P Gentilini

O Yazoghli-Marzouk

V Delmotte

Y Descantes
email: yannick.descantes@ifsttar.fr

Determination of the water content of fillerised fine aggregates in the saturated surface dry state

Keywords: fine aggregate, sand, cement concrete, water absorption, test method, experimental setup, drying

An experimental setup designed to capture the saturated surface dry (SSD) state of porous fine aggregates incorporating significant amounts of fines is presented. This setup is validated upon testing fine aggregate sources with different water absorption values up to 5.6 %, and at least 8 % of fines. Test duration is found to lie in the 120-to-240-minute-range, depending on test sample initial mass and water content. Several procedures for calculating the water content in SSD state are presented and discussed.

One of these procedures uses the drying theory to fit curves depicting water content shift as a function of drying time and identify breakpoints. A more straightforward strategy is focused on the transition point between retained and free water on charts representing the variations of water content as a function of relative humidity. In both cases, fairly repeatable and discriminant water content values in SSD state are obtained.

. Moreover the lack of reliability of water absorption values determined in the presence of fines according to the cone test procedure is verified. Eventually, several perspectives are pointed out to improve the setup which could easily be automated.

Introduction

Short term and long term performances of cement concrete are known to depend on effective-water-to-cement ratio (W eff /C), where effective water refers to water available for cement hydration. Effective water may be defined as the total mass of water contained in the cement concrete (W tot ) less the mass of water retained by the aggregate fractions. For a given aggregate source, this 'retained water' is known to increase with decreasing size fraction, especially in the fine fractions 0/4 or 0/5 mm. The maximum retained water is routinely though disputably [START_REF] Kasemchaisiri | A method to determine water retainability of porous fine aggregate for design and quality control of fresh concrete[END_REF] assessed by the 'water absorption' value (WA) -mass of water penetrating the voids of particles during a prescribed period of time, routinely 24h, expressed as a percentage of the oven-dried mass of the aggregate sample [START_REF] Cen | Tests for mechanical and physical properties of aggregates -Part 6: Determination of particle density and water absorption[END_REF]. For a long time, the large quantities of fine aggregates needed for cement concrete production have been purveyed almost exclusively by natural fine aggregates (NFA) from alluvial or limestone deposits, whose WA values are known to be low (< 1%). In the recent years, the shortage of alluvial sources and waste storage capacity limitations have given rise to a worldwide interest in abundant alternative fine aggregate sources such as recycled concrete fine aggregates (RCFA).

However, their use is still limited due to high WA values in the range 6-10 % [START_REF] Rilem | RILEM recommendation I2I-DRG Guidance for demolition and reuse of concrete and masonry. Specifications for concrete with recycled aggregates[END_REF][START_REF]STAR 217-PRE Progress of Recycling in the Built Environment[END_REF]. Indeed, adjusting the W eff /C ratio of fresh cement concrete becomes much more difficult when fine aggregates with high WA value are incorporated, since they may add or subtract water allotted for cement hydration, thus impact its short term workability as well as mechanical and durability performances in hardened state [START_REF] Larrard | Concrete Mixture Proportioning: A Scientific Approach[END_REF] [START_REF] Zhao | Influence of fine recycled concrete aggregates on the properties of mortars[END_REF]. Khatib [START_REF] Khatib | Properties of concrete incorporating fine recycled aggregate[END_REF] reported the good workability of fresh cement concrete incorporating 100% RCFA with additional water to reach W tot /C = 0.5 (no plasticizer); however, he also noted a compressive strength drop down to 35% after 28 days of curing and a drying shrinkage increase up to 50% when compared with a reference cement concrete incorporating NFA exclusively. Reducing the W tot /C ratio upon using an admixture allows both to maintain sufficient workability and prevent compressive strength drop, but concrete properties in hardened state are still impacted: splitting resistance may decrease by 30%, water absorption and sorptivity may increase by 46% and 70% respectively with respect to corresponding values of the reference cement concrete. As a consequence, external fluids may penetrate the concrete microstructure and introduce deleterious substances such as carbon dioxide and chloride ions, causing concrete carbonation and steel corrosion respectively [START_REF] Evangelista | Durability performance of concrete made with fine recycled concrete aggregates[END_REF].

The water absorption of alternative fine aggregates such as RCFA having significant implication on W/C ratios for concrete mixtures, determining it accurately is critical [START_REF]STAR 217-PRE Progress of Recycling in the Built Environment[END_REF]. Therefore, most test methods consist of drying a pre-saturated sample until achievement of the state under which particles surface is free of vaporizable moisture but their accessible pores remain saturated with water, called saturated-surface-dry state (SSD, see Figure 1), then in promptly weighing the sample. To capture the SSD state, some methods focus on the visual detection of test portion color change when particles become free of surface vaporizable moisture [START_REF] Kandhal | An Evaluation of the Bulk Specific Gravity for Granular Materials[END_REF][START_REF] Krugler | Improving the Precision of Test Methods Used in VMA Determination[END_REF]. Others track the slump of a cone-shaped test portion [START_REF] Cen | Tests for mechanical and physical properties of aggregates -Part 6: Determination of particle density and water absorption[END_REF][START_REF]C 128-07a: Standard Test Method for density, relative density ([END_REF][START_REF] Aashto | Standard test method of test for specific gravity and absorption of fine aggregate[END_REF] or the flow of a test sample off a tilted masonry trowel [START_REF] Krugler | Improving the Precision of Test Methods Used in VMA Determination[END_REF] which occur when between-particles surface tension forces disappear. samples as a function of drying time, such as electrical resistivity reflecting the presence of between-particles water bridges [START_REF] Lin | Determination of SSD condition of fine aggregates using AC impedance spectroscopy[END_REF]. A few test methods attempt to detect the end of the saturation phase while soaking a dry test portion, either from direct measurement of the volume of absorbed water [START_REF] Saxer | A direct Method of Determining Absorption and Specific Gravity of Aggregates[END_REF], or from detecting the reflection signal of infra-red light rays when particles surface becomes moist [START_REF] You | Measuring the Specific Gravities of Fine Aggregates: An Automated Procedure[END_REF]. Unfortunately, most of these test methods are unsuitable for fine aggregates such as RCFA, which may incorporate up to 10-13% of fines (particles passing the 0.063 mm sieve) [START_REF] Dre Bourgogne | Catalogue des matériaux de substitution en Bourgogne[END_REF][START_REF] Gomes | Mechanical Performance of Structural Concrete with the Incorporation of Coarse Recycled Concrete and Ceramic Aggregates[END_REF].

Others spot slope breakpoints on curves measuring physical characteristics of test

Indeed, Rogers et al [START_REF] Rogers | Fine aggregate density testing, what is the right way to do? 15th[END_REF] have reported a 100% overestimation of WA values assessed from methods using the cone slump criterion [START_REF] Cen | Tests for mechanical and physical properties of aggregates -Part 6: Determination of particle density and water absorption[END_REF][START_REF]C 128-07a: Standard Test Method for density, relative density ([END_REF][START_REF] Aashto | Standard test method of test for specific gravity and absorption of fine aggregate[END_REF], which they allotted to fines adhering to larger particles and forming clusters, thus trapping part of the formerly free water into their inner structure and causing earlier achievement of the sample 'SSD' state than in the absence of fines. Similarly, methods focused on test portion color change yield scattered results in the presence of fines, since homogenously drying a sample becomes delicate [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF].

Promising WA values of fine aggregates were measured using test methods derived from thermodynamic [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF][START_REF] Dana | Experimental Moisture Determination for Defining Saturated Surface Dry State of Highway Aggregates[END_REF] or industrial drying approaches [START_REF] Mechling | Mesure de l'absorption d'eau des additions minérales des bétons par vaporométrie (In French)[END_REF]. The former consists in air drying a pre-saturated fine aggregate sample placed into a rotating drum closed at both ends by 0.075 mm square mesh screens, while recording the temperature gradient between drum inlet and outlet as well as relative humidity at drum outlet. The drying phase is stopped and the sample is weighed when the temperature gradient or relative humidity curves evidence breakpoints similar to those shown on Fig. 2, presumably representative of SSD state according to the authors. The latter assimilates the drying of soaked aggregates at rest in a chamber to the evaporation of water from a porous medium at constant air temperature. Fig. 3 depicts sample mass reduction through evaporation as a function of time, which consists of three well-known phases [START_REF] Charreau | Séchage. Théorie et calculs[END_REF]:

-a transition phase corresponding to the drying chamber conditioning;

-then, a linear part depicting the evaporation of surface water at a constant rate monitored by environmental conditions (temperature and relative humidity) until achievement of the SSD state;

-Eventually, a phase at decreasing mass reduction rate evidencing the evaporation of absorbed water from aggregate pores.

Though promising, these two test methods have drawbacks: the drum method is biased by a loss of fines through the screens and disassembly operations which delay the weighing once SSD state is achieved; the drying chamber is unsuitable for uniformly drying polydisperse fine aggregates at rest due to crusting effects [START_REF] Charreau | Séchage. Théorie et calculs[END_REF]. Fig. 3. Water content (left) and evaporation rate (right) in a porous material [START_REF] Mechling | Mesure de l'absorption d'eau des additions minérales des bétons par vaporométrie (In French)[END_REF].

