M Souley

A Thoraval

Nonlinear mechanical and poromechanical analyses: comparison with analytical solutions

Keywords: Nonlinear behavior, elastoplasticity, hydromechanical coupling, code verification, analytical solutions

The validation of computational schemes for modeling multiphysic phenomena in porous geomaterials is a key importance in geoenvironmental applications. This paper focuses on the validation of nonlinear and coupled hydromechanical schemes by comparing the results of numerical predictions performed with COMSOL and the mathematical solutions. Firstly, we simulate the nonlinear mechanical behavior of rock mass around a deep cylindrical hole. The second example treats on the simulation of the well-known one-dimensional consolidation of a saturated porous rock column. Finally a validation of poroelastic calculations related to a cylindrical hole excavation in a saturated porous media is achieved.

Introduction

Waste storage problems, as well as the natural resource management (water, gas or hydrocarbons), explain the increased interest of the scientific community to study the multiphysic behavior of rock masses. Typically, the long-term behavior of the underground excavations is a social and economic challenge particularly in the contexts of post-mining or radioactive waste storage. Despite the constraints imposed by geological considerations, attention has been directed by developing mathematical and numerical models of the thermo-hydro-mechanical behavior of saturated geological media [START_REF] Tsang | Coupled Processes Associeated with Nuclear Waste Repositories[END_REF][START_REF] Tsang | Coupled thermo-mechanical hydro-chemical processes in rock fractures[END_REF][START_REF] Bear | Flow and Contaminant transport in Fractured Rock[END_REF][START_REF] Selvadurai | Computational modelling of isothermal consolidation of fractured media[END_REF][START_REF] Jing | DEOCOVALEX -An international cooperative research project on mathematical models of coupled THM processes for safety analysis of radioactive waste repositories[END_REF], Stephanson et al. 1996, Rutquist et al. 2001). Those numerical modelings are currently used to understand and forecast the complex behavior of the rock mass around the underground cavities. In order to accurately perform these multiphysic modelings at large space and time scales, it is necessary to check the capacity of these codes, to precisely reproduce the correct response in the case of some problems for which analytical solutions exist in the field of geomechanics. This is an important step before evaluating the ability of the code to correctly reproduce laboratory or in situ measurements or specific experiments.

This paper focuses on comparing the results of numerical predictions with mathematical solutions. The code COMSOL is used in the framework of nonlinear mechanics and hydromechanical couplings of saturated porous media through three verification cases.

The first is focused on the nonlinear mechanical behavior. The second concerns the hydromechanical modeling of the onedimensional consolidation, where a column of fluid-saturated porous medium of finite length is subjected to a surface load. The third case involves the excavation of a deep cylindrical hole in a saturated porous rock and subjected to an anisotropic stress field.

Theoretical background and COMSOL implementation

The classical dealing on the process of consolidation in one-dimension inducing isothermal fluid flow through a deformable porous medium was firstly addressed by Terzaghi (1925) and later developed and generalized by [START_REF] Biot | General theory of threedimensional consolidation[END_REF] in three dimensions. The theory assumes linear elasticitity to characterize the mechanical behavior of the porous solid and Darc's law for fluid flow through the porous medium. Within the framework of an extension to poroplasticity, [START_REF] Coussy | Mechanics of porous continua[END_REF] has shown the existence of a plastic effective stress when the volumetric plastic strain ( v p ) and plastic porosity ( p ) are related by a linear form. This plastic effective stress is, in general case, different to the elastic ones and results in defining a plastic Biot coefficient (scalar or tensor).

The constitutive equations (equilibrium equation, mechanical constitutive behavior, law of effective stresses according to Biot's concept and fluid diffusivity equation which combines Darcy's law and fluid mass conservation) are expressed in a general way as follows: [START_REF] Bear | Flow and Contaminant transport in Fractured Rock[END_REF] where , ( 0 , 0 ) are total and effective The yield function defines the stress combination for which plastic flow takes place. These functions are represented by one or more limiting surfaces in a generalized stress space with points below or on the surface being characterized by an incremental elastic or plastic behavior, respectively. The flow rule specifies the direction of the plastic strain increment vector as that normal to the potential surface: it is called associated if the potential and yield functions coincide FG , and non-associated otherwise.

0 0 0 . und f d p int f f f f m f = C b p p I tr pk S p b Q tt gg g
The implementation of elastoplastic material in COMSOL is based on the additive decomposition of total strain (assumption of small strains). Thus, the stress-strain relationship is given by the second equation of system [START_REF] Bear | Flow and Contaminant transport in Fractured Rock[END_REF]. The plastic strain tensor p and a vector of state parameters describe the state of the plastic strain. The evolution of these variables is governed by the following rate equations:

, , , , and

p p p G G [2]
where is the plastic multiplier determined by the complementary conditions (also called consistency rule): Mohr-Coulomb or Hoek and Brown criterions generally used in geomechanics present the disadvantage of being expressed according to the extreme stresses (minor and major: 3 , 1 ). This leads to difficulties related to their numerical implementation in the case of complex stress paths (particularly in the vicinity of corners).

, , 0 0 , , 0 , , 0 0 , , 0 ,
To avoid that, it is better to use criteria which utilize the three principal stresses. For instance, the Drucker-Prager shear criterion is represented in the principal stress space (σ 1 , σ 2 , σ 3 ) by a cone with axis along the octahedric line (σ 1 = σ 2 = σ 3 ). The Mohr-Coulomb shear criterion, characterized by the cohesion, C, and friction angle, φ, is represented by an irregular hexagonal pyramid. The parameters q φ and k φ (intervening in the Drucker-Prager criterion) can be adjusted so that the Drucker-Prager cone will either pass through the outer or the inner edges of the Mohr-Coulomb pyramid.

For instance, the inner adjustment allows expressing the Drucker-Prager yield function as follows: m m sin C cos F F p q q q k q sin sin [START_REF] Detournay | Poroelastic Response of a Borehole in a Non-Hydrostatic Stress Field[END_REF] where q is the deviatoric (von Mises) stress; m is the mean stress /3 tr . Finally, for seek of simplicity we consider a standard material (G = F). Also, in order to compare to analytical solution, we assume an elastic perfectly plastic behavior.

Nonlinear mechanical validation

Firstly, the problem concerns the determination of stresses and displacements distribution around a cylindrical hole in an infinite elastoplastic medium subjected to initial stresses. The medium is assumed to be linearly elastic, perfectly plastic, with a failure surface defined by the Drucker-Prager criterion (Eq. 4) with the associated flow rules leading to a maximum of material dilatancy.

An isotropic in situ stress state, 0 , exists with stresses equal to -13.7 MPa (compressive stresses are negative). It is assumed that the problem is symmetric with respect to both the horizontal and vertical axes. The hole radius , r 0 , is 3 m and is assumed to be small compared to the length of the cylinder. This permits the use of the plane-strain condition, even if the problem is, in this case, 1D and then could be solved with axisymmetric configuration. The geometrical model and boundary conditions used in COMSOL are shown on figure 9 without the hydraulic ones. The input parameters are : E 0 (Young modulus) = 5800 MPa; 0 (Poisson ratio) = 0.3; C = 1 MPa and φ = 25°.

When the failure surface is defined by the Mohr-Coulomb criterion, the analytical solution for this problem is provided in terms of stresses and displacements by [START_REF] Salencon | Contraction Quasi-Statique D'une Cavité a Symétrie Sphérique Ou Cylindrique Dans Un Milieu Elastoplastique[END_REF] for both associated plastic flow and non-associated plastic flow. The yield zone radius (or plastic radius), r p , is given analytically by:

1 1 0 0 1 2 1 1 p K q p p q p p C K rr C K pi K [5]
where p i is the internal pressure at the hole wall (considered as null here); .

The author also proposes the distribution of radial and orthoradial stresses as well as radial displacement in the two domains: plastic (r r p ) and elastic (r>r p ), where r is the distance from the hole center. For more details, the reader could consult Salençon (1969).

Figures 1 and2 show a direct comparison between COMSOL results and the analytical solution along a radial line. The small differences between the numerical and analytical results is essentially related to numerical scheme: (a) use of the inner cone to adjust the Mohr-Coulomb pyramid, and (b) use of the three principal stresses rather than the two extreme stresses.

In a second step, we consider now an anisotropic stress state of: x0 = -16.1 MPa; y0 = -12.7 MPa; a0 = -12.4 MPa, leading to an averaged mean stress of -13.7 MPa.

In this case, there is no analytical solution in the literature. We will thus compare the numerical results with the previous analytical solution by assuming an isotropic stress equal to a mean stress of -13.7 MPa. Figure 4 shows a direct comparison between COMSOL results (in terms of radial displacement along horizontal and vertical lines) and the analytical solution assuming an isotropic initial stress state of -13.7 MPa. It is interesting to note that the radial displacement obtained from the analytical solution is the average of the predicted radial displacements in the horizontal and vertical directions.

