and cell-associated metal concentration). When bivalves were fed Heterocapsa triquetra, Emiliania huxleyi and Isochrysis galbana, AE of Mn, Co and Zn was strongly influenced by the phytoplankton species and by the metal considered. In contrast, when fed one given phytoplankton species previously exposed to different concentrations of Co, phytoplankton-associated Co load had no influence on the AE and on the retention time of the metal in both bivalves. Metals ingested with I. galbana displayed generally the highest AE in both bivalve species, except for Mn in clams for which the highest AE was observed for H. triquetra. Influence of food quantity was investigated by exposing bivalves to different cell densities of I. galbana (5  103, 104 or 5  104 cell ml1). As for food quality, food quantity was found to influence AE of Mn, Co and Zn, the highest AE being observed when bivalves were fed the lowest cell density. Overall, results indicate that the two bivalve species are able to adjust their feeding strategies according to the food conditions prevailing in their environment. Ó 2010 Elsevier Ltd. All rights reserved.

1. Introduction Changes in coastal ecosystem functioning due to anthropogenic metal inputs is a worldwide issue of concern especially as metals are not biodegradable and enter biogeochemical cycles (Tessier and Turner, 1995). In the coral reef lagoon of New Caledonia, metal contamination is a critical problem in relation with its extreme biodiversity (Labrosse et al., 2000). Indeed, the lagoon is subject to an increasing environmental pressure imposed by urban development and intensive mining activities. In addition, the use of hydrometallurgic process employing heated and pressured sulphuric acid (lixiviation) has been recently developed in New Caledonia and is expected to be implemented at industrial scale early 2010 (Goro-Nickel, 2001, 2003). Such a process will provide new poten* Corresponding author. Present address: International Atomic Energy Agency, Technical Cooperation Department, Division for Africa, Wagramer Strasse 5, PO Box 100, A-1400 Vienna, Austria E-mail address: [email protected] (M. Warnau). 1 Present addresses: IRD, Universidad Autonoma Metropolitana Iztapalapa, Departamento de Hidrobiología, Col. Vicentina C.P. 09340, Iztapalapa, DF, Mexico and Université de la Méditerranée, UMR CNRS 6535 LOPB, F-13007 Marseille, France. 0025-326X/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi:10.1016/j.marpolbul.2010.06.034

tial to exploit laterite soils that display lower nickel (Ni) contents than garnierite ores currently used in pyrometallurgic plants, such as at the Société Le Nickel, and will allow recovering the cobalt (Co) as a by-product (Mihaylov et al., 2000; Dalvi et al., 2004). However, the Ni and Co extraction based on lixiviation is an unselective process that may result in additional discharges of by-product metals such as chromium (Cr), iron (Fe), manganese (Mn) or zinc (Zn) (Goro-Nickel, 2001; Baroudi et al., 2003). Although long lasting contamination exists in New Caledonia (Laganier, 1991; Ambatsian et al., 1997) with high levels of metals reported in coastal marine sediments (e.g., Fernandez et al., 2006), few data on contamination levels in marine organisms and possible local marine ecosystem impairments are available so far in the open literature (e.g., Monniot et al., 1994; Dalto et al., 2006; Hédouin et al., 2008a,b; Metian and Warnau, 2008; Chouvelon et al., 2009). Therefore, programmes for monitoring possible impact of the land-based mining activities in the New Caledonia lagoon are needed. Such programmes should largely rely on the use of biomonitor species, as already developed and implemented in temperate areas (e.g., US and EU Mussel Watches; see e.g., Goldberg et al., 1983; Warnau and Acuña, 2007; Thébault et al., 2008). Indeed,

