Deck
email: deck@mines.u-nancy.fr

A L Olivier

Heib
email: heib@mines.u-nancy.fr

HOMAND Marwan

Françoise

Taking the soil-structure interaction into account in assessing the loading of a structure in a mining subsidence area

Keywords: mining subsidence, soil-structure interaction, numerical methods, equivalent stiffness

Underground mining of raw materials is often the cause of ground movements at the surface. Whether planned or accidental, such movements can cause considerable damage to structures located within the area of influence of underground mining works. Examples are the recent subsidences that took place at the end of the 1990s in the Lorraine iron mining field. A better understanding of how ground surface movements can be imparted to the supported structure and damage it is necessary. Indeed, it is too often considered that damage depends only on ground strain and no account has been taken of soil-structure interaction phenomena, which may affect considerably the structural behaviour.

The stiffness of a structure is quantified in comparison to that of the ground as regards the various movements of the ground surface. This investigation highlights situations in which ground movements are integrally imparted to a structure. When this is not the case, the resulting complex soil-structure interaction phenomena is analysed. For this purpose, a finiteelement software is used to generate models incorporating the ground material and a supported rigid structure. The ground movements are broken down into two basic movements in order to highlight the impact and the relative importance of one of theses movements : ground curvature and horizontal strain. Structural stresses are quantified for different mechanical properties of the ground and the structure, as well as for different amplitudes of ground movement. This investigation made it possible to devise a methodology for analyzing structures in mining subsidence areas in order to determine cases in which soilstructure interaction phenomena must be considered. The investigation made it possible to hierarchically organize the ground and structure parameters, the variability of which has a significant effect on the behaviour of the structure affected by mining subsidence.

Introduction

The industrial need for large quantities of raw materials at an acceptable cost has led to large underground mines and quarries. Because of the extraction methods, such underground mining works create underground voids which may cause mining subsidence phenomena, i.e. significant movements at ground surface. This sometimes results in serious damage to structures built in the area of influence of such movements. Mining subsidence is planned in the case of mining methods that incorporate the caving-in of the created cavities as work proceeds ("caving-in" method in coal mines, for example).

On the other hand, mining subsidence is of a highly accidental nature when it takes place over mines and quarries that use methods based on abandoned rooms and pillars. Indeed, in the latter case, the operator has deliberately left in place natural or artificial pillars sized to withstand the weight of the overburden. Recent cases of mining subsidence that have taken place in the Lorraine iron mining area (France) denote the hazard of such mining works when left abandoned.

The cases of subsidence in Lorraine led public authorities to carry investigations over the entire Lorraine iron mining field. These investigations highlighted the existence of about 2000 hectares of urbanised areas undermined by abandoned works consisting of rooms and pillars. A hierarchy of the hazard was derived : from the risk of caving-in to the risk of structures being damaged by planned subsidence. The second part is the topic of this paper. Which methodology can be proposed to predict the damage likely to affect a structure located in a hazard area, in which considerable strata movements may occur? A more fundamental question may be formulated: are ground level movements imparted integrally to the structure or does this involve soil-structure interaction phenomena caused by significant differences in stiffness between the ground and the structures? As long as the ground movements are imparted to the structure integrally, a geotechnical engineer can inform a civil engineer of the predicted importance of such movements. The latter can then apply these movements to the foundations of a structure in order to assess their impact. If, on the other hand, there is strong interaction between the ground and the structure, neither the geotechnical engineer nor the civil engineer can assess the structure loading.

The ground movements describe caused by a mining cave-in were first described. Then a method is presented to characterise the relative stiffness of a structure and of the ground, in terms of the surface movements generated during a subsidence. Finally the unfavourable case of a strong interaction between the ground and the structure is assumed to investigate the relative effect of different types of ground movement on the structure behaviour.

Figure 1 : Description of ground surface movement as a result of a mining subsidence.

Description of the phenomenon

Mining subsidence often produces significant horizontal and vertical movements at the ground surface (Figure 1). The maximum value "Sm" of the vertical subsidence is usually considered as a characteristic of the trough. However, the horizontal strain of the ground, its curvature and its slope, are the three movements loading structure and causing structural damage. The maximum values observed for these parameters can be disastrous for a structure if the movements are imparted integrally (Wagner and Schumann [START_REF] Wagner | Surface effects of total coal-seam extraction by underground mining methods[END_REF]). The measurement of these parameters entails significant difficulties either when a site of mining subsidence is instrumented, or in a case where cave-in has not yet taken place and prediction is regarded.

The real measurements of movement often reveal that the vertical movement is in agreement with its theoretical value, but the slope and the horizontal strain deviate slightly from theory and the curvature even more so, as shown in Figure 2 to be compared with Figure 1. The increasing difference between theory and practice can be readily appreciated since the slope, strain and curvature are in reality the primary or secondary derivatives of the displacement. The differences between the theoretical and real displacement are then considerably amplified. When movement predictions are involved, this difficulty entails the presence of a large number of empirical methods for predicting vertical displacements, a smaller number of such methods for predicting strains and virtually no method developed for the curvature or slope (Whittaker et Reddish [START_REF] Whittaker | Subsidences : Occurrence, Prediction, Control[END_REF]).

Horizontal scale Legend 1 : vertical subsidence 2 : ground strain area, the structure is sometimes considered to be rigid, making possible to estimate the distribution of vertical stress in the ground (Rausch [4] and Kratzsch [5]). On the other hand, Boscardin and Cording [START_REF] Boscardin | Building response to excavation-induced settlement[END_REF] consider the structure to be flexible and to behave like an isostatic beam whose curvature is imposed by that of the ground and for which the horizontal strain of the lower fibre of the beam is imposed by the horizontal strain of the ground. With regard to horizontal strains, Speck and Bruhn [START_REF] Speck | Non-uniform subsidence ground movement and resulting surfacestructure damage[END_REF] merely consider the development of shear stress at the soilstructure interface or the increase in the horizontal stresses on buried parts of the structure. Kratzsch [START_REF] Kratzsch | Mining Subsidence Engineering[END_REF] recalls a former experimental investigation carried out on a physical model, reproducing the failure occurring in the ground because differences in horizontal strain. [START_REF] Kratzsch | Mining Subsidence Engineering[END_REF] a _L Boscardin et Cording (1989) Treshold values of £ 5tmcture (mm/m) < 0,5 for negligible damage 0,5 à 0,75 for very slight damage 0,75 à 1,5 for slight damage 1,5 à 3 for appreciable to severe damage. > 3 for severe damage. "Integrally" means that the structure is loaded by displacements, "not at all" means that the structure is loaded by stresses, whereas "partially" indicates the existence of complex soil-structure interaction phenomena.

-20 -i -1 -25 1 _2 1 strain subsides (mnVm) (m)

Characterisation of the relative stiffness of a structure compared to the ground

The investigation of soil-structure interaction require a clear definition of stiffness. Two stiffness parameters are involved : a first stiffness parameter characterises the behaviour of the structure as compared with that of the ground as regards surface curvature ; a second parameter characterises the compared behaviour as regards the ground horizontal strain. We propose to characterise the ground stiffness and the structure stiffness as regards these two parameters of ground movement in order to determine the situations in which one or the other of these parameters is integrally transmitted to the structure. The reasoning is based on calculating the mechanical energy required either to impose the ground curvature on a structure, or to impose a structure horizontally on an initially curved ground. The scenario calling for the lowest energy is considered the more likely.

Figure 5 A detailed development of the calculation is done for the case of a concave area. A similar approach can be adopted for the case of a convex area. The ground is modelled by spring elements (Figure 6). adopting the Winkler model. The latter assumes that the spring elements do not interact between each other. Each spring is thus characterised simply by its stiffness "K". A number of other more complex models (Henry [START_REF] Henry | The design and construction of engineering foundations[END_REF]) have been developed, which could be adopted if necessary. The difficulty with such a model is to select a value for the stiffness "K". If it is assumed that there is a rigid substratum at a depth "h", it is possible to estimate the stiffness as a function of "h" and the ground mechanical properties (Figure 6). If there is no rigid substratum at depth, the height "H" that is required to define the springs stiffness can be estimated from knowledge of the initial settlement under a uniformly loaded foundation. Milovic [START_REF] Milovic | Stresses and displacements for shallow foundations[END_REF] proposes, for example, an estimation of this settlement in different situations. The main stages in calculating the force and the work required to deform the ground are presented in Figure 6. If it is assumed that the structure length "L" is negligible compared to the ground curvature "R", the formulas for the force and the work of the deformation forces " Wj " are greatly simplified. To calculate the structural deformation work "W 2 ", only the work of the bending moments is considered, the work of the shear force being neglected. By imposing a constant curvature along the structure, a constant moment is also imposed. For a unit length in the third dimension, the work "W 2 " can then be calculated easily with equation 1.

E.I 9R

/ ->x 2i-ound ground (equ.l)

Where "E" is the Young's modulus of the structure, "I" the structure inertia and "Rground" the radius of curvature of the ground imposed on the structure. This equation assumes the classical hypothesis of trifling influence of shear work of the beam theory. radius that the structure can take according to its loading "Q" is calculated half-way along the structure length, assuming that the structure is simply supported at its two extremities (concave area) or at its centre (convex area).

An identical radius of curvature of 8EI/qL 2 is obtained.

Concave area

M=EI/R

Figure 8 : Methodology for estimating whether the ground curvature is imparted to the structure (Yokel and al., [START_REF] Yokel | Housing construction in areas of mine subsidence[END_REF]).