Figure 5 illustrates the variation of tangential and radial stresses. The evolution of the predicted curves is qualitatively in adequacy with that we already analytically discuss.

Poromechanical validation 4.1 One dimensional consolidation

We consider the problem of a onedimensional column of 20 m height of a saturated poroelastic medium. Initially, stresses and pressure are assumed to be equal to zero. No displacements are assumed on boundaries except on the top limit where a constant surface load, z0 = 10 5 Pa, is applied. During phase 1, no flux boundaries are applied (undrained condition). During phase 2, the impervious boundary condition at the top is replaced by a zero pressure condition (drained condition).

The soil matrix is homogeneous and behaves elastically; the isotropic Darcy's transport law is assumed. The applied pressure is initially carried by the fluid (during phase 1) but, as time goes on (during phase 2), the fluid drains through the top of layer surface, transferring the load to the soil matrix. The solution to this one-dimensional consolidation problem is derived in the framework of Biot theory by solving Eq. 1. [START_REF] Detournay | Fundamentals of Poroelasticity[END_REF].

The pore pressure value, p 0 , reached at the end of phase 1, can be derived from the fluid constitutive law considering undrained conditions and using the one-dimensional mechanical constitutive law, say finally the Skempton's equation:

0 0 2 0 4 / 3 z bM p K G b M [6]
where K 0 is the drained bulk modulus; G is the shear modulus; M is the Biot's modulus. The closed-form solution from Detournay and Cheng (1993) is expressed in terms of vertical displacement (or subsidence) u z and pore pressure as follows: For the numerical application, we consider a layer of soil of large lateral extent, and thickness L = 20 m. The drained Young modulus E 0 is 3435 MPa, and the Poisson coefficient 0 is 0.12. The density of the dry rock ρ is 2750 kg/m 3 , the density of water ρ f is 1000 kg/m 3 , the water bulk modulus K f is 2000 MPa, the intrinsic permeability k int is 5 10 -20 m 2 . The Biot modulus, M, and coefficient, b, are 5112 MPa and 0.6, respectively. Also, gravity is set to 9.81 m/s 2 .

The contours of subsidence (vertical displacement) at selected fluid-flow time corresponding to 10 10 s (or ~317 years) are plotted in figure 6. A maximum of 0.56 mm is reached at the top. The numerical solution for pore pressure and vertical displacement, along the column are evaluated and compared to the analytical solution at four different times (10 7 , 10 8 , 10 9 and 10 10 s). The results are plotted versus the distance, d, from the top of surface (corresponding to d = 0).

The simulation results are in very good agreement with the analytical solution for pore water pressure dissipation (figure 7), timedependent surface displacement (figure 8), and also stresses (not presented herein).

Poroelastic response of a borehole

A borehole is excavated in a saturated porous rock subject to an anisotropic in situ stress field. The borehole boundary is free to drain and is exposed to atmospheric pressure. The initial pore pressure field is p 0 . The problem is analyzed assuming plane-strain conditions and instantaneous drilling of the borehole. The numerical simulation allowed to capture the poroelastic effects taking place during the short time response of the system. The initial and boundary conditions (mechanic and hydraulic) are summarized in figure 9. The hydromechanical parameters are the same than in the previous section. The two-dimensional poroelastic solution for a borehole in an anisotropic stress field can be found in [START_REF] Detournay | Poroelastic Response of a Borehole in a Non-Hydrostatic Stress Field[END_REF]. The general solution is derived in the Laplace transform domain and results in the time domain can be obtained using a numerical inversion technique. The solution is formulated by superposition of asymptotic solutions for three loading modes: (1) a far-field isotropic stress: this loading mode produces the classical Lamé solution in elasticity (known also as Kirsh solution); (2) an initial pore pressure distribution that induces short-time asymptotic solution for stresses, pore pressure and displacements; and (3) a far-field stress deviator. The radial, tangential and shear stresses (σ r , σ θ , σ rθ ) in polar coordinates, pore pressure (p), and radial and tangential displacements (u r , u θ ) induced by each loading mode are formulated. The equations relating to the fundamental solutions of these 3 load patterns can be consulted in [START_REF] Detournay | Poroelastic Response of a Borehole in a Non-Hydrostatic Stress Field[END_REF].

Due to the instantaneous undrained response in an anisotropic initial stress field, over-pressures develop in the direction of the initial minor stress, and under-pressures in the direction of the initial major stress, in accordance with the analytical solution. Indeed in relation to the anisotropy of the initial stress state, the instantaneous response induced a variation (increase and reduction, respectively in the horizontal and vertical directions) of the mean stress, which in return by Skempton effect, involves a variation (positive and negative) of the pore pressure. It is not the case when the initial stress state is isotropic. Figure 10 presents the contours of pore pressure after t = 10 6 s of drainage. Even at this time, one can notice the presence of these over-pressures (not yet dissipated) in the horizontal direction and under-pressures in the vertical direction inside the solid mass. Finally, the contours of total displacement resulting to the coupled hydro-mechanical effects are illustrated in figure 11 at t = 10 6 s. Isochrones of pore pressure variation with distance from the hole wall are compared to the analytical solution in figure 12 for t = 10 6 s, and the direction θ = 0 and 90° (with respect to the x-axis). The steep radial gradient of pore pressure developing at the borehole wall is illustrated in this figure. This gradient is associated with a rapid drainage of fluid near the borehole which influences the apparent stiffness properties of the rock.

The radial displacement versus the distance from the hole wall at θ = 0 and 90° is compared to the analytical solution in figure 13 for a simulation time of 10 6 s. Figure 14 summarizes the profiles of radial and orthoradial stresses between numerical and analytical solution.

From these figures, we can notice that the results of the COMSOL simulations show a very good agreement with the analytical predictions.

Concluding remarks

Numerical modeling are currently used to understand and forecast the complex rock mass behavior around the underground structures where several physics and their couplings are present. In order to accurately perform these multiphysic modelings at large scales, it is necessary to check the capacity of these codes, to correctly reproduce the phenomena through some simple stress load paths. This paper presents this exercise of validation where numerical simulations were performed with COMSOL First, we simulate the nonlinear mechanical behavior (elastoplasticity) of rock mass around a deep cylindrical hole and successfully compare with the analytical solution (Salençon 1969).

The second example treats on the simulation of one-dimensional consolidation of a column of saturated porous rock in the framework of poroelasticity. The simulation results are in very good agreement with the analytical solution [START_REF] Detournay | Fundamentals of Poroelasticity[END_REF] in terms of pore pressure dissipation, time-dependent subsidence and stresses.

Thirdly, a borehole is excavated in a saturated porous rock subject to an anisotropic in situ stress field. Here also, the fields of displacement, pore pressure and stresses numerically predicted are successfully compared with those obtained by the semianalytical solution proposed [START_REF] Detournay | Poroelastic Response of a Borehole in a Non-Hydrostatic Stress Field[END_REF].

Next stage of this work concerns in particular the following applications: (a) study of water effects on the stability of slopes and underground cavities, (b) dimensioning of CO 2 storage sites. These applications require complementary developments (such as poroplasticity of saturated and unsaturared porous media) which will have to also be validated.

Figure 1 .

 1 Figure 1. Radial displacement along a radial line.

Figure 2 .

 2 Figure 2. Radial and tangential stresses along a radial line.

Figure 3

 3 Figure 3 represents the contours of the von Mises deviatoric stress. In relation to the development of plastic zone (resulting from stress corrections) the maximum deviatoric stress is reached at the interface between elastic and plastic domains.Figure4shows a direct comparison between COMSOL results (in terms of radial displacement along horizontal and vertical lines) and the analytical solution assuming an isotropic initial stress state of -13.7 MPa. It is interesting to note that the radial displacement obtained from the analytical solution is the average of the predicted radial displacements in the horizontal and vertical directions.Figure5illustrates the variation of tangential and radial stresses. The evolution of

Figure 3 .

 3 Figure 3. Contours of von Mises stress : anisotropic initial stresses.

Figure 4 .

 4 Figure 4. Radial displacement along horizontal ( =0°) and vertical ( =90°) lines compared to mean analytical solution : anisotropic initial stresses.

Figure 5 .

 5 Figure 5. Tangential and radial stresses along horizontal ( =0°) and vertical ( =90°) lines compared to mean analytical solution: anisotropic initial stresses.

2 ˆ

 2 , is the fluid diffusivité [m /s].

Figure 6 .

 6 Figure 6. Contours of cummulative subsidence at the end of phase 2 (at t = 10 10 s).

Figure 7 .

 7 Figure 7. Profile of pore pressure as a function of distance from the top surface for flow time ranged between 10 7 and 10 10 s.

Figure 8 .

 8 Figure 8. Profile of subsidence as a function of distance from the top surface for flow time ranged between 10 7 and 10 10 s.

