Anh Minh Tang

Prof Yu-Jun Cui
email: cui@cermes.enpc.fr

Guy Richard

Pauline Défossez

A study on the air permeability as affected by compression of three French soils

Keywords: air permeability, confined uniaxial compression test, air-filled porosity, degree of saturation, vertical stress, void ratio

Soil air permeability is one of the most important parameters which govern the aeration in agricultural soils and thus has a significant effect on the plant growth and crop production. Therefore, it appears important, when analysing the effect of soil compaction due to agricultural machinery, to correlate air permeability with soil capacity parameters such as air-filled porosity, degree of saturation, water content, etc. In the present work, the relationship between air permeability, soil capacity parameters and vertical stress was analysed by performing confined uniaxial compression tests accompanied by air permeability measurements. Three French soils having different textures were studied. Tests were performed on remoulded and undisturbed soils, at various initial dry bulk densities and water contents. For the remoulded soils, the air permeability has been found strongly correlated with the applied vertical stress for sandy loam; by contrast, no obvious correlation could be established for clay. As far as the undisturbed soils are concerned, the air permeability could be correlated with the air-filled porosity for sandy loam and siltyclayey loam but also no evident correlation could be established for clay. Examination of an existing model predicting the air permeability from the air-filled porosity using one tortuosity/connectivity parameter showed that this parameter varies in a small range for sandy soils and in a larger range for clayey soils.

Introduction

Prevention of soil degradation is an important issue in the context of intensive agriculture and forest exploitation. Compaction by traffic has been identified as a major process that affects the production and the environment by changing the soil structure and the physical properties of soils. It changes the mechanical strength, water and gas transports and thus affects the root and shoot growth. It changes also soil nitrogen and carbon cycles and increases soil erosion due to water flow [START_REF] Soane | Soil compaction problems in world agriculture[END_REF]. Quantifying the soil damage by compaction is therefore of importance when establishing strategies for farming and forest management on a local scale and for environmental protection measures on a larger scale. The evaluation of the soil compaction effects on soil physical properties is generally based on the consideration of the changes in soil mechanical strength, aeration and hydraulic properties (Horn et al., 1995;[START_REF] Kozlowski | Soil compaction and growth of woody plants[END_REF][START_REF] Lipiec | Quantification of compaction effects on soil physical properties and crop growth[END_REF][START_REF] Schäfer-Landefeld | Effects of agricultural machinery with high axle load on soil properties of normally managed fields[END_REF]Hemmat and Adamchuk, 2008). Different approaches have been proposed to assess soil degradation due to compaction using relations between soil compaction parameters and soil capacity parameters such as air-filled porosity, degree of saturation, water content, etc: (i) Håkansson (1990) described the soil compactness in terms of relative soil porosity variations; (ii) [START_REF] Koolen | Soil deformation under compressive forces[END_REF] examined the soil sensitivity to compaction and proposed various compaction criteria in terms of variations of soil strength parameters such as the pre-compression pressure; (iii) [START_REF] Horn | Impact of modern forest vehicles on soil physical properties[END_REF] and [START_REF] Mosaddeghi | Suitability of pre-compression stress as the real critical stress of unsaturated agricultural soils[END_REF] investigated the relationships between applied stress and soil air permeability; (iv) Håkansson and Lipiec (2000) analysed soil compaction using relations between soil capacity parameters and air permeability. Note that after Horn and Kutilek (2009), a capacity parameter defines a general status, while an intensity parameter includes dynamic aspects over time and space. Goss and Ehlers (2010) presented their disagreement about these definitions, arguing that both intensity and capacity properties can vary in both space and time. In the present work, the term "capacity parameter" is adopted and it defines a general status, i.e., the composition of a given volume but not the internal structure and function (as proposed by Horn and Kutilek, 2009); at the same time, it is admitted that the capacity parameters can vary with time (following Goss and Ehlers, 2010).

Laboratory studies on air permeability have shown its dependency on various soil parameters related to the capacity parameters, such as the degree of saturation [START_REF] Seyfried | Use of air permeability to estimate infiltrability of frozen soil[END_REF][START_REF] Juca | Gas permeability of a compacted soil used in a landfill cover layer[END_REF], the water content [START_REF] Sanchez-Giron | Response of five types of soil to simulated compaction in the form of confined uniaxial compression tests[END_REF] and the air-filled porosity [START_REF] Olson | Comparison of three techniques to measure unsaturated-zone air permeability at Picatinny Arsenal, NJ[END_REF][START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF]. In general, the air permeability is lower at a higher degree of saturation with a lower air-filled porosity. Based on the experimental data of compacted silty soil, Delage et al. (1998) concluded that airfilled porosity is the unique parameter affecting the air permeability. [START_REF] Moon | Effectiveness of compacted soil liner as a gas barrier layer in the landfill final cover system[END_REF] found that the air permeability of compacted soils depends on the compaction energy as well as the moisture content at moulding; the lowest value of air permeability being at the optimum moisture content (maximum dry unit weight). Studies on undisturbed and repacked soils have shown significant effects of the soil structure and pore-space characteristics on the air permeability [START_REF] O'sullivan | Shear effects on gas transport in soil[END_REF][START_REF] Moldrup | Tortuosity, diffusivity, and permeability in the soil liquid and gaseous phases[END_REF][START_REF] Tuli | Comparison of air and water permeability between disturbed and undisturbed soils[END_REF]Dörner and Horn, 2006). It has been found that the air permeability was greatly reduced for repacked soils. As far as the anisotropy is concerned, the air permeability measured in the vertical direction has been found higher than in the horizontal direction due to the presence of biopores and vertical cracks. For further analysis about the air permeability dependency on the water and air contents, [START_REF] Tuli | Comparison of air and water permeability between disturbed and undisturbed soils[END_REF] fitted the experimental data to the model proposed by [START_REF] Mualem | A new model for predicting the hydraulic conductivity of unsaturated porous media[END_REF] and analyzed the fitting parameters. Similar studies were performed by [START_REF] Seyfried | Use of air permeability to estimate infiltrability of frozen soil[END_REF], [START_REF] Moldrup | Tortuosity, diffusivity, and permeability in the soil liquid and gaseous phases[END_REF], [START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF], [START_REF] Kamiya | A new method for the measurement of air permeability coefficient of unsaturated soil[END_REF]Dörner andHorn (2006), showing that it is appropriate to evaluate the changes in soil pores structure based on the measurement of air permeability.

In addition to the soil capacity parameters, the effect of applied stress has been also reported by various authors. [START_REF] Mosaddeghi | Suitability of pre-compression stress as the real critical stress of unsaturated agricultural soils[END_REF] noted that cyclic loading is not always accompanied by significant irreversible strain but it could decrease the air permeability by one order of magnitude. The effects of stress state on air permeability were also reported by Horn et al. (1995) and [START_REF] Sanchez-Giron | Response of five types of soil to simulated compaction in the form of confined uniaxial compression tests[END_REF]. Other factors affecting air permeability have been also observed: the matric suction [START_REF] Samingan | A flexible wall permeameter for measurements of water and air coefficients of permeability of residual soils[END_REF] and the wetting/drying process [START_REF] Kamiya | A new method for the measurement of air permeability coefficient of unsaturated soil[END_REF]. Even if the matric suction can be related to the capacity parameters (i.e. degree of saturation), it is usually considered as a stress parameter (see Gens and Alonso 1992). On the whole, it has been observed that the air permeability coefficient decreases when the stress increases or when the matric suction decreases; the relationships between air permeability coefficient and suction showed hysteresis in drying and wetting processes.

It can be concluded that air permeability has been recognised as one of the most appropriate parameters for soil compaction assessment. Nevertheless, examination of the studies mentioned above shows that the conclusions made by the authors were based on the results obtained on either one soil or a limited range of water content and porosity. The present work aims at analysing the effects of soil capacity parameters on air permeability for three French soils of various textures, with a large range of water content and porosity. Air permeability was measured in oedometer cell using the technique developed by [START_REF] Yoshimi | Compression of partially saturated cohesive soils[END_REF] and Delage et al. (1998). Emphasis was put on the effects of various parameters: soil type (sandy loam, silty clay loam and clay), vertical stress (15 -800 kPa), initial dry bulk density (0.98 -1.66 Mg m -3 ) and initial water content (14.0 -40.5 %). A comprehensive analysis of the obtained data was made and allowed identifying the most relevant parameters which affect the soil air permeability.

Materials and method

The studied soils were taken from three sites in France: (i) Le Breuil; (ii) Avignon; (iii) Epernay. Le Breuil is an experimental forest site located in the Morvan (47°18'N, 4°4'E, centre of France) where monospecific plantation have been conducted since thirty years (oaks, beech, spruce and Douglas fir); it involves a sandy loam (Dystric Cambisol). The Avignon site is a sugar beet field (43°55'N, 4°53'E, south of France). The soil is calcareous with a silty clay loam texture (Calcaric Cambisol). The Epernay site is an experimental site managed by the CIVC -Technical Institute for Champagne Wine (49°N, 3°56'E, east of France) and the soil involved is calcareous with a clay texture (Calcaric Cambisol). Some physical and chemical properties of the studied soils are presented in Table 1. The soil properties were determined following the French Standard for Geotechnical Engineering: the particle density was determined using water pycnometer on soil sieved at 2 mm; the Atterberg limits were determined on soil sieved at 0.4 mm; and the blue value was determined using the Methylene blue absorption method on soil sieved at 0.5 mm. The organic carbon content is 82.8 g.kg -1 for Le Breuil soil, 10.2 g.kg -1 for Avignon soil and 16.8 g.kg -1 for Epernay soil. Soil texture was classified following FAO-UNESCO (1974) system (after [START_REF] Jones | Vulnerability of subsoils in Europe to compaction: a preliminary analysis[END_REF] and USDA classification that are based on the particle size distribution. According to FAO classification, the texture of the tested soils varies from medium to fine. This classification was in good agreement with the plasticity indexes and the blue values, i.e., the finer the soil texture the higher the plasticity index and the larger the blue value. Note that the physical and chemical properties of the soils presented in Table 1 were determined from a mixture of soil taken from a layer of 60 cm in thickness. The variability of these properties within the depth (in the range of 0 -60 cm) was therefore not considered in this study.

Remoulded samples were tested for the topsoil layer (0-30 cm) depth which is frequently tilled whereas undisturbed samples were tested for the subsoil layer (30-60 cm depth) where compaction is persistent. For the preparation of remoulded samples, the topsoil was airdried, crushed and passed through a 2-mm sieve. It was then wetted by spraying distilled water to achieve the desired water content and then stocked in a hermetic box for 24 hours for water homogenization. Finally, the soil was poured directly into the oedometer cell and manually compacted.