This methodology can be compared with that proposed by Yokel and al. [START_REF] Yokel | Housing construction in areas of mine subsidence[END_REF] and shown in Figure 8. The latter sought to characterise the structure critical length at which the actual weight of the structure is sufficient for it to assume the ground curvature. This is equivalent to the above estimation of the radius of curvature, taking l cr =L. A difference can be noted in the concave area. Indeed, Yokel and al. [START_REF] Yokel | Housing construction in areas of mine subsidence[END_REF] consider that not only the structure is on two supports (corresponding to the moment M and the force V in Figure 8), but in addition the displacement of the extremity is imposed (A=l" cr /2R). Neither of the approaches is completely realistic since it is impossible with a uniformly distributed loading to have a curvature which is constant, and thus equal to the ground curvature, along the structure. However, both approaches produce fairly realistic results.

Estimation of the stiffnesses as regards ground horizontal strain

Figure 9 schematically illustrates the approach for estimating of the stiffnesses as regards ground horizontal strain. The ground and the structure are modelled by two springs in parallel. As far as a horizontal movement is concerned, the behaviour of the ground will not be disrupted by the presence of a structure as long as the stiffness of the structure is low compared to that of the ground.

Model adopted :

-ground displacement Figure 9 Approach to compare the stiffness of a structure against that of the ground with regard to horizontal strain of the ground.

Stiffness of the structural elements "El" calculated by taking the product of the Young's modulus of the material "E" and the section inertia "I" of the considered element.

The type of link with the ground and which depends on the type of foundation or the presence of basements.

The links were considered to be articulated, embedded or with a stiffness to be determined in order to allow the model to be applied to various local structures.

The stiffness of the various structures are calculated analytically ; results are shown in Figure 10. An numerical investigation carried out to characterise the stiffness of a solid wall, has underlined the very strong stiffness of such a structure compared with the frame structure stiffness, with values ten to five hundred times higher.

The stiffness of the structure is modelled by different categories of frame structures as summarised in Figure 10. The parameters are as follows:

Dimensions of the structure: height "h" and length "L", as well as the ratio "a" = h/L. For all the investigations, the ground is considered to be elastoplastic: Young's modulus "E terrain ", Poisson's ratio 0,25, zero cohesion, angle of friction 30° and angle of dilation 10°F or all the calculations, the structure is considered to be elastic: Young's modulus "E structure ", Poisson's ratio 0.2

The ground stiffness is more complex to estimate analytically. It involves determining the ground area which takes part in its stiffness as regards the horizontal stress that is investigated. For this purpose, the surface area of the ground that is disturbed by the presence of the structure is examined. It is considered that the more a ground point is subjected to the specific weight of the structure, the more a change of state of this point is likely to generate a change of state of the structure. This methodology makes use of the "Maxwell-Betti" theorem used in structure analysis.

This estimation is carried out by a numerical investigation with the CESAR-LCPC V 3.2.4 finite element software 1 (Figure 11). A ground domain (100m* 100m) loaded by a constant horizontal strain at depth is considered.

In addition, the horizontal displacement is blocked for a number of nodes at the upper border, over a length "L/2", in order to simulate a complete interaction between the ground and a structure of length " L ". The strain field is found to be disturbed by the presence of the structure (nodes blocked in displacement) over a depth approximately equal to half the length of the structure. As the calculation was carried out elastically, the ground 1 Informations about CESAR-LCPC V. 3.2.4 are available on the web : http://www.lcpc.fr. This software is developped by the "Laboratoire Centrale des Ponts et Chaussées". Principales features are computation of underground works, civil engineering structures, seepage and heat tranfert. stiffness was presumably overestimated. It can thus be assumed that the portion of the ground which takes part in stressing the structure is distributed, at most, over a depth approximately equal to the structure length. More specifically, the result of the numerical investigation highlights a fairly regular variation in the horizontal displacements under the structure. It appears that the contribution of the ground to its own stiffness increases with the proximity to the ground surface. This results in a ground stiffness which can be estimated to half its Young's modulus (Figure 11).

As the real phenomenon is three-dimensional, a unit extension must be considered to compare the two elements. The stiffnesses of the structural elements "El" must thus be homogenised for a unit extension of the structure. If the stiffness of the structure is low as compared to that of the ground (figures 10 and 11), the structure is considered to be flexible and the horizontal movement of the ground is integrally transmitted to the structure. If, on the other hand, the stiffness of the structure is high as compared to that of the ground, the structure is considered to be rigid. The ground movement is then necessarily strongly disrupted by the presence of the structure, and this calls for a detailed investigation of the soil-structure interaction phenomena. For this purpose, the following section investigates the behaviour of a rigid structure of the load-bearing-wall type, with regard to mining subsidence.

Stress-strain investigation

This investigation is intended to highlight the overall behaviour of a solid-wall type structure, i.e.

structure that is very rigid as compared to the ground. For this purpose, a set of numerical simulations was carried out in order to investigate the behaviour of a structure with regard to each basic loading components: horizontal strain and curvature. The effect of the slope was not investigated since the slope, as a rigid body movement of the structure, produces very little stress in the structure.

The finite element software CESAR-LCPC was used with six-node triangular elements, eight-node quadrangular elements and six-node interface elements.

The latter elements make it possible to take into account a friction type behaviour as well as an interface separation.

In all of the simulations, the ground behaviour was considered to be elastoplastic with a Mohr-Coulomb failure criteria ; the structure was considered elastic. The results shown below correspond to the structure and the soil-structure interaction before structure damage. This choice is justified considering the very high mechanical strength of the structure in comparison with that of the ground.

Curvature (concave or convex)

"around = 0 tO +/-7500 m fixed values of mechanical properties ; then the influence a variation of these properties was analysed (Table 1). A description of all the investigations is first presented before results are discussed.

• Reference investigation: the consolidation of the ground under its own weight and the weight of the structure is investigated, with no additional loading.

• Investigation of the ground curvature: the effect of a concave and convex curvature of the ground with a radius of +/-1000 m is investigated.

• Investigation of the horizontal strain of the ground: the effect of a tensile and compressive strain with an intensity of+/-5 mm/m is investigated.

• Ground properties (elastoplastic): Young's modulus "Eground" 100 MPa, Poisson's ratio 0.25, cohesion 0, friction angle 30° and dilation angle 10°.

• Structure properties (elastic): Young's modulus of the ground "E stracture " is 5000 MPa and its Poisson's ratio is 0.2.

Curvature analysis

The effect of the ground curvature on a structure is easy to understand as illustrated in Figure 3 The analysis of the vertical stress under the structure shows that the disturbances are greater for a concave curvature than for a convex curvature. The vertical stress in the ground, underneath the area on which the structure rests preferentially, increase by 60% in convex areas and by about 100% in concave areas. In addition, a structure is more likely to be found partially disconnected from the ground in the concave area than in the convex area. This support the intuitive approach adopted by Kratzsch [START_REF] Kratzsch | Mining Subsidence Engineering[END_REF],

who suggested that the structure remains horizontal as compared to that of Boscardin and Cording [START_REF] Boscardin | Building response to excavation-induced settlement[END_REF], who suggested that the curvature of the ground is transmitted to the structure. It appears that the separation is actually not related to the bending flexibility of the structure but instead to the vertical flexibility of the ground.

Strain analysis

In the same way as for curvature, the effect of a horizontal strain of the ground on a structure has often been grasped qualitatively (Figure 4). The main idea adopted so far has been to assume that the horizontal strain of the ground only produces a uniform horizontal strain in the structure. The investigation presented here provides a visualisation and a quantitative analysis of the behaviour of a structure under such a load. The results are shown in Figure 15 and 16.

•

The failure points in the model vary differently according to the strain direction. A generalised failure in the model can be observed in the tensile area, which can be readily understood given that the strain tends to decrease the horizontal minor stress whereas the vertical major stress remains unchanged. In compression areas, however, a confined area is observed under the structure, similarly to that obtained with the reference investigation (structure resting on a flat soil without strain) and which is similar to the cone of ground which shape is preserved under a vertically loaded foundation.

•

The stress field in the model shows that a compressive strain generates a stress concentration in the ground under the centre of the structure as well as bending stresses in the structure. In a tensile area, however, the stress field seems to be relatively little modified in the ground and in the structure.

•

The variation in vertical stress under the structure provides a good representation of the overall behaviour.

The compressive strain leads the structure to rest preferentially on its middle section. The stress increase is then about 40%, i.e. an value comparable to that observed for the curvature. In tensile areas, however, a rehomogenisation of the vertical stress is observed under the structure.

•

The horizontal stress in the ground increases only slightly in tensile areas. In compressive areas, however, a penetration of the horizontal stress under the structure can be observed and no increase in these stresses is observed towards the outside of the structure. Indeed, the presence of the free surface does not allow a significant increase in the horizontal stress as the vertical stress is very low. It is of interest to compare this result against one of the remedial actions that is proposed to minimise the effect of the strain: the digging of trenches around the periphery of the structure is often mentioned as a technical solution.

The stress on the ground surface proves to be low on account of the adopted failure criterion (Mohr-Coulomb).

Therefore, such a solution does not completely cancel the effect of the strain.

A shearing phenomenon appears naturally under the structure. Contrary to what is often assumed (Kratzsch, [START_REF] Kratzsch | Mining Subsidence Engineering[END_REF], the value of the shear stress is not constant. In a compressive area, the variation in the shear stresses is more complex than in a tensile area. This phenomenon is associated with the distribution of the vertical stress which varies significantly along the structure.