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the main advantage of the biomonitoring approach compared to direct measurement in water or sediment is to provide a direct and time-integrated assessment of the metal fraction that is actually available to the organisms (bioavailable fraction) (e.g., Phillips, 1991; Coteur et al., 2003; Danis et al., 2004; Metian et al., 2008b). In this context, both experimental and field studies have recently identified the oyster Isognomon isognomon and the clam Gafrarium tumidum as promising candidates biomonitoring metal contamination in New Caledonia lagoon waters (Metian et al., 2005; Hédouin et al., 2006, 2007, 2008a; Chouvelon et al., 2009). It is now well established that food is often a dominant pathway for metal bioaccumulation in marine invertebrates and that food composition and/or quantity can strongly influence metal assimilation efficiency (AE) (e.g., Borchardt, 1983; Riisgard et al., 1987; Wang and Fisher, 1999a). Furthermore, feeding processes such as filtration rate are flexible in marine filter-feeding organisms and may be adapted according to the changes in environmental conditions such as food quantity and/or composition (e.g., Widdows and Donkin, 1992; Navarro and Iglesias, 1993). For example, Cd assimilation in the mussel Mytilus edulis is inversely related to food quantity (Borchardt, 1983). In the scallop Pecten maximus, food is the main bioaccumulation pathway for Ag (98%) when diet is composed of Bacillariophyceae phytoplankton whereas dietary contribution drops below 40% when the scallop is fed Prymnesiophyceae phytoplankton (Metian et al., 2008a). Furthermore, heterorhabdic bivalves (those which gills are composed of two different filament types) are also able to select the particles that they are ingesting (Ward et al., 1998), which results in a preferential ingestion of nutritionallyrich particles that may also affect metal influx from food (e.g., Bayne, 1993; Wang and Fisher, 1997). The objective of this study was thus to investigate the possible influence of food quality (i.e., phytoplankton species) and quantity on the assimilation efficiency of three metals of concern in New Caledonia lagoon waters (Co, Mn and Zn) in the oyster I. isognomon and the clam G. tumidum. The variations in the feeding conditions that were considered are: (1) the phytoplankton species used as food, (2) the phytoplankton density and (3) the metal concentration associated with phytoplankton. Radiotracer techniques were used to enhance the detection sensitivity of metals and to allow for measuring metal flux at environmentally realistic contaminant concentrations (Warnau and Bustamante, 2007).

2. Materials and methods 2.1. Collection and acclimation The organisms (n = 100 per species) were collected by SCUBA diving in Maa Bay (oysters I. isognomon) or by hand-picking in Dumbea Bay (clams G. tumidum) in October 2003. Both locations are located 15–20 km north of Nouméa City, New Caledonia. Body size is known to affect bioaccumulation of metals in marine organisms (e.g., Boyden, 1974; Warnau et al., 1995); hence, according to previous preliminary studies (Metian, 2003; Hédouin et al., 2006, 2008a), only individuals with a shell longer than 70 mm (I. isognomon) or a shell wider than 35 mm (G. tumidum) were used in the experiments. After collection, clams and oysters clams were shipped to IAEA-MEL premises in Monaco, where they were acclimated for 2 months to laboratory conditions (open circuit aquarium; water renewal: 30% h1; salinity: 36 p.s.u.; temperature T° = 25 ± 0.5 °C; pH 8.0 ± 0.1; light/dark cycle: 12 h/12 h) simulating the conditions prevailing in the New Caledonia lagoon. During acclimation, bivalves were fed phytoplankton using the Prymnesiophyceae Isochrysis galbana (104 cells ml1). Recorded mortality was lower than 5% over the acclimation period.