For the preparation of undisturbed samples, a core sampler of 70-mm high, 150 mm in inner diameter and 1 mm in thickness, was pushed vertically into the subsoil layer (30-60 cm depth) in the field. The soil cylinders were then wetted by spraying distilled water to achieve the desired water content and then covered by a plastic film for 24 hours for water homogenization. Finally, the soil specimen (70 mm in diameter and 20 mm in height) was trimmed directly from the cylinder and inserted into the oedometer cell. When a drying process was involved, the soil cylinder was air-dried for 2 hours, and then covered by plastic film for 6 hours. The procedure was repeated until the desired water content was achieved. This drying process allowed the soil to be prevented from any dry-cracking during the preparation. Finally, as for the wet samples, the soil specimen was prepared by trimming and inserted into the oedometer cell.

The experimental setup developed by Delage et al. (1998) was used for the measurement of soil air permeability. The soil sample was installed in an oedometer cell. The basis of the cell was connected to a tank of large volume (V = 3120 cm 3 ) and a U-shaped manometer. When measuring the air permeability, an initial low value of air pressure was applied in the tank (p = 6 kPa, that corresponds to a water column of 0.60 m high); that induces an air flow through the soil specimen from the basis of the cell toward the upper surface. Delage et al. (1998) recommended an air pressure lower than 8 kPa for this method in order to ensure the validity of Darcy's law. The reduction of the air pressure in the tank was recorded as a function of the elapsed time, enabling the calculation of air permeability (see Delage et al. 1998 for more details).

After the installation of the soil specimen, a low vertical stress (15 kPa) was applied to ensure a good contact between the piston and the soil surface. The vertical stress was incrementally applied to 30,50,100,200,300,600,800 kPa. Each stress level was maintained for 5 min and the vertical displacements were recorded at the end of each step using a LVDT (linear variable differential transformer) to an accuracy of ±0.001 mm. Note that the short loading duration of 5 min was defined based on the values generally adopted when studying the compaction behaviour of agricultural soils under loading by a tractor vehicle [START_REF] Keller | Soil precompression stress. II. A comparison of different compaction tests and stress -displacement behaviour of the soil during wheeling[END_REF]Saffih-Hdadi et al., 2009). After 5 min, the piston was blocked to keep the soil volume constant and the measurement of air permeability was performed. Note that the piston blockage did not cancel the applied stress. After the air permeability measurement, the piston was un-blocked and the soil was further loaded. The test was stopped when no air flow was observed during the air permeability measurement or when the maximum load (800 kPa) was applied. At the end of each test, the soil sample was taken out of the cell and its dimension was measured using a calliper to an accuracy of ±0.001 mm. Its final water content was determined by oven-drying at 105 °C for 24 hours. The final void ratio and the degree of saturation were then determined. The initial void ratio was back-calculated using the final void ratio and the total displacement measured.

A total of 27 samples were prepared: 9 undisturbed samples of the subsoil layers and 18 remoulded samples of the topsoil layers. Their initial parameters (void ratio, e i ; water content, w i ; dry bulk density, ρ i ; degree of saturation, S ri ; air-filled porosity, V a /V) are presented in Table 2. Note that the degree of saturation S r is defined as the ratio of the volume of pore-water to the volume of void; it can be calculated from the water content (w), void ratio (e), particle density (ρ s ) and the density of water (ρ w ) as follows: (1)

The air-filled porosity V a /V, the degree of saturation S r and void ratio e are related by the following equation:

e S e V V r a + - = 1 ) 1 ( (2)
Tests on the remoulded specimens were performed on the soil from Le Breuil that has the lowest clay content and on the soil from Epernay that has the highest clay content. Nine tests were performed on each soil: three values of w i were considered; for each value of w i , the soil was compacted at three dry densities ranging from 0.98 to 1.47 Mg.m -3 . In the case of undisturbed specimens, four values of w i were considered for the soil from Avignon, three values of w i for the soil from Le Breuil, and two values of w i for the soil from Epernay.

Results and discussion

Experimental results

The results from the tests on the remoulded soil from Le Breuil are presented in Fig. 1. The air permeability k a is plotted versus air-filled porosity V a /V (Fig. 1a on a linear scale and Fig. 1c on a logarithmic scale), void ratio e (Fig. 1b), degree of saturation S r (Fig. 1d), and vertical stress σ v (Fig. 1e). Note also that the air permeability coefficient is usually plotted on a logarithmic scale when it involves a wide range of values [START_REF] Kamiya | A new method for the measurement of air permeability coefficient of unsaturated soil[END_REF] and on a linear scale when it involves a narrow range [START_REF] Seyfried | Use of air permeability to estimate infiltrability of frozen soil[END_REF][START_REF] Moldrup | Tortuosity, diffusivity, and permeability in the soil liquid and gaseous phases[END_REF]. In this work, both scales were used in order to have a complete view of the relationship between the air permeability coefficient and the air-filled porosity. The e-logσ v plot (Fig. 1f) corresponds to the compression curve. V a /V and S r were calculated by assuming that the gravimetric water content was constant during each test. This assumption was justified by measuring the mass of soil specimen before and after each test.

For each test, it can be observed that compression by increasing σ v decreased e, V a /V and increased S r . In addition, k a decreased when σ v was increasing (Fig. 1e). The k a -V a /V plots of all tests (Fig. 1a) have similar shape: at the beginning of the compression with large values of V a /V and k a decreased drastically with a large slope dk a /d( V a /V) while V a /V remained almost constant upon compression; at higher σ v (Fig. 1f), the soil volume decreased significantly (both e and V a /V decreased) leading to a significant decrease of the slope dk a /d(V a /V). This shape can be also observed when plotting k a with other capacity parameters as e (Fig. 1b) and S r (Fig. 1d).

The compression curves shown in Fig. 1f evidence the effect of density on its compressibility. Indeed, the curves show that the looser the soil (the larger the initial void ratio) the lower the pre-compression stress, which corresponds to the stress where irrecoverable plastic strain started to take place upon further loading (Défossez and Richard, 2002;Gregory et al., 2006;[START_REF] Pereira | Soil susceptibility to compaction by wheeling as a function of some properties of a silty soil as affected by the tillage system[END_REF][START_REF] Tang | Analysing the form of the confined uniaxial compression curve of various soils[END_REF]. Considering for example the results of test K17 (thick lines in Fig. 1): loading from the initial vertical stress to 50 kPa (close to the pre-compression value) did not change significantly the void ratio (e decreased from 1.39 to 1.35, Fig. 1f). Further loading significantly decreased the void ratio by following a larger slope in the e-logσ v plot. The first part of the compression curve (where the vertical stress is lower than 50 kPa) corresponds to the first part of the k a -V a /V plot (where V a /V remains constant, and is equal to 0.4). The slope of the k a -V a /V curve changes significantly for V a /V lower than 0.39 (k a lower than 6.39 x 10 -10 m 2 ). These values of point of maximum curvature are shown in Table 3 for all the tests. It can be noted that the points of maximum curvature on the k a -V a /V plot (Fig. 1a) corresponds to that of the e-logσ v plot (Fig. 1f). The similarity between the two relationships, k a versus capacity parameters and these parameters versus logσ v , explains the quasi-linear relationship between k a and logσ v observed in Fig. 1e. Application of linear regression over the whole stress range showed that the slopes dk a /dlogσ v of all tests are similar, ranging from -2.7×10 -10 (m 2 ) (test K18) to -4.3×10 -10 (m 2 ) (test K20), the mean value being -3.6×10 -10 (m 2 ).

The results of the remoulded soil from Epernay are presented in Fig. 2. The overall trend observed previously on the remoulded soil from Le Breuil can be also observed: the compression (with increase of σ v ) decreased e and V a /V, increased S r and decreased k a .

Nevertheless, unlike the remoulded soil from Le Breuil, the shapes of k a -V a /V plots of remoulded soil from Epernay are not similar (Fig. 2a). Moreover, the range of k a of the soil from Epernay (k a = 5×10 -12 to 2×10 -9 m 2 ) was larger than that of the soil from Le Breuil (k a = 1×10 -10 to 1×10 -9 m 2 ). When analysing the k a -logσ v plots (Fig. 2e), the linear trend was not as clear as in the case of Le Breuil (Fig. 1e). In addition, the range of the slope dk a /dlogσ v was larger, from -3.1×10 -12 (m 2 ) (test K02) to -3.0×10 -9 (m 2 ) (test K04). This difference can be attributed to the larger difference in the initial void ratio between the soil from Epernay (e i ranges from 0.82 to 1.73) and the soil from Le Breuil (e i ranges from 0.9 to 1.39; see Fig. 2f and Table 2).

Fig. 3 presents the results of the undisturbed soil from Le Breuil. Three values of water content were considered (w = 16.9%, 21.4%, and 22.5% for tests K15, K11, and K14 respectively, Table 2), corresponding to an initial air-filled porosity V a /V ranging from 0.20 to 0.35 (Fig. 3a) and an initial degree of saturation S r ranging from 30 to 60% (Fig. 3d). The k a -e plots of the three tests were almost identical (Fig. 3b); the k a -V a /V plots (Fig. 3a,c) and the k a -S r plots (Fig. 3d) were similar. Again this similarity can be attributed to the close values of initial void ratio of the three tests (e i ranges from 0.98 to 1.16, see Table 2) and to the similarity of the e-logσ v curves (see Fig. 3f). In addition, the shape of the k a -V a /V plots (Fig. 3a) and the k a -e plots (Fig. 3b) was also similar to that of remoulded soils from Le Breuil (Fig. 1a,b): at the beginning of the compression, k a decreased drastically while the volume change remained small; at higher stresses, all changes in k a and capacity parameters (V a /V and e) upon compression were significant. Furthermore, the relationship between k a and logσ v of the undisturbed soil from Le Breuil was no longer linear (Fig. 3e). Compared to the remoulded soil, the undisturbed soil presents a larger range of k a (k a = 5×10 -12 to 2.5×10 - 9 m 2 ).

In Fig. 4, the results of the undisturbed soil from Avignon are presented. Four values of water content in the range of 17.7 -22.9% were considered (Table 2). The e-logσ v plots show two distinct families (Fig. 4f). However, the k a -V a /V plots of all tests (Fig. 4a) are almost identical. This is because the difference in initial void ratio between the four tests is quite limited (e i ranges from 0.63 to 0.76, see Table 2) and moreover the shape of the four elogσ v curves are quite similar. The effect of initial void ratio can be clearly identified in other plots as the k a -e plots (Fig. 4b), logk a -V a /V plots (Fig. 4c) and logk a -S r plots (Fig. 4d).