Figure 9 .

 9 Figure 9. COMSOL model : geometry and boundary conditions of 2D consolidation.

Figure 10 .

 10 Figure 10. COMSOL model : contours of pore pressure for 2D consolidation (t=10 6 s).

Figure 11 .

 11 Figure 11. COMSOL model : contours of total displacement for 2D consolidation (t=10 6 s).

Figure 12 .

 12 Figure 12. Pore pressure variation with the distance from the hole wall at = 0° and 90° (t = 10 6 s).

Figure 13 .

 13 Figure 13. Radial displacement variation with the distance from the hole wall at = 0° and 90° (t = 106 s).

Figure 14 .

 14 Figure 14. Radial and tangential stresses variation with the distance from the hole wall at = 0° and 90° (t = 10 6 s).


C Rollin

As transport characterization from the soil to the groundwater: an original laboratory study

Keywords: arsenic transport, laboratory column, soil

Heavy metals are major soil pollutants since a lot of former industrial soils are polluted by these contaminants. In the context of risk assessment of contaminated sites, they are of particular concern because of their toxicity toward human beings. Nevertheless, the vadose zone of the soil is not taken into consideration in this kind of study, although this is where the pollution enters the soil. That is why mechanisms responsible for trace element release in the unsaturated zone of the soil have to be understood. In addition, tools and methods to measure and put forward interactions between contaminants and the solid matrix have to be provided. Therefore, we selected a soil from a wood preserving facility site (contaminated by As) and decided to follow As release as it was the only trace element measured on site at a concentration in groundwater above the EU guidelines for drinking water quality. We designed an original laboratory set up: two columns were filled with 2 sub-samples of the As contaminated soil (202 and 253 mg/kg) and As release was studied as it could occur on site. Two main phenomena were simulated during these experiments: rain water infiltration and an increase in the water table level. After the results of the two experiments, it was shown that As concentrations at the outlet of the two columns were constant over time. Given that and the results of the sequential extraction carried out on this soil, As release could mainly occur from the soluble and exchangeable parts. This will be modeled in a future work in order to confirm this hypothesis.

Introduction

In industrialized countries, a lot of brownfields polluted with trace metals have been identified. In France, site management strategies are based on a site specific approach. This corresponds to a combined approach between field investigations and modeling to understand and predict pollutant migration through the soil. In these studies the chemistry and hydrology of the vadose zone of the soil are not considered although the pollutants are entering the soil from the unsaturated zone. That is why it seems necessary to have a good knowledge of the mechanisms responsible for heavy metal retention and transfer from the vadose zone of the soil to the water table, in order to put forward potential risks for groundwater. In the case of trace metals, mechanisms controlling their mobility in soils are relatively well known but the predominance of one over another is still unclear. Most of the time, pollutant transport is evaluated at the laboratory scale with batch or column experiments. Nevertheless, these systems are of small dimensions, the soil used is generally sieved, and these studies are mainly carried out under saturated flow conditions (Michel, 2009). We therefore may wonder whether these conditions are representative of what occurs in the field. That is why experiments at a pilot scale are necessary to assess heavy metal transport at the field scale. But these kinds of experiments are rather time consuming and quite expensive, and as a consequence cannot be used when doing risk assessment studies. Therefore, the aim of this work was to design an original laboratory experiment able to represent trace metal transport as it occurred on site. This study focused on As release as it was the trace element released on site at concentrations above the EU guidelines for drinking water quality. This will help us in putting forward mechanisms responsible for As migration in this soil.

Materials and methods

Polluted soil sample

This work was carried out in the frame of the CAPHEINE project (Characterization of trace element transfer in the vadose zone of the soil), funded by ADEME (French Environment and Energy Management Agency). The polluted soil was sampled on an in-service wood preserving facility site, located in the northeast of France. The wood preserving activity for phone and power line poles started in 1929. Different preserving techniques were used so far, meaning that different kinds of chemicals were applied and may have contaminated soil and groundwater. The main chemicals used were chromated copper arsenate compounds (CCA) and creosote distillates. Soil samples were taken between 0 and 6 m depth, where the main pollution source was supposed to be localized. The surface of this area was 100 m 2 (8 m x 12 m) and soil was sampled with a helical auger. The soil was analyzed with a portable X-ray fluorescence analyzer. Arsenic was detected between 2.5 and 3 m depth, that is why 21 kg of soils were collected at this depth with a helical auger, placed in plastic bags and stored in a cooler before being analyzed in the laboratory. These samples were homogenized by quartering and separated in 2 sub-samples (S1 and S2). A small quantity of each sub-sample was dried, sieved, grinded and submitted to a sequential and a total extraction. As distribution in these soils was assessed using a slightly modified sequential extraction according to the Tessier protocol [START_REF] Tessier | Sequential extraction procedure for the speciation of particulate trace metals[END_REF]. This modified protocol was published by Denys [START_REF] Denys | Bioaccessibility, solid phase distribution, and speciation of Sb in soils and in digestive fluids[END_REF]. These chemical extractions were done on three replicates. Total concentrations for As were 202 and 253 mg/kg for S1 and S2, respectively. As was found in soluble and exchangeable form (about 8 %) and mainly bound to manganese and iron oxides (more than 50 %). In addition, soil physico-chemical parameters such as grain size distribution, pH, TOC, CEC, and total trace element content were determined on each fraction.

Experimental set up

Two laboratory columns were used in this study. They were all the same size and were made of transparent PVC and composed of 3 parts (Figure 1). The middle part was filled with 5.4 kg of As polluted soil. This part was 30 cm high and 12 cm in diameter. The lower part of the set up was composed of a funnel.

Figure 1. Laboratory column.

Experimental strategy

Two kinds of experiments were conducted with two columns. The first one was an infiltration column (C1) and was filled with the sub-sample S1. Cycles of rain water infiltration and drying periods were simulated: DI water was applied at the top of the column thanks to a peristaltic pump, and samples were collected at the bottom. Note that between the upper and middle parts, a plate was sealed; it was composed of holes, and pipes were set in these holes, so that the soil in the middle part was homogeneously fed. The water fluxes were controlled and adjusted to the fluxes recorded on site. The influence of the flowrate (between 0.04 and 0.25 mL/min), the feed duration (between 2 and 10 days), and the drying period (between 6 and 90 days) was studied. As the flowrate was very low, the column ran under unsaturated conditions. The second column (C2) was filled with sub-sample S2 and saturated upward with calcium nitrate (2.5 10 -3 M) thanks to a peristaltic pump. The flowrate varied between 0.02 and 0.1 mL/min and was adjusted to the water table level increase speed measured on site. When the soil column was fully saturated, the flow was stopped, the pump was disconnected and water was collected at the bottom of the column. Several cycles were applied to the column and the flowrate influence on As release was studied. pH, conductivity, redox potential, TOC and As content were measured at the outlet of both columns.

Results and discussion

At the outlet of C1, As concentrations were constant over time and were mainly around 3 000 µg/L. No influence of the flowrate, the feed duration and the drying period on As release was noticed. The same trend was observed for C2: As concentrations at the outlet of the column did not show any particular evolution over time and were mainly around 2000 µg/L, with no influence of the flowrate. These results indicated that As release in this soil was not rate limited. Therefore, we could hypothesize that it resulted from a thermodynamic equilibrium and that As could mainly come from the soluble and exchangeable parts.

Conclusions

In this work, As release was studied at the laboratory scale. An original laboratory column helps us to identify phenomenon responsible for As release in the unsaturated zone of the soil. In a future work As release in these soil columns will be modelled in order to confirm the hypothesis of As coming from soluble and exchangeable parts. This study paves the way for a better understanding of trace element behavior in the vadose zone of the soil during risk assessment studies and gives a new approach to study this part of the soil.


C D Barillot

C O Sarde

E Tarnaud

N Cochet

V Bert

Phytoextraction process optimization: characterization of the soil bacteria flora associated to the hyperaccumulator Arabidopsis halleri

Keywords: Arabidopsis halleri, bacteria diversity, bioaugmentation, phytoextraction, PGPB, trace elements

Phytotechnologies are microbial-assisted techniques that use living plants for the treatment of contaminated sites. Among these, phytoextraction based on hyperaccumulator plants such as Arabidopsis halleri, may be an option to remove trace elements in soil. One method to optimize Arabidopsis halleri's phytoextraction is the inoculation of plant roots by plant growth promoting bacteria (PGPB). In this study, we analyzed the total bacterial diversity in a Zn and Cd contaminated industrial soil (Auby, France) with restriction fragment length polymorphism (RFLP). Preliminary results showed a large bacterial diversity in Auby's soil. Cultivable bacteria were isolated and characterized for their PGPB traits (ACC deaminase activity, siderophores and Indol-Acetic-Acid (IAA) productions). Bacteria that will answer positively to the three PGPB tests will be preferentially selected, and inoculated to Arabidopsis halleri roots. Bacteria effects on plant biomass and/or accumulating yield will be presented.