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2.2. Radiotracers and counting Investigated elements (Co, Mn and Zn) were introduced into the experimental microcosms as radiotracers of high specific activity, purchased from Amersham, UK (57Co in 0.1 M HCl, T½ = 271.8 d) and Isotope Product Lab., USA (54Mn in 0.1 M HCl, T½ = 312.2 d; 65 Zn in 0.5 M HCl; T½ = 243.9 d). Radioactivity was measured using a high-resolution c-spectrometer system composed of three Germanium N- or P-type detectors (EGNC 33-195-R, EurysisÒ) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (InterwinnerÒ 6). The radioactivity of the samples was determined by comparison with standards of known activities and of appropriate geometry. Measurements were corrected for counting efficiency, background and physical radioactive decay. Counting times were adapted to obtain counting rates with propagated errors less than 5% (Rodriguez y Baena et al., 2006a). 2.3. Experimental procedures 2.3.1. Testing the influence of Co concentration in food I. galbana cells from an axenic stock culture were re-suspended into four erlenmeyer flasks (light/dark cycle: 12 h/12 h at 25 °C). Each flask contained 500 ml sterile-filtered seawater enriched with f/2 nutrients without EDTA and Si (Guillard, 1975). Flasks were spiked with four increasing Co concentrations (0, 5, 50, and 500 ng l1) and phytoplankton was allowed to grow under these conditions for 6 d. Added Co concentrations were realized using increasing amount of Co(NO3)2 (synthesis quality, Merck) and a fixed activity of the corresponding radiotracer 57Co (2.5 kBq l1, corresponding to 0.13 ng Co l1). The range of concentrations selected covers those encountered in the New Caledonia lagoon waters (Fernandez et al., 2002; Goro-Nickel, 2004). After 6 d of incubation, cell density increased from 103 to 1.5  106 cell ml1. The cells were gently filtered (1 lm-mesh size, NucleporeÒ Polycarbonate filters) and re-suspended in clean seawater. The radioactivity of the radiolabelled I. galbana in each flask was c-counted before and after the filtration. The radioactivity of algal cells used in feeding experiments was not significantly different among the different flasks, with an average calculated activity of 0.49 ± 0.14 lBq cell1. For each added Co concentration, four groups of nine oysters (shell length from 71 to 94 mm) and four groups of nine clams (shell width from 35 to 40 mm) were placed in four aquaria containing 16 l of 0.45-lm filtered natural seawater (close circuit aquaria constantly aerated; other parameters as previously described). Oysters were acclimated for one week to these conditions and seawater was renewed daily. Bivalves from each aquarium were then allowed to feed for 2 h on one out of the four batches of previously radiolabelled I. galbana (104 cell ml1) (pulse-chase feeding method; see e.g., Warnau et al., 1996b). Empty shells were placed as control in each aquarium to check for any direct uptake of radiotracers from seawater due to possible recycling from phytoplankton cells during the 2-h feeding period (Metian et al., 2007). These control shells were radioanalysed at regular intervals of time. At the end of the feeding period, all organisms were c-counted and open circuit conditions were restored (water renewal rate: 30% h1; salinity: 36 p.s.u.; T = 25 ± 0.5 °C; pH 8.0 ± 0.1; light/dark cycle: 12 h/12 h). From that time on, all individuals were ccounted at different time intervals over a 25-d period in order to determine the whole-body depuration kinetics of the radiotracers ingested with food. Throughout the depuration period, bivalves were fed daily for 1 h non-radiolabelled phytoplankton (I. galbana, 104 cell ml1).

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2.3.2. Testing the influence of phytoplankton species Two batches of nine oysters (shell length from 73 to 90 mm) and two batches of nine clams (shell width from 35 to 44 mm) were placed in two aquaria containing 16 l of 0.45-lm filtered natural seawater (close circuit aquaria constantly aerated; other parameters as previously described). Clams and oysters were acclimated to these conditions for 1 week (daily seawater renewal) and then fed either radiolabelled Emiliania huxleyi or Heterocapsa triquetra (104 cell ml1) for 2 h (pulse-chase feeding) in order to asses the possible influence of the phytoplankton species on metal assimilation efficiency and retention capacity in the bivalves. Both phytoplankton species occur naturally in several bays of the New Caledonia lagoon where the clams and oysters are living (Jacquet et al., 2006). For radiolabelling phytoplankton species, experimental approaches conducted on I. galbana were applied to the Prymnesiophyceae E. huxleyi and to the Dinophyceae H. triquetra. Cells from axenic stock cultures were re-suspended in two different erlenmeyer flasks (103 cell ml1), containing 4.5 l sterile-filtered seawater enriched with f/50 for E. huxleyi and enriched with f/2 nutrients without EDTA and Si for H. triquetra (Guillard, 1975). The two cultures were spiked with 5 kBq l1 of 54Mn, 57Co and 65Zn, corresponding to 3.6 ng Mn l1, 25 ng Co l1 and 60 ng Zn l1. The cultures were then incubated for 6 d at 25 °C (light/dark cycle: 12 h/12 h). After incubation, the cell densities were 7  105 cell ml1 for E. huxleyi and 1.6  105 cell ml1 for H. triquetra. The cells were then gently filtered, re-suspended in clean seawater and c-counted as described above (Section 2.3.1). The radioactivity of algal cells used in the feeding experiments was 0.26 ± 0.18 lBq cell1 for E. huxleyi and 0.96 ± 0.11 lBq cell1 for H. triquetra for 54Mn, 2.1 ± 0.8 and 20.8 ± 12.1 lBq cell1 for 57Co and 3.2 ± 1.3 and 3.3 ± 0.1 lBq cell1 for 65Zn, respectively. Empty bivalve shells were used as controls for possible metal recycling and whole-body depuration kinetics of radiotracer ingested with the food were determined in both bivalve species as described in Section 2.3.1.