•

The structure is much less affected by the strain imposed by the ground than if such strain is imparted integrally. Indeed, a strain of +/-0.005 mm/m has the effect of a horizontal stress of 25 MPa in the structure.

Instead of such a value, the compressive area causes a compressive stress of -0.2 MPa in the lower edge and a tensile stress of 0.1 MPa in the upper edge ; the structure is thus bent. To a lesser extent, the same phenomenon is observed in a tensile area since the latter tensile horizontal stain generates tensile stresses of 0.04 MPa in the lower edge and -0.02 MPa in the upper edge. The compressive stress distributed in the ground and the direction of the shear stress are both responsible for the bending that is observed.

Sensitivity analysis

A sensitivity analysis was carried out to evaluate the effect of the mechanical properties of the ground and of the structure on the results discussed above. Whole of the investigation are presented in Table 1 and the main observations are discussed below:

• All of the calculations showed that the mechanical properties of the structure have no effect on the results. In reality the structure is much more rigid than the ground in all of the calculations: The Young's modulus of the structure is at least five times greater than that of the ground, and the calculation for plane strains leads to an overestimation of the structure stiffness.

• An increase of the ground Young's modulus leads to an increase of the phenomena described above. In a curved area, it can be seen that the disconnected length of the structure increases with a higher modulus of elasticity of the ground (100 MPa) and disappears completely with a lower modulus (30 MPa).

•

The horizontal stress in the structure increases and show a bending moment which increases as the ground stiffness increases.

Irrespective of the selected mechanical properties, the stress state in the ground and in the structure is more disrupted in a concave area than in a convex area.

An increase of the plastic strain value is observed in the model with increasing of the ground modulus of elasticity. In particular, the analysis of a compressive strain with a ground Young's modulus of 300 MPa gives clearly inconsistent results. The model thus produced does not make it possible to describe the behaviour of a ground domain subject to large plastic strain.

An overall picture of the numerous quantitative results that was obtained is provided by series of value of tensile stress in the structure observed in the different models. Figure 17 The ground movements that apply a larger load to the structure are, in order of importance: a concave curvature, a convex curvature, a compressive strain and a tensile strain. It can also be seen that the ground mechanical properties have a relative greater influence on the effect of a curvature than on the effect of horizontal strain.

Using the set of results shown above, diagrams can be constructed of the behaviour of a structure in a curvature area or in a strain area. These models are shown in Figure 19 and 21. These figures show the location of slippage lines and the distribution of the vertical stress in the ground and under the structure at various depths. A comparison with Figure 3, 4 and 20 reveals that some aspects are similar, in particular, the slip lines proposed by Speck and Bruhn [START_REF] Speck | Non-uniform subsidence ground movement and resulting surfacestructure damage[END_REF] or Kratzsch [START_REF] Kratzsch | Mining Subsidence Engineering[END_REF] are fairly similar to that shown in Figure 21. This confirms our results, since the slippage lines have been observed experimentally on scale models in sand. The horizontal stress observed along the middle section of the structure is also highlighted. The stress is shown as a function of a parameter "a" for the single purpose of being able to make comparisons between them. This state of stress corresponds to the investigation presented in detail in Figure 13 to 16 (E ground =100 MPa, E s nucture=5000 MPa). It is possible to adjust this state of stress, for other cases, using the synthetic results of Figure 17 and 18. This distribution of stress may be compared to the distribution proposed by Kratzsch [START_REF] Kratzsch | Mining Subsidence Engineering[END_REF] and shown in Figure 20. The difference is, however, that his approach superimposes the effects of curvature and horizontal strain.

In view of the adopted failure criteria and of the extent of the plasticity that is observed, superimposing the effects of basic stresses is not possible for deducing the actual loading. A global model must then be used to load a structure simultaneously by a curvature and a strain (Deck andal., [START_REF] Deck | Study of consequences of mining subsidence on buildings by numerical modelling (Etude des conséquences des affaissements miniers par la modélisation numérique)[END_REF]). The investigation of the mechanical work needed to deform the ground of the structure in a curved area made it possible to distinguish the cases in which the curvature is certainly imparted to the structure, from the cases in which it is not. By considering the structure specific weight, it is also possible to estimate whether the structure is partially disconnected or whether it rests fully on the ground.

Modelling a structure by beam elements assembled together by hinged joints of variable rigidity makes it possible to evaluate a structure ability to be deformed horizontally. It is possible to compare the resulting structure stiffness which results from the ground stiffness.

This method makes it possible to distinguish cases in which the ground horizontal strain is imparted to the structure, from other cases.

The behaviour of a flexible structure with regard to the ground may be investigated by means of structure calculation software, imposing the ground displacements to the structure. The phenomena at work when the structure is rigid and such transmission is not complete, was investigated by considering the behaviour of a rigid structure in a curved area and in a strained area.

Numerical modelling has provided comparison between the effects of the different loadings and the effects of the ground mechanical properties.

•

The investigation of the ground curvature is consistent with the behaviour from intuitive models devised to date.

•

The strain investigation shows that this aspect is more complex than assumed until now. In particular, strain causes a bending moment in the structure that is not negligible compared with the one induced in the curvature area. Tensile strain also produces immediate rupture in the ground which prevents the stress from being imparted to the structure.

•

We propose overall behaviour models of a structure in a curvature area and in a strain area. The latter provides a better understanding of the phenomenon and a better assessment of the effectiveness of the preventive measures that are proposed to protect buildings.

•

The overall behaviour model that we propose in a tensile area suggests that the structure stability cannot be guaranteed unless the ground is secured. However, such instability cannot be described by the numerical model that was used.

6 Bibliography

Figure 2 :

 2 Figure 2: Example of a mining cave-in in South Africa: vertical displacement and horizontal strain, Merwe [3],

Figure 3 :

 3 Figure 3: Behaviour of a structure in a curvature area

Figure 4 :

 4 Figure 4 : Behaviour of a structure in a horizontal strain

Figure 5 :

 5 Figure 5 : Various possible behaviours of a structure in a concave curvature area

Figure 6 :

 6 Figure 6 : Calculation of force and work required to deform the ground.

Figure 7 :

 7 Figure 7 : Methodology for estimating whether the ground curvature is imparted to the structure.

Figure 7 Figure 7 .

 77 Figure 7 then summarises the method and the results. For a given ground radius of curvature and structure length, it is possible to compare the different works W] and W 2 . The lowest indicates the most probable deformation mode, which corresponds to test No. 1 in Figure 7. According to the selected scenario, one then must determine whether the actual weight of the structure is sufficient to allow the structure or the ground to become

Figure 10 :i

 10 Figure 10 : Horizontal stiffnesses calculated for the various types of structures modelled.

Figure 11 :

 11 Figure 11 : Horizontal stiffness of the ground. Blow up shows a portion of the ground increasing its own stiffness.

Table 1 :

 1 Description of the various numerical investigations carried out Investigation of the effect of the various stresses for constant mechanical properties. Purpose Investigation in a ground curvature area Effect in a ground horizontal strain area Mechanical properties taken into account Eground: 100 MPa Estructure : 5000 MPa E^: 100 MPa Estructm-e : 5000 MPa Movements investigated Concave and convex radius of curvature: +/-7500, +/-5000, +/-2500 et +/-1000 m Compressive and tensile strain: +/-10, +/-5, +/-2.5, +1-1 and +/-0.5 mm/m Effect of mechanical properties for a constant stress Purpose Investigation in a ground curvature area Effect in a ground horizontal strain area Mechanical properties taken into account Eground: 30, 100 and 300 MPa E^ure: 1500, 5000, 10000 and 15000 MPa E g round: 30, 100 and 300 MPa Estructure: 1500, 5000, 10000 and 15000 MPa Stresses investigated Concave radius of curvature: -1000 m Convex radius of curvature: 1000 m Compressive strain. -5 mm/m Tensile strain: 5 mm/m

Figure 12 :

 12 Figure 12 : Description of the models used to investigate

  that shows different diagrams of the operating mechanism in a curvature area. A concentration of vertical stresses at the ends of the structure is expected in a concave area and at the centre in the convex area in the centre. The schematic diagrams of Figure 3 can be compared to the results obtained with numerical modellings, Figure 13 and 14. These figures respectively show the results in the entire model and along two sections shown in figure 12 and located horizontally at a depth of 15 cm in the ground (section "BC") and vertically in the centre of the structure (section "AB"). Interesting aspects of these results are discussed below. Failure area differs from the failure area obtained in the reference investigation. The plastic points are concentrated perpendicularly to the areas on which the structure rests preferentially. These points are thus deduced from the high values of the vertical stress. Failure which occur at the right hand edge of the model is the consequence of the mesh size in these area and of the zero cohesion of the ground.The representation of the principal stresses highlights remarkably the stress redistribution in the ground. Stresses observed in the structure reflect a bending movement, consistent with the ground curvature. However, the structure curvature is never as high as the ground curvature. The structure stiffness is such that it retains a horizontal geometry.

Figure 13 :

 13 Figure 13 : Stress fields and failure areas in a curvature area (+/-1000m)

Figure 14 :Figure 15 :c

 1415 Figure 14 : Stresses along a horizontal section under the structure and along a vertical section in the centre of the structure in a curvature area (+/-1000m)

Figure 17 :

 17 Figure 17 : Tensile stress observed in the structure for

Figure 18 :

 18 Figure 18 : Tensile stress observed in the structure for different loadings and for different values of the ground Young's modulus.