Introduction

Phytoextraction, a microbial-assisted plant technology usable for the treatment of contaminated sites, exploits natural physiological traits of certain plants able to accumulate high levels of metals in their aerial tissues. The success of phytoextraction is strongly determined by the amount of plant biomass and the concentration of metals in plant tissues. Arabidopsis halleri, a hyperaccumulator plant characterized by its exceptional Zn and Cd concentrations in its aerial parts, has a relatively small biomass, limiting its use for phytoextraction process. However, some soil microorganisms described in literature, such as PGPB, are able to significantly increase plant biomass and/or metal uptake through the secretion of specific compounds such as organic acids, siderophores and/or phytohormones as well as through enzymatic activities such as ACC deaminase [START_REF] Zhuang | New advances in plant growth-promoting rhizobacteria for bioremediation[END_REF][START_REF] Lebeau | Performance of bioaugmentation-assisted phytoextraction applied to metal contaminated soils: A review[END_REF]. Therefore, rhizospheric microorganisms represent a potential valuable tool for the improvement of A. halleri mediated phytoextraction. The aim of this work is to study the microbial diversity associated to the roots of Arabidopsis halleri growing on a Zn and Cd polluted soil by RFLP and to identify PGPB strains that may be potentially involved in plant biomass production and/or plant Zn and Cd accumulation. The effect on selected PGPB bacteria on the A. halleri's phytoextraction process will be studied.

Materials and methods

Three different soil samples (A, B and C) were collected from an industrial site contaminated with Zn and Cd located in Auby (59, France). Each sample was chosen to reflect a different degree of soil contamination (ranging from 2700 ppm to 36 000 ppm Zn of FW polluted soil; Niton analyser). For each sample, three sub-fractions of soil were defined and collected independently relatively to their distance to A. halleri's roots (global, rhizospheric and rhizoplan soils, respectively). Total DNA extraction was performed for the nine bacterial samples using Power soil DNA kit (MoBio industries). A 1300 bp fragment of the 16S rDNA gene was amplified by PCR. Generated amplicons were ligated in pGEMt Easy cloning vector (Promega) to generate specific minilibraries. For each sample, 100 clones were analysed by RFLP with Rsa I restriction enzyme (Invitrogen). Sequences generated were compared using the BLAST algorithm in Ribosomal Database Project. Cultivable bacteria were spread on LB-agar plates to estimate global bacterial density. Individual clones were tested for the presence of PGPB traits (ACC deaminase activity, IAA and siderophore productions). Positive PGPB for the three criteria will be preferentially selected (in progress). Arabidopsis halleri seedlings will be further inoculated with selected PGPB to see the potential effect on either biomass and/or metal accumulation. Plant growth as well as accumulation will be monitored regularly.

Results

RFLP profiles exhibited a large diversity in the nine soil samples analysed (Figure 1). No significant differences in visual profile distributions could be detected according to soil pollution level or root distance. In order to confirm this result, 50 clones issued from different samples and presenting a RFLP profile resemblance were sequenced, assuming that they may belong to the same OTU. Indeed, among 50 sequences, some could be assigned to an identical genus: Flavobacterium (8), Aquicella (7), Pseudomonas (3), Rhodoplanes (3). However, sequence identities observed were 95% in average and none of the clone but two belonged to the same species. A sample of each clone library is actually sequenced and analysed. Bacterial densities were estimated after spreading on LB-agar medium 100 µl of diluted bacterial suspension and after 72 h of incubation at 25°C. The results are expressed in log CFU per gram of soil dry weight. For each soil sample a significant increased of bacterial density was observed when the root distance decreased: G< R< RP. The degree of pollution seemed also to influence significantly (p<0.05). The density of resident flora, following a gradient A>B>C (Figure 2).

Discussion

RFLP analysis showed a large diversity of the total flora present in Zn and Cd contaminated soils. RsaI restriction analysis alone was sufficient to efficiently distinguish the clones at species level. This was confirmed when sequencing the clones presenting the closest RFLP profile. The excellent discrimination power of RsaI on 16S amplicon is in good agreement with data published by [START_REF] Moyer | A computer-simulated restriction fragment length polymorphism analysis of bacterial small-subunit rRNA genes: Efficacy of selected tetrameric restriction enzymes for studies of microbial diversit in nature[END_REF], who recommanded RsaI as one of the best enzyme for RFLP analysis. Considering the high diversity observed even on the most polluted soil, it would be tempting to conclude that pollution seems to have little impact on endogenous bacteria populations. However, since a clear restriction of cultivable flora diversity was observed when pollution increased, this may not be true. We were not able to identify predominant species, cultivable or not, in examinated soil. This can be explained by the fact that heavy metals are neither metabolized nor used by endogenous strains, as it is the case for organic pollutants [START_REF] Taok | Monitoring the impact of hdrocarbon contamination and nutrient addition on microbial density, activity, and diversity in soil[END_REF]. However, if no dominant species were found, some uncultivable genus such as the recently identified genus Aquicella [START_REF] Santos | Gamma-proteobacteria Aquicella lusitana gen. nov., sp. Nov., and Aquicella siphonis sp. Nov. infect protozoa and require activated charcoal for growth in laboratory media[END_REF] seems to be more present. A higher cultivable bacterial density was also observed around the roots in all soils. This observation is in accordance with other studies and is generally explained by the root exudates that create a favorable microenvironment for bacterial survival and growth. On another hand, preliminary results obtained for classical PGPB tests allowed us to identify bacteria that are able to produce IAA, siderophores and/or presenting a significant ACC deaminase activity. Some of these bacteria will be tested in a bioaugmentation experiment to see their potential impact on phytoextraction. The presence of bacteria harboring PGPB traits, together with the higher cultivable density found in close vicinity of the roots is in favor of regular reciprocal exchanges between plants and microflora.

Figure 1 :

 1 Figure 1: Seventeen RFLP profiles obtained with RSAI restriction enzyme for sample A, global fraction of soil (AG) and revealed with Sybr Green stain I (Sigma).

Figure 2 :

 2 Figure 2: Estimation of bacterial densities for each soil A, B, and C split in global (G: white), rhizospheric (R: grey) and rhizoplan (RP: black) soils. The number of CFU data were analysed by analysis of variance (two-ways ANOVA). For each sample, values with different small letters are significantly different (p<0.05). Values with different capital letters are significantly different (p<0.05) (means +/-SD). The LB-agar plates allowed us to isolate 240 bacteria strains that presented different morphological aspects. PGPB tests such as activity ACC deaminase, siderophores and acid indolacetic-3 productions, were performed.


G Uzu

J J Sauvain

A Baeza

M Riediker

M Sánchez

S Hohl

S Val

K Tack

S Denys

P Pradère

C Dumat

Pulmonary and gastric lead burden assessment for lead-recycling plant workers

Keywords: bioaccessibility, DTT, lead, PM, oxidative stress, toxicity

As a function of PM size (PM 10-2.5 , PM 2.5-1 and PM 1 ) and origin (furnace, refining and channeled emissions), lead toxicity and bioaccessibility assessment were performed in a lead recycling plant using complementary biologic and chemical acellular tests. According to their origin, process PM displays differences in metal contents, granulometry and percentage of inhalable fraction. Lead gastric bioaccessibility was relatively low (maximum 25%) in comparison with previous available studies, though due to their high total lead concentrations, significant metal quantities could reach the circulatory system. Whatever their origin, finest PM 1 particles induced the greatest pro-inflammatory response from human bronchial epithelial cells. Moreover, this biological response was correlated with the acellular DiThioThreitol (DTT) assay, suggesting some biological predictive value for such acellular test.

Introduction

Among numerous epidemiological studies showing that atmospheric particulate matter (PM) increases respiratory and cardiovascular pathologies in urban areas, many deal with PM from transport. But, the fate and effects of PM from industry enriched with metals and emitted in the atmosphere, focused in the present work, was poorly studied [START_REF] Uzu | Characterization of lead-recycling facility emissions at various workplaces: major insights for sanitary risks assessment[END_REF]. PM of different size (PM 10-2.5 , PM 2.5-1 and PM 1 ) and origin (furnace, refining and channeled emissions), lead toxicity and bioaccessibility assessment was performed in a lead recycling plant using complementary biological and chemical acellular tests.

