F Deschaux-Beaume

F Schmidt

N Fre ´ty

J C Boyer

C Levaillant

Failure prediction for ceramic dies in the hot-forging process using FEM simulation

Keywords: Finite-element method, Fracture prediction, Ceramics, Forging

A fracture prediction criterion for brittle materials has been introduced in the POLLUX finite-element code, in order to predict the risk of rupture of ceramic tools during a forging operation. The POLLUX code, developed by INSA (Lyon) especially to simulate forging operations, is presented. The fracture model is based on the weakest link theory and Weibull analysis. Two different criteria were chosen in order to characterise the stress state, considering the tensile normal stresses. Comparison between the simulation results and the analytical calculations, in a simple compression case, enables the validation of the numerical model. Applications are presented, in which the design of ceramic forging tools is realized using the failure prediction software. A run-strategy of the program is proposed in order to improve the design of the forging tools.

Introduction

In the aircraft industry, the development of engines with higher performance, has led to the introduction of new titanium and nickel base materials, requiring forming techniques that are quite different from those used for more conventional products. The isothermal forging of superplastic materials has appeared as the most appropriate forming process of some titanium alloys and nickel-base superalloys. This technique should allow the production of near-net-shape pieces directly from simple-shape preforms. However, superplastics properties are achieved only at high temperature and low strain-rate for these alloys. For instance, in the case of some nickel-base alloys, superplasticity is reached at temperatures exceeding 1373 K, which means that the material for the forging dies, which will be used at the same temperature as the workpiece, must be heat resistant.

The isothermal forging of advanced alloys has been carried out using metal dies (Mo based), which requires a controlled atmosphere system in order to limit the oxidation of the dies. The use of ceramic dies should be a less expensive way, as the very good oxidation resistance of these materials does not require a controlled atmosphere. Advanced ceramics are also attractive for their physical and mechanical properties, such as their high-temperature strength, their light weight and excellent erosion resistance. However, ceramics are inherently brittle, and the lack of ductility leads to low fracture toughness and large variations in observed fracture strength. The usual design techniques are not suited to ceramic components. The wide variation of material strength is due to the nature and distribution of intrinsic microscopic flaws, that are unavoidably present as a result of material manufacturing operations. Failure in ceramics usually initiates at a single weakest flaw when the local stress reaches a critical value. Statistics and reliability analysis are therefore necessary to predict the failure of brittle material components as a function of service time.

Statistical fracture models, based on the weakest-link theory, have been developed previously. This theory assumes that the structure is analogous to a chain with many links. Each link may have a different limiting strength. When a load is applied to the structure such that the weakest link fails, then the structure fails.

The first probabilistic approach used to account for the scatter in fracture strength of brittle materials was introduced by Weibull [START_REF] Weibull | A statistical distribution function of wide appli-cability[END_REF]. This model requires statistical parameters to phenomenologically describe the failure response of a material. These statistical parameters, depending on the material characteristics, are usually determined from uniaxially-loaded, simple-geometry samples. In order to predict the material behaviour in other stress states using statistical parameters from uniaxial tests, Weibull proposed the calculation of the risk of rupture considering the maximal tensile principal stress or averaging the tensile normal stress in all directions. However, several investigators have shown that these models could lead to unsafe estimates of failure probability, and another model was introduced, based on the assumption that the principal stresses act independently [START_REF]Statistical approach to brittle fracture[END_REF]. In this method, the reliability of a component under multi-axial stresses is the product of the reliability of the individual principal stresses acting independently.

In another model, Batdorf [START_REF] Batdorf | A statistical theory for the fracture of brittle structures subjected to nonuniform polyaxial stresses[END_REF] combines the weakestlink theory and linear elastic fracture mechanics. Conventional fracture mechanics analysis assumes that both the size of the critical crack and its orientation respective to the applied load determine the fracture stress. The Batdorf theory includes the calculation of the combined probability of the critical flaw being within a particular size range and being located and oriented so that it may cause fracture. In addition to the statistical parameters determined, as for the Weibull model, by simple uniaxial tests, other parameters characterising the flaw orientation as a function of the stress directions must be calculated. These parameters can be difficult to determine for complex shapes and stress states.

Except in some simple cases, it is necessary, for predicting the risk of failure of ceramics structures with these different models, to use numerical simulation. Some studies have been conducted previously in which brittle fracture models were coupled to finite-element simulations to calculate the failure probability of particular ceramic structures. Berdin [START_REF] Berdin | Etude expe ´rimentale et nume ´rique de la rupture des mate ´riaux fragiles[END_REF] introduced both the Weibull and Batdorf models in the post processor of the Zebulon finite-element program, to predict the failure of particular disks under biaxial stresses. The statistical parameters of both models were determined from four point bending tests. In this study, results of the Weibull analysis for fractured disks were in better agreement with the experimental results than those of the Batdorf analysis.

Lamon [START_REF] Guillaumat | Simulation statistique-probabiliste du comportement me ´canique non-line ´aire d'un composite 2D SiC/ SiC[END_REF] has also developed a numerical model of statistical fracture analysis of ceramic composites in the FLAG post processor of a commercial finite-element program named MARC. The simulation, based on the Weibull approach and on the properties of different composite constituents, has led to the computation of the distribution of failure probabilities, and the global failure probability of a unit cell, as a function of strain.

However, most of the research in this field has been realised by the National Aeronautics and Space Administration [START_REF] Gyekenyesi | SCARE: a postprocessor program to MSC/ NASTRAN for reliability analysis of structural ceramic components[END_REF][START_REF] Gyekenyesi | Surface flaw reliability analysis of ceramic components with the SCARE finite element postprocessor program[END_REF][START_REF] Nemeth | Designing ceramic components for durability[END_REF]. In the late 1970s, NASA initiated the development of the SCARE computerised design program for the reliability evaluation of ceramic structures. SCARE can be coupled to the commercial finite-element programs ANSYS, ABAQUS, NASTRAN and COSMOS, and use the output data from these programs. The Weibull and Batdorf fracture models are present in the code, but simulation results show that the selected criterion has no significant effect on fracture prediction [START_REF] Nemeth | Designing ceramic components with the CARES computer program[END_REF]. Nevertheless, the finite-element programs quoted above are not convenient for the simulation of the forging process, where large strains take place in the workpiece.

The objective of this study is to develop a finite-element model in order to be able to predict the failure of ceramic forging dies as a function of the service conditions, in order to improve the design of these components. This numerical model has been introduced into the POLLUX finite-element code, developed previously by INSA Lyon [START_REF] Michel | Elasto-visco-plastic finite element analysis of a cold upsetting test and stress state validation by residual stress measurements[END_REF]. The Weibull model has been chosen to describe the fracture characteristics of ceramic materials, because the statistical parameters of this model are easy to determine experimentally. Both the maximal principal stress and the principle of independent actions criteria are available in the model. One of the principal characteristics of the program is that the fracture probability of the tools is calculated at each increment. This calculation is run at the same time increment as the stress tensor evaluation. The limit fracture probability, which is chosen by the user, controls the end of the simulation.

Software description

The finite-element code POLLUX has been developed in order to predict the stress state in the workpiece and the tools during a forging process. The thermo-elasto -visco-plastic formulation is based on a displacement approach. The model uses a separated implicit integration scheme of the constitutive law [START_REF] Michel | Elasto-visco-plastic finite element analysis of a cold upsetting test and stress state validation by residual stress measurements[END_REF] and is presented in the following sections.

Displacement formulation

Thermo-elasto-plastic constituti6e law

The Cauchy stress tensor | can be deduced from the elastic strain tensor m e , itself deduced from the difference between the total strain m t , the plastic strain m p , and the thermal strain m th , and the elastic behaviour of the material represented by the fourth-order tensor C, which can be temperature dependant:

| =C:m e =C:(m t -m p -m th ) (1)

Equilibrium equation for a displacement approach

Consider a continuum media V, with an equilibrium stress field | at time t, subjected to a loading step for time dt. The displacement field occurring during dt is the solution of the equilibrium equation applied to the loading increment. A weak formulation with the test function w of the equilibrium equation on the continuum media V and the introduction of the elasto-plastic constitutive law give the following equation:

& V (C:Dm t ): grad w d6 = & S DF a .w ds+ & V (C:Dm p ): grad w d6 (2)
The variation D is for the total time step dt, the tension variation DF a , applied on the surface S, is obtained with the boundary conditions, and the final stress state will be deduced from the balanced stress variation.

Constituti6e equation

The plastic response of the material is described using a classical Von Mises yield condition. The determination of the variation of the stress state is based on the normality principle of the plastic flow applied to a small strain variation:

lm p =u.n (3)
where n is the unitary tensor normal to the yield stress surface:

n= 3 2 • s | eq (4)
s is the deviatoric stress tensor, | eq is the equivalent Von Mises stress and u is the equivalent plastic strain deduced from the projection of the tensorial elastoplastic constitutive equation onto the normal to the yield stress surface.