For instance, in Fig. 4b, the tests K13 and K27 show that k a varied from 0 to 5×10 -10 (m 2 ) while e varied in the range from 0.52 to 0.62 for K13 and from 0.63 to 0.75 for K27. Similar observations can be made when comparing K16 and K26 in Fig. 4b. In Fig. 4c and Fig. 4d, k a is plotted on a logarithmic scale, that makes the difference between the data clearer in the range of low k a values (k a <10 -11 m 2 ). When applying linear regression over the whole stress range to the k a -logσ v plots (Fig. 4e), the slope dk a /dlogσ v obtained showed variations ranging from -1.5×10 -10 (m 2 ) (test K16) to -4.6×10 -10 (m 2 ) (test K27).

The results of the two tests of the undisturbed soil from Epernay are presented in Fig. 5. Except the similar linear relationship observed on the k a -logσ v plot with a slope dk a /dlogσ v of about -1.9×10 -10 m 2 (Fig. 5e), other curves show significant difference between the two tests. The large difference between the initial void ratio of the two tests (e i ranges from 1.05 to 1.22, see Fig. 5f and Table 2) can also explain this difference. When comparing the results of remoulded and undisturbed soil from Epernay, it was observed that the orders of magnitude of k a are similar: k a = 5×10 -12 -1×10 -9 m 2 for V a /V = 0 -0.2, e = 0.8 -1.2, S r = 65 -100%.

The effect of σ v on k a has been observed in several studies. Horn et al. (1995) reported the study of [START_REF] Semmel | Auswirkungen kontrollierter Bodenbelastungen auf das Druckfortpflanzungsverhalten und physikalisch-mechanische Kenngrijssen von Ackerboden[END_REF] on the stress-dependent changes in air permeability observed from tests on soil samples from 0.4-m depth in a Luvisol derived from loess, at a matric suction of 6 kPa. Upon additional loading, it was observed that k a decreased sharply as soon as the precompression stress value (100 kPa) was exceeded. In the work of [START_REF] Mosaddeghi | Suitability of pre-compression stress as the real critical stress of unsaturated agricultural soils[END_REF], k a was measured on five remoulded soils from sandy loam to clay at four matric suctions (10, 20, 50 and 80 kPa) and three axial stresses (200, 400, and 600 kPa). The results showed that cyclic loading was not always accompanied by significant irrecoverable strain but could result in up to 10 times decrease in air permeability at low matric suctions. Linear regression was applied to the relationship between the logarithm of k a and the logarithm of vertical stress. In the present work, it was observed that the shape of k a -σ v relationship is different from that described by [START_REF] Semmel | Auswirkungen kontrollierter Bodenbelastungen auf das Druckfortpflanzungsverhalten und physikalisch-mechanische Kenngrijssen von Ackerboden[END_REF] and Mossaddeghi et al. (2007) and a satisfactory linear relationship can be obtained on a semi-logarithmic scale. This difference may be explained by the test conditions: in the study of [START_REF] Semmel | Auswirkungen kontrollierter Bodenbelastungen auf das Druckfortpflanzungsverhalten und physikalisch-mechanische Kenngrijssen von Ackerboden[END_REF] and Mossaddeghi et al. (2007), the soil was compressed under constant suction condition while in the present work the tests were carried out at constant water contents thus decreasing suctions. Sanchez-Giron et al. (1998) also measured k a in confined uniaxial compression conditions. The measurements were performed on five remoulded soils having various textures at four vertical stresses (50, 100, 200, and 400 kPa) and five water contents (5, 10, 15, 20, and 25%). The results showed that the effects of water content and applied stress on the compaction process (thus, on the air permeability changes) depend on soil texture. This is in agreement with the results obtained in the present work where the behaviour of remoulded soil from Epernay (Fig. 2) was found to be significantly different from that of Le Breuil (Fig. 1).

Evaluation of the Moldrup et al. (2003) model

To further analyse the experimental data, the model proposed by [START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF] was evaluated by determining the single tortuosity/connectivity parameter involved in the model, η. This model is expressed by the following equation:

η ε ε ) / ( / * * = a a k k (3) where ε = V a /V, * a
k and ε * are reference point values of air permeability and air-filled porosity at a given soil matric potential.

Eq. ( 3) can be re-written as follows:

) / ( ) ( * * η η ε ε a a k k = (4)
In the work of [START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF], the parameter ε * was determined from the point corresponding to a suction of 100 cm of water (10 kPa) on the water retention curve. The parameter * a k was the measured value of k a corresponding to this value of ε * . The tortuosity/connectivity parameter, η, was fitted from the slope of the soil-water retention curve plotted in a log(θ) -log (s) coordinate system (θ is the volumetric water content and s is the soil suction). In the present work, Eq. ( 4) was used to fit the experimental data in order to determine η. As an example, the results of the test K01 are shown in Fig. 6 4)). The results of test K01 gave a value of η equal to 15.6 with a coefficient of determination (R 2 ) of 0.985. The values of these two parameters, η and R 2 , were determined for all tests and are summarised in Table 4. 4 shows that the parameter η of remoulded soil from Le Breuil (K17 -K25) varies from 1.91 to 3.69 while that of remoulded soil from Epernay (K02 -K10) varies from 0.49 to 5.09. For test K08, only one value was available, making impossible the determination of the parameter. For undisturbed soils, η varies from 1.59 to 3.99 for the soil from Breuil, from 2.49 to 6.74 for the soil from Avignon. In the case of the soil from Epernay, a value of 1.11 was obtained from test K12 and a very high value (15.6) was obtained from test K01. Note that in the work of [START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF], η varies from 1 to 3 for various undisturbed volcanic ash soils in Japan. The larger range of variations of parameter η for the clay from Epernay reflects its more complex mechanism for air transfer compared to the sandy soil from Le Breuil. In other words, it seems difficult to describe the air permeability variations of one soil using a unique value of η. Gens and Alonso (1992) also reported that the hydro-mechanical behaviour of high plasticity unsaturated soils (clays) is significantly different from that of low plasticity unsaturated soils (sands and silts). Clayey soils having various hydric states (water content) show different properties at the level of soil aggregates (compressibility and cohesion between aggregates). Thus the soil structure change upon mechanical loading (compression) of clayey soils strongly depends on the hydric state. On the contrary, the microstructural properties of low plasticity unsaturated soils (sands and silts) are significantly less sensitive to the hydric changes. That explains also why predictive models are usually applied to sandy soils only [START_REF] Poulsen | Predicting Air Permeability in Undisturbed, Subsurface Sandy Soils from Air-Filled Porosity[END_REF][START_REF] Moldrup | Tortuosity, diffusivity, and permeability in the soil liquid and gaseous phases[END_REF][START_REF] Tuli | Comparison of air and water permeability between disturbed and undisturbed soils[END_REF]Dörner and Horn, 2006). It is worth mentioning that there are a large difference in organic carbon content between the three soils studied (82.8 g.kg -1 for Le Breuil, 10.2 g.kg -1 for Avignon and 16.8 g.kg -1 for Epernay). This parameter would have also an effect on soil structure and then on the air permeability. Dexter (2004a) proposed a soil physical parameter, S, as an index of soil physical quality. Examination of various experimental results showed that S was higher when the organic carbon content was higher. In addition, Dexter (2004b) showed a higher hydraulic conductivity at higher S. These observations are in agreement with the present study where the soil from Le Breuil that has the highest organic carbon content showed higher air permeability.

Examination of Table

Conclusion

In an attempt to study the relationships between soil capacity parameters and the air permeability coefficient, air permeability tests were carried out on three soils at various textures, densities, water contents and types (remoulded soils from the topsoil layer and undisturbed soils from the subsoil layer). Tests at various densities and water contents were carried out. For each test performed, the air permeability coefficient k a was plotted versus various parameters (V a /V, e, S r , and σ v ) in order to examine the relationship between compaction and soil aeration. Note that relations between air permeability and the capacity parameters (V a /V, e, S r ) allow analyse of the processes involved in air transfer. The relations between k a and the applied stress were established in order to link the effect of applied stress with the soil physical properties.

The effects of various parameters on k a observed in the present work are in agreement with previous studies found in the literature: k a decreases when V a /V decreases (O' Sullivan et al., 1999;[START_REF] Olson | Comparison of three techniques to measure unsaturated-zone air permeability at Picatinny Arsenal, NJ[END_REF] or S r increases (Delage et al., 1998;[START_REF] Samingan | A flexible wall permeameter for measurements of water and air coefficients of permeability of residual soils[END_REF][START_REF] Juca | Gas permeability of a compacted soil used in a landfill cover layer[END_REF].

The air permeability of the remoulded sandy loam (from Le Breuil) has a clearer correlation with the vertical stress than with other soil capacity parameters. For undisturbed soils, the air permeability of sandy loam (from Le Breuil) and silty clay loam (from Avignon) has a good correlation with soil capacity parameters. No unique correlation between the studied parameters (air permeability, void ratio, air-filled porosity, vertical stress, and degree of saturation) has been observed for both the remoulded and undisturbed clay from Epernay.

Evaluation of the model proposed by [START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF] using the tests results showed that the tortuosity/connectivity parameter, η, varies in a narrow range (from 1.91 to 3.69) for the remoulded soil from Le Breuil, from 0.49 to 5.09 for the remoulded soil from Epernay, from 1.59 to 3.99 for the undisturbed soil from Le Breuil, from 2.49 to 6.74 for the undisturbed soil from Avignon, and from 1.11 to 15.6 for the undisturbed soil from Epernay. It appears then difficult to describe the air permeability variations using only one parameter for clayey soils as the soil from Epernay, especially in the undisturbed state. It is finally worth noting that the present study was limited to three soil textures, from sandy to clayey soil. Further studies should be performed on other soils in order to refine the analysis. In addition, it would be interesting to further investigate the effects of water retention capacity, organic carbon content, and soil heterogeneity with depth, etc. Table 1. Some physical and chemical properties of studied soils (determined from a mixture of soils taken from the depth of 0 -60 cm). Table 2. Initial conditions of the tests performed (k a : air permeability coefficient; V a /V: airfilled porosity; e i : initial void ratio; S ri : initial degree of saturation; ρ i : initial dry density; w: water content; Ep.: Epernay; Br: Le Breuil; Av.: Avignon) Table 3: Results of the remoulded soil from Le Breuil -values corresponding to the points of maximum curvature in the k a -V a /V plots and e-logσ v plots (k a : air permeability coefficient;

List of Tables

V a /V: air-filled porosity; e: void ratio; σ v : vertical stress)

List of Figures

Fig. 1. Results of the remoulded soil from Le Breuil (k a : air permeability coefficient; V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ v : vertical stress; ρ i : initial dry density; w: water content) Fig. 2. Results of the remoulded soil from Epernay (k a : air permeability coefficient; V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ v : vertical stress; ρ i : initial dry density; w: water content) Fig. 3. Results of the undisturbed soil from Le Breuil (k a : air permeability coefficient; V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ v : vertical stress; w: water content) Fig. 4. Results of the undisturbed soil from Avignon (k a : air permeability coefficient; V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ v : vertical stress; w: water content) Fig. 5. Results of the undisturbed soil from Epernay (k a : air permeability coefficient; V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ v : vertical stress; w: water content) Fig. 6. Results of test K01 -Comparison between the experimental results and the best fitted curve using the model of [START_REF] Moldrup | Air permeability in undisturbed volcanic ash soils: predictive model test and soil structure fingerprint[END_REF].

