Ismael Mejía
email: ismael.mejia@mines-nantes.fr

Mario Südholt
email: mario.sudholt@mines-nantes.fr

STRUCTURED AND FLEXIBLE GRAY-BOX COMPOSITION: APPLICATION TO TASK RESCHEDULING FOR GRID BENCHMARKING

Keywords: Software Composition, Software Engineering, Distributed Software

The evolution of complex distributed software systems often requires intricate composition operations in order to adapt or add functionalities, react to unanticipated changes to security policies, or do performance improvements, which cannot be modularized in terms of existing services or components. They often need controlled access to selected parts of the implementation, e.g., to manage exceptional situations and crosscutting within services and their compositions. However, existing composition techniques typically support only interface-level (black-box) composition or arbitrary access to the implementation (gray-box or white-box composition). In this paper, we present a more structured approach to the composition of complex software systems that require invasive accesses. Concretely, we provide two contributions, we (i) present a small kernel composition language for structured gray-box composition with explicit control mechanisms and a corresponding aspect-based implementation; (ii) present and compare evolutions using this approach to gray-box composition in the context of two real-world software systems: benchmarking of grid algorithms with NASGrid and transactional replication with JBoss Cache.

INTRODUCTION

The evolution of large-scale distributed software systems often requires the unanticipated introduction of new functionalities or the modification of existing ones. Such evolution tasks are often inherently difficult because of two fundamental problems. First, the compositions cannot be expressed only in terms of the interfaces of the involved systems (non-invasive modifications) but also imply changes to some (typically limited) parts of the corresponding implementations. Second, the compositions often involve functionalities that are not well modularized in the existing systems or in the resulting composed system. Such composition problems occur frequently in legacy ERP systems that, e.g., to cope with new security requirements imposed by changing legal frameworks, such as the Sarbanes-Oxley act in the U.S. (such evolution problems for SAP AG's SOA infrastructure are considered, e.g. in the CESSA1 research project). As a concrete real-world example (that we will consider in more detail later on), we have studied NASA's NASGrid benchmarking infrastructure for computational grids [START_REF] Frumkin | Nasgrid benchmarks: a tool for grid space exploration[END_REF]. This benchmark is used to time grid computations that may execute on different communication topologies. Fig. 1 shows the main components, shown with gray background, of the NASGrid benchmarking frameworks: three sets of classes that respectively provide a benchmarking interface, exception handling (principally of network conditions), and management of the graph structure representing the graph communication topology (the remainder of the system being constituted essentially by routines for numerical algorithms, called computational tasks in the figure).

NASGrid basically executes computations on distributed nodes and forwards intermediate results according to communication dependencies defined in terms of a topology graph. Grid computations are aborted in the case of exceptions, such as severe network errors; task rescheduling in the case of exceptions is not supported. We have investigated an evolution of NASGrid to add this useful functionality that fits well with existing, frequently long-running, grid applications. Our analysis of the existing code base has shown that the extension of NASGrid by task rescheduling partially requires modifications to the existing interfaces (i.e., sets of public classes and methods that are marked by disks in Fig. 1). However, the extension also requires some access to the NASGrid implementation because the necessary modifications as a whole are crosscutting with respect to the existing structure of NASGrid (the corresponding classes are marked by stars in Fig. 1).

Performing such evolutions using mainstream languages or development methods is highly difficult and error prone: (i) the crosscutting nature of such evolutions involve a potentially large number of modifications that have to be carefully synchronized; (ii) structural means and semantic properties should be supported in order to control the effects of invasive modifications to implementations.

In this paper we present an approach of structured invasive, i.e., gray-box, composition that supports accesses to interfaces and implementations through compositions of basic programming patterns for invasive access, resulting in gray-box compositions whose degree of invasiveness and their impact on an implementation can be controlled explicitly and flexibly. Furthermore, these operators allow crosscutting functionalities that are part of the subsystems to be expressed modularly.

Concretely, we present two contributions: First, we introduce in section 2 a kernel language for invasive composition that enables explicit and expressive compositions of invasive distributed patterns [START_REF] Benavides | Invasive patterns for distributed programs[END_REF] (henceforth simply called invasive patterns). Invasive patterns and compositions thereof provide flexible control of gray-box compositions and support the modularization of crosscutting functionalities using aspect-oriented programming techniques [START_REF] Kiczales | Aspect oriented programming[END_REF]. We also briefly present an implementation of this kernel language using the AWED system [START_REF] Benavides | Explicitly distributed AOP using AWED[END_REF], (AWED website, 2010) for explicitly-distributed AOP. Second, in section 3 we present and evaluate how our approach supports an evolution scenario of NASGrid that add task rescheduling. This extension is non-trivial, interacting with the original application at 28 places and is modularly implemented by an aspect and four new ordinary classes. Finally, we briefly compare the corresponding composition properties with those of two other case studies we have performed as part of previous work: a less invasive evolution of NASGrid for checkpoint introduction; and a highly invasive evolution of JBoss Cache, a middleware for transactional replication of data in distributed systems.

Our results show that invasive compositions allow a whole space of evolutions that require invasive modifications to be expressed while maintaining much higher control of the impact of invasive modifications, and this for systems requiring from moderately invasive to highly invasive accesses. As to our knowledge, no other approach to gray-box composition has been applied to such a range of evolution scenarios nor provides a comparable level of control of effects.

STRUCTURED AND FLEXIBLE INVASIVE COMPOSITION

Evolution scenarios as discussed in the previous section require three essential requirements to be addressed: R1) Enable (modifications to) the coordination of distributed communications and computations. R2) Support modularization of crosscutting functionalities that are subject to evolution tasks. R3) Provide structural and property-based control over modifications, in particular invasive ones. From a general point of view, we address these three issues as follows: we exploit invasive patterns as basic abstractions in order to express coordination and communication requirements of distributed applications that involve crosscutting functionalities. We introduce a composition language over patterns that enables the definition of structured and flexible pattern compositions whose effects may be controlled, e.g., by limiting invasive accesses to contexts defined by event sequences. In the remainder of this section we briefly revisit the notion of invasive patterns for distributed programming and then define a kernel language for the flexible composition of such patterns. Finally, we show how such pattern compositions can be implemented in terms of the AWED system [START_REF] Benavides | Explicitly distributed AOP using AWED[END_REF], a system for distributed aspects.

Invasive Patterns

Invasive patterns [START_REF] Benavides | Invasive patterns for distributed programs[END_REF] have been introduced as generalizations of standard parallel and distributed programming patterns. Fig. 2 shows the three invasive patterns we consider: a gather, a farm and a pipelining pattern. All of these patterns match sequences of execution events (illustrated by the dotted curves) over calls to interface methods or methods called in the implementation. These sequences are matched on one or several source nodes, construct data (using a computation represented by the filled rectangle on the source nodes) that is sent to a number of one or several target nodes and integrated into the computations there (as represented by the filled rectangle on the target side). Invasive patterns allow quantifying over sets of source and target nodes, in particular, the event sequences that trigger actions as well as the actions themselves.

Fig. 2. Invasive Patterns

Invasive patterns provide basic support for the three requirements mentioned above: as frequently used patterns for distributed programming, they support distribution coordination (R1); modularization of crosscutting functionalities (R2) can be achieved by means of aspects for the definition of event sequences (history-based pointcuts in AOP-speak) and actions (advice in AOP-speak) that compute data to be transferred from source to target nodes and that integrate data into target computations. Finally, some control over accesses and computations is provided by their fixed overall structure.

A kernel language for non/invasive composition

In this paper, we introduce a composition language over invasive patterns in order to fully address the requirements for evolution tasks. We strive, in particular, for a language that enables flexible compositions of patterns to handle more complex crosscutting functionalities and provides better control over, possibly invasive, modifications performed by pattern compositions.

Fig. 3. Kernel language for invasive composition

Fig. 3 presents the essentials of our kernel language for invasive composition. Some remarks on notation: non-terminals, such as Op or P start with an upper case letter and are set in italic font; lexical categories, such as e are denoted by lower case, italic letters; terminals, such as if set in typewriter font. X denotes finite sequences of expressions of non-terminal X. (A more elaborate version of the language that supports, e.g., repetitions in form of regular expressions is in preparation but not needed for the extension of the NASGrid application by dynamic task rescheduling considered in this paper).

The intuition behind this core language is as follows: operators match contexts that trigger sequences of adaptations. Contexts are built from event sequences that may be guarded. Adaptations include simple manipulations enabling the insertion of glue code, such as communication statements, but also potentially complex pattern compositions built from the three invasive patterns introduced above.