Equilibrium equation sol6er

Sol6ing algorithm

To solve the equilibrium equation a constant operator strategy is used. The stiffness matrix of the problem is constituted with the elastic coefficients of the material and is integrated on the geometry at time t. Then the inverse stiffness matrix has to be computed only once per increment, which leads to shorter computation times. The total variation D for time step dt is cut into small iterative variations l, the equilibrium equation being written for an iteration k in the following way:

& V 1 2 C:(grad T la k +grad la k ) : grad w d6 = & S DF a k .w ds+ & V (C:Dm pk ): grad w d6 (5)
The second part of the equation is deduced from the variation of the solution at the previous iteration with the implicit integration of the constitutive law, the tension boundary at the surface S being introduced only for the first iteration. The convergence of the resolution is achieved when the residual unbalance is lower than a very small scalar.

Discrete decomposition

A triangular finite-element with quadratic interpolation functions is used for the geometry and the displacement, the stress and the strain fields being linear on each element:

a k = N b j .a j ( j from 1 to 6) (6)
The weak function is chosen as the displacement interpolation base, and the continuous equilibrium can be replaced in the approximation space by the following discrete system:

K ij .la k j =lf k i ( 7
)
K ij = & V 1 2 C:(grad T N b j +grad N b j ) : grad N b i d6 (8) lf k i = & S lF a k .N b i ds+ & V (C:lm pk ): grad N b i d6 (9)

Contact and friction law

The contact and friction conditions are imposed with a penalty method. Eqs. ( 7) -( 9) are written in a local coordinate system for all the concerned nodes. In order to keep a constant stiffness matrix in an increment of time dt, the formulation must be explicit. If a boundary condition changes at iteration k of increment i the calculation is stopped. The matrix is then modified and the calculation restarts at the first iteration of increment i.

As the displacement of the tool la 0 is imposed, the unknown displacement is limited to a relative displacement of the node la r so that Eq. ( 5) becomes:

(K ij -K fij ).la k j = lf k j ( 10
)
with:

lf k i = & S lF a k .N b i ds+ & S l~f la 0 la r .N b i ds + & V (C:lm pk ): grad N b i d6 (11)
K fij = -& S l~f 1 la r .N b i .N b j .ds ( 12
)
where ~f is the tangential friction stress.

The tool surfaces are modelled with linear elements on which different friction laws can be defined. The Tresca friction law, the Coulomb-Orowan friction law and the plastic wave model are available [START_REF] Baillet | A friction model for closed-die forging FEM simulation[END_REF].

Thermo-mechanical coupling

Energy-balance law

Considering an axisymmetric domain V, the temperature field must satisfy the energy-balance equation:

z.c. dT dt =div k.grad T+ Q ( 13
)
where z is the density, c is the heat capacity, k is the heat conductivity, T is the temperature and Q is the internal source.

Internal heat sources

In the metal-forming case there are two kinds of internal sources that are deduced from the mechanical results: a fraction of the plastic strain power and the friction power at the workpiece -die interfaces. Absorption-reflection: according to the Kirchoff law, the absorption and reflection coefficients are equal in the same direction and frequency. Extending this law to every direction of space and every frequency of the signal spectrum:

a = m i and r =(1 -m) i ( 16
)
where i is the incident flux, a is the absorbed flux and r is the reflected flux.

Stress state in the tools

Hypothesis

As the strains are supposed small, the tool meshes remain constant from the start to the end of the simula-tion, and are totally independent of the thermal meshes. The radial displacements of the tool nodes that are at the press boundary are usually free but can also be stopped.

Sol6ing algorithm

The discrete decomposition is also a triangular finite element composed of six nodes and three integration points and the solving algorithm used to find the tool stress state is similar to that described for the workpiece.

When the strain and the stress states are known for each Gauss point of the workpiece, they are extrapolated at the tool-workpiece interface nodes. As the tool border nodes are different from those of workpiece, the normal stress and the tangential stress are interpolated linearly and used as the loading conditions for the tool-solving algorithm.

Failure-prediction routine

Brittle fracture modelling

The statistical nature of the fracture of ceramics is connected to the presence of flaws in the material. Indeed, the failure of ceramic structure depends on the size, on the orientation and on the position in the structure of the critical flaw which initiates the fracture. Because this flaws distribution is impossible to determine, some statistical functions are used to describe the strength variability of a structure.

In his theory, Weibull associates a strength | c with each elementary volume V e . The strength of the material is characterised by the distribution function of | c , denoted F(|), which is defined as the number of all elementary volumes having | c 5 |, divided by the total volume. This function also gives the probability P of randomly choosing an elementary volume having a strength equal to or less than |, or also the probability that this volume fails under the stress |:

P(| c 5|)= F(|) ( 17
)
Any distribution function may be written in the form:

F(|) =1 -exp(-(|)) (18)
where (|) must be a positive, non-decreasing function, vanishing at a value | u , which is not necessarily equal to zero. The advantage of this equation appears when the failure probability of the structure constituted by all the elementary volumes, using the weakest link theory, is considered. If it is supposed that the total volume V fails if any one of its parts fails, then the probability of non failure, under an uniform uniaxial load |, is equal to:

(1 -P) n =exp( -n(|)) (19)
where n is the number of elementary volumes of the structure (V =nV e ).

The failure probability P n of the structure takes then the simple form:

P n =1 -exp( -n(|)) (20)
Weibull has chosen a power form for the function (|), which is the most simple function satisfying the general conditions previously described:

(|)= |-| u | 0 m ( 21
)
where | u , | 0 and m are parameters depending on the material characteristics.

The distribution function obtained has no theoretical basis, but experience has shown that in many cases it gives a good description of the strength fracture reliability of brittle materials. The threshold stress | u is usually taken as equal to zero. The two other parameters can then be identified easily by comparison with results of fracture tests.

In the case of non uniform uniaxial load |, the failure probability takes the integral form:

P =1-exp -1 V e & V | | 0 m dV (22)
This expression can be generalised to multi-axial stress states. Two strategies are possible: the first consists in considering an equivalent stress that characterises the loading at each point; the second consists in supposing the independence of action of each principal tensile stress [START_REF]Statistical approach to brittle fracture[END_REF].

Weibull, taking up the first approach, proposed to calculate an equivalent stress by averaging the normal tensile stress in all directions. This model leads to a complex integral formulation, which may increase the computation time. In order to obtain shorter computation times, only the maximal normal stress has been considered in the present model, as has been done by other investigators [START_REF] Berdin | Etude expe ´rimentale et nume ´rique de la rupture des mate ´riaux fragiles[END_REF]. This variable is also the maximal principal stress at each point of the structure.

In the second approach, the fracture probability depends on all of the tensile principal stresses. The independence of action of the principal stresses means that reliability of the structure is the product of the reliability of the individual principal stresses acting independently. The fracture probability is then given by the equation:

P= 1-exp -1 V e & V | 1 | 0 m + | 2 | 0 m + | 3 | 0 m dV (23)
where | 1 , | 2 and | 3 are the positive principal stresses or take the zero value for the compressive principal stresses.

It is interesting to note that in the both of the retained models, the compressive stresses are not taken into account, it being supposed that fracture is always the result of tensile stresses. The present authors have made this choice because the compressive fracture strength of ceramics is three or four times higher than the tensile strength, so that it is very unlikely that a structure would fail under compressive stresses.

Numerical introduction of the model

Both models previously described have been introduced into POLLUX. The architecture and basic computational element of the failure prediction program are described in Fig. 1. First, the thermomechanical analysis is achieved, leading to the calculation of the stress tensor on each point of the tools, which latter are supposed to be perfectly elastic. Then, the failure probabilities of the tools are determined, using the Weibull parameters and criteria chosen by the user.

The discrete decomposition and the Gauss quadrature of the models gives, if V e is arbitrary taken equal to unity:

P = 1-exp -% iel % igs | 1 | 0 m V(igaus) (24)
for the maximal principal stress criteria (criteria 1), and:

P= 1-exp -% iel % igs | 1 | 0 m + | 2 | 0 m + | 3 | 0 m V(igaus) (25)
for the principle of independent actions (criteria 2), in which: | 1 , | 2 , | 3 are the principal stresses in the Gauss point igaus of element iel (| 1 ] | 2 ] | 3 ) or equal to zero for compressive principal stress, where V(igaus) is the volume associated to the Gauss point igaus.

The elementary failure probability DPr, or local risk of rupture, is also calculated at each Gauss point, and interpolated to the nodes (Fig. 1). This variable corresponds to the fracture probability of an elementary volume of constant size equal to unity, supporting the stress state of the point considered. The values can be stored in a data file for graphical display of the structure critical regions. Another data file, containing the failure probability of overall structure as a function of time or tools displacement, is also generated. At the end of each increment, the program compares the failure probability of the tools to the maximal probability permitted, chosen by the user. The computation is stopped when this limit or the maximal displacement of the tools is achieved.