Table 1. Some physical and chemical properties of studied soils (determined from a mixture of soils taken from the depth of 0 -60 cm). V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ σ σ σ v : vertical stress; w:

Site

water content) 0.13 0.14 0.15 0.16 0.17

  . The experimental results were best fitted with a power function (k a = aε b ; where a and b are the two fitting parameters; a = b following the Eq. (

Fig. 1 .Fig. 2 .Fig. 3 .Fig. 4 .Fig. 5 .

 12345 Fig. 1. Results of the remoulded soil from Le Breuil (k a : air permeability coefficient;V a /V: air-filled porosity; e: void ratio; S r : degree of saturation; σ σ σ σ v : vertical stress; ρ ρ ρ ρ i : initial dry density; w: water content)

Fig. 6 .

 6 Fig. 6. Example of a comparison between the experimental results and the best fitted curve using the model of Moldrup et al. (2003). Test K01 (Soil from Epernay; undisturbed, e i = 1.05; w i = 26.2%; ρ i = 1.31 Mg.m -3 ; S ri = 67%; V a /V = 0.17).

Table 4 :

 4 Fitting parameters following the model ofMoldrup et al.

	(2003)

Table 3 : Results of the remoulded soil from Le Breuil -values corresponding to the points of maximum curvature in the k a -V a /V plots and e-logσ σ σ σ v plots (k a : air permeability coefficient; V a /V: air-filled porosity;

 3

	e: void ratio; σ σ σ σ v : vertical stress)
	Test No. k a (10 -10 m 2 )	V a /V e	σ v (kPa)
	K17	6.39	0.39 1.35 50
	K18	4.06	0.29 1.02 200
	K19	5.04	0.25 0.90 200
	K20	7.25	0.43 1.34 50
	K21	4.58	0.32 0.97 200
	K22	4.77	0.29 0.88 200
	K23	5.40	0.32 1.24 100
	K24	4.21	0.21 0.93 200
	K25	3.71	0.18 0.85 200

Acknowledgements

This work was carried out under the project "Soil degradation due to compaction" with the financial support of (1) the « ANR-Agence Nationale de la Recherche -The French National Research Agency » under the « Programme Agriculture et Développement Durable», project « ANR-05-PADD-013 », (2) the Ministry in charge of Environment under the programme GESSOL2 "Impact des pratiques agricoles sur le sol et les eaux ». The authors are also grateful for the technical assistance of D. Boitez and F. Bornet.

Brooks, R.H., Corey, A.T., 1966. Properties of porous media affecting fluid flow. J. Irrig. Drain. Div. Proc. ASCE. 92(IR 2), 61-88. Défossez, P., Richard, G., 2002. Models of soil compaction due to traffic and their evaluation. Soil Till. Res. 67, 41 -64. Delage, P., Cui, Y. J., De Laure, E., 1998. Air flow through an unsaturated compacted silt. Proceedings of the 2 nd International Conference on Unsaturated Soils, Beijing, Vol. 1, pp. 563 -568. Dexter, A. R., 2004a. Soil physical quality. Part I. Theory, effects of soil texture, density and organic matter, and effects on root growth. Geoderma 120, 210 -214. Dexter, A. R., 2004b. Soil physical quality. Part III. Unsaturated hydraulic conductivity and general conclusions about S-theory. Geoderma 120, 227 -239. Dörner, J., Horn, R., 2006. Anisotropy of pore functions in structured Stagnic Luvisols in the Weichselian moraine region in N Germany. J. Plant Nutr. Soil Sci. 169, 213 -220. FAO-UNESCO, 1974. FAO-UNESCO Soil Map of the World, vol. 1, Legend. UNESCO, Paris. Gens, A., Alonso, E. E., 1992. A framework for the behaviour of unsaturated expansive clays. Can. Geotech. J. 29, 1013 -1032. Goss, M. J., Ehlers, W., 2010. Comment on the Editorial "The intensity-capacity concept -How far is it possible to predict intersity values with capacity parameters" [R. Horn, M. Kutilek, Soil Till. Res. 103 (2009) 1 -3]. Soil Till. Res. 106, 349 -350. Gregory, A.S., Whalley, W.R., Watts, C.W., Bird, N.R.A., Hallet, P.D., Whitmore, A.P., 2006. Calculation of the compression index and precompression stress from soil compression test data. Soil Till. Res. 89, 45 -57. Håkansson, I., 1990. A method for characterizing the state of compactness of the plough layer. Soil Till. Res. 16, 105 -120. Håkansson, I., Lipiec, J., 2000. A review of the usefulness of relative bulk density values in studies of soil structure and compaction. Soil Till. Res. 53, 71 -85. Hemmat, A., Adamchuk, V.I., 2008. Sensor systems for measuring soil compaction: Review and analysis. Computers and Electronics in Agriculture 63, 89 -103. Horn, R., Kutilek, M., 2009. The intensity-capacity concept -How far is it possible to predict intensity values with capacity parameters. Soil Till. Res. 103, 1 -3. Horn, R., Domzal, H., Slowinska-Jurkiewicz, A., van Ouwerkerk, C., 1995. Soil compaction processes and their effects on the structure of arable soils and the environment. Soil Till. Res. 35,[23][24][25][26][27][28][29][30][31][32][33][34][35][36]


Justo ; Lobato
email: justo.lobato@uclm.es

Pablo Cañizares

Diego Úbeda

F Javier Pinar

José J Linares

Optimization of the Microporous Layer for a Polybenzimidazole-based High Temperature PEMFC. Effect of carbon content

Keywords: Carbon, High Temperature, ESA, Performance, PBI, Microporus Layer Carbon, ESA, High temperature, Microporous layer, PBI, Performance

Optimization of the Microporous

be the most suitable for both automobile and stationary applications [4].

The membrane-electrode assembly (MEA) is the main part of PEMFCs. It consists of a proton exchange membrane located between two catalyst layers and a gas diffusion layer (GDL) backing each one. The most important function of the GDL is to distribute the reactant gases over the catalyst layer and to remove the generated products out of the fuel cell [5,6]. More information about the characteristics and manufacturing processes of GDLs can be found in an interesting review by Cindrella et al. [7]. With the aim of improving water management in PEMFCs, GDLs have been impregnated with a hydrophobic polymer and a micro-porous layer (MPL) has been added on one side of the GDL [8]. Ramasamy et al. have demonstrated mass transfer improvement when these treatments are applied, in terms of both increasing limiting current as well as reducing mass transfer resistance [9]. MPL characteristics depend on the carbon type, the polymeric adhesive used, the solvent used to prepare the solution and on the composition. Moreover, the deposition techniques and the solvent evaporation rates have an important role on the final MPL properties [7,[START_REF] Mathias | Handbook of Fuel Cells[END_REF][START_REF] Yan | [END_REF]. Once the MPL is deposited, the sintering process is an important step. The objective of this method is the right distribution of the polymeric adhesive over the entire surface and the removal of some remaining solvent [12]. In this work, GDLs containing MPL with different carbon contents were prepared to be used in a high temperature PEMFC operating above 100 ºC. In order to optimize the amount of carbon in the MPL, the GDLs were physically characterised and the electrodes prepared with those GDLs were electrochemically characterised in a single fuel cell.

Experimental

Black carbon Vulcan XC72-R and PTFE were used as carbon material and polymeric adhesive, respectively. Isopropyl alcohol was the solvent used to prepare the ink that was deposited by air spraying onto the commercial GDL (Toray Graphite paper TGPH-120. 0.35 mm) with a Teflon content of 10 %, chosen from a previous work [6].

The Teflon content in the MPL was also 10 % [13] and the carbon amount was in the range 0.5-4.0 mg cm -2 .

Physical characterisation of the gas diffusion media

Mercury porosimetry was used to determine the porosity of the samples. This also enabled the evaluation of pore size distribution. The equipment used for the determination was a Micromeritics Auto Pore IV 9500 Hg porometer.

Permeability was determined by using Darcy's law [6,10.13]. Permeability coefficient (k, m 2 ) can be calculated by using the following expression: Permeability was evaluated with a home-made apparatus that was designed and made in-house. In this set up, air is forced to flow through the GDM and pressure drop is measured with a water column. Circular-shaped samples of 10 cm diameter were utilized for the measurements [13].

∆P l µ υ k ⋅ ⋅ = (1)

Fuel cell measurements

The preparation of a membrane-electrode-assembly (MEA) can be briefly described as follows. On top of each gas diffusion media, the catalytic layer formed by 0.5 mg/cm 2 of platinum from 20% Pt on Vulcan XC-72R carbon black (ETEK-Inc, USA) and 0.5 mg/cm 2 PBI (from a 5% PBI solution in N,N'-dimethylacetamide) was deposited with an aerograph (N 2 as carrier gas). The gas diffusion electrodes were then cured in an oven for 2 hours at 190 ºC and soaked with a 10% wt. H 3 PO 4 solution until a load of 30 mg•cm -2 was obtained, leaving them for at least 4 days in order to achieve complete impregnation. Polybenzimidazole membranes, produced according to the procedure described elsewhere [14,15], were immersed in an 80% H 3 PO 4 bath (doping level of 6.7) and then removed and blotted with filter paper to remove the superficial acid. Hotpressing to obtain the MEA was carried out by placing the membrane between the electrodes. A load of 1 tonne (minimum applicable using our equipment) was subsequently applied at 130 ºC for 15 minutes. A press for the preparation of IR pellets (Graseby Specac, United Kingdom) was modified and adapted for the membrane- Measurements were taken using an Autolab PGSTAT 30 Potentiostat/Galvanostat equipped with a Current Booster (20 A). The cell was firstly conditioned at a fixed potential of 0.5 V and 125 ºC for 24 hours, with the system attaining a steady current after several hours, as reported elsewhere [16,17]. Once this period had elapsed, polarization curves were recorded in a potentio-dynamic polarization mode [6]. The potential was swept between the cell open circuit voltage and 0 V at 1 mV s -1 . The cell was always operated at 125 ºC and atmospheric pressure. Hydrogen (99.999% pure, Praxair, Spain) was fed into the cell at a flow rate of 200 ml/min, whereas oxygen (99.999% pure, Praxair, Spain) was fed.