2.3.3. Testing the influence of cellular density Three groups of nine oysters (shell length from 71 to 92 mm) and three groups of nine clams (shell width from 36 to 45 mm) were placed in three aquaria containing 16 l of 0.45-lm filtered natural seawater (close circuit aquaria constantly aerated; other parameters as previously described), and acclimated for 1 week (daily seawater renewal) during which time their food was prepared. To do this, cells of I. galbana from an axenic stock culture were re-suspended in an erlenmeyer flask containing 4.5 l sterilefiltered seawater enriched with f/2 nutrients without EDTA and Si. The culture was then spiked with 5 kBq l1 of 54Mn, 57Co and 65 Zn and incubated for 6 d at 25 °C (light/dark cycle: 12 h/12 h). After incubation, the cell density had increased from 103 to 1.4  106 cell ml1. Three sub-samples of 58, 115 and 580 ml of the culture were then gently filtered and re-suspended in clean seawater. These three batches were prepared to obtain final cell density of 5  103, 104 and 5  104 cell ml1 in the 16-l exposure aquaria. The radioactivity of the radiolabelled I. galbana was measured before and after the cellular filtration. The radioactivity of algal cells ranged from 1.11 to 1.80 lBq cell1 for 54Mn, 0.83 to 1.37 lBq cell1 for 57Co, 2.69 to 4.38 lBq cell1 for 65Zn. Each group of clams and oysters was then fed for 2 h one of the radiolabelled I. galbana batches (5  103, 104 or 5  104 cell ml1). Whole-body depuration kinetics of the radiotracers ingested with the food were then followed as described in Section 2.3.1 and controls (empty shells) were placed in the aquaria for assessing possible radiotracer recycling.

2.4. Data analysis Depuration of the radiotracers was expressed as the percentage of remaining radioactivity (radioactivity at time t divided by initial radioactivity measured in the organisms just after the feeding period  100) (Warnau et al., 1996b; Rodriguez y Baena et al., 2006b). Depuration kinetics for all experiments were fitted using kinetic models and statistical methods as described by Warnau et al. (1996a,b) and Lacoue-Labarthe et al. (2008). Depuration kinetics were always best fitted by a double-component exponential equation (decision based on F test and ANOVA tables for two fitted model objects):

At ¼ A0s ekes t þ A0l ekel t where ke is the depuration rate constant (d1), At and A0 are the remaining activities (%) at time t (d) and 0, respectively, and ‘s’ and ‘l’ are the subscripts for the short-lived and long-lived components. The short-lived component represents the loss of the radiotracer fraction that remains associated with the faeces and is rapidly eliminated with them, whereas the long-lived component describes the loss of the radiotracer fraction that is actually absorbed by the organism and slowly eliminated (Whicker and Schultz, 1982; Warnau et al., 1996b). The long-lived component allows assessing the assimilation efficiency (AE) of the radiotracer ingested with food (AE = A0l). Also, for each exponential component (s and l), a biological half-life can be calculated (Tb½s and Tb½l) from the corresponding depuration rate constant (kes and kel) according to the relation Tb½ = ln 2/ke. Constants of the models and their statistics were estimated by iterative adjustments of the model and Hessian matrix computation using the nonlinear curve-fitting routines in the StatisticaÒ 5.2.1 software. Differences among the estimated kinetic parameters for the different feeding conditions were tested using comparison tests of the means and possible trends linking metal concentrations to cell densities were assessed using simple linear regression techniques (Zar, 1996). The level of significance for statistical analyses was always set at a = 0.05. 3. Results Depuration kinetics of the radiotracers were followed in the organisms which ingested enough food to display sufficient radioactivity to be accurately counted. Most oysters met this requirement; however some clams displaying very low activities were discarded. No activity was detected on control shells, indicating that no detectable recycling of phytoplankton-associated tracers occurred in the experimental microcosms. 3.1. Effect of Co concentration in phytoplankton Fitting of the whole-body depuration kinetics of 57Co in oysters fed Co-loaded I. galbana by a double-exponential model was quite satisfactory (R2: 0.86–0.90) for all the food-associated Co concentrations tested (Table 1, Fig. 1). The major fraction (80–85%) of the total radioactivity in oysters was rapidly lost (Tb½s < 1 d) whereas the long-lived component accounted for only 15–20% of the 57Co ingested with food that was eliminated with a biological half-life (Tb½l) ranging from 13 to 25 d. Similarly, the fit of the whole-body depuration of 57Co in clams was quite good (R2: 0.27–0.64) for all the Co concentrations tested (Table 1, Fig. 1). However, the estimated AE of 57Co ingested with food was much higher than in oysters (i.e., 76–84%) and this fraction was retained with a Tb½ ranging from 36 to 39 d. In both bivalve species, no significant difference (p always >0.05) was found among the estimated kinetic parameters (Aos,