In order to determine the water absorption of fine aggregates incorporating fines, the present paper introduces an improved experimental setup designed to capture the SSD state and measure the water content in this state. Section 2 describes the experimental setup and the test program designed for its validation. Section 3

investigates the presence of breakpoints on various curves representing recorded data and discusses methods for assessing the water content in SSD state.

Experimental setup and test program a. Experimental setup

The experimental setup used to capture the SSD state is inspired from early development by Dana et al [START_REF] Dana | Experimental Moisture Determination for Defining Saturated Surface Dry State of Highway Aggregates[END_REF] and subsequent development by Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF]. As shown in Fig. 4, it is composed of a hair dryer that blows warm air at one end of a rotating drum, which is mounted on caster wheels and driven by a belt through a step motor. The drum incorporates flights to stir the test sample in order to achieve reasonably uniform drying, as well as screens at drum inlet (0.063 mm opening) and outlet to reduce aggregates loss. Temperature is measured by two probes located at either ends of the drum and relative humidity is captured by a hygrometry probe located at drum outlet. However, three major improvements have been implemented: first, both the screen blinding and loss of fines issues reported by Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF] have been sorted out using respectively an outlet screen with a wider opening (0.5 mm), and a fines collector consisting of a simple vortex system equipped with a cup, in which fines will remain trapped till the end of the test (see Fig. 4); second, no more drum removal is needed for weighing as the full experimental setup is mounted on a balance, thus allowing continuous weight monitoring to ± 1 g; third, the air dryer is fed by a voltage regulator to monitor the blown air temperature to roughly 70°C, that is not too high to avoid removing crystallized water in the mortar attached to the aggregate [START_REF] Tam | New approach in measuring water absorption of recycled aggregates[END_REF][START_REF] Mindess | Concrete[END_REF] though sufficiently high to keep the drying time reasonable. The total mass of unloaded experimental setup mounted on the balance is 13.1 kg.

b. Test program

Tested materials were sampled from three different sources, crushed sand-lime alluvium, crushed limestone and recycled concrete. Fig. 5 depicts the mean grading curve of each material tested, all of them incorporating at least 8% fines. Table 1 summarizes the densities and water absorption values determined for each material according to EN 1097-6 clause 9. The selected materials cover a reasonably wide range of WA values, from 0.8 for NFA up to 5.6% for RCFA, and the corresponding densities vary conversely as already noticed elsewhere [START_REF] Khatib | Properties of concrete incorporating fine recycled aggregate[END_REF][START_REF] Poon | Feasible use of recycled concrete aggregates and crushed clay brick as unbound road sub-base[END_REF]. Although the test procedure depicted in EN 1097-6 clause 9 prescribes fines discarding, a second set of samples were tested with their fines, supplemented with methylene blue (MB) tests performed on 0/2 mm samples in accordance with EN 933-9 [START_REF] Cen | Tests for geometrical properties of aggregates -Part 9: Assessment of fines -Methylene blue test[END_REF] to check the fines quality. As expected [START_REF] Rogers | Fine aggregate density testing, what is the right way to do? 15th[END_REF], water absorption values in the presence of fines are systematically higher than those measured without fines, although MB values suggest low clay contents.

For each material source, three 0/4 mm test samples were prepared with random dry masses and initial moisture contents as mentioned in Table 2. Moisture content differences were obtained upon immersing each sample for at least 24h (96 h for RCFA, see Table 1) and then letting it drain at room temperature for some time.

Samples were subsequently introduced into the experimental setup and air dried until their masses reach equilibrium. Meanwhile, room, drum inlet and outlet temperatures were recorded every second, as well as relative humidity. Every minute, the drum rotation was stopped for a few seconds in order to record the mass of the loaded experimental setup. Once the mass equilibrium was achieved, each sample was further oven-dried at 105°C until constant mass in order to assess its dry mass according to EN 1097-5. First, the test duration is comprised between 120 and 240 min, which seems much longer than the test duration reported by Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF] with 500-g-samples.

However, no objective comparison may be conducted since Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF] discarded particles below 0.6 mm and provided no information on initial water content (the lower the quicker the test) or inlet air temperature (the higher the quicker the test).

Next, the residual water content at the end of the non-linear phase is zero except for RCFA, whose residual water content ranges between 1.4 and 1.9 %. This observation suggests that, as expected, the moderate drying temperature used (70°C) efficiently prevents the dehydration of hydrates, with part of the water remaining trapped into micropores or mesopores of the cement paste [START_REF] Khoury | Modelling of heated concrete[END_REF][START_REF] Zhang | Dehydration kinetics of Portland cement paste at high temperature[END_REF][START_REF] Gong | Empirical Estimation of Pore Size Distribution in Cement , Mortar , and Concrete[END_REF]. This aspect will not be investigated further as it falls beyond the scope of the present paper.

Furthermore, despite the absence of a transition phase that may be explained by a rather low thermal inertia of the experimental setup, any curve in Fig. 6 evidences a linear phase depicting the evaporation of surface water at constant rate. This phase is followed by a non-linear one at decreasing evaporation rate, which corresponds to the evaporation of absorbed water. These similarities with the drying theory suggest using it in order to assess the SSD state separating these two phases.

b. SSD assessment from water content versus drying time curves

Given the experimental setup used to dry a test sample, assessing its SSD state from the water content versus time curve according to the drying theory principles requires the following three assumptions be made: first, the stirring action of flights inside the rotating drum ensures fairly homogenous drying; second, as long as free water (i.e. not retained by the aggregate) may evaporate, the air/aggregate exchange surface has a constant area that remains saturated; last, variations of the laboratory temperature, relative humidity and air generator temperature are neglected.

Under these assumptions, the evaporation rate during the linear drying velocity phase is controlled exclusively by the air/aggregate exchange surface area and the thermodynamic characteristics of the humid air inside the rotating drum (relative humidity and temperature). Hence it may be expressed as [START_REF] Charreau | Séchage. Théorie et calculs[END_REF]:

= - = - = - for W ≥ WSSD ( 1
)
where W is the test sample water content (%), h is the thermal transfer coefficient between air and test sample surface (W.m -2 .K -1 ), A is the air/test sample exchange surface area (m 2 ), T a -T s is the mean difference between air and test sample surface temperatures inside the rotating drum (in K, T s being equal to the air humid temperature), L v is the latent heat of vaporization at test sample surface temperature (J.kg -1 ), M s is the test sample dry mass (kg), a and ρ app are the specific surface (m 2 /m 3 ) and apparent density (kg/m 3 ) of the test sample respectively, and C 1 is a positive constant (%.s -1 ). Observe from equation ( 1) that the slope of this linear phase shall be material dependent, since different materials generally have different grading curves, hence different specific surfaces, and apparent densities. Furthermore, for a given material source, samples with significantly higher masses shall yield significantly lower absolute slopes, since A/M s ratio in a cylinder decreases when M s increases (this property, valid as long as the test sample fills no more than half of the rotating drum, may be mathematically demonstrated upon observing that A may be approximated by the horizontal free surface area of the test sample at rest in the same cylinder without flights). Notice that curves depicted in Fig. 6 meet these qualitative predictions.

Still under the same assumptions, the capillary theory applicable to granular test samples allows to express the evaporation rate during the non-linear drying velocity phase as [START_REF] Charreau | Séchage. Théorie et calculs[END_REF]:

= - - for W ≤ WSSD (2)
Table 3. Fit of linear and non-linear phases of drying curves and water content values.

sand-lime alluvium limestone recycled concrete I.4). For limestone, the standard deviation is just below 0.5 due to the relatively high value calculated for test sample #1.

#1 #2 #3 #1 #2 #3 #1 #2 #3 C 1 (%.
|[C 1 -C 2 (W-W res )]/C 1 | 15% 2% 16% 11% 19% 15% 15% 15% 20% W res (%) 0.1 0.1 0.0 0.0 0.1 0.1 1.4 1.9 1.5 W SSD (%) 2. 9 2.3 2.5 3.2 2.5 2.3 6.5 7.0 6.7 W trans (%) 2.7 2.2 2.7 3.5 2.7 2.8 6.4 7

c. SSD assessment from temperature or relative humidity versus time curves

Dana et al and Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF][START_REF] Dana | Experimental Moisture Determination for Defining Saturated Surface Dry State of Highway Aggregates[END_REF] have suggested that the SSD state could be captured upon detecting breakpoints on curves depicting temperature gradient ('inletoutlet' temperature) or relative humidity as a function of time. In order to check this statement, Fig. 7 represents for each material source tested (one test sample per material) temperature gradient and relative humidity variations as a function of time.