F
Measurement of the electrochemical surface area (ESA) of the electrodes was carried out by cyclic voltammetry (CV) running N 2 (200 ml/min) through the cathode and H 2 (134 ml/min) through the anode at room temperature with a potential range from 0 V approx. to 1.4 V approx. versus standard hydrogen electrode (SHE) and a sweep rate of 20 mV/s. The electrochemical impedance spectra were recorded using the frequency response analysis (FRA) module of the potentiostat/galvanostat at a potential of 0.3 V.

Frequency ranged from 10 KHz down to 10 mHz, with a potential wave of 0.01 V. Moreover, the carbon amount is higher the lower is the macroporosity until a value of 2 mg carbon cm 2 is reached. From this value, the macroporosity does not decrease, which means that no more carbon particles are introduced into the gas diffusion layer.

Results and discussion

Respecting the meso and micro pores region, it can be said that as the carbon content increases, the meso and micropores generated are higher, which leads to a porosity increase in this range of pore sizes [18].

Figure 1

From pore size distribution data, porosity, tortuosity and mean pore diameter can be estimated. Figure 2 shows those parameters versus carbon content. It can be seen that the overall porosity decreases with the carbon deposited on the microporous layer. This can be due to the decrease of the macroporosity of the carbon support when the microporous layer is deposited and then introduced into the carbon support of the gas diffusion. On the other hand, it can be due to the microporous layer intrinsic microporosity, which leads to a decrease of the overall porosity. The same comment can be made for the case of mean pore diameter. The microporous layer carbon can block part of the macropores of the gas diffusion layer, leading to a decrease in the pore size.

However, at carbon load values higher than 2 mg cm -2 the decrease in the pore size is lower. With respect to tortuosity, it can be observed that this parameter increases linearly with the carbon content of the microporous layer. This effect was the expected

Gas and water vapour permeability

The variation of permeability of the different gases present in a fuel cell (H 2 , O 2 and air), as well as water vapour with respect to the carbon content of the MPL is shown in Figure 3. It must be remarked that in the experimental system, water produced in the cathode will be in vapour state because it operates at temperature above 100 ºC.

Moreover, the corresponding value of the GDL is also shown. In all cases, it can be seen that the deposition of a thicker microporous layer (higher carbon content) leads to a permeability decrease. As it was observed in the previous figures, the microporosity increase and the macroporosity decrease hinder the gas flow through the electrode and hence from the mass transfer point of view, the deposition of a microporous layer would not be beneficial. indicates the platinum load in the catalyst layer; therefore electrodes ESA (m 2 /g Pt) can be easily estimated [19].

Cyclic voltammetry

It can be observed, independently of the MPL carbon content, that the deposition of a MPL is beneficial because in all cases the value of ESA was higher than when a MPL was not deposited. Elimination of the microporous layer, which acts as supporting layer of the catalyst, facilitates that part of the catalyst sites penetrate deep inside the backing of the gas diffusion layer and thus making those sites inaccessible to the gas or the proton. Moreover, it is seen that the available area of Pt sites does not increase from 2 mg cm -2 to 4 mg cm -2 . This could be explained as follows. In the pore size distribution, it has been seen that the volume of macropores (related to the carbon support) does not decrease when the carbon loading in the MPL increases in the same range, so that the MPL has fully completed the protective role of the catalytic layer. Thus, any extra carbon content above 2 mg cm -2 does not have any positive effect from the point of view of ESA.

Fuel cell test. Cathode performance

In order to evaluate cathode performance, a set of experiments at 125 ºC was carried out using cathodes with different carbon content in the MPL. In the anode, carbon content was in all cases equal to 1 mgcm -2 . To evaluate the effect of the different carbon content on the fuel cell performance oxygen stoichiometry was changed from 4 to 1.5. An air Deleted: Moreover, it is seen that the available area of Pt sites reaches its maximum value at 2 mg carbon cm -2 , approx. Further increase beyond this value does not increase the electrochemical surface area. It seems that at 2 mg cm -2 the microporous layer performs its role in protecting the catalyst layer making the catalyst sites more accessible to the reactants. flow was also used (oxygen stoichiometry equals to 4). Figure 5 shows the corresponding polarisation curves. In all cases it can be observed that the deposition of a thicker MPL leads to a performance increase up to a carbon amount of 2 mgcm -2 . The lower performance, independently of the comburent used, detected on the cell incorporating the electrode without the supporting layer, MPL, can be explained by the reduction of available catalyst sites [20] as it was observed previously by the cyclic voltammetry analysis.

Figure 5.

With the aim of obtaining more information about the fuel cell performance using cathodes containing MPL with different carbon contents, AC impedance spectroscopy was used to diagnose the fuel cell under the operation conditions above commented.

Figure 6 shows the impedance spectra and Table 1 shows the values of the different parameters obtained from it and the equivalent circuit used in this work, which is the same to other used by Zhang et al. for a PBI-based high temperature PEMFC [21]. R o is the high-frequency resistance which represents the ohmic resistance of the fuel cell that is dominated by the membrane resistance. R ct is the charge transfer resistance related to the fuel cell reaction kinetics, contributed by both the cathodic ORR and anodic HOR processes. Finally, R mt is the resistance related to mass transfer processes, contributed by the diffusion of H 2 and O 2 to catalyst sites and the proton transfer resistance within the catalyst layers [21].

Figure 6

Table 1 AC impedance analysis also confirms the existence of an optimal MPL carbon amount in the cathode electrode for all oxygen stoichiometries studied as well as the beneficial 1 it can be better noticed the effect of the microporous layer carbon content on the fuel cell performance. In the activation polarization region (low current densities) it can be observed that the presence of a thicker MPL is beneficial because the active surface area increases with the thickness of the MPL till a value of carbon content of 2 mg cm -2 . From this value on, there is not any performance increase in this polarization curve region and the R ct values are very similar from that point. On the other hand, the R mt values are higher when the O 2 stoichiometry decreases or when air is used as comburent. The effect of carbon load is more noticeable in these poor oxygen available conditions, where any improvement of the catalytic activity will have a big repercussion on the oxygen reduction reaction. Under these conditions, the R ct values for the GDL with 4 mg cm -2 of carbon increase with respect to those of the 2 mg cm -2 .

Regarding the ohmic resistance, it can be observed that the slope of the polarisation curves and the Ro values decrease again until the carbon content value of 2 mg cm -2 is reached. A good distribution of the different layers, carbon support (GDL), microporous layer (MPL), and catalyst layer allow to reduce the fuel cell ohmic polarisation and to improve the transit of reactant or product gases. Even the improvement of the catalyst activity could help to decrease R o [22,23]. Another factor that is helpful to decrease the Ro is the membrane hydration, which improves the membrane conductivity. A different level of membrane hydration could be occurring with the different MPL prepared;

without MPL, ohmic resistance is higher than with MPL due to a MPL could retain water, which increases the conduction mechanism. Higher R o values when using a carbon load of 4 mg cm -2 can be consequence of a thicker MPL than the resulting when Finally, the same comments stated above can be valid for the case of the concentration polarization (high current densities). The R mt values decrease as the carbon content increases till a value of 2 mgcm -2 . Higher carbon contents are not recommended because of some diffusion problems through the thicker layer. Moreover, it can be noticed that the decrease of the O 2 availability affects mainly to the mass transfer properties. Thus, the R mt variations are higher when a low O 2 stoichiometry or air as comburent is used.

Fuel cell test. Anode performance

In this case, fuel cell performance was studied changing the MPL carbon content of the anodes. Two different H 2 stoichiometries were selected (1 and 4 for 1 Acm -2 ). Figure 7 shows the polarisation curves. The same trends can be observed than those for the cathode study. There is an optimum level of carbon in the MPL which corresponds to the value of 2 mgcm -2 . Nevertheless in this case, the differences are not so high because the electrode that actually controls fuel cell performance is the cathode. without MPL, (--) 0.5 mg cm -2 , (-•-) 1 mg cm -2 , (-••-) 2 mg cm -2 , (--) 4 mg cm -2 .

5 where

 5 υ is the air flux (m s -1 ), µ is the air viscosity (Pa s), l is the thickness of the sample (m), and ∆P is the pressure drop across the substrate (Pa).

3. 1 7 Figure 1

 171 Figure1shows pore size distributions of the GDL with different carbon loads in MPLs and, solely to compare, with no MPL. Two ranges of the specific pore volume are shown in this figure; Figure1ashows the macropores and Figure1bthe micro and mesopores region. It can be observed that the deposition of a MPL in the electrode structure leads to a decrease in the number of macropores of the gas diffusion layers.

  carbon amount increases, its thickness is higher and the gas flow through that layer is then more difficult.

Figure 2 .

 2 Figure 2.

Figure 3 .

 3 Figure 3.

Figure 4

 4 Figure 4 shows the cyclic voltammograms of the electrodes prepared with MPL with different carbon contents. In figure 4b the values of the electrochemical surface area (ESA) calculated from the voltammograms are shown. They were given by the following formula:

Figure 4 .

 4 Figure 4.

  cm -2 , as well as the lower performance of this assembly which further reduces the level of hydration of this membrane.

Figure 7 4FIGURE 1 .

 71 Figure 7

FIGURE 2 .

 2 FIGURE 2. Parameters from Hg-porosimetry vs carbon load. a) Porosity; b) Mean pore diameter; c) Tortuosity.

FIGURE 3 .

 3 FIGURE 3. Permeability measurements vs carbon content of the MPL. a) H 2 ; b) O 2 ; c) Air; d) water vapour

FIGURE 4 .FIGURE 5 .

 45 FIGURE 4. Cyclic voltammetry analysis. a) Cyclic voltammograms of the electrodes with different carbon contents in the MPL. Scan rate 20 mV/s. b) Values of ESA vs carbon content in the MPL. (-) without MPL, (--) 0.5 mg cm -2 , (-•-) 1 mg cm -2 , (-••-) 2 mg cm -2 , (--) 4 mg cm -2 .

FIGURE 6 . 2 FIGURE 7 .

 627 FIGURE 6. Impedance spectra for the different gas diffusion layers. a) O 2 stoichiometry equals 4; b) O 2 stoichiometry equals 1.5; c) air. Carbon content in the

  Figure 1

Figure 4 Page

 4 Figure 4

Table 1 .