Figure 19 :

 19 Figure 19 : Behaviour of a structure with regard to a ground curvature.

Figure 20 :Figure 21 :

 2021 Figure20: Horizontal stress in a structure subjected to a curvature and a strain(Kratzsch,[START_REF] Kratzsch | Mining Subsidence Engineering[END_REF]).


Wilfried Sanchez

Olivier Palluel

Laurent Meunier

Marina Coquery

Jean-Marc Porcher

Selim Ait-Aissa
email: selim.ait-aissa@ineris.fr

Sélim Aït-Aïssa

Copper-induced oxidative stress in three-spined stickleback : relationship with hepatic metal levels

Keywords: bioaccumulation, biomarkers, copper, liver, oxidative stress, three-spined stickleback

Introduction

Mining activity, domestic waste emission or application of fertilizers and pesticides induce contamination of aquatic ecosystems by heavy metals. Among them, copper is a widespread pollutant found in surface waters at concentrations up to 100 µg/L [START_REF] Roy | Environmental contaminants encyclopedia : copper entry[END_REF]. It is also a particular pollutant because it is an essential trace element for living organisms, used as a cofactor for structural and catalytic properties of a variety of enzymes involved in the biological processes of growth, development and maintenance [START_REF] Turnlund | Fish bioaccumulation and biomarkers in environmental risk assessment: a review[END_REF]. Although this metal is a required element, high concentrations appear to be toxic to freshwater organisms with no observed effect concentrations comprised between 4 and 120 µg.L -1 as reported for 14 fish species [START_REF] Grosell | Sodium turnover rate determines sensitivity to acute copper and silver exposure in freshwater animals[END_REF]. The freshwater predicted no effect concentration (PNEC) was recently evaluated for copper and the value of 1.6 µg.L -1 was proposed [START_REF] Grnc | Risque pour l'environnement -Evaluation des risques associés aux rejets chimiques des installations nucléaires du Nord-Cotentin[END_REF]. Copper toxicity varies with water chemistry, temperature and fish species and induces various damages that can lead to the death of the organisms.

At the cellular level, copper can interfere with several metabolic pathways and thereby induce different cellular responses. This metal has been well described as a promoter of oxidative stress by catalysing the formation of highly reactive oxygen species (ROS), such as HO ° radical through the Haber-Weiss reaction [START_REF] Matés | Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology[END_REF] and generating peroxidation of membrane lipids [START_REF] Chan | Copper (II)-catalyzed lipid peroxidation in liposomes and erythrocyte membrane[END_REF] and DNA alterations [START_REF] Ozawa | DNA single strand breakage by copper (II) complexes and hydrogen peroxide at physiological conditions[END_REF]. Copper also binds thiol containing molecules such as glutathione (GSH) or metallothioneins (MT) where it is trapped [START_REF] Roesijadi | Metallothionein and its role in toxic metal regulation[END_REF]. Direct interaction of copper with proteins can be the source of enzyme inhibition such as cytochrome P450 associated monooxygenase activities [START_REF] Kim | Differential effect of copper II on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase : inhibition of cytochrome P450-catalysed reactions by copper II ion[END_REF].

This study was conducted to characterise the response of several hepatic biomarkers, including oxidative stress and xenobiotic transformation biomarkers, in three-spined stickleback (Gasterosteus aculeatus L.) exposed to sublethal copper concentrations and to determine whether these responses are related to copper accumulation in the target organ.

Three-spined stickleback is present in all aquatic ecosystems in Northern hemisphere and is an abundant fish species in both unpolluted and polluted areas. Recently, this fish has been used as a model organism to study bioaccumulation of organic compounds [START_REF] Andersson | Bioaccumulation of selected PCBs in zebrafish, three-spined stickleback and arctic char after three different routes of exposure[END_REF][START_REF] Falandysz | Polychlorinated naphthalenes in three-spined stickleback Gasterosteus aculeatus from the Gulf of Gdansk[END_REF] and metals [START_REF] Bervoets | Accumulation of metals in the tissues of three spined stickelback (Gasterosteus aculeatus) from natural fresh waters[END_REF], as well as biochemical biomarker responses to various xenobiotics such as pesticides [START_REF] Sturm | Different sensitivity to organophosphates of acetylcholinesterase and butyrylcholinesterase from three-spined stickleback (Gasterosteus aculeatus): application in biomonitoring[END_REF], organics compounds [START_REF] Holm | Effects of exposure to food contaminated with PBDE, PCN or PCB on reproduction, liver morphology and cytochrome P450 activity in the three-spined stickleback, Gasterosteus aculeatus[END_REF] or environmental estrogens and androgens [START_REF] Katsiadaki | Detection of environmental androgens: a novel method based on enzyme-linked immunosorbent assay of spiggin, the stickleback (Gasterostus aculeatus) glue protein[END_REF][START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF]. To our knowledge, no study has investigated the effect of metals on the biomarker responses in three-spined stickleback. Hence, as its uses as a sentinel species for aquatic pollution biomonitoring has been suggested [START_REF] Handy | Histopathologic biomarkers in three spined sticklebacks, Gasterosteus aculeatus, from several rivers in Southern England that meet the freshwater fisheries directive[END_REF][START_REF] Pottinger | The three-spined stickleback as an environmental sentinel: effects of stressors on whole-body physiological indices[END_REF], there is still a need to characterise biomarker responses to widespread environmental contaminants in this fish species.

The biomarkers that we measured were enzymatic [superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx)] and non enzymatic (GSH) antioxidants, as well as two biotransformation enzymes [7-ethoxyresorufin-O-deethylase (EROD) and glutathione-Stransferase (GST)] involved in oxidative metabolism of xenobiotics. Furthermore, copper measurement in liver was used to link metal accumulation with biochemical responses. In addition, hepatic zinc content was measured in order to evaluate whether MT induction occurred during our experiment.

Materials and methods

Exposure protocol

Male and female adult sticklebacks (3.4-6.8 cm) were collected in outdoor artificial streams during spring 2003 and maintained in denitrated water (ionic composition : K, 3.0 mg.L -1 ; Mg, 11.7 mg.L -1 ; Na, 10.9 mg.L -1 ; Si, 5.8 mg.L -1 ; Ca, 55.5 mg.L -1 ; Al<0.01 mg.L -1 ; Fe<0.02 mg.L -1 ; PO 4 3-, <0.1 mg.L -1 ; NH 4 + , 0.3 mg.L -1 ; Cl -, 58 mg.L -1 ; NO 3 -, 0.45 mg.L -1 ; SO 4 2-, 11.7 mg.L -1 ; alkalinity, 367 mg.L -1 as CaCO 3 ; pH, 8.35 ± 0.05) for 3 weeks prior to experimentation. Fish were randomly distributed in four groups of 30 fish and exposed to waterborne copper sulphate in semi-static conditions with complete water renewal and food supply every 3 days. The concentrations of copper were chosen to be representative of environmental concentrations encountered in polluted freshwaters (25 and 100 µg Cu.L -1 ) with an additional higher concentration (200 µg Cu.L -1 ) that was expected to exert subtoxic events. The control group received no added copper. Water copper concentrations in each aquarium were controlled by inductively coupled plasma optical emission spectrometry (Ultima 2, Jobin Yvon) and showed that actual copper concentrations were stable between two water changes (data not shown) and throughout the experiment (table 1). The water temperature was 13.5°C ± 0.4°C and the photoperiod was a light/dark cycle of 8/16 hours.

Sampling, biometrical and biochemical analysis

At day 0, 4, 8, 12 and 21, six fish per concentration were randomly sampled. Fish were sacrificed, measured and weighed. Liver, gonads and spleen were dissected and weighed to calculate the corresponding somatic index according to the following equation : organ somatic index = (organ weight / fish weight) x 100.

Liver was homogenized in 200 µL of ice-cold phosphate buffer (100 mM, pH 7.8) supplemented with 20% v/v glycerol and 0.2 mM phenylmethylsulfonyl fluoride as a protease inhibitor. The homogenate was centrifuged at 10,000 g, 4°C, for 15 min and the supernatant was used for biochemical assays. Total proteins were determined using the method of [START_REF] Bradford | A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding[END_REF] with bovine serum albumin (Sigma) as a standard. SOD, CAT and GPx were assessed according to the methods of [START_REF] Paoletti | A sensitive spectrophotometric method for the determination of superoxide dismutase activity in tissue extracts[END_REF], [START_REF] Babo | In vitro effects of thiram on liver antioxidant enzyme activities of rainbow trout (Oncorhynchus mykiss)[END_REF] and [START_REF] Paglia | Studies on the quantitative and qualitative characterization of erythrocyte glutathione peroxidase[END_REF] respectively, using purified bovine enzymes (Sigma) as standards. For GST activity determination, chlorodinitrobenzene was used as substrate [START_REF] Habig | Glutathione S-Transferases. The first enzymatic step in mercapturic acid formation[END_REF] and purified GST from equine liver (Sigma) as a standard. GSH and oxidised glutathione (GSSG) concentrations were measured by the spectrophotometric method of [START_REF] Vandeputte | A microtiter plate assay for total glutathione and glutathione disulfide contents in cultured/isolated[END_REF]. All measures were carried out at room temperature in microtiter plates, using a microplate reader (Power Wave x -Bio-Tek instruments). EROD activity was determined by a fluorimetric method in black microplates [START_REF] Flammarion | Statistical analysis of cyprinid ethoxyresorufin-O-deethylase data in a large french watershed[END_REF] using a microplate spectrofluorimeter reader (Victor 2 Wallac, Perkin-Elmer).