Depending on test sample initial mass and water content, as well as heat generator fluctuations (±1.5 °C), these curves acquired every second while both the heat generator and rotating drum were operating appear more or less noisy. To mitigate the noise, mobile means over 60 s (corresponding to the weighing period) have been represented on each chart. These curves are clearly very different from those reported by Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF] (see Fig. 2). Despite a slight downward trend visible mainly on relative humidity curves, the temperature gradient curves and, above all, the relative humidity curves evidence fairly clear drop breakpoints. These are located on each curve past the vertical dotted line depicting the achievement of the SSD state as calculated from the corresponding water content versus drying time curves. The temperature gradient drop is caused by test samples warming once free surface water has totally evaporated.

However, temperature charts on Fig. 7 show that this warming is offset by 5-25 min. from the SSD state, which may be explained by the presence of water trapped in fines porosity and the fairly high specific heat of aggregates (about 1000 J.Kg -1 .K -1 ). Contrariwise, Fig. 7 suggests that drop on relative humidity curves occurs more promptly once the SSD state is achieved, which tends to confirm the assumption of homogenous drying. Upon sampling every 60s and preserving only the 4 lowest frequencies, Fourier fits performed on relative humidity versus time curves yield smooth curves whose last local maxima just before the drops superimpose remarkably well with corresponding SSD states.

d. SSD assessment from water content versus relative humidity curves

In order to improve the straightforwardness and possibly the accuracy of SSD state assessment, the two physical properties found to yield the most accurate assessment of the SSD state among those recorded were represented one as a function of the other. Hence, for each material tested, Fig. 8 depicts water content variations as a function of relative humidity. Although the inlet temperature is kept reasonably constant all through the drying operations, with the temperature gradient fairly constant until achievement of the SSD state (see Fig. 7), curves depicted on Fig. 8 should not be confused with actual water vapour desorption isotherms as defined by IUPAC [START_REF] Sing | INTERNATIONAL UNION OF PURE COMMISSION ON COLLOID AND SURFACE CHEMISTRY INCLUDING CATALYSIS * REPORTING PHYSISORPTION DATA FOR GAS / SOLID SYSTEMS with Special Reference to the Determination of Surface Area and Porosity[END_REF]. Indeed, actual water vapour desorption isotherms are usually determined for a discrete set of relative humidity values, which are successively imposed during several days using various saturated salt solutions until test sample mass achieves an equilibrium [START_REF] Baroghel-Bouny | Water vapour sorption experiments on hardened cementitious materials[END_REF]. Here, drying operations are quick (less than 4 hours), which partly explains curves fluctuations, and no static equilibrium

Conclusion and perspectives

An improved experimental setup was introduced in order to capture the SSD state of porous fine aggregates incorporating significant amounts of fines, such as recycled concrete fine aggregates. This setup was used to test three fine aggregate sources with different water absorption values up to 5.6 % (measured in the absence of fines) and at least 8 % of fines. Test duration was found to lie in the range 120-240 minutes, which suggests differences from the shorter duration reported by Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF]. Nevertheless, curves depicting water content shift as a function of drying time were found similar to those reported in the drying theory literature. As a consequence, the drying theory was used to fit these curves and a calculation procedure was explained in full detail to assess the SSD state. Fairly repeatable and discriminant water content values in SSD state were obtained using this procedure. As expected, these water absorption values were found significantly higher than those determined in the absence of fines according to the usual cone test [START_REF] Cen | Tests for mechanical and physical properties of aggregates -Part 6: Determination of particle density and water absorption[END_REF]. Furthermore, the lack of reliability of water absorption values determined in the presence of fines according to the cone test procedure was verified. Besides, as reported by Dana et al and Kandhal et al [START_REF] Kandhal | Measuring Bulk Specific Gravity of Fine Aggregates: Development of a New Test Method[END_REF][START_REF] Dana | Experimental Moisture Determination for Defining Saturated Surface Dry State of Highway Aggregates[END_REF], curves representing temperature gradient or relative humidity shift as a function of time were found to evidence drop breakpoints, however the latter appeared to be located closer to actual SSD state than the former. Eventually, a more straightforward strategy for assessing the water content in SSD state was suggested, which consists in identifying the transition point between retained and free water on charts representing the variations of water content as a function of relative humidity.

Although the experimental setup was found able to assess the water content of fine aggregates incorporating fines in SSD state -even in the case of recycled concrete fine aggregates thanks to moderate drying -, several perspectives should be pointed out.

First, recorded curves tend to be noisy, which causes results scattering that could be avoided by using a more stable hot air generator setup. Second, test duration could be reduced upon optimizing the sample mass. Next, automation of the device would make no difficulty. Eventually, more theoretical work is needed to model the full outline of water content versus relative humidity curves determined using the experimental setup.

FreeFig. 1 .

 1 Fig. 1. Schematic view of an aggregate particle for various water content values.

Fig. 2 .

 2 Fig. 2. Temperature gradient versus time (left) and relative humidity versus time

Fig. 4 .

 4 Fig. 4. The experimental set-up used for the determination of SSD state.

Fig. 5 .

 5 Fig. 5. Grading curves of tested materials.

3 .

 3 Fig.6represents the variations of the water content of all tested samples as a

Fig. 6 .

 6 Fig. 6. Water content as a function of time for all tested samples.

  range between 2 and 20%. These low deviations to zero might reflect minor diffusion phenomena, whose study falls beyond the scope of the present paper.

Fig. 7 .

 7 Fig. 7. Temperature gradient (top figures) and relative humidity (bottom figures)

Fig. 8 .

 8 Fig. 8. Water content as a function of relative humidity (3 test samples/material). 325

Table 1 .

 1 Densities, water absorption and MB values of tested sands.

			sand-lime alluvium	limestone	recycled concrete
			#1	#2		#1	#2
	immersion time (h)	24		24	96
		ρ ρ ρ ρ ssd (t/m 3 )	2.68	2.67	2.61	2.41	-
	no fines
		WA 24 (%)	0.8	1.1	2.2	5.6	-
		WA 24 (%)	1.5	-	5.4	7.1	7.3
	with fines	ρ ρ ρ ρ ssd (t/m 3 )	2.65	-	2.50	2.34	2.36
		MB (g/kg)	0.8	-	0.9	-	-

Table 2 .

 2 Dry masses and initial moisture contents of the test samples.

		sand-lime alluvium		Limestone		recycled concrete
		#1	#2	#3	#1	#2	#3	#1	#2	#3
	Immersion time (h)		24			24			96
	Dry mass (g)	444.1 940.2 430.8 422.0 590.8 909.8 618.4 408.4 371.6
	Initial moisture
	content (%)	20.0 16.6 18.6 21.2 20.3 18.7 16.1 39.3 39.7

Table 3

 3 also gathers water content values calculated for each test sample from equation[START_REF]STAR 217-PRE Progress of Recycling in the Built Environment[END_REF]. Mean water content values in SSD state for sand-lime alluvium, limestone and RCFA are 2.6, 2.7 and 6.7 % respectively. As expected, observe that these values are higher than corresponding WA 24 values reported in Table1in the absence of fines. With fines, comparison between W SSD and WA 24 values is chancy, which reflects the irrelevance of the cone slump test in the presence of fines. For any material source but limestone, the standard deviation of W SSD values is about 0.3 %, which is close to the 0.2 % repeatability standard deviation determined on natural fine aggregates in the absence of fines (see EN 1097-6, Table

Acknowledgements

This paper was written in memory of Patrick Gentilini, who initiated this research.

The authors thank Florian Huchet, Maxime Piton, Lauredan Le Guen and Alexis Cothenet from IFSTTAR for helpful conversations.

where C 2 is a positive constant (s -1 ) and W res stands for the residual water content of test sample at the end of the drying operation.

Upon integration, equations ( 1) and (2) yield the following system of equations:

Where W 0 is the water content at t=0 according to the linear fit, t SSD is the drying time when W = W SSD and C 3 is a constant. For each curve depicted on Fig. 6, constants C 1 , W 0 , C 2 and C 3 may easily be determined upon fitting the relations of system 3 to their respective phases using the least squares method. Eventually, W SSD may be calculated upon eliminating the time in system (3) at t = t SSD and solving the following equation (for example using the Newton-Raphson's method):

For each curve depicted on Fig. 6, Table 3 summarizes the calculated values of constants C 1 , W 0 , C 2 and C 3 as well as two coefficients of determination R² reflecting the quality of linear and non-linear fits. With R² values of 1.00, the linear fits quality is excellent, and the non-linear fits are also very good with R² values ranging from 0.84 up to 0.99. A consequence of the capillary theory applied to the drying of fine aggregates is that the rate of evaporation should vary continuously between the linear and non-linear phases. This may be checked upon equalizing equations ( 1) and ( 2) when W=W SSD [START_REF] Charreau | Séchage. Théorie et calculs[END_REF],

which yields the following relation:

For each tested sample, Table 3 displays the values of state is reached. Nevertheless, the outline of curves depicted in Fig. 8 looks similar to that of water vapour desorption isotherms: a fairly flat phase at the end of the drying operation (relative humidity about 10-15%) corresponds to minor water content shift; this phase is preceded by a steep one evidencing important water content change at higher relative humidity (typically about 15 to 25% depending on material tested). The transition between these two phases may well be seen as the drying point beyond which water becomes much harder to eliminate from the material. In other words, this point is representative of the retained water content assessed by the SSD state. In order to precisely locate this transition point on each curve shown in Fig. 8, relative humidity values in the range [5 %; 25 %] may be fit using the mathematical expression of the B.E.T model [START_REF] Brunauer | Gases i n Multimolecular Layers[END_REF]:

where HR is relative humidity and C and X 12 are constants. As shown in Fig. 8, fit quality is very good (R² in the range [0.84; 0.97]) for all test samples but alluvium sample #3 (R²=0.66). Fit parameters C and X 12 range in [-4.5; -1] and [-0.005; -0.249] respectively (note that these parameters would have positive values for actual water vapour adsorption isotherms, since parameter X 12 for example denotes a water content). The transition point sought may be defined as the point where the fit significantly differs from actual curve, which may be identified graphically. For each curve depicted in Fig. 8, a blue arrow spots this transition point, whereas the intersection point of two perpendicular dotted lines highlights the achievement of the SSD state as determined from Table 3. Observe that the transition point provides a very good assessment of the SSD state, as confirmed by the last three rows of Table 3.