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Table 1 Assimilation efficiency (AE, %), depuration rate constant (kel, d1) and biological half-life (Tb½l, d) of 57Co in the oyster Isognomon isognomon and the clam Gafrarium tumidum fed radiolabelled Isochrysis galbana (104 cell ml1) previously exposed to four increasing Co concentrations (n = 9 oysters per concentration tested, n = 6 clams for 0 and 5 ng l1 and n = 8 clams for 50 and 500 ng l1). ASE: asymptotic standard error; R2: determination coefficient. Species

Co concentration added (ng l1)

AE ± ASE

kel ± ASE

Tb½l ± ASE

a

*

*

R2

I. isognomon

0 5 50 500

15.8 ± 7.0 19.6 ± 5.4c 16.6 ± 6.2b 14.7 ± 6.0a

0.032 ± 0.036 0.054 ± 0.028* 0.050 ± 0.036* 0.027 ± 0.033*

22 ± 24 13 ± 7* 14 ± 10* 25 ± 30*

0.87 0.88 0.86 0.90

G. tumidum

0 5 50 500

77.2 ± 3.9d 77.4 ± 3.9d 75.7 ± 3.9d 84.1 ± 5.9d

0.018 ± 0.006b 0.019 ± 0.006c 0.018 ± 0.006b 0.019 ± 0.007a

37 ± 11b 36 ± 10c 39 ± 12b 36 ± 13a

0.64 0.27 0.51 0.51

Significance of the estimated parameters: a p < 0.05. b p < 0.01. c p < 0.001. d p < 0.0001. * Not significant (p > 0.05).

Fig. 1. Influence of phytoplankton-associated Co concentrations on whole-body depuration kinetics of 57Co in the oyster Isognomon isognomon and the clam Gafrarium tumidum fed radiolabelled Isochrysis galbana (104 cells ml1). A (%): remaining activity (%) ± SD (n = 9 oysters; n = 6 clams for 0 and 5 ng l1 and n = 8 clams for 50 and 500 ng l1).

kes, Aol, kel) determined for the four different food-associated Co concentrations. 3.2. Effect of phytoplankton species In oysters, depuration kinetics of 54Mn, 57Co and 65Zn ingested with I. galbana (ISO), E. huxleyi (EMI) or H. triquetra (HET) (104 cell ml1) were best described by a double-exponential model (R2: 0.23–0.63 for ISO, 0.11–0.83 for EMI and 0.57–0.92 for HET) (Table 2, Fig. 2). No significant difference was found among estimated Tb½ for all radiotracers and all phytoplankton species tested. In addition, no significant difference was found between AEs for Co and Mn in oysters fed EMI and HET, and for Zn in oysters fed ISO and EMI. In contrast, significant differences (p < 0.02) among AEs were observed for Co and Mn (ISO > HET = EMI, p < 0.004) and for Zn (ISO = EMI > HET, p < 0.02). In clams, fitting of the whole-body depuration of the radiotracers ingested with I. galbana, E. huxleyi or H. triquetra (104 cell ml1) were generally somewhat better than in the oyster (R2: 0.47–0.98 for ISO, 0.47–0.93 for EMI and 0.61– 0.89 for HET) (Table 2, Fig. 2). Tb½ of 54Mn was significantly longer when it was assimilated from HET than from EMI or ISO (p = 0.03 and 0.004, respectively). Significant differences were also observed among AEs calculated for Co, Mn and Zn ingested with the three phytoplankton strains (Mn: ISO < EMI = HET, p < 0.03; Co: ISO = EMI > HET, p < 0.0004; and Zn: ISO P EMI P HET, p = 0.04).