Chemical analysis

Hepatic copper and zinc concentrations were determined after digestion of homogenized liver using suprapure nitric acid (Carlo Erba) at 120 °C for at least 2 h (1 mL of nitric acid per 20 µL of sample). The volume was then adjusted to 20 mL with de-ionized water. Metal concentrations were determined by inductively coupled plasma optical emission spectrometry (Ultima 2, Jobin Yvon). Quality assurance controls based on certified reference materials of the National Research Council of Canada (DOLT-2, dogfish liver) were carried out to validate this procedure.

Statistics

All statistical calculations were performed with SPSS 10.1 software. Since data were not normally distributed (Lilliefors' test) and/or exhibited heterogeneous variances (Levene's test), non-parametric tests (Kruskall-Wallis ANOVA and Mann-Whitney U test) were performed (significant for p<0.05). Data are reported as means ± standard error.

Results

During this experiment, low mortality was observed for fish exposed to 200 µg.L -1 . Four out of 30 fish died during the first five days and one died at day 16. This mortality prevented the analysis of hepatic copper concentration and biomarkers at day 21. At this concentration, the surviving fishes appeared stressed as indicated by a dark colour, a lower mobility and a loss of appetite. Those behaviour alterations were not observed in the other groups.

A significant bioaccumulation of copper was observed in liver of fish exposed to 200 µg.L -1 for 8 and 12 days and to 100 µg.L -1 for 12 days (Fig. 1A). At day 21, a trend is noted for copper accumulation at 100 µg.L -1 although this was not statistically significant. An increase of zinc concentration in liver was noticed after 12 days of exposure to 100 and 200 µg.L -1 , whereas a transient increase of zinc level was noticed after 4 days at 25 µg.L -1 (Fig. 1B).

No significant variation of somatic indexes was observed for any copper concentration nor any sampling time (data not shown), whereas several differences were observed at the biochemical level.

As seen in Fig. 2, a rapid and transient induction of antioxidant enzymes was measured within the first week of copper exposure. SOD activity was clearly increased at day 4, although this effect was statistically significant only at 25 µg.L -1 and returned to the basal level at day 8 (Fig. 2A). CAT activity was characterised by a general increase for all copper treatments after 4 days (Fig. 2B). This increase was maintained up to 12 days in fish exposed to 200 µg.L -1 .

GPx activity showed a significant increase after 8 days in fish exposed to 200 µg.L -1 and returned to the basal level at day 12 (Fig. 2C).

In control fish, total and oxidized GSH were characterised by an important variation of the basal level during the experiment (Fig. 3). Nevertheless, some effects of copper can be seen for this biomarker. A significant decrease of both total and oxidised GSH content was noticed in fish exposed to 200 µg.L -1 after 4 and 8 days of exposure (Fig. 3A and3B). This depletion was not accompanied by an impairment of glutathione redox status as no effect on GSSG/GSH ratio was noticed (not shown). After 12 days of exposure, this effect was reversed and fish exposed to the highest copper concentration presented a total glutathione level significantly higher than control fish.

For EROD activity measured in fish exposed 21 days to 100 µg.L -1 , a significant inhibition was observed as compared to the control group (Fig. 4A). For the other concentrations and sampling time, no significant variation of this enzyme was detected as compared to the control group. The hepatic GST activity did not vary significantly with respect to either copper concentration or time of exposure (Fig. 4B).

Discussion

Despite the low mortality observed after 4 days at the highest copper concentration, our exposure conditions were clearly sublethal. For three-spined stickleback exposed to copper sulphate, the 50 % lethal concentrations (LC50) were reported to be 2.78, 1.96 and 1.59 mg Cu.L -1 after 24, 48 and 72 hours respectively [START_REF] Svecevicius | Acute toxicity of copper to common freshwater fishes of Lithuania[END_REF], which are much higher than the concentrations used in our study. However, comparison is rather difficult since copper toxicity is highly dependent on several abiotic factors, such as pH, water hardness and alkalinity that influence copper speciation. The physico-chemical forms in which the metal is present in the aquatic environment (free ionic Cu 2+ , dissolved inorganic or organic complexes, associated with particular matter) will indeed determine its bioavailability for the organisms. In our study, the slightly basic conditions (pH 8.35) together with the elevated water hardness (367 mg.L -1 as CaCO 3 ) and very low dissolved organic carbon (DOC) suggest that a major fraction of the copper in the water is present as inorganic complexes (mainly CuCO 3 ) and only a small fraction as free Cu 2+ . Nevertheless, our results

showed that these inorganic copper complexes are also bioavailable to the organisms since significant accumulation and biochemical responses to copper were observed.

At day 12, copper did accumulate at similar levels in the liver of fish exposed either to 100 or 200 µg.L -1 (Fig. 1). Copper concentrations in liver measured during this laboratory exposure are in accordance with the levels observed in three-spined stickleback sampled in the field [START_REF] Bervoets | Accumulation of metals in the tissues of three spined stickelback (Gasterosteus aculeatus) from natural fresh waters[END_REF]. This may indicate that saturation for copper accumulation occurred right from 100 µg.L -1 and is in agreement with the fact that, in rainbow trout (Oncorhynchus mykiss), the time to half saturation for copper in the liver is 6 days [START_REF] Mcgeer | Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout 2: tissue specific metal accumulation[END_REF]. At day 12, the bioconcentration factor (BCF = ([Cu] exposed -[Cu] control ) / [Cu] water ) reached 401 and 208 in the 100 and 200 µg.L -1 groups-exposed fish respectively, which are in the same order of magnitude to those previously reported for zebrafish (Brachydanio rerio) exposed for 14 days to copper [START_REF] Paris-Palacios | Biochemical and (ultra)structural hepatic perturbations of Brachydanio rerio (Teleostei, Cyprinidae) exposed to two sublethal concentrations of copper sulfate[END_REF]. These relatively low BCF, as compared to those reported for other metals such as Cd, may reflect the existence of homeostatic regulation of copper levels in the liver [START_REF] Mcgeer | Effects of chronic sublethal exposure to waterborne Cu, Cd or Zn in rainbow trout 2: tissue specific metal accumulation[END_REF].

The biomarker responses in stickleback were in accordance with the known capacity of copper to promote oxidative stress and to modulate enzymatic and non enzymatic antioxidant expressions in fish tissues, as shown by a rapid and transient increase of SOD, CAT and GPx enzymes and by a decrease of total GSH. In fish, antioxidant enzymes have been shown to be either induced or inhibited by copper, depending on the dose, the species and/or the route of et al., 2000). Conversely, in carp, CAT and GST were inhibited after 96 hours of exposure to 100 and 250 µg Cu.L -1 as CuSO 4 (Dautremepuits et al., 2002). Using intra-peritoneal injection of copper, SOD inhibition was reported in carp after 48 hours (Varanka et al., 2001), while this enzyme was rapidly induced in gilthead seabream (Sparus aurata; [START_REF] Pedrajas | Oxidative stress in fish exposed to model xenobiotics. Oxidatively modified forms of Cu,Zn-superoxide dismutase as potential biomarkers[END_REF].

In stickleback, the rapid response of both enzymatic and non enzymatic antioxidants is in line with the view that copper is able to act through different oxidative mechanisms, including generation of ROS and direct interaction with intracellular thiols.

SOD are metalloenzymes that play a key role in the defence against ROS by transforming superoxide anions into hydrogen peroxide (Yim et al., 1993). The total SOD activity that we measured in the post-mitochondrial fraction corresponds mainly to the Cu,Zn-SOD activity, the major isoform in the cytosol, nucleus and peroxisomes. Thus, the rapid and marked drop in SOD activity at day 4 may result from direct binding of the metal to the enzyme, as previously suggested in fish [START_REF] Pedrajas | Oxidative stress in fish exposed to model xenobiotics. Oxidatively modified forms of Cu,Zn-superoxide dismutase as potential biomarkers[END_REF]. In addition, induction of CAT and GPx indicated that copper induce (hydro)peroxides species in the liver. It is likely that the resulting H 2 O 2 produced from increased SOD activity was the source for subsequent increase of CAT activity after the fourth day of copper exposure (Fig. 2B). Moreover, the known capacity of copper to oxidize membrane lipids in fish [START_REF] Pedrajas | Oxidative stress in fish exposed to model xenobiotics. Oxidatively modified forms of Cu,Zn-superoxide dismutase as potential biomarkers[END_REF], together with the role of GPx in the detoxification of lipoperoxidation products [START_REF] Matés | Effects of antioxidant enzymes in the molecular control of reactive oxygen species toxicology[END_REF] may also account for the observed increase of this enzyme at the day 8 (Fig. 2C). Unfortunately, the induction of lipid damages by copper was not measured in this study to confirm this hypothesis.