Model testing

In order to assess the validity of the numerical model developed, the software has been tested for the case of a ceramic cylinder under compression loading, considering an elastic cylindrical workpiece of radius R and height h, between two vertical planes (Fig. 2). A constant velocity 6 0 is prescribed on the upper plane, whilst the lower plane has no displacement in the vertical direction. The shear stress ~(~\0) between the planes and the workpiece is assumed to be constant, except for near to the symmetry axis, where it increases in a linear manner with the radius r.

The failure probability of the cylinder, computed by the software as a function of the tool displacement, has been compared to the results of an analytical calculation. For this validation, the following changes have been provided to the failure prediction program: the failure prediction calculation is run in the workpiece instead of in the tools; the stress state in the cylinder being essentially compressive, the absolute value of the principal stress has been considered for the calculation of the failure probability.

Analytical calculation

Basic hypothesis

It is supposed that the flow is axisymmetric, and that the axial displacement is independent of r:

z = h Í Ã Ã Á Ä w= -w 0 = -6 0 .t 0 5r 5m m5 r5R | rz = ~r m | rz = ~(28) r =R | rr = 0 (29) 4.1

.3. Solution

The resolution of the average equilibrium equations, using the average of the values of the stresses and of the radial displacement, gives the following expressions for the average stress | zz :

0 5r5m | zz (r) = w (1-w) hr 2 m +m + (1-2w) 6 m 3 R 2 - 4 3 (2 -w)R -Ew 0 h (30a) m5r 5R | zz (r) = w (1-w) h 2r + (1 -2w) 6 m 3 R 2 - 4 3 (2 -w)R -Ew 0 h (30b) if (h/R) 1, it is assumed that: |(r,z) $|(r) (31)
In addition, if ~ | rr , | qq , | zz , then | rr , | qq and | zz can be considered as the principal stresses at each point of the cylinder, and | zz as the maximal principal stress. The failure probability of the cylinder can then be expressed, using the criteria 1 as:

P= 1 -exp -2yh & R 0 | zz (r) | 0 m r dr (32)
The value of m is obtained by identification with the stress field given by numerical computation.

The integral

& R 0 | zz (r) | 0 m r dr
having no analytical solution, has been evaluated numerically, for different values of w 0 , using an adaptive recursive Simpson rule [START_REF] Forsythe | Computer Methods for Mathematical Computations[END_REF].

Numerical results

Analytical and numerical values of the stress | zz as a function of radius r are compared on Fig. 3, for a displacement of the upper plane w 0 equal to 0.03 mm. The analytical calculation gives a slightly lower stress (1%) than that of the numerical computation, but nevertheless it gives a good approximation.

Fig. 4 represents the evolution of failure probability calculated with the finite-element program (criteria 1), and with the analytical expression for the stress | zz . The two curves are relatively close, nevertheless, the analytical calculation give significantly lower values than the numerical computation, which is in accordance with the difference observed in the stress values and with the value of the m parameter (m=12).

Application: design of ceramic forging dies

The software has been developed in order to be able to predict the reliability of ceramic tools during a forging operation, the resulting information enabling the improvement of the design of the tools.

Two data types are accessible: (i) the overall failure probability of the tool as a function of its displacement, or of time, the reliability of the tool during one operation being deduced from the maximal value of this probability; (ii) the elementary failure probability, or local risk of rupture at a given time of the forging process, which allows the identification of the tool critical regions.

These two items of information are required for the designing of the tools. The design technique using failure prediction software consists of three steps: in the first step, the user defines the tool initial geometry and reliability, according to the type and the geometry of the forged piece; in the second step, the software calcu-lates the overall failure probability of the tools, using the initial geometry, where if the corresponding reliability is consistent with the defined reliability, then the geometry is convenient. On the contrary, if the calculated reliability is lower than the chosen reliability, then the third step consists in modifying the geometry of the tools, according to the local risk of rupture. These data enable the user to identify the critical zones of the tools which must be modified. It is then possible, by local change in the tool geometry, to increase the reliability without modifying in a significant manner the final shape of the forged piece.

This technique has been applied to a simple case of isothermal forging. The workpiece material is of Astroloy (nickel-base alloy) and the forging dies are of refractory ceramic.

Forging modelling

The running of the POLLUX code with the failure prediction program requires the creation of three files. The first file contains the geometry of the initial workpiece and of the tools. As described earlier, the first step in the design method must be the definition of the tool initial geometry, according to the shape of the final piece. The second file contains the data of the mechanical and thermal behaviour of the different materials, the initial and boundary conditions (temperature, tools displacement, etc.), and the friction conditions between the workpiece and the tools. The third file contains the fracture parameters of the tools material. The following sections present the data contained in these different files.

Geometry

For this example, a simple geometry has been chosen for the final piece (Fig. 5). According to this shape, the tools geometry has been defined (Fig. 6). The lower tool is flat with an axisymmetric geometry, like the final piece, whilst the engraving of the upper tool corresponds to the shape of the piece to be obtained. The radius in the concave fillet of the engraving was not indicated in the geometry of the piece, and has been taken as 3 mm. The workpiece has a cylindrical initial geometry, with a height of 50 mm and a diameter of 50 mm.

Material beha6iour and initial and boundary conditions

Astroloy material has superplastic behaviour at temperatures above 1323 K, and at low strain rates. The simulation of an isothermal forging operation at the temperature of 1373 K, with a downwards speed of the upper tool of 0.1 mm min -1 (the lower tool remains fixed), has been chosen. Heat transfer can be neglected, the temperature of the tools and the workpiece being assumed to be constant during the operation.

The mechanical behaviour of the tools is supposed to be perfectly elastic during the forging operation. Young's modulus and the Poisson coefficient are respectively equal to 262 GPa and 0.3 at the forging temperature.

The rheological behaviour of the workpiece is assumed to be viscoplastic in the process range temperature. A constitutive model has been proposed by Soucail for Astroloy material [START_REF] Soucail | Mode ´lisation du soudage par friction inertielle du superalliage a `base de nickel astroloy[END_REF]:

| = K(T).m ¯; n ( 33
)
where | is the Von Mises equivalent stress (MPa); m ¯; is the equivalent strain rate (s -1 ); K(T) is a proportionality constant, taken as equal to 119 at 1373 K; and n is the strain-rate sensitivity factor, taken as equal to 0.19. Friction between the tools and the workpiece is modelled by the Coulomb law, with a coefficient v of 0.1.

Fracture parameters of material tools

The Weibull parameters describing the fracture characteristics of the used ceramic have been determined according to the results of four point bending tests, run at 1373 K. The | 0 and m values are respectively equal to 400 MPa and 12, and are assumed to be constant during the whole of the forging process. These data have been deduced from quasi-uniaxial stress-state tests, which makes it difficult to choose the multi-axial stress-state criteria. Thus, it is assumed arbitrarily that the maximal principal stress criteria (criteria 1) is more suitable for the present ceramic material.

A tool reliability of 0.9999 has been chosen, which means that the failure probability of the tools must be always lower than 10 -4 during the process cycle.

Failure prediction results

To obtain a forging piece with a near net shape, the final displacement of the upper tool has been fixed at 30 mm. The initial and final configurations of the simulation computation are presented in Fig. 7.

Owing to its being flat, the lower tool is subjected to essentially only compressive stresses, so that its failure probability is very low compared to that of the upper tool. In the latter, the engraving leads to the occurrence of stress concentration during forging, which increases the failure probability. Fracture-prediction software has thus been used for the upper tool only.

The evolution of the overall fracture probability of the upper tool is represented in Fig. 8 (r=3) as a function of the vertical displacement of the tool. This probability increases swiftly, and overshoots the fixed limit before the end of the forging operation. The stress state to which the upper tool is subjected is too severe to preserve the fixed reliability. The tool geometry must be then modified to improve the reliability, according to indications given by the local risk of rupture distribution (Fig. 9).

The graphical analysis of local failure probability allows the identification of regions sustaining the more critical stress states. According to this distribution, the maximal local risk is localised around the bottom radius of the engraving. The stress concentration in this zone is too high.

The stress field around a corner being related to the value of its radius, the increasing of the bottom radius of the engraving leads to a decrease of the stress concentration factor, and correspondingly to a decrease of failure probability of the piece.

A second tool geometry can then be defined, with a radius in the concave fillet engraving of 5 mm instead of 3 mm. The overall fracture probability corresponding to this new geometry is represented as a function of tool displacement in Fig. 8 (r = 5): the global risk does not exceed 10 -4 , which is in agreement with the fixed reliability.

Conclusions

A fracture-prediction software for brittle materials has been introduced into the POLLUX finite-element code, in order to be able to predict the risk of rupture of ceramic tools during a forging operation. Comparison between the code results and the results of the analytical calculation in a simple compression case has led to the validation of the implementation of the numerical model. The use of this software allows the improvement of the design of ceramic forging tools. The utilisation strategy is presented for a forging example of a simple shape piece.