Manfred Haider

Marco Conter

Reinhard Wehr

Fabienne Ulf Sandberg

Fabienne Anfosso

Project ROSANNE: rolling

Keywords: resistance, skid resistance, and noise emission measurement standards for road surfaces tyre/road noise, pavement, road surface, noise measurement methods

come   L'archive ouverte pluridisciplinaire

INTRODUCTION

The major challenge of the European road networks is to provide efficient transportation while maintaining high standards with regard to road safety, public health and environmental impact. Road administrations have to achieve cost-effective management of their networks while facing requirements to contribute to a reduction in accident rates, noise pollution of res idential areas and greenhouse gas emissions of road vehicles. Road surface parameters play a key role in the options available on a national and international level to achieve these des irable results.

Previous research, especially in the TYROSAFE project [START_REF]TYROSAFE -Tyre and Road Surface Optimisation for Skid Resistance and Further Effects[END_REF], has shown that controlling skid resistance, noise emission and rolling resistance of pavements enables road administrations to make a beneficial contribution to making road transport safer and greener. The same research also shows that assessment methods and policies for these three road surface parameters vary greatly across Europe; if at all available. In the case of skid resistance, for instance, not even the physical property that is measured is exactly the same. In the case of rolling resistance there is not even any generally recognized measurement method available. In the case of noise emission, the two main measurement methods available provide different indicators whose correlation has not yet been established.

This leads to the situation that while their importance is recognized, the exchange of expertise and good practice among EU countries faces considerable barriers. For this reason any steps forward in developing and harmonising the assessment of skid resistance, noise emission and rolling r esistance Inter-noise 2014 will contribute to the reduction of accidents, noise pollution and greenhouse gas emission. Additionally, it will reduce trade barriers, e.g. for the supply of road surfacing materials and the provision of road survey services, and foster innovation in the road construction sector. Standardization of these methods could prove even more beneficial, provided the necessary pre-normative research is carried out.

PROJECT OVERVIEW

Project structure

ROSANNE started in November 2013 and will run for a time period of three years. The main objective of ROSANNE is to develop and/or harmonize measurement methods for skid resistance, noise emission and rolling resistance of road pavements as a preparation for standardization. An overview of the work package structure of ROSANNE is given in Figure 1. As road surface texture is the main influence parameter of the pavement on these three surface properties under consideration, special focus will be laid on the interrelations of the texture in work package 4.

Figure 1 -Work package structure of ROSANNE This paper will focus on the noise emission properties of road surfaces ; i.e., work package 2. Its main aim is to consider the existing measurement methods (SPB and CPX) for providing a harmonised characterization method for pavement noise emission properties. An evaluation of its viability for acceptance testing, monitoring and compatibility with environmental noise calculation methods like CNOSSOS-EU will also be carried out. Remaining problems with the measureme nt methods, such as reference tyres and the influence of temperature will also be addressed.

Noise emission properties of road surfaces

In recent years low-noise road surfaces have become more and more accepted as an effective means of road traffic noise abatement which has some key advantages over other options like noise barriers. Low-noise pavements reduce tyre/road noise at the source and the noise reduction does not depend on the relative position of noise source and receiver. Moreover, they do not provide a visual barrier. Significant and beneficial noise reduction up to 10 dB(A) can be achieved in addition to other noise source limitations on vehicles and tyres. The European Environmental Noise Directive 2002/49/EC (END) (5) obliges the Member States to create noise maps around the major roads and inside the main agglomerations and to accompany them with action plans which could employ low-noise pavements as a key element. Therefore it is crucial to correctly characterize the acoustic performance of pa vements for approval testing, life-cycle monitoring and in connection with environmental noise calculation methods.

Road authorities need such a characterization method in order to introduce specifications in the tenders for road surface renewals. Furthermore, road manufacturers also need a characterization method to compare their products, develop quieter products and thus increase their competitiveness. Functional requirements such as planned here will also foster innovations. Because the noise performance of a given type of pavement can be difficult to reproduce from one construction site to another, it is also very important for road authorities to have a measuring method for checking the actual noise reduction once the new pavement is laid down. Also because noise properties of road surfaces are not stable over time, there is a need for road owners to monitor the acoustic performances of their network. Finally, in order to draw the strategic noise maps as requested by END and to design appropriate action plans, the responsible authorities need accurate and comparable input data related to the noise quality of the road surfaces. The acoustic properties of low-noise road surfaces have been or are being investigated in European projects like SILVIA [START_REF]SILVIA -Sustainable Road Surfaces for Traffic Noise Control, EU FP5 project[END_REF], SILENCE [START_REF]SILENCE -Quieter Surface Transport in Urban Areas, EU FP6 project[END_REF] or PERSUADE [START_REF]PERSUADE -Poroelastic Road Surface: an Innovation to Avoid Damages to the Environment -EU FP7 project[END_REF], among others.

The key standards for determining the pavement influence on road traffic noise emission are ISO 11819-1 (Statistical Pass-By or SPB method) and ISO/DIS 11819-2 (Close ProXimity or CPX method). While the SPB method relies on recording the sound pressure levels and speeds of passing-by vehicles at the roadside, CPX uses trailers or self-powered vehicles fitted with microphones close to the tyre/road contact of selected reference tyres. These methods offer complementary advantages and are continually improved by the responsible ISO working group, which is ISO/TC 43/SC 1/WG 33. The few existing national noise characterization procedures typically use different variants of one or both methods.

Characterization methods for noise properties of pavements used for approval testing, monitoring or the determination of input values for noise calculation procedures are available in some European countries at the national level. However, they use a variety of methodologies, based on several measurement methods and different references. Input road data for environmental noise calculations are currently often linked to the various national noise calculation schemes, and exhibit other national specifics, which makes results in general incomparable. However, in the common noise calculation method envisaged in the END and defined to date within the CNOSSOS-EU report, road surface noise corrections are introduced as input parameters in the road traffic noise emission model. A common European methodology to determine these road surface noise corrections is under discussion. It is of importance to check the applicability of this methodology to the more general purpose of road surface classification and its consistency with other envisaged methods.

Within the European project SILVIA, finished in 2005, a first proposal for such a common European noise characterisation methodology was presented. The data it delivered were as far as possible suitable for adaptation for input into national noise cal culation schemes and the HARMONOISE/IMAGINE noise prediction model (9, 10) which was, at the time, expected to be the future European harmonized noise prediction model. The SILVIA project concluded before any validation of the characterization methodology was performed and there has, at best, only been a very limited adoption of the principles of the methodology.

DEVELOPMENT OF A NOISE CHARACTERIZATION METHOD

To achieve the objective of producing a useful method for characterizing the noise emission properties of road surfaces, it is necessary to settle some remaining technical issues with the basic SPB and CPX standards; primarily the temperature influence on tyre/road noise. For the CPX method, additionally, reference tyres must be specified. It may also be necessary to define additional parameters and topics not treated within the current measurement standards, such as details related to the lateral road position of measurement, required lengths for measurements (sample size), frequency or period of measurement, etc. Finally, a generalized method for describing the noise emission properties of road surfaces shall be developed on the basis of these two measurement methods , or based on only one of them (in the latter case, probably the CPX method) . The compatibility with the noise calculation models shall be investigated and validated. An outline of the work structure is depicted in Figure 2.