Materials and Methods

Various size fractions of PM were separated and characterized according to [START_REF] Uzu | Study of lead phytoavailability for atmospheric industrial micronic and sub-micronic particles in relation with lead speciation[END_REF][START_REF] Uzu | Foliar Lead Uptake by Lettuce Exposed to Atmospheric Fallouts[END_REF][START_REF] Uzu | Characterization of lead-recycling facility emissions at various workplaces: major insights for sanitary risks assessment[END_REF]. Size distribution (laser granulometry), total lead concentration (ICP-OES after acid digestion) and reactivity were studied. Bioacessibility was measured according to Unified Barge Method (UBM) [START_REF] Cave | Preliminary assessment of a unified bioaccessibility method for Arsenic in soils[END_REF]. DTT Assay was performed according to [START_REF] Sauvain | Nanoparticle reactivity toward dithiothreitol[END_REF]. In vitro assays of cytotoxicity and inflammation were performed according to [START_REF] Val | Carbon black and titanium dioxide nanoparticles induce proinflammatory responses in bronchial epithelial cells: Need for multiparametric evaluation due to adsorption artifacts[END_REF].

Results and Discussion

As determined by their origin, process PM presented differences in metal contents, granulometry and percentage of inhalable fraction. Percentages of soluble lead in gastric fluids (pH~2) ranged from 10 to 16% for PM tot and 15 to 23% for PM 2.5-1. Positive dose-dependent relations were observed for GM-CSF cytokine release by 16HBE14o-cells after 24h exposure to PM (pro-inflammatory response). At the lowest non-cytotoxic dose (5 µgPM.cm -2 ), PM 1 let appear the most important GM-CSF induction potential, suggesting that this fraction strongly contribute to pro-inflammatory effects. Considering PM 1 , the most prone to interact with bronchial cells, Emissions showed a higher pro-inflammatory response than Refining and Furnace at non cytotoxic concentrations. Irrespective of PM origin, GM-CSF secretion increased in parallel to total lead content and was inversely related to lead solubility. Moreover, this biological response was correlated with the acellular DiThioThreitol (DTT) assay, suggesting some biological predictive value for such acellular tests. Knowing PMtot concentrations at Furnace (average 350 µg.m-3) and Refining (average 125 µg.m-3), together with size distributions and lead contents in each fraction, we evaluated the lung and gastric burden due to inhalation of such lead-rich particles. Results clearly indicate that ingestion is the most important source of bioavailable lead. Total lead deposited in lungs during 8 hour shift is 6 time lower than the inhaled fraction (compare 92 µg total Pb deposited in lungs to 539 µg total Pb ingested for Furnace). When looking for the bioaccessible Pb 2+ fraction, this ratio increases to 11-16 (compare 5 µg Pb 2+ in lung to 54 µg Pb 2+ ingested for Furnace). The Pb 2+ fraction in lungs is a minimum, knowing that internalized PM is found in phagolysosomes where low pH values can favor their dissolution.

Conclusions

Taken together, our data suggest that biological behavior for such lead-rich PM could be driven by a combination of both lead solubility (related to speciation) and particulate effect. Finally, according to pro-inflammatory effect, DTT test and PM size distribution, Emissions and Furnace PM were found to be the most of concern for health. These findings are promising in a view to combine different acellular and cellular assays as recommended by REACH legislation.

  Figure 1. Gastric and gastro-intestinal bioaccessibility values for PM tot and PM 2.5-1 . Results are expressed as the percentage of the soluble lead and the total lead content. *different from the respective PM tot for gastrical values.


Jean Christophe Le Coze

Romuald Périnet
email: romuald.perinet@ineris.fr

Nicolas Herchin
email: nicolas.herchin@gdfsuez.com

Philipe Louys
email: philippe.louys@grtgaz.com

Observing resilience within a large technical system

This paper introduces some of the results of an empirical field study within a large technical system (high pressure gas transmission network), exploited by one company, in which safety results from a combination of anticipation and resilience obtained by a 'mixture' of individual expertise, team coordination and organisational context favouring the tasks carried out at the 'sharp end'. High pressure gas transmission presents itself as a network of pipelines for which one threat are 'external aggressions', such as damages occurring when tractors dig, for instance, trenches for civil, municipal or urban engineering projects. In a first section, some elements of definition of 'large technical system' (LTS) is provided; it introduces some of the specific features met in these types of systems. In a second section, after a presentation of the methodological and theoretical elements, a description of the safety measures designed within this LTS for prevention of external aggression is offered. In a third section, a first and heuristic (for further developments) model of resilience, inferred from observations and interviews, and inspired from the literature on 'macro cognition' and 'collective mindfulness' is presented. Finally, in a last section, some thoughts about the translation from description to action ('engineering') of resilience (whether creating, maintaining or improving resilience) are suggested.

HIGH PRESSURE GAS TRANSMISSION AS A LARGE TECHNICAL SYSTEM (LTS)

This paper presents some of the results of an empirical study investigating the way in which safety is produced in normal operation within a 'large technical system' (LTS) in charge of high pressure gas transmission. The analytical category of LTS was developed about twenty years ago to describe numerous infrastructures (networks) that appeared to share common features. One pioneer in the field is the historian of technology Hughes (1983) who studied electricity network, from its invention to its wide spread throughout society. This work allowed in the following years to spark interest in the research community and to gather other researchers already partly involved in this area (two important conferences were held in the eighties on this subject, leading to the publication of two books, Mayntz, Hughes, 1988, La Porte, 1991). In all these contributions, it is made explicit that beyond the electrical case studied by Hughes, it is interesting to identify a much wider category of systems, which, according to Joerges (1988,24) '(1) are materially integrated, or 'coupled' over large spans of space and time, quite irrespective of their particular cultural, political, economic and corporate make-up, and (2) support or sustain the functioning of very large numbers of other technical systems, whose organisations they thereby link.' For this author, examples of LTS can, as a consequence, be 'integrated transport systems, telecommunication systems, water supply systems, some energy systems, military defense systems, urban integrated public works', etc.

Of course, with a safety perspective, a very well studied case of LTS comes to mind: aviation. From pilot's psychological models elaborated by cognitive scientists to cabin crew communication and coordination issues (leading to crew resource management programs) as well as atc studies (air traffic control) by high reliability organization researchers, there is a very large literature dedicated to the safety aspects of this LTS. However, other LTS have not been granted as much attention as aviation has been granted in the past decades of research on safety, at least to the authors knowledge. One can think of electrical grid or gas transmission networks, although serious safety issues are also associated with them. A 'black out' can have severe indirect consequences on the exploitation of highly hazardous technological installations. A recent incident in a nuclear power plant in Sweden in 2006 demonstrated this when its diesel generators failed to start as expected for cooling down the reactor's core, following a 'black out' of the electrical network supplying energy. Although an indirect consequence, the centrality of such a LTS, requires that its functioning becomes an integrated part of prevention of major hazard. As far as high pressure gas transmission networks are concerned, they can have direct (as well as indirect) consequences. One direct is the loss of containment of pressurized gas, leading, potentially, to fires.

THE THREAT OF 'EXTERNAL AGGRESSION'

One of the specificity of these LTS in general is that they are opened to potential 'external aggressions', voluntary or not. Pipelines are not confined within the boundaries of an industrial site. They cover a very wide geographical area, and represent in France a total of 32 000 kms. As a result, one specific safety activity that has been considered in this research is the identification and prevention by the exploiting company of 'external aggressions' on gas pipelines. For a LTS such as high pressure gas transmission network, any urban, municipal or civil engineering (umce) work carried out nearby pipelines and requiring drilling or digging with tractors is a potential threat to their integrity.

Ghislenghien's disaster (2004)

The accident of Ghislenhien in Belgium in 2004 (24 persons killed, 132 severely injured) demonstrated what type of scenario could lead to major consequences. This accident was caused by a pipeline's structure weakened following an 'external aggression', which would have been caused, from a 'sharp end' point of view and according to the current explanations, by a truck involved in umce nearby. After hitting the pipeline without noticing and/or informing the operating company of the pipelines about it, the incident remained totally unknown until the pressure increased in the pipeline and that the weakened structure, where the tractor hit, failed to contain the rising pressure. It created a high pressure gas leak, and then to a huge flame when ignited by a source of energy. This disaster is an illustration of the challenge faced by companies managing high pressure gas transmission networks for identifying, locating and assessing any works being performed nearby their pipelines. In this study, the company involved implements many levels of preventive measures ranging from national initiatives to local ones. The empirical observations have been restricted to local measures in this study. At this level, prevention is based on a decentralised mode of organising. For a given territory, a 'sector' (composed of less than 10 individuals, in this case study, it was 6) is in charge of the maintenance and the surveillance of pipelines. The sector selected by the company for the study was considered as a 'good' one, with good results and with a good image within the organisation. Located in a complex urban context of a major city, it requires a high level of interaction between many different actors including engineering contracting and subcontracting companies, municipal employees, architects, etc.