The fracture-prediction software requires, however, the knowledge of experimental data about the fracture behaviour of the ceramic material used. These data enable the choice of the failure criteria that is more convenient for the material, and the identification model parameters. Two failure criteria are already available. Future developments forecast the introduction of other criteria, including shear stresses, and the differentiation between two types of defects (surface defaults and volume defects, for instance). In addition, fracture prediction tests are currently being performed on computer-designed samples in order to further validate the chosen numerical model.

2. 5 . 3 .

 53 Prescribed boundary heat fluxes 2.5.3.1. Con6ection. The prescribed boundary heat convection flux is a function of the heat transfer coefficient h(T) and the difference between the surface temperature T and the room temperature T : q=h(T).(T-T ) (14) 2.5.3.2. Radiation. An evolutive closed-cavity model with current view factors is used [12]. Radiation emission: each point along the boundary of a cavity C emits a flux proportional to the temperature to the fourth power, to the Stefan-Boltzmann constant | and to the surface emissivity m: =m.|.T 4 (15)

Fig. 1 .

 1 Fig. 1. Computational elements of the failure-prediction routine.

Fig. 2 .

 2 Fig. 2. A cylindrical workpiece pressed between two planes with uniform friction.

Fig. 3 .

 3 Fig. 3. Evolution of the stress | zz as a function of the radius r. w(z) (26)

Fig. 4 .

 4 Fig. 4. Evolution of the failure probability of the cylinder as a function of the displacement of the upper tool.

Fig. 7 .

 7 Fig. 7. Initial and final configuration of the finite-element simulation.

Fig. 5 .

 5 Fig. 5. Final geometry of the forged piece (dimensions: mm).

Fig. 6 .

 6 Fig. 6. Geometry of the tools (dimensions: mm).

Fig. 8 .

 8 Fig. 8. Evolution of the fracture probability of the upper tool as a function of its displacement, for two different engraving concave fillet radii.

Fig. 9 .

 9 Fig. 9. Local risk of rupture (%) of the upper tool around the engraving fillet radius.

Acknowledgements

The ceramic material was provided by Norton Desmarquest (Evreux F27), the help of Mr Cales and Mr Main being gratefully acknowledged. Information on POLLUX software can be supplied by Dr J.C. Boyer.


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International Amphibian Micronucleus Standardized Procedure (ISO 21427-1) for In Vivo Evaluation of Double-Walled Carbon Nanotubes Toxicity and Genotoxicity in Water

Keywords: double-walled carbon nanotubes, toxicity, genotoxicity, amphibian larvae, Xenopus laevis, Raman analysis

Considering the important production of carbon nanotubes (CNTs), it is likely that some of them will contaminate the environment during each step of their life cycle. Nevertheless, there is little known about their potential ecotoxicity. Consequently, the impact of CNTs on the environment must be taken into consideration. This work evaluates the potential impact of well characterized double-walled carbon nanotubes (DWNTs) in the amphibian larvae Xenopus laevis under normalized laboratory conditions according to the International Standard micronucleus assay ISO 21427-1:2006 for 12 days of half-static exposure to 0.1-1-10 and 50 mg L 21 of DWNTs in water. Two different endpoints were carried out: (i) toxicity (mortality and growth of larvae) and (ii) genotoxicity (induction of micronucleated erythrocytes). Moreover, intestine of larvae were analyzed using Raman spectroscopy. The DWNTs synthetized by catalytic chemical vapor deposition (CCVD) were used as produce (experiment I) and the addition of Gum Arabic (GA) was investigated to improve the stability of the aqueous suspensions (experiment II). The results show growth inhibition in larvae exposed to 10 and 50 mg L 21 of DWNTs with or without GA. No genotoxicity was evidenced in erythrocytes of larvae exposed to DWNTs, except to 1 mg L 21 of DWNTs with GA suggesting its potential effect in association with DWNTs at the first nonacutely toxic concentration. The Raman analysis confirmed the presence of DWNTs into the lumen of intestine but not in intestinal tissues and cells, nor in the circulating blood of exposed larvae.

INTRODUCTION

Carbon nanotubes (CNTs), a man-made form of carbon, are one dimensional nanoscale objects, characterized by exceptional properties, in relation with their nanosize. They are allotropes of carbon and their structure can be described as a graphene sheet rolled up to form a cylinder. There are two main types of CNTs [START_REF] Bethune | Cobalt-catalyzed growth of carbon nanotubes with single-atomic-layerwalls[END_REF][START_REF] Iijima | Single-shell carbon nanotubes of 1-nm diameter[END_REF]: single-walled CNTs (SWNTs) and multiwalled CNTs (MWNTs) depending on the number of concentric walls. Among the MWNTs, double-walled carbon nanotubes (DWNTs) are at the frontier between SWNTs and MWNTs. Their morphology is very close to SWNTs. CNTs have a diameter from 1 nm and a length up to tens of lm or more, giving them a very high aspect ratio. Their specific surface area is generally important and can theoretically reach to 1310 m 2 g 21 in the case of closed SWNTs.

Since their discovery in 1991 by Iijima [START_REF] Iijima | Helical microtubules of graphitic carbon[END_REF][START_REF] Hata | Water-assisted highly efficient synthesis of impurity-free single-walled carbon nanotubes[END_REF], interest in CNTs has grown rapidly due to their unique physical (mechanic, electronic, thermal) and chemical properties. CNTs represent one of the fastest developing nanoparticle materials. Applications of CNTs are numerous including TV screens (flat-screens), sport equipments (bike frame, baseball bat, tennis rackets), and tires [START_REF] Baughman | Carbon nanotubes-the route toward applications[END_REF]. Some others are in preparations such as in paints and composite materials in general, special technical clothes, medical, and pharmaceutical products.

Because of their increasing production, use, and applications, it is likely that some of them will get into the environment during each step of their life cycle (production, use, and disposal), especially in the aquatic compartment which concentrates all kinds of pollution. The presence of CNT-contaminated waste could lead in the near future to ecotoxicity problems. CNTs releases may come from (i) different point sources in relation with their production (manufacturing, wastewater effluents), landfills, and (ii) nonpoint sources corresponding to their use and application until their end of life, such as wet deposition from the atmosphere, storm-water runoff, groundwater, surface water leakage, and attrition of products containing CNTs. Consequently, CNTs must receive considerable attention as new, unknown, and potentially hazardous materials. Nevertheless, to our knowledge, there is little known about their potential ecotoxicity, especially on aquatic organisms, which are likely to enter the human food chain [START_REF] Farre | Ecotoxicity and analysis of nanomaterials in the aquatic environment: Review[END_REF]. Only few studies on different aquatic organisms exposed to CNTs are available. All of them indicated that exposure to CNTs generally lead to biological disorders at different levels, usually above 10 mg L 21 .

Until now, amphibians have not yet been really used to characterize the potential toxic effects of CNTs in the aquatic medium. Nevertheless, amphibians are well-known environmental health warning organisms due to their bipha-sic life cycle, permeable eggs, skin, and gills [START_REF] Gauthier | The amphibian micronucleus test, a model for in vivo monitoring of genotoxic aquatic pollution. Review paper[END_REF]. Their specific physiology makes them particularly sensitive to the presence of contaminants in the water, influencing their behavior, so that they are more and more used as monitoring systems for water quality assessment [START_REF] Bridges | Comparative contaminant toxicity: Are amphibian larvae more sensitive than fish?[END_REF][START_REF] Gauthier | Biomonitoring of the genotoxic potential (micronucleus assay) and detoxifying activity (EROD induction) in the river Dadou (France), using the amphibian Xenopus laevis[END_REF].

To our knowledge, only two studies (Mouchet et al., 2007a;Mouchet et al., 2007b;[START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF] are devoted to the assessment of the potential genotoxic effects of CNTs on amphibian larvae in vivo.

Among toxic actions, genotoxic effects may durably affect the aquatic ecosystems and the presence of genotoxic compounds in water can also have repercussions on nonaquatic species, via food chains, or simply as a result of drinking water. The interaction of genotoxic compounds with DNA initially may cause structural changes in the DNA molecule. Unrepaired damage can generate other cell lesions and thus lead to tumor formation [START_REF] Vuillaume | Reduced oxygen species, mutation, induction and cancer initiation[END_REF][START_REF] Malins | A novel DNA lesion in neoplastic livers of feral fish: 2, 6-diamino-4hydroxy-5-formamidopyrimidine[END_REF]. A number of tests have been developed to assess the genotoxic potency of water samples, using either plants or aquatic animals (see for review [START_REF] Jaylet | Micronucleus test using peripheral red blood cells of amphibian larvae for detection of genotoxic agents in freshwater pollution[END_REF].