Reduced glutathione is considered as a first line of cellular defence against metals by chelating and detoxifying them, scavenging oxyradicals and participating in detoxification reactions catalysed by glutathione peroxidases [START_REF] Sies | Glutathione and its role in cellular functions[END_REF]. The effects that we observed on this parameter, i.e. inhibition of total GSH (both reduced and oxidised GSH) without any impairment of GSSG/GSH ratio, is in line with the known ability of copper to interact with GSH forming stable GS-Cu(I) binding complexes. Besides, as proposed by [START_REF] Canesi | Heavy metals and glutathione metabolism in mussel tissues[END_REF], it is likely an inhibitory effect on GSH regenerating systems (e.g. GSH reductase or GSH synthetase), which was not assessed in our study, that could have contributed to the observed depletion of total GSH. The fact that we did not observe any impairment of GSSG/GSH ratio by copper does not reach previous report in gilthead seabream fish exposed by copper intra peritoneal injection [START_REF] Rodriguez-Ariza | Rapid determination of glutathione status in fish liver using high-performance liquid chromatography and electrochemical detection[END_REF]. Finally, although the mechanism by which copper inhibited GSH in fish is not fully understood and remains complex, this rapid depletion may have contributed to the early toxicity observed at the higher concentrations as suggested by [START_REF] Conners | Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica)[END_REF].

Interestingly, biochemical responses were more rapid than copper accumulation, suggesting the involvement of differential mechanisms in copper metabolism and accumulation. Cellular copper metabolism involves copper intake by GSH and subsequent formation of GS-Cu(I) complex, from which the metal is further transferred to metallothionein (MT) apoproteins where it is stored [START_REF] Freedman | The role of glutathione in copper metabolism and toxicity[END_REF][START_REF] Conners | Effects of glutathione depletion on copper cytotoxicity in oysters (Crassostrea virginica)[END_REF]. The GS-Cu(I) pool is the source for the synthesis of other metalloenzymes such as SOD or cytochrome oxidase, as well as for ROS production through GS-Cu(I) oxidation [START_REF] Freedman | The role of glutathione in copper metabolism and toxicity[END_REF]. In our study, antioxidant responses occurred within the first week of exposure and then recovered concomitantly with copper accumulation in the liver (Fig. 1 and3). This indicates that at day 8 other detoxification systems, such as MTs, have been induced to replace GSH in the uptake and sequestration of copper [START_REF] Freedman | The role of glutathione in copper metabolism and toxicity[END_REF]. Moreover, the role of MTs in cellular protection against oxidative stress (Viarengo et al., 2000) argues in the way that these proteins could have been involved in response to copper in three-spined stickleback. This hypothesis is strengthened by the finding that copper accumulation was accompanied by increased hepatic zinc concentrations. In fact, a significant positive correlation was observed between these two metals (r 2 = 0.596, p<0.001, Spearman correlation). The sequestration of metals such as cadmium and copper occurs by displacement of zinc from their binding sites, leading to a release of free zinc that could induce de novo MT synthesis [START_REF] Hollis | Tissue-specific cadmium accumulation, metallothionein induction, and tissue zinc and copper levels during chronic sublethal exposure in juvenile rainbow trout[END_REF][START_REF] Roesijadi | Metallothionein and its role in toxic metal regulation[END_REF]. Relationships between liver metal content and MT expression have been described in aquatic organisms [START_REF] Hollis | Tissue-specific cadmium accumulation, metallothionein induction, and tissue zinc and copper levels during chronic sublethal exposure in juvenile rainbow trout[END_REF] but further studies on MT response to copper in stickleback will be necessary to confirm these hypotheses for this fish species.

EROD inhibition by metals has been reported in several in vitro and in vivo studies on fish [START_REF] Ghosh | Impact of copper on biomonitoring enzyme ethoxyresorufin-o-deethylase in cultured catfish hepatocytes[END_REF][START_REF] Stien | Effect of copper chloride in vitro and in vivo on the hepatic EROD activity in the fish Dicentrarchus labrax[END_REF]. In our study, we detected an inhibition of EROD activity in fish exposed to 100 µg.L -1 for 21 days (Fig. 4). This effect, which involves direct interaction of copper with the enzyme leading to a conformational change of the protein [START_REF] Kim | Differential effect of copper II on the cytochrome P450 enzymes and NADPH-cytochrome P450 reductase : inhibition of cytochrome P450-catalysed reactions by copper II ion[END_REF], may reflect cellular toxicity induced by an increased content of metal in the cell [START_REF] Stien | Effect of copper chloride in vitro and in vivo on the hepatic EROD activity in the fish Dicentrarchus labrax[END_REF]. Although this effect could be considered as minor regarding the responses of the other biomarkers to copper, EROD inhibition should be taken into account when using it as a biomarker of exposure in fish exposed to a mixture of chemicals, i.e. containing both organic and metallic compounds, which mostly occurs in environmental pollution.

In summary, this dynamic study showed that waterborne copper induced several biochemical responses in three-spined stickleback, in a transient manner, suggesting the occurrence of adaptive processes. Rapidly after the beginning of the exposure, antioxidant biomarkers were induced in order to compensate the oxidative stress generated by the metal. In a second phase, copper accumulation was correlated to a recovery of GSH and a return of antioxidant

  exposure. Zebrafish submitted to 40 and 140 µg Cu.L -1 as CuSO 4 presented a clear-cut induction of CAT and GST within two weeks of waterborne copper exposure (Paris-Palacios

Fig. 4 .

 4 Fig. 4. Effect of copper treatment on biotransformation enzyme activities : EROD (A) and

Fig

  Fig. 1.

  enzymes to the basal level, suggesting that other detoxification mechanisms, such as MTs, have been involved to allow fish adaptation to copper. This study shows the suitability of three-spined stickleback as a model fish species to study oxidative stress biomarker responses to waterborne metal exposure. Field studies that evaluate several biomarkers, including oxidative stress biomarkers, EROD and vitellogenin, are under progress to determine the potential of stickleback as an indicator species of sublethal stress in multipollution contexts. Effect of copper treatment on hepatic copper (A) and zinc (B) concentration. Data represent the means ± S.E. of measurements on six fish per group except for the 100 µg.L -1 on day 21 (n=5). *, statistically different from t 0 control (P<0.05).

	cells: performance study of a new miniaturized protocol. Cell Biol. Toxicol. 10, 415-421. Varanka, Z., Rojik, I., Varanka, I., Nemcsok, J., Abraham, M., 2001. Biochemical and morphological changes in carp (Cyprinus carpio L.) liver following exposure to Fig. 1. Fig. 2. Effect of copper treatment on hepatic SOD (A), catalase (B) and GPx (C) activities.
	copper sulfate and tannic acid. Comp. Biochem. Physiol. C 128, 467-478. Data represent the means ± S.E. of measurements on six fish per group except for the 100
	Viarengo, A., Burlando, B., Ceratto, N., Panfoli, I., 2000. Antioxidant role of µg.L -1 on day 21 (n=5). *, statistically different from t 0 control (P<0.05).
	metallothioneins: A comparative overview. Cell. Mol. Biol. 46, 407-417. Fig. 3. Effect of copper treatment on activities of total glutathione (A) and oxidized
	Yim, M., Chock, P., Stadtman, E., 1993. Enzyme function of copper, zinc superoxide glutathione (B) content. Data represent the means ± S.E. of measurements on six fish per
	dismutase as a free radical generator. J. Biol. Chem. 268, 4099-4105. group except for the 100 µg.L -1 on day 21 (n=5). *, statistically different from 0 µg.L -1 control
	(P<0.05).

Table 1

 1 Actual copper concentrations measured in the water throughout the experiment a .

	Nominal copper	Measured copper
	concentration (µg.L -1 )	concentration (µg.L -1 )
	0	<0.05
	25	24.6 ± 1.5
	100	91.6 ± 2.8
	200	176.8 ± 2.3
	a : measurements were performed in water sampled 2 hours after each water renewal. Data are
	mean ± SE (n=7).

Acknowledgements

We wish to thank Jean-Pierre Blanquet for the measure of physico-chemical parameters of the water and Dominique Hervin for technical assistance in fish maintenance. This work was supported by the French Budget Civil de la Recherche et du Développement (BCRD 00-102).


Frédéric Y Bois

Keywords: PGME isomers, reproductive toxicology, developmental toxicity PGME reproductive toxicity E Lemazurier et al

Propylene glycol monomethyl ether. A three-generation study of isomer B effects on reproductive and developmental parameters in rats

Emmanuel Lemazurier, Anthony Lecomte, Franck Robidel,

1 Propylene glycol monomethyl ether. A 3-generation study of isomer effects on reproductive and developmental parameters in rats.

Emmanuel Lemazurier (*) , Anthony Lecomte, Franck Robidel and Frédéric Y. Propylene glycol monomethyl ether (PGME) is widely used as a solvent in numerous commercial products. Its chemical synthesis leads to the formation of 2 isomers, and , the later being usually present in the range of 0.5% to 1.5%. Isomer has been shown to be of low toxicity. Isomer raises concerns as to its reproductive and developmental effects.

We evaluated the reproductive and developmental toxicity of two different commercial mixes of PGME (Mix A 99% isomer and 0.5% isomer Mix B 98.5% isomer and 1.5% isomer ) on Sprague Dawley rats. The use of two mixes allowed us to differentiate between isomer and isomer effects. Male and female rats were exposed through drinking water to mixes A or B during a gametogenesis cycle (64 days for males and 15 days for females) to 0%, 2%, 5%, 10% and 15% (v/v) of each mix. These animals (F 0 ) and the three following generations (F 1 , F 2 and F 3 ) were followed.

We observed a statistically significant decrease in the number of pups in isomer treatedanimals of generation F 1 and a non dose-related variation of the sex ratio in F 1 and F 2

Introduction

Propylene glycol monomethyl ether (PGME) is a colorless liquid, miscible with water and many organic solvents. This makes it useful for a wide variety of solvent applications in the manufacture of lacquers, paints, dyes, inks, cleaning agents and liquid soaps, etc.