The grammar defines four main syntactic categories: (evolution) programs Prog, operators Op, contexts Ctx and adaptations Adap. (Evolution) programs are sequences over evolution operations (instantiations of invasive patterns or pattern compositions). An operator is defined as a pair of a context and an adaptation. Contexts consist of sequences of guarded events (cf. G), i.e., events that may be matched on specific hosts or under specific conditions (represented by B, the nature of which is unspecified here; typically we expect conditions of limited expressiveness to support property analysis and verification).

Adaptations come in two forms: sequences of (i) possibly guarded events that represent (computation or communication) glue code potentially triggered on specific hosts and under specific conditions; (ii) structured adaptations in form of pattern compositions P that are pairs of sequences of operators. Such a pattern, say (s,t) denotes adaptations on sources s and targets t, typically the extraction of data on sources that are send for further handling to the targets. Note that patterns and pattern compositions may form both the context and adaptation parts of the operators.

Our language directly supports very flexible pattern compositions. As a simple example (that has been applied for NASGrid task rescheduling) consider an application of a farm-pattern followed by a gather pattern. The farm will match an event sequence on one node, extract information, send and inject it into a number of target nodes. The gather pattern will then monitor for event sequences on its source nodes that, in its simplest case, are the target nodes of the farm pattern, extract information on the source nodes of the gather pattern and inject them in its target node.

Implementation using distributed aspects and AWED

The AWED system [START_REF] Benavides | Explicitly distributed AOP using AWED[END_REF]) provides an aspect model for distributed systems that provides means for the monitoring of sequences of events, history-based pointcuts in AOP-speak, that occur on different (groups of) hosts. Such event sequences are described in terms of guarded finite-state systems; AWED also provides various means to trigger actions, advice in AOP, where the corresponding pointcutdefining event sequences are matched.

The language above can be implemented using AWED in terms of event sequences that define the, interface level or implementation-level, context (Ctx in the above grammar) and use actions to define adaptations (Adap). Invasive patterns (farm, gather and pipeline) are then implemented as pairs of aspects corresponding to source and target computations of the patterns. Pattern compositions, say p 2 ѻ p 1 , are implemented by aspects that match end-marking events in p 1 and trigger execution of p 2 . We have implemented task rescheduling for NASGrid using AWED this way.

Fig. 4 shows the main component of the implementation of the task rescheduling aspect. Here, the pointcut taskRescheduling (lines 4-11) defines a mixed interface/implementation-level context that identifies exception occurrences (state EXCEPTION) and, possibly repeated, choices of alternative available hosts (state LOOKUP). The second advice (lines 18-27) chooses an alternative and restarts the benchmark (i.e., triggers a farm pattern that sends info to the successor nodes of the current one). Overall, this language provides flexible structured invasive access through pattern compositions that may be subjected to explicit control through predefined compositions and the precise definition of application contexts by means of event sequences.

Implementing composition of invasive patterns. Fig. 5 shows an interface we have developed that represents a subset of the above language that makes explicit invasive patterns and pattern compositions. The pattern composition constructors enable building of compositions from simple operators (constructor op), sequences of compositions (seq), and compositions of farm and gather patterns (the latter two being expressible as sequences and are necessary for the task rescheduling example).

STRUCTURED AND FLEXIBLE INVASIVE COMPOSITION

In this section we consider the implementation of evolution scenarios using invasive patterns in the context of two real-world software systems of medium size, the NASGrid application (ca. 21 KLOC) and the JBoss Cache middleware for distributed caching under transactional control (ca. 50 KLOC). Concretely, we present the task rescheduling evolution for NASGrid in more detail, especially its use of invasive composition and how our language can be used to exert control over invasive modifications. Furthermore, we briefly compare the composition characteristics of the task rescheduling case study with two other evolution scenarios that we have previously performed using invasive patterns but without explicit support for the composition of patterns. This comparison shows that compositions of invasive patterns allow to cover a whole range of evolution scenarios, from limited invasive ones to highly invasive ones and that the flexible pattern compositions our language supports simplify such evolution tasks.

Invasive composition for NASGrid task rescheduling

NASA's NASGrid benchmark allows to time grid applications that are deployed on different hardware topologies; communication paths taken as part of a grid application are represented in NASGrid using a topology graph. Each computation on a node is modeled using an individual worker thread that executes some numerical computation, using building blocks, such as LU matrix decomposition and Fourier transforms (FT). These computational tasks are supervised by a coordinator thread which forwards the results to other nodes as defined by the topology graph.

The main obstacle for adding task rescheduling as a strategy for improving fault tolerance in the case of network or node failures, is the static topology representation and benchmark execution in NASGrid. More concretely, in terms of the NASGrid system architecture shown in Fig. 1, the graph manipulation part does not accommodate topology changes, and the benchmarking part does not allow to probe the status of network connections, or to test the availability of remote hosts, or to modify the routing of data between nodes. Our extension to NASGrid introduces these features and exploits them when exceptional situations occur. In order to achieve that goal we have to extend the interfaces (of interfaces and classes marked disks in the diagrams) and the implementation of the classes (that are marked by stars in the figure) at multiple points. Note that a almost all disks or stars represent several modifications within the same interface or class. Overall, NASGrid has to be modified at 28 locations to extend it modularly by the task rescheduling functionality.

In order to give a concrete idea of which code manipulations are involved in invasive accesses and pattern compositions, let us first have a look at the code excerpt shown in Fig. 6. This excerpt shows the NASGrid code for localization and propagation of data between nodes. In case that a remote node is unavailable (lines 14-17) no reaction is taken and the exception is only passed along. However, as this method makes explicit the data on the real successor nodes of the current node (lines 4-8), we have to access it to update the new node information with the corresponding data after rescheduling. We have extended NASGrid with the help of invasive composition operators implemented with sequences in AWED as presented in Fig. 4. This required the extension of two interfaces: Benchmark to enable dynamic task rescheduling, and DGraph to permit the dynamic modification of the graph. We also used controlled invasive accesses to inject new code for coordination that corresponds to the identification of the precise context in which exceptional situations have to be handled by means of a sequence of events, and then a farmGather composition operator (cf. 5). Fig. 7 illustrates the resulting compositional algorithm (which gives a high level view of the patterns involved to modularize the task scheduling functionality): when a benchmarking execution fails (represented by the dashed circle) the exception is matched, and an execution of a farm pattern that sends a request to all successor nodes is triggered. We then use a gather pattern to collect the availability and load average information of all successor nodes and proceed to select the best available node (bold node in the figure) and reschedule the computation.

Concretely, using our AWED implementation this is performed using the rescheduling aspect shown in Fig. 4, lines 4-11, we first identify exceptional situations as a context in which a correct initialization (state CONFIG) and the start of a concrete benchmark (START) is followed by a relevant exception (EXCEPTION) to the method Benchserver.PutArcData. We then get the list of successor nodes (state LOOKUP) that are admissible by the topology of the grid application as defined by the user through the class Nodemanager. At that point, the second advice is applied (lines 18-27) that applies the farmGather composition in order to choose the best alternative and invasively modify the graph topology by a call to the method adapter.updateGraphDefinition. Finally, we restart the benchmarking operation proper (RESTART).

The implementation of NASGrid consists of 20490 LOC. The task rescheduling concern is implemented as a whole module using the concepts of invasive operators using 391 lines of code that correspond to the TaskReschedulingAspect in AWED (97 LOC) and three auxiliary classes DFGAdapter, NodeManager and TaskUtility (294 LOC). These classes perform the dynamic graph manipulation and manage the task relocation to the new nodes. Overall we therefore achieve a concise, fully modularized and compositional implementation of the extension of NASGrid by task rescheduling, Furthermore, the composition shown in Fig. 7 provides very precise control on the contexts in which invasive modifications are performed and thus enable, in principle, to model check properties over the event sequences defining such compositions (this is however future work).

Finally, note that the overhead of task rescheduling basically consists, for each exceptional situation, in 1. A sequence of a small number of locally executed instructions up to the exceptional situation, followed by 2. A small number of parallel executions of sequences of two message exchanges for the farmGather composition and 3. a small number of local instructions to reschedule the benchmark). This overhead is clearly negligible compared to the execution of the benchmark itself in almost all use cases (i.e., unless exceptional situations abound, a case that should very rarely constitute a reasonable application of NASGrid).

Degrees of invasive composition

In previous work we have applied invasive patterns (without support for pattern composition as introduced here) to two other evolution scenarios, an extension of NASGrid for checkpointing and an extension of the replication strategy of JBoss Cache, an infrastructure for replication under transactional control that is part of the JBoss Application Server.