In amphibian larvae, as in most eukaryotes, genome mutations may result in the formation of micronuclei, which are a consequence of chromosome fragmentation or malfunction of the mitotic apparatus. The micronucleus test (MNT) has been widely used with many amphibian species (Pleurodeles waltl, Ambystoma mexicanum, and Xenopus laevis) in the laboratory [START_REF] Gauthier | The amphibian micronucleus test, a model for in vivo monitoring of genotoxic aquatic pollution. Review paper[END_REF][START_REF] Ferrier | Genotoxicity tests in amphibians-A review[END_REF][START_REF] Gauthier | Biomonitoring of the genotoxic potential (micronucleus assay) and detoxifying activity (EROD induction) in the river Dadou (France), using the amphibian Xenopus laevis[END_REF][START_REF] Mouchet | Comparative study of the comet assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene. ethyl methanesulfonate, and methyl methanesulfonate: Establishment of a positive control in the amphibian comet assay[END_REF]Mouchet et al., , 2006aMouchet et al., ,b, 2007a,b),b). The sensitivity and reliability of the MNT to detect chromosomal and/or genomic mutations makes it a good method to analyze the potential cytogenetic damage caused by pure substances for instance [START_REF] Jaylet | Micronucleus test using peripheral red blood cells of amphibian larvae for detection of genotoxic agents in freshwater pollution[END_REF][START_REF] Gauthier | The amphibian micronucleus test, a model for in vivo monitoring of genotoxic aquatic pollution. Review paper[END_REF][START_REF] Mouchet | Comparative study of the comet assay and the micronucleus test in amphibian larvae (Xenopus laevis) using benzo(a)pyrene. ethyl methanesulfonate, and methyl methanesulfonate: Establishment of a positive control in the amphibian comet assay[END_REF]Mouchet et al., , 2006aMouchet et al., ,b, 2007a)). This method has been standardized on Xenopus laevis in French [START_REF] Afnor | Association franc ¸aise de normalisation (the French National Organization for quality regulations)[END_REF] and International (ISO, 2006) recommendations. One of the key functions of such biomarkers (micronucleus) is to provide an ''early warning'' signal of significant biological effects (changes at the genetic/molecular level) with suborganism (molecular, biochemical, and physiological) responses preceding those occurring at higher levels of biological organization such as cellular, tissue, organ, whole-body levels, and in fine at population level. In this way, the use of the MNT may provide an important tool for the prediction of the potential long-term effects on amphibians in the environment.

The aim of the present work is to contribute to the ecotoxicological assessment of the potential impact of CNTs using the standardized method ISO 21427-1 (ISO, 2006). Xenopus larvae were exposed to DWNTs at concentrations ranging from 0.1 mg L 21 (to mimic potential environmental doses) to 50 mg L 21 (which may represent an accidental release, and optimize the observation of the potential toxic effects), with and without Gum Arabic (GA), a natural polysaccharide which acts as a dispersant, under controlled laboratory conditions to evaluate two different endpoints after 12 days of exposure: (i) toxicity on larvae (mortality and growth) and (ii) genotoxicity as the expression of the clastogenic and/or aneugenic effects observed in erythrocytes in the running blood. Then, the presence of DWNTs was investigated in the larvae using traditional microscopy methods (photonic and electronic), but also by Raman spectroscopy to confirm the presence of DWNTs.

MATERIALS AND METHODS

Preparation of CNTs Samples

DWNTs were prepared by catalytic chemical vapor deposition (CCVD) by decomposition of a H 2 À ÀCH 4 mixture over an MgO-based catalyst [START_REF] Flahaut | Gram-scale CCVD synthesis of double-walled carbon nanotubes[END_REF]. The carbon content of the as-produced CCVD product was about 7.8 wt %, as determined by elemental analysis (flash combustion). Assuming that all the carbon is present in the form of CNTs [START_REF] Flahaut | Synthesis of single-walled carbon nanotube-Co-MgO composite powders and extraction of the nanotubes[END_REF], it is thus possible to calculate the amount of as-produced CCVD product corresponding to a given amount of CNTs. CNTs were then obtained by treating the required amount of CCVD product with a concentrated aqueous hydrochloric acid (HCl) solution. After washing with deionized water until neutrality, the CNTs were maintained in wet conditions to limit aggregation. A sample was taken and dried for further characterizations (elemental analysis, BET, Raman spectroscopy, SEM, TEM, XRD). The carbon content of the CNTs sample was about 90 wt %, as obtained by elemental analysis. This corresponds to more than 97.7 mol % of carbon, assuming that the sample contains mainly Co and C. The remaining Co was assumed to be present only as carbon-encapsulated nanoparticles [START_REF] Flahaut | Synthesis of single-walled carbon nanotube-Co-MgO composite powders and extraction of the nanotubes[END_REF][START_REF] Flahaut | CCVD Synthesis and Characterization of cobalt-encapsulated nanoparticles[END_REF]. The BET (Brunauer Emmett Teller) specific surface area measured was between 800 and 900 m 2 g 21 . Raman analysis (k 5 488 nm, not shown) revealed that the ratio between the intensity of the D and G bands was close to 10%, corresponding to a good structural quality of the CNTs. Analysis of the radial breathing modes (which frequency can be easily associated to the diameter of the CNTs) indicated the presence of CNTs with diameters ranging from 0.7 to 2.2 nm. Figure 1(a) shows a representative FEG-SEM (Field Emission Gun-Scanning Electron Microscopy) image of the raw CNTs sample (as-produced CCVD product), showing a very high density of CNTs bundles, with extensive branching. Their diameter (bundles) typically ranged between 10 and 20 nm but numerous individual CNTs were also present. No carbon nanofibre (a typical by-product of CCVD methods) was observed in the sample. HRTEM (High Resolution Transmission Electron Microscopy) observation was performed on the CNTs after elimination of the catalyst by HCl washing [Fig. 1(b)] and revealed clean CNTs surfaces; as suggested by SEM, the CNTs are mainly iso-lated, or gathered into small bundles. The CNTs obtained in those conditions contain about 80% DWNTs, together with about 15% single-wall carbon nanotubes (SWNTs), and about 5% triple-walled carbon nanotubes. The outer diameter of DWNTs is typically ranging between 1 and 3 nm. The concentration of CNT in suspension in water after dispersion was monitored and published in a previous work [START_REF] Datsyuk | Double-walled carbon nanotube dispersion via surfactant substitution[END_REF].

Xenopus Rearing and Breeding

The Xenopus males were injected with 50 IU of PMSG 500 (Pregnant Mare's Serum Gonadotropin, Intervet, France) and the females with 750 IU of HCG (Human Chorionic Gonadotropin, Organon, France) to induce spawning. Each pair was then placed together in normal tap water filtered through active charcoal at (22 6 2)8C. Twenty four hours later, the pair was separated and viable eggs were maintained in an aquarium also containing normal tap water filtered through active charcoal at 20-228C, until they reached a development stage appropriate for experimentation. The larvae were fed every day on dehydrated aquarium fish food. Two different hatches were used in the present work (one for experiment I and the other for the experiment II).

Exposure Conditions

The exposure was performed according to the French Standard AFNOR NF T90-325 [START_REF] Afnor | Association franc ¸aise de normalisation (the French National Organization for quality regulations)[END_REF] and the International Standard 21427-1 (ISO, 2006). Two independent experiments (I and II) were conducted, in semistatic exposure conditions consisting in a daily renewal of the medium during the 12 days of exposure. In both experiments, Xenopus larvae were exposed for 12 days to the same DWNTs concentrations i.e., 0.1, 1, 10, and 50 mg L 21 of DWNTs without GA (experiment I) or with GA (experiment II). The choice of adding a surfactant is justified by both the stabilization of the exposure media and the limitation of the size of the aggregates of CNTs. Moreover, CNTs which could end up in the environment would be likely to have adsorbed natural compounds such as natural sugars present into the aquatic compartment. Primarily used in the food industry as a stabilizer, GA was used because it is composed of polysaccharides which are secreted by most of photosynthetic organisms into the environment.

The amphibian larvae were exposed in reconstituted water (RW) (distilled tap water to which nutritive salts were added [294 mg L 21 CaCl 2 .2H 2 O, 123.25 mg L 21 MgSO 4 .7H 2 O, 64.75 mg L 21 NaHCO 3 , 5.75 mg L 21 KCl]). Xenopus exposure began on larvae at stage 50 of the Xenopus development table [START_REF] Nieuwkoop | Normal Tables of Xenopus laevis (Daudin)[END_REF]. For a given experiment, the larvae were taken from the same hatch to reduce interanimal genetic variability within each experiment. Larvae were exposed in groups of 20 animals (100 mL/larva) in 2 L pyrex crystallising dishes containing either the control medium (negative and positive controls) or the test medium (0.1-1-10 and 50 mg L 21 of DWNTs with or without GA), or the GA control (50 mg L 21 ). An initial stock suspension of CNTs with or without GA containing the total amount of CNTs required for the 12 days of exposure was prepared by adding RW to the corresponding amount of wet DWNTs. Each day, DWNTs stock suspensions (with or without GA) were homogenized by bath sonication for 20 min before taking the required volume to obtain the target concentration by dilution in RW (final volume: 2L). The final suspensions of CNTs were then homogenized by mechanical stirring (Ultraturax) for 5 min at 9500 rpm. In the case of the addition of GA, the concentration was 50 mg L 21 . The negative control (NC) was the RW alone. The positive control (PC) was monohydrated cyclophosphamide (CP,, Sigma France) in RW at 20 mg L 21 (ISO, 2006). CP is a standard indirect mutagen requiring metabolic activation in liver prior to becoming effective. Positive control was systematically performed in each experiment to check the responsiveness of the amphibian larvae. The larvae were submitted to a natural dark cycle at 22.08C 6 28C during the 12 days of exposure. They were fed every day on dehydrated aquarium fish food.