Commercial PGME typically contains at least 98.5% 1-methoxy-2-propanol ( isomer), and only a small amount of 2-methoxy-1-propanol ( isomer) [START_REF] Carney | Assessment of adult and neonatal reproductive parameters in Sprague-Dawley rats exposed to propylene glycol monomethyl ether vapors for two generations[END_REF].

An ECETOC report has reviewed studies up to 1995 on different PGME mix effects on development and reproduction by the oral, inhalation and subcutaneous routes of exposure (European chemical Industry Ecology and Toxicology Center [ECETOC], 1995).

Developmental studies with 100% of PGME isomer evidenced in rats and rabbits different skeletal malformations and variations but not with PGME mix containing 99.5% isomer .

Reproductive effects were also assessed in a 2-generation-rats inhalation study conducted with 0, 300, 1000 and 3000 ppm of PGME (98% isomer , 2% isomer ) for 6 hours/day, 5 days/week prior to mating and 6 hours/day, 7 days/week during mating, gestation and lactation [START_REF] Carney | Assessment of adult and neonatal reproductive parameters in Sprague-Dawley rats exposed to propylene glycol monomethyl ether vapors for two generations[END_REF]. These concentrations corresponded to 0, 400, 1300 and 4000 mg/kg/day. That study did no evidence any treatment-related effects at 300 ppm of PGME.

The no-observable-effect level for reproductive/neonatal effects was 1000 ppm, and that for parental toxicity was 300 ppm. The profile of generally low developmental or reproductive toxicity for PGME contrasts that of ethylene glycol monomethyl ether (EGME) and ethylene glycol monoethyl ether (EGEE), which are teratogenic and cause reproductive effects, and in particular testicular toxicity [START_REF] Hardin | Reproductive toxicity of the glycol ethers[END_REF][START_REF] Ecetoc | The toxicology of glycol ethers and its relevance to man[END_REF]. Two structural features seem to be key determinants of glycol ether reproductive and developmental toxicity [START_REF] Carney | Significanceof 2-methoxypropionic acid formed from bpropylene glycol monomethyl ether: integration of pharmacokinetic and developmental toxicity assessments in rabbits[END_REF]: (1) carbon chain length (inversely related to toxicity), and (2) the presence of a primary alcohol moiety. Glycol ethers containing primary alcohol moieties (e.g. EGME) are metabolized by alcohol dehydrogenases to the corresponding alkoxy acids established as the proximate developmental/reproductive toxicants [START_REF] Hardin | Reproductive toxicity of the glycol ethers[END_REF][START_REF] Ecetoc | The toxicology of glycol ethers and its relevance to man[END_REF]. Glycol ethers containing secondary alcohol moieties (e.g., PGME) are poor substrates for alcohol dehydrogenases and do not form appreciable quantities of alkoxy acid metabolites (Miller et al., 1983). Nevertheless, the PGME isomer has been shown to be metabolized, into the corresponding alkoxy acid metabolite [START_REF] Carney | Significanceof 2-methoxypropionic acid formed from bpropylene glycol monomethyl ether: integration of pharmacokinetic and developmental toxicity assessments in rabbits[END_REF].

In order to further evaluate the potential reproductive and neonatal toxicity of two different commercial mixes of PGME (Mix A 99% isomer and 0.5% isomer , Mix B 98.5% isomer and 1.5% isomer ) on Sprague Dawley rats, a 3-generation oral reproduction study was conducted. In addition to the standard parameters of a 2-generation test guidelines (U.S. EPA, 1999;OECD, 1983), behavior, demeanor, gross necroscopy, histology, weight, feed and water consumption, estrous cycle length and normality, litter size, sperm analysis, hormonal assay were also evaluated in adult and litters. Parental generation was exposed during a gametogenesis cycle and the effects on reproductive and developmental parameters of the F 0 , F 1 , F 2 and F 3 generations were evaluated.

Materials and methods

Test material

The PGME used in this study was obtained from Acros Organics (France). Two grades of purity were used: (1) PGME mix A: 99.5% propylene glycol monomethyl ether and 0.5% propylene glycol monomethyl ether and (2) PGME mix B: 98.5% propylene glycol monomethyl ether and 1.5% propylene glycol monomethyl ether

Animals

The study was conducted in accordance with French State Council guidelines for the care and

Experimental design

Groups of 13 male and 13 female rats were exposed to 0%, 2%, 5%, 10% and 15% PGME mix A or B (v/v) via drinking water for one gametogenesis cycle (64 days for males and 15 days for females respectively). Bottles were weighted each week in order to calculate the quantity of each mix drank by the rats. Treatment of the first parental generation F 0 rats, called P 1 when bred, began at approximately 6 weeks of age. After exposure, P 1 rats were mated (one male:one female) to produce the F 1 litters. Ten males and females per group were randomly selected from the F 1 weanlings and assigned to new groups to become the second parental generation (P 2 ). This second parental generation and followings were not treated. The P 2 adults were then mated to produce the F 2 litters. Ten males and females per group were randomly selected from the F 2 weanlings and assigned to new groups to become the third parental generation (P 3 ). The P 3 adults were then mated to produce the F 3 litters which were sacrificed on gestational day 19.

Breeding procedures

Breeding of the F 0 /P 1 adults started after 64 days (for males) and 15 days (for females) of treatment. Each female was placed with a single male from the same exposure group (10 females and 10 males) or with a non-exposed partner (3 treated females and 3 non-treated males or 3 non-treated females and 3 treated males) until pregnancy occurred or 2 weeks had elapsed. During each breeding period, daily vaginal lavage samples were evaluated for the presence of sperm, as an indication of mating. The day on which sperm were detected or a vaginal plug was observed in situ was considered as day 0 of gestation. If mating did not occur during the 2 weeks, the animals were separated without further opportunity to mate. For the F 1 /P 2 and F 2 /P 3 mating, cohabitation of male and female littermates was avoided. Three males and females from F 1 /P 2 and F 2 /P 3 litters born from treated groups were mated with nonexposed females and males, respectively.

Parental data

All adult rats were observed daily for changes in behavior, demeanor, or overt indications of toxicity. Rats found dead or moribund were submitted to a complete pathologic examination in an effort to determine the cause of death. F 0 /P 1 rats were weighted weekly during the treatment period. Feed and water consumption were measured, the latter allowing the calculation of an ingested dose. Estrous cycle length and normality were evaluated daily by vaginal lavage (Cooper et al., 1993) for all F 0 /P 1 , F 1 /P 2 and F 2 /P 3 females starting at the beginning of the cohabitation period of the F 1 /P 2 , F 2 /P 3 , and F 3 .

Litter data

All litters were examined as soon as possible after delivery. The following parameters were recorded for each litter: total litter size on the day parturition was initiated (day 0), the number of live and dead pups on days 0, 1, 4, 7, and 21 postpartum, the sex and the weight of each pup on days 1, 4, 7, 14, 21 of lactation. Any visible physical abnormalities or changes in demeanor in the neonates were recorded during the lactation period. All pups found dead, or pups that were euthanized in moribund condition, were examined to the extent possible for defects and/or cause of death and preserved in neutral, phosphate-buffered 10% formalin.

Sperm analysis

Sperm analysis was performed on F 0 /P 1 , F 1 /P 2 and F 2 /P 3 males. Sperm motility was first assayed. A dilution medium was prepared as follows: . bovine serum albumin (BSA) was added at 0.5% w/v to medium 199 with Hank's salts and L-glutamine (Sigma, St Quentin Fallavier, France). After BSA was dissolved, the solution was brought to pH 7.4 with 0.1N

NaOH. The left cauda epididymis from all males was used for sperm motility analysis. It was placed into a Petri dish containing 2 ml of warmed dilution medium (37°C) for 2 min on a heating plate. The cauda was held with forceps and one stab was made with a scalpel blade.

The Petri dish containing the concentrated sperm sample was gently swirled to evently distribute the non-diffused sperm. Ten µl of this solution was deposed on slide 80µ, double chamber (Hamilton-Thorne Research, Berverly, MA, USA) heating at 37°C on a heating plate. Sperm samples were then analyzed for motion using the HTM-IVOS (Hamilton-Thorne

Research, Berverly, MA, USA, user's manual version 12) (Appendix 1). Motion parameters measured are given in Appendix 2.

The sperm count per gram of testis and epididymis was also evaluated. Left testis was used to determine that number. Testis tunica albuginea was removed and testicular parenchyma was weighted and re-suspended with 15 ml of SMT medium (0.9% NaCl, 0.01% methiolate, 0.05% triton X-100). The mix was then crushed with an Ultra Thurax, medium speed during 30 seconds, twice. The solution was waved with a Vortex at maximal speed during 1 minute before a 1 ml sample of the solution was placed for 1 minute in an ultra sound bath in order to desegregate possible remains. After a new agitation with the vortex at medium speed during 30 seconds twice, 15 µl were deposited on a Malassay cell for reading under microscope with a 10 x lens. The epididymis was treated in the same way as the testis.

Hormonal assays

Testosterone (T) and 17 -estradiol (E2) levels were assayed with Enzyme Linked ImmunoSorbent Assay technique (ELISA) according to supplier protocol (R et D System, Lille, France). Intra-assay and inter-assay precision were 6.7% and 8%, respectively for E2 and 9.5% and 11.7%, respectively for T. The linearity calculated by (observed hormonal level/expected hormonal level) x 100 after dilutions of one assayed neat sample was 98% for E2 and 96% for T. The sensitivity of the assays was 10.1 pg/ml for E2 and 3.8 pg/ml for T.