Checkpointing in NASGrid. We have shown how a different reliability property of NASGrid can be improved upon via checkpointing introduction [START_REF] Benavides | Aspect-based patterns for grid programming[END_REF]. A checkpointing algorithm for error recovery defines a protocol to create checkpoints (snapshots of the distributed states), and guarantees the global consistency by returning to a previously-recorded state in case of failure. As we have to only add the snapshot creation and recovery actions, the degree of invasive access required is limited. It is restricted to just the context definition that triggers the snapshot creation, and also includes invasive access to the unexposed data structure in order to create its backup and play it back as part of the recovery.

Evolution of the JBoss Cache replication strategy. We have also shown how to extend the replication strategy of JBoss Cache, an infrastructure that replicates data within a cluster of distributed nodes [START_REF] Benavides | Invasive patterns for distributed programs[END_REF]. The cache ensures that data replication is consistent with the transactional control over independent accesses to a distributed database. The replication and transaction functionalities are heavily crosscutting within the JBoss Cache implementation (accounting for more than 500 LOC in total scattered over a code base of around 50 KLOC). This refactoring scenario required a high level of invasive access but both concerns, replication and transactional behavior has been fully modularized using invasive patterns (once again without explicit pattern compositions). Fig. 8 shows the degrees of crosscutting diagrams for the three examples. Since we have been able to perform all three evolutions in a fully modular way using invasive patterns, this provides solid evidence that our approach scales from applications that are using limited invasiveness and crosscutting to applications with concerns that are highly invasive and crosscutting.

To conclude the discussion of applications of our approach, we briefly discuss if and how we can exploit the additional control we provide through the composition language introduced in this paper. Let us consider the checkpointing introduction and replication strategy extension scenarios. In the following we briefly describe these extensions and compare how the approach presented here can improve on the previous solutions. First, the pattern applications used in the NASGrid checkpointing evolution can straightforwardly be expressed using our pattern composition language and the resulting additional control would allow, e.g., to reason over the correctness properties of checkpoint-based recovery (which is, admittedly rather simple in this case anyway due to the limited invasive nature). In the case of the JBoss Cache replication evolution, the additional control provided by our composition language is crucial in order to provide crucial correctness properties, such as the absence of certain race conditions during replication. This is also future work.

RELATED WORK

Our work is mainly related to three types of work: (i) other approaches to gray-box composition of software entities, (ii) approaches that use aspects in order to invasively manipulate software entities, mostly components, and (iii) work that advocates the use of patterns for distributed programming. None of these approaches, however, provides such flexible compositions of patterns for invasive composition that can be controlled precisely using a composition language. Because of space constraints we only discuss a few most relevant works. The probably best-known analysis of gray-box composition and an approach relying on code entities with holes as basic building blocks has been presented by [START_REF] Aßmann | Invasive Software Composition[END_REF]. Composition can be controlled by standard abstraction mechanisms such as component parameterization. This approach is however less structured than explicit pattern compositions and supports less precise control than ours. [START_REF] Lorenz | Aspectual collaborations: Combining modules and aspects[END_REF] present a model for so-called aspectual collaboration in which aspects can be used to invasively modify software entities, mostly classes. Such aspect-based approaches provide no support in form of compositions of basic entities we do, and support only very coarse-grained control over invasive modifications. Patterns for distributed programming and massively programming [START_REF] Cole | Algorithmic skeletons: structured management of parallel computation[END_REF], [START_REF] Schmidt | OO design patterns for concurrent, parallel, and distributed systems[END_REF] have been mostly used as design patterns for non-invasive programming. Our previous work on invasive distributed patterns provide patterns as programming abstractions that can be composed manually but without support of a flexible composition language.

CONCLUSION AND PERSPECTIVES

In this paper we have made the case for more expressive and structured means for flexible gray-box compositions of distributed software systems. We have introduced a kernel language for structured flexible gray-box composition that enables to concisely define and precisely control pattern composition. We have sketched an aspect-based implementation of this language and applied it to a non-trivial extension of the NASGrid system for grid benchmarking. Finally, we have provided evidence that our approach scales from moderately to highly crosscutting applications. This work paves the way to the investigation of a (formallydefined) theory of invasive composition. In the long term, the quest for a set of operators that is complete with respect to a large number of evolution scenarios should be undertaken.

Fig. 1 .

 1 Fig. 1. NASGrid: application structure and scheduling-relevant code parts

Fig. 4 .

 4 Fig. 4. Implementation of task rescheduling in NASGrid using AWED

Fig. 6 .

 6 Fig. 6. Class BenchServer (fragment) task rescheduling Fig. 7. NASGrid invasive composition

Fig. 8 .

 8 Fig. 8. Crosscutting diagrams for the three evolution case studies

This work has been supported by the CESSA project (ANR 09-SEGI-002-01, see cessa.inria.gforge.fr).


S Aït-Aïssa

S Laskowski

N Laville

J.-M Porcher

F Brion

Anti-androgenic activities of

Keywords: reporter gene assay, pesticides, androgen and glucocorticoid receptors, antiandrogenic potency

Introduction

A number of environmental chemicals, the so-called endocrine disruptive chemicals (EDCs), can alter the normal functioning, synthesis and metabolism of endogenous hormones, and thereby affect growth, development and reproduction in wildlife and humans [START_REF] Colborn | Pesticides--how research has succeeded and failed to translate science into policy: endocrinological effects on wildlife[END_REF]. EDCs are supposed to be active through several molecular mechanisms of action, among which the binding to and subsequent (in)activation of nuclear receptors, including the estrogen (ER) and androgen receptors (AR), has been identified as an important mechanism that mediates endocrine disrupting effects. These receptors are transcriptional factors that play crucial role in the regulation of target gene expression in physiological processes like development, sex differentiation, reproduction, as well as neuro-endocrine system functioning.

A large diversity of environmentally occurring chemicals, including natural and synthetic steroids, pharmaceuticals, industrial and phytopharmaceutical chemicals, have been described as ligands of nuclear receptors [START_REF] Vos | Health effects of endocrine-disrupting chemicals on wildlife, with special reference to the European situation[END_REF]. Among them, the risks for wildlife and human health posed by pesticides is currently a major cause for concern [START_REF] Colborn | Pesticides, Sexual Development, Reproduction, and Fertility: Current Perspective and Future Direction[END_REF][START_REF] Mckinlay | Endocrine disrupting pesticides: Implications for risk assessment[END_REF]. Pesticides encompass a large panel of chemical classes that are widely used in both agricultural and non-agricultural applications, and wildlife and human may be exposed through multiple routes including atmosphere, water, occupational, domestic and food consumption. While certain environmental pesticides (e.g. vinclozolin and methoxychlor) or pesticide metabolite (e.g. p,'p-DDE) have been early identified as potent anti-androgen and/or estrogen mimicking compounds [START_REF] Kelce | Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist[END_REF][START_REF] Leblanc | At the cutting edge -Pesticides: Multiple mechanisms of demasculinization[END_REF], the evaluation of currently used pesticides is of more recent concern besides [START_REF] Andersen | Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro[END_REF][START_REF] Kojima | Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells[END_REF][START_REF] Lemaire | Effect of organochlorine pesticides on human androgen receptor activation in vitro[END_REF]. [START_REF] Mckinlay | Endocrine disrupting pesticides: Implications for risk assessment[END_REF] recently listed 127 pesticides as having endocrine disrupting properties through various modes of molecular actions in in vitro or in vivo systems. Although some of the listed pesticides were banned or restricted for use, they are still found in the aquatic environment [START_REF] Baugros | Multiresidue analytical methods for the ultra-trace quantification of 33 priority substances present in the list of REACH in real water samples[END_REF][START_REF] Kinani | Bioanalytical characterisation of multiple endocrine-and dioxin-like activities in sediments from reference and impacted small rivers[END_REF]. In addition, many pesticides that are authorised for use are now detected in water bodies and may pose a risk to exposed population [START_REF] Comoretto | Pesticides in the Rhone river delta (France): Basic data for a field-based exposure assessment[END_REF][START_REF] Ifen | Pesticides in waters, 2005 data[END_REF].

However, there is still a lack of information on the ED potency of a number of currently used pesticides. In this context, the use of in vitro screening assays that inform on specific EDrelated mechanisms of action can provide rapid and relevant toxicological information in order to characterise ED property of not yet evaluated pesticides.