Toxicity

Acute toxicity (death or abnormal behavior) of larvae exposed to CNTs was examined for 12 days according to the standardized recommendations [START_REF] Afnor | Association franc ¸aise de normalisation (the French National Organization for quality regulations)[END_REF]ISO, 2006) by visual inspection compared to NC. Abnormal behavior corresponding to reduced and/or stopped growth of larvae, reduced food intake and abnormal motility. The visual inspection of the size of larvae was completed by measuring the size of each larva at the beginning of the exposure (Time 0 5 t 0 ) and at the end of the exposure (Time 12 days 5 t 12 ). For this, larvae were preliminarily anesthetized (Tricay ¨ne methane sulfonate, MS 222, Sandoz) and photographed (Leica, France). The measure was then performed on photograph of each larva using the Mesurim image analysis software [START_REF] Madre | Logiciel Mesurim[END_REF]. Statistical analyses were performed using SimagStat 3.1. Nonparametric tests were preferred because of (i) nonnormality, (ii) and/or nonequivalence of variances and (iii) samples size (n \ 30). For each time of exposure (t 0 and t 12 ), the data (size of larvae expressed in cm) were compared to (i) confirm that there is not a significant difference at t 0 between larval size from a same condition on one hand [i.e., larval size was in accordance with the stage 50 of the development stage table [START_REF] Nieuwkoop | Normal Tables of Xenopus laevis (Daudin)[END_REF]] and between larvae size from the different conditions on the other hand, (ii) conclude at t 12 about the significant difference between the different conditions compared to NC and (iii) conclude about the significant difference for a given condition between t 0 and t 12 . Kruskal-Wallis test (variance analyze on ranks) was performed to compare between all conditions, followed by Dunn's (same size of sample) or Dunnet's (different size of sample) test to isolate the group(s) that differ(s) from the others using a multiple comparison procedure, with unpaired date, versus the negative control group. The Mann-Whitney U-test was performed to compare on the basis of two and two conditions.

Graphic representations are proposed, based on the growth rate calculated as follow:

[((mCdt8Xt 12 2 mCdt8Xt 0 ) 2 (mCdt8NCt 12 2 mCdt8NCt 0 ))/(mCdt8NCt 12 2 mCdt8NCt 0 )] 3 100] where mCdt8Xt 12 and mCdt8Xt 0 represent the mean value of the size of larvae exposed to the condition X and measured at respectively t 12 and t 0 , mCdt8NC t12 and mCdt8NC t0 represent the mean value of the size of larvae exposed to the condition NC and measured at respectively at t 12 and t 0 . Statistics analyses are realized on the size of larvae (mean value) measured at respectively, t 12 and t 0 . Asterisks are written on the graphic representation based on the growth rate when a significant different size of larvae compared to the negative control group (mean value) is concluded.

Micronucleus Test, Genotoxicity Assay

Formation of micronuclei is the consequence of chromosome fragmentation or malfunction of the mitotic apparatus and may result in genome mutations. In both cases, entire or fragmented chromosomes can no longer migrate to the cellular poles in the anaphase of the cell cycle resulting in a little clump of chromatin, called a micronucleus, near the principal nucleus in the cytoplasm of the daughter cells. Thus, clastogenic compounds and spindle poisons both lead to an increase in the number of micronucleated cells. At the end of exposure, a blood sample was obtained from each anesthetized larva (MS222, Sandoz, France) by cardiac puncture with heparinized micropipettes (20% solution at 5000 IU mL 21 , Sigma, France). After fixing in methanol and staining with hematoxylin (Sigma, France), the smears were screened under the microscope (oil immersion lens, 15003). The number of erythrocytes that contained one micronucleus or more (micronucleated erythrocytes, MNE) was determined in a total sample of 1000 erythrocytes per larva. All slides were scored blinded by only one individual. Since micronucleus frequency was not normally distributed, median values and quartiles were calculated instead of means [START_REF] Mcgill | Variations of box plots[END_REF]. For each group of animals, the results (number of micronucleated erythrocytes per thousand, MNE %) obtained for each larva were arranged in increasing order of magnitude. The medians and quartiles were then calculated. The statistical method used to compare the medians consists in determining the theoretical medians of samples of size n (where n ! 7) and their 95% confidence limits expressed by M 6 1.57 3 IQR/Hn, where M is the median and IQR is the Inter-Quartile Range (upper quartile -lower quartile) [START_REF] Mcgill | Variations of box plots[END_REF]. The difference between the theoretical medians of the test groups and the theoretical median of the negative control group is significant to within 95% certainty if there is no overlap. In this case, the induction of micronucleus in exposed larvae, compared to the control, is considered as a genotoxic response.

Raman Spectrometry Analysis

Raman spectrometry analysis was performed on an intestine sample of larvae exposed to a concentration of 100 mg L 21 (higher than the maximum 50 mg L 21 concentration investigated in this study). Exposure at this concentration was carried out only for the analysis with the Raman spectrometry to increase the chances of tissue contamination. Such a concentration is clearly toxic for Xenopus [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF]. Histological preparation of intestine (semithin sections of 1 lm of depth) was observed by Raman analysis. The Raman spectra were recorded on a Renishaw spectrometer with a green laser excitation (514.5 nm). The presence of CNTs was evidenced using the G' 2D band of CNTs at a wave number of about 2675 cm 21 . This allowed avoiding any interference with the biological matrix. The laser power was kept at 25 mW with an objective magnification of 503 (spot size about 3-lm diameter), leading to about 0.25 mW lm 22 on the sample. No particular care was taken to avoid heating effect, as only the intensity was used to evidence the CNTs presence and location.

RESULTS

Toxicity

The results show no mortality of the larvae whatever the experimental condition and the experiment (I and II), except at 10 and 50 mg L 21 without GA, where 5 and 15% of mortality were respectively observed in experiment I (Table I). The visual inspection of the acute toxicity in larvae was confirmed by measurements of the size of the larvae and shows that larvae exposed in presence of 10 and 50 mg L 21 of DWNTs with (experiment II) or without GA (experiment I) have reduced size compared to the NC in a dose dependant manner (Table I, Fig. 2). In contrast, larvae exposed to 0.1 and 1 mg L 21 of DWNTs do not show any sign of toxicity compared to the NC. Furthermore, the results show that the growth rate decreases in a dose dependant manner in larvae of both experiments (Fig. 2).

Genotoxicity

The median value of MNE % for the negative control was 6.0 6 1.2 in experiment I [Fig. 3(a)] and 1.5 6 0.4 Fig. 3(b)]. The positive control showed significantly higher MNE % as compared to the NC group in experiments I and II (30.0 6 4.7 and 10.5 6 1.6, respectively). Larvae exposed to 50 mg L 21 of DWNTs with or without GA, were not punctured because of the toxicity (growth inhibition and lack of cell divisions) observed at this concentration. Indeed, genotoxic effects are usually expressed at subtoxic concentrations of the tested substance, following DNA damage, micronucleus induction is tributary to cellular division and hence to the mitotic index of the red blood cells. In experiment I, the results indicate no genotoxicity via micronucleus induction in erythrocytes of Xenopus larvae, whatever the DWNTs concentration tested without GA. No genotoxicity was also observed in larvae exposed to GA alone (2%). In contrast, a significant micronucleus induction was observed in larvae exposed to 1 mg L 21 of DWNTs in presence of GA (3%).