Analysis and comparison of existing noise measurement methods

For the characterization of the noise emission properties of road surfaces, the standards Inter-noise 2014 ISO 11819-1 (SPB method) and ISO 11819-2 (CPX method) are the most common measurement methods in Europe. In USA, they use a variant of the CPX method, called the OBSI method (on-board sound intensity). While both the SPB and the CPX methods aim at providing a method for measuring the influence of road surfaces on traffic noise, their approach differs. While the SPB method measures vehicle noise, it relies on the representativity of the current traffic collective at the measu rement site, which is a spot. On the other hand the CPX method provides the opportunity to measure tyre/road noise over an arbitrary length of the pavement, and also how its properties vary along the road. The SPB method has the disadvantage of providing only a spot measurement, as well as it can be used only where the acoustical environment near the spot is undisturbed and non -reflecting, and it is often difficult to find the traffic volumes making the measurement practical. The CPX method measures only a part of the noise from the road traffic, namely tyre/road noise, and for a limited set of test tyres (two); however, tyre/road noise is mostly the dominating type of noise when noise properties of pavements are of interest. The CPX method relies on the control of tyre parameters as tyre type, age, rubber hardness, etc., whose impact on the generated noise are being investigated. As already mentioned, these methods offer complementary advantages and a first task has been started in the course of ROSANNE to investigate on the correlation between both method and to possibly establish a relationship. One of the most substantial influences on both methods is the temperature, which can be measured as air, surface and/or tyre temperature. Which of these parameters that are the most suitable to provide the basis for a correction factor will be investigated in the course of ROSANNE in close cooperation with ISO/TC 43/SC 1/WG 27 and CEN/TC 227/WG5. Another important aspect in studying the two available methods is their correlation, advantages/disadvantages, problems, uncertainties, and their performance for non -standard pavement textures. The latter may be, for example, tined, ground or polished surfaces, other directional textures, poroelastic surfaces, and surfaces based on extremely small maximum aggregate sizes. The use of texture indicators to harmonize and/or supplement the methods will be studied.

Compatibility with CNOSSOS-EU and national noise calculation methods

National noise calculation methods in Europe currently differ in their complexity to take different road surface types into account. In the road traffic noise part of the Common Noise Assessment Methods in Europe (CNOSSOS-EU), the effect of the road surface on road noise emission is introduced in the calculation through a correction factor, the determination of which is to be defined in the coming implementation phase of CNOSSOS-EU. However, proposals have been considered, based on the outputs of the HARMONOISE and IMAGINE project, in which SEL (Sound Exposure Level) indicators are measured instead of L Amax and at a 3 m high position instead of 1.2 m in the SPB method. Furthermore, this correction factor is intended to represent the no ise performance not only in new condition but also over the lifetime of the pavement. There is a need to check the equivalence or at least the consistency between indicators and approaches. A comparison of SPB, CPX and CNOSSOS-EU approaches is necessary, as well as an identification of possible relationships. Also, to minimise barriers for future implementation, the compatibility with existing national prediction methods has to be considered. As a final step in this part of ROSANNE, measurements will be carried out in the future to verify the applicability of the characterization method developed.

CONCLUSION

In this paper, the currently ongoing EU FP7 project ROSANNE is presented. One of its main goals is to develop and harmonise measurement methods for skid resistance, noise emission and rolling resistance. The harmonised measurement framework for describing the emission of road surfaces will be based on the existing methods described in ISO 11819-1 and/or ISO/DIS 11819-2. Final remaining technical issues of these measurement methods, such as the temperature influence and reference tyres, will be solved during the project in close cooperation with the concerned ISO and CEN working groups. Special focus will be laid on its compatibility with the Common Noise Assessment Methods in Europe (CNOSSOS-EU) and national noise calculation methods.

ROSANNE started in November 2013 and some measurements have already been made, but results are not available when this is written. The reader may find more information o n the project website www.rosanneproject.eu.

Figure 2 -

 2 Figure 2 -Development process of the noise characterization method

manfred.haider@ait.ac.at

ulf.sandberg@vti.se

fabienne.anfosso@ifsttar.fr

ACKNOWLEDGEMENTS

The work described in this paper is part of the ROSANNE project funded by the European Commission in the Seventh EU Framework Programme, with national contributions by the partners or their sponsors.


Sandrine Bayle

Joel Chopineau

Benoit Roig

Dr Denis Habauzit
email: denis.habauzit@univ-rennes1.fr

Estrogen Receptor preparation effects on the receptor-DNA interaction by surface plasmon resonance

Keywords: Surface plasmon resonance, Estrogen, Estrogen receptor, Dimerization, Temperature

published or not. The documents may come

Estrogen receptor preparation effects on the receptor-DNA interaction by surface plasmon resonance

Introduction

Since three decades several publications have aimed to understand the molecular mechanisms by which estrogen receptor alpha (ERα) bind with small palindromic consensus sequence known as estrogen response element (ERE) in the presence of the ER natural ligand 17-β-estradiol (E2) [START_REF] Druege | Introduction of estrogenresponsiveness into mammalian cell lines[END_REF][START_REF] Klinge | Estrogen receptor interaction with estrogen response elements[END_REF][START_REF] Kumar | The estrogen receptor binds tightly to its responsive element as a ligandinduced homodimer[END_REF]. The first assessments were mainly based on the use of band shift, and then the emergence of new technologies, such as surface plasmon resonance [START_REF] Cheskis | Estrogen Receptor Ligands Modulate Its Interaction with DNA[END_REF], fluorescence anisotropy [START_REF] Ozers | Equilibrium binding of estrogen receptor with DNA using fluorescence anisotropy[END_REF][START_REF] Ozers | The dissociation rate of estrogen receptor alpha from the consensus estrogen response element[END_REF], quartz crystal microbalance [START_REF] Su | Combinational application of surface plasmon resonance spectroscopy and quartz crystal microbalance for studying nuclear hormone receptorresponse element interactions[END_REF], gold nanoparticles [START_REF] Tan | Gold-nanoparticle-based assay for instantaneous detection of nuclear hormone receptor-response elements interactions[END_REF], silicon nanowire [START_REF] Zhang | Highly sensitive and reversible silicon nanowire biosensor to study nuclear hormone receptor protein and response element DNA interactions[END_REF], have permit to characterize the interaction properties of ERα towards ERE. Moreover knowledge in E2/ER/ERE interaction is used for the development of new screening methods in drug discovery [START_REF] Rich | Kinetic analysis of estrogen receptor/ligand interactions[END_REF] and also for the monitoring the presence of endocrine disrupting chemicals (EDC) in environment [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF][START_REF] Seifert | In vitro analysis of xenoestrogens by enzyme linked receptor assays (ELRA)[END_REF].

Previous papers have described ER/ERE interaction for several purposes [START_REF] Habauzit | SPR-based biosensors: a tool for biodetection of hormonal compounds[END_REF] by using Surface Plasmon Resonance as analytical tool. The first one is the molecular mechanism by which ER bind with E2 [START_REF] Rich | Kinetic analysis of estrogen receptor/ligand interactions[END_REF][START_REF] Seifert | In vitro analysis of xenoestrogens by enzyme linked receptor assays (ELRA)[END_REF], form a dimer [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF][START_REF] Jisa | Kinetic analysis of estrogen receptor homo-and heterodimerization in vitro[END_REF] and then interact alone or with cofactor on ERE [START_REF] Cheskis | Estrogen Receptor Ligands Modulate Its Interaction with DNA[END_REF][START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF][START_REF] Li | Regulation of bcl-2 transcription by estrogen receptor-α and c-Jun in human endometrium[END_REF][START_REF] Song | Study sequence rules of estrogen receptor α-DNA interactions using dual polarization interferometry and computational modeling[END_REF][START_REF] Song | Spacer effect of cooperative binding of estrogen receptor α and specificity protein 1 to composite DNA: A surface plasmon resonance study[END_REF]. This knowledge was used for the determination of interaction properties (i.e interaction constant) and recently for environmental purpose (i.e detection or quantification) [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF][START_REF] Gao | Assessment of estrogenic activity of perfluoroalkyl acids based on ligandinduced conformation state of human estrogen receptor[END_REF][START_REF] Wong | A new bisphenol A derivative for estrogen receptor binding studies with surface plasmon resonance[END_REF]. But a discrepancy in the experimental conditions reported in the literature can be noticed [START_REF] Habauzit | Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology[END_REF] and few publications have detailed the biochemical parameters used and the influence of the parameters choice.

On the whole, this study aims to show the outstanding of ER preparation initial steps, generally not described in the literature, but that were found very fundamental for a reproducibility of the analytical method.

Three steps are necessary, ER is thawed, then ER is mixed with E2, and finally ER/E2 is injected for interaction with ERE. An overnight incubation at 4°C is required for ER dimerization [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF]. In our previous paper, we compared various experimental conditions usually employed for the determination of ER/ERE interaction properties [START_REF] Habauzit | Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology[END_REF] such as the effects of solvent in the estrogenic compounds solubilisation, the temperature and the time for the E2/ER incubation. Three parameters could deeply modify the ER binding level towards ERE [START_REF] Habauzit | Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology[END_REF]. A decrease of ER/ERE binding level was clearly observed depending on the temperature and time. Furthermore, incubation at different times and temperatures induce a decrease of the ER binding level, due to a thermal deactivation of the active ER form. In this context, the present paper aims to study the influence of the temperature of ER thaw and the presence of two additional compounds, which are known to avoid protein damages (bovine serum albumin (BSA) and protease inhibitor cocktail). These two last assessed parameters have (1) potential aspecific interaction with E2 and the surface or prevention of ER enzymatic degradation by competition (BSA) and have [START_REF] Klinge | Estrogen receptor interaction with estrogen response elements[END_REF] prevent ER enzymatic degradation by competition and involvement in ER stability (protease inhibitor cocktail).