In a previous study, we have reported that several currently used pesticides were able to interfere with steroid hormone biosynthesis by altering aromatase enzymatic activity and gene expression in a human placental cell line [START_REF] Laville | Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line[END_REF]. In order to gain further information on their ED potency, these compounds (listed in Table 1) were tested in the present study for their ability to alter the transcriptional activity of the androgen receptor (AR) in the MDA-kb2 reporter cell line [START_REF] Wilson | A novel cell line, MDAkb2, that stably expresses an androgen-and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists[END_REF]. This cell line is derived from MDA-MB-453 cells that were stably transfected by the luciferase reporter gene driven by the MMTV promoter. In these cells, this promoter is up-regulated by two endogenous nuclear receptors, AR and glucocorticoid receptors (GR). After calibration of the reporter system in our test conditions using reference AR and GR ligands, the agonistic and AR antagonist actions of pesticides were screened.

Materials and methods

Chemicals

Flutamide, testosterone, 5 -dihydrotestosterone (DHT), androstenedione, dexamethasone, RU846 and pesticides (listed in Table 1) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France), except azimsulfuron (DuPont de Nemours, France) and sodium arsenite (Alpha Aesar, France). Stock solutions (10 mM) of chemicals were made up in dimethylsulfoxide (DMSO) and stored at -20°C.

Cell culture and reporter gene assay

The MDA-kb2 cell line (ATCC #CRL-2713) was obtained from LGC Promochem (Molsheim, France). The cells were routinely grown at 37°C under humidified air atmosphere in phenol red containing L15 Medium (Sigma) supplemented with 10 % foetal calf serum (FCS), 1 % (v/v) non essential amino acids, penicillin/streptomycin (50 U/ml each) and 0.1 mg/ml geneticin (Invitrogen, France). The assay procedure for chemical testing is given as follows, accordingly to preliminary assays done in our laboratory in order to optimise assay conditions. Briefly, the cells were seeded at a density of 10 4 cells per well of 96-well white opaque tissue culture plates, and left to incubate for 24 hours at 37°C. Then, for each plate, the cells were dosed in triplicates with solvent alone (negative control), 1 nM and 0.1 nM DHT (as positive controls for AR agonism), 0.1 nM DHT plus 1 µM flutamide (as positive control for AR antagonism), and a range of concentrations of test chemical administrated either alone (for AR/GR agonistic activity) or in the presence of 0.1 nM DHT in culture medium (for AR antagonistic activity). A screening approach was first used by exposing cells to 1 and 10 µM of test chemicals in order to identify active chemicals. After confirmation of biological effect in second experiments, active compounds were further tested using larger range of concentration (0.01 to 10 µM, up to 30 µM for fenarimol) to establish complete dose-response curves allowing determination of median effective concentration (see section 2.4.). Overall, each chemical has been tested in at least three independent experiments. The final solvent concentration in the medium never exceeded 0.1 % (v/v). At this concentration, DMSO did not alter either luciferase activity or cell viability. After 24 h incubation at 37°C, the medium was removed and replaced by 50 µl of phenol red free medium containing 3.10 4 M D-luciferin (Sigma) and the luminescence signal in living cells was read after 5 minutes with a microtiter plate luminometer (µBeta, Wallac). Results were expressed as percent of maximal luciferase induction by 10 nM DHT. Anti-androgenic activity was examined after co-exposure of the cells to both 0.1 nM DHT and the test chemical. At this concentration, DHT induced about half-maximal luciferase activity in our experiments.

Cytotoxicity assay

The cellular viability was assessed in independent experiments by using the 3-(4,5dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, as described by [START_REF] Mosmann | Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays[END_REF], with slight modifications. Briefly, cells were seeded at density of 10 4 cells/well in 96-wells culture plates, left to grown for 24 hours, and exposed in triplicates to 1 and 10 µM of test chemicals for 18 hours. After exposure, culture medium was removed and 0.5 mg/ml MTT dissolved in culture medium was added to the wells. After two hours of incubation at 37°C, the medium was removed and the purple formazan product was dissolved by isopropanol addition. Plates were read at 570 nm against a 660 nm reference wavelength with a microplate spectrophotometer reader (BioTek Instruments, France). Cell viability was expressed as a percentage of the value measured in untreated control cells (solvent control).

Data analysis

Statistical significance of the effects of chemicals was assessed by using the non parametric Mann-Whitney test. A value of p < 0.05 was considered significant. The SPSS™ software version 10.1 for Windows was used for the statistical analysis. For reporter gene response, dose-response curves were modelled by using the Regtox 6.3 Microsoft Excel macro [START_REF] Vindimian | REGTOX : macro Excel™ for dose-response modelling[END_REF], which allows calculation of EC 50 (concentration that induces 50 % of maximal response) and IC 50 (concentrations that inhibits 50 % of maximal response). The relative anti-androgenic potency (RAAP) was determined as the ratio of IC 50 value of flutamide to that of test compound.

Results

Activity of reference AR and GR ligands

In order to calibrate the use of the MDA-kb2 cell line in our laboratory conditions, reference AR and GR ligands were first tested. Typical dose-response curves that were obtained for AR agonists and antagonists are shown in Figures 1 and2, respectively. In these experiments, DHT was the most potent AR agonist, followed by testosterone (T), methyltestosterone (MT) and androstenedione (AD), in a decreasing potency order. Maximum fold-induction of luciferase by AR full agonists varied from 5 to 10 across experiments, while the GR agonist dexamethasone induced luciferase 50 to 80 times as compared to untreated control cells (not shown). The EC 50 and the ranges of fold-induction of luciferase determined in our study for both well-known AR and GR ligands were in line with those previously published using the same cellular model (summarised in Table 2, references therein). These results validated the use of this cell model in our experimental conditions, which involves slight differences in the assay procedure (i.e. detection of luciferase activity in living cells) comparing to that previously described for the use of this reporter cell line for the testing of chemicals [START_REF] Wilson | A novel cell line, MDAkb2, that stably expresses an androgen-and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists[END_REF].

(Anti-)androgenic activity of pesticides

At 10 µM, no significant cytotoxicity was observed after exposure to individual pesticides, as determined by the MTT assay (data not shown). When tested alone for their agonist activity, none of the pesticides significantly induced the luciferase activity in MDA-kb2 cells (data not shown). Conversely, 14 out of 30 pesticides behaved as AR antagonists as they were able to significantly inhibit DHT-induced luciferase in a concentration dependent manner. Active compounds were arbitrarily grouped in two groups of chemicals according to their inhibitory potencies, i.e. as moderate (chemicals that inhibited less than 50 % of DHT-induced luciferase at 10µM) (Figure 3), and potent anti-androgenic pesticides (chemicals that inhibited more than 50% of DHT-induced luciferase at 10µM) (Figure 4). Their IC 50 ranged from 0.19 µM for vinclozolin, the most potent pesticide, up to 12 µM for fenarimol (Table 1). By regards to their relative anti-androgen potency (RAAP), most of the pesticides were found 3 (o,p'-DDE) to 100 (fenarimol) times less potent than the reference anti-androgen flutamide (Table 1), except for vinclozolin and the methoxychlor metabolite HPTE, that were more or equally potent than flutamide, respectively. Interestingly, bupirimate and fipronil were newly identified as potential antiandrogens.

It is noteworthy that flutamide is a moderate anti-androgen but is metabolised in vivo in hydroxyflutamide (HF), a stronger anti-androgen. Using this compound as a reference to determine RAAP of a given compound could be misleading as it is not known whether MDA-kb2 cells can metabolise it. However, Ma et al. (2002) reported that HF was about ten times more potent than flutamide suggesting that metabolism of flutamide in this cell system has minor influence on its anti-androgenic activity. Nonetheless, in the present study, RAAP based on flutamide as a reference should first be viewed as an index allowing relative ranking of active chemicals rather than as an absolute value of their intrinsic anti-androgenic activity.

Discussion

By using the MDA-kb2 cell line established by [START_REF] Wilson | A novel cell line, MDAkb2, that stably expresses an androgen-and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists[END_REF], we were able to assess the ability of various selected pesticides to interact with the human AR transcriptional pathway. This study shows the usefulness of the MDA-kb2 cell line to detect AR antagonistic property of several pesticides. The main outcomes of this study are:

-the suitability of the MDA-kb2 cell line to detect anti-androgenicity of pesticides in a screening approach, -the anti-androgenicity of 15 out of the 30 tested pesticides from various chemical classes, among which several were previously poorly or not described as such, -the newly identified anti-androgenic property of two currently used pesticides, fipronil and bupirimate.