Raman Spectrometry Analysis

We have performed a Raman line scan to localize the CNTs. The signal from the tissues is composed of several bands around 1450 cm 21 and a very intense one at 1630 cm 21 (Fig. 4). As CNTs present no Raman signal at this frequency, the peak at 1630 cm 21 was used as an indication of the presence of the tissues. In the range where D (for defect induced band) or G' 2D (overtone of D band) bands are present, no other band associated to organic material was visible and consequently, fitting these bands is a very good way to know where the CNTs are located. We have also fitted the G band and compared all signals coming from the tubes. To fit properly the G band, we should fix all parameters, including the signal coming from the tissues, and leave only the intensities free. We obtain the same results than with G' 2D band but the zero intensity is less clean (very small residual intensity sometimes). We consider that both approaches are correct but we prefer to keep G' 2D intensity as in earlier work [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF]. Figure 4(b) shows a histological section of the intestine of Xenopus exposed to 100 mg L 21 of DWNTs. The lumen and the intestinal wall (villi and intestinal cells) are clearly visible. On the one hand, the intensity of the G' 2D band strongly decreases from the lumen to the intestinal wall. On the other hand, the intensity of the signal corresponding to the Xenopus biological matrix increases from the lumen to the intestinal wall [Fig. 4(c)]. The presence of CNTs was clearly identified; thanks to Raman spectroscopy. However, it is unlikely that all the black material visible on the light microscopy image would correspond to DWNTs [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF].

DISCUSSION

Considering the global planned production of CNTs on the ton scale, and their integration in everyday-life products, it is likely that some of them will enter the environment during their product's life cycle (manufacture, use, and disposal). Their widespread use thus constitutes a potential risk of exposure for all living organisms in the environment. Curiously, one of the most important areas of interest in environmental risk assessment of such new materials, i.e., the ecotoxicological field of research, remains uninvestigated. Very few data have been published on the impact of CNTs on aquatic organisms (see for review [START_REF] Templeton | Life-cycle effects of single-walled carbon nanotubes (SWNTs) on an estuarine meiobenthic copepod[END_REF][START_REF] Cheng | Effect of carbon nanotubes on developing zebrafish (Danio rerio) embryos[END_REF][START_REF] Helland | Reviewing of the environmental and human health knowledge base of carbon nanotubes[END_REF][START_REF] Roberts | In vivo biomodification of lipidcoated carbon nanotubes by Daphnia magna[END_REF][START_REF] Smith | Toxicity of single walled carbon nanotubes on rainbow trout (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other physiological effects[END_REF][START_REF] Kennedy | Factors influencing the partitioning and toxicity of nanaotubes in the aquatic environment[END_REF]Mouchet et al., 2007a[START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF][START_REF] Petersen | Ecological uptake and depuration of carbon nanotubes by Lumbriculus variegates[END_REF].

Toxicity

Toxicity (mainly growth inhibition) was clearly observed in Xenopus larvae exposed to 10 and 50 mg L 21 of DWNTs with or without GA according to the classical standardized test procedure of the amphibian micronucleus assay (ISO, 2006). Low mortality rate (5 and 15%) was observed at 10 mg L 21 and at the highest DWNTs concentration (50 mg L 21 ), respectively in larvae exposed without GA. Nevertheless, this mortality rate is not strong enough to be considered as significant. The toxicity observed in Xenopus larvae from 10 mg L 21 in both experiments is in agreement with previous results on Xenopus larvae exposed to the same type of DWNTs in static conditions [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF].

To explain the general toxicity observed in Xenopus larvae exposed to DWNTs, as already suggested in a previous study [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF], different hypothesis were proposed considering the different levels of observations in larvae (binocular, photonic, or electronic microscopy). The visual inspection of the larvae under the binocular after exposure to DWNTs, whatever the concentration, shows black material inside gill suggesting first that toxicity may be mediated by branchial obstruction generating gaseous exchanges perturbation and anoxia. Recently, other authors demonstrated the link existing between the presence of CNTs in water and the apparition of respiratory pathologies in aquatic organisms. For instance, [START_REF] Smith | Toxicity of single walled carbon nanotubes on rainbow trout (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other physiological effects[END_REF] showed that exposure of juvenile trout (Oncorhynchus mykiss) for up to 10 days to dispersed SWNTs (prepared in sodium dodecyl sulfate supported by a sonication step) caused respiratory toxicity (a dose-dependant rise in ventilation rate) and gill pathologies (edema, altered mucocytes, hyperplasia).

Moreover, in amphibian larvae, ''black masses'' were also observed into the intestine lumen after dissection, suggesting that toxicity may be also mediated by intestinal obstruction due to the DWNTs ingested from the water exposure. In the same way, some other authors also observed absorption of CNTs in intestine of organisms such as trout exposed to SWNTs [START_REF] Smith | Toxicity of single walled carbon nanotubes on rainbow trout (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other physiological effects[END_REF], daphnia exposed to coated SWNTs [START_REF] Roberts | In vivo biomodification of lipidcoated carbon nanotubes by Daphnia magna[END_REF], oligochaetes exposed to 14 C labeled SW and MWNTs [START_REF] Petersen | Ecological uptake and depuration of carbon nanotubes by Lumbriculus variegates[END_REF] and crustaceans exposed to raw and oxidized MWNTs [START_REF] Kennedy | Factors influencing the partitioning and toxicity of nanaotubes in the aquatic environment[END_REF] inducing different kind of toxicity, via inflammatory processes for example in trout [START_REF] Smith | Toxicity of single walled carbon nanotubes on rainbow trout (Oncorhynchus mykiss): Respiratory toxicity, organ pathologies, and other physiological effects[END_REF] or mortality and immobilization in crustacean [START_REF] Kennedy | Factors influencing the partitioning and toxicity of nanaotubes in the aquatic environment[END_REF]. In amphibian larvae, the microscopy observations of intestine cross sections in control and exposed larvae shows that black masses are clearly identifiable with the food intake in the lumen of the gut in exposed Xenopus larvae, whereas food is homogenously distributed into the lumen of nonexposed larvae suggesting thirdly a possible competition between DWNTs and nutritive compounds of the food. Nevertheless, neither histological preparations, nor the observation of the preparation for TEM observations revealed black masses in epithelial or in chorionic tissues of the instestin cutting. [START_REF] Petersen | Ecological uptake and depuration of carbon nanotubes by Lumbriculus variegates[END_REF] also observed that the detected 14 C labeled SWNTs and MWNTs were in association with sediments remaining in the gut of L. variegatus and were not readily adsorbed into organism tissues. In the present work, the Raman analysis carried out on the gut of exposed larvae confirms the presence of DWNTs in the lumen of the intestine, in agreement with the previous study [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF], but the abrupt vanishing of the signal of DWNTs [G' 2D band, Fig. 4(c)] in tissues allows to conclude to their absence in intestinal wall (villi and cells), suggesting that DWNTs do not cross the intestinal barrier. The Raman imaging technique is very sensitive and allows assessing without doubt that the intestine of CNT-exposed animals contains CNTs, as already suggested by the simple visual inspection and/or the TEM observation. Although this analysis seems to be necessary to confirm the presence of CNTs in organisms, only one other author, to our knowledge, carried out the Raman analysis to confirm that black material was indeed CNTs in the gut of D. magna [START_REF] Roberts | In vivo biomodification of lipidcoated carbon nanotubes by Daphnia magna[END_REF].

In the present work, the observed toxicity in larvae was globally the same with or without GA. Gum Arabic (GA), is exempt of demonstrated toxicity in in vivo studies in mammals [START_REF] Melnick | Chronic effects of agar, guar gum, gum arabic, locustbean gum, or tara gum in F344 rats and B6C3F 1 mice[END_REF][START_REF] Collins | Study of the teratogenic potential of gum arabic[END_REF]. The presence of GA can help to improve dispersion of CNTs. In absence of larvae, suspension of DWNTs was stabilized by the addition of GA. However, suspensions were rapidly destabilized in presence of the amphibians probably due both to the ingestion of GA by larvae and consequently to decrease in GA concentration in the water, as well as the modification of exposure conditions (pH). Similarly, [START_REF] Roberts | In vivo biomodification of lipidcoated carbon nanotubes by Daphnia magna[END_REF] have shown that D. magna exposed to SWNTs suspensions stabilized with a surfactant (lysophosphatidylcholine) were able to ingest the coated SWNTs through normal feeding behavior and to use the surfactant coating as a food source. Their study also provides some evidence of biomodification of a carbon-based nanomaterial by an aquatic organism. This is a very important point to be taken into account when dealing with ecotoxicity evaluation of CNTs in the environment, since CNTs which could end-up in the environment are likely to have been functionalized (deliberately or not) and/or to have adsorbed natural compounds such as natural sugars and organic matter etc., which are present into the aquatic compartment.

Genotoxicity

Only two studies concern genotoxicity of CNTs are available to our knowledge. Both are in vitro studies. [START_REF] Szendi | Lack of genotoxicity of carbon nanotubes in a pilot study[END_REF], using a pilot study in rat, show that oral exposure to SW and MWNTs did not increase urinary mutagenicity in rats as investigated using Ames test and that no genotoxicity effect was found using the in vitro micronucleus and sister chromatid exchange assays. [START_REF] Zhu | DNA damage induced by multiwalled carbon nanotubes in mouse embryonic stem cells[END_REF] found that MWNTs can accumulate and induce apoptosis in mouse ES cells and activate the tumor suppressor protein p53 within 2 h of exposure. Among the studies available on the impact of CNTs in vivo none focuses on genetic effects, and especially in aquatic organisms.