Materials and methods

Reagents

Chemicals, Tris buffer, tween 20, methanol 17β-estradiol (E2), NaCl, MgCl2, bovine serum albumin (BSA) and EDTA free protease inhibitor cocktail for use with mammalian cell and tissue extract were analytical grade and purchase from Sigma-Aldrich (Saint Quentin Fallavier, France). Human recombinant estrogen receptor alpha (ERα), and estrogen response element (ERE) sequence were obtained as previously described [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF].

ER preparation and incubation period in ice or ambient temperature

ERα was stored at -80°C until their use and was then thaw on ice or at ambient temperature (22 °C) during 1 hour (Fig. 1). ER stock solution was diluted in TNMT buffer (50 mM Tris HCl, 150 mM NaCl, 10 mM MgCl2, 0.05% Tween 20, pH 7.5) to a final concentration of 100 nM, and then equally mix were stored either on ice or at ambient temperature for one hour (Fig. 1) with TNMT containing different concentrations of E2 previously dissolved in methanol. Methanol concentration was 0.1% in TNMT buffer. The final concentration of ER and E2 was 50 nM and between 1 nM and 100 nM respectively.

Preparation of BSA and protease inhibitor solution and mixing step with ER

BSA and anti-protease cocktail were prepared in TNMT buffer. ER solution (with or without E2) was prepared with anti-protease cocktail (1/2000 of TNMT) or BSA (final concentration of 1 mg/ml). Finally all ER preparations were incubated at least overnight before their analysis on Biacore instrument.

SPR experiments

Experiments were performed at 25 °C on Biacore 1000 apparatus. After ERE immobilisation, ER samples were injected on the sensor chip with a flow rate of 20 µl/min during 2 min. After the injection the dissociation phase was followed during 180 s. ER binding level was measured 24 s after each injection. The surface was regenerated with 0.1% of SDS solution followed by 2 min in running buffer.

Results

Incubation temperature effects on the ER/ERE binding level

During manipulations implementing the ER / E2 / ERE interaction, several steps were required to set the temperature and time such as the ER thawing step and the incubation step of ERα protein with E2. The effect of these steps was evaluated by the measurement of the ER binding level on ERE in the presence of E2 concentration ranging from 10 -9 to 10 -7 M. As shown in Fig. 2, modification of temperature during these steps modify the ER binding in the presence of low concentration of E2 (10 -9 M) with an average of 1 247 ± 13 RU (mixing ambient) and 989 ± 9 RU (mixing ice) for ER protein thaw on ice and 669 ± 26 RU (mixing ambient) and 789 ± 53 RU (mixing on ice) for protein thaw at ambient temperature respectively (Fig. 2). Such discrepancies are also observed for high E2 concentration (10 -7 M) with an average of 2 097 ± 109 RU (mixing ambient) and 1 232 ± 91 RU (mixing ice) for ER protein thaw at ambient temperature and 1 131 ± 80 RU (mixing ambient) and 1 154 ± 97 RU (mixing on ice) for protein thaw on ice respectively. Three conditions (thawing ice/mixing ice; thawing ambient /mixing ice; thawing ambient/mixing ambient) reach the same level of ER binding in the presence of high concentration of E2. Considering the ratio of the signal ER with 10 -7 M E2 / signal ER with 10 -9 M E2 increase due to the increase of E2 concentration, the values are respectively 1.7, 1.2, 1.7 and 1.5 (Fig. 2 A-B) depending upon the step used.

Thawing ER protein on ice instead of ambient temperature induces a higher level of the basal ER binding level (Fig. 2A). But these differences are less pronounced for higher E2 concentration; the influence of the presence of E2 in the sample is lower when incubation was performed on ice, the ratio reaches 1.2 and 1.5 instead of 1.7 for room temperature incubation. Previously, high concentration of E2 was showed to be linked with ER dimerization [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF]. The whole results ask on the ER state during the experiment and the ER binding level may not represent the same event as previously published. As the ER binding level decreases with the temperature used during the sample preparation, the question of the level of degradation of the protein ER is raised at ambient temperature is worth mentioning.

Effect of Protease inhibitor and BSA on ER/ERE binding level

No clear difference in the ER binding profiles is observed between thaw ice / mix ice and thaw ambient / mix ice. It may be due to the fact that during the ambient thaw step the temperature is not ambient but between -80°C and ambient temperature (Fig. 3). On the contrary, the combination with the second temperature step seems to be critical for ER binding level. Indeed, the ambient temperature after initial thaw step at ambient temperature produces a decrease of the ER binding level in the absence of E2 (Fig. 3 A-B). This decrease of around 25% is in the same order as those previously described after incubation at 21°C during two hours [START_REF] Habauzit | Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology[END_REF].

This decrease seems to demonstrate the thermal inactivation of ER is characterized by a decrease in the ER binding level. Moreover, the presence of a low amount of E2 prevents ER from this inactivation phenomena probably by maintaining ER in its active conformation. Indeed, high E2 concentration permits ER dimerization after overnight incubation (characterized by ligand dependent increase in ER binding level). Therefore the decrease of ER binding level on ERE after an ambient thawing could be linked either to a degradation of ER by proteolysis or by the influence of temperature on the protein folding. That decrease in binding level is never observed when ER is conserved on ice. Therefore, protease inhibitor cocktail and/or BSA were added to ER solutions (Fig. 3). The incubation was then performed in the same conditions.

The addition of protease inhibitor cocktail decreases the ER binding on ERE sequence. However, E2 induced an increase of the ER binding ratio that remain in the same range as after ice incubation with respectively 1.2 (Thawing ice /mixing ambient) and 1.5 (Thawing ambient /mixing ambient). Even if the protease inhibitor cocktail is EDTA free, the cocktail composition deeply prevents ER from binding to ERE as well as the ligand dependent dimerization of ER. The presence of BSA did not modify the ER binding level when the protein is incubated at ambient temperature regardless of the E2 concentration. The addition of BSA did not increase the SPR signal showing that there are neither interaction between ER and BSA nor aspecific BSA binding on the surface.

Fig. 3. Effect of conditions on the ER binding level onto ERE, either by comparison of the ER without E2 or with low or high concentration of E2 (A -B) or by studying the E2 dose effect on the ER binding level (C -D).

Discussion

Dimerization of ER is obtained in the presence of high concentrations of E2, but a decrease of the ER binding level was observed as a consequence of a thermal inactivation of ER [START_REF] Habauzit | Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology[END_REF]. Therefore, to decipher the role of time and thermal effect on ER activity, two critical parameters were assessed. The first one is the temperature of ER aliquot thaw. The second parameter is the temperature during ER/E2 mixing. The thawing step in ice and the mixing step at ambient temperature are necessary to obtain a high ER binding level. Then ER have to be incubated overnight at 4°C. The other conditions, ER stabilization with protease inhibitor cocktail or BSA, were also studied to show if the decrease of ER binding level is not linked to the ER enzymatic degradation. Indeed these two substances could inhibit the enzymatic degradation of ER.

Except the protease inhibitor treated samples, all samples showed an increase of the ER binding level depending on E2 concentration [START_REF] Habauzit | Determination of estrogen presence in water by SPR using estrogen receptor dimerization[END_REF][START_REF] Habauzit | Impact of biochemical design on estrogen receptor/estrogen response element interaction by surface plasmon resonance technology[END_REF]. This phenomena illustrates the importance of the 4°C overnight incubation that permits the dimerization process to occur. But the ER binding level magnitude as well as the ratio (ER high E2/ ER low E2) are linked with the temperature step occurring before the overnight incubation at 4°C. Less difference is observed for the sample thawed on ice and mixed at ambient temperature. Here the ambient mix seems to improve the ER binding capacity by a 60% increase of the binding level. It may illustrate the ER refolding by the combination of the temperature and the overnight incubation. And low concentration of E2 seems to mimic this effect by increasing ER binding level by 25% for samples thawed on ice and incubated on ice. Conversely, for ambient incubation conditions no differences were observed with or without low concentration of E2. Interestingly, anti-protease cocktail inhibit drastically the ER binding level. It is merely due to one component of the anti-protease cocktail that modifies the ER dimerization and binding, unlike BSA that did not modify the binding level. These two conditions underline that the decrease of the ER binding level is related to the temperatures of thawing and of mixing and not to an enzymatic degradation.

Conclusion

ER binding level to ERE depends on the physical and biochemical parameters used for the preparation of the samples and those used during the interaction measurements. Up to now, no works have tried to compare these parameters effects on the protein binding level on ERE sequence. Here we have shown that these conditions (temperature used for ER thaw, temperature used for the mixing step and adjunction of BSA and protease inhibitor cocktail) modify interactions and underline that some other effects could affect the protein binding such as thermal inactivation as previously shown. Therefore, measurement of affinity constants should not been performed before the critical biochemical parameters established. Indeed, our data showed that depending upon the protocol used for the study of ER binding properties, the dimerization and thermal denaturation could be equally studied making difficult to determine what phenomena are measured. In conclusion, for biosensor development the best parameters are the ER thaw in ice followed by incubation at ambient temperature without BSA or protease inhibitor cocktail. Finally such biochemical parameters therefore deeply modify biosensor answer.