In first experiments using reference AR and GR ligands, we could establish a very good adequacy between our results and those previously published by using the same cellular model (Table 2). Moreover, the use of a slightly divergent procedure assay (i.e. detection of luciferase activity in living cells) than that one originally described by [START_REF] Wilson | A novel cell line, MDAkb2, that stably expresses an androgen-and glucocorticoid-responsive reporter for the detection of hormone receptor agonists and antagonists[END_REF] did not yield divergent results when comparing the IC 50 values for the reference compounds. In a practical way, the detection of luciferase in living cells has been previously reported for the assessment of luciferase activity in other reporter cell lines [START_REF] Balaguer | Reporter cell lines are useful tools for monitoring biological activity of nuclear receptor ligands[END_REF][START_REF] Cosnefroy | A stable fish reporter cell line to study estrogen receptor transactivation by environmental (xeno)estrogens[END_REF][START_REF] Creusot | Evaluation of an hPXR reporter gene assay for the detection of aquatic emerging pollutants: screening of chemicals and application to water samples[END_REF][START_REF] Pillon | Binding of estrogenic compounds to recombinant estrogen receptor alpha : Application to environmental analysis[END_REF] and can thus be recommended to enhance the screening capacity of stable luciferase reporter assays using MDA-kb2 cells. This comforted the validity of the MDA-kb2 assay in our laboratory conditions and allowed us to further explore the effects of pesticides of environmental interest.

When tested alone, none of the studied pesticides significantly induced the luciferase activity in MDA-kb2 cells (data not shown), which is not an unexpected finding. Indeed, the lack of AR agonistic activity by the tested pesticides is in line with the frequently reported absence of in vitro androgenic potency for a large range of organic environmental contaminants, such as pesticides from various chemical classes [START_REF] Kojima | Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells[END_REF][START_REF] Lemaire | Effect of organochlorine pesticides on human androgen receptor activation in vitro[END_REF][START_REF] Xu | Evaluation of androgen receptor transcriptional activities of some pesticides in vitro[END_REF], benzophenone derivatives [START_REF] Molina-Molina | Profiling of benzophenone derivatives using fish and human estrogen receptor-specific in vitro bioassays[END_REF], alkyphenols and parabens [START_REF] Satoh | Androgenic and antiandrogenic effects of alkylphenols and parabens assessed using the reporter gene assay with stably transfected CHO-K1 cells (AR-EcoScreen System)[END_REF]. Additionally, by using the in vitro AR-EcoScreen reporter gene assay, [START_REF] Araki | Screening for androgen receptor activities in 253 industrial chemicals by in vitro reporter gene assays using AR-EcoScreen (TM) cells[END_REF] reported that only two out of 253 tested industrial chemicals exerted slight AR agonist activity.

Conversely to the absence of agonistic effects, fifteen out of 30 tested pesticides were able to inhibit in a concentration dependent manner the DHT-induced luciferase in MDA-kb2 cells (Figure 2, Table 1). Vinclozolin and HPTE, which are well-described antagonists of the AR in various cellular models [START_REF] Andersen | Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro[END_REF][START_REF] Kelce | Environmental hormone disruptors -Evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites[END_REF], were the most active compounds with experimental IC 50s equal to (HPTE) or lower (vinclozolin) than the reference AR antagonist flutamide. The 12 other active pesticides presented lower affinities for the AR than flutamide, with relative potencies ranging from 31.5 % for o,p'-DDE to 4.3 % for fenarimol. Some of them, such as o,p'-DDT, o,p'-DDE, endosulfan, methoxychlor, fenarimol or prochloraz, are well known EDCs and their in vitro anti-androgenic potency has been previously reported, albeit in different cellular models, which confirms our results (see Table 1 for references). HPTE was more potent than its parent compound methoxychlor to act as an AR antagonist in MDA-kb2 cells. HPTE is a di-hydroxylated metabolite that is generated in vivo by demethylation of methoxychlor by cytochrome P450 monooxygenases (van den [START_REF] Van Den Berg | Role of metabolism in the endocrine-disrupting effects of chemicals in aquatic and terrestrial systems[END_REF]. Thus, the comparison of their effects in MDA-kb2 cells suggest that this cell line does not bioactivate such xenobiotic compounds, likely due to low phase I metabolic capacities. To our knowledge however, no information is available regarding xenobiotic metabolic of this cell line.

In MDA-kb2 cells, few recent studies have reported the AR antagonist activity of pesticides. Tamura et al (2006) reported the anti-androgenic activities for fenarimol and methoxychlor in this cell line, although with higher IC 50 than in our study. Chlordane was not active in our study, confirming its lack of anti-AR activity recently reported in MDA-kb2 cells (Tamura et al, 2006), while it has been shown to slightly inhibit R1881-induced luciferase activity in other cell models, like the stable reporter PALM cells [START_REF] Lemaire | Effect of organochlorine pesticides on human androgen receptor activation in vitro[END_REF] and CHO using a transient transfection assay [START_REF] Kojima | Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells[END_REF]. These discrepancies may be due to the use of different cellular models, which have been largely unexplored for in vitro AR-luciferase assays.

Permethrin and cypermethrin were ineffective to exert either estrogenic or (anti)androgenic in rat in vivo [START_REF] Kunimatsu | Lack of (anti-) androgenic or estrogenic effects of three pyrethroids (esfenvalerate, fenvalerate, and permethrin) in the Hershberger and uterotrophic assays[END_REF]. Other authors recently reported the ability of the two pesticides to partially inhibit DHT-induced luciferase at 10 µM in an androgen-responsive reporter cell line [START_REF] Xu | Evaluation of androgen receptor transcriptional activities of some pesticides in vitro[END_REF]. In our study, no effect was observed at 10 µM for both pesticides.

Fipronil and bupirimate were newly identified as possible anti-androgen chemicals. Fipronil is a phenylpyrazole insecticide and is currently authorised for use in many applications in Europe and other areas. Apart from its potency to interfere with the AR signalling pathway, fipronil is also able to activate pregnane X receptor (PXR) activity in vitro [START_REF] Lemaire | Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system[END_REF]. Since it has been well described as both environmental (surface waters, soil) and food chemical contaminant, the present data reinforce the need to consider environmental hazard due to fipronil with regards to its endocrine disrupting potential. Bupirimate is a pyrymidine fungicide that acts by inhibiting fungal growth through interference with auxines. To our knowledge, almost no data are available regarding its endocrine disrupting potency. It has only been described as a potent PXR activator in vitro [START_REF] Lemaire | Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system[END_REF]. We show here that it may also potentially interfere with the AR signalling pathway.

Overall, it is noted that several of identified active chemicals only partially inhibited DHTinduced luciferase at high concentrations, hence posing the question of possible false positives. Although no cytotoxic effect was noted at the highest tested concentration and no alteration of basal luciferase expression was observed in cell exposed to pesticides alone, it cannot be excluded that some of the observed effects could be related to unspecific inhibition of reporter enzyme independently from the AR signalling pathway. Further experiments, such as AR competitive binding assay, would thus be useful to confirm the data reported in the present study and elucidate underlying mechanisms.

In summary, this study shows the suitability of the MDA-kb2 cell line for a screening purpose to assess anti-androgenic property of chemicals, including pesticides. Among the pesticides that were found active in this study, several are still in use despite data on their endocrine disrupting potencies are very recent or scarce. It is however acknowledged that the present assay describes potential (anti)androgens and does not necessarily inform on global endocrine disruptive potential of test chemical. In addition, weakly active pesticides in vitro are likely to exert limited anti-androgenic effect in vivo. However, together with other previous studies that addressed the effects of this set of molecule on other molecular targets of EDCs [START_REF] Laville | Modulation of aromatase activity and mRNA by various selected pesticides in the human choriocarcinoma JEG-3 cell line[END_REF][START_REF] Lemaire | Identification of new human pregnane X receptor ligands among pesticides using a stable reporter cell system[END_REF], our present results strengthen the need to further explore their endocrine disruptive effects in vivo, especially for chemical that are able to alter multiple targets for EDCs. a: RAAP: relative anti-androgenic potency; A : described as either agonist or antagonist for the AR; A-: AR antagonist; n.e. : non effective pesticide; no data : no data could be found in literature.

b: Reported IC50 were derived from single experiments (Figures 3 and4); anti-androgenic activity of pesticides have been confirmed in at least three independent experiments. c: 1: [START_REF] Kelce | Environmental hormone disruptors -Evidence that vinclozolin developmental toxicity is mediated by antiandrogenic metabolites[END_REF]; 2: (Sohoni and Sumpter 1998); 3: [START_REF] Andersen | Effects of currently used pesticides in assays for estrogenicity, androgenicity, and aromatase activity in vitro[END_REF]; 4: [START_REF] Kelce | Persistent DDT metabolite p,p'-DDE is a potent androgen receptor antagonist[END_REF]; 5: [START_REF] Kojima | Screening for estrogen and androgen receptor activities in 200 pesticides by in vitro reporter gene assays using Chinese hamster ovary cells[END_REF]; 6: [START_REF] Tamura | Interaction of organophosphate pesticides and related compounds with the androgen receptor[END_REF]; 7: [START_REF] Lemaire | Effect of organochlorine pesticides on human androgen receptor activation in vitro[END_REF], 8: [START_REF] Sultan | Environmental xenoestrogens, antiandrogens and disorders of male sexual differentiation[END_REF]; 9: [START_REF] Yamada | Functional genomics may allow accurate categorization of the benzimidazole fungicide benomyl: lack of ability to act via steroid-receptor-mediated mechanisms[END_REF]; 10: [START_REF] Xu | Evaluation of androgen receptor transcriptional activities of some pesticides in vitro[END_REF]; 11: [START_REF] Scippo | Recombinant human estrogen, androgen and progesterone receptors for detection of potential endocrine disruptors[END_REF].