OBSERVING RESILIENCE IN LTS

Elements of methodology and theory

The approach retained for describing, understanding and explaining the activity of a team within a sector involved in prevention of 'external aggression' (through the angle of resilience) relied on participant observations and interviews. Interviews were conducted with the manager of the team and then the team members, with a focus on three of them involved, directly or indirectly, in prevention of 'external aggression'. We saw them sometimes twice, in order to come back on some aspects missed in the first interview. These interviews were performed at times collectively but also individually, and oriented on specific topics, according on the function of the interviewee. Questions covered different topics including task complexity, expertise required (technical, relational) for handling work situations, relationship at work between employees and management, training, information flows about work issues and incidents, etc. Participant observations of activities within and outside the sector premises were performed, consisting in taking into account technological interfaces, communications and coordination between employees, the different steps followed for performing tasks, while interacting with employees from umce companies. When possible, some observed activities were questioned 'on the spot' (as someone learning his job would probably do). For example, following a decision that seemed to rely on a judgment that was unclear from an outsider point of view, it was asked to the employee, when possible, to explain about the rationale behind his decision.

The main purpose of these series of interviews and observations was to get close to the specificity of the work activity at the 'sharp end'. When interviews and observations were done separately by researchers, feedback sessions were organised between the observers in order to cross data, and discuss interpretations and hypotheses. Five days were spent observing and interviewing. This 'immersion' within the activity of the sector was prepared few weeks before, by a description and understanding of the more global (regulatory, economical, regulatory and political) context of this LTS. Several meetings with knowledgeable engineers and managers in this domain allowed the team of observers to get to know the context. Theoretical background for this study includes disciplines such as engineering, psychology, cognitive science, management and sociology applied to safety. The polysemous notion of resilience [START_REF] Hollnagel | Resilience engineering: concepts and precepts[END_REF] is defined in this research, at the 'team level' of investigation considered in this paper, as the adaptations (or tradeoffs) of individuals to situations within their technical and relational context, in normal operation. Resilience requires to be thought as a product of the interaction between technological, cognitive and social features.

Main safety measures for preventing external aggressions

In order to prevent 'external aggressions' on pipelines, a number of 'defense in depth' are implemented, ranging from technical to national level: Technical: increased resistance of pipelines, mechanical protection, pipelines external (yellow) signalization.

Regulatory: demand of information about pipeline location by companies intending to dig (DR 'demande de renseignements'), declaration of start of projects (DICT 'déclaration d'intention de commencement de travaux')

Operational: answer to demands of information (DR), risk assessment at work site following DICT by companies digging for umce work, surveillance of work sites and pipelines.

Organisational: training of organisation's employees, information to municipalities and civil, urban or municipal engineering companies about the regulation (DR, DICT), incident analysis at national level.

It is the operational type of measures that were investigated in this case study, leaving in the background, although equally important but not discussed in this paper but partly investigated in this study.

Some results

A task requiring constant adaptations

One very explicit feature of the task of preventing 'external aggressions' appeared to be the ability of some 'home made' experts to adapt to daily variations of work constraints. Whereas ideally, the task was supposed to be in the form of sequential steps, (figure 1), it turned out to be of a very different kind.

Figure 1 : a sequential task, in theory

In theory, preventing 'external agression' consists in receiving an information request (a 'DR') by a company intending to perform umce work on a given area, asking the organisation exploiting the pipelines about the presence or not of a pipeline nearby planned umce work. If it is a positive answer, then this company must warn the organisation (through a 'DICT') about the intended date of start so that an employee of the LTS exploitation can come and assess the situation on site. The employe of the LTS locates then the pipeline (using a specific equipment), indicates to the umce company some safety measure to be taken given the specific situation. This agent must then wait for the end of the project to identifty any damages that would have been caused to the pipeline. All this is represented in figure 2. This is however the theory. In reality, it is difficult to implement all these steps sequentially. First, there are other activities not included, such as responding to invitation from umce companies to assist them before projects start, in order to get to know where the pipelines are. But there are also emergency engineering work that are to be treated immediately (such as for example water leak to be fixed by water enginnering companies which necessitates sometimes digging close to high pressure gas pipelines). Secondly, companies performing engineering work do not always warn ahead and call the very same day that they start. As it is not planned, solutions must be made to deal with the situation, and to establish priorities among schedule. Instead of a linear sequence of activities, observations quickly reveal a different adaptive type of task, involving multiple parallel activities (figure 2).

DR treatment DICT treatment

Site visit

Sites surveillance

Pipelines surveillance

Emergency work

External agression prevention activity

Meeting ahead of projects

GRT Gaz Work site surveillance

Figure 3.a task involving parallel activities

From a collective to an individual expertise

All these activities cover a wide range of different aspects and skills: administrative, relational and technical. Depending on the time of year, workload varies. In the most intensive of these periods, tradeoffs must be made between all these activities in order to allocate resources (time, expertise) for what they consider as the umce works nearby to pipelines deserving the closest watch. The coordination between the members of the team is then very important. Defined for example by Weick as one dimension of 'collective mindfulness' (Weick, 2001), this collective side of the activity revealed in this case study interesting features among which a mix of stability and flexibility (Wildaavsky, 1988). In this team, it appeared that individual expertise was at the heart of the decision making process, when circumstances pressed for adaptive patterns. While not in the hand of the manager of the team (who hasn't got a strong experience in the field of 'external aggression' prevention), great flexibility was granted to one of the individual in the team who was acknowledged as the most expert in this area.

Organising his own schedule, he is in charge of balancing several factors for deciding in real time about the priorities when tradeoffs must be made. Of course, he relies on written procedures provided by the organisation but, given the specificity of local urban contexts, he has to adapt this procedure in order to achieve what seems to him to be a satisficing response (Simon, 1957) to his local constraints and unplanned demands. His choices have an impact on the team workload, as he can, at times, when it is required; ask a colleague (or even his manager) to replace him for a planned visit. While he is replaced, he is then able to deal with an unexpected situation, such as an emergency engineering work in a sensitive area for which he wishes to be present to ensure close supervision. This employee commented this situation as an unusual one, as he could 'give order to his own boss'. Whereas probably in many cases one could imagine the problem of such a situation, in this team, it was not an issue. A balance had been found between hierarchy and expertise, introducing here a key issue of power in teams (and more widely, in organisations, Le [START_REF] Le Coze | BP Texas city accident : weak signals or sheer power ? 3 rd international symposium on resilience engineering[END_REF]. This is one side of the collective expertise of the team, which partly results from the managerial style of the team leader, willing to have an expert of the team leading some decisions for the rest of team, although not a manager.

Beyond this collective side of expertise, and as a complement, what has proved also very interesting to identify and to investigate are elements of decision making process on which this individual expertise relies. Without attempting to produce a final model of such a complex topic as decision making, the strategy in this study was rather to see how collected data during interviews and observations could be exploited in order to indicate some of the key dimension of this individual expertise. In this respect, the (methodological and theoretical) approach was close to principles of 'macro cognition' or 'cognition in the wild', considering cognition to be the understood as individuals ('cleverly') adapting within their work context. Rather than expecting to fully understand cognition only through experiments (and also normative frameworks), field studies ('in the wild') become one input for the understanding of how cognition proceeds in face of complex and dynamic environments instead of simple and static ones.

Towards a heuristic model of resilience in the domain of 'external aggression'

One first step was to group together many of the data that this expert processed in his activity. A first set of six groups of key dimensions were identified. They combine many different sides implied by 'external aggression' prevention, among which 'itinerary', 'urban geography', 'pipeline', 'urban features', 'political context' and 'engineering'.

Each group refers to specific topics, such as rush hour, depth of pipelines, umce companies, municipalities' services etc (figure 4). It is clear from this description that only expertise acquired by individual from many years of experience is in a position to feel comfortable with the decision making process involved. One reason for having a specialised 'expert' in this team and to afford him flexibility of decision is to find an answer to this problem of knowing sufficiently well the area, the umce companies (see groups in figure 4) etc, in order to elaborate appropriate choices in real time when needed. One way to suggest a first model of resilience was as a consequence to introduce the notion of expertise at the heart of it, based on a combination of collective and individual expertise. For describing expertise, Klein's approach proved useful [START_REF] Klein | Sources of power[END_REF][START_REF] Klein | Streetlights and Shadows. Searching for the keys to adaptive decision making[END_REF]. Synthethised through four key features: mental simulations, intuition, mastery of time horizon and knowledge by expert of his own limit, they were illustrated by some of the stories collected during observations and interviews. For example, cases of 'intuition' or 'mental simulations' were used as illustration of how expertise shaped decisison making. Combining the various features of the activity together led to the followin model (figure 5). Based on a wide range of data collected by the expert on many different topics (figure 4), divided in categories of 'global context' and 'specific context', individual expertise combined with collective expertise allow the team to adjust in real time their schedule in order to maximise the chance to be at 'the right place at the right time'. Tasks transfer to team members

Urban geography

Buildings

'being at the right place at the right time'

Figure 5. A heuristic model of resilience.