The present results indicate no genotoxicity via micronucleus induction in Xenopus larvae exposed for 12 days in presence of pristine DWNTs (without GA), whatever their concentration in the water. This result is in good agreement with the results of the previous studies on amphibian larvae (Xenopus and Axolotl) exposed to the same type of pristine DWNTs in suspension in water (Mouchet et al., 2007a[START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF]. Several hypotheses can be proposed to explain this lack of genotoxic response: (i) erythrocytes are not adequate or sensitive targets, (ii) micronuclei induction at high concentrations ([10 mg L 21 ) would be masked by the toxicity expression, (iii) micronuclei induction is not a relevant biomarker for CNTs, since micronuclei are non repairable mutation, (iv) CNTs are present as bundles instead of individual nanoparticles, and are thus too large to penetrate into the cells, and finally (v) CNTs used in both studies do not lead to genotoxicity in amphibians.

As indicated by the Raman analysis carried out on the gut of exposed Xenopus, and on circulating blood of Xenopus and Axoltl exposed to DWNTs (data not shown), DWNTs do not cross the intestinal barrier. These data support an indirect cytotoxic effect for DWNTs. Genotoxicity effects are usually expressed at subtoxic concentrations of the tested substance. Then, in this case, genotoxicity expression can be masked or limited by the expression of the cytotoxicity. In the present case, the mitotic index of intoxicated larvae exposed to 10 mg L 21 of DWNTs was reduced compared to the index in larvae exposed to lower CNTs concentrations and to the negative control (data not shown). [START_REF] Szendi | Lack of genotoxicity of carbon nanotubes in a pilot study[END_REF] have shown mitotic inhibition, a possible cytotoxic effect, in the human lymphocyte cultures upon treatment with SWNTs. Concerning the biomarkers, further investigations must be carried out since genetic damages such as oxidative stress was highlighted by some authors as a potential way of toxicity. For example, in the case of in vitro studies, the increase in intracellular reactive oxygen species (ROS) was explained by the metal traces associated with the commercial nanotubes [START_REF] Pulskamp | Carbon nanotubes show no sign of actute toxicity but induce intracellular reactive oxygen species in dependence on contaminants[END_REF]. In our experiments, the metals particles (Co) associated to the purified DWNTs used are supposed to be biologically inert [START_REF] Flahaut | CCVD Synthesis and Characterization of cobalt-encapsulated nanoparticles[END_REF] and could thus explain the negative genotoxic effects observed, if it is assumed that the potentially genotoxic effects observed are ROS mediated.

Nevertheless, the present results revealed genotoxicity in larvae exposed to 1 mg L 21 in presence of GA, whereas no genotoxicity was observed in larvae exposed to a lower (0.1 mg L 21 ) or a higher concentration (10 mg L 21 ). No genotoxicity was observed in larvae exposed to GA alone which is in agreement with experiments in mammals for which GA is exempt of evidenced toxicity [START_REF] Melnick | Chronic effects of agar, guar gum, gum arabic, locustbean gum, or tara gum in F344 rats and B6C3F 1 mice[END_REF][START_REF] Collins | Study of the teratogenic potential of gum arabic[END_REF]. GA is a natural polysaccharide stabilizer. It acts as a dispersant agent and can promote the bioavailability of CNTs to larvae. Maybe the agglomerates of CNTs are smaller and/or fewer in presence of GA. This concentration of 1 mg L 21 of DWNTs is the higher non toxic concentration and the first concentration at which toxicity would not mask genotoxicty. One other hypothesis would be in relation with the association of GA and DWNTs at 1 mg L 21 . Indeed, the results [Fig. 3(b)] show an increased MNE % (median value) in the case of larvae exposed to 1 mg L 21 of DWNTs in presence of GA even if there is no significant genotoxic response. Moreover, GA being a sugar, exposure to 50 mg L 21 (high concentration) could induce several metabolic disorders which may act in a synergetic way with DWNTs. Work is in progress to develop a dispersion protocol limiting the amount of added surfactant [START_REF] Datsyuk | Double-walled carbon nanotube dispersion via surfactant substitution[END_REF].

One can note that direct comparison of the present results with the literature must be carried out with care because toxic and genotoxic results are likely to depend on the administration route (peritoneal injection for rodent and water exposure for aquatic organism), exposure conditions of aquatic species in relation with their biology and physiology. The behavior of CNTs also depends on their intrinsic structure (number of walls, diameter, etc.) and synthesis route but also on their surface chemistry (pristine or functionalized), which plays an important role on their ability to form stable suspensions.

CONCLUSION

The present work evaluates the eco(geno) toxicity of DWNTs in amphibian Xenopus larvae in controlled laboratory conditions (ISO, 2006) according to two different endpoints after 12 days of exposure: toxicity and genotoxicity as the expression of the clastogenic and/or aneugenic effects observed in erythrocytes of the running blood. The results highlight the potential risk of the DWNTs used in this study, since (i) toxicity was observed in larvae exposed to DWNTs to 10 and 50 mg L 21 with or without GA and (ii) genotoxicity was observed in larvae exposed to 1 mg L 21 of DWNTs with GA. Even if DWNTs were evidenced in the larvae (gills and lumen) using microscopy method as already observed in previous study [START_REF] Mouchet | Characterisation and in vivo ecotoxicity evaluation of double-wall carbon nanotubes in larvae of the amphibian Xenopus laevis[END_REF], and Raman analysis confirmed their presence into the lumen of intestine, but not in intestinal cells suggesting that intestinal barrier is not crossed. Since DWNTs are ingested by the larvae, one can not exclude the possibility that DWNTs may be found later in the food chain, once released into the environment. Considering the increasing use of CNTs in commercial products, this study emphasizes fur-ther needs to study ecotoxicity of this nanomaterial and highlights that assessing the risks of the CNTs requires a better understanding of their toxicity, bioavailability and behavior into the environment.

Fig. 1 .

 1 Fig. 1. (a) FEG-SEM (field emission gun-scanning electron microscopy) and (b) TEM (transmission electron microscopy) images of raw CCVD (catalytic chemical vapor decomposition) sample. Both the inner and outer wall of the DWNTs are clearly visible on (b).

Fig. 3 .

 3 Fig. 3. Results of the micronucleus assay in larvae exposed according an half-static exposure to 0.1, 1, and 10 mg L 21 of DWNTs in experiment I (without GA, (a)) and II (with GA, (b)). NC: negative control, PC: positive control (cyclophosphamide, CP 20 mg L 21 ). Genotoxicity is expressed as the values of the medians (number of micronucleated erythrocytes per thousand, MNE %) and their 95% confidence limits. The 50 mg L 21 of DWNTs has not been evaluated because of the high acute toxicity. The hatched stick indicates a genotoxic response compare to the NC. (a) Experiment I without GA and (b) Experiment II with GA.

Fig. 2 .

 2 Fig. 2. Growth rate of Xenopus larvae exposed to 0.1, 1, 10, and 50 mg L 21 of DWNTs. (a) Experiment I without GA and (b) Experiment II with GA. ' '*' ' corresponds to a significant different size of larvae compared to the negative control group (mean value). NC: Negative Control; GAC: Gum Arabic Control (50 mg L 21 )

Fig. 4 .

 4 Fig. 4. (a) Compared Raman spectra of pristine DWNT and Xenipus intestine material; (b) Light microscopy image of an histological section of Xenopus intestine exposed to 100 mg L 21 of DWNTs ; (c) Horizontal line scan Raman analysis (see (a)) showing the variation of the intensity of DWNTs. (G' 2D ) * and Xenopus tissues ^. [Color figure can be viewed in the online issue, which is available at wileyonlinelibrary.com.]

TABLE I .

 I Results of acute toxicity in larvae exposed to 0.1, 1, 10, and 50 mg L 21 554 of DWNTs in experiment I (without GA) and II (with GA)

	DWNTs Concentrations (mg L 21 ) 2 GA
	Experiment I	NC		0.1 1	10	50
	Mortality	0%		0% 0%	5% 15%
	Visual inspection			-	-	*	***
	DWNTs concentrations (mg L 21 ) 1 GA (50 mg L 21 )
	Experiment II	NC GAC 0.1 1	10	50
	Mortality	0%	0%	0% 0%	0%	0%
	Visual inspection			-	-	*	***
	in experiment II [

''-'': No sign of acute toxicity compared to the negative control group (visual inspection). ''*'': reduced or stopped size, anaemia signs compared to the negative control group (visual inspection). The number of asterisks is function of the intensity of increasing effects. NC: Negative Control; GAC: Gum Arabic Control (50 mg L 21 558 of GA alone).

The authors acknowledge Elodie Sarremejean and Guillaume Bernard for their technical help, Pr A. Peigney of the CIRIMAT for SEM characterization of pristine DWNTs and Georges Merlina of EcoLab for fruitful discussion.