Figure 1 :Figure 2 :

 12 Figure 1: Luciferase induction by the reference AR ligands. MDA-kb2 cells were exposed for 18 h to testosterone (T), 5 -dihydrotestosterone (DHT), 17 -methyltestosterone (MT) and androstenedione (ATD). Data are representative of at least three independent experiments.

Table 1 :

 1 Dose-response curves of luciferase inhibition by the most active pesticides in MDA-kb2 cells. The cells were co-exposed for 18 hours to both 0.1 nM DHT and various concentrations of pesticides. Calculated IC 50 s are reported in Table1. *: different from solvent control (p<0.05). Data are representative of at least three independent experiments. Summary of anti-androgenic potency of pesticides tested in MDA-kb2 cells and comparison with previously published activities using other in vitro biological models a .

			140%			120%
			120%	Methoxychlor	100%		Endosulfan
			80% 100%		*	80%		*
			60%		*	60%
			40%			40%
			20%			20%
			0%			0%
			1E-07	1E-06	1E-05	1E-07	1E-06	1E-05
			140%			120%
	% of transactivation	(100%: DHT 0.1 nM)	120% 0% 20% 40% 60% 80% 100% 120% 1E-07	1E-06	Diuron 1E-05 *	0% 20% 40% 60% 80% 100% 1E-07 120%	1E-06	1E-05 Fipronil * *
			100%	Fenarimol	100%	Metolachlor
			80%	*		80%		*
			60%			60%
			40%		*	40%
			20%			20%
			0%			0%
			1E-06	1E-05	1E-04	1E-07	1E-06	1E-05
				120%		Propiconazole
				100%
				80%
				60%			*
				40%
				20%
				0%
				1E-07	1E-06	1E-05
					Concentration (M)
	Figure 3: Dose-response curves of luciferase inhibition by moderately active pesticides in
	MDA-kb2 cells. The cells were co-exposed for 18 hours to both 0.1 nM DHT and various
	concentrations of pesticides. Calculated IC 50 s are reported in Table 1. *: different from
	solvent control (p<0.05). Data are representative of at least three independent experiments.

Acknowledgements

This study was supported by grants from the French Ministry of Ecology and Sustainable Development (P189-DRC03-08 and the "NEMO" project).

 [START_REF] Korner | Interlaboratory comparison of four in vitro assays for assessing androgenic and antiandrogenic activity of environmental chemicals[END_REF]c [START_REF] Ma | UV filters with antagonistic action at androgen receptors in the MDA-kb2 cell transcriptional-activation assay[END_REF] d [START_REF] Satoh | Androgenic and antiandrogenic effects of alkylphenols and parabens assessed using the reporter gene assay with stably transfected CHO-K1 cells (AR-EcoScreen System)[END_REF]


Laetitia Hédouin

Marc Metian

Thomas Lacoue-Labarthe

Renaud Fichez

Paco Bustamante

Michel Warnau
email: warnaumichel@yahoo.com

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

Keywords: Mollusks, Isognomon isognomon, Gafrarium tumidum, New Caledonia, Radiotracer, Feeding

à la diffusion de documents scientifiques de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

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 [START_REF] Tessier | Metal speciation and bioavailability in aquatic systems[END_REF]. In the coral reef lagoon of New-Caledonia, metal contamination is a critical problem in relation with its extreme biodiversity [START_REF] Labrosse | New Caledonia[END_REF]. 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- [START_REF] Goro-Nickel | Projet Goro Nickel[END_REF]Nickel, , 2003)). Such a process will provide new potential 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;[START_REF] Dalvi | The past and the future of nickel laterites[END_REF]. 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 chromiun (Cr), iron (Fe), manganese (Mn) or zinc (Zn) [START_REF] Goro-Nickel | Projet Goro Nickel[END_REF][START_REF] Baroudi | Analyse critique de l'acceptabilité du niveau de rejet de manganèse dans le milieu marin, Rapport final Goro-Nickel[END_REF].

Although long lasting contamination exists in New Caledonia [START_REF] Laganier | Erosion, transport and sedimentation processes associated with opencast mining in New Caledonia: interactions with weather and climate[END_REF][START_REF] Ambatsian | High metal inputs to closed seas: the New Caledonia lagoon[END_REF] with high levels of metals reported in coastal marine sediments (e.g. [START_REF] Fernandez | A combined modelling and geochemical study of the fate of terrigenous inputs from mixed natural and mining sources in a coral reef lagoon (New Caledonia)[END_REF], 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;[START_REF] Dalto | Muddy-bottom meiofauna responses to metal concentrations and organic enrichment in New Caledonia South-West Lagoon[END_REF]Hédouin et al., 2008a,b;Metian and Warnau, 2008;[START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF]. 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. [START_REF] Goldberg | Mussel Watch: 1977-1978 results on trace metals and radionuclides[END_REF][START_REF] Warnau | Is there a need for developing an integrated basin-wide monitoring network to enhance sustainable development in the Mediterranean Sea?[END_REF][START_REF] Thébault | 137 Cs baseline levels in the Mediterranean and Black Sea: a cross-basin survey of the CIESM Mediterranean Mussel Watch Programme[END_REF]. Indeed, 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;[START_REF] Coteur | Echinoderms as bioindicators, bioassays, and impact assessment tools of sediment-associated metals and PCBs in the North Sea[END_REF][START_REF] Danis | Contaminant levels in sediments and asteroids (Asterias rubens L., Echinodermata) from the Belgian coast and Scheldt estuary: polychlorinated biphenyls and heavy metals[END_REF]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 [START_REF] Metian | Use of radiotracer techniques to study subcellular distribution of metals and radionuclides in bivalves from the Noumea Lagoon, New Caledonia[END_REF][START_REF] Hédouin | this issue. Metal and metalloid bioconcentration capacity of two tropical bivalves for monitoring the impact of land-based mining activities in the New Caledonia lagoon Hédouin[END_REF][START_REF] Hédouin | Nickel bioaccumulation in bivalves from the New Caledonia lagoon: seawater and food exposure[END_REF]Hédouin et al., , 2008a;;[START_REF] Chouvelon | Hg concentrations and related risk assessment in coral reef crustaceans, molluscs and fish from New Caledonia[END_REF].

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., [START_REF] Borchardt | Influence of food quantity on the kinetics of cadmium uptake and loss via food and seawater in Mytilus edulis[END_REF][START_REF] Riisgard | Accumulation of cadmium in the mussel Mytilus edulis: kinetics and importance of uptake via food and seawater[END_REF]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., [START_REF] Widdows | Mussels and environmental contaminants: bioaccumulation and physiological aspects[END_REF]Navarro and Iglesias, 1993). For example, Cd assimilation in the mussel Mytilus edulis is inversely related to food quantity [START_REF] Borchardt | Influence of food quantity on the kinetics of cadmium uptake and loss via food and seawater in Mytilus edulis[END_REF]. 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 2 different filament types) are also able to select the particles that they are ingesting [START_REF] Ward | Particle sorting in bivalves: in vivo determination of the pallial organs of selection[END_REF], which results in a preferential ingestion of nutritionally-rich particles that may also affect metal influx from food (e.g., [START_REF] Bayne | Feeding physiology of bivalves: time-dependence and compensation for changes in food availability[END_REF][START_REF] Wang | Modeling metal bioavailability for marine mussels[END_REF].

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

Isognomon isognomon and the clam Gafrarium 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 [START_REF] Warnau | Radiotracer techniques: a unique tool in marine ecotoxicological studies[END_REF].