Compliance with ethical standards

Conflict of interest: The authors declare that they have no competing interest

Fig. 1 .

 1 Fig. 1. Summary of experiment realized

Fig. 2 .

 2 Fig. 2. Thawing and mixing temperature effect on ER binding level. Mean of the ER binding level after thawing onto ice (A) or thawing at ambient temperature (B). (C) to (E): Temporal sensor response to sample containing different concentrations of E2; thawing on ice -Mixing ice (C) or mixing at ambient temperature (E) and thawing at ambient temperature -mixing ice (D) or mixing ambient (F). The data are shifted along the X-axis.


Bert De Coensel

Kang Sun

Weigang Wei

Timothy Van Renterghem

Matthieu Sineau

Carlos Ribeiro

Arnaud Can

Pierre Aumond

Catherine Lavandier

Dick Botteldooren

Dynamic Noise Mapping based on Fixed and Mobile Sound Measurements

Keywords: no. 43.50.Rq, 43.50.Lj, 43.50.Qp

This work introduces an approach for calculating dynamic noise maps on the basis of fixed as well as mobile sound measurements. The proposed approach extends the temporal and spatial resolution of the traditional noise map that is based on long-term equivalent levels, and is therefore well suited for the prediction of indicators that are more closely related to the perception of the acoustic quality of the urban environment. The approach uses a model based interpolation technique, and accounts for the presence of sources that are generally not considered in noise maps. On the one hand, data from fixed noise measurement stations is used to continuously adapt the parameters underlying the map to temporal changes due to variations in source strength and propagation conditions. On the other hand, data from mobile noise measurements is used to increase the spatial resolution of the map. As an illustration of this approach, the construction of a dynamic map of the 13th district of Paris is presented. A network of twenty-four fixed, intelligent sound measurement devices is installed in the area. Next to this, mobile sound measurements are performed at regular periods inside the case study area. For this, five measurement devices are carried by researchers performing walks through the area, logging instantaneous 1/3-octave band levels as well as GPS data, such that trajectories can be fully reconstructed afterwards.

Introduction

The European Environmental Noise Directive [1] has led to the implementation of strategic noise mapping in many European cities since 2002. Strategic noise maps have become an important noise policy instrument for identifying black spots, and for the assessment of the effects of urban noise exposure on inhabitants [START_REF] Miedema | Annoyance from transportation noise: relationships with exposure metrics DNL and DENL and their confidence intervals[END_REF]. Nevertheless, measurement campaigns often show significant discrepancies between calculated and measured acoustical indicators, in particular at highly shielded urban zones, mainly due to simplifying assumptions made within the applied source and prop-(c) European Acoustics Association agation models. Moreover, although strategic noise maps certainly have their merits, they fail to capture sounds that are less easy to predict, and the temporal dynamics of the sound environment is not at all included. The latter might be quite important to evaluate sleep disturbance and noise annoyance [START_REF]Burden of disease from environmental noise: quantification of healthy life years lost in Europe[END_REF].

The increasing availability of low-cost computing devices, microphones, and (wireless) internet access forms a technological push for the use of distributed sound measurement networks. Consequently, the latter are increasingly being applied to validate and improve the accuracy of traditional noise maps that are based on long-term equivalent levels. In addition, acoustic sensor networks can be useful to improve the spatial and temporal resolution of such noise maps. If real-time measurement data is available from a relatively dense network of sound measurement stations, one can use this data to create so called dynamic noise maps, which are periodically updated (e.g. every 10 minutes). In previous efforts in creating such dynamic noise maps, it was assumed that the propagation of sound is independent of time, and that the time-varying sound level at the receiver location can be obtained by only updating source powers [START_REF] Geréb | Real-time updating of noise maps by sourceselective noise monitoring[END_REF]. Another method consisted of interpolating between the noise levels measured instantaneously at many immission points using mobile measurement stations [START_REF] Cho | Noise mapping using measured noise and GPS data[END_REF][START_REF] Bennett | Environmental noise mapping using measurements in transit[END_REF]. The accuracy of these methods strongly depends on the density of the measurement network, and because errors in the propagation model (e.g. due to meteorological effects) are not accounted for, the spatial accuracy is usually poor.

In this paper, a general model-based interpolation technique for calculating dynamic noise maps is presented, in which source and propagation parameters are tuned simultaneously. The approach makes use of a combination of fixed as well as mobile sound measurements, to increase the temporal and spatial resolution of a basic noise map. The proposed method is an extension of earlier work [START_REF] Wei | Van Renterghem: Monitoring sound exposure by real time measurement and dynamic noise map[END_REF] in which only fixed measurement stations were used. In Section 2, the general methodology is presented. In Section 3, a case study that will illustrate the proposed approach is presented. This case study is currently still being carried out in the 13th district of the city of Paris, in the framework of the grafic project.

Methodology

Sound level estimation

The proposed methodology for dynamic noise mapping relies on a sound source emission and propagation model that is periodically updated based on sound level measurements, which can be performed both by fixed or by mobile measurement stations. Figure 1 shows an overview of the proposed methodology. In general, the model estimates the equivalent sound pressure level at a particular location x and at time t, for each 1/3-octave band with center frequency f , by summing the contributions L f,i,j (x, t) of all modelled sources i = 1, . . . , N s through all propagation paths j = 1, . . . , N h,i between source i and location x:

L eq,f (x, t) = 10 log 10 Ns i N h,i j 10 [L f,i,j (x,t)]/10 (1)
The contribution at x of source i through propagation path j is calculated on the basis of an estimate of the sound emission of the source and an estimate of the attenuation along the propagation path,

L f,i,j (x, t) = L W,f,i (t) -A f,i,j (x, t) (2)
The estimates of sound emission and attenuation are each composed of two parts: a part that is obtained through sound source and propagation modelling [START_REF] Wei | Van Renterghem: Monitoring sound exposure by real time measurement and dynamic noise map[END_REF], and a relatively small time-varying correction that incorporates all factors that are not taken into account in the model, and thus makes up for the difference between model and measurement:

L W,f,i (t) = L W,f,i (t) + f,i (t -1) (3)
A f,i,j (x, t) = A f,i,j (x) + δ f,i,j (t -1) (4)
Modelled values for the sound emission L W,f,i (t) could simply be long-term average values. However, diurnal pattern effects on sound source emissions can be taken into account; therefore the time dependence is explicitly noted. Values can be spatially adjusted based on detailed mobile sound measurements (see Section 2.3), or could even be obtained from a detailed temporal simulation model for particular sound sources, such as a microscopic traffic simulation model coupled with an emission model for the case of urban road traffic noise [START_REF] Coensel | Botteldooren: The influence of traffic flow dynamics on urban soundscapes[END_REF].

Modelled values for the propagation attenuations A f,i,j (x) are to be pre-calculated using a propagation model, which should include the direct path between source and receiver, as well as paths including reflections and diffractions, and should account for the effect of atmospheric turbulence [START_REF] Wei | Van Renterghem: Monitoring sound exposure by real time measurement and dynamic noise map[END_REF]. Within the current implementation, this propagation calculation is time-independent, but in future, temporal atmospheric effects on sound propagation, e.g. obtained through meteo measurements or a meteo model, could be accounted for.

Both corrections and δ can be functions of frequency and time. Values are obtained by minimizing the squared error between estimated sound pressure levels and measured values L eq,f (x meas , t), obtained through a number of fixed measurement stations. Inspired by the least mean squares (LMS) algorithm that is popular in signal processing, the minimization is not resolved explicitly at every time step, but on average over a longer time period, hereby assuming that and δ change only slowly with time over the course of a day.

It should be obvious that a model that aims to predict instantaneous sound pressure levels, e.g. on a second-by-second basis, will most likely fail. A suitable aggregation period has to be chosen, which balances stability and repeatability with a sufficient degree of dynamics. For this work, a 10-minute temporal interval was chosen. The equivalent sound pressure level L Aeq,10min includes the total sound energy received at a measurement location; every sound source contributes to this quantity independently.

Reducing degrees of freedom

As model parameters are to be tuned based on measurements, the number of degrees of freedom that can be resolved is determined by the number of measurement stations and the assumptions on temporal coherence on the time scale considered in the model. From equations 3 and 4, it is clear that, for each frequency band and for each receiver, the number of unknown equals N s , and the number of unknown δ equals Ns i=1 N h,i . This makes that the system of equations obtained from matching calculations with a limited number of observations will be strongly under-determined. Moreover, many sources and propagation paths have only little influence on a particular immission point, which makes the system also ill-conditioned. Drastically reducing the number of degrees of freedom is therefore necessary.