 1 1 mg cm -2 , ( ) 2 mg cm -2 , ( ) 4 mg cm -2 . Resistance values obtained from the Nyquist plot (Figure6)

	Page 16 of 26

Wiley-VCH Fuel Cells

, ( ) 2 mg cm -2 , ( ) 4 mg cm -2 .

Acknowledgments

The authors want to thank the Ministry of Education and Science of the Spanish Government, the Junta de Comunidades de Castilla-La Mancha and the enterprise CLM-H2 for the financial support through the projects CTM2007-60472 and PBI08-151-2045, respectively. The Spanish Government (Ministerio de Ciencia e Innovación) is also thanked for the grant AP2007-02713 awarded to D. Úbeda.


MSc, MPhil M L Molendijk
email: molendijkml@fsw.leidenuniv.nl

Baa Bus

Ph Spinhoven

Bwjh Penninx

G Kenis

J Prickaerts

R C Oude Voshaar

B M Elzinga

Serum Levels of Brain-Derived Neurotrophic Factor in Major Depressive Disorder: State -Trait Issues, Clinical Features, and Pharmacological Treatment

Keywords: , method, results, discussion): 3489 depression, brain-derived neurotrophic factor, antidepressants, BDNF

Recent evidence supports 'the neurotrophin hypothesis of depression' in its prediction that Brain-Derived Neurotrophic Factor (BDNF) is involved in depression. However, some key questions remain unanswered, including whether abnormalities in BDNF persist beyond the clinical state of depression, whether BDNF levels are related to the clinical features of depression, and whether distinct antidepressants affect BDNF levels equally. We addressed these questions and investigated serum BDNF levels in 962 depressed patients, 700 fully remitted persons (≥ 6 months) and 382 healthy controls. We found serum BDNF levels to be low in antidepressant free depressed patients relative to controls (P=.007) and to depressed patients who were treated with an antidepressant (P=.001). BDNF levels of fully remitted persons (whether unmedicated or treated with an antidepressant) were comparable to those of controls.

Analyzing the sample of antidepressant free depressed patients showed that BDNF levels were unrelated to the core clinical features of depression such as its severity or first versus a recurrent episode. The antidepressant associated up-regulation of serum BDNF in depressed patients was confined to SSRIs (P=.003) and St. John's wort (P=.03). Our results suggest that low serum levels of BDNF are a state abnormality that is evident during depression and normalizes during remission. Increases in serum levels of BDNF during antidepressant treatment appear to be confined to some antidepressants and do not parallel clinical characteristics, such as the severity of depressive symptoms.

Introduction

Brain-Derived Neurotrophic Factor (BDNF) is a neurotrophin that has been linked to the viability of neurons in brain circuits that regulate emotion, memory, learning, sleep, and appetite. [START_REF] Duman | Neuronal plasticity and survival in mood disorders[END_REF][START_REF] Sutton | Dendritic protein synthesis, synaptic plasticity, and memory[END_REF] The neurotrophin hypothesis of depression is based on these functions of BDNF and postulates that depression results from stress induced decreases in BDNF expression and that antidepressants are efficacious because they increase BDNF expression. [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF][START_REF] Duman | A molecular and cellular theory of depression[END_REF] Consistent with this hypothesis are the findings that depression is accompanied by decreased central and peripheral levels of BDNF [START_REF] Dwivedi | Altered gene expression of brain-derived neurotrophic factor and receptor tyrosine kinase B in postmortem brains of suicide victims[END_REF][START_REF] Sen | Serum brain-derived neurotrophic Factor, depression, and antidepressant medications: meta-analyses and implications[END_REF][START_REF] Brunoni | A systematic review and meta-analyses of clinical studies on major depression and BDNF levels: implications for the role of neuroplasticity in depression[END_REF] and that antidepressants elicit an increase in BDNF levels in animal models for depression [START_REF] Angelucci | BDNF in schizophrenia, depression and corresponding animal models[END_REF] and in depressed humans. [START_REF] Sen | Serum brain-derived neurotrophic Factor, depression, and antidepressant medications: meta-analyses and implications[END_REF][START_REF] Brunoni | A systematic review and meta-analyses of clinical studies on major depression and BDNF levels: implications for the role of neuroplasticity in depression[END_REF] Together with the latency of weeks before antidepressants become clinically effective, [START_REF] Nemeroff | Treatment of mood disorders[END_REF] these observations shaped the hypothesis that the efficacy of antidepressants depend on neuroadaptive changes that are brought about by changes in BDNF signaling. [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF] Taken together, there is reason to believe that BDNF is involved in depression and in antidepressant action. Results inconsistent with the neurotrophin hypothesis, however, also have been reported. There are, for example, studies that did not detect alternations in BDNF in depressed persons or in the course of treatment with an antidepressant. [START_REF] Basterzi | Effects of fluoxetine and venlafaxine on serum brain-derived neurotrophic factor levels in depressed patients[END_REF][START_REF] Matrisciano | Changes in BDNF serum levels in patients with major depression disorder (MDD) after 6 months treatment with sertraline, ecitalopram, or venlafaxine[END_REF][START_REF] Ziegenhorn | Serun Neurotrophins -a study on the time course and influencing factors in a large old sample[END_REF] Additionally, some questions remain unanswered so that the neurotrophin hypothesis is at best incomplete. [START_REF] Groves | Is it time to reassess the BDNF hypothesis of depression?[END_REF] A major question that largely remains to be answered is whether low BDNF levels persist beyond the clinical state of depression. [START_REF] Trajkovska | Whole blood BDNF levels in healthy twins discordant for affective disorder: Association to life events and neuroticism[END_REF][START_REF] Monteleone | Decreased levels of brainderived neurotrophic factor in both depressed and euthymic patients with unipolar depression and in euthymic patients with bipolar I and II disorders[END_REF] A second question is whether BDNF levels are related to the clinical features of depression, such as a first versus a recurrent episode. [START_REF] Lee | Decreased plasma BDNF level in depressive patients[END_REF][START_REF] Karege | Decreased serum brain-derived neurotrophic factor in major depressed patients[END_REF] Yet a third outstanding question is whether all classes of antidepressants affect BDNF levels equally. [START_REF] Sen | Serum brain-derived neurotrophic Factor, depression, and antidepressant medications: meta-analyses and implications[END_REF][START_REF] Brunoni | A systematic review and meta-analyses of clinical studies on major depression and BDNF levels: implications for the role of neuroplasticity in depression[END_REF] We studied, cross-sectionally, serum BDNF levels of depressed patients, remitted depressed persons, and never depressed persons. Our efforts had three concerns: (1) to compare serum BDNF levels of antidepressant free and antidepressant treated current and fully remitted depressed patients and never depressed persons, (2) to explore the associations between some of the core clinical features of depression and serum BDNF levels, and (3) to evaluate the association between the use of several distinct classes of antidepressants and serum BDNF levels.

Subjects and Methods

Subjects and sample collection

Subjects were from the Netherland Study of Depression and Anxiety (NESDA). Full details on the rationale, objectives, and protocol of NESDA are described in a previous paper by Penninx et al. [START_REF] Penninx | NESDA Research Consortium. The Netherlands Study of Depression and Anxiety (NESDA): rational, objectives and methods[END_REF] In brief, NESDA is a prospective cohort study (N = 2981) that recruited subjects in mental health care, primary care, and in the general population. Included were persons with a depressive and/or an anxiety disorder, persons with a depressive and/or an anxiety disorder in remission, and persons without a history or current depressive or anxiety disorders. Persons who were diagnosed with psychotic-, bipolar I or II -, obsessive-compulsive-or severe alcohol use disorder were not eligible. DSM-IV diagnoses [START_REF]Diagnostic and Statistical Manual of Mental Disorders[END_REF] were assigned on the basis of responses to the Composite International Diagnostic Interview 2.1 (CIDI) lifetime version [START_REF] Wittchen | Crosscultural feasibility, reliability and sources of variance of the Composite International Diagnostic Interview (CIDI). The multicentere WHO/ADAMHA field trials[END_REF] that was administered by trained interviewers. At baseline, participants provided blood samples, underwent a medical examination, and gave written informed consent for the study that was approved by the Ethical Committees of the participating institutes.

Our study enrolled 2044 persons (68.6% of the NESDA sample). Based on diagnosis, antidepressant use and the availability of BDNF data we created 5 groups; antidepressant free depressed patients (n=541), antidepressant treated depressed patients (n=421), antidepressant free remitted depressed persons (n=539), antidepressant treated remitted depressed persons (n=161), and healthy persons who served as controls (n=382). Depressed patients met the criteria for a depressive episode within the last 6 months (n=541). The majority of these patients had a current diagnosis of depression (n=388), but some (n=153) had a diagnosis of depression 1-6 months prior to baseline and did not fulfill all criteria in the past month. Persons who were in full remission of depression were diagnosed with MDD somewhere in their lives, but had been free of depression and anxiety during at least 6 months. Persons were included in the control group when they had: (1) no lifetime mood or anxiety disorders, (2) no documented family history of depression or anxiety, and (3) a low score (≤ 14) on the Inventory of Depressive Symptoms (IDS). [START_REF] Rush | The Inventory of Depressive Symptomatology (IDS): psychometric properties[END_REF]

Antidepressants

Data on the use of antidepressants was acquired through drug container observation and self-report. Use of an antidepressant was defined as intake of minimally the daily dose as recommended by the World Health Organization 22 during the last month on at least 50% of the days. We coded for the use of Selective Serotonin Reuptake Inhibitors (SSRIs), Serotonin-Norepinephrine Reuptake Inhibitors (SNRIs), Tricyclic Antidepressants (TCAs), Noradrenergic and Specific Serotonergic Antidepressants (NaSSAs), and St. John's wort (Hypericum perforatum). The duration of use was expressed in months.

Clinical features of MDD

All subjects were characterized on the symptom severity of depression using the IDS. [START_REF] Rush | The Inventory of Depressive Symptomatology (IDS): psychometric properties[END_REF] Patient samples were further characterized on having a first or a recurrent depressive episode, the presence of comorbid anxiety, the age at onset of depression, the recency of depression, the chronicity of depression, and on the presenceof suicide ideation. The CIDI interview [START_REF] Wittchen | Crosscultural feasibility, reliability and sources of variance of the Composite International Diagnostic Interview (CIDI). The multicentere WHO/ADAMHA field trials[END_REF] served as source of information on the presence of a first or a recurrent depressive episode, the presence of a comorbid anxiety, age at onset of depression (ie., the age at which the first episode occurred), and the recency of depression (ie., fulfilling criteria in the past month versus fulfilling criteria in the past 6 months but not in the past month). Depression was considered chronic if symptoms had been present ≥ 24 months during the last 5 years, which was assessed using the Life Chart method. [START_REF] Lyketsos | The life chart interview: a standardized method to describe the course of psychopathology[END_REF] The scale for suicide ideation [START_REF] Beck | Assessment of suicidal intention: the Scale for Suicide Ideation[END_REF] was used to examine the presence (yes vs. no) of suicide ideation during the past week.