Materials and Methods

Collection and Acclimation

The organisms (n = 100 per species) were collected by SCUBA diving in Maa Bay (oysters Isognomon isognomon) or by hand-picking in Dumbea Bay (clams Gafrarium tumidum) in October 2003. Both locations are located 15 to 20 km north of Nouméa City, New Caledonia. Body size is known to affect bioaccumulation of metals in marine organisms (e.g., [START_REF] Boyden | Trace elements contents and body size in molluscs[END_REF][START_REF] Warnau | Allometry of heavy metal bioconcentration in the echinoid Paracentrotus lividus[END_REF]; hence, according to previous preliminary studies [START_REF] Metian | Bioaccumulation des métaux lourds chez 4 espèces marines du lagon de Nouvelle Calédonie: Caractérisation de leur potentiel bioindicateur pour le monitoring des activités minières locales[END_REF][START_REF] Hédouin | this issue. Metal and metalloid bioconcentration capacity of two tropical bivalves for monitoring the impact of land-based mining activities in the New Caledonia lagoon Hédouin[END_REF]Hédouin et al., , 2008a)) were fed phytoplankton using the Prymnesiophyceae Isochrysis galbana (10 4 cells ml -1 ).

Recorded mortality was lower than 5% over the acclimation period.

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 ( 57 Co in 0.1M HCl, T ½ = 271.8 d) and Isotope Product Lab., USA ( 54 Mn in 0.1M HCl, T ½ = 312.2 d; 65 Zn in 0.5M HCl; T ½ = 243.9 d).

Radioactivity was measured using a high-resolution γ-spectrometer system composed of three Germanium -N or P type-detectors (EGNC 33-195-R, Eurysis ® ) connected to a multi-channel analyzer and a computer equipped with a spectra analysis software (Interwinner ® 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).

Experimental Procedures

Testing the influence of Co concentration in food

Isochrysis galbana cells from an axenic stock culture were resuspended into 4 erlenmeyer flasks (light/dark cycle: 12 hrs/12 hrs at 25°C). Each flask contained 500 ml sterile-filtered seawater enriched with f/2 nutrients without EDTA and Si [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF]. Flasks were spiked with 4 increasing Co concentrations (0, 5, 50, 500 ng l -1 ) and phytoplankton was allowed to grow under these conditions for 6 d. Added Co concentrations were realized using increasing amount of Co(NO 3 ) 2 (synthesis quality, Merck) and a fixed activity of the corresponding radiotracer 57 Co (2.5 kBq l -1 , corresponding to 0.13 ng Co l -1 ). The range of concentrations selected covers those encountered in the New Caledonia lagoon waters [START_REF] Fernandez | The fate of metals in New Caledonia[END_REF][START_REF] Goro-Nickel | Annexe 2.1 IRD, « Rapport intermédiaire -Caractéristiques physicochimiques des eaux » -mars 2004[END_REF]. After 6 d of incubation, cell density increased from 10 3 to 1.5 x 10 6 cell ml -1 . The cells were gently filtered (1 µm-mesh size, Nuclepore ® Polycarbonate filters) and resuspended in clean seawater. The radioactivity of the radiolabelled I. galbana in each flask was γ-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 µBq cell -1 .

For each added Co concentration, 4 groups of 9 oysters (shell length from 71 to 94 mm) and 4 groups of 9 clams (shell width from 35 to 40 mm) were placed in 4 aquaria containing 16 l of 0.45-µm 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 hrs on one out of the 4 batches of previously radiolabelled

Isochrysis galbana (10 4 cell ml -1 ) (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-hr feeding period [START_REF] Metian | Interspecific comparison of Cd bioaccumulation in European Pectinidae (Chlamys varia and Pecten maximus)[END_REF]. These control shells were radioanalysed at regular intervals of time.

At the end of the feeding period, all organisms were γ-counted and open circuit conditions were restored (water renewal rate: 30% hr -1 ; salinity: 36 p.s.u.; T° = 25 ± 0.5°C; pH = 8.0 ± 0.1; light/dark cycle: 12 hrs/12 hrs). From that time on, all individuals were γ-counted 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 hr non-radiolabelled phytoplankton (Isochrysis galbana, 10 4 cell ml -1 ).

Testing the influence of phytoplankton species

Two batches of 9 oysters (shell length from 73 to 90 mm) and 2 batches of 9 clams (shell width from 35 to 44 mm) were placed in 2 aquaria containing 16 l of 0.45µm 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 (10 4 cell ml -1 ) for 2 hrs (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 [START_REF] Jacquet | Response of phytoplankton communities to increased anthropogenic influences (southwestern lagoon, New Caledonia)[END_REF].

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 (10 3 cell ml -1 ), 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 [START_REF] Guillard | Culture of phytoplankton for feeding marine invertebrates[END_REF]. The two cultures were spiked with 5 kBq l -1 of54 Mn, 57 Co and 65 Zn, corresponding to 3.6 ng Mn l -1 , 25 ng Co l -1 and 60 ng Zn l -1 . The cultures were then incubated for 6 d at 25°C (light/dark cycle: 12 hrs/12 hrs). After incubation, the cell densities were 7 x 10 5 cell ml -1 for E. huxleyi and 1.6 x 10 5 cell ml -1 for H. triquetra. The cells were then gently filtered, resuspended in clean seawater and γ-counted as described above (section 2.3.1.). The radioactivity of algal cells used in the feeding experiments was 0.26 ± 0.18 µBq cell -1 for E. huxleyi and 0.96 ± 0.11 µBq cell -1 for H. triquetra for for 65 Zn, 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.

Testing the influence of cellular density

Three groups of 9 oysters (shell length from 71 to 92 mm) and 3 groups of 9 clams (shell width from 36 to 45 mm) were placed in 3 aquaria containing 16 l of 0.45µm filtered natural seawater (close circuit aquaria constantly aerated; other parameters as previously described), and acclimated for one week (daily seawater renewal) during which time their food was prepared.

To do this, cells of I. galbana from an axenic stock culture were resuspended in an erlenmeyer flask containing 4.5 l sterile-filtered seawater enriched with f/2 nutrients without EDTA and Si. The culture was then spiked with 5 kBq l -1 of 54 Mn, 57 Co and 65 Zn and incubated for 6 d at 25°C (light/dark cycle: 12 hrs/12 hrs). After incubation, the cell density had increased from 10 3 to 1.4 x 10 6 cell ml -1 . Three sub-samples of 58, 115 and 580 ml of the culture were then gently filtered and resuspended in clean seawater. These 3 batches were prepared to obtain final cell density of 5 x 10 3 , 10 4 and 5 x 10 4 cell ml -1 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 µBq cell -1 for 54 Mn, 0.83 to 1.37 µBq cell -1 for 57 Co, 2.69 to 4.38 µBq cell -1 for 65 Zn.

Each group of clams and oysters was then fed for 2 hrs one of the radiolabelled I.

galbana batches (5 x 10 3 , 10 4 or 5 x 10 4 cell ml -1 ). 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.

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. where k e is the depuration rate constant (d -1 ), A t and A 0 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 [START_REF] Whicker | Radioecology: nuclear energy and the environment[END_REF]Warnau et al., 1996b). The long-lived component allows assessing the assimilation efficiency (AE) of the radiotracer ingested with food (AE = A 0l ). Also, for each exponential component (s and l), a biological half-life can be calculated (T b½s and T b½l )

from the corresponding depuration rate constant (k es and k el ) according to the relation T b½ = ln2 / k e .

Constants of the models and their statistics were estimated by iterative adjustments of the model and Hessian matrix computation using the nonlinear curvefitting routines in the Statistica® 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 [START_REF] Zar | Biostatistical analysis[END_REF]. The level of significance for statistical analyses was always set at α = 0.05.

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.

Effect of Co Concentration in Phytoplankton

Fitting of the whole-body depuration kinetics of 57 Co 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 (T b½s < 1d) whereas the long-lived component accounted for only 15-20% of the 57 Co ingested with food that was eliminated with a biological half life (T b½l ) ranging from 13 to 25 d.

Similarly, the fit of the whole-body depuration of 57 Co 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 57 Co ingested with food was much higher than in oysters (i.e., 76-84%)

and this fraction was retained with a T b½ ranging from 36 to 39 d.

In both bivalve species, no significant difference (p always > 0.05) was found among the estimated kinetic parameters (A os , k es , A ol , k el ) determined for the 4 different food-associated Co concentrations.

Effect of Phytoplankton Species

In oysters, depuration kinetics of 54 Mn, 57 Co and 65 Zn ingested with I. galbana (ISO), E. huxleyi (EMI) or H. triquetra (HET) (10 4 cell ml -1 ) 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 T b½ 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 fro 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 (10 4 cell ml -1 ) were genrally 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). T b½ of 54 Mn 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 ≥ EMI ≥ HET, p = 0.04).