'ENGINEERING' RESILIENCE

Beyond the attempt to model or theorise on resilience within this LTS, there are also expectations for 'engineering' solutions. This 'engineering' side is the question of how to practically enhance, support, create or maintain resilience. This study is a start. The simple and heuristic models proposed consist in some indications about what direction to pursue. One key outcome of this case study is for the work of 'external aggression' prevention by experts to access a better visibility in the organisation. Comparisons between teams should provide more data and reference points to move forward. But other concrete applications of this initial model could be translated in current incident investigations which do not explicitly rely on this type of model acknowledging the collective and individual expertise behind the scene. One other concrete use is training of managers, but also of experts in sectors. The ability to better contextualize indicators with qualitative data is also one possible prospect along with possibilities of adapting audits for them to reflect the findings of this study, rather than strict compliance with rules which is a very difficult objective for teams in such a complex and open environment. A case study on normal operation with the intent to identify and describe resilient properties of individual and teams can lead to useful and practical outcomes. However, such a translation remains part of a research problem, when model and practical ideas need to be tested to prove their relevance, implying there again strong tradeoffs between purposes of description and transformation. A good description does not necessarily lead to practical tools to help transformation (Le [START_REF] Coze | Is resilience engineering realist or constructivist ? 3 rd international symposium on resilience engineering[END_REF].

CONCLUSION

This study presented here had the purpose of observing some of the features of resilience and to imagine ways of being practical in order to promote, maintain or create resilience. After describing the specificity of LTS, and associating high pressure gas transmission network to this category, the paper introduced the threat of 'external aggressions' as one activity to be studied. LTS are opened systems that need to be managed in order to cope with this specific threat. The study relied on interviews and observations for approaching what could be seen as the resilient properties of team dealing with the prevention of 'external aggressions'. The paper shows that a collective and an individual expertise, allowing flexibility and quality of decision making to be obtained, is necessary. A heuristic model, as a first attempt to capture characteristic of resilience within this LTS, has been suggested. It can serve as a basis for 'engineering' resilience, and will be completed in the future through comparisons with other sectors.

Figure 4 .

 4 Figure 4.Expertise at the center of many different topics


François Brion
email: francois.brion@ineris.fr

Yann Le Page

Benjamin Piccini

Sok-Keng Tong

Bon-Chu Chung

Olivier Kah

Brain Aromatase (Cyp19a1b) is a highly sensitive gene to estrogens and xeno-estrogens

PhD thesis in 2001 at the University of Metz investigating the effect of estrogens on the ER-regulated expression of vitellogenin and its use as biological marker of estrogenic compounds in fish. Since this date, he worked at INERIS still focusing on the effects of EDCs on the expression of hormone-regulated genes involved in key reproductive processes in fish. He coordinated a project of the National Research Program on Endocrine Disruptors on the molecular interactions between ER and AhR and their reproductive effects in fish and together with Olivier Kah he is working on the effects of EDCs at the central nervous system in zebrafish (NEED project of the National Research Agency). Since 2009, he also coordinates the NEMO project which aims to set-up a large panel of new in vitro and in vivo mechanism-based screening tools for studying EDCs effects in the zebrafish (Danio rerio). He supervised three thesis in the field of endocrine disruption in fish and is (co)-author of 29 peer-review articles.

Introduction

It is now well established that a number of natural and man-made compounds referred to as Endocrine Disrupting Chemicals (EDCs) interfere with the endocrine system of animals, including wildlife and humans. A growing number of environmental compounds have been proved or suspected to act as endocrine disruptors. EDCs belong to structurally and functionally diverse group of chemicals and include, among others, natural and synthetic steroids, phyto-and myco-estrogens, alkylphenols, phtalates, bisphenolA, polybrominateddiphenyl ethers (PBDE's), several pesticides, dioxins and polycyclic aromatic hydrocarbons (PAHs). In a regulatory perspective, these compounds have been defined as exogenous substances or mixture that alter function(s) of the endocrine system and consequently cause adverse health effects in an intact organism, or its progeny, or (sub)populations [START_REF] Who | Global Assesment of the States-of-the-Science of Endocrine Disruptors[END_REF]. They have been also defined according to their multiple modes of action on the endocrine system as exogenous agents that interfere with the production, release, transport, metabolism, binding, action, or elimination of natural hormones in the body responsible for the maintenance of homeostasis and the regulation of developmental processe [START_REF] Kavlock | Research needs for the risk assessment of health and environmental effects of endocrine disruptors: a report of the U.S. EPA-sponsored workshop[END_REF].

Because a large proportion of potential EDCs end up in surface waters, aquatic species are particularly vulnerable to their potential adverse effects. In fact, endocrine disruption has been demonstrated to occur in wildlife, particularly in aquatic species [START_REF] Tyler | Endocrine disruption in wildlife: a critical review of the evidence[END_REF] and there is strong evidence supporting a causal linkage between exposure and effect (Jobling et al., 1998;[START_REF] Sanchez | Adverse effects in wild fish living downstream from pharmaceutical manufacture discharges[END_REF]. Until now, most of the EDCs effects reported on fish been concerned their peripheral actions such as gonadal development, oocyte development, egg production and fertilization, sperm count and liver vitellogenin synthesis, an estrogendependent yolk precursor(for review see, e.g. [START_REF] Hotchkiss | Fifteen years after "Wingspread"--environmental endocrine disrupters and human and wildlife health: where we are today and where we need to go[END_REF]. However, the reproductive axis also includes upper levels of regulation inthe central nervous system (CNS)

that is likely to be targeted by a wide range of EDCs. For instance, we recently shown that the synthetic estrogen ethinylestradioland the alkyphenolic compound nonylphenol disrupt the ontogeny of GnRH system by inducing in a concentration-dependent manner the number of GnRH3 neurons in zebrafish embryos and larvae (Vosges et al., ;[START_REF] Vosges | 17alphaethinylestradiol disrupts the ontogeny of the forebrain GnRH system and the expression of brain aromatase during early development of zebrafish[END_REF].

These effects were observed as early as 5 days post-fertilisation and occurred at low and environmentally relevant concentrations. Furthermore, the effects ofnonylpenol and ethinylestradiol were shown to be blocked by the pure estrogen receptors (ERs) antagonist ICI 182-780, demonstrating the involvement of functional ERs in mediating their effects (Vosges et al.). The effects of EDCs upon neuroendocrine systems of fish are not limited to the GnRH neurons. For instance, other studies have shown the effects of several EDCs on the dopaminergic and serotoninergic circuits in fish (for a review see (Le [START_REF] Page | Neuroendocrine effects of endocrine disruptors in teleost fish[END_REF]).

Another target of the central nervous system of fish known to be affected by EDCs is brain aromatase B, encoded by the cyp19a1b gene. In all vertebrates, cytochrome P450 aromatase (Cyp19a1) converts C19 androgens into C18 estrogens, important hormones involved in the control of many important physiological processes, notably reproduction. In vertebrates, with the notable exception of the pigs, aromatase is produced by a single Cyp19a1 gene, whose tissue specific expression is driven by alternative promoters [START_REF] Simpson | Aromatase cytochrome P450, the enzyme responsible for estrogen biosynthesis[END_REF]. Conversely, in many teleosts studied so far, including the model species zebrafish (Danio rerio), two structurally distinctcyp19a genes are found, cyp19a1a and cyp19a1b. These two genes generate two structurally and functionally different isoforms referred to as Aromatase A (AroA), predominantly expressed in gonads and Aromatase B (AroB), mainly found in neuronal tissues and responsible for the well-known exceptional aromatase activity of the brain of fish [START_REF] Pasmanik | Aromatase and 5 alpha-reductase in the teleost brain, spinal cord, and pituitary gland[END_REF]Gonzalez and Piferrer, 2003).

Expression of the cyp19a1b in the brain is regulated by estrogens [START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF] and accumulating data acknowledge aromatase B as a highly and sensitive target of estrogens and an outstanding biomarker of xenoestrogens exposure. In the developing and adult brain, aromatase B expression is located exclusively in radial glial cells which are stem cells as they are capable of giving birth to newneurons. Given the key role of these cells in neurogenesis, modulation of brain aromatase expression due to EDCs raises the question of their potentialeffect on neurogenesis.

Regulation of cyp19a1b expression in the brain of fish.

The structure of the cyp19a1bgene pointed out the presence of a well-conserved estrogenresponse element (ERE) and a half ERE located within the 500bp of the cyp19a1b proximal promoter [START_REF] Tchoudakova | Promoter characteristics of two cyp19 genes differentially expressed in the brain and ovary of teleost fish[END_REF][START_REF] Tong | Analysis of zebrafish cyp19 promoters[END_REF]. Transactivation experiments using zebrafish cyp19a1b linked to luciferase revealed that, in the presence of estrogen receptors, 17β-estradiol(E2) up-regulates cyp19a1b. This up-regulation was however highly dependent on the cell context. Indeed, while no or weak activity of the promoter following E2 treatment was observed in most cell lines studied (e.g., Hela, CHO), a dramatic luciferase activity was reported in cell line providing a glial or a neuro-glial cell context, i.e. U251-MG.