3.3. Effect of cellular density When oysters were fed 104 and 5  104 cells ml1 of radiolabelled I. galbana, whole-body depuration kinetics of 54Mn, 57Co and 65Zn were fitted with R2 ranging from 0.23 to 0.63 and 0.38 to 0.60, respectively (Table 2, Fig. 3). No significant difference in Tb½ and AE between cell densities was found for Co. In contrast, significant differences in AE were found for Mn and Zn, with higher AE calculated at the low cell density (p = 0.001 and 0.0003, respectively). For clams, examination depuration kinetics of the radiotracers (R2: 0.33–0.65 at 5  103 cell ml1 and 0.47–0.98 at 104 cell ml1) indicated that Tb½ was not significantly different between the two food densities for all three radiotracers (Table 2, Fig. 3). However, when fed the low cell density, clams incorporated Co, Mn and Zn with significantly higher AE (p = 0.003, 0.047 and 0.0003, respectively). 4. Discussion During the last two decades, dietary pathway has been increasingly recognized as a major source of contaminant accumulation in marine invertebrates (e.g., Wang et al., 1996; Reinfelder et al., 1998; Wang and Fisher, 1999b). The assimilation efficiency (AE) and retention time (Tb½) are the critical parameters in assessing and modelling the dietary uptake of contaminants and numerous studies have been devoted to assess these parameters in different

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Table 2 Assimilation efficiency (AE, %), depuration rate constant (kel, d1) and biological half-life (Tb½l, d), of 54Mn, 57Co and 65Zn in the oyster Isognomon isognomon and the clam Gafrarium tumidum fed radiolabelled Emiliania huxleyi (104 cell ml1), Heterocapsa triquetra (104 cell ml1) and Isochrysis galbana (104 cell ml1 and 5  104 cell ml1 for I. isognomon; 5  103 cell ml1 and 104 cell ml1 for G. tumidum) (n = 8 oysters; n = 7 clams per phytoplankton species tested). ASE: asymptotic standard error; R2: determination coefficient. Species I. isognomon

Phytoplankton strain E. huxleyi

H. triquetra

I. galbana

G. tumidum

E. huxleyi

H. triquetra

I. galbana

Cell density (cells ml1) 4

Isotope

AE ± ASE

Tb½l ± ASE

R2

10 104 104

Mn Co 65 Zn

34 ± 6.5 22 ± 5.6c 70 ± 6.5d

0.028 ± 0.015 0.039 ± 0.021* 0.0002 ± 0.007*

24 ± 13 18 ± 10* 2783*

0.74 0.83 0.11

104 104 104

54

Mn Co 65 Zn

20 ± 2.6d 21 ± 4.1d 51 ± 3.2d

0.025 ± 0.012a 0.050 ± 0.020a 0.006 ± 0.005*

28 ± 13a 14 ± 6a 123 ± 109*

0.92 0.88 0.57

104 104 104 5  104 5  104 5  104

54

Mn Co 65 Zn 54 Mn 57 Co 65 Zn

90 ± 5.6d 55 ± 7.1d 76 ± 4.1d 41 ± 7.8d 37 ± 21* 52 ± 3.5d

0.010 ± 0.005a 0.026 ± 0.010b 0.015 ± 0.004b 0.015 ± 0.014* 0.027 ± 0.043* 0.010 ± 0.005a

70 ± 32a 26 ± 10b 45 ± 13b 47 ± 46* 26 ± 42* 70 ± 34a

0.23 0.63 0.36 0.38 0.51 0.60

104 104 104

54

Mn Co 65 Zn

39 ± 4.3d 80 ± 3.5d 42 ± 2.3d

0.051 ± 0.011d 0.010 ± 0.004a 0.014 ± 0.004b

14 ± 3d 70 ± 26a 48 ± 14b

0.92 0.47 0.93

104 104 104

54

Mn Co 65 Zn

56 ± 23a 41 ± 3.1d 33 ± 7.3d

0.021 ± 0.026* 0.014 ± 0.006a 0.005 ± 0.016*

34 ± 10* 49 ± 9a 143 ± 474*

0.61 0.89 0.71

104 104 104 5  103 5  103 5  103

54

22 ± 3.7d 73 ± 2.7d 51 ± 3.8d 72 ± 17.9c 87 ± 6.1d 90 ± 3.0d

0.044 ± 0.015b 0.010 ± 0.003b 0.013 ± 0.006a 0.046 ± 0.021a 0.002 ± 0.005* 0.011 ± 0.003c

16 ± 5b 68 ± 21b 55 ± 25a 15 ± 7a 416 ± 135* 61 ± 16c

0.65 0.33 0.52 0.98 0.47 0.68

57

57

57

57

57

Mn Co 65 Zn 54 Mn 57 Co 65 Zn 57

d

kel ± ASE

54

*

*

Significance of the estimated parameters: a p < 0.05. b p < 0.01. c p < 0.001. d p < 0.0001. * Not significant (p > 0.05).