BDNF protein measurements

Fifty millilitre of blood was withdrawn into vacuum tubes between 07:30 and 09:30 a.m. after an overnight fast. Following blood collection, serum was separated and stored at -85 Celsius until it was assayed. BDNF protein levels were measured using the Emax Immuno Assay system from Promega according to the manufacturer's protocol (Madison, WI, USA), in 1 laboratory (Maastricht University) by 1 technician who was blind to diagnoses. Undiluted serum was acid treated since this reliably increased the detectable BDNF in a dilution-dependent way. Greiner Bio-One high affinity 96-well plates were used. Serum samples were diluted 100 times and the absorbency was read in duplicate using a Biorad Benchmark microplate reader at 450 nm. The intra-and inter-assay coefficients of variation were found to be within 3% and 9% respectively. Four persons had BDNF values that were below the reliable detection threshold of 1.56 ng/ml. These values were set at the lower detection limit.

Positive outliers (mean + 3 STDs, n=6) were trimmed to the mean + 3 STDs value.

There were no differences between persons with missing and non-missing BDNF with regard to gender (P=.71), age (P=.67), and diagnoses (P=.33).

Covariates

Potential variance due to gender, age, and educational level was controlled for in all analyses. Additionally, we controlled for Body Mass Index (BMI), physical activity, and smoking since these variables are associated with BDNF [START_REF] Suwa | Serum brainderived neurotrophic factor level is increased and associated with obesity in newly diagnosed female patients with type 2 diabetes mellitus[END_REF][START_REF] Rojas-Vega | Acute BDNF and cortisol response to low intensity exercise and following ramp incremental exercise to exhaustion in humans[END_REF][START_REF] Kim | Increased plasma brain-derived neurotrophic factor levels in chronic smokers following unaided smoking cessation[END_REF] and mood. [START_REF] Simon | Association between obesity and psychiatric disorders in the US adult population[END_REF][START_REF] Harris | Physical Activity, exercise coping, and depression in a 10-year cohort study of depressed patients[END_REF][START_REF] Kendler | Smoking and major depression[END_REF] Data on weight and height was collected and BMI was calculated (weight/height 2 ).

Information on physical activity was gathered using the International Physical

Activity Questionnaire [START_REF] Craig | International physical activity questionnaire: 12-country reliability and validity[END_REF] and expressed as number of met-minutes (ie., the ratio of the amount of energy expenditure during activity to the energy expenditure at rest).

Smoking status was dichotomized as current versus non-smoker. Time of the morning blood withdrawal and duration of serum storage were controlled for since BDNF levels vary according to variation on these variables. [START_REF] Begliuomini | Plasma brain-derived neurorophic factor daily variations in men: correlation with cortisol circadian rhythm[END_REF][START_REF] Trajkovska | Measurements of brain-derived neurotrophic factor: methodological aspects and demographical data[END_REF]

Statistical analyses

All computations were performed in SPSS version 17.0 (SPSS, Chicago, Illinois). BDNF values were controlled for basic covariates in all analyses. Effect sizes on pairwise comparisons were presented as Cohen's d. [START_REF] Cohen | Statistical power analysis for the behavioral sciences[END_REF] A 2-tailed α level of .05 was used to determine statistical significance.

Analysis of Variance (ANOVA) was used to compare BDNF levels of antidepressant free depressed patients and antidepressant treated depressed patients, antidepressant free patients and antidepressant treated persons who were in remission (≥ 6months), and controls. Post-hoc tests between the groups were performed following a significant F-statistic using Tukey tests.

A multivariable regression analysis was used to identify whether the clinical features of depression were associated with BDNF levels. Regression was performed in patient groups in which the mean BDNF level deviated significantly from the control group. Pearson correlation coefficients between predictors and BDNF levels were also calculated. Basic covariates were entered in the first step of regression. In the second step the clinical features of depression were entered. The regression model was fit using method enter. Tolerance of the predictors and normality of error variances was verified.

To establish whether the use of an antidepressant effected BDNF levels equally in current and remitted depression, a 2 (currently depressed vs. depression in (full) remission) × 2 (antidepressants; yes or no) ANOVA was performed. Potential antidepressant specific associations between the use of SSRI, TCA, SNRI, NaSSA, and St. John's wort and BDNF levels were evaluated by contrasting BDNF levels of persons who used 1 of these agents against the BDNF level of the antidepressant free persons. Analyses were repeated with the severity of depressive symptoms and the duration of antidepressant use as covariates.

Results

Demographics and clinical features

Demographical and clinical features among the 5 groups are given in Table 1.

ANOVA and χ-square tests showed that, compared to controls, depressed and remitted persons were more likely to be female, to be older, to have received fewer years of education, and to smoke. BMI was higher in current and remitted antidepressant treated depressed persons compared to controls and to antidepressant free depressed and remitted persons. The amount of physical activity was low in the antidepressant treated currently depressed group relative to the other groups. Post-hoc comparisons on demographical and clinical features between the current and remitted depressed groups are given in Table 1.

BDNF levels in persons with current or remitted depression and controls

An ANOVA model showed a main effect of diagnostic status on serum levels of BDNF (F 1, 1578 =4.09, P=.01). Pair-wise comparisons (see Figure 1) indicated that serum BDNF levels were low in antidepressant free depressed patients compared to controls (d=0. 19), to antidepressant free persons who were in full remission (d=0.15), and to antidepressant treated depressed patients (d=0.23). BDNF levels of antidepressant free persons who were in full remission and depressed patients who were treated with an antidepressant were comparable to those of controls.

BDNF and the clinical features of MDD

The exploration of the association between the clinical features of MDD and serum BDNF was restricted to the antidepressant free currently depressed group, since BDNF levels in this group were low relative to controls.

Pearson's correlation showed that female gender and being in the early remission phase of depression (1-6 months) versus having a current episode were negatively associated with serum BDNF. Age, BMI, age at onset of MDD, and the presence of comorbid anxiety were positively associated to serum BDNF (Table 2).

Basic covariates were entered in the first step of the multivariable regression analysis, followed by the clinical features that were entered in step two. Tolerance of the predictors was high (all > 0.70) indicating that our individual predictors were not redundant with one another. Error variances were normally distributed. Results of the first step showed that gender and age were significant predictors of BDNF levels.

Women had lower levels of BDNF compared to men (β=-0.10, P=.02) and older patients had higher levels of BDNF (β=0.11, P=.002) compared to younger patients.

Results of the second step showed that none of the clinical features (listed in Table 2)

was significantly associated with serum BDNF. Gender and age preserved its significance. BMI emerged as a significant (positive) predictor of serum BDNF.

Table 2 presents the results of the second step of the regression analysis.

BDNF and the use of antidepressants

A 2 (currently depressed vs. depression in (full) remission) × 2 (antidepressant use; yes vs. no) ANOVA showed that diagnostic status interacted with antidepressant use (F 1, 1578 =4.19, P=.03) indicating that the use of an antidepressant during a depressive episode was associated with higher BDNF levels whereas in the remission phase the use of an antidepressant did not show such an association (see Figure 2). Main effects of diagnostic status and antidepressant use were not observed.

To uncover potential differences between various classes of antidepressants we compared BDNF levels of depressed patients who used SSRIs, SNRIs, TCAs, NaSSAs, or St. John's wort among each other and with BDNF level of antidepressant free depressed patients. This analysis was restricted to the currently depressed group since the effect of the use of an antidepressant on serum BDNF levels was confined to this group. In this group, 67% (n = 282) used and antidepressant for longer then 12 weeks. We observed a main effect of group (F 5, 941 =4.29, P<.001). Post-hoc comparisons (see Figure 2) showed that, relative to not using an antidepressant, the use of SSRIs (d=0.39) and St. John's wort (d=0.63) was associated with high levels of BDNF. The use of a NaSSA was associated with low levels of BDNF relative to SSRI (d=0.54) and St. John's wort (d=0.85) use. Analyses were run with and without covarying for the severity of depressive symptoms and for the duration of antidepressant use. These analyses revealed a similar pattern of results. Furthermore, serum BDNF levels were unrelated to treatment duration (r=-0.02, P=.65) which might suggests that our findings were not driven by duration of antidepressant use.

Discussion

Largely in accord with previous findings [START_REF] Sen | Serum brain-derived neurotrophic Factor, depression, and antidepressant medications: meta-analyses and implications[END_REF][START_REF] Brunoni | A systematic review and meta-analyses of clinical studies on major depression and BDNF levels: implications for the role of neuroplasticity in depression[END_REF] and with the neurotrophin hypothesis of depression [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF][START_REF] Duman | A molecular and cellular theory of depression[END_REF] our data showed that serum BDNF levels were low in antidepressant free depressed patients compared to healthy controls. Our data further showed that BDNF levels were low in depressed patients who were not on antidepressant medication compared to antidepressant free persons who were in full remission and that BDNF levels of this latter group were comparable to those of controls. Herewith we establish as one of the first 14 that low levels of BDNF in serum are a state characteristic for depression. In line with one study that reported low levels of BDNF in euthymic patients [START_REF] Monteleone | Decreased levels of brainderived neurotrophic factor in both depressed and euthymic patients with unipolar depression and in euthymic patients with bipolar I and II disorders[END_REF] we found that patients who were in early remission (1-6 months) had serum BDNF levels that were comparable to those of currently depressed patients. Thus serum BDNF levels remain low after clinical improvement has set in.

This could indicate that low levels of BDNF are a consequence of depressive symptoms that persist into early remission. Alternatively, the low levels of BDNF during early remission might also represent a scar of a depressive episode. These explanations could not be fully elucidated in the current study and longitudinal designs clearly are essential to understand this issue.