Furthermore, deletions or mutations studies indicated that integrity of the ERE is absolutely required for the estrogenic regulation [START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF]. But ERs are not sufficient to drive cyp19a1b expression in the brain of fish in the presence of estrogens. In fact, it was shown that a sequence, referred to as GxRE located between -277 and -257 bp from the initiation start site, plays an important role in the cell specific regulation of the cyp19a1b gene and its E2-regulation (Le [START_REF] Page | Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene[END_REF]. The cyp19a1b regulation requires glial specific factor(s) that bind to a glial x responsive element (GxRE)acting synergistically with the ERE sequences (Le [START_REF] Page | Characterization of a cis-acting element involved in cell-specific expression of the zebrafish brain aromatase gene[END_REF].This mechanism confers to the cyp19a1b gene a high sensitivity to estrogens and a cell-specific expression in radial glial cells (figure 1). In agreement with in vitro studies, it isnow well-established that short-term exposure of zebrafish embryos 17beta-estradiol (E2) for two days causes a strong increase in cyp19a1b mRNAs, Cyp19a1b protein expression in radial glial cells, and aromatase activity in the head.

All these effects can be blocked by an excess of ICI 182 780, indicating the requirement of functional estrogen receptors (ERs) in mediating the effect of E2 on cyp19a1b expression [START_REF] Menuet | Expression and estrogen-dependent regulation of the zebrafish brain aromatase gene[END_REF]. In contrast, the cyp19a1a gene that encodes gonadal aromatase is poorly sensitive to estrogens at this stage of development [START_REF] Hinfray | Brain and gonadal aromatase as potential targets of endocrine disrupting chemicals in a model species, the zebrafish (Danio rerio)[END_REF][START_REF] Cheshenko | Expression of Zebra Fish Aromatase cyp19a and cyp19b Genes in Response to the Ligands of Estrogen Receptor and Aryl Hydrocarbon Receptor[END_REF].

Effect of endocrine disruptors on cyp19a1b expression.

The In addition to potent agonist ligands of the estrogen receptors, studies in zebrafish have demonstrated that weak estrogenic compounds such as genistein and zearalenone, individually or in combination are able to modulate cyp19a1b gene transcription at its promoter level. (Le [START_REF] Page | Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system[END_REF]. In vivo,, the alkylphenolic compound NP is able to induce cyp19a1b mRNA levels [START_REF] Kazeto | Effects of endocrine disrupting chemicals on the expression of CYP19 genes in zebrafish (Danio rerio) juveniles[END_REF] and aromatase B protein expression in a concentration-dependent manner in radial glial cells of developing embryos, this effect being blocked by ICI 182-780 (Vosges et al.).

These data provide the rationale for using the cyp19a1b gene as an outstanding biomarker for xeno-estrogens. Recently, a transgenic zebrafish that expresses green fluorescent protein (GFP) driven by the zebrafish brain aromatase cyp19a1b promoter was generated [START_REF] Tong | A cyp19a1b-gfp (aromatase B) transgenic zebrafish line that expresses GFP in radial glial cells[END_REF]. This cyp19a1b-GFP transgenic line faithfully mimics the expression and the regulation of endogenous AroB expression in radial glial cells. Based on the use of the cyp19a1b-GFP transgenic, we developed and validated a novel in vivo assay to detect and to quantify the estrogenic potencies of chemicals. In our assay, zebrafish embryos are exposed from 0 to 5 days post-fertilization to chemical substances and GFP expression is quantified in vivo using fluorescence microscopy and image analysis (figure 3). Several estrogenic compounds belonging to various chemical families have been tested and includes natural and synthetic estrogens, phyto and myco-estrogens, alkylphenolic compounds several pesticides, etc. We showed that exposure of tg(cyp19a1b-GFP) embryos to various xeno-estrogens induces GFP expression in a concentration-dependent manner and demonstrated the sensitivity of the assay as evidence by the extremely low concentration of EE2 required to induce GFP expression. For instance, concentrations of EE2 as low as 5 pM are sufficient to up-regulate GFP expression in zebrafish embryos. In addition to xenoestrogens, we have evaluated the effects of several Aryl hydrocarbon Receptor (AhR) ligands. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), or Benzo[a]Pyrene (B[a]P) did not affect GFP expression in vivo. However, when tg(cyp19a1b-GFP) embryo were co-exposed TCDD and E2, we have shown the attenuation of estrogen-induced up-regulation of GFP expression, showing that TCDD has anti-estrogenic effect which is in agreement with previously reported data on the expression of zebrafish aromatase cyp19a1b gene in response to TCDD [START_REF] Cheshenko | Expression of Zebra Fish Aromatase cyp19a and cyp19b Genes in Response to the Ligands of Estrogen Receptor and Aryl Hydrocarbon Receptor[END_REF].

Our data demonstrate the usefulness of the transgenic cyp19a1b-GFP as a reliable, sensitive and rapid in vivo estrogenic screening assay that could, together with fish-specific estrogenic in vitro assays (Le [START_REF] Page | Assessment of xenoestrogens using three distinct estrogen receptors and the zebrafish brain aromatase gene in a highly responsive glial cell system[END_REF]Cosnefroy et al., In press), enhance the efficiency and accuracy of EDCs testing strategies for environmental hazard and risk assessment of chemical substances.

Conclusion

Recent studies have now identified a number of brain targets for xeno-estrogen commonly present in environmentally relevant concentrations in surface waters. The most prominent effect of EDCs, in zebrafish is their impact on the cyp19a1b gene that encodes aromatase B in the brain. Detailed molecular and whole animal studies in wild-type and transgenic cyp19a1b-GFP zebrafish demonstrated the extreme sensitivity of the cyp19a1b gene to xeno-estrogens. A striking feature of fishes is that aromatase expression is restricted to radial glial cells that behave as progenitors in developing and adult zebrafish. The consequences of disruption of cyp19a1b gene in radial glial cells are still unknown but may have important functional implications. Given that our recent studies demonstrated that estrogens modulate proliferative activity of radial glia progenitors, it is likely that (xeno)estrogen have similar activity and thus the potential outcome requires thorough investigations. In any case, those studies have led to the development of a very sensitive in vivo assay that makes use of cyp19a1b-GFP transgenic embryos.

  studies mentioned above clearly demonstrate the estrogenic regulation of the brain aromatase cyp19a1b gene in radial glial cell context both in vitro and in vivo and revealed that aromatase B is very sensitive to estrogens through ER-dependent mechanisms. Many environmental compounds belonging to various chemical families have been shown to bind and activate ERs. Among EDCs, synthetic steroidal estrogens (including the pharmaceutical 17α-ethinylestradiol) are some of the major and most potent estrogenic contaminants in the aquatic environment. Recently, we precisely documented the spatio-temporal expression of AroB within the central nervous system during the early development of control and EE2exposed zebrafish. The strong and precocious up-regulation of AroB expression was observed in the brain of EE2-treated larvae as early as 5 dpf for EE2 concentration as low as 0.02 nM (figure2). At 5 dpf, AroB-immunoreactive cells were located in the caudal olfactory bulbs, the telencephalon, the preoptic region, the optic tecta, the rostral hypothalamus and caudal hypothalamus and bordering the fourth ventricle up to the spinal cord. This expression pattern of AroB immunoreactivity in the brain remained unchanged but the intensity of AroB immunoreactivity increased during development demonstrating that upregulation of AroB was time-dependent and EE2 concentration-dependent. This study confirms and further illustrates the extreme sensitivity of the zebrafish brain aromatase to (xeno)-estrogens by demonstrating that short exposures to low and environmentally relevant concentrations of EE2 are able to strongly induce the expression of AroB in radial glial cells in early life stages.

Figure 1 .

 1 Figure 1. Schematic representation of regulation of the cyp19a1b gene by estradiol in radial glial cells. AhR: binding site for the aryl hydrocarbon receptor; CRE: item responses to the cyclic AMP ERE: estrogen response element; GxRE: response element factor glial-specific (Gx) TATA: TATA box; E2: estradiol, T: testosterone; X: xenoestrogens. Adapted from Mouriec et al. (2008).

Figure 2 .Figure 3 .

 23 Figure 2. Photomicrographs of expression of AroB-ir cells (red) on transversal sections of 5dpf old zebrafish larvae control and exposed to nM and 0.5nM of EE2 at the level of the preoptic area (200× magnification). Blue: Hoechst staining. Scale bars = 20µm.

Acknowledgements:

The ANR NEED (CES-2008-11) (to O.K. and F.B), the Post-Grenelle grant NEMO (to F.B.and O.K.) supported this research.