Fig. 2. Influence of phytoplankton species (Isochrysis galbana, Emiliania huxleyi and Heterocapsa triquetra; 104 cells ml1) used as food on whole-body depuration kinetics of 54 Mn, 57Co and 65Zn in the oyster Isognomon isognomon (n = 9 for I. galbana and 8 for E. huxleyi and H. triquetra) and the clam Gafrarium tumidum (n = 8 for I. galbana and 7 for E. huxleyi and H. triquetra). A (%): remaining activity (%) ± SD.

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Fig. 3. Influence of phytoplankton cell density (5  103, 104 or 5  104 cells ml1) on whole-body depuration kinetics of 54Mn, 57Co and 65Zn in the oyster Isognomon isognomon (n = 9 for 104 cells ml1 and 5  104 cells ml1) and the clam Gafrarium tumidum (n = 6 for 5  103 cells ml1 and n = 8 for 104 cells ml1) fed radiolabelled Isochrysis galbana. A (%): remaining activity (%) ± SD.

marine organisms (e.g., Wang et al., 1996; Warnau et al., 1996b, 1999; Pan and Wang, 2008). However, as almost a rule in tropical environments (e.g., Phillips, 1991; Chong and Wang, 2000; Metian et al., 2005), only very few data are yet available regarding AE and Tb½ parameters for organisms from New Caledonia (e.g., Hédouin et al., 2006, 2007). Ideally, the concentrations of metals in the tissues of a biomonitor species should reflect those occurring in the ambient environment. This essential criterion has been previously experimentally assessed for I. isognomon and G. tumidum for the dissolved pathway (Hédouin et al., 2007, 2010). Exposures to a range of dissolved concentrations of As, Cr, Co, Cd, Mn, Ni and Zn indicated that, over a realistic range of concentrations, these elements were generally bioconcentrated in direct proportion to their concentration in seawater (ibid.). The results presented here are complementary with these previous studies as they expand the available knowledge regarding metal accumulation in I. isognomon and G. tumidum to the dietary pathway. When ingested with phytoplankton previously exposed to a range of increasing Co concentration (up to 500 ng added Co l1) Co was shown to be assimilated in the same proportion (AE) and retained with similar relative strength (Tb½) whatever the foodassociated Co concentration was (see Fig. 1 and Table 1). The experimental conditions were designed to cover the whole range of Co concentrations that can be encountered in New Caledonia waters, from pristine up to extremely contaminated areas (Fernandez et al., 2002; Goro-Nickel, 2004). Similar trends have been previously reported by Chong and Wang (2000) who observed that concentration of Cd, Cr and Zn in sediment had little effect on the assimilation efficiency of sediment-bound metals in the green mussel Perna viridis and in the Manila clam Ruditapes philippinarum. However, the response to metal concentration variation in ingested food appears to depend on the element as well as on the species investigated. Indeed, whereas AE of Se in the mussel

M. edulis was not affected by the Se concentration in the ingested diatoms (Thalassiosira pseudonana), AE of Zn and Cd did, respectively, decrease and increased with increasing contamination of the diatom used as food (Wang and Fisher, 1996). Nevertheless, along with data obtained from exposures to increasing dissolved Co concentrations in the same species (Hédouin et al., 2010), the present results on Co AE indicate that in the field Co concentrations in both I. isognomon and G. tumidum would be reflecting the level of Co in their environment, both in the dissolved and particulate phases. Whereas food quality and quantity were shown to have limited influence on the retention time of Co, Mn and Zn in clam and oyster tissues, metal AE generally differed according to the feeding conditions. Metals were generally better assimilated when bivalves were fed I. galbana than the two other phytoplankton species (E. huxleyi or H. triquetra). I. galbana cells have comparable cell length (c.l.: 4– 6 lm) and cell width (c.w.: 2–4 lm) than E. huxleyi cells (c.l.: 3– 4 lm; c.w.: 3–4 lm), but are much smaller than H. triquetra cells (c.l.: 20–28 lm; c.w.: 14–18 lm). Hence, the differences and similarities in AE observed among the feeding conditions indicate that phytoplankton size would not be the major factor driving metal AEs in the two bivalves. Bivalves are able to feed selectively on particles of different size and of different nature (various phytoplankton species as well as inorganic particles) (e.g., Newell et al., 1989) and species-related selectivity and/or dietary preferences could at least partly explain the specific differences observed in AE. Alternatively or complementarily, specific difference in metal speciation such as storage of the metal under bioavailable forms in the cytoplasm of the phytoplankton cells (e.g., Reinfelder and Fisher, 1991; Wang et al., 1996; Metian et al., 2008a) could also explain the AE differences that were observed. Food availability is another key factor that is well known to influence feeding behaviour of filter-feeding bivalves (e.g., Bayne et al., 1987; Bayne, 1993; Pan and Wang, 2008). Generally, filter-