We were unable to replicate the earlier findings that a higher depression severity [START_REF] Karege | Decreased serum brain-derived neurotrophic factor in major depressed patients[END_REF][START_REF] Shimuzu | Alternations of serum levels of Brain-Derived Neurotrophic Factor (BDNF) in depressed patients with or without antidepressants[END_REF] , having a recurrent compared to a first episode of MDD [START_REF] Lee | Decreased plasma BDNF level in depressive patients[END_REF] , and the occurrence of suicide ideation [START_REF] Deveci | Serum BDNF levels in suicide attempters related to psychosocial stressors: a comparative study with depression[END_REF][START_REF] Kim | Low plasma BDNF is associated with suicidal behavior in major depression[END_REF] are accompanied by lower levels of BDNF. In fact, we even found that the early remission phase, which was accompanied by a lower symptom severity of depression (mean IDS scores were 22.4 ± 11.4 vs. 32.4 ± 12.1 in early remitted and currently depressed patients respectively), was associated with somewhat lower BDNF levels compared to the current depressive state. The other clinical features (i.e., age at onset of depression, the presence of comorbid anxiety, and the chronicity of depression) also were unrelated to serum BDNF in multivariable analyses. These findings, given the size of the current cohort, give us confidence in excluding the clinical features of depression as potential correlates of serum BDNF levels. This might be an important conclusion, since it hints that other (than specifically depression related) factors may be at play in the relative fall of BDNF levels during a depressive episode. Interestingly, being male and having a higher BMI were found to be positively associated with BDNF among antidepressant free depressed patients. Although these findings were unsought, they parallel the results of some previous studies [START_REF] Karege | Decreased serum brain-derived neurotrophic factor in major depressed patients[END_REF][START_REF] Monteleone | Circulating brain-derived neurotrophic factor is decreased in women with anorexia and bulimia nervosa but not in women with binge eating disorder: relationships to co-morbid depression, psychopathology and hormonal variables[END_REF][START_REF] Nakazato | Decreased levels of serum brain-derived neurotrophic factor in female patients with eating disorders[END_REF] and they give ground to interesting hypotheses. For example, since weight loss is a prime behavioral abnormality of depression [START_REF]Diagnostic and Statistical Manual of Mental Disorders[END_REF] and often a residual symptom in early remission [START_REF] Paykel | The clinical interview for depression development, reliability and validity[END_REF][START_REF] Paykel | Residual symptoms after partial remission: an important outcome in depression[END_REF] it could be that, alternations in BDNF levels are mediated by (transient) changes in eating behavior during, or in the aftermath of, a depressive episode. Likewise, weight gain is a documented side effect of antidepressant treatment [START_REF] Kachur | Antidepressant-induced weight gain[END_REF][START_REF] Antilla | A review of the pharmacological and clinical profile of mirtazapine[END_REF] and thus the absence of weight loss could potentially explain the absence of a relative fall of BDNF in depressed patients during treatment with an antidepressant.

Alternative factors that have been proposed to underlie the low levels of BDNF during depression are exposure to stressful life events. Two studies, for example, found that adverse life events are associated with lower peripheral BDNF levels within a depressed and bipolar patient samples. [START_REF] Kauer-Sant'anna | Traumatic life events in bipolar disorder: Impact on BDNF levels and psychopathology[END_REF][START_REF] Grassi-Oliveira | Low plasma Brainderived neurotrophic factor and childhood physical neglect are associated with verbal memory impairment in major depression -acpreliminary report[END_REF] Therefore, it seems worthwhile to integrate a wider range of variables, notably (early) adverse life events, but also genetic variants and their interactions with environmental variables [START_REF] Gatt | Interactions between BDNF val66met polymorphism predicts brain and arousal pathways to syndromal depression and anxiety[END_REF] in models that study the link between BDNF and depression.

Additionally, we found that serum BDNF levels were higher in antidepressant treated patients compared to patients who were antidepressant free. This finding largely is in accord with previous findings. [START_REF] Sen | Serum brain-derived neurotrophic Factor, depression, and antidepressant medications: meta-analyses and implications[END_REF][START_REF] Brunoni | A systematic review and meta-analyses of clinical studies on major depression and BDNF levels: implications for the role of neuroplasticity in depression[END_REF] We were able to expand previous findings by showing that the use of an antidepressant is associated with increased serum BDNF during a depressive episode but not during remission. This suggests that antidepressant induced increases in BDNF occur in a disease state when BDNF functioning might be defective and not in remission when BDNF functioning is normalized. Additionally, we found the increase in serum BDNF levels to be a specific associate of the use of SSRIs and St. John's wort and not of the use of SNRIs, TCAs, or NaSSAs. Although not directly confirmed, this finding might be explained by increased availability of extra-synaptic levels of serotonin. It is known that serotonin stimulates the expression of BDNF. [START_REF] Mattson | BDNF and 5-HT: a dynamic duo in age related neuronal plasticity and neurodegenerative disorders[END_REF][START_REF] Martinowich | Interaction between BDNF and serotonin: role in mood disorders[END_REF] In line with this we found the highest BDNF levels in patients who were treated with an agent that generally leads to an increase in the availability of serotonin (ie., SSRIs and St John's wort). [START_REF] Mann | The medical management of depression[END_REF][START_REF] Gaster | St John's wort for depression[END_REF] Furthermore, we found the lowest levels of BDNF in patients who were treated with agents that have little or no impact on the availability of serotonin (ie., NaSSAs). [START_REF] Antilla | A review of the pharmacological and clinical profile of mirtazapine[END_REF][START_REF] Kent | NaSSAs, and NaRIs: new agents for the treatment of depression[END_REF] Nevertheless, this antidepressant specific finding seems at odds with the specific prediction of the neurotrophin hypothesis, stating that increases in BDNF levels are a key mediator for an antidepressant response to occur. [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF] According to this prediction, one might expect that antidepressants that are known to be about equally efficacious in the treatment of the symptoms of depression [START_REF] Gaster | St John's wort for depression[END_REF][START_REF] Kent | NaSSAs, and NaRIs: new agents for the treatment of depression[END_REF][START_REF] Berton | New approaches to antidepressant drug discovery[END_REF] would have similar effects on serum BDNF levels. Yet another finding that seems hard to reconcile with the neurotrophin hypothesis is that the group of depressed persons who used antidepressants (prolonged and frequently) had the highest BDNF levels, but also the highest symptom severity of depression. This suggests, to our belief that increases in peripheral BDNF levels do not parallel clinical effectiveness, or at least have no direct effects on the depression characteristics such as its severity. Such a conclusion on the absence of direct effects could also be drawn on the findings that the severity of a depressive episode was unrelated to serum BDNF levels and that persons who were in early remission had similar levels of BDNF yet marked lower levels of depression severity compared to depressed patients. Caution, however, is warranted when interpreting our findings on the associations between the use of an antidepressant and serum levels of BDNF because our subjects were not randomly assigned to the various drugs (or no drug) conditions. Thus our findings might be confounded by indication. An additional limitation of our study is that we relied on data that were collected in a single wave, precluding any form of causality. Furthermore, we measured serum levels of BDNF and assume that these measurements mirror the amount of BDNF in the brain. This assumption is validated on preclinical work that showed that cortical and peripheral levels of BDNF are correlated [START_REF] Sartorius | Correlations and discrepancies between serum and brain tissue levels of neurotrophins after electroconvulsive treatment in rats[END_REF][START_REF] Karege | Postnatal developmental profile of brain derived neurotrophic factor in rat brain and platelets[END_REF][START_REF] Klein | Blood BDNF concentrations reflect brain-tissue BDNF levels across species[END_REF] but remains complicated, because in addition to neurons, several other tissues serve as sources of BDNF in serum. [START_REF] Karege | Postnatal developmental profile of brain derived neurotrophic factor in rat brain and platelets[END_REF] However, various strengths of our study seem evident and these include the use of multivariable techniques and the large sample size (that relates positive to all previous studies and to two previous metaanalyses [START_REF] Sen | Serum brain-derived neurotrophic Factor, depression, and antidepressant medications: meta-analyses and implications[END_REF][START_REF] Brunoni | A systematic review and meta-analyses of clinical studies on major depression and BDNF levels: implications for the role of neuroplasticity in depression[END_REF] as well).

In conclusion, we believe that our data indicate that low levels of BDNF in blood serum are a state characteristic of depression and thus an abnormality that is evident during the clinical state and the early remission phase of depression but not when the symptoms of depression are in full remission. Our findings further suggest that some of the core clinical features of depression are unrelated to serum levels of BDNF.

Finally, increases in serum levels of BDNF appear to be a specific pharmacological effect of a subset of antidepressants that does not parallel depression characteristics such as the severity of depression. Reuptake Inhibitor; TCA, Tricyclic Antidepressant. [START_REF] Duman | Neuronal plasticity and survival in mood disorders[END_REF] Mean met-minutes (ie., ratio of energy expenditure during activity to energy expenditure at rest) divided by 1000. [START_REF] Sutton | Dendritic protein synthesis, synaptic plasticity, and memory[END_REF] Symptoms were considered chronic if they were present for at least 24 months during the last 5 years. [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF] Included social phobia, panic disorder with and without agoraphobia, agoraphobia, and generalized anxiety disorder. A Indicates a statistical significant difference (at P < .05) between the antidepressant treated and antidepressant free current MDD groups. B Indicates a statistical significant difference (at P < .05) between the antidepressant treated and antidepressant free remitted MDD groups.

Figure 1 .Figure 2 .

 12 Figure 1. Plotted are mean serum BDNF levels by diagnoses and antidepressant use. Error bars reflect the standard error of the mean. * Denotes statistical significance at P < .05. ** Denotes statistical significance at P <.01.

Table 1

 1 Demographic and clinical characteristics (percentages (%) or mean ± standard deviation) of participants by depression diagnosis (never, current, and remitted) and antidepressant use (yes versus no).

Table 2

 2 Results of correlation and multiple regression analyses of demographical and clinical characteristics with serum levels of BDNF in antidepressant free patients with MDD. Abbreviations: IDS, Inventory of Depressive Symptoms; MDD, Major Depressive Disorder; NaSSA, Noradrenergic and Specific Serotonergic Antidepressant; SNRI, Serotonin and Norepinephrine Reuptake Inhibitor; SSRI, Selective Serotonin

b In minutes from 06:00 AM. c The presence of a current (1 month) versus an early remission (1 to 6 months of remission) diagnosis. * Denotes statistical significance of the univariate correlation at P < .05. ** Denotes statistical significance of the univariate correlation at P < .01.

Acknowledgement:

We want to thank Robin Struijk (Maastricht University) for determining serum levels of BDNF in our sample.

1 Mean met-minutes (ie., ratio of energy expenditure during activity to energy expenditure at rest) divided by 1000.

2 Symptoms were considered chronic if they were present for at least 24 months during the last 5 years. [START_REF] Duman | A neurotrophic model for stress-related mood disorders[END_REF] Included social phobia, panic disorder with and without agoraphobia, agoraphobia, and generalized anxiety disorder.

A Indicates a statistical significant difference (at P < .05) between the antidepressant treated and antidepressant free current MDD groups B Indicates a statistical significant difference (at P < .05) between the antidepressant treated and antidepressant free remitted MDD groups