Effect of Cellular Density

When oysters were fed 10 4 and 5 x 10 4 cells ml -1 of radiolabelled I. galbana, whole-body depuration kinetics of 54 Mn, 57 Co and 65 Zn 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 T b½ 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

x 10 3 cell ml -1 and 0.47-0.98 at 10 4 cell ml -1 ) indicated that T b½ 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).

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;[START_REF] Reinfelder | Trace element trophic transfer in aquatic organisms: a critique of the kinetic model approach[END_REF]Wang and Fisher, 1999b). The assimilation efficiency (AE)

and retention time (T b½ ) 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 marine organisms (e.g., Wang et al., 1996;Warnau et al., 1996b[START_REF] Warnau | Biokinetics of radiocobalt in the asteroid Asterias rubens (echinodermata): sea water and food exposures[END_REF]Pan and Wang, 2008). However, as almost a rule in tropical environments (e.g., Phillips, 1991;[START_REF] Chong | Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum[END_REF][START_REF] Metian | Use of radiotracer techniques to study subcellular distribution of metals and radionuclides in bivalves from the Noumea Lagoon, New Caledonia[END_REF], only very few data are yet available regarding AE and T b½ parameters for organisms from New Caledonia (e.g. [START_REF] Hédouin | this issue. Metal and metalloid bioconcentration capacity of two tropical bivalves for monitoring the impact of land-based mining activities in the New Caledonia lagoon Hédouin[END_REF][START_REF] Hédouin | Nickel bioaccumulation in bivalves from the New Caledonia lagoon: seawater and food exposure[END_REF].

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, this issue). 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 l -1 ) Co was shown to be assimilated in the same proportion (AE) and retained with similar relative strength (T b½ ) whatever the foodassociated Co concentration was (see Fig. 1 andTable1). 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 [START_REF] Fernandez | The fate of metals in New Caledonia[END_REF][START_REF] Goro-Nickel | Annexe 2.1 IRD, « Rapport intermédiaire -Caractéristiques physicochimiques des eaux » -mars 2004[END_REF]). Similar trends have been previously reported by [START_REF] Chong | Bioavailability of sediment-bound Cd, Cr and Zn to the green mussel Perna viridis and the Manila clam Ruditapes philippinarum[END_REF] 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 Mytilus 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., this issue), 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 µm) and cell width (c.w.: 2-4 µm) than E. huxleyi cells (c.l.: 3-4 µm; c.w.: 3-4 µm), but are much smaller than H. triquetra cells (c.l.: 20-28 µm; c.w.: 14-18 µm). 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. [START_REF] Bayne | Feeding and digestion by the mussel Mytilus edulis L. (Bivalvia: Mollusca) in mixtures of silt and algal cells at low concentration[END_REF][START_REF] Bayne | Feeding physiology of bivalves: time-dependence and compensation for changes in food availability[END_REF]Pan and Wang, 2008). Generally, filter-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 [START_REF] Jin | Filtration dynamics of the zebra mussel Dreissena polymorpha[END_REF][START_REF] Dong | Environmental factors affecting the feeding physiological ecology of Manila clam, Ruditapes philippinarum[END_REF][START_REF] Zhuang | Influence of size, habitat and food concentration on the feeding ecology of the bivalve, Meretrix meretrix Linnaeus[END_REF]. 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. [START_REF] Wang | Modeling metal bioavailability for marine mussels[END_REF]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., [START_REF] Thomann | Steady-state model of biota sediment accumulation factor for metals in two marine bivalves[END_REF]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. Figure 3. Influence of phytoplankton cell density (5 x 10 3 , 10 4 or 5 x 10 4 cells ml -1 ) on whole-body depuration kinetics of 54 Mn, 57 Co and 65 Zn in the oyster Isognomon isognomon (n = 9 for 10 4 cells ml -1 and 5 x 10 4 cells ml -1 ) and the clam Gafrarium tumidum (n = 6 for 5 x 10 3 cells ml -1 and n = 8 for 10 4 cells ml -1 ) fed radiolabelled Isochrysis galbana. A(%): remaining activity (%) ± SD. Gafrarium tumidum fed radiolabelled Emiliania huxleyi (10 4 cell ml -1 ), Heterocapsa triquetra (10 4 cell ml -1 ) and Isochrysis galbana (10 4 cell ml -1 and 5 10 4 cell ml -1 for I. isognomon; 5 10 3 cell ml -1 and 10 4 cell ml -1 for G. tumidum) (n = 8 oysters; n = 7 clams per phytoplankton species tested). ASE: asymptotic standard error; R²: determination coefficient.

Acknowledgements

Captions to Figures

Species Phytoplankton strain

Cell density (cells ml

  Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: qualitative and quantitative aspects Laëtitia Hédouin, Marc Metian, Thomas Lacoue-Labarthe, Renaud Fichez, Paco Bustamante, Michel Warnau To cite this version: Laëtitia Hédouin, Marc Metian, Thomas Lacoue-Labarthe, Renaud Fichez, Paco Bustamante, et al.. Influence of food on the assimilation of selected metals in tropical bivalves from the New Caledonia lagoon: qualitative and quantitative aspects. Marine Pollution Bulletin, 2010, 61 (7-12), pp.568-575. 10.1016/j.marpolbul.2010.06.034. hal-00511997

(

  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):A t = A 0s e -k es t + A 0l e -k el t

Figure 1 .

 1 Figure 1. Influence of phytoplankton-associated Co concentrations on whole-body depuration kinetics of 57 Co in the oyster Isognomon isognomon and the clam Gafrarium tumidum fed radiolabelled Isochrysis galbana (10 4 cells ml -1 ). A(%): remaining activity (%) ± SD (n = 9 oysters; n = 6 clams for 0 and 5 ng l -1 and n = 8 clams for 50 and 500 ng l -1 ).

Figure 2 .

 2 Figure 2. Influence of phytoplankton species (Isochrysis galbana, Emiliania huxleyi and Heterocapsa triquetra; 10 4 cells ml -1 ) used as food on whole-body depuration kinetics of 54 Mn, 57 Co and 65 Zn in the oyster Isognomon isognomon (n = 9 for I. galbana and 8 for E.

Fig

  Fig. 1.

Table 1 .

 1 Assimilation efficiency (AE, %), depuration rate constant (k el , d -1 ) and biological half-life (T b½l , d) of 57 Co in the oyster Isognomon isognomon and the clam Gafrarium tumidum fed radiolabelled Isochrysis galbana (10 4 cell ml -1 ) previously exposed to four 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)

	increasing Co concentrations (n = 9 oysters per concentration tested, n = 6 clams for 0 and 5
	ng l -1 and n = 8 clams for 50 and 500 ng l -1 ). ASE: asymptotic standard error; R²:
	determination coefficient.
		Co	concentration
	Species	added (ng l -1 )	AE ± ASE k el ± ASE	T b½l ± ASE	R 2
	I. isognomon 0		15.8 ± 7.0 a 0.032 ± 0.036* 22 ± 24*	0.87
		5		19.6 ± 5.4 c 0.054 ± 0.028* 13 ± 7*	0.88
		50		16.6 ± 6.2 b 0.050 ± 0.036* 14 ± 10*	0.86
		500		14.7 ± 6.0 a 0.027 ± 0.033* 25 ± 30*	0.90
	G. tumidum	0		77.2 ± 3.9 d 0.018 ± 0.006 b	37. ± 11 b	0.64
		5		77.4 ± 3.9 d 0.019 ± 0.006 c	36 ± 10 c	0.27
		50		75.7 ± 3.9 d 0.018 ± 0.006 b	39 ± 12 b	0.51
		500		84.1 ± 5.9 d 0.019 ± 0.007 a	36 ± 13 a	0.51

Table 2 .

 2 Assimilation efficiency (AE, %), depuration rate constant (k el , d -1 ) and biological half-life, (T b½l , d) of 54 Mn, 57 Co and 65 Zn in the oyster Isognomon isognomon and the clam

Mn, 2.1 ± 0.8 and 20.8 ± 12.1 µBq cell -1 for 57 Co and 3.2 ± 1.3 and 3.3 ± 0.1 µBq cell -1

The authors thank J.L. Teyssié (IAEA-MEL) for valuable technical advices and 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.

MW is an

Honorary Senior Research Associate of the National Fund for Scientific Research (NFSR, Belgium) and holds a 2008-09 Invited Expert position at LIENSs (CNRS-Université 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.