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feeders can adjust their filtration rate to ambient phytoplankton density and thereby are able to maintain a stable ingestion rate even at high food concentrations (Jin et al., 1996; Dong et al., 2000; Zhuang and Wang, 2004). Although no conclusion on the influence of this adaptive feeding behaviour could be directly drawn from our results, it is clear that food availability notably influenced the AE of the metals examined in I. isognomon and G. tumidum. It is nowadays well documented that the dietary pathway is an important contributor to the global bioaccumulation of metals in marine organisms (e.g., Wang and Fisher, 1997; Metian et al., 2008a). Since the present study has shown that the feeding behaviour of I. isognomon and G. tumidum is influenced by the feeding conditions (quality and/or quantity of food), it is strongly recommended that future studies take into account these parameters so as to refine the prediction of biodynamic models (e.g., Thomann et al., 1995; Metian et al., 2008a; Pan and Wang, 2008). The consideration of such data is also needed to explain bioaccumulation data obtained in the framework of biomonitoring programmes. For example, Bendell-Young and Arifin (2004) demonstrated the influence of mussel feeding behaviour on their predicted tissue concentrations in Cd, especially under conditions of highly variable quantity and quality of suspended particles. In conclusion, our experimental results suggest that food quality (phytoplankton composition) and quantity (cell density) may play a significant role in the assimilation of metals ingested with food in I. isognomon and G. tumidum. Because of the major importance of the dietary contribution to global metal bioaccumulation in marine organisms, it is thus recommended to pay great attention to factors influencing AE. This would help refining both bioaccumulation model predictions and interpretation of data from field surveys and biomonitoring programmes. Acknowledgements The authors thank O. Pringault (IRD-Nouméa) for critical reading of the manuscript. LH was beneficiary of a PhD grant (CIFRE, France) supported by the Goro-Nickel Company, New Caledonia. M.W. is an Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium) and has benefited from a 2008-2009 Invited Expert position at LIENSs (CNRSUniversité de La Rochelle), supported by the Conseil Régional de Poitou-Charentes. This work was supported by the IAEA, the French PNEC Programme (Chantier ‘‘Nouvelle-Calédonie”) and IRD. The IAEA is grateful for the support provided to its Marine Environment Laboratories by the Government of Monaco. References Ambatsian, P., Fernex, F., Bernat, M., Parron, C., Lecolle, J., 1997. High metal inputs to closed seas: the New Caledonia lagoon. Journal of Geochemical Exploration 59, 59–74. Baroudi, H., Bureau, J., Rollin, C., 2003. Analyse critique de l’acceptabilité du niveau de rejet de manganèse dans le milieu marin. Rapport final Goro-Nickel INERIS Institut National de l’Environnement Industriel et des Risques, 37p (in French). Bayne, B.L., 1993. Feeding physiology of bivalves: time-dependence and compensation for changes in food availability. In: Dame, R.F. (Ed.), Bivalve Filter Feeders in Estuarine and Coastal Ecosystem Processes. Springer-Verlag, Heidelberg, pp. 1–24. Bayne, B.L., Hawkins, A.J.S., Navarro, E., 1987. Feeding and digestion by the mussel Mytilus edulis L. (Bivalvia: Mollusca) in mixtures of silt and algal cells at low concentration. Journal of Experimental Marine Biology and Ecology 111, 1–22. Bendell-Young, L.I., Arifin, Z., 2004. Application of a kinetic model to demonstrate how selective feeding could alter the amount of cadmium accumulated by the blue mussel (Mytilus trossolus). Journal of Experimental Marine Biology and Ecology 298, 21–33. Borchardt, T., 1983. Influence of food quantity on the kinetics of cadmium uptake and loss via food and seawater in Mytilus edulis. Marine Biology 79, 67–76. Boyden, C.R., 1974. Trace elements contents and body size in molluscs. Nature 251, 311–314.

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