Statistical evaluation

Statistical significance was performed using the S-PLUS2000 software. Parental data were analyzed by ANOVA linear regression. Lost, pre-coital delay and pups number values were regressed on isomers or PGME concentrations. Sex ratio differences were tested using a binomial distribution test [START_REF] Steel | Principles and Procedures of Statistics[END_REF] .

Results

PGME intake

Table 1 gives the calculated isomer PGME and isomer PGME dose ingested by F 0 /P 1 generation treated with PGME mix A and mix B. The total dose of isomer PGME ingested by rats treated with PGME mix A and mix B was about the same (Table 1A and1B). As expected, the dose of isomer PGME was higher for rats treated with PGME mix B (Table 1A and1B) than those treated with PGME mix A.

Parental data

The main effects observed on F 0 /P 1 males and females were a decrease in weight (20% to 50%) related to a decrease in food intake (data not shown 1 and2). No change in number of lost pups was noticed in F 0 /P 1 generation (Figures 1 and2). The effects observed appear at lower PGME beta concentrations than PGME alpha concentrations. Parents of the F 1 /P 2 and F 2 /P 3 generations did not show any trouble (both F 1 /P 2 male and female issued from F 0 /P 1 treated animals or only F 1 /P 2 male or female issued from F 0 /P 1 treated animals bred with a non treated animal).

Those generations did not have a 15% PGME mix A treated group because of the mortality observed in this group in F 0 /P 1 generation.

Litter data

A diffuse effect on the distribution of male and female pups and on sex ratio was observed.

Even though no statistical effect was evidenced using a binomial distribution test, the sex ratio was inverted in both F 1 /P 2 and F 2 /P 3 generations without any relation to treatment when both F 0 /P 1 male and female as well male or female were treated with mix A or B PGME. Due to a technical problem, we did not register sex ratio data concerning the animals issued from F 0 /P 1 females treated with PGME 5% and bred with a non-treated male. But that effect was not significant for none of the treated groups (data not shown).

The main observed effect was a decrease in the number of male pups per litter, directly responsible in most cases of the inversion of the sex ratio. We also observed a decrease in both male and female numbers per litter as well as an increase of female number, without inversion of the sex ratio. None of these effects evidenced any dose relation. But that effect was not significant for none of the treated groups (data not shown).

Sperm analysis

The sperm count per gram of epididymis in F 0 /P 1 generation was strongly disturbed by beta PGME treatment (Figures 3 and4).

In F 0 /P 1 epididymis, we evidenced a statistically significant dose-related decrease in sperm count of 56% in animals treated with 1,120 mg/kg/d of PGME alpha and 16. 4). No significant effect was observed in F 1 /P 2 or F 2 /P 3 epididymis or testis (Data not shown).

Data collected on percentage of motile sperm, VCL, VAP, VSL did not show any variations in any of treated group when compared to control.

Hormonal assay

Hormonal assays did not show any variations in estradiol or testosterone concentration in F 0 /P 1 , F 1 /P 2 male or female groups (data not shown).

Discussion

The goal of this work was to evaluate effects of 2 mixes of commercial PGME (99.5% isomer , 0.5% isomer and 98.5% isomer , 1.5% isomer ) on rat reproductive and developmental features. We chose a 3-generation test study in which only the first parental generation (F 0 /P 1 ) was treated during a whole gametogenesis cycle in order to evaluate possible persistent effects. We chose to treat animals via drinking water to ensure that the treatment continuously covered a whole gametogenesis cycle, 24 h a day, 7 days a week. In the case of rats, a cycle of gametogenesis extends on approximately 60 days in male and 15 days in female (OCDE, 1998). Isomer and PGME intake by rats were obtained from measurements of water consumption. Even though the chosen PGME concentrations were high (between 1,000 and 5,000 mg/kg/day for PGME and 5 to 8 mg/kg/day for PGME ), they were still in accordance with those used in previous works (ECETOC report 1995).

These exposure doses are much higher than those encountered during occupational exposures (about 2 mg/kg/day of PGME according to Devanthery et al., 2000). PGME typical exposure should be about a hundred times lower since PGME mixes contains only 0.5 to 1.5% isomer .

Among the tested generations, F 0 /P 1 was the most affected, showing a decrease in litter size above about 3,000 mg/kg/d PGME and 16.75 mg/kg/d PGME (Figures 1 and2). This generation also evidenced an increase in pre-coital delay above about 3,000 mg/kg/d PGME and 19 mg/kg/d PGME The decrease in litter size was not related to a postpartum effect since number of lost pups did differ between the various PGME dose groups assayed after birth. This decrease in litter size was therefore related to implantation loss.

These results are in accordance with numerous publications on the subject (ECETOC, 1995).

In [START_REF] Carney | Assessment of adult and neonatal reproductive parameters in Sprague-Dawley rats exposed to propylene glycol monomethyl ether vapors for two generations[END_REF] sex ratios did not differ statistically between controls and litters issued from parents treated with 0, 400, 1300 and 4000 mg/kg/day of PGME (98% isomer , 2% isomer ) prior to mating and during mating, gestation and lactation. In our work, the sex ratio of F 1 /P 2 and F 2 /P 3 litters was also not affected. However, when both parents or one of the parents were treated with PGME mix A or B a slight but non-significant increase in the number of males in the litters was observed. If this effect were to be confirmed, it would imply that treatment with PGME and mixes have a specific effect on the number of male in litters, directly related to the sex chromosome. So far, no evidence has been provided of a specific effect of PGME or any glycol ethers on sex chromosomes. El [START_REF] El-Zein | Exposure to ethylene glycol monomethyl ether: clinical and cytogenetic findings[END_REF] show a genotoxic effect of EGME in human: Mexican children, issued from women strongly exposed to EGME before or during pregnancy, evidenced the same malformations than those observed in Turner syndrome. The X chromosome has been shown to be responsible for this syndrome [START_REF] Noonon | Noona Syndrome: an update and review for the primary pediatrician[END_REF]. Furthermore, the observed malformations were strongly correlated to chromosomal aberrations in blood cells. It would be of interest to check whether PGME and mixes could be responsible for X chromosome aberrations leading to the increase of male numbers in subsequent generations.

The most relevant results of our work came from sperm analysis. We showed a large decrease (> 50%) in sperm counts in epididymis of F 0 /P 1 generation treated with PGME mix and (Figures 3 and4). Sperm count in F 0 /P 1 epididymis decrease was statistically significant for PGME isomer doses as low as 14.55 mg/kg/d and this effect was related to treatment. We did not use data on percentage of progressively motile sperm, ALH, BCF, linearity and straightness because these have been shown to be no relevant in toxicology study (Kato et al., 2001). The mechanism could involve the bioconversion of isomer into MPA [START_REF] Carney | Significanceof 2-methoxypropionic acid formed from bpropylene glycol monomethyl ether: integration of pharmacokinetic and developmental toxicity assessments in rabbits[END_REF] and raises concern about MPA persistance and reprotoxicity. [START_REF] Carney | Significanceof 2-methoxypropionic acid formed from bpropylene glycol monomethyl ether: integration of pharmacokinetic and developmental toxicity assessments in rabbits[END_REF] showed that, in rabbits, PGME isomer was totally and rapidly bioconverted into 2-methoxypropionic acid (MPA), which in turn had a relatively long elimination half time (33-44 hours). Our results show that high daily exposure to a commercial PGME formula is harmful to male sperm in rats, and that this effect is directly related to the small amount (0.5% or 1.5%) of isomer in the mixture.

In this work, we found a NOEL of 11.5 mg/kg/d for PGME -induced sperm count decrease in rats in case of a continuous exposure to "commercial pure" PGME (isomer 99.5%; isomer 0.5%). The suspected effect on the sex ratio will require further work in order to assess the possibility of persistent effects of PGME exposure through a genotoxic mechanism.

  Bois.Laboratoire de Toxicologie expérimentale, Institut National de l'Environnement Industriel et des Risques, INERIS DRC TOXI, Parc technologique ALATA BP2, F-60550 Verneuil en Halatte, France (*) Corresponding author: E. Lemazurier, INERIS DRC/TOXI Parc technologique Alata BP 2 60550 Verneuil en Halatte, France tel +33 (0) 3 44 55 62 64, fax +33 (0)3 44 55 66 05, Email : emmanuel.lemazurier@ineris.fr

  use of laboratory animals (decree n°87-849, October the 19 th 1987) and was approved by the Institutional Animal Care and Use Committee at the INERIS. Animals were housed in the INERIS animal-care unit, a facility accredited by the Departmental Direction of Veterinary Services. Animals were kept in a HEPA-filtered, mass air-displacement room with a 12-h light-dark cycle at 20-24°C and relative humidity of 40-70%. Animals had access ad libitum to deionized water and rodent chow (AO4C10, UAR, Epinay sur Orge, France). Time-mated, 6-to 7-week-old, nulliparous Sprague Dawley male and female rats were obtained from IFFA CREDO laboratories (L'Arbresles, France). Animal allocation to treatment groups was done with body weight randomization to ensure unbiased weight distribution among groups. Animals were group-housed, up to 6 per cage, by treatment in polycarbonate cages Makrolon type 3 (UAR, Epinay sur Orge, France).

Acknowledgements

This work was supported by a grant from the French Ministry of Ecology and Sustainable Development (BCRD INERIS/DRC AP 2000, convention number 00 -102).