Inspired by land-use regression modelling, sources are categorized according to their type, and a single correction is applied to the emission of all sources within the same category. To be able to resolve the solution of , the number of source categories should therefore typically be less than the number of measurement locations. Road traffic sound sources can for example be grouped by road (section), and grouped further according to vehicle category, traffic intensity, or the speed limit. If long-term measurements are available, e.g. from fixed measurement stations located within the study area, an alternative statistical approach can be used, whereby road sections are clustered based on sound level indicators (equivalent levels, percentile levels, average spectrum etc.), for example using Kohonen maps [START_REF] Coensel | Classification of soundscapes based on their dynamics[END_REF][START_REF] Brocolini | Discrimination of urban soundscapes through Kohonen map[END_REF].

To be able to resolve the solution of δ, propagation paths are grouped into three categories: (i) the 2D path, consisting of direct line-of-sight propagation and reflections or diffractions in the horizontal plane; (ii) the diffraction path over roof tops, and (iii) the scattered path, i.e. the contribution to the immission due to turbulence scattering, mainly important in shielded areas. It is expected that the 2D path estimate is relatively accurate, whereas the diffraction path might be slightly inaccurate but time-independent, and the scattered path may change significantly over time. Therefore, it is important to keep these three corrections separate. Paths within the same category are then adjusted by a single value of δ.

Although in principle, there are no fundamental constraints in performing the fit to measurements on a per frequency basis, this additional degree of freedom jeopardizes the uniqueness of the solution and could easily lead to over-fitting. Therefore, in the current implementation, the corrections and δ are still performed independent of frequency.

Temporal and spatial resolution

The use of both fixed and mobile sound measurements allows to extend the temporal and spatial resolution of the noise map based on long-term equivalent levels. On the one hand, fixed measurement stations that are placed at strategic locations within a study area [START_REF] Botteldooren | The internet of sound observatories[END_REF] allow to periodically correct for temporal changes in source emissions and propagation attenuations, according to the methodology described above, thus resulting in a dynamic noise map. The larger the number of fixed measurement stations, the larger the number of temporal effects that can be accounted for. On the other hand, mobile sound measurements, for which the goal is to cover the complete study area with good spatial resolution, allow to better characterize the spatial distribution of the various sound sources within the study area. For example, mobile sound measurements can be carried out along the different roads within a study area, with the goal of better characterizing the spatial properties of the emission of road traffic. This would e.g. allow to account for the influence of vehicle stop-and-go behaviour on sound levels near intersections [START_REF] Coensel | Logghe: Microsimulation based corrections on the road traffic noise emission near intersections[END_REF]. The measurement duration needed to characterize the emission of road traffic at a particular location for different times of the day will obviously depend on the the traffic intensity [START_REF] Brocolini | Optimization of the acoustic measurement duration in urban environment through Kohonen self-organizing map[END_REF].

Paris case study

Study area

The 13th district of Paris is situated to the south of the city center, on the left bank of the River Seine, centered around the Place d'Italie roundabout. It has an area of about 7.15 km 2 , and has about 180.000 inhabitants, part of which live in dwellings inside highrise appartment buildings. Figure 2 shows a noise map (L den ) of the area (courtesy of the city of Paris). Most exposed façade noise levels exceed 55 dB(A) for the vast majority of dwellings inside the study area. However, inner courtyards, which provide lower noise levels, are available for at least part of the dwellings inside the area, thanks to the typical rowhouse architectural style.

Fixed measurement stations

The environmental sound inside the study area is monitored using 24 cost-effective ASAsense measurement stations, placed at the façade of the dwellings of volunteers living in the area. Figure 3 shows an example of such a measurement station, placed on the windowsill. These devices consist of a single board computer, equiped with a CF card, a sound card, an ethernet card and a microphone, all put inside a weatherproof housing. The devices are fully plug-and-measure: installation only involves connecting the device to the power outlet and to the internet (the volunteers agreed to make their internet connection available); there are no buttons or displays on the measurement station.

The measurement stations log 1/3-octave band levels with a temporal resolution of 125 ms. Data is sent over the internet to the server infrastructure located in Ghent, Belgium, where the data is further processed. The data communication has some robustness built-in: if the internet connection fails, the devices save their data internally, and send the data in one batch once the internet connection is back online. Further processing of recorded sound levels is carried out using a server-side agent-based approach. For all data available at each location, a range of acoustical indicators is calculated on a 10-minute basis. These indicators include L Aeq,10min , needed for the dynamic mapping introduced in Section 2, as well as percentile levels, the number of sound events, indicators for the temporal and spectral structure of the sound [START_REF] Coensel | Botteldooren: The quiet rural soundscape and how to characterize it[END_REF], psychoacoustic indicators, etc. More details on the general sensor network architecture can be found in [START_REF] Botteldooren | The internet of sound observatories[END_REF][START_REF] Coensel | Botteldooren: Smart sound monitoring for sound event detection and characterization[END_REF].

Recruitment of participants within the 13th district of Paris was carried out by Bruitparif. Figure 4 shows an overview of the locations of the fixed measurement stations (blue stars), scattered over the study area. As of March 1, 2015, the measurement stations have already been active for about 6 months, resulting in about 120.000 hours of sound level data. The grafic project aims to gather at least one full year of measurement data for dynamic mapping purposes.

Mobile measurement stations

Continuous, mobile sound level measurements are performed using five dedicated measurement stations, carried by researchers performing walks through the study area. The devices are specially tailored for mobile measurements: they are mounted in a backpack, and are powered by a battery pack [START_REF] Filipan | Soundscape quality indicators for city parks, the Antwerp case study[END_REF][START_REF] Filipan | Botteldooren: Identifying and recognizing noticeable sounds from physical measurements and their effect on soundscape[END_REF]. Instantaneous 1/3-octave band levels are logged in the same way as with the fixed measurement stations. In addition, instantaneous GPS data is logged, such that the walks can be fully reconstructed afterwards. While measuring, participating researchers carrying the backpacks are asked not to disturb the acoustic environment; care is taken not to record footsteps and other undesirable sounds. As such, the mobile meaurements allow to perform a detailed spatial characterization of the various sound sources within the study area, increasing the spatial resolution of the noise map as described in Section 2.

Mobile sound measurements are still being carried out; to date about half of the study area has been covered, and about 24 h of mobile data has been gathered. Figure 4 shows an overview of the preliminary mobile measurement results. In order to smooth out the measurement data and to compensate for jitter in the GPS signal, results (sound levels and GPS locations) are aggregated over 10-second intervals.

Conclusion and perspectives

In this paper, a method to dynamically update noise maps based on fixed and mobile measurements is proposed. The model starts from reasonable good sound source and propagation models. Within the proposed approach, mobile measurements covering the full study area are used to increase the spatial resolution of the base map, whereas the data from a network of fixed measurement stations is used to create a dynamic noise map, which is periodically updated. The least mean squares method is applied for continuously tuning model parameters. To avoid an underdetermined system, the number of degrees of freedom is reduced by grouping the sources and propagation paths into broad categories. Source power and propagation attenuation are hereby corrected per category through a small offset that is dynamically adjusted.

The efficiency of the proposed method is currently being evaluated through a case study in the 13th district of Paris, France, carried out in the framework of the grafic project. On the one hand, a network of twenty-four fixed sound measurement devices is installed in the area. On the other hand, mobile sound measurements using five portable measurement devices are being performed at regular times inside the study area, logging instantaneous 1/3-octave band levels as well as GPS data. Measurement data collection is still ongoing; once sufficient data is available, the next step will be to group modelled sound sources into broad categories. Road segments, to which road traffic noise sources are mapped, will be grouped based on an a priori classification of roads, but the use of automated clustering algorithms based on noise indicators measured along the road will be investigated, in order to obtain a categorization that fits more closely to the emission properties along the roads inside the study area.

Figure 1 .

 1 Figure 1. General overview of the proposed methodology. For simplicity, indices f , i and j have been removed.

Figure 2 .

 2 Figure 2. Static noise map (distribution of L den ) of the study area.

Figure 3 .

 3 Figure 3. Example of a fixed measurement station, placed on the windowsill of a dwelling.

Figure 4 .

 4 Figure 4. Overview of the locations of the 24 fixed measurement stations, each marked with a blue star. The colored lines show the preliminary measurement results (LAeq,10s) for the mobile measurements. The color scale is the same as in Figure 2.

DE COENSEL, Bert, SUN, Kang, WEI, Weigang, VAN RENTERGHEM, Timothy, SINEAU, Matthieu, RIBEIRO, Carlos, CAN, Arnaud, AUMOND, Pierre, LAVANDIER, Catherine, BOTTELDOOREN, Dick, 2015, Dynamic noise mapping based on fixed and mobile sound measurements, EURONOISE 2015, MAASTRICHT, PAYS-BAS, 2015-06-01, 6p

Acknowledgement

The work described in this paper is supported by the French Environment and Energy Management Agency (Agence de l'Environnement et de la Maîtrise de l'Énergie, ADEME) under grant agreement number 